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Presentations and Media

Presentations and Media (1,992, 1,385 invited, 1,561 abstracts or articles published)

1. Zhang, H., A. P. Arkin and T. C. Hazen. 2023. Improving Environmental Contamination Monitoring Through Microbial Genomics with Machine Learning and Mechanistic Knowledge. The University of Michigan MTV annual meeting
2. Wu, Xiaoqin, Sara Gushgari-Doyle, Lauren M. Lui, Andrew J. Hendrickson, Yina Liu, Sindhu Jagadamma, Torben N. Nielsen, Nicholas B. Justice, Tuesday Simmons, Nancy J. Hess, Dominique C. Joyner, Terry C. Hazen, Adam P. Arkin and Romy Chakraborty. 2023. Distinct Depth-Discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone. Applied and Environmental Microbiology abstract
   Microbial assembly and metabolic potential in the subsurface critical zone (SCZ) are substantially impacted by subsurface geochemistry and hydrogeology, selecting for microbes distinct from those in surficial soils. In this study, we integrated metagenomics and geochemistry to elucidate how microbial composition and metabolic potential are shaped and impacted by vertical variations in geochemistry and hydrogeology in terrestrial subsurface sediment. A sediment core from an uncontaminated, pristine well at Oak Ridge Field Research Center in Oak Ridge, Tennessee, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone, was used in this study. Our results showed that subsurface microbes were highly localized and that communities were rarely interconnected. Microbial community composition as well as metabolic potential in carbon and nitrogen cycling varied even over short vertical distances. Further analyses indicated a strong depth-related covariation of community composition with a subset of 12 environmental variables. An analysis of dissolved organic carbon (DOC) quality via ultrahigh resolution mass spectrometry suggested that the SCZ was generally a low-carbon environment, with the relative portion of labile DOC decreasing and that of recalcitrant DOC increasing along the depth, selecting microbes from copiotrophs to oligotrophs and also impacting the microbial metabolic potential in the carbon cycle. Our study demonstrates that sediment geochemistry and hydrogeology are vital in the selection of distinct microbial populations and metabolism in the SCZ.IMPORTANCE In this study, we explored the links between geochemical parameters, microbial community structure and metabolic potential across the depth of sediment, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone. Our results revealed that microbes in the terrestrial subsurface can be highly localized, with communities rarely being interconnected along the depth. Overall, our research demonstrates that sediment geochemistry and hydrogeology are vital in the selection of distinct microbial populations and metabolic potential in different depths of subsurface terrestrial sediment. Such studies correlating microbial community analyses and geochemistry analyses, including high resolution mass spectrometry analyses of natural organic carbon, will further the fundamental understanding of microbial ecology and biogeochemistry in subsurface terrestrial ecosystems and will benefit the future development of predictive models on nutrient turnover in these environments. In this study, we explored the links between geochemical parameters, microbial community structure and metabolic potential across the depth of sediment, including the shallow subsurface, vadose zone, capillary fringe, and saturated zone. Our results revealed that microbes in the terrestrial subsurface can be highly localized, with communities rarely being interconnected along the depth.
3. Sa’ad Abd Ar Rafie, D. Williams, D. C. Joyner, J. Franklin and T. C. Hazen. 2023. Impact of Pyrogenic Carbon on Forest soils: a lab-scale study on the effect of burn temperature and fuel types on post-fire soil microbial community. MICROBE ASM 2023 abstract
   Fires are a natural ecological process in forest biomes whose regular occurrence is essential to maintain ecological processes and biodiversity conservation. In the southeastern US region's pine and hardwood forests, high-severity fire events have been more frequent over the past few decades. Burn severity refers to how organic matter is affected by fire, both aboveground and belowground. Microbial communities in the soil regulate the nutrient pool and play a key role in maintaining the soil's ecological processes. There is a critical knowledge gap regarding microbiome response to fire across burn severity gradients and prior history of burning. Fire-induced changes in soil chemistry result in a lower bioavailability of Carbon and, consequently, lower microbial biomass production. Post-fire changes in soil microbial characteristics can be explained by the transformation of the Soil Organic Carbon (SOC) to Pyrogenic Carbon (PyC) generated by high-severity forest fires. For this project, we conducted a lab-scale study comparing soil nutrient uptake and geochemistry after different fuel treatments (i.e., different PyC sources): hardwood slash and pine slash in charred and uncharred form, and no additional fuel treatment. PyC was generated from the slash fuel types to simulate fuel load treatments observed in prescribed burning scenarios of temperate deciduous forests. For details, see Figure 1 attached. We hypothesized that (I) exposure to PyC affects the metabolic capacity of the soil microbiome and affects soil organic carbon dynamics, and (II) microbial community composition in fire-impacted soils shows significant shifts upon the addition of varying quantities and sources of PyC. Preliminary results from our pilot study show significant variation in microbial DNA concentration under different experimental conditions. Burnt soil was observed to be more basic and indicated a shift in pH from unburnt. Minor changes were observed for porewater anions and organic acids across fuel types and temperatures, while more significant changes were noticed in the case of cations like Ca2+ and K+. Variation in PyC sources (slash fuel) and burn temperature resulted in significant geochemical variation in the 16srRNA microbiome structure.
4. Diliya U. Murtazina, Andrew D. Putt, Daniel E. Williams, Nathan R. Cooley, Dominique C. Joyner, Jizhong Zhou, Peter J. Walian, Terry C. Hazen, Paul D. Adams and Adam P. Arkin. 2023. Colloidal biological and chemical transport at an extreme groundwater nitrate site. MICROBE ASM 2023 abstract
   Adsorption of several heavy metals and radionuclides to the surface of colloids poses a major threat in the subsurface environment from pore water migration (Li et al., 2010). Underestimating the migration of colloids and colloid-facilitated dissolved pollutants impacts the risk to the groundwater system (Wang et al., 2020). Using extracted DNA cleaned (260-280 nm = 1.8-2.0; 260/230 = 2-2.2) and DNA (diluted to 0.2 ng/μl in 10mM Tris pH 7.5-8.5 or molecular grade water), we amplify and sequence the genomic DNA. Isolation of eDNA from solid and liquid environmental samples follows the methods of Bairoliya et al. (2022). qPCR and Amplicon sequencing (16S) are used to determine microbial community structure and dominant genes. This method helps us to detect if colloids have bacteria and/or genetic elements that can transfer further downstream in pore water than they can from the regular hydrological flow. We test several hypotheses: if colloidal aggregates of DNA can transfer downstream in pore water and if Rhodanobacter spp. and Patescibacteria spp. can transfer downstream as a colloid in pore water. This enables the nitrate reductase gene to transfer downstream via the pore water providing a source of nitrate reduction for bacteria that have not been exposed to high nitrate levels in the DOE highly contaminated legacy sites that we are studying. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231
5. Michael, J., A. Putt, Y. Yang, B. Adams, K. McBride, K. Lowe, Y. Fan, D. Ning, S. Jagadamma, D. Klingeman, D. Joyner, J. Van Nostrand, Y. Fu, T. C. Hazen and J. Zhou. 2023. Contributed. Deterministic processes govern reproducible geochemical and microbial successional patterns in the subsurface after carbon source amendment. SIMB 2023 Annual Meeting abstract
   Carbon source injections to contaminated groundwater are commonly used to mitigate environmental contamination by stimulating native microbial communities to transform contaminants in situ. For uranium contamination, carbon amendments create reducing conditions to convert soluble uranium (VI) to insoluble uranium (IV), limiting its mobility in water. However, the anoxic reducing environment generated by microbial activity is transient, and thus repeated treatments are often necessary. The reproducibility of these injections is not fully elucidated, particularly on longer timescales. We evaluated the impact of two injections of emulsified vegetable oil performed eight years apart at Oak Ridge, TN, USA, to understand the consistency of the geochemical and microbial response. Both injections resulted in similar geochemical and microbial successional patterns. Moreover, microbial taxa responded at roughly the same time points in both injections, including Geobacter, Desulfovibrio, and members of the phylum Comamonadaceae that are well established in uranium, nitrate, and sulfate reduction. Both injections induced a transition from stochastic to deterministic assembly of microbial taxonomic and phylogenetic structures. We conclude that geochemical and microbial successions after carbon amendment are reproducible, likely owing to the dominant role of deterministic processes in shaping community composition.
6. MacGregor, H., K. Ash, I. Fukai, A. Arkin and T. C. Hazen . 2023. Microbial Biosensors for Detecting Nuclear Fuel Cycle Activities in the Environment.. The University of Michigan MTV annual meeting
7. Li, Ye, K. T. Ash, Dominique C. Joyner, Daniel E. Williams, I. Alamilla, P. J. McKay, C. Iler, B. M. Green, F. Kara-Murdoch, C. M. Swift and Terry C. Hazen. 2023. Decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA in raw sewage from university dormitories. Frontiers in Microbiology 14. abstract
   Introduction: Although severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA has been frequently detected in sewage from many university dormitories to inform public health decisions during the COVID-19 pandemic, a clear understanding of SARS-CoV-2 RNA persistence in site-specific raw sewage is still lacking. To investigate the SARS-CoV-2 RNA persistence, a field trial was conducted in the University of Tennessee dormitories raw sewage, similar to municipal wastewater. Methods: The decay of enveloped SARS-CoV-2 RNA and non-enveloped Pepper mild mottle virus (PMMoV) RNA was investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw sewage at 4 degrees C and 20 degrees C. Results: Temperature, followed by the concentration level of SARS-CoV-2 RNA, was the most significant factors that influenced the first-order decay rate constants (k) of SARS-CoV-2 RNA. The mean k values of SARS-CoV-2 RNA were 0.094 day(-1) at 4 degrees C and 0.261 day(-1) at 20 degrees C. At high-, medium-, and low-concentration levels of SARS-CoV-2 RNA, the mean k values were 0.367, 0.169, and 0.091 day(-1), respectively. Furthermore, there was a statistical difference between the decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA at different temperature conditions. Discussion: The first decay rates for both temperatures were statistically comparable for SARS-CoV-2 RNA, which showed sensitivity to elevated temperatures but not for PMMoV RNA. This study provides evidence for the persistence of viral RNA in site-specific raw sewage at different temperature conditions and concentration levels.
8. Li, Y., K. Ash, D. C. Joyner, D. E. Williams, I. Alamilla, P. McKay, C. Iler, B. Green, F. Kara-Murdoch, C. Swift and T. C. Hazen. 2023. Defining the methodological approach for wastewater-based epidemiological studies—Surveillance of SARS-CoV-2. MICROBE ASM 2023 abstract
   Background: Wastewater-based epidemiology (WBE) demonstrates the potential for COVID-19 community transmission monitoring. We report the outcomes of a raw wastewater surveillance program at the University of Tennessee Knoxville, a large urban university with more than 7,000 students living in on-campus dormitories, fraternities, and sororities. Surveillance was conducted at the building level once weekly throughout the university’s one-and-a-half academic years. However, WBE results can be significantly impacted by the sampling strategies (i.e., grab vs. composite). Method: Samples were collected from buildings across the Knoxville campus using a conventional autosampler. The autosampler was programmed to collect a 400ml sample every 1h, 2h, 4h, 6h, and 8h over 24h. In addition, we selected several 2h and 4h composite samples from one 24h time period, aliquoted equal volume from each of them, and pooled each of them to create one 24h composite sample, respectively. RNA from SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV) were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw wastewater. Results: There was no statistically significant difference between the mean concentration of SARS-CoV-2 from 2h and 4h composite samples over a 24h period and the corresponding 24h composite sample for SARS-CoV-2 and PMMoV. The variation from 1h composite samples of PMMoV was more significant than the variation observed in 4h, 6h, and 24h composite samples, not for SARS-CoV-2. Conclusion: Although 24h composite samples decreased variability compared to other less time composite samples, especially for the pathogenic virus, like SARS-CoV-2, it saves a lot of money and time to surveillant the infections within a community.
9. Jinwoo Im, Andrew Putt, Kathleen F Walker, Dominique C. Joyner, James Marquis, Lauren M Lui, Dipankar Dwivedi, Alex Carr, Yupeng Fan, Jennifer Goff, Kristopher Hunt, Jonathan Michael, Farris Poole, Yajiao Wang, Michael W. W. Adams, Nitin S. Baliga, David A. Stahl, Jizhong Zhou, Matthew W. Fields, Terry C. Hazen, Michelle E Newcomer, Adam P. Arkin and Paul D. Adams. 2023. Three-Dimensional High Spatial Resolution Simulation for Groundwater Flow and Nitrogen Transport under Rainfall Perturbations in the Subsurface of Area 3. Genomics Science Program abstract
   Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Uranium and nitrate contaminant transport in Area 3 near the S3 ponds at Oak Ridge National Lab are investigated through 3D field-scale modeling and simulation. We leverage a recently acquired Cone Penetration Testing (CPT) dataset which provides the hydraulic conductivity field of Area 3 with a high spatial resolution as input data to the numerical subsurface model. The CPT data shed light on local heterogeneity of subsurface materials, significantly decreasing the uncertainty due to limited sampling points. By further using 27 well survey data collected by the ENIGMA SFA (e.g., meteorological, hydrological, microbial, and geochemical datasets), a hydrogeological model is built on PFLOTRAN and run on the high performance computing system, NERSC. Generalized stoichiometries are used for biogeochemical reactions related to nitrogen cycling. Computational results of the 3D field-scale simulation show: 1) different flow and transport regimes depending on subsurface materials, 2) impacts of rainfall events on nitrous oxide emission, 3) influential controls of flow conditions through sensitivity analysis enabling a full treatment of the ModEx approach to designing and implementing the Subsurface Observatory (SSO). The results help us understand nitrogen cycling in Area 3 and determine the location of the ENIGMA Subsurface Observatory site. Furthermore, the results will be compared to omics-informed modeling and simulation as planned in the FICSME framework (Lui et al., 2021). References 1. Lui LM, Majumder EL-W, Smith HJ, Carlson HK, von Netzer F, Fields MW, Stahl DA, Zhou J, Hazen TC, Baliga NS, Adams PD and Arkin AP (2021) Mechanism Across Scales: A Holistic Modeling Framework Integrating Laboratory and Field Studies for Microbial Ecology. Front. Microbiol. 12:642422. doi: 10.3389/fmicb.2021.642422. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
10. Griffiths, Z., A. Putt, M. Campa, D. C. Joyner, I. J. Miller, O. Pelz, N. Garajayeva, M. Ceccopieri, P. Gardinali and T. C. Hazen. 2023. Comparing the Response of the Indigenous Microbial Community to Native Crude Oil Amendment in Oxic versus Hypoxic Conditions. MICROBE ASM 2023 abstract
    The Caspian Sea is the world’s largest landlocked waterbody which lies between Europe and Asia. This region is particularly known for its large-scale oil reserves, pipeline and drilling activities which has contributed to the environmental decline of the waterbody. In addition to pollution from the petroleum industry, drainage from various river basins brings an influx of residential, industrial, and agricultural effluents that induce eutrophication and hypoxic conditions in deeper, colder waters, creating an oxygen gradient. This presents a unique opportunity to unlock the potential of the biodegradative processes carried out by the indigenous microbial community. Some microorganisms can metabolize crude oil found in seawater and marine sediments as an energy source via biodegradation. This process subsequently reduces the concentration of this contaminant in the environment. We believe that these indigenous microbes possess different metabolic capabilities to degrade oil as they adapted to declining oxygen concentrations and temperatures with increasing depths. Hence, community structure and composition will vary with depth. This experiment was conducted using seawater collected from surface waters (~25m) and deep waters (~350m) in the Caspian Sea. Microcosms were set up to observe the indigenous microbial reaction after a 60ppm native crude oil amendment over 115 days. Surface water microcosms were kept at 28ºC in an aerated area while deep water microcosms were kept at 8ºC under anaerobic conditions. These different conditions represent the temperature and oxygen gradient in this waterbody as we try to simulate the indigenous community response to this contaminant. DNA was extracted and amplified from these different microcosms and sequenced using the Illumina MiSeq. Bioinformatic analysis was performed to track changes in the abundance of taxa present and biodiversity over different time points to show the progression of community structure. We compared the biodiversity and taxa observed for oxic and hypoxic conditions to make inferences about possible adaptation strategies to the oxygen variation in each microcosm. All microcosms showed the presence of hydrocarbon-degrading phyla whose presence is consistent with other reports from oil-enriched environments. However, distinct communities were observed in oxic vs hypoxic microcosms. Orders of Bacteria related to sulphate reduction and nitrogen cycling were found in hypoxic microcosms, indicating a possible mechanism for the anaerobic biodegradation of crude oil.
11. Gall, Izzie. 2023. Hazen Digs Deep into Y-12’s Past—and Future. Recent News Department of Civil and Environmental Engineering
12. Yupeng Fan, Daliang Ning, Zhili He, Ping Zhang, Andrea M. Rocha, Ya Zhang, Joy D. Van Nostrand, Liyou Wu, Dwayne A. Elias, David B. Watson, Michael W. W. Adams, Matthew W. Fields, Eric J. Alm, Terry C. Hazen, Paul D. Adams, Adam P. Arkin and Jizhong Zhou. 2023. The linkage between microbial taxonomic and functional diversities is weakened in a uranium-polluted aquifer. MICROBE ASM abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Abstract: Microbial taxonomic diversity usually declines with increasing heavy metal stress. However, few studies have examined whether the phylogenetic and functional diversities vary similarly to taxonomic diversity along stress gradients. Here, we investigated bacterial communities in a polluted aquifer in Oak Ridge, Tennessee, USA that has a large gradient of uranium concentrations. Both taxonomic and phylogenetic α-diversities were decreased in the most polluted wells. In contrast, the decrease in functional α-diversity was modest and statistically insignificant, showing a better buffering capacity to environmental stress. Differences in functional composition, sometimes called β-diversity, were enlarged under metal pollution, while convergent functional composition was observed in unpolluted wells. Relative abundances of most carbon degradation genes were decreased in polluted wells, but those of many genes associated with nitrogen cycling, sulfur cycling and metal homeostasis were increased. Environmental variables had a much higher explanatory power in functional composition than taxonomic and phylogenetic compositions, suggesting that niche selection favored microbial functionality. Together, we demonstrate that microbial functionality is more tolerant to stress than taxonomy, and extend the Anna Karenina Principle based on Leo Tolstoy’s assertion in that microbial community adapts to a stressful environment in its own way.
13. Romy Chakraborty, Brandon C Enalls, Mon Oo Yee, Amrita Bhattacharyya, Sara Gushgari-Doyle and Terry C Hazen. 2023. Crystalline Iron Oxides Stimulate Methanogenesis Under Sulfate Reducing Conditions in the Terrestrial Subsurface . Goldschmidt2023: Redox processes and NOM as drivers of biogeochemical reactions in natural and engineered ecosystems
14. Ash, K. T., Y. Li, I. Alamilla, D. C. Joyner, D. E. Williams, P. J. McKay, B. M. Green, C. Iler, S. E. DeBlander, C. M. North, F. Kara-Murdoch, C. M. Swift and T. C. Hazen. 2023. SARS-CoV-2 raw wastewater surveillance from student residences on an urban university campus. Frontiers in Microbiology 14. abstract
    The COVID-19 pandemic brought about an urgent need to monitor the community prevalence of infection and detect the presence of SARS-CoV-2. Testing individual people is the most reliable method to measure the spread of the virus in any given community, but it is also the most expensive and time-consuming. Wastewater-based epidemiology (WBE) has been used since the 1960s when scientists implemented monitoring to measure the effectiveness of the Polio vaccine. Since then, WBE has been used to monitor populations for various pathogens, drugs, and pollutants. In August 2020, the University of Tennessee-Knoxville implemented a SARS-CoV-2 surveillance program that began with raw wastewater surveillance of the student residence buildings on campus, the results of which were shared with another lab group on campus that oversaw the pooled saliva testing of students. Sample collection began at 8 am, and the final RT-qPCR results were obtained by midnight. The previous day's results were presented to the campus administrators and the Student Health Center at 8 am the following morning. The buildings surveyed included all campus dormitories, fraternities, and sororities, 46 buildings in all representing an on-campus community of over 8,000 students. The WBE surveillance relied upon early morning "grab" samples and 24-h composite sampling. Because we only had three Hach AS950 Portable Peristaltic Sampler units, we reserved 24-h composite sampling for the dormitories with the highest population of students. Samples were pasteurized, and heavy sediment was centrifuged and filtered out, followed by a virus concentration step before RNA extraction. Each sample was tested by RT-qPCR for the presence of SARS-CoV-2, using the CDC primers for N Capsid targets N1 and N3. The subsequent pooled saliva tests from sections of each building allowed lower costs and minimized the total number of individual verification tests that needed to be analyzed by the Student Health Center. Our WBE results matched the trend of the on-campus cases reported by the student health center. The highest concentration of genomic copies detected in one sample was 5.06 x 10(7) copies/L. Raw wastewater-based epidemiology is an efficient, economical, fast, and non-invasive method to monitor a large community for a single pathogen or multiple pathogen targets.
15. Ash, K. T., Y. Li, I. Alamilla, D. Joyner, D. Williams, P. McKay, B. Green, C. Iler, S. Deblander, C. North, F. Kara-Murdoch, C. M. Swift, and T. C. Hazen. 2023. SARS-CoV-2 Raw Wastewater Surveillance from Student Residences on an Urban Univ. Campus. ASM Microbe 2023
16. Ash, K., I. Fukai, H. MacGregor, A. P. Arkin and T. C. Hazen. 2023. Microbial Biosensors for Detecting Nuclear Fuel Cycle Activities in the Environment. The University of Michigan MTV annual meeting
17. Ash, K., I. Fukai, H. MacGregor, A. Arkin, and T. C. Hazen. 2023. Microbial Biosensors For Detecting Nuclear Fuel Cycle Activities In The Environment. ASM Microbe 2023
18. Wu, X., S. Gushgari Doyle, T. Neilsen L. Lui, N. Hess Y. Liu, S. Jagadamma, T. C Hazen, A. P. Arkin and R. Chakraborty. 2022. Distinct Depth-discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone. Goldschmidt2022
19. Walker, K. F., E. R. Dixon, D. C. Joyner, K. A. Lowe, F. L. Poole, X. Ge, M. P. Thorgersen, D. Ning, Y. Fan, J. P. Michael, J. D. Van Nostrand, L. M. Lui, X. Wu, J. L. Goff, M. W. W. Adams, R. Chakraborty, D. A. Elias, R. L. Wilpiszeski, J. Zhou, M. W. Fields, A. P. Arkin, P. D. Adams and T. C. Hazen. 2022. Diurnal and Seasonal Fluctuations within 27 Contaminated Subsurface Wells. ASM Microbe 2022 abstract
    Spatiotemporal variability of groundwater in contaminated wells is one of the major determining factors of geochemistry and microbial communities within the subsurface. This survey of 27 wells at DOE’s Oak Ridge Reservation (ORR) Y-12 Complex in Tennessee, set out to obtain diurnal and seasonal fluctuations within three contamination levels (low, moderate, and high) of nitrate and heavy metals. Using RockWorks, we created 3D geochemical models to study groundwater’s geochemical patterns and changes across all three areas. Samples were taken from 27 previously established groundwater wells, four days a week, for 17 weeks (70 days total) from July to December 2019 to build diurnal and seasonal time series of geochemistry and microbial communities. In-field geochemical measurements were taken using In-Situ Aqua TROLL 600s, including dissolved oxygen, pH, conductivity, oxidation-reduction potential, and nitrate concentration. Samples were collected from each well to measure 52 metals, anions, organic acids, and total organic/inorganic carbon/nitrogen. Results show diurnal and seasonal changes in geochemistry with wide variations between each well and area. Subsurface geochemistry was greatly affected by rainfall events, as seen after two months of regional drought. One well in each area (3 wells total) was selected for microbial analysis. We sampled groundwater (unattached) and sediment (attached) microbial communities. Groundwater was filtered through 8µm and 0.2µm filters for 16S rRNA and metagenomic analysis. In each well, 18 unamended sediment traps were placed for a time series analysis of the attached microbial community and sediment geochemistry. Results of all analyses are linked to groundwater flow vectors (Geotech Colloidal Borescope) on-site weather data (HOBO RX300 Weather Station) to produce a correlation analysis between source water and flow paths, groundwater geochemistry, weather events, and levels of contamination on a spatiotemporal scale. With this data, we aim to establish a predictive model between groundwater geochemistry and microbial communities to inform future groundwater and sediment sampling in a planned subsurface observatory. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
20. Putt, Andrew D., Sa'ad Abd Ar Rafie and Terry C. Hazen. 2022. Large-Data Omics Approaches in Modern Remediation. Journal of Environmental Engineering 148.
21. Putt, Andrew D., Erin R. Kelly, Kenneth A. Lowe, Miguel, Jr. Rodriguez and Terry C. Hazen. 2022. Effects of Cone Penetrometer Testing on Shallow Hydrogeology at a Contaminated Site. Frontiers in Environmental Science 9. abstract
    Penetration testing is a popular and instantaneous technique for subsurface mapping, contaminant tracking, and the determination of soil characteristics. While the small footprint and reproducibility of cone penetrometer testing makes it an ideal method for in-situ subsurface investigations at contaminated sites, the effects to local shallow groundwater wells and measurable influence on monitoring networks common at contaminated sites is unknown. Physical and geochemical parameters associated with cone penetrometer testing were measured from a transect of shallow groundwater monitoring wells adjacent to penetrometer testing. For wells screened above the depth of cone refusal, the physical advancement and retraction of the cone had a significant effect (p < 0.01) on water level for several pushes within 10 meters of a monitoring well, and a measured increase in specific conductivity. No effect on geochemistry or water level was observed in continuous monitoring data from wells screened below the depth of cone refusal, but variability in specific conductivity from these wells during penetration testing was only a fraction of the natural variation measured during precipitation events. Continuous measurements of specific conductivity and water level demonstrated that the effects of penetration testing have limited spatial and temporal distributions with a null effect post-testing.
22. Peng, Mu, Dongyu Wang, Lauren M. Lui, Torben Nielsen, Renmao Tian, Megan L. Kempher, Xuanyu Tao, Chongle Pan, Romy Chakraborty, Adam M. Deutschbauer, Michael P. Thorgersen, Michael W. W. Adams, Matthew W. Fields, Terry C. Hazen, Adam P. Arkin, Aifen Zhou and Jizhong Zhou. 2022. Genomic Features and Pervasive Negative Selection in Rhodanobacter Strains Isolated from Nitrate and Heavy Metal Contaminated Aquifer. Microbiology Spectrum 10. abstract
    Rhodanobacter species dominate in the Oak Ridge Reservation (ORR) subsurface environments contaminated with acids, nitrate, metal radionuclides, and other heavy metals. To uncover the genomic features underlying adaptations to these mixed-waste environments and to guide genetic tool development, we sequenced the whole genomes of eight Rhodanobacter strains isolated from the ORR site. The genome sizes ranged from 3.9 to 4.2 Mb harboring 3,695 to 4,035 protein-coding genes and GC contents approximately 67%. Seven strains were classified as R. denitrificans and one strain, FW510-R12, as R. thiooxydans based on full length 16S rRNA sequences. According to gene annotation, the top two Cluster of Orthologous Groups (COGs) with high pan-genome expansion rates (Pan/Core gene ratio) were "replication, recombination and repair" and "defense mechanisms." The denitrifying genes had high DNA homologies except the predicted protein structure variances in NosZ. In contrast, heavy metal resistance genes were diverse with between 7 to 34% of them were located in genomic islands, and these results suggested origins from horizontal gene transfer. Analysis of the methylation patterns in four strains revealed the unique 5mC methylation motifs. Most orthologs (78%) had ratios of nonsynonymous to synonymous substitutions (dN/dS) less than one when compared to the type strain 2APBS1, suggesting the prevalence of negative selection. Overall, the results provide evidence for the important roles of horizontal gene transfer and negative selection in genomic adaptation at the contaminated field site. The complex restriction-modification system genes and the unique methylation motifs in Rhodanobacter strains suggest the potential recalcitrance to genetic manipulation. IMPORTANCE Despite the dominance of Rhodanobacter species in the subsurface of the contaminated Oak Ridge Reservation (ORR) site, very little is known about the mechanisms underlying their adaptions to the various stressors present at ORR. Recently, multiple Rhodanobacter strains have been isolated from the ORR groundwater samples from several wells with varying geochemical properties. Using Illumina, PacBio, and Oxford Nanopore sequencing platforms, we obtained the whole genome sequences of eight Rhodanobacter strains. Comparison of the whole genomes demonstrated the genetic diversity, and analysis of the long nanopore reads revealed the heterogeneity of methylation patterns in strains isolated from the same well. Although all strains contained a complete set of denitrifying genes, the predicted tertiary structures of NosZ differed. The sequence comparison results demonstrate the important roles of horizontal gene transfer and negative selection in adaptation. In addition, these strains may be recalcitrant to genetic manipulation due to the complex restriction-modification systems and methylations.
23. Paradis, Charles J., John I. Miller, Ji-Won Moon, Sarah J. Spencer, Lauren M. Lui, Joy D. Van Nostrand, Daliang Ning, Andrew D. Steen, Larry D. McKay, Adam P. Arkin, Jizhong Zhou, Eric J. Alm and Terry C. Hazen. 2022. Sustained Ability of a Natural Microbial Community to Remove Nitrate from Groundwater. Groundwater 60:99-111. abstract
    Microbial-mediated nitrate removal from groundwater is widely recognized as the predominant mechanism for nitrate attenuation in contaminated aquifers and is largely dependent on the presence of a carbon-bearing electron donor. The repeated exposure of a natural microbial community to an electron donor can result in the sustained ability of the community to remove nitrate; this phenomenon has been clearly demonstrated at the laboratory scale. However, in situ demonstrations of this ability are lacking. For this study, ethanol (electron donor) was repeatedly injected into a groundwater well (treatment) for six consecutive weeks to establish the sustained ability of a microbial community to remove nitrate. A second well (control) located upgradient was not injected with ethanol during this time. The treatment well demonstrated strong evidence of sustained ability as evident by ethanol, nitrate, and subsequent sulfate removal up to 21, 64, and 68%, respectively, as compared to the conservative tracer (bromide) upon consecutive exposures. Both wells were then monitored for six additional weeks under natural (no injection) conditions. During the final week, ethanol was injected into both treatment and control wells. The treatment well demonstrated sustained ability as evident by ethanol and nitrate removal up to 20 and 21%, respectively, as compared to bromide, whereas the control did not show strong evidence of nitrate removal (5% removal). Surprisingly, the treatment well did not indicate a sustained and selective enrichment of a microbial community. These results suggested that the predominant mechanism(s) of sustained ability likely exist at the enzymatic- and/or genetic-levels. The results of this study demonstrated the in situ ability of a microbial community to remove nitrate can be sustained in the prolonged absence of an electron donor.
24. Li, Y., K. Ash, D. C. Joyner, D. E. Williams, I. Alamilla, P. McKay, C. Iler, B. Green, F. Kara-Murdoch, C. Swift, F. Löffler and T. C. Hazen. 2022. Decay of SARS-CoV-2 RNA at 4° and 20°C in raw university student residence for wastewater-based epidemiology. ASM Microbe 2022 abstract
    Background: Wastewater-based epidemiology (WBE) demonstrates the potential for COVID-19 community transmission monitoring. We report the outcomes of a raw wastewater surveillance program at the University of Tennessee Knoxville, a large urban university with more than 7,000 students living in on-campus dormitories, fraternities and sororities. Surveillance was conducted at the building level on a once-weekly schedule throughout the university’s one and half academic years. However, data on the stability of SARS-CoV-2 RNA in raw wastewater is needed to interpret WBE results. Method: Samples were collected from buildings across the Knoxville campus. We selected the samples in which the detection of SARS-CoV-2 was around 10^2, 10^3, and 10^4 viral copies/L to investigate the decay rates of RNA from SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV) by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw wastewater stored at 4° and 20°C. A 15ml wastewater was collected on day 0 (the day that we collected samples) and on days 3, 7, 14, 21, 28, 35, and 40. Results: There was no statistically significant difference among different concentration levels of SARS-CoV-2 RNA decay. Decay rate constants for the temperatures showed that SARS-CoV-2 were less sensitive to elevated temperatures in raw wastewater. Conclusion: SARS-CoV-2 RNA is likely to persist at least one month in raw wastewater, which can provide a reliable detection for WBE application.
25. Im*, J., A. Putt, K. Walker, J. Marquis, L. Lui, A. Carr, Y. Fan, J. Goff, K. Hunt, J. Michael, F. Poole, Y. Wang, M. Adams, N. S. Baliga, D. Stahl, J. Zhou, M. W. Field, T. C. Hazen, A. P. Arkin and M. E. Newcomer. 2022. Incorporation of Microbial Communities into Reactive Transport Modeling of Nitrogen in Subsurface Systems under Rainfall Perturbations. AGU Fall Meeting
26. Im, J., A. Putt, K.F. Walker, L.M. Lui, A. Carr, K. Hunt, Y. Wang, Y.J. Fan, J.P. Michael, J.L. Goff, F.L. Poole, J. Marquis, M.W.W. Adams, J. Zhou, M.W. Fields, T.C. Hazen, A.P. Arkin and M. Newcomer. 2022. Incorporation of microbial communities into reactive transport modeling of nitrogen in subsurface systems under rainfall perturbations. ENIGMA Monthly Seminar abstract
    Interests in microbial communities in subsurface systems are growing as one of the main contributors to global N2O emissions. However, N2O production under microbial communities in subsurface systems is still poorly estimated. This is because microbial community composition and its activity depend on site-specific environmental conditions, such as rainfall perturbations and heterogeneous hydraulic conductivity. In the modeling of the N2O production in subsurface systems, it is necessary to incorporate the changes of the microbial community in response to hydrological perturbations and the subsequent biogeochemical processes leading to the N2O production. In order to achieve this type of integrated modeling, we develop a reactive transport model of nitrogen in the subsurface system for the ENIGMA Subsurface Observatory site. The model utilizes the FICSME framework (Lui et al., 2021) as the foundation for developing an effective reactive transport code in conjunction with a module utilizing omics data. The model is parameterized and calibrated by many different types of data collected by the ENIGMA SFA (e.g., meteorological, hydrological, microbial, geophysical, and geochemical datasets). As a novel capability to the modeling community, our work incorporates omics data into the model, connecting the microbial community to nitrogen cycling in the meso-field scale subsurface system, and allows the microbial community to evolve in response to geochemical and flow conditions. This study predicts N2O emissions under extreme climatic conditions as well.
27. Hazen, T. C., Y. Li, K. Ash, D. C. Joyner, D. E. Williams, P. McKay, I. Alamilla and C. North. 2022. Monitoring of Wastewater for COVID at the University of Tennessee campus during the pandemic and wastewater epidemiological studies. Health section of the Tennessee Environmental Conference
28. Hazen, T. C., Y. Li, K. Ash, D. C. Joyner, D. E. Williams, P. McKay, I. Alamilla and C. North. 2022. Raw wastewater surveillance for SARS-CoV-2 in the University of Tennessee student residential buildings. Global EnviroSummit
29. Hazen, T. C.. 2022. Environmental Systems Biology: The Whole is Greater than the Sum of its Parts – Team Science.. Microbial Insights
30. Hazen, T. C.. 2022. Hazen Lab Studies for EPS Alumni Board Meeting. Earth and Planetary Sciences Alumni Board Spring Meeting Strong Hall pdf
31. Hazen, T. C.. 2022. PAWES Protecting and Advancing Water, Environment and Sustainability an ERC with SPARKS? Lighting Talk. SPARKS (Seeking Partnerships to Advance Research, Knowledge, and Science) UT Office of Research, Innovation and Economic Development. abstract pdf
    Led by the University of Tennessee with partners at Colorado State University and the University of Puerto Rico, the PAWES Engineering Research Center (ERC) planning grant, funded by the National Science Foundation, is integrating community perspectives and concerns into research initiatives to develop innovative solutions for optimizing wastewater treatment and water conservation with energy security and rural and tribal workforce development. The PAWES ERC planning grant has helped identify unique team capabilities for development of biosensors to track pathogens (e.g., COVID-19) and chemicals (e.g., antiviral drugs) in sewage, allowing for better epidemiological studies. Data from wastewater treatment plants around the world could be collected on a common server/platform and a big data approach using machine learning and artificial intelligence could be used to track and alert about potential epidemics in real time.
32. Hazen, T. C.. 2022. Repsol Oil Spill in Peru.
33. Hazen, T. C.. 2022. Exxon Valdez vs. Deepwater Horizon and Consderations for Peru Repsol Spill. Webinar pdf
34. Harik, Ann-Marie G., Zabrenna Griffiths and Terry C. Hazen. 2022. Omics of oil biodegradation. Current Opinion in Chemical Engineering 36. abstract
    Omics studies (metagenomics, transcriptomics, metabolomics, proteomics) for marine oil biodegradation research increased rapidly after the 2010 Deepwater Horizon (DWH) accident in the Gulf of Mexico. Since then, it has been demonstrated how omics techniques can be used to model and better understand pre-spill environments, monitoring during a spill and post-spill. Data that encompass everything from the ecosystem to the molecular level are needed for understanding the complicated process of petroleum biodegradation in marine environments. Consequently, using omics for monitoring oil in the ocean will help in developing more robust systems models and would make responses to spills much more defensible in terms of risks to the environment and people. Omics is enabling for a Systems Biology approach to oil spills which allows a search for hidden interactions and attributes at different trophic levels because 'the whole is greater than the sum of its parts'.
35. Goff, Jennifer L., Elizabeth G. Szink, Michael P. Thorgersen, Andrew D. Putt, Yupeng Fan, Lauren M. Lui, Torben N. Nielsen, Kristopher A. Hunt, Jonathan P. Michael, Yajiao Wang, Daliang Ning, Ying Fu, Joy D. Van Nostrand, Farris L., II Poole, John-Marc Chandonia, Terry C. Hazen, David A. Stahl, Jizhong Zhou, Adam P. Arkin and Michael W. W. Adams. 2022. Ecophysiological and genomic analyses of a representative isolate of highly abundant Bacillus cereus strains in contaminated subsurface sediments. Environmental Microbiology 24:5546-5560. abstract
    Bacillus cereus strain CPT56D-587-MTF (CPTF) was isolated from the highly contaminated Oak Ridge Reservation (ORR) subsurface. This site is contaminated with high levels of nitric acid and multiple heavy metals. Amplicon sequencing of the 16S rRNA genes (V4 region) in sediment from this area revealed an amplicon sequence variant (ASV) with 100% identity to the CPTF 16S rRNA sequence. Notably, this CPTF-matching ASV had the highest relative abundance in this community survey, with a median relative abundance of 3.77% and comprised 20%-40% of reads in some samples. Pangenomic analysis revealed that strain CPTF has expanded genomic content compared to other B. cereus species-largely due to plasmid acquisition and expansion of transposable elements. This suggests that these features are important for rapid adaptation to native environmental stressors. We connected genotype to phenotype in the context of the unique geochemistry of the site. These analyses revealed that certain genes (e.g. nitrate reductase, heavy metal efflux pumps) that allow this strain to successfully occupy the geochemically heterogenous microniches of its native site are characteristic of the B. cereus species while others such as acid tolerance are mobile genetic element associated and are generally unique to strain CPTF.
36. Goff, Jennifer L., Lauren M. Lui, Torben N. Nielsen, Michael P. Thorgersen, Elizabeth G. Szink, John-Marc Chandonia, Farris L., II Poole, Jizhong Zhou, Terry C. Hazen, Adam P. Arkin and Michael W. W. Adams. 2022. Complete Genome Sequence of Bacillus cereus Strain CPT56D-587-MTF, Isolated from a Nitrate-and Metal-Contaminated Subsurface Environment. Microbiology Resource Announcements 11. abstract
    Bacillus cereus strain CPT56D-587-MTF was isolated from nitrate- and toxic metal-contaminated subsurface sediment at the Oak Ridge Reservation (ORR) (Oak Ridge, TN, USA). Here, we report the complete genome sequence of this strain to provide genomic insight into its strategies for survival at this mixed-waste site.
37. Goddard, David. 2022. Hazen Offers Expertise to Peruvian Officials, Scientists in Wake of Oil Spill. Recent News Department of Civil and Environmental Engineering
38. Fukai, I., K. Ash, D. Williams, C. Young, M. Duff, E. Pettit, H. Vermeulen, M. Cooke, D. Klingeman, K. Lowe, A. Faiia, D. Bernstein, A. P. Arkin and T. C. Hazen. 2022. Microbial Signatures of Nuclear Fuel Cycle Activities in the Environment. MTV DOE NNSA 2022 University Program Review (UPR) invited by Sean Stave abstract
    Environmental sensors that can detect and monitor fuel cycle activities are critical for use in verifying nuclear arms nonproliferation and supporting the safe development of global nuclear energy resources. The detection of undeclared nuclear fuel cycle activities currently presents a significant challenge for nonproliferation technology, with wide-area environmental sampling (WAES) considered to be one of the most promising approaches for identifying clandestine materials/operations. As part of the Monitoring Technology and Verification (MTV) Consortium, the Savannah River Site (SRS) and The Oak Ridge Reservation (ORR) have been selected as field sites of interest to collect samples from environments exposed to specific nuclear activities and materials. The goal of sample collection and analysis is to investigate the potential for deconvolution of nuclear fuel cycle activities from contamination signatures preserved in microbial communities, plants, and the surrounding geochemical environment. This presentation will describe the results of 16s rRNA microbial community analysis at each site, including initial characterization of soil, plant, and surface water samples from the SRS and ORNL’s High Flux Isotope Reactor (HFIR). Results from this work will be used to assess whether characteristic microbiomes can be correlated to geochemical signatures in environments associated with specific stages of the nuclear fuel cycle. Characterization of microbial-geochemical signatures at these sites may further aid in the development of technologies for monitoring and detection of proliferation-sensitive fuel cycle activities.
39. Fukai, I., K. Ash, D. Williams, C. Young, M. Duff, E. Pettit, H. Vermeulen, M. Cooke, D. Klingeman, K. Lowe, A. Faiia, D. Bernstein, A. P. Arkin and T. C. Hazen. 2022. Microbial Signatures of Nuclear Fuel Cycle Activities in the Environment. MTV DOE NNSA 2022 University Program Review (UPR) abstract
    Environmental sensors that can detect and monitor fuel cycle activities are critical for use in verifying nuclear arms nonproliferation and supporting the safe development of global nuclear energy resources. The detection of undeclared nuclear fuel cycle activities currently presents a significant challenge for nonproliferation technology, with wide-area environmental sampling (WAES) considered to be one of the most promising approaches for identifying clandestine materials/operations. As part of the Monitoring Technology and Verification (MTV) Consortium, the Savannah River Site (SRS) and The Oak Ridge Reservation (ORR) have been selected as field sites of interest to collect samples from environments exposed to specific nuclear activities and materials. The goal of sample collection and analysis is to investigate the potential for deconvolution of nuclear fuel cycle activities from contamination signatures preserved in microbial communities, plants, and the surrounding geochemical environment. This presentation will describe the results of 16s rRNA microbial community analysis at each site, including initial characterization of soil, plant, and surface water samples from the SRS and ORNL’s High Flux Isotope Reactor (HFIR). Results from this work will be used to assess whether characteristic microbiomes can be correlated to geochemical signatures in environments associated with specific stages of the nuclear fuel cycle. Characterization of microbial-geochemical signatures at these sites may further aid in the development of technologies for monitoring and detection of proliferation-sensitive fuel cycle activities.
40. Chen, Si, Yongfeng Wang, Huicai Cheng, Terry C. Hazen, Chunguang He and Qiang He. 2022. Identification of Propionate-Degrading Microbial Populations in Methanogenic Processes for Waste Treatment: Methanosaeta and Methanoculleus. Environmental Engineering Science 39:202-211. abstract
    Methanogenic processes have great potential in the sustainable treatment of organic wastes with the production of methane as a renewable source of energy. However, the broader application of methanogenic processes has been hindered by process instability frequently encountered during fluctuations in operational conditions. The accumulation of organic acids, particularly propionate, is considered to be an important cause of process instability. Therefore, to gain an understanding of microbial responses during process instability, it is imperative to identify microbial populations involved in the utilization of elevated levels of propionate. In this study, microbial community analysis showed that bacterial populations from the orders of Syntrophobacterales and Clostridiales were the primary syntrophic partners in anaerobic conversion of propionate. Archaeal populations associated with Methanosaeta and Methanoculleus dominated the propionate-degrading communities enriched in methanogenic batch bioreactors. The involvement of Methanosaeta and Methanoculleus in anaerobic conversion of propionate was further supported by the close correspondence between elevated propionate and increased population abundance of Methanosaeta and Methanoculleus in continuous anaerobic digesters treating animal waste. Subsequent testing using additional methanogenic batch bioreactors revealed that the dominance of Methanosaeta and Methanoculleus populations was linked to the anaerobic degradation of elevated levels of propionate and butyrate, but not the conversion of formate, acetate, or methanol into methane. The identification of microbial populations specifically linked to anaerobic conversion of elevated propionate in this study provided much needed insight for the understanding of microbial processes relevant to process stability in methanogenic waste treatment.

41. Campa, Maria Fernanda, Jeremy R. Chen See, Lavinia V. Unverdorben, Olivia G. Wright, Kimberly A. Roth, Jonathan M. Niles, Daniel Ressler, Ella M. S. Macatugal, Andrew D. Putt, Stephen M. Techtmann, Timothy L. Righetti, Terry C. Hazen and Regina Lamendella. 2022. Geochemistry and Multiomics Data Differentiate Streams in Pennsylvania Based on Unconventional Oil and Gas Activity. Microbiology Spectrum abstract
    Unconventional oil and gas (UOG) extraction is increasing exponentially around the world, as new technological advances have provided cost-effective methods to extract hard-to-reach hydrocarbons. While UOG has increased the energy output of some countries, past research indicates potential impacts in nearby stream ecosystems as measured by geochemical and microbial markers. Here, we utilized a robust data set that combines 16S rRNA gene amplicon sequencing (DNA), metatranscriptomics (RNA), geochemistry, and trace element analyses to establish the impact of UOG activity in 21 sites in northern Pennsylvania. These data were also used to design predictive machine learning models to determine the UOG impact on streams. We identified multiple biomarkers of UOG activity and contributors of antimicrobial resistance within the order Burkholderiales. Furthermore, we identified expressed antimicrobial resistance genes, land coverage, geochemistry, and specific microbes as strong predictors of UOG status. Of the predictive models constructed (n = 30), 15 had accuracies higher than expected by chance and area under the curve values above 0.70. The supervised random forest models with the highest accuracy were constructed with 16S rRNA gene profiles, metatranscriptomics active microbial composition, metatranscriptomics active antimicrobial resistance genes, land coverage, and geochemistry (n = 23). The models identified the most important features within those data sets for classifying UOG status. These findings identified specific shifts in gene presence and expression, as well as geochemical measures, that can be used to build robust models to identify impacts of UOG development. IMPORTANCE The environmental implications of unconventional oil and gas extraction are only recently starting to be systematically recorded. Our research shows the utility of microbial communities paired with geochemical markers to build strong predictive random forest models of unconventional oil and gas activity and the identification of key biomarkers. Microbial communities, their transcribed genes, and key biomarkers can be used as sentinels of environmental changes. Slight changes in microbial function and composition can be detected before chemical markers of contamination. Potential contamination, specifically from biocides, is especially concerning due to its potential to promote antibiotic resistance in the environment. Additionally, as microbial communities facilitate the bulk of nutrient cycling in the environment, small changes may have long-term repercussions. Supervised random forest models can be used to identify changes in those communities, greatly enhance our understanding of what such impacts entail, and inform environmental management decisions. The environmental implications of unconventional oil and gas extraction are only recently starting to be systematically recorded. Our research shows the utility of microbial communities paired with geochemical markers to build strong predictive random forest models of unconventional oil and gas activity and the identification of key biomarkers.
42. Alcime, P., S. Abd Ar Rafie* and T. C. Hazen. 2022. Biogeochemical Responses of Burns. TN UTK REU NSF CEE summer students
43. Abd Ar Rafie, S., M. F. Campa-Ayala, C. Rawn, J. Keum and T. C. Hazen. 2022. Methanotrophs And Methane Hydrates for Wastewater Desalination: Forming Methane Hydrates in Salt Solutions. ASM Microbe 2022 abstract
    Background: Gas hydrates are clathrate compounds in which gas is trapped inside a water crystal structure. A number of publications and patents, devoted to using gas hydrates as a method of desalination, exist where in the initial water is excluded upon hydrate formation, leaving behind solid gas hydrates and concentrated brine. Flowback and produced wastewater (FPW), from the fracking industry, is loaded with salts, organics, and dissolved solids: the economical removal of which is difficult. The overarching goal of this project is to determine the feasibility of methane hydrates for FPW desalination where historically wasted methane can be used to synthesize the hydrates. After dissociation, the methane released from the hydrates can be used to feed the methanotrophic bacteria to remove the complex organics present in FPW. This sub-project investigated how different hydrate formation conditions and salt content can affect hydrate nucleation; and whether we can propose the formation of homogenous hydrates. Understanding the difference in hydrate formation will help us determine optimal conditions to promote homogeneous nucleation in our system and eventually help better engineer a wastewater desalination system. Methods: A 300 ml Parr pressure vessel was used to synthesize the hydrates over 3 to 4 weeks, with temperature dropping from 10 °C to -20 °C. Three different hydrate nucleation conditions, were used to generate samples: one sample was generated using methane, DI water and SnoMax, and a second sample had an additional 1% NaCl. A third sample had the same constituent ingredients as the second sample but underwent freeze thaw cycles to promote more efficient hydrate formation. Low Temperature X-ray Diffraction (LTXRD) data for the samples were obtained at Oak Ridge National Laboratory (ORNL). Phase fractions of hydrate and ice were refined by Rietveld analysis from the data using the General Structural Analysis System (GSAS-II). Results: LTXRD data were collected on the three samples. Diffraction patterns obtained show that gas hydrates were present in each of the samples tested. Hydrate decomposition curves showed regions of rapid and slowed decomposition and all three samples had a similar decomposition scheme across the experimental temperature range of 100 K to 270K. Conclusions: LTXRD experiments confirm that methane hydrates can be formed under saline conditions providing proof of concept necessary for us to answer questions pertaining to the exclusion or inclusion of the salt, during and after hydrate formation.
44. Zhang, A. D. M. Needham A. E. Kazakov W. Zheng S. Zhao Y. Yin D. A. Weitz T. C. Hazen E. J. Alm N. S. Baliga A. M. Deutschbauer J.-M. Chandonia M. W. Fields T. R. Northen J. D. Wall M. W. W. Adams M. Auer K. Bender G. Butland R. Chakraborty D. A. Elias P. S. Novichkov A. Mukhopadhyay G. E. Siuzdak D. A. Stahl P. J. Walian J. Zhou A. P. Arkin P. D. Adams. 2021. Targeted Isolation Using Field-Informed Approaches. Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Single-cell sequencing yields information about strain-level differences within a population that can hardly be obtained by metagenome sequencing. We profiled 22 high-quality and high novelty single cell genomes (with >97% complete and < 1% contamination) from an ENIGMA groundwater well GW462 by droplet microfluidics (Microbe-seq). We observed high strain dynamics and functional diversity in these single cell genomes. We identified CRISPR loci with 154 unique spacers in 4 of 22 single cell genomes, indicating intense infections of bacteriophage and coevolution of bacterial host and phage. We observed high functional diversity across single cell genomes in pathways involving antimicrobial resistance, energy metabolisms (especially nitrogen metabolisms and methane metabolisms), and xenobiotics degradation. This indicates distinct strategies of adaptation and diversification among bacterial species in ENIGMA environment. Moreover, we found a high diversity of Burkholderiales species in nitrogen metabolisms involving denitrification and nitrification. All single cell genomes were imported into KBase (narrative 65855), as good references for environmental microbiology research in ENIGMA This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
45. Walker, K. F., E. R. Dixon, D. C. Joyner, K. A. Lowe, F. L. Poole, X. Ge, M. P. Thorgersen, D. Ning, Y. Fan, J. P. Michael, Y. Fu, R. Tian, Y. Wang, J. D. Van Nostrand, L. M. Lui, X. Wu, M. W. W. Adams, R. Chakraborty, D. A. Elias, R. L. Wilpiszeski, J. Zhou, M. W. Fields, A.P. Arkin, P. D. Adams and T. C. Hazen. 2021. Diurnal and Seasonal Fluctuations with the Subsurface: A 17-Week Survey of Groundwater and Sediment in 27 Contaminated Wells. World Microbe Forum abstract
    Diurnal and Seasonal Fluctuations with the Subsurface: A 17-Week Survey of Groundwater and Sediment in 27 Contaminated Wells K. F. Walker1,2*, E. R. Dixon1,3, D. C. Joyner1,3, K. A. Lowe1, F. L. Poole4, X. Ge4, M. P. Thorgersen4, D. Ning5, Y. Fan5, J. P. Michael5, Y. Fu5, R. Tian5, Y. Wang5, J. D. Van Nostrand5, L. M. Lui6, X. Wu6, M. W. W. Adams4, R. Chakraborty6, D. A. Elias1, R. L. Wilpiszeski1, J. Zhou5,6, M. W. Fields7, A.P. Arkin6, P. D. Adams6, and T. C. Hazen1,2,3,6 1Oak Ridge National Laboratory, Oak Ridge, TN; 2Bredesen Center of Interdisciplinary Research and Education, Knoxville, TN; 3Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN; 4University of Georgia, Athens, GA; 5University of Oklahoma, Norman, OK; 6Lawrence Berkeley National Laboratory, Berkeley, CA; and 7Montana State University, Bozeman, MO. http://enigma.lbl.gov Background ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Spatiotemporal variability of groundwater within contaminated wells greatly effects the geochemistry and microbial communities present. This survey of 27 wells at the DOE Oak Ridge Reservation Y-12 Complex in Oak Ridge, Tennessee, set out to obtain diurnal and seasonal fluctuations within three levels (low, moderate, and high) of nitrate and heavy metal contamination. With this data, we created 3D geochemical models using RockWorks of these areas to study changes within the attached and unattached microbial communities in relation to groundwater geochemistry. Methods Measurements were gathered in 27 previously established groundwater wells, four days a week, for the span of 17 weeks (70 days total) spanning from July to December 2019 to build both diurnal and seasonal time series of geochemistry. In-field geochemical measurements were obtained using In-Situ Aqua TROLL 600s, including dissolved oxygen (DO), pH, Conductivity, oxidation-reduction potential (ORP), and nitrate concentration. Samples were then taken from each well to study 52 metals, anions, organic acids, and total organic and inorganic carbon/nitrogen. Throughout the study, results show diurnal and seasonal changes in geochemistry with wide variations between each well and levels of contamination. The subsurface geochemistry was also greatly effected by rainfall events, which was evident after two months of regional drought conditions. Additionally, one well in each level of contamination (3 wells total) was selected to complete a microbial analysis by sampling for groundwater (unattached) and sediment (attached) microbial communities. Groundwater was filtered through 8um and 0.2um filters for 16S rRNA and metagenomic analysis (420 filters). In each of the three wells, 18 unamended sediment traps were placed to complete a time series analysis of the attached microbial community’s sediment geochemistry. Results Results of all analyses are linked to groundwater flow vectors (using the Geotech Colloidal Borescope) on-site weather data (using a HOBO RX300 Weather Station) to produce a correlation analysis between source water and flow paths, groundwater geochemistry, weather events, and levels of contamination on a spatiotemporal scale. With this data, we aim to establish a predictive model between groundwater geochemistry and microbial communities to inform future ENIGMA groundwater and sediment sampling in a planned subsurface observatory. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
46. TCE. 2021. Faculty Recognized for Cited Research . Tickle College of Engineering abstract
    Faculty Recognized for Cited Research January 12, 2021 Accolades, Faculty, Press Releases, Research Highlights Having your work cited by peers as they conduct their own studies is a sign of quality, respect, and success for researchers around the world, regardless of discipline. Stanford University recently undertook the task of ranking scientists based on the number of citations that they’ve received throughout the course of their careers, coming up with a list of more than 150,000 people representing the top 2 percent of cited researchers in the world. Of those, 54 current or former faculty members of the Tickle College of Engineering made the list, with citations stretching 56 years from 1964 through 2020. “We are very proud of the long, successful track record that our faculty have built in establishing themselves as thought leaders in their respective fields,” said Janis Terpenny, dean of the Tickle College of Engineering and Wayne T. Davis Dean’s Chair at UT. “This recognition is well earned, and I congratulate all who were so honored.” Three research areas—electrical and electronic engineering, energy, and materials—are represented by seven faculty members on the list, highlighting the college’s particular strengths in those areas. A total of 20 different research thrusts are represented by college faculty. Of those, Professor William Weber (MSE), UT-Oak Ridge National Laboratory (ORNL) Governor’s Chair for Nuclear Materials Steven Zinkle (NE), UT-ORNL Distinguished Scientist and Director of the Joint Institute for Neutron Sciences Takeshi Egami (MSE), Emeritus Professor Bimal Bose (EECS), and UT-ORNL Governor’s Chair for Biorefining Arthur Ragauskas (CBE) have the five highest citation scores of college faculty on the list. College faculty to make the list are: Name Citation Score Subject Field (According to the Study) William Weber 4.347263403 Applied Physics Steven Zinkle 4.25430464 Energy Takeshi Egami 4.242280269 Applied Physics *Bimal Bose 4.135627941 Electrical and Electronic Engineering Arthur Ragauskas 4.116155545 Biotechnology Peter Liaw 3.969945018 Materials Lynne Parker 3.898299644 Industrial Engineering and Automation Matthew Mench 3.875465867 Energy Suresh Babu 3.848551838 Materials Stephen Paddison 3.827805571 Chemical Physics Zhanhu Guo 3.818935532 Nanoscience and Nanotechnology Fei Wang 3.775698798 Electrical and Electronic Engineering Audris Mockus 3.762920991 Software Engineering Tony Schmitz 3.694875324 Industrial Engineering and Automation Fangxing Li 3.672033827 Energy Brian Wirth 3.67015446 Energy Terry Hazen 3.669806272 Microbiology Charles Melcher 3.664219728 Nuclear and Particle Physics Leon Tolbert 3.655955897 Electrical and Electronic Engineering Husheng Li 3.61673215 Networking and Telecommunications David Mandrus 3.605225121 Applied Physics Thomas Zawodzinski 3.599202616 Energy *William Steele 3.569713952 Chemical Engineering Kurt Sickafus 3.560936495 Applied Physics James Morris 3.548177851 Materials Jack Parker 3.518366925 Environmental Engineering Kevin Tomsovic 3.496244222 Energy *Jack Weitsman 3.490938214 Materials Easo George 3.452878314 Materials Asad Khattak 3.444287116 Logistics and Transportation Yanfei Gao 3.418754707 Materials Philip Rack 3.405158385 Nanoscience and Nanotechnology Bamin Khomami 3.379815642 Polymers David Greene 3.297119725 Logistics and Transportation *Lawrence Townsend 3.280453831 Aerospace and Aeronautics Baoshan Huang 3.227440713 Logistics and Transportation Uday Vaidya 3.225393219 Materials Kenneth Kihm 3.222164727 Mechanical Engineering and Transports Kai Sun 3.208281744 Electrical and Electronic Engineering *John Landes 3.158810413 Mechanical Engineering and Transports Hairong Qi 3.151821661 Artificial Intelligence and Image Processing Yilu Liu 3.105780467 Energy Abhijeet Borole 3.105021967 Biotechnology *Jay Frankel 3.098944456 Mechanical Engineering and Transports Aly Fathy 3.092129923 Networking and Telecommunications Michael Vose 3.07750385 Artificial Intelligence and Image Processing Mohamed Mahfouz 3.063438756 Networking and Telecommunications *Carl Lundin 3.054516951 Mechanical Engineering and Transports Mongi Abidi 3.036729033 Artificial Intelligence and Image Processing *R. D. Krieg 2.986432176 Civil Engineering Benjamin Blalock 2.920227773 Electrical and Electronic Engineering Daniel Costinett 2.880611807 Electrical and Electronic Engineering Hua Bai 2.785889712 Electrical and Electronic Engineering *Denotes emeritus or retired status “As scientists and researchers, we broaden our understanding of the world and of our disciplines by building on the work of our peers,” said UT Knoxville Vice Chancellor for Research Deborah Crawford. “To have your contributions cited by fellow scientists is incredibly rewarding, and it demonstrates the important discoveries we are making every day here at UT.” UT has 156 faculty members on the list, with Gore Hunger Professor of Environmental Science Daniel Simberloff, who ranks number 706 in the world, as the university’s highest ranked professor.
47. See, Jeremy R. Chen, Olivia Wright, Lavinia V. Unverdorben, Nathan Heibeck, Stephen M. Techtmann, Terry C. Hazen and Regina Lamendella. 2021. Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures. Jove-Journal of Visualized Experiments abstract
    Hydraulic fracturing (HF), commonly called "fracking", uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking's impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.
48. Putt, A., P. Pineda, I. Alamilla, A. Salim, A. P. Arkin, P. D. Adams and T. C. Hazen. 2021. Response of Filterable Microbes to a Beta-Cyclodextrin Injection. World Microbe Forum abstract
    Response of Filterable Microbes to a Beta-Cyclodextrin Injection A. Putt1, P. Pineda1, I. Alamilla1, A. Salim1, A. P. Arkin3,4, P. D. Adams3,4 and T. C. Hazen1,2 1University of Tennessee, Knoxville; 2Oak Ridge National Lab, Oak Ridge, 3Lawrence Berkeley National Lab, Berkeley; 4University of California at Berkeley. http://enigma.lbl.gov Background The filterable microbial community is diverse and under sampled due to their passage though commonly used 0.2 µm pores. Function is derived from assembled genomes, but a limited number of field and laboratory experiments restricts our understanding of how the filterable community responds to stress. This work aims to address which filterable microbes form surface biofilms, how the filterable microbial community responds to a carbon-limited system, and the injection of the cyclic starch-derived Beta-cyclodextrin common in pharmaceuticals Methods Urban stream water filtered through a 0.2 µm PES membrane was loaded on triplicate Phenotype Microarray PM2 plates where metabolic activity to several simple sugars and Beta-cyclodextrin was measured. Twelve microcosms were constructed from the 0.2 µm filtrate (single-filtered) and twelve by re-filtering the filtrate through a second 0.2 µm membrane (twice-filtered). Half of the microcosms were injected with Beta-cyclodextrin to an internal concentration of 0.1 ppm and the microcosms were injected with an equal volume of sterile deionized water. Microcosms were destructively sampled 0, 10, and 24 days post-injection with biomass collected from bottle walls, 0.2 µm, and 0.1 µm PES filters and 16S rRNA sequenced using an Illumina MiSeq. Anions, cations, organic acids, pH, and direct cell counts were also measured. Results Nitrate and sulfate were reduced in all microcosms. The injected microcosms showed increased cell counts and the production of acetate and lactate. A slight decrease in the 0.2 µm filter diversity and attached community was measured between the carbon-limited control and the injected samples with an overall decrease in diversity and evenness. The injection caused a significant decrease in the 0.1µm filter diversity and evenness although the carbon-limited and starting community showed no change in diversity. The most abundant 0.2 µm and biofilm ASVs (primer match percent) were Burkholderiales (88%) and Moraxellaceae (86%). The most abundant 0.1 µm ASVs include Parcubacteria (6.1%), Flavobacteraceae (85%), and Methylophilaceae (91%). Conclusions Even with a highly mis-matching primers, Parcubacteria was highly abundant although Burkholderiales was overall in greatest abundance particularly in the injected samples. Importantly, several unknown filterable isolates were cultured from the samples for further work and an Adonis test showed the number of times the water was filtered and the injection had a significant effect on the diversity. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
49. Pineda, P., I. Alamilla, A. Salim, A. Putt and T. C. Hazen. 2021. Comparison of Bacterial DNA Extraction from Stream Water. World Microbe Forum abstract
    Comparison of Bacterial DNA Extraction from Stream Water P. Pineda1, I. Alamilla1, A. Salim1, A. Putt1 and T. C. Hazen1,2 1University of Tennessee, Knoxville; 2Oak Ridge National Lab, Oak Ridge ASM Track: Urban Water Microbiology (AES09) Presentation Type: Poster Background The widespread availability of off-the-shelf DNA extraction kits provides a uniform product with diverse applications. However, the approach of eluting DNA from a biomass source is varied. This project compares the relative efficiency in time, skill, and cost per ng of DNA for the following methods: elution from syringe filters of variable pore sizes, filter cutting, and extracting biofilm DNA from bottle walls. Methods Twelve 100 mL anaerobic stream water microcosms in-which six were injected with 0.1 ppb Beta-cyclodextrin, and six controls were injected with sterile water were incubated on a shaker at 8°C for 10 days. The water was then filtered through sterile in-line 0.2 µm and 0.1 µm PES syringe filters. Six filters were washed with elution buffer and vortexed for 2 minutes and another six were cut open. Attached biofilms of twelve bottles were extracted using a sterile swab on half and then vortexed with elution buffer and beads. DNA concentrations were measured on a Qubit fluorometer and 16S rRNA amplicons were sequenced on an Illumina MiSeq. Results Filter cutting yielded the highest DNA recovery where the 0.2 µm mean = 18.2 ng/µl, and 0.1 µm mean = 0.5 ng/µl. Filter washing produced eluted and retained biomass. The 0.2 µm eluted biomass was double that of the retained biomass which ranged from 0.2 to 5.4 ng/µl, and the retained biomass was <0.05 to 3.5 ng/µl. The 0.1 µm filter DNA averaged 0.1 ng/µl but had many below detection samples. The bead beating biomass DNA extraction had slightly higher recovery than the swab method. Conclusions Based on the 0.2 µm DNA which had the most consistent extraction results, the filter cutting method was the cheapest but also required the most time and skill. Filter elution is ~50% as effective and costs five-times more per ng of DNA but had the fastest average prep time. Bead beating and swab methods respectively cost nine and ten-times more than filter cutting. Biomass methods do show promise in that they increased the overall DNA yield by 20% in the control samples, and work is ongoing to measure diversity by method and determine if either method has an effect on the resulting diversity.
50. Ning, D., Y. Fan, L. M. Lui, J. P. Michael, Y. Fu, J. D. Van Nostrand, R. Tian, Y. Wang, K. F. Walker, E. R. Dixon, A. D. Putt, D. E. Williams, D. C. Joyner, K. A. Lowe, F. L. Poole, X. Ge, M. P. Thorgersen, M. W. W. Adams, R. Chakraborty, X. Wu, D. A. Elias, R. L. Wilpiszeski, J. Zhou, M. W. Fields, T. C. Hazen, A. P. Arkin and P. D. Adams. 2021. Physical size matters in groundwater bacterial community assembly. Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. A central issue in microbial ecology is to identify key drivers shaping community assembly and functioning, which is also fundamental for ecological modeling. Various physical-chemical and microbial drivers have been extensively investigated, including climate (e.g. temperature), chemistry (e.g. pH, carbon sources), microbial functional genes, etc. However, a basic trait, physical size of cells or the particles they attached, was rarely studied. Considering the porous habitats in subsurface, physical size should be particularly important in groundwater microbial community assembly and functioning. Small-size microbes (e.g. ultramicrobacteria) can be easier to pass through soil pores along groundwater flow; but when flow rate is limited, they may be more difficult to escape from absorption, trapping, or flocculation. In addition, small size generally leads to higher specific surface area and slower growth rate (longer division period) of bacteria. Thus, we hypothesize that physical size plays an essential role in the variation of microbial diversity, assembly, and functioning. The groundwater in the Oak Ridge Integrated Field Research Challenge site (FRC, Oak Ridge, TN) has large geochemical gradients and diverse subsurface conditions for microbial dispersal. In the spring of 2019, three areas under different contamination levels, three groundwater wells in each area, were selected for 9-week bi-weekly sampling from March to May. Each groundwater sample was filtered in succession through 10-μm filter mainly for large-size and particle-attached bacteria (so-called ‘large’), 0.2-μm mainly for normal-size free-living bacteria (so-called ‘medium’), and 0.1-μm for small free-living bacteria (so-called ‘small’). The results of bacterial communities from 16S rRNA gene sequencing well supported our hypothesis. The large, medium, and small-size bacteria counted for 7%, 62%, and 31% of the DNA concentrations on average, and the small-size portion was higher than 20% (up to 32% ) in some well in May, indicating these three types were all not negligible. The size significantly affected bacterial diversity and community structure. Alpha diversity of small-size bacteria (Shannon 3.8±1.2) was generally lower than medium (5.4±1.2) and large-size bacteria (5.1±1.1). The size difference explained a substantial proportion (24.4%, P<0.001; higher than location and time) in the variation of bacterial alpha diversity (Shannon index), and proportion (8.4%, P<0.001; similar to location and time) of beta diversity (Bray-Curtis index). The location, which reflects the influence of contamination and/or spatial distance, showed obvious effect on the alpha (21% and 29%) and beta diversity (26% and 29%) of large and medium-size bacteria, but had much lower impact on small-size bacteria (14% of alpha and 10% of beta). While large and medium-size bacteria from different areas always showed different community structure, small-size bacteria from different areas became convergent after late Apr. The results might be related to better dispersal of small-size bacteria and increased precipitation in late April and May. The size also affected the dominant phylogeny and some key functional species. While alpha- and/or beta-Proteobacteria generally dominated in large and medium-size bacteria, the phylum Bacteroidetes significantly increased or even predominate in small-size bacteria after late April, mainly attributed to the genus Hydrotalea. Sulfate-reducing bacteria, a relevant functional group in this site, were mainly detected in the orders Syntrophobacterales and Desulfobacterales and Class Thermodesulfovibrio in both large and medium-size bacteria, but were nearly undetectable in small-size bacteria. A newly developed framework based on phylogenetic-bin-level null model analysis (iCAMP) was applied to explore the different assembly mechanisms of bacteria with different sizes. Based on the results, dispersal limitation generally played more important roles in large-size (46±5%) than in medium (38±6%) and small-size bacteria (23±9%). In contrast, selection was obviously more influential in small (31±12%) and medium (23±5%) than in large-size bacteria (18±2%). The major assembly processes, selection, dispersal limitation, and drift, all showed dramatically higher (1.5-4.0 folders) temporal variations in small (CV 0.39-0.45) than in medium (CV 0.17-0.29) and large-size bacteria (CV 0.10-0.19). In conclusion, the size of cells or attached particles is essential in shaping groundwater microbiome. Bacteria with larger sizes or particle-attached bacteria were more affected by dispersal limitation and showed more variation among different locations but higher resistance in temporal dynamics; in contract, smaller free-living bacteria are more sensitive to temporal change of environmental conditions, and can be easier to migrate under adequate flow rate. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
51. Lui, L. M., E. L-W. Majumder*, H. J. Smith*, H. K. Carlson, F. Von Netzer, N. Nielsen, M. Peng, X. Tao, A. Zhou, M. Price, J. V. Kuehl, A. J. Hendrickson, V. Trotter, S. Gushgari-Doyle, J. Valenzuela, A. Otwell, K. Hunt, A. Carr, K. Walker, E. Dixon, F. Poole, M. Thorgersen, X. Ge, M. W. W. Adams, E. J. Alm, N. S. Baliga, J.-M. Chandonia, A. M. Deutschbauer, D. A. Elias, M. W. Fields, T. C. Hazen, T. R. Northen, A. Mukhopadhyay, G. E. Siuzdak, D. A. Stahl, P. J. Walian, J. Zhou, R. Chakraborty, A. P. Arkin and P. D. Adams. 2021. Mechanism across scales: integrating laboratory and field studies for microbial ecology as illustrated by the ENIGMA SF. Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA - Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Abstract Over the last century, leaps in technology for imaging, sampling, detection, high-throughput sequencing and ‘omics analyses have revolutionized microbial ecology to enable rapid acquisition of extensive datasets for microbial communities across ever-increasing temporal and spatial scales. The present challenge is capitalizing on our enhanced abilities of observation and integrating diverse data types from different scales, resolutions and disciplines to reach a causal and mechanistic understanding of how microbial communities transform and respond to perturbations in the environment. This type of causal and mechanistic understanding will make predictions of microbial community behavior more robust and actionable in addressing microbially-mediated global problems. To discern drivers of microbial community assembly and function, we recognize the need for coordinated, model-driven experiments that integrate the analysis of genomics data, biogeochemical parameters, and ecological and physical forces to rates of microbial growth at specific locations. To link processes and factors from the gene scale to the ecosystem scale for subsurface microbiology, ENIGMA, a U.S. Department of Energy Science Focus Area, seeks to understand the biogeochemical and microbial processes in the Oak Ridge Reservation (ORR). To accomplish this we are using a coordinated inter-laboratory framework to link processes and factors from the gene scale to the ecosystem scale. ENIGMA coordinates multiple studies at the field scale, mesocosm scale, and molecular/species level and has major research thrusts aimed at field surveys, laboratory and bioreactor studies of isolates, syncoms, enrichments, improved isolation methods, genetic tool development, and bioinformatics analyses and tools. We describe how these ENIGMA efforts are being utilized to characterize and build a predictive understanding of the microbial subsurface communities of ORR and how we are generalizing this integrated approach to be applicable to other study systems and environments.
52. Lui, Lauren M., Erica L. W. Majumder, Heidi J. Smith, Hans K. Carlson, Frederick von Netzer, Matthew W. Fields, David A. Stahl, Jizhong Zhou, Terry C. Hazen, Nitin S. Baliga, Paul D. Adams, Adam P. Arkin and Enigma Consortium. 2021. Mechanism Across Scales: A Holistic Modeling Framework Integrating Laboratory and Field Studies for Microbial Ecology. Frontiers in Microbiology 12. abstract
    Over the last century, leaps in technology for imaging, sampling, detection, high-throughput sequencing, and -omics analyses have revolutionized microbial ecology to enable rapid acquisition of extensive datasets for microbial communities across the ever-increasing temporal and spatial scales. The present challenge is capitalizing on our enhanced abilities of observation and integrating diverse data types from different scales, resolutions, and disciplines to reach a causal and mechanistic understanding of how microbial communities transform and respond to perturbations in the environment. This type of causal and mechanistic understanding will make predictions of microbial community behavior more robust and actionable in addressing microbially mediated global problems. To discern drivers of microbial community assembly and function, we recognize the need for a conceptual, quantitative framework that connects measurements of genomic potential, the environment, and ecological and physical forces to rates of microbial growth at specific locations. We describe the Framework for Integrated, Conceptual, and Systematic Microbial Ecology (FICSME), an experimental design framework for conducting process-focused microbial ecology studies that incorporates biological, chemical, and physical drivers of a microbial system into a conceptual model. Through iterative cycles that advance our understanding of the coupling across scales and processes, we can reliably predict how perturbations to microbial systems impact ecosystem-scale processes or vice versa. We describe an approach and potential applications for using the FICSME to elucidate the mechanisms of globally important ecological and physical processes, toward attaining the goal of predicting the structure and function of microbial communities in chemically complex natural environments.
53. Li, Y., K. Ash, D. C. Joyner, D. E. Williams, C. Iler, I. Alamilla, P. McKay, B. Green, F. Kara-Murdoch, C. Swift, F. Löffler and T. C. Hazen. 2021. Decay of SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV) RNA in raw wastewater to inform application in wastewater-based epidemiology of the University of Tennessee student residential buildings. World Microbe Forum abstract
    Decay of SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV) RNA in raw wastewater to inform application in wastewater-based epidemiology of the University of Tennessee student residential buildings. Y. Li, K. Ash, D. C. Joyner, D. E. Williams, C. Iler, I. Alamilla, P. McKay, B. Green, F. Kara-Murdoch, C. Swift, F. Löffler, and T. C. Hazen Background: Wastewater-based epidemiology (WBE) demonstrates the potential for COVID-19 community transmission monitoring. We report the outcomes of a raw wastewater surveillance program at the University of Tennessee Knoxville campus, a large urban university with >7,000 students living in on-campus dormitories, fraternities and sororities. Surveillance was conducted at the building level on a once-weekly schedule throughout the university’s academic year. However, data on the stability of SARS-CoV-2 RNA in raw wastewater are needed to interpret WBE results. Method: Samples were collected from 47 buildings across the Knoxville campus. We selected the samples in which the detection of SARS-CoV-2 was above 10^2 viral copies/L to investigate the decay rates of RNA from SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV) by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in raw wastewater stored at 4° and 20°C. Results: There was no statistically significant difference between RNA decay of SARS-CoV-2 and PMMoV for the same sample. Decay rate constants for the temperatures showed that SARS-CoV-2 were less sensitive to elevated temperatures in raw wastewater. Conclusion: SARS-CoV-2 RNA is likely to persist at least one month in raw wastewater to provide a reliable detection for WBE applicatio
54. Kelly, E. R., A. Putt, K. Walker, D. C. Joyner, K. Lowe, M. Rodriguez Jr, M. W. Fields, R. Chakraborty, X. Wu, D. Stahl, T. Lie, M. W. W. Adams, F. Poole, P. J.Walian, J. Zhou, J. Van Nostrand, T. R. Northen, J.-M. Chandonia, A. P. Arkin, P. D. Adams and T. C. Hazen. 2021. Cone Penetrometer 3-D Characterization of DOE Legacy Site for Hydrological, Geological and Biogeochemistry Subsurface Observatories. World Microbe Forum abstract
    Cone Penetrometer 3-D Characterization of DOE Legacy Site for Hydrological, Geological and Biogeochemistry Subsurface Observatories E. R. Kelly1*, A. Putt1, K. Walker1, D.C. Joyner1, K. Lowe2, M. Rodriguez Jr2, M. W. Fields3, R. Chakraborty4, X. Wu4, D. Stahl5, T. Lie5, M. W. W. Adams6, F. Poole6, P. J.Walian4, J. Zhou5, J. Van Nostrand5, T. R. Northen4, J.-M. Chandonia4, A. P. Arkin4,8, P. D. Adams4,8, and T. C. Hazen1,2 1University of Tennessee, Knoxville; 2Oak Ridge National Lab, Oak Ridge, 3Montana State University, Bozeman; 4Lawrence Berkeley National Lab, Berkeley; 5University of Washington, Seattle; 6University of Georgia, Athens; 7University of Oklahoma, Norman ; 8University of California at Berkeley. http://enigma.lbl.gov Background The Cone Penetrometer field study overarching aim was to get a detailed view of lithology of the subsurface in Area 3 next to the S-3 ponds disposal site to select the best sites for installing subsurface observatories for follow-up studies. Additional aims were to: 1. Create a groundwater flow model of Area 3 that shows nitrate concentration, 2. Analyze impacts of groundwater rate and flow direction on geochemical parameters and nitrate concentration in Area 3, 3. Analyze impact of subsurface lithology on nitrate concentration in Area 3, and 4. Analyze impact of subsurface lithology on DOC concentration in Area 3. Methods Over 16 days, a 131-push cone penetrometer grid was completed across the 2,600 square meters of Area 3 with subsurface lithology mapped from 1m to 11m below ground surface (mean=5.9m). In addition, 34 sediment samples were collected near select pushes. Each 0.6m core was collected within 2 to 9.5m below ground surface and consisted primarily of clay. Cores were subdivided into 292 subsamples for: metals, C, N, DOC, DON, biomass, pH, nitrate, nitrite, isotopic fractionation, 16S/18S sequencing, Geochip, respiration, and metabolomics. Groundwater in wells in this area were also measured during the cone penetrometer activity for: DO, pH, temperature, conductivity, redox, salinity, nitrate, depth to water and vector for groundwater flow and rate with colloidal boroscope. Results The cone penetrometer survey provided a detailed view of the unconsolidated sediment layers of Area 3. This study was conducted to evaluate the interaction between groundwater, different sediment types, and biogeochemistry in order to identify the locations of the future subsurface observatories for ENIGMA. Sediment types, cone pressures, and geochemical data can be mapped to find the extent of subsurface sediment layers with the intention of identifying pathways of flow and recharge. Water levels, colloidal borescope vectors, and geochemical measurements were gathered in combination with the stratigraphy data to investigate the possible locations for future multi-level subsurface observations. Because of the large number of wells (102) in Area 3 it allowed measurements before, during and after cone penetrometer pushes near these wells. Conclusions This demonstrated that the cone penetrometer pushes had no effect on adjacent well water level, vector for flow direction and rate of flow or for DO, pH, temperature, conductivity, redox, salinity, and nitrate. To our knowledge this is the first time this has been demonstrated for a cone penetrometer survey. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
55. Joyner, D. C. and T. C. Hazen. 2021. Managing Your Graduate Career: Guidelines for Success. World Microbe Forum abstract
    Managing Your Graduate Career: Guidelines for Success Authors: Dominique C Joyner, Terry C Hazen INTRODUCTION According to ASM, a PhD in microbiology is proposed to take three to eight years. The expectation is four years. A guide is proposed, from a project management viewpoint, that gives each graduate student the tools they need to manage their graduate career. The old thinking of high motivation and lots of hard work just doesn't work these days, this unfortunately seems to result in burnout. Having a plan and a framework will keep the big picture in focus and will give latitude for the agility and flexibility required to reach the finish line. METHODS 1) MOTIVATION: This is not a 9-5, that comes later. 2) WORK WITH YOUR TEAM: You selected a committee, now have them fulfill their commitment to you. Do not plan to do your graduate work in a vacuum. 3) SCOPE CREEP: Get your proposal written and approved by your committee before you go too far down the road and stay in your lane. 4) COLLABORATE: You will expand your capabilities by collaborating with others, do this early on so that you aren’t waiting for product. Be sure to be in control of your primary analysis. 5) TIMELINE: Meet your milestones, being agile and creative will be the key to your success. 6) POSITIVITY: Don't go down the drain of defeat, every graduate student thinks it hard, It is! RESULTS and CONCLUSION Planning a graduate career framework and sticking to it while utilizing the resources available will lead to success.
56. Hunt, K. A. , A. E. Otwell, S. Bowman, S. D. Wankel, K. F. Walker, E. R. Dixon, M. Rodriguez, K. A. Lowe, D. C. Joyner, A. Carr, L. Lui, T. Nielsen, N. S. Baliga, T. C. Hazen, D. A. Stahl, A. P. Arkin and P. D. Adams. 2021. Resolving Biotic and Abiotic Controls of Nitrous Oxide Flux in a Subsurface Site Contaminated with High Nitrate Concentrations. Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Abstract: Linking field observations with laboratory studies, and vice versa, is essential for advancing predictive understanding of environmental systems and for stewardship of those systems. We are developing tools and identifying systems that capture phenomena observed in the field in a reproducible, minimally disruptive, and dissectible manner (see poster by Smith et. al.). In turn, those observations serve to direct more refined laboratory studies designed to more completely resolve the roles of microbial ecology and abiotic processes in observable systems-level processes (see poster by Valenzuela et. al.). Microbial activity in the field can be a challenge to quantify due to spatial and temporal heterogeneity and the cost/methodological constraints of real time observations without perturbation. Stable isotopic analysis of biogeochemically active substrates and products can circumvent some of these limitations. The field research center (FRC) at Oak Ridge, TN contains a site that has been contaminated with low pH (3-7), heavy metal laden nitrate (~10 g/l) for decades from historical activities. To understand the microbial processes of this site, we analyzed stable isotopes of ground water as well as dissolved nitrate and nitrous oxide collected from a total of 27 different wells over 3 different areas. These isotopic analyses are well suited for this investigation given the strong process signals (denitrification vs nitrification vs dilution) imprinted on δ15N and δ18O of different nitrogen species. Previous measurements of nitrous oxide flux indicated major subsurface production of nitrous oxide, which are supported here by δ15N, δ18O, and site preference of nitrous oxide samples analyzed from groundwater. Analyses revealed a variable distribution of activity across the site (horizontally and with depth) and implicated both denitrification and chemodenitrification in nitrous oxide production. These data are being contextualized by complementary molecular, biological, and chemical characterization of groundwater and sediment traps recovered from the 27-well survey, together contributing to a framework for developing a more predictive understanding of biotic and abiotic controls of local and systems-level processes. High concentrations and fluxes of nitrous oxide measured in the subsurface without associated surface emissions indicated significant microbial activity driven by both the production and consumption of this high energy electron acceptor. Two populations of nitrous oxide reducers (clade I and II) were observed to be stratified with depth, a distribution suggested to be controlled by either nitrous oxide concentration or response to inhibitory factors, including competition for oxygen. To understand these processes and populations, nitrous oxide reducers are being isolated and will provide field relevant kinetic and physiological parameters to evaluate the roles of the activities and variable distribution of clade I and II nitrous oxide reducers in controlling nitrous oxide emissions from the subsurface (see poster by Gushgari-Doyle et. al.). This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
57. Hazen, T. C. Invited. . September 16, 2021. Charlotte, NC. . 2021. Wastewater Based Epidemiology during the Covid-19 Pandemic at the University of Tennessee, Knoxville. Global ENVIRO Summit https://www.envirosummit.com/ abstract
    The University of Tennessee rapidly mobilized the labs and collection protocols for SARS CoV-2 (Covid-19) testing of wastewater in 42 buildings on campus that were residences of students at the start of the Fall 2020 semester (this was approved by the Institutional Biosafety Committee as Level 2E). This included dormitories, sororities, and fraternities for >7,500 students, testing of all 42 buildings was done weekly from September 1, 2020 until June 2, 2021. The purpose of this research was to determine which buildings at UTK (especially dorms) may have SARS CoV-2 virus signals in the wastewater from the building, i.e. Wastewater Based Epidemiology (WBE). This is simply a relative abundance test using rapid RT qPCR, so that we can determine if further testing is needed. Positive outcomes were not handled by the WBE team (pooled surveillance testing by floor or section of the building followed by individual testing by the student health center for CLIA certified testing and Contact-Tracing). SARS CoV-2 has been detected in wastewater by many others and WBE. This surveillance lasted until Covid-19 was no longer a significant health threat at the UTK campus. The WBE testing was tracked against active cases and self-isolations for residents on campus and compared to the local county pandemic numbers. While the number of cases and SARS CoV-2 signals in the buildings was highest in September and October it subsided in November and only increased slightly when the students returned to campus in January. By April the signals in the wastewater and the number of positive cases significantly declined. During the spring semester the number of positive cases in Knox County was significantly higher than the UTK campus. Since the sampling team was dressed out (Tyvek suits) during sampling they were visible and may have acted as a deterrent for students avoiding testing. Wastewater testing of campus residential buildings was a helpful tool as one component of a broader program that includes pooled saliva, diagnostic tests, health screening, and contact tracing. Details and benchmarks will be discussed.
58. Hazen, T. C., E. R. Kelly*, A. Putt, K. Walker, D. C. Joyner, K. Lowe, M. Rodriguez Jr, M. W. Fields, R. Chakraborty, X. Wu, D. Stahl, T. Lie, M. W. W. Adams, F. Poole, P. J. Walian, J. Zhou, J. Van Nostrand, T. R. Northen, J-M. Chandonia, A. P. Arkin and P. D. Adams. 2021. Cone Penetrometer 3-D Characterization of Y-12 Site to Determine the Hydrological, Geological and Biogeochemistry Best Sites for ENIGMA Subsurface Observatories. Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. The Cone Penetrometer field study overarching aim was to get a detailed view of lithology of the subsurface in Area 3 next to S-3 ponds disposal site to select the best sites for installing subsurface observatories for follow-on ENIGMA studies. Additional aims were to: 1. Create groundwater flow model of Area 3 that shows nitrate concentration, 2. Analyze impacts of groundwater rate and flow direction on geochemical parameters and nitrate concentration in Area 3, 3. Analyze impact of subsurface lithology on nitrate concentration in Area 3, and 4. Analyze impact of subsurface lithology on DOC concentration in Area 3. Over 16 days, a 131-push cone penetrometer grid was completed across the 2,600 square meters of Area 3 with subsurface lithology mapped from 1m to 11m below ground surface (mean=5.9m). In addition, 34 sediment samples were collected near select pushes. Each 0.6m core was collected within 2 to 9.5m below ground surface and consisted primarily of clay. Cores were subdivided into 292 subsamples for: metals, C, N, DOC, DON, biomass, pH, nitrate, nitrite, isotopic fractionation, 16S/18S sequencing, Geochip, respiration, and metabolomics. Groundwater in wells in this area were also measured during the cone penetrometer activity for: DO, pH, temperature, conductivity, redox, salinity, nitrate, depth to water and vector for groundwater flow and rate with colloidal boroscope. The cone penetrometer survey provided a detailed view of the unconsolidated sediment layers of Area 3. This study was conducted to evaluate the interaction between groundwater, different sediment types, and biogeochemistry in order to identify the locations of the future subsurface observatories for ENIGMA. Sediment types, cone pressures, and geochemical data can be mapped to find the extent of subsurface sediment layers with the intention of identifying pathways of flow and recharge. Pushes were driven to refusal by stiff fine grained material or rock. In the northern section of Area 3, refusal was reached around 4m and the layers most likely contributing to flow are those high in sand and gravel with detectable radiation measured on an in-field Geiger counter. Moving southward, the distance to refusal increased linearly with some pushes extending to 12m indicating a southward slope in the direction of a low-order surface stream. The well-defined gravel layers of the north appear to be mixed in the southern and central section’s where mixed sand layers are abundant. The sands include gravely sands, silty sands, and clayey sands with shifting sedimentary composition indicating heterogeneity within the layers. Initial models of the lithology suggest these layers are connected although the central section had a reduced number of pushes due to existing infrastructure. The cone penetrometer study also revealed a large number of localized discontinuous clay and silty clay lenses with limited horizonal and vertical extent. This high-resolution study of the sediment types will benefit our future investigations and current understanding of the transport, storage, and fate of both organic and inorganic substrates in Area 3.Water levels, colloidal borescope vectors, and geochemical measurements were gathered in combination with the stratigraphy data to investigate the possible locations for future multi-level subsurface observations. Because of the large number of wells (102) in Area 3 it allowed measurements before, during and after cone penetrometer pushes near these wells. This demonstrated that the cone penetrometer pushes had no effect on adjacent well water level, vector for flow direction and rate of flow or for DO, pH, temperature, conductivity, redox, salinity, and nitrate. To our knowledge this is the first time this has been demonstrated for a cone penetrometer survey. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
59. Hazen, T. C., I. Fukai, A. P. Arkin, E. Alm and Henrietta Dulai. 2021. Environmental Surveillance for Biological Traces of Radionuclide Sources. MTV Monthly Seminar pdf
60. Hazen, T. C.. 2021. Biosensors for Detecting Nuclear Production Activity in the Environment (Consortium for Monitoring, Technology, and Verification (MTV)). Office of Defense Nuclear Nonproliferation Research and Development, University Program Review (UPR) 2021 Meeting
61. Hazen, T. C.. 2021. After Chat June 21 3:45-4:30PM. “Environmental Systems Biology: The Whole is Greater than the Sum of it’s Parts – Team Science” ASM Environmental Microbiology Award. World Microbe Forum, Online, June 20-24, 2021. American Society for Microbiology and Federation of European Microbiological Societies. World Microbe Forum abstract
    Previous studies at the Y-12 S-3 Ponds legacy contamination site demonstrated the microbial community structure significantly predicted 18 different geochemical parameters in the ground water. These included contaminants from the nuclear production activities done at Y-12 more than 40 years ago ie. U, pH, nitrate, Cr, Sr, Pb, Cd, etc. We are further refining these models using higher resolution metagenomic and metatranscript analysis for our Random Forest, Monte Carlo models. We are also sampling groundwater and sediment both horizontally and vertically along groundwater plume transects to determine if these models also predict when these contaminants first appeared. In the near future we plan on doing similar studies of contaminant plumes from P and R nuclear reactor basins and canals at the Savannah River Site. We will also add on sampling of surface vegetation and microbial rhizosphere to see if this also allows significant prediction of nuclear production activities. Several other sites are also being considered for sampling including around the High Flux Isotope Reactor at ORNL and sites at Los Alamos, Hanford, Idaho.
62. Hazen, T. C.. 2021. Careers in National Labs.
63. Hazen, T. C.. 2021. Environmental Systems Biology: The Whole is Greater than the Sum of it’s Parts – Team Science. World Microbe Forum abstract
    Hazen, T. C. Invited. Environmental Systems Biology: The Whole is Greater than the Sum of it’s Parts – Team Science Depts. Civil & Environmental Engineering, Microbiology, Earth & Planetary Sciences, The University of Tennessee, Knoxville, TN 37996-1605 and Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6342 The whole is greater than the sum of its parts. By using an environmental systems biology approach to our greatest environmental problems and cross-linkage of systems at all levels providing multiple lines of evidence involving environmental observations, laboratory testing, microcosm simulations, hypothesis refinement, field testing and validation, and multiple iterations of this circle, we will be able to make new theories and paradigms for changing pristine and contaminated environments. Pollution is everywhere. Changing environments are everywhere. Microbes are also everywhere, and many have the ability to degrade environmental contaminants and dramatically alter biogeochemistry related to climate change. Understanding how these microbial communities work to degrade environmental contaminants and alter biogeochemistry will enable us to use these microbes to clean up the pollution and alter long-term and short-term changes to the environment. Understanding, monitoring, and controlling the environment with biogeochemical processes, i.e., an environmental systems biology approach. By using an environmental systems approach, we make sure we know of any “fatal flaws” in the approach, get a much better handle on life-cycle cost analysis, and can grade an engineered solution into a natural solution. Examples of this approach and their outcomes will be given for nuclear reactor thermal effluent effects; fish and alligator epizootiology related to nitrate, pulp mill, cortical steroids, and animal immune response; Legionella in cooling towers and thermal effluents; fecal coliforms in the tropics; Vibrio in coral reefs and tropical rain forests; chlorinated solvents in groundwater; oil in marine environments; deep subsurface environments that may suggest ways to determine life on other planets; oil spill natural recovery; nuclear legacy site natural attenuation and predictions of nuclear activity; landfill control; metal contamination of soil and groundwater; and phosphorus dynamics in tropical soils.
64. Harik, A-M., T. C. Hazen, D. C. Joyner and S. Rafie. 2021. Imaging and Analysis of Methanotroph Induced Bioaggregation in Sand. World Microbe Forum abstract
    Sand aggregation in recent decades has seen a trend toward more bio-based aggregation research, research in the field was initially seen in microbial ureolysis and calcite precipitation to aggregate sand introduced by Ferris et al. in the 90s. However this method has significant downfalls, the main of which is the release of ammonia as a byproduct. In looking for alternative bioaggregation methods, inspiration was taken from nature where biofilm can be found aggregating sand surfaces, biofilm contents and structure vary widely; however, a key feature in biofilm is extracellular polysaccharides (EPS) - which are sugars produced by microbes. The goal of the work is to understand the potential to utilize methanotrophically produced EPS for surface stability of sand. For the initial bench scale experiments, three methanotrophic cultures known to produce eps were chosen and applied to sand and sampled at different timepoints. Sand for the experiment was purchased U.S. Silica 100 mesh sand that was subsampled, washed and dried in preparation. Methanotrophic species were grown in Nitrate Mineral Salts media, a minimal media; the media without any cultures was also applied as one of the control conditions. Time points were imaged and tested for eps estimates, cell counts, and dry sieving analysis. In this presentation, the imaging and structure of aggregates and eps bonds between sand will be the focus; looking at the adhesive bond structures, location and failure methods in the thin aggregate layers produced. Some of which differed between microbial cultures and time points. Imaging was done in a scanning electron microscope with no coating, after oven drying samples.
65. Gushgari-Doyle, S., M. O. Yee, J. V. Kuehl, H. J. Smith, M. P. Thorgersen, X. Ge, A. E. Otwell, T. L. Lie, K. A. Hunt, M. W. W. Adams, E. J. Alm, N. S. Baliga, J.-M. Chandonia, A. M. Deutschbauer, D. A. Elias, M. W. Fields, T. C. Hazen, T. R. Northen, A. Mukhopadhyay, G. E. Siuzdak, D. A. Stahl, P. J. Walian, J. Zhou, R. Chakraborty, A. P. Arkin and P. D. Adams. 2021. Targeted Isolation Using Field-Informed Approaches. Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Outline: Isolation of microorganisms representative of key environmental metabolisms is essential to developing a fundamental understanding of ecological processes., yet, the scientific community estimates that we are able to culture fewer than 2% of microorganisms on Earth in the laboratory.1 To recover field-relevant isolates from Oak Ridge Reservation Field Research Center (ORR FRC), we combined several statistical analyses using our environmental and sequencing metadata to A) identify high-priority targets for isolation based on abundance, community correlation, and other metrics, B) select samples and enrichments with increased probability to yield those targets, and C) inform growth medium composition as well as enrichment and isolation approaches. We have employed these targeted isolation techniques (in addition to high-throughput, untargeted approaches- ENIGMA SFA poster by Kuehl et al.) to not only increase isolate diversity in the ENIGMA culture catalogue, but also to recover key isolates from the fieldsite that are suspected to play integral roles in carbon and nitrogen cycling in the terrestrial subsurface. Here, we present several isolation and enrichment successes resulting from this targeted approach, including organisms exhibiting nitrate- and sulfate-reducing metabolisms, complex carbon-transforming metabolisms, high metal tolerance, and acidophiles. Several of our isolates are novel and rarely cultivated/ previously uncultivated clades. We also present ongoing efforts in enrichment and isolation of ammonia oxidizing- and nitrous oxide reducing- microorganisms (see ENIGMA SFA poster by Hunt et al.). In addition, we have several ongoing efforts enriching and isolating for metabolisms and characteristics representative of field observations at the ORR FRC. The results of this work will facilitate development of novel, tractable genetic systems, community interaction studies, and environmental simulations to connect phenotype and genotype to field observations. References 1. Steen, A. D. et al. High proportions of bacteria and archaea across most biomes remain uncultured. ISME J. 13, 3126–3130 (2019). This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
66. Griffths, Z., A. Putt, M. Campa, D. Joyner, J. Miller, O. Pelz, N. GaraJayeva, P. Gardinali and T. C. Hazen. 2021. Observing the Indigenous Microbial Community Response to Crude Oil Amendment in Aerobic and Anerobic Conditions. World Microbe Forum abstract
    The Caspian Sea is the world’s largest landlocked waterbody that lies between Europe and Asia. This region is particularly known for its oil reserves, pipeline and drilling activities and the large scale of this industry has contributed to the environmental decline of the waterbody. In addition to this pollution from the petroleum industry, drainage from various river basins brings an influx of untreated sewage, industrial and agricultural effluents that induce eutrophication and anoxic conditions in deeper, colder waters. Weak tidal forces in this waterbody compact this issue and encourages the persistence of these pollutants. However, this also presents a unique opportunity to unlock the potential of the biodegradative processes carried out by the indigenous microbial community. Some microorganisms are able to use crude oil found in seawater and sediments as an energy source, subsequently reducing its concentration in the environment, via biodegradation. We believe that these indigenous microbes will have different metabolic strategies to degrade oil as they adapted to declining oxygen concentrations with increasing depths. Hence, community structure and composition will vary with depth. This experiment was conducted using seawater taken from surface waters (~25m) and deep waters (~350m) collected from the Caspian Sea. Microcosms were set up and sampled to observe the indigenous microbial reaction after a 60ppm crude oil amendment over a period of 115 days. Surface water microcosms were kept at 28ºC in an aerated area while deep water microcosms were kept at 4ºC under anaerobic conditions. These different depths represent the temperature and oxygen gradient found in this waterbody as we try to simulate the indigenous community response to this contaminant. DNA was extracted and amplified from these different microcosms and sequencing data using Illumina MiSeq, 16S rRNA analysis was performed using Mothur and R Studio. We will analyze changes in the abundance of taxa present and biodiversity over different time points to show the progression of community structure over the experiment. We will compare the biodiversity and taxa observed for aerobic and anerobic conditions and use this to make any inferences about possible adaptation strategies to the oxygen differences in each microcosm. All microcosms showed the presence of hydrocarbon-degrading phyla (Proteobacteria, Firmicutes and Bacteroidetes whose presence is consistent with other reports from oil-enriched environments. Orders of Bacteria related to sulphate reduction and nitrogen cycling were found in anoxic microcosms spiked with crude oil, indicating a possible mechanism for the anaerobic biodegradation of crude oil.
67. Fukai, I. and T. C. Hazen. 2021. Germ Anti-Warfare: Evaluating Microbial Biosensors for Nuclear Arms Nonproliferation. 9th Annual Oak Ridge Postdoctoral Association (ORPA) Research Symposium abstract
    Development of robust environmental biosensors for monitoring nuclear fuel cycle activities is needed to strengthen global nonproliferation safeguards and support development of peaceful nuclear technologies. Prior research has shown that correlations between microbial genomes and geochemical parameters can be used to monitor and predict concentrations of uranium, strontium and other associated nuclear materials at radionuclide-contaminated sites. The sensitivity of potential microbial biosensors to proliferation-related activities of the nuclear fuel cycle and the spatial-temporal limitations of their application have not been constrained. It's hypothesized that characteristic microbiomes may be associated with current and historical uranium enrichment activities, nuclear reactor operations, and spent fuel storage/reprocessing. These characteristic microbiomes could inform development of sensor technologies for rapid detection and screening of special nuclear materials, source materials and their origins. To test these hypotheses, environmental samples are being collected from lateral and vertical contamination gradients associated with specific fuel cycle activities at Oak Ridge National Laboratory, the Y12 National Security Complex, and the Savannah River Site. Data from each site will be integrated to determine how long potential biosensors retain fuel cycle signatures, the lateral and vertical extent of their application, and their ability to differentiate between peaceful and arms-related fuel cycle activities.
68. Fukai, I. and T. C. Hazen. 2021. Evaluation Of Microbial Biosensors With Applications In Nuclear Arms Nonproliferation. World Microbe Forum abstract
    Control/Tracking Number: 2021-A-5851-MICROBE Activity: Abstract Current Date/Time: 3/18/2021 2:37:12 PM Evaluation Of Microbial Biosensors With Applications In Nuclear Arms Nonproliferation Author Block: I. Fukai, T. Hazen; Univ. of Tennessee, Knoxville, TN Abstract: Background. Development of robust environmental biosensors capable of monitoring nuclear fuel cycle activities is critical for supporting nuclear arms nonproliferation and peaceful development of nuclear energy. Correlations between microbial sequences and geochemical parameters have been used in previous studies to predict concentrations of uranium, strontium and other key environmental signatures at radionuclide-contaminated sites (Smith et al., 2015). The spatial and temporal limitations of microbial biosensors and their sensitivity to di!erent stages of the nuclear fuel cycle are unknown. To address these knowledge gaps, data from nuclear legacy sites was collected and analyzed to determine how long biosensors retain fuel cycle signatures, the lateral and vertical limits of their application, and their ability to di!erentiate between peaceful and arms-related nuclear activities. Methods. Samples were collected from Oak Ridge National Laboratory (ORNL) and the Y12 National Security Complex in Tennessee, and The Savannah River Site (SRS) in South Carolina. In addition to their historical roles in U.S. nuclear weapons manufacturing, these nuclear legacy sites currently support a variety of nuclear research, defense, and fuel cycle activities, making them ideal locations for evaluating biosensor resolution and field deployment. Surface water, groundwater, and sediment samples were collected from lateral and vertical contamination gradients, and from uncontaminated areas to provide background data at each site. Microbial analysis included 16s rRNA amplicon analysis, metagenomics and transcriptomics to characterize the composition of biosensor communities, gene functions, and gene expression in environments associated with di!erent stages of the nuclear fuel cycle. Results. Initial comparison of data from contaminated and background areas suggests that specific stages of the nuclear fuel cycle may be associated with characteristic microbiomes. Distinct microbial biosensors were identified in association with enriched uranium activities at Y12, nuclear reactor fission products at ORNL and SRS, and plutonium activities at SRS. Data from legacy waste disposal areas at each site also suggest biosensors may retain these signatures for as long as 35 years. Conclusions. Preliminary results suggest characteristic microbiomes may be associated with specific stages of the nuclear fuel cycle, with the potential for microbial biosensors to be valuable components of nuclear arms non-proliferation technologies. Acknowledgments/ References: Smith et al. 2015. Natural bacterial communities serve as quantitative geochemical biosensors. mBio 6(3):e00326-15. doi:10.1128/mBio.00326- 15. This work was funded by in part by the Consortium for Monitoring, Technology, and Verification under Department of Energy National Nuclear Security Administration award number DE-NA0003920. . Author Disclosure Information: I. Fukai: None. T. Hazen: None. ASM Sub-track/FEMS Topic (Complete): AES06 Bioremediation/Biodegradation and Biotransformation Keyword (Complete): metagenomics ; 16S RNA Presentation Preference (Complete): Presentation Preference: iPoster only This abstract submission describes a bioinformatic tool or method (Required): No Would you like to be considered as an oral abstract presenter in the CPEP Rapid Fire Track Hub?: No
69. Campa, M. F., J. Chen See, L. Unverdorben, O. Wright, K. A Roth, J. M. Niles, D. Ressler, E. Macatugal, A. Putt, S. M. Techtmann, T. C. Hazen and R. Lamendella. 2021. Geochemistry, land coverage, and multiomics data differentiate streams in Pennsylvania based on unconventional oil and gas activity. World Microbe Forum abstract
    Title page: Geochemistry, land coverage, and multiomics data differentiate streams in Pennsylvania based on unconventional oil and gas activity Authors: Maria Fernanda Campa*1,2, Jeremy Chen See*3, Lavinia Unverdorben3, Olivia Wright3, Kimberly A Roth3, Jonathan M. Niles4, Daniel Ressler4, Ella Macatugal5, Andrew Putt1,2, Stephen M. Techtmann6, Terry C. Hazen#1,2, Regina Lamendella#3 Affiliations: 1 University of Tennessee, Knoxville, TN 2 Oak Ridge National Laboratory, Oak Ridge, TN 3 Juniata College, Huntingdon, PA 4 Susquehanna University, Selinsgrove, PA 5 University of Guam, Mangilao, GU 6 Michigan Technological University, Houghton, MI *co-first authors #co-corresponding authors Abstract: Understanding the impact of unconventional oil and gas activity (UOG) on surrounding streams ecosystems is of environmental and ecological importance. To determine the impact UOG activity has on ecosystems, this study measured geochemistry, total and active microbial community composition, expressed functional gene profiles, and expressed antimicrobial resistance genes and utilized these data to design predictive machine learning models for UOG impacts to streams. Specifically, 16S rRNA amplicon sequencing (ASVs), metagenomics (DNA), metatranscriptomics (RNA), geochemistry, and trace element analyses were used to determine the effect of UOG activity in 21 streams in northern Pennsylvania, U.S. The taxonomic Order Burkholderiales was identified as a biomarker of UOG activity and contributor to the antimicrobial resistance profile in the streams. UOG status could be predicted based on antimicrobial resistance genes, geochemistry, land coverage, and microbial communities. Together, these measurements yielded stronger predictive models for UOG status compared to total microbial composition and metatranscriptomic alone. Important predictors of UOG included active rifampin and macrolide resistance genes. Geochemical measurements of barium, bromide, and zinc contributed significantly to the variation in total bacterial communities associated with UOG status in sediments. This study identified shifts in gene presence and expression, as well as geochemical measures that can be used to build robust models to identify impacts of UOG development. Machine learning models can be developed using different types of high-resolution data to assess and quantify the environmental impact of UOG development, help identify impacted sites, and inform data driven environmental management decisions
70. Ash, K. T., Y. Li, D. C. Joyner, D. E. Williams, I. Alamilla, P. McKay, B. Green, C. Iler, F. Kara-Murdoch, C. Swift, F. Löffler and T. C. Hazen. 2021. Miles Away From Ordinary: Raw Wastewater Surveillance For The Novel Sars-cov-2 Virus On The University Of Tennessee - Knoxville Campus. World Microbe Forum abstract
    Background:Wastewater-based surveillance for the SARS-CoV-2 virus has become an incredibly useful tool in the fight against the Covid-19 pandemic. Analysisof untreated wastewater can reveal the extent to which a community is affected, and which areas of that community are of the most concern. On a universitycampus, information as to which buildings are currently experiencing viral infections is invaluable to campus health officials. Methods:Samples were collectedfrom over 40 buildings across campus, including student dorms, fraternities, and sororities. The first samples were collected on September 4th, 2020, andcontinue to the present day. This time frame represents a significant portion of the 2020 fall semester, during which time the campus was closed and classeswere moved online. For most buildings, we collected a single sample per week, which ranged from 50 to 500 mL. For the buildings with the highest populations,we would perform a 24-hour sampling per week. The analysis process for each sample began with a two-hour pasteurization at 60°C, followed by centrifugationand filtering to remove heavy sediments. RNA was extracted with the Qiagen QIAamp Viral RNA mini kit. The presence of SARS-CoV-2 was determined using theqPCR assay developed by the CDC, which targets two locations corresponding to the N-capsid protein. Results:To date, we have collected and analyzed 1079samples with ~34% of those being reported as a positive detection. Out of 49 sampling locations, 3 buildings account for half of all the positive detections and 5buildings never had a sample which tested positive. Detections averaged at about 10^3 viral copies/L, with our highest detections at 10^5 viralcopies/L.Conclusion:Our method has proven to be a reliable method of detection for SARS-CoV-2 in wastewater and could prove useful for future community-based disease analysis.
71. Ash, K. T., I. Alamilla, Y. Li, D. C. Joyner, D. E. Williams, P. J. McKay, B. M. Green, C. Iler, S. E. DeBlander, F. Kara-Murdoch, C. M. Swift and T. C. Hazen. 2021. Coding-Complete Genome Sequence of a SARS-CoV-2 Variant Obtained from Raw Sewage at the University of Tennessee-Knoxville Campus. Microbiology Resource Announcements 10. abstract
    Reported here is a coding-complete genome sequence of a SARS-CoV-2 variant obtained from raw wastewater samples at the University of Tennessee-Knoxville campus. This sequence provides insight into SARS-CoV-2 variants that circulate on large college campuses but remain mostly undetected.
72. Alhajjar, Rehab K., Ryan B. Ghannam, Jeremy R. Chen See, Olivia G. Wright, Maria Fernanda Campa, Terry C. Hazen, Regina Lamendella and Stephen M. Techtmann. 2021. Comparative study of the effects of biocides and metal oxide nanoparticles on microbial community structure in a stream impacted by hydraulic fracturing. Chemosphere 284. abstract
    Our study goal was to investigate the impact of biocides and nanoparticles (NPs) on the microbial diversity in a hydraulic fracturing impacted stream. Biocides and NPs are known for their antimicrobial properties and controlling microbial growth. Previous work has shown that biocides can alter the microbial community composition of stream water and may select for biocide-resistant bacteria. Additional studies have shown that nanoparticles can also alter microbial community composition. However, previous work has often focused on the response to a single compound. Here we provide a more thorough analysis of the microbial community response to three different biocides and three different nanoparticles. A microcosm-based study was undertaken that exposed stream microbial communities to either biocides or NPs. Our results showed a decrease in bacterial abundance with different types of nanoparticles, but an increase in microbial abundance in biocide-amended treatments. The microbial community composition (MCC) was distinct from the controls in all biocide and NP treatments, which resulted in differentially enriched taxa in the treatments compared to the controls. Our results indicate that NPs slightly altered the MCC compared to the biocide-treated microcosms. After 14 days, the MCC in the nanoparticle-treated conditions was similar to the MCC in the control. Conversely, the MCC in the biocidetreated microcosms was distinct from the controls at day 14 and distinct from all conditions at day 0. This finding may point to the use of NPs as an alternative to biocides in some settings.
73. Rafie. S. A.A., K. P. Hoyt, M. R. Schubert, M. T. Kerr, L. R. Blentlinger, A. M. Faiia, A. Szynkiewicz, J. F. Franklin, S. P. Horn and T. C. Hazen. 2021. Soil bacterial response to prescribed fires in a southern Appalachian clear cut with fuel manipulation. World Microbe Forum abstract
    Soil bacterial response to prescribed fires in a southern Appalachian clear cut with fuel manipulation Saad A.A. Rafie1*, Kevin P. Hoyt2, Martin R. Schubert3, Matthew T. Kerr4, Luke R. Blentlinger4, Anthony M. Faiia5, Anna Szynkiewicz5, Jennifer F. Franklin6, Sally P. Horn4, Terry C. Hazen1,5,7,8,9 1 Department of Civil and Environmental Engineering, University of Tennessee, Knoxville TN 2 Forest Resources AgResearch and Education Center, Oak Ridge Forest 3 Forest Resources AgResearch and Education Center, Cumberland Forest 4 Department of Geography, University of Tennessee, Knoxville TN 5 Department of Earth & Planetary Sciences, University of Tennessee, Knoxville, TN 6 Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Knoxville TN 7 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 8 Institute for a Secure and Sustainable Environment, Knoxville, TN 9 Department of Microbiology, University of Tennessee, Knoxville, TN Background: Prescribed fires are used by land managers across the southern Appalachian region, and wildfires also occur, usually set by people but in some cases by lightning. Multiple large wildfires occurred during a severe drought in October and November 2016, heightening concern over the possibility that global climate change would increase fire frequency in the region. The current importance and possible future increase in fire frequency in the southern Appalachian region make it critical to understand how burning affects biogeochemical characteristics of soils. This project investigated the postfire soil microbiome, PAH concentrations, bulk carbon and nitrogen contents, bulk δ13C and δ15N, and pore-water chemistry in a former clear-cut site located in the Ridge and Valley section of the Southern Appalachian region in Tennessee. Methods: Pre- and post-burn soil samples were collected across a period of fifteen months, from plots with varying woody fuels added and analyzed for cell counts (Acridine-Orange direct counts), community shifts (16s rRNA sequencing), biological activity (dehydrogenase assay), bulk C-N contents and bulk stable isotope compositions (13C/12C and 15N/14N), and pore-water chemistry (ICP-MS). Results: No PAHs were detected in our postfire samples. Soil microbial communities clustered significantly (p< 0.005) by added fuel type and time since the burn event. Measurable differences were observed for the bulk N content and lower δ15N, pre- and post-fire, across varying fuel types, suggesting that these parameters could be an indicator of fire-related changes in the soil. Pore-water chemistry was relatively unchanged in post-burn samples, with minor differences seen across fuel types or collection time. Conclusions: Considering the fires were of visibly uneven intensity, the minimal differences seen between the analyzed variables imply that the low severity prescribed burn had a minimal effect on the soil chemistry.
74. Wilpiszeski, Regina L., Caitlin M. Gionfriddo, Ann M. Wymore, Ji-Won Moon, Kenneth A. Lowe, Mircea Podar, Sa'ad Rafie, Matthew W. Fields, Terry C. Hazen, Xiaoxuan Ge, Farris Poole, Michael W. W. Adams, Romy Chakraborty, Yupeng Fan, Joy D. Van Nostrand, Jizhong Zhou, Adam P. Arkin and Dwayne A. Elias. 2020. In-field bioreactors demonstrate dynamic shifts in microbial communities in response to geochemical perturbations. Plos One 15. abstract
    Subsurface microbial communities mediate the transformation and fate of redox sensitive materials including organic matter, metals and radionuclides. Few studies have explored how changing geochemical conditions influence the composition of groundwater microbial communities over time. We temporally monitored alterations in abiotic forces on microbial community structure using 1L in-field bioreactors receiving background and contaminated groundwater at the Oak Ridge Reservation, TN. Planktonic and biofilm microbial communities were initialized with background water for 4 days to establish communities in triplicate control reactors and triplicate test reactors and then fed filtered water for 14 days. On day 18, three reactors were switched to receive filtered groundwater from a contaminated well, enriched in total dissolved solids relative to the background site, particularly chloride, nitrate, uranium, and sulfate. Biological and geochemical data were collected throughout the experiment, including planktonic and biofilm DNA for 16S rRNA amplicon sequencing, cell counts, total protein, anions, cations, trace metals, organic acids, bicarbonate, pH, Eh, DO, and conductivity. We observed significant shifts in both planktonic and biofilm microbial communities receiving contaminated water. This included a loss of rare taxa, especially amongst members of theBacteroidetes,Acidobacteria,Chloroflexi, andBetaproteobacteria, but enrichment in the Fe- and nitrate- reducingFerribacteriumand parasiticBdellovibrio. These shifted communities were more similar to the contaminated well community, suggesting that geochemical forces substantially influence microbial community diversity and structure. These influences can only be captured through such comprehensive temporal studies, which also enable more robust and accurate predictive models to be developed.
75. Walker, K. F., E. R. Dixon, D. C. Joyner, K. A. Lowe, F. L. Poole, X. Ge, M. P. Thorgersen, D. Ning, Y. Fan, J.P. Michael, Y. Fu, R. Tian, Y. Wang, J.D. Van Nostrand, L.M. Lui, X. Wu, K.J. Davis, M.W.W. Adams, R. Chakraborty, D. A. Elias, R. L. Wilpiszeski, J. Zhou, M.W. Fields, T. C. Hazen, A.P. Arkin and P.D. Adams. 2020. Spatiotemporal Dynamics of Groundwater and Sediment: Geochemistry, Microbial Communities and Activities in a Contaminated Aquifer. 2020 Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Spatiotemporal variability of groundwater levels and sources could greatly impact the geochemistry and the drift, dispersal, and selection of associated microbial communities. As a pilot study, one uncontaminated site and two contaminated sites (Area 2 and Area 3) at the DOE Oak Ridge Reservation Y-12 Complex in Oak Ridge, Tennessee, were selected for bi-weekly sampling. Three groundwater wells at each site were sampled for geochemistry and microbial activity measurements. Groundwater was also filtered in succession through 10 μm, 0.2 μm and 0.1μm filters for assessment of microbial communities (16S and metagenomic). After completing five sampling time points from March to May 2019, the preliminary results of bacterial communities revealed distinct succession patterns affected by contamination and cell size. For the uncontaminated site and Area 2, total microbial cell counts ranged between 5 to 18 x 106 cells /ml and the counts for Area 3 averaged between 2 to 8 x 106 cells/ml. Cell counts for most contaminated site (Area 3) were more consistent over time when compared to the other two sites. Total microbial activity was assessed via the uptake of 3H-leucine. Activity measurements changed temporally and were an order of magnitude higher for uncontaminated wells compared to the contaminated wells (1-3 x 10-5 ng C/cell/d versus 0.6-8 x 10-6 ng C/cell/d, respectively). The changes in total microbial activity did not always correlate to changes in microbial cell numbers. These results suggest that not all microbes were active over the same times and places (i.e., mechanisms of dispersal and selection were likely impacting different populations spatiotemporally). The bacterial communities from contaminated wells (Area 2 and 3) were similar in diversity and structure compared to those in uncontaminated wells. The unique ESVs This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231 respective to each well (contaminated or uncontaminated) were typically lower in abundance compared to ESVs detected across areas and wells. The results supported environmental filtering, particularly in contaminated wells, for unique, low-abundance populations. In addition, detected ESVs from the 0.1μm filters showed decreased relative abundance of area-specific species over time, but ESVs from larger fractions (0.2μm to 10μm) did not. While larger-size bacteria from different wells always showed different community structure, small-size bacteria from different areas became convergent after late April. This implicates the influence of migration, corresponding to precipitation changes in April and May that coincided with observed changes in the dO18 values of groundwater. While a- and/or b-Proteobacteria generally dominated in larger-size bacteria, the phylum Bacteroidetes significantly increased or even predominated in the small-size fraction after late April, mainly attributed to the genus Hydrotalea. Sulfate-reducing bacteria, a relevant functional group at this site, belonged to the orders Syntrophobacterales and Desulfobacterales, which were nearly undetectable in small-size bacteria. In contrast, Rhodanobacter, a dominant genus in contaminated wells of this site, was detectable in different size fractions and showed decreased relative abundance after late April. These preliminary data demonstrated the value of more frequent sampling for an in-depth time series analysis. Starting in July 2019 a comprehensive, high-resolution time series survey of 27 wells was carried out to obtain diurnal and seasonal fluctuations within three levels (mild, moderate, and high) of nitrate and heavy metal contamination. With this data, we aim to model these areas and study changes within the attached and unattached microbial communities in relation to groundwater geochemistry. Measurements were gathered from 27 previously established groundwater wells four days/week over the span of 17 weeks (70 days total, July to December) to build both diurnal and seasonal time series. In-field geochemical measurements were obtained for dissolved oxygen (DO), pH, conductivity, oxidation-reduction potential (ORP), and nitrate concentration. Samples were also taken for metals, anions, organic acids, and total organic and inorganic C/N. Preliminary results show diurnal and seasonal changes in geochemistry with wide variations between each well and levels of contamination. Additionally, one well in each level of contamination (3 wells total) was selected to complete a “deep-dive” analysis by sampling for microbial communities in groundwater (unattached) and sediment (attached). Groundwater was filtered through 8μm and 0.2μm filters for 16S rRNA and metagenomic analysis for a total of 420 filters. In each of the three “deep-dive” wells, 18 unamended sediment traps were deployed throughout the sampling period in order to complete a time series soil analysis. The attached microbial communities and soil geochemistry will be compared to the unattached communities and groundwater geochemistry. Results for each stage of analysis will be linked to groundwater flow vectors and on-site weather data. With this data, we aim to establish a predictive systems model to understand potential distribution of microbial communities and associated activities in the shallow subsurface.
76. Tian, Renmao, Daliang Ning, Zhili He, Ping Zhang, Sarah J. Spencer, Shuhong Gao, Weiling Shi, Linwei Wu, Ya Zhang, Yunfeng Yang, Benjamin G. Adams, Andrea M. Rocha, Brittny L. Detienne, Kenneth A. Lowe, Dominique C. Joyner, Dawn M. Klingeman, Adam P. Arkin, Matthew W. Fields, Terry C. Hazen, David A. Stahl, Eric J. Alm and Jizhong Zhou. 2020. Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity. Microbiome 8. abstract
    Background The newly defined superphylum Patescibacteria such as Parcubacteria (OD1) and Microgenomates (OP11) has been found to be prevalent in groundwater, sediment, lake, and other aquifer environments. Recently increasing attention has been paid to this diverse superphylum including > 20 candidate phyla (a large part of the candidate phylum radiation, CPR) because it refreshed our view of the tree of life. However, adaptive traits contributing to its prevalence are still not well known. Results Here, we investigated the genomic features and metabolic pathways of Patescibacteria in groundwater through genome-resolved metagenomics analysis of > 600 Gbp sequence data. We observed that, while the members of Patescibacteria have reduced genomes (similar to 1 Mbp) exclusively, functions essential to growth and reproduction such as genetic information processing were retained. Surprisingly, they have sharply reduced redundant and nonessential functions, including specific metabolic activities and stress response systems. The Patescibacteria have ultra-small cells and simplified membrane structures, including flagellar assembly, transporters, and two-component systems. Despite the lack of CRISPR viral defense, the bacteria may evade predation through deletion of common membrane phage receptors and other alternative strategies, which may explain the low representation of prophage proteins in their genomes and lack of CRISPR. By establishing the linkages between bacterial features and the groundwater environmental conditions, our results provide important insights into the functions and evolution of this CPR group. Conclusions We found that Patescibacteria has streamlined many functions while acquiring advantages such as avoiding phage invasion, to adapt to the groundwater environment. The unique features of small genome size, ultra-small cell size, and lacking CRISPR of this large lineage are bringing new understandings on life of Bacteria. Our results provide important insights into the mechanisms for adaptation of the superphylum in the groundwater environments, and demonstrate a case where less is more, and small is mighty.
77. Satinover, Scott J., Miguel, Jr. Rodriguez, Maria F. Campa, Terry C. Hazen and Abhijeet P. Borole. 2020. Performance and community structure dynamics of microbial electrolysis cells operated on multiple complex feedstocks. Biotechnology for Biofuels 13. abstract
    Background Microbial electrolysis is a promising technology for converting aqueous wastes into hydrogen. However, substrate adaptability is an important feature, seldom documented in microbial electrolysis cells (MECs). In addition, the correlation between substrate composition and community structure has not been well established. This study used an MEC capable of producing over 10 L/L-day of hydrogen from a switchgrass-derived bio-oil aqueous phase and investigated four additional substrates, tested in sequence on a mature biofilm. The additional substrates included a red oak-derived bio-oil aqueous phase, a corn stover fermentation product, a mixture of phenol and acetate, and acetate alone. Results The MECs fed with the corn stover fermentation product resulted in the highest performance among the complex feedstocks, producing an average current density of 7.3 +/- 0.51 A/m(2), although the acetate fed MECs outperformed complex substrates, producing 12.3 +/- 0.01 A/m(2). 16S rRNA gene sequencing showed that community structure and community diversity were not predictive of performance, and replicate community structures diverged despite identical inoculum and enrichment procedure. The trends in each replicate, however, were indicative of the influence of the substrates.Geobacterwas the most dominant genus across most of the samples tested, but its abundance did not correlate strongly to current density. High-performance liquid chromatography (HPLC) showed that acetic acid accumulated during open circuit conditions when MECs were fed with complex feedstocks and was quickly degraded once closed circuit conditions were applied. The largest net acetic acid removal rate occurred when MECs were fed with red oak bio-oil aqueous phase, consuming 2.93 +/- 0.00 g/L-day. Principal component analysis found that MEC performance metrics such as current density, hydrogen productivity, and chemical oxygen demand removal were closely correlated. Net acetic acid removal was also found to correlate with performance. However, no bacterial genus appeared to correlated to these performance metrics strongly, and the analysis suggested that less than 70% of the variance was accounted for by the two components. Conclusions This study demonstrates the robustness of microbial communities to adapt to a range of feedstocks and conditions without relying on specific species, delivering high hydrogen productivities despite differences in community structure. The results indicate that functional adaptation may play a larger role in performance than community composition. Further investigation of the roles each microbe plays in these communities will help MECs to become integral in the 21st-century bioeconomy to produce zero-emission fuels.
78. Salim, A. A., A. Putt and T. C. Hazen. 2020. Learning and growing as a Scholar : My Experience as an Undergraduate Researcher. 1794 UTK Annual Scholars Showcase one of 20 finalists pdf
79. Salim, A. A., P. Pineda, I. Alamilla, A. Putt and T. C. Hazen. 2020. A Novel approach for Characterizing the Ultra-Micro Size-Fraction Community. EURēCA Undergraduate UTK Annual Research Meeting pdf
80. Needham, D. M., A. Zhang, J-M. Chandonia, D. Chivian, L.M. Lui, W. Zheng, S. Zhao, Y. Yin, D.A. Weitz, T.C. Hazen, P.S. Novichkov, J. Zhou, E.J. Alm, A.P. Arkin and P.D. Adams. 2020. Integrating data and algorithms from the ENIGMA project into KBase. 2020 Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. We aim to obtain novel genomes with high quality (of completeness and contamination) from ENIGMA samples through single-cell sequencing and integrate them into KBase as good references for not-yet cultured bacteria in natural environments. As sequencing becomes less expensive, researchers are turning from 16S rRNA surveys to metagenomics in order to add a new level of functional and phylogenetic resolution to their sequence-based analyses. Metagenomic data harbors additional layers of data on population structure, strain dynamics, and genome evolution that cannot be inferred from 16S alone. Yet, powerful and user-friendly tools for the analysis of this data are not publicly available. Microbial communities across nearly all biological systems, from sediment to groundwater, are diverse, dynamic ecosystems comprised of genetically diverse populations. Such diversity can be broad, as between species, as well as, within populations, as in strains of species. Typical metagenomic approaches explore this diversity in a manner than loses information the genomes of individual cells. In contrast, characterizations at the individual cell level yield information about interactions between organisms, such as between bacteria and phage, as well as strain level differences within a population. However, Public available genomes are good resources as reference for functional and taxonomy annotation, while most of them are culturable species from host-associated environments. Genomes of good quality and novelty are lacking to serve as references for not-yet cultured bacteria in natural environments. Thus, population genetic and evolutionary data analysis tools within the KBase environment and single cell sequencing of ENIGMA samples could have an outsize impact on environmental microbiology research. Here we report new functions that we add to the KBase environment to catalyze metagenomic data analysis. First, we have built a standard and comprehensive set of reference genomes to which metagenomic reads can be compared. We have designed the pipeline to compare metagenomes to references and tested it in our samples. We built the estimators of strain level nucleotide diversity, and even inference of strain genomes. We designed the tools to study within-population genome rearrangements and mutations. We published one compact tool, the meta_decoder (https://github.com/caozhichongchong/meta_decoder), that automatically identifies and compares the bacterial strains, mobile genetic elements, and phase variation across samples. We have tested meta_decoder using simulated datasets and we are now applying it to ENIGMA genomes and metagenomes. We profiled 15,343 single genomes by droplet microfluidics (Microbe-seq) of an enigma groundwater sample GW-FW-305. We assembled many genomes, including 16 high quality bacterial genomes, > 75% completion and < 5% genomic redundancy. Several genomes were >97% complete and < 1% redundant, characteristics that are unusual for traditional metagenomes from a single sample. We uploaded these novel genomes with high quality (of completeness and contamination) into KBase as good references for not-yet cultured bacteria in natural environments. We collaborate with ENIGMA data management team (John-Marc Chandonia) and KBase team (Dylan Chivian). The ENIGMA data we use is the Pseudomonas genomes from Lauren M. Lui (Arkin Lab). This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
81. Moon, Ji-Won, Charles J. Paradis, Dominique C. Joyner, Frederick von Netzer, Erica L. Majumder, Emma R. Dixon, Mircea Podar, Xiaoxuan Ge, Peter J. Walian, Heidi J. Smith, Xiaoqin Wu, Grant M. Zane, Kathleen F. Walker, Michael P. Thorgersen, Farris L., II Poole, Lauren M. Lui, Benjamin G. Adams, Kara B. De Leon, Sheridan S. Brewer, Daniel E. Williams, Kenneth A. Lowe, Miguel, Jr. Rodriguez, Tonia L. Mehlhorn, Susan M. Pfiffner, Romy Chakraborty, Adam P. Arkin, Judy D. Wall, Matthew W. Fields, Michael W. W. Adams, David A. Stahl, Dwayne A. Elias and Terry C. Hazen. 2020. Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer. Chemosphere 255. abstract
    The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated areas. This study processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater. Fresh core sediments were compared by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including pollutants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate- and nitrate-reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of potentially hazardous elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments. (C) 2020 The Author(s). Published by Elsevier Ltd.
82. Miller, John I., Stephen Techtmann, Dominique Joyner, Nagissa Mahmoudi, Julian Fortney, James A. Fordyce, Nargiz GaraJayeva, Faig S. Askerov, Claudio Cravid, Maarten Kuijper, Oliver Pelz and Terry C. Hazen. 2020. Microbial Communities across Global Marine Basins Show Important Compositional Similarities by Depth. Mbio 11. abstract
    The environmental surveys following the 2010 Deepwater Horizon (DWH) spill identified a variety of hydrocarbon-degrading microorganisms, and laboratory studies with field-collected water samples then demonstrated faster-than-expected hydrocarbon biodegradation rates at 5 degrees C. Knowledge about microbial community composition, diversity, and functional metabolic capabilities aids in understanding and predicting petroleum biodegradation by microbial communities in situ and is therefore an important component of the petroleum spill response decision-making process. This study investigates the taxonomic composition of microbial communities in six different global basins where petroleum and gas activities occur. Shallow-water communities were strikingly similar across basins, while deepwater communities tended to show subclusters by basin, with communities from the epipelagic, mesopelagic, and bathypelagic zones sometimes appearing within the same cluster. Microbial taxa that were enriched in the water column in the Gulf of Mexico following the DWH spill were found across marine basins. Several hydrocarbon-degrading genera (e.g., Actinobacteria, Pseudomonas, and Rhodobacteriacea) were common across all basins. Other genera such as Pseudoalteromonas and Oleibacter were highly enriched in specific basins. IMPORTANCE Marine microbial communities are a vital component of global carbon cycling, and numerous studies have shown that populations of petroleum-degrading bacteria are ubiquitous in the oceans. Few studies have attempted to distinguish all of the taxa that might contribute to petroleum biodegradation (including, e.g., heterotrophic and nondesignated microbes that respond positively to petroleum and microbes that grow on petroleum as the sole carbon source). This study quantifies the subpopulations of microorganisms that are expected to be involved in petroleum hydrocarbon biodegradation, which is important information during the decision-making process in the event of a petroleum spill accident.
83. Miller, J. I., Zabreena Griffiths, Stephen Techtmann, Julian Fortney, Nagissa Mahmoudi, Dominique Joyner, Jiang Liu, Scott Olesen, Eric Alm, Adolfo Fernandez, Piero Gardinali, Nargiz GaraJayeva, Faig S. Askerov, Odd Gunnar Brakstad, Oliver Pelz, Maarten Kuijper and Terry C. Hazen. 2020. Microbial Community Structure and Oil Biodegradation in a Hypoxic Marine Environment. SETAC SciCon SETAC Europe 30th Annual Meeting
84. Mahmoudi, Nagissa, Shane M. Hagen, Terry C. Hazen and Andrew D. Steen. 2020. Patterns in extracellular enzyme activity and microbial diversity in deep-sea Mediterranean sediments. Deep-Sea Research Part I-Oceanographic Research Papers 158. abstract
    Deep-sea sediments are populated by diverse microbial communities that derive their nutritional requirements from the degradation of organic matter. Extracellular hydrolytic enzymes play a key role in the survival of microbes by enabling them to access and degrade complex organic compounds that are found in seafloor sediments. Despite their importance, extracellular enzymatic activity is poorly characterized at water depths greater than a few hundred meters where physical properties, such as pressure and temperature, create a unique environment for influencing enzyme behavior. Here, we investigated microbial communities and enzyme activities in surface sediment collected at four sampling stations in the central Mediterranean Sea at water depths ranging from 800 to 2200 m. Fluorometric assays revealed that extracellular hydrolytic activity varied according to substrate type and water depth which suggests that the distributions of these enzymes within this basin are not homogenous. Furthermore, enzyme activities indicated substantial demand for phosphomonoesters and proteins, with measurable but much lower demand for polysaccharides. Barcoded amplicon sequencing of bacterial and archaeal SSU genes revealed that microbial communities varied across sampling stations and some groups displayed water-depth related trends. Our results demonstrate that heterotrophic capabilities of microbes in deep-sea Mediterranean sediments can differ substantially even within the same region.
85. Lui, L. M., H.J. Smith, F. von Netzer, K.B. De León, E.L-W. Majumder, J.V. Kuehl, F. Song, A. Sczesnak, T. Nielsen, M.P. Thorgesen, X. Ge, F.L. Poole, B.P. Bowen, S.M. Kosina, C.J. Paradis, K.F. Walker, K.A. Lowe, D.C. Joyner, Jr. M. Rodriquez, B.A. Adams, D. Williams, J-W. Moon, J.D. Van Nostrand, D. Ning, Y. Fu, W. Shi, Y. Li, D.J. Curtis, Y. Fan, L. Wu, R. Tian, G.M. Zane, A.B. Aaring, X. Wu, A.E. Kazakov, J-M. Chandonia, P.S. Novichkov, P.J. Walian, R. Chakraborty, M.W.W. Adams, J. Zhou, T.R. Northen, J.D. Wall, D.A. Stahl, D.A. Elias, T.C. Hazen, M.W. Fields, A.P. Arkin and P.D. Adams. 2020. Core Values: Spatial Variation in Microbial Function, Activity, and Community Assembly in Groundwater and Sediment from a Contaminated Subsurface Aquifer . 2020 Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) uses a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Primary motivations for studying the subsurface are to expand what is known about Earth’s microbial diversity and the subsurface microorganisms under low nutrient conditions that significantly impact C, S, N, P and mineral cycles. However, on an ecosystem scale, there is limited information regarding the exact relationship between microbial diversity, environmental parameters, and biogeochemical processes between subsurface groundwater and porous media (i.e., sediment). The Department of Energy ENIGMA Scientific Focus Area seeks to map the causal interactions that constrain microbial community assembly, functionality, and dispersal in chemically and physically complex environments. We recently initiated an in depth study of microbial distribution and activity throughout sediment and water compartments in the shallow subsurface at the Oak Ridge Field Research Center, a site of nuclear weapon development during the Manhattan Project. We hypothesize that strong gradients of pH, heavy metals, nitrate, and other contaminants at the site influence the distribution, structure, and activity of microbial communities. We performed large-scale analysis of two sediment cores and associated groundwater for which we produced depth-index data sets of physical, chemical, bulk biological and sequencing measurements. One core (466 cm) was from a region outside the area of heavy chemical contamination and the other core (815 cm) from within the contaminated area. We divided the cores into ~23 cm segments for processing, resulting in 56 segments which allowed a finer-grained analysis of the vertical transect as compared to other subsurface studies. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231 We observed differences in diversity and distribution of dominant metabolisms between the heavily contaminated and less contaminated cores. The heavily contaminated core was less diverse as measured by 16S amplicon sequencing. Approximately 250 exact sequence variants (ESVs) accounted for half the observed reads in the heavily contaminated core as compared to 660 from the uncontaminated core, suggesting strong selective pressure from contamination. In general, there is little overlap in ESVs between the two cores (~300-350 meters apart). Analysis of ESV distribution and inferred functional potential using FAPROTAX suggests that the less contaminated core has strongly ordered communities with well-defined functional zones for nitrification, denitrification, methanogenesis, and sulfate reduction, and that these functions are carried out by specific diverse subcommunities differentially distributed with depth. There was a clear interplay among communities mediating denitrification, methanogenesis, and sulfate reduction within the variably saturated zone. These metabolisms are also correlated with the presence of key chemical constraints, such as uranium, nitrate, and pH, not just location in the core. Conversely, the heavily contaminated core has a far more heterogenous population structure with little evident intersegmental transfer (i.e., little mixing between adjacent layers). Within the heavily contaminated core we observed two distinct cosmopolitan communities: (1) a large and diverse community enriched in denitrifying organisms and (2) a less diverse highly abundant community not clearly enriched in any metabolism. We found that the ENIGMA isolate collection is not representative of all of the organisms in the cores, but in some cases we have greater than 80% coverage of the strains present in a subcommunity. Based on the isolates we have and the strong chemical and physical correlates to specific community compositions, we are currently in the process of generating enrichments and synthetic communities representative of each of the identified subcommunities. From activity assays, we identified a competitive exclusion of sulfate reduction and denitrification; species representative of these metabolisms are found and enriched in different locations in the core. This exclusion was further supported by shotgun metagenomics. From 22 groundwater and 91 sediment shotgun metagenomes, we have successfully circularized 8 genomes and have over 50 genomes with >98% completeness and <2% contamination. We are tracking these genomes with depth in the core and analyzing the potential functional roles of these organisms in denitrification and sulfate reduction. To distinguish intact and potentially viable cells from “relic” DNA, we used complementary culture-independent methods to determine respiratory rates and identify the active fractions of microbial assemblages from groundwater and sediment. Groundwater from the less contaminated area had higher diversity and three- to four-fold higher activity than heavily contaminated samples. Additionally, in the less contaminated area, the activity on a per cell basis was two to three-fold greater for planktonic cells compared to particle associated organisms, with small cells (<0.1μm) contributing up to 19% of total activity. Conversely, in heavily contaminated aquifers, activity was greater for sediment-associated cells. To understand the distribution of the active ESVs across the ORNL landscape we are analyzing the top active ESVs and checking their abundance in historic 16S rRNA sequence data from nearly 100 wells from ORFRC. This study integrates over 12 measures of microbial community composition, activity, and environmental controls to provide new insights into how contamination impacts the distribution of activity between attached and planktonic populations in subsurface microbial communities. The application and refinement of in situ measurements spanning different scales will aid in the development predictive frameworks for understanding large scale biogeochemical cycling from groundwater environments.
86. Lui, L. M., T. Nielsen, H.J. Smith, F. von Netzer, E.L-W. Majumder, J.V. Kuehl, F. Song, A. Sczesnak, M.P. Thorgesen, X. Ge, F.L. Poole, C.J. Paradis, K.F. Walker, K.A. Lowe, D.C. Joyner, D. Ning, Jr. M. Rodriquez, A.B. Aaring, B.A. Adams, D. Williams, J.D. Van Nostrand, G.M. Zane, M.W.W. Adams, J. Zhou, R. Chakraborty, J.D. Wall, D.A. Stahl, T.C. Hazen, M.W. Fields, AP Arkin and PD Adams. 2020. A Method for Circularizing Microbial Genomes from Metagenomics Data . 2020 Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Metagenomics facilitates the study of the genetic information from unculturable microbes and complex microbial communities, but achieving complete microbial genomes (i.e., circular) from metagenomics data is difficult because of inherent qualities of short read sequencing data and assembly and binning methods currently available. To our knowledge, only 62 circularized genomes have been assembled from metagenomics data despite the thousands of datasets that are available. We believe that circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a method to achieve circularized genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one copy of the ribosomal RNA genes. From our work, we present 42 CPR genomes and one Margulisbacteria genome from 19 published datasets and from ENIGMA sequencing of sediment and groundwater samples from Oak Ridge National Lab Field Research Center. We demonstrate findings that would likely be difficult without circularized genomes, including that ribosomal genes are likely not operonic in the majority of CPR, diverged forms of RNase P in CPR, and presence of megaplasmids in the datasets. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
87. Hunt, K. A., A.V. Carr, K.F. Walker, E.R. Dixon, M. Rodriguez Jr, K.A. Lowe, D.C. Joyner, A.E. Otwell, S.D. Wankel, N.S. Baliga, T.C. Hazen, D.A. Stahl, A.P. Arkin and P.D. Adams. 2020. High nitrous oxide emissions from a nitrate contaminated subsurface indicate significant metabolic activity.. 2020 Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Linking field observations with laboratory studies and vice versa is essential for advancing predictive understanding of environmental systems and for stewardship of those systems. However, some environments that provide critical ecosystem services, such as the subsurface and groundwater systems, are extremely difficult to sample and monitor in real-time, and doing so is both expensive and invasive. Thus, nondestructive approaches to process analyses are essential tools for connecting lab and environment. To this end, we are developing protocols that use the flux and isotopic composition of the greenhouse gas nitrous oxide to resolve alternative biotic (e.g., nitrification and denitrification) and abiotic processes, such as iron catalyzed reduction of nitrite, that contribute to its emission from the subsurface. ENIGMA has developed a program to connect measurements of nitrous oxide to biologically mediated processes through field observations (For more information on ENIGMA field observations see “Spatiotemporal Dynamics of Groundwater and Sediment: Geochemistry, Microbial Communities and Activities in a Contaminated Aquifer” by Walker et. al.) and laboratory simulations (For more information on ENIGMA lab to field plan see “A Multi-Laboratory Effort to Use Synthetic Communities to Discover, Characterize, and Dissect Key Microbial Processes Relevant to Field Observations” by Valenzuela et. al.), thereby establishing a noninvasive metric for quantifying activity without destructive sampling. Contamination by nitrogen species is a concern in many terrestrial and aquatic environments impacted by past and current human activities, including release associated with intensive agriculture and industrial activity, and from wastewater treatment. This contamination has been shown to lead to altered plant, animal, and microbial communities and to increased production of the greenhouse gas nitrous oxide, primarily through either nitrification or denitrification. The subsurface of the Field Research Center (FRC), near Oak Ridge National Lab in Tennessee, has been contaminated with low pH (3-7), heavy metal laden nitrate (~10 g/l) for decades. To understand how this contamination has influenced subsurface processes we are investigating environmental variables influencing nitrous oxide emissions, considering both biotic and abiotic contributions to this important greenhouse gas. By using flux analysis and isotopic characterization of nitrous oxide, combined with complementary molecular and chemical characterization of multiple observation wells, we anticipate developing a more predictive understanding of the controlling variables. Current analyses are focused on wells positioned at different depths and spanning a range of pH, nitrate contamination, and metals contamination to resolve biotic and abiotic sources of production and to identify controlling environmental variables. Initial data sets have revealed that subsurface fluxes are orders of magnitude higher than those observed in other systems, including agricultural soils and the marine oxygen minimal zone. In contrast, the observed surface fluxes are in the range observed for other sites, indicating additional consumptive processes within the vadose zone that mitigate surface emissions. Funding statement: ENIGMA is a Scientific Focus Area Program at Lawrence Berkeley National Laboratory and is supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231
88. Hazen, T. C.. 2020. SARS CoV-2 Watewater Survielance at the University of Tennessee Knoxville. Optimists Club
89. Gushgari-Dolye, S. A. P. Arkin L. M. Lui R. Chakraborty T. C. Hazen X. Wu. 2020. Functional Diversity of Arthrobacter Strains Across the Dynamic Capillary Fringe and Adjacent Sediment Zones. AGU Annural Meeting
90. Ge, Xiaoxuan, Michael P. Thorgersen, Farris L., II Poole, Adam M. Deutschbauer, John-Marc Chandonia, Pavel S. Novichkov, Sara Gushgari-Doyle, Lauren M. Lui, Torben Nielsen, Romy Chakraborty, Paul D. Adams, Adam P. Arkin, Terry C. Hazen and Michael W. W. Adams. 2020. Characterization of a Metal-Resistant Bacillus Strain With a High Molybdate Affinity ModA From Contaminated Sediments at the Oak Ridge Reservation. Frontiers in Microbiology 11. abstract
    A nitrate- and metal-contaminated site at the Oak Ridge Reservation (ORR) was previously shown to contain the metal molybdenum (Mo) at picomolar concentrations. This potentially limits microbial nitrate reduction, as Mo is required by the enzyme nitrate reductase, which catalyzes the first step of nitrate removal. Enrichment for anaerobic nitrate-reducing microbes from contaminated sediment at the ORR yielded Bacillus strain EB106-08-02-XG196. This bacterium grows in the presence of multiple metals (Cd, Ni, Cu, Co, Mn, and U) but also exhibits better growth compared to control strains, including Pseudomonas fluorescens N2E2 isolated from a pristine ORR environment under low molybdate concentrations (<1 nM). Molybdate is taken up by the molybdate binding protein, ModA, of the molybdate ATP-binding cassette transporter. ModA of XG196 is phylogenetically distinct from those of other characterized ModA proteins. The genes encoding ModA from XG196, P. fluorescens N2E2 and Escherichia coli K12 were expressed in E. coli and the recombinant proteins were purified. Isothermal titration calorimetry analysis showed that XG196 ModA has a higher affinity for molybdate than other ModA proteins with a molybdate binding constant (K-D) of 2.2 nM, about one order of magnitude lower than those of P. fluorescens N2E2 (27.0 nM) and E. coli K12 (25.0 nM). XG196 ModA also showed a fivefold higher affinity for molybdate than for tungstate (11 nM), whereas the ModA proteins from P. fluorescens N2E2 [K-D (Mo) 27.0 nM, K-D (W) 26.7 nM] and E. coli K12[(K-D (Mo) 25.0 nM, K-D (W) 23.8 nM] had similar affinities for the two oxyanions. We propose that high molybdate affinity coupled with resistance to multiple metals gives strain XG196 a competitive advantage in Mo-limited environments contaminated with high concentrations of metals and nitrate, as found at ORR.
91. Ge, Xiaoxuan, Michael P. Thorgersen, Farris L., II Poole, Adam M. Deutschbauer, John-Marc Chandonia, Pavel S. Novichkov, Paul D. Adams, Adam P. Arkin, Terry C. Hazen and Michael W. W. Adams. 2020. Draft Genome Sequence of Bacillus sp. Strain EB106-08-02-XG196, Isolated from High-Nitrate-Contaminated Sediment. Microbiology Resource Announcements 9. abstract
    Bacillus sp. strain EB106-08-02-XG196 was isolated from a high-nitrate- and heavy metal-contaminated site at the Oak Ridge Reservation in Tennessee. We report the draft genome sequence of this strain to provide insights into the genomic basis for surviving in this unique environment.
92. Fuqai, I. and Terry C. Hazen. 2020. Biosensors for Detecting Nuclear Production Activity in the Environment. MTV abstract
    Previous studies at the Y-12 S-3 Ponds legacy contamination site demonstrated the microbial community structure significantly predicted 18 different geochemical parameters in the ground water. These included contaminants from the nuclear production activities done at Y-12 more than 40 years ago ie. U, pH, nitrate, Cr, Sr, Pb, Cd, etc. We are further refining these models using higher resolution metagenomic and metatranscript analysis for our Random Forest, Monte Carlo models. We are also sampling groundwater and sediment both horizontally and vertically along groundwater plume transects to determine if these models also predict when these contaminants first appeared. In the near future we plan on doing similar studies of contaminant plumes from P and R nuclear reactor basins and canals at the Savannah River Site. We will also add on sampling of surface vegetation and microbial rhizosphere to see if this also allows significant prediction of nuclear production activities. Several other sites are also being considered for sampling including around the High Flux Isotope Reactor at ORNL and sites at Los Alamos, Hanford, Idaho.
93. Chakraborty, R., Xiaoqin Wu, Dominique C. Joyner, Terry C Hazen, Ria Gracielle Malana, Adam P. Arkin and Paul D. Adams. 2020. Applying Stable Isotopes for Source Fingerprinting of Dissolved Organic Nitrogen in Groundwater. 2020 Genomic Sciences Program (GSP) Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. In this project, we aim to: 1) differentiate the multiple sources of dissolved organic nitrogen (DON) in FRC contaminated groundwater via isotopic and elemental analysis; 2) quantify the contributions of DON in FRC contaminated groundwater during four successive seasons using stable isotope analysis in R (SIAR) mixing model; 3) verify the impact of source on labile fractions of DON in groundwater. Abstract DON constitutes a major pool of dissolved N in many aquatic ecosystems, playing an important role in biogeochemical cycling of both carbon (C) and N. Our recent work on natural organic matter at FRC indicated that dissolved organic nitrogen (DON) contributes to more than 50% of dissolved N pool in uncontaminated sediments, serving as an important source of C and N for microbes when labile C is limiting. Although considerable research has been carried out on dissolved inorganic N (DIN) transformation at FRC, the role of DON is largely unknown and has been overlooked thus far. Variations in source determine the quantity and quality of DON, therefore greatly affecting the microbial turnover of DON, and resultant microbial community structure. Stable isotope signature at natural abundance is a powerful tool in source fingerprinting of bulk C and N in soil and sediment, as well as nitrate in aquatic environments. However, only a handful of studies have been reported on isotopic survey of DON in marine, lake, and soil environments, none available for groundwater. To obtain baseline information of DON in Oak Ridge FRC groundwater, we investigated the quantity and quality of dissolve organic matter (DOM) in groundwater during an ENIGMA 2- month sampling campaign, carried out in spring 2019 (mid March to mid May) at both FRC uncontaminated background area and nitrate contaminated area. Both DON and DOC content in nitrate-contaminated wells during late campaign period (mid April to mid May) was significantly lower (p < 0.05) than those during early campaign period. This suggests the existence of a temporal fluctuation of DON quantity in FRC groundwater especially at nitrate-contaminated area, and the fluctuation can likely be explained as dilution by water from melting existing snowpack on ground. DOM quality in groundwater changed during this period as well. We used the ratio of ON to OC (ON/OC) as an indicator of DOM quality. During late campaign period (mid April to mid May), the ON/OC decreased in nitrate-contaminated groundwater, indicating that water input from melting snowpack potentially introduces different types of DOM (N-poor molecules) to groundwater. Following up on these observations we are developing a reliable analytical method to measure N isotope signature of trace level of DON in groundwater. Currently, there is no well-established method for directly measuring δ15N-DON in environmental water sample. We are testing solidphase extraction based methods to concentrate trace level of DON from groundwater and to eliminate inorganic N such as nitrate and ammonium. We are testing different sorbents, and will apply Elemental Analyzer-Isotope Ratio Mass Spectrometry (EA-IRMS) for measuring δ15NDON in the solid sorbents. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
94. Walker, K. F., K. A. Lowe, B. G. Adams, E. R. Dixon, D. C. Joyner, M. Rodriguez Jr., M. W. Fields, D. A. Elias, T. C. Hazen, A. P. Arkin and P. D. Adams. 2019 . ENIGMA: Long-Term Continuous Monitoring Gives Insight into Patterns Between Groundwater and Weather Events. DOE 2019 Genomic Sciences Program Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. The main goals of this project are to use long-term continuous monitoring to understand the fluctuations seen in the microbial communities and geochemistry of groundwater over time, including diurnal, seasonal and annual time scales. Abstract: To take an environmental systems approach to studying the microbial communities found in groundwater and sediment, we began long-term continuous monitoring as a way to understand how ecosystem phenomena were playing a role in these microscopic environments. To do this, we deployed two down-well groundwater monitoring units (In-Situ AquaTroll600) and set up two meteorological stations (HOBO U30) within contaminated and uncontaminated research field sites at Y-12 National Security Complex in Oak Ridge, Tenn. After monitoring the groundwater and weather at these sites, we have seen interesting trends within the data. There are natural diurnal fluctuations in the groundwater elevation, relative humidity and air temperature. We also see increases in groundwater elevation and changes in groundwater geochemistry (dissolved oxygen, pH, conductivity) following heavy rain events. However, there is a distinct delay between the detected rainfall and a rise in the groundwater elevation. We will begin monthly sampling of groundwater in the spring to detect the fluctuations in microbial community and geochemistry in these sites. Our ENIGMA team members are developing a sampling plan for sediment coring in the coming months. With the continued use of these units and the collection of groundwater and sediment, we are hoping to capture the extended, especially annual and seasonal, relationships between all of these parameters and their effect on the microbial community. This data is also being used to guide future scientific focus on meteorological events of interest, seasonal focus for sampling and geochemical variations over time. Funding Statement: This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
95. Ning, D., R. Tian, J. D. Van Nostrand, L. Wu, P. Zhang, W. Shi, L. Wu, Y. Zhang, Y. Yang, D. J. Curtis, Z. He, M. B. Smith, A. M. Rocha, C. S. Smillie, S. W. Olesen, C. J. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, D. A. Elias, K. L. Bailey, R. A. Hurt Jr., S. P. Preheim, M. C. Sanders, J. Yang, M. A. Mueller, W. A. Lancaster, B. J. Vaccaro, F. L. Poole II, M. W. Fields, E. J. Alm, T. C. Hazen, M. W. W. Adams, P. D. Adams, A. P. Arkin and J. Zhou. 2019 . ENIGMA: Assembly Mechanism of Subsurface Microbial Community under Stress Gradient and Adaptation of Super Phylum Patescibacteria with Genome Simplicity. DOE 2019 Genomic Sciences Program Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies mission is to support the development of laboratory and computational tools that link the molecular functions within individual microbes to the integrated activities of microbial communities as they interact with their environment. Our goal is to understand how human activity associated with energetic processes - in particular, contamination and climate change - is affecting the ecology of critical soil, groundwater, and aquifer systems. Community assembly mechanism under a stress gradient is a fundamental question in ecology. Classical stress gradient theory discussed the roles of different deterministic forces under different degrees of stresses, however we still know little about the relative importance of various stochastic and deterministic processes along stress gradients. The groundwater in the Oak Ridge Integrated Field Research Challenge site (FRC, Oak Ridge, TN) has large geochemical gradients and has been comprehensively surveyed, providing a rare opportunity to examine ecological processes and drivers shaping subsurface microbial diversity. Groundwater samples were taken from 98 wells that covered the geochemical diversity across the site. The 16S rDNA were sequenced for all samples by Illumina MiSeq and metagenomic shotgun sequencing were performed for 12 representative samples by Illumina HiSeq. We applied various approaches to disentangle the ecological processes controlling community assembly. Since obvious limitation of previous methods, we developed two new approaches to infer community assembly mechanisms. The first one is a general framework to estimate ecological stochasticity with a new index, normalized stochasticity ratio (NST). Another one is a quantitative framework to estimate relative influence of major ecological processes, respecting the fact that different microorganisms can be under different assembly mechanisms. We tested the index with simulated communities and demonstrated substantial improvement of quantitative and qualitative performance of the new approaches. We applied these new approaches to investigate how ecological stochasticity and different ecological processes varied along stress gradients at FRC. The results suggested a decrease of ecological stochasticity with the increase of environmental stress, consistent with multivariate analysis. The influence of heterogeneous selection sharply increased around 3 fold from low-stress to high-stress wells, related to chemical stresses imposed by abnormal pH, nitrate, carbon limitation, uranium, and some other metals. Dispersal limitation appeared as the most influential process (42% on average), corresponding to the strong limitation of microbial migration in groundwater system. Dispersal limitation becomes less important under higher stresses, in accordance with the connectivity of groundwater among highly contaminated wells, and thus significantly correlated with pH and some metals in the supernatant rather than pellet-associated metals. In addition to community assembly mechanism variation, we also observed members from an uncultivated superphylum Patescibacteria, which was found prevalent in subsurface environments, but the mechanisms for its prevalence are not known. The genomic features and metabolisms of this super phylum were investigated through genome-resolved metagenomics analysis. While the members of Patescibacteria had reduced genomes (~1 Mbp) exclusively, our results demonstrated they retained functions essential to growth and reproduction such as genetic information processing. Surprisingly, they have sharply reduced redundant and unessential functions of metabolisms, cellular activities and stress responses. The Patescibacteria have ultra-small cell sizes and simplified membrane structures including flagellar assembly, transporters and two-component systems. Despite the lack of CRISPR viral defense, the bacteria could have alternative strategies to evade predation such as lacking phage receptors in membranes, which may explain the lack of phage-related proteins detected in the genomes. By establishing the linkages between bacterial features and the groundwater environmental conditions, we noticed that the adaptation of Patescibacteria to the environment could drive the features of a reduced genome, ultra-small cell size and lack of CRISPR viral defense. Altogether, the contamination stress gradient significantly switched the microbial community assembly mechanism and the special environment of FRC groundwater led to adaption of the superphylum Patescibacteria with genome simplicity. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231
96. Mayes, M. A., Julia Brenner, Jana Phillips, Ryan K. Quinn, Carla López Lloreda, Brian Yudkin, Maria Fernanda Campa, Debjani Sihi, Yang Song, Terry C. Hazen, Jianqiu Zheng, Christine O’Connell, Whendee Silver and Brent Newman. 2019 . Topographic Controls over Greenhouse Gas Emissions from Puerto Rican Tropical Rainforest Soils. DOE 2019 Genomic Sciences Program Annual Principal Investigator (PI) Meeting abstract
    Humid tropical forests emit greenhouse gases including methane into the atmosphere, and a large proportion of tropical gas emissions are due to soil microbial respiration. Landscape position exerts key controls over emissions of carbon dioxide and methane, and the proportion of redox-active compounds like nitrate, sulfate, and iron in soil water. I will present lab- and field-based measurements of soils from a 6-point valley to ridgetop transect in the El Yunque National Forest in Puerto Rico, over the time frame before and after Hurricanes Irma and Maria. Surface soils were collected seasonally to determine basic soil characteristics and 16S ribosomal RNA gene sequencing. Soil water was collected following rainstorms using rhizon samplers at depths of 10 and 30 cm and analyzed for anions, cations, pH, and dissolved organic carbon and nitrogen. Soils were also incubated to quantify carbon dioxide (CO2) and methane (CH4) emissions under oxic, anoxic, and fluctuating conditions. Finally, field-scale measurements of CO2 and methane CH4 were performed resulting in strong topographic gradients in gas emissions. This poster will summarize key findings from a variety of observations with a focus on building a comprehensive understanding of differences as a function of topographic gradient, in order to build and test a model that considers both the geochemistry and microbiology of the soil environment.
97. Lui, L. M., H. J. Smith, F. von Netzer, K. B. De León, E. L-W. Majumder, J. V. Kuehl, F. Song, A. Sczesnak, T. Nielsen, M. P. Thorgesen, X. Ge, F. L. Poole II, B. P. Bowen, S. M. Kosina, C. J. Paradis, K. F. Walker, K. A. Lowe, D. C. Joyner, M. Rodriquez Jr, B. Adams, D. Williams, J.-W. Moon, J. D. Van Nostrand, D. Ning, Y. Fu, W. Shi, Y. Li1, D. J. Curtis, Y. Fan, L. Wu, R. Tian, G. M. Zane, A. B. Aaring, X. Wu, A. E. Kazakov, J.-M. Chandonia, P. S. Novichkov, P. J. Walian, R. Chakraborty, M. W. W. Adams, J. Zhou, T. R. Northen, J. D. Wall, D. A. Stahl, D. A. Elias, T. C. Hazen, M. W. Fields, A. P. Arkin and P. D. Adams. 2019 . ENIGMA: Core Values: Large-Scale Analysis of Environmental Constraints on Microbial Community Assembly, Activity, and Dispersal in Groundwater and Sediment from a Contaminated Subsurface Aquifer. DOE 2019 Genomic Sciences Program Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Abstract: Subsurface microorganisms play important roles in mediating major biogeochemical cycles, but only in recent years have studies shed light on their population structure, biogeography, and metabolisms present. The Department of Energy ENIGMA Scientific Focus Area seeks to map the causal interactions that constrain microbial community assembly and dispersal in chemically and physically complex environments. Recently, we initiated a study of microbial communities in the shallow subsurface of a contaminated aquifer at the Oak Ridge Field Research Center, a site of nuclear weapon development during the Manhattan Project. We hypothesize that strong gradients of pH, heavy metals, nitrate, and other contaminants at the site influence the distribution, structure, and activity of microbial communities. To study community assembly mechanisms, we performed large-scale analysis of two sediment cores and associated groundwater for which we produced depth-index data sets of physical, chemical, bulk biological and sequencing measurements. One core is considered uncontaminated (466 cm), and the other core (815 cm) is contaminated by chemicals from man-made processes. We divided the cores into ~23 cm segments for processing, resulting in 56 segments which allowed us to do a finer-grained analysis of the vertical transect as compared to other subsurface studies. The contaminated core is much less diverse as 250 exact sequence variants (ESVs) from 16S amplicon sequencing account for 50% of observed reads as compared to 660 from the uncontaminated core, suggesting strong selective pressure from contamination. Initial analysis of ESV location suggests that there is little mixing and dispersal along a core. In general, there is little overlap in ESVs between the two cores (~300-350 meters apart). Many of the chemical and physical measures are strongly dependent on depth and highly colinear; these colinear groups are strongly predictive of the ESV group distribution and their measured activity although not all observed groups are well predicted. Currently, we are processing shotgun metagenomics data to compare taxonomy and genes to our other data. From the water and sediment data, we have over 50 genomes with >98% completeness and <2% contamination. We are tracking these genomes through the vertical length of the core and analyzing the potential functional roles of these organisms. This study integrates over 12 measures of microbial community composition, activity, and environmental controls to provide new insights into subsurface microbial communities. Funding statement: ENIGMA is a Scientific Focus Area Program at Lawrence Berkeley National Laboratory and is supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
98. Ge, X., M. P. Thorgersen, F. L. Poole II, E. L. Majumder, G. M. Zane, K. B. De León, J. Moon, C. J. Paradis, F. von Netzer, D. A. Stahl, J. M. Chandonia, P. S. Novichkov, A. M. Deutschbauer, J. D. Wall, T. C. Hazen, M. W. W. Adams, A. P. Arkin and P. D. Adams. 2019 . ENIGMA: Characterization of Microbial Strains from Contaminated Groundwater and from Contaminated Sediments Using Environmental Concentrations of Metals at the Oak Ridge Reservation. DOE 2019 Genomic Sciences Program Annual Principal Investigator (PI) Meeting abstract
    Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. The goal is to identify mechanisms that enable the growth of microbial communities in nitrate-, metal-contaminated and molybdenun-limited environments. A strategy was employed to isolate microorganisms from the ORR contaminated environment using environmentally relevant enrichment conditions. Strains of interest were then selected from the isolates using high throughput growth and biochemical assays. Mechanisms of resistance are being characterized in the strains of interest using genome sequencing, RB-TnSeq, metallomics and metabolomics approaches, in order to understand the role that they play in communities within this special ORR contaminated environment. Abstract: The contamination plumes surrounding the S-3 ponds at Oak Ridge Reservation (ORR) are an extreme environment that has elevated concentrations of nitrate (up to 233 mM) and multiple metals including Al (up to 20.7 mM), Mn (up to 3.1 mM), U (up to 576 μM), and Ni (up to 157 μM). We hypothesize that microorganisms that live in this hostile environment have nitrate reduction and metal resistance mechanisms with unique properties that enable them to survive here. The goal of this project is to isolate microorganisms under environmentally relevant conditions from ORR contaminated groundwater and sediment, and to characterize the nitrate reduction and metal resistance properties of these isolates. To this end, comprehensive elemental analysis of over 50 elements was performed on two sediment cores, one outside and one within the contamination plumes, revealing differences in concentrations and populations of metals between the two locations. A mix of multiple metals approximating the contaminated environment was then used in a series of high-throughput enrichments to isolate microorganisms from both ORR groundwater and sediment. In depth characterization of selected isolates have uncovered a diversity in pH preference, carbon source utilization, metal tolerance, and nitrate utilization properties between the isolates. This includes metal resistant isolates capable of concentrating multiple metals on the cell surface, and an isolate, XG196, that is capable of efficiently reducing nitrate when growing in low pM concentrations of molybdenum, a metal required in the catalytic center of nitrate reductase. Further in-depth characterization of the isolates is underway using multiple avenues including the development of a genetic system for MT124, construction of RB-TnSeq libraries for MT58 and other isolates, and genome sequencing of the strains. Also, compilation of the sequencing and characterization data in KBase will aid in further analysis of the isolates for pathways of interest. Finally, This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231 understand the role and impact of these isolates at ORR, a cloud-based charting and mapping to fully system called Google Fusion Tables was created by integration of ENIGMA’s previous 100- survey topography, geochemical, biological and isolate-specific data streams. This could be used to rapidly analyze isolates, sequencing, and/or geochemical data using custom geospatial maps in a very user-friendly way.
99. Zhang, A. N., J.-M. Chandonia, S. Zhao, A. E. Kazakov, P. S. Novichkov, W. Zheng, D. A. Weitz, N. S. Baliga, A. M. Deutschbauer, M. W. Fields, T. C. Hazen, T. R. Northen, J. D. Wall, M. W. W. Adams, M. Auer, K. Bender, G. Butland, R. Chakraborty, D. A. Elias, A. Mukhopadhyay, A. K. Singh, G. E. Siuzdak, D. A. Stahl, P. J. Walian, J. Zhou, E. J. Alm, A. P. Arkin and P. D. Adams. 2019. Building a reference-based metagenomics workflow in Kbase. ENIGMA Annual Retreat
100. Zeng, L., D. Parkinson, D. A. Elias, R. Chakraborty, T. C. Hazen and P. J. Walian. 2019. Characterization of Oak Ridge Reservation Sediment by Micro-Computerized Tomography. ASM Microbe 2019 abstract
     A key aim of the Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) Department of Energy scientific focus area program, is to develop fundamental understanding of functional mechanisms utilized by microbial communities in the unique environment of the ENIGMA Field Research Center (FRC) at the Oak Ridge Reservation (ORR). This knowledge is expected to facilitate development of models capable of predicting the response of these communities to changes in the environment. To obtain details of the geophysical context in which these communities and other biological constituents are found, as well as insight into their distribution, we are using micro-computerized tomography (micro-CT) to image and characterize the structure of intact sediment samples isolated from the FRC-ORR. Here we describe methods used for sample preparation, data collection and processing. We also present results derived from a study of sediment core samples from several regions and depths within the FRC-ORR. Using the micro-CT beamline at the Lawrence Berkeley National Laboratory, Advanced Light Source, sample volumes of about 2 mm3 can be surveyed at a resolution of ~0.7 mm. With this configuration, a range of particle types and sizes can be discerned. Observable features include metals and clay minerals, water pore regions, plant and fungal matter, larger microorganisms and candidate cell clusters. Sediment porosity can be estimated from analysis of the collected image volumes. The tunable X-ray source of the micro-CT beamline can be used to help identify select elements within a sediment sample. Results from these studies will be integrated with the other biophysical, biochemical and genomic measurements obtained from corresponding sediment samples by other teams within the ENIGMA program. Future work will include modification of sediment holders to improve moisture retention and reduce sample movement, and the investigation of reagents to enhance the contrast of biological constituents within a sediment sample.
101. Zeng, L, V. Munoz, D. Joyner, A. Putt, X. Wu, R. Chakraborty, T. C. Hazen and P. J. Walian. 2019. Image-based Characterization of OR.R-FRC Integrated Survey Groundwater. ENIGMA Annual Retreat
102. Zelaya, Anna J., Albert E. Parker, Kathryn L. Bailey, Ping Zhang, Joy Van Nostrand, Daliang Ning, Dwayne A. Elias, Jizhong Zhou, Terry C. Hazen, Adam P. Arkin and Matthew W. Fields. 2019. High spatiotemporal variability of bacterial diversity over short time scales with unique hydrochemical associations within a shallow aquifer. Water Research 164. abstract
     Understanding microbial community structure and function within the subsurface is critical to assessing overall quality and maintenance of groundwater; however, the factors that determine microbial community assembly, structure, and function in groundwater systems and their impact on water quality remains poorly understood. In this study, three shallow wells (FW301, FW303, FW305) in a non contaminated shallow aquifer in the ENIGMA-Oak Ridge Field Research Center (Oak Ridge, TN) were sampled approximately 3 times a week over a period of three months to measure changes in groundwater geochemistry and microbial diversity. It was expected that the sampled microbial diversity from two historic field wells (FW301, FW303) would be relatively stable, while diversity from a newer well (FW305) would be less stable over time. The wells displayed some degree of hydrochemical variability over time unique to each well, with FW303 being overall the most stable well and FW301 being the most dynamic based upon dissolved oxygen, conductivity, and nitrate. Community analysis via ss-rRNA paired-end sequencing and distribution-based clustering revealed higher OTU richness, diversity, and variability in groundwater communities of FW301 than the other two wells for diversity binned over all time points. Microbial community composition of a given well was on average > 50% dissimilar to any other well at a given time (days), yet, functional gene diversity as measured with GeoChip remained relatively constant. Similarities in community structure across wells were observed with respect to the presence of 20 shared bacterial groups in all samples in all wells, although at varying levels over the tested time period. Similarity percentage (SIMPER) analysis revealed that variability in FW301 was largely attributed to low abundance, highly-transient populations, while variability in the most hydro chemically stable well (FW303) was due to fluctuations in more highly abundant and frequently present taxa. Additionally, the youngest well FW305 showed a dramatic shift in community composition towards the end of the sampling period that was not observed in the other wells, suggesting possible succession events over time. Time-series analysis using vector auto-regressive models and Granger causality showed unique relationships between richness and geochemistry over time in each well. These results indicate temporally dynamic microbial communities over short time scales, with day-to-day population shifts in local community structure influenced by available source community diversity and local groundwater hydrochemistry. (C) 2019 Elsevier Ltd. All rights reserved.
103. Xue, J., E. L.-W. Majumder, T. Huan, E. M. Forsberg, A. D. Putt, T. C. Hazen, J. D. Wall and G. Siuzdak. 2019. Activity of Sulfur-Containing Metabolites in Regulating Bacterial Respiration. ENIGMA Annual Retreat
104. Wu, X., L. Lui, Y. Liu, N. Justice, T. Simmons, T. Nielsen, S. Jagadamma, N. J. Hess, T. C. Hazen, A. P. Arkin and R. Chakraborty. 2019. Insights into the Depth-resolved Geochemical Constraints on Microbial Community Structure and Metabolic Potential for Carbon Cycling in Shallow Subsurface Sediment. AGU Fall Meeting.
105. Wiscovitch Russo, R., Y. Narganes-Storde, T. C. Hazen and G. A. Toranzos. 2019. 16SrRNA comparative analysis of Vieques and Guayanilla coprolites. ASM Microbe 2019 abstract
     Background: Huecoid and Saladoid were pre-Columbian indigenous cultures of Puerto Rico. The Huecoid and Saladoid cultures coexisted in Sorcé Vieques, whereas the Tecla I Site in Guayanilla was solely inhabited by the Saladoid Culture. Both sites are estimated at a distance of 140km from each other. Methods: The cores of five human coprolites (dated approximately 1,400 to 1,800 years before present) were used for DNA extraction followed by amplicon based sequencing the V4 region using the Caporaso 16S rRNA primers, data analysis was performed using QIIME, R graphical interphase and MEGAN resulting in the representative fecal microbiome profiles. Results: Significant differences were observed between Vieques and Guayanilla microbiomes. The coexisting cultures of Vieques resulted in similar profiles with Actinobacteria being the most abundant phylum. The Saladoid culture from Guayanilla observed a higher abundance of Firmicutes, overall the fecal microbial profile was distinct from that of Vieques cultures. Interestingly, some non-enteric opportunistic pathogens were present in some coprolites, namely; Bulkholderia spp., Chlamidiales, and Leptospirales. Treponema sequences were also found, most likely belonging to T. denticola. According to Shannon Diversity Index, the Huecoid culture has the highest diversity whereas the Saladoid from Vieques and Guayanilla had a low diversity. Principal Coordinate Analysis and UPGMA hierarchical clustering method positioned the Vieques microbiome apart from that of Guayanilla. Conclusion: The differences in microbiome profiles between the inhabiting cultures of Vieques and Guayanilla can potentially be attributed to the cultures life-style adapted according to the geographic region. Most importantly, the data from one specific ethnic group from two different geographical sites showing different microbiomes indicate the plasticity in the human gut microbiome.
106. Wilpiszeski, R. L., Caitlin M. Gionfriddo, Ann M. Wymore, Ji-Won Moon, Kenneth A. Lowe, Mircea Podar, Dominique C. Joyner, Craig C. Brandt, Anthony V. Palumbo, Matthew W. Fields, Terry C. Hazen, Judy D. Wall, Nitin S. Baliga, David A. Stahl, Michael W. W. Adams, Farris Poole III, Romy Chakraborty, Yupeng Fan, Joy D. Van Nostrand, Jizhong Zhou, Dwayne A. Elias, Adam P. Arkin and Paul D. Adams. 2019. ENIGMA: Using in-field bioreactors to monitor microbial community dynamic shifts with geochemical perturbations. DOE 2019 Genomic Sciences Program Annual Principal Investigator (PI) Meeting abstract
     Project Goals: ENIGMA -Ecosystems and Networks Integrated with Genes and Molecular Assemblies use a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Abstract Subsurface microbial communities mediate the transformation and fate of redox-sensitive compounds including organic matter, metals and radionuclides. Few studies have explored how changing geochemical conditions influence the composition of groundwater microbial communities. As part of the ENIGMA Environmental Ark campaign, we used 1L in-field bioreactors receiving background and contaminated well water from the Department of Energy site at the Oak Ridge Reservation, TN to test the effect of abiotic forces on microbial community structure. (For additional reactor studies see “Dissecting microbial nitrogen cycling in the subsurface using tailored reactor schemes” by Hunt et. al., and “Metabolomics and Transcriptomics for Environmental Systems Biology: Molecular Mechanisms of Reduced Sulfur Caused Growth Inhibition of Field-Isolated Nitrate-Reducing Bacterium” by Majumder et. al.) Planktonic and biofilm microbial communities were initialized with background water to establish communities in triplicate control reactors and triplicate test reactors. All were fed filtered water from the background site for 18 days. On day 18, three reactors were switched to filtered water from a contaminated well, enriched in total dissolved solids relative to the background site, particularly chloride, nitrate, uranium, and sulfate. Biological and geochemical data were collected throughout the experiment, including planktonic and biofilm DNA for 16S rRNA amplicon sequencing, cell counts, total protein, anions, cations, trace metals, organic acids, bicarbonate, pH, Eh, DO, and conductivity. We observed significant shifts in both planktonic and biofilm microbial communities receiving contaminated water. This included a loss in diversity, especially amongst members of the Bacteroidetes, Acidobacteria, Chloroflexi, and Betaproteobacteria, but enrichment in the Fe(III)-reducing Ferribacterium and parastitic Bdellovibrio. These shifted communities were more similar to the contaminated well community, suggesting that geochemical influences on microbial community structure are substantial. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
107. Targeted_News_Service. 2019. Using Tiny Organisms to Unlock Big Environmental Mysteries.
108. States_News_Service_(United_States). 2019. Using Tiny Organisms to Unlock Big Environmental Mysteries.
109. Smith, H. J., M. W. Fields, L. M. Lui, R. Miller, D. C. Joyner, F. von Netzer, A. D. Putt, T. C. Hazen, A. P. Arkin and P. D. Adams. 2019. ENIGMA: Novel Bio-Signatures and Activity in Fractionated Groundwater from Uncontaminated and Contaminated Sites. DOE 2019 Genomic Sciences Program Annual Principal Investigator (PI) Meeting abstract
     Project Goals: For any environment, it is possible to predict the temporal changes in biogeochemistry with some precision, for some length of time, given geochemical and biological inputs. A fundamental goal in the field of microbial ecology is to link the activity of specific microorganisms to processes occurring within an ecosystem. This project aims to identify the drivers of community structure and succession by quantifying activity and identifying the metabolically active fraction of microbial communities from both pristine and contaminated groundwater and sediment from the Field Research Center (FRC) at Oak Ridge National Laboratory (ORNL). Recent work has shown the existence of ultra-small bacteria (100-300 nm) in groundwater but no work has confirmed in situ activity, and the ultra-small bacteria could display very different mass transport and activity distributions in porous media flow. Therefore, field observation and characterization are needed to determine the functional role and activity of ultra-small bacteria that could specialize in specific activity and distributions as well as potential metabolic interactions. Moreover, microorganisms that can alter size in response to nutrient levels need to be differentiated from microorganisms that remain small. Total cell numbers, translationally-active cell numbers with bioorthogonal non-canonical amino acid tagging (BONCAT), and microbial activity (3H-Leucine incorporation) were investigated for both low biomass pristine and contaminated groundwater (0.2 and 0.1 m). In addition, metagenomics characterization was performed for 0.1 m fraction of the pristine groundwater. Operational taxonomic units (OTUs) of 16S rRNA amplicons from contaminated FRC groundwater collected ~15 feet below ground surface (FW215) showed a high relative abundance of Acidobacteria, Actinobacteria, Bacteroides, and Proteobacteria in the 0.1µm bacterial community. Among the 0.1µm size fraction collected in the shallow FRC aquifer (FW215) Crenarchaeota and Eurarchaeota demonstrated the highest relative abundance among identified archaeal OTUs. The plot identified as area 2 in the FRC has also been subject to repeated bioimmobilization injections using emulsified vegetable oil as a biostimulant. In a recent injection, phyla AC1, Gemmatimonadetes, OP8, WS2, WS3, and WWE1 collected from five shallow wells downgradient of the injection demonstrated notable increases in absolute OTU abundance among the 0.1µm community. Total cell numbers (0.2 m filter) for pristine groundwater (GW271) were 1.3 x 106 +/- 4.4 x 105 cells/ml, whereas, for contaminated groundwater the total cell abundances were 7.3 x 105 +/- 3.4 x 104 and 4.6 x 105 +/- 8.4 x 104 cells/ml for FW115-24 and FW106, respectively. Abundances of smaller cells (0.1 m filter) were highest for the pristine groundwater 6.40 x 104 +/- 2.1 x 104 cells/ml, while abundances for the contaminated wells were 6.7 x 102 +/- 1.1 x 101 and 6.3 x 102 +/- 6.5 x 101 cells/ml for FW115-24 and FW106, respectively. The results demonstrated that cell numbers for the 0.2 m fraction were approximately an order of magnitude higher for the pristine groundwater compared to the contaminated groundwater (106 v. 105). Cell numbers for the small fraction (0.1 m fraction) were also at least an order of magnitude higher for the pristine well compared to the contaminated groundwater (104 v. 102). For pristine groundwater activity, the small cell fraction (0.1 m) made up almost 20% of total BONCAT activity (per ml), and the small cell fraction had roughly 3-fold greater activity on a per cell basis. When pristine groundwater was compared to the contaminated, there was a drastic reduction in the BONCAT activity and the contaminated groundwater was between 172-769-fold less. Additionally, the rate of leucine incorporation (3H-leucine) on a per cell basis for the 0.2 m fraction in pristine groundwater was 172 and 8,000 times greater than the contaminated groundwater (FW115 and FW106, respectively). While the overall activity for contaminated wells was low (0.5-2.0 ng C/ml/d), between 25 and 57% of the total activity was from ultra-small microbes (0.1 m fraction). Moreover, for all tested groundwater (pristine and contaminated), the 0.1 m fraction had higher activity on a per cell basis than the 0.2 m fraction. Overall, for both size fractions, activity was lower (both per volume and per cell) in contaminated groundwater compared to pristine groundwater.
110. Serrano Matos, Y., A. Gonzalez, A. Rivera, D. Williams, T. C. Hazen and G. A. Toranzos. 2019. Prophage and CRISPR Sequences Detected in Enterococci Isolates From Soils and Waters with Low Anthropogenic Disturbances. Annual Biomedical Research Conference for Minority Students (ABRCMS) 2019 abstract
     Bacteriophages are the most abundant entities and are important vectors of genetic exchange between bacterial cells. Studies in clinical setting have shown bacteriophage ability to transfer virulence genes to bacteria, yet little is known of the role of bacteriophages in the environment and much less about their genomic content. The purpose of this study was to determine the possible transfer of virulence traits among bacteria in pristine ecosystems; we also looked for mechanisms to stop or control phage infection looking at CRISPRs sequences in the isolates. This study allow us to better understand the role of bacteriophages in the diversification of chromosomal DNA and contribution of genes involved in the biological processes of bacterial cells. We hypothesize that the host range of bacteriophages is the most crucial role in the dispersal of genes that could help in the survival and fitness of bacteria in pristine environments. We performed Whole Genome Sequencing (WGS) on fourteen Enterococcus spp. isolates from soil and water of El Yunque rainforest in Puerto Rico, followed by bioinformatic software for quality control, assembly of draft genomes, characterization of DNA, detection of virulence and Antibiotic Resistance Genes (ARG). For the detection of CRISPR sequences web-based programs PHASTER and CRISPRs CAS were used. Among the 14 Enterococci isolates, 47 prophage regions were detected. Of all the prophage regions we saw varying degrees of conservation with 4 questionable regions, 10 intact, and 33 incomplete genomes. The average percent similarity to known prophage sequences in the database of the interface was 29.60%. Data show that the morphology of the complete prophage regions will include a tail. Results also indicated the presence of 30 CRISPR arrays in (with different level of evidence), some with CAS system Type IIA (3) in the same contig as the palindromic sequences and spacers. The screening of virulence genes and resistance genes in the prophage regions resulted in 4 prophage regions harboring Csp gene, a virulent trait that confers resistance to thermal shock in nucleic acids. However, no antibiotic resistance genes were found in the prophage regions. Furthermore, we found in our isolates, prophage regions with the potential of transferring virulent traits in the environment and CRISPRs sequences which we continue to study in order to understand their role in the immunity of the bacteria cell. The presence of lysogens, supports the hypothesis that phages may be key in the resulting genomic heterogenicity in genome architecture (at least amongst members of the same genus). Our data clearly demonstrate that lysogenic bacteriophages have a wide host range and may be promiscuous leading to a favorable gene exchange in the environment.
111. Serrano Matos, Y., A. Gonzalez, A. Rivera, D. Williams, T. C. Hazen and G. A. Toranzos. 2019. Enterococcus spp. and environment as a reservoir of genes for the survival and fitness. ASM Microbe 2019 abstract
     Enterococci are used as water quality indicator bacteria in recreational waters, because they are sturdy microorganisms able to survive in harsh conditions. Yet, at our lab, we have also isolated Enterococcus spp. from uncontaminated ecosystems as part of the microbiota. Pathogenic enterococci are a concern for public health because of their apparent acquisition of antibiotic resistance genes and virulence factors that may be responsible for nosocomial infections. We have documented the presence of Enterococcus spp. in pristine soils and waters in the rainforest of Puerto Rico and through the use of whole genome sequencing (WGS) we characterized these pristine environment isolates. We are proposing that the environment plays a role as a reservoir for resistance and virulence genes. However, this does not make sense evolutionarily unless these genes have other functions for the survival and fitness in the environment. WGS was done on eighteen isolates identified as Enterococcus faecalis (12), Enterococcus gallinarum (5) and Enterococcus hirae (1). Antibiotic resistance genes for vancomycin (Van C) and macrolides (lsa A) were found in all the isolates, except E. hirae. Surprisingly, we also identified the presence of virulence factor genes in 15 of the 18 isolates (e.g. tpx, ace, hylA, hylB, gelE, cyl operon and others). MLST profiles resulted in the identification of 5 variable ST . The prophage regions found in the isolates belong to groups that infect other bacterial genera, thus clearly indicating the promiscuity of these lysogens. The characterization of environmental Enterococcus spp. from pristine sites lead us to conclude that the environmental microbiota contains antibiotic resistance as well as virulence factor genes. We need to determine what other possible functions these genes may have, since there is no evolutionary pressure for these genes be present in the environment.
112. ScienceDaily. 2019. Scientists hit pay dirt with new microbial research technique. ScienceDaily
113. ScienceDaily. 2019. Using Tiny Organisms to Unlock Big Environmental Mysteries.
114. Satinover, Scott, Maria F. Campa, Terry C. Hazen and Abhijeet P. Borole. 2019. Flowback Water Treatment and Hydrogen Production using Microbial Electrolysis Cells. ASM Microbe 2019 abstract
     Flowback water is a primary waste of early time oil and gas extraction that has significant salinity and organic content, but little reuse. Here, we demonstrate flowback water degradation in Microbial Electrolysis Cells (MECs) at varying conductivities (20, 30, and 40 mS/cm2) fed in batch organic loading, using inoculum sources from an anaerobic digester, previously reported MECs [1], and raw flowback water. We then compared the performance of matured MECs using acetate at the same conductivity and organic loading as determined by chemical oxygen demand. At all carbon loadings, acetate fed MECs outperformed produced water MECs, however performance across salinities did not appear to vary as significantly. Average current density reached 3.4 ± 0.02 A/m2 using acetate at 40 mS/cm2 while produced water only reached 0.71 ± 0.02 A/m2 under the same loading conditions. The largest hydrogen productivity reached 3.32 ± 0.32 L/L-day at 30 mS/cm2 using acetate, while produced water only made 0.67 ± 0.0 L/L-day at these conditions. 16s rRNA gene amplicon sequencing was used to characterize microbial anode community structure used in the studies for produced water at 40 and 30 mS/cm2. For all cases, Geobacter was found to be the dominant genus amongst replicate MECs, exceeding 33% of operational taxonomic units detected under the conditions tested. Other genera detected included Paludibacter (greater than 4% but less than 15%) and Blvii28 (greater than 10% but less than 15%). By comparison, the dominant genus in the produced water used in this study was Paenibacillus (59% of OTUs). Geobacter represented only 2% of OTUs found in the flowback water.
115. Salim, A., Ti. Tran, A. Putt and T. C. Hazen. 2019. Ultramicrobacteria Candidate Genome Database Project. UT’s Exhibition of Undergraduate Research and Creative Achievement (EUReCA)
116. Rafie, Saad Abd Ar, Kevin P. Hoyt, Martin R. Schubert, Sally P. Horn and Terry C. Hazen. 2019. Changes in Microbial Community Structure after Controlled Burn Treatment of Temperate Hardwood and Mixed Forests. ASM Microbe 2019 abstract
     The occurrence of extraordinary wildfires has become more frequent over the past few years and raises concern regarding the economic and ecological consequences of these events. The recent ones in California (2018) almost overwhelmed fire control measures while leaving behind swaths of scorched land. Controlled burns are a viable option for fire management, but its ecological impacts are not much different. The soil microbial community helps maintain the balance in nutrient cycling and regenerative processes thereby playing a crucial role in forest ecosystems. A disturbance, natural or enforced, like a fire, causes abrupt physicochemical changes to the topsoil that affect and change the microbial community structure. The goal of this study was to understand the changes in soil microbial community structure following a prescribed fire in a temperate hardwood forest site and relating it to physicochemical changes observed within the topsoil. Pre- and post-burn soil samples were collected and analyzed for soil pH, moisture, carbon and nitrogen content. The microbial community was assessed using generation 16S rRNA amplicon sequencing, using the qiime2 platform to process the data. Using different types of slash fuel for the same burn treatment caused similar responses in the microbial community structure, but there appears to be a more significant difference in plots with hardwood slash. PERMANOVA analysis (to understand beta-diversity group significance) shows that there are significant differences (p <= 0.001) observed between plots with hardwood slash (HS) and plots having pinewood slash (PS) or no additional fuel added. Alpha diversity metrics show that greater species richness (p <= 0.02) for the HS treatment compared to PS, hinting that the use of HS could result in a greater number of community members surviving. PCoA analysis (weighted Unifrac) also shows separate clustering of HS treatment. The findings demonstrate that the type of slash fuel used has an impact on microbial community structure within the forest soil.
117. Putt, A. D., K. McBride, B. G. Adams and T.C. Hazen. 2019. Ten-year investigation of the subsurface microbiome in a variably saturated contaminant pathway with two carbon-amendments. Geological Society of America Annual Meeting and Exposition
118. Putt, A.. 2019. Biostimulation of Ultramicrobacteria Consortia to aid in Uranium Bioimmobilization. Threee Minute Thesis abstract
     Ultramicrobacteria (UMB) exhibit a small cell volume (<0.1µm3) and unique metabolic pathways. Uncultured UMB genomes have been identified in a wide array of environments making UMB some of the most prolific organisms on the planet. Metabolic pathways identified from some UMB suggest their role in the biostimulation of the surrounding microbial community. Effective biostimulation is valuable for bioimmobilization of heavy metals and radionuclides like those found at our sample site on the Y-12 complex in Oak Ridge, TN. The tested bioimmobilization treatment utilizes a solution of soybean oil, lactate, and vitamins known as Emulsified Vegetable Oil (EVO) to stimulate reducing conditions.1,040L of a 20% EVO solution was injected into a contaminated aquifer and monitored from 8 wells for one-year. Groundwater collections were filtered through 0.2µm pore membranes to collect the larger microbial size-fraction and UMB organisms were captured on 0.1µm pore membranes. Geochemistry measurements of the samples revealed decreases in aqueous uranium, rapid sulfate reduction, and acetate production in the days following the injection. Fifty days post-injection a 2log fold change p<0.001 of collected 16s rRNA amplicon operational taxonomic units identified Geobactereacae and sulfate reducing taxa like Desulfobacteraceae, Desulfovibrionaceae, and Desulfobulbaceae as dominant taxa with a log increase post-injection. Members of the UMB phyla OD1 were dominant among UMB and were notably dominant in the 0.2µm biomass. A Pearson correlation with Bonferroni corrected p-values p<0.001 also showed strong correlations between decreases in uranium and increases in UMB with metabolic pathways related to biostimulation indicating activity of UMB with the treatment.
119. Paradis, Charles J., Larry D. McKay, Edmund Perfect, Jonathan D. Istok and Terry C. Hazen. 2019. Push-pull tests for estimating effective porosity: expanded analytical solution and in situ application (vol 26, pg 381, 2018). Hydrogeology Journal 27:437-439.
120. Paradis, Charles J., Emma R. Dixon, Lauren M. Lui, Adam P. Arkin, Jack C. Parker, Jonathan D. Istok, Edmund Perfect, Larry D. McKay and Terry C. Hazen. 2019. Improved Method for Estimating Reaction Rates During Push-Pull Tests. Groundwater 57:292-302. abstract
     The breakthrough curve obtained from a single-well push-pull test can be adjusted to account for dilution of the injection fluid in the aquifer fluid. The dilution-adjusted breakthrough curve can be analyzed to estimate the reaction rate of a solute. The conventional dilution-adjusted method assumes that the ratios of the concentrations of the nonreactive and reactive solutes in the injection fluid vs. the aquifer fluid are equal. If this assumption is invalid, the conventional method will generate inaccurate breakthrough curves and may lead to erroneous conclusions regarding the reactivity of a solute. In this study, a new method that generates a dilution-adjusted breakthrough curve was theoretically developed to account for any possible combination of nonreactive and reactive solute concentrations in the injection and aquifer fluids. The newly developed method was applied to a field-based data set and was shown to generate more accurate dilution-adjusted breakthrough curves. The improved dilution-adjusted method presented here is simple, makes no assumptions regarding the concentrations of the nonreactive and reactive solutes in the injection and aquifer fluids, and easily allows for estimating reaction rates during push-pull tests.
121. Newswise. 2019. Using Tiny Organisms to Unlock Big Environmental Mysteries.
122. LongRoom News. 2019. Scientists hit pay dirt with new microbial research technique. LongRoom News
123. Miller, John I., Stephen Techtmann, Julian Fortney, Nagissa Mahmoudi, Dominique Joyner, Jiang Liu, Scott Olesen, Eric Alm, Adolfo Fernandez, Piero Gardinali, Nargiz Garajayeva, Faig S. Askerov and Terry C. Hazen. 2019. Oil Hydrocarbon Degradation by Caspian Sea Microbial Communities. Frontiers in Microbiology 10. abstract
     The Caspian Sea, which is the largest landlocked body of water on the planet, receives substantial annual hydrocarbon input from anthropogenic sources (e.g., industry, agriculture, oil exploration, and extraction) and natural sources (e.g., mud volcanoes and oil seeps). The Caspian Sea also receives substantial amounts of runoff from agricultural and municipal sources, containing nutrients that have caused eutrophication and subsequent hypoxia in the deep, cold waters. The effect of decreasing oxygen saturation and cold temperatures on oil hydrocarbon biodegradation by a microbial community is not well characterized. The purpose of this study was to investigate the effect of oxic and anoxic conditions on oil hydrocarbon biodegradation at cold temperatures by microbial communities derived from the Caspian Sea. Water samples were collected from the Caspian Sea for study in experimental microcosms. Major taxonomic orders observed in the ambient water samples included Flavobacteriales, Actinomycetales, and Oceanospirillales. Microcosms were inoculated with microbial communities from the deepest waters and amended with oil hydrocarbons for 17 days. Hydrocarbon degradation and shifts in microbial community structure were measured. Surprisingly, oil hydrocarbon biodegradation under anoxic conditions exceeded that under oxic conditions; this was particularly evident in the degradation of aromatic hydrocarbons. Important microbial taxa associated with the anoxic microcosms included known oil degraders such as Oceanospirillaceae. This study provides knowledge about the ambient community structure of the Caspian Sea, which serves as an important reference point for future studies. Furthermore, this may be the first report in which anaerobic biodegradation of oil hydrocarbons exceeds aerobic biodegradation.
124. Miller, J. I., S. M. Techtmann, J. Fortney, N. Mahmoudi, D. C. Joyner, J. Liu, S. Olesen, E. Alm, A, Fernandez, P. Gardinali, N. GaraJayeva, F. S. Askerov and T. C. Hazen. 2019. Potential for rapid microbial biodegradation of petroleum hydrocarbons in hypoxic marine environments. International Petroleum Environmental Conference Annual Meeting
125. Majumder, E. L. W., T. Huan, E. M. Forsberg, A. D. Putt, T. C. Hazen, J. D. Wall and G. Siuzdakn. 2019. Activity of sulfur-containing metabolites in regulation bacterial respiration. ASM Microbe 2019 abstract
     Although consisting of only 0.03% of the mass of the Earth, the element sulfur acts in essential functions in all levels of life on Earth. From the biogeochemical sulfur cycle to intracellular redox regulation, sulfur-containing metabolites are drivers of respiration and environmental conditioning. However, the full extent of the population of sulfur-containing metabolites, their activities and their distribution in different species or environments is unknown. In this work, we elucidated the profile of sulfur-containing metabolites of four bacteria growing in different respiration modes from untargeted metabolomics workflows. Escherichia coli was grown in aerobic and fermentation modes. Pseudomonas stutzeri RCH2 was grown in aerobic and nitrate-reducing conditions. Desulfovibrio vulgaris Hildenborough and Desulfovibrio desulfuricans ND132 were grown under sulfate-reducing conditions. We traced the biosynthetic routes of metabolites with stable isotope 34S addition. We observed that low-molecular weight thiols like glutathione were in high abundance during aerobic respiration, but virtually absent in anaerobic respiration modes. Then, we probed the functional activity of some of the sulfur-containing metabolites on key metabolic pathways. We explored the network that bacteria use to regulate gene translation with sulfur relay, methionine salvage and tRNA thiolation.
126. Lux, T. and T Baurick. 2019. Coastal News Roundup: Were The Chemicals Used To Clean Up BP Oil Spill Harmful?.
127. Kothari, Ankita, Yu-Wei Wu, John-Marc Chandonia, Marimikel Charrier, Lara Rajeev, Andrea M. Rocha, Dominique C. Joyner, Terry C. Hazen, Steven W. Singer and Aindrila Mukhopadhyay. 2019. Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes. Mbio 10. abstract
     Naturally occurring plasmids constitute a major category of mobile genetic elements responsible for harboring and transferring genes important in survival and fitness. A targeted evaluation of plasmidomes can reveal unique adaptations required by microbial communities. We developed a model system to optimize plasmid DNA isolation procedures targeted to groundwater samples which are typically characterized by low cell density (and likely variations in the plasmid size and copy numbers). The optimized method resulted in successful identification of several hundred circular plasmids, including some large plasmids (11 plasmids more than 50 kb in size, with the largest being 1.7 Mb in size). Several interesting observations were made from the analysis of plasmid DNA isolated in this study. The plasmid pool (plasmidome) was more conserved than the corresponding microbiome distribution (16S rRNA based). The circular plasmids were diverse as represented by the presence of seven plasmid incompatibility groups. The genes carried on these groundwater plasmids were highly enriched in metal resistance. Results from this study confirmed that traits such as metal, antibiotic, and phage resistance along with toxin-antitoxin systems are encoded on abundant circular plasmids, all of which could confer novel and advantageous traits to their hosts. This study confirms the ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses. IMPORTANCE Plasmidomes have been typically studied in environments abundant in bacteria, and this is the first study to explore plasmids from an environment characterized by low cell density. We specifically target groundwater, a significant source of water for human/agriculture use. We used samples from a well-studied site and identified hundreds of circular plasmids, including one of the largest sizes reported in plasmidome studies. The striking similarity of the plasmid-borne ORFs in terms of taxonomical and functional classifications across several samples suggests a conserved plasmid pool, in contrast to the observed variability in the 16S rRNA-based microbiome distribution. Additionally, the stress response to environmental factors has stronger conservation via plasmid-borne genes as marked by abundance of metal resistance genes. Last, identification of novel and diverse plasmids enriches the existing plasmid database(s) and serves as a paradigm to increase the repertoire of biological parts that are available for modifying novel environmental strains.
128. Kothari, A., H. Liu, V. Trotter, A. Tang, D. Soneja, H. Zhang, J.-M. Chandonia, D. Cirri, H. Carlson, T. C. Hazen, H. Carlson, N. Daliang, F. Poole, M. W. W. Adams, J. Zhou, A. Deutschbauer and A. Mukhopadhyay. 2019. Mining Newly Discovered Native Plasmids for Genetic Modification of Intractable Environmental Strains. ENIGMA Annual Retreat
129. Kazakov, A. E., L. M. Lui, P. S. Novichkov, N. B. Justice, T. B. Simmons, S. J. Spencer, C. J. Paradis, K. A. Lowe, A. M. Rocha, S. Jagadamma, D. C. Joyner, A. Aaring, X Wu, R. Chakraborty, T. C. Hazen, P. D. Adams and A. P. Arkin. 2019. Functional characterization of nitrogen cycle genes in shotgun metagenomic datasets. ASM Microbe 2019 abstract
     High-throughput sequencing after direct DNA extraction from environmental samples is a powerful approach for culture-independent analysis of microbial communities in natural environments. However, functional analysis of terrestrial metagenomic samples is a challenging task due to high complexity of microbial communities and presence of uncharacterized taxa. In this project, we developed Fama computational pipeline for focused analysis of shotgun metagenomes. This pipeline profiles genetic potential of microbial communities for selected functions and recovers functional genes of interest. Since outcome of functional analysis depend greatly on quality of reference dataset, we developed three reference libraries of functional proteins: (i) proteins related to nitrogen cycle, (ii) carbohydrate-active enzymes, (iii) universal single-copy proteins. These reference libraries are based on SEED, KEGG and CAZy DB annotations, with additional manual curation. Fama pipeline includes modules for preprocessing of sequence reads, functional profiling and gene assembly. The preprocessing module performs adapter trimming, quality-based trimming and filtering by length. The functional profiling module runs two sequential similarity searches. The first search against compact database of reference proteins identifies sequence reads with some similarity to proteins of interest. Such reads are selected for subsequent search against large database of reference proteomes. The second search finds best hit for each of the reads, and number of reads for each function of interest is estimated. The gene assembly module reconstructs gene sequences from selected reads, maps reads to assembled genes and runs similarity search for functional and taxonomic assignment of the predicted genes. We applied Fama pipeline to six sediment samples collected at different depths in Oak Ridge Field Research Center. We analyzed genetic potential of nitrogen cycle processes, including nitrate reduction, denitrification and nitrification. We observed a dramatic difference for denitrification, ammonification and nitrification genes with depth, with highest abundance of nitrification genes in deeper samples. Predominant genes for nitrate/nitrate oxidoreductase in those samples cannot be reliably mapped to any known taxa, and thus comprising a taxonomic 'dark matter'. Concomitant presence of ammonia monooxygenases from Thaumarchaeota suggests possible interaction between ammonia-oxidizing archaea and nitrifying bacteria in the sediment.
130. Kazakov, A. E., L. M. Lui, P. S. Novichkov, N. B. Justice, T. B. Simmons, S. J. Spencer, C. J. Paradis, K. A. Lowe, A. M. Rocha, S. Jagadamma, D. C. Joyner, A. Aaring, X. Wu, R. Chakraborty, E. J. Alm, T. C. Hazen, A. P. Arkin and P. D. Adams. 2019. Functional profiling of nitrogen cycle genes and taxonomic characterization of EB106 and EB271 sediment core samples. ENIGMA Annual Retreat
131. Johnston, Eric R., Minjae Kim, Janet K. Hatt, Jana R. Phillips, Qiuming Yao, Yang Song, Terry C. Hazen, Melanie A. Mayes and Konstantinos T. Konstantinidis. 2019. Phosphate addition increases tropical forest soil respiration primarily by deconstraining microbial population growth. Soil Biology & Biochemistry 130:43-54. abstract
     Tropical ecosystems are an important sink for atmospheric CO2; however, plant growth is restricted by phosphorus (P) availability. Although soil microbiota facilitate organic P turnover and inorganic P mobilization, their role in carbon-phosphorus coupled processes remains poorly understood. To advance this topic, soils collected from four sites representing highly weathered tropical soils in the El Yunque National Forest, Puerto Rico were incubated with exogenous PO43- under controlled laboratory conditions. P amendment increased CO2 respiration by 14-23% relative to control incubations for soils sampled from all but the site with the greatest total and bioavailable soil P. Metatranscriptomics revealed an increase in the relative transcription of genes involved in cell growth and uptake of other nutrients in response to P amendment. A new methodology to normalize gene expression by population-level relative (DNA) abundance revealed that the pattern of increased transcription of cell growth and division genes with P amendment was community-wide. Soil communities responsive to P amendment possessed a greater relative abundance of alpha-glucosyl polysaccharide biosynthesis genes, suggestive of enhanced C storage under P-limiting conditions. Phosphorylase genes governing the degradation of alpha-glucosyl polysaccharides were also more abundant and increased in relative transcription with P amendment, indicating a shift from energy storage towards growth. Conversely, microbial communities in soils nonresponsive to P amendment were found to have metabolisms tuned for the phosphorolysis of labile plant-derived substrates, such as beta-glucosyl polysaccharides. Collectively, our results provided quantitative estimates of increased soil respiration upon alleviation of P constraints and elucidated several underlying ecological and molecular mechanisms involved in this response.
132. Johnson, J.. 2019. Dispersants one of many oil cleanup tools, panel says. Chemical and Engineering News
133. Hunt, K. A., A. E. Otwell, J. Hardwicke, F. von Netzer, E. J. Alm, N. S. Baliga, A. M. Deutschbauer, M. W. Fields, T. C. Hazen, T. R. Northen, J. D. Wall, M. W. W. Adams, M. Auer, K. Bender, G. Butland, R. Chakraborty, J.-M. Chandonia, D. A. Elias, P. S. Novichkov, A. Mukhopadhyay, G. E. Siuzdak, P. J. Walian, J. Zhou, A. P. Arkin, P. D. Adams and D. A. Stahl. 2019. Field nitrous oxide as a tractable indicator for subsurface biotic and abiotic processes and testable lab simulations. ENIGMA Annual Retreat
134. Hazen, T. C., Maria Campa and Scott Satinover. 2019. Fracking: the Good, Bad, and Ugly. Seniors for Creative Learning, O’Connor Senior Center
135. Hazen, T. C., A. P. Arkin, E. Alm and D. Graham. 2019. Structured Learning in Microbial Ecology Model. Kickoff MTV Meeting NNSA University of Michigan
136. Hazen, T. C., A. P. Arkin, E. Alm and D. Graham. 2019. SLiME – Structured Learning in Microbial Ecology Model. Biosensing Kickoff Meeting NNSA Georgia Tech.
137. Hazen, Terry C.. 2019. Environmental Systems Biology of the S-3 Ponds at Oak Ridge Reservation. Environmental Engineering Departmental Seminar, University of Tennessee
138. Hazen, T. C.. 2019. Hazen Researches Effects of Fracking on Water Health. Tennessee Engineer 20:17.
139. Hazen, T. C.. 2019. UT-ORNL Governor’s Chair selected by Clarivate Analytics as being among the world’s most cited researchers for 2018. Tennessee Engineer 20:40.
140. Hazen, T. C.. 2019. UT-ORNL Governor’s Chair Program Benefits Society. Tennessee Engineer 20:31.
141. Hazen, T. C.. 2019. Be an Influencer: Involvement in professional organizations gives scientists a unique opportunity to influence their field while at the same time building the networks that help them advance their careers. ORNL Today
142. Hazen, T. C.. 2019. Be an Influencer: Involvement in professional organizations gives scientists a unique opportunity to influence their field while at the same time building the networks that help them advance their careers. ORNL Today abstract
     Be an influencer Involvement in professional organizations gives scientists a unique opportunity to influence their field while at the same time building the networks that help them advance their careers. That's the pitch Terry Hazen gives when asked why he's excited about being an active member of no less than seven professional organizations. He also holds three fellowships and serves as president of the ORNL–UTK chapter of Sigma Xi, the Scientific Research Honor Society. Terry is a UT–ORNL Governor's Chair Professor in the Biosciences Division and has appointments in multiple University of Tennessee- Knoxville departments and research programs, as well as with the Bredesen Center for Interdisciplinary Research and Graduate Education. His professional memberships mirror his many interests and the fields he's influencing: civil and environmental engineering, microbiology, hydrology, chemistry, ecology and parasitology, among others. Throughout his career, after becoming a member, Terry began suggesting symposia topics and helped recruit speakers. Organizing symposia allowed him to influence the research areas his professional societies explored. Along the way, Terry built a network of prominent scientists, collaborating on research projects with some of them, and he was increasingly asked to present at meetings himself. The immediate benefits to his career were obvious: he gained visibility in his field and he had steady work. As he became recognized in his field, Terry was asked to review research proposals for federal-level agencies including the National Science Foundation, the National Institutes of Health, the Environmental Protection Agency and the departments of Energy and Agriculture. This has given him two advantages. First, Terry has had front-row-seat access to all the new research being proposed nationwide—revealing emerging and "hot" areas of scientific investigation—and, second, he's been in a position to advise program-level decision
143. Hazen, T. C.. 2019. What Microbes can do for You. https://www.ornl.gov/news/terry-hazen-what-microbes-can-do-you
144. Hazen, T. C.. 2019. BP Angola Cruise Microbial Ecology. BP Angola
145. Gordon, E., A. M. Harik and T. C. Hazen. 2019. Impact of Methane Pulse Frequency on TCE Degradation as Conducted by Methanosarcina quisquiliarum. UT’s Exhibition of Undergraduate Research and Creative Achievement (EUReCA)
146. Gonzalez, A., R. Wiscovitch, T. C. Hazen and G. A. Toranzos. 2019. Pristine Environments and the Escherichia coli pangenome: a gene tool box for survival. ASM Microbe 2019 abstract
     Escherichia coli is present in the gastrointestinal tract of warm-blooded mammals as part of the normal microbiota. This genus which has commensal and pathogenic strains has been reported as part of the environmental microbiome in tropical soils. We report the first description of 9 genome sequences of environmental isolates of E. coli from a truly pristine ecosystem. This data is a unique opportunity to decipher the genetic tools of the environmental strains. Whole-genome analysis is an essential step to improve our understanding of genetic diversity and genome dynamics of non-clinical E. coli populations. Results highlight the presence of 5 MLST sequence types and 4 that seem to belong to 'new' sequence types. Pangenome analysis show that the environmental isolates are a gene reservoir of an estimated total of 9682 gene families, of which <2500 genes are shared among all nine isolates (core genes). Bioinformatic tools have also revealed a complex identification of the resistome providing resistance to antibiotics, biocides and heavy metals. Resistance genes to Fosfomycin, aminoglycosides and beta-lactams were present, which could be a concern in terms of public health. We uncovered an arsenal of genes for resistance to biocides and heavy metals with a variety of mechanism (e.g., binding protein, regulators, membrane transporter, enzymes, efflux pumps, heat shock proteins and outer membrane lipoprotein). The presence of a diverse set of genes associated with pathogenicity have been identified. Although little is known about the role of E. coli in the environment, we describe the pangenome of environmental isolates from non-impacted (pristine environments) and revealed the unexpected presence of genes associated with pathogenicity; this is an important step towards a better understanding of the environmental microbiome and an opportunity for the future development of possible countermeasures against emerging pathogenic microorganisms.
147. Goddard, David. 2019. Examining the safety of using dispersants in oil spill clean ups. Homeland Security News Wire
148. Goddard, David. 2019. Hazen Co-Authors Study on Oil Spill Clean-Up Safety. Tennessee Today
149. Goddard, David. 2019. News story from Big News Network on Tuesday 16 April 2019. Big News Network
150. Goddard, David. 2019. Is it safe to use dispersants in oil spill cleanups. ANI
151. Goddard, David. 2019. Minimal risk associated with the use of dispersants. The Asian Age
152. Goddard, David. 2019. Minimal risk associated with the use of dispersants. TDECCAN Chronicle
153. Goddard, David. 2019. New report examines the safety of using dispersants in oil spill clean ups. Tennessee Today
154. Goddard, David. 2019. New report examines the safety of using dispersants in oil spill clean ups. News story from Eurasia Review on Tuesday 16 April 2019
155. Goddard, David. 2019. New report examines the safety of using dispersants in oil spill clean ups. News story from Eurasia Review on Tuesday 16 April 2019
156. Goddard, David. 2019. New report examines the safety of using dispersants in oil spill clean ups. Lab Manager
157. Goddard, David. 2019. New report examines the safety of using dispersants in oil spill clean ups.
158. Goddard, David. 2019. New report examines the safety of using dispersants in oil spill clean ups.
159. Gionfriddo, C. M., R. L. Wilpiszeski, A. M. Wymore, Ji-Won Moon, M. Podar, A. V. Palumbo, M. W. Fields, Hazen T. C, J. D. Wall, N. S. Baliga, D. A. Stahl, X. Ge, F. Poole II, M. W. W. Adams, R. Chakraborty, Y. Fan, J. Van Nostrand, J. Zhou, A. P. Arkin and D. A. Elias. 2019. Using In-field Bioreactors to Monitor Microbial Community Dynamic Shifts with Geochemical Perturbations. Metabolites in Regulating Bacterial Respiration. ENIGMA Annual Retreat
160. Geissberger, T., A. Putt, Sa’ad Abd Ar Rafie, D. C. Joyner and T. C. Hazen. 2019. What Microbes are in Recreational and Urban Park Streams of Knoxville? . UT’s Exhibition of Undergraduate Research and Creative Achievement (EUReCA)
161. Geissberger, T., A. Putt, Sa’ad Abd Ar Rafie, D. C. Joyner and T. C. Hazen. 2019. What Microbes are in Recreational and Urban Park Streams of Knoxville?. 7th Annual Watershed Symposium.
162. Ge, Xiaoxuan, Brian J. Vaccaro, Michael P. Thorgersen, Farris L., II Poole, Erica L. Majumder, Grant M. Zane, Kara B. De Leon, W. Andrew Lancaster, Ji Won Moon, Charles J. Paradis, Frederick von Netzer, David A. Stahl, Paul D. Adams, Adam P. Arkin, Judy D. Wall, Terry C. Hazen and Michael W. W. Adams. 2019. Iron- and aluminium-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle. Environmental Microbiology 21:152-163. abstract
     Anthropogenic nitrate contamination is a serious problem in many natural environments. Nitrate removal by microbial action is dependent on the metal molybdenum (Mo), which is required by nitrate reductase for denitrification and dissimilatory nitrate reduction to ammonium. The soluble form of Mo, molybdate (MoO42-), is incorporated into and adsorbed by iron (Fe) and aluminium (Al) (oxy) hydroxide minerals. Herein we used Oak Ridge Reservation (ORR) as a model nitrate-contaminated acidic environment to investigate whether the formation of Fe- and Al-precipitates could impede microbial nitrate removal by depleting Mo. We demonstrate that Fe and Al mineral formation that occurs as the pH of acidic synthetic groundwater is increased, decreases soluble Mo to low picomolar concentrations, a process proposed to mimic environmental diffusion of acidic contaminated groundwater. Analysis of ORR sediments revealed recalcitrant Mo in the contaminated core that co-occurred with Fe and Al, consistent with Mo scavenging by Fe/Al precipitates. Nitrate removal by ORR isolate Pseudomonas fluorescens N2A2 is virtually abolished by Fe/Al precipitate-induced Mo depletion. The depletion of naturally occurring Mo in nitrate- and Fe/Al-contaminated acidic environments like ORR or acid mine drainage sites has the potential to impede microbial-based nitrate reduction thereby extending the duration of nitrate in the environment.
163. Ge, X., M. P. Thorgersen, F. L. Poole II, E. L. Majumder, A. M. Deutschbauer, J.-M. Chandonia, P. S. Novichkov, G. Siuzdak, P. D. Adams, A. P. Arkin, T. C. Hazen and M. W. W. Adams. 2019. Characterization of Metal Resistance and Molybdenum Utilization Capability of New Microbial Strains Isolated from Contaminated Oak Ridge Sediments. ENIGMA Annual Retreat
164. Freiesleben, Simon. 2019. Rapport sår tvivl om skadeligheden af kemikalier fra oprydningen efter Deepwater Horizon . Ingenioren
165. EurekAlert!. 2019. Using Tiny Organisms to Unlock Big Environmental Mysteries.
166. Dixon, E. R., K. F. Walker, D. Williams and T. C. Hazen. 2019. Modeling Dynamic Geochemical Processes: How Diurnal and Seasonal Water Table Fluctuations Influence Contaminated Groundwater Geochemistry. AGU Fall Meeting.
167. Dixon, E. R., K. F. Walker and T. C. Hazen. 2019. Modeling Dynamic Geochemical Processes: How Water Table Fluctuations Influence RedOx Conditions in the Presence of Contamination. Geological Society of America Annual Meeting and Exposition
168. Dijkgraaf, Arjen. 2019. Actieve bodembacterie is er gloeiend bij. C2W
169. Couradeau, Estelle, JoeIle Sasse, Danielle Goudeau, Nandita Nath, Terry C. Hazen, Ben P. Bowen, Romy Chakraborty, Rex R. Malmstrom and Trent R. Northen. 2019. Probing the active fraction of soil microbiomes using BONCAT-FACS. Nature Communications 10. abstract
     The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.
170. Cornwall, W.. 2019. Do chemicals that disperse oil spills make the problem worse? Probably not, new study finds.. 2019.
171. Chen See, J., N. Ulrich, H. Nwanosike, C. McLimans, V. Tokarev, J. Wright, M. F. Campa, T. C. Hazen, C. Grant, J. Niles, S. Brewer and R. Lamendella. 2019. Antibiotic Resistant Bacteria Associated with Hydraulic Fracturing. ASM Microbe 2019
172. Campa, Maria Fernanda, Amy K. Wolfe, Stephen M. Techtmann, Ann-Marie Harik and Terry C. Hazen. 2019. Unconventional Oil and Gas Energy Systems: An Unidentified Hotspot of Antimicrobial Resistance?. Frontiers in Microbiology 10. abstract
     Biocides used in unconventional oil and gas (UOG) practices, such as hydraulic fracturing, control microbial growth. Unwanted microbial growth can cause gas souring, pipeline clogging, and microbial-induced corrosion of equipment and transportation pipes. However, optimizing biocide use has not been a priority. Moreover, biocide efficacy has been questioned because microbial surveys show an active microbial community in hydraulic fracturing produced and flowback water. Hydraulic fracturing produced and flowback water increases risks to surface aquifers and rivers/lakes near the UOG operations compared with conventional oil and gas operations. While some biocides and biocide degradation products have been highlighted as chemicals of concern because of their toxicity to humans and the environment, the selective antimicrobial pressure they cause has not been considered seriously. This perspective article aims to promote research to determine if antimicrobial pressure in these systems is cause for concern. UOG practices could potentially create antimicrobial resistance hotspots under-appreciated in the literature, practice, and regulation arena, hotspots that should not be ignored. The article is distinctive in discussing antimicrobial resistance risks associated with UOG biocides from a biological risk, not a chemical toxicology, perspective. We outline potential risks and highlight important knowledge gaps that need to be addressed to properly incorporate antimicrobial resistance emergence and selection into UOG environmental and health risk assessments.
173. Campa, Maria Fernanda, Stephen M. Techtmann, Mallory P. Ladd, Jun Yan, Megan Patterson, Amanda Garcia de Matos Amaral, Kimberly E. Carter, Nikea Ulrich, Christopher J. Grant, Robert L. Hettich, Regina Lamendella and Terry C. Hazen. 2019. Surface Water Microbial Community Response to the Biocide 2,2-Dibromo-3-Nitrilopropionamide, Used in Unconventional Oil and Gas Extraction. Applied and Environmental Microbiology 85. abstract
     Production of unconventional oil and gas continues to rise, but the effects of high-density hydraulic fracturing (HF) activity near aquatic ecosystems are not fully understood. A commonly used biocide in HF, 2,2-dibromo-3-nitrilopropionamide (DBNPA), was studied in microcosms of HF-impacted (HF+) versus HF-unimpacted (HF-) surface water streams to (i) compare the microbial community response, (ii) investigate DBNPA degradation products based on past HF exposure, and (iii) compare the microbial community response differences and similarities between the HF biocides DBNPA and glutaraldehyde. The microbial community responded to DBNPA differently in HF-impacted versus HF-unimpacted microcosms in terms of the number of 16S rRNA gene copies quantified, alpha and beta diversity, and differential abundance analyses of microbial community composition through time. The differences in microbial community changes affected degradation dynamics. HF-impacted microbial communities were more sensitive to DBNPA, causing the biocide and by-products of the degradation to persist for longer than in HF-unimpacted microcosms. A total of 17 DBNPA by-products were detected, many of them not widely known as DBNPA by-products. Many of the brominated by-products detected that are believed to be uncharacterized may pose environmental and health impacts. Similar taxa were able to tolerate glutaraldehyde and DBNPA; however, DBNPA was not as effective for microbial control, as indicated by a smaller overall decrease of 16S rRNA gene copies/ml after exposure to the biocide, and a more diverse set of taxa was able to tolerate it. These findings suggest that past HF activity in streams can affect the microbial community response to environmental perturbation such as that caused by the biocide DBNPA. IMPORTANCE Unconventional oil and gas activity can affect pH, total organic carbon, and microbial communities in surface water, altering their ability to respond to new environmental and/or anthropogenic perturbations. These findings demonstrate that 2,2-dibromo-3-nitrilopropionamide (DBNPA), a common hydraulic fracturing (HF) biocide, affects microbial communities differently as a consequence of past HF exposure, persisting longer in HF-impacted (HF+) waters. These findings also demonstrate that DBNPA has low efficacy in environmental microbial communities regardless of HF impact. These findings are of interest, as understanding microbial responses is key for formulating remediation strategies in unconventional oil and gas (UOG)-impacted environments. Moreover, some DBNPA degradation by-products are even more toxic and recalcitrant than DBNPA itself, and this work identifies novel brominated degradation by-products formed.
174. Campa, Maria Fernanda, Sheridan Brewer, Benjamin H. Allen, Robert Murdoch, Stephen M. Techtmann, Regina Lamendella and Terry C. Hazen. 2019. Comparative genomics elucidate the bacterial mechanism of resistance to the biocide 2-2-dibromo-3-nitrilopropionamide. ASM Microbe 2019 abstract
     The biocide 2-2-dibromo-3-nitrilopropionamide (DBNPA) is commonly used in many industries including unconventional oil and gas (UOG) production. Biocides are used in the hydraulic fracturing fluids used in UOG to prevent microbial induced corrosion of equipment and gas souring. Biocides are one of the most toxic chemicals used in UOG production, and there are rising concerns of increased microbial resistance. Comparative genomics of environmental strains was used to elucidate the microbial mechanism of DBNPA resistance. UOG impacted stream water was used to construct microcosms with either DBNPA or glutaraldehyde. After 56 days incubation, water from the microcosms was spread onto Nutrient medium agar plates at ~21C for 5-14 days aerobically, colonies were then transferred to either Nutrient agar + 100 mg/L DBNPA or Nutrient agar + 100 mg/L. The resistant colonies were transferred and cultured by increasing stepwise concentrations. The resistant strains chosen for whole genome sequencing were from the Paenibacillus and Bacillus genera. Members of those genera have been repeatedly found in UOG fluids and may have a role in microbial induced corrosion. Five selected isolates were sequenced on an Illumina HiSeq-2500 1TB and assembled using the IMG pipeline. The assembled reads were uploaded to KBase and annotated using the RAST pipeline. The genomes were compared using OrthoMCL using the genome of a publicly available Paenibacillus strain as reference. A total of 13,014 ortholog clusters were detected, 2,110 of which were shared among the six genomes. The pangenome was queried to only focus on orthologs present in all isolates except the ones enriched with glutaraldehyde and the reference genome. This step removed what is expected to be core functional genes and resistance genes not specific to DBNPA, such as those coding for ABC efflux pumps, which are shared among glutaraldehyde and DBNPA resistant strains. This effort yielded 13 ortholog clusters that were present only in DBNPA resistant isolates. These 13 ortholog clusters encode functions relevant to conferring resistance to a xenobiotic stressor. Their putative functions include mobile elements (recombinase and terminase), efflux pumps, and possible enzymatic deactivation of the biocide. For example, the major facilitator superfamily had not been detected in DBNPA or UOG related biocide use. These findings provide a first look into the potential mechanism of DBNPA resistance. This information may serve to formulate optimized biocides that target these efflux pumps.
175. Brand, S. and M. Chung. 2019. Scientists hit pay dirt with new microbial research technique. AAAS/EurekAlert!
176. Brand, S. and M. Chung. 2019. Scientists hit pay dirt with new microbial research technique - A better method for studying microbes in the soil will help scientists understand large-scale environmental cycles. SeedQuest
177. Brand, S. and M. Chung. 2019. Scientists hit pay dirt with new microbial research technique. Phys.Org
178. Brand, S. and M. Chung. 2019. Scientists hit pay dirt with new microbial research technique. Lab Manager
179. Brand, S. and M. Chung. 2019. Scientists hit pay dirt with new microbial research technique. Newswise
180. Brainard, J.. 2019. Oil dispersants did little harm to ocean, academy says. Science 302:106.
181. Arkin, A. P., T. C. Hazen, E. Alm and D. Graham. 2019. Modeling of Biological and Abiotic Data to Predict Nuclear Process Marker Presence and Age. Kickoff MTV Meeting NNSA University of Michigan
182. Alm, E., T. C. Hazen, A. P. Arkin and D. Graham. 2019. Environmental Surveillance for Radionuclide Sources. MTV Kickoff Meeting University of Michigan
183. Yao, Qiuming, Zhou Li, Yang Song, S. Joseph Wright, Xuan Guo, Susannah G. Tringe, Malak M. Tfaily, Ljiljana Pasa-Tolic, Terry C. Hazen, Benjamin L. Turner, Melanie A. Mayes and Chongle Pan. 2018. Community proteogenomics reveals the systemic impact of phosphorus availability on microbial functions in tropical soil. Nature Ecology & Evolution 2:499-509. abstract
     Phosphorus is a scarce nutrient in many tropical ecosystems, yet how soil microbial communities cope with growth-limiting phosphorus deficiency at the gene and protein levels remains unknown. Here, we report a metagenomic and metaproteomic comparison of microbial communities in phosphorus-deficient and phosphorus-rich soils in a 17-year fertilization experiment in a tropical forest. The large-scale proteogenomics analyses provided extensive coverage of many microbial functions and taxa in the complex soil communities. A greater than fourfold increase in the gene abundance of 3-phytase was the strongest response of soil communities to phosphorus deficiency. Phytase catalyses the release of phosphate from phytate, the most recalcitrant phosphorus-containing compound in soil organic matter. Genes and proteins for the degradation of phosphorus-containing nucleic acids and phospholipids, as well as the decomposition of labile carbon and nitrogen, were also enhanced in the phosphorus-deficient soils. In contrast, microbial communities in the phosphorus-rich soils showed increased gene abundances for the degradation of recalcitrant aromatic compounds, transformation of nitrogenous compounds and assimilation of sulfur. Overall, these results demonstrate the adaptive allocation of genes and proteins in soil microbial communities in response to shifting nutrient constraints.
184. WVLT. 2018. Puerto Rican students find temporary home for research in East Tennessee. WVLT News abstract
     KNOXVILLE, Tenn. (WVLT) -- Students visiting from Puerto Rico have made the University of Tennessee their home for the past few weeks after Hurricane Maria's destruction affected their laboratory. "The worst part was our second generator of electricity failed from the very beginning because a big tree fell into the room and everything was crushed," visiting graduate student Benjamin Mercado said. That ceiling fell in, and students said there was no way to save specimens they had been working on. "So we didn't have the conditions to do our work there, so we had to stop doing the experiments that we were doing there," student Yadeliz Serrano said. East Tennessee professor Terry Hazen taught in Puerto Rico years ago and was concerned about graduate students trying to complete their research. He used his connections at the University of Tennessee and Oak Ridge National Laboratory. A combination of help from the facilities and national science grants brought the top students to East Tennessee to use the labs. "So they got exposure to that, could be sequencing, proteomics and all that," UT Governor's Chair Professor Hazen said. "So I reached out to some of the scientists in Oak Ridge, and they agreed to help them, too."
185. Wu, Xiaoqin, Liyou Wu, Yina Liu, Ping Zhang, Qinghao Li, Jizhong Zhou, Nancy J. Hess, Terry C. Hazen, Wanli Yang and Romy Chakraborty. 2018. Microbial Interactions With Dissolved Organic Matter Drive Carbon Dynamics and Community Succession. Frontiers in Microbiology 9. abstract
     Knowledge of dynamic interactions between natural organic matter (NOM) and microbial communities is critical not only to delineate the routes of NOM degradation/transformation and carbon (C) fluxes, but also to understand microbial community evolution and succession in ecosystems. Yet, these processes in subsurface environments are usually studied independently, and a comprehensive view has been elusive thus far. In this study, we fed sediment-derived dissolved organic matter (DOM) to groundwater microbes and continually analyzed microbial transformation of DOM over a 50-day incubation. To document fine-scale changes in DOM chemistry, we applied high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and soft X-ray absorption spectroscopy (sXAS). We also monitored the trajectory of microbial biomass, community structure and activity over this time period. Together, these analyses provided an unprecedented comprehensive view of interactions between sediment-derived DOM and indigenous subsurface groundwater microbes. Microbial decomposition of labile C in DOM was immediately evident from biomass increase and total organic carbon (TOC) decrease. The change of microbial composition was closely related to DOM turnover: microbial community in early stages of incubation was influenced by relatively labile tannin-and protein-like compounds; while in later stages the community composition evolved to be most correlated with less labile lipid-and lignin-like compounds. These changes in microbial community structure and function, coupled with the contribution of microbial products to DOM pool affected the further transformation of DOM, culminating in stark changes to DOM composition over time. Our study demonstrates a distinct response of microbial communities to biotransformation of DOM, which improves our understanding of coupled interactions between sediment-derived DOM, microbial processes, and community structure in subsurface groundwater.
186. Woo, Hannah L. and Terry C. Hazen. 2018. Enrichment of Bacteria From Eastern Mediterranean Sea Involved in Lignin Degradation via the Phenylacetyl-CoA Pathway. Frontiers in Microbiology 9. abstract
     The degradation of allochthonous terrestrial organic matter, such as recalcitrant lignin and hemicellulose from plants, occurs in the ocean. We hypothesize that bacteria instead of white-rot fungi, the model organisms of aerobic lignin degradation within terrestrial environments, are responsible for lignin degradation in the ocean due to the ocean's oligotrophy and hypersalinity. Warm oxic seawater from the Eastern Mediterranean Sea was enriched on lignin in laboratory microcosms. Lignin mineralization rates by the lignin-adapted consortia improved after two sequential incubations. Shotgun metagenomic sequencing detected a higher abundance of aromatic compound degradation genes in response to lignin, particularly phenylacetyl-CoA, which may be an effective strategy for marine microbes in fluctuating oxygen concentrations 16S rRNA gene amplicon sequencing detected a higher abundance of Gammaproteobacteria and Alphaproteobacteria bacteria such as taxonomic families Idiomarinaceae, Alcanivoraceae, and Alteromonadaceae in response to lignin. Meanwhile, fungal Ascomycetes and Basidiomycetes remained at very low abundance. Our findings demonstrate the significant potential of bacteria and microbes utilizing the phenylacetyl-CoA pathway to contribute to lignin degradation in the Eastern Mediterranean where environmental conditions are unfavorable for fungi. Exploring the diversity of bacterial lignin degraders may provide important enzymes for lignin conversion in industry Enzymes may be key in breaking down high molecular weight lignin and enabling industry to use it as a low-cost and sustainable feedstock for biofuels or other higher-value products.
187. Walker, K. F., B. G. Adams, K. Lowe, M. Rodriquez, D. C. Joyner and T. C. Hazen. 2018. Long-Term Continuous Weather and Groundwater Monitoring at Y-12 for Suggesting ENIGMA Field and Lab Studies. ENIGMA SAC and Retreat abstract
     In order to to take an environmental systems approach to other levels of complexity like the microbial community structure/function, gene transfer, degradation pathways and geochemistry found in groundwater and sediment, we needed a way to understand how ecosystem phenomena were playing a role in these microscopic environments. Therefore, we deployed two down-well groundwater monitoring units (AquaTroll 600) and set up two meteorological stations (Onset HOBO U30) above ground at a contaminated (Area 2) and an uncontaminated site (Background Site) at Y-12. After monitoring for 105 days at the Background Site and 88 days at Area 2, we have seen interesting correlations within the data. There are natural diurnal fluctuations in the groundwater elevation, relative humidity and air temperature. We also see increases in groundwater elevation and changes in groundwater chemistry (dissolved oxygen, pH, conductivity) following heavy rain events. With the continued use of these units, we are hoping to capture the extended, especially annual and seasonal, relationships between all of these parameters. This data can also be used to guide future scientific focus on various times of the year, meteorological events of interest and geochemical variations over time.
188. von Netzer, F., K. A. Hunt, J. Valenzula, A. Otwell, S. Turkarslan, N. S. Baliga, J.-W. Moon, K. Lowe, M. Rodriguez, D. Elias, D. C. Joyner, C. Paradis, S. Pfiffner, D. Williams, K. Fitzgerald, S. Brewer, B. Adams, T. C. Hazen, E. L.-W. Majumder, G. M. Zane, J. D. Wall, D. Ning, J. Zhou, M. T. Thorgensen, X. Ge, M. W.W. Adams, L. Lui, R. Chakraborty, H. Carlson, A. Deutschbauer, D. Vuono, K. Meinhardt, D. A. Stahl, A. P. Arkin and P. D. Adams. 2018. Coupling of Field- and Lab-based Experiments to resolve controls of Nitrate Respiration Pathway Partitioning at the Oak Ridge Shallow Aquifer. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     The Oak Ridge site has a long history of research on biogeochemical impacts of contamination. Past studies largely focused on the fate and transport of radioisotopes, heavy metals, halogenated organic compounds, and mercury. Less studied is the consequence of the disposal of heavy metal laden nitric acid reaching nitrate concentrations of up to 2 g/l, acidifying large regions of this shallow aquifer. Thus, the combined impact of nitrate and lowered pH on microbial respiration and associated processes in this oxygen-limited environment is not well constrained in the literature. Microbially-driven nitrate respiration can operate through different pathways, resulting in either removal of nitrate through gaseous metabolites or retention through dissimilatory nitrate reduction to ammonium (DNRA). In this project, we focus on biotic and abiotic controls of nitrate transformation through field assays and experiments in lab systems. A grand challenge is to understand the function of microbial communities in the field within the limitations of available sampling methods. Therefore, we use a two-tier approach by combining field data and model laboratory systems. In the field, we survey the potential for nitrate respiration through the acetylene block method and nitrate isotope fractionation. For experiments with model isolates and communities in the lab, we are characterizing different reactor system formats but since biology in the subsurface is a combination of sediment-associated biofilms and planktonic organisms in pore water, we here focus on fluidized bed reactor (FBR) technology. Fluidization of sediment selects for both planktonic and attached populations, which can be challenging in planktonic chemostats, thus better emulating subsurface conditions while avoiding physical heterogeneities, such as channeling, that develop in packed-bed reactors. Abiotic characterization of FBR fluidization has shown that particles of less than 100 μm in diameter have impractically low fluidization velocities while particles larger than 300 μm in diameter require excessive flow rates to fluidize. We have established controlled fluidization under both oxic and anoxic conditions, and are evaluating the influence of medium composition, substratum size, composition as well as mass per reactor volume on biomass accumulation and activity. FBR operating conditions are initially refined with monocultures of Desulfovibrio vulgaris to select for both attached and planktonic populations or primarily attached populations. These studies are also evaluate the influence of physical parameters (particle size/composition, shear stress, and surface area) on colonization and factors controlling the partitioning between planktonic and attached organisms, a common issue in the field. These data inform the range of operating conditions needed to develop more complex reactor communities, now being evaluated by reactor colonization by Desulfovibrio spp. syntrophically coupled with different hydrogenotrophic methanogens. A simple acetylene block test serves to measure active regions of nitrate respiration in the field, identifying active source material for the FBR experiments. Using a model mixture of carbon sources to stimulate nitrate respiration with either biomass from filtered groundwater or sediment in native sterile groundwater, we trace the formation of nitrous oxide and ammonium over relatively short incubation periods in different incubations setups. Those studies are complemented by stable isotope fractionation data to constrain abiotic and biotic sinks/sources of nitrate and its transformation products, including nitrous oxide. Initial acetylene block and nitrate isotope fractionation data are consistent with a significant abiotic source of nitrous oxide in the highly contaminated area EB106 while biotic processes dominate at lower nitrate concentrations in both groundwater and sediment fractions in a region of lower contamination (EB271). DNRA and denitrification were significant processes at EB271, with DNRA more prominent in the vadose zone and denitrification in the transition zone between capillary fringe and saturated zone. The field data will guide FBR design and operation, as needed to identify and quantify variables governing microbial community dynamics, such as activity, resilience, and persistence as they relate to different respiratory processes at the Oak Ridge site. While the FBR reactor configuration may better emulate the subsurface environment through retention of both planktonic and attached microbial populations than standard liquid cultures, we anticipate that a more fully predictive understanding of variables controlling field site processes will derive from comparative studies of different reactor formats, including ongoing complementary studies of both field chemostats and packed-bed columns. Another connection to the field is the use isotopic fractionation signatures, as well as the metabolic and thermodynamic modeling of reactors operated under field relevant conditions. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
189. vbio. 2018. Es gibt mehr und vielfältigere plastikfressende Bakterien, als bisher angenommen.
190. US_Fed_News_(United_States). 2018. UT OFFERS CHANCE FOR PUERTO RICO STUDENTS TO CONTINUE WORK. US Fed News (United States) abstract
     "KNOXVILLE, Tenn., March 5 -- The University of Tennessee issued the following news release: Hurricane Maria was the 10th most powerful storm on record when"
191. US_Fed_News_(United_States). 2018. UT Researches Effects of Fracking on Water Health. Environment https://news.utk.edu/2018/10/12/ut-researches-effects-of-fracking-on-water-health/. abstract
     A new three-year study involving University of Tennessee-Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology Terry Hazen will look at how aquatic microbial communities are impacted by biocides associated with hydraulic fracking. “Fracking is something that has really changed the energy industry, but its environmental impacts are largely disputed,” Hazen said. “For example, biocides are used to help keep machinery and equipment protected against microbial corrosion, but that exposure can make the microbes resistant to the chemicals.” Terry Hazen, head of the Institute for a Secure and Sustainable Environment and joint UT–Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology. Terry Hazen, head of the Institute for a Secure and Sustainable Environment and joint UT–Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology. Led by Gina Lamanendalla, Steve Techtmann, and Maria Campa, the project will look at how microbial communities are affected and consider the possibility of alternative biocides while ensuring that fracking can continue to be used to gain natural resources. Lamanendalla and Techtmann are former postdoctoral fellows of Hazen’s who are now faculty members at Juniata College and Michigan Technological University, respectively. Campa is a postdoctoral fellow with Hazen in the Methane Center in UT’s Institute for a Secure and Sustainable Environment. Hazen’s team wants to clarify whether the use of biocides could lead to antibiotic-resistant strains of microbes, what the impact of the biocides is on the overall environment, and help pinpoint biocides that can continue to be used with greatest effect on equipment and the least impact on environment. Students of UT-ORNL Governor’s Chair for Environmental Biotechnology Terry Hazen take water samples in Pennsylvania. Students of UT-ORNL Governor’s Chair for Environmental Biotechnology Terry Hazen take water samples in Pennsylvania. The study will specifically observe a set of streams in Pennsylvania that are near active hydro fracking sites. Those will be compared to streams not within active fracking areas. Additionally, Hazen’s team hopes to look at what systems, biologically speaking, actively resist biocides and biocide-resistant strains. “We’ll conduct our study over many years to help give us a better picture of what the long-term impact might be,” Hazen said. “This work could help develop future contamination-detection techniques.” The project is backed by the National Science Foundation, with funding set to run through summer 2021.
192. Ulrich, Nikea, Veronica Kirchner, Rebecca Drucker, Justin R. Wright, Christopher J. McLimans, Terry C. Hazen, Maria F. Campa, Christopher J. Grant and Regina Lamendella. 2018. Response of Aquatic Bacterial Communities to Hydraulic Fracturing in Northwestern Pennsylvania: A Five-Year Study. Scientific Reports 8. abstract
     Horizontal drilling and hydraulic fracturing extraction procedures have become increasingly present in Pennsylvania where the Marcellus Shale play is largely located. The potential for long-term environmental impacts to nearby headwater stream ecosystems and aquatic bacterial assemblages is still incompletely understood. Here, we perform high-throughput sequencing of the 16 S rRNA gene to characterize the bacterial community structure of water, sediment, and other environmental samples (n = 189) from 31 headwater stream sites exhibiting different histories of fracking activity in northwestern Pennsylvania over five years (2012-2016). Stream pH was identified as a main driver of bacterial changes within the streams and fracking activity acted as an environmental selector for certain members at lower taxonomic levels within stream sediment. Methanotrophic and methanogenic bacteria (i.e. Methylocystaceae, Beijerinckiaceae, and Methanobacterium) were significantly enriched in sites exhibiting Marcellus shale activity (MSA+) compared to MSA-streams. This study highlighted potential sentinel taxa associated with nascent Marcellus shale activity and some of these taxa remained as stable biomarkers across this five-year study. Identifying the presence and functionality of specific microbial consortia within fracking-impacted streams will provide a clearer understanding of the natural microbial community's response to fracking and inform in situ remediation strategies.
193. Thorgersen, M. P., X. Ge, F. L. Poole, G. M. Zane, E. M. Forsberg, A. Deutschbauer, R. Chakraborty, T. C. Hazen, A. Mukhopadhyay, G. Siuzdak, J. D. Wall, J. Chandonia, P. Novichkov, M.W.W. Adams, A. P. Arkin and P. D. Adams. 2018. Characterization of Microorganisms Resistant to Multiple Metals from the Contaminated Environment at the Oak Ridge Reservation. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     The metal resistance campaign of ENIGMA is focused on investigating molecular mechanisms of microbial metal resistance. One of the defining characteristics of the Oak Ridge Reservation (ORR) environment is the presence of nitrate and mixed metal industrial waste with concentrations many metals over 1,000 times elevated in contaminated areas compared to pristine groundwater. Several metal centric high throughput anaerobic enrichments from contaminated ORR groundwater and sediment have been conducted including those using media containing a suite of metals at concentrations based on the ORR environment. These isolates are all resistant to metals such as uranium, aluminum, manganese and nickel (≥ 100 μM) when grown under nitrate-reducing growth conditions. After high-throughput preliminary screening, isolates that have unusual metal resistance properties indicative of novel mechanisms will be selected for in depth characterization by an array of lab processes and technologies as part of the metal resistance campaign pipeline including; genome sequencing, screening for genetic tractability, random barcode TN-Seq, DNA affinity purification sequencing, and global isotopic metabolomics. All characterization data can then be uploaded to KBase where new tools developed to analyze generic data sets are being used to organize, analyze, and eventually distribute results to the public. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
194. University of Tennessee. 2018. UT Home to Several Most Cited Researchers. State News Service https://news.utk.edu/2018/12/12/ut-home-to-several-most-cited-researchers/. abstract
     "The following information was released by the University of Tennessee: When peers cite a faculty member's research, it's a solid indication of the respect they"
195. University of Tennessee. 2018. UT Home to Several Most Cited Researchers. UT NEWS https://news.utk.edu/2018/12/12/ut-home-to-several-most-cited-researchers/. abstract
     When peers cite a faculty member’s research, it’s a solid indication of the respect they have attained in their field. Clarivate Analytics annually calculates the world’s most cited researchers, and the 2018 list features eight people who work with the UT Knoxville campus in some capacity. Those selected from UT this year are: Sheng Dai, professor of chemistry; his work focuses on synthesizing and characterizing materials used in energy applications. Terry C. Hazen, UT-Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology; he researches microbial ecology, water health, bioenergy, and the environment. David Mandrus, Jerry and Kay Henry Endowed Professor in materials science and engineering; he leads a group focused on discovering and characterizing new quantum materials. Art J. Ragauskas, UT-ORNL Governor’s Chair for Biorefining; his work deals with bioenergy, biofuels, and green chemical engineering. Gerald Tuskan, adjunct professor in the UT Institute of Agriculture; his work involves the study of plants for bioenergy. All five hold joint appointments with ORNL. Also selected were ORNL’s Sergei Kalinin, Richard Norby, and Bobby Sumpter, all of whom work with the ORNL-UT Bredesen Center for Interdisciplinary Research and Graduate Education.
196. Targeted_News_Service. 2018. Highly Cited Researchers list includes 15 from ORNL. Targeted News Service (United States) https://www.ornl.gov/news/highly-cited-researchers-list-includes-15-ornl. abstract
     "The U.S. Department of Energy's Oak Ridge National Laboratory issued the following news release: Fifteen scientists at the Department of Energy's Oak Ridge National Laboratory"
197. States_News_Service(United_States). 2018. UT OFFERS CHANCE FOR PUERTO RICO STUDENTS TO CONTINUE WORK. States News Service (United States) abstract
     "The following information was released by the University of Tennessee: Benjamin Mercado works on sample as part of the Hazen Lab, the group working under Dr. Terry C. Hazen”
198. States_News_Service(United_States). 2018. UT Researches Effects of Fracking on Water Health. Environment https://news.utk.edu/2018/10/12/ut-researches-effects-of-fracking-on-water-health/. abstract
     A new three-year study involving University of Tennessee-Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology Terry Hazen will look at how aquatic microbial communities are impacted by biocides associated with hydraulic fracking. “Fracking is something that has really changed the energy industry, but its environmental impacts are largely disputed,” Hazen said. “For example, biocides are used to help keep machinery and equipment protected against microbial corrosion, but that exposure can make the microbes resistant to the chemicals.” Terry Hazen, head of the Institute for a Secure and Sustainable Environment and joint UT–Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology. Terry Hazen, head of the Institute for a Secure and Sustainable Environment and joint UT–Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology. Led by Gina Lamanendalla, Steve Techtmann, and Maria Campa, the project will look at how microbial communities are affected and consider the possibility of alternative biocides while ensuring that fracking can continue to be used to gain natural resources. Lamanendalla and Techtmann are former postdoctoral fellows of Hazen’s who are now faculty members at Juniata College and Michigan Technological University, respectively. Campa is a postdoctoral fellow with Hazen in the Methane Center in UT’s Institute for a Secure and Sustainable Environment. Hazen’s team wants to clarify whether the use of biocides could lead to antibiotic-resistant strains of microbes, what the impact of the biocides is on the overall environment, and help pinpoint biocides that can continue to be used with greatest effect on equipment and the least impact on environment. Students of UT-ORNL Governor’s Chair for Environmental Biotechnology Terry Hazen take water samples in Pennsylvania. Students of UT-ORNL Governor’s Chair for Environmental Biotechnology Terry Hazen take water samples in Pennsylvania. The study will specifically observe a set of streams in Pennsylvania that are near active hydro fracking sites. Those will be compared to streams not within active fracking areas. Additionally, Hazen’s team hopes to look at what systems, biologically speaking, actively resist biocides and biocide-resistant strains. “We’ll conduct our study over many years to help give us a better picture of what the long-term impact might be,” Hazen said. “This work could help develop future contamination-detection techniques.” The project is backed by the National Science Foundation, with funding set to run through summer 2021.
199. Smith, H. J., A. J. Zelaya, K. B. De Leon, R. Chakraborty, D. A. Elias, T. C. Hazen, A. P. Arkin, A. B. Cunningham and M. W. Fields. 2018. Impact of hydrologic boundaries on microbial planktonic and biofilm communities in shallow terrestrial subsurface environments. Fems Microbiology Ecology 94. abstract
     Subsurface environments contain a large proportion of planetary microbial biomass and harbor diverse communities responsible for mediating biogeochemical cycles important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the saturated zone, capillary fringe and vadose zones, microorganisms can reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the contribution of each phase to subsurface processes is largely unresolved. Here, we synthesize current information on the diversity and activity of shallow freshwater subsurface habitats, discuss the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and geological gradients, and discuss how biofilms may be constrained within shallow terrestrial subsurface aquifers. We suggest that merging traditional activity measurements and sequencing/-omics technologies with hydrological parameters important to sediment biofilm assembly and stability will help delineate key system parameters. Ultimately, integration will enhance our understanding of shallow subsurface ecophysiology in terms of bulk-flow through porous media and distinguish the respective activities of sessile microbial communities from more transient planktonic communities to ecosystem service and maintenance.
200. Smith, H. J., A. Zelaya, I. Miller, D. C. Joyner, E. Couradeau, T. R. Northen, T. C. Hazen, M. W. Fields, A. P. Arkin and P. Adams . 2018. Linking Activity to Phylogeny in Groundwater/Soil Ecosystems. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     Project Goals: ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) uses a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. A fundamental goal in the field of microbial ecology is to link the activity of specific microorganisms to processes occurring within an ecosystem. This project aims to identify the drivers of community structure and succession by quantifying activity and identifying the metabolically active fraction of microbial communities from both pristine and contaminated groundwater and sediment from the Field Research Center (FRC) at Oak Ridge National Laboratory (ORNL). It is broadly accepted that free-living and attached communities have distinct microbial community compositions; however, due to sampling constraints there are significantly fewer studies that have simultaneously looked at the activity of microorganisms in both the sediment and groundwater fractions. Historically, it has been shown that not only are there greater densities of total cells (90-99.99% of the microbial biomass in porous aquifers) but there are also higher proportions of active cells associated with sediment compared to free-living groundwater cells. An explanation for differences in activities between attached and free-living populations is likely due to differences in the densities of cell abundances. However, it remains unresolved whether free-living cells in porous subsurface habitats are in fact metabolically slower or if the explanation for these differences in activities is merely based upon numbers respective to a given volume. Recently, it has been proposed that microbial competition selects against rapid growth in attached populations and that bulk-phase populations have faster growth rates . These findings offer a unique and contradictory perspective as to the role of free-living organisms compared to sediment biofilms which alter our current understanding of dispersal and colonization in porous environments as well as the distribution of microbial activities. Using a combination of complementary culture-independent methods, activity rates and the active microbial assemblages were determined for groundwater and sediment associated cells from pristine and contaminated aquifers. Bioorthogonal non-canonical amino acid tagging (BONCAT) and Propidium Monoazide (PMA) were used to differentiate the contributions between different DNA pools (DNA from viable cells with intact cellular membranes versus extracellular or "relic" DNA) for two groundwater wells representing geochemical extremes. BONCAT samples were microscopically evaluated and sorted for amplicon sequencing (BONCAT-Seq). Pseudomonadacea and Comamonadaceae were the dominant active assemblages for pristine groundwater, while Xanthomonadaceae and Nocardiaceae dominated contaminated groundwater. A greater diversity of active organisms was observed in background sites. For pristine wells, 1,268 OTUs were observed on average, with between 8.5 and 26% of identified OTUs being translationally active for 24 and 72 h of incubation, respectively. On average 346 OTUs were observed for contaminated groundwater, with between 60 and 66% of observed OTUs being active for 24 and 72 h, respectively. With PMA analyses, the pristine groundwater showed average higher richness (4,958 OTUs) than contaminated groundwater (3,886 OTUs). For communities captured on 0.2 μM filters, OTU richness was similar with and without PMA treatment for pristine groundwater, and these results indicated that most sampled populations captured within this fraction did not have compromised cellular membranes and were viable. A similar trend was observed for contaminated groundwater, and OTU richness was similar between PMA treated and non-treated samples. Ordination analysis demonstrated that samples formed tight clusters that were primarily separated by well and secondarily by filter size. These results corroborated BONCAT analyses in that a significant portion of groundwater populations appear to be viable in terms of non-compromised membranes for both pristine and contaminated groundwater. In pristine wells activity on a per cell basis was two to three-fold greater for planktonic cells compared to particle associated organisms, with small cells (<0.1um) contributing up to 19% of total activity. Conversely, in contaminated samples, activity was greater for sediment associated cells. We observed two to three orders of magnitude lower cell specific growth for sediment associated cells compared to planktonic groundwater cells. However, combining activity measurements, cellular abundances, porosity, and the degree of saturation, the biological activity of planktonic groundwater cells and sediment associated cells in a cubic meter of the saturated subsurface was estimated. The activity estimated to corresponding sediment associated cells accounted for up to 99% of the activity within a cubic meter of the saturated subsurface. Using a combination of methods, we show that the majority of planktonic populations in pristine aquifers are highly active and consist of intact cells. While attached populations have slower rates on a per cell basis, the sediment biofilms are responsible for the majority of the activity within a shallow aquifer. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
201. See, Jeremy R. Chen, Nikea Ulrich, Hephzibah Nwanosike, Christopher J. McLimans, Vasily Tokarev, Justin R. Wright, Maria F. Campa, Christopher J. Grant, Terry C. Hazen, Jonathan M. Niles, Daniel Ressler and Regina Lamendella. 2018. Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania. Frontiers in Microbiology 9. abstract
     Unconventional oil and gas (UOG) extraction, also known as hydraulic fracturing, is becoming more prevalent with the increasing use and demand for natural gas; however, the full extent of its environmental impacts is still unknown. Here we measured physicochemical properties and bacterial community composition of sediment samples taken from twenty-eight streams within the Marcellus shale formation in northeastern Pennsylvania differentially impacted by hydraulic fracturing activities. Fourteen of the streams were classified as UOG+, and thirteen were classified as UOG based on the presence of UOG extraction in their respective watersheds. One stream was located in a watershed that previously had UOG extraction activities but was recently abandoned. We utilized high-throughput sequencing of the 16S rRNA gene to infer differences in sediment aquatic bacterial community structure between UOG+ and UOG streams, as well as correlate bacterial community structure to physicochemical water parameters. Although overall alpha and beta diversity differences were not observed, there were a plethora of significantly enriched operational taxonomic units (OTUs) within UOG+ and UOG samples. Our biomarker analysis revealed many of the bacterial taxa enriched in UOG+ streams can live in saline conditions, such as Rubrobacteraceae. In addition, several bacterial taxa capable of hydrocarbon degradation were also enriched in UOG+ samples, including Oceanospirillaceae. Methanotrophic taxa, such as Methylococcales, were significantly enriched as well. Several taxa that were identified as enriched in these samples were enriched in samples taken from different streams in 2014; moreover, partial least squares discriminant analysis (PLS-DA) revealed clustering between streams from the different studies based on the presence of hydraulic fracturing along the second axis. This study revealed significant differences between bacterial assemblages within stream sediments of UOG+ and UOG streams and identified several potential biomarkers for evaluating and monitoring the response of autochthonous bacterial communities to potential hydraulic fracturing impacts.
202. Christopher Salvemini. 2018. UT fracking study to break new ground, may lead to antibiotic-resistant microbes. Knoxville News Sentinel
203. Ribicic, Deni, Roman Netzer, Terry C. Hazen, Stephen M. Techtmann, Finn Drablos and Odd Gunnar Brakstad. 2018. Microbial community and metagenome dynamics during biodegradation of dispersed oil reveals potential key-players in cold Norwegian seawater. Marine Pollution Bulletin 129:370-378. abstract
     Oil biodegradation as a weathering process has been extensively investigated over the years, especially after the Deepwater Horizon blowout. In this study, we performed microcosm experiments at 5 degrees C with chemically dispersed oil in non-amended seawater. We link biodegradation processes with microbial community and metagenome dynamics and explain the succession based on substrate specialization. Reconstructed genomes and 16S rRNA gene analysis revealed that Bermanella and Zhongshania were the main contributors to initial n-alkane breakdown, while subsequent abundances of Colweilia and microorganisms closely related to Porticoccaceae were involved in secondary n-alkane breakdown and beta-oxidation. Cycloclasticus, Porticoccaceae and Sponsiiabcteraceae were associated with degradation of mono- and poly-cyclic aromatics. Successional pattern of genes coding for hydrocarbon degrading enzymes at metagenome level, and reconstructed genomic content, revealed a high differentiation of bacteria involved in hydrocarbon biodegradation. A cooperation among oil degrading microorganisms is thus needed for the complete substrate transformation.
204. Putt, A. D., K. McBride, B. G. Adams, N. Daliang, P. J. Walian, K. Lowe, J. Zhou and T. C. Hazen. . 2018. Long-term full-scale field study of repeated Emulsified Vegetable Oil Injection for bioimmobilization of heavy metals with new focus on hydrogeology, ultramicrobacteria, and memory response. ENIGMA SAC and Retreat abstract
     In 2009 an Emulsified Vegetable Oil (EVO) amendment was injected into the Y-12 fractured saprolite clay aquifer as a bioremediation treatment for the leading edge of a nitrate, heavy metal, and radionuclide contamination plume. EVO injections have been shown to immobilize uranium and some heavy metals, but the limits of this approach are still being studied. This research aims to identify similarities in geochemical and biological changes as a way to identify the predictability of a microbial community response, and the overall effectiveness of this treatment in immobilizing uranium. Collected groundwater was monitored for metals, radionuclides, pH, specific conductance, dissolved oxygen, anions, cations, organic acids, cell density, and changes in hydraulic conductivity. The microbial community was divided into two size fractions: the ≥ 0.2 µm bacterial community, and the ultramicrobacterial (UMB) and ultra-small bacterial (USB) communities which were captured on 0.1 µm pore membranes. Bacterial DNA was extracted using modified methods for low DNA concentrations and 16s rRNA amplicons were sequenced on an Illumina MiSeq. The injection of a 20% EVO to groundwater mixture was monitored closely for 134 days. The area underwent rapid sulfate reduction and acetate production indicated by organic acid and anion data collected using high pressure ion chromatography. Inductively coupled plasma mass spectrometry measured iron, uranium, and manganese reduction that occurred from day 7 to day 50 post-injection. Acridine orange direct counts showed an increase in cell density of the microbial community following the injection and 16S rRNA amplicon data identified distinct bacterial consortia. Transmission electron microscopy (TEM) yielded images of candidate UMB organisms from the study site. TEM methodology improvements led to the identification of UMB in low concentrations from within environmental samples as well as the imaging of bacteria in an EVO solution. Hydraulic conductivity data demonstrated the persistence of EVO, providing evidence that EVO temporarily alters the physical parameters of the injection site. The changes to the microbial community consortia in this study differed from the previous findings providing inconclusive evidence of a predictable response, but there are some indications of a relationship between the 0.1 and 0.2 µm microbial communities. Current data do not indicate an identifiable change to the planktonic 0.1 µm community like was seen in the 0.2 µm community. Continuing investigations at the site are underway looking for methods to better understand, identify and classify the potential relationship between the 0.1 and 0.2 µm communities.
205. Printed_Electronics_Now. 2018. Highly Cited Researchers list includes 15 from ORNL. Printed Electronics Now https://www.printedelectronicsnow.com/contents/view_breaking-news/2018-12-04/highly-cited-researchers-list-includes-15-from-ornl/. abstract
     Honored for production of multiple highly cited papers ranking in the top 1% by citations during 2006-2016. Fifteen scientists at the Department of Energy’s Oak Ridge National Laboratory were named in the 2018 Highly Cited Researchers list compiled by Clarivate Analytics. The list recognizes researchers for exceptional performance, demonstrated by the production of multiple highly cited papers ranking in the top one percent by citations for field and year in Web of Science during the 11-year period 2006-2016. This year’s listing includes the following researchers from ORNL: Ilias Belharouak of the Energy and Transportation Science Division; Miaofang Chi of the Center for Nanophase Materials Sciences; Sheng Dai of the Chemical Sciences Division; Terry Hazen, University of Tennessee (UT)-ORNL Governor’s Chair for Environmental Biotechnology; Juan Carlos Idrobo of the Center for Nanophase Materials Sciences; Sergei Kalinin of the Center for Nanophase Materials Sciences; David Mandrus of the Materials Science and Technology Division; Michael McGuire of the Materials Science and Technology Division; Michael Naguib of the Chemical Sciences Division; Richard Norby of the Environmental Sciences Division; Arthur Ragauskas, UT-ORNL Governor’s Chair for Biorefining; Brian Sales of the Materials Science & Technology Division; Bobby Sumpter of the Center for Nanophase Materials Sciences; Peter Thornton of the Environmental Sciences Division; Gerald Tuskan, director of the Center for Bioenergy Innovation The list “is a contribution to the identification of that small fraction of the researcher population that contributes disproportionately to extending the frontier and gaining for society knowledge and innovations that make the world healthier, richer, sustainable and more secure,” according to Clarivate.
206. PhysORG. 2018. Researchers reveal how microbes cope in phosphorus-deficient tropical soil. PhysORG
207. Paradis, Charles J., Ji-Won Moon, Dwayne A. Elias, Larry D. McKay and Terry C. Hazen. 2018. In situ decay of polyfluorinated benzoic acids under anaerobic conditions. Journal of Contaminant Hydrology 217:8-16. abstract
     Polyfluorinated benzoic acids (PBAs) can be used as non-reactive tracers to characterize reactive mass transport mechanisms in groundwater. The use of PBAs as non-reactive tracers assumes that their reactivities are negligible. If this assumption is not valid, PBAs may not be appropriate to use as non-reactive tracers. In this study, the reactivity of two PBAs, 2,6-difluorobenzoic acid (2,6-DFBA) and pentafluorobenzoic acid (PFBA), was tested in situ. A series of two single-well push-pull tests were conducted in two hydrogeologically similar, yet spatially distinct, groundwater monitoring wells. Bromide, 2,6-DFBA, and PFBA were added to the injection fluid and periodically measured in the extraction fluid along with chloride, nitrate, sulfate, and fluoride. Linear regression of the dilution-adjusted breakthrough curves of both PBAs indicated zero-order decay accompanied by nitrate and subsequent sulfate removal. The dilution-adjusted breakthrough curves of chloride, a non-reactive halide similar to bromide, showed no evidence of reactivity. These results strongly suggested that biodegradation of both PBAs occurred under anaerobic conditions. The results of this study implied that PBAs may not be appropriate to use as non-reactive tracers in certain hydrogeologic settings, presumably those where they can serve as carbon and/or electron donors to stimulate microbial activity. Future studies would benefit from using ring-C-14-labeled PBAs to determine the fate of carbon combined with microbial analyses to characterize the PBA-degrading members of the microbial community.
208. Paradis, Charles J., Larry D. McKay, Edmund Perfect, Jonathan D. Istok and Terry C. Hazen. 2018. Push-pull tests for estimating effective porosity: expanded analytical solution and in situ application. Hydrogeology Journal 26:381-393. abstract
     The analytical solution describing the one-dimensional displacement of the center of mass of a tracer during an injection, drift, and extraction test (push-pull test) was expanded to account for displacement during the injection phase. The solution was expanded to improve the in situ estimation of effective porosity. The truncated equation assumed displacement during the injection phase was negligible, which may theoretically lead to an underestimation of the true value of effective porosity. To experimentally compare the expanded and truncated equations, single-well push-pull tests were conducted across six test wells located in a shallow, unconfined aquifer comprised of unconsolidated and heterogeneous silty and clayey fill materials. The push-pull tests were conducted by injection of bromide tracer, followed by a non-pumping period, and subsequent extraction of groundwater. The values of effective porosity from the expanded equation (0.6-5.0%) were substantially greater than from the truncated equation (0.1-1.3%). The expanded and truncated equations were compared to data from previous push-pull studies in the literature and demonstrated that displacement during the injection phase may or may not be negligible, depending on the aquifer properties and the push-pull test parameters. The results presented here also demonstrated the spatial variability of effective porosity within a relatively small study site can be substantial, and the error-propagated uncertainty of effective porosity can be mitigated to a reasonable level (< +/- 0.5%). The tests presented here are also the first that the authors are aware of that estimate, in situ, the effective porosity of fine-grained fill material.
209. Paradis, C. J., T. C. Hazen, A. P. Arkin and P. D. Adams. 2018. In situ demonstration of sustained adaptation of a natural microbial community to transform substrates. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     Project Goals: The goals of this project are to: (1) demonstrate the exposure history dependence of microbial mediated substrate transformation rates in groundwater at the field scale and (2) elucidate the microbial mechanism(s) which control the exposure history dependence of microbial mediated substrate transformation rates Prior exposure of a natural microbial community to a substrate can result in the increased potential of the community to transform the substrate; this phenomenon is known as adaptation. Adaptation is thought to play an important role in biogeochemical cycling at the ecosystem scale and has been demonstrated at the laboratory scale. However, in situ demonstrations of the magnitude and duration of adaptation are lacking. Ethanol was used as a substrate and was injected into a groundwater well (substrate treatment) for six consecutive weeks to establish adaptation. A second well (substrate control) was not injected with ethanol during this time. The substrate treatment demonstrated adaptation for microbial-mediated oxidation of ethanol to acetate and reduction of nitrate and sulfate as evident by sequential and significant increases in zero-order reaction rates. Both wells were then monitored for six additional weeks under natural conditions. During the final week, ethanol was injected into both wells. The substrate treatment demonstrated sustained adaptation as evident by significantly higher reaction rates than the substrate control. Surprisingly, the selective enrichment of a microbial community within the first six weeks of the substrate treatment was not sustained after the six-week absence of ethanol, as revealed by analysis of planktonic DNA. These results demonstrated that adaptation can be induced and sustained with no apparent enrichment of a select microbial community. This suggests that the predominant mechanisms of adaptation may exist at the enzymatic- and/or genetic-levels. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231.
210. Office_of_Science_U.S._Department_Energy_(United_States). 2018. Researchers Reveal How Microbes Cope in Phosphorus-deficient Tropical Soil. Oak Ridge National Laboratory News abstract
     OAK RIDGE, Tenn., Jan. 22, 2018 – A team led by the Department of Energy’s Oak Ridge National Laboratory has uncovered how certain soil microbes cope in a phosphorus-poor environment to survive in a tropical ecosystem. Their novel approach could be applied in other ecosystems to study various nutrient limitations and inform agriculture and terrestrial biosphere modeling. Phosphorus is a critical nutrient for global biological processes, such as collecting the sun’s energy during photosynthesis and degrading plant debris and soil organic matter. Most tropical ecosystems endure long-term weathering that leaches phosphorus from soil. The ORNL-led team set out to discover how soil microbial communities respond to the lack of phosphorus and other nutrient deficiencies at the molecular level. Melanie Mayes/Oak Ridge National Laboratory, U.S. Dept. of Energy Soil scientists from Oak Ridge National Laboratory uncovered how certain microbes cope in a phosphorus-poor environment to survive in a tropical ecosystem. Credit: Melanie Mayes/Oak Ridge National Laboratory, U.S. Dept. of Energy (hi-res image) They collected soil samples at the Smithsonian Tropical Research Institute in the Republic of Panama, an experimental field site with phosphorus-rich plots and unfertilized control plots. “This was the perfect place to test the optimal foraging theory, which is a model that helps predict an organism’s behavior when searching for resources,” said Chongle Pan, ORNL senior staff scientist and joint associate professor at the University of Tennessee. “We learned how this theory plays out when applied to microbial communities as they compete for nutrients.” The team analyzed the behaviors of many genes and proteins, and in the phosphorus-deficient, untreated soil, they found an increased number of genes responsible for producing phosphorus-acquiring enzymes. They also discovered more than 100 genes that work to pull phosphorus from phytate, which is a complex organic compound found in plant tissue. “Finding so many genes to break apart and transport such a complex molecule tells us that microbes are hungry for phosphorus in untreated soil,” said Melanie Mayes, an ORNL senior staff scientist who studies multi-scale environmental processes. Conversely, she noted that when phosphorus was plentiful, more genes needed to acquire complex carbon compounds were present. “The microbial community prioritizes the breakdown of the most needed nutrients, focusing efforts on the most limiting element to balance their overall nutritional needs,” she said. The team ran each soil sample through a series of rigorous and comprehensive analyses. The DOE Joint Genome Institute conducted deep sequencing of the soils’ metagenomes, or genetic material recovered directly from the soil. ORNL then used mass spectrometry and metaproteomics to identify more than 7,000 proteins in each sample. ORNL’s Titan supercomputer quickly analyzed the large amounts of metagenomics and metaproteomics data, comparing microbial activities in phosphorus-rich and -poor soils. Environmental Molecular Sciences Laboratory scientists further characterized the soils’ organic matter at Pacific Northwest National Laboratory. These unique tools working together enabled one of the deepest proteogenomics studies done on soil microbial communities, according to Pan. The ORNL-led team plans to continue their research to characterize the ecology and evolution of soil microbial communities in nutrient-poor environments, which has applications in agriculture and terrestrial biosphere modeling worldwide. Additionally, Mayes and her team are incorporating metagenomics information into nutrient cycling models under a DOE Early Career Research Program Award. Results from their three-year study titled, “Community Proteogenomics Reveals the Systemic Impact of Phosphorus Availability on Microbial Functions in Tropical Soil,” were published in Nature Ecology & Evolution. The paper’s coauthors included Qiuming Yao, Zhou Li, Yang Song, Melanie A. Mayes and Chongle Pan of ORNL; S. Joseph Wright and Benjamin L. Turner of the Smithsonian Tropical Research Institute; Terry C. Hazen, University of Tennessee-ORNL Governor’s Chair for Environmental Biotechnology; Xuan Guo of UT; Susannah G. Tringe of the DOE Joint Genome Institute; and Malak M. Tfaily and Ljiljana Paša-Tolic of Pacific Northwest National Laboratory. The research was supported by the Laboratory Directed Research and Development program at ORNL. Metagenomic sequencing was conducted by the DOE Joint Genome Institute and soil organic matter analyses were performed using Fourier-transform ion cyclotron resonance mass spectrometry by PNNL’s Environmental Molecular Sciences Laboratory, both DOE Office of Science User Facilities. This work also leveraged the Oak Ridge Leadership Computing Facility, a DOE Office of Science User Facility. ORNL is managed by UT-Battelle for DOE’s Office of Science. The Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time. For more information, please visit http://science.energy.gov/.
211. Ning, D., Y. Deng, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, Y. Fu, D. J. Curtis, Y. Li, Y. Fan, M. B. Smith, A. M. Rocha, C. S. Smillie, S. W. Olesen, C. J. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, K. L. Bailey, R.A. Hurt Jr., S.P. Preheim, M.C. Sanders, J. Yang, M. A. Mueller, W. A. Lancaster, B. J. Vaccaro, F. L. Poole II, S. Brooks, D. B. Watson, A. Aaring, B. Adams, S. Brewer, K. De Leon, K. Fitzgerald, G. X. Ge, C. Hans, S. Kosina, L. Lui, E. Majumder, J.-W. Moon, A. Ottwell, S. Pfiffner, H. Smith, M. Thorgersen, S. Turkarslan, F. von Netzer, D. Williams, S. X. Wu, G. Zane, A. Zelaya, E. J. Alm, N. S Baliga, A. M. Deutschbauer, M. W. Fields, T. C. Hazen, T. R. Northen, J. D. Wall, M.W.W. Adams, R. Chakraborty, J.-M. Chandonia, D. A. Elias, D. A. Stahl, P. J. Walian, J. Zhou, A. P. Arkin and P. D. Adams. 2018. Ecological Stochasticity in Subsurface Microbial Community Assembly under Stress Gradient: Application of A General Quantitative Framework. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     Project Goals: ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) uses a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Our goal is to understand how human activity associated with energetic processes - in particular, contamination and climate change - is affecting the ecology of critical soil, groundwater, and aquifer systems. Despite decades of debate, many fundamental questions regarding the ecological drivers of community assembly remain unanswered. Classical stress gradient theory in ecology discussed the role of different deterministic forces (abiotic filtering, biotic interactions, etc.) under different degrees of stresses, however, we still know little about the basic question how deterministic versus stochastic forces vary along stress gradients, particularly in microbial ecology. The groundwater in the Oak Ridge Integrated Field Research Challenge site (FRC, Oak Ridge, TN) has large geochemical gradients and has been comprehensively surveyed, providing a rare opportunity to examine ecological processes and drivers shaping subsurface microbial diversity. Groundwater samples were taken from 98 wells that covered the geochemical diversity across the new index (NST) showed obviously higher accuracy and precision, of which the coefficients were over 0.9 in most simulated scenarios. However, all approaches showed limited performance at large spatial scale or under very high environmental noise. We then applied the new index to an empirical study on groundwater microbial community succession in response to emulsified vegetable oil (EVO) injection at FRC, with expected trend of ecological stochasticity has been supported by various evidences. The new index revealed that community assembly processes were shifted from deterministic to highly stochastic post-EVO input, and that, as EVO is consumed, the groundwater communities gradually returned to be more deterministic similar to pre-EVO injection. Null model algorithms and community similarity metrics showed strong effects on quantitatively estimating ecological stochasticity, among which preferred algorithm and metrics were suggested based on reasonability of the results. Then, we applied this new index to investigate how ecological stochasticity varies along stress gradients at FRC. The results suggested obvious decrease of ecological stochasticity by the increase of environmental stress. Furthermore, we explored depth profile of ecological stochasticity based on a pilot study of sediment bacterial communities in a contaminated well and a background well. We found obvious variation of ecological stochasticity from vadose to saturated layers related to both selection and dispersal limitation. Across different empirical datasets we tested, the new index generally can correct the overestimation of stochasticity by previous approaches to some extent, and revealed the obvious effects of environmental stress on the role of ecological stochasticity in governing underground microbiome. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
212. Newswise. 2018. Researchers Reveal How Microbes Cope in Phosphorus-deficient Tropical Soil. Newswise
213. Science Newsline. 2018. Researchers Reveal How Microbes Cope in Phosphorus-deficient Tropical Soil. Science Newsline
214. News(India). 2018. United States : Highly Cited Researchers list includes 15 from ORNL. UT NEWS https://news.utk.edu/2018/12/12/ut-home-to-several-most-cited-researchers/. abstract
     When peers cite a faculty member’s research, it’s a solid indication of the respect they have attained in their field. Clarivate Analytics annually calculates the world’s most cited researchers, and the 2018 list features eight people who work with the UT Knoxville campus in some capacity. Those selected from UT this year are: Sheng Dai, professor of chemistry; his work focuses on synthesizing and characterizing materials used in energy applications. Terry C. Hazen, UT-Oak Ridge National Laboratory Governor’s Chair for Environmental Biotechnology; he researches microbial ecology, water health, bioenergy, and the environment. David Mandrus, Jerry and Kay Henry Endowed Professor in materials science and engineering; he leads a group focused on discovering and characterizing new quantum materials. Art J. Ragauskas, UT-ORNL Governor’s Chair for Biorefining; his work deals with bioenergy, biofuels, and green chemical engineering. Gerald Tuskan, adjunct professor in the UT Institute of Agriculture; his work involves the study of plants for bioenergy. All five hold joint appointments with ORNL. Also selected were ORNL’s Sergei Kalinin, Richard Norby, and Bobby Sumpter, all of whom work with the ORNL-UT Bredesen Center for Interdisciplinary Research and Graduate Education.
215. ORNL News. 2018. Researchers reveal how microbes cope in phosphorus-deficient tropical soil. ORNL News
216. Technology Networks. 2018. Multi-Pronged Analysis Uncovers How Microbes Survive in Phosphorus-Poor Environments. Technology Networks
217. Miller, J. I., S. M. Techtmann, J. Fortney, N. Mahmoudi, D. C. Joyner, J. Liu, S. Olesen, E. Alm, A. Fernandez, P. Gardinali and T. C. Hazen. 2018. Anoxic hydrocarbon degradation in Caspian Sea. ASM Microbe 2018 abstract
     Background: Many geochemical and hydrological studies of the Caspian Sea exist, but information on its microbial community is sparse. It is a closed basin (riverine inflow, no outflow) with large inputs of anthropogenic and natural hydrocarbons (eg, from mud volcanoes). These factors combine with hypoxia in the deep water creating a unique niche, the effects of which on the microbial community have not been investigated. Methods: Ambient water was collected at 6 sites and 4 depths for analysis and microcosm experiments. Community structure of all samples was analyzed by 16S rRNA gene amplicon sequencing. Microcosm experiments were amended with 100 ppm oil hydrocarbons or no oil (control). On ship microcosms were maintained with atmospheric headspace, and destructively sampled at 0, 24, and 72 hours. In lab microcosms were monitored for CO2 respiration under under nitrogen vs atmospheric headspace and destructively sampled at 0, 3, and 17 days for sequencing and hydrocarbon analysis by GC/MS. Results: The ambient community was dominated by Gammaproteobacteria suggestive of oil-degraders. Deep sites were co-dominated by Crenarchaeota suggesting nutrient limitation or microaerophilic conditions. On ship microcosm communities differed by depth of sample origin but not by oil amendment, suggesting oil biodegradation may be influenced by environmental factors eg, oxygen concentration. In lab microcosm communities were dominated by Gammaproteobacteria, including enrichment of known hydrocarbon degraders (e.g., Oceanospirillacea) by 17 days. Oil amended microcosms respired more CO2 than controls (no oil). Oxic and anoxic microcosms degraded similar fractions of total hydrocarbons, but anoxic microcosms degraded substantially more 4 ring PAHs (100% vs 10%). The approximate half-life for total hydrocarbons was 11 days (anoxic) and 15 days (oxic). Conclusion: The half-life of the oil hydrocarbons is similar to other marine environments, but it is unexpected that anoxic loss exceeds oxic. Relative to surface waters, the microbial community in deep waters may have improved oil hydrocarbon biodegradation as an adaptation to the low oxygen, low nutrient environment.
218. McBride, K., S. Jagadamma, N. Daliang, J.-W. Moon, C. Paradis, D. C. Joyner, T. Melhorn and T. C. Hazen. 2018. Do microbes have memory? Repeated exposure to emulsified vegetable oil may increase degradation ability of native microbial communities. ASM Microbe 2018 abstract
     Background: Microbial "memory response" is the idea that microbial communities will degrade a substrate more rapidly if it has been exposed previously. In order to test this concept, anaerobic microcosm experiment was conducted for 150 days using sediment and groundwater from a low-contamination aquifer at the Oak Ridge Field Research Center which had been previously amended with an emulsified vegetable oil (EVO) injection years before. Four groundwater wells from the same site were used to create the microcosms-two of the wells were directly downstream from the previous injection of EVO, and the other two were upstream and unexposed to EVO. All microcosms were amended with EVO, and changes in both microbial communities and geochemical properties were compared to see if the rate of degradation was faster in those that had already been exposed to EVO. Gas chromatography was used to measure CO2 and CH4 production in the microcosms at several time points, while ion chromatography measured levels of acetate, nitrate, and sulfate in the water. ICP-MS was also utilized to measure trace metals found in the water and sediment. To analyze microbial communities, DNA was extracted from both microcosm sediment and groundwater followed by 16S rRNA sequencing. Results showed that after EVO addition, CH4 and CO2 were produced in both upstream and downstream samples at the same rate; similarly, nitrate and sulfate were also consumed at the same rate. However, acetate formed by EVO degradation was produced more rapidly and in much higher abundance in downstream wells. 16S data indicated that the relative abundance of known sulfate-reducing taxa, including those from the family Desulfobacteraceae, Desulfovibrionaceae, Geobacteraceae, and Desulfobulbaceae, were highest for all samples 30 days after EVO amendment, however, abundance was higher in downstream samples. Detrended correspondent analysis of OTU tables show that throughout all time points, there is a significant difference in the taxonomic community between upstream and downstream wells. This data indicates that perhaps degradation occurs at the same rate in both previously exposed and un-exposed samples, however the abundance of relevant degrading-species-and therefore overall reduction ability-may be higher in the previously exposed samples. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231.
219. McBride, K., S. Jagadamma, N. Daliang, J.-W. Moon, C. Paradis, D. Joyner, T. Mehlhorn and T. C. Hazen. 2018. Do microbes have memory? Repeated exposure to emulsified vegetable oil may increase degradation ability of native microbial communities. ISME17 abstract
     Microbial “memory response” is the idea that microbial communities will degrade a substrate more rapidly if it has been exposed to it previously—a novel concept which could increase efficiency of many bioremediation techniques. In order to observe a memory response, anaerobic microcosms were constructed with groundwater and sediment that had been previously exposed and unexposed to an injection of emulsified vegetable oil (EVO). The goal was to see which microcosm would degrade EVO faster. Microcosm groundwater and sediment were collected from the Oak Ridge Field Research Center (Oak Ridge, TN) downstream (exposed), and upstream (unexposed) from the previous injection of EVO. Anaerobic microcosms were amended with EVO and monitored for changes in microbial community and geochemistry. Gas chromatography measured CO2 and CH4 production, while ion chromatography measured levels of electron acceptors. DNA was extracted from sediments and groundwater, then analyzed by 16S rRNA sequencing. After EVO addition, gas production and electron acceptor consumption were the same rate in both samples. Although acetate production, a byproduct of degradation, was more rapid in exposed samples. 16S data indicated the relative abundance of sulfate-reducing taxa increased and peaked 30 days after amendment but was higher in exposed samples. Detrended correspondent analysis of OTU tables show a significant difference in the taxonomic population relative abundances between both samples throughout the experiment. Results indicate that degradation occurs at the same rate in both samples, but the abundance of relevant degrading species—and therefore overall degradation ability—may be higher in the previously exposed samples.
220. Marietou, Angeliki, Roger Chastain, Felix Beulig, Alberto Scoma, Terry C. Hazen and Douglas H. Bartlett. 2018. The Effect of Hydrostatic Pressure on Enrichments of Hydrocarbon Degrading Microbes From the Gulf of Mexico Following the Deepwater Horizon Oil Spill. Frontiers in Microbiology 9. abstract
     The Deepwater Horizon oil spill was one of the largest and deepest oil spills recorded. The wellhead was located at approximately 1500 m below the sea where low temperature and high pressure are key environmental characteristics. Using cells collected 4 months following the Deepwater Horizon oil spill at the Gulf of Mexico, we set up Macondo crude oil enrichments at wellhead temperature and different pressures to determine the effect of increasing depth/pressure to the in situ microbial community and their ability to degrade oil. We observed oil degradation under all pressure conditions tested [0.1, 15, and 30 megapascals (MPa)], although oil degradation profiles, cell numbers, and hydrocarbon degradation gene abundances indicated greatest activity at atmospheric pressure. Under all incubations the growth of psychrophilic bacteria was promoted. Bacteria closely related to Oleispira antarctica RB-8 dominated the communities at all pressures. At 30 MPa we observed a shift toward Photobacterium, a genus that includes piezophiles. Alphaproteobacterial members of the Sulfitobacter, previously associated with oil-degradation, were also highly abundant at 0.1 MPa. Our results suggest that pressure acts synergistically with low temperature to slow microbial growth and thus oil degradation in deep-sea environments.
221. Marietou, Angeliki, Roger Chastain, Felix Beulig, Alberto Scoma, Terry C. Hazen and Douglas H. Bartlett. 2018. The Effect of Hydrostatic Pressure on Enrichments of Hydrocarbon Degrading Microbes From the Gulf of Mexico Following the Deepwater Horizon Oil Spill (vol 9, pg 808, 2018). Frontiers in Microbiology 9.
222. Lui, L. M., H. K. Carlson, A. W. Sczesnak, O. Erbilgin, M. De Raad, V. Mutalik, A. Kazakov, X. Wu, A. Aaring, J. Voriskova, J. Kuehl, M. Price, D. Chivian, N. B. Justice, T. Simmons, K. De Leon, D. C. Joyner, H. J. Smith, P. S. Novichov, A. M. Deutschbauer, R. Chakraborty, T. R. Northen, J. D. Wall, T. C. Hazen, M.W. Fields, A. P. Arkin and P. D. Adams. 2018. Integration of metagenomics and consortia data to study microbial interactions and community assembly. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     Project Goals: ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) uses a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. ENIGMA uses a systems biology approach to build a predictive understanding how phenomena at the genetic, community, ecological, and environmental level influence microbial community assembly and activity. Sampling of sediment and groundwater microbial communities from the Oakridge National Laboratory Field Research Center (ORNL FRC) are coordinated with measurements of biogeochemical parameters to provide a rich set of samples, isolates, and data for integrated analysis across ENIGMA labs. Three categories characterize our efforts to mechanistically understand microbial community assembly: (1) direct analysis of isolate interactions and use of genetic tools to study them, (2) enrichment of communities under controlled laboratory conditions, and (3) analysis of amplicon and whole genome shotgun sequencing data. Each of these types of analyses is informed by the biogeochemical measurements from the field site and functional and genetic fitness characterization of isolates (see other ENIGMA posters). We study microbial interactions directly with synthetic communities and are developing novel microfluidics and isolation methods to directly observe interacting cells. Work with a consortium of Pseudomonas spp., the most common type of isolate from the FRC, has revealed that the type of carbon source available can influence the production of inhibitory secondary metabolites. Analysis of metabolomics data (NIMS and RP-LC-MS) derived from spent media provides candidate inhibitory compounds and the program MAGI is used to predict genes that produce them. Growth of transposon mutant libraries in spent media can provide information about which genes lead to susceptibility to these compounds. Preliminary data from experiments with combined carbon sources suggest that there is a “tipping point” in the proportion of a particular carbon source that is needed for an isolate to produce an inhibitory effect on other microbes. Currently, we are developing methods to contain sediment particles or transposon mutant libraries in microfluidics droplets to observe microbial interactions and irreducible communities. We are also developing a method that studies flocs as we hypothesize that in ORNL FRC groundwater direct interactions of microbes are likely to occur within flocs of two or more cells. We are collecting flocs for identification of its members as well as for isolations and enrichments that will be used to reconstruct these floc communities in the laboratory. To provide a means to edit microbes in a group and dissect ecosystem function, we also have a discovery project to find bacteriophages at ORNL FRC. Cultivation of enrichments provides a means to study sample communities under field relevant conditions and to study how ecological and environmental selection pressures in the same experiment affect community assembly. Building from experiments of isolates obtained with specific carbon sources, an experiment with 293 microcosms inoculated with ORNL FRC groundwater with varied type and number of sugars as a carbon source suggests that (1) a higher diversity of carbon sources leads to higher species diversity and (2) some carbon sources have a higher selection effect on the community structure than others. To study sulfate and nitrate reduction, two important metabolic activities at ORNL FRC, we are developing techniques for cultivation of enriched microbial communities under sulfate and nitrate reducing conditions. Enrichments are initiated under anoxic conditions using a variety of field relevant carbon sources. Additionally, we are using field geochemical information to guide media design. Finally, we are using amplicon and whole genome metagenomics shotgun sequencing to study community structure across ORNL FRC and to study predicted microbial functional profiles in conjunction with field data. Comparison of the 16S rDNA sequences that were most abundant at the field site (either in groundwater or sediment) to the sequences of >1,000 isolates from the site informs our isolation efforts. We predict that that many of the uncultivated yet abundant microorganisms are aerobic chemolithoautotrophs or slow-growing facultative heterotrophs that utilize more recalcitrant carbon. We have also developed a computational pipeline to do a fast, focused analysis of nitrogen cycle related genes in metagenomics data and used it to analyzed six samples that represented sections of a 20 foot sediment core. This pipeline can be adapted to other functional roles. This pipeline yielded a view of how nitrification changes with depth and predicts a consortium of an ammonia-oxidizing thaumarchaeon and a nitrite-oxidizing bacterial species. In addition to this computational pipeline, we are working with KBase to integrate other metagenomics tools for fast analysis of other ENIGMA samples. Currently we are working on metagenomics of two more cores and accompanying groundwater from ORNL FRC that are coordinated with biogeochemical data and activity data. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
223. Labor_Praxis. 2018. Hoffnung beim Kampf gegen Plastikmüll?.
224. LABO. 2018. Plastikfressende Bakterien: mehr und vielfältigere Arten als bisher angenommen.
225. Kothari, A., Y.-W. Wu, M. Charrier, L. Rajeev, A. Rocha, C. Paradis, T. C. Hazen, S. Singer and A. Mukhopadhyay. 2018. Identification and characterization of a mercury resistance plasmid shows latent functions harbored in pristine groundwaters. ISME17 abstract
     Native plasmids constitute a major category of extrachromosomal DNA elements responsible for harboring and transferring genes important for survival and fitness. We optimized a cultivation-free method targeting plasmid DNA across a range of sizes and abundance to examine the plasmidome of ground water samples from several pristine wells proximal to a superfund waste site. We made two interesting observations. One, the taxonomic distribution of bacteria showed greater similarity across samples based on their plasmidome sequences, relative to that based on 16S rRNA sequences. Second, candidates providing resistance to metals (copper, zinc, cadmium, arsenic, mercury) and antibiotics were particularly abundant. Deep sequencing was used to assemble several circular plasmids that further confirmed that the metal resistance genes, such as the highly conserved merR, merTPFA genes, are plasmid-encoded. To experimentally validate their function, we synthesized the most abundant plasmid containing the mer genes, and used a model bacterial system to demonstrate increased mercury resistance. Our results show that functional Hg2+ resistance is present on plasmids even though the ground water analysis does not reveal detectable levels of mercury. This study shows the unique ecological role of the plasmidome in maintaining the latent capacity of a microbiome, enabling rapid adaptation to environmental stresses.
226. Knoxville_News-Sentinel. 2018. UT focuses on search for two vice chancellors. Knoxville News-Sentinel abstract
     "The University of Tennessee, Knoxville is advertising for a vice chancellor for communications while it conducts an internal search for a vice chancellor for research."
227. Kimbrel, Jeffrey A., Nicholas Ballor, Yu-Wei Wu, Maude M. David, Terry C. Hazen, Blake A. Simmons, Steven W. Singer and Janet K. Jansson. 2018. Microbial Community Structure and Functional Potential Along a Hypersaline Gradient. Frontiers in Microbiology 9. abstract
     Salinity is one of the strongest environmental drivers of microbial evolution and community composition. Here we aimed to determine the impact of salt concentrations (2.5, 7.5, and 33.2%) on the microbial community structure of reclaimed saltern ponds near San Francisco, California, and to discover prospective enzymes with potential biotechnological applications. Community compositions were determined by 16S rRNA amplicon sequencing revealing both higher richness and evenness in the pond sediments compared to the water columns. Co-occurrence network analysis additionally uncovered the presence of microbial seed bank communities, potentially primed to respond to rapid changes in salinity. In addition, functional annotation of shotgun metagenomic DNA showed different capabilities if the microbial communities at different salinities for methanogenesis, amino acid metabolism, and carbohydrate-active enzymes. There was an overall shift with increasing salinity in the functional potential for starch degradation, and a decrease in degradation of cellulose and other oligosaccharides. Further, many carbohydrate-active enzymes identified have acidic isoelectric points that have potential biotechnological applications, including deconstruction of biofuel feedstocks under high ionic conditions. Metagenome-assembled genomes (MAGs) of individual halotolerant and halophilic microbes were binned revealing a variety of carbohydrate-degrading potential of individual pond inhabitants.
228. Johnston, E. R., M. Kim, J. K Hatt, J. R Phillips, Q. Yao, Y. Song, C. Pan, T. C. Hazen, M. A. Mayes and K. T. Konstantinidis. 2018. Phosphate addition increases tropical soil respiration primarily by deconstraining microbial population growth. ASM Microbe 2018 abstract
     Tropical ecosystems are an important sink for anthropogenic CO2 emissions; however, sustained uptake is increasingly restricted by phosphorus (P) availability. Soil microbiota facilitate the turnover of organic P and carbon (C) compounds and scavenge P from inorganic forms, but their role in tropical C-P-coupled biogeochemistry remains poorly understood. To advance this topic, soils collected from four sites in the El Yunque National Forest in Puerto Rico were incubated with exogenous PO43-. P-amendment increased CO2 respiration by 13.6-23.3% relative to control incubations for soils taken from all sites except the most P-rich one (no difference compared to its control). RNA-seq analysis revealed the increased relative transcription of genes involved in the biosynthesis of essential cell infrastructure components (purines, pyrimidines, phospholipids), cell division, and nutrient uptake and assimilation. A novel methodology assessing population-level gene expression by integrating companion metagenomic and metatranscriptomic datasets revealed that the trend of enhanced growth/respiration was community-wide. P-limited microbial communities possessed a greater abundance of genes for the biosynthesis of α-glucosyl polysaccharides, reflecting a community-wide adaptation to store excess organic carbon substrates under poor growth conditions (e.g., limiting phosphorus). Phosphorolysis genes governing the degradation of α-glucosyl polymers were more abundant in low-P soils and also had increased relative expression with P-amendment; these functions likely regulate the metabolism of stored organic substrates in soils with scarce P-availability. Collectively, our results provide quantitative estimates of increased CO2 respiration upon an alleviation of P-constraints and elucidated the underlying ecological and molecular mechanisms involved.
229. Inovations_report. 2018. Es gibt mehr und vielfältigere plastikfressende Bakterien, als bisher angenommen.
230. idw. 2018. Es gibt mehr und vielfältigere plastikfressende Bakterien, als bisher angenommen.
231. Hazen, T. C., S. M. Techtmann, J. Fortney, D. Joyner, O. G. Brakstad, N. Mahmoudi, S. Pfiffner, J. Liu, S. Olesen, E. Alm, A. Fernandez, P. Gardinali, D. Ning, J. Zhou and T. Linley. 2018. The ecology of oil-degrading microbiome in six deep sea basins worldwide. ISME17 abstract
     The Deepwater Horizon oil spill resulted in oil and gas being dispersed at 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT. This has also enabled comparative data for risk assessment and microbial community structure on several other potential and existing deep-water drilling sites around the world (Eastern and Central Mediterranean, Caspian Sea, Great Australian Bight, and off the coast of Angola) that have been examined in the same way over the past 6 years. Microbial community structures were very similar above 200m and below 200m. The half-life of oil from each basin ranged from 11-16 days, whereas the in the Gulf of Mexico it was 1.2-6.1 days. Mineralization rates were however, much lower for all basins then the Gulf of Mexico. Limiting nutrients and adaption to natural oil seeps were major factors controlling these differences.
232. Hazen, T. C., D. C. Joyner, A.-M. Harik and A. Putt. 2018. Governor's School Students. Governor's School Students
233. Hazen, T. C.. 2018. Environmental Systems Biology . NASA Life Detection/Microbial Monitoring Workshop.
234. Hazen, T. C.. 2018. Environmental Systems Biology Approach to Bioremediation. Bioremediation Workshop abstract
     Pollution is everywhere. Microbes are also everywhere and many have the ability to degrade environmental contaminants. Understanding how these microbial communities work to degrade environmental contaminants will enable us to use these microbes to clean up the pollution. Understanding, monitoring and controlling the environment with biological processes, i.e. environmental systems biology approach to bioremediation (the need is everywhere). By using an environmental systems approach to bioremediation, we make sure we know of any ‘fatal flaws’ in the approach, get a much better handle on life cycle cost analysis and can grade an engineered solution into a natural attenuation solution. The whole is greater than the sum of its parts. Using an environmental systems biology approach to bioremediation and cross linkage of systems at all levels providing multiple lines of evidence involving environmental observations, laboratory testing, microcosm simulations, hypothesis refinement, field testing and validation and multiple iterations of this circle we will be able to make new theories and paradigms for bioremediation of contaminated environments.
235. Hazen, T. C.. 2018. ENIGMA GeoDD campaign. ENIGMA SAC and Retreat
236. T. C. Hazen. 2018. Bioremediation. Bioremediation
237. T. C. Hazen. 2018. Laboratory Safety. Nuclear Engineering Department Seminar
238. Hazen, T. C.. 2018. Oil Bioremediation of Soil and Sediment Worldwide. China Petroleum Company
239. Hazen, T. C.. 2018. Structured learning in microbial ecology using microbial community structure to predict geochemistry for food, water and energy. Modelling the Nexus of Food, Energy and Water Systems
240. T. C. Hazen. 2018. Moderator: Managing Stress: Microbial Community Responses to Environment Challenges. ASM Microbe 2018
241. Hazen, T. C.. 2018. Microbial Community Structure Predicts Groundwater Geochemistry. University of Puerto Rico RICE Program abstract
     At the Department of Energy's Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0 µm pre-filter and 0.2 µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry and even contaminants in marine environments.
242. T. C. Hazen. 2018. Contrasting Ecosystem Responses Between EVOS and DWH. Alaska Oil Spill Technology Symposium.
243. T. C. Hazen. 2018. Panel discussion on professional organizations representing biological, environmental, and earth sciences. Lunch & Learn Series ORNL
244. Haag, Tyra. 2018. UT Announces Search for Two Vice Chancellors. University of Tennessee Campus News abstract
     Searches for UT’s next vice chancellor for research and vice chancellor for communications are now underway. Both positions serve on the chancellor’s cabinet. An internal search for the vice chancellor for research has begun, with a goal of having a new leader in place by late October. This position serves as the chief research officer of the university and works to promote research and economic development activities, particularly in collaboration with Oak Ridge National Laboratory through UT–Battelle and with other research and development institutions in the state and region. Stacey Patterson, vice president for research at the UT System, will chair the search. Members of the search committee include Christine Boake, associate dean for research and facilities, College of Arts and Sciences; Michelle Buchanan, deputy for science and technology, Oak Ridge National Laboratory; Mark Dean, John Fisher Distinguished Professor, Tickle College of Engineering; Terry Hazen, Governor’s Chair for Environmental Biotechnology; Diane Kelly, director of the School of Information Sciences, College of Communication and Information; Jean Mercer, assistant vice chancellor and director, Office of Sponsored Projects, Office of Research and Engagement; Marisa Moazen, executive director, undergraduate research, Office of Research and Engagement; Jeff Pappas, director of the School of Music, College of Arts and Sciences; Hollie Raynor, interim assistant dean for research, College of Education, Health, and Human Sciences; and Tami Wyatt, associate dean of research and Torchbearer Professor, College of Nursing. Robert Nobles has been serving as interim vice chancellor for research since August 1 and will continue to serve until the position is filled. A national search is underway for the next vice chancellor for communications. This position is the university’s chief communications and marketing officer and advises the chancellor and other senior leaders on communications-related matters. The position oversees the Office of Communications and Marketing and WUOT-FM 91.9, the university’s public radio station. Vice Chancellor for Student Affairs Vincent Carilli has been serving in this role unofficially since May and will continue to do so until the position is filled. Mike Wirth, dean of the College of Communication and Information, is chairing the search. Committee members include Lola Alapo, public information officer for public safety; Chip Bryant, vice chancellor for advancement; Tiffany Carpenter, associate vice president for communications and marketing for the UT System; Fabrizio D’Aloisio, assistant vice provost for enrollment management and director of undergraduate admissions; Tyra Haag, director of media relations in the Office of Communications and Marketing; Sadie Hutson, assistant dean for graduate programs in the College of Nursing; Haley Paige, vice president of the Student Government Association; and Donna Thomas, senior associate athletics director and chief of staff for the Athletics Department. The committee’s review of candidate materials for the vice chancellor for communications will begin in the coming weeks with the intent to bring candidates to campus in late September or early October for public forums. Brooke Swart, executive recruiter in UT Human Resources, is coordinating both searches. Position descriptions are available online.
245. Goddard, David. 2018. UT Researches Effects of Fracking on Water Health. . Knoxville, TN. UT News. http://news.utk.edu/2018/10/12/ut-researches-effects-of-fracking-on-water-health/ . UT News
246. EurekAlert!. 2018. Researchers Reveal How Microbes Cope in Phosphorus-deficient Tropical Soil. EurekAlert
247. Christensen, Geoff A., JiWon Moon, Allison M. Veach, Jennifer J. Mosher, Ann M. Wymore, Joy D. van Nostrand, Jizhong Zhou, Terry C. Hazen, Adam P. Arkin and Dwayne A. Eliasl. 2018. Use of in-field bioreactors demonstrate groundwater filtration influences planktonic bacterial community assembly, but not biofilm composition. Plos One 13. abstract
     Using in-field bioreactors, we investigated the influence of exogenous microorganisms in groundwater planktonic and biofilm microbial communities as part of the Integrated Field Research Challenge (IFRC). After an acclimation period with source groundwater, bioreactors received either filtered (0.22 mu M filter) or unfiltered well groundwater in triplicate and communities were tracked routinely for 23 days after filtration was initiated. To address geochemical influences, the planktonic phase was assayed periodically for protein, organic acids, physico-/geochemical measurements and bacterial community (via 16S rRNA gene sequencing), while biofilms (i.e. microbial growth on sediment coupons) were targeted for bacterial community composition at the completion of the experiment (23 d). Based on Bray-Curtis distance, planktonic bacterial community composition varied temporally and between treatments (filtered, unfiltered bioreactors). Notably, filtration led to an increase in the dominant genus, Zoogloea relative abundance over time within the planktonic community, while remaining relatively constant when unfiltered. At day 23, biofilm communities were more taxonomically and phylogenetically diverse and substantially different from planktonic bacterial communities; however, the biofilm bacterial communities were similar regardless of filtration. These results suggest that although planktonic communities were sensitive to groundwater filtration, bacterial biofilm communities were stable and resistant to filtration. Bioreactors are useful tools in addressing questions pertaining to microbial community assembly and succession. These data provide a first step in understanding how an extrinsic factor, such as a groundwater inoculation and flux of microbial colonizers, impact how microbial communities assemble in environmental systems.
248. Chen, Chunyi, Gang Pan, Wenqing Shi, Feng Xu, Stephen M. Techtmann, Susan M. Pfiffner and Terry C. Hazen. 2018. Clay Flocculation Effect on Microbial Community Composition in Water and Sediment. Frontiers in Environmental Science 6. abstract
     Clay-based flocculation techniques have been developed to mitigate harmful algal blooms; however, the potential ecological impacts on the microbial community are poorly understood. In this study, chemical measurements were combined with 16S rRNA sequencing to characterize the microbial community response to different flocculation techniques, including controls, clay flocculation, clay flocculation with zeolite, and clay flocculation with O-2 added zeolite capping. Sediment bacterial biomass measured by PLFA were not significantly altered by the various flocculation techniques used. However, 16S rRNA sequencing revealed differences in water microbial community structure between treatments with and without zeolite capping. The differences were related to significant reductions of total nitrogen (TN), total phosphorus (TP) and ammonia (NH4+) concentration and increase of nitrate (NO3-) concentration in zeolite and O-2 loaded zeolite capping. The relative abundance of ammonia oxidizing bacteria increased four-fold in zeolite capping microcosms, suggesting zeolite promoted absorbed ammonia removal in the benthic zone. Zeolite-capping promoted bacteria nitrogen cycling activities at the water-sediment interface. Potential pathogens that are usually adapted to eutrophic water bodies were reduced after clay flocculation. This study demonstrated clay flocculation did not decrease bacterial populations overall and may reduce regulatory indicators and pathogenic contaminants in water. Zeolite capping may also help prevent nutrients from being released back into the water thus preventing additional algal blooms. IMPORTANCE Despite the effectiveness of clay flocculation for removing harmful algal blooms as highlighted in numerous studies, the potential ecological impacts on the microbial community have rarely been investigated. Characterization of clay flocculation treated algal bloom water and sediment microbial community provides new insights into the ecological impacts of this algal bloom controlling technology.
249. Chakraborty, R., X. Wu, L. Wu, Y. Liu, P. Zhang, J. Zhou, N. Hess and T. C. Hazen. 2018. Microbial interactions with dissolved organic matter drive carbon dynamics and community succession in groundwater. ISME17 abstract
     Understanding the interaction between natural organic matter (NOM) and microbial communities is fundamental to predicting the links between carbon flux, microbial community structure and function in ecosystems. Yet, these key processes are mostly studied disconnected as separate units, independent of each other, and a meaningful comprehensive view has been elusive thus far. Insight into mechanisms of microbially mediated carbon cycling from NOM is of deep interest to microbial ecologists, and an approach combining multidisciplinary tools is imperative to connect carbon to microbial communities. In this study, we fed sediment-derived dissolved organic matter (DOM) to aquifer groundwater microbes and continually analyzed microbial transformation of DOM over a 50-day incubation. To document fine-scale changes in DOM chemistry, we applied high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). We also monitored the trajectory of microbial biomass, community structure and activity over this time period. With depletion of labile C and relative increase of recalcitrant C pool during incubation, microbial community shifted towards organisms that might possess a greater affinity for less labile C metabolism. The changes in microbial community structure and function, coupled with the contribution of microbial products to DOM pool affected the further transformation of DOM, culminating in stark changes to DOM composition over time.
250. Chakraborty, R., M. W. Fields, X. Wu, T. C. Hazen, H. J. Smith, T. R. Northen, Y. Liu, N. Hess, P. Zhang, J. Zhou, A. P. Arkin and P. D. Adams. 2018. Natural Organic Matter Dynamics and ExoMetabolomics for Microbial Cultivation from the Shallow Subsurface at the Oak Ridge FRC. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     Project Goals: ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) uses a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. Natural organic matter (NOM) is central to microbial food webs and microbially mediated NOM transformations determine much of the carbon (C) flux in subsurface environments. However, little is known about the molecular signatures from differnet C pools, the microbial activities/populations that regulate NOM turnover, nor the exometabolic markers for this activity. The goal of this project is to study the interactions between NOM (extracted from the field site) and native microbial communities present in uncontaminated and contaminated environments at Oak Ridge Field Research Center, TN. Water-soluble NOM was extracted from sediment samples collected from two background uncontaminated sites. The amount of inorganic C in extracted NOM decreased significantly with depth. Extracted NOM was used as the sole source of carbon in controlled lab incubations. Lignin, lipid, and protein levels were similar for the different depth intervals, while the relative proportions of carbohydrate, tannin, and amino sugars declined with depth and condensed aromatics increased with depth. The nitrogen content of the extractable sediment NOM compounds varied between 13 to 28%, and the deeper sediment intervals contained more S than surface intervals. Groundwater NOM was compared to sediment-extracted NOM for an uncontaminated and contaminated well (well 106 v. 271). For groundwater, the NOM is dominated by amino acid-like fluorescence, and the fluorescence is approximately 60-fold greater in the contaminated well. The spectra also indicate the formation of more recalcitrant compounds in the sampled uncontaminated groundwater. For the sediment-extracted NOM, we used the humification index (HIX) as a measure of the complexity and the condensed (aromatic) nature of the NOM. The HIX indicates a drastic decrease in humic-like fluorescence for depths >2m for both cores compared to the shallower depths (<2m). The deeper depths had mainly amino acid-like fluorescence. NOM was extracted from the uncontaminated samples and incubated with groundwater as the inoculum. We monitored the trajectory of microbial biomass, respiration, community structure and activity over the course of the incubation. To document changes in organic matter chemistry, we applied high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and soft X-ray absorption spectroscopy (sXAS). Together, these analyses provided a greater view of NOM degradation by indigenous microbes. Transformation of NOM continued even after depletion of the labile C pool, and microbial populations shifted from presumptive copiotrophic to presumptive oligotrophic microorganisms that most likely possess greater affinity for diverse non-labile carbon. The C pool shifted during incubation, the proportion of lignin in cultures increased while overall protein levels declined. In addition, Geochip was used to identify the changes of microbial communities and expression of functional genes during transformation of the NOM, and putative gene sequences associated with chitin and lignin degradation increased after the depletion of more labile carbon. Futhermore, exometabolomic methods were developed to characterize ninety-six metabolites from sediment samples that included amino acids, carbohydrates (acid/alcohol), carboxylic acids, and nucleosides. The extraction procedure focused specifically on potentially accessible for microbial metabolites that may be present in sediment/soils. The water extractable organic carbon metabolites (WEOCs), were first qualitatively characterized using both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS). Application of these complementary technologies provided orthogonal confirmation of metabolite identification while also expanding the analytical scope of the study. From these data, a subset of metabolites was selected for absolute quantification to assist in formulating a set of defined media that approximate the composition and quantity of metabolites within the sediments. One composition was demonstrated to support the growth of 25 phylogenetically diverse isolates out of 30 tested from the Oak Ridge Field Research Center (ORFRC), and detailed time series characterization of the substrate preferences are underway. Thus, the characterization of NOM and exometabolites in groundwater and sediment is described and then used to construct a defined medium for use with indigenous populations for the assessment of substrate utilization and microbial interactions. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
251. Campa, Maria Fernanda, Stephen M. Techtmann, Caleb M. Gibson, Xiaojuan Zhu, Megan Patterson, Amanda Garcia de Matos Amaral, Nikea Ulrich, Shawn R. Campagna, Christopher J. Grant, Regina Lamendella and Terry C. Hazen. 2018. Impacts of Glutaraldehyde on Microbial Community Structure and Degradation Potential in Streams Impacted by Hydraulic Fracturing. Environmental Science & Technology 52:5989-5999. abstract
     The environmental impacts of hydraulic fracturing, particularly those of surface spills in aquatic ecosystems, are not fully understood. The goals of this study were to (1) understand the effect of previous exposure to hydraulic fracturing fluids on aquatic microbial community structure and (2) examine the impacts exposure has on biodegradation potential of the biocide glutaraldehyde. Microcosms were constructed from hydraulic fracturing-impacted and nonhydraulic fracturing-impacted streamwater within the Marcellus shale region in Pennsylvania. Microcosms were amended with glutaraldehyde and incubated aerobically for 56 days. Microbial community adaptation to glutaraldehyde was monitored using 16S rRNA gene amplicon sequencing and quantification by qPCR. Abiotic and biotic glutaraldehyde degradation was measured using ultra-performance liquid chromatography-high resolution mass spectrometry and total organic carbon. It was found that nonhydraulic fracturing-impacted microcosms biodegraded glutaraldehyde faster than the hydraulic fracturing-impacted microcosms, showing a decrease in degradation potential after exposure to hydraulic fracturing activity. Hydraulic fracturing-impacted microcosms showed higher richness after glutaraldehyde exposure compared to unimpacted streams, indicating an increased tolerance to glutaraldehyde in hydraulic fracturing impacted streams. Beta diversity and differential abundance analysis of sequence count data showed different bacterial enrichment for hydraulic fracturing impacted and nonhydraulic fracturing-impacted microcosms after glutaraldehyde addition. These findings demonstrated a lasting effect on microbial community structure and glutaraldehyde degradation potential in streams impacted by hydraulic fracturing operations.
252. Campa, M. F., S. M. Techtmann, C. Gibson, X. Zhu, M. Patterson, A. Garcia de Matos Amaral, N. Ulrich, S. R. Campagna, C. J. Grant, R. Lamendella and T. C. Hazen. 2018. Hydraulic fracturing impacted aquatic microbial community response to the biocides glutaraldehyde and 2-2-dibromo-3-nitrilopropionamice. ISME 2018 abstract
     Hydraulic fracturing (HF) is a method of hydrocarbon extraction that uses a mixture of pressurized water and chemicals to create fractures in non-permeable strata. HF produces large volumes of hyper-saline wastewater, containing chemicals such as biocides. Handling of HF wastewater is of environmental concern. Microcosms of stream water impacted by a HF spill (HF+) and not impacted by HF (HF-) were used to understand difference in natural attenuation after an HF spill. Preceding a 56-day aerobic incubation two sets of microcosms were spiked with either the biocide glutaraldehyde (GA) or other with the biocide 2-2-dibromo-3-nitrilopropionamide (DBNPA). 16S rRNA amplicon sequencing revealed that GA and DBNPA caused different responses in microbial community structure and degradation potential. GA HF+ microcosms experienced a smaller log fold reduction in 16S rRNA gene copies/mL than GA HF- microcosms, this was opposite for DBNPA. Alpha diversity of the HF+ microbial community showed a higher tolerance for GA and DBNPA. GA had a half-life of 51.9 d in HF+ and 33.8 d in HF- microcosms, while the abiotic controls did not exhibit degradation of GA, demonstrating GA degradation was microbially mediated. DBNPA had a half-life of 70.0 d in HF+ and 17.5 d in HF- microcosms, whereas the abiotic microcosms had a half-life of 30.0 d in HF+ and 8.12 d in HF- microcosms. This demonstrates biotic-abiotic interactions were at play. These findings revealed a lasting effect on microbial community structure and suppressed degradation potential in streams impacted by HF spills.
253. Campa, M. F., S. M. Techtmann, C. Gibson, X. Zhu, M. Patterson, A. Garcia de Matos Amaral, N. Ulrich, S. R. Campagna, C. J. Grant, R. Lamendella and T. C. Hazen. 2018. A comparison of the impacts of the biocides Glutaraldehyde and DBNPA on aquatic microbial community structure and degradation potential. ASM Microbe 2018 abstract
     Hydraulic fracturing (HF) is a method of hydrocarbon extraction that employs a mixture of pressurized water, chemicals, and sand to create fractures in hydrocarbon rich underground shale. Large volume of extremely saline fluids with chemicals, sand, and hydrocarbons flows back to the surface. Potential spills and mishandling of this wastewater are of environmental concern. Biocides are one of the most toxic chemicals used in HF. Biocides are added to prevent biocorrosion of equipment and gas souring. To understand the effect different biocides have on aquatic microbial community structure and degradation potential after a HF fluids spill, two sets of microcosms were constructed using stream water impacted (HF+) and not impacted (HF-) by Marcellus shale HF operations. Microcosms were incubated aerobically at ambient temperature for 56 days. One set of microcosm was spiked with glutaraldehyde (GA), the most commonly used biocide in HF operations, and the second set was spiked with 2-2-dibromo-3-nitrilopropionamide (DBNPA), the second most commonly used biocide in HF operations. Microbial community response to the biocides was monitored every two weeks using next-generation 16S rRNA amplicon sequencing. Biotic and abiotic GA degradation was measured every week using Liquid Chromatography- coupled with Exactive Quadrupole-Orbitrap Mass Spectrometry, and DBNPA degradation was measured using High Performance Liquid Chromatography. GA and DBNPA caused similar responses in microbial community structure and degradation potential, but GA caused a more pronounced response. HF+ experienced a smaller log fold reduction in 16S rRNA gene copies/mL in GA microcosms, but HF- in DBNPA microcosms experiences a smaller log reduction after addition of DBNPA but by day 56 HF+ was more enriched than HF-. Furthermore, more members of the microbial community of HF+ were able to tolerate GA as showed by higher alpha diversity, which was similar to DBNPA. Beta diversity and differential expression analysis for sequence count data showed different bacterial enrichment after GA and DBNPA addition for HF+ and HF- microcosms. However, the phylogenetically different response between HF+ and HF- microcosms was different between GA .
254. Cabage, Bill. 2018. Highly Cited Researchers list includes 15 from ORNL. ORNL Review https://www.ornl.gov/news/highly-cited-researchers-list-includes-15-ornl. abstract
     Honored for production of multiple highly cited papers ranking in the top 1% by citations during 2006-2016. Fifteen scientists at the Department of Energy’s Oak Ridge National Laboratory were named in the 2018 Highly Cited Researchers list compiled by Clarivate Analytics. The list recognizes researchers for exceptional performance, demonstrated by the production of multiple highly cited papers ranking in the top one percent by citations for field and year in Web of Science during the 11-year period 2006-2016. This year’s listing includes the following researchers from ORNL: Ilias Belharouak of the Energy and Transportation Science Division; Miaofang Chi of the Center for Nanophase Materials Sciences; Sheng Dai of the Chemical Sciences Division; Terry Hazen, University of Tennessee (UT)-ORNL Governor’s Chair for Environmental Biotechnology; Juan Carlos Idrobo of the Center for Nanophase Materials Sciences; Sergei Kalinin of the Center for Nanophase Materials Sciences; David Mandrus of the Materials Science and Technology Division; Michael McGuire of the Materials Science and Technology Division; Michael Naguib of the Chemical Sciences Division; Richard Norby of the Environmental Sciences Division; Arthur Ragauskas, UT-ORNL Governor’s Chair for Biorefining; Brian Sales of the Materials Science & Technology Division; Bobby Sumpter of the Center for Nanophase Materials Sciences; Peter Thornton of the Environmental Sciences Division; Gerald Tuskan, director of the Center for Bioenergy Innovation The list “is a contribution to the identification of that small fraction of the researcher population that contributes disproportionately to extending the frontier and gaining for society knowledge and innovations that make the world healthier, richer, sustainable and more secure,” according to Clarivate.
255. ASCE First Bell. 2018. UT Professor participates in Fracking Research. FIRST BELL ASCE
256. Beattie, Rachelle E., Wyatt Henke, Maria F. Campa, Terry C. Hazen, L. Rex McAliley and James H. Campbell. 2018. Variation in microbial community structure correlates with heavy-metal contamination in soils decades after mining ceased. Soil Biology & Biochemistry 126:57-63. abstract
     Microorganisms play vital roles in Earth's biogeochemical cycles. Identifying disturbances in microbial communities due to anthropogenic contamination can provide insights into the health of ecosystems. Picher, Oklahoma, was the site of large-scale mining operations for Pb, Zn, and other heavy metals until the mid-1950s, operating within the Tri-State Mining District (TSMD) of Missouri, Kansas and Oklahoma. Although mining ceased decades ago, high concentrations of heavy metals (> 1000 ppm) remain in area soil and water systems. Previously, we mapped metal concentrations on samples collected from mine tailings in Picher and along cardinal-direction transects within an 8.05-km radius of the town. To elucidate changes in microbial community structure due to regional metal contamination, 16S rRNA gene sequences and qPCR calculations of total Bacteria and Archaea were analyzed against these metal concentrations. Bacteria were negatively and significantly correlated with Pb, Cd, Zn, and Mg; however, Archaea was only significantly and positively correlated with pH. Illumina sequencing of 16S rRNA genes showed significant differences in microbial communities of chat and west transect samples. Comparison of soil chemistry with community structure indicated that Al, Pb, Cd, and Zn significantly impacted community composition and distribution of individual OTUs. Mapping the distribution of heavy-metal contamination and microbial communities in these soils represents the first step in understanding effects of long-term, heavy-metal contamination at a basic trophic level.
257. Arkin, A. P., D.A. Stahl, E. J. Alm, N. S. Baliga, A. M. Deutschbauer, M. W. Fields, T. C. Hazen, T. R. Northen, J. D. Wall, M.W.W. Adams, M. Auer, K. Bender, G. Butland, R. Chakraborty, J.-M. Chandonia, D. A. Elias, P. S. Novichkov, A. Mukhopadhyay, G. E. Siuzdak, P. J. Walian, J. Zhou and P. D. Adams. 2018. ENIGMA Science Focus Area. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     Project Goals: ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) uses a systems biology approach to understand the interaction between microbial communities and the ecosystems that they inhabit. To link genetic, ecological, and environmental factors to the structure and function of microbial communities, ENIGMA integrates and develops laboratory, field, and computational methods. The goal of Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) scientific focus area is to develop an unprecedented mechanistic and predictive understanding of the assembly, activity, stability and resilience of complex microbial communities in variable conditions. Established in 2009, ENIGMA is a collaborative consortium of 23 investigators at fourteen institutions located across the country and is led by principals at the Lawrence Berkeley National Laboratory. We are focused on sediment and groundwater communities and the processes that impact (and are impacted by) anthropogenic contaminants resulting from legacy nuclear programs at the Oak Ridge National Laboratory (ORNL). At the Oak Ridge Reservation (ORR) we perform sophisticated field experiments to measure the natural and anthropogenically perturbed dynamics of these geochemical processes and microbial community assembly and activity. From these we infer the chemical, physical and microbial interactions most predictive of these dynamics and estimate the ecological forces, both stochastic. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
258. Alm, E. J., S. J. Spencer, V. K. Mutalik, T. C. Hazen, J.-M. Chandonia, J. Zhou, R. Chakraborty, A. Deutchbauer, A. P. Arkin and P. D. Adams. 2018. Strain isolation, genome sequencing, and functional genomics reveals adaptive evolution of a Pseudomonas population at a human impacted field site. Genomic Sciences Program Annual PI Meeting https://www.orau.gov/gsp2018/2018-GSP-Abstract-Book.pdf. abstract
     Project Goals: Whole genome sequencing of environmental strains can reveal recent adaptive changes, making it a powerful tool to understand how environmental conditions impact the microbial genomes in those environments. Here we aimed to combine whole genome sequencing with functional genomics of field isolates to build a mechanistic understanding of natural selection at a human impacted groundwater site. Microbial communities in groundwater ecosystems are inherently difficult to study due to sampling challenges paired with highly dynamic microenvironments. A number of recent efforts have characterized and mined the vast bacterial diversity within groundwater via amplicon surveys and shotgun metagenomics, but these data sources cannot reveal dynamic or historical changes without high-resolution sampling. Here we identify recent microbial adaptation within groundwater sites using isolation and whole genome sequencing, and combined these insights with functional genomic experiments directly measuring the contribution of genes to fitness under controlled laboratory conditions. We isolated 139 Pseudomonas strains, that grouped into 15 lineages, and found that each lineage contained strains from multiple sampling sites across a broad geographic region. SNP analysis confirmed that recent mutational changes were impacting strains in individual sampling sites, despite a fast-flowing aquifer, and a two-component system showed evidence of positive selection. We also searched for gene gain and loss within strain isolates, and found evidence of transcriptional gene excision within otherwise clonal isolates at a single location. We next sought to investigate the genes identified from genome sequencing with high-throughput in vitro fitness measurements. Transposon-based mutagenesis was used to generate a saturating library of knock out mutants which were grown across hundreds of conditions. Fitness measurement revealed that genes in the signaling pathway identified by genome analysis as undergoing adaptation were also among the most likely to be selected for across a variety of in vitro conditions. These data suggest it may be possible to not only identify the genes under selection, but the environmental factors driving that selection by combinging sequencing with high-throughput functional genomics. Funding statement: ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH1123
259. Yao, Q., Z. Li, Y. Song, S. J. Wright, X. Guo, A. Biswas, S. G. Tringe, T. C. Hazen, B. L. Turner, M. Mayes and C. Pan. 2017. Characterizing microbe response to P availability in Panama soils by long term fertilization. DOE JGI user meeting
260. von Netzer, F., D. Gorman-Lewis, E. Shock, S. Turkarslan, C. E. Arens, A. W. Thompson, N. S. Baliga, A. Zhou, J. Zhou, A. Aaring, R. Chakraborty, J. W. Moon, D. Elias, D. C. Joyner, T. C. Hazen, H. Smith, M. Fields, F. Poole, M. W. W. Adams, H. Carlson, A. Deutschbauer, D. Vuono, K. Meinhardt, D. A. Stahl, A. P. Arkin and P. D. Adams. 2017. Understanding the thermodynamic Foundations of microbial Growth Efficiencies in the Lab and Field. 2017 Genomic Sciences Program Annual PI Meeting. abstract
     Project Goals: A key element of microbial growth and therefore microbial community assembly is how microbes partition the available resources between energy required for maintenance and growth. Using microcalorimetry and thermodynamic modelling, we gained a quantitative proxy for microbial growth efficiency under different growth conditions. As the next step, we aim to apply our methods to address the ecological framework guiding the partitioning of denitrification pathways at the Oak Ridge National Laboratory Field Research Site (FRC); through the establishment of microbial activity assays, isotope fractionation analysis as well as establishment of mass balances and stoichiometry for representative nitrate respiring isolates. The assembly of microbial communities is determined by many factors, with the environment setting the stage via availability of electron acceptors, donors and carbon sources as well as with physical/chemical parameters such as temperature and salinity. In this framework microbes have to adapt to either stable or dynamic conditions and to either compete or share resources for survival. Also, bacteria need to balance constantly the division of available energy between maintenance of basic cellular functions and growth. Therefore, microbes with the most favorable ratio between maintenance and growth requirements should be more competitive compared to microbes with higher energy demands for maintenance and growth. Maintenance energy levels as a proxy for microbial competitiveness are usually measured in chemostats near zero growth conditions. Here, we attempt to capture maintenance energies via microcalorimetry and a metabolite-dependent thermodynamic model as a quantitative proxy for microbial growth efficiency. Microcalorimetry offers a direct and highly sensitive method to assess the enthalpy-related terms of microbial growth in relation to the potential energy supplied by growth substrates. This in turn allows for a quantitative description of growth and maintenance in thermodynamic terms under different growth conditions, based on the comparison of the metabolite profile at the start and end of growth. We have examined the influence of temperature stress, simulated environmental dynamics and adaptation to salt stress on the growth efficiencies of different strains of Desulfovibrio vulgaris Hildenborough and D. alaskensis G20. These analyses quantified the cost of maintenance (survival) in relationship to increasingly suboptimal growth conditions, the cost of regulation in a fluctuating environment and the reduction in maintenance costs realized through adaptive evolution. We aim to apply our growth efficiency and maintenance proxy to field conditions by addressing the ecological framework guiding the partitioning of denitrification pathways at the Oak Ridge National Laboratory Field Research Site (FRC). This site has a long contamination history with nitrate among a large variety of other contaminants. Thermodynamic modeling based on mass balances and stoichiometries of site-relevant nitrate respiring isolates will be associated with in situ measurements of nitrate respiration and nitrate stable isotope fractionation analyses to develop a predictive framework for microbial community assembly and activity. As the next step, we are cultivating available nitrate respiring isolates (Rhodanobacter, Acidovorax and Pseudomonas spp.) under different C/N ratios and the application of the thermodynamic model established with metabolite profiles, we aim to record the energy requirements under different biogeochemical conditions. These profiles can then out into context to actual biogeochemical field conditions as characterized by nitrate specific acetylene-block activity assays and the metabolic history as traced by nitrate stable isotope fractionation. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231
261. Smith, H. J., A. Zelaya, I. Miller, D. Joyner, T. C. Hazen, M. W. Fields, A. P. Arkin and P. D. Adams. 2017. Temporal Variability and Microbial Activity in Groundwater Ecosystems. 2017 Genomic Sciences Program Annual PI Meeting. abstract
     Project Goals: A fundamental goal in the field of microbial ecology is to link the activity and structure of microbial populations and communities to processes occurring within an ecosystem. This project aims to identify the drivers of community structure and succession by identifying the metabolically active fraction of microbial communities from both pristine and contaminated groundwater habitats at the Field Research Center (FRC) at Oak Ridge National Laboratory (ORNL). It is hypothesized that community function is independent of phylogeny and that functionality will be altered as a result of environmental perturbations. The use of geochemically distinct wells in combination with the enumeration and sequencing of translationally-active microorganisms, activity assays, carbon utilization profiles, and geochemical measurements will allow for the elucidation of the mechanisms shaping community structure and function in terms of turnover of natural organic matter and major contaminants (e.g., NO3 -). Multiple assay comparisonswill be used to achieve an accurate characterization of the active fraction of groundwater microbial communities and will ultimately be applied to continuous sediment cores from both contaminated and pristine locations. Abstract: Saturated subsurface environments are estimated to contain approximately 40% of the prokaryotic biomass on Earth, and due to the complexity of these habitats they support highly diverse microbial communities. In addition, it is estimated that over 98% of the Earth’s consumable and available freshwater is in the subsurface as groundwater. However, the factors that determine microbial community assembly, structure, and function in groundwater systems and the impact on water quality and contaminant transport remain poorly understood. Three non-contaminated background wells were sampled for groundwater geochemistry and microbial diversity approximately 3 times a week over a period of three months. Community analysis via ss-rRNA paired-end sequencing and distribution-based clustering revealed temporal differences in richness, diversity, and variability in the groundwater communities. Microbial community composition of a given well was on average >50% dissimilar to any other well at a given time point. Similarities in community structure across wells were observed with respect to the presence of 20 cosmopolitan populations in all samples in all wells; however, wells differed in the relative abundances of these taxa. Similarity percentage (SIMPER) analysis revealed that temporal variability was explained by lowly abundant and transient populations or more highly abundant and frequently present taxa in a sample-dependent manner.
262. Paradis, C. J., T. C. Hazen, A. P. Arkin and P. D. Adams. 2017. Exposure History Dependence of Microbial Mediated Substrate Transformation Rates in Groundwater. 2017 Genomic Sciences Program Annual PI Meeting. abstract
     Project Goals: The goals of this project are to: (1) demonstrate the exposure history dependence of microbial mediated substrate transformation rates in groundwater at the field scale and (2) elucidate the microbial mechanism(s) which control the exposure history dependence of microbial mediated substrate transformation rates The rates at which natural microbial communities can transform a substrate in groundwater have been shown to increase after repeated exposures to the substrate; herein referred to as the “memory effect”. The objectives of this study were to determine: (1) how long the memory effect can last and (2) how the memory effect can alter the structure and function of natural microbial communities. Ethanol substrate was injected into a single groundwater test well for six consecutive weeks in order to establish a memory effect. The groundwater control well, located up-gradient of the test well, was not injected with ethanol during this time. The rate of ethanol removal in the test well was negligible the first week whereas subsequent rates were significant. The test and control wells will be monitored for six additional weeks under ambient conditions. Ethanol substrate will then be injected into both test and control wells in order to determine: (1) if the test well retained its memory effect and (2) if the rate of ethanol removal in the control well is negligible. Here we present the hydrological, geochemical, and microbiological data and analyses in hand from the study site and the experimental well pair. This includes: (1) the direction and magnitude of groundwater velocity, (2) the effective porosity of the groundwater system, (3) diffusive mass transport in the experimental well pair, (4) the rates of ethanol removal in the test well, (5) the extent of nitrate, sulfate, and uranium removal in the test well, (6) the extent of limiting metal nutrient and/or co-factor removal in the test well, (7) microbial community structure (16S rRNA sequencing) at the study site, and (8) microbial community function (GeoChip) at the study site. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231.
263. Paradis, C., L. M. McKay and T. C. Hazen. 2017. Exposure History Dependence of Microbial Mediated Substrate Transformation in Groundwater. National Groundwater Association annual meeting
264. Ning, D., J. Zhou, Z. He, P. Zhang, J. D. Van Nostrand, L. Wu, R. Tian, E. J. Alm, T. C. Hazen, D. Elias, M. W. Fields, M. W. W. Adams, R. Chakraborty, D. Stahl, J. Wall, A. P. Arkin and P. D. Adams. 2017. Stress mediates relative importance of deterministic and stochastic assembly in groundwater microbial communities. 2017 Genomic Sciences Program Annual PI Meeting. abstract
     Project Goals: Understanding the mechanisms controlling community diversity, distribution and succession is a central, but poorly understood, issue in ecology, particularly in microbial ecology. Although both stochastic and deterministic processes are believed to play roles in shaping community diversity and distribution, their relative importance is hotly debated. The importance of ecological stochasticity in shaping microbial community composition and structure is far less appreciated. Moreover, despite recent intensive studies on ecological community assembly, the factors mediating the relative importance of deterministic vs stochastic processes in shaping community composition and structure remain elusive. Thus, the major goal of this study is to illustrate the relative roles of deterministic and stochastic processes in shaping community structure and the factors controlling their relative importance. To determine whether and how environmental factors mediate community assembly processes, about 100 wells representative of no or low, medium, high and extremely high stress were sampled and more than 200 environmental variables were measured. Null model analysis based on phylogenetic diversity of 16S rRNA gene revealed that the groundwater microbial communities at control or low stress wells without contamination were largely stochastic (~67%). As environmental stresses increased, the communities became less and less stochastic, with 41% of stochasticity at the extremely stressed wells. Also, quantitative analysis showed that variable selection (24~49%) and dispersal limitation (25~57%) played dominant roles while homogeneous selection (7.6~10%), homogeneous dispersal (0~1.6%), and undominated (or drift) (3.9~14%) play minor roles. Environmental stresses had strong positive correlation with variable selection (r=0.96), and negative correlations (r=-0.93) with dispersal limitation. Interestingly, drift (e.g. undominated) were higher at both low (8.6%) and extremely (14%) stressed wells than medium (4.7%) and high (3.9%) stressed wells. The spatial patterns of various processes were consistent with spatial distributions of various contaminants. In addition
265. Mayes, M. A., Y. Song, Q. Yao, C. Pan, T. C. Hazen, X. Yang, G. Wang, Z. Li, A. Biswas, B. Turner, S. J. Wright, S. G. Tringe and P. Thornton. 2017. Linking Meta-omics with the Microbial ENzyme Decomposition Model. 2017 DOE TES/SBR Joint Investigators Meeting abstract
     Microbes are increasingly included in soil carbon decomposition models, but it is widely recognized that one functional group may be insufficient to represent the diversity of substrates. Microbial metagenomics, metaproteomics, metatranscriptomics, and other related techniques provide important insight into microbial genes and activities, but it remains unclear how to include such detailed information in models of any scale. We provide a solution to this complex problem and demonstrate its application using a pilot study from the Gigante Fertilization Experiment near Barro Colorado Island, Panama. Soils were collected from control and phosphorus (P) addition plots and analyzed for metagenomics, metaproteomics, phosphatase enzyme activities, and CO2 production during incubation experiments. Fertilized soils exhibited around 30% more CO2 release than control soils and had greater microbial biomass than control soils. Control soils exhibited greater monophosphoesterase and diphosphoesterase activities, and had significantly more genes coding for the production of phytase, phospholipase, and exoribonuclease phosphomonoesterase than P addition soils. We also observed differences in genes for carbon decomposition and the reduction of nitrogen and sulfur, and results were consistent between metagenomic and metaproteomic analyses. We incorporated the enzyme functions, the P cycle (Yang et al. 2013, 2016), and a continuum carbon decomposition scheme into the Microbial ENzyme Decomposition model (Wang et al. 2013, 2014, 2015). Model results were able to match the patterns of CO2 evolution in control and P addition soils, indicating the improved model provides a reasonable pathway for including meta-omic information into soil nutrient cycling models. Future work will focus on including anaerobic decomposition pathways.
266. Mayes, M., Y. Song, Q. Yao, C. Pan, T. C. Hazen, G. Wang, X. Yang, Z. Li, A. Biswas, J. Wright, B. Turner, E. Johnston, M. Kim, K. Konstantinidis, P. Thornton, M. Tfaily, L. Pasa-Tolic and S. G. Tringe. 2017. Linking Proteogenomics with a Soil Carbon Decomposition Model. Multi-omics for Microbiomes EMSL Conference
267. Ling, F., J. Friedman, S. Zhao, M. B. Smith, A. M. Rocha, C. J. Paradis, J. Zhou, T. C. Hazen, E. J. Alm, A. P. Arkin and P. D. Adams. 2017. Microbes at the blurred boundary of natural and built environments. 2017 Genomic Sciences Program Annual PI Meeting. abstract
     Project Goals: This project aims to better understand the assembly of low-diversity communities in a groundwater ecosystem with genomic tools. Humans today spend more time working, living, and recreating in the manmade environment than the natural environment. However, the boundary between built and natural environments isn’t always clear. The Oak Ridge Field Research Center (FRC) presents a case where past anthropogenic contamination to the natural environment caused by uranium enrichment influences the current built environment through the channel of water supply. Microbes living in this environment have been shown to be useful as quantitative biosensors of contamination. In the present study, we found that microbial communities at the FRC sites exhibited an unusual range of diversity, which correlates to the performance of the microbial sensor. We show that the variation in diversity is poorly explained by chemical gradients or cell count, yet well explained by migration when viewed from a dispersal-limited community assembly perspective. Our ongoing work applies shotgun sequencing to infer strain-level diversity that can provide further insights on the community assembly processes. Funding statement. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231. Fangqiong Ling is supported by Alfred P. Sloan Foundation Microbiology of the Built Environment Postdoctoral Fellowship
268. Lewis, A. J., M. F. Campa, T. C. Hazen and A. P. Borole. . 2017. Unlocking Renewable Hydrogen from Biomass via Emergent Electroactive Biofilms. American Society for Microbiology Microbe 2017 abstract
     In microbial electrolysis, organic compounds are converted to electrons, protons, and CO by a microbial community in the anode, and the electrons and protons are used to produce H at the cathode [1, 2]. In order to harness the evolved functionality of nature to generate renewable products from biomass sources in engineered systems, a better understanding of the synergy between microbes within reactor communities is necessary. Here we investigate an MEC employing an enriched microbial consortium [3] for conversion of a switchgrass-derived bio-oil aqueous phase (BOAP) into H . Approach to understanding community interactions: 1.Conversion of BOAP (complex fermentable compounds) under poised conditions, 2.Conversion of acetic acid (main substrate for electroactive bacteria) under poised conditions 3.Conversion of BOAP under open-circuit conditions to assess fermentative conversion, while restricting exoelectrogenesis Microbial community characterization was also carried to provide insights into the relative changes in fermentative, methanogenic and exoelectrogenic populations during these experiments. A maximum H productivity of 9.35 ± 1.73 L/L-d was achieved with BOAP and increased 3-fold to 27.6 ± 5.29 L/L-d using pure acetic acid, indicating that fermentation was the limiting step. The anode microbial community displayed simultaneous conversion of compounds present in BOAP, but at differing rates. Open-circuit stimulus-response conditions led to acetic acid production at efficiency of 68.3%, while subsequent operation as MEC with closed-circuit resulted in a Coulombic efficiency above 80%. Microbial groups including Bacteroidetes, Firmicutes and several Proteobacteria persisted when fed with BOAP and diminished when pure acetic acid was used, while Geobacteraceae were enriched with both substrates, demonstrating the separate fermentative and exoelectrogenic roles played by these microbes, respectively. The results provide evidence that the anode microbial community uses a division of labor strategy and syntrophic interactions for emergent functionality in converting BOAP. This enables the utilization of normally toxic and recalcitrant compounds at appreciable rates, generating a significant source of reducing power that could be directed to produce other high value products in addition to H . This study serves to provide a foundation from which to build on for understanding biocomplexity in bioelectrochemical systems for conversion of biomass-derived streams to renewable products.
269. Johnston, E. R., M. Kim, J. K. Hatt, J. R. Phillips, Q. Yao, Y. Song, C. Pan, T. C. Hazen, M. A. Mayes and K. T. Konstantidis. 2017. Phosphate addition increases CO2 respiration in tropical soils primarily by promoting microbial growth. Multi-omics for Microbiomes EMSL Conference
270. T. C. Hazen. 2017. Phenotypic Microarray Provides Functional Verification of Genotype. American Society for Microbiology Microbe 2017 abstract
     16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
271. Hazen, T. C.. 2017. Is the Solution to Pollution Dilution i.e. Intrinsic Remediation? Oil Spills, Solvents, Metals, and Radionuclides (OH MY!!!!). University of Tennessee Honors Faculty Lecture Series, Chancellor's Honors Program
272. T. C. Hazen. 2017. ENIGMA Project: Lab to Field and Back - Environmental System Microbiome. 2017 DOE TES/SBR Joint Investigators Meeting
273. T. C. Hazen. 2017. Environmental Justice and Disposal of Toxic Waste. Integrating Environment and Health, 17th National Conference and Global Forum on Science, Policy and the Environment. http://www.ncseconference.org/
274. Harik, A., S. M. Techtmann and T. C. Hazen. 2017. Water Swap: Control of Geochemistry Versus Microbial Community Composition on Hydrocarbon Degradation. American Society for Microbiology Microbe 2017 abstract
     Although the ideal for the oil industry is a lack of oil spills and a reliance on preventative measures, the industry must also be prepared for the worst-case scenarios. Current spill response plans are general and can be lacking site and microbial community specific responses. In the water swap experiment the relative control biogeochemistry plays on the microbial degradation of hydrocarbons is looked at in comparison to the role of the pre-spill microbial community composition. Our hypothesis is that the rates of oil biodegradation are primarily controlled by the geochemistry and nutrient limitations. Media simulating near bottom and surface ocean water will be used. Microbial communities will be grown in each type of water then switched to the other, comparing their hydrocarbon degradation rates in both. If microbial community structure is shown to control degradation rates then drilling locations whose communities are not capable of high rates of degradation will need a more extensive spill response plan, where as communities that are better equipped to handle a spill may allow for a smaller response. If geochemistry is found to control oil degradation rates that spill response procedures should include amending with necessary nutrients; further studies will be needed to identify key nutrients.
275. Chakraborty, R., X. Wu, T. C. Hazen, Y. Liu, N. Hess, M. W. Fields, P. Zhang, L. Wu, J. Zhou, Q. Li, W. Yang, A. P. Arkin and P. D. Adams. 2017. Microbial Interactions with Natural Organic Matter Extracted from the Oak Ridge FRC. 2017 Genomic Sciences Program Annual PI Meeting. abstract
     Project Goals: Natural organic matter (NOM) is central to microbial food webs and microbially mediated NOM transformations determine much of the carbon (C) flux in subsurface environments. However, little is known about the molecular signature of this pool of C and the microbial activities that regulate NOM turnover are still poorly resolved. The goal of this project was to study the interactions between NOM (extracted from the field site) and native microbial communities present in groundwater at a background site (FW305) at Oak Ridge Field Research Center, TN. Water-soluble NOM was extracted from sediment samples collected from the background uncontaminated site, and the extraction efficiencies were 3.2% for organic carbon and 1.6% for inorganic carbon. Extracted NOM was used as the sole source of carbon in controlled lab incubations, and groundwater from FW305 well served as the microbial inoculum. Subsamples were harvested at several time points during a 50 day incubation for both chemical and microbial analyses. Results indicated a rapid decrease of total organic carbon within the first 1.5 days, concomitant to a rapid burst in CO2, and increased in cell numbers. 16S rRNA gene amplicon sequencing suggested that Massilia spp dominated in the original inoculum, and gradually decreased to below 5% after 50 days, while Azospirillum spp and Cupriavidus spp gradually increased from < 5% to 20-30%. Advanced chemical techniques including FTICR-MS and sXAS were used to characterize the C pool that included NOM metabolites and microbial byproducts produced during the incubation period. The molecular mass of C pool generally ranged from 200-600. Relative abundance of compounds with mass around 400-600 increased with incubation time and were considered to be produced from microbial activities. Also, the C pool shifted during incubation, the proportion of lignin in cultures increased, while proteins decreased. Further, GeoChip was used to identify the changes of microbial communities and expression of functional genes during transformation of the NOM. In conclusion, the data clearly showed that microbial community present at Oak Ridge FRC responded to NOM from the site, and that the community shifted to reveal dominant members in sequence over time in response to transformation of the different functional groups in NOM.
276. Campa, M. F., S. M. Techtmann, C. Gibson, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella and T. C. Hazen. . 2017. Outstanding Abstract Award Presentation: The Impacts of the Biocide Glutaraldehyde on Community Structures and Degradation Potential in Streams Impacted by Hydraulic Fracturing. American Society for Microbiology Microbe 2017 abstract
     Hydraulic fracturing (HF) involves injecting chemicals, sand, and water into shale formations to create fractures and release hydrocarbons. Large volumes of the hyper-saline fluids used return to the surface as waste. Potential spills of this wastewater raise concerns about the environmental impact these fluids may impose. Biocides are one of the main chemicals of concern used in HF fluids. Biocides are added to prevent biocorrosion of equipment and gas souring. To understand the effect of biocides on the environmental microbial community after a HF surface spill, microcosms were inoculated using stream water impacted and not impacted by Marcellus shale HF operations. Microcosms were incubated aerobically at ambient temperature for 56 days using glutaraldehyde (GA), the most commonly used biocide in HF operations. The microbial community adaptation to biocide was monitored every two weeks using next-generation16S rRNA amplicon sequencing, and abiotic and biotic GA degradation was measured every week using Liquid Chromatography- coupled with Exactive Quadrupole-Orbitrap Mass Spectrometry. 16S rRNA amplicon sequencing showed three methanotrophic taxa enriched in the HF impacted sites after the addition of GA. These taxa were the genera Beijerinckia, Methylobacterium, and Methylosinus. Over time, HF impacted sites kept a higher Simpson alpha diversity than the HF not-impacted sites. Presumably, HF-impacted sites have more organisms capable of tolerating the biocides. The difference of alpha diversity between HF-impacted and HF-not-impacted sites suggests there is a long lasting effect in the microbial population after a HF spill. Furthermore, HF impacted experienced a smaller log reduction of bacterial 16S rRNA gene copy number, that can be attributed to less bacteria dying off. HF not impacted sites show more “enrichment” afterwards, but this could be due to the new carbon sources provided by dead microbes. In 56 days there was not a statistically significant abiotic degradation of GA. However, overtime HF not impacted sites, experienced more biotic degradation of GA as compared to the HF impacted sites. Showing there is a decrease in degradation potential after GA exposure, even though more members of the microbial community are able to tolerate the biocide. These findings show there are lasting effects in microbial community structure and degradation potential in streams impacted by HF operations.
277. Campa, M. F., S. M. Techtmann, C. Gibson, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella and T. C. Hazen. . 2017. The Impacts of the Biocide Glutaraldehyde on Community Structures and Degradation Potential in Streams Impacted by Hydraulic Fracturing. American Society for Microbiology Microbe 2017 abstract
     Hydraulic fracturing (HF) involves injecting chemicals, sand, and water into shale formations to create fractures and release hydrocarbons. Large volumes of the hyper-saline fluids used return to the surface as waste. Potential spills of this wastewater raise concerns about the environmental impact these fluids may impose. Biocides are one of the main chemicals of concern used in HF fluids. Biocides are added to prevent biocorrosion of equipment and gas souring. To understand the effect of biocides on the environmental microbial community after a HF surface spill, microcosms were inoculated using stream water impacted and not impacted by Marcellus shale HF operations. Microcosms were incubated aerobically at ambient temperature for 56 days using glutaraldehyde (GA), the most commonly used biocide in HF operations. The microbial community adaptation to biocide was monitored every two weeks using next-generation16S rRNA amplicon sequencing, and abiotic and biotic GA degradation was measured every week using Liquid Chromatography- coupled with Exactive Quadrupole-Orbitrap Mass Spectrometry. 16S rRNA amplicon sequencing showed three methanotrophic taxa enriched in the HF impacted sites after the addition of GA. These taxa were the genera Beijerinckia, Methylobacterium, and Methylosinus. Over time, HF impacted sites kept a higher Simpson alpha diversity than the HF not-impacted sites. Presumably, HF-impacted sites have more organisms capable of tolerating the biocides. The difference of alpha diversity between HF-impacted and HF-not-impacted sites suggests there is a long lasting effect in the microbial population after a HF spill. Furthermore, HF impacted experienced a smaller log reduction of bacterial 16S rRNA gene copy number, that can be attributed to less bacteria dying off. HF not impacted sites show more “enrichment” afterwards, but this could be due to the new carbon sources provided by dead microbes. In 56 days there was not a statistically significant abiotic degradation of GA. However, overtime HF not impacted sites, experienced more biotic degradation of GA as compared to the HF impacted sites. Showing there is a decrease in degradation potential after GA exposure, even though more members of the microbial community are able to tolerate the biocide. These findings show there are lasting effects in microbial community structure and degradation potential in streams impacted by HF operations.
278. M. F. Campa and T. C. Hazen. 2017. Phenotypic Microarray for Fracking studies. American Society for Microbiology Microbe 2017 abstract
     Hydraulic fracturing (HF) involves injecting chemicals, sand, and water into shale formations to create fractures and release hydrocarbons. Large volumes of the hyper-saline fluids used return to the surface as waste. Potential spills of this wastewater raise concerns about the environmental impact these fluids may impose. Biocides are one of the main chemicals of concern used in HF fluids. Biocides are added to prevent biocorrosion of equipment and gas souring. To understand the effect of biocides on the environmental microbial community after a HF surface spill, microcosms were inoculated using stream water impacted and not impacted by Marcellus shale HF operations. Microcosms were incubated aerobically at ambient temperature for 56 days using glutaraldehyde (GA), the most commonly used biocide in HF operations. The microbial community adaptation to biocide was monitored every two weeks using next-generation16S rRNA amplicon sequencing, and abiotic and biotic GA degradation was measured every week using Liquid Chromatography- coupled with Exactive Quadrupole-Orbitrap Mass Spectrometry. 16S rRNA amplicon sequencing showed three methanotrophic taxa enriched in the HF impacted sites after the addition of GA. These taxa were the genera Beijerinckia, Methylobacterium, and Methylosinus. Over time, HF impacted sites kept a higher Simpson alpha diversity than the HF not-impacted sites. Presumably, HF-impacted sites have more organisms capable of tolerating the biocides. The difference of alpha diversity between HF-impacted and HF-not-impacted sites suggests there is a long lasting effect in the microbial population after a HF spill. Furthermore, HF impacted experienced a smaller log reduction of bacterial 16S rRNA gene copy number, that can be attributed to less bacteria dying off. HF not impacted sites show more “enrichment” afterwards, but this could be due to the new carbon sources provided by dead microbes. In 56 days there was not a statistically significant abiotic degradation of GA. However, overtime HF not impacted sites, experienced more biotic degradation of GA as compared to the HF impacted sites. Showing there is a decrease in degradation potential after GA exposure, even though more members of the microbial community are able to tolerate the biocide. These findings show there are lasting effects in microbial community structure and degradation potential in streams impacted by HF operations.
279. Yongquist, E., S. M. Hagan, S. M. Techtmann and T. C. Hazen. 2016. Genomic Diversity of Pseudoalteromonas spp. from Geographically Distant Deep Marine Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     The 32 closely related Pseudoalteromonas spp. we isolated were found in different environments not previously sampled throughout the world’s deep oceans. The isolates’ 16S rRNA gene sequences were more than 99% similar, while the genomic makeup was less than 99% similar. The environmental parameters of these locations vary greatly and little is known about how adaptation to growth under different environmental conditions affects the genomic makeup of very closely related bacteria. To address this, we sought to investigate what physical conditions affect the genomic diversity of 10 closely related oil-degrading Pseudoalteromonas strains from three distant deep marine locations. We wanted to determine how physical and chemical factors of the environment contribute to the genomic differences of the 10 Pseudoalteromonas isolates. This was done by comparing the phenotypes of each strain to one another. We compared the optimal growth temperature, along with the carbon, nitrogen, and osmolyte metabolisms of each isolate in this experiment. We hypothesize that the different physical and chemical environmental factors are influencing the genomic differences of the Pseudoalteromonas species. This work will help better understand how diverse a bacterial species is and how the environment is contributing to driving these differences.
280. Wu, X., T. C. Hazen, P. M. Fox, P. S. Nico, Q. Li, W. Yang, Y. Liu, N. J. Hess, P. Zhang, Y. Qin and J. Zhou. 2016. Interactions between Natural Organic Matter and Native Microbes in the Oak Ridge FRC Groundwater. AGU Fall Meeting abstract
     Natural organic matter (NOM) is central to microbial food webs; however, little is known about the interplay between the physical and chemical characteristics of the carbon in NOM and its turnover by microbial communities. Microbial activity changes NOM’s structure and properties, which may further influence the bioavailability of NOM. The change of NOM may reversely affect the microbial community structure as well. To date, our understanding of these interactions is insufficient, and it is critical to identify the role of NOM to carbon turnover, structure of microbial community and to the metabolic potential of that community. In this study, we aimed to study the interactions between NOM and native microbial communities present in groundwater at a background site (FW305 well) at Oak Ridge Field Research Center, TN. The total organic carbon and inorganic carbon in FW305 deep sediment samples were 0.071% and 0.011%, respectively. Water-soluble NOM was extracted from these sediment samples, the extraction efficiencies were 3.2% for organic carbon and 1.6% for inorganic carbon. The extracted NOM was then provided as the sole carbon source to native microbes present in groundwater. Subsamples were harvested several times from these incubations during a 50-day study. 16S rRNA gene amplicon sequencing and Geochip were used to identify the changes of microbial communities and expression of functional genes during transformation of the NOM. Several advanced chemical techniques including FTICR-MS and NEXAFS were used to characterize the C pool (i.e., NOM metabolites and microbial byproducts). Preliminary data clearly showed that microbial community responded to NOM, and shifted as functional groups in NOM transformed. Further detailed metabolite and gene-based analysis to elucidate these changes is currently being conducted.
281. Woo, H. L. and T. C. Hazen. 2016. Using high throughput sequencing methods to identify keystone bacterial species in recalcitrant terrestrial organic matter transformation. 3rd Annual Southeastern Biogeochemistry Symposium abstract
     The microbial transformation of terrestrial organic matter, particularly recalcitrant lignin and hemicellulose, is a significant but poorly understood phenomenon. 16S rRNA gene amplicon sequencing and metagenomic sequencing are relatively inexpensive molecular tools to quickly assess microbial diversity and functional genes in response to different carbon sources. Using sequencing and bioinformatics, we aim to elucidate the diversity and metabolic potential of bacterial communities subsisting on lignin and hemicellulose in laboratory cultures. Seawater microcosms were incubated with an added concentration of purified lignin or xylan. CO2 respirometry and enzyme assays showed high microbial activity on both substrates. Using an Illumina MiSeq platform, both amended cultures and unamended controls were sequenced for 16S rRNA gene amplicons and metagenomics. Reads were annotated using Qiime and MG-Rast. Annotated data was compared in detail using phyloseq, vegan, and DESeq2 in R. Several species belonging to phylum of Proteobacteria, Flavobacteria, and Firmicutes significantly increased in abundance when lignin or hemicellulose was added. Groups of functional genes related to carbohydrates and aromatic catabolism significantly increased in abundance. Our methods may uncover important species that have been previously overlooked for terrestrial organics degradation.
282. Ulrich, N., C. McLimans, W. Bernard, J. R. Wright, M. F. Campa, T. C. Hazen and R. Lamendella. 2016. Metagenomics, metatranscriptomics, and single cell sequencing of microbial communities associated with hydraulic fracturing. International Symposium for Microbial Ecology
283. Thorgersen, M. P., B. J. Vaccaro, W. A. Lancaster, F. L. Poole, A. E. Kazakov, L. Rajeev, M. Garber, G. M. Zane, M. N. Price, K. M. Wetmore, A. M. Rocha, T. Mehlhorn, P. S. Novichkov, A. M. Deutschbauer, A. Mukhopadhyay, J. D. Wall, R. Chakraborty, T. C. Hazen, M. W. W. Adams, A. P. Arkin and P. D. Adams. 2016. Microbial responses to toxic metals in the Oak Ridge Reservation environment. DOE Genomic Sciences Contractor Annual Meeting abstract
     Project Goals: The environmentally relevant isolation of several metal-resistant microorganisms is described along with the characterization of a novel uranium-binding complex from a strain isolated from the ORR site. Abstract: The metal resistance campaign is focused on investigating molecular mechanisms to microbial metal resistance. One of the defining characteristics of the Oak Ridge Reservation (ORR) environment is the presence of mixed industrial waste and the effect this waste has on the groundwater microbial community. Metals are a key component of the mixed waste with concentrations of uranium, aluminum, manganese, cadmium and cobalt in contaminated groundwater wells over 1,000 times greater than those in pristine background wells. The metal resistance campaign is exploring the effects of metal toxicity on the ORR groundwater community in several ways. Strains will be described that were isolated from ORR groundwater in media that contain metal concentrations similar to those present in the contaminated environments. The physiology of metal toxicity was studied through measurement of genomewide gene fitness under copper, zinc, chromium and uranium toxicity using the model organism Pseudomonas stutzeri RCH2. In addition, the properties of a uranium-binding protein complex from the ORR isolate Pelosinus fermentans UFO1 will be presented.
284. Spencer, S. J., M. V. Tamminen, A. M. Rocha, T. C. Hazen, E. J. Alm, A. P. Arkin and P. D. Adams. 2016. Assays for spatial structure and transdomain dynamics in environmental communities. DOE Genomic Sciences Contractor Annual Meeting abstract
     Project Goals: Our group focuses on biotechnology development that moves the field of microbial ecology toward complete genomic awareness. Specifically, we use bulk water-in- oil emulsion droplets combined with tailored molecular biology to provide a more comprehensive microscale view of transdomain ecological players and their functional capacity within complex environments. The bulk activity of microbial communities is composed of the additive effect of microscale interactions between bacteria, viruses, and eukaryotes coexisting within a dynamic environment. These microscale competitive or mutualistic exchanges bridge between the foundational principles of ecology and the global activity of microbial communities that we observe in bulk assays. Before we are able to understand, model, or perturb systems at the macroscopic scale, we need improved methods at the resolution of individual cells. Our group recently developed an emulsion-based droplet assay termed epicPCR (emulsion, paired isolation, and concatenation PCR) to physically link functional genes with phylogenetic indicators within single cells. Here we expand upon this platform to map the physical associations of bacteria with each other and with eukaryotic hosts. We are beginning to assay biofilm and host-prey structures by capturing small aggregates of cells in nanoliter droplets, then physically linking segments of the 16S rRNA gene between cells. In biofilms a preliminary untargeted assay, trying to link every cell with every other adjacent cell, highly favored only the most abundant strains present. We’ve now redesigned primer sets that anneal to specific phyla of interest and their physical partners, a semi-targeted version of the assay. With this approach we’ve recovered library contructs enriched for cells as rare as 1 in 10,000 within complex biofilms. In parallel to assays of bacterial proximity, we’ve refined the same droplet methodology to capture eukaryotes with their bacterial symbionts and prey. Sequenced co-aggregations between eukaryotes and bacteria in both wastewater and lake water are enriched for predators, heterotrophs, and known symbionts. We plan to apply this approach to Oak Ridge FRC samples, and in preparation we’ve completed standard eukaryotic and bacterial sequencing from control wells. These structural assays in combination will provide novel microscale data about the complex interchanges connecting bacteria with each other and their broader ecological context. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231
285. Song, Y., Q. Yao, G. Wang, X. Yang, C. Pan, E. Johnston, M. Kim, K. Konstantinidis, T. C. Hazen and M. Mayes. 2016. Integrating “omics” data into a biogeochemical model: A new model scheme to predict climate feedbacks from microbial function in tropical ecosystems. AGU Fall Meeting abstract
     Soil microorganisms and their activities, which play a significant role in regulating carbon (C) and nutrient biogeochemical cycles, are highly responsive to changes in climate. The diversity of microorganisms, however, complicates the explicit representation of microbial and enzymatic processes in biogeochemical or earth system models. Uncertainties in accounting for microbial diversity therefore limits our ability to incorporate microbial functions into models. However, ‘omics technology provides abundant information to identify the structure and function of the microbial community and strengthens our ability to understand microbially-mediated C and nutrient cycles and their climate feedbacks. We collected soils from control and phosphorus (P) fertilized plots at the Gigante Peninsula long-term fertilization experiment at the Smithsonian Tropical Research Institute in Panama, an ecosystem where P limitation constrains primary productivity and microbial activities. We monitored effects P addition on soil carbon decomposition with respiration measurements and investigated the responsible microbial mechanisms with metagenomics, metatranscriptomics, metaproteomics, and enzyme activity assays. We integrated the P dynamics into the C-N coupled Microbial Enzyme Decomposition (MEND) model. We integrated the ‘omics data with the new microbially-enabled C-N-P model to examine the mechanistic responses of soil microbial activity and heterotrophic respiration to P availability. Our finding indicates that increases in soil P availability can alter both the abundance and activity of enzymes related to soil carbon decomposition and P mineralization in the tropical soil, leading to increased CO2 emissions to the atmosphere. Integrating the ‘omics data into the biogeochemical model enabled scaling of complex ecosystem functions from genes to functional groups to enable predictions of microbial controls on C, N and P cycles.
286. Rocha, A. M., B. Adams, C. Paradis, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. M. Klingeman, D. B. Watson, D. C. Joyner, S. Jagadamma, J. L. Fortney, J. J. Zhou, J. D. Van Nostrand, M. W. W. Adams, R. Chakraborty, D. Elias, E. J. Alm, T. C. Hazen, A. P. Arkin and P. D. Adams. 2016. Temporal Variation in Groundwater Microbial Community Structure: Implications for Groundwater Monitoring. DOE Genomic Sciences Contractor Annual Meeting abstract
     Project goals: The overarching goal of the Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) field microbiology component is to understand the interactions between environmentally relevant microbial communities and their environment. As part of this, we must understand the mechanisms that may potentially impact microbial community structure, function, and activity. At the DOE Oak Ridge field site, groundwater wells are subject to temporal and seasonal changes in groundwater hydrology and geochemistry, which may result in temporal bias in microbial community monitoring data. Here, we characterize the microbial community response to temporal variability to determine to what extent variation in groundwater geochemistry and hydrology impacts microbial community structure and function. Large-scale groundwater sampling events, such as those associated with biomonitoring, may span the course of several weeks or months. Based on the sampling methods and time-scales involved, microbial community and geochemical data may contain significant temporal bias, as well as, biases attributed to external factors. In this study, we characterized the temporal dynamics of microbial groundwater communities at the background site of the Oak Ridge Field Research Center in Oak Ridge, TN to determine (1) if, and to what extent, temporal and/or seasonal variation of the groundwater geochemistry affects the microbial community structure and (2) to assess the impact of groundwater flow and transport of geochemical constituents on the microbial structure. To determine how resilient microbial communities are to daily and weekly changes in groundwater chemistry, the temporal dynamics of microbial communities from six groundwater wells were monitored and compared to geochemical and hydrological measurements. Of the six wells, we physically and chemically cleaned four wells to remove biofilm and attached particulates from the well casing. The remaining two wells that weren’t cleaned served as controls. All wells were sampled prior to cleaning to establish a baseline microbial community profile. Post-cleaning, each well was sampled a total of twelve times from December 1, 2014 – January 12, 2015. For each well and time-point, groundwater samples were collected for geochemical and microbial community analyses using low flow sampling methods. Nucleic acids were collected by sequentially filtering water through a 10μm pre-filter and 0.2μm-membrane filter and then extracted using a Modified Miller method. A total of 6,959 OTUs were identified across all six wells and size fractions. Of the OTUs Proteobacteria represented a significant portion of the taxa. Analyses of microbial community data indicate overall diversity of the taxa did not vary significantly during time-course sampling. However, significant shifts in the population were observed between cleaning treatments and during sampling time point 01/5/15 for select wells. Additionally, daily and weekly variation in the relative OTU abundance within each well was detected. Throughout the study, groundwater geochemical measurements were relatively stable. However, shallow wells varied in concentrations of Na+, K+, Ca2+, HCO3 -, and CO3 -2 following rain events. The geochemical values for these ions are consistent with distinct differences in water types between deep and shallow wells. The stability of the geochemical measurements may indicate that groundwater chemistry is not a dominant factor in the observed daily and weekly variances, but rather contributes to taxonomic differences observed between well depths. However, further analysis of geochemical shifts at higher resolutions is necessary to understand the full impact of geochemistry on microbial response. Furthermore, analyses of groundwater results indicate that the pumping/sampling of wells did not contribute to sampling bias. Overall, results demonstrate that groundwater microbial community data contain temporal biases. As such caution must be used when designing large-scale sampling events. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231
287. D. Ribic, O. G. Brakstad, R. Netzer, T. C. Hazen and F. Drabløs. 2016. From simple to complex degradation of hydrocarbons. A concept of metagenome succession in oil-amended microcosms. MaCuMBA Conference abstract
288. Paradis, C., N. Mahmoudi, S. Jagadamma, D. Driver, K. O’Dell, S. Schaeffer and T. C. Hazen. 2016. Response of soil respiration and microbial community structure to simulated heavy precipitation and drought in a Lexington silt loam. Oak Ridge Institute for Science and Education (ORISE) Summer 2016 Graduate, Post Graduate, Employee Participant, and Faculty Poster Session
289. Paradis, C., N. Mahmoudi, S. Jagadamma, D. Driver, K. O’Dell, S. Schaeffer and T. C. Hazen. 2016. Response of soil respiration and microbial community structure to simulated heavy precipitation and drought in a Lexington silt loam. International Symposium for Microbial Ecology abstract
     Mean annual precipitation and consecutive dry day lengths are projected to increase in the Southeastern United States and cause dramatic changes to the natural soil moisture regimes of non-irrigated land. Changes to moisture content can strongly affect the production of soil-respired carbon dioxide (CO2) and the structure of soil microbial communities and are thought to be climate-zone and soil-type specific. The objective of this study was to simulate heavy precipitation and drought stress under highly-controlled laboratory conditions to determine the response of soil-respired CO2 and microbial community structure in a Lexington silt loam. Soil samples were collected from a non-tilled, non-fertilized, non-cover crop plot in West Tennessee during a period of normal weather conditions. Batch microcosms were constructed in triplicate with variable water addition per 6-day drying cycles to simulate ambient, heavy, and drought moisture conditions for 37 days. Simulated heavy precipitation resulted in substantially increased soil-respired CO2 and notable shifts in microbial community structure towards taxa that are structurally resistant to increased osmotic pressure (Firmicutes Clostridia) and capable of oxygen-limited metabolic activity (Delta-proteobacteria). Simulated drought resulted in substantially decreased soil-respired CO2 and a shift in microbial community structure (increase in Actinobacteria and decrease in Acidobacteria) towards those previously observed in arid and semi-arid climate zones. The results of this laboratory study yielded valuable insights as to the directions in which soil-respired CO2 and microbial communities may shift at the ecosystem scale in response to a changing climate.
290. Ning, D., J. Wang, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, M. B. Smith, A. M. Rocha, S. W. Olesen, C. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, S. M. Techtmann, D. C. Joyner, D. Elias, K. L. Bailey, R. A. Hurt, S. P. Preheim, M. C. Sanders, M. A. Mueller, D. B. Watson, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, E. J. Alm, T. C. Hazen, A. Lancaster, B. J. Vaccaro, F. L. Poole, M. W. Adams and J. Zhou. 2016. Disentangling Ecological Processes and Drivers In Subsurface Microbial Community Assembly In A Nuclear Waste Site. International Symposium for Microbial Ecology
291. Ning, D., J. Wang, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, M. B. Smith, A. M. Rocha, S. W. Olesen, C. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, S. M. Techtmann, D. C. Joyner, D. Elias, K. L. Bailey, R. A. Hurt, S. P. Preheim, M. C. Sanders, M. A. Mueller, D. B. Watson, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, E. J. Alm, T. C. Hazen, A. Lancaster, B. J. Vaccaro, F. L. Poole, M. W. Adams and J. Zhou. 2016. Disentangling Ecological Processes And Drivers In Subsurface Microbial Community Assembly In A Nuclear Waste Site. ASM Microbe Annual Meeting abstract
     Background: A central issue in ecology is understanding the processes shaping biodiversity. The groundwater in Oak Ridge Integrated Field Research Challenge site provides a rare opportunity to examine ecological processes and drivers shaping subsurface microbial diversity.Methods: Groundwater samples from 98 wells were analyzed for 205 environmental variables, as well as 16S rRNA genes by an Illumina MiSeq sequencer. After the phylogenetic signal was determined, the influence of selection was estimated based on beta nearest taxon index (βNTI). Then, turnovers not dominated by selection were analyzed using the Raup-Crick metric (RC) based on Bray-Curtis index to estimate the influence of dispersal limitation (RC>0.95) and homogenizing dispersal (RC<-0.95).Results: Across the entire site, both variable selection (44.6%) and dispersal limitation (46.3%) were important in shaping microbial diversity. Environment variables were used to fit the models with βNTI and RC using distance-based redundancy analysis. The model of βNTI identified 25 significant principal coordinates (PCs) imposing selection, with high loading of heavy metals, pH, nitrate, and DO. The model of RC identified 10 significant PCs imposing dispersal limitation, with high loading of spatial eigenvectors or metals in pellets. The spatial distribution of ecological processes showed the areas with little dispersal limitation for the microbial communities were those areas having low elevation, good diffusion of contaminants or a high level of dissolved oxygen.Conclusions: while contaminants and oxygen availability imposed obvious selection on subsurface microbial communities, the microbial spatial turnover was also largely affected by dispersal limitation, which may reflect actual underground dispersal conditions.
292. McBride, K., A. Rossi, H. Woo, J. Wang, N. Labbe and T. C. Hazen. 2016. Xylan-degrading Bacteria Isolated and Characterized from Eastern Mediterranean Sea. National Council Undergraduate Research Annual Meeting abstract
     Hemicellulose xylan is an important structural component within plant cell walls that is difficult to degrade. There is relatively little information on the diversity of bacteria that produce xylan-degrading enzymes, or the necessary conditions to maximize enzyme production. Additional knowledge about xylanase producing bacteria could lead to more efficient ways to break down plant material for biofuels. Marine environments were sampled because they are rich in hemicellulosic biomass. This study aims to identify xylan-degrading bacteria by sequencing isolates from Eastern Mediterranean Sea, and measuring their growth rates on xylan media. Isolates were cultivated from a laboratory incubation of xylan-amended seawater. A set of ninety isolates were sub-cultured several times on synthetic seawater agar with xylan as the sole carbon source. All strains were then identified by 16S rRNA gene sequencing. Isolates were closely related to Halomonas, Pseudomonas, Joostella, Glaciecola and Janibacter. None of the species found are well-known xylan degraders. Growth curve data and preliminary metabolic tests with xylan show that several isolates have high xylanase producing ability. Understanding more about xylan degradation could find more cost effective ways to produce biofuels.
293. McBride, K., A. Rossi, H. Woo, J. Wang, N. Labbe and T. C. Hazen. 2016. Xylan-degrading Bacteria Isolated and Characterized from Eastern Mediterranean Sea. ASM Microbe Annual Meeting abstract
     Hemicellulose xylan is an important structural component within plant cell walls that is difficult to degrade. There is relatively little information on the diversity of bacteria that produce xylan-degrading enzymes, or the necessary conditions to maximize enzyme production. Additional knowledge about xylanase producing bacteria could lead to more efficient ways to break down plant material for biofuels. Marine environments were sampled because they are rich in hemicellulosic biomass. This study aims to identify xylan-degrading bacteria by sequencing isolates from Eastern Mediterranean Sea, and measuring their growth rates on xylan media. Isolates were cultivated from a laboratory incubation of xylan-amended seawater. A set of ninety isolates were sub-cultured several times on synthetic seawater agar with xylan as the sole carbon source. All strains were then identified by 16S rRNA gene sequencing. Isolates were closely related to Halomonas, Pseudomonas, Joostella, Glaciecola and Janibacter. None of the species found are well-known xylan degraders. Growth curve data and preliminary metabolic tests with xylan show that several isolates have high xylanase producing ability. Understanding more about xylan degradation could find more cost effective ways to produce biofuels.
294. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2016. Microbial Community Changes and Crude Oil Biodegradation in Different Deep Oceans. International Symposium for Microbial Ecology abstract
     Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil and dispersant in various deep-sea basins around the world where oil exploration is anticipated (Eastern and Central Mediterranean Sea, Great Australian Bight and Caspian Sea). In-lab microcosm experiments were set up aerobically to study the microbial respiration, community changes and oil biodegradation. Microbial respiration followed a similar pattern in all of these basins. The treatment of oil and dispersant had the highest CO2 production. The amendment of oil lead to a higher CO2 accumulation compared to control. However, they were all much lower than the Gulf of Mexico (GOM). What’s more, oil biodegradation occurs in all of the sites. The total organic carbon revealed that a big portion of oil was degraded in the first several days, which was consistent with the GC-MS results. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil and dispersant can simplify the community and speed up the oil biodegradation. In particular, the relative abundance of Proteobacteria increased drastically while the relative abundance of archaea decreased. In addition, although oil and dispersant drive the community into a similar endpoint, different bacterial groups were found associating with oil and dispersant.
295. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2016. Microbial Community Response and Crude Oil Biodegradation in Different Deep Oceans. 3rd Annual Southeastern Biogeochemistry Symposium abstract
     Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern and Central Mediterranean Sea, Great Australian Bight and Caspian Sea). In this study, microcosms were set up aerobically with three different treatments: seawater, seawater + oil and seawater + oil + oil dispersant. Samples were taken at three time points for the analysis of oil degradation by fluorescence and GC-MS, and microbial community changes by16S rRNA sequencing. CO2 evolution followed a similar pattern among off the basins. The treatment of seawater + oil + dispersant had the highest CO2 production. The amendment of oil lead to a more CO2 accumulation than seawater treatment. However, it’s much higher in the Gulf of Mexico (GOM) than other oceans. What’s more, the dissolved organic mater analysis revealed that application of oil dispersant lead to better oil degradation, which was consistent with the GC-MS results. Oil biodegradation appears to occur rapidly in all of the sites. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil amendment affected how quickly the diversity decreased. The relative abundance of Proteobacteria increased drastically while the relative abundance of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
296. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2016. Microbial Community Response and Crude Oil Biodegradation in Different Deep Oceans. ASM Microbe Annual Meeting abstract
     Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern and Central Mediterranean Sea, Great Australian Bight and Caspian Sea). In this study, microcosms were set up aerobically with three different treatments: seawater, seawater + oil and seawater + oil + oil dispersant. Samples were taken at three time points for the analysis of oil degradation by fluorescence and GC-MS, and microbial community changes by16S rRNA sequencing. CO2 evolution followed a similar pattern among off the basins. The treatment of seawater + oil + dispersant had the highest CO2 production. The amendment of oil lead to a more CO2 accumulation than seawater treatment. However, it’s much higher in the Gulf of Mexico (GOM) than other oceans. What’s more, the dissolved organic mater analysis revealed that application of oil dispersant lead to better oil degradation, which was consistent with the GC-MS results. Oil biodegradation appears to occur rapidly in all of the sites. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil amendment affected how quickly the diversity decreased. The relative abundance of Proteobacteria increased drastically while the relative abundance of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
297. Kothari, A., Y.-W. Wu, M. Charrier, L. Rajeev, A. M. Rocha, T. C. Hazen, P. S. Dehal, D. Chivian, S. J. Spencer, E. J. Alm, S. Singer, A. Mukhopadhyay, A. P. Arkin and P. D. Adams. 2016. Extrachromosomal Plasmid DNA Project. DOE Genomic Sciences Contractor Annual Meeting abstract
     Project Goals: The extrachromosomal plasmid DNA project is one of the discovery projects funded by ENIGMA. It is a short term, high impact, investigatory effort to study the plasmid populations of the ENIGMA wells. The goal of this project is to exclusively explore the prevalence of plasmid DNA in these communities. Additionally, the project aims at studying the relevant functional genes that are typically encoded on the plasmids, conferring advantageous traits to the host, in these communities. Plasmids are autonomously replicating extra-chromosomal genetic elements that often act as mediators of horizontal gene transfer in the environment. Plasmids host and distribute non-essential genes, independent of the host’s chromosome, thereby benefitting the host bacteria in certain specific environmental conditions. Native plasmids typically range from 2 kb to 250 kb in size and have been shown to be present in 10-30% of the cultivated isolates from varied environments. The best-characterized wells at the Oakridge site are now documented to contain several hundred bacterial strains, many of which are likely to contain plasmids. This is the first study to selectively isolate and analyze the plasmid population from these sites. To optimize a robust method that isolates a range of plasmid sizes, we developed a model system comprising of three strains containing plasmids of sizes – 5kb, 48kb and 202kb in equal proportions, and tested the potential of various alkaline hydrolysis based methods to isolate plasmids from the serial dilutions of the mixed population. The presence of each plasmid was determined by targeting a unique plasmid borne gene via qPCR. In order to get rid of genomic DNA contamination, the isolated DNA was subjected to Plasmid-Safe-ATP-Dependent DNase and the lack of genomic DNA contamination was confirmed using degenerate primers targeting the 16s rRNA coding sequence. The total plasmid DNA thus obtained was amplified using Phi29 DNA polymerase to generate high-quality template for use in DNA sequencing. To increase the sensitivity of the plasmid isolation procedure, the extraction procedures and Phi29 amplification conditions were optimized. Subsequently, plasmid DNA was isolated from the wells GW460 and GW456 and subjected to deep sequencing using the Illumina paired-end protocol. The reads obtained were trimmed using Trimmomatics, assembled using IDBA-UD (Iterative De Bruijn graph Assembler for reads with Highly Uneven Sequencing Depth) and subjected to MG-RAST to produce gene calls, functional annotations and taxonomic classification. A total of 42543 (including 130 circular contigs) and 32313 contigs (including 760 circular contigs) above 2kb size were detected from the wells GW456 and GW460, respectively. These encode several known plasmid associated genes along with genes involved in secondary metabolism, antibiotic resistance, metal resistance, and nitrogen metabolism, to name a few. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
298. Kothari, A., Y.-W. Wu, M. Charrier, L. Rajeev, A. M. Rocha, T. C. Hazen, P. Dehal, D. Chivian, S. Spencer, E. Alm, S. Singer and A. Mukhopadhyay. 2016. Plasmidome studies reveal a variety of horizontally transferred functions within the microbial communities at the Oak Ridge Field Research sites. International Symposium for Microbial Ecology abstract
     The Oak Ridge Field Research Center, in Oak Ridge, TN, has contaminated and uncontaminated areas/wells where research can be conducted on the microbial communities present in the groundwater and sediment. Plasmids host and distribute non-essential genes, independent of the host’s chromosome, thereby benefitting the host bacteria in certain specific environmental conditions. The best-characterized wells at the Oakridge site are now documented to contain several hundred bacterial strains, many of which are likely to contain plasmids. This is the first study to selectively isolate and analyze the plasmid population from these sites. Plasmid isolation was optimized using a model system comprising of three strains containing plasmids of three different sizes (5, 48 and 202 kb). Subsequently, plasmid DNA was isolated from the groundwater of two background wells GW460 and GW456. Genomic DNA was eliminated with the use of a plasmid-safe-DNase, the plasmid DNA was amplified with Phi29 DNA polymerase, and subjected to deep sequencing (Illumina). A total of 42543 (including 67 circular contigs) and 32313 contigs (including 545 circular contigs) greater than 2kb size were assembled from the wells GW456 and GW460, respectively. These encode several known plasmid- associated genes such as those involved in plasmid replication and mobilization. They also encode a variety of other functions, such as genes involved in secondary metabolism, antibiotic resistance, heavy metal resistance, and nitrogen metabolism,. The plasmid-represented functions were comparable in both wells. Interestingly, the most abundant circular contigs (plasmids) from both wells contained genes annotated to be involved in mercury uptake and resistance, along with plasmid mobilization and replication genes. We discuss these and other findings from the plasmidome analysis.
299. Kim, M., E. R. Johnston, T. C. Hazen, M. A. Mayes, C. Pan, Q. Yao and K. T. Konstantinidis. 2016. Response of Soil Microbial Communities to Phosphorus in Tropical Ecosystems. March 12, 2016. Knoxville, TN. 3rd Annual Southeastern Biogeochemistry Symposium. 3rd Annual Southeastern Biogeochemistry Symposium
300. Justice, N. B., A. Sczesnak, T. C. Hazen and A. P. Arkin. 2016. Unraveling Community Assembly and Organism Interactions with Large Scale Enrichment Culturing. International Symposium for Microbial Ecology abstract
     Bacterial population structures are central to explaining microbial ecosystem function and properties. However, the ecological forces that shape community structures—including species interactions—are myriad and complex, leaving gaps in our ability to understand and predict microbial community structure and functioning. Here we examine microbial community assembly, uncover species interactions, and examine the influence of abiotic factors in microbial community structure by systematically varying the number of organisms founding each of ~1,000 enrichment cultures started from a single groundwater inoculum. We inoculated the groundwater (containing ~37,000 cells mL-1) into both aerobic and anaerobic nitrate-reducing cultures that spanned five dilutions (10-1-10-5) and, following incubation, community structures were evaluated with 16S gene amplicon sequencing. As expected, species richness decreased with increasing inoculum dilution as low abundance individuals were removed. Aerobic and anaerobic communities varied in community composition and taxonomic membership, especially at high inoculum concentrations. Using a most probable number technique, we estimated abundance (as cultivable units/mL of each taxon) of each taxon in the initial sample in aerobic and anaerobic enrichment conditions, and approximated that only ~5-7% of cells from the initial inoculum were cultured. The initial estimated abundances of each OTU were used to develop a null model of community assembly which, compared to measured data, was used to bin organisms as putative strong or weak competitors. Although strong competitors were rare (<5% of cultivated taxa), they drastically shaped community structures when present. Finally, we calculated co-occurrence probabilities for abundant taxa to infer putative positive or negative interspecific interactions amongst organisms. Nearly twice as many interactions were detected in anaerobic samples as aerobic samples, with many of the negative interactions pointing to antagonistic relationships between species of the Bacillaceae with species of Oxalobacteraceae, Paneibacillaceae, and Pseudomonadaceae. Together, this novel approach allows us to show how abiotic and biotic factors interact to structure microbial communities.
301. He, Z., P. Zhang, L. Wu, A. Rocha, Q. Tu, Z. Shi, Y. Qin, J. Wang, D. Curtis, J. Van Nostrand, L. Wu, D. Elias, D. Watson, M. Adams, M. Fields, E. Alm, T. C. Hazen, P. Adams, A. Arkin and J. Zhou. 2016. Microbial Functional Diversity Predicts Groundwater Contamination and Ecosystem Functioning. ASM Microbe Annual Meeting abstract
     Background: Anthropogenic activities have significantly impacted the biosphere of Earth due to contamination of air, water and soil environments, thus decreasing biodiversity and destabilizing ecosystem functions. However, little is known about how environmental contamination affects the biodiversity of groundwater microbial communities and its feedbacks to ecosystem functioning.Methods: We used a comprehensive functional gene array (GeoChip 5.0) to analyze the functional diversity of groundwater microbial communities from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide range of uranium, nitrate and other contaminant concentrations, as well as pH and dissolved gases (e.g., CO2, CH4, N2O).Results: It is hypothesized that the functional diversity would decrease as environmental contamination (e.g., uranium, nitrate) increased, or at low or high pH; however, specific populations capable of utilizing or resistant to those contaminants would increase. Thus, it would be possible to predict groundwater contamination and ecosystem functioning using those key microbial functional genes. Our results indicate that the functional richness/diversity significantly (p < 0.05) decreased as uranium (but not nitrate) increased in groundwater, and about 5.9% of specific key functional populations (e.g., dsrA, cytochrome genes, nirK, norB, nosZ, napA) significantly (p < 0.05) increased as uranium or nitrate increased. Also, we showed that microbial functional diversity could successfully predict uranium and nitrate contamination and ecosystem functioning.Conclusions: This study provides new insights into our understanding of the effects of environmental contamination on groundwater microbial communities and the potential for predicting environmental contamination and ecosystem functioning.
302. T. C. Hazen and S. M. Techtmann. 2016. Metagenomic Applications in Environmental Monitoring and Bioremediation. The 2016 China-US Joint Annual Symposium “International Nexus of Food, Energy, Water, and Soil”
303. T. C. Hazen and G. S. Sayler. 2016. Environmental Systems Approaches to Bioremediation of Contaminated Sites. The 2016 China-US Joint Annual Symposium “International Nexus of Food, Energy, Water, and Soil”
304. T. C. Hazen. 2016. Phenotypic Microarray Provides Functional Verification of Genotype. American Society for Microbiology Microbe 2016 abstract
     16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
305. T. C. Hazen. 2016. Deep Water Horizon Engineering Lessons’ Learned. Engineering Fundamentals College of Engineering
306. T. C. Hazen. 2016. Lessons from Deep Water Horizon?. Microbial Ecology
307. T. C. Hazen. 2016. Life in the Slow Lane: Limits to Life in the Subsurface. Micro 606 abstract
     A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced using a combination of Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Moorella thermoacetica), as well as other known sulfate reducers and thermophilic organisms. D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea. Additionally, study of the deep subsurface has offered access to the simplest community yet studied by environmental genomics, perhaps consisting of just a single species that is capable of performing all of the tasks necessary for life.
308. T. C. Hazen. 2016. Life in the Slow Lane: Limits to Life in the Subsurface. Philosophical Society of the Oak Ridge Institute for Continued Learning abstract
     A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced using a combination of Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Moorella thermoacetica), as well as other known sulfate reducers and thermophilic organisms. D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea. Additionally, study of the deep subsurface has offered access to the simplest community yet studied by environmental genomics, perhaps consisting of just a single species that is capable of performing all of the tasks necessary for life.
309. T. C. Hazen. 2016. Oil bioremediation. Cenovus
310. T. C. Hazen. 2016. Systems Biology of Oil Biodegradation in 5 Deep Marine Basins, Implications from Deep Water Horizon?. University of Tennessee, Department of Biochemistry & Cellular and Molecular Biology Seminar
311. T. C. Hazen. 2016. NSF GRFP to Microbiology Graduate Students. University of Tennessee
312. T. C. Hazen. 2016. Methane: The New Paradigm. UT Science Forum
313. T. C. Hazen. 2016. Phenotypic Microarray Provides Functional Verification of Genotype. American Society for Microbiology Microbe 2016 abstract
     16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
314. T. C. Hazen. 2016. Paradigm change? Predicting water geochemistry from microbial community structure. American Ecological Engineering Society Annual Meeting abstract
     At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry.
315. T. C. Hazen. 2016. Oil Biodegradation Potential in Deep Marine Basins Worldwide. 23rd International Petroleum Environmental Conference (IPEC) abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water.  Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels.  Furthermore, this degradation took place without significant oxygen depletion.  Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common.  The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment and microbial community structure on several other potential deep-water drilling sites around the world (Eastern and Central Mediterranean, Caspian Sea, Great Australian Bight, and off the coast of Angola) all of which are quite different then the Gulf of Mexico.
316. Terry C. Hazen. 2016. Oil Biodegradation in Marine Environments. Environmental Writing Course
317. Hazen, T. C.. 2016. Microbial Community Structure Predicts Groundwater and Marine Geochemistry. . . . Seminar Department of Biological Sciences, Michigan Technological University
318. Hazen, T. C.. 2016. Laboratory Safety. Lunch and Learn University of Tennessee
319. T. C. Hazen. 2016. Deepwater Horizon Oil Spill. Seminar to 12th grade science classes at Lincoln International Academy. abstract
     The microbial transformation of terrestrial organic matter, particularly recalcitrant lignin and hemicellulose, is a significant but poorly understood phenomenon. 16S rRNA gene amplicon sequencing and metagenomic sequencing are relatively inexpensive molecular tools to quickly assess microbial diversity and functional genes in response to different carbon sources. Using sequencing and bioinformatics, we aim to elucidate the diversity and metabolic potential of bacterial communities subsisting on lignin and hemicellulose in laboratory cultures. Seawater microcosms were incubated with an added concentration of purified lignin or xylan. CO2 respirometry and enzyme assays showed high microbial activity on both substrates. Using an Illumina MiSeq platform, both amended cultures and unamended controls were sequenced for 16S rRNA gene amplicons and metagenomics. Reads were annotated using Qiime and MG-Rast. Annotated data was compared in detail using phyloseq, vegan, and DESeq2 in R. Several species belonging to phylum of Proteobacteria, Flavobacteria, and Firmicutes significantly increased in abundance when lignin or hemicellulose was added. Groups of functional genes related to carbohydrates and aromatic catabolism significantly increased in abundance. Our methods may uncover important species that have been previously overlooked for terrestrial organics degradation.
320. Harik, A-M. and T. C. Hazen. 2016. Methanotrophically Mediated Bioaggregation to Control Sand Dust. ASM Microbe Annual Meeting abstract
     Sand and Sand dust events are commonplace throughout many parts of the world and they pose a threat to public health, infrastructure, and daily life. Current techniques to control sand dust are limited, expensive, and not environmentally friendly. Some traditional techniques have been outlawed in the US, among other countries, due to concerns about groundwater contamination. Looking towards safer methods current research has begun to focus on bacteria and their byproducts as a substitute for chemical aggregates. We believe that methanotrophic bacteria - bacteria that can live off of methane - and the sugars they excrete could be applied to the sand surface where they will act as a glue to form a stable layer. Methanotrophic bacteria are known for producing excessive amounts of sugars known as extracellular polysaccharides, these sugars are currently used in the pharmaceutical and food additive industries. While methanotrophic bacteria are not the only type of bacteria that can produce sugars they are unique in a few useful ways; the first being that their targeted growth can be encouraged in the environment with the addition of methane gas, other bacteria will not be able to utilize the methane and will not change in abundance. This is useful when applying the bacteria to sand to encourage the bacteria we want to grow - thus stabilizing the sand - but not others; other bacteria with non-specific sources of carbon would not easily be able to have such targeted growth. A second useful aspect of these bacteria is that they are capable of breaking down many pollutants, this is important because the reactors used to grow the bacteria can have a second purpose of destroying environmental contaminants. The production of sugars is not unique to any one species of methanotrophic bacteria, allowing us the possibility to utilize native bacteria from each location’s sand preventing the introduction of new species to the environment. This project is still in the early lab scale stage, but results so far have been promising.
321. Curtis, D., P. Zhang, Z. He, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2016. Microbial Populations Influencing Metal and Nitrogen Cycling are Structured Along Contaminant Gradients at a Nuclear Legacy Site. International Symposium for Microbial Ecology abstract
     Microbial communities perform key roles in biogeochemical cycling, which in turn, is an important component of ecosystem function in Earth’s biomes. As such, how anthropogenic contaminants impact microbial community function is a critical question. In this study, the structure and response of subsurface functional microbial populations to uranium and nitrate contamination were evaluated. Groundwater samples were collected along uranium and nitrate gradients and extracted DNA was analyzed with a comprehensive functional gene array. For analysis, samples were categorized as high, moderate and low concentration based on the EPA’s maximum contaminant levels for nitrate (10 mg/L; LN, MN, HN) and uranium (0.03 mg/L; LU, MU, HU). Ordination plots revealed clustering of samples by the level of nitrate or uranium present and genes from known metal and uranium reducing genera were detected in all sample groups. Dissimilatory tests with cytochrome and hydrogenase genes identified significant differences between LU-HU groups and between LU-HU and MU-HU groups, respectively, while nitrate reduction genes showed significant differences among all groups. The abundance of cytochrome, dsrAB and hydrogenase genes decreased from LU to HU, while the abundance of nitrate reduction genes was highest in the MN samples. Canonical correspondence analysis indicated that in addition to NO3- and U, pH and SO42- exerted strong influences on the subsurface community structure, irrespective of whether samples were grouped by nitrate or uranium. These findings point to impacts in biogeochemical cycling within the subsurface with potential downstream effects on ecosystem function.
322. Curtis, D., P. Zhang, Z. He, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2016. Microbial Communities Promoting Metal Reduction are Structured Along the Uranium Gradient at a Nuclear Legacy Site. ASM Microbe Annual Meeting abstract
     Prominent pH, nitrate and uranium gradients characterize the Oak Ridge Integrated Field Research Center and provide a setting to understand how the unique subsurface geochemistry influences microbial structure and function. This body of work aims to identify the effect of the uranium gradient on structuring the dissimilatory metal reducing bacteria (DMRB), which influence metal reduction in the subsurface. Samples were grouped into low (LU, <0.03 mg/L, 11 wells), moderate (MU, 0.03-1 mg/L, 15 wells) and high U (HU, >1 mg/L, 15 wells) categories. The community structure and functional potential were discerned through data generated via hybridizing groundwater community DNA to a functional gene array (GeoChip 5.0). Samples did not cluster tightly when relating site characteristics to community assemblage using ordination; however dissimilarity tests did reveal significant differences among groups of samples (p<0.05). Significant differences were observed between HU and LU samples when focusing on cytochrome genes while differences between HU-LU and HU-MU samples were found to be significant when using hydrogenase genes as indicators. DMRB including known uranium reducing genera (Anaromyxobacter, Desulfovibrio, Geobacter) were detected in all groups of samples. Their abundance was found to decrease along the uranium gradient. Sulfate, nitrate and pH were the primary geochemical variables influencing the community structure based on the total repertoire of functional genes detected. These results point to structured functional populations within the subsurface and further our understanding the impact of such gradients on DMRB involved biogeochemical cycling.
323. Chakraborty, R., X. Wu, S. Jagadamma, T. C. Hazen, N. Justice, S. Jenkins, T. R. Northen, M. W. Fields, P. Fox, P. Nico, A. P. Arkin and P. D. Adams. 2016. The Properties of and Microbial Interactions with Natural Organic Matter Extracted from Oak Ridge FRC. DOE Genomic Sciences Contractor Annual Meeting abstract
     Project Goal: Natural organic matter (NOM) availability and transformations determines much of the carbon (C) flux in subsurface environments. However, the molecular signature of this pool of C is largely unknown, and the microbial activities that regulate NOM turnover are still poorly resolved. The goal of this discovery project was to ascertain physical and chemical characteristics of NOM, using minimally destructive techniques that retains the originality of the material, and to use it as a C source in enrichments to study it’s turnover by microbial communities at Oak Ridge FRC. Discovery projects are short term, high impact, investigatory efforts to drive changes in science or technological capability that deeply impact the program in some way. NOM was obtained by extracting FW305 and FW306 sediment samples from different depths using MilliQ-water via shaking and sonication. Using this extraction, total organic C in NOM was 0.4% in the surficial layers and dropped to 0.15% in the deeper layers. The amount of inorganic C in extracted NOM decreased significantly with depth. Results from UV and FTIR analyses showed that extracted NOM mainly contained aromatic and unsaturated compounds in shallower depths and mostly polysaccharides in deeper sediments. HPSEC was used to study the molecular weight distribution of the NOM and a stirred ultrafiltration cell was used to fractionate the extracted NOM based on molecular weight. Extracted NOM was provided as the sole C source to microbes present in background well waters of Oak Ridge FRC. Analysis of the enriched microbial community, and transformed NOM metabolites was carried out. Several fine-scale chemical techniques including FTIR, LC-TOF-MS, and Orbitrap were used to characterize the metabolites, and 16S rRNA sequencing and metatranscriptomics were used to identify the changes in microbial communities in these enrichments. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
324. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella, C. Grant and T. C. Hazen. 2016. Environmental Microbial Community Tolerance and Adaptation to Biocides Use in Hydraulic Fracturing Operations. ACS Annual Meeting abstract
     Hydraulic fracturing (HF) in the process of injecting a cocktail of chemicals, sand, and water into an underground rock formation to create fractures and release natural gas. Commonly, biocides are added to prevent biofouling of equipment and prevent microbial growth during gas extraction and gas souring. During extraction some of the injected water and chemicals returns to the surface; this flowback has been accidentally released into the environment, potentially exposing streams to HF chemicals such as biocides. To understand the effect biocides have in steams, microcosms were inoculated using stream water impacted by HF operations (three streams), and as control pristine stream water from the same area were used. Two groups of microcosms were set using the two more commonly used biocides, glutaraldehyde and DBNPA. The microcosms were incubated at ambient temperature over a period of 8 weeks. The microbial community adaptation to biocide was tracked by sampling every two weeks and performing 16s rRNA amplicon sequencing as compared to a biological control (no biocide added). Chemical degradation of the biocide was tracked every week by HPLC, as compared to an autoclaved control to track abiotic degradation. Finally, RNA was sampled at time zero and at the end of the experiment to understand what transcripts were upregulated by the presence of biocide, and hence allow the bacteria to adapt and tolerate the biocide. Our aim is that the results of this experiment will help understand the pathways of biocide resistance and the effect they have in the native microbial community.
325. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella, C. Grant and T. C. Hazen. 2016. Environmental Microbial Community Tolerance and Adaptation to Biocides Use in Hydraulic Fracturing Operations. ASM Microbe Annual Meeting abstract
     Hydraulic fracturing (HF) in the process of injecting a cocktail of chemicals, sand, and water into an underground rock formation to create fractures and release natural gas. Commonly, biocides are added to prevent biofouling of equipment and prevent microbial growth during gas extraction and gas souring. During extraction some of the injected water and chemicals returns to the surface; this flowback has been accidentally released into the environment, potentially exposing streams to HF chemicals such as biocides. To understand the effect biocides have in steams, microcosms were inoculated using stream water impacted by HF operations (three streams), and as control pristine stream water from the same area were used. Two groups of microcosms were set using the two more commonly used biocides, glutaraldehyde and DBNPA. The microcosms were incubated at ambient temperature over a period of 8 weeks. The microbial community adaptation to biocide was tracked by sampling every two weeks and performing 16s rRNA amplicon sequencing as compared to a biological control (no biocide added). Chemical degradation of the biocide was tracked every week by HPLC, as compared to an autoclaved control to track abiotic degradation. Finally, RNA was sampled at time zero and at the end of the experiment to understand what transcripts were upregulated by the presence of biocide, and hence allow the bacteria to adapt and tolerate the biocide. Our aim is that the results of this experiment will help understand the pathways of biocide resistance and the effect they have in the native microbial community.
326. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral and T. C. Hazen. 2016. Stream water microbial population resistance to biocides used in hydraulic fracturing fluids. International Symposium for Microbial Ecology abstract
     Hydraulic fracturing (HF) has grown very fast in the past several years, with an estimated 700% increase since 2007. During the process of HF a cocktail of chemicals, sand, and water are injected into a shale formation to create fractures, which enable release of gas. In most HF operations, biocides are added to prevent biofouling of equipment and prevent growth of subsurface microbes during gas extraction in order to avoid gas souring. During, extraction some of the injected water returns to the surface carrying with it many of injected chemicals including the remaining biocides. It has been reported that flowback water has been accidentally released into the environment potentially exposing streams to HF chemicals such as biocide. To understand the effect biocides may have in case of HF flowback water surface spills, microcosms were inoculated using stream water impacted by Marcellus shale HF operations (three streams), and as HF not impacted stream water from the same Marcellus shale area (three streams) was used. Two groups of microcosms were set using the two more commonly used biocides: glutaraldehyde (100 ppm concentration) and DBNPA (125 ppm concentration). The microcosms were incubated aerobically at ambient temperature over a period of 8 weeks. The microbial community adaptation to biocide was tracked by sampling every two weeks and performing 16s rRNA amplicon sequencing as compared to a biological control (no biocide added). Chemical degradation of the biocide was tracked every week by HPLC (for DBNPA) and GC-FID (for Glutaraldehyde) as compared to an autoclaved control to track abiotic degradation of the biocides. Preliminary data for the Glutaraldehyde microcosms shows the genus Methylobacterium in enriched in both the HF impacted and not impacted sites, while the biological control shows no enrichment of the genus Methylobacterium. Simpson alpha diversity analysis, shows evenness and similarity of the microbial population decrease drastically in the HF not impacted sites as compared to the HF impacted sites, presumably because the HF affected sites have more members of the microbial community able to tolerate the biocides. Preliminary data for DBNPA, shows an enrichment of an unclassified genus. Simpson alpha diversity analysis shows a decrease in evenness and richness of the HF not impacted sites at week two, but then the alpha diversity increases at the experiment continues. DBNPA quantification shows higher degradation of the biocide in HF not impacted sites as compared to the abiotic control, showing that biodegradation is taking place. However, the HF impacted sites, show less degradation of DBNPA as compared to the abiotic control. This may indicate that HF spill and the amendment of biocide inhibit the microbial population capable of degrading the DBNPA. The aim of this on-going study is to understand the pathways of biocide resistance and the effect they have in the native microbial community in case of a HF fluids spill into the environment.
327. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. 3rd Annual Southeastern Biogeochemistry Symposium abstract
     Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
328. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
329. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. National Council Undergraduate Research Annual Meeting abstract
     Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
330. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Microbial Communities Promoting Metal Reduction are Structured Along the Uranium Gradient at a Nuclear Legacy Site. ASM Microbe Annual Meeting abstract
     Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
331. Bailey, R. E., W. A. Henke, C. T. Davis, M. F. Campa, T. C. Hazen, A. W. Johnson, N. O. Hoilett, L. R. McAliley and J. H. Campbell. 2016. Heavy-metal contamination and its effects on microbial community structure in soils near Picher, OK, within the Tar Creek Superfund Site. The Extreme Science and Engineering Discovery Environment (XSEDE16)
332. Zhang, P., A. Rocha, Z. He, J. Van Nostrand, E. Alm, T. C. Hazen, D. Elias, M. Fields, A. Arkin, P. Adams and J. Zhou. 2015. Impacts of Environmental Contaminants on Diversity of Groundwater Microbial Communities at a U(VI)-contaminated Aquifer. American Society for Microbiology Annual Meeting abstract
     Microbial diversity in groundwater ecosystems has not been well studied but can be impacted by different geochemical conditions such as contaminant type and level and pH. As part of the Global Survey at the US DOE Oak Ridge site, groundwater samples were collected from 69 wells that cover a large scale gradient of contaminant and pH levels at this site, with U(VI) ranging from 0.002 to 55.3 mg/L, nitrate ranging from 0.1 to 11648 mg/L, and pH ranging from 3 to 10.5. Groundwater functional communities were analyzed using a comprehensive functional gene microarray (GeoChip 5.0), and phylogenetic communities were analyzed using Illumina sequencing of 16 rRNA genes. The results indicated that both functional and phylogenetic diversity and structure of groundwater microbial communities were significantly affected by different geochemical characteristics. The community differences were largely correlated with the differences in U(VI), nitrate, pH, dissolved organic carbon and sulfate in the groundwater. The microbial functional and phylogenetic diversity significantly decreased with U(VI) concentration, while high nitrate concentrations showed less inhibition on the microbial diversity. Both functional and phylogenetic diversity was highest at neutral pH and decreased with increased and decreased pH levels. Our results also suggested that although high contaminant levels and pH inhibited functional and phylogenetic diversity, the metabolic potential of indigenous groundwater microbial communities was less impacted than phylogenetic diversity. This study improves our understanding of the diversity of groundwater microbial communities across a large scale of contaminant and pH levels at this site.
333. Yongquist, E., S. Techtmann and T. C. Hazen. 2015. Genomic Diversity of Pseudoaltermonas species from Geographically Distant Marine Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Microbes are extremely diverse and capable of catalyzing many functions. Currently, microbial species are defined though characterization of the microbe’s metabolism and through sequencing of 16S rRNA. Recent studies suggest that microbes classified as the same species through 16S rRNA sequencing may demonstrate high genetic diversity. The goal of the study is to identify how much genomic diversity exists within a single species from different marine locations. Differences in the physical and chemical parameters of these locations may select for particular populations of microbes capable of growing in these environments. We isolated 99 microbial strains from the coasts of Australia, Angola, and Bermuda. Out of those isolates, we chose 32 Pseudoalteromonas spp. that have greater than 99% 16S rRNA identity. We will sequence the entire genomes of the 32 strains and compare their genomes to better understand the genomic heterogeneity within this species. This work will result in a better understanding of biogeographic patterns in marine microbial species. We hope that this work will demonstrate the utility of performing whole genome sequencing to differentiate between closely related taxa.
334. Wu, X., S. Jagadamma, A. Lancaster, M. Adams, T. C. Hazen, N. Justice and R. Chakraborty. 2015. Microbial Interactions with Natural Organic Matter Extracted from the Oak Ridge FRC. American Geophysical Union Annual Meeting abstract
     Natural organic matter (NOM) is central to microbial food webs; however, little is known about the interplay between the physical and chemical characteristics of NOM and its turnover by microbial communities based upon biotic and abiotic parameters (e.g., biogenic precursors, redox state, bioavailability). Microbial activity changes the structures and properties that influence further bioavailability of NOM. To date, our understanding of these interactions is insufficient, and indigenous microbial activities that regulate NOM turnover are poorly resolved. It is critical to identify NOM characteristics to the structure and composition of microbial communities and to the metabolic potential of that community. Towards that end, sediment samples collected from the background area well FW305 (Oak Ridge Field Research Center, Oak Ridge, TN) were tested for NOM extraction methods that used three mild solvents, e.g., phosphate buffered saline (PBS), pyrophosphate, and MilliQ-water. MilliQ-water was finally chosen for extracting sediment samples via shaking and sonication. Groundwater from well FW301 was used as an inoculum to which the extracted NOM was added as carbon sources to feed native microbes. To identify the specific functional groups of extracted NOM that are bioavailable to indigenous microbes, several techniques, including FTIR, LC-MS, EEM, were applied to characterize the extracted NOM as well as the transformed NOM metabolites. 16S rDNA amplicon sequencing was also performed to identify the specific microbial diversity that was enriched and microbial isolates that preferentially grew with these NOM was also cultivated in the lab for future detailed studies.
335. Wu, X., S. Jagadamma, T. C. Hazen, T. Northen, M. Fields and R. Chakraborty. 2015. Microbial Interactions with Native Natural Organic Matter at Contaminated Sites from Oak Ridge FRC. American Society for Microbiology Annual Meeting abstract
     Natural organic matter (NOM) is central to microbial food webs. Physical and chemical characteristics of NOM differ based on their biogenic precursors as well as the redox state and biogeochemistry of the specific environment where they are present. Microbial activity may cause degradation, oxidation, or reduction of NOM, changing the structures and properties that influence their further bioavailability. So far our understanding of these interactions are insufficient, and indigenous microbial activities that regulate NOM turnover are poorly resolved. Therefore, in order to connect NOM to the structure of the microbial community and to the metabolic potential of that community, our goal was to extract indigenous NOM using minimal destructive techniques. The extracted NOM will then be used to feed microbes (as electron donor/acceptor/carbon source). Sediment samples were collected from the background area well FW305 of our field site- Oak Ridge Field Research Center (FRC),TN. We developed methods that using three mild extracting solvents (pH 6.5–7.0), e.g., phosphate buffered saline (PBS), pyrophosphate, and warm de-ionized water with shaking or mild sonication as extraction method were tested. We also tested the efficacy of glass beads as a physical abrasive. The extracted dissolved organic carbon (DOC) was in the range of 8.8–28 mg/L. Sonication with PBS could extract higher levels of DOC and total iron of 1mM when compared with other tested methods. The extracted NOM was fractionated using molecular weight cut-off filters (100 kDa). Groundwater from well FW305 was used as an inoculum to which the fractionated NOM was added to feed native microbes to identify the microbial isolates/community that preferentially grew with these NOM. Analysis of the enriched microbial community, and transformed NOM metabolites was also carried out. Advanced characterization of extracted NOM and transformed NOM will be performed using FTIR and NMR techniques to identify the specific functional groups of NOM that indigenous microbes are interacting with.
336. Wright, J. R., D. Marabello, J. McDermott, W. Wang, T. Macbeth, M. F. Campa, D. C. Joyner, T. C. Hazen and R. Lamendella. 2015. Microbial Community Structure and Function Associated with Dichloromethane Contaminated Groundwater. American Society for Microbiology Annual Meeting
337. Woo, H. L. and T. C. Hazen. 2015. Using high throughput sequencing methods to identify keystone bacterial species in recalcitrant terrestrial organic matter transformation. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
338. Woo, H., K. O'Dell, S. Techtmann and T. C. Hazen. 2015. What Happens to Lignin in the Ocean? Evidence of Bacterial Lignin Degradation in Marine Microcosms. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     The microbial transformation of allochthonous terrestrial organic matter, particularly recalcitrant aromatic lignin, is a significant but poorly understood phenomenon in the marine ecosystem. We aim to elucidate the diversity and metabolic potential of a marine bacterial community subsisting on lignin. Seawater from the oxic and ultra-oligotrophic Mediterranean Sea, near the Egyptian Nile Delta, was amended with lignin and incubated for 2 weeks. Microbial activity was assessed by CO2 respirometry and enzyme assays. Community structure and metabolic potential of the microcosms were assessed using 16S rRNA gene amplicon sequencing, and metagenomic sequencing. Lignin amendment caused higher respiration rates and oxidative enzyme activity in comparison to the control. Halomonas, Idiomarina, Thalassospira spp. were several of the dominant OTUs in the microbiome. The metagenome of the lignin-amended microcosm had higher abundance of genes encoding information storage and processing, and aromatic catabolism. Conversely, the unamended control metagenome had signs of starvation conditions, such as higher abundance of genes encoding desiccation stress, spore protection, and plasmid function that were not found in the lignin-amended metagenome. We believe the positive effect of lignin on microbial activity and abundance of aromatic catabolism genes supports the notion of lignin degradation by marine microbes
339. Woo, H., K. O'Dell, S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2015. What happens to lignin in the ocean? Evidence of Lignin Transformation from Marine Microcosms. American Society for Microbiology Annual Meeting abstract
     The microbial transformation of allochthonous terrestrial organic matter, particularly recalcitrant aromatic lignin, is a significant but poorly understood phenomenon in the marine ecosystem. The genetic basis of microbial lignin degradation is of high interest because of its potential application to lignocellulosic biofuel and lignin valorization. In this study, we aim to use metagenomics to elucidate the metabolic potential of a marine bacterial community subsisting on lignin. Marine seawater from the oxic and ultra oligotrophic Eastern Mediterranean, near the Egyptian Nile Delta outlet, was amended with insoluble Organosolv lignin and incubated for 2 weeks. During the incubation, microbial activity was assessed by CO2 respirometry and oxidative enzyme assays. The community structure and metabolic potential of the microcosms were assessed using selective cultivation of lignin-degrading isolates, 16S rRNA gene amplicon (microbiome) sequencing, and metagenomic sequencing. Lignin amendment caused higher rates of respiration and oxidative enzyme activity in comparison to the control. Cultivated isolates of Halomonas, Idiomarina, Thalassospira spp. from the lignin amended microcosm possessed oxidative activity. The strains represented several of the dominant OTUs in the microbiome. The metagenome of the lignin amended microcosm had higher abundance of the broad COG categorizes, particularly information storage and processing, and genes encoding aromatic catabolism. Conversely, the unamended control metagenome had signs of starvation conditions, such as desiccation stress, spore protection, plasmid function that were not found in the lignin amended metagenome. We believe the positive effect of lignin on the microbial activity and abundance of aromatic catabolism genes supports the notion of marine lignin degradation by microbes.
340. Whitt, K., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2015. Abundance and Diversity of Thaumarcheaota in Four Ocean Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Microbes are the most prevalent living organisms in the world. They are found in almost every environment on earth, and are essential for the cycling of nutrients, such as carbon and nitrogen, in the environment. The nitrogen cycle is essential to life as it converts nitrogen into forms that can be used as nutrients for life. While much is known about the nitrogen cycle in soils, less is known about the marine nitrogen cycle. Recently, Thaumarchaeota, members of the Domain Archaea have been found to be major contributors to the nitrogen cycle in marine environments. Very little is known about the diversity and distribution of Thaumarchaeota in the world’s oceans. This study aims to determine the abundance and diversity of Thaumarcheaota in four ocean basins. We performed qPCR to determine the copy number of genes from Thaumaracheaotes and used statistical analyses to determine differences in gene abundance between basins. Additionally, we investigated the correlations between gene abundance and different environmental factors. 16S rRNA sequences of Thaumarchaeotes was also used to investigate the diversity of Thaumarcheaotes in different environments. Preliminary data shows certain Thaumaracheaotes are specific to particular marine locations and their abundance is related to different environmental factors.
341. Wang, J., P. Zhang, L. Wu, Z. He, M. B. Smith, A. M. Rocha, C. S. Smillie, S. W. Oleson, C. J. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, S. P. Preheim, M. S. Sanders, J. Yang, M. A. Mueller, S. Brooks, D. B. Watson, M. W. W. Adams, W. A. Lancaster, F. L. Poole, E. Dubinsky, D. A. Elias, P. D. Adams, A. P. Arkin, M. W. Fields, E. J. Alm, T. C. Hazen and J. Zhou. 2015. Free-living and particle-attached bacterial communities of groundwater along multiple environmental gradients. American Society for Microbiology Annual Meeting abstract
     We characterized the groundwater bacterial communities of 93 wells from the Oak Ridge Integrated Field-scale Research Challenge site with two habitat fractions presenting different species dispersal traits: free-living (0.22-um filter) and particle-associated (10-um filter). Along the multiple environmental gradients (including pH and contaminants), environmental filtering significantly controlled community spatial variations, as indicated by the significant correlations between community variations and environmental changes, and the non-significant distance-decay relationships for both communities. The dominant effect of environmental filtering was further supported by the significantly lower phylogenetic relatedness than null expectations for all communities, and the species partitioning between the two habitats was mainly controlled by nitrate, but not pH, uranium or spatial factors. For the free-living communities, the diversity was more constrained by spatial factors (latitude or longitude) than their counterpart particle-associated communities, while the latter was more affected by environmental variables (e.g., magnesium, dissolved oxygen and nitrate). Unexpectedly, we found there were no significant differences in the diversity between these two habitats, contrasting with our prediction that particles would provide more niches (e.g., microenvironments in nutrients) to support more species than the surrounding groundwater. Furthermore, the particle-associated communities had a significantly higher spatial variation than the free-living ones, indicating the former have a higher retardation rate and lower dispersal rate. Collectively, our results showed that the multiple environmental filters were dominant in controlling the bacterial communities in the studied contaminated aquifer, but the dispersal effects also played important roles in partitioning the species distribution between different habitat fractions.
342. Ulrich, N., J. Wright, A. Rosenberger, T. C. Hazen, M. Campa, D. C. Joyner and C. J. Grant. 2015. A Temporal Analysis of Impacts of Unconventional Natural Gas Extraction on Microbial Communities in Headwater Stream Ecosystems in Northwestern Pennsylvania. American Society for Microbiology Annual Meeting
343. Techtmann, S., S. Stelling, D. Joyner, S. Uttukar, A. Harris, N. Alshibli, S. Brown and T. C. Hazen. 2015. Phenotypic and Genomic Heterogeneity among Colwellia psychrerythraea Strains from Distant Deep-Sea Basins. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     16S rRNA sequencing is routinely used to investigate the diversity of prokaryotes in environmental settings. Evidence suggests that microbes with nearly identical 16S rRNA genes can have genotypic heterogeneity. To better understand the diversity within a single microbial species, we characterized the phenotypic and genomic heterogeneity of three strains of Colwellia psychrerythraea. Colwellia are psychrophilic heterotrophic marine bacteria ubiquitous in cold ecosystems. We have recently isolated two Colwellia strains: ND2E from the Eastern Mediterranean and GAB14E from the Great Australian Bight. The 16S rRNA of these two strains are greater than 98.2% identical to the type strain C. psychrerythraea 34H, which was isolated from Arctic sediments. Carbon source utilization profiles for these strains were determined using the Biolog phenotype microarrays. The carbon source utilization profiles were distinct with less than half of the carbon sources being shared between all three strains. Whole genome sequencing revealed that the genomes of these three strains were quite diverse with some genomes having up to 1600 unique genes. These findings suggest that a single microbial species can exhibit substantial phenotypic and genomic heterogeneity. This diversity must be taken into account when trying to interpret 16S rRNA sequencing data from complex environmental microbial communities.
344. Techtmann, S., S. Stelling, D. Joyne, S. Uttukar, A. Harris, N. Alshibli, S. Brown and T. C. Hazen. 2015. Phenotypic and Genomic Heterogeneity among Colwellia psychrerythraea Strains from Distant Deep-Sea Basins. American Society for Microbiology Annual Meeting abstract
     16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
345. Rocha, A. M., T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. M. Klingeman, D. B. Watson, D. C. Joyner, J. L. Fortney, S. Jagadamma, J. J. Zhou, J. D. Van Nostrand, M. W. W. Adams, F. L. Poole, W. A. Lancaster, R. Chakraborty, D. Elias, P. D. Adams, A. P. Arkin, E. J. Alm and T. C. Hazen. 2015. Temporal Variation in Groundwater Geochemistry and Microbial Community Structure at Oak Ridge Field Site. Genomics Sciences Program Contractor-Grantee Meeting abstract
     Project goals: The goal of the ENIGMA field microbiology component is to identify key microbial populations and determine the community events and mechanisms of these populations that impact and control environmental activities of interest, ultimately predicting how perturbations of the environment may affect community structure and function. From these results, we can develop models that can be applied to microbial populations overlaid with geochemical parameters and engineering controls. An example of such a model is described in the 100-well survey where we demonstrated the ability to utilize natural bacterial communities as in situ environmental sensors that capture environmental perturbations and elucidate key systems biology features (Smith et al., submitted). Here, we expand upon the survey to capture the microbial community response to temporal changes in the groundwater geochemistry to (1) provide for temporal tuning of predictive models, and (2) to determine if and to what extent geochemical variation in groundwater affects microbial community, activity, and genetic diversity along different well depths. Across aquatic and terrestrial environments, numerous studies have sought to characterize key microbial communities and to identify factors that drive changes in microbial community structure and activity. While these studies enable us to further understand and potentially uncover key correlations between the composition of microbial communities and their environment, information regarding temporal community dynamics is often limited or in many cases lacking. One such example is the Oak Ridge Field Research Center (ORFRC) where there has been a large focus to characterize the spatial distribution of groundwater and soil microbial communities across different geochemical transects (e.g. Uranium-Nitrate-pH). In this timeseries study, we aim to bridge this gap by capturing the spatio- and temporal variation of geochemistry on the overall structure, function, and genetic diversity of the groundwater microbial communities in the groundwater wells at the ORFRC. Here, we present our findings from two pilot studies conducted during November 2013 and from November 2014 - January 2015, respectively, at the ORFRC background field site. One of our main objectives of the temporal study is to determine how resilient (or volatile) microbial communities are to daily and weekly changes in groundwater geochemistry. To capture changes in microbial community structure and geochemical constituents, we initially sampled two deep and two shallow groundwater wells over the course of three weeks during November 2013. For each well and time-point, groundwater samples were collected for geochemical and microbial communities analyses. Nucleic acids were collected by filtering water through a 10.0μm pre-filter and 0.2μm membrane filter and then extracted using a Modified Miller method. Results from the study showed that geochemical measurements across all the wells remained fairly stable over the course of the study. However, a decrease in pH and increase in conductivity measurements was observed in the shallow wells during small rain events. Unlike the geochemistry, the 16S rRNA sequencing of the microbial community structure within each well varied on a daily basis in both the 0.2 μm and 10.0 μm size fraction. Statistical analysis of the 16S data using Adonis indicated that there were statistically significant differences (p-value = 0.0001) in the community structure between wells throughout the study. Nonmetric multidimensional scaling analysis of the community structure did not show distinct differences between communities present in shallow versus deep well depth. However, analysis indicated that communities present in wells FW-300 (shallow) and GW-460 (deep) were much more variable throughout the time course. Due to the stability of the geochemistry in the 2013 pilot study, we hypothesize that the variation in microbial communities is a result of both a sampling effect and from the inclusion of communities associated with biofouling in the well casing and sloughing off during sampling. To determine if the daily variation in groundwater microbial community profiles in the first study were naturally occurring within the aquifer or if (and to what extent) the variation in community structure is a result of factors associated with biofouling or sampling artifacts, we sampled six wells from the background site from November 2014 – January 2015. Of the six wells, we physically and chemically cleaned four wells to remove biofilm and attached particulates from the well casing. The remaining two wells that weren’t cleaned, served as controls. Prior to cleaning, all wells were sampled to establish a baseline microbial community profile. Postcleaning, each well was sampled a total of twelve times. For each well and time-point, groundwater samples were collected for geochemical and microbial community analyses. Currently, the microbial and geochemical data are still being analyzed although the preliminary results indicate geochemical variation in response to rain events during the course of the study. Overall, results from both pilot studies suggest evidence of geochemical and microbial response within select wells in response to rain events. Findings from these will enable ENIGMA campaigns for more specialized questions on microbial community structure, provide for temporal tuning on environmental models, and further our understanding of the natural temporal variations versus external factors, such as biofouling or sampling-related effects on microbial communities. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231
346. Rocha, A. M., T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. M. Klingeman, D. B. Watson, D. C. Joyner, J. L. Fortney, S. Jagadamma, B. Detienne, B. Adams, J. J. Zhou, J. D. Van Nostrand, M. W. W. Adams, F. L. Poole, W. A. Lancaster, R. Chakraborty, E. J. Alm and T. C. Hazen. 2015. The Impact of Groundwater Well Disinfection on Microbial Community Response and Regrowth. American Society for Microbiology Annual Meeting abstract
     Hydrogeological studies have shown that groundwater microbial communities and/or geochemical measurements can be biased by a number of external factors, such as, well construction, well maintenance and sampling methodologies. In a recent study at the Oak Ridge Field Research Center’s (ORFRC) background site, the temporal variation of groundwater microbial communities was monitored during November 2013. Findings showed little to no fluctuation in overall geochemistry. However, 16S rRNA sequencing of the microbial community structure varied on a daily basis. Due to the stability of the geochemistry, we hypothesize that the variation in microbial communities is a result of both a pumping effect and from the inclusion of communities associated with biofouling in the well casing and sloughing off during sampling. In this study our goal is to determine if the daily variation in groundwater microbial community profiles are naturally occurring within the aquifer or if (and to what extent) the variation in community structure is a result of factors associated with biofouling or sampling artifacts. To determine the impact of biofouling on the temporal variation of the groundwater microbial community structure we sampled six wells at the ORFRC background site. Of these, we physically and chemically cleaned four wells to remove biofilm and attached particulates from the well casing. The remaining two wells that weren’t cleaned served as controls. All wells were sampled prior to cleaning to establish a baseline microbial community profile. Post-cleaning, each well was sampled a total of twelve times from December 2014 – January 2015. For each well and time-point, groundwater samples were collected for geochemical and microbial community analyses. Nucleic acids were collected by sequentially filtering water through a 10µm pre-filter and 0.2µm-membrane filter. Currently, the microbial and geochemical data are still being analyzed although the preliminary results indicate geochemical variation in response to rain events during the course of the study. Here, we present the temporal variation in microbial response to the well disinfection. Results of this study will further our understanding of the natural temporal variations versus external factors, such biofouling or sampling-related effects on microbial communities.
347. Paradis, C., N. Mahmoudi, D. Driver, K. O’Dell, J. Fortney, S. Jagadamma, S. Schaeffer and T. C. Hazen. 2015. Soil Microbial Respiration and Community Structure in Response to Severe Drought and Precipitation Events. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     There is strong evidence of an increasing trend of severe drought and precipitation events in the Unites States (US) due to greenhouse gas emissions. However, there is a considerable knowledge gap between these severe weather events and soil microbial respiration. The objective of this study was to quantify soil microbial respiration and assess microbial community structure under ambient and heavy weekly precipitation drying/wetting cycles and continuous drought conditions. Laboratory microcosms were constructed using soil native to the southeastern US. Soil moisture and CO2 were measured periodically during the 6-week experiment. Soil microbial DNA (16S rRNA) was extracted and analyzed before and after incubation. The rate of CO2 production in the drought treatment was substantially less compared the ambient and heavy moisture treatments after 2 weeks. The cumulative CO2 production in the ambient and heavy moisture treatments were similar. The rate of CO2 production was substantially higher immediately after wetting of the drying soil (wetting effect) in the ambient treatment compared to the heavy moisture treatment. The microbial community taxa was similar among the treatments before and after incubation. These results suggested that extended drought may result in decreased respiration and the wetting effect becomes negligible during frequent heavy precipitation. Further, our study suggested that severe drought or precipitation events may simply decrease or increase respiration, respectively, of taxa already present rather than select for specific microbial communities.
348. McBride, K. R., C. Chen and T. C. Hazen. 2015. Assessing Ecological Impact of Clay Flocculation Techniques by Measuring Microbial Community Structure. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Clay flocculation is a recently developed technique that restores water systems experiencing harmful algal blooms; but the ecological impact of flocculation on the quality of these water systems has not been thoroughly studied. This project aims to assess the ecological impact of different flocculation treatments by measuring microbial community structure. In order to do this, sedimentary columns treated with three different types of flocculation were tested to observe microbial community composition. For this, one of the methods used was 16s RNA sequencing, which gave a general profile of microbial diversity; the other method was Phospholipid Fatty Acid (PLFA) analysis, which measures biomass in relation to the community structure. The results of the 16s RNA sequencing showed no significant variation in microbial composition between the different flocculation treatments and control. Preliminary PLFA results suggest small changes in the biomass between different techniques. Results of this project entail that flocculation may still have an impact on microbial community structure, which could lead to alteration of nutrient levels in sediment and affect overall water quality. This study can help broaden our understanding of potential ecological impact of clay flocculation on water and in sedimentary environments.
349. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2015. Microbial Community changes and Crude Oil Biodegradation and Microbial Community Changes in Deep Oceans. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
350. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2015. Microbial Activity and Community Changes to Crude Oil in Deep Oceans. American Society for Microbiology Annual Meeting abstract
     Oil spills such as the Deepwater Horizon Oil spill in the Gulf of Mexico have the potential to drastically impact marine systems. However, offshore oil and gas prospecting is expanding with increased demand for petroleum. As a result, the risk of oil contamination in the marine system remains a huge concern to the public and governments around the world. Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern Mediterranean Sea, Central Mediterranean Sea, Great Australian Bight and Southeastern Atlantic Ocean). In this study, microcosms were set up aerobically in three different conditions: seawater, seawater + oil and seawater + oil + oil dispersant (Corexit). Different treatments were set up with 125ml serum bottles and connected to a Micro-OxyMax respirometer, to constantly measure CO2 levels. Samples were taken at three time points for the analysis of oil degradation by fluorescence and GC/MS analysis and microbial community changes by16S rRNA sequencing. CO2 evolution followed a similar pattern in all of the basins sampled. The treatment of seawater + oil + Corexit had the highest CO2 production.. The amendment of oil lead to a higher CO2 accumulation than seawater only treatment. The CO2 accumulation of seawater samples from the Eastern Mediterranean and Great Australian Bight are higher than the Southeastern Atlantic Ocean and Central Mediterranean Sea. However, they were all much lower than the data from the Gulf of Mexico (GOM). What’s more, the fluorescence of dissolved organic mater revealed that most oil in the microcosms was degraded in the first several days, which was consistent with the GC-MS results. Oil biodegradation appears to occur rapidly in all of the sites, but much slower than what was observed in GOM. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil amendment affected how quickly the diversity decreased. The relative abundance of Proteobacteria increased significantly, while the relative abundance of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Southeastern Atlantic Ocean and Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
351. Liu, J., J. Fortney, S. Techtmann, D. Joyner and T. C. Hazen. 2015. Crude Oil Biodegradation and Microbial Community Changes in Deep Oceans. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     Crude oil contamination in the ocean from oil spills becomes a huge concern to the public and governments. Very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. In this study, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern Mediterranean Sea, Central Mediterranean Sea and Great Australian Bight). Microcosms were set up aerobically in three different conditions: seawater, seawater + oil and seawater + oil + oil dispersant (Corexit). CO2 evolution followed a similar pattern in all of the basins sampled. The treatment of seawater + oil + Corexit had the highest CO2 production. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. The relative abundance of Proteobacteria increased drastically while the population of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
352. Lamendella, R., J. Wright, N. Weit, S. Rummel, T. C. Hazen, M. Fernanda Campa, D. C. Joyner and C. Grant. 2015. Microbial Community Structure of a Passive Abandoned Coal Mine Remediation System In Pennsylvania. American Society for Microbiology Annual Meeting
353. Johnston, E. R., Z. Li, A. Harris, B. L. Turner, S. J. Wright, C. Pan, K. T. Konstantinidis, T. C. Hazen and M. A. Mayes. 2015. Predicting climate feedbacks: metabolic response of soil microbial communities to phosphorus and oxygen availability in tropical ecosystems. American Society for Microbiology Annual Meeting abstract
     Soil microbial communities exert significant control over release of greenhouse gases (GHGs) through degradation of soil organic matter (SOM). Global environmental change is expected to substantially impact these processes, especially in humid tropical forests, where a predicted warmer and wetter weather pattern will likely lead to depletion of O2 below-ground. Phosphorus (P) limitation, a defining feature of many tropical soil systems, may also constrain plant productivity and SOM turnover. Tropical soils remain understudied, substantially limiting our predictive ability of GHG emissions from such ecosystems. In this study, soils were collected from the Gigante Peninsula fertilization experiment at the Smithsonian Tropical Research Institute in Panama. Soils were sampled from 40x40 m P fertilized and unfertilized plots in lowland and upland zones and incubated at 26°C under aerobic and anaerobic conditions in the laboratory. A Micro-Oxymax respirometer was used to continuously measure several gases (CO2, CH4, H2S, H2, and O2). A second treatment was employed to compare how those fluxes were affected by addition of P to the incubation experiments. Microbial gene expression under different incubation conditions was investigated through RNA-seq on Illumina HiSeq platform. Illumina HiSeq and PacBio scaffold sequencing from metagenomes was used to assess community functional attributes between plots and for the assembly of dominant microbial populations. Early results reveal that P addition significantly increased SOM turnover and CO2 release in unfertilized plots compared to fertilized plots. This suggests that P availability constrained SOM degradation only in the unfertilized plots. DNA and RNA sequencing was used to assess these results by detecting the presence and activity, respectively, of genes related to phosphorus acquisition and organic carbon oxidation. We will also report on our efforts to incorporate omics-derived information to models of GHG emissions from these tropical soils.
354. Jagadamma, S., C. J. Paradis, S. W. Olesen, A. M. Rocha, D. C. Joyner, J. L. Fortney, D. B. Watson, D. Elias, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, P. Zhang, R. Chakraborty, M. Fields, M. W.W. Adams, J. Zhou, E. J. Alm and T. C. Hazen. 2015. The Memory Effect: Investigating the exposure-history dependence of electron donor biodegradation rates in groundwater. American Society for Microbiology Annual Meeting abstract
     Biodegradation rates of electron donors in groundwater are well known to depend on numerous site-specific physical, chemical and biological parameters. However, emerging scientific evidence suggests that the exposure history of a groundwater zone to an electron donor can have a prolonged and positive effect on its biodegradation rate, referred to here as “memory effect”. The goal of this study was to investigate the long-term memory effect in a groundwater zone previously exposed to an electron donor. The study site is located on the Oak Ridge Reservation in East Tennessee and is contaminated with nitrate and uranium. In 2009, emulsified vegetable oil (EVO) was pulse-injected into a contaminated groundwater zone in order to stimulate in–situ microbial-mediated nitrate and uranium reduction coupled to EVO oxidation. EVO was rapidly biodegraded and concentrations have been below detectable limits since 2010. In this study, we tested whether the planktonic microbial community from groundwater wells with and without previous EVO exposure exhibited a memory effect. We hypothesized that EVO biodegradation rates would be higher in wells with previous exposure versus those without due to sustained enrichment of EVO-degrading functional genes within genomes. This study specifically aimed to answer the following questions: (i) Can a long-term memory effect be detected? (ii) If detectable, is the memory effect significant? (iii) How long can the memory effect last? and (iv) What mechanism(s) are likely responsible for the memory effect? The first phase of this study is in progress and aimed to determine if the memory effect is detectable. We are currently assessing the planktonic microbial community structure in groundwater from wells with and without an EVO exposure history using 16S rRNA sequencing. If the microbial community structure has returned to its pre-EVO state, we will conduct a second EVO injection in wells with and without previous exposure to investigate the in-situ memory effect. The results of the initial EVO exposure in 2009 and the current (2015) state of the planktonic microbial community structure will be presented here. Investigating the magnitude, duration and mechanism(s) of the memory effect may provide more accurate prediction of the fate of contaminants in environments with various electron donor exposure histories.
355. He, Z., P. Zhang, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, D. Curtis, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2015. Microbial Functional Genes Predict Groundwater Contamination and Ecosystem Functioning. American Society for Microbiology Annual Meeting
356. Hazen, T. C., M. B. Smith, A. M. Rocha, C. S. Smillie, S. W. Olesen, C. Paradis, L. Wu, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, D. A. Elias, K. L. Bailey, R. A. Hurt, S. P. Preheim, M. C. Sanders, J. Yang, M. A. Mueller, S. Brooks, D. B. Watson, P. Zhang, Z. He, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, J. Zhou and E. J. Alm. 2015. Microbial Community Structure Predicts Groundwater Geochemistry. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
357. Hazen, T. C.. 2015. International Scientific Advisory Committee. 3rd Conference of Phenotype MicroArrays
358. Hazen, T. C.. 2015. Chair: Genotype/Phenotype. 3rd Conference of Phenotype MicroArrays
359. Hazen, T. C.. 2015. Phenotypic and Genomic Heterogeneity among Colwellia psychrerythraea Strains from Distant Deep-Sea Basins. 3rd Conference of Phenotype MicroArrays abstract
     16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
360. T. C. Hazen. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. Seminar Nanjing University abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
361. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. Seminar Shenyang China Agriculture Institute
362. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. Seminar Beijing China Agriculture Institute
363. T. C. Hazen. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. European Bioremediation Conference VI abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
364. T. C. Hazen. 2015. Oil biodegradation in five deepwater basins around the world. SINTEF Sea Lab
365. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. University of Tennessee Alumni Reception
366. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. University of Tennessee Sigma Xi
367. T. C. Hazen. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. Georgia Tech, School of Earth and Atmospheric Sciences & Biology Seminar abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
368. Terry C. Hazen. 2015. "OMICS" the Fantasy is Over: We need multiple lines of evidence. RemTEC
369. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. College of Engineering Distinguished Lecture abstract
     Good: The US is expected by many to achieve energy independence by 2016 and to surpass Saudi Arabia as the top producer of oil by 2020. These changes have been largely due to Shale Fracking for gas and oil. We have seen a rapid decrease in the price of oil and gasoline at the pump in the last few months reaching 5 year lows. This has in turn stimulated the economy. The combustion of CH4 produces about half the amount of CO2 relative to coal and a third as much as oil, suggesting reduced overall CO2 emissions. Methane has other attendant environmental benefits including less SO2, NOx, and volatile organic carbon emissions produced during combustion relative to other forms of carbon. Methane can also be a cheap feedstock for valuable products, including remediation of other toxic organics. Bad: Methane is 24 times more potent as a greenhouse gas then carbon dioxide. The recent Intergovernmental Panel on Climate Change report (IPCC 2014) suggests that greenhouse gas emissions must be substantially curbed over the next 35 years with overall fuel switching from coal to gas and greater reliance on nuclear and renewable energy sources. However the US EPA predicts methane emissions from oil and gas activities are projected to grow 4.5% from 2011 to 2018. Ugly: Aging infrastructure in cities and oil/gas production/distribution facilities is a major source of fugitive methane emissions and it is increasing dramatically with more reliance on cheap natural gas. The public in places has become furious about fracking activities with some cities, counties and states banning all or some of fracking activities. Informed decisions are sorely missing.
370. Hazen, T. C.. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. Biology Department Seminar Series, Central Michigan University
371. Harik, A.-M., S. Techtmann, J. Fortney and T. C. Hazen. 2015. Water Swap. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     To determine change in microbial community structure and hydrocarbon degradation rates when placed in foreign waters, as compared to ambient waters. Microbial directed biodegradation has a large impact on hydrocarbon degradation; from naturally occurring oil seeps and from spills. The microbial communities from two separate Angola ocean waters, one surface and one deep water, were removed via 0.22 μm filter then either placed back in their original water source or placed into the other depth’s water by flipping the filter and filtering the water back through it. 16S rRNA sequencing and GC analysis are being performed, we expect there to be differences in microbial community structure and hydrocarbon degradation rates of the communities in their ambient waters versus their swapped waters. If it is found that microbial community structure plays a much larger role than nutrients on biodegradation than oil drilling locations whose microbial communities are not capable of high rates of oil biodegradation will need to have a more extensive spill response than those locations whose microbial communities are well equipped. However, if nutrients are found to be the major factor then spill response procedures can include supplementing the waters with necessary nutrients.
372. S. Hagen, S. Techtmann and T. C. Hazen. 2015. Quantifying Extracellular Enzyme Activity In Deep-Sea Sediment From the Mediterranean Sea Through The Use Of Fluorometric Assays. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     The Mediterranean Sea is home to a diverse community of heterotrophic microbes responsible for cycling much of the organic carbon that enters its waters. The subset of those organisms that utilize macromolecules produce extracellular enzymes as a means of carbon degradation. However, this hydrolytic community is poorly characterized at water column depths greater than a few hundred meters where physical properties, such as pressure and temperature, create a unique environment for influencing enzyme behavior. Here we describe enzyme activities of a suite of hydrolases in surface sediment collected at four sampling stations in the Mediterranean Sea at water depths ranging from 800-2200m. In total, nine enzymes were studied- three peptidases, one esterase, and five glycosidases. Fluorometric assays revealed alkaline phosphatase and aminopeptidase to be active on the magnitude of 100x the other enzymes studied here. Furthermore, assays revealed a significant correlation between activity and depth for a majority of the studied community, indicating adaptation to environmental conditions. A qualitative assessment of the data also showed difference in the activity of enzymes from cores taken from the same site, suggesting variability in hydrolytic potential over a short (approximately 1 m) spatial scale.
373. Hagen, S., N. Mahmoudi, A. Steen and T. C. Hazen. 2015. Quantifying Extracellular Enzyme Activity in Deep-Sea Sediment from the Mediterranean Sea through the use of Fluorometric Assays. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     The Mediterranean Sea is home to a diverse community of heterotrophic microbes responsible for cycling much of the organic carbon that enters its waters. The subset of those organisms that utilize macromolecules produce extracellular enzymes as a means of carbon degradation. However, this hydrolytic community is poorly characterized at water column depths greater than a few hundred meters where physical properties, such as pressure and temperature, create a unique environment for influencing enzyme behavior. Here we describe enzyme activities of a suite of hydrolases in surface sediment collected at four sampling stations in the Mediterranean Sea at water depths ranging from 800-2200m. In total, nine enzymes were studied- three peptidases, one esterase, and five glycosidases. Fluorometric assays revealed alkaline phosphatase and aminopeptidase to be active on the magnitude of 100x the other enzymes studied here. Furthermore, assays revealed a significant correlation between activity and depth for a majority of the studied community, indicating adaptation to environmental conditions. A qualitative assessment of the data also showed difference in the activity of enzymes from cores taken from the same site, suggesting variability in hydrolytic potential over a short (approximately 1 m) spatial scale.
374. Savannah Gillman. 2015. UT, ORNL: partners in science. The Daily Beacon
375. Garcia de Matos Amaral, A., M. F. Campa and T. C. Hazen. 2015. Community Structure of Fracking Flowback water from Marcellus shale of Pennsylvania. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Hydraulic fracking is an important process for shale gas extraction. It has caused an exponential growth of gas extraction in the United States, and is helping the country get a step closer to energy independence. However, this process has also raised questions about its impact on the environment. Millions of gallons of water are produced as waste from the process, increasing water stress in many areas that are already experiencing drought. Bioremediation is a possible way to treat and reuse the produced wastewater. Particularly, intrinsic bioremediation may be aided through the characterization and understanding of microorganisms present into the flowback water. The aim of this study is to investigate raw and treated flowback from the Marcellus shale of Pennsylvania, and identifying all the microorganisms present and understanding their physiology. To do this DNA was extracted from the samples, and 16S rRNA gene amplicons sequencing was performed. The molecular and metagenomics techniques used will aid to explore and help to understand the physiology and metabolism of them the microbes present This knowledge will be used to suggest intrinsic bioremediation capabilities of the microorganisms and treat the wastewater generated through the fracking process.
376. Fortney, J. L., J. Liu, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2015. Oil Biodegradation in Oxygen Minimum Zones. American Society for Microbiology Annual Meeting
377. Fitzgerald, K., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2015. Diversity and Distribution of Archaeal amoA Genes in Geochemically Distinct Marine Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Microbes are essential in the cycling of nutrients within the environment. Since the discovery of ammonia-oxidizing archaea (AOA), it is now understood that Thaumarchaeota may play an important role in the nitrogen cycle within marine environments. AOA are mainly responsible for the nitrogen cycle's first step, which is oxidation of ammonia to nitrite. However, investigation of AOA's diversity and distribution has only recently begun. In this study, we aimed to understand the diversity and distribution of the Archaeal amoA gene, which is required for ammonia oxidation. We used high-throughput next-generation sequencing to investigate the amoA diversity in 59 samples taken from three marine basins at varying depths. Our preliminary data suggest that diversity of Archaeal amoA is low and the relative abundance varies by basin. We will investigate if there are distinct populations of amoA genes within each basin and the phylogenetic diversity of amoA in our samples. These basins are geochemically distinct and grant the opportunity to understand how geochemical variables affect the diversity and distribution of amoA. The study will lead to a greater understanding of AOA in distinct marine basins.
378. Elias, D. A., A. J. King, K. L. Bailey, S. P. Preheim, M. S. Robeson II, T. R. Chowdhury, B. R. Crable, R. A. Hurt Jr., A. C. Somenahally, S. Techtmann, T. Mehlhorn, K. A. Lowe, A. M. Rocha, A. Zelaya, M. W. Fields, A. P. Arkin, J.-M. Chandonia, T. C. Hazen, E. J. Alm, J. Zhou, T. J. Phelps, C. C. Brandt, S. D. Brown, M. Podar, M. W. W. Adams, D. B. Watson and P. D. Adams. 2015. Groundwater-fed Bioreactors Show Distinct Colonization and Community-wide Response Dynamics to Perturbations. Genomics Sciences Program Contractor-Grantee Meeting abstract
     Laboratory bioreactors have long been used for investigating the characteristics of a microorganism or simple synthetic community. However, for studying natural or in-situ microbial communities, discontinuous “snapshot in time” sampling has mainly occurred. In this study an in-field bioreactor system was developed to temporally monitor and manipulate the in-situ microbial community while maintaining the in-situ community structure. Three above ground, in-field reactors were continuously fed microaerobic (0.2% O2) groundwater directly from an existing well at the Oak Ridge Field Research Center, Oak Ridge, TN, for 12 weeks. Each bioreactor contained 800 ml of groundwater and 8 replicate biofilm coupons filled with sterilized site sediment to monitor both the planktonic and biofilm communities. Gas influx was varied from aerobic (weeks 1-7, & 9) to anaerobic (weeks 8 & 10) to confirm that manipulation of bioreactor microorganisms was tractable. Samples from the incoming groundwater and from each bioreactor were taken every two days to match the dilution rate of the reactors. Each sample was analyzed by 16S rRNA sequencing at an average of 10,000 reads and key biogeochemical properties were measured including pH, dissolved oxygen, ORP, conductivity, 12 organic acids 14 anions and 3 sugars. At every third time point 53 different metals were also measured. Community structure and diversity was highly similar across all three bioreactors according to 16S rRNA sequencing, representing 30-65% of the groundwater OTUs overall and 50-85% of high abundance groundwater OTUs. Biofilm coupons captured a unique subset of the groundwater OTUs but on average were only 27% similar to groundwater and 48% similar to the bioreactor planktonic samples. Community beta-diversity patterns indicated bioreactors were more different to the groundwater than expected if no growth was occurring, thereby suggesting growth in the reactors. Correlations between organic acid profiles and bacterial clades revealed that the metabolic function was conserved across all three bioreactors and the in-situ groundwater community. Transitions to anoxic conditions and subsequent lowering of the pH at weeks 7 and 10 resulted in strong, repeatable bacterial community and individual clade shifts toward the groundwater composition. However, not all bacterial groups in the bioreactors mirrored those in the groundwater. In fact, known metal- and organic acid-metabolizing clades increased in abundance in the bioreactors when the incoming groundwater increased in solute concentration despite no change in the incoming clade abundance. Similarly, co-occurrence relationships of OTUs known for syntrophic and predatorprey interactions were observed over time in the bioreactors. This type of in-field bioreactor system allows for discreet temporal monitoring of microbial community structure and function simultaneously while allowing community responses to be determined during the testing of new strategies for environmental amendment or restoration on a small and affordable scale. Keywords. Bioreactor, Microbial Community, Groundwater, Diversity, Anaerobic, Time Course Funding Statement: The work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
379. Detienne, B. L., A. M. Rocha, S. Pfiffner, S. Jagadamma and T. C. Hazen. 2015. Microbial Community Structure and Abundance in Uranium and Nitrate Contaminated Groundwater. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Based on geochemical and geographical differences between groundwater wells, the distribution and abundance of microbial communities in each well could vary drastically. At the Department of Energy’s Oak Ridge field site, 243-acres of contaminated area exist. Contaminants in this site include nitrate and uranium with concentrations ranging from <0.05 -14400ppm and <0.005 - 55ppm, respectively. In order to understand relationship of microbial communities to different concentrations of uranium, groundwater wells were sampled for geochemical and microbial analyses. The goal of this study is to characterize microbial community structure and diversity of uranium and nitrate contaminated wells versus non-contaminated wells. Microbial diversity and biomass were determined using phospholipid fatty acid (PLFA). To measure PLFAs, groundwater was filtered through 0.2 μm pore size Sterivex filter, immediately frozen, and stored at -80°C. Lipids were extracted using the modified Bligh-Dyer method. Currently, PLFA results are being analyzed. However, preliminary PLFA data indicates community diversity in uranium contaminated wells have greater microbial diversity compared to non-contaminated wells. Additionally stress indicators for Gram-negative bacteria were identified in a small fraction of the wells. Results from this study indicate that groundwater microbial communities can vary in respect to the geochemistry and environmental stress factors at the site.
380. Curtis, D., P. Zhang, Z. He, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2015. Changes induced in the subsurface microbial community by the nitrate gradient at a nuclear legacy site. American Society for Microbiology Annual Meeting abstract
     It is important to understand the impact of contamination events on subsurface microbial community-induced biogeochemical cycling. The Oak Ridge Integrated Field Research Center (OR-IFRC) is a nuclear legacy site characterized by a contamination plume emanating from the former S-3 ponds. Wastes within the plume have contributed to significant pH, nitrate and uranium gradients across the site. This work aims to identify the effect of the nitrate gradient on the subsurface microbial community involved in nitrogen cycling. DNA extracted from groundwater was utilized to study the functional composition of microbial communities from low (LN, 1-5mg/L, 13 wells), moderate (MN, 10-100mg/L, 12 wells) and high NO3- (HN, >100mg/L, 13 wells) areas using a functional gene array (GeoChip 5.0). Detrended correspondence analysis revealed that samples were not generally clustered based on the nitrate concentration, though significant differences (p<0.05) between LN-MN, LN-HN and MN-HN groups were observed using dissimilatory tests. Canonical correspondence analysis indicated NO3- strongly influenced the community structure only in HN samples. The abundance of genes contributing to denitrification and nitrate reduction was higher in the MN group relative to the LN and HN groups. A significant decrease in abundance of genes contributing to denitrification (narG, nirS, norB) and dissimilatory nitrate reduction to ammonia (nrfA) was evident between MN-HN groups (p<0.05). The findings presented offer an insight into both the structure of and the factors influencing the subsurface microbial communities in relation to a large nitrate gradient.
381. Chen, C., G. Pan, W. Shi, F. Xu, S. M. Techtmann, S. M. Pfiffner and T. C. Hazen. 2015. How does clay flocculation of harmful algal blooms affect microbial community composition in water and sediment. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
382. Chen, C., T. C. Hazen, G. Pan, W. Shi and F. Xu. 2015. Impact of Clay Flocculation of Algal Blooms on Pond Microbial Community. American Society for Microbiology Annual Meeting abstract
     16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
383. Chen, C., T. C. Hazen, G. Pan, W. Shi and F. Xu. 2015. Impact of Clay Flocculation of Algal Blooms on Pond Microbial Community. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     One of the major environmental concerns of clay flocculation in harmful algal blooms control is the ecological impact of the clay and modified clay. Here we investigated the impact of the clay flocculation technique on the microbial composition in water samples collected in June 2014 at eutrophic ponds in Datong, China. Successional changes in the microbial community structure due to the clay flocculation for algal cells removal were determined by deep sequencing of 16S rRNA genes. Quantitative PCR was used to quantify the biomass in water samples. In addition, total phosphate, soluble reactive phosphate, total nitrate, NH4-N, and NO3-N were measured. The 16S data revealed that microbial community structure significantly changed after clay flocculation in pond water. Water samples were dominated by Actinobacteria, Spartobacteria, Betaproteobacteria, Acidimicrobiia, and Synechococcophycideae before clay flocculation, whereas Actinobacteria, Sphingobacteriia and Betaproteobacteria were dominant during the first 10 days after flocculation, and Actinobacteria decreased dramatically in the following 5 days. Deltaproteobacteria as well as Gammaproteobacteria and Bacilli were dominant in water samples on the 11th day and 15th day. In addition, the total coliform test results indicated a decrease of indicator bacteria after clay flocculation. This study provides an ecological impact assessment of clay flocculation for harmful algae blooms in pond water.
384. Chakraborty, R., A. Pettenato, X. Wu, S. Jagadamma, T. C. Hazen, M. Fields, T. Northen, S. Jenkins, W. A. Lancaster, M. W. W. Adams, A. P. Arkin and P. D. Adams . 2015. Microbial Interactions with Native Natural Organic Matter in Groundwater and Sediment from the Oak Ridge FRC. Genomics Sciences Program Contractor-Grantee Meeting abstract
     Project Goal: Natural organic matter (NOM) is central to microbial food webs; however, little is known about the interplay between physical and chemical characteristics of NOM and it’s turnover by microbial communities in groundwater and sediments of Oak Ridge Field Research Center (FRC). Microbe mediated molecular and geochemical mechanisms control the flow of carbon that support evolution and maintenance of a community within a given environment. To gain insight into the cycling of carbon, and how the turnover of NOM regulates the development of indigenous microbial community at the FRC, it is critical to extract NOM, identify its characteristics, and correlate this to the structure and composition of microbial communities and to the metabolic potential of that community. In this FY15 Discovery project, we focused on NOM from the background well, FW305. Preliminary experiment initiated with FW305 sediment enriched with 10ppm fulvic acid (as part of the Microparticles Campaign), showed rapid evolution of CO2. Isolations from this fulvic acid enriched samples yielded strains belong to Sphingomonas, Pseudomonas, Undibacterium, Rugamonas, Rhodococcus genera. The same sediment sample (FW305) was tested for NOM extraction methods using three mild solvents, e.g., phosphate buffered saline (PBS), pyrophosphate, and warm de-ionized water with shaking or mild agitation. The pH was kept close to in-situ conditions, 6.5–7.0. We also tested the efficacy of glass beads as a physical abrasive to aid in extraction. The dissolved organic carbon (DOC) in the extracted NOM was in the range of 8.8–28 mg/L. Sonication with PBS extracted higher levels of DOC, while shaking with pyrophosphate extracted higher levels of metals such as iron, zinc, cobalt and manganese when compared with other tested methods. A spectrophotometric scan of the sample with highest DOC content showed high absorbance between 300-380nm, indicative of presence of functional groups with high aromaticity such as carboxylic and phenolic groups. The extracted NOM was fractionated using molecular weight cut-off filters (100 kDa). In ongoing experiments, we are testing existing isolates from FW 305 for their ability to transform the NOM, and identify the transformation product. In addition, we will add this fractionated NOM to feed native microbes present in the groundwater of well FW305 to identify the microbial isolates/community that preferentially grew with this NOM. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
385. Campa, M. F., S. M. Techtmann, S. Brewer, A. Garcia de Matos Amaral, J. Wright, N. Ulrich, R. Lamandella and T. C. Hazen. 2015. Hydraulic Fracturing Flowback Water: A Look into the Subsurface Microbial Community and Intrinsic Bioremediation. 2nd Annual Southeastern Biogeochemistry Symposium abstract
     Treatment and reuse of hydraulic fracturing flowback water is the best alternative to handle the enormous volume of wastewater produced. Intrinsic bioremediation and bioaugmentation are efficient ways to treat the high salinity and chemical content of this water. The microbial community in flowback water can provide an insight into what facilitates bioremediation. Thus, raw and treated flowback waters from the Marcellus Shale were collected. The microbial community from the flowback water, and how it changes at different stages of the treatment process was determined by sequencing of 16S rRNA amplicons. Furthermore, aerobic and anaerobic bacteria with physiologies of interest were isolated. Samples were plated on marine broth and ORN7a supplemented with oil. High growth of halophilic bacteria as well as the presence of sulfate reducing bacteria was observed. The isolates will be sequenced (16S rRNA amplicon) and compared to the microbial community structure data. The isolates that are most representative of the community will be selected for high-throughput phenotypic microarray analysis. The study is currently in progress. Characterizing the microbial community structure of the flowback water and understanding its function will help develop and optimize a bioremediation strategy so that flowback water can be inexpensively treated and reused.
386. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella, C. Grant and T. C. Hazen. 2015. Environmental microbial community tolerance and adaptation to biocides use in hydraulic fracturing operations. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
387. Campa, M. F., S. Techtmann, S. Brewer, A. Garcia de Matos Amarral, K. Manz, K. Carter, R. Lamandella and T. C. Hazen. 2015. Flowback Water: A Look into the Subsurface Microbial Community and Intrinsic Bioremediation. American Society for Microbiology Annual Meeting abstract
     Hydraulic fracturing is growing exponentially, causing a 702% increase in shale gas production in the U.S. since 2007. This process is water intensive, requiring between 3.3 to 27m3 of water per million cubic meters of gas extracted. As this process keeps growing, and water scarcity issues continue to arise throughout the world, questions keep rising on how to efficiently deal with the flowback water (which is up to 40% of the total injected water). The goal of this investigation is to study the microbial community from six flowback water samples and harness intrinsic bioremediation capabilities to develop an inexpensive and efficient method to treat the flowback water for reuse. Flowback water contains over 700 different chemicals; we will select a couple of contaminants of interest (COI) by Gas Chromatography- Mass Spectrometry (GC-MS). In this study, we collected raw flowback and treated flowback water from a fracking site at the Marcellus Shale. The first aim of the study is to determine the microbial community from the flowback water, and how it changes at different stages of the treatment process. This is being done using 16S rRNA amplicon sequencing using Illumina MiSeq. The second aim is to isolate aerobic and anaerobic bacteria with physiologies of interest. To do this, marine broth plates and ORN7a plus oil plates were inoculated at room temperature and 37°C. The marine broth plates are meant to mimic the high salinity environment in the subsurface and the ORN7a + oil plates were used to isolate hydrocarbon degraders. Preliminary observations showed high growth of halophilic bacteria and the presence of sulfate reducing bacteria. The isolates will be sequenced and compared to the microbial community 16S rRNA amplicon data. The isolates that are most representative of the community will be selected for high-throughput phenotypic microarray, OmniLog, analysis. This will be used to do kinetic and metabolic studies of COI present in the flowback water and the isolates. The study is currently in progress. Characterizing the microbial community structure of the flowback water and understanding its function will help develop and optimize a bioremediation strategy, so that flowback water can be inexpensively treated and reused.
388. Brown, Randall. 2015. Hazen Encourages UT-ORNL Internships. @COE e-newsletter abstract
     Dr. Terry Hazen, UT-Oak Ridge National Laboratory (ORNL) Governor's Chair Professor in the Department of Civil Engineering, discussed the advantages of UT-ORNL internships in a recent article in the Daily Beacon.
389. Brewer, S. S., M. F. Campa, S. M. Techtmann, J. L. Fortney and T. C. Hazen. 2015. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed recently and is projected to keep growing. With the increase of this unconventional drilling method, concerns have been proposed about environmental safety and dangers to human health. Because this method involves injecting fluid between 1-3 km deep into the Earth and the fluid produced from the well after drilling is often reused in other hydraulic fracturing operations. The goal of this study is to identify novel organisms that might have bioremediation capabilities for the toxic flowback water and compare microbial communities isolated from fracking water samples in anaerobic conditions. Water samples from Pennsylvania include six different collections of produced (flowback) water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown under anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. DNA was extracted, and 16S rRNA gene sequences were used to identify the isolated microbes, and the microbial communities were characterized by 16S rRNA gene amplicon Illuminia sequencing. The physiological conditions of some significant isolated microbes were further characterized by the Omnilog phenotypic microarray system. Early results show presence of numerous anaerobic microbes including sulfate reducers.
390. Brewer, S. S., M. F. Campa, A. G. Amaral, S. M. Techtmann, J. L. Fortney, K. Fitzgerald and T. C. Hazen. 2015. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. Tennessee Experimental Learning Symposium (TELS)
391. Brewer, S., S. M. Techtmann, N. Mahmoudi, D. Niang, S. Pfiffner and T. C. Hazen. 2015. Co-extraction of DNA and PLFA from Soil Samples using Bligh and Dyer PLFA Extraction and Modified Miller DNA Extraction. Posters at the Tennessee Capitol. http://ugresearch.utk.edu/activities/posters-at-the-capitol/posters-at-the-capitol-2015/
392. Adams, B. G., A. M. Rocha, C. Paradis and T. C. Hazen. 2015. Geochemical Response to Temporal Variations in Groundwater Head. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
393. Adams, B. G., A. M. Rocha, S. Jagadamma, C. Paradis and T. C. Hazen. 2015. Impact of Temporal Variations of Hydrology on Groundwater Geochemistry. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     The Department of Energy’s (DOE) Oak Ridge field research uncontaminated site is located within the Bear Creek Valley (BCV) in Oak Ridge, TN. At the site there are 6 groundwater monitoring wells of multiple depths ranging from 20-71ft deep. Previous hydrologic and geochemical studies have identified the presence of two geochemically distinct groundwater zones, as well as, fluctuations in the gradients between flow in shallow and deep wells. Although we know that fluctuations occur in the height of the hydraulic head, it is unclear how these variations affect the geochemistry in shallow versus deep wells. The goals of this study are to (1) determine the direction and magnitude of the horizontal and vertical hydraulic gradients, and (2) determine how the stability (or instability) of the hydraulic head impacts groundwater geochemistry. Use of In-Situ Aqua TROLL® 200 CTD Logger allowed for continual monitoring of the following physical parameters; conductivity, dissolved oxygen, and water level/pressure. Using water level/pressure, we determined direction and magnitude of the horizontal and vertical hydraulic gradients. By combining geochemical data and time-series hydrologic data from the 6 wells, we hope to better understand the geochemical response to variations in hydraulic gradient.
394. 2015. Hazen Addresses Wastewater Treatment Idea.
395. 2015. Yeast-Filled Fibers Could Treat Polluted Wastewater.
396. 2015. Department of Energy Honors Rocha as Part of Series.
397. 2015. Postdoctoral Fellow Honored for Helping Advance Latinas in Tech Field.
398. 2015. Terry Hazen Discusses Gulf Recovery on WBIR. WBIR TV News
399. 2015. Latinas at Tech Giants. Diversity News
400. 2015. Bacteria the newest tool in detecting environmental damage. ENVASS
401. 2015. The Promise of Water Security. Southeast Green
402. 2015. Hazen to Lead UT’s Institute for a Secure and Sustainable Environment. Southeast Green
403. 2015. Hazen to Lead UT’s Institute for a Secure and Sustainable Environment. Tennessee Today
404. 2015. Using Microbial Communities to assess Environmental Contamination. TerraDaily
405. 2015. Bacterial communities serve as ready-made biosensors of environmental contaminants. mBioblog
406. 2015. Bacteria the newest tool in detecting environmental damage. Eurekalert
407. 2015. Bacteria the newest tool in detecting environmental damage. Tennessee Today
408. 2015. Bacteria the newest tool in detecting environmental damage. SciGuru
409. 2015. Using microbial communities to assess environmental contamination. LBNL Newscenter
410. 2015. Microbes serve as markers for environmental contamination. Science News
411. 2015. Bacteria the newest tool in detecting environmental damage. ScienceDaily
412. 2015. Using microbial communities to assess environmental contamination. PhysOrg
413. 2015. Bacteria the newest tool in detecting environmental damage. Science World
414. 2015. Bacterial communities can act as precise biosensors of environmental damage. Biology News Net
415. 2015. Microbes serve as markers for environmental damage. Weird4U.com
416. 2015. Bacterial communities can act as precise biosensors of environmental damage. Kalen2Utech
417. 2015. Microbes serve as markers for environmental damage. Lastminutestuff
418. 2015. Bacteria the newest tool in detecting environmental damage. AZ News
419. 2015. Bakteri bias jadi alat deteksi polusi lingkungan. Antara News
420. 2015. Using microbial communities to assess environmental contamination. R&D Magazine
421. 2015. Microbes serve as markers for environmental contamination. AnchorFree
422. 2015. Using microbial communities to assess environmental contamination. Lab Manager
423. 2015. Bacteria the newest tool in detecting environmental damage. Yourwebapps
424. 2015. Bacteria detects presence of Pollutants, Contamination. American Laboratory
425. 2015. Using Microbial Communities to assess Environmental Contamination. NZ Health Tec
426. Zhang, P., Z. He, J. D. Van Nostrand, L. Wu, D. Curtis, T. C. Hazen, D. E. Elias, M. W. Fields, A. P. Arkin, P. D. Adams and J. Zhou. 2014. Impacts of Environmental Contaminants on Functional Diversity of Groundwater Microbial Communities at a U(VI)-Contaminated Aquifer. ENIGMA SFA Annual Retreat abstract
     Abstract: Microbial diversity in groundwater ecosystems has not been well studied yet. The functional diversity of groundwater microbial communities at heavy metal contaminated sites would be impacted by different geochemical characteristics such as contaminants and pH. As part of the Global 100 Well Survey at the Oak Ridge site, groundwater samples were collected from seven groups of wells with different geochemical characteristics at this site. The first four groups primarily concern the effects of contaminants (U(VI) and nitrate) on microbial diversity: (1) low contaminants (nitrate<2 mg/L and U(VI)<0.01 mg/L) and neutral pH (6.5-7.2), (2) light contaminants (nitrate 4-36 mg/L and U(VI) 0.1-0.2 mg/L) and also neutral pH, (3) moderate contaminants (nitrate 5.5-1471 mg/L and U(VI) 0.1-1.5 mg/L) and neutral pH, and (4) high contaminants (nitrate 2692-11648 mg/L and U(VI) 3.8-55 mg/L) and low pH (3-5.2). The remaining three groups with low contaminants address possible effects of other geochemical characteristics such as groundwater pH: (5) acidic pH (5.2-6.7), (6) high pH (8.9-9.8), and (7) extremely high pH (9.2-10.5). Groundwater microbial communities were analyzed using a comprehensive functional gene microarray (GeoChip 5.0). The results indicated that the groundwater microbial community functional diversity and structure was significantly different among these seven different groups of wells. Such community differences were largely correlated with the differences in U(VI), nitrate, pH, dissolved organic carbon and sulfate in the groundwater. The microbial functional diversity significantly decreased with U(VI) concentrations. Overall, our results suggested that high contaminants and low pH appeared to inhibit the biodiversity and metabolic potential of indigenous groundwater microbial communities. This study improves our understanding of the functional diversity and metabolic potential of groundwater microbial communities across a large scale of contaminant and pH levels at this site.
427. Zhang, P., Z. He, J. D. Van Nostrand, L. Wu, D. Curtis, T. C. Hazen, D. E. Elias, M. W. Fields, A. P. Arkin, P. D. Adams and J. Zhou. 2014. Impacts of Environmental Contaminants on Functional Diversity of Groundwater Microbial Communities at a U(VI)-Contaminated Aquifer. International Symposium of Microbial Ecology (ISME 15) abstract
     Abstract: Microbial diversity in groundwater ecosystems has not been well studied yet. The functional diversity of groundwater microbial communities at heavy metal contaminated sites would be impacted by different geochemical characteristics such as contaminants and pH. As part of the Global 100 Well Survey at the Oak Ridge site, groundwater samples were collected from seven groups of wells with different geochemical characteristics at this site. The first four groups primarily concern the effects of contaminants (U(VI) and nitrate) on microbial diversity: (1) low contaminants (nitrate<2 mg/L and U(VI)<0.01 mg/L) and neutral pH (6.5-7.2), (2) light contaminants (nitrate 4-36 mg/L and U(VI) 0.1-0.2 mg/L) and also neutral pH, (3) moderate contaminants (nitrate 5.5-1471 mg/L and U(VI) 0.1-1.5 mg/L) and neutral pH, and (4) high contaminants (nitrate 2692-11648 mg/L and U(VI) 3.8-55 mg/L) and low pH (3-5.2). The remaining three groups with low contaminants address possible effects of other geochemical characteristics such as groundwater pH: (5) acidic pH (5.2-6.7), (6) high pH (8.9-9.8), and (7) extremely high pH (9.2-10.5). Groundwater microbial communities were analyzed using a comprehensive functional gene microarray (GeoChip 5.0). The results indicated that the groundwater microbial community functional diversity and structure was significantly different among these seven different groups of wells. Such community differences were largely correlated with the differences in U(VI), nitrate, pH, dissolved organic carbon and sulfate in the groundwater. The microbial functional diversity significantly decreased with U(VI) concentrations. Overall, our results suggested that high contaminants and low pH appeared to inhibit the biodiversity and metabolic potential of indigenous groundwater microbial communities. This study improves our understanding of the functional diversity and metabolic potential of groundwater microbial communities across a large scale of contaminant and pH levels at this site.
428. Zeng, L., R. Csencsits, C. Petzold, T. C. Hazen, F. Poole, W. A. Lancaster, M. Adams, M. Fields, D. Stahl, H. Nikaido, B. Jap and P. Walian. 2014. Membrane-based Mechanisms in Stress Response and Community Interactions. ENIGMA SFA Annual Retreat abstract
     Project Goals: In support of our long-standing interest in understanding mechanisms central to stress response and community organization at the molecular level, a primary aim of our project has been the identification and analysis of proteins key to these processes. Particular emphasis is placed on the study of membrane-based systems. Core objectives stemming from this aim involve the isolation and characterization of protein complexes using biochemical and biophysical methods, elucidation of corresponding functional mechanisms, and the development of molecular models and testable hypotheses. Additional goals include supporting objectives of the ENIGMA campaigns, particularly those relevant to microbial communities and interaction mechanisms, metalloprotein function, and the development of technologies for functional screening. Abstract: A central theme of our studies has been the identification and characterization of membranebased molecular mechanisms utilized in the response to environmental conditions, and the establishment and maintenance of communities. In addition to the characterization of targeted protein function, we have been studying growth condition-associated changes in protein abundance, protein-protein interactions, post-translational modifications, and mutations leading to altered function. Recent work has focused on developing antibodies useful for studying the transition from planktonic to biofilm community organization, chromatographic approaches for separating lipidated from non-lipidated proteins, characterization of outer membrane-based metal-binding proteins (where metals can be essential for stabilizing protein conformation, protein-protein interactions, and may play a role in environmental sensing), and investigating the composition and functional properties of vesicles (found to facilitate the protected extracellular transfer of a range of molecules including proteins and nucleic acids; evidence is mounting that this process can support cellular housekeeping, offensive and defensive scenarios, horizontal gene transfer, and biofilm formation). Through these studies we are developing models of how certain membrane proteins (e.g. DVU1422, 3104, 1012 and 0799) factor in community interactions, membrane stability, stress response and outer membrane vesicle production. Sequence homologies suggest that many of the findings derived from these efforts may be applicable across a range of bacteria.
429. Yilmaz, S., M. Smith, E. J. Alm, D. A. Elias, T. C. Hazen, A. P. Arkin, A. K. Singh and P. D. Adams. 2014. Single Cell Genomics Applications in ENIGMA. Genomics Sciences Program Contractor-Grantee Meeting abstract
     Project Goals: We are developing a versatile single cell genomics pipeline that can respond to the needs of a multi-institutional program like ENIGMA. Our pipeline utilizes FISH (fluorescence in situ hybridization) for targeting species of interest, FACS (fluorescence activated cell sorting) for high throughput isolation of single cells, and MDA (multiple displacement amplification) for production of sufficient DNA for genome sequencing. At present, this pipeline is being used for a number of collaborative projects in ENIGMA. Single cell sequencing is a powerful tool for the analysis of uncultivated microorganisms. Current culture-independent, population based techniques (i.e., metagenomics) relying on pooled nucleic acids from communities of microorganisms can independently measure metabolic activity and the species present, but cannot link the activity deterministically to the species. In an attempt to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities, we developed a high-throughput approach to study uncultivable microorganisms one cell at a time. Our approach includes isolation of individual cells by cell sorting, followed by whole genome amplification and sequencing. This pipeline is being utilized to analyze groundwater samples from DOE bioremediation sites (e.g., Hanford 100H, Oak Ridge FRC) to identify keystone organisms and link their functions to species as well as to estimate the level of horizontal gene transfer within the community; to isolate and identify viruses in deep subsurface groundwater, and investigate their role in microbial community structure and function; to assess the composition of bioaggregates in environmental samples with the ultimate goal of verifying the stereotypical configurations of microorganisms. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
430. Woo, H. L., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Lignin-Degrading Microbes of the Eastern Mediterranean Sediment and Seawater. International Symposium of Subsurface Microbiology 2014 (ISSM 14).
431. Woo, H. L., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Investigating Lignin Degradation Potential in the Hypersaline Eastern Mediterranean Deep-Sea Basin. American Society for Microbiology Annual Meeting abstract
     The fate of terrestrial organic carbon (terrOC) in the ocean is unclear as only a fraction of the estimated input is found buried in the sediments, thereby suggesting that the rest is degraded. Since terrOC consists of lignin among other recalcitrant organics, the largely unknown microbial diversity of deep-sea saline environments should be able to degrade lignin even in the presence of ionic liquid, a solvent with salt-like properties used by the lignocellulosic biofuel industry. A better understanding of these microbes can aid the discovery of enzymes that can turn lignin waste into a higher value feedstock as well as inform models of terrOC cycling in the ocean. To obtain a simplified lignolytic microbial community, hypersaline oxic Eastern Mediterranean surface water and sediment were collected from a deep basin near the Nile Delta and incubated aerobically in bottles with insoluble lignin for 2 weeks, during which carbon dioxide was monitored. Ionic liquid, 1-ethyl-3-methylimidazolium acetate, was also added to some treatments as a stressor. The taxonomic diversity was then assessed using 16S rRNA gene amplicon sequencing and the lignolytic enzyme activity was tested using the model lignin L-3,4-dihydroxyphenylalanine. Microcosms with lignin had 10-fold higher respiration rates within 1 day and higher lignolytic activity than the unamended controls. Adding 0.5% ionic liquid did not negatively impact respiration rates. Sequencing revealed that ionic liquid did not alter the community structure either, as Halomonas remained the dominant at 30% relative abundance. Sequencing also revealed that the water and sediment microcosms have different microbial communities with lignin despite having similar respiration rates; Novosphingobium dominated the sediment while Idiomarina dominated the water community. Ionic liquid in combination with lignin caused the Crenarchaeota to increase 10-fold; Archaea have not been well-studied for lignin degradation or ionic liquid tolerance before. Our findings show that the deep-sea has a novel microbial diversity with lignin degradation potential that is unhindered by ionic liquid.
432. Woo, H., S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Investigating lignin degradation potential by microbial communities in the deep-ocean. International Symposium of Microbial Ecology (ISME 15) abstract
     The fate of terrestrial organic carbon (terrOC) in the ocean is unclear as only a fraction of the estimated input is found buried in the sediments, thereby suggesting that the rest is degraded. Since terrOC consists of lignin among other recalcitrant organics, the largely unknown microbial diversity of deep-sea saline environments should be able to degrade lignin even in the presence of ionic liquid, a solvent with salt-like properties used by the lignocellulosic biofuel industry. A better understanding of these microbes can aid the discovery of enzymes that can turn lignin waste into a higher value feedstock as well as inform models of terrOC cycling in the ocean. To obtain a simplified lignolytic microbial community, hypersaline oxic Eastern Mediterranean surface water and sediment were collected from a deep basin near the Nile Delta and incubated aerobically in bottles with insoluble lignin for 2 weeks, during which carbon dioxide was monitored. Ionic liquid, 1-ethyl-3-methylimidazolium acetate, was also added to some treatments as a stressor. The taxonomic diversity was then assessed using 16S rRNA gene amplicon sequencing and the lignolytic enzyme activity was tested using the model lignin L-3,4-dihydroxyphenylalanine. Microcosms with lignin had 10-fold higher respiration rates within 1 day and higher lignolytic activity than the unamended controls. Adding 0.5% ionic liquid did not negatively impact respiration rates. Sequencing revealed that ionic liquid did not alter the community structure either, as Halomonas remained the dominant at 30% relative abundance. Sequencing also revealed that the water and sediment microcosms have different microbial communities with lignin despite having similar respiration rates; Novosphingobium dominated the sediment while Idiomarina dominated the water community. Ionic liquid in combination with lignin caused the Crenarchaeota to increase 10-fold; Archaea have not been well-studied for lignin degradation or ionic liquid tolerance before. Our findings show that the deep-sea has a novel microbial diversity with lignin degradation potential that is unhindered by ionic liquid.
433. Voordeckers, J. M., P. Zhang, Z. Shi, Y. Deng, J. D. Van Nostrand, L. Wu, Z. He, T. C. Hazen, D. A. Elias, M. M. Fields, A. P. Arkin, P. D. Adams and J. Zhou. 2014. Effects of Uranium Contamination on the Metal Homeostasis Genes of Groundwater Microbial Communities. American Society for Microbiology Annual Meeting
434. Trexler, R., C. Solomon, C. Brislawn, E. McClure, A. Grube, T. C. Hazen, M. Keddache, C. Grant and R. Lamendella. 2014. The Response of Freshwater Aquatic Microbial Communities to Marcellus Shale Natural Gas Extraction. American Society for Microbiology Annual Meeting abstract
     At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
435. Techtmann, S. M., K. Ayers, J. L. Fortney, D. C. Joyner, S. M. Pfiffner and T. C. Hazen. 2014. The Eastern Mediterranean Microbial Community is Strongly Stratified by Water Mass. American Society for Microbiology Annual Meeting abstract
     The Mediterranean is a semi-enclosed sea at the intersection of the European, Asian, and African continents. The waters of the Eastern Mediterranean are characterized by high salinity, elevated deep-water temperatures and ultra oligotrophic conditions. Several studies have sought to characterize the microbial community of the Eastern Mediterranean. However, few studies have applied next-generation sequencing to understanding the relationship between community structure and geochemistry of these waters. Here we characterize the geochemistry and microbial community at various depths throughout the water column at five stations in the Eastern Mediterranean. The geochemistry of the water column is stratified by depth, corresponding to three prominent water masses. The carbon and nutrient levels significantly vary between these water masses. The microbial community was characterized using a combination of high-throughput DNA sequencing and PLFA analysis. These analyses demonstrate that the microbial community in these waters is also highly stratified by depth with the largest differences occurring between the surface water and the deeper water masses. However more subtle differences exist between the intermediate water mass and the deep water mass. The diversity of the microbial community is lowest in the surface waters and increases with depth. The dominant bacterial phyla in the surface waters are Cyanobacteria, Proteobacteria (Gamma- and Alpha-) and Bacteriodetes. In the deeper water masses, in addition to Alpha- and Gammaproteobacteria, the Deltaproteobacteria, Chlorflexi, and Planctomycetes are also significant members of population. Archaea make up a much smaller proportion of the microbial community in the surface waters (<1% of recovered reads) whereas they are more dominant in the deeper waters (28 - 50% of recovered reads). Thaumarchaeaota related to Nitrosopumilis dominate the Archaeal community in the deeper water masses. Indicator species analysis revealed the presence of distinct taxa in the different water masses. These indicator taxa may be microbial signatures for these important water masses. The distinct geochemistry of the water masses in the Eastern Mediterranean may act to structure the microbial community allowing for unique populations to thrive in these adjacent water masses.
436. Techtmann, S., K. Ayers, J. L. Fortney, D. C. Joyner, S. Pfiffner and T. C. Hazen. 2014. The structure and function of the eastern Mediterranean microbial community is strongly stratified by water mass. International Symposium of Microbial Ecology (ISME 15)
437. Stelling, S., S. Techtmann and T. C. Hazen. 2014. Comparison of oil degrading Bacteria in the Gulf of Mexico and Eastern Mediterranean Sea. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
438. Song, R., A. Zhou, Z. He, J. D. Wall, A. P. Arkin, T. C. Hazen and J. Zhou. 2014. Evolution and Adaptation of Desulfovibrio vulgaris Hildenborough to Elevated Temperature: Fitness and Trade-Offs. American Society for Microbiology Annual Meeting
439. Rocha, A. M., M. Smith, C. Smillie, J. L. Fortney, S. M. Techtmann, D. C. Joyner, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, J. H. Campbell, S. Pfiffner, K. Ayers, C. Paradis, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, J. Zhou, M. W. W. Adams, A. Lancaster, P. D. Adams, A. P. Arkin, E. J. Alm and T. C. Hazen. 2014. Microbial Community Structure Predicts Groundwater Geochemistry. Genomics Sciences Program Contractor-Grantee Meeting abstract
     Project goals: ENIGMA’s Field Microbiology component works with microbial communities in the environment to investigate how biological structure and function relate to critical biochemical conditions/changes, such as the immobilization of toxic metals and the flow of carbon and nitrogen. The goal is to identify key microbial populations and determine the community events and mechanisms of these populations that impact and control environmental activities of interest, ultimately predicting how perturbations of the environment may affect community structure. From these results, we can develop models that can be applied to microbial populations overlaid with geochemical parameters and engineering controls. Development of such a model and key findings from a 100-well survey at the Department of Energy’s Oak Ridge Field site is described below. Specifically, we demonstrate the ability to utilize microbial community assembly from independent groundwater environments to accurately predict the geochemistry and elucidate key systems biology features. One of the primary objectives of the ENIGMA Environmental Core Field Microbiology Component is to design an efficient field sampling study that will maximize the geochemical diversity of the study site and provide for enhanced resolution of microbial communities and geochemical associations. At the Department of Energy’s Oak Ridge field research site, 243-‐acres of contaminated area is located within the Y-‐12 plant area of responsibility of the Oak Ridge Reservation (ORR). Here, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 100 groundwater wells in order to (1) characterize key microbial populations at geochemically distinct locations and (2) identify associations between environmental gradients and microbial communities. We hypothesize that differences in geochemistry underlie differences in microbial community structure across the groundwater wells. In order to identify microbe-environment associations, a statistically informed experimental design was developed by ENIGMA’s computational core. To optimize geochemical diversity and identify wells where environmental factors are uncorrelated, k-medians clustering algorithm was used to group 818 wells into 100 clusters using 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering 4-L of water through a 10.0μm pre-filter and 0.2μm-membrane filter and then extracted using a Modified Miller method. Community genomic DNA yields from the 0.2μm and the 10.0μm filters range from 0.096- 8.5μg and 0.096-22μg, respectively. Cell counts for the field samples ranged from 103-106 cells/mL and were consistent with biomass estimates from phospholipid fatty acid analysis (PLFA). Initial analysis of PLFA data suggests stress indicators for Gram Negative populations, such as those associated with pollutants and nutrient limitation, are present in 15% of the wells sampled. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. However, initial analysis indicates there are some organisms unique to the 10.0μm size fraction. A total of 27 wells, which can be classified into 7 groups based on pH and contaminant concentrations, were analyzed with the newest GeoChip 5.0_180K to evaluate functional differences and potentials of microbial communities under different environmental conditions. Our results indicated that the groundwater microbial community functional structure was significantly different among seven different groups of wells, and such community differences were largely correlated with the differences in pH, U(VI), nitrate, dissolved organic carbon (DOC) and sulfate in the groundwater. The microbial functional diversity significantly decreased with Ur concentrations. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. Using the synthetic learning in microbial ecology (SLiME) algorithm and the large independent dataset, we are able to predict the geochemistry from the 16S rRNA. Additionally, using the relationship between the geochemistry and microbiology, the critical OTUs that geochemistry can be identified. Overall, results from this study indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the geochemistry. Such predictions may provide the ability of microbial monitoring for natural attenuation at legacy sites and be enabling for ENIGMA for more specialized questions on microbial community and network structure and function. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02- 05CH11231.
440. Rocha, A. M., C. Smillie, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, C. Paradis, K. Bailey, D. Joyner, J. L. Fortney, S. Pfiffner, J. J. Zhou, J. D. Van Nostrand, L. Wu, P. Zhang, D. Curtis, D. Xu, D. Elias, M. W. Adams, A. Lancaster, R. Chakraborty, A. P. Arkin, E. J. Alm and T. C. Hazen. 2014. Temporal Variation in Groundwater Geochemistry Has a Dominant Effect on Microbial Community Structure at the Oak Ridge Field Research Site. ENIGMA SFA Annual Retreat abstract
     Across the DOE Oak Ridge Reservation (ORR), there have been a number of studies aimed at characterizing groundwater microbial community assemblages, activity, and functional diversity at along various geochemical gradients. Results of these studies have led towards further understanding of geochemical-microbial associations, as well as, elucidating microbial response to environmental stress factors. Often these studies focus on the spatial distribution of microbial communities rather than the temporal variability of both microbial assemblages and groundwater chemistry. If variability in groundwater chemistry plays a dominant effect on the assembly and activity of groundwater microbial communities, then key geochemical-microbial associations may be missed if the temporal dynamics of a system is not considered. In this study, our overall goal is to determine to what extent, temporal variation of the groundwater geochemistry effects microbial community structure, function, and genetic diversity in groundwater wells along different depths and geochemical transects. Here, we present the findings from our initial time-series study. To determine how resilient microbial communities are to daily and weekly changes in groundwater chemistry, we sampled 2 shallow and 2 deep wells at the ORR background site three times per week for 3 weeks. For each well and time-point, groundwater samples were collected for geochemical and microbial communities analyses. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Cell abundance in the deep wells was an order of magnitude higher than in the shallow wells. Initial PLFA community structure indicates a predominately dominant Gram-negative community. Conductivity and pH field measurements within the deep wells remained stable over time. However, a decrease in pH was indicated in the shallow wells after a rain event. Similarly, dissolved CO2 estimates were higher in the shallow wells compared to the deep wells. Overall, results from this study suggest evidence of geochemical and microbial response within shallow wells response to rain events.
441. Rocha, A. M., C. Smillie, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, C. Paradis, K. Bailey, D. Joyner, J. L. Fortney, S. Pfiffner, J. J. Zhou, J. D. Van Nostrand, L. Wu, P. Zhang, D. Curtis, D. Xu, D. Elias, M. W. Adams, A. Lancaster, R. Chakraborty, A. P. Arkin, E. J. Alm and T. C. Hazen. 2014. Temporal Variation in Groundwater Geochemistry Has a Dominant Effect on Microbial Community Structure at the Oak Ridge Field Research Site. American Society for Microbiology Annual Meeting abstract
     Across the DOE Oak Ridge Reservation (ORR), there have been a number of studies aimed at characterizing groundwater microbial community assemblages, activity, and functional diversity at along various geochemical gradients. Results of these studies have led towards further understanding of geochemical-microbial associations, as well as, elucidating microbial response to environmental stress factors. Often these studies focus on the spatial distribution of microbial communities rather than the temporal variability of both microbial assemblages and groundwater chemistry. If variability in groundwater chemistry plays a dominant effect on the assembly and activity of groundwater microbial communities, then key geochemical-microbial associations may be missed if the temporal dynamics of a system is not considered. In this study, our overall goal is to determine to what extent, temporal variation of the groundwater geochemistry effects microbial community structure, function, and genetic diversity in groundwater wells along different depths and geochemical transects. Here, we present the findings from our initial time-series study. To determine how resilient microbial communities are to daily and weekly changes in groundwater chemistry, we sampled 2 shallow and 2 deep wells at the ORR background site three times per week for 3 weeks. For each well and time-point, groundwater samples were collected for geochemical and microbial communities analyses. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Cell abundance in the deep wells was an order of magnitude higher than in the shallow wells. Initial PLFA community structure indicates a predominately dominant Gram-negative community. Conductivity and pH field measurements within the deep wells remained stable over time. However, a decrease in pH was indicated in the shallow wells after a rain event. Similarly, dissolved CO2 estimates were higher in the shallow wells compared to the deep wells. Overall, results from this study suggest evidence of geochemical and microbial response within shallow wells response to rain events.
442. Rocha, A., S. Pfiffner, K. Ayers, J. L. Fortney, S. Techtmann, D. C. Joyner, J. Van Nostrand, P. Zhang, A. Lancaster, J. Zhou, D. Watson, M. Adams, R. Chakraborty, A. Arkin, E. Alm and T. C. Hazen. 2014. Phospholipid fatty acid analysis for characterization of a microbial community structure across a geochemically diverse watershed. International Symposium of Microbial Ecology (ISME 15)
443. Rajan, S. S., N. Flournoy, M. J. Beazley, R. J. Martinez, T. C. Hazen and P. A. Sobecky. 2014. Application on Marine Bacterial Populations. American Society for Microbiology Annual Meeting
444. Pettenato, A., M. Schicklberger, J. Ray, A. M. Deutschbauer, T. C. Hazen, A. P. Arkin and R. Chakraborty. 2014. Nitrate-Contaminated Groundwater at Oakridge FRC. American Society for Microbiology Annual Meeting abstract
     The groundwater at U.S. Department of Energy’s Field Research Center (FRC) in Oak Ridge contains high plumes of uranium, technetium, nitrate, volatile organic compounds and has a pH gradient from 3-10. Nitrate concentrations in the groundwater ranges from 0 to 14,000mg/L, cell counts varied from 103 cells/ml to 2 X 106 cells/ml. We investigated the diversity and metabolism of nitrate-reducing bacteria isolated from several groundwater wells across these geochemical gradients. A wide range of media was used for the isolations and lactate, acetate, glycerol, simple sugars or simple fatty acids were used as carbon and electron donors. More than 200 diverse clonal isolates were obtained and identified by 16S-rDNA sequencing with representatives from Pseudomonas, Castellaniella, Rhodanobacter, Intrasporangium, Aquaspirillum, Variovorax, Duganella, Delftia, Chromobacter, Cupriavidus genera among others. Isolated strains utilized different pathways for nitrate reduction (nitrogen or ammonia as end products) as demonstrated using physiological and molecular methods, and some of the strains such as Intrasporangium strain GW247B1, grew robustly in very high concentrations of nitrate (300mM). Several nitrate-reducers exhibited chemolithotrophic metabolisms and oxidized Iron and dissolved humics. More than a dozen different denitrifying Pseudomonas species were obtained. These Pseudomonas strains significantly differed in their ability to detoxify nitrate. Preliminary comparative analysis of their genomes revealed that each strain contained circa 1000 genes distinct from each other, while sharing approximately 3000 ‘core’ genes. We present results from our ongoing detailed genotypic and phenotypic characterization of several of such nitrate-reducing strains from FRC.
445. Paradis, C. J., S. Jagadamma, J. L. Fortney, T. Mehlhorn, D. B. Watson, L. D. McKay and T. C. Hazen. 2014. Hydrogeologic Characterization of a Groundwater System for Investigating Ethanol Biodegradation Rates as a Function Exposure History. ENIGMA SFA Annual Retreat abstract
     Project Goals: The goals of this project are to (1) characterize electron donor biodegradation rates as a function of groundwater system exposure history at the field scale, (2) elucidate the responsible mechanism(s) at the laboratory scale and (3) numerically model both data sets in a groundwater system using a relatively rapid and simple microbial-mediated redox reaction. We hypothesize that the degradation rate of a pulse injection of ethanol is significantly greater when the reaction zone has been previously exposed to ethanol, even after the physical and chemical conditions of the groundwater reaction zone have returned to their initial conditions between exposures. We further hypothesize that the mechanism responsible for the memory effect is either that (1) microbes capable of degrading ethanol are enriched following pulse exposures and remain enriched (although possibly dormant) or (2) pathways for ethanol metabolism are upregulated more rapidly following repeated pulse exposures of ethanol. Abstract: Experimental field studies are critical to elucidating in situ physical, chemical and biological processes. Rigorous characterization of such processes under ambient conditions and prior to controlled perturbation(s) is of utmost importance. However, characterization of environmental systems can be incredibly challenging, time consuming and costly due to considerable temporal variability and spatially heterogeneity. The objective of this study was to rapidly and rigorously characterize the fundamental physical and chemical conditions of a groundwater system using manual and real-time digital in situ monitoring equipment prior to push-pull experimentation. Future push-pull experiments will be focused on characterizing ethanol biodegradation rates and mechanisms as a function of the groundwater system exposure history to the ethanol. The groundwater system is shallow, unconfined and consists of 3 to 4 meters of unconsolidated fill followed by 3 meters of intact saprolite; monitoring wells are screened across the water table and the fill/saprolite contact. The direction, magnitude and temporal variability of the horizontal hydraulic gradient was determined by manual depth to water measurements and geospatial analysis in ArcMap. The magnitude and spatial variability of the hydraulic conductivity was determined by manual flow and drawdown measurements during low-flow steady-state pumping and was visualized in ArcMap. Groundwater quality parameters (temperature, pH, dissolved oxygen, oxidation-reduction potential, and electrical conductivity) were monitored in real-time during low-flow pumping though a standard multimeter with a flow through cell. These results suggested that the horizontal hydraulic gradient (dh/dl) was temporally stable (220±4o at 3.5±0.5%) and may indicate stable groundwater flow direction and magnitude. These results also suggested that wells FW003, FW218, FW219, FW226 and FW230 may not be sufficiently permeable (K≤0.08 m/day) to facilitate push-pull experimentation. Lastly, the groundwater quality results suggested that temperature (24 to 30oC) and pH (cicrumneutral) were relatively stable compared to dissolved oxygen, oxidation-reduction potential and electrical conductivity; variability in these latter parameters may be due to heterogeneity of the groundwater system. This study demonstrated that rapid and rigorous characterization of the fundamental physical and chemical conditions of groundwater systems is feasible using only in situ monitoring equipment combined with manual measurements of groundwater head and flow rates during low-flow pumping. This study also established a robust pre-experimental data set to facilitate optimal push-pull test design, application and data interpretation.
446. Paradis, C. J., S. Jagadamma, J. L. Fortney, T. Mehlhorn, J. C. Parker, D. B. Watson, L. D. McKay and T. C. Hazen. 2014. The Memory Effect: In situ electron donor biodegradation rates as a function of exposure history in a shallow groundwater system. Geological Society of America Annual Metting abstract
     Microbial-mediated redox reactions in groundwater systems can result in significant degradation or immobilization of contaminants that are harmful to the environment and human health. Groundwater conditions can be perturbed to stimulate native microbial communities via the addition of electron donors, electron acceptors, and/or nutrients in order to increase the rates of contaminant transformation. The degradation rate of an electron donor are well known to vary widely between study sites depending on several site-specific factors including the physical characteristics of the solid subsurface media, the chemical species and concentrations of electron acceptors, and the structure and function of the microbial community. Recent scientific studies have suggested that the electron donor degradation rate can vary significantly within a single monitoring well based on the short-term exposure history of the groundwater system. Moreover, groundwater remediation practitioners suggest that the electron donor degradation rate can increase as a function of exposure history even after the physical and chemical conditions of the groundwater system have returned to their initial, pre-electron donor addition state; we refer to this as the “memory effect”. The objective of this study is to adequately characterize and control the physical and chemical groundwater system conditions before, during and after repeated electron donor exposures in order to scientifically describe the memory effect. The experimental field site is located at the Oak Ridge Field Research Center in Oak Ridge, Tennessee where previous scientific studies focused on electron donor additions for reduction and immobilization of radionuclides. The most recent electron donor addition was in 2005 and the site has since been under natural conditions. This study will use the single well push-pull method to repeatedly expose a shallow and unconfined aquifer to ethanol (electron donor) and nitrate (electron acceptor). The biodegradation rate of ethanol under nitrate-reducing conditions will be compared between groundwater wells with and without a short-term exposure history. The memory effect described here may have broader implications on other electron donors that are contaminants such as crude oil and its refined products.
447. Mahmoudi, N., M. S. Robeson, S. M. Techtmann, S. M. Pfiffner, S. C. Stelling, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Diversity and Function of Microbial Communities in the Caspian Sea. American Society for Microbiology Annual Meeting abstract
     The Caspian Sea is the world’s largest enclosed body of water and accounts for 40% of the total lacustrine waters of the world. Approximately 130 rivers drain into the Caspian Sea making riverine inputs the primary source of water and nutrients into this system. As a result, the salinity of the Caspian Sea differs significantly from other bodies of water and is approximately a third the salinity of seawater. Furthermore, excessive inputs of organic matter and nutrients via runoff over the last few decades have led to permanent hypoxic conditions in bottom waters with oxygen concentrations of 5% to 7%. The Caspian Sea has been the subject of extensive long-term studies into the geochemical and hydrological context; however, little work has been done to characterize the microbial communities in this unique marine system using the latest molecular techniques. The low salinity levels combined with anoxic conditions could provide niches for distinct microbial communities in seafloor sediments. We investigated the diversity and function of microbial communities in sediments using a combination of high throughput DNA sequencing, Geochip, and PLFA analysis. Sediment cores were collected at bottom depths of 141m, 205m and 600m from the southern Caspian Sea in July 2013. Gammaproteobacteria and Deltaproteobacteria were found to be dominant bacterial groups across all sediment samples. Within Delaproteobacteria, Desulfobacterales was a commonly observed group indicating that sulfate reduction may be a primary metabolism in these sediments. Crenarchaeota, specifically Marine Benthic Group B and Cenarchaeales, were found to be the dominant Archaeal groups across sediment samples. Cell densities ranged from 9.1x106 to 9.1x107 and were found to decrease with core depth. Concurrently, the metabolic function of these microbial communities is being investigated using Geochip functional microarray analysis. Our study is one of the first to provide a comprehensive characterization of microbial communities in the Caspian Sea using both DNA and lipid-based approaches.
448. Mahmoudi, M., M. Robeson, T. Porter, S. Pfiffner, S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Sedimentary microbiomes in the Caspian Sea. International Symposium of Microbial Ecology (ISME 15)
449. Liu, J., S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen*. 2014. Microbial respiration and community changes to crude oil in deep Eastern Mediterranean and Great Australian Bight. International Symposium of Microbial Ecology (ISME 15)
450. Liu, J., S. Techtmann, J. Fortney, D. Joyner and T. C. Hazen. 2014. Oil-Induced Changes in the Structure and Function of the Eastern Mediterranean Sea Microbial Community. American Society for Microbiology Annual Meeting abstract
     The 2010 oil spill in the Gulf of Mexico brought intense interest in oil contamination and microbial biodegradation in the deep ocean. We have been researching the co-relationship among geochemical characteristics, microbial community structure and oil degradation in deep basins. Eastern Mediterranean sea has some potential for oil production. It is deeper than Gulf of Mexico and considered as one of the most oligotrophic regions on the earth, in which the primary productivity is phosphorus limited. What’s more, the bottom temperature in this region is between 12 to 14℃, which is much warmer than many other deep basins. Very limited research has been done on the microbial community and its changes according to oil in this area. Eastern Mediterranean sea waters from different sites and various depths were collected and studied using a systems biology approach. The indigenous microbial communities can be grouped according to different depths rather than locations and archaea accounted for more than 20% of the bacterial community in deep water samples. On-ship perturbation tests were carried out to examine the changes of microbial community when exposed to crude oil and dispersant. In general, oil played a role that decreased the diversity of the microbial community. The abundance of archaea decreased during the incubation and it disappeared faster when there was more crude oil, but the population of Oceanspirillales increased in the appearance of oil and dispersant. In addition, microrespirometry experiments were used to study the CO2 accumulation from microorganisms exposed to oil, dispersant and different nutrient conditions. The addition of dispersant and phosphate increased CO2 accumulation, which was much higher than the addition of iron. However, compared with Gulf of Mexico, CO2 accumulation was very low. What’s more, many molecular and systems biology tools, like geochip, are applying to target functional genes and microbial activity from communities and isolates. These results will broaden our knowledge on the microbial community in Eastern Mediterranean sea and make us better bioremediation method for oil contamination in this area.
451. Jansson, J. K., J. Kimbrel, N. Ballor, H. Woo, T. Ruegg, T. C. Hazen, M. P. Thelen, B. A. Simmons and S. W. Singer. 2014. Halophilic Communities as a Source for Novel Lignocellulolytic Enzymes. Genomics Sciences Program Contractor-Grantee Meeting abstract
     Project Goals: Characterize halophilic communities from saline environments as sources of novel halophilic microorganisms, genes, and enzymes for biofuel feedstock deconstruction. Selectively enrich microbial populations from complex microbial communities from saline environments on biofuel feedstocks (Miscanthus, Pine and Eucalyptus) to obtain candidates potentially capable of deconstruction of feedstocks under high salinity conditions. Describe the metabolic potential and gene expression patterns in both natural saline communities and feedstock enrichments by sequencing and screening of metagenomes, metatranscriptomes and metaproteomes. Use functional metagenomics to express a library of genes that potentially represent novel mechanisms for deconstruction of biomass that are currently underrepresented in gene catalogues. Formulate (by synthesis, cloning and expression of genes characterized above) and verify activity of a cocktail of halophilic enzymes for deconstruction of biomass in the presence of ionic liquids. Lignocellulose presents a challenge to next generation biorefineries due to its recalcitrance to microbial degradation. Ionic liquid (IL)-based pretreatment has been successful in preparing biomass for enzyme saccharification, but the most common ILs used for pretreatment inhibit many downstream enzymatic and microbial processes mediated by mesophilic enzymes. Halophiles, by definition, are adapted to high-salt environments and are thus a potential source for IL tolerant enzymes. Here we sought to discover & recover novel lignocellulolytic enzymes from environmental and feedstock-enriched halophilic bacterial communities. We collected both liquid and sediment samples from different saline environments in Puerto Rico and San Francisco including salt flats, saltern ponds and turtle grass beds. For each of the environmental samples we obtained 16S rRNA gene sequences, metagenomes and metaproteomes. The data revealed an increase in relative abundance of haloarchaea and genes and proteins implicated in a hypersaline lifestyle with increasing salinity. In addition, a fosmid library was constructed in an expression vector for high throughput functional metagenomics screening using the robotics platform at JBEI. Samples from a turtle grass bed (3.5% salinity) and a high salinity saltern pond (33.2% salinity) were selected for enrichment on the potential biofuels feedstocks: miscanthus (M), eucalyptus (E) or pine (P) under aerobic and anaerobic conditions and followed through three 2-‐week passages. At the end of each passage cells were harvested, specific enzyme activities were measured and DNA, RNA and proteins were extracted. Data collected include enzyme activities for B glucosidase, cellobiohydrolase and xylanase, 16S rRNA gene sequences, metagenomes metatranscriptomes and metaproteomes. We found that enzyme activity was typically highest after the first passage, with the aerobic turtle grass enrichments having consistent activity on each feedstock. After the third passage, metagenomes were constructed and binned using MaxBin, a binning algorithm developed at JBEI. Bins were subsequently searched against the CAZY/dbCAN HMMs. In addition, expressed transcripts from 11 metatranscriptomes were identified by either alignment to the reference metagenomes or de novo assembled. To date we have identified over 1000 expressed candidate carbohydrate active enzymes from the enrichments and obtained reconstructed genomes for >100 feedstock-enriched archaea/bacteria. Heterologous expression of a diverse collection of 29 putative glycoside hydrolases is ongoing. The next step will be to validate and incorporate these candidate enzymes into a halophilic deconstruction enzyme mixture with high activity and IL tolerance. This work conducted by the Joint Bio Energy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DEAC02-05CH11231.
452. Hemme, C. L., Q. Tu, Z. Shi, Y. Qin, W. Gao, Y. Deng, J. D. Van Nostrand, L. Wu, Z. He, S. J. Green, L. Rishishwar, O. Prakash, P. S. G. Chain, S. Tringe, M. W. Fields, R. Chakraborty, A. M. Deutchbauer, I. K. Jordan, J. E. Kostka, E. M. Rubin, J. M. Tiedje, A. P. Arkin, T. C. Hazen and J. Zhou. 2014. Metagenomic Analysis of Pristine and Stressed Groundwater Communities. ENIGMA SFA Annual Retreat abstract
     Metagenomes were sequenced from two sites at the Oak Ridge Integrated Field Research Center (OR-IFRC). The first site, FW301, is from the IFRC background area and represents a pristine groundwater system. The second site, FW106, is from Area 3 at the base of a contaminant plume originating from the S3 waste disposal ponds (see Hemme et al. 2010 ISME J. 4 660-672). The FW106 community experiences constant exposure to high levels of nitric acid, uranium, organic compounds, etc. Previous analyses of the FW106 metagenome show a low-diversity community dominated by denitrifying γ-proteobacteria species. Analysis also suggested the possibility of lateral transfer of genes related to geochemical resistance. To complement these analyses and to address outstanding questions regarding lateral gene transfer and nutrient cycling, the FW106 metagenome was compared to the pristine FW301 metagenome, other environmental metagenomes, and the genomes of Rhodanobacter isolates from IFRC sites.
453. Hemme, C. L., S. J. Green, L. Rishishwar, O. Prakash, R. Chakraborty, A. M. Deutschbauer, J. D. Van Nostrand, L. Wu, Z. He, I. Jordan, T. C. Hazen, A. P. Arkin, J. E. Kostka and J. Zhou. 2014. Lateral Gene Transfer and Gene Duplication Contribute to Overabundance of Geochemical Resistance Genes in Uranium-Contaminated Groundwater Communities. American Society for Microbiology Annual Meeting
454. He, Z., P. Zhang, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, D. Curtis, J. D. Van Nostrand, L. Wu, E. Alm, M. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. Adams and J. Zhou. 2014. Functional genes of groundwater microbial communities predict contamination and ecosystem functioning. ENIGMA SFA Annual Retreat abstract
     Project Goals: This project aims to understand the biodiversity, composition, structure, dynamics, and assembly mechanisms of groundwater microbiomes and their relationships with environmental factors, especially across a large range of concentrations of uranium, nitrate and pH. Advanced metagenomic approaches are used to survey groundwater microbiomes from 100 wells located at Oak Ridge Field research Center. This study is focused on how environmental contamination affects the biodiversity of groundwater microbial communities and their feedbacks to ecosystem functioning using functional gene arrays and other technologies. The results provide insights into our understanding of dimensions of biodiversity of groundwater microbiomes and their relationships with environmental contamination, and the potential to predict contaminants in the environment towards bioremediation of contaminated sites. Abstract Various contaminants in groundwater, such as heavy metals (e.g., uranium), nitrate, and extreme pH, present numerous threats to the environment and human health. Although studies have shown these contaminants affect groundwater microbial communities, how the overall microbial community functional diversity, composition, structure and function vary and change across a large contaminant gradient is not well understood. It would be interesting to be able to predict environmental contamination and ecosystem functioning bases on the microbial community diversity, composition and structure, or vice versa. It is hypothesized that the functional diversity would decrease as environmental contamination (e.g., increased uranium and nitrate) increases, or as pH is too low or too high; however, specific populations capable of utilizing the available contaminants would increase. In this study, groundwater microbial communities were collected from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN) representing a large range of uranium, nitrate and other contaminant concentrations as well as pH and analyzed using a new version of GeoChip 5.0. The results indicated that the functional diversity significantly decreased as uranium and nitrate increased in the groundwater, particularly at high concentrations, or pH was low or high. About 5% of specific key functional populations significantly (p < 0.05) increased as uranium and/or nitrate increased in the groundwater, including dsrA for sulfur (S) reduction, cytochrome and hydrogenase genes for metal reduction, napA and nrfA for dissimilatory nitrogen (N) reduction, nasA and nirA/B for assimilatory N reduction, nirK, nirS and nosZ for denitrification. These genes may indicate the potential of bioremediation of the contaminated groundwater. Additionally, we used a machine learning method, random forests, to predict uranium and nitrate contamination in groundwater using these key microbial functional genes. Our results showed a high specificity and sensitivity and a low error rate when out-of-bagging (OOB) estimation was used, suggesting it is possible to predict environmental contamination and ecosystem functioning using key microbial functional genes. Overall, this study provides new insights into our understanding of the effects of environmental contamination on groundwater microbial communities and their feedbacks to ecosystem functioning.
455. Hazen, T. C., A. M. Rocha, M. Smith, C. Smillie, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, C. Paradis, K. Bailey, D. Joyner, J. L. Fortney, S. Pfiffner, J. J. Zhou, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, D. Curtis, D. Xu, D. Elias, M. Adams, F. Poole, R. Chakraborty, A. P. Arkin and E. Alm. 2014. Microbial Community Structure Predicts Groundwater Geochemistry. International Symposium of Subsurface Microbiology 2014 (ISSM 14). abstract
     At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry.
456. Terry C. Hazen. 2014. "OMICS" the Fantasy is Over: We need multiple lines of evidence. Microbial Insights Webinar
457. Hazen, T. C.. 2014. Harnessing metagenomics in oil-spill cleanup: lessons from the Deepwater Horizon spill. Genome Canada “Genomics: the Power and the Promise” abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
458. Hazen, T. C. . 2014. Deepwater Horizon Oil Spill: How resilient is the Gulf of Mexico?. Scripps Institution of Oceanography abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
459. Hazen, T. C.. 2014. Deepwater Horizon Oil Spill: Deepwater oil-degrading bacterial communities. Seminar Norwegian University of Science and Techonology (NTNU) abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
460. Hazen, T. C.. 2014. Deepwater Horizon Oil Spill: Deepwater oil-degrading bacterial communities. Seminar SINTEFF abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
461. Hazen, T. C.. 2014. How many punches can Mother Nature take in the Gulf of Mexico: Focus on Deepwater Horizon Oil Spill & Science and the Media. Mic/Nite University of Tennessee abstract
     At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
462. Hazen, T. C.. 2014. Omics and Geochemistry: the ENIGMA 100-Well Survey. TES/SBR Joint Investigators Meeting abstract
     At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
463. Hazen, T. C.. 2014. ENIGMA at ORNL. Fourth Biennial Southeastern In Situ Soil and Groundwater Remediation Conference 2014.
464. Hazen, T. C.. 2014. Comparing and Contrasting Petroleum Degrading Microbial Communities in Deep-Sea Environments.. MedRem Conference abstract
     Very large reservoirs of oil and gas have recently been discovered in the Eastern Mediterranean, and other deep-sea environments around the world, making this important developing interest for oil and gas prospecting. Using a systems biology approach we have been comparing and contrasting these sites at the molecular level all the way up to the ecosystem level because “the whole is greater then the sum of it’s parts”. These studies are also bridging our ‘response phase” research on the Deep Water Horizon oil spill in the Gulf of Mexico. These studies will shed light on the microbial community structure at different deep-sea sites and will help to clarify how unique physicochemical parameters could affect the microbial response to an oil spill. Practical information will be gained regarding the conditions needed to elicit robust oil biodegradation. This work will expand our understanding of the native microbial communities and their hydrocarbon-degrading potential and how they function at a systems biology level.
465. Handley, K. M., O. U. Mason, T. C. Hazen, J. Gilbert and J. Jansson. 2014. Genomic Insights into Uncultivated Microbial Communities Associated with Spatially Distinct Oil Polluted Marine Sediments. American Society for Microbiology Annual Meeting
466. Elias, D. A., M. W. W. Adams, R. Chakraborty, M. W. Fields, T. C. Hazen, J. Zhou, T. Northern, N. Baliga, J.-M. Chandonia, A. P. Arkin and P. D. Adams. 2014. Natural and Synthetic Ecology in ENIGMA: Determining the links between Microbial Community Structure and Function. Genomics Sciences Program Contractor-Grantee Meeting abstract
     Project Goals: The overarching goal of the Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) is to understand environmentally-relevant microbial community structure and function through a series of integrated field-to- laboratory campaigns. The Natural and Synthetic Ecology campaign is designed as an interdisciplinary platform to elucidate the fundamental ties between structure and function, as well as determine the environmental influences on these ties. Our initial focus has been the development of in-field bioreactors as a method of capturing temporal fluctuations in the in-situ community due to either natural or induced influences. Going forward we will determine the influence of naturally-occurring carbon sources, invasive species and selected pressures on microbial activities such as nitrate- and metal- reduction. One of the most difficult aspects of studying microbial ecology is determining and understanding the fundamental ties between microbial community structure (the organism biodiversity and their relative abundances that comprise a given microbial community) and the observed functions (the detectable biochemical activities that support survival of the observed species). Although microorganisms are important in controlling the fate of contaminants in the subsurface, information on the basis of how why microbial communities respond to contaminants is lacking. Hence, it is important to characterize microbial communities, establish linkages between biodiversity and function, and study interactions between different species. As a part of the overall ENIGMA goal to link genotypes to phenotypes, the overall objective of this campaign is to obtain deep understanding of the composition, structure, function, activity and interaction of subsurface microbial communities at DOE contaminated sites (i.e. Oak Ridge Integrated Field Research Center). We have developed a bioreactor system for manipulating and temporally monitoring the in-situ microbial community in the field so as to maintain the in-situ community structure. Community structure was measured through sequencing, PCR and qPCR for selected genes, cell counts and total protein before and after the cells entered the bioreactor system. Temporal community function was qualified by alterations in the concentration of 53 metals, 12 organic acids, 14 anions and 4 sugars, pre- and post- bioreactor exposure. Near future experimental plans include determining an adequate naturally-occurring carbon source that will allow for an increase in total biomass with minimal alteration to the relative abundances of the major phylogenetic groups within the community. Finally, in order to establish the environmental relevance of new field isolates from this site, a pilot study using the bioreactor system will be conducted in collaboration with the Metals Metabolism campaign of ENIGMA. The bioreactors will use synthetic groundwater mimicking the geochemistry of the ORNL wells and will be inoculated with groundwater supplemented with various Mo concentrations since a current hypothesis is that a lack of Mo may inhibit nitrate-reduction. The experimental duration is expected to be 30 days with temporal measurements of metals (53 elements), metabolites, 16S rRNA to determine changes in community structure, and by qPCR of key denitrification genes. End-point samples will be used for the isolation and characterization of new denitrifying strains. This work is highly collaborative, involving several ENIGMA campaigns including the 100 Well Survey, Microparticle Mesogenomics, Microbial Isolations and Characterizations and, Metals with down the line benefits to the Predictive Biology and Printable Worlds campaigns. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02- 05CH11231.
467. Driver, D. A., K. O’Dell, C. J. Paradis, N. Mahmoudi, J. L. Fortney, S. M. Schaeffer and T. C. Hazen. 2014. Soil Microbial Respiration and Biomass as a Function of Soil Moisture Content in a Lexington Silt Loam from West Tennessee. Geological Society of America Annual Metting
468. Deng, Y., P. Zhang, Z. He, Z. Shi, Y. Qin, J. D. Van Nostrand, L. Wu, M. Fields, C. W. Schadt, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin and J. Zhou. 2014. Network dynamics of groundwater microbial community succession during uranium bioremediation and new network analysis approaches. ENIGMA SFA Annual Retreat abstract
     In recent years, the inference of species/population interactions and their relevant network analyses have evoked substantial interests in microbial ecology, but few studies have focused on the dynamic changes of microbial community networks in response to substrate amendments or environmental perturbations. In this study, an improved functional molecular ecological network (fMEN) approach was used to explore the interaction of groundwater microbial community succession during uranium bioremediation with emulsified vegetable oil (EVO) amendment. Samples were collected at 0, 4, 17, 31, 80, 140 and 269 days after a onetime EVO injection and examined with a functional gene array (GeoChip 4.0). Our results showed the species/population interaction was dynamically altered after EVO amendment. Competition among microbial species was notable in groundwater microbial communities after EVO injection, especially through EVO-stimulated microorganisms (e.g., sulfate-reducing bacteria) that restrained the growth of other species, resulting in the decline of microbial community richness and diversity. Also, microbial interactions were dynamically switched during the microbial community succession but the changes appeared to be time-lagging. In addition, seven keystone microorganisms were identified, which may play important roles in uranium bioremediation. This study provides new insights into our understanding of the dynamics of microbial interactions during the groundwater microbial community succession in response to EVO amendment.
469. Curtis, D., P. Zhang, J. D. Van Nostrand, A. M. Rocha, T. C. Hazen and J. Zhou. 2014. Reduction in U(VI) Concentration Influences the Subsurface Microbial Community during the Titration of Highly Acidic Sediments. American Society for Microbiology Annual Meeting
470. Chen, C., T. C. Hazen and S. Techtmann. 2014. The response of microbial community structure to clay flocculation of harmful algae blooms. International Symposium of Microbial Ecology (ISME 15)
471. Bailey, K. L., B. R. Crable, R. A. Hurt, II M. S. Robeson, S. Techtmann, D. A. Stahl, T. C. Hazen, A. P. Arkin, J. Chandonia, T. Northen, M. W. Fields, E. J. Alm, J. Zhou, M. W. W. Adams and D. A. Elias. 2014. Reproducibility of a Groundwater Microbial Community in Replicate Bioreactors. ENIGMA SFA Annual Retreat abstract
     Project Goals: To develop a well-head bioreactor system for temporally monitoring microbial community structure without impacting the microbial community in-situ. Abstract: One of the most difficult aspects of studying microbial ecology is determining and understanding the fundamental ties between[1] microbial community structure and observed functions. Although microorganisms are important in controlling contaminant fate in the subsurface, knowledge of how/why microbial communities respond to contaminants is lacking[2]. Hence, it is important to characterize microbial communities, establish linkages between biodiversity and function, and study interactions between different species. We have developed a bioreactor system for manipulating and temporally monitoring the microbial community without disturbing the community structure in-situ. Three above ground, in-field reactors were fed groundwater from well FW305 at the Oak Ridge Field Research Center, Oak Ridge, TN for 11 weeks. Each bioreactor contained 8 replicate biofilm coupons filled with sterilized sediment obtained from the FRC. The microbial communities from the planktonic and biofilm portions of the reactors were compared to the groundwater community for structure and function. Community structure was measured through sequencing and total protein before and after the cells entered the bioreactor system. Temporal community function was qualified by alterations in the concentration of 53 metals, 9 organic acids, 7 anions and 3 sugars, pre- and post- bioreactor exposure. The findings of this initial experiment will directly impact the number of biological replicates that will be required going forward with future reactor studies at this site.
472. Bailey, K., R. A. Hurt, T. R. Chowdhury, M. S. Robeson II, S. Techtmann, T. Mehlhorn, A. Zelaya, M. W. Fields, A. P. Arkin, S. D. Brown, M. Podar, D. A. Stahl, T. C. Hazen, J. Zhou, T. J. Phelps, M. W. W. Adams, D. B. Watson and D. A. Elias. 2014. Reproducibility of a Groundwater Microbial Community in Replicate Bioreactors. June, 2014. Goldschmidt, Sacramento CA. Goldschmidt Conference
473. Alshbli, N., S. M. Techtmann, Y. M. Piceno, L. M. Tom, G. L. Andersen and T. C. Hazen. 2014. 16S rRNA Microarray Analysis of Microbial Communities in Hydrocarbon-Containing Deep-Sea Environments. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
     At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
474. 2014. 2013 Research Annual Report. “Mitigating Oil Spills”. 2013 Research Annual Report
475. J. Zhou, Y. Deng, P. Zhang, K. Xue, J. D. Van Nostrand, Y. Yang, Z. He, D. A. Stahl, T. C. Hazen, J. M. Tiedje and A. P. Arkin . 2013. Stochasticity, Succession and Environmental Perturbations in Fluidic Ecosystems. American Society for Microbiology Annual Meeting abstract
     Unraveling the drivers of community structure, succession, and resilience in response to environmental change is a central issue, but poorly understood in ecology. Although the relative importance of ecological processes (deterministic vs stochastic) in shaping community structure is intensively examined, very little is known about the mechanisms controlling ecological succession, a phenomenon that a community undergoes more or less orderly and predictable changes following disturbance. To understand the relative importance of stochastic and deterministic processes in mediating microbial community succession, here, we examined the responses of a groundwater microbial community to a large change in carbon resource, the addition of slow-release emulsified vegetable oil (EVO) for sustained uranium immobilization at a contaminated site. A novel general theoretical framework was developed to conceptualize the relationships between stochasticity, succession and environmental perturbations. Our results revealed that the groundwater microbial communities diverged substantially after EVO amendment, but subsequently converged to new community structures very similar to, but distinct from, the initial states, indicating the resilience and adaptation of the groundwater microbial communities. Consistent with the proposed conceptual framework but contradictory to the conventional wisdom, null model analyses revealed that the community succession responding to EVO amendment was primarily controlled by stochastic rather than deterministic processes. To our knowledge, this is the first study to explicitly demonstrate the resilience of microbial communities at a field scale and the importance of stochastic processes in mediating ecological succession. Elucidating the mechanisms controlling community structure, succession and resilience is fundamental to biodiversity preservation, ecosystem restoration and environmental management.
476. P. Zhang, R. Chakraborty, J. Van Nostrand, Z. He, D. Curtis, Y. Deng, T. C. Hazen, A. Arkin and J. Zhou. 2013. Diversity of Microbial Functional Communities during Long-term Cr(VI) Immobilization Stimulated with a Slow-release Substrate in the Hanford Aquifer. American Society for Microbiology Annual Meeting abstract
     The sustainability of reduction has been one of the greatest interests during design of in-situ subsurface strategies for bioremediation of toxic metals. A one-time amendment of slow-release glycerol polylactate (HRCa) stimulated long-term bioreduction of Cr(VI) at the DOE Hanford site. To understand the microbial functional communities that mediated this dramatic Cr(VI) immobilization, a comprehensive functional gene array was used to analyze the diversity and changes of groundwater microbial communities after HRCa. The results showed that the abundance of a wide diversity of functional genes significantly increased after HRCa. HRCa first stimulated various genes for HRC acetogenesis, bacteriophage replication and lysis, and degradation of microbial biomass. Also, HRCa stimulated most genes involved in reduction pathways of nitrate, Fe(III), Cr(VI), and sulfate. Additionally, many genes for retaining of C, N, S, P nutrients (e.g. N2 and CO2 fixation) in the ecosystem, Cr(VI) resistance, and transformations of organic and metal contaminants were highly enriched. These genes increased immediately after HRCa and were further enriched after one year, resulting in a dramatically altered community functional structure, which exhibited enhanced physiological ecological potential of better utilizing C, N, P, S nutrients, Cr(VI) reduction and resistance, and adaption to the contaminated environment. These community composition and structure changes were closely correlated with levels of electron donors, Cr(VI), Fe(II), sulfate, and nitrate in the groundwater. Cr(VI) reduction appeared mediated enzymatically and indirectly through Fe(II) and H2S production, and Fe(III)-, sulfate-, and nitrate-reducing bacteria could play important roles. This study provides insights into the diversity of microbial functional communities and geochemical factors important to long-term Cr(VI) immobilization in-situ.
477. S. Yilmaz, Y. K. Light, R. J. Meagher, T. C. Hazen, A. P. Arkin and A. K. Singh. 2013. Single-cell Analysis Platforms for Uncultivable Microorganisms. American Society for Microbiology Annual Meeting abstract
     Single cell sequencing is emerging as a powerful tool for the analysis of uncultivated environmental microorganisms. Current culture-independent, population based techniques (i.e., metagenomics) relying on pooled nucleic acids from lysed bacteria can independently measure metabolic activity and the species present, but cannot link the activity deterministically to species. We are developing high-throughput tools for studying microorganisms one cell at a time, allowing us to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities. Our approach includes FISH-based identification of desired species, enrichment by cell sorting, followed by single-cell encapsulation, whole genome amplification and sequencing. We are utilizing this pipeline to analyze water samples from DOE bioremediation sites (e.g., Oak Ridge FRC) to identify keystone organisms and link their functions to species; to analyze the oral microbiome for targeted sequencing of rare species; to analyze human gut communities to estimate the level of horizontal gene transfer within the community; and to analyze an evolving coculture to determine the genetic, metabolic, and structural basis of the evolution of new social networks that are more efficient, stable, and productive than ancestral networks.
478. S. M. Techtmann, J. L. Fortney, D. C. Joyner, A. M. Rocha, T. D. Linley and T. C. Hazen. 2013. Hydrocarbon Degrading Bacteria in the Warm Oligotrophic Deep Eastern Mediterranean. Second International Symposium on Bioremediation and Sustainable Environmental Technologies abstract
     Very large reservoirs of oil and gas have recently been discovered in the Eastern Mediterranean, making this area an important developing region for oil and gas prospecting. Many of the physical properties of the Eastern Mediterranean are unique compared to other deep-sea hydrocarbon basins. The Mediterranean is very deep with an average depth of 2500 m. Additionally, the water temperature in the deepest parts of the Mediterranean is very warm (between 12.5 and 14.5°C). In contrast, the temperature of Atlantic waters at similar latitudes and comparable depths are typically below 4°C. Furthermore, the Eastern Mediterranean has been reported to be extremely oligotrophic with a N:P ratio of 27.4 (typically N:P ratios are <16). All of these factors will affect the microbial community structure and may potentially select for a unique complement of oil-degrading microbes in these deep warm waters. The oil-degrading capacity of the microbial community in the deep Eastern Mediterranean is unknown. If a spill of crude oil were to occur in the Eastern Mediterranean, the proper approach for using bioremediation to assist in spill clean up is unclear due to the unique characteristics of this environment. To that end, the hydrocarbon degrading potential of the microbial community from various sites in the Eastern Mediterranean was examined. Lab microrespirometry experiments were performed to examine the effect of oil, dispersant, and various nutrient amendments on oil biodegradation. The addition of oil and oil plus COREXIT 9500 both stimulated respiration. Microbial cell numbers increased in the oil and oil plus dispersant treatments. The effect of these treatments on the microbial community structure is currently being examined using molecular techniques. A variety of hydrocarbon degrading isolates were also obtained from the Eastern Mediterranean waters sampled in this study. Furthermore, molecular techniques are being employed to probe both the phylogenetic diversity and functional diversity of the native microbial community and the hydrocarbon degraders present. This study will shed light on the natural microbial community structure of the Eastern Mediterranean and will help to clarify how the unique physicochemical parameters of the Eastern Mediterranean could affect the microbial response to an oil spill.
479. S. M. Techtmann, J. L. Fortney, D. C. Joyner, A. M. Rocha, T. D. Linley and T. C. Hazen. 2013. Hydrocarbon Degrading Bacteria in the Warm Oligotrophic Deep Eastern Mediterranean. American Society for Microbiology Annual Meeting abstract
     Very large reservoirs of oil and gas have recently been discovered in the Eastern Mediterranean, making this area an important developing region for oil and gas prospecting. Many of the physical properties of the Eastern Mediterranean are unique compared to other deep-sea hydrocarbon basins. The Mediterranean is very deep with an average depth of 2500 m. Additionally, the water temperature in the deepest parts of the Mediterranean is very warm (between 12.5 and 14.5°C). In contrast, the temperature of Atlantic waters at similar latitudes and comparable depths are typically below 4°C. Furthermore, the Eastern Mediterranean has been reported to be extremely oligotrophic with a N:P ratio of 27.4 (typically N:P ratios are <16). All of these factors will affect the microbial community structure and may potentially select for a unique complement of oil-degrading microbes in these deep warm waters. The oil-degrading capacity of the microbial community in the deep Eastern Mediterranean is unknown. If a spill of crude oil were to occur in the Eastern Mediterranean, the proper approach for using bioremediation to assist in spill clean up is unclear due to the unique characteristics of this environment. To that end, the hydrocarbon degrading potential of the microbial community from various sites in the Eastern Mediterranean was examined. Lab microrespirometry experiments were performed to examine the effect of oil, dispersant, and various nutrient amendments on oil biodegradation. The addition of oil and oil plus COREXIT 9500 both stimulated respiration. Microbial cell numbers increased in the oil and oil plus dispersant treatments. The effect of these treatments on the microbial community structure is currently being examined using molecular techniques. A variety of hydrocarbon degrading isolates were also obtained from the Eastern Mediterranean waters sampled in this study. Furthermore, molecular techniques are being employed to probe both the phylogenetic diversity and functional diversity of the native microbial community and the hydrocarbon degraders present. This study will shed light on the natural microbial community structure of the Eastern Mediterranean and will help to clarify how the unique physicochemical parameters of the Eastern Mediterranean could affect the microbial response to an oil spill.
480. Techtmann, S. and T. C. Hazen. 2013. Can Mother Nature Take a Punch? The Science of the Big Gulf Oil Spill. Harden Valley Academy
481. A. C. Somenahally, J. J. Mosher, Jr. R. A. Hurt, T. J. Phelps, S. D. Brown, M. Podar, A. V. Palumbo, T. C. Hazen, A. P. Arkin and D. A. Elias . 2013. Chromium as a Geochemical Determinant of Microbial Community Structure and Function. American Society for Microbiology Annual Meeting abstract
     Background: As part of a continuing effort to facilitate metal-reduction/remediation with in-situ microbial communities, this study focused on influence of the contaminating metal Chromium on microbial community structure and function. Methods: Metal contaminated groundwater was stimulated with lactate for 15 weeks in duplicate continuous-flow anaerobic reactors with 0, 0.1, or 3 mg/L Cr(VI) simultaneously to give a stable, enriched microbial communities. Temporal analyses included GC, HPLC, 16S pyrosequencing, metal-reduction assays, metal uptake, small metabolites, metagenomics and metatranscriptomics. At the experiment conclusion, the consortia were used to attempt to obtain isolates by traditional serial dilutions or fluorescence-activated cell sorting (FACS). Results: Previous experiments using no Cr(VI) revealed a dominance of Pelosinus spp. within the final community and subsequent isolates were capable of reducing U(VI), CrVI) and Fe(III). Here, temporal metal-reduction showed increased Cr(VI)-reduction rates in chemostats with higher Cr(VI). There were no obvious differences in trends of H2 or CH4 and genomic analyses are ongoing. Conclusions: Cultivation and metabolic characterization of organisms from a stable, enriched community allows for a deeper understanding of the community metabolism as a whole. Comprehensive investigations such as these allow the evaluation of remediation strategies and identify which community members are important for bioremediation.
482. A. M. Rocha, M. Smith, C. Smillie, J. L. Fortney, S. M. Techtmann, D. C. Joyner, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. B. Watson, J. H. Campbell, E. Alm, A. P. Arkin and T. C. Hazen. 2013. Global Survey of the Department of Energy’s Oak Ridge Field Research Site. 1st Annual Postdoc Research Symposium, Oak Ridge National Laboratory
483. A. M. Rocha, J. L. Fortney, S. M. Techtmann, D. C. Joyner, T. L. Mehlhorn, J. Earles, K. A. Lowe, D. B. Watson, J. H. Campbell, E. Alm, M. Smith, A. P. Arkin and T. C. Hazen. 2013. Geochemical diversity and microbial-environmental associations of uranium-contaminated groundwater at Oak Ridge field research sites. American Society for Microbiology Annual Meeting abstract
     The physical and geochemical nature of the groundwater environment is important towards understanding the taxonomic, genetic and functional diversity of the microbial communities. Within various groundwater environments, key geochemical transects may provide constraints on microbial activities and community composition. At the Department of Energy’s Oak Ridge field research site, 243-acres of contaminated area is located within the Y-12 plant area of responsibility of the Oak Ridge Reservation. Here, over 800 groundwater wells each containing different geochemical properties are present. The goal of this study is to identify key geochemical transects where microbial communities and activities can be assessed. To maximize the geochemical diversity and to enhance the resolution of microbial-geochemical associations, 100 contaminated groundwater wells containing key geochemical features from were surveyed. Wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. Within each cluster, sites for sampling were chosen randomly or by accessibility to the well. For each well in situ groundwater parameters, including temperature, pH, dissolved oxygen, conductivity, and oxidation-reduction potential were measured. Additionally, unfiltered and filtered groundwater samples were collected for geochemical and microbial analysis. Initial analysis of groundwater samples shows a pH and nitrate range of 2.9-10 and 0.09-14,000ppm, respectively, with the lowest nitrate concentrations corresponding to higher pH values (>7). Comparison of background sites show that pH values were similar, ranging 6.5-7.16. However, there are notable differences in nitrate concentrations, with FW301 being ten-fold higher (36.3 ppm) than FW300 (3.65 ppm) and FW303 (3.96 ppm). Analysis of nucleic acids collected from 4L of filtered groundwater, indicated low DNA yields (237-423 ng gDNA) at the background sites. Further analysis of the geochemical and microbial communities across the contaminated wells will provide a better understanding of the geochemical transects present across the field site. These transects will allow for elucidation of relationships between the environment and the genetic and functional diversity of the community.
484. D. B. Bowen De Leon, B. D. Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen and M. W. Fieldss. 2013. Injection of Nitrate as a Competing Electron Acceptor during Stimulation for Cr(VI) Reduction Alters the Microbial Population in Groundwater and Surrogate Sediment. American Society for Microbiology Annual Meeting abstract
     The Hanford 100-H site is a chromium-contaminated site in which stimulation for Cr(VI) reduction using a polylactate compound resulted in depletion of terminal electron acceptors such oxygen, nitrate, and sulfate, and a significant decrease in soluble Cr(VI). Nitrate, common in DOE metal and radionuclide waste sites, results in denitrification, changes in redox conditions, and inhibition of sulfate-reducing organisms, targeted during stimulation for Cr(VI) reduction. The purpose of this study was to study the microbial population dynamics during a simulated episodic nitrate event to better understand the resilience and sustainability of Cr(VI) reduction. Five days following a lactate injection to stimulate Cr(VI) reduction, 55 gallons of potassium nitrate (5,000 ppm) was injected. The lactate injection resulted in reduction of Cr(VI) to below detection; however, following the nitrate injection, Cr(VI) rebounded to concentrations higher than before lactate injection. Nitrate was consumed or dispersed within 13 days. The planktonic and biofilm microbial populations were monitored by groundwater filtration and surrogate sediment sampling in wells upstream and downstream of the lactate and nitrate injections. Pyrosequencing results demonstrated a drastic shift in the injection well of a shift from metal-reducing organism to denitrifiers in both the planktonic and biofilm communities (namely Thauera and Thiobacillus, respectively). This shift was not observed downstream of the injection, indicating that nitrate was consumed before reaching downstream wells. In the injection well, with the exception of the Thiobacillus increasing to ~60% relative abundance, the sediment community was stable compared to the groundwater community where many of the pre-nitrate dominant members became rare or absent post-nitrate injection. SparCC correlation analyses resulted in highly significant (p < 0.001) and strong (cor ≤ -0.8) negative correlations between sulfate- and metal-reducers and denitrifiers in sediment. This was not observed in groundwater. These data indicate that an episodic nitrate event would result in an increase of Cr(VI) in groundwater and an alteration in the microbial population at or near the point-source.
485. Kemsley, J. 2013. After The Deepwater Horizon Disaster. Chemical and Engineering News
486. J. Huang, A. Pettenato, M. Schicklberger, A. M. Deutschbauer, A. M. Rocha, D. B. Watson, T. C. Hazen, A. P. Arkin and R. Chakraborty. 2013. Physiology Of Nitrate-reducing Anaerobes Isolated From Background And Nitrate-contaminated Groundwater At Oakridge FRC. American Society for Microbiology Annual Meeting abstract
     The U.S. Department of Energy’s Field Research Center (FRC) in Oak Ridge includes 243-acres of contaminated area, the groundwater contains high plumes of uranium, technetium, nitrate, volatile organic compounds and has a pH gradient from 3-10. The goal of this project was to investigate the diversity of nitrate-reducers present across these geochemical gradients. Groundwater samples collected from several wells were incubated at 25°C in the dark under anaerobic conditions with nitrate as electron acceptor. Several compounds including lactate, acetate, glycerol, simple sugars or simple fatty acids were used as carbon and electron donors. More than 50 colonies were picked and identified by 16S-rDNA sequencing. Clonal isolates obtained from the background sites mostly belonged to Pseudomonadales (Gamma-Proteobacteria) or Burkholderiales (Beta-Proteobacteria). Bacterial isolates obtained from the high nitrate-contaminated site mostly belonged to Actinomycetales (Actinobacteria), Neisseriales and Burkholderiales (Beta-Proteobacteria). Few Pseudomonas strains from the background site preferred nitrite over nitrate as the electron acceptor. Phenotypic studies with these isolates show a differential response in their growth rates and ability to reduce nitrate. Intrasporangium calvum strain GW247B1 grew robustly at nitrate concentration of 200mM with lactate as sole carbon and electron source, Sinobacter strain FW507F5 could only grow with 10mM nitrate under the same conditions. Chromobacterium strain FW507F1 also showed growth at nitrate concentrations beyond 180mM and was most versatile in its ability to utilize different carbon compounds as electron donors. Whole-genome sequence of several of these isolates is underway which will provide a better understanding of the predominant nitrate removal mechanisms in these diverse environmental strains.
487. Hienz, W.. 2013. Four Faculty Named AAAS Fellows. Tennessee Today
488. C. L. Hemme, Q. Tu, Z. Shi, Y. Qin, J. D. Van Nostrand, L. Wu, Z. He, M. W. Fields, T. C. Hazen, J. M. Tiedje and J. Zhou. 2013. Metagenomic Analysis of Pristine Groundwater Suggests Robust Community Capable of Efficient Geochemical Cycling. American Society for Microbiology Annual Meeting abstract
     To better understand the patterns of nutrient flow in healthy versus stressed groundwater ecosystems, the metagenome of a pristine groundwater community (well FW301) was sequenced and compared to multiple metagenomes including a previously characterized stressed groundwater metagenome FW106 which is chronically exposed to high concentrations of nitric acid and heavy metals. The phylogenetic and metabolic diversity of the pristine groundwater sample was comparable to analyzed temperate soil and surface freshwater metagenomes and was much higher and more diverse than that of the stressed groundwater metagenome. The FW301 community is dominated by proteobacterial species with Pseudomonadales, Burkholdariales and other Betaproteobacterial lineages representing the major phylotypes. FW301 shows robust nitrogen and sulfur cycles complete with ammonia oxidation, nitrification, denitrification, sulfate reduction and sulfur oxidation activities and a capacity for cycling of carbon monoxide and metabolism of aromatic compounds. The FW301 community displays a robust community metabolism that allows for efficient cycling of key elements such as carbon, nitrogen and sulfur. In contrast, the loss of metabolic diversity coupled with inhibitory concentrations of nitrate and sulfate at FW106 has resulted in truncated geochemical cycles in which carbon, nitrogen and sulfur are utilized inefficiently and significant pools of these elements may be lost to the community. The results suggest that the diverse pristine community encodes multiple metabolic and phylogenetic redundancies that allow for rapid adaptation to changing ecological conditions, while the stressed community is highly adapted to the contaminated groundwater environment but likely has a lower capacity to adapt to changing ecological conditions.
489. Hazen, T. C. and R. Prince. 2013. Marine Oil Spills. Second International Symposium on Bioremediation and Sustainable Environmental Technologies
490. Hazen, T. C.. 2013. Systems Biology Approach to an Ecological Disaster Deepwater Horizon Oil Spill. UTK Micro310 abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
491. Hazen, T. C.. 2013. Systems Biology Approach to an Ecological Disaster Deepwater Horizon Oil Spill. Clean Gulf 2013 abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
492. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. CSIRO Seminar abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
493. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. CSIRO Seminar abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
494. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Harbin Institute of Technology Seminar abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
495. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to Oil Contaminated Beaches, Marshes, and Sediment. Institute of Soil Science, Chinese Academy of Sciences, China‐US Ecopartnership “Frontiers in Environmental Research” abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite significant efforts to protect hundreds of miles of beaches, wetlands and estuaries from the Deepwater Horizon oil spill, oil began washing up on the Gulf Coast in early May 2010. We determined the temporal response of the autochthonous microbial communities to the oil on a heavily-impacted beach on Elmer’s Island, Louisiana, Mobile Bay, Alabama, and on Pensacola Beach, Florida. We also studied sediment cores from around the Macondo Well. Analysis of deep 16S rRNA gene pyrotag sequence data revealed that the oil-contaminated samples were dominated by members of the Alpha- and Gammaproteobacteria and that there was a succession in the microbial community over time. Our combined 16S rRNA and metatranscriptome sequence data revealed a rapid response of the natural microbial community to oil contaminants, including prevalence of bacteria with the capacity to degrade oil. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
496. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to Oil Contaminated Beaches, Marshes, and Sediment. China‐US Ecopartnership for Environmental Sustainability abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite significant efforts to protect hundreds of miles of beaches, wetlands and estuaries from the Deepwater Horizon oil spill, oil began washing up on the Gulf Coast in early May 2010. We determined the temporal response of the autochthonous microbial communities to the oil on a heavily-impacted beach on Elmer’s Island, Louisiana, Mobile Bay, Alabama, and on Pensacola Beach, Florida. We also studied sediment cores from around the Macondo Well. Analysis of deep 16S rRNA gene pyrotag sequence data revealed that the oil-contaminated samples were dominated by members of the Alpha- and Gammaproteobacteria and that there was a succession in the microbial community over time. Our combined 16S rRNA and metatranscriptome sequence data revealed a rapid response of the natural microbial community to oil contaminants, including prevalence of bacteria with the capacity to degrade oil. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
497. Hazen, T. C.. 2013. A Systems Biology Approach to Biotransformation of Heavy Metals and Radionuclides in Groundwater. China‐US Ecopartnership for Environmental Sustainability abstract
     Environmental biotechnology encompasses a wide range of characterization, monitoring and control or remediation technologies that are based on biological processes. Recent breakthroughs in our understanding of biogeochemical processes and genomics are leading to exciting new and cost effective ways to monitor and manipulate the environment. Indeed, our ability to sequence an entire microbial genome in just a few hours is leading to similar breakthroughs in characterizing proteomes, metabolomes, phenotypes, and fluxes for organisms, populations, and communities. Understanding and modeling functional microbial community structure and stress responses in subsurface environments has tremendous implications for our fundamental understanding of biogeochemistry and the potential for natural attenuation or bioremediation of contaminated sites. Monitoring techniques that inventory and monitor terminal electron acceptors and electron donors, enzyme probes that measure functional activity in the environment, functional genomic microarrays, phylogenetic microarrays, metabolomics, proteomics, and quantitative PCR are also being rapidly adapted for studies in environmental biotechnology. Integration of all of these new high throughput techniques using the latest advances in bioinformatics and modeling will enable break-through science in environmental biotechnology. A review of these techniques with examples from field studies and lab simulations for biotransformation of heavy metals and radionuclides in groundwater will be discussed.
498. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. China‐US Ecopartnership for Environmental Sustainability abstract
     The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
499. Terry C. Hazen. 2013. Gulf of Mexico Has Surprising “Innate” Ability to Cleanse Oil Spills. CBS Radio
500. Terry C. Hazen. 2013. Big Idea Professor Develops ‘SuperChip’ to Speed Up Lab Results. Tennessee Today
501. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. ACS annual meeting
502. Hazen, T. C.. 2013. A Systems Biology Approach to Understanding Metal/Radionuclide Contaminated Sites. RemTEC annual meeting
503. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Clemson University
504. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Gulf of Mexico Oil Spill and Ecosystem Science Conference http://gulfofmexicoconference.org
505. K. L. Bailey, J. G. Moberly, T. J. Phelps, A. M. Rocha, H. Woo, M. Podar, S. D. Brown, Z. K. Yang, M. M. Drake, T. C. Hazen, A. P. Arkin, A. V. Palumbo and D. A. Elias. . 2013. Transcriptomic and Proteomic Analysis of Geobacter sulfurreducens PCA and Desulfovibrio vulgaris Hildenborough Co-cultures. 1st Annual Postdoc Research Symposium, Oak Ridge National Laboratory
506. K. L. Bailey, J. G. Moberly, T. J. Phelps, M. Podar, S. D. Brown, Z. K. Yang, M. M. Drake, T. C. Hazen, A. P. Arkin, A. V. Palumbo and D. A. Elias. 2013. Transcriptomic and Proteomic Analysis of Geobacter sulfurreducens PCA and Desulfovibrio vulgaris Hildenborough Co-cultures. American Society for Microbiology Annual Meeting abstract
     Synthetic community systems consisting of model organisms are essential to elucidating the mechanisms that mediate microbial community structure and function. These microbial communities form ecosystem foundations, drive biogeochemical processes, and are relevant for biotechnology and bioremediation. A model, metal-reducing community was constructed to study microbial cell to cell interactions, cell signaling and competition for resources. Co-cultures of Geobacter sulfurreducens PCA and Desulfovibrio vulgaris Hildenborough grown in the presence and absence of sulfate were grown in 5 L chemostats and physiological and metabolic analyses (GC, HPLC, species specific fluorescent antibody staining) were performed as well as specially designed multispecies microarrays to follow gene expression changes in the various cultivation conditions. Additionally, comprehensive AMT tag proteomics was used for a protein level assessment of the model community. Lactate oxidation by D. vulgaris was sufficient to support G. sulfurreducens via the excretion of H2 and acetate. Fumarate was utilized by G. sulfurreducens and reduced to succinate and malate. Both transcriptomic and proteomic data reveal significant differences amongst/between the syntrophic and competitive growth of these bacteria. Steady state community cultivation coupled to state-of-the-science analyses such as described herein will allow for a comprehensive, system biology level assessment of a metal-reducing microbial community and may further our understanding interspecies communication for syntrophy or direct competition.
507. 2013. Top Thirteen “In the News” Stories for 2013. Tennessee Today
508. 2013. ORNL’s Keller, Babu, Hazen elected AAAS fellows. Oak Ridge Today
509. 2013. Radio Interview. Deepwater Horizon: Gulf of Mexico 'deep-cleaned' itself. BBC Radio Live 5
510. 2013. TV Interview. Gulf of Mexico Has Surprising “Innate” Ability to Cleanse Oil Spills. CNN TV
511. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. American Chemical Society
512. 2013. Gulf of Mexico Has Greater-Than-Believed Ability to Self-Cleanse Oil Spills. ScienceDaily
513. 2013. Deepwater Horizon Gulf of Mexico 'deep-cleaned' itself. BBC News
514. 2013. Gulf of Mexico cleans itself naturally from Deepwater Horizon blowout. Ottawa Citizen
515. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. Phys.Org.
516. 2013. UT Professor's Research Shows Gulf of Mexico Resilient After Spill. Tennessee Today
517. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. Science Codex
518. 2013. Study Oil-Eating Bacteria Mitigated Deepwater Horizon Oil Spill. US News and World Report
519. 2013. An explosion in oil-munching bacteria made fast work of BP oil spill, scientist says. The Times Picayune
520. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. Bartle Doo
521. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. e! Science News
522. 2013. Study Oil-Eating Bacteria Mitigated Deepwater Horizon Oil Spill. BrothersJudd
523. 2013. Gulf of Mexico Has Surprising “Innate” Ability to Cleanse Oil Spills. Softpedia
524. 2013. Environment Is the Gulf of Mexico resilient to oil spills? . Summit County Citizens Voice
525. 2013. Deepwater Horizon Gulf of Mexico 'deep-cleaned' itself. LiverpoolWired
526. 2013. Gulf Of Mexico Bacteria Found To Break Down And Consume Crude Oil. RedOrbit
527. 2013. Oil From Deepwater Horizon Spill Broken Down By Hungry Ocean Bacteria, Researcher Says . Nature World News
528. 2013. Deepwater Horizon: Gulf of Mexico 'deep-cleaned' itself. EdinburghWired
529. 2013. Oceans clean themselves. Leader-Post
530. 2013. DID THE GULF OF MEXICO SELF-CLEAN AFTER DEEPWATER DISASTER?. FastCompany
531. 2013. Deepwater Horizon Gulf of Mexico 'deep-cleaned' itself . GlasgowWired
532. 2013. Microbes ate Macondo oil in Gulf of Mexico . UPI
533. 2013. An explosion in oil-munching bacteria made fast work of BP oil spill, scientist says. DemocraticUnderground
534. 2013. Oil-Eating Bacteria Fixed The Deepwater Horizon Disaster, But They May Not Help Next Time. FastCoexist
535. 2013. Gulf of Mexico Can Self-Cleanse Oil Spills. Laboratory Equipment
536. 2013. Gulf of Mexico can ‘self-deep-clean’. AfroEdge
537. 2013. Nature’s Key Gulf Cleanup Role After Oil Spill Touted by Researchers. NewsMax
538. 2013. Deepwater Horizon: Gulf of Mexico ‘deep-cleaned” itself. Random News
539. 2013. Research shows Gulf of Mexico resilient after spill. R&D Magazine
540. 2013. Self-Healing. The k2p blog
541. 2013. Gulf of Mexico Has Greater-Than-Believed Ability to Self-Cleanse Oil Spills. VibesForum
542. 2013. UT experts: BP oil spill gone from deep ocean, but remains in marshes . Knoxville Sentinel
543. Zhou, J., Z. He, Y. Deng, A. Zhou, P. Zhang, Q. Tu, J. Van Nostrand, H. Yu, Z. Shi, J. Voordeckers, Y. Lee, R. Song, L. Wu, M. W. Fields, D. A. Stahl, J. D. Wall, T. C. Hazen, A. P. Arkin and P. D. Adams. 2012. ENIGMA Environmental Metagenomics-Enabled Understanding Of Microbial Communities At DOE Contaminated Sites. DOE Genomic Science Meeting abstract
     Project Goals: Although high throughput sequencing and associated (meta)genomic technologies provide an avenue to determine genetic and organismal diversity of an ecosystem, linking the genetic/population diversity to phenotypic diversity across different organizational levels (e.g., molecular, cellular, populations, communities and ecosystems) is extremely challenging. As a part of the overall ENIGMA goal, the ultimate aim of this project is to utilize (meta)genomic technologies to better understand the mechanistic connections between molecular-level interactions/processes and community-level processes/functions. The following three specific objectives have been pursued: (i) To determine adaptation and molecular mechanisms of Desulfovibrio vulgaris Hildenborough (DvH) in response to multiple environmental stresses; (ii) To understand microbial community functional diversity at U/Cr-contaminated sites and develop high-throughput functional gene arrays (FGAs) for microbial community analysis; and (iii) To determine the responses, interactions, mechanisms and dynamics of groundwater/sediment microbial communities to U/Cr contamination and bioremediation treatments. Long-term experimental evolution of DvH. To better understand the mechanistic connections between molecular-level functions and community-level processes, experimental evolution has been carried out to determine molecular mechanisms of DvH in response to high salinity. Significantly increased salt resistance was observed in evolved DvH (eDvH) with increased biomass, higher growth rate and shorter lag phases. Whole genome sequencing of eDvH at 1200 generations revealed specific point mutations and deletions. Their contribution to increased salt resistance has been proven by mutagenesis and phenotype analyses. Glu and Ala significantly increased in eDvH. After 5000 generations, the final biomass and growth rate of eDvH in the medium with high salinity was similar to that in the medium without extra salt. To further investigate the dynamics of evolution, repeatability of the evolution, whole genome sequencing, fitness assay and site-directed mutagenesis are in progress. GeoChip-based metagenomic technology development. We have developed the GeoChip 4.0 series (4.0-4.2) for characterizing microbial communities. The GeoChip 4.0 series are manufactured based on the NimbleGen microarray format. For example, GeoChip 4.0 contains 120,054 distinct probes, and covers 200,393 coding sequences for 539 gene families in different microbial functional processes. The StressChip subset contains 22,855 probes covering 79,628 gene sequences for 46 genes involved in microbial responses to environmental stresses (e.g., temperature, osmolarity, oxidative status, nutrient limitation). The specificity, sensitivity and quantification of the developed GeoChip 4.0 series were evaluated computationally and experimentally. High specificity was observed for both synthesized oligonucleotides and genomic DNA from pure strains; the sensitivity was estimated to be 0.5 µg of DNA; the log(signal intensity) vs. log(DNA concentration) was highly correlated (R = 0.925). All the results showed that the GeoChip 4.0 series are specific, sensitive, and quantitative tools for characterizing microbial communities. GeoChip applications. GeoChips have been used to study groundwater microbial communities to examine sustained reduction of contaminants using slow-degrading/slow-hydrolysis e-donors. At the Oak Ridge site, a one-time injection of emulsified vegetable oil (EVO) was used to examine U(VI) bioreduction and immobilization. Samples collected from the control and treatment wells (W1-7) were analyzed using GeoChip 3.0. Acetate, from EVO biodegradation, stimulated NO3-, Mn(IV), Fe(III), SO42-, and U(VI) bioreduction in W1-7 and increased functional gene diversity. After EVO depletion, functional gene diversity declined. Fe(III)- and sulfate-reducing bacteria could play key roles in U(VI) reduction, whereas acetogens, denitrifiers and methanotrophs could be important for e-donor production and maintaining favorable reducing conditions. At the Hanford site, a one-time injection of poly-lactate was used to test Cr(VI) bioreduction. The groundwater microbial communities were monitored for 390 days using GeoChip 4.0. Cr(VI) was effectively reduced and functional gene diversity increased. Fe(III)- and sulfate-reducing bacteria could play key roles in Cr(VI) reduction, whereas denitrifiers could be important for maintaining reducing conditions. Metagenomic sequencing. We have sequenced or resequenced metagenomes and dominant isolates from Oak Ridge FRC wells FW106 (contaminated with uranium, nitric acid, organics, and mercury) and FW301 (pristine). The FW106 metagenome was previously sequenced, and the results suggest extensive lateral transfer of metal resistance and organic compound metabolism genes. To extend these analyses, the metagenomes of FW106 and FW301, and genomes of multiple isolates of the dominant Rhodanobacter strain found in FW106 were sequenced or resequenced using Illumina sequencing technology. We are currently conducting comparative analyses of FW106 to FW301 to identify ecological trends observed between pristine and highly stressed groundwater communities. Furthermore, we are comparing the FW106 metagenome to Rhodanobacter isolates to confirm predicted lateral transfer events. Molecular ecological network analysis. A novel random matrix theory-based approach has been developed to construct molecular ecological networks (MENs) based on GeoChip hybridization or high-throughput sequence data. Various mathematic and statistical tools and methods have been integrated into a comprehensive MEN analysis pipeline (MENAP). We have applied this approach to construct and analyze MENs from the Oak Ridge EVO experiment described above. Functional MENs were constructed from three GeoChip datasets: (i) Early EVO injection (≤31 days), (ii) Days 80 to 140, and (iii) Control well (7 time points) and pre- injection (0 day). All three constructed functional MENs posed general network characteristics (scale-free, small world and modularity), and the topology of these functional MENs was distinctly different, suggesting that the interactions among different microbial functional groups/populations in each community were dynamically altered during uranium bioremediation. Additionally, the changes in network structure were significantly correlated with environmental geochemical dynamics and EVO concentrations. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
544. Zhou, A., Z. He, E. Baidoo, K. Hillesland, M. P. Joachimiak, J. K. Baumoh, P. Benke, A. Mukhopadhyay, G. M. Zane, P. S. Deha, J. D. Wall, A. P. Arkin, D. Stahl, T. C. Hazen, J. Zhou. 2012. Molecular basis to adaption to salt in Desulfovibrio vulgaris in an evolutionary context. Annual Meeting of the American Society for Microbiology abstract
     High salinity is a key environmental stressor Desulfovibriovulagris often faces in its natural habitats. The molecular basis of its adaptation to elevated salinity in an evolutionary context is unknown. A long-term evolution experiment of 12 replicatepopulations under control or salt stress conditions has been set up. Salt resistance/tolerance was evaluated by growth on media containing higher concentrations of NaCl. The fitness change of evolved D. vulgarispopulations was determined by competition with the ancestor. Molecular basis of adaptation to elevated NaClwas further revealed by whole genome sequencing, global transcriptional profiling and metabolite assays. A rapid phenotypic adaptation to elevated NaCl could be observed as early as 100 generations and then gradual increases of salt resistance/tolerance. SNPs and insertion/deletions were identified in evolved D. vulgarisThe contribution of these mutations to salt resistance/tolerance wasnot only supported by the linkage between phenotype and genotype, but also by phenotype of the site-directed mutants.Parallelism of adaptive evolution was observed at about 5000 generations. Consistent data from transcriptomics and the metabolite assay demonstrated many changes in gene transcription and accumulation of metabolites in evolved clones under non-stress conditions. Increased transcription of genes involved in amino acid synthesis and transport, exclusion of Na+ and energy metabolism was detected in the evolved D. vulgaris. Interestingly, different sets of organic solutes may be responsible for releasing low or high salt stress. In summary, D. vulgaris quickly adapted to salt stress through genetic changes, gene expression changes and accumulation of organic solutes. Glu, Ala and Asp might be major players for coping with NaCl stress; Gln, GB and Gly might play important role at low NaCl stress.
545. Zhang, P., W-M. Wu, J. Van Nostrand, Y. Deng, Z. He, D. Curtis, T. Gihring, G. Zhang, C. Schadt, D. Watson, P. Jardine, C. Criddle, S. Brooks, T. Marsh, J. Tiedje, T. C. Hazen, J. Zhou. 2012. Dynamic Changes of Microbial Communities in Response to Stimulation with Emulsified Vegetable Oil for U(VI) Reduction at a Contaminated Aquifer. Annual Meeting of the American Society for Microbiology abstract
     To examine the microbial functional communities stimulated with a slow-biodegrading electron (e) donor for sustainable U(VI) reduction, a one-time injection of emulsified vegetable oil (EVO) was conducted at a U(VI)-contaminated aquifer. Groundwater samples were collected from an up-gradient control well and seven down-gradient wells (W1-7) to monitor functional gene changes of indigenous microbial communities over a 269-day period using GeoChip-based metagenomics technology. Acetate was detected as one of the intermediates from EVO biodegradation that stimulated bioreduction of NO3-, Mn(IV), Fe(III), SO42-, and U(VI) for at least 140 days in W1-7. During this period, the functional gene diversity increased, with significant enrichments of genes involved in acetogenesis, methanogenesis, methane oxidation, denitrification, dissimilatory nitrate reduction, metal reduction, and sulfate reduction, while the community in the control well did not show a significant change. Fe(III)- and sulfate-reducing bacteria, including Geobacter, Anaeromyxobacter, Desulfovibrio and Desulfotomaculum, could play key roles in the sustainable U(VI) reduction, whereas acetogens, denitrifiers and methanotrophs could be important for the production of e-donors and maintaining reducing conditions favorable for U(VI) reduction. After EVO was depleted, the functional gene diversity declined and became more similar to the pre-injection levels by day 269. Dynamic changes of the functional microbial communities were highly correlated with changes of acetate, NO3-, Mn, Fe, SO42-, and U(VI). This study provides the first in-situ evidence that a one-time injection of a slow-biodegrading e-donor effectively stimulated functional communities and sustainable U(VI) reduction. The dynamic changes of the communities were primarily associated with the availability of the e-donor and e-acceptors in the aquifer.
546. Yilmaz, S., P. Liu, R. J. Meagher, Y. K. Light, A. P. Arkin, T. C. Hazen, A. K. Singh. 2012. Single‐cell Analysis Platforms for Genomic Analysis of Uncultivable Environmental Microbes. Annual Meeting of the American Society for Microbiology abstract
     Current metagenomic techniques (e.g., microarray or 16S rRNA sequencing) relying on pooled nucleic acids from lysed bacteria can independently measure metabolic activity and the species present, but cannot link the activity deterministically to species. We are developing high-throughput tools for studying bacteria one cell at a time, allowing us to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities. Our approach includes FISH-based identification of desired species, enrichment by cell sorting, followed by single-cell encapsulation, whole genome amplification and sequencing. Encapsulation of bacteria in nanoliter plugs in particular allows us to scale down conventional (microliter-volume) assays, such as WGA, into much smaller reaction volumes better suited to the size of an individual microbe. We are using this pipeline to analyze water samples from DOE bioremediation sites (e.g., Hanford) to identify keystone organisms and link their functions to species. Furthermore, we are also using our single-cell genomics pipeline to complement the metagenomic sequencing efforts in ENIGMA. Metagenomic sequencing typically fails to achieve complete assembly and metabolic reconstruction of individual genomes in a complex community. Single-cell sequencing, together with metagenomics, makes it possible to assemble genomes of novel uncultivated organisms.
547. Yilmaz, S., P. Liu, R. J. Meagher, Y. Light, A. P. Arkin2 T. C. Hazen, A. K. Singh, and P. D. Adams. 2012. Single-cell Analysis Platforms for Genomic Analysis of Uncultivable Environmental Microbes. DOE Genomic Science Meeting abstract
     Project Goals: We are developing a pipeline for single cell genomics that utilizes FISH (fluorescence in situ hybridization) for targeting species of interest, FACS (fluorescence activated cell sorting) for high throughput isolation of single cells, and MDA (multiple displacement amplification) for production of sufficient DNA from single cells for genome sequencing. This pipeline is being used for a number of collaborative projects in ENIGMA. Current metagenomic techniques (e.g., microarray or 16S rRNA sequencing) relying on pooled nucleic acids from lysed bacteria can independently measure metabolic activity and the species present, but cannot link the activity deterministically to species. We are developing high-throughput tools for studying bacteria one cell at a time, allowing us to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities. Our approach includes FISH-based identification of desired species, enrichment by cell sorting, followed by single-cell encapsulation, whole genome amplification and sequencing. Encapsulation of bacteria in nanoliter plugs in particular allows us to scale down conventional (microlitervolume) assays, such as WGA, into much smaller reaction volumes better suited to the size of an individual microbe. We are using this pipeline to analyze water samples from DOE bioremediation sites (e.g., Hanford) to identify keystone organisms and link their functions to species. Furthermore, we are also using our single-cell genomics pipeline to complement the metagenomic sequencing efforts in ENIGMA. Metagenomic sequencing typically fails to achieve complete assembly and metabolic reconstruction of individual genomes in a complex community. Single-cell sequencing, together with metagenomics, makes it possible to assemble genomes of novel uncultivated organisms. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
548. Walian, P. J., S. Allen, Max Shatsky, Lucy Zeng, Evelin D. Szakal, Haichuan Liu, Bonita Lam, Jil T. Geller, Kristina L. Hillesland, Steven C. Hall, Susan J. Fisher, Matthew W. Fields, David A. Stahl, T. C. Hazen, Steven E. Brenner, Adam M. Deutschbauer, Trent R. Northen, John-Marc Chandonia, H. Ewa Witkowska, Mark D. Biggin, Bing K. Jap, and Paul D. Adams. 2012. ENIGMA Biotechnology: Membrane Protein Complexes—Their Roles in Desulfovibrio vulgaris Stress Response and in the Establishment and Maintenance of Communities. DOE Genomic Science Meeting abstract
     Project Goals: Key aims of this project are—to develop a system for the high-throughput isolation and identification of membrane protein complexes, optimizing this process for effectiveness across a range of sample types including planktonic cultures and biofilms; to apply this system in the study of DOE relevant microbes such as Desulfovibrio vulgaris in order to detect and characterize changes in their membrane protein complexes brought about by environmental stressors, and through the role of these proteins in the establishment and maintenance of communities. A central goal of the ENIGMA consortium is to develop robust molecular-level models capable of predicting how target microbes respond to a range of environmental conditions. In support of this goal, our interests within ENIGMA have centered on the dynamic role of membrane protein complexes in this process. Cell membranes represent the “front-line” of cellular defense and the interface between a cell and its environment. Significant changes in response to environmental conditions are expected to take place through the proteins situated within these membranes. Membrane protein-associated changes may occur in the form of abundance level, protein-protein interactions, post-translational modifications and even mutations. To understand some of the earliest and perhaps most critical responses to stress, characterization of these changes on a molecular level is needed. The study of membrane proteins presents a major challenge in protein biochemistry; to address this we have developed a unique high-throughput process for the isolation and identification of untagged membrane protein complexes that features mild, but effective, detergent solubilization, liquid chromatography and native electrophoresis methods. We have been applying this system in two main areas of investigation, one of which has been our work on developing a D. vulgaris membrane protein complex database covering standard and stressed growth conditions, and the second to characterize the roles of selected membrane proteins in the establishment and maintenance of communities. Our study of membrane protein complexes in the outermembrane of D. vulgaris grown under standard conditions is complete and we are at an advanced stage with the innermembrane component. An interactome of proteins identified in D. vulgaris outer-membrane preparations is in the final stages of refinement. These preparations have yielded 69 outer-membrane protein identifications (which is over 80% of the number expected); 90% of these proteins were found to be in complexes. The most prevalent categories of proteins detected were the lipoproteins, and proteins with non-specific annotations (hypothetical and conserved hypothetical). This compendium of D. vulgaris outer-membrane protein complexes will serve as an essential reference for the detection and characterization of environment-driven changes in these proteins. Processing of outer-membrane proteins from stressed D. vulgaris cultures (including growth to stationary phase, and growth under elevated levels of nitrate or NaCl) has recently been completed. Initial analysis of stress-associated changes in outer-membrane protein abundance suggests that for many proteins there are significant differences between these changes and the changes in expression level inferred from mRNA experiments. Efforts on the preparation of the inner-membrane protein interactome and completion of the analysis of stress-induced changes occurring in the outer-membrane proteins of D. vulgaris are on-going. In addition to our studies on large-volume planktonic monocultures, we have refined the methods employed in the pipeline so that they may be used to process samples derived from more native-like sources. Recent improvements made in pipeline sensitivity and resolution are now allowing us to work successfully with relatively small cell samples such as biofilms. We anticipate that through additional optimization of the system, we will be able to process yet smaller samples, not only cultured in the lab but obtained directly from field sites. To assess the potential for discovery from such sources, we have begun pilot studies on biofilm samples. Early results contain evidence of protein changes occurring during the transition from stationary phase to biofilms, suggesting that this will be a productive direction for future studies of microbial communities. Recent work by the Stahl group on adaptive evolution experiments with co-cultures of Methanococcus maripaludis and D. vulgaris, has identified mutations of soluble and membrane protein genes likely to be important in establishing the syntrophic mutualism between these species. These results suggest a large influence of acquired membrane protein mutations leading to improved growth rates within this co-culture community. We have begun to process membranes from clonal isolates to characterize changes in the D. vulgaris membrane protein population. Interestingly, the most abundant protein of the D. vulgaris outer-membrane (DVU_0799) is also the most consistently mutated protein in these experiments. Therefore, a key goal of ours will be to purify and functionally characterize those mutated membrane proteins found to play a role in facilitating improved rates of growth. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
549. Walian, P., S. Allen, M. Shatsky, L. Zeng, E. Szakal, H. Liu, B. Lam, J. Geller, K. Hillesland, S. Hall, S. Fisher, M. Fields, D. Stahl, T. C. Hazen, S. Brenner, J‐M. Chandonia, E. Witkowska, M. Biggin, B. Jap. 2012. Membrane Protein Complexes of Desulfovibrio vulgaris‐ Changes in Response to Stress and the Establishment of Communities. Annual Meeting of the American Society for Microbiology abstract
     Background: Cell membranes represent the “front-line” of cellular defense and the interface between a cell and its environment. Significant changes in response to environmental conditions are expected to take place through the proteins situated within these membranes. Membrane protein-associated changes may occur in the form of abundance level, protein-protein interactions, post-translational modifications and even mutations. To characterize these changes we have developed a processing pipeline for membrane proteins and have chosen as our initial subject the Gram-negative sulfate-reducing bacterium, D. vulgaris. Methods: We are utilizing a high-throughput process for the isolation and identification of untagged membrane protein complexes that features mild, but effective, detergent solubilization, liquid chromatography and native electrophoresis methods. Protein identification is accomplished by mass spectrometry analysis of in-gel samples. Bioinformatics methods are used to assess complex stoichiometry and prepare interactome maps. Results: Processing of D. vulgaris membrane proteins from cultures grown under standard and stressed conditions (including growth to stationary phase, and growth under elevated levels of nitrate or NaCl) has been completed. An analysis of stress-associated changes detected in the outer-membrane proteins of D. vulgaris will be presented. In addition to supporting our studies on large-volume planktonic monocultures, we have refined the methods employed in the pipeline so that they would be more effective in processing samples derived from smaller, more native-like, sources. Preliminary results obtained from biofilm and other small-volume samples will be discussed. Conclusions: The catalog of D. vulgaris outer-membrane protein complexes prepared from these studies will serve as an essential reference for the detection and characterization of environment-driven changes in these proteins. Recent improvements in pipeline sensitivity and resolution are helping us to work successfully with relatively small cell samples such as biofilms which in turn will permit characterization of the roles of membrane proteins in the establishment and maintenance of communities.
550. Spier, C. L., W. T. Stringfellow, T. C. Hazen and M. Conrad. 2012. An investigation into the distribution of hydrocarbons in sediments and the subsurface water column after the 2010 explosion of the Macondo 252 Deepwater Oil Rig. 2012 Ocean Sciences Meeting. abstract
     The explosion of the Deepwater Horizon oil platform on April 20, 2010 resulted in the third largest oil spill in history. We investigated the distribution and chemical composition of hydrocarbons surrounding the spill site. A complete set of hydrocarbon data were acquired from the NOAA and BP, including data from 16 research missions. Several hydrocarbon plumes were identified including near-surface plumes (0.5 to 200m), a small mid-depth plume (850-880m), and a large deepwater plume between approximately 1000 and 1400m below surface. The vertical, lateral, and temporal distribution of hydrocarbons within the water column was investigated, and we found significant differences in the chemical composition of the plumes. The distribution of hydrocarbons remaining in sediments between August and October, 2010 was investigated. All sediment samples with total polycyclic aromatic hydrocarbons (PAHs) concentrations exceeding chronic toxicity limits were located less than 3.2km from the wellhead. All sediment samples with concentrations above the mean pre-spill PAH levels (>600µg/kg), based on 2006 and 2009 survey’s by the Minerals Management Service in the Deep Gulf of Mexico, were found within 12km of the wellhead.
551. Spier, C. L., W. T. Stringfellow, T. C. Hazen and M. Conrad. 2012. An investigation of hydrocarbons sampling distribution in subsurface sediment and water samples after the 2010 deepwater horizon oil spill and the relationship between contamination in sediments and the water column. 8th National Monitoring Conference abstract
     The explosion of the Deepwater Horizon oil platform on April 20, 2010 resulted in the third largest oil spill in history. We investigated the distribution and chemical composition of hydrocarbons surrounding the spill site. A complete set of hydrocarbon data were acquired from the NOAA and BP, including data from 16 research missions. Several hydrocarbon plumes were identified including near-surface plumes (0.5 to 200m), a small mid-depth plume (850-880m), and a large deepwater plume between approximately 1000 and 1400m below surface. The vertical, lateral, and temporal distribution of hydrocarbons within the water column was investigated, and we found significant differences in the chemical composition of the plumes. The distribution of hydrocarbons remaining in sediments between August and October, 2010 was investigated. All sediment samples with total polycyclic aromatic hydrocarbons (PAHs) concentrations exceeding chronic toxicity limits were located less than 3.2km from the wellhead. All sediment samples with concentrations above the mean pre-spill PAH levels (>600µg/kg), based on 2006 and 2009 survey’s by the Minerals Management Service in the Deep Gulf of Mexico, were found within 12km of the wellhead.
552. Schmidt, C.. 2012. Exxon Valdez Vs. Deepwater Horizon ES&T’s Top Feature Article 2011. Envrionmental Science & Technology 46:3603-3604. pdf
553. Mircea Podar, Jennifer J. Mosher, Steven D. Brown, Dominique C. Joyner, Roseann Csencsits, Tommy J. Phelps, Kenneth H. Downing, Terry C. Hazen, Adam P. Arkin, Anthony V. Palumbo and Dwayne A. Elias. 2012. A Functional Genomic Characterization of Metal-reducing Pelosinus spp. isolated from Cr(VI) Contaminated Groundwater. Annual Meeting of the American Society for Microbiology abstract
     Background As part of a continuing effort to stimulate metal-reduction/remediation with natural microbial communities at sites contaminated by human activities, this study focused on physiological and functional genomics characterization of metal-reducing bacterial isolates from U(VI) and Cr(VI) contaminated groundwater at Hanford, WA. Four strains of Pelosinus spp. were obtained from chemostatic lactate-enrichments of the indigenous microbial community. Methods Each strain was isolated from a stable, enriched microbial community using fluorescence-activated cell sorting (FACS). Inter-strain characterization included metal-reduction assays (HFO, Fe(III), U(VI) and Cr(VI)), growth on >180 carbon sources via Omnilog, morphological TEM characterization, and metal uptake. Additionally, the genomes of all four strains as well as the type strain Pelosinus fermentans R7 have been sequenced. Results While all strains are capable of Fe(III)-reduction, only some were able to reduce Cr(VI) and one could reduce U(VI). Growth characteristics showed that two strains are highly similar to the type strain while the other two were similar to each other but physiologically different from the type strain and also accumulate polyphosphates. Each species appears morphologically similar overall with multiple flagella and all have identical SSU rRNA genes. At the genomic level, while there are large regions with identical sequence, islands where the sequences diverged and with different gene contents were identified. Conclusion Cultivation and metabolic characterization of isolated organisms from a stable, enriched community allows for a deeper understanding of the community metabolism as a whole. Comprehensive investigations such as these allow the evaluation of remediation strategies and identify which community members are important for bioremediation. The closely related genomic sequences suggest these strains have diverged relatively recently. Identification of genes responsible for the physiological differences should shed light into mechanisms of adaptation to changing environments and niche selectivity. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
554. Piceno, Y., L. Tom, F. Reid, S. Borglin, J. Fortney, D. Joyner, A. Pettenato, T. C. Hazen, C. Spier, W. Stringfellow, J. Wong and G. Andersen. 2012. Microbial community structure differences associated with elevated hydrocarbon concentrations in sediment near and far from the Deepwater Horizon MC-252 wellhead. International Symposium of Microbial Ecology (ISME 14) abstract
     The Deepwater Horizon oil spill in the Gulf of Mexico released large amounts of oil into the water column and may have affected sediments nearby. Sediment cores were collected at distances from less than 1 km to over 250 km from the MC-252 wellhead during September-October 2010. Cores were sectioned at 0-1 cm and 4-5 cm. DNA was extracted, amplified for 16S rRNA genes, and analyzed by PhyloChip. Hydrocarbon concentrations were measured in adjacent cores. Microbial communities in the 0-1 cm horizon in cores with elevated hydrocarbon concentrations were hypothesized to have greater relative abundances of microbes associated with hydrocarbon degradation. Such differences were not predicted for the 4-5 cm horizon communities. For 0-1 cm sediment horizons, results suggested moderate differences in PhyloChip profiles between sample groups. Families with increased relative abundances in the samples with the highest hydrocarbon concentrations were Rhodobacteraceae, Flavobacteraceae, Desulfobulbaceae, Helicobacteraceae, Lachnospiraceae, and Campylobacteraceae. Members of some ofthese families (e.g., Sulfitobacter, Polaribacter, and Arcobacter) have been found in seawater amended with crude oil, while others (e.g., Sulfurovum and members of the Desulfobulbaceae) have been proposed to grow syntrophically under benzene-degrading conditions. The families with higher relative abundances in samples with low or no hydrocarbons were Acidobacteriaceae, Rhodospirillaceae, various Deltaproteobacteria (e.g., Syntrophobacteraceae), Sinobacteraceae, and Chromatiaceae. As hypothesized, there was no evidence of differences in the 4-5 cm horizon. These results suggest the effects of elevated hydrocarbon concentrations on the microbial community were primarily restricted to the upper few cm of the sediment.
555. Jennifer J. Mosher, Tommy J. Phelps, Mircea Podar, Steven D. Brown, Terry C. Hazen, Adam P. Arkin, Anthony V. Palumbo, Boris A. Faybishenko and Dwayne A. Elias. 2012. Determination of the Influence of Chromium on Microbial Community Structure and Function. Annual Meeting of the American Society for Microbiology abstract
     Background: As part of a continuing effort to facilitate metal-reduction/remediation with in-situ microbial communities, this study focused on influence of the contaminating metal Chromium on microbial community structure and function. Methods: Metal contaminated groundwater was stimulated with lactate for 15 weeks in duplicate continuous-flow anaerobic reactors with 0, 0.1, or 3 mg/L Cr(VI) simultaneously to give a stable, enriched microbial communities. Temporal analyses included GC, HPLC, 16S pyrosequencing, metal-reduction assays, metal uptake, small metabolites, metagenomics and metatranscriptomics. At the experiment conclusion, the consortia were used to attempt to obtain isolates by traditional serial dilutions or fluorescence-activated cell sorting (FACS). Results: Previous experiments using no Cr(VI) revealed a dominance of Pelosinus spp. within the final community and subsequent isolates were capable of reducing U(VI), CrVI) and Fe(III). Here, temporal metal-reduction showed increased Cr(VI)-reduction rates in chemostats with higher Cr(VI). There were no obvious differences in trends of H2 or CH4 and genomic analyses are ongoing. Conclusion: Cultivation and metabolic characterization of organisms from a stable, enriched community allows for a deeper understanding of the community metabolism as a whole. Comprehensive investigations such as these allow the evaluation of remediation strategies and identify which community members are important for bioremediation.
556. James G. Moberly, Tommy J. Phelps, Mircea Podar, Steven D. Brown, Zamin K. Yang, Meghan M. Drake, Terry C. Hazen, Adam P. Arkin, Anthony V. Palumbo and Dwayne A. Elias. 2012. Development of a Model Microbial Community for a Systems Biology Level Assessment of Metal-reduction. Annual Meeting of the American Society for Microbiology abstract
     BACKGROUND. One of the largest experimental gaps is between the simplicity of pure cultures and the complexity of open environmental systems, particularly in metal-contaminated areas. These microbial communities form ecosystem foundations, drive biogeochemical processes, and are relevant for biotechnology and bioremediation. METHODS. A model, metal-reducing community was constructed to study microbial cell to cell interactions, cell signaling and competition for resources. The microbial community was comprised of the metal-reducing Desulfovibrio vulgaris Hildenborough and Geobacter sulfurreducens PCA along with Methanococcus maripaludis S2 so as to study complete carbon reduction and maintain a low hydrogen partial pressure. Cultivation used 5L chemostats and analysis included physiological and metabolic analyses (GC, HPLC, species specific fluorescent antibody staining) as well as specially designed multispecies microarrays to follow gene expression changes in the various cultivation conditions. State-of -the-science comprehensive AMT tag proteomics was also used for a protein level assessment of the model community. RESULTS. Preliminary data revealed that lactate oxidation by D. vulgaris was sufficient to support both G. sulfurreducens and M. maripaludis via the excretion of H2 and acetate. Fumarate was utilized by G. sulfurreducens and reduced to succinate and malate. Methane was quantified, suggesting acetate and H2 concentrations were sufficient for M. maripaludis. CONCLUSIONS. Steady state community cultivation coupled to state-of-the-science analyses such as described herein will allow for a comprehensive, system biology level assessment of a metal-reducing microbial community and may further our understanding interspecies communication for syntrophy or direct competition.
557. Meyer, B., J. Kuehl, A. Deutschbauer, M. Price, A. Arkin, T. C. Hazen, D. Stah. 2012. Alternative Electron Transfer Systems in Desulfovibrio‐Methanogen Assemblies: Case Study of Desulfovibrio alaskensis str. G20‐Methanogen Cocultures Grown on Lactate. Annual Meeting of the American Society for Microbiology abstract
     In the absence of inorganic electron acceptors, the complete degradation of organic matter relies on the cooperative activities between phylogenetically and metabolically distinct microbial groups, e.g. fermenting, acetogenic bacteria and hydrogenotrophic, methanogenic archaea that are obligately linked through metabolite exchange in syntrophic association. To extend understanding of the genetic and metabolic basis of this common mutualism, we investigated two-member communities composed of alternative acetogenic Desulfovibrio (Dv.) species and methanogen pairings. These studies demonstrated that (1) different pairings vary significantly in their growth characteristics (e.g. growth rate, biomass yield and population dynamics) reflecting differences in the syntrophy-associated enzyme systems of Dv. spp. and (2) certain Dv. species interact differently with varying methanogenic partners in coculture. To investigate these observations in greater detail, we grew Dv. alaskenis str. G20 in combination with either Methanococcus maripaludis or Methanospirillum hungatei in chemostats under various dilution rates. Whole genome microarray-based transcriptional analysis of Dv. alaskensis str. G20 grown at high dilution rates in coculture with Methanococcus maripaludis showed a significant up-regulation of genes encoding periplasmic formate dehydrogenases, Ni-Fe hydrogenases, and membrane-bound, quinone-interacting complexes Qrc and Qmo relative to monoculture growth. The transcriptional patterns were distinct from previously studied Dv. vulgaris str. Hildenborough indicating that Dv. alaskensis str. G20 uses an alternative electron transfer system for syntrophic growth. In addition, the elevated levels of formate measured in the chemostats pointed to its importance as a mediator of electron exchange in Dv. alaskensis str. G20 syntrophic cocultures in contrast to the solely hydrogen exchange-based system of Dv. vulgaris str. Hildenborough. Further analyses revealed that gene expression of syntrophically grown Dv. alaskensis str. G20 varied with both growth rate and the methanogenic partner. As opposed to the existence of a conserved core of genes common to Dv. spp., the study points to considerable plasticity in the genetic and metabolic basis of the syntrophic lifestyle.
558. Meyer, B., K. L Hillesland, J. Flowers, N. Pinel, N. Elliott, J. D. Wall, M. Joachimiak, A. Zhou, J. Zhou, J. Kuehl, A. Deutschbauer, M. Price, Z. He, A. Mukhopadhyay, E. Baidoo, T. Northern, N. Baliga, M. Biggin, M. Dong, P. Walian, A. Singh, S. Yilmaz, D. Elias, M. Fields, H. Garcia-Martin, T. C. Hazen, A. P. Arkin, and D. A. Stahl. 2012. ENIGMA Laboratory Evolutionary and Ecological Origins of Community Assembly, Stability, and Efficiency. DOE Genomic Science Meeting abstract
     Project Goals: A goal of DOE and ENIGMA is to understand and ultimately predict microbial community assembly and the adaptive response of communities to environmental change. To address these questions, we are examining assemblies of simple two-member communities composed of a secondary fermenter like Desulfovibrio species and hydrogenotrophic methanogens. This type of association (syntrophy) is representative of a trophic interaction sustaining both pollutant transformation and organic matter mineralization in many anoxic environments typical of the subsurface. To study the metabolic versatility and specificity of these assemblies, we first investigated the association of twelve different pairings of Dv. species and two methanogenic species (Methanococcus maripaludis and Methanospirillum hungatei). The results demonstrated that different Desulfovibrio-methanogen pairings vary significantly in their growth characteristics, most notably in their ability to ferment lactate at elevated hydrogen levels, presumably reflecting differences in their syntrophy-associated enzyme systems (e.g., hydrogenases and electron transfer complexes). Those studies now serve to direct a systems-level approach to the study of common and divergent features of community interaction: focusing on the genetic and metabolic signatures of efficient species interaction, major determinants of community stability, and the capacity for these communities to improve through adaptive evolution. Abstract: Comparative studies have so far shown that both the electron transfer system and the mediator for electron transfer differ among Desulfovibrio species. For example, comparison of different assemblies grown in chemostats under various dilution rates demonstrated the importance of formate as major mediator of electron exchange in Dv. alaskensis strain G20 syntrophic cocultures in contrast to the hydrogen exchange-based system of Dv. vulgaris strain Hildenborough. Notably, the transcript analyses revealed that gene expression during syntrophic growth of Dv. alaskensis str. G20 also varies with both growth rate and the methanogenic partner. These conclusions were subsequently confirmed using a tagged-transposon Dv. alaskenis G20 mutant mini-library (1200 strains) to examine the relative fitness of different insertion mutants grown syntrophically in chemostats. Complementary studies are examining the adaptive evolutionary response of the two species to syntrophic growth. Ongoing laboratory evolution experiments of 24 replicated lines have so far documented a remarkable capacity for rapid improvement in the stability and efficiency of this mutualism after only 1000 generations of cooperative growth. The genetic basis of improved cooperation is now being examined by genome resequencing, initially of twelve of the pairings at 1000 generations using both Illumina and SOLiD next generation sequencing platforms and microarrays. Since some lines have evolved to obligate syntrophy, the history of their adaptive evolution will be reconstructed using single cell genome sequencing of earlier generations. The first mutations in Dv. vulgaris to become fixed in multiple lines were in an outer membrane porin (DVU0799), suggesting that amino acid replacements near the outer face of this porin alters the flux of metabolites and/or substrates. Other notable and frequent mutations were in genes implicated in EPS synthesis and regulation of lactate metabolism. Collaborative biophysical studies with ENIGMA members are now exploring altered function of the different porin mutants and mutants implicated in EPS synthesis. Of particular note was the replacement in all evolved lines of six amino acids in the sensory PAS domain of a histidine kinase (DVU3022) implicated in the regulation of lactate metabolism. The high frequency replacement with an identical short amino acid sequence having predicted sensory function suggests a novel phase variation-like mechanism of adaptive response. Together these ongoing studies point to both common and divergent mechanisms of interspecies interaction, and offer a framework to better resolve genetic, structural, and metabolic features contributing to stability and efficiency of community assembly. Funding This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
559. Mason, O. U., Terry C. Hazen, T. Woyke and J. K. Jansson. 2012. OMics analyses of the deep-sea microbial community response to the Deepwater horizon oil spill. ACS Spring Meeting.
560. Lee, S., D. Tarjan, J. T. Geller, M. E. Singer, C. Wu, T. Torok, T. C. Hazen, N. J. Hillson, A. P. Arkin. 2012. Assessing and Mitigating the Biological Risks of Genetically Modified Bacteria in the Environment. Annual Meeting of the American Society for Microbiology abstract
     We are developing a means for understanding and assessing the biological risks and fates of genetically modified bacteria and their genetic elements in the environment. We are using as our model waste effluent such as might be contaminated by the failure of standard safety protocols for industrial bioreactors. This study seeks to estimate the risks associated with this model failure scenario by investigating the feasibility of horizontal gene transfer (HGT) of genetically engineered genes in model sewage reactors that simulate the conditions and microbial communities found in wastewater treatment plants. Here, we present results on the transmissibility and stability of fitness conferring genetic material between engineered strains of E. coli and an activated sludge microbial community sampled from the EBMUD wastewater treatment plant in Oakland, CA. To confer fitness in a controlled environment, we are identifying an exotic carbon source whose catabolism in E. coli is only possible through exogenous genetic material. The stabilities of broad-host range plasmids containing carbon catabolic genes, co-expressed with fluorescent protein markers, are being evaluated in E. coli grown in an artificial sewage minimal medium supplemented with the exotic carbon source. Further, we are characterizing the rate of enrichment of plasmid-bearing strains co-cultured with a wild-type E. coli strain. The transmissibility of these plasmids to the microbial community is being studied using a linked pair of bioreactors that allowed for the exchange of nucleic acids but not cells. One bioreactor cultures the engineered E. coli while the other cultures the activated sludge microbial community. Plasmid prevalence is quantified over time in both chambers using qPCR and FACS. Together this work establishes methodologies for assessing and mitigating the risks of future large-scale microbial metabolic engineering projects, including those extending beyond the bioreactor.
561. Lancaster, W. A., Israel Scott, Brian Vacarro, Angeli Lal Menon, Farris L. Poole, Jil Geller, Jennifer J. Mosher, T. C. Hazen, Dwayne A. Elias, Michael W.W. Adams, Adam P. Arkin and Paul Adams. 2012. ENIGMA Microbial Physiology Assimilatory and Dissimilatory Metallomics of Desulfovibrio vulgaris and Pelosinus Strain A11. DOE Genomic Science Meeting abstract
     Project Goals: The goals of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) project are to understand at a molecular systems biology level the microbial communities at DOE sites contaminated with heavy metals or radionuclides with sufficient detail to predictively model interactions within microbial and community processes that drive complex geochemistry in key environments. We expect to define biological principles governing selection of microbial community function and composition in given environments. While some heavy metals are environmental contaminants, metals in general have a very positive role in biological systems as they afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and greatly impact protein stability. Consequently, metal-containing proteins play key roles in virtually all biological processes. However, the full complement of metalloproteins within a given cell cannot be predicted solely from bioinformatic analyses of a genome sequence since metal coordination sites are diverse and poorly recognized. Hence it is not possible to predict the number and types of metals that a microorganism utilizes and how these might vary with the growth conditions. Determining the metals that are taken up during microbial growth and the metal content of fractionated native biomass can provide insight into these issues and, using coupled MS/MS analyses with extensive fractionations, can reveal completely new aspects of metal metabolism (1). Herein we report comparative metallomic analyses of the model microbe, Desulfovibrio vulgaris strain Hildenborough (DvH), and a newly described microbe, Pelosinus strain A11, which was recently isolated from an enrichment of a groundwater sample from the Hanford site by Elias and coworkers at ORNL. DvH was grown on lactate under sulfate-reducing conditions in a 600-liter metal fermenter and in a glass 5-liter fermenter and the nature of metals assimilated were compared. Pelosinus A11 was also grown at the 600-liter scale on fructose in the absence of sulfate and the assimilated metals were determined using ICP-MS (53 elements). Both organisms assimilated 20 or so metals into their cytoplasmic fractions, but the types and amounts were species specific. For example, DvH assimilated cobalt, cadmium and tungsten into high molecular weight complexes (>3 kDa) but Pelosinus A11 did not. In contrast to DvH, Pelosinus A11 assimilated vanadium, copper and uranium into >3kDa complexes. A number of additional factors affecting metal metabolism were analyzed, including growth with and without added chromium or uranium to study assimilation and dissimilatory reduction of these metals. The results will be presented in terms of the ranges and types of metals assimilated by DvH and Pelosinus A11 and the metals available in the organisms’ natural environments, including Hanford groundwater. Reference 1. Cvetkovic, A., Menon, A. L., Thorgersen, M., Scott, J. W., Poole, F. L., Jenney, F. E., Lancaster, W. A., Praismann, J. L., Shanmukh, S., Vaccaro, B., Trauger, S. A., Kalisiak, E., Apon, J. V., Siuzdak, G., Yannone, S. M., Tainer, J. A. and Adams, M. W. W. (2010) Microbial metalloproteomes are largely uncharacterized. Nature 466, 779-782 This work was conducted as part of the ENIGMA project and was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
562. Lamendella, R., S. E. Borglin, R. Chakraborty, T. C. Hazen, J. K. Jansson. 2012. Microbial Community Dynamics on an oil contaminated beach following the Deepwater Horizon Oil Spill. Society of Industrial Microbiology Annual Meeting abstract
     Despite significant efforts to protect hundreds of miles of beaches, wetlands and estuaries from the Deepwater Horizon oil spill, oil began washing up on the Gulf Coast in early May 2010. In this study, we aimed to determine the temporal response of the autochthonous microbial communities to the oil on a heavily-impacted beach on Elmer’s Island, Louisiana. Analysis of deep 16S rRNA gene pyrotag sequence data revealed that the oil-contaminated samples were dominated by members of the Alpha- and Gammaproteobacteria and that there was a succession in the microbial community over time. We also sequenced RNA extracted from the samples to determine which microbes and functions were active. Our combined 16S rRNA and metatranscriptome sequence data revealed a rapid response of the natural beach microbial community to oil contaminants, including prevalence of bacteria endowed with the functional capacity to degrade oil. By correlation to hydrocarbon data from the same samples, we determined that the oil originating from the Deepwater Horizon oil spill underwent further degradation by indigenous microbial consortia on the beach.
563. Lamendella, R., S. E. Borglin, R. Chakraborty, T. C. Hazen, J. K. Jansson. 2012. Metatranscriptomics of an oil contaminated beach following the Deepwater Horizon Oil Spil. Annual Meeting of the American Society for Microbiology abstract
     The Deepwater Horizon oil spill represents one of the largest environmental catastrophes in the history of the oil industry. There are few molecular surveys describing the response of active indigenous microbial populations in oil-polluted coastal ecosystems. In this study, we aimed to determine the impact of the oil spill on the microbial community dynamics and gene expression over time on a heavily oil-polluted beach in Louisiana. Additionally, we aimed to determine which members of the microbial community and which functional genes were responsible for hydrocarbon degradation. We took samples (n=162) from Elmer’s Island, LA, over three time periods in June 2010, and determined the bacterial community structure by 16S rRNA gene pyrotag sequencing. The pyrotag data was analyzed using the QIIME annotation pipeline. Metadata collected from each sample included hydrocarbon composition, cell counts, sample depth, and location. RNA was extracted and sequenced from one sample from each collection date using the Illumina HiSeq platform. Metatranscriptomic data was assembled using the CLC Genomics Assembly tool, contigs were submitted to MG-RAST for annotation, and statistical analyses were performed in STAMP v2.0. The pyrotag data revealed a higher relative abundance of specific proteobacterial groups and reduced amounts of several other bacterial taxa, including actinobacteria in heavily oiled samples. Community structure was highly correlated to hydrocarbon concentration, extent of oil biodegradation, and bacterial diversity. The metatranscriptomic data revealed that the Rhodobacterales and other alphaproteobacteria were more active at the two earlier sampling points, whereas members of the Firmicutes and Actinobacteria were more active at the later sampling period. Additionally, an increased abundance of transcripts coding for proteins involved in carbon metabolism, respiration, and cell signaling, were identified in the more heavily oiled samples from the first two timepoints. By contrast, expressed genes for nitrogen metabolism were more elevated in the lesser contaminated sample. These findings illustrate successional and functional changes occurred in the beach microbial communities during biodegradation of the oil contaminants following the Deepwater Horizon oil spill.
564. Lamendella, R., S. E. Borglin, R. Chakraborty, T. C. Hazen, and J. K. Jansson. 2012. Microbial Community Dynamics on an oil contaminated beach following the Deepwater Horizon Oil Spill. ACS Spring Meeting.
565. Hemme, C. L., Y. Deng, T. J. Gentry, M. W. Fields, L. Wu, S. Green-Tringe, D. B. Watson, Z. He, P. Chain, T. C. Hazen, J. M. Tiedje, E. M. Rubin, J. Zhou. 2012. Metagenomic Insights into Evolution of a Heavy Metal-Contaminated Groundwater Microbial Community. Annual Meeting of the American Society for Microbiology abstract
     The 30% of global freshwater reserves located in subsurface streams and aquifers represent a critical source of freshwater for human consumption and irrigation. Significant portions of natural groundwater reserves are currently contaminated by natural or anthropogenic means. The introduction of contaminants limits the use of groundwater without extensive processing, contributes to human disease, and has a significant negative effect on local ecosystems. Comprehensive characterization of microbial communities in natural systems remains a challenge due to their extremely high diversity and the as-yet uncultivated status of the vast majority of environmental microorganisms. Metagenomics and associated technologies have revolutionized the study of microbial diversity, adaptation and evolution. Low-complexity microbial communities from extreme environments such as acidic geothermal hot springs and contaminated sites are ideal for high-resolution, in-depth metagenomics studies. In this study, a microbial community from highly uranium-contaminated groundwater was sequenced using a random shotgun sequencing-based strategy and compared to a related pristine groundwater metagenome with the goal of addressing the following questions: (i) How does anthropogenic environmental change such as contamination affect groundwater microbial community diversity and structure? (ii) How do microbial communities adapt to severe environmental changes such as heavy metal contamination? (iii) What ecological trends are expected to manifest in stressed microbial ecosystems? Results indicate an overabundance of contaminant resistance genes (e.g. mercuric resistance, heavy metal ion efflux, etc.) in the stressed metagenome as well as an order of magnitude loss of species, metabolic and allelic diversity. Analysis further shows that carbon monoxide metabolism may be a key metabolic process in pristine groundwater systems that has been almost entirely lost in the stressed system. The cumulative results suggest that expected ecological trends for stressed ecosystems (reduced nutrient turnover, lower diversity, etc.) are consistent for stressed groundwater systems.
566. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. AGU
567. Hazen, T. C.. 2012. A Systems Biology Approach to the Deepwater Horizon Oil Spill, the 2nd largest marine oil spill in the world. South Central China University abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
568. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Key Lab on Pollution Ecology & Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
569. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. The 2012 China-US Joint Symposium “Land Use, Ecosystem Services, and Sustainable Development” abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
570. Hazen, T. C.. 2012. Cover The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Environmental Microbiology 14.
571. Hazen, T. C.. 2012. Science and the Media: perspectives for the Scientist. Wake Forest University, Perspectives in Biology Symposium
572. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Wake Forest University, Perspectives in Biology Symposium
573. Hazen, T. C.. 2012. Dispersants and Oil, What We Learned from the Deepwater Horizon Disaster. University of Tennessee, Earth and Planetary Sciences Seminar
574. Hazen, T. C.. 2012. Deepwater Horizon Oil Spill Ecological Disaster, Engineering Disaster, Science of Opportunity!!!. University of Tennessee, College of Engineering, Board of Advisors
575. Hazen, T. C.. 2012. Dispersants and Oil, What We Learned from the Deepwater Horizon Disaster.. Rice University abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
576. Terry C. Hazen. 2012. Can Mother Nature Take a Punch The Deepwater Horizon Incident. Technical Society of Knoxville
577. Terry C. Hazen. 2012. A Systems Biology Approach to the Deepwater Horizon Oil Spill. University of Aberdeen, OceanLab
578. Hazen, T. C. 2012. Fate and distribution of Deepwater Horizon oil. University of Wisconsin at Milwaukee
579. Terry C. Hazen. 2012. Metagenomics and the Environment. Society of Industrial Microbiology Annual Meeting
580. Hazen, T. C. 2012. Ecogenomics of the Deepwater Horizon Spill. Univiversity of California, Davis abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
581. Hazen, T. C. 2012. Ecogenomics of the Deepwater Horizon Spill. Univiversity of Tennessee, Genomics Sciences Program abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
582. Hazen, T. C. 2012. Fate and distribution of Deepwater Horizon oil. Gordon Research Conference on Oceans and Human Health
583. Hazen, T. C. 2012. Deepwater Horizon Oil Spill Ecogenomics. ACS Spring Meeting.
584. Hazen, Terry C. 2012. Can Mother Nature Take a Punch: the Gulf Oil Spill. University of Tennessee, Science Forum. abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill. This team-science approach suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
585. Hazen, T. C., M. W. Fields, J. Zhou, J. Van Nostrand, D. A. Elias, J. J. Mosher, S. D. Brown, T. J. Phelps, M. Podar, A. V. Palumbo, R. A. Hurt, A. Singh, E. Alm, M. B. Smith, D. C. Joyner, R. Chakraborty, B. Faybishenko, K. DeLeon, J. Geller, B. Lam, T. Torok, J. Fortney, S. E. Borglin, D. Stahl, J. Wall, A. P. Arkin, and P. D. Adams. 2012. ENIGMA Environmental Overview: Field to Lab to Field. DOE Genomic Science Meeting abstract
     Project Goals: ENIGMA working hypotheses: 1) Key transects in the environment provide constraints on community composition and activity that are discernable at multiple scales. The uranium/nitrate/pH gradient may provide one such transect and we will examine the communities and activities in different in situ (and laboratory simulated) uranium/chromium contaminated environments. 2) These environmental constraints change in time due in part to the structure and function of these communities and causal relationships may be discovered and quantified. 3) Community structure is established and maintained by varying factors that include transient populations, niche diversification, optimized interactions, and resource competition. We will be attempting to dissect these by field perturbation and time series studies and laboratory simulations of the environment. 4) There are important stable communities that achieve the above, which we can dissect at the level of molecular interactions, and we will drive toward isolation of key organisms, discovery of key activities and interactions, and dissect cellular networks that controls when and how these activities are expressed. The implications of the above hypotheses suggest: 1) There is a core set of metabolic factors that are the direct effectors of metal-reduction in soil and while exact species may vary, the molecular network will be relatively constant. This effort includes the identification of the biomolecular determinants of metal reduction in key populations that directly and/or indirectly interact with other populations that have programmed responses to important environmental parameters. 2) There are particular variants of these that adapt them for different metals and different concentrations and these are deployed differently in planktonic and attached communities over time during the reductive and reoxidative processes following stimulation. 3) At a particular site there are core, relatively stable sub-communities of microbes whose interspecific interactions are responsible for the stable reduction (and ultimate reoxidation) of metals. There is also a core community structure of necessary functional classes of microbes that form a stable “food web” to near optimally exploit the available energy in the environment. For example at the Hanford 100-H study site, soil samples differed from the corresponding groundwater (even at the phyla level) and were more diverse (p=0.001). While many of the populations were observed in both groundwater and surrogate sediments, the respective matrices appeared to enrich for particular OTUs. Results do not indicate a large shift in dominant organisms in soil from pre- to post- injection, and this may be due to the organisms remaining dominant from the first stimulation. However, a prevalence of core genera and rare genera were observed across 34 samples while urban and rural genera were less abundant. Background and Significance. ENIGMA is planning on shifting the principle field focus from Hanford to Oak Ridge. For the last 12 years ORNL has been characterizing, monitoring, and conducting field experiments at DOE’s BER ORNL Field Research site. The focus has been on elucidating the mechanisms and efficacy of bioreduction and bioimmobilization of U, one of the DOE’s most common waste site contaminant. This has involved a number of field studies including pump tests, hydrological modeling, characterization of sediment and groundwater and amendments of ethanol, bromide, and nitrate, etc. More than 300 wells have been established and characterized and are available for analysis in a searchable database Research and Design • Overarching Driver: microbial community structure and function in both in situ environments and constructed consortia - environment to the laboratory. • Elucidate structure to function during key biogeochemical transformations - immobilization of metals • Determine key succession events and mechanisms - stability in the context of geochemical and thermodynamic constraints • Parameterization at phylogenetic and functional level in conjunction with key biogeochemical variables that together, impact and control activities of interest (e.g., metal-reduction; N flow; C flow) • Identify key populations, directly and/or indirectly related to activities of interest • in situ and laboratory consortia will be used to explicate levels of biological organization from populations to proteins • models will be developed using various bioinformatic tools (e.g., AdaptML, random matrix theory, multivariate statistics) commonly used for genes and proteins but applied to populations overlaid with geochemical parameters and engineering controls. The current tasks for the Environmental Core are as follows: Task 1. Optimize omics protocols for environmental samples. Task 2. Use existing data from the ORNL FRC database to design efficient field sampling studies that maximize the geochemical diversity of study sites. This strategy is expected to enhance the resolution of associations between microbial communities and key geochemical features. Toward this end, we have developed a Monte Carlo search algorithm to optimize site selection for geochemical diversity. To this end we are currently looking for 100 wells that we can do a metagenome analysis in cooperation with the existing a planned SBR IFRC program at ORNL FRC. Overarching principle for Field Studies and Field Linked Resources: Establish as a team with the cooperation of the ORNL IFRC the hypotheses, experiments, sampling, analyses, and schedule as a test plan, including all protocols, amounts, and responsibilities. Prioritize everything so that time, money, shipping, sample limitations, and unforeseen contingencies will not limit delivery of key milestones. This includes 50%, 75%, and 95% evaluations of test plans by team prior to execution, and fatal-flaw analyses at each step. We are currently statistically trying to maximize the geochemical diversity of study sites from the ORNL FRC database. This strategy is expected to enhance the resolution of associations between microbial communities and key geochemical features. Toward this end, we have developed a Monte Carlo search algorithm to optimize site selection for geochemical diversity. We are currently considering a 100 well survey for metagenomics, this is still under planning with the ORNL IFRC. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
586. Hazen, T. C. 2012. Ecogenomics of the Deepwater Horizon Spill. 2012 Ocean Sciences Meeting abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
587. Hazen, Terry C. 2012. Ecogenomics enables a new systems biology understanding of the Deepwater Horizon oil spill. Genome Alberta Hydrocarbon Metagenomics Workshop. abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
588. Hazen, Terry C. 2012. Horizon Oil Spill Degradation of oil by indigenous deep water microbes. Manitoba Environmental Industries Association annual workshop abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
589. Hazen, Terry C.. 2012. Ecogenomics enables a new systems biology understanding of the Deepwater Horizon oil spill. University of Alaska, Fairbanks. abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
590. Hazen, Terry C. 2012. Can Mother Nature Take a Punch Systems Biology of the Gulf Oil Spill. University of Tennessee, Civil & Environmental Engineering. abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
591. Hazen, T. C. 2012. What happened to the Gulf Oil Spill: A Systems Biology Approach. Biosciences Division, Oak Ridge National Lab. abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
592. Goodheart, D., T. C. Hazen and M. K. Firestone. 2012. Deconstructing the Microbial Community Degrading Plant Material in a Wet, Tropical Forest. International Symposium of Microbial Ecology (ISME 14) abstract
     The lower montane tropical forest in the Luquillo Experimental Forest in Puerto Rico has the highest litter decomposition rate measured to date. This soil undergoes frequent oxic/anoxic fluctuations lasting from days to weeks, caused by rain events. The persistent fluctuating environmental condition in the Luquillo Experimental Forest creates two disparate conditions (oxic and anoxic) under which distinct metabolic processes function. The persistent fluctuation in the environmental conditions could enable the rapid litter decomposition by coupling the oxic and anoxic decomposition processes. The goals of this research are to: i) determine the abundance and identity of the bacterial and fungal communities degrading different components of plant material (whole lignocellulose, cellulose and lignin) and ii) assess the metabolic potential of the microbial community under each environmental condition and substrate in a wet, tropical forest soil. To address these goals, soil from the Luquillo Experimental Forest was used in microcosm studies with 13C-labeled plant material added (either Avena barbata -whole lignocellulose analog, cellulose, or vanillin-lignin analog) under either continuous oxic, continuous anoxic or four day oxic/anoxic fluctuation. Gas samples were taken and triplicate Sample (with substrate) and Control (no substrate) soil jars were destructively sampled over time. DNA was extracted and used for qPCR of the bacterial 16S and the fungal ITS, 16S pyrotags and GeoChip 4 analyses. As expected, cumulative CO2 and CH4 were greatest in the oxic and anoxic conditions, respectively, for each substrate. The cumulative CO2 and CH4 from the fluctuating condition of each substrate were similar to the cumulative CO2 and CH4 of the oxic condition. Fungal abundance increased under oxic conditions and dramatically decreased under anoxic conditions with cellulose as the substrate, suggesting fungi are the main agents of decomposition under oxic conditions for cellulose in this soil. The abundance of bacteria does not change in response to environmental condition. However, 16Spyrotag data shows that the structure of the bacterial community altered in response to the environmental condition and substrate. We used the GeoChip to assess the functional capacity of the microbial community under each environmental condition and substrate. The relative abundances of carbon degradation genes altered in response to environmental condition and substrate. In particular, cellulases and hemicellulases showed the greatest changes in relative abundance due to the presence of substrate and environmental conditions, respectively. The abundance, structure and metabolic capacity of the microbial community altered in response to both the environmental conditions and the substrate. This dynamic response could drive the observed rapid decomposition rate in this wet, tropical forest soil.
593. Gilbert, D.. 2012. Waves of Berkeley Lab responders deploy omics to track Deepwater Horizon cleanup microbes. EurekAlert http://www.eurekalert.org/pub_releases/2012-06/dgi-wob061512.php
594. Geller, J., S. Lee, D. Tarjan, C. Wu, T. Torok, T. C. Hazen, A. P. Arkin, and N. J. Hillson. 2012. ELSI Pilot Assessing and Mitigating the Risks of Large-Scale Metabolic Engineering. DOE Genomic Science Meeting abstract
     Project Goals: Establish methodologies for assessing and mitigating the risks of future large-scale metabolic engineering microbial projects, including those extending beyond the bioreactor. The DOE EERE-funded Advanced Biofuel Process Development Unit (ABPDU) houses two 300 liter microbial fermentation tanks. In the extremely unlikely event that the ABPDU’s post-fermentation microbicidal protocol (e.g. base treatment and neutralization) should catastrophically fail, broth harboring viable genetically engineered micro-organisms could be purged directly to downstream waste-water treatment processes. This Ethical, Legal and Social Implications (ELSI) pilot study seeks to quantitate the risks associated with this scenario, by measuring the viability of the engineered microbes (and perhaps more importantly, their embedded genes) in mock sewage reactors that mimic the conditions and microbial communities found in real-world waste water treatment plants. Furthermore, investigation of how differential genetic backgrounds (e.g. gene deletions) impact survival and gene transmission to sewage sludge communities will guide subsequent forwardengineering efforts to further reduce risk. This pilot study establishes methodologies (leveraging only recently available technologies) for assessing and mitigating the risks of future large-scale metabolic engineering microbial projects, including those extending beyond the bioreactor.
595. Elias, D. A., J. J. Mosher, T. J. Phelps, M. Podar, R. A. Hurt, J. H. Campbell, M. M. Drake, J. G. Moberly, C. W. Schadt, S. D. Brown, T. C. Hazen, A. P. Arkin, A. V. Palumbo, B. A. Faybishenko, and P. D. Adams. 2012. ENIGMA Environmental: Succession of Hanford Groundwater Microbial Communities During Lactate Amendment and Electron-acceptor Limitation. DOE Genomic Science Meeting abstract
     Project Goals Bioremediation strategies involving in-situ microbial stimulation for Cr(VI) - reduction/immobilization are ongoing, but determining their relative success is complex. By using controlled laboratory conditions, the influence of individual variables on the successful community structure, dynamics and the metal-reducing potential can be studied. The goal of the current work was to determine the impact of lactate stimulation during sulfate limitation on the succession of a native microbial community. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI) contaminated groundwater from the Hanford, Washington 100-H area and incubated for 95 days to obtain stable, enriched communities. The microbial community structure shifted with a significant loss of diversity. Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp. with small levels of other organisms including methanogens. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Isolation efforts attained four new strains of Pelosinus spp. Three of the 4 Pelosinus strains were capable of Cr(VI)- reduction and one also reduced U(VI). Under the tested conditions of limited sulfate, it appears that the sulfate-reducers, including Desulfovibrio spp., were outcompeted even though they are capable of fermentative growth. These results suggest that lesser-known organisms, such as Pelosinus spp., may play a more important role in metal-reduction than currently suspected. Currently, a similar bioreactor experiment is underway and builds from the above findings. Hanford groundwater was enriched in duplicate bioreactors with lactate and; 1) no Cr(VI) to emulate the earlier work and establish a baseline, 2) 0.1 mg/L Cr(VI) to reflect the low plume concentrations nearing the Columbia river and 3) 3.0 mg/L Cr(VI) to reflect the source metal concentration. Multiple levels of analysis are now underway after a 105 day experiment. These include temporal measurements of; 16S rRNA gene pyrosequencing, gas and organic acid quantification by GC and HPLC, Geochip, Phylochip, metagenomics, metatranscriptomics, metaproteomics, soluble Fe(III), HFO, Cr(VI) and U(VI) reduction assays, metal uptake characteristics for 36 metals, small metabolite quantification, and fluorescent antibody cell counts for species of Desulfovibrio, Geobacter, Methanococcus, and Pelosinus. These results will help to determine if and how the added complexity of Cr(IV) will influence the microbial community structure and metabolism as well as revealing the any acclimation of the community to Cr and quantifying the relative reduction potential. Acknowledgement This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02- 05CH11231. Oak Ridge National Laboratory is managed by University of Tennessee UTBattelle LLC for the Department of Energy under Contract No. DE-AC05-00OR22725
596. Dubinsky, E. A., Y. M. Piceno, F. C. Reid, L. M. Tom, Terry C. Hazen and G. L. Andersen. 2012. Microbial Community Composition in a Deep Water Oil Plume and Dissolved Oxygen Anomalies. 2012 Ocean Sciences Meeting. abstract
     In the aftermath of the Deepwater Horizon blowout, a dilute hydrocarbon plume was observed between 1,100 and 1,300 M below the surface. We collected water samples at plume depth on three ships from May 25 to August 26, 2010. Samples from transects starting near the source followed the prevailing current at this depth. No detectable hydrocarbons were observed at plume depth by the end of July, however, multiple samples taken in August had small reductions in DO suggesting microbial respiration and oxygen consumption. Microbial communities in plume samples were distinctive from non-plume with increased relative abundance of potential hydrocarbon-degrading gammaproteobacteria and Vibrio. Community compositions in August samples with reduced DO were similar to plume communities with a few notable differences. Taxa from the Methylococcaceae were more abundant in the late season, reduced DO samples. This supports the hypothesis that methanotrophs bloom late in plume succession in response to methane in water column. These results demonstrate that microbial communities at depth respond to trace amounts (<1 ppb) of hydrocarbon and microbial communities can be effective biosensors for hydrocarbon
597. Dubinsky, E. A., L. Tom, F. Reid, S. Borglin, K. Chavarria, J. Fortney, D. Joyner, J. Kuehl, R. Lamendella, H. Lim, O. Mason, Y. Piceno, K. Wetmore, C. Wu, T. C. Hazen, G. L. Andersen. 2012. Succession and persistence of hydrocarbon-degrading microbial communities following the Deepwater Horizon Oil Spill. Annual Meeting of the American Society for Microbiology abstract
     The Deepwater Horizon oil spill created large plumes of dispersed oil deep in the Gulf of Mexico and stimulated growth of indigenous bacteria that can degrade hydrocarbons at cold temperatures. We tracked microbial community composition during and after the spill to determine relationships between microbial dynamics, and hydrocarbon and dissolved-oxygen concentrations. Oil plumes were dominated by a few species of Gammaproteobacteria, but community structure varied over time and was dependent on hydrocarbon concentration and composition. Pelagibacter were the most negatively impacted group of bacteria in oil plumes. Dissolved oxygen anomalies persisted over six weeks after well containment and remained dominated by plume bacteria despite the absence of petroleum hydrocarbons. Methanotrophs were also enriched in lingering oxygen anomalies, consistent with reports that methane consumption lagged behind the biodegradation of other hydrocarbon inputs.
598. Dubinsky, E. A., L. M. Tom, F. Reid, S. Borglin, K. Chavarria, J. Fortney, D. Joyner, J. Kuehl, R. Lamendella, H.- C. Lim, R. Mackelprang, O. U. Mason, Y. Piceno, K. Wetmore, C. Wu, T. C. Hazen, G. L. Andersen. 2012. Microbial community composition as a highly sensitive biosensor for oil spills in the deep ocean. ACS Spring Meeting.
599. Dhaeseleer, P. D., J. Gladden, J. Park, A. Redding, C. Petzold, M. Allgaier, D. Chivian, S. Singer, T. C. Hazen, and B. Simmons. 2012. Metagenomics, Metabolic Reconstruction, and High-Resolution Proteomics of Biomass Degradation in a Thermophilic Bacterial Community. DOE Genomic Science Meeting. abstract
     Project Goals: The Microbial Communities group at JBEI aims to develop a fundamental understanding of how microbial communities degrade targeted biomass feedstocks, and to utilize a targeted, function-based screening approach to genomics and proteomics to identify, isolate, and characterize new enzymes that are capable of efficiently degrading lignocellulosic feedstocks. Focusing on a thermophilic switchgrass-adapted enrichment community yields an order of magnitude more useful enzyme sequences compared to our previous work on a more complex community, and the resulting enzymes are more likely to be well suited to our targeted feedstock, pretreatment, and processing conditions. Combining enzymatic assays, metagenomics, zymography, MS proteomics, and metabolic modeling provides a multidimensional view of the internal functioning of this highly active biomass degrading bacterial community. A microbial enrichment culture with high biomass degrading activity was selected for metagenomic sequencing, annotated using JGI’s IMG/M system, and binned into phylogenetic groups. Metabolic reconstructions were generated using Pathway Tools, allowing us to assign metabolic roles to the different members of the bacterial community. High resolution MS metaproteomics by EMSL was mapped to the community members to analyze differential expression of their metabolic pathways and identify highly expressed biomass degrading enzymes.
600. DeAngelis, K., C. Dylan, S. Blake, T. C. Hazen and S. Whendee. 2012. Shifting dynamics of bacteria and fungi during litter decomposition in wet tropical forest soils. International Symposium of Microbial Ecology (ISME 14) abstract
     The rates of decomposition in wet tropical forest soils are extremely fast compared to other biomes, with rapid turnover of labile carbon pools. Fast rates of decomposition are curious considering the low and fluctuating redox potentials. Oxygen limitation coupled to high rates of decomposition suggests that anaerobic or facultative decomposing bacteria are prevalent and active decomposers in tropical forest soils, but the communities involved are poorly understood. This study was intended to test the hypothesis that litter decomposition rates and communities would vary across a rainfall gradient in the Luquillo Experimental Forest LTER in Puerto Rico USA. We employed large-scale microbial community sequencing in combination with measurements of enzyme rates and chemistry of decomposing switchgrass litter and adjacent soil samples over a two-year period at 1, 4, 13, 30, 60 and 96 weeks. The experiment was designed to capture both short- and long-term temporal trends in decomposition, as well as evaluate the roles of rainfall (low and high elevation) and soil oxygen availability (low and high redox) among four sites in the Luquillo Experimental Forest. The low elevation sites had the lowest C:N ratios after two years and the most mass lost. Though the high elevation, low redox site had the least mass lost, it also had the highest litter enzyme activity. Partial small subunit ribosomal RNA genes were sequenced with universal primers for bacteria, archaea and eukaryotes, yielding 40,850 unique taxa after quality filtering, dereplication and clustering. Community profiles based on phylogenetic (UniFrac) distance showed that during early decomposition (weeks 1-13), bacterial litter communities were distinct from soil, while fungal soil and litter communities were similar; during later decomposition (weeks 30-96), bacterial soil and litter communities were indistinguishable while fungal litter communities were distinct from soil. A relatively small set of dominant taxa was highly enriched on litter (>1% relative abundance) during early-stage decomposition, including Beta- and Gammaproteobacteria, Firmicutes and Stramenopiles. Fungi appeared among the dominant taxa after 60 weeks decomposition. Richness estimates revealed that in the high elevation sites, litter selected for a much less diverse community compared to soil. Measures of phylogenetic structure showed that bacteria were more clustered in soil than litter, while the opposite was true for fungi. This suggests that net negative interactions (like competition or predation) are involved to bacterial litter colonization, while net positive interactions (like phenotypic attraction or spatial isolation) are involved in fungal litter colonization. Taken together, these data reveal a succession of fungi, bacteria, and their likely eukaryotic predators that conspire to accomplish decomposition under low and fluctuating redox conditions, as well as strategies for microbial decomposition in low redox potential tropical forest soils.
601. De Leon, K. B., B. D. Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen and M. W. Fields. 2012. Microbial population dynamics in groundwater and surrogate sediments during HRC® biostimulation for Cr(VI)-reduction. International Symposium of Microbial Ecology (ISME 14) abstract
     The Hanford 100-H site is a chromium-contaminated site that has been designated by the Department of Energy Environmental Management as a field study site for in situ chromium reduction. In August 2004, the first injection of hydrogen release compound (HRC®) resulted in an increase of microorganisms and a reduction of soluble chromium(VI) to insoluble chromium(III). Little is understood about the microbial community composition and dynamics during stimulation. The aim of this study is to compare microbial communities of groundwater and soil samples across time and space during a second injection of HRC®. A second injection occurred November 2008 and geochemical data collected throughout the study showed an overall decrease in nitrate, sulfate, and chromium(VI). Spatial and temporal water and soil samples (n=34) were collected pre-and post-injection from four wells at the field site. Soil columns constructed from stainless steel mesh were lined with nylon mesh and filled with Hanford soils from the 100-H site. The soil columns were used to represent not only the microbes flowing through the soil via groundwater, but the microbes that require a matrix in order to grow. DNA was extracted from each of the samples and the V1V2 region of the 16S rRNA gene was sequenced via multiplex pyrosequencing. Soil sample populations differed from the corresponding groundwater (even at the phyla level) and were more diverse (p=0.001). While many of the populations were observed in both groundwater and surrogate sediments, the respective matrices appeared to enrich for particular OTUs. Of 667 total genera, 141 and 69 were unique to groundwater and soil, respectively. Genera observed only in the sediment included Marinomonas while genera observed only in the groundwater included Desulfonauticus, Desulfomicrobium, and Syntrophobacter. Pseudomonas, Acidovorax, Clostridium, and Herbaspirillum were dominant regardless of sample type. Results do not indicate a large shift in dominant organisms in soil from pre- to post- injection, and this may be due to the organisms remaining dominant from the first stimulation. Correlation analyses of genera were done for each sample type using SparCC. Metal-reducing organisms such as Geobacter, Desulfovibrio, and Geothrix were correlated in soil while possible fermenting bacteria such as Clostridium, Pelotomaculum, and Pelosinus were correlated in groundwater. For each well, HRC® injection resulted in increased diversity, but the greatest changes during stimulation occurred in the populations of mid-dominance either between wells or across time. These organisms could be important to consider as possible indicator species in future work that includes targeted isolations to better understand the mechanisms of microbial interactions.
602. De Leon, K. B., B. D Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen, and M. W. Fields, and Paul D. Adams. 2012. ENIGMA Microbial Community Dynamics in Groundwater and Surrogate Sediments During HRC¿ Biostimulation of Cr(VI)-Reduction. DOE Genomic Science Meeting abstract
     Project Goals: The elucidation of bacterial community dynamics for both groundwater and sediment-associated communities over time and space during biostimulation for chromate reduction in relation to geochemical variables. The Hanford 100-H site is a chromium-contaminated site that has been designated by the Department of Energy Environmental Management as a field study site for in situ chromium reduction. In August 2004, the first injection of hydrogen release compound (HRC®) resulted in an increase of microorganisms and a reduction of soluble chromium(VI) to insoluble chromium(III). Little is understood about the microbial community composition and dynamics during stimulation. The aim of this study is to compare microbial communities of groundwater and soil samples across time and space during a second injection of HRC®. A second injection occurred November 2008 and geochemical data collected throughout the study showed an overall decrease in nitrate, sulfate, and chromium(VI). Spatial and temporal water and soil samples (n=34) were collected pre-and post-injection from four wells at the field site. Soil columns constructed from stainless steel mesh were lined with nylon mesh and filled with Hanford soils from the 100-H site. The soil columns were used to represent not only the microbes flowing through the soil via groundwater, but the microbes that require a matrix in order to grow. DNA was extracted from each of the samples and SSU rDNA gene fragments was sequenced via multiplex pyrosequencing. Sequences were refined by length, primer errors, and Ns, and sequences with a high percentage of low Phred quality score values were removed. Python scripts were developed to filter the pyrotag data with respect to quality scores, and the filtering technique was validated with environmental samples. Soil samples differed from the corresponding groundwater (even at the phyla level) and were more diverse (p=0.001). While many of the populations were observed in both groundwater and surrogate sediments, the respective matrices appeared to enrich for particular OTUs. Predominant populations for the sediments were Psuedomonas, Acidovorax, Clostridium, Aquaspirillum, Methylibium, Anaeromyxobacter while predominant populations for groundwater were Psuedomonas, Pleomorphomonas, Ramlibacter, Arthrobacter, and Herbaspirillum. Genera observed only in the sediment included Marinomonas while genera observed only in the groundwater included Desulfonauticus, Desulfomicrobium, and Syntrophobacter. Results do not indicate a large shift in dominant organisms in soil from pre- to post- injection, and this may be due to the organisms remaining dominant from the first stimulation. However, a prevalence of core genera and rare genera were observed across 34 samples while urban and rural genera were less abundant. There was a shift from Acidovorax to Aquaspirillum from upstream (non-stimulated) to downstream soil both pre- and post-injection. Surrogate soil samples indicated similar changes in the soil community in the injection (Well 45) and downstream (Well 41) wells across time, while water samples seem to indicate more of a pre- and post-injection grouping instead of gradual changes across time. Furthermore, while post-injection soil samples indicate a continuing dominance of Aquaspirillum, corresponding water samples indicate Pseudomonas as a dominant genus. For each well, HRC® injection resulted in increased diversity, but the greatest changes during stimulation occurred in the populations of mid-dominance either between wells or across time. These organisms could be important to consider as possible indicator species in future work. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
603. Cheng, X., W. Reindl, K. Deng, B. Bowen, B. Lai, J M. Gladden, S. W. Singer, A. Wong, T. C. Hazen, B. Fox, K. Sale, B. A. Simmons, A. K. Singh, J. Keasling, P. D. Adams, and T. R. Northen . 2012. Nanostructure-Initiator Mass Spectrometry (NIMS): High Throughput Enzyme Activity Assays for Biofuel Development.. DOE Genomic Science Meeting abstract
     Project Goals: This project meets an urgent need for a highly specific activity screening approach and offers tremendous potential for the high-throughput identification and optimization of industrial enzymes and enabling application of biological approaches utilizing large libraries. The efficient deconstruction of lignocellulosic biomass into biofuels represents a critical and formidable challenge. JBEI is addressing this challenge using a multifaceted approach that is highly dependent on enzyme discovery, optimization and synthetic biology. The optimization of deconstruction processes requires technologies for the high throughput screening and identification of glycoside hydrolase activities. The high sensitivity, specificity, and resolution of mass spectrometry make it well suited for the analysis of sugar molecules. However, the low throughput of conventional GC/MS and LC/MS precludes implementation for screening purposes. Here we present a multiplexed approach based on nanostructure-initiator mass spectrometry (NIMS) that allows for the rapid analysis of several glycolytic activities in parallel under diverse assay conditions. By forming colloids, it was possible to perform aqueous reactions in microwell plates despite the substrate analogs’ hydrophobic perfluorinated tags. Our assay can be used both for the characterization of known enzymes (pH and temperature profiles, kinetic studies, ionic liquid tolerance), and the identification of yet unknown activities, even from complex biological samples (environmental and enrichment cultures). We are now integrating this assay with acoustic printing resulting in a 100-fold increase in throughput.
604. Chandonia, J.-M., Maxim Shatsky, Ming Dong, Haichuan Liu, Lee Yang, Jil T. Geller, Megan Choi, Barbara Gold, Nancy L. Liu, Marjon Khairy, Sonia Reveco, Tom R. Juba, Bonita R. Lam, Evelin D. Szakal, Simon Allen, Sunil Kumar, Farris L. Poole, Steven E. Brenner, Steven C. Hall, Susan J. Fisher, Michael Adams, T. C. Hazen, Judy D. Wall, Swapnil Chhabra, Jian Jin, H. Ewa Witkowska, Adam P. Arkin, Gareth P. Butland, Mark D. Biggin, and Paul D. Adams. 2012. ENIGMA Microbiology Physiology: Accurate, High-Throughput Identification of Stable Protein Complexes in Desulfovibrio vulgaris. DOE Genomic Science Meeting abstract
     Project Goals: Desulfovibrio vulgaris has been selected as a model bacterium for intensive study by ENIGMA because it can reduce heavy metals and radionuclide contaminants present in the soil at many DOE sites, rendering the contaminants insoluble. ENIGMA seeks to model, at a molecular systems level, how this and similar bacteria respond to natural and human induced changes in their environment and how this alters their ability to stabilize contaminants in the soil. A component of our strategy is to develop and use high throughput pipelines to purify and characterize soluble protein complexes. We expect that these interaction data will improve our ability to produce accurate metabolic and regulatory models of key members of microbial communities. The group led by Mark Biggin has developed a novel method for identification of stable, soluble protein complexes in microbes. In a small-scale pilot study, we showed that many protein complexes survive intact through a series of orthogonal chromatographic methods, with complex components having correlated elution profiles. These profiles were measured with the aid of mass spectrometry (MS) and iTRAQ reagents (Dong et al., 2008). We developed statistical and machine learning methods to analyze a full-scale data set, which were required in order to obtain biologically meaningful results due to the high potential for false positives (FP) caused by co-elution of proteins that are not part of a complex. Our methods were tuned using a manually curated gold standard (GS) set. As a first high-throughput study, we demonstrated this technique in identifying a highprecision subset of stable protein complexes in Desulfovibrio vulgaris. We have shown that our predicted network of interactions is significantly enriched in pairs with similar functional annotations. The quantitative information from elution profiles allowed us to develop a statistical model to estimate the false discovery rate in our predictions; because this varies according to how “crowded” the eluted fractions are, we are able to identify a subset of hundreds of highly reliable (i.e., with very low false discovery rate) interactions, as well as a much larger set of interactions that can be predicted with known false discovery rates. Advantages of the tagless approach include not requiring a mutant library (needed for alternative tag-based approaches such as TAP), and a false discovery rate comparable to TAP. The group led by Gareth Butland has identified a number of protein complexes using TAP. We have developed an automated pipeline for synthesis of tagged gene constructs in collaboration with Swapnil Chhabra (Chhabra et al., 2011). To date, over 700 pulldowns (comprising more than 600 unique D. vulgaris strains) have been subject to analysis. In these experiments, more than 10,000 interactions were detected with over 1,000 distinct prey proteins. Using curated GS datasets (as in the tagless analysis), we filtered out ubiquitous proteins and other likely FP, resulting in a set of high-confidence interactions. A number of these interactions have been reciprocally confirmed, using strains in which the original prey protein was tagged and used as bait. Preliminary analysis of the data have identified several novel complexes, including multiple paralogous versions of the DnaJK-GrpE chaperone complex, each of which is bound to a small protein that may act as an allosteric regulator. References 1. Chhabra SR, Butland GP, Elias D, Chandonia JM, Fok OY, Juba T, Gorur A, Allen S, Leung CM, Keller K, Reveco SA, Zane GM, Semkiw ES, Prathapam R, Gold B, Singer M, Ouellet M, Szakal ED, Jorgens D, Price MN, Witkowska HE, Beller HR, Arkin AP, Hazen TC, Biggin MD, Auer M, Wall JD, Keasling JD. 2011. Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome. Applied and Environmental Microbiology 77:7595-7604. 2. Dong M, Yang LL, Williams K, Fisher SJ, Hall SC, Biggin MD, Jin J, Witkowska HE. 2008. A “tagless” strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking. J Proteome Res 7:1836-1849. ENIGMA is a Lawrence Berkeley National Laboratory Scientific Focus Area Program supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics:GTL Foundational Science through contract DE-AC02- 05CH11231.
605. Chakraborty, R., Y. M. Piceno, F. C. Reid, S. E. Borglin, E. A. Dubinsky, L. M. Tom, T. C. Hazen, G. L. Andersen. . 2012. Microbial Community Structure and Hydrocarbon Degradation by Isolates Obtained from Different Depths in the Aftermath of the Deepwater Horizon Spill in the Gulf of Mexico. Annual Meeting of the American Society for Microbiology abstract
     The deepwater horizon spill resulting from the exploded drilling rig released over 3 million barrels of crude oil into the Gulf of Mexico. Bio-Sep beads amended with volatile hydrocarbons from MC-252 oil were used to create hydrocarbon-amended traps for attracting oil-degrading microbes in situ. Traps were placed on a drilling rig about 600-m from the original MC-252 oil spill site at four depths: 46-m, 205-m, 1,096-m and 1,509-m. These traps were harvested at different time points to study hydrocarbon degradation, microbial community structure, and to isolate oil-degrading bacteria. While the extractable organics did not show a significant over all decrease, there was a loss of short chain alkanes (
606. Chakraborty, R., S. E Borglin, B. Faybishenko, P. Dehal, A. P. Arkin, T. C. Hazen, M. W. Fields, J. Geller, J. Fortney, D. Joyner, M. Conrad, and P. D. Adams. 2012. Effect of Nitrate Stress on Metal-reducing Microbes and Results of Nitrate push/pull Field Tests at Hanford 100H. DOE Genomic Science Meeting abstract
     Project Goal: The ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) project seeks to elucidate the mechanisms and key processes that enable microorganisms and their communities to function in metal-contaminated soil sites. One goal is to understand the effect of environmental stressors that enable ENIGMA-relevant microorganisms to thrive in such environments. Abstract: As part of the ongoing investigation of sustainable bioremediation of Cr(VI) in groundwater at the Hanford 100H area, we performed groundwater biostimulation tests by injecting Hydrogen Release Compound (HRC ) and three lactate (17mM) injection experiments. To investigate the response of resident microbes to nitrate stress, a push-pull test was then conducted by injecting 55 gals of groundwater (collected from the background well) with KNO3 (nitrate concentration 5,000 ppm) in October, 2010. After one day, pumping began from the same well, and lasted for 16 days. As a result of nitrate injection, total biomass decreased and sulfate concentration increased, but the sulfide and iron concentrations dropped. During pumping, the nitrate concentration decreased about 3 orders of magnitude. PLFA data showed biomass on the order of 107 cells/ml prior to push pull, and dropping off to 105 cells/ml during the test, but recovering toward the end back to 107 cells/ml by the end. Biomarker lipids indicate a shift toward monoenoics indicating an increase in gram negative bacteria and decrease in branched lipids (gram postive) and branched monoenoic (sulfate reducers). We discuss the field test results in details and elucidate the effect of nitrate stress on environmentally relevant microbes Geobacter metallireducens and Desulfovibrio vulgaris as observed through controlled lab experiments. The lab studies and the field study together will help in understanding the overall fate of microbes under changing environmental conditions in the field and the key cellular mechanisms impacted by such stress conditions. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
607. Butland, G. P., S. R. Chhabra, B. Gold, N. L. Liu, S. Reveco, T. R. Juba, J. D. Wall, B. R. Lam, J. T. Geller, T. C. Hazen, M. Choi, M. D. Biggin, E. D. Szakal, S. Allen, H. Liu, H. E. Witkowska, J‐M. Chandonia. 2012. High Throughput Identification of Protein Complexes from Desulfovibrio vulgaris by a Tandem Affinity Purification Pipeline. Annual Meeting of the American Society for Microbiology abstract
     Desulfuvibrio vulgaris is a sulfate reducing bacterium (SRB) found in DOE sites contaminated with heavy metals or radionuclides and can reduce many of the contaminants to an insoluble form. Environmental change or human intervention can alter the chemical environment in the subsoil, which in turn affects which species predominate as well as microbial physiology. It is therefore critical to learn how such changes affect SRBs and their interaction with other members of the community. We have chosen Desulfovibrio vulgaris to address these questions in molecular detail as it is one of the dominant SRBs found at DOE sites. Most cellular processes in D. vulgaris are mediated by multiple proteins interacting with each other in the form of multi-protein complexes and not by individual proteins acting in isolation. In order to accurately model cellular processes in this organism and its response to stress, our goal is to develop a comprehensive knowledgebase of protein complexes and protein-protein interactions using high throughput tandem affinity purification (TAP). Currently, we have generated 1588 unique TAP-tagged strains, of which 866 with correctly integrated TAP-tagged chromosomal fusions have been subject to TAP analysis and have had their composition analyzed by mass spectrometry. In ~80% of these analyses, the bait was verified to be present by gel-free mass spectroscopy. Despite many bottlenecks including larger culture volumes and strict anaerobiosis associated with working with D. vulgaris, we have obtained throughput statistics, data quality and success rates similar to those previously reported for E. coli. Detected high-confidence interactions cover a range of biological processes including energy conservation, protein secretion, protein folding, and cofactor biosynthesis with both novel and previously predicted interactions. We are now in a position to conduct a system-wide analysis of all stable protein-protein interactions in D. vulgaris and to target how these change in response to stresses typically occurring in the subsoil of contaminated sites for a select set of stress response genes.
608. Boxall, B. 2012. Gulf currents aided breakdown of oil after BP spill, study says. http://www.miamiherald.com/2012/01/09/2581760/gulf-currents-aided-breakdown.html
609. Baidoo, E. E., S. Yilmaz, J. Geller, T. C. Hazen, A. K. Singh, J. D. Keasling. 2012. Differential Analysis of Metabolic Intermediates from Desulfovibrio vulgaris Hildenborough and Methanococcus maripaludis under Syntrophic Growth Conditions. Annual Meeting of the American Society for Microbiology abstract
     Metabolite profiling experiments are normally conducted on the monoculture of microorganisms. However, microorganisms in their natural habitats rarely live alone as they are part of ecosystems that are largely made up of microbial communities forming a network that is responsible for processing and exchanging material. Initial steps towards characterizing the metabolisms of individual microorganisms from their respective communities are being performed at the most basic level in the form of laboratory co-culture experiments. In general, metabolite concentrations from the co-culture are compared to the concentrations of the monoculture. However, monoculture conditions are not a true reflection of the natural environment; and although co-culture studies facilitate accurate characterization of the metabolisms of syntrophic partners, metabolite profiling still has some major challenges. The greatest being the determination of the origin of each metabolite. One way of achieving this is to physically separate the co-culture prior to metabolite analysis. For this purpose, we have designed a dual chamber bioreactor in which the chambers containing monocultures of Desulfovibrio vulgaris Hildenborough (D. vulgaris) and Methanococcus maripaludis (M. maripaludis) are separated by a membrane filter that allows the free passage of nutrients from one population to another. In the absence of sulfate, sulfate reducing bacteria (SRB) such as D. vulgaris species ferment organic acids and alcohols, producing hydrogen, acetate and carbon dioxide. Under these conditions they are reliant upon hydrogen and acetate-scavenging methanogens to convert the aforementioned compounds to methane. This syntrophy increases the chances of survival of SRBs in environments that are limited in electron acceptors. The metabolic versatility of D. vulgaris is highlighted by the complexity of energy metabolism in this SRB and, hence, requires further study. To this end, a combination of LC-MS and CE-MS technologies are being used to provide metabolite data for the accurate characterization of the central metabolisms of D. vulgaris and M. maripaludis under syntrophic growth conditions. Further, this approach is being used to determine metabolite exchange between the organisms.
610. Auer, M. A. Gorur, P. Arbeleaz, N. Baliga, D. A. Ball, M. Biggin, J. M. Chandonia, S. Chhabra, R. Csencsits, K. H. Downing, M. W. Fields, J. T. Geller, R. Glaeser, T. C. Hazen, G. L. Hura, T. Juba, B. Lam, C. M. Leung, J. Liphardt, J. Malik, J. P. Remis, S. Reveco, J. Tainer, A. Tauscher, J. Wall, A. Deutschbauer, T. Northen, A. Arkin, and P. D. Adams. 2012. ENIGMA Biotechnology: Biofilm Imaging: From Protein Complexes to Intact Microbial Communities. DOE Genomic Science Meeting abstract
     Project Goals: The ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) project aims to elucidate the mechanisms and key processes that enable microorganisms and their communities to function in metal-contaminated soil sites. The ENIGMA Biotechnology Component focuses its efforts on providing crosscutting technologies that will support all other ENIGMA components with a particular focus on biological imaging at different scales of bacteria and microbial communities. These data will help to develop models of microbial community activity and principles of community organization in an effort to predict the role that microbial species and their interactions play in the dynamics of geochemical transformations in a changing environment. Microbial physiology is inherently a multiscale biological process that coordinates complex processes such as extracellular metal reduction and response to environmental stresses and competing species. Bacteria often assemble into sustainable communities that allow individual bacteria to coordinate their respective behavior and thus optimizing the efficiency of biological processes, which may enhance the chances for species survival. ENIGMA is addressing the complexity of multiscale spatiotemporal biofilm organization through a combination of expertise in traditional structural biology and modern multimodal imaging. SAXS (Rambo et al. 2010) and single particle cryo-EM are proven technologies to determine protein complex stoichiometry and shape, allowing the fitting of high-resolution structures into the intermediate resolution density envelope (Han et al. 2009). Cryo-electron tomography of bacterial whole mount samples can detect intra- and extracelluar specializations e.g. those important for metal reduction. Cryo-EM analysis is complemented by widefield 2D section TEM and advanced 3D SEM imaging approaches (FIB/SEM and SBF/SEM) of cryopreserved, freeze substituted and resin-embedded samples. With these novel EM imaging approaches, we have begun to examine large areas and volumes of biofilms in DvH and other soil bacteria. We have found outer membrane vesicles, vesicle chains and cell-cell connections (Palsdottir et al. 2009, Remis et al. 2010, Remis et al. submitted), as well as compartmentalization of metal precipitation (Auer, unpublished observation). These observations suggest a an intricate set of interactions and possibly coordination of function between community members. X-ray and EMbased imaging approaches are complemented by tag-based labeling of proteins both at the light and electron microscopy level, and allow the study of cell-to-cell variations in protein abundance and protein localization (Chabra et al. 2010). Advanced optical super-resolution imaging methods (including PALM and STORM) allow high precision localization and counting (Betzig et al 2006). Further integration of small molecule mass spectrometry imaging, while at a somewhat larger size scale, promises to link structural observation and protein localization with metabolic activity of biofilm regions. Through the integrated application of these imaging modalities ENIGMA is deconstructing a mechanistic understanding of biofilm function. Acknowledgement: This work conducted by ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. References 1. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006 313 (5793): 1642–1645 2. Chhabra SR, Butland G, Elias DA, Chandonia JM, Fok OY, Juba TR, Gorur A, Allen S, Leung CM, Keller KL, Reveco S, Zane GM, Semkiw E, Prathapam R, Gold B, Singer M, Ouellet M, Szakal ED, Jorgens D, Price MN, Witkowska HE, Beller HR, Arkin AP, Hazen TC, Biggin MD, Auer M, Wall JD, Keasling JD. Generalized schemes for highthroughput manipulation of the Desulfovibrio vulgaris genome. Appl Environ Microbiol. 2011 Nov;77(21):7595- 604 3. Han BG, Dong M, Liu H, Camp L, Geller J, Singer M, Hazen TC, Choi M, Witkowska HE, Ball DA, Typke D, Downing KH, Shatsky M, Brenner SE, Chandonia JM, Biggin MD, Glaeser RM. Survey of large protein complexes in D. vulgaris reveals great structural diversity. Proc Natl Acad Sci U S A. 2009 Sep 29;106(39):16580-5. 4. Palsdottir H, Remis JP, Schaudinn C, O’Toole E, Lux R, Shi W, McDonald KL,Costerton JW, Auer M. Three-dimensional macromolecular organization of cryofixed Myxococcus xanthus biofilms as revealed by electron microscopic tomography. J Bacteriol. 2009 Apr;191(7):2077-82 5. Rambo RP, Tainer JA. Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. Curr Opin Struct Biol. 2010 Feb;20(1):128-37. 6. Remis JP, Costerton JW, Auer M. Biofilms: structures that may facilitate cell-cell interactions. ISME J. 2010 Sep;4(9):1085-7 7. Remis JP, Wei W, Gorur A, Allen S, Witkowski HE, Costerton JW, Auer M Bacterial Social Networks: Targeted Delivery of Outer Membrane Proteins via Membrane Vesicle Chains, submitted
611. Andersen, G. L., Y. M. Piceno, F. C. Reid, R. Chakraborty, S. E. Borglin, E. A. Dubinsky, L. M. Tom, H.-Y. N. Holman, T. C. Hazen. 2012. Microbial community structure and in situ MC-252 oil degradation at different depths in the Gulf of Mexico. ACS Spring Meeting.
612. 2012. Business Bulletins. KnoxvileBiz.com
613. Zhou, A., Z. He, E. Baidoo, K. Hillesland, M. P. Joachimiak, J. Baumohl, P. Benke, A. Mukhopadhyay, P. S. Dehal, A. P. Arkin, D. Stahl, T. C. Hazen, and J. Zhou. 2011. Transcriptomics and Metabolites Assay of Salt-Adapted Desulfovibrio vulagris Hildenborough in Experimental Evolution. ASM annual Meeting.
614. Zhang, P., W-M. Wu, J. Van Nostrand, Y. Deng, Z. He, T. Gihring, G. Zhang, C. Schadt, D. Watson, P. Jardine, C. Criddle, S. Brooks, T. Marsh, J. Tiedje, T. C. Hazen, and J. Zhou. 2011. Diverse Functional Genes of Microbial Community Stimulated with Emulsified Vegetable Oil for in situ U(VI) Reduction. ASM annual Meeting.
615. Yoon, S. H., D. J. Reiss, J. C. Bare, D. Tenenbaum, M. Pan, J. Slagel, S. Lim, M. Hackett, A.-L. Menon, M. W.W. Adams, A. Barnebey, S. M. Yannone, J. A. Leigh, and N. S. Baliga. PI: N. S. Baliga, Co-PIs: J. A. Leigh, M. Hackett, W. Whitman, P. Adams, A. P. Arkin, T. C. Hazen, M. W.W. Adams, G. Hura, S. M. Yannone, S. Holbrook, G. Siuzdak, and J. A. Tainer. 2011. Parallel Evolution of Transcriptome Structure During Genome Reorganization. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
616. Yong, E. 2011. Bacteria ate up all the methane that spilled from the Deepwater Horizon well. http://blogs.discovermagazine.com/notrocketscience/2011/01/06/bacteria-ate-up-all-the-methane-that-spilled-from-the-deepwater-horizon-well/
617. Yarris, L. 2011. Lessons learned from the two worst oils spills in U.S. history. http://www.physorg.com/news/2011-08-lessons-worst-oils-history.html
618. Walian, P. J., S. Allen, L. Zeng, E. Szakal, S. C. Hall, S. J. Fisher, R. Santos, B. Lam, J. T. Geller, T. C. Hazen, J. M. Chandonia, H. E. Witkowska, M. D. Biggin, and B. K. Jap. 2011. High-throughput Pipeline for the Purification and Identification of Desulfovibrio vulgaris Membrane Protein Complexes. ASM annual Meeting.
619. Walian, P. J., S. Allen, L. Zeng, E. D. Szakal, H. Liu, S. C. Hall, S. J. Fisher, R. Santos, B. Lam, J. T. Geller, T. C. Hazen, J.-M. Chandonia, H. E. Witkowska, M. D. Biggin, and B. K. Jap. 2011. High-Throughput Pipeline for the Purification and Identification of Desulfovibrio vulgaris Membrane Protein Complexes. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
620. Voigt, E. 2011. Invasion of the Oil-Eating Microbes. Odyssey January 2011 pg 16-18.
621. Vergano, D. 2011. Study: Undersea bugs ate natural gas released in oil spill. http://www.usatoday.com/tech/science/environment/2011-01-06-gulf-oil-spill-methane-natural-gas-bacteria_N.htm
622. Vastag, B. 2011. Bacteria devoured methane gas from gulf oil spill, scientists say. http://www.washingtonpost.com/wp-dyn/content/article/2011/01/06/AR2011010603570.html
623. Tu, Q., Y. Deng, Z. He, H. Yu, Y. Qin, A. Zhou, J. Xie, Z. Lu, J. Voordeckers, Y. Lee, K. Xue, J. Van Nostrand, L. Wu, Y. Jiang, T. C. Hazen, P. Adams, and J. Zhou. 2011. Development of Metagenomic Technologies for Analyzing Microbial Communities. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
624. Spier, C.L., W.T. Stringfellow, E. Sonnenthal, M. Conrad, and T. C. Hazen. 2011. The distribution of hydrocarbons in surface and deepwater plumes during the MC252 oil spill in the Gulf of Mexico. American Geophysical Union Fall Meeting. abstract
     The explosion of the Deepwater Horizon oil platform on April 20, 2010 resulted in the third largest oil spill in history. We investigated the distribution and chemical composition of hydrocarbons surrounding the spill site. A complete set of hydrocarbon data were acquired from the NOAA and BP, including data from 16 research missions. Several hydrocarbon plumes were identified including near-surface plumes (0.5 to 200m), a small mid-depth plume (850-880m), and a large deepwater plume between approximately 1000 and 1400m below surface. The vertical, lateral, and temporal distribution of hydrocarbons within the water column was investigated, and we found significant differences in the chemical composition of the plumes. The distribution of hydrocarbons remaining in sediments between August and October, 2010 was investigated. All sediment samples with total polycyclic aromatic hydrocarbons (PAHs) concentrations exceeding chronic toxicity limits were located less than 3.2km from the wellhead. All sediment samples with concentrations above the mean pre-spill PAH levels (>600µg/kg), based on 2006 and 2009 survey’s by the Minerals Management Service in the Deep Gulf of Mexico, were found within 12km of the wellhead.
625. Sobecky, P. A., M. Beazley, R. J. Martinez, S. S. Rajan, J. Powell, Y. Piceno, G. L. Andersen, L. Tom, T. C. Hazen, J. D. Nostrand, J. Zhou, and B. Mortazav. 2011. Coastal Alabama Bacterial Community Responses to the Deepwater Horizon Oil Spill. SIM annual meeting
626. Raloff, J. 2011. Bacteria binged on BP oil but didn?t grow. http://www.sciencenews.org/view/generic/id/333086/title/Bacteria_binged_on_BP_oil_but_didnt_grow__
627. Raloff, J. 2011. Methane from BP spill goes missing. http://www.sciencenews.org/view/generic/id/68461/title/Methane_from_BP_spill_goes_missing
628. Rajan, S., R. J. Martinez, M. J. Beazley, Y. Piceno, G. L. Andersen, T. C. Hazen, P. A. Sobecky, and B. Mortazavi. 2011. Coastal Alabama Microbial Responses to the Deepwater Horizon Oil Spill. ASM annual Meeting.
629. Mosher, J. J., T. J. Phelps, S. L. Carroll, M. M. Drake, C. W. Schadt, M. Podar, S. D. Brown, T. C. Hazen, A. P. Arkin, A. V. Palumbo, B. A. Faybishenko, and D. A. Elias. 2011. Isolation of metal reducing organisms from lactate-enriched contaminated groundwater. ASM annual Meeting.
630. Mosher, J. J., J. G. Moberly, C. W. Schadt, T. J. Phelps, M. Podar, S. D. Brown, A. V. Palumbo, M. W.W. Adams, D. A. Stahl, K. L. Hillesland, J. D. Wall, M. W. Fields, T. C. Hazen, and D. A. Elias. 2011. Characterization of Naturally Occurring and Model Microbial Communities. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy
631. Morrison, J. 2011. Dirty Work. American Way Magazine.
632. Moberly, J. G., T. J. Phelps, C. W. Schadt, M. Podar, S. D. Brown, Z. K. Yang, M. M. Drake, T. C. Hazen, A. P. Arkin, A. V. Palumbo, and D. A. Elias. 2011. Development of a Model Microbial Community for a Systems Biology Level Assessment of Metal-reduction. ASM annual Meeting.
633. Mason, O. U., T. C. Hazen, and J. R. Jansson. 2011. Metagenomic, Metatranscriptomics and single cell genomic analysis of the deep-sea microbial community response the to Deep Water Horizon oil Spill. SIM annual meeting
634. Mason, O. U., R. Lamendella, J. Hultman, R. Mackelprang, S. Borglin, L. M. Tom, E. A. Dubinsky, J. Fortney, T. C. Hazen, and J. K. Jansson. 2011. Metagenomic analysis of the deep-sea microbial community response to the Deepwater Horizon oil spill. ASM annual Meeting.
635. Mason, O. U., T. C. Hazen, P. Chain, E. A. Dubinsky, J. Fortney, J. Han, J. Hultman, R. Lamendella, R. Mackelprang, L. M. Tom, S. G. Tringe, T. Woyke, E. M. Rubin, and J. K. Jansson. 2011. Omics analyses of the deep-sea microbial community response to the Deepwater Horizon Oil Spill. DOE Joint Genome Institute annual user meeting.
636. Martinez, R. J., C. H. Wu, M. J. Beazley, G. L. Andersen, T. C. Hazen, M. Taillefert, and P. A. Sobecky. 2011. Microbial Phosphatase Activity Involved in Subsurface Uranium Sequestration. ASM annual Meeting.
637. Lovett, R. A. 2011. Why Did Huge Oil Plumes Form After the Gulf Spill?. http://news.nationalgeographic.com/news/2011/04/110420-gulf-oil-spill-anniversary-plumes-dispersants-science-nation/
638. Liu, P., R. J. Meagher, Y. Light, S. Yilmaz, R. Chakraborty, A. P. Arkin, T. C. Hazen, and A. K. Singh. 2011. Microfluidic Fluorescence in situ Hybridization and Flow Cytometry Microdevice for Environmental Microbial Detection. ASM annual Meeting.
639. Liu, P., R. J. Meagher, Y. K. Light, S. Yilmaz, R. Chakraborty, A. P. Arkin, T. C. Hazen, and A. K. Singh. 2011. Microfluidic Tools for Single-Cell Genomic Analysis of Environmental Bacteria. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
640. Lamendella, R., S. Borglin, J. Hultman, O. U. Mason, F. Reid, J. Fortney, K. Wetmore, J. Kuehl, H.- C. Lim, T. C. Hazen, and J. Jansson. 2011. Impact of the Deepwater Horizon Oil Spill on beach microbial community dynamics. ASM annual Meeting.
641. Kaufman, L. 2011. Nearly a year after spill, Gulf studies yield more than damage. http://www.bendbulletin.com/article/20110412/NEWS0107/104120407/
642. Joyner, D. C., R. Chakraborty, S. E. Borglin, D. H. Long, and T. C. Hazen. 2011. High Throughput Metabolic Phenotype Profiling of Oil and Dispersant Degrading Consortia from the MC252 Oil Spill in the Gulf of Mexico. ASM annual Meeting.
643. Hulick, K. 2011. Learning from Disasters Exxon-Valdez vs. Deepwater Horizon. Odyssey January 2011 pg 24-26.
644. Hu, P., C. H. Wu, T. Desantis, P. Jasrotia, H. Woo, K. Kearcher, S. Meiss, T. Torok, L. D. Taylor, W. Overholt, S. Green, G. L. Andersen, J. E. Kostka, and T. C. Hazen. 2011. Validation of MycoChip ? A Microarray for Fungal Community Studies. ASM annual Meeting.
645. Hotz, R. L. 2011. Microbes Devoured Methane From BP Spill, Study Says. http://online.wsj.com/article/SB10001424052748703730704576065942040672186.html?mod=googlenews_wsj
646. Hillesland,, K. L., B. Meyer, N. Pinel, N. Elliott, M. Joachimiak, J. Kuehl, A. Deutschbauer, A. Zhou, Z. He, J. Zhou, D. Elias, T. C. Hazen, A. P. Arkin, and D. A. Stahl. 2011. Adaptive Evolution and Physiology of Nascent Microbial Mutualisms. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
647. Hazen, T. C., B. Faybishenko, H. R. Beller, E. Brodie, E. L. Sonnenthal, C. I. Steefel, J. Larsen, M. E. Conrad, J. N. Christensen, S. T. Brown, D. C. Joyner. S. E. Borglin, J. Geller, R. Chakraborty, P. Nico, P. Long, D. Newcomer, and E. Armtzen. 2011. Comparison of field groundwater biostimulation experiments using polylactate and lactate solutions at the Chromium-contaminated Hanford 100-H Site. American Geophysical Union Annual Meeting.
648. Hazen, T. C. 2011. NOAA Marine Microbes Workshop. NOAA.
649. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. University of North Carolina Wilmington Planet Ocean Seminar Series.
650. Hazen, T. C. 2011. Deepwater Horizon Oil Spill - popular media vs. science lessons learned. University of North Carolina Wilmington Planet Ocean Seminar Series.
651. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. IPEC Conference.
652. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Kazakhstan National Library, US State Department Visit to Kazakhstan.
653. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. The Euroasian National University, US State Department Visit to Kazakhstan.
654. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Kazakhstan National Center for Biotechnology, US State Department Visit to Kazakhstan.
655. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Caspian State University of Technologies and Engineering, US State Department Visit to Kazakhstan.
656. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Aktau Region Akimat, US State Department Visit to Kazakhstan.
657. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Atyrau Region Akimat, US State Department Visit to Kazakhstan.
658. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Atyrau Institute of Oil and Gas, US State Department Visit to Kazakhstan.
659. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. American Club, US State Department Visit to Kazakhstan.
660. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. West Kazakhstan State University
661. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. West Kazakhstan Technical University
662. Hazen, T. C. 2011. Ecogenomics enables a new systems biology understanding of the Deepwater Horizon oil spill. UC-Berkeley, Department of Plant and Microbial Biology.
663. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume. Goldschmidt Conference. abstract
     The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
664. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. BP Headquarters.
665. Hazen, T. C. 2011. A Systems Biology Approach?to the Deepwater Horizon Oil Spill ??an?example of Team Science for ecological disasters.. Grimes Distinguished Lecturer Program, Gulf Coast Research Lab.
666. Hazen, T. C. 2011. The Gulf Oil Spill. SIM annual meeting.
667. Hazen, T. C. 2011. Metagenome and single cell sequencing. SIM annual meeting.
668. Hazen, T. C., B. Faybishenko, H. Beller, E. Brodie, E. Sonnenthal, C. Steefel, J. Larsen, M. Conrad, M. Bell, J. Christensen, S. Brown, D. Joyner, S. Borglin, J. Geller, R. Chakraborty, P. Nico, P. Long, D. Newcomer, and E. Arntzen. 2011. Comparison of Field Groundwater Biostimulation Experiments Using Polylactate and Lactate Solutions at the Chromium Contaminated Hanford 100-H Site. The International Symposium on Bioremediation and Sustainable Environmental Technologies.
669. Hazen, T. C. 2011. The Gulf Oil Spill. The International Symposium on Bioremediation and Sustainable Environmental Technologies.
670. Hazen, T. C. 2011. The Gulf Oil Spill. JBEI Seminar Program.
671. Hazen, T. C. 2011. A Systems Biology Approach to the Deepwater Horizon Oil Spill ? an example of Team Science for ecological disasters. Pacific Northwest National Laboratory Frontiers Lecture Series.
672. Hazen, T. C. 2011. Microbial Response to the Deepwater Horizon Oil Spill in the Gulf of Mexico. BAGECO2011 Meeting.
673. Hazen, T. C. 2011. Impact Of BP Spill May Not Be Catastrophic. KTVU http://www.ktvu.com/news/27986393/detail.html
674. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Tulane University
675. Hazen, T. C. 2011. Microbial Ecogenomic Response to the Deepwater Horizon Oil Spill in the Gulf of Mexico - Deepwater Horizon & Beyond. ASM annual Meeting.
676. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. REMTEC.
677. Hazen, T. C. 2011. Microbes Cope with Calamity: Gulf Oil Spill. JGI@Lesher: The Deal with Carbon: How Earth?s Mighty Microbes Respond
678. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Darden Lecture, Department of Biology, University of Alabama.
679. Hazen, T. C. 2011. Panel 1. The Interpretation of Scientific Data for Disaster. Response After the Spill: A Dialogue Between Students and Policymakers. Roosevelt Institute, University of Georgia.
680. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Department of Microbiology, University of Georgia.
681. Hazen, T. C. 2011. The Gulf Oil Spill ? Ecogenomics and Ecoresilience Genomics of Energy & Environment. DOE Joint Genome Institute annual user meeting
682. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Science Night for Orinda Intermediate School.
683. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. Presentation arranged by the Center for Safe Energy: for the Environment and Energy ministers of the Republic of Kazakhstan.
684. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. University of Wisconsin.
685. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. Purdue University Sigma Xi meeting.
686. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. AAAS annual meeting.
687. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Luncheon Seminar to Ex-LBNL employees.
688. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Regional Science Council Seminar Series, EPA region 9.
689. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. Stanford University.
690. Grossman, E. 2011. Murky Waters.
691. Grimes, D. J., T. C. Hazen, S. McClellan, M. Sogin, and J. B. Paul. 2011. Vibrios and petroleum biodegradation ? capable but contributory ?. SIM annual meeting.
692. Gorur, A., C. M. Leung, A. Tauscher, D. Jorgens, S. Reveco, J. Remis, B. Lam, J. T. Geller, T. C. Hazen, T. Juba, S. Chhabra, J. Wall, M. Biggin, K. H. Downing, and M. Auer. 2011. High Throughput Subcellular Protein Expression and Localization Studies in the Anaerobic Sulfate Reducer Desulfovibrio vulgaris. ASM annual Meeting.
693. Gorur, A., C. M. Leung, S. Chhabra, T. Juba, A. Tauscher, S. Reveco, J. P. Remis, B. Lam, J. T. Geller, T. C. Hazen, M. Biggin, J. M. Chandonia, K. H. Downing, J. Wall, and M. Auer. 2011. Subcellular Localization of Proteins in the Anaerobic Sulfate Reducer Desulfovibrio vulgaris via SNAP-Tag Labeling and Photoconversion. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
694. Goldenberg, S. 2011. Has BP really cleaned up the Gulf oil spill? April 14. http://www.guardian.co.uk/environment/2011/apr/13/deepwater-horizon-gulf-mexico-oil-spill
695. Geller, J. T., H. Woo, D. C. Joyner, S. Kendall, and T. C. Hazen. 2011. Microfluidic Studies of Nitrate Stress on Shewanella oneidensis Biofilms. ASM annual Meeting.
696. Forrester, M. 2011. Study: Undersea bugs ate natural gas released in oil spill. http://video-game-schools.freeawx.com/study-undersea-bugs-ate-natural-gas-released-in-oil-spill/
697. Fancher, L. 2011. Mother Nature Cleans up Gulf Oil Spill. Walnut Creek Patch
698. Dickey Zakaib, G.. 2011. Role of bacteria in Gulf oil spill under the microscope.
699. DHaeseleer, P. and A. M. Redding-Johanson, C. J. Petzold, P. I. Benke, M. Allgaier, D. C. Chivian, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, and S. W. Singer J. M. Gladden. 2011. Metagenomics, Proteomics, and Metabolic Reconstruction of a Thermophilic Feedstock-adapted Bacterial Community. ASM annual Meeting.
700. DHaeseleer, P. and A. M. Redding-Johanson, C. J. Petzold, P. I. Benke, M. Allgaier, D. C. Chivian, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, and S. W. Singer J. M. Gladden. 2011. Metagenomics, Proteomics, and Metabolic Reconstruction of a Thermophilic Feedstock-Adapted Bacterial Community. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy
701. DeAngelis, K. M., P. D'Haeseleer, J. Fortney, S. Borglin, W. L. Silver, and T. C. Hazen. 2011. Metagenomics of anaerobic lignocellulolytic feedstock-adapted consortia derived from tropical forest soils. SIM annual meeting
702. DeAngelis, K. M., J. Fortney, S. Borglin, W. Silver, and T. C. Hazen. 2011. Feedstock-Adapted Anaerobic Consortia Derived from Tropical Forest Soils. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy
703. DeAngelis, K. M., W. L. Silver, and T. C. Hazen. 2011. Anaerobic deconstruction of switchgrass by tropical soil-derived feedstock adapted consortia. DOE Joint Genome Institute annual user meeting.
704. De Leon, K. B., B. D. Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen, J. Zhou, and M. W. Fields. 2011. Microbial Community Dynamics from Groundwater and Surrogate Sediments During HRC¿ Biostimulation for Cr(VI)-Reduction. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
705. Conrad, M. E., M. Bill, W.T. Stringfellow, S. E. Borglin, O. U. Mason, E. A. Dubinsky, Y. M. Piceno, J. L. Fortney, L. M. Tom,? K. L. Chavarria, R. Lamendella, D. C. Joyner, K. Wetmore, J. Kuehl, R. Mackelprang, C. Wu, J. Lim, F. Reid, and T. C. Hazen. 2011. Isotopic evidence for microbial oxidation of dissolved methane in the Gulf of Mexico oil spill deep plume. Goldschmidt Conference.
706. Chhabra, S., B. Gold, N. L. Liu, S. Reveco, T. R. Juba, J. D. Wall, B. R. Lam, J. T. Geller, T. C. Hazen, M. Choi, M. D. Biggin, E. D. Szakal, S. Allen, H. Witkowska, J-M. Chandonia, and G. P. Butland. 2011. Engineering Desulfovibrio vulgaris Hildenborough for High Throughput Tandem Affinity Purification of Protein Complexes. ASM annual Meeting.
707. Chhabra,, S., M. Auer, G. Butland, J.-M. Chandonia, T. C. Hazen, J. D. Wall, E. Witkowska, D. Elias, M. Adams, M. Fields, J. Liphardt, G. Hura, and D. Stahl. 2011. High Throughput Production and Analysis of Genetically Engineered Desulfovibrio vulgaris Strains via Homologous Recombination. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
708. Chandonia, J-M., M. Dong, M. Shatsky, H. Liu, S. E. Brenner, L. Yang, T. C. Hazen, J. T. Geller, M. Choi, E. D. Szakal, J. Jin, H. E. Witkowska, A. P. Arkin, and M. D. Biggin. 2011. Accurate, High-Throughput Identification of Stable Protein Complexes Using a Tagless Strategy. ASM annual Meeting.
709. Chandonia, J.-M., M. Dong, M. Shatsky, H. Liu, L. Yang, T. C. Hazen, J. T. Geller, M. Choi, E. D. Szakal, S. Allen, S. E. Brenner, S. C. Hall, S. J. Fisher, S. Kumar, F. L. Poole, M. Adams, J. Jin, H. E. Witkowska, A. P. Arkin, and M. D. Biggin. 2011. Accurate, High-Throughput Identification of Stable Protein Complexes in Desulfovibrio vulgaris using a Tagless Strategy. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
710. Chakraborty, R., T. C. Hazen, S. E. Borglin, and D. C. Joyner. 2011. Degradation and oil and dispersant by microbes isolated from the Gulf of Mexico in the aftermath of MC252 oil spill. SIM annual meeting.
711. Chakraborty, R., S. E. Borglin, D. H. Long, D. C. Joyner, and T. C. Hazen. 2011. Interaction of MC252 oil and COREXIT with isolates and enrichments from Gulf of Mexico. ASM annual Meeting.
712. Chakraborty, R., D. Joyner, B. A. Faybishenko, M. Fields, T. Torok, G. L. Andersen, and T. C. Hazen. 2011. Integrated Microbiological Approaches to Characterize Cr(VI)-Reducing Microbial Community at the DOE Hanford 100H Site. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
713. Cassidy, G. 2011. One Year Later: Did Bacteria Save Oil and Gas Stocks?. http://www.benzinga.com/trading-ideas/long-ideas/11/04/1011228/one-year-later-did-bacteria-save-oil-and-gas-stocks
714. Butland, G. P., S. R. Chhabra, B. Gold, N. L. Liu, S. Reveco, T. R. Juba, J. D. Wall, B. R. Lam, J. T. Geller, T. C. Hazen, M. Choi, M. D. Biggin, E. D. Szakal, S. Allen, H. Liu, H. E. Witkowska, and J.-M. Chandonia. 2011. High Throughput Identification of Protein Complexes from Desulfovibrio vulgaris by a Tandem Affinity Purification Pipeline. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
715. Borglin, S. E., Y. Piceno, D. C. Joyner, J. Fortney, and T. C. Hazen. 2011. Analysis of microbial community structure and alkane composition in Mississippi Canyon oil spill using phospholipid fatty acid analysis. ASM annual Meeting.
716. Bohan, S. 2011. Lawrence Berkeley Lab scientists tinker with microbes to battle climate change. Contra Costa Times
717. Berwyn, B. 2011. Microbial cleanups touted for major oil spills. http://summitcountyvoice.com/2011/08/20/microbial-cleanups-touted-for-major-oil-spills/
718. Baelum, J., S. E. Borglin, J. L. Fortney, R. Lamendella, O. U. Mason, M. Bill, M. E. Conrad, S. Malfatti, S. Tringe, H.-Y. Holman, T. C. Hazen, and J. K. Jansson. 2011. Simulations of the microbial community response to the Deepwater Horizon Oil spill in the Gulf of Mexico using a microcosm approach. BAGECO2011 Meeting.
719. Baelum, J., S. Borglin, J. L. Fortney, R. Lamendella, O. U. Mason, M. Bill, M. E. Conrad, H-Y. Holman, S. A. Malfatti, S. Tringe, T. C. Hazen, and J. K. Jansson. 2011. Microcosm Simulations of the Microbial Community Response to the Deepwater Horizon Oil Spill in the Gulf of Mexico. ASM annual Meeting.
720. Anthony, L. 2011. UC researcher reflects on Gulf oil spill.
721. Andersen, G. L., E. Dubinsky, Y. M. Piceno, L. Tom, K. Sublette, T. Z. DeSantis, T. C. Hazen, and S. E. Borglin. 2011. Response of petroleum-degrading microbial communities at different depths to the Deepwater Horizon oil spill. SIM annual meeting.
722. 2011. Lessons learned from the two worst oils spills in US history: Microbes matter. http://www.bayoubuzz.com/louisiana-local-news/bp-oil-spill/323275-lessons-learned-from-the-two-worst-oils-spills-in-us-history-microbes-matter
723. 2011. Microbes? role in oil spills investigated. http://www.istockanalyst.com/business/news/5370015/microbes-role-in-oil-spills-investigated
724. 2011. Berkeley Lab Scientist Wins PNNL?s Outstanding Lecture Award. Today at Berkeley Lab, http://today.lbl.gov/2011/06/17/berkeley-lab-scientist-wins-pnnl?s-outstanding-lecture-award/
725. 2011. Bacteria. The Current, Canadian Broadcast Company
726. 2011. Bacteria devoured methane gas from gulf oil spill, scientists say. http://berkeleylabreport.blogspot.com/2011/01/bacteria-devoured-methane-gas-from-gulf.html
727. Zhang, P., W. Wu, J. Van Nostrand, Y. Deng, Z. He, T. Gihring, G. Zhang, C. Schadt, D. Watson, P. Jardine, S. Brooks, T. Marsh, J. Tiedje, T. C. Hazen, and J. Zhou. 2010. Microarray-based characterization of microbial community functional structure during in situ biostimulation at a uranium-contaminated aquifer. ISME 13 ? 13th International Symposium on Microbial Ecology>
728. Zhang, P., W. Wu, J. D. Van Nostrand, Y. Deng, Z. He, T. Gihring, G. Zhang, C. W. Schadt, D. Watson, P. Jardine, S. Brooks, T. L. Marsh, J. M. Tiedje, T. C. Hazen, and J. Zhou. 2010. Geochip-based analysis of metabolic diversity of microbial communities during in situ biostimulation at a uranium-contaminated aquifer. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
729. Yarris, L. 2010. Caution Required for Gulf Oil Spill Clean-up. www.articleant.com
730. Yarris, L. 2010. Caution Required for Gulf Oil Spill Clean-up. Berkeley Lab News Center.
731. Wurth, J. 2010. Professor's expertise might help with cleanup of future oil spills. The News Gazette. http://www.news-gazette.com/news/environment/2010-08-08/professors-expertise-might-help-cleanup-future-oil-spills.html
732. Wu, C. H., E. A. Dubinsky, J. Hulls, S. R. Osman, T. C. Hazen, and G. L. Andersen. 2010. Temporal dynamics of cattle and human fecal microbial communities in fresh and marine waters. ISME 13 ? 13th International Symposium on Microbial Ecology.
733. Woo, H. L., K. M. DeAngelis, T. C. Hazen, and B. A. Simmons. 2010. Isolation of lignin- and cellulose- degrading bacteria from tropical soils for biofuel feedstock deconstruction. Annual Meeting of the Society for Industrial Microbiology.
734. Woo, H. L., K. M. DeAngelis, T. C. Hazen, and B. A. Simmons. 2010. Isolation of Aerobic Lignin- and Cellulose- degrading Bacteria from Tropical Soils from Biofuel Feedstock Deconstruction. Annual meeting of the American Society for Microbiology Meeting.
735. Wells, G. F., C. H. Wu, Y. M. Piceno, B. Eggleston, E. L. Brodie, T. Z. DeSantis, G. L. Andersen, T. C. Hazen, C. A. Francis, and C. S. Criddle. 2010. Immigration as a driver of microbial community structure: sloughing from a biofilm bioreactor is linked to downstream-activated sludge community dynamics. ISME 13 ? 13th International Symposium on Microbial Ecology.
736. Waters, K. 2010. Oil Spill Eaten By RidX? June 22.
737. Walian, P. J., S. Allen, L. Zeng, E. Szakal, E. Johansen, H. Liu, S. C. Hall, S. J. Fisher, M. E. Singer, J. T. Geller, S. Lin, T. C. Hazen, H. E. Witkowska, M. D. Biggin, and B. K. Jap. 2010. Pipeline for Large-scale Purification and Identification of Desulfovibrio vulgaris Membrane Protein Complexes. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
738. Vilcaez, J., L. Li, S. S. Hubbard, and T. C. Hazen. 2010. Biodegradation of Deep-Sea Oil Spill at the Gulf of Mexico: an Estimate of Half Life Time. AGU annual meeting.
739. Vieru, T. 2010. Berkeley Lab Scientists on Hot to Clean Gulf Oil Spill. Sci Pry.
740. Van Nostrand, J. D., L. Wu, P. Waldron, P. Zhang, Y. Deng, Z. He, W. Wu, S. Carroll, C. Schadt, A. Palumbo, D. Watson, C. Criddle, P. Jardine, T. C. Hazen, and J. Zhou. 2010. Applications of GeoChip for analysis of different microbial communities. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
741. Van Nostrand, J., P. Waldron, D. Watson, Z. He, L. Wu, P. Jardine, T. C. Hazen, and J. Zhou. 2010. Geochip-based analysis of groundwater microbial communities across a gradient of pH, heavy metal, and nitrate contamination. ISME 13 ? 13th International Symposium on Microbial Ecology.
742. Tunnicliffe, H. 2010. BP plugs one leak, containment dome ready. www.tcetoday.com.
743. Trevedi, B. 2010. Feature Story ? The Right Bugs. www.miller-mccune.com.
744. Timberlake, S. C., M. P. Joachimiak, D. Joyner, R. Chakraborty, J. K. Baumohl, P. S. Dehal, A. P. Arkin, T. C. Hazen, and E. J. Alm. 2010. Conservation of Modules but not Phenotype in Bacterial Stress Responses. Annual meeting of the American Society for Microbiology Meeting.
745. Tierney, J. 2010. BP's Detergents Might Cause More Harm.
746. Tasker, F. 2010. BP's Gulf leak boosts interests in oil-eating microbes.
747. Smith, M. 2010. Bugs in the Water.
748. Singer, S. W., J. M. Gladden, P. D'haeseleer, M. Allgaier, D. C. Chivian, T. C. Hazen, J. S. VanderGheynst, P. Hugenholtz, and B. A. Simmons. 2010. Targeted Discovery of Enzymes From Enriched Microbial Consortia for High Temperature Saccharification of Ionic-Liquid Pre-Treated Biomass. American Institute of Chemical Engineers annual meeting.
749. Singer, S. W., J. M. Gladden, M. Allgaier, A. P. Reddy, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, and P. Hugenholtz. 2010. Targeted Enzyme Discovery in Thermophilic Feedstock-adapted Microbial Communities. 32nd Symposium on Biotechnology for Fuels and Chemicals.
750. Singer, S., J. Gladden, M. Allgaier, A. Reddy, J. VanderGheynst, T. C. Hazen, P. Hugenholtz, P. D'haeseleer, B. Simmons. 2010. Targeted enzyme discovery in thermophilic feedstock-adapted microbial communities using proteogenomic and biochemical techniques. ISME 13 ? 13th International Symposium on Microbial Ecology.
751. Siegel, R. P. 2010. How to Clean Up the Oil. www.triplepundit.com.
752. Schoof, R. 2010. Despite Gulf cleanup efforts, nature will have to do most it. http://www.mcclatchydc.com/2010/07/16/97702/despite-gulf-cleanup-efforts-nature.html
753. Reddy, A. P., M. Allgaier, J. M. Gladden, S. W. Singer, P. Hugenholtz, B. A. Simmons, T. C. Hazen, and J. S. VanderGheynst. 2010. Characterization of the activity of thermophilic microbial communities on bioenergy feedstocks. 32nd Symposium on Biotechnology for Fuels and Chemicals.
754. Reddy, A. P., M. Allgaier, J. M. Gladden, S. Singer, P. Hugenholtz, B. Simmons, T. C. Hazen, and J. S. VanderGheynst. 2010. Enrichment of highly efficient thermophilic microbial communities active on switchgrass and corn stover in a high-solids environment. 239th ACS National Meeting & Exposition.
755. Raval, A. 2010. The right spill response: cautious optimism about the Gulf?s future.
756. Piceno, Y. M., D. Venkateswaran, L. Tom, S. Chaudhuri, M. Vu, T. C. Hazen, R. Chakraborty, G. L. Andersen. 2010. Optimization of extraction techniques for microbial community analysis of MEOR samples. Annual meeting of the American Society for Microbiology Meeting.
757. Paine, A. 2010. Tennesseans donate hair, pantyhose to sop up oil spill.
758. Outzen, R. 2010. Doing nothing is better than dispersants. http://ricksblog.biz
759. Ott, R., K. Arnold, J. Hofmeister, T. Hazen, and K. M. Yeager. 2010. Plan B in the Gulf. The New York Times (www.nytimes.com)
760. Northen, T., W. Reindl, K. Deng, J. Gladden, S. Singer, A. Singh, T. C. Hazen, B. Simmons, P. Adams, and J. Keasling. 2010. High Throughput Multiplexed GlycoChip Enzymatic Assays for Biofuels Development. American Institute of Chemical Engineers annual meeting.
761. The Medical Muckraker. 2010. Did oil-gobbling bacteria rid the Gulf of BP?s submarine oil plumes?.
762. Mosher, J. J., M. M. Drake, S. L. Carroll, Z. K. Yang, C. W. Schadt, S. D. Brown, M. Podar, T. C. Hazen, A. P. Arkin, T. J. Phelps, A. V. Palumbo, B. A. Faybishenko, and D. A. Elias. 2010. Lactate enrichment of uranium and chromium contaminated Hanford groundwater samples. ISME 13 ? 13th International Symposium on Microbial Ecology.
763. Mosher, J. J., M. M. Drake, S. L. Carroll, Z. K. Yang, C. W. Schadt, S. D. Brown, M. Podar, T. C. Hazen, A. P. Arkin, T. J. Phelps, A. V. Palumbo, B. A. Faybishenko, and D. A. Elias. 2010. Microbial community dynamics of lactate enriched Hanford groundwaters. Annual meeting of the American Society for Microbiology Meeting.
764. Morgan, C. 2010. When will spill be cleaned up? Maybe never. Brandenton. http://www.bradenton.com/2010/08/02/2474305/recovery-test-of-technology-nature.html
765. Morgan, C. 2010. When will spill be cleaned up? Maybe never. Kansas City Star. http://www.kansascity.com/2010/07/31/2119839/when-will-spill-be-cleaned-up.html
766. Morgan, C. 2010. When will spill be cleaned up? Maybe never. The Republic. http://www.therepublic.com/view/story/OILSPILL-CLEANUP_2910483/OILSPILL-CLEANUP_2910483/
767. Metzner, J. 2010. Pulse of the Planet.
768. Mason, O. U., A. Probst, E. Dubinsky, Y. Piceno, L. Tom, J. Fortney, R. Lamendella, T. C. Hazen, and J. Jansson. 2010. The microbial community of a dispersed, deep-sea oil plume. ISME 13 ? 13th International Symposium on Microbial Ecology.
769. Mason, O. U., E. A. Dubinsky, Y. M. Piceno, A. T. Iavarone, L. M. Tom, T. C. Hazen, and J. K. Jansson. 2010. Omics analyses of resident microbes in extreme environments such as petroleum reservoirs and deep sea oil plumes. Annual Meeting of the Society for Industrial Microbiology.
770. Mason, O., A. Iavarone, L. Tom, S. Borglin, R. Chakraborty, T. C. Hazen, and J. Jansson. 2010. Pressure assisted omics analyses of resident microbes in petroleum reservoirs. ISME 13 ? 13th International Symposium on Microbial Ecology.
771. Martinez, R., M. Beazley, C. Wu, G. Andersen, T. C. Hazen, M. Taillefert, and P. Sobecky. 2010. Microbial phosphatase activity involved in subsurface uranium sequestration. ISME 13 ? 13th International Symposium on Microbial Ecology.
772. Lowe, M. 2010. Tactics and Techniques for Tackling the Oil Spill. Magblog.audubon.org.
773. Liu, H., M. Dong, L. L Yang, E. D Szakal, S. Allen, S. C. Hall, S. J. Fisher, T. C. Hazen, J. T. Geller, M. E Singer, J. Jin, M. D. Biggin, and H. E. Witkowska. 2010. Development and Refinement of an iTRAQ-Based Tagless Strategy for High-Throughput Purification and Identification of Soluble Protein Complexes. 58th ASMS Conference on Mass Spectrometry.
774. Khudyakov, J. I., K. M. DeAngelis, H. Woo, S. Borglin, T. C. Hazen, and M. Thelen. 2010. Ionic liquid tolerance in Enterobacter cloacae, a lignocellulolytic bacterium isolated from tropical rain forest soil. Annual Meeting of the Society for Industrial Microbiology.
775. Kaufman, R. 2010. 3 Future Oil-Spill Fighters: Sponges, Superbugs, and Herders. National Geographic Daily News.
776. Joyner, D. C., Hazen, T. C. 2010. Phenotypic MicroArray for bioenergy applications. Florence Conference on Phenotype Microarray Analysis of Microorganisms.
777. Joyner, D. C., J. L. Fortney, R. Chakraborty, and T. C. Hazen. 2010. Adaptation the Biolog OmniLog Phenotype MicroArray plate technology to profile the strict metal reducing anaerobe Geobacter metallireducens. Annual meeting of the American Society for Microbiology Meeting.
778. Joachimiak, M. P., R. Chakraborty, A. Zhou, J. L. Fortney, J. T. Geller, Z. He, J. Wall, J. Zhou, A. P. Arkin, T. C. Hazen, J. D. Keasling and S. R. Chhabra. 2010. Revisiting modes of energy generation in sulfate reducing bacteria. Annual meeting of the American Society for Microbiology Meeting.
779. Joachimiak, M., R. Chakraborty, A. Zhou, J. L. Fortney, Z. He, P. Dehal, M. R. Price, J. Wall, J. Zhou, A. P. Arkin, T. C. Hazen, J. D. Keasling, and S. R. Chhabra. 2010. A revised bioenergetic model of Desulfovibrio vulgaris strain Hildenborough. Annual meeting of the American Society for Microbiology Meeting.
780. Hubbard, S. S., J. Ajo-Franklin, H. Beller, E. Brodie, J. Chen, J. Christensen, M. Conrad, D. DePaolo, B. Faybishenko, S. Finsterle, T. C. Hazen, M. Kowalsky, E. Sonnenthal, N. Spycher, C. Steefel, T. Tokunaga, J. Wan, K. Williams, Y. Wu, M. Denham, Y. Fujita, L. Li, and P. Long. 2010. Overview of the LBNL Sustainable Systems SFA. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
781. Holman, H.-Y., E. Wozei, L. R. Comolli, S. A. Ball, S. E. Borglin, M. W. Fields, T. C. Hazen, and K. H. Downing. 2010. Real-Time monitoring of Chemical Environment in Cells during Stress-Adaptive Response. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
782. Hazen, T. C. and P. Bayer. 2010. Systems Environmental Microbiology: Innovative Approaches to Understand Cellular and Microbial Community Activity and Function. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
783. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. Science@Cal.
784. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. LBNL invited press conference for AGU annual meeting.
785. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. Contra Costa Watershed Forum.
786. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. Vibrios in the Environment 2010.
787. Hazen, T. C. 2010. The Gulf of Mexico Oil Spill - Rescue from Microbiology?. Danish Society for Microbiology annual meeting.
788. Hazen, T. C. 2010. Oil Spill Aftermath. Discover Cal Lecture Series.
789. Hazen, T. C. 2010. LBNL Oil Spill Research. Town Hall Office of Chief Financial Officer LBNL.
790. Hazen, T. C. 2010. Biosciences in Earth Sciences Division. LBNL Scientific Advisory Committee.
791. Hazen, T. C. 2010. Oil Spill Aftermath. Discover Cal Lecture Series.
792. Hazen, T. C., R. M. Atlas, D. J. Grimes, J. Spain, and J. M. Suflita. 2010. Microbes and Oil Spills Mini-Colloquium. American Academy of Microbiology.
793. Hazen, T. C. 2010. Deep Water Horizon Oil Spill - Intrinsic Bioremediation or Mother Natures? Abilities to Cleanup Our Messes. City College of San Francisco.
794. Hazen, T. C. 2010. MEHR Program Ecogenomics Core Review. Energy Biosciences Institute, University of California at Berkeley.
795. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. Civil and Environmental Engineering, University of California at Berkeley.
796. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. Northern California Science Writers Association Dinner.
797. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. LBNL Open House.
798. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. BP Deep Sea Oil Release Water Column Meeting.
799. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. National Commission on the BP Deepwater Horizon Oil Spill and Offshore Drilling.
800. Hazen, T. C. 2010. Ecogenomics and biogeochemistry enables understanding of the Deepwater Horizon disaster in the Gulf of Mexico. University of Granada.
801. Hazen, T. C. 2010. Phenotypic MicroArray for bioenergy applications. Florence Conference on Phenotype Microarray Analysis of Microorganisms.
802. Hazen, T. C. 2010. The Gulf of Mexico Oil Spill. Florence Conference on Phenotype Microarray Analysis of Microorganisms.
803. Hazen, T. C. 2010. Undersea oil-eating bacteria. http://www.sciencefriday.com/program/archives/201008275
804. Hazen, T. C. 2010. Microbial ecology of the 2010 Deepwater Horizon Oil Spill. ISME 13 ? 13th International Symposium on Microbial Ecology.
805. Hazen, T. C. 2010. The Spill and Ecogenomics. ISME 13 ? 13th International Symposium on Microbial Ecology.
806. Hazen, T. C. 2010. Gulf spill biodegradation rates and ecogenomics. NOAA National Science Response Team.
807. Hazen, T. C. 2010. Systems Biology: The New Frontier for Environmental Biotechnology. Army Sponsored Microbial Data Integration Workshop.
808. Hazen, T. C. 2010. Gulf spill biodegradation rates and ecogenomics. National Incident Response JAG.
809. Hazen, T. C. 2010. Environmental Microbiology ? Deconstruction Ecogenomics for Biofuels. Annual Meeting of the Society for Industrial Microbiology.
810. Hazen, T. C. 2010. Environmental Microbiology - In Situ Bioremediation and Ecogenomics. Annual Meeting of the Society for Industrial Microbiology.
811. Hazen, T. C. 2010. Gulf Oil Spill, Bioremediation the Hope and the Hype. LBNL Center for Science and Engineering Education.
812. Hazen, T. C. 2010. Gulf Oil Spill, Update. LBNL Earth Sciences Division Town Hall meeting.
813. Hazen, T. C. 2010. Forum on BP Oil Disaster. Mt. Diablo Peace & Justice Center.
814. Hazen, T. C. 2010. Systems Biology Approach to Bioremediation: Omics and Hydrobiogeochemical Processes. Goldschmidt 2010: Earth, Energy and the Environment.
815. Hazen, T. C., E. Dubinsky, and O. U. Mason. 2010. Deepwater Horizon Oil Spill. LBNL chapter of Institute for Nuclear and Particle Astrophysics (INPA)
816. Hazen, T. C. 2010. Extreme Caution Required for Gulf Oil Spill Clean-up. Axis of Logic.
817. Hazen, T. C. 2010. Ecogenomics with MicrobesOnline and KnowledgeBase. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
818. Hazen, T. C. 2010. A Systems Biology Approach to the Environment Using Ecogenomics. Earth Sciences Division Review.
819. Hazen, T. C., G. Anderson, S. Borglin, E. Brodie, S. van Dien, M. Fields, J. Fortney, J. Geller, E. Hendrickson, K. L Hillesland, H.-Y. Holman, J. Leigh, T. Lie, D. Joyner, R. Chakraborty, D. Elias, A. Mukhopadhyay, C. Schadt, D. Stahl, S. Stolyar, C. Walker, J. Wall, Z. Yang, H.-C. Yen, G. Zane, J. Zhou. 2010. Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers ENIGMA:VIMSS:ESPP. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
820. Hazen, T. C. 2010. Systems Biology (Integration of the Omics, Bioinformatics, and Biogeochemistry): The New Frontier for Environmental Biotechnology. Central South China University
821. Hazen, T. C. 2010. Life in the Slow Lane: Ecogenomics in Extreme Environments - Implications for Enhanced Oil Recovery. Harbin Institute of Technology
822. Hazen, T. C. 2010. Systems Biology (Integration of the Omics, Bioinformatics, and Biogeochemistry): The New Frontier for Environmental Biotechnology. Harbin Institute of Technology
823. Hazen, T. C. 2010. Targeted Enzyme Discovery using Metagenomics. Harbin Institute of Technology
824. Harrell, A. 2010. Your Hair Is Not Enough to Combat Major Oil Spill. SFweekly, blogs.sfweekly.com.
825. Han, B. G., H. Liu, M. Dong, M. Shatsky, S. E. Brenner, P. Arbelaez, J. Malik, D. Typke, T. C. Hazen, J. T. Geller, H. J. Sterling, L. Yang, M. Choi, E. D. Szakal, S. Allen, S. C. Hall, Susan J. Fisher, E. R. Williams, J.-M. Chandonia, J. Jin, H. E. Witkowska, R. M. Glaeser, M. D. Biggin. 2010. High Throughput Identification, Purification and Structural Characterization of Soluble Protein Complexes in Desulfovibrio vulgaris. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
826. Gupta, S. 2010. Gulf spill: Is the oil lurking underwater?.
827. Gupta, S. 2010. Deep oil in Gulf appears to have vanished.
828. Grimes, D. J., T. C. Hazen, and S. McLellan. 2010. Marine Bioremediation: The Microbial Response to the Deepwater Horizon Incident. International Marine Biotechnology Convention 2010.
829. Graffis, M. 2010. Caution Required for Gulf Oil Spill Clean-up. misc.activism.progressive.
830. Goodheart, D. B., W. L. Silver, T. C. Hazen, and M. K. Firestone. 2010. The Diversity and Activity of Methanogens in a Wet, Tropical Forest during Plant Decomposition. ISME 13 ? 13th International Symposium on Microbial Ecology.
831. Goldenberg, S. 2010. Gulf oil spill: White House accused of spinning report. Guardian. http://www.guardian.co.uk/environment/2010/aug/05/oil-spill-white-house-accused-spin
832. Gladden, J. M., A. M. Reddy, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, P. Hugenholtz, and S. W. Singer. 2010. Targeted enzyme discovery in feedstock-adapted microbial communities. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
833. Geller, J. T., S. E. Borglin, J. L. Fortney, B. R. Lam, T. C. Hazen, and M. D. Biggin. 2010. Large-Scale, Continuous-Flow Production of Stressed Biomass (Desulfovibrio vulgaris Hildenborough). Annual meeting of the American Society for Microbiology Meeting.
834. Fowler, P. 2010. Oil Cleanup Could Be Worse For Environment Than Actual Spill, Expert Says. www.newsroomamerica.com.
835. Fortney, J. L., K. M. DeAngelis, Y. Chavarria, W. Silver, and T. C. Hazen. 2010. Anaerobic feedstock-adapted consortia and isolates from Puerto Rico tropical forest soils. Annual meeting of the American Society for Microbiology Meeting.
836. Dubinsky, E. A., G. L. Andersen, M. E. Conrad, O. U. Mason, Y. M. Piceno, W. T. Stringfellow, J. Zhou, and T. C. Hazen. 2010. Response of petroleum-degrading microbial communities to the Deepwater Horizon oil spill at the surface and in the deep. Annual Meeting of the Society for Industrial Microbiology.
837. Dubinsky, E., G. L. Andersen, M. E. Concord, O. U. Mason, Y. Piceno, W. T. Stringfellow, J. Zhou, and T. C. Hazen. 2010. Response of Petroleum-Degrading Microbial Communities to the Deepwater Horizon Oil Spill at the Surface and in the Deep. Sustainable Approaches to Remediation of Contaminated Land (SARCL-2010) and Contaminated Site Management (CSM-2010).
838. Dubinsky, E., T. DeSantis, Y. Piceno, O. Mason, N. Singh, A. Probst, D. Joyner, R. Chakraborty, T. C. Hazen, G. Andersen. 2010. Phylochip assay finds deepwater oil plume enrichment of psychrophilic oil-degrading bacteria. ISME 13 ? 13th International Symposium on Microbial Ecology.
839. DeAngelis, K. M., M. Allgaier, Y. Chavarria, J. Fortney, P. Hugenholtz, B. Simmons, K. Sublette and and T. C. Hazen W. L. Silver. 2010. Trapping Lignin Degrading Microbes in Tropical Forest Soil. Annual meeting of the American Society for Microbiology Meeting.
840. DeAngelis, K. M., M. Allgaier, P. D?haeseleer, J. L. Fortney, P. Hugenholtz, B. Simmons, and T. C. Hazen. 2010. Analysis of anaerobic lignocellulose decomposing consortia from Puerto Rico tropical forest soils. ISME 13 ? 13th International Symposium on Microbial Ecology.
841. DeAngelis, K. M., M. Allgaier, W. L. Silver, Y. Chavarria, J. Fortney. P. Hugenholtz, B. Simmons, K. Sublette, and T. C. Hazen. 2010. Trapping lignin-degrading microbes in tropical forest soil. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
842. De Leon, K., D. Newcomer, B. Faybishenko, B. Ramsay, T. C. Hazen, and M. Fields. 2010. Microbial community dynamics from groundwater and surrogate sediments during HRC¿ stimulation at a chromium contaminated field site. ISME 13 ? 13th International Symposium on Microbial Ecology.
843. Christensen, J. N., E. Sonnenthal, S. T. Brown, M. E. Conrad, L. Yang, S. Mukhopadhyay, C. I. Steefel, B. Faybishenko, and T. C. Hazen. 2010. Using Cr Isotopic Measurements Together with Reactive Transport Modeling to Monitor Stimulated Bio-containment at the 100H Test Site, Hanford, Washington. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
844. Chhabra, S., G. Butland, D. Elias, S. Reveco, V. Fok, B. Gold, T. Juba, J.-M. Chandonia1, E. Witkowska, T. C. Hazen, J. Wall, and J. Keasling. 2010. Protein Complex Analysis Project (PCAP): Large-scale identification of protein-protein interactions in Desulfovibrio vulgaris using tandem-affinity purification. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
845. Chakraborty, R., and T. C. Hazen. 2010. Microbial community changes during sustained Cr(VI) reduction at the 100H site in Hanford, WA. Annual Meeting of the Society for Industrial Microbiology.
846. Chakraborty, R., E. L. Brodie, B. Faybishenko, Y. M. Piceno, L. Tom, S. Choudhuri, H. R. Beller, J. Liu, T. Torok, D. C. Joyner , P. E. Long, D. R. Newcomer, G. L. Andersen, and T. C. Hazen. 2010. Microbial community changes during sustained Cr(VI) reduction at the 100H site in Hanford, WA. Annual meeting of the American Society for Microbiology Meeting.
847. Chakraborty, C., J. Fortney, A. Zhou, M. Joachimiak, A. Mukhopadhyay, S. Borglin, Z. He, A. P. Arkin, J. Zhou, and T. C. Hazen. 2010. Investigation of osmotic stress response in the anaerobic metal-reducing microbe Geobacter metallireducens, strain GS -15. ISME 13 ? 13th International Symposium on Microbial Ecology.
848. Byrne-Bailey, K. G., K. C. Wrighton, R. A. Melnyk, T. C. Hazen, and J. D. Coates. 2010. The First Genome Sequence of a Gram-Positive Bacterium Isolated from a Microbial Fuel Cell: Thermincola potens strain JR. Joint Genome Institute Users Meeting.
849. Borglin, S. E., O. U. Mason, E. Dubinsky, J. Fortney, R. Lamendella, D. Joyner, Y. Piceno, and T. C. Hazen. 2010. Analysis of microbial community structure in crude oil and oil spill samples using phospholipid fatty acid analysis. Annual Meeting of the Society for Industrial Microbiology.
850. Borglin, S., O. U. Mason, E. Dubinsky, J. Fortney, R. Lamendella, D. Joyner, Y. Piceno, and T. C. Hazen. 2010. Analysis of Microbial Community Structure in Crude Oil and Oil Spill Samples Using Phospholipid Fatty Acid Analysis. Sustainable Approaches to Remediation of Contaminated Land (SARCL-2010) and Contaminated Site Management (CSM-2010).
851. Ball, D. A., S. Chhabra, D. Elias, V. Fok, J. T. Geller, A. Gorur, T. C. Hazen, D. Jorgens, T. Juba, A. Leung, J. Remis, m. E. Singer A. Tauscher, J. Wall, M. Auer, and K. H. Downing. 2010. Towards localization of functionality in Desulfovibrio vulgaris by electron microscopy. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
852. Arkin, A. P., E. Baidoo, K. Bender, P. I. Benke, A. Deutschbauer, M. Fields, T. C. Hazen, Z. He, D. C. Joyner, J. Keasling, K. Keller, E. G. Luning, A. Mukhopadhyay, L. Rajeev, J. Ray, J. D. Wall, G. Zane, A. Zhou, and J. Zhou. 2010. Laboratory models for the study of community interaction, functional stability, and survival. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
853. Arkin, A. P., E. Baidoo, P. Dehal, D. Elias, M. Fields, J. Geller, T. C. Hazen, Z. He, K. Hillesland, J. Keasling, K. Keller, M. Keller, L. Krumholz, B. Meyer, L. Miller, J. Mosher, A. Mukhopadhyay, A. Palumbo, T. Phelps, M. Podar, L. Rajeev, A. Redding, C. Schadt, D. Stahl, S. Stolyer, A. Venkateswaren, C. Walker, J. Wall, Z. Yang, G. Zane, A. Zhou, and J. Zhou. 2010. Laboratory models for the study of community interaction, functional stability, and survival. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
854. Allgaier, M., A. Reddy, J. I. Park, N. Ivanova, P. D?haeseleer, S. Lowry, R. Sapra, T. C. Hazen, B. A. Simmons, J. S. VanderGheynst, and P. Hugenholtz. 2010. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost microbial community. ISME 13 ? 13th International Symposium on Microbial Ecology.
855. Adame, J. 2010. Clean-up role for microbes is sought. Abilene Reporter News.
856. ……. 2010. Gulf oil spill: White House accused of spinning report.
857. ……. 2010. Despite BP efforts to clean Gulf, nature will do most of it.
858. .... 2010. BP Watch: Hiding the oil.
859. ---------. 2010. Hazen et. al. Deep-sea oil plume enriches psychrophilic oil-degrading bacteria.
860. -------. 2010. Lab Scientist Pens New York Times Op-Ed on Gulf Spill. Today at Berkeley Lab.
861. -------. 2010. Cleaning Hazardous Waste with Green Microbes. Today at Berkeley Lab.
862. -----. 2010. 3 Future Oil-Spill Fighters: Sponges, Superbugs, and Herders. Berkeley Lab Media Report.
863. -----. 2010. Plan B in the Gulf. Bloomberg Surveillance.
864. -----. 2010. Plan B in the Gulf. www. Almendron.com.
865. -----. 2010. Oil spill. KCBS all news radio.
866. -----. 2010. Gulf Oil Spill Solution ? Donated Hair? May 6. Today at Berkeley Lab.
867. -----. 2010. Deepwater Horizon Spill Detergents Could Make Bad Situation Worse. www.outlookseries.com.
868. -----. 2010. Disperse and Conquer. www.mnn.com.
869. -----. 2010. Chemical Detergents May Make Gulf Oil Disaster Worse, Say Experts. www.foxnews.com.
870. -----. 2010. Extreme caution required in Gulf Oil-spill clean up: Berkeley expert. www.thaindian.com.
871. -----. 2010. 5-May. www.thaindian.com.
872. -----. 2010. Caution Required for Cleanup of Oil Spill. Today at Berkeley Lab.
873. -----. 2010. Caution urged in oil spill cleanup. www.upi.com/Science_News.
874. -----. 2010. Caution Required for Gulf Oil Spill Clean-Up, Bioremediation Expert Says. www.sciencedaily.com.
875. Zhou, J. and Terry C. Hazen. 2009. High Throughput Genomic Technologies for Complex Microbial Community Analysis. Annual meeting of the American Society for Microbiology
876. Zhou, A. and M. J. Joachimiak, P. S. Dehal, A. Pl Arkin, K. Hillesland, D. Stahl, J. Wall, Terry C. Hazen, J. Zhou Z. He. 2009. Genetic Adaptation to Salt Stress during the Long-Term Evolution of Desulfovibrio vulgaris Hildenborough. Annual meeting of the American Society for Microbiology abstract
     In order to understand the molecular mechanism of salt adaptation, a long-term evolution experiment was carried out under controlled laboratory conditions with Desulfovibrio vugaris Hildenborough, a sulfate reducing bacteria which has been recognized as a model environmental organism. With the recent advances in genome sequencing and highthroughput genomic technologies, it is possible to now link sub-cellular molecular/metabolic processes with population-level processes. Control and stressed lines (6 lines each, from a single colony-based pure culture) grown in the LS4D medium (control) and LS4D with 100 mM NaCl (stressed) were transferred every 48 hrs using one to one hundred dilution. The phenotypic response of salt tolerance was tested periodically by monitoring the growth of all cell lines in LS4D supplemented with 250 mM NaCl. The results demonstrated that the adaptation to salt stress is a dynamic process. Enhanced salt tolerance of stressed lines was observed at 300 generations and became more obvious with the increasing number of generations. De-adaptation of cell lines (500, 1000 and 1200 generations) by removal of salt stress did not affect the increased salt tolerance, indicating that the observed phenotype changes were due to genetic changes instead of physiological adaptation. Further, results of the de-adaptation experiment suggested the dynamic trend of genetic adaptation and that the genetic mutation may have become stable by 1000 generations. Gene expression profiles of the 500 and 1000 generation samples were examined using the D. vulagris whole genome microarray and the microarray data confirms the phenotypic test results. Genes involved in energy production and conversion and signal transduction mechanisms were among the gene categories with the most genes up-regulated. Whole genome sequencing of selected colonies is underway to identify beneficial genetic mutations in the D. vulgaris genome.
877. Zhou, A., Z. He, M. P. Joachimiak, P. S Dehal, A. P. Arkin, K. Hillesland, D. Stahl, J. Wall, Terry C. Hazen and J. Zhou. 2009. The molecular mechanism of adaptation to salt stress revealed by the long-term evolution of Desulfovibrio vulgaris Hildenborough. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
878. Zhou, A., Z. He, G. M. Zane, C. Hemme, Y. Chen*, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou. 2009. Study of Global Gene Regulation by CRP/FNR in Desulfovibrio vulgaris Hildenborough. Annual meeting of the American Society for Microbiology abstract
     Characterization of transcriptional regulatory networks is essential for understanding cellular processes. CRP/FNR regulators are DNA binding proteins which function as positive transcription factors. There are four (DVU2547, DVU0379, DVU3111 and DVU2097) CRP/FNR homologues in the Desulfovibrio vulgaris Hildenborough genome. D. vulgaris is an obligate anaerobe and has been used as a model organism of sulfate-reducing bacterium (SRB) for studying the energy metabolism, sulfur cycling, and biocorrosion. Knowledge about the gene regulation by these regulators is limited. Evidence from other bacteria demonstrates that CRP/FNR regulators function in response to a broad spectrum of intracellular and exogenous signals such as oxidative and nitrosative stress, nitric oxide, carbon monoxide or temperature. Microarray data from D. vulgaris showed that the transcript levels of Crp/Fnr were altered in response to nitrate, nitrite, heat shock, and oxygen stresses. In order to understand the function of D. vulgaris CRP/FNR, knockout mutants for all four CRP/ FNR proteins were generated. The physiological studies of the mutants included the utilization of various electron donors and acceptors, response to different stressors, etc. The global gene regulation by these regulators was investigated by transcriptomic analysis with mutants and wildtype grown under regular and stressed conditions. Recombinant proteins for all four CRP/FNR were obtained and polyclonal antibodies were generated. The transcription data will be confirmed by in vivo evidence of the gene regulation via chromatin immunoprecipitation-chip (ChIP-chip). Then the CRP/FNR binding motif can be predicted by computational analysis. Future studies will focus on the understanding how those homologues respond to different environmental conditions.
879. Wu, L. Y. and Y. Q. Luo, J. D. Van Nostrand, R. A. Sherry, Z. L. He, Terry C. Hazen, J. Z. Zhou J. P. Xie. 2009. The Responses of the Underground Microbial Communities of Grassland to the Global Warming and Different Land-use Practices Revealed by GeoChip and Pyrosequencing Analyses. Annual meeting of the American Society for Microbiology abstract
     Responses of microbial communities to warming (2oC) and clipping of grasses in grassland soils at the Kessler Farm Field Laboratory in McClain County, Oklahoma, were revealed by GeoChip and pyrosequencing analyses. Samples were collected from a long-term (eight years) experiment involving four treatments (replicated on 6 plots): warmed-unclipped (WU), warmed-clipped (WC), unwarmed-clipped (UC), and unwarmedunclipped( UU). DNA was extracted using a freezegrind method. 16S rDNA was PCR amplified from each sample using tagged primer pairs, and mixed equally for pyrosequencing. 240,000 16S rRNA gene sequences (70,000,000 bp total) were obtained and 12,000 OTUs were identified based on a 97% similarity cutoff. OTUs were identified from each treatment condition: WU (3926), WC (4399), UC (2931), and UU (3328). 23 phyla or phylum equivalents were identified, although 98% of the OTUs were from only 10 phyla. Comparisons were made between treatments using response ratios based on the OTUs identified. The treatments UC and UU had the highest percentage of OTUs in common (44.3%), while WU and UC had the least (36.2%). Of the shared OTUs, 23% had significantly different quantities between WC and UU, while only 21.5% did between WU and WC. When compared to UU, UC has more OTUs that were significantly more abundant; while warmed samples have more OTUs that are less abundant. However, the warmed samples have more unique OTUs than the unwarmed samples. These results indicate that both warming and clipping altered the microbial communities in the grassland soils. Clipping seems to have increased the population of dominant species while warming decreased the abundance of the dominant species. To study the functional diversity and dynamics of the microbial communities, communities were analyzed with GeoChip 3. Cluster analysis of the GeoChip data showed that replicate samples clustered based on warming and then on clipping, indicating obvious differences in the microbial communities under warming and clipping treatments. Results of DCA showed similar relationships. More statistical analyses of the pyrosequencing and GeoChip data are underway. Acknowledgments/References: OBC
880. Wu, L. Y., J. D. Van Nostrand, T. J. Gentry, Z. J. Huang, C. W. Schadt, W. M. Wu, D. Watson, M. W. Fields, C. S. Criddle, J. Tiedje, Terry C. Hazen, J. J. Zhou. 2009. Microbial Community Dynamics and the Effect of Geochemistry in Uranium Bioremediation Revealed by Functional Gene Array Analysis. Annual meeting of the American Society for Microbiology abstract
     Composition and dynamics of the functional microbial communities were examined in a pilot-scale system of in situ bioremediation and immobilization of U(VI) at a highly contaminated aquifer at the U. S. DOE’s Field Research Center, Oak Ridge, TN. Samples were collected from three monitoring wells (FW101-2, 102-2, and 102-3) during a period of active U(VI) reduction (days 161-719) and were studied using GeoChip 2, a functional gene array containing >24,000 probes covering 10,000 functional genes. About 20% of functional genes on the GeoChip 2 were detected. Higher percentages of cytochrome C (25-31%), metal resistant (20-25%), and methane oxidation genes (29% in FW101-2) were detected. Detrended correspondence analysis showed that the microbial communities were more similar withinwells while changed along the process of the U(VI) bioremediation. Microbial populations, as indicated by gene numbers, increased slowly during the early phase of operation due to ethanol injections, reached a peak late around 100 days and then decreased and stabilized. The microbial community structure continued to changeover the course of these experiments. Cytochrome genes in FW102-2 increased from day163 (9.4%) to 184 (12.0%) then decreased and stabilized after day248. Nitrogen cycling genes showed a similar trend, while sulfate reducing genes changed little. The populations of six metal reducing microorganisms (e.g., Desulfovibrio desulfuricanse G20 and Anaeromyxobacter dehalogenans 2CP-C) shifted during the process as U(VI) decreased and followed similar patterns in all three wells. The results of canonical correspondence analyses indicate that the microbial communities were significantly affected by geochemistry including COD, nitrate, sulfate, and pH. Variance portioning analysis revealed that the effect of COD on the microbial communities was independent from other geochemical factors. Metal test results showed that the effects of geochemistry on microbial communities changed at different stages of U(VI) bioremediation. Acknowledgments/References: DOE
881. Wu, C. H. and L. C. Van De Werfhorst, T. Z. DeSantis, E. L. Brodie, Terry C. Hazen, G. L. Andersen, P. A. Holden B. Sercu. 2009. Bacterial Biogeography of an Urban Creek Impacted with Fecal Pollution. Annual meeting of the American Society for Microbiology abstract
     High levels of fecal bacteriological contamination in coastal waters are often attributed to nearby human development. Tracking inland sources of fecal bacteria can be a significant challenge. To address this in Santa Barbara, CA, a multi-phase study has been conducted in an urban coastal creek that discharges into an impacted beach frequently posted with warnings. TRFLP and high-density microarray (PhyloChip) were used to characterize bacterial community composition during dry weather at several locations during three consecutive days. For comparison, human fecal and two sewage samples were analyzed as well. Spatial and temporal variations in bacterial community composition were characterized. The Mantel test was used to compare the TRFLP and the PhyloChip data, and a significant correlation (r=0.5881) was obtained. There is a positive significant correlation (r=0.6188) between the environmental variables similarity matrix and the community similarity matrix. In addition, a strong positive significant correlation (r=0.8132) was determined between the geographical distance of the sites and day 3 of the community composition, but not for day one or two. PhyloChip analysis enabled identification of transient and resident bacterial communities over the three-day sampling period. Bacterial community profiles comparing different habitat types (ocean, lagoon, creek and fecal) indicated that more than 50% of the community composition in ocean, lagoon and creek samples consists of Proteobacteria. Fecal samples had higher percentages of Clostridia and Bacilli than all water samples. When only fecal-associated OTUs (taxa common to all three sewage/fecal samples) were considered, grouping according to location was stronger, and relatedness to sewage remained similar. The microarray metagenomics approach enabled the characterization of bacterial communities in a sewageimpacted urban creek. The results from this study enhance our understanding of bacterial community fluxes over short period of time and small spatial scale, and will guide further development of fecal contamination detection with the PhyloChip.
882. Wu, C. H. and J. Chou, M. Bill, J. Henriksen, K. E. Wright, E. L. Brodie, G. L. Andersen, Terry C. Hazen, Y. Fujita, M. E. Conrad B. R. Lam. 2009. Microbial metabolism of triethylphosphate, a potential phosphate source for radionuclide mineralization. American Geophysical Union Annual Meeting abstract
     Significant quantities of metals and radionuclides contaminate unsaturated zones at several sites in the western U.S. In many cases, this contamination has migrated to groundwater, sometimes decades after being released into the subsurface. A potentially useful approach for immobilizing radionuclides such as uranium and strontium in the vadose zone is precipitation with microbially-generated phosphate. Triethylphosphate (TEP) is a low-toxicity organophosphate that can be vaporized and delivered to the vadose zone. Microbes can catalyze TEP degradation, leading to the release of inorganic phosphate that can then lead to the precipitation of phosphate minerals. These minerals are typically highly stable and poorly soluble under environmental conditions. Sequestration in phosphate minerals is a promising strategy for mitigating radionuclide transport in the environment. To examine the feasibility of this strategy, we set up lab-scale incubation experiments with TEP-amended synthetic groundwater inoculated with vadose zone-derived mixed cultures from the Idaho National Laboratory (INL), and sediment slurries using solids from the Hanford Reservation in Washington (U.S. Department of Energy facilities with significant radionuclide contamination in the vadose zone). The amount of phosphate released in the cultures was monitored, and the microbial communities were characterized with a high-density microarray (PhyloChip). Significant biodegradation of TEP was observed in the experiments with the synthetic groundwater amended with 5 mM TEP. Phosphate concentrations in live cultures steadily increased to >0.25 mM after 13 months with no phosphate accumulated in killed controls. Surprisingly, no evidence for phosphate mineral precipitation was observed, contrary to expectations based on equilibrium considerations. Studies are underway to investigate potential kinetic inhibition of precipitation under these conditions. Cell counts increased by approximately one order of magnitude during that period. Significant decreases in the d13C values of dissolved inorganic carbon in the live cultures were observed, indicating the microbial community was respiring the carbon in the TEP. In contrast, no significant accumulation of phosphate was observed in the sediment slurries with 5 mM TEP, most likely due to phosphate adsorption to the solids. Microbial community identification indicated that organisms in the families of Xanthomonadaceae, Crenotrichaceae and Comamonadaceae were enriched by the addition of TEP. Further characterization of radionuclide-biota interactions would lead to enhanced understanding of the fate and transport of these contaminants in the subsurface.
883. Walian, P. J. and L. Zeng, E. Szakal, E. Johansen, S. C. Hall, S. J. Fisher, M. E. Singer, C. Park, Terry C. Hazen, H. E. Witkowska, M. D. Biggin, B. K. Jap S. Allen. 2009. Protein Complex Analysis Project (PCAP): Isolation and Identification of Membrane Protein Complexes from D. vulgaris. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
884. Van Nostrand, J. D. and M. Xu, W. Wu, L. Wu. Z. He, Y. Deng, C. Hemme, D. Watson, C. Criddle, P. Jardine, Terry C. Hazen, J. Zhou P. J. Waldron. 2009. Improvements to GeoChip 3.0 and Application for Microbial Community Analysis. 4th Annual DOE-ERSP PI Meeting
885. Van Nostrand, J. D. and P. Waldron, Ye Deng, Z. He, W. Wu, S. Carroll, C. Schadt, A. Palumbo, D. Watson, C. Criddle, P. Jardine, Terry C. Hazen, J. Zhou L. Wu. 2009. Applications of GeoChip for analysis of different microbial communities. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
886. Van Nostrand, J. D., P. J. Waldron*, W. Wu, L. Wu, Y. Deng, J. Carley, Z. He, C. S. Criddle, P. Jardine, Terry C. Hazen, J. Zhou. 2009. GeoChip-based Analysis of Functional Microbial Communities in a Bioreduced Uranium-contaminated Aquifer during Nitrate Exposure. Annual meeting of the American Society for Microbiology abstract
     A pilot-scale system at the U.S. DOE’s Field Research Center in Oak Ridge, TN was established for biostimulation of subsurface U(VI) reduction by injection of ethanol. The system was able to reduce U(VI) to a level below EPA drinking water standards. In this study, stability of the bioreduced area during oxidation by nitrate was examined. Introduction of nitrate to the reduced area resulted in U(IV) oxidation but subsequent removal of the nitrate and injection of ethanol resulted in reduction of the U(VI) back to the low level. Geochip 2.0, a comprehensive 50mer microarray containing probes for genes involved in the geochemical cycling of N, S, and C, metal resistance and contaminant degradation, was used to monitor the dynamics of the groundwater microbial community structure and function before, during, and after reoxidation of U(IV) by nitrate. The numbers of functional genes detected decreased 10- to 20- fold following the increase in nitrate concentration and were slow to recover after nitrate levels decreased. The relative abundance of each functional gene group was similar prior to the increase in nitrate level, then the relative proportion of nitrogen fixation genes increased from 1-2% to over 20%. By the end of the study period, the relative abundance of all gene groups returned to the original values. Detrended correspondence analysis indicated that communities before and immediately following introduction of nitrate were similar. A shift in community structure occurred during the period of nitrate elevation and for several months afterwards; however, by day 1607, approximately 200 days after nitrate exposure and then injection of ethanol, the communities had begun to recover and were more similar to the initial community. Canonical correspondence analysis indicated that Fe(II), pH, U, and nitrate were the most important environmental variables in controlling community structure (p=0.04). These results demonstrate that introduction of elevated nitrate levels dramatically affected the microbial community structure; however, the community was able to recover.
887. Van Nostrand, J. D., P. Waldron, D. B. Watson, Z. He, L. Wu, P. Jardine, Terry C. Hazen, J. Zhou. 2009. GeoChip Analysis of Groundwater Microbial Communities across a Gradient of pH, Heavy Metal, Nitrate Contamination. Annual meeting of the American Society for Microbiology abstract
     The Field Research Center (FRC) site of the U.S. DOE ERSP (Environmental Remediation Science Program) at Oak Ridge, TN, is contaminated with radionuclides, heavy metals, and other contaminants due to historical U enrichment activities. To examine the impact of contaminant level on microbial communities, five contaminated and one uncontaminated well, providing a gradient of groundwater nitrate, pH and U concentrations, were sampled. DNA from these samples was analyzed with a comprehensive functional gene microarray (GeoChip 2.0) containing probes for >10,000 genes involved in carbon, sulfur, and nitrogen cycling, contaminant degradation and metal resistance and reduction. Results of principle component analysis of functional genes showed that the most contaminated wells clustered together and had the greatest gene overlap. Gene abundance correlated with contaminant levels. Higher percentages of nitrogen fixation genes were detected in wells with lower nitrate concentrations, while the percentage of nitrate reduction genes generally decreased with decreasing nitrate. Wells with elevated sulfate concentrations had a greater percentage of genes dedicated to sulfate reduction, and higher signal intensities for dsrAB genes than the background, indicating a greater abundance of those genes. The total signal intensity of metal resistance and reduction genes in contaminated wells was greater than the background, indicating that metal-related genes were more prevalent in the contaminated wells. CCA indentified pH, Sr, U, Cs, and NO3 as the most important environmental variables in determining community structure. This study provides an overall view of the functional genes present in a highly contaminated environment, and shows that the contaminant level has significant effects on bacterial community structure.
888. Sobecky, P. A., R. J. Martinez, M. J. Beazley, K. Salome, C. Wu, Terry C. Hazen, G. L. Andersen, S. M. Webb, M. Taillefert. 2009. Uranium Immobilization by the Activities of Microbial Phosphatases. 4th Annual DOE-ERSP PI Meeting
889. Skerker, J., A. Deutschbauer, J. Mar, K. Wetmore, M. Price, P. Dehal, J. Baumohl, I. Dubchak, Terry C. Hazen, A. P. Arkin. 2009. A systems biology approach for optimizing biofuel production in Zymomonas mobilis. Energy Biosciences Institute Annual Retreat
890. Skerker, J., A. Deutschbauer, P. Novichkov, A. Gerasimova, J. Mar, K. Wetmore, J. Baumohl, M. Price, P. Dehal, J. Kuehl, C. Wu, I. Dubchak, Terry C. Hazen, A. P. Arkin. 2009. An idiosyncratic view of Fuel Synthesis Challenges. Energy Biosciences Institute Annual Retreat
891. Reddy, A. P. and P. Hugenholtz, B. A. Simmons, Terry C. Hazen, J. VanderGheynst M. Allgaier. 2009. Tracking Microbial Community Changes during Decomposition of Switchgrass. 31st Symposium on Biotechnology for Fuels and Chemicals, Society for Industrial Microbiology
892. Ray, J. and A. Deutschbauer, K. Keller, J. Robertson, G. Zane, M. Price, S. Chhabra, J. Wall, A. Arkin, Terry C. Hazen, J. Keasling, A. Mukhopadhyay E. Luning. 2009. Study of Two-component Signal Transduction Systems in Desulfovibrio vulgaris Hildenborough. Annual meeting of the American Society for Microbiology abstract
     Two-component systems, comprised of histidine kinase (HK) and response regulator (RR) proteins, represent the primary and ubiquitous mechanism in bacteria for initiating cellular response towards a wide variety of environmental conditions. In D. vulgaris, more than 60 such systems have been predicted, but remain mostly uncharacterized. The ability of D. vulgaris to survive in its environment is undoubtedly linked with the activity of genes modulated by these two-component signal transduction systems. Using recently developed methods for in vitro confirmation of specific phosphotransfer from HKs to their cognate RRs a high throughput mapping of two-component systems is possible. This is aided by in silico methods to predict candidate partners for HKs or RRs that are either ORFans or have no proximal genes that may serve as their cognate partners. Additional corroboration of these predictions may be found from microarray data in our VIMSS database. The requirement of highly pure, active proteins for these high throughput phosphotransfer assays were fulfilled by generating expression vectors for all RRs and the soluble portions of HKs using the Gateway® Cloning system. His-tagged HKs and the corresponding cognate RRs have been purified under native conditions and used successfully in in vitro phosphotransfer mapping for selected two-component systems in D. vulgaris. The immediate applications of this research are for two-component systems discovered to be involved in specific stress studies. Availability of a library of RRs and HKs also allows us to test a variety of broader questions experimentally. For example, do the many highly homologous HKs in D. vulgaris, still maintain specific interactions with one or few RRs? Beyond testing such hypothesis, this library of purified proteins can also be used to map entire two-component signal transduction cascades. In addition, the availability of a library of HK knockout mutants allowed us to follow-up on conclusions from the in vitro phosphotransfer studies. Results from the in vitro assays and follow up studies are presented in this poster.
893. Ramsay, B. and S. Carroll, A. Lapidus, J. C. Detter, C. Han, M. Land, L. Hauser, Terry C. Hazen, A. Arkin, A. Beliaev, R. Sanford, F. Löeffler, M. W. Fields C. Hwang. 2009. Characterization of Metal-Reducing Communities and Isolates from Uranium-Contaminated Groundwater and Sediments. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
894. Mukhopadhyay, A. and J. Ray, A. Deutschbauer, K. Keller, J. Robertson, G. Zane, M. Price, S. Chhabra, J. Wall, A. P. Arkin, Terry C. Hazen, J. Keasling E. Luning. 2009. ESPP2: Study of Two component signal transduction systems in Desulfovibrio vulgaris Hildenborough. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
895. Miller, L. D. and J. Mosher, M. Drake, Z.K. Yang, M. Rodriguez, S.D. Brown, T. J. Phelps, M. Podar, A. V. Palumbo, C. W. Schadt, M. Keller, D. C. Joyner, Terry C. Hazen, S. Stolyar, K. Hillesland, D.A. Stahl A. Venkateswaran. 2009. Development and Analysis of Multispecies Consortia to Study Microbial Community Stress and Survival. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
896. Meagher, R. J. and Y. K. Light, P. Dehal, Terry C. Hazen, A. P. Arkin, A. K. Singh M. Z. Hadi. 2009. Microfluidic tools for single-cell genomic analysis of environmental bacteria. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
897. Martinez, R. J. and C. H. Wu, G. L. Andersen, Terry C. Hazen, M. Taillefert, P. A. Sobecky M. J. Beazley. 2009. Microbial Phosphatase Activity Involved in Subsurface Uranium Sequestration. Annual meeting of the American Society for Microbiology abstract
     Soils and groundwater contaminated with heavy metals and radionuclides remain a legacy of Cold War nuclear weapons development. Due to the widespread contamination of soils and groundwater, in situ sequestration of heavy metals and radionuclides has proven to be a cost-effective strategy for remediation. We are currently investigating a remediation approach that utilizes the phosphatase activity of bacterial strains extant within the contaminated soils of the DOE Field Research Center (ORFRC) in Oak Ridge, TN, for in situ contaminant sequestration. We have previously demonstrated the accumulation of PO4 3- and concomitant mineralization of U(VI) using pure cultures grown under both oxic and anoxic conditions with glycerol phosphate as the sole C and P source and NO3 -, the sole N source and terminal electron acceptor in the absence of oxygen. To determine the microbial diversity of U(VI) and NO3 - contaminated ORFRC Area 2 soils as well as the microbial community response to exogenous organophosphate additions under oxic and anoxic growth conditions, soil slurry experiments were conducted at pH 5.5. Incubations were conducted for 36 days at 25ÅãC with 10 mM G2P and 15mM NO3 - as the sole C, P and N sources, respectively. Under oxic growth conditions, greater than 4 mM soluble PO4 3- was measured at the end of the slurry incubations and NO2 - was not detected. Preliminary data obtained for anoxic soil slurry incubations indicated an accumulation of greater than 1mM PO4 3- as well as the accumulation and subsequent removal of NO2 -. Following triplicate incubations, total DNA was extracted from the slurries and 16S diversity analyzed with a high-density oligonucleotide microarray (PhyloChip). Our soil slurry studies demonstrate the efficacy of organophosphate-mediated sequestration of U(VI) driven by the diverse microbial community extant within ORFRC contaminated subsurface soils. Thus, a strategy which employs microbial hydrolysis of organophosphates could complement current remediation approaches.
898. Martinez, R. J. and C. W., Terry C. Hazen, G. L. Andersen, M. Taillfert, P. A. Sobecky, Patricia K. Salome. 2009. Uranium Immobilization by the Activities of Microbial Phosphatases. Geological Society of America Annual Meeting
899. Liu, H. and N. Khainovski, M. Dong, E. D. Szakal, M. Choi, S. Allen, Terry C. Hazen, J. T. Geller, M. E. Singer, P. Walian, B. Jap, S. C. Hall, S. J. Fisher, H. E. Witkowska, J. Jin, M. D. Biggin L. Yang. 2009. Protein Complex Analysis Project (PCAP): Introduction of Iterative MS/MS Acquisition (IMMA) to the MALDI LC MS/MS Workflow To Enable High Throughput Protein Complex Identification using Tagless Strategy. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
900. Kozina, C. L. and D. Joyner, K. L. Sale, D. S. Reichmuth, Terry C. Hazen, R. Sapra A. S. Pawate. 2009. Metabolic engineering of a novel thermophilic ethanologen Geobacillus thermoglucosidasius M10EXG for enhanced ethanol production. 31st Symposium on Biotechnology for Fuels and Chemicals, Society for Industrial Microbiology abstract
     The thermophilic bacterium Geobacillus thermoglucosidasius (Gth) M10EXG is a facultative anaerobe that has an optimal growth temperature of 60 oC. It can metabolize both C5 (xylose) and C6 (glucose) sugars and is tolerant to 10% ethanol, making it an attractive candidate for industrial bioethanol production from lignocellulosic biomass. However, in order to maximize the production of ethanol from the fermentative pathway, it is essential to understand the fermentative metabolism, operational pathways, and the flux through the different fermentative pathways. We have completed a metabolic analysis of the growth of Gth M10EXG using both xylose and glucose under varying concentrations of oxygen. As expected, ethanol production is detected only under anaerobic conditions using either xylose or glucose as the sole carbon source. Furthermore, metabolic flux analysis of the anaerobic and aerobic growth using glucose as the sole carbon source shows that 0.6 mol lactate, 0.9 mol acetate, 0.4 mol ethanol, and 1.0 mol formate are produced per mole of glucose metabolized. With recent genome sequencing and metabolic flux analysis completed, we have targeted both lactate and formate production pathways for modification to increase ethanol production. We present results from the metabolic engineering of the aforementioned pathways and the effect on ethanol production.
901. Knierim, B. and S. Singh, D. Jorgens, M. Zemla, K. DeAngelis, A. Reddy, J. VanderGheynst, Terry C. Hazen, B. Holmes, R. Sapra, B. Simmons, P. Adams, M. Auer L. Prak. 2009. Electron Microscopic Imaging a JBEI. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
902. Knierim, B. and S. Singh, D. Jorgens, M. Zemla, K. DeAngelis, A. P. Reddy, J. VanderGheynst, Terry C. Hazen, B. M. Holmes, R. Sapra, B. A. Simmons, P. D. Adams, M. Auer L. Prak. 2009. Electron Microscopic Imaging at JBEI. 31st Symposium on Biotechnology for Fuels and Chemicals, Society for Industrial Microbiology
903. Kang, S. and J. Van Nostrand, Z. He, L. Wu, D. A. Stahl, Terry C. Hazen, J. Zhou H. L. Gough. 2009. Controlling Factors of Sediment Microbial Communities at the Metal Contaminated Freshwater Lake (Lake DePue). Annual meeting of the American Society for Microbiology abstract
     Microbes are known for their versatility under different and even certain extreme environmental conditions. The effect of extreme environments on microbial distribution is of special interest. Lake DePue (Illinois, USA) was subjected to metal contamination for about 80 years from adjacent zinc smelting activities. Sediments were collected in triplicate from five areas of the lake along a transect from near a creek inlet to the main body of the lake. GeoChip II, with over 10,000 microbial functional genes, was used to investigate metal impact on the microbial communities and their distributions. In addition to comparison of the overall microbial community, three microbial communities of functional subgroups, defined by genes relevant to metal contamination (S cycling, metal resistance & reduction, and C cycling genes) were examined. While most environmental variables were spatially structured, the pore water metal concentration, total and three subgroup microbial communities were not spatially structured except for a very significant single patch at ~250 m in microbial communities. Polyphasic approaches identified 2-4 metals (e.g., zinc, arsenic, lead etc) were the likely controlling factors for predicting different microbial communities. While geographic distance between areas was held constant, selected pore water metals (Zn, As and Pb) showed fairly significant correlations with microbial communities (P < 0.099) except for the S cycling community. Canonical correspondence analysis (CCA) models with the same sets of pore water metals were also very significant based on Monte Carlo permutation (P < 0.013) except for the S cycling community. Canonical variance partitioning analysis (VPA) results indicated that selected pore water metals explained more variances than geographic distance, and the significance of the pore water metals effect was much higher in all microbial communities. In conclusion, microbial communities in sediment from Lake DePue varied significantly with the level of metal contamination.
904. Hubbard, S. S. and H. Beller, E. Brodie, J. Chen, J. Christensen, M. Conrad, M. Denham, D. DePaolo, B. Faybishenko, S. Finsterle, Y. Fujita, Terry C. Hazen, M. Kowalsky, L. Li, P. Long, P. Nico, E. Sonnethal, N. Spycher, C. Steefel, T. Tokunaga, J. Wan, K. H. Williams, Y. Wu J. Aho-Franklin. 2009. LBNL Sustainable Systems SFA. 4th Annual DOE-ERSP PI Meeting
905. Hemme, C. L. and T. J. Gentry, M. W. Fields, L. Wu, K. Barry, S. Green-Tringe, D. B. Watson, Z. He, Terry C. Hazen, J. M. Tiedje, E. M. Rubin, J. Zhou Y. Deng. 2009. Analysis of a Microbial Metagenome from a Pristine Groundwater Ecosystem. Annual meeting of the American Society for Microbiology abstract
     Previous analyses of a microbial metagenome from uranium and nitric-acid contaminated groundwater (FW106) showed significant environmental effects resulting from the rapid introduction of multiple contaminants. Effects include a massive loss of species and strain biodiversity, accumulation of toxinresistant genes in the metagenome and lateral transfer of toxin resistance genes between community members. To better understand these results in an ecological context, a second metagenome from a pristine groundwater system located along the same geological strike was sequenced and analyzed (FW301). It is hypothesized that FW301 approximates the ancestral FW106 community based on phylogenetic profiles and common geological parameters; however, even if is not the case, the datasets still permit comparisons between healthy and stressed groundwater ecosystems. Complex carbohydrate metabolism has been almost entirely lost in the stressed ecosystem. In contrast, the pristine system encodes a wide diversity of complex carbohydrate metabolism systems, suggesting that carbon turnover is very rapid in the healthy groundwater system. FW301 encodes many (~160+) carbon monoxide dehydrogenase genes while FW106 encodes none. This result suggests that the community is frequently exposed to oxygen from aerated rainwater percolating into the subsurface, with a resulting high rate of carbon metabolism and CO production. When oxygen levels fall, the CO then serves as a major carbon source for the community. FW301 appears to fix CO2 via the reductive carboxylase (reverse TCA) cycle and possibly by acetogenesis, activities; these activities are lacking in the heterotrophic FW106 system which relies exclusively on respiration of nitrate and/or oxygen for energy production. Overall comparative analysis suggests that the introduction of contaminants is accompanied by a decrease in biodiversity, loss of nutrient cycling, less metabolic diversity, increased respiration and dominance by r-strategists. These results are consistent with trends predicted for stressed ecosystems. Acknowledgments/References: The authors would like to thank Dr. Fares Najar and Dr. Bruce Roe for providing sequencing services, and Dr. Tommy Phelps and Dr. Christopher W. Schadt assisted in groundwater sampling. This research was supported by The United States Department of Energy under the Environmental Remediation Science Program (ERSP), and Genomics: GTL program through the Virtual Institute of Microbial Stress and Survival (VIMSS; http://vimss.lbl.gov), Office of Biological and Environmental Research, Office of Science, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52- 07NA27344, and Los Alamos National Laboratory under contract No. DE- AC02-06NA25396. Oak Ridge National Laboratory is managed by University of Tennessee UT-Battelle LLC for the Department of Energy under contract DE-AC05-00OR22725
906. He, Z., A. Zhou*, Q. He, A. Mukhopadhyay, E. Baidoo, M. Joachimiak, C. L. Hemme, P. Benke, A. M. Redding, M. M. Fields, D. A. Stahl, J. D. Keasling, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou. 2009. Desulfovibrio vulgaris Hildenborough responses to salt and H2O2 stresses. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
907. He, Z., Y. Deng, J. D. Van Nostrand, L. Wu*, C. L. Hemme, T. J. Gentry, J. Liebich, Q. Tu, A. P. Arkin, Terry C. Hazen, J. Zhou. 2009. Development and Applications of GeoChip 3.0 for Analysis of Microbial Community Structures, Compositions, Potential Functions. Annual meeting of the American Society for Microbiology abstract
     Functional gene arrays (FGAs), or GeoChip constructed with key genes involved in various biological and geochemical processes have been widely used to analyze microbial communities. Based on GeoChip 2.0, a new generation of Geo- Chip (GeoChip 3.0) has been developed. GeoChip 3.0 has several new features in terms of coverage, design, and data analysis. GeoChip 3.0 covers approximately 50,000 gene sequences for 306 gene families, including functional genes involved in antibiotic resistance and energy metabolism. The phylogenic marker GyrB has been added, which allows us to obtain additional phylogenetic information and to analyze more diverse environmental samples. Sequence-specific, exclusive group-specific, and inclusive group-specific probes with different specificities were selected for probe design. These various types of probes greatly increase gene coverage.Three strategies have been implemented for data analysis. First, a universal standard has been implemented so that data normalization and comparison of samples from different sites, time points, or laboratories can be conducted. Second, a genomic standard is used to quantitatively analyze gene abundance. Third, a software package (including databases) has been developed for sequence retrieval, probe design, information storage, and especially, data analysis and automatic updates, which greatly facilitates the management and analysis of complicated GeoChip data sets. GeoChip 3.0 has been used for analyses of responses of microbial communities to elevated CO2 and increased temperature. The results showed that autotrophic CO2 and N2 fixation by microorganisms increased significantly in response to elevated CO2. This also demonstrates that GeoChip can provide insights into biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, global climate change, ecosystem management, and environmental cleanup and restoration. It is also particularly useful for providing direct linkages of microbial genes/populations to ecosystem processes and functions. Acknowledgments/References: This work was supported by the US Department of Energy under the Genomics: GTL Program through the Virtual Institute of Microbial Stress and Survival (VIMSS, http://vimss.lbl.gov), by Environmental Remediation Science Program (ERSP), Office of Biological and Environmental Research, Office of Science, and by the Oklahoma Center for Advancement of Science and Technology (OCAST) under Oklahoma Applied Research Support (OARS) Project AR062-034.
908. He, Q. and D. C. Joyner, M. Joachimiak, M. N. Price, Z. K. Yang, H.-C. B. Yen, C. L. Hemme, R Chakraborty, W. Chen, M. M. Fields, D. A. Stahl, J. D. Keasling, M. Keller, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou Z. He. 2009. Impact of Elevated Nitrate on Sulfate-Reducing Bacteria: Implications of inhibitory mechanisms in addition to osmotic stress. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
909. Hazen, Terry C. and B. A. Simmons. 2009. Systems Biology Approaches to Environmental Microbial Adaptations for Bioenergy Products. Society for Industrial Microbiology Annual Meeting abstract
     Environmental biotechnology encompasses a wide range of characterization, monitoring and control or bioenergy technologies that are based on biological processes. Recent breakthroughs in our understanding of biogeochemical processes and genomics are leading to exciting new and cost effective ways to monitor and manipulate the environment and potentially produce bioenergy fuels. Indeed, our ability to sequence an entire microbial genome in just a few hours is leading to similar breakthroughs in characterizing proteomes, metabolomes, phenotypes, and fluxes for organisms, populations, and communities. Understanding and modeling functional microbial community structure and stress responses in subsurface environments has tremendous implications for our fundamental understanding of biogeochemistry and the potential for making biofuel breakthroughs. Monitoring techniques that inventory and monitor terminal electron acceptors and electron donors, enzyme probes that measure functional activity in the environment, functional genomic microarrays, phylogenetic microarrays, metabolomics, proteomics, and quantitative PCR are also being rapidly adapted for studies in environmental biotechnology. Integration of all of these new high throughput techniques using the latest advances in bioinformatics and modeling will enable break-through science in environmental biotechnology. A review of these techniques with examples from field studies and lab simulations will be discussed.
910. Hazen, T. C. and S. Singer, J. VanderGheynst, P. M. D’haeseleer, M. P. Thelen, K. DeAngelis, A. Reddy, M. Allgaier, J. Fortney, G. Andersen, T. DeSantis, E. Brodie, C. Wu, D. Goodheart, M. Firestone, W. Silver, B. Simmons P. Hugenholtz. 2009. JBEI Microbial Communities Deconstruction Research Activities. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
911. Hazen, T. C. and J. Keasling, A. Mukhopadhyay, S. Chhabra, J. T. Geller, M. Singer, D. Joyner, L. Camp, T. Torok, J. Wall, D. Elias, M. D. Biggin H.-Y. Holman. 2009. Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes during Stress Response in Desulfovibrio vulgaris: Microbiology Subproject. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
912. Hazen, T. C. and S. Borglin, E. Brodie, S. van Dien, M. Fields, J. Fortney, J. Geller, E. Hendrickson, K. L Hillesland, H.-Y. Holman, J. Leigh, T. Lie, J. Jacobsen, D. Joyner, R Chakraborty, M. Keller, A. Mukhopadhyay, C. Schadt, D. Stahl, S. Stolyar, C. Walker, J. Wall, Z. Yang, H.-C. B. Yen, G. Zane, J. Zhou G. Anderson. 2009. Applied Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers VIMSS:ESPP. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
913. Hazen, T. C. and B. Faybishenko. 2009. Update on Groundwater Issues across the DOE Complex. 4th Annual DOE-ERSP PI Meeting
914. Hazen, T. C. and H. Beller, E. Brodie, S. S. Hubbard, J. Peterson, E. Sonnenthal, C. Steefel, L. Yang, J. Larsen, M. Conrad, J. Christensen, S. Brown, D. Joyner, S. Borglin, J. Geller, R. Chakraborty, P. Nico, T. Tokunaga, J. Wan, M. Firestone, P. Long, D. Newcomer, L. N’Guessan B. Faybishenko. 2009. Field-Scale Investigations of Cryptic Growth and Memory Response Hypotheses at the Chromium Contaminated Hanford 100-H Site. 4th Annual DOE-ERSP PI Meeting
915. Hazen, Terry C. and G. L. Andersen. 2009. A Systems Biology Approach to Environmental Biotechnology using Ecogenomics. National University of Singapore
916. Hazen, Terry C. and G. L. Andersen. 2009. LBNL SuperChip Grand Challenge. Science and Technology, U. S. Department of Homeland Security
917. Hazen, Terry C.. 2009. Systems Biology the New Frontier for Bioenergy. National University of Singapore
918. Hazen, Terry C. 2009. A Systems Biology Approach to Environmental Biotechnology using Ecogenomics. International Society for Microbial Ecology and International Water Association Special Colloquia
919. Hazen, Terry C. 2009. Bioremediation: the Hope and the Hype. Manitoba Environmental Industry Association Meeting
920. Hazen, Terry C. 2009. A Systems Biology Approach to Bioremediation. Manitoba Environmental Industry Association Meeting
921. Hazen, Terry C. 2009. Hanford 100H Cr Bioremediation. DOE EM RO conference on 100 area Cr
922. Hazen, Terry C. 2009. Systems Biology for Bioenergy. DOE Bioenergy Research Centers Biweekly Conference Call
923. Hazen, Terry C.. 2009. Environmental Biotechnology – a Tour through the ‘Omics’. AEHS annual west coast meeting abstract
     Environmental biotechnology encompasses a wide range of characterization, monitoring and control or remediation technologies that are based on biological processes. Recent breakthroughs in our understanding of biogeochemical processes and genomics are leading to exciting new and cost effective ways to monitor and manipulate the environment. Indeed, our ability to sequence an entire microbial genome in just a few hours is leading to similar breakthroughs in characterizing proteomes, metabolomes, phenotypes, and fluxes for organisms, populations, and communities. Understanding and modeling functional microbial community structure and stress responses in subsurface environments has tremendous implications for our fundamental understanding of biogeochemistry and the potential for natural attenuation or bioremediation of contaminated sites. Monitoring techniques that inventory and monitor terminal electron acceptors and electron donors, enzyme probes that measure functional activity in the environment, functional genomic microarrays, phylogenetic microarrays, metabolomics, proteomics, and quantitative PCR are also being rapidly adapted for studies in environmental biotechnology. Integration of all of these new high throughput techniques using the latest advances in bioinformatics and modeling will enable break-through science in environmental biotechnology. A review of these techniques with examples from field studies and lab simulations will be discussed.
924. Hazen, Terry C.. 2009. Systems Biology: The New Frontier for Bioenergy. NanoFocus and Bioenergy Oklahoma EPSCoR Annual State Conference 2009
925. Hazen, Terry C.. 2009. Systems Biology: The New Frontier for Bioenergy. Department of Microbiology, University of Oklahoma
926. Hazen, Terry C. 2009. Metagenomics of Soil Rain Forest. Terragenome Workshop
927. Hazen, Terry C.. 2009. Life in the Slow Lane: Ecogenomics of an Extreme Environment. BAGECON 10 International Conference abstract
     A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced using a combination of Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Moorella thermoacetica), as well as other known sulfate reducers and thermophilic organisms. D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea. Additionally, study of the deep subsurface has offered access to the simplest community yet studied by environmental genomics, perhaps consisting of just a single species that is capable of performing all of the tasks necessary for life.
928. Hazen, Terry C., B. K. Fouke, J. D. Coates. 2009. EBI Microbial Enhanced Hydrocarbon Recovery. Energy Biosciences Institute Annual Retreat
929. Hazen, Terry C.. 2009. EBI Microbial Enhanced Hydrocarbon Recovery. Energy Biosciences Institute Annual Retreat
930. Hazen, Terry C. 2009. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. Master Universitario in Biotecnologia, University of Granada
931. Hazen, Terry C. 2009. Life in the Slow Lane: Ecogenomics in Extreme Environments - Implications for Enhanced Oil Recovery. University of California at Berkeley, Energy Biosciences Institute Seminar
932. Hazen, Terry C., B. Faybishenko, P. Jordan. 2009. Characterization of a Contaminant Inventory at DOE Sites, as a Tool for Selecting Monitoring and Remediation Technologies. American Geophysical Union Annual Meeting abstract
     The U.S. Department of Energy (DOE) is responsible for the remediation and long-term stewardship of one of the world’s largest groundwater contamination portfolios, with a significant number of plumes containing various contaminants, and considerable total mass and activity. The frequency of occurrence and ranking of contaminants in groundwater plumes is one of the main criteria needed for decision-making related to planning and prioritizing the types of basic research and the development of site characterization, monitoring, and remedial approaches. Using the data from 60 DOE sites, including 221 groundwater plumes, collected in the DOE Groundwater Database (GWD), we evaluated the frequency of occurrence of specific contaminants and their associations, plume volumes, contaminant maximum concentrations, masses, and isotope activities. Contaminants detected in groundwater at 60 DOE sites and facilities can be categorized into the following eight generic contaminant groups: chlorinated hydrocarbons (chlorinated ethenes), fuels and fuel components (i.e., petroleum/fuel hydrocarbons), explosives, metals, radioactive isotopes (excluding tritium), tritium, sulfates, and nitrates. The most common are plumes containing two (29.4% of all plumes in the GWD) and three (29%) contaminant groups. The most frequent binary combinations of contaminant groups are those of mixed waste, including chlorinated hydrocarbons and tritium—35% and metals and isotopes—28% of all plumes. Our results were compared to the data from 18 DOE sites and 91 plumes, collected in 1992, to illustrate the progress in site characterization and remediation over the past decade. The analysis of contaminant inventory and plume characteristics should be helpful in establishing priorities for basic research needs, which will enable cost-effective and efficient application of new characterization, monitoring, modeling, and remediation technologies.
933. Hazen, Terry C., E. Sonnenthal, S. Mukhopadhyay, C. Steefel, P. Long, B. Faybishenko. 2009. Field and Numerical Modeling Study of Reductive Bioimmobilization of Cr (VI) in Groundwater at Hanford 100H Site. American Geophysical Union Annual Meeting abstract
     The 2004-2008 field experiment at the Hanford 100-H Site showed that a single injection of the hydrogen release compound (HRC)—a mixture of slow release glycerol polylactate, fast disassociating lactic acid, and glycerol into groundwater— stimulated an increase in biomass and a depletion of terminal electron acceptors, resulting in a significant decrease in soluble Cr (VI). The Cr (VI) concentration remained below the background concentration in the downgradient pumping/monitoring well, and below the detection limit in the injection well, for more than 3 years after the initial HRC injection in 2004. Reaction-transport modeling of the field experiment was performed to elucidate reaction pathways and rates of biogeochemical processes governing Cr (VI) bioimmobilization. Field observation data were used to develop a 3-D hydraulic and reaction-transport-isotopic model, simulated using the TOUGHREACT code. This model was used to assess the degradation kinetics of the HRC, the effects of lactate and acetate-induced bioreduction of Cr and Fe, and the effects on Cr and Sr isotopic compositions in the fluids and mineral phases (calcite dissolution in the acidic plume and precipitation at the fringes). The 2008 experiment involved reinjection of HRC into the same zones as the earlier injection and likely resulted in dissolution of previously precipitated Cr (Fe) hydroxides and additional calcite dissolution in sediments outside of the wellbore. Detailed results of the integrated field and modeling study will be presented.
934. Faybishenko, B., Terry C. Hazen and S. S. Hubbard. 2009. Application of Innovative Bioremediation Technologies for Metals and Radionuclides in Soils and Groundwater. IAEA International Conference on Remediation of Land Contaminated by Radioactive Material Residues abstract
     Bioremediation of metals and radionuclides is a relatively new approach for cleaning up metal and radioactively soils and groundwater at contaminated sites. The presentation will include: (1) a review of the main types of bioremediation technologies—biotransformation, bioaccumulation/bisorption, biodegradation using chelation, biovolatilization, and natural attenuation—as well as an application of multiple treatment methods; (2) a discussion of the promising results of ongoing research and case studies from field tests, demonstrations, and full-scale implementation of different bioremediation technologies at several contaminated sites, along with a summary of biogeochemical criteria needed to implement these technologies for cleaning up metals and radionuclides in soils and groundwater; and (3) a series of innovative in situ site characterization and long-term monitoring methods associated with these technologies. We will also emphasize the need for implementing a combination of both in situ and ex situ remediation strategies.
935. Faybishenko, B., Terry C. Hazen. 2009. Multiple Factor Analysis and k-Means Clustering-Based Classification of the DOE Groundwater Contaminant Database. American Geophysical Union Annual Meeting abstract
     A proper classification of the plume characteristics is critical for selecting the most suitable characterization, monitoring, and remediation technologies. To perform a statistical analysis of the different groundwater plume characteristics, we used the DOE Groundwater Database, including 221 groundwater plumes located at 60 DOE sites. To classify the plume characteristics, we used a multiple factor analysis (MFA), including a principal component analysis (PCA) of quantitative plume characteristics and a multiple correspondence analysis (MCA) of qualitative plume characteristics. The input parameters used for the statistical analysis are: the presence of eight types of contaminant groups—chlorinated hydrocarbons, fuels, explosives, sulfates, nitrates, metals, tritium, and radioisotopes; a number and associations of contaminant groups; a contamination severity index (based on the association of contaminant groups and complexity of remediation); contaminant mass and plume volumes; groundwater depth and velocities; and climatic conditions. The input variables are also partitioned into the active and supplementary plume characteristics. Statistical results include the evaluation of the correlation matrix between the groups of variables and individual plume characteristics. From the results of the MFA, the first four factors can be used to describe the variability of the basic plume characteristics. The contaminant severity index and the number of contaminant groups provide a major contribution to the 1st factor; the types of contaminant groups and carbon tetrachloride concentrations provide the major contribution to the 2nd factor. The contribution of the supplementary data (climate and plume depth and velocity) is insignificant. The presence of radioactive contaminants is mostly related to the 1st factor; the presence of sulfates, and to a lesser degree the presence of nitrates and metals, is related to the 2nd factor. The strongest relationship is, as expected, between the types of contaminant groups and the contamination severity. The relationships between contaminant groups and the plume depth and velocity, and contaminant groups and climate are weak, and there is no a significant relationship with the plume volumes. To visualize the contribution of different factors, the results of MFA calculations are presented using two- and three-dimensional maps. Using the first four factors for the basic plume characteristics, a k-means cluster analysis was applied to classify the plumes into respective clusters. These results can be used to plan characterization, monitoring, and modeling of contaminant behavior at contaminated sites, and to design appropriate remediation technologies.
936. Elias, D. A. and M. D. Biggin, G. Butland, S. Chhabra, A. Fagorala, Terry C. Hazen, D. Jorgans, D. C. Joyner, T. R. Juba, M. Perez, J. P. Remis, A. Tauscher, J. D. Wall M. Auer. 2009. Protein Complex Analysis Project (PCAP): Localization of Multi-Protein Complexes through SNAP-Tag Labeling. Annual meeting of the American Society for Microbiology abstract
     The broad goal of this subproject of PCAP is to develop tag-based labeling approaches for highthroughput subcellular localization of proteins in microorganisms of interest to DOE. Our goal is to determine the abundance, the spatial organization and relative locations of proteins within Desulfovibrio vulgaris Hildenborough (DvH) cells in pure cultures, as well as in DvH in microbial communities under both routine laboratory and