Hazen Lab Masthead
Lab Director

Dr. Terry C. Hazen
UT/ORNL Governor's Chair Professor

Dept. of Civil & Environmental Engineering
Dept. of Microbiology
Dept. of Earth and Planetary Sciences
Genome Science and Technology
Bredesen Center

The University of Tennessee
Office: 325 John D. Tickle Engineering Building
Office: 851 Neyland Drive
Office: 507 SERF
Lab: 729 SERF
Knoxville, Tennessee 37996-2313
Cell: 707-631-6763
Phone: 865-974-7709
E-mail: tchazen@utk.edu

Oak Ridge National Laboratory
Office: 327 JIBS
Lab: 316 JIBS


(420 published, 1 in press, 14 submitted, 10 in preparation, 18,180 citations, ISI H-index=69, Research Gate H-index=79 for 22,793 citations Google Citations)  Note: ISI Journal Impact Factor for 2022, citations as of 05/03/2024
Endnote XML file    Abstracts:

  1. Rafie,S. A. A., L. R. Blentlinger, A. D. Putt, D. E. Williams, D. C. Joyner, M. F. Campa, M. J. Schubert, K. P. Hoyt, Sally P. Horn, J. A. Franklin and T. C. Hazen. 2024. Impact of prescribed fire on soil microbial communities in a Southern Appalachian Forest clear-cut. Frontiers in Microbiology 15. abstract pdf

    Escalating wildfire frequency and severity, exacerbated by shifting climate patterns, pose significant ecological and economic challenges. Prescribed burns, a common forest management tool, aim to mitigate wildfire risks and protect biodiversity. Nevertheless, understanding the impact of prescribed burns on soil and microbial communities in temperate mixed forests, considering temporal dynamics and slash fuel types, remains crucial. Our study, conducted at the University of Tennessee Forest Resources AgResearch and Education Center in Oak Ridge, TN, employed controlled burns across various treatments, and the findings indicate that low-intensity prescribed burns have none or minimal short-term effects on soil parameters but may alter soil nutrient concentrations, as evidenced by significant changes in porewater acetate, formate, and nitrate concentrations. These burns also induce shifts in microbial community structure and diversity, with Proteobacteria and Acidobacteria increasing significantly post-fire, possibly aiding soil recovery. In contrast, Verrucomicrobia showed a notable decrease over time, and other specific microbial taxa correlated with soil pH, porewater nitrate, ammonium, and phosphate concentrations. Our research contributes to understanding the intricate relationships between prescribed fire, soil dynamics, and microbial responses in temperate mixed forests in the Southern Appalachian Region, which is valuable for informed land management practices in the face of evolving environmental challenges.

  2. Ning, D. L., Y. J. Wang, Y. P. Fan, J. J. Wang, J. D. Van Nostrand, L. Y. Wu, P. Zhang, D. J. Curtis, R. M. Tian, L. R. Lui, T. C. Hazen, E. J. Alm, M. W. Fields, F. Poole, M. W. W. Adams, R. Chakraborty, D. A. Stahl, P. D. Adams, A. P. Arkin, Z. L. He and J. Z. Zhou. 2024. Environmental stress mediates groundwater microbial community assembly. Nature Microbiology 9:22. abstract pdf
    Community assembly describes how different ecological processes shape microbial community composition and structure. How environmental factors impact community assembly remains elusive. Here we sampled microbial communities and >200 biogeochemical variables in groundwater at the Oak Ridge Field Research Center, a former nuclear waste disposal site, and developed a theoretical framework to conceptualize the relationships between community assembly processes and environmental stresses. We found that stochastic assembly processes were critical (>60% on average) in shaping community structure, but their relative importance decreased as stress increased. Dispersal limitation and 'drift' related to random birth and death had negative correlations with stresses, whereas the selection processes leading to dissimilar communities increased with stresses, primarily related to pH, cobalt and molybdenum. Assembly mechanisms also varied greatly among different phylogenetic groups. Our findings highlight the importance of microbial dispersal limitation and environmental heterogeneity in ecosystem restoration and management.
  3. Michael, Jonathan P., Andrew D. Putt, Yunfeng Yang, Benjamin G. Adams, Kathryn R. McBride, Yupeng Fan, Kenneth A. Lowe, Daliang Ning, Sindhu Jagadamma, Ji Won Moon, Dawn M. Klingeman, Ping Zhang, Ying Fu, Terry C. Hazen and Jizhong Zhou. 2024. Reproducible responses of geochemical and microbial successional patterns in the subsurface to carbon source amendment. Water Research 255:121460. abstract pdf
    Carbon amendments designed to remediate environmental contamination lead to substantial perturbations when injected into the subsurface. For the remediation of uranium contamination, carbon amendments promote reducing conditions to allow microorganisms to reduce uranium to an insoluble, less mobile state. However, the reproducibility of these amendments and underlying microbial community assembly mechanisms have rarely been investigated in the field. In this study, two injections of emulsified vegetable oil were performed in 2009 and 2017 to immobilize uranium in the groundwater at Oak Ridge, TN, USA. Our objectives were to determine whether and how the injections resulted in similar abiotic and biotic responses and their underlying community assembly mechanisms. Both injections caused similar geochemical and microbial succession. Uranium, nitrate, and sulfate concentrations in the groundwater dropped following the injection, and specific microbial taxa responded at roughly the same time points in both injections, including Geobacter, Desulfovibrio, and members of the phylum Comamonadaceae, all of which are well established in uranium, nitrate, and sulfate reduction. Both injections induced a transition from relatively stochastic to more deterministic assembly of microbial taxonomic and phylogenetic community structures based on 16S rRNA gene analysis. We conclude that geochemical and microbial successions after biostimulation are reproducible, likely owing to the selection of similar phylogenetic groups in response to EVO injection.
  4. Li, Ye, K. T. Ash, D. C. Joyner, D. E. Williams and T. C. Hazen. 2024. SARS-CoV-2 virus in Raw Wastewater from Student Residence Halls with concomitant 16S rRNA Bacterial Community Structure changes. medRxiv 2024.02.11.24302582. pdf
  5. Li, Y, K. T. Ash, I. Alamilla, D. C. Joyner, D. E. Williams, P. J. McKay, B. M. Green, S. E. DeBlander, C. M. North, F. Kara-Murdoch, C. M. Swift and T. C. Hazen. 2024. COVID-19 Trends at The University of Tennessee: Predictive Insights from Raw Sewage SARS-CoV-2 Detection and Evaluation and PMMoV as an Indicator for Human Waste. medRxiv pdf
  6. Li,Ye, Kurt Ash, Isablla Alamilla, Dominique Joyner, Daniel Edward Williams, Peter J. McKay, Brianna Green, Sydney DeBlander, Carman North, Fadime Kara-Murdoch, Cynthia Swift and Terry C. Hazen. 2024. COVID-19 trends at the University of Tennessee: predictive insights from raw sewage SARS-CoV-2 detection and evaluation and PMMoV as an indicator for human waste. Frontiers in Microbiology 15. abstract pdf

    Wastewater-based epidemiology (WBE) has become a valuable tool for monitoring the prevalence of SARS-CoV-2 on university campuses. However, concerns about effectiveness of raw sewage as a COVID-19 early warning system still exist, and it’s not clear how useful normalization by simultaneous comparison of Pepper Mild Mottle Virus (PMMoV) is in addressing variations resulting from fecal discharge dilution. This study aims to contribute insights into these aspects by conducting an academic-year field trial at the student residences on the University of Tennessee, Knoxville campus, raw sewage. This was done to investigate the correlations between SARS-CoV-2 RNA load, both with and without PMMoV normalization, and various parameters, including active COVID-19 cases, self-isolations, and their combination among all student residents. Significant positive correlations between SARS-CoV-2 RNA load a week prior, during the monitoring week, and the subsequent week with active cases. Despite these correlations, normalization by PMMoV does not enhance these associations. These findings suggest the potential utility of SARS-CoV-2 RNA load as an early warning indicator and provide valuable insights into the application and limitations of WBE for COVID-19 surveillance specifically within the context of raw sewage on university campuses.

  7. Hunt, Kristopher A., Alex V. Carr, Anne E. Otwell, Jacob J. Valenzuela, Kathleen S. Walker, Emma R. Dixon, Lauren M. Lui, Torben N. Nielsen, Samuel Bowman, Frederick von Netzer, Ji-Won Moon, Christopher W. Schadt, Miguel Rodriguez Jr, Kenneth Lowe, Dominique Joyner, Katherine J. Davis, Xiaoqin Wu, Romy Chakraborty, Matthew W. Fields, Jizhong Zhou, Terry C. Hazen, Adam P. Arkin, Scott D. Wankel, Nitin S. Baliga and David A. Stahl. 2024. Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide. Environmental Science & Technology abstract pdf
    The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.
  8. Wu, X. Q., S. Gushgari-Doyle, L. M. Lui, A. J. Hendrickson, Y. N. Liu, S. Jagadamma, T. N. Nielsen, N. B. Justice, T. Simmons, N. J. Hess, D. C. Joyner, T. C. Hazen, A. P. Arkin and R. Chakraborty. 2023. Distinct Depth-Discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone. Applied and Environmental Microbiology 89:14. abstract pdf
    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.
  9. Thorgersen, M. P., J. L. Goff, F. L., II Poole, K. F. Walker, A. D. Putt, L. M. Lui, T. C. Hazen, A. P. Arkin and M. W. W. Adams. 2023. Mixed nitrate and metal contamination influences operational speciation of toxic and essential elements. Environmental Pollution 338:122674. abstract pdf
    Environmental contamination constrains microbial communities impacting diversity and total metabolic activity. The former S-3 Ponds contamination site at Oak Ridge Reservation (ORR), TN, has elevated concentrations of nitric acid and multiple metals from decades of processing nuclear material. To determine the nature of the metal contamination in the sediment, a three-step sequential chemical extraction (BCR) was performed on sediment segments from a core located upgradient (EB271, non-contaminated) and one downgradient (EB106, contaminated) of the S-3 Ponds. The resulting exchangeable, reducing, and oxidizing fractions were analyzed for 18 different elements. Comparison of the two cores revealed changes in operational speciation for several elements caused by the contamination. Those present from the S-3 Ponds, including Al, U, Co, Cu, Ni, and Cd, were not only elevated in concentration in the EB106 core but were also operationally more available with increased mobility in the acidic environment. Other elements, including Mg, Ca, P, V, As, and Mo, were less operationally available in EB106 having decreased concentrations in the exchangeable fraction. The bioavailability of essential macro nutrients Mg, Ca, and P from the two types of sediment was determined using three metal-tolerant bacteria previously isolated from ORR. Mg and Ca were available from both sediments for all three strains; however, P was not bioavailable from either sediment for any strain. The decreased operational speciation of P in contaminated ORR sediment may increase the dependence of the microbial community on other pools of P or select for microorganisms with increased P scavenging capabilities. Hence, the microbial community at the former S-3 Ponds contamination site may be constrained not only by increased toxic metal concentrations but also by the availability of essential elements, including P.
  10. Li,Ye, Kurt T. Ash, Dominique C. Joyner, Daniel E. Williams, Isabella Alamilla, Peter J. McKay, Chris Iler and Terry C. Hazen. 2023. Evaluating various composite sampling modes for detecting pathogenic SARS-CoV-2 virus in raw sewage. Frontiers in Microbiology 14. abstract pdf
    Inadequate sampling approaches to wastewater analyses can introduce biases, leading to inaccurate results such as false negatives and significant over- or underestimation of average daily viral concentrations, due to the sporadic nature of viral input. To address this challenge, we conducted a field trial within the University of Tennessee residence halls, employing different composite sampling modes that encompassed different time intervals (1 h, 2 h, 4 h, 6 h, and 24 h) across various time windows (morning, afternoon, evening, and late-night). Our primary objective was to identify the optimal approach for generating representative composite samples of SARS-CoV-2 from raw wastewater. Utilizing reverse transcription-quantitative polymerase chain reaction, we quantified the levels of SARS-CoV-2 RNA and pepper mild mottle virus (PMMoV) RNA in raw sewage. Our findings consistently demonstrated that PMMoV RNA, an indicator virus of human fecal contamination in water environment, exhibited higher abundance and lower variability compared to pathogenic SARS-CoV-2 RNA. Significantly, both SARS-CoV-2 and PMMoV RNA exhibited greater variability in 1 h individual composite samples throughout the entire sampling period, contrasting with the stability observed in other time-based composite samples. Through a comprehensive analysis of various composite sampling modes using the Quade Nonparametric ANCOVA test with date, PMMoV concentration and site as covariates, we concluded that employing a composite sampler during a focused 6 h morning window for pathogenic SARS-CoV-2 RNA is a pragmatic and cost-effective strategy for achieving representative composite samples within a single day in wastewater-based epidemiology applications. This method has the potential to significantly enhance the accuracy and reliability of data collected at the community level, thereby contributing to more informed public health decision-making during a pandemic.
  11. Li, Y., K. T. Ash, D. C. Joyner, D. E. Williams, I. Alamilla, P. J. McKay, C. Iler, B. M. Green, F. Kara-Murdoch, C. M. Swift and T. C. Hazen. 2023. Decay of enveloped SARS-CoV-2 and non-enveloped PMMoV RNA in raw sewage from university dormitories. Frontiers in Microbiology 14:8. abstract pdf
    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.
  12. Kokate, P. P., E. Bales, D. Joyner, T. C. Hazen and S. M. Techtmann. 2023. Biogeographic patterns in populations of marine Pseudoalteromonas atlantica isolates. Fems Microbiology Letters 370:9. abstract pdf
    Intra-specific genomic diversity is well documented in microbes. The question, however, remains whether natural selection or neutral evolution is the major contributor to this diversity. We undertook this study to estimate genomic diversity in Pseudoalteromonas atlantica populations and whether the diversity, if present, could be attributed to environmental factors or distance effects. We isolated and sequenced twenty-three strains of P. atlantica from three geographically distant deep marine basins and performed comparative genomic analyses to study the genomic diversity of populations among these basins. Average nucleotide identity followed a strictly geographical pattern. In two out of three locations, the strains within the location exhibited >99.5% identity, whereas, among locations, the strains showed <98.11% identity. Phylogenetic and pan-genome analysis also reflected the biogeographical separation of the strains. Strains from the same location shared many accessory genes and clustered closely on the phylogenetic tree. Phenotypic diversity between populations was studied in ten out of twenty-three strains testing carbon and nitrogen source utilization and osmotolerance. A genetic basis for phenotypic diversity could be established in most cases but was apparently not influenced by local environmental conditions. Our study suggests that neutral evolution may have a substantial role in the biodiversity of P. atlantica.
  13. https://cee.utk.edu/natures-wrath-environmental-experts-home-damaged-by-tornado/. 2023. Nature’s Wrath: Environmental Expert’s Home Damaged by Tornado. Civil & Environmental Engineering
  14. Griffiths,Z. G., Andrew D. Putt, J. I. Miller, Maria Fernanda Campa, Dominique C. Joyner, O. Pelz, Nargiz Garajayeva, M. Ceccopieri, P. Gardinali and Terry C. Hazen. 2023. Comparing the response of the indigenous microbial community to crude oil amendment in oxic versus hypoxic conditions. Frontiers in Microbiomes 2. abstract pdf
    IntroductionThe Caspian Sea is the world’s largest landlocked saline lake which lies between Europe and Asia. This region is particularly known for its large-scale oil reserves, pipelines, and drilling activities, which have contributed to the environmental decline of this lake. 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. The temperature and oxygen stratification in this environment has presented a unique opportunity to investigate the potential of the biodegradative processes carried out by the indigenous microbial community. We believe these indigenous microbes possess different metabolic capabilities to degrade oil as they adapted to declining oxygen concentrations and temperatures with increasing depths over a prolonged period. Hence, community structure and composition will vary with depth.MethodsMicrocosms were set up to observe the indigenous microbial reaction after a 60 ppm native crude oil amendment over 115 days. Surface water microcosms were incubated at 28ºC and aerated while deep water microcosms were incubated at 8ºC under anaerobic conditions. These two environmental conditions represent the temperature and oxygen extremes along the gradient and were selected as we try to simulate the indigenous community’s response to this oil contamination. DNA was extracted and amplified from these microcosms and sequenced. 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.ResultsAll 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 versus hypoxic microcosms.ConclusionOrders of Bacteria related to sulfate and nitrogen cycling were found in hypoxic microcosms, indicating a possible mechanism for the anaerobic biodegradation of crude oil. GC-MS analysis of initial and final microcosms also provided evidence of degradation of hydrocarbon fractions in both warm, oxic and cold, hypoxic conditions.
  15. Goff, J. L., L. M. Lui, T. N. Nielsen, F. L. Poole II, H. J. Smith, D. C. Joyner, K. F. Walker, T. C. Hazen, M. W. Fields, A. P. Arkin and M. W. W. Adams. 2023. Mixed waste contamination selects for a mobile genetic element population enriched in multiple heavy metal resistance genes. bioRxiv 2022.04.05.481366. abstract pdf
    Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal contaminated regions of the Oak Ridge Reservation (ORR) subsurface. This meta-mobilome was compared to one derived from samples collected from unimpacted regions of the ORR subsurface. We assembled 1,615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.Competing Interest StatementThe authors have declared no competing interest.
  16. 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 14:9. abstract pdf
    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.
  17. Zhou Jason Shi, Naijia Xiao, Daliang Ning, Renmao Tian, Ping Zhang, Daniel Curtis, Joy D. Van Nostrand, Liyou Wu, Terry C. Hazen, Andrea M. Rocha, Zhili He, Adam P. Arkin, Mary K. Firestone and Jizhong Zhou. 2022. EcoFun-MAP: An Ecological Function Oriented Metagenomic Analysis Pipeline. bioRxiv 2022.04.05.481366.
  18. Shi, Zhou Jason, Naijia Xiao, Daliang Ning, Renmao Tian, Ping Zhang, Daniel Curtis, Joy D. Van Nostrand, Liyou Wu, Terry C. Hazen, Andrea M. Rocha, Zhili He, Adam P. Arkin, Mary K. Firestone and Jizhong Zhou. 2022. EcoFun-MAP: An Ecological Function Oriented Metagenomic Analysis Pipeline. bioRxiv 2022.04.05.481366. abstract pdf
    Annotating ecological functions of environmental metagenomes is challenging due to a lack of specialized reference databases and computational barriers. Here we present the Ecological Function oriented Metagenomic Analysis Pipeline (EcoFun-MAP) for efficient analysis of shotgun metagenomes in the context of ecological functions. We manually curated a reference database of EcoFun-MAP which is used for GeoChip design. This database included ∼1,500 functional gene families that were catalogued by important ecological functions, such as carbon, nitrogen, phosphorus, and sulfur cycling, metal homeostasis, stress responses, organic contaminant degradation, antibiotic resistance, microbial defense, electron transfer, virulence and plant growth promotion. EcoFun-MAP has five optional workflows from ultra-fast to ultra-conservative, fitting different research needs from functional gene exploration to stringent comparison. The pipeline is deployed on High Performance Computing (HPC) infrastructure with a highly accessible web-based interface. We showed that EcoFun-MAP is accurate and can process multi-million short reads in a minute. We applied EcoFun-MAP to analyze metagenomes from groundwater samples and revealed interesting insights of microbial functional traits in response to contaminations. EcoFun-MAP is available as a public web server at http://iegst1.rccc.ou.edu:8080/ecofunmap/.Competing Interest StatementThe authors have declared no competing interest.
  19. Putt, A. D., S. Rafie and T. C. Hazen. 2022. Large-Data Omics Approaches in Modern Remediation. Journal of Environmental Engineering 148:02522001-3. abstract pdf
    Forum papers are thought-provoking opinion pieces or essays founded in fact, sometimes containing speculation, on a civil engineering topic of general interest and relevance to the readership of the journal. The views expressed in this Forum article do not necessarily reflect the views of ASCE or the Editorial Board of the journal.
  20. Putt, Andrew D., Erin R. Kelly, Kenneth A. Lowe, Miguel Rodriguez and Terry C. Hazen. 2022. Effects of Cone Penetrometer Testing on Shallow Hydrogeology at a Contaminated Site. Frontiers in Environmental Science 9. abstract pdf
    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.
  21. Peng, M., D. Y. Wang, L. M. Lui, T. Nielsen, R. M. Tian, M. L. Kempher, X. Y. Tao, C. L. Pan, R. Chakraborty, A. M. Deutschbauer, M. P. Thorgersen, M. W. W. Adams, M. W. Fields, T. C. Hazen, A. P. Arkin, A. F. Zhou and J. Z. Zhou. 2022. Genomic Features and Pervasive Negative Selection in Rhodanobacter Strains Isolated from Nitrate and Heavy Metal Contaminated Aquifer. Microbiology Spectrum 10:17. abstract pdf
    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.
  22. 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 pdf
    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.
  23. Harik, A. M. G., Z. Griffiths and T. C. Hazen. 2022. Omics of oil biodegradation. Current Opinion in Chemical Engineering 36:6. abstract pdf
    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'.
  24. Goff, J. L., E. G. Szink, M. P. Thorgersen, A. D. Putt, Y. P. Fan, L. M. Lui, T. N. Nielsen, K. A. Hunt, J. P. Michael, Y. J. Wang, D. L. Ning, Y. Fu, J. D. Van Nostrand, F. L. Poole, J. M. Chandonia, T. C. Hazen, D. A. Stahl, J. Z. Zhou, A. P. Arkin and M. 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 pdf
    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.
  25. Goff, J. L., L. M. Lui, T. N. Nielsen, M. P. Thorgersen, E. G. Szink, J. M. Chandonia, F. L. Poole, J. Z. Zhou, T. C. Hazen, A. P. Arkin and M. 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 11:3. abstract pdf
    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.
  26. Chen, S., Y. F. Wang, H. C. Cheng, T. C. Hazen, C. G. He and Q. He. 2022. Identification of Propionate-Degrading Microbial Populations in Methanogenic Processes for Waste Treatment: Methanosaeta and Methanoculleus. Environmental Engineering Science 39:202-211. abstract pdf
    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.

  27. Campa, M. F., J. R. C. See, L. V. Unverdorben, O. G. Wright, K. A. Roth, J. M. Niles, D. Ressler, E. M. S. Macatugal, A. D. Putt, S. M. Techtmann, T. L. Righetti, T. C. Hazen and R. Lamendella. 2022. Geochemistry and Multiomics Data Differentiate Streams in Pennsylvania Based on Unconventional Oil and Gas Activity. Microbiology Spectrum 15. abstract pdf
    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.
  28. See, J. R. C., O. Wright, L. V. Unverdorben, N. Heibeck, S. M. Techtmann, T. C. Hazen and R. Lamendella. 2021. Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures. Jove-Journal of Visualized Experiments 17. abstract pdf
    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.
  29. Putt, A. D., E. R. Kelly, K. A. Lowe, Jr. M. Rodriguez and T. C. Hazen. 2021. Effects of Cone Penetrometer Testing on Shallow Hydrogeology at a Contaminated Site. EarthArXiv abstract pdf
    Penetration testing is a popular and instantaneous technique for subsurface mapping, contaminant tracking, and the determination of soil characteristics. While the small footprint and reproducibly 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 upgradient and down-gradient of CPT activity. The physical act of advancing and retracting a piezocone had a significant effect on specific conductivity and water level but no effect on dissolved oxygen or pH. While cone penetrometer effects were significant and detectable, the variability induced by CPT activity was only a fraction of the natural variation caused by precipitation events. Therefore, we concluded that CPT effects are less than those of natural event-driven variation in clayey and silty unconsolidated residuum.
  30. Charles J. Paradis, 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. 2021. Sustained ability of a natural microbial community to remove nitrate from groundwater. bioRxiv 2021.05.27.446013.
  31. Nayfach, S., S. Roux, R. Seshadri, D. Udwary, N. Varghese, F. Schulz, D. Y. Wu, D. Paez-Espino, I. M. Chen, M. Huntemann, K. Palaniappan, J. Ladau, S. Mukherjee, T. B. K. Reddy, T. Nielsen, E. Kirton, J. P. Faria, J. N. Edirisinghe, C. S. Henry, S. P. Jungbluth, D. Chivian, P. Dehal, E. M. Wood-Charlson, A. P. Arkin, S. G. Tringe, A. Visel, T. Woyke, N. J. Mouncey, N. N. Ivanova, N. C. Kyrpides, E. A. Eloe-Fadrosh and Img M. Data Consortium. 2021. A genomic catalog of Earth's microbiomes (Nov, 10.1038/s41587-020-0718-6, 2020). Nature Biotechnology 39:521-521. abstract pdf
    Correction to: Nature Biotechnology https://doi.org/10.1038/s41587-020-0718-6, published online 9 November 2020. In the version of this article initially published, four people were missing from the alphabetical list of IMG/M Data Consortium members: Lauren V. Alteio of the Centre for Microbiology and Environmental Systems Science, University of Vienna, Vienna, Austria; Jeffrey L. Blanchard of the Biology Department, University of Massachusetts Amherst, Amherst, MA, USA; Kristen M. DeAngelis of the Department of Microbiology, University of Massachusetts Amherst, Amherst, MA, USA; and William Rodriguez-Reillo of the Research Computing Division, Harvard Medical School, Boston, MA, USA. The error has been corrected in the PDF and HTML versions of the article. *A list of authors and their affiliations appears online.
  32. Lui, L. M., E. L. W. Majumder, H. J. Smith, H. K. Carlson, F. von Netzer, M. W. Fields, D. A. Stahl, J. Z. Zhou, T. C. Hazen, N. S. Baliga, P. D. Adams, A. P. Arkin and Enigma Consortium. 2021. Mechanism Across Scales: A Holistic Modeling Framework Integrating Laboratory and Field Studies for Microbial Ecology. Frontiers in Microbiology 12:15. abstract pdf
    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.
  33. Si Chen, Yongfeng Wang, Huicai Cheng, Terry C. Hazen, Chunguang He and Qiang He. 2021. Identification of Propionate-Degrading Microbial Populations in Methanogenic Processes for Waste Treatment: Methanosaeta and Methanoculleus. Environmental Engineering Science abstract pdf
    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.
  34. 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:e0104921-e0104921. abstract pdf
    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.
  35. Alhajjar, R. K., R. B. Ghannam, J. R. Chen See, M. F. Campa, O. G. Wright, T. C. Hazen, R. Lamendella and S. 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 284:131255. abstract pdf
    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.
  36. Wilpiszeski, R. L., C. M. Gionfriddo, A. M. Wymore, J. W. Moon, K. A. Lowe, M. Podar, S. Rafie, M. W. Fields, T. C. Hazen, X. X. Ge, F. Poole, M. W. W. Adams, R. Chakraborty, Y. P. Fan, J. D. Van Nostrand, J. Z. Zhou, A. P. Arkin and D. A. Elias. 2020. In-field bioreactors demonstrate dynamic shifts in microbial communities in response to geochemical perturbations. bioRxiv 2020.04.16.044594.
  37. Wilpiszeski, R. L., C. M. Gionfriddo, A. M. Wymore, J. W. Moon, K. A. Lowe, M. Podar, S. Rafie, M. W. Fields, T. C. Hazen, X. X. Ge, F. Poole, M. W. W. Adams, R. Chakraborty, Y. P. Fan, J. D. Van Nostrand, J. Z. Zhou, A. P. Arkin and D. A. Elias. 2020. In-field bioreactors demonstrate dynamic shifts in microbial communities in response to geochemical perturbations. Plos One 15:18. abstract pdf
    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.
  38. Tian, R. M., D. L. Ning, Z. L. He, P. Zhang, S. J. Spencer, S. H. Gao, W. L. Shi, L. W. Wu, Y. Zhang, Y. F. Yang, B. G. Adams, A. M. Rocha, B. L. Detienne, K. A. Lowe, D. C. Joyner, D. M. Klingeman, A. P. Arkin, M. W. Fields, T. C. Hazen, D. A. Stahl, E. J. Alm and J. Z. Zhou. 2020. Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity. Microbiome 8:15. abstract pdf
    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.
  39. 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 13:169-169. abstract pdf
    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 10L/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±0.51A/m2, although the acetate fed MECs outperformed complex substrates, producing 12.3±0.01A/m2. 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. Geobacter was 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±0.00g/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 genusappeared to correlated to these performance metricsstrongly, 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.
  40. Nayfach, Stephen, Simon Roux, Rekha Seshadri, Daniel Udwary, Neha Varghese, Frederik Schulz, Dongying Wu, David Paez-Espino, I. Min Chen, Marcel Huntemann, Krishna Palaniappan, Joshua Ladau, Supratim Mukherjee, T. B. K. Reddy, Torben Nielsen, Edward Kirton, Jose P. Faria, Janaka N. Edirisinghe, Christopher S. Henry, Sean P. Jungbluth, Dylan Chivian, Paramvir Dehal, Elisha M. Wood-Charlson, Adam P. Arkin, Susannah G. Tringe, Axel Visel, Tanja Woyke, Nigel J. Mouncey, Natalia N. Ivanova, Nikos C. Kyrpides, Emiley A. Eloe-Fadrosh and Img M. Data Consortium. 2020. A genomic catalog of Earth's microbiomes. Nature biotechnology abstract pdf
    The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth's continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.
  41. National Academies of Sciences, Engineering, and Medicine. 2020. The Use of Dispersants in Marine Oil Spill Response. 410. abstract pdf
    Whether the result of an oil well blowout, vessel collision or grounding, leaking pipeline, or other incident at sea, each marine oil spill will present unique circumstances and challenges. The oil type and properties, location, time of year, duration of spill, water depth, environmental conditions, affected biomes, potential human community impact, and available resources may vary significantly. Also, each spill may be governed by policy guidelines, such as those set forth in the National Response Plan, Regional Response Plans, or Area Contingency Plans. To respond effectively to the specific conditions presented during an oil spill, spill responders have used a variety of response options—including mechanical recovery of oil using skimmers and booms, in situ burning of oil, monitored natural attenuation of oil, and dispersion of oil by chemical dispersants. Because each response method has advantages and disadvantages, it is important to understand specific scenarios where a net benefit may be achieved by using a particular tool or combination of tools. This report builds on two previous National Research Council reports on dispersant use to provide a current understanding of the state of science and to inform future marine oil spill response operations. The response to the 2010 Deepwater Horizon spill included an unprecedented use of dispersants via both surface application and subsea injection. The magnitude of the spill stimulated interest and funding for research on oil spill response, and dispersant use in particular. This study assesses the effects and efficacy of dispersants as an oil spill response tool and evaluates trade-offs associated with dispersant use.
  42. Moon, J. W., C. J. Paradis, D. C. Joyner, F. von Netzer, E. L. Majumder, E. R. Dixon, M. Podar, X. X. Ge, P. J. Walian, H. J. Smith, X. Q. Wu, G. M. Zane, K. F. Walker, M. P. Thorgersen, F. L. Poole, L. M. Lui, B. G. Adams, K. B. De Leon, S. S. Brewer, D. E. Williams, K. A. Lowe, M. Rodriguez, T. L. Mehlhorn, S. M. Pfiffner, R. Chakraborty, A. P. Arkin, J. D. Wall, M. W. Fields, M. W. W. Adams, D. A. Stahl, D. A. Elias and T. C. Hazen. 2020. Characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer. Chemosphere 255:14. abstract pdf
    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.
  43. Miller, J. I., S. Techtmann, D. Joyner, N. Mahmoudi, J. Fortney, J. A. Fordyce, N. GaraJayeva, F. S. Askerov, C. Cravid, M. Kuijper, O. Pelz and T. C. Hazen. 2020. Microbial Communities across Global Marine Basins Show Important Compositional Similarities by Depth. Mbio 11:12. abstract pdf
    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.
  44. Mahmoudi, N., S. M. Hagen, T. C. Hazen and A. D. Steen. 2020. Patterns in extracellular enzyme activity and microbial diversity in deep-sea Mediterranean sediments. Deep-Sea Research Part I-Oceanographic Research Papers 158:7. abstract pdf
    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.
  45. Ge, X. X., M. P. Thorgersen, F. L. Poole, A. M. Deutschbauer, J. M. Chandonia, P. S. Novichkov, S. Gushgari-Doyle, L. M. Lui, T. Nielsen, R. Chakraborty, P. D. Adams, A. P. Arkin, T. C. Hazen and M. 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 11:15. abstract pdf
    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.
  46. Ge, X. X., M. P. Thorgersen, F. L. Poole, A. M. Deutschbauer, J. M. Chandonia, P. S. Novichkov, P. D. Adams, A. P. Arkin, T. C. Hazen and M. W. W. Adams. 2020. Draft Genome Sequence of Bacillus sp. Strain EB106-08-02-XG196, Isolated from High-Nitrate-Contaminated Sediment. Microbiology Resource Announcements 9:2. abstract pdf
    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.
  47. Ge, Xiaoxuan, Michael P. Thorgersen, Farris L. 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. Data_Sheet_1_Characterization of a Metal-Resistant Bacillus Strain With a High Molybdate Affinity ModA From Contaminated Sediments at the Oak Ridge Reservation.PDF. Figshare 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 ( Copyright: CC BY 4.0
  48. Hazen T. C.. 2020. Lessons from the 2010 Deepwater Horizon Accident in the Gulf of Mexico. Hydrocarbons, Oils and Lipids: Diversity, Origin, Chemistry and Fate. Handbook of Hydrocarbon and Lipid Microbiology 847-864. abstract pdf
    The 2010 Deepwater Horizon (DWH) accident in the Gulf of Mexico had many unique aspects to it not seen in previous marine spills. Indeed, research related to the DWH response phase, Natural Resource Damage Assessment, Gulf of Mexico Research Initiative (GoMRI), National Academy of Sciences, US agencies: NOAA, EPA, Fish & Wildlife, DOE, and Coast Guard have made this the most studied marine oil spill in the world. There are many oil biodegradation lessons learned from this experience and these will undoubtedly continue for many years.
  49. Zelaya, A. J., A. E. Parker, K. L. Bailey, P. Zhang, J. Van Nostrand, N. Daliang, D. A. Elias, J. Zhou, T. C. Hazen and M. W. Fields. 2019. High spatiotemporal variability of bacterial diversity over short time scales with unique geohydrochemistry associations within a shallow, pristine aquifer scales with unique geohydrochemistry associations within a shallow, pristine aquifer. Water Research 164. abstract pdf
    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 noncontaminated 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 hydrochemically 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.
  50. Paradis, Charles J., Larry D. McKay, Edmund Perfect, Jonathan D. Istok and Terry C. Hazen. 2019. Correction: Push-pull tests for estimating effective porosity: expanded analytical solution and in situ application. Hydrogeology Journal 27:437-439. abstract pdf
    In the recent article by Paradis et al. (2018), an error was made when developing a conceptual model and an analytical solution to describe the one-dimensional displacement of the center of mass of a non-reactive tracer during a single-well injection, drift, and extraction test (push-pull test).
  51. 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 pdf
    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 ratio of the concentrations of the non‐reactive and reactive solutes in the injection fluid versus 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 non‐reactive 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 non‐reactive and reactive solutes in the injection and aquifer fluids, and easily allows for estimating reaction rates during push‐pull tests.
  52. Moon, J.-W., C. J. Paradis, D. C. Joyner, F. von Netzer, E. L. Majumder, E. Dixon, M. Podar, G. Xiaoxuan, P. J. Walian, H. J. Smith, X. Wu, G. M. Zane, K. S. Walker, M. P. Thorgersen, F. L. Poole II, L. Lui, B. G. Adams, K. B. De León, S. S. Brewer, D. E. Williams, K. A. Lowe, M. Rodriguez, Jr., T. L. Mehlhorn, S. M. Pfiffner, R. Chakraborty, A. P. Arkin, J. D. Wall, M. W. Fields, M. W. W. Adams, D. A. Stahl, D. A. Elias and T. C. Hazen. 2019. Integrated characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer. bioRxiv abstract pdf
    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 environments. To provide site assessment for a larger study, we processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater from the ENIGMA Field Research Site at the United States Department of Energy (DOE) Oak Ridge Reservation (ORR). We compared fresh core sediments by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including contaminants, 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. This environmental systems approach provided detailed site-specific biogeochemical information from the various properties of subsurface media to reveal the influences of solid, liquid, and gas phases. 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. This suggested that groundwater predominantly flowed through preferential paths with high flux and little mixing with water in the interstices of sediment particles, which could impact microbial activity. The abundant clay minerals with high surface area and high water-holding capacity of micro-pores of the fine clay rich layer suggest suppression of nutrient supply to microbes from the surface. 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 highly toxic 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.
  53. 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. Data_Sheet_1_Oil Hydrocarbon Degradation by Caspian Sea Microbial Communities.PDF. Figshare 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. Copyright: CC BY 4.0
  54. Miller, J. I., S. Techtmann, J. Fortney, N. Mahmoudi, D. Joyner, J. Liu, S. Olesen, E. Alm, A. Fernandez, P. Gardinali, N. Garajayeva, F. S. Askerov and T. C. Hazen. 2019. Oil Hydrocarbon Degradation by Caspian Sea Microbial Communities. Frontiers in Microbiology 10:15. abstract pdf
    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.
  55. Kothari, A., Y. W. Wu, J. M. Chandonia, M. Charrier, L. Rajeev, A. M. Rocha, D. C. Joyner, T. C. Hazen, S. W. Singer and A. Mukhopadhyay. 2019. Plasmid DNA analysis of pristine groundwater microbial communities reveal extensive presence of metal resistance genes. bioRxiv 113860.
  56. Kothari, A., Y. W. Wu, J. M. Chandonia, M. Charrier, L. Rajeev, A. M. Rocha, D. C. Joyner, T. C. Hazen, S. W. Singer and A. Mukhopadhyay. 2019. Large Circular Plasmids from Groundwater Plasmidomes Span Multiple Incompatibility Groups and Are Enriched in Multimetal Resistance Genes. Mbio 10:15. abstract pdf
    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.
  57. Johnston, E. R., M. Kim, J. K. Hatt, J. R. Phillips, Q. M. Yao, Y. Song, T. C. Hazen, M. A. Mayes and K. T. Konstantinidis. 2019. Phosphate addition increases tropical forest soil respiration primarily by deconstraining microbial population growth. Soil Biology & Biochemistry 130:43-54. abstract pdf
    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.
  58. Hazen, Terry C.. 2019. Environmental Systems Biology Approach to Bioremediation. Understanding Terrestrial Microbial Communities 103-127. abstract pdf
    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., an environmental systems biology approach to bioremediation, answer the need which 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. By 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.
  59. Ge, X. X., B. J. Vaccaro, M. P. Thorgersen, F. L. Poole, E. L. Majumder, G. M. Zane, K. B. De Leon, W. A. Lancaster, J. W. Moon, C. J. Paradis, F. von Netzer, D. A. Stahl, P. D. Adams, A. P. Arkin, J. D. Wall, T. C. Hazen and M. W. W. Adams. 2019. Iron- and aluminium-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle. Environmental Microbiology 21:152-163. abstract pdf
    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.
  60. Couradeau, E., J. Sasse, D. Goudeau, N. Nath, T. C. Hazen, B. Bowen, R. Chakraborty, R. R. Malmstrom and T. R. Northen. 2019. Probing the active fraction of soil microbiomes using BONCAT-FACS. Nature Communications 10:10. abstract pdf
    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.
  61. Campa, M. F., A. K. Wolfe, S. M. Techtmann, A. M. Harik and T. C. Hazen. 2019. Unconventional Oil and Gas Energy Systems: An Unidentified Hotspot of Antimicrobial Resistance?. Frontiers in Microbiology 10:9. abstract pdf
    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.
  62. Campa, M. F., S. M. Techtmann, M. P. Ladd, J. Yan, M. Patterson, A. G. D. Amaral, K. E. Carter, N. Ulrich, C. J. Grant, R. L. Hettich, R. Lamendella and T. 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 85:18. abstract pdf
    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.
  63. Yao, Q. M., Z. Li, Y. Song, S. J. Wright, X. Guo, S. G. Tringe, M. M. Tfaily, L. Pasa-Tolic, T. C. Hazen, B. L. Turner, M. A. Mayes and C. L. Pan. 2018. Community proteogenomics reveals the systemic impact of phosphorus availability on microbial functions in tropical soil. Nature Ecology & Evolution 2:499-509. abstract pdf
    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.
  64. Wu, X. Q., L. Y. Wu, Y. Liu, P. Zhang, Q. H. Li, J. Z. Zhou, N. J. Hess, T. C. Hazen, W. L. Yang and R. Chakraborty. 2018. Microbial Interactions With Dissolved Organic Matter Drive Carbon Dynamics and Community Succession. Frontiers in Microbiology 9:1-12. abstract pdf
    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.
  65. Woo, H. L. and T. C. Hazen. 2018. Enrichment of Bacteria From Eastern Mediterranean Sea Involved in Lignin Degradation via the Phenylacetyl-CoA Pathway. Frontiers in Microbiology 9:1-11. abstract pdf
    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.
  66. Ulrich, N., V. Kirchner, R. Drucker, J. R. Wright, C. J. McLimans, T. C. Hazen, M. F. Campa, C. J. Grant and R. Lamendella. 2018. Response of Aquatic Bacterial Communities to Hydraulic Fracturing in Northwestern Pennsylvania: A Five-Year Study. Scientific Reports 8:12. abstract pdf
    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.
  67. 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 94:16. abstract pdf
    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.
  68. See, J. R. C., N. Ulrich, H. Nwanosike, C. J. McLimans, V. Tokarev, J. R. Wright, M. F. Campa, C. J. Grant, T. C. Hazen, J. M. Niles, D. Ressler and R. Lamendella. 2018. Bacterial Biomarkers of Marcellus Shale Activity in Pennsylvania. Frontiers in Microbiology 9:13. abstract pdf
    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.
  69. Ribicic, D., R. Netzer, T. C. Hazen, S. M. Techtmann, F. Drablos and O. G. 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 pdf
    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.
  70. Paradis, C. J., J. W. Moon, D. A. Elias, L. D. McKay and T. C. Hazen. 2018. In situ decay of polyfluorinated benzoic acids under anaerobic conditions. Journal of Contaminant Hydrology 217:8-16. abstract pdf
    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.
  71. Paradis, C. J., L. D. McKay, E. Perfect, J. D. Istok and T. C. Hazen. 2018. Push-pull tests for estimating effective porosity: expanded analytical solution and in situ application. Hydrogeology Journal 26:381-393. abstract pdf
    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.
  72. Marietou, A., R. Chastain, F. Beulig, A. Scoma, T. C. Hazen and D. 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 9:1-11. abstract pdf
    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.
  73. Lewis, A. J., M. F. Campa, T. C. Hazen and A. P. Borole. 2018. Unravelling biocomplexity of electroactive biofilms for producing hydrogen from biomass. Microbial Biotechnology 11:84-97. abstract pdf
    Leveraging nature's biocomplexity for solving human problems requires better understanding of the syntrophic relationships in engineered microbiomes developed in bioreactor systems. Understanding the interactions between microbial players within the community will be key to enhancing conversion and production rates from biomass streams. Here we investigate a bioelectrochemical system employing an enriched microbial consortium for conversion of a switchgrass-derived bio-oil aqueous phase (BOAP) into hydrogen via microbial electrolysis (MEC). MECs offer the potential to produce hydrogen in an integrated fashion in biorefinery platforms and as a means of energy storage through decentralized production to supply hydrogen to fuelling stations, as the world strives to move towards cleaner fuels and electricity-mediated transportation. A unique approach combining differential substrate and redox conditions revealed efficient but rate-limiting fermentation of the compounds within BOAP by the anode microbial community through a division of labour strategy combined with multiple levels of syntrophy. Despite the fermentation limitation, the adapted abilities of the microbial community resulted in a high hydrogen productivity of 9.35L per L-day. Using pure acetic acid as the substrate instead of the biomass-derived stream resulted in a three-fold improvement in productivity. This high rate of exoelectrogenesis signifies the potential commercial feasibility of MEC technology for integration in biorefineries.
  74. Kimbrel, J. A., N. Ballor, Y. W. Wu, M. M. David, T. C. Hazen, B. A. Simmons, S. W. Singer and J. K. Jansson. 2018. Microbial Community Structure and Functional Potential Along a Hypersaline Gradient. Frontiers in Microbiology 9:1-15. abstract pdf
    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.
  75. He, Z. L., P. Zhang, L. W. Wu, A. M. Rocha, Q. C. Tu, Z. Shi, B. Wu, Y. J. Qin, J. J. Wang, Q. Y. Yan, D. Curtis, D. L. Ning, J. D. Van Nostrand, L. Y. Wu, Y. F. Yang, D. A. Elias, D. B. Watson, M. W. W. Adams, M. W. Fields, E. J. Alm, T. C. Hazen, P. D. Adams, A. P. Arkin and J. Z. Zhou. 2018. Microbial Functional Gene Diversity Predicts Groundwater Contamination and Ecosystem Functioning. Mbio 9:15. abstract pdf
    Contamination from anthropogenic activities has significantly impacted Earth's biosphere. However, knowledge about how environmental contamination affects the biodiversity of groundwater microbiomes and ecosystem functioning remains very limited. Here, we used a comprehensive functional gene array to analyze groundwater microbiomes from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide pH range and uranium, nitrate, and other contaminants. We hypothesized that the functional diversity of groundwater microbiomes would decrease as environmental contamination (e.g., uranium or nitrate) increased or at low or high pH, while some specific populations capable of utilizing or resistant to those contaminants would increase, and thus, such key microbial functional genes and/or populations could be used to predict groundwater contamination and ecosystem functioning. Our results indicated that functional richness/diversity decreased as uranium (but not nitrate) increased in groundwater. In addition, about 5.9% of specific key functional populations targeted by a comprehensive functional gene array (GeoChip 5) increased significantly (P < 0.05) as uranium or nitrate increased, and their changes could be used to successfully predict uranium and nitrate contamination and ecosystem functioning. This study indicates great potential for using microbial functional genes to predict environmental contamination and ecosystem functioning. IMPORTANCE Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology.
  76. Hazen, T. C. and S. M. Techtmann. 2018. Oil Biodegradation in Deep Marine Basins. Handbook of Hydrocarbon and Lipid Microbiology Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Biodegradation and Bioremediation. abstract pdf
    Nine important hydrocarbon (oil) basins where offshore petroleum leases have been licensed are compared. These nine basins (Gulf of Mexico, Eastern Mediterranean’s Nile Deep-Sea Fan, Central Mediterranean and the Sirte Basin, North Sea, Caspian Sea, Angola, Trinidad and Tobago, Great Australian Bight, and Brazil’s Amazonian Deep-Sea Basin) are geographically separated and are impacted by very different water masses. The geochemical parameters of these basins are quite distinct, for example, salinities ranging from 39 psu in the Eastern Mediterranean to 12 psu in the Caspian. Additionally, parameters like temperatures of the bottom water are also very distinct, with the temperature in the deep water of the Eastern Mediterranean being between 12 °C and 14 °C and the temperature of the deep water in the North Sea being −2 °C. Each basin represents a unique ecosystem in which distinct microbes may thrive. These distinct environmental parameters may act to constrain the extent of hydrocarbon degradation in these basins. Another potential constraint on hydrocarbon degradation is the extent of natural hydrocarbon seeps in the area. Though many basins have similar if not 16S rRNA identical strains of oil-degrading bacteria, Colwellia psychrerythraea from different basins showed that a mixture of natural selection and neutral evolution has contributed to the divergence of these. Most if not all deep ocean basin microbial communities are dominated by Thaumarchaeota below 200 m. These microaerophilic, ammonium oxidizer, psychrophiles are very adapted to an oligotrophic lifestyle, and though many in this group will degrade oil, they are rapidly outcompeted by other bacteria in oil or high hydrocarbon intrusions, thus the virtual “canary in the coal mine.” Cometabolic biodegradation of oil is well documented but could be an important natural attenuation mechanism for oil in deep marine basins with episodic methane seeps. Microbial community structure can also predict concentrations of oil in deep basins. Many other synergistic effects require more research in environmental systems biology in deep marine basins.
  77. Hazen, Terry C.. 2018. Bioremediation. Microbiology of the Terrestrial Subsurface 247-266. pdf
  78. Hazen, Terry C.. 2018. Lessons from the 2010 Deepwater Horizon Accident in the Gulf of Mexico. Hydrocarbons, Oils and Lipids: Diversity, Origin, Chemistry and Fate. Handbook of Hydrocarbon and Lipid Microbiology 1-19. abstract pdf
    The 2010 Deepwater Horizon (DWH) accident in the Gulf of Mexico had many unique aspects to it not seen in previous marine spills. Indeed, research related to the DWH response phase, Natural Resource Damage Assessment, Gulf of Mexico Research Initiative (GoMRI), National Academy of Sciences, US agencies: NOAA, EPA, Fish & Wildlife, DOE, and Coast Guard have made this the most studied marine oil spill in the world. There are many oil biodegradation lessons learned from this experience and these will undoubtedly continue for many years.
  79. Hazen, Terry C.. 2018. In Situ: Groundwater Bioremediation. Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Biodegradation and Bioremediation 1-18. abstract pdf
    In situ groundwater bioremediation of hydrocarbons has been used for more than 40 years. Most strategies involve biostimulation; however, recently bioaugmentation have been used for dehalorespiration. Aquifer and contaminant profiles are critical to determining the feasibility and strategy for in situ groundwater bioremediation. Hydraulic conductivity and redox conditions, including concentrations of terminal electron acceptors, are critical to determine the feasibility and strategy for potential bioremediation applications. Conceptual models followed by characterization and subsequent numerical models are critical for efficient and cost-effective bioremediation. Critical research needs in this area include better modeling and integration of remediation strategies with natural attenuation.
  80. Hazen, Terry C.. 2018. Cometabolic Bioremediation. Consequences of Microbial Interactions with Hydrocarbons, Oils, and Lipids: Biodegradation and Bioremediation 1-15. abstract pdf
    Cometabolic bioremediation is probably the most underappreciated bioremediation strategy currently available. Cometabolism strategies stimulate only indigenous microbes with the ability to degrade the contaminant and cosubstrate, e.g., methane, propane, toluene, and others. This highly targeted stimulation insures that only those microbes that can degrade the contaminant are targeted, thus reducing amendment costs, well and formation plugging, etc. Cometabolic bioremediation has been used on some of the most recalcitrant contaminants, e.g., PCE, TCE, MTBE, TNT, dioxane, atrazine. Methanotrophs have been demonstrated to produce methane monooxygenase, an oxidase that can degrade over 1000 compounds. Cometabolic bioremediation also has the advantage of being able to degrade contaminants to trace concentrations, since the biodegrader is not dependent on the contaminant for carbon or energy. In the Gulf of Mexico and in the Arctic Tundra, we have recently found that natural attenuation can be a cometabolic process also. Increasingly we are finding that in order to protect human health and the environment that we must remediate to lower and lower concentrations, especially for compounds like endocrine disrupters and trace organics, thus cometabolism may be the best and may be the only possibility that we have to bioremediate some contaminants.
  81. Danso, D., C. Schmeisser, J. Chow, W. Zimmermann, R. Wei, C. Leggewie, X. Z. Li, T. Hazen and W. R. Streit. 2018. New Insights into the Function and Global Distribution of Polyethylene Terephthalate (PET)-Degrading Bacteria and Enzymes in Marine and Terrestrial Metagenomes. Applied and Environmental Microbiology 84:13. abstract pdf
    Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly alpha- and beta-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes. Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil. IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases.
  82. Couradeau, Estelle, Joelle Sasse, Danielle Goudeau, Nandita Nath, Terry C. Hazen, Benjamin P. Bowen, Rex R. Malmstrom and Trent R. Northen. 2018. Study of Oak Ridge soils using BONCAT-FACS-Seq reveals that a large fraction of the soil microbiome is active. bioRxiv abstract pdf
    The ability to link soil microbial diversity to soil processes requires technologies that differentiate active subpopulations of microbes from so-called relic DNA and dormant cells. Measures of microbial activity based on various techniques including DNA labelling has suggested that most cells in soils are inactive, a fact that has been difficult to reconcile with observed high levels of bulk soil activities. We hypothesized that measures of in situ DNA synthesis may be missing the soil microbes that are metabolically active but not replicating, and we therefore applied BONCAT (Biorthogonal Non Canonical Amino Acid Tagging) i.e. a proxy for activity that does not rely on cell division, to measure translationally active cells in soils. We compared the active population of two soil depths from Oak Ridge (TN) incubated in the same condition for up to seven days. Depending on the soil, a maximum of 25-70% of the cells were active, accounting for 3-4 million cells per gram of soil, which is an order of magnitude higher to previous estimates. The BONCAT positive cell fraction was recovered by fluorescence activated cell sorting (FACS) and identified by 16S rDNA amplicon sequencing. The diversity of the active fraction was a selected subset of the bulk soil community. Excitingly, some of the same members of the community were recruited at both depths independently from their abundance rank. On average, 86% of sequence reads recovered from the active community shared >97% sequence similarity with cultured isolates from the field site. Our observations are in line with a recent report that, of the few taxa that are both abundant and ubiquitous in soil, 45% are also cultured - and indeed some of these ubiquitous microorganisms found to be translationally active. The use of BONCAT in soil provides evidence that a large portion of the soil microbes can be active simultaneously. We conclude that BONCAT coupled to FACS and sequencing is effective for interrogating the active fraction of soil microbiomes in situ and provides new perspectives to link metabolic capacity to overall soil ecological traits and processes.
  83. Christensen, G. A., J. Moon, A. M. Veach, J. J. Mosher, A. M. Wymore, J. D. van Nostrand, J. Z. Zhou, T. C. Hazen, A. P. Arkin and D. A. Elias. 2018. Use of in-field bioreactors demonstrate groundwater filtration influences planktonic bacterial community assembly, but not biofilm composition. Plos One 13:20. abstract pdf
    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.
  84. Chen, C. Y., G. Pan, W. Q. Shi, F. Xu, S. M. Techtmann, S. M. Pfiffner and T. C. Hazen. 2018. Clay Flocculation Effect on Microbial Community Composition in Water and Sediment. Frontiers in Environmental Science 6:10. abstract pdf
    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.
  85. Campa, M. F., S. M. Techtmann, C. M. Gibson, X. J. Zhu, M. Patterson, A. G. D. Amaral, N. Ulrich, S. R. Campagna, C. J. Grant, R. Lamendella and T. 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 pdf
    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.
  86. Beattie, R. E., W. Henke, M. F. Campa, T. C. Hazen, L. R. McAliley and J. 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 pdf
    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.
  87. Zhang, P., Z. L. He, J. D. Van Nostrand, Y. J. Qin, Y. Deng, L. Y. Wu, Q. C. Tu, J. J. Wang, C. W. Schadt, M. W. Fields, T. C. Hazen, A. P. Arkin, D. A. Stahl and J. Z. Zhou. 2017. Dynamic Succession of Groundwater Sulfate-Reducing Communities during Prolonged Reduction of Uranium in a Contaminated Aquifer. Environmental Science & Technology 51:3609-3620. abstract pdf
    To further understand the diversity and dynamics of SRB in response to substrate amendment, we sequenced genes coding for the dissimilatory sulfite reductase (dsrA) in groundwater samples collected after an emulsified vegetable oil (EVO) amendment, which sustained U(VI)-reducing conditions for one year in a fast-flowing aquifer. EVO amendment significantly altered the composition of groundwater SRB communities. Sequences having no closely related-described species dominated (80%) the indigenous SRB communities in nonamended wells. After EVO amendment, Desulfococcus, Desulfobacterium, and Desulfovibrio, known for long-chain-fatty-acid, short-chain-fatty-acid and H-2 oxidation and U(VI) reduction, became dominant accounting for 7 +/- 2%, 21 +/- 8%, and 55 +/- 8% of the SRB communities, respectively. Succession of these SRB at different bioactivity stages based on redox substrates/products (acetate, SO4-2, U(VI), NO3-, Fe(II), and Mn(II)) was observed. Desulfovibrio and Desulfococcus dominated SRB communities at 4-31 days, whereas Desulfobacterium became dominant at 80-140 days. By the end of the experiment (day 269), the abundance of these SRB decreased but the overall diversity of groundwater SRB was still higher than non-EVO controls. Up to 62% of the SRB community changes could be explained by groundwater geochemical variables, including those redox substrates/products. A significant (P < 0.001) correlation was observed between groundwater U(VI) concentrations and Desulfovibrio abundance. Our results showed that the members of SRB and their dynamics were correlated significantly with slow EVO biodegradation, electron donor production and maintenance of U(VI)-reducing conditions in the aquifer.
  88. Wright, J., V. Kirchner, W. Bernard, N. Ulrich, C. McLimans, M. F. Campa, T. Hazen, T. Macbeth, D. Marabello, J. McDermott, R. Mackelprang, K. Roth and R. Lamendella. 2017. Bacterial Community Dynamics in Dichloromethane-Contaminated Groundwater Undergoing Natural Attenuation. Frontiers in Microbiology 8:15. abstract pdf
    The uncontrolled release of the industrial solvent methylene chloride, also known as dichloromethane (DCM), has resulted in widespread groundwater contamination in the United States. Here we investigate the role of groundwater bacterial communities in the natural attenuation of DCM at an undisclosed manufacturing site in New Jersey. This study investigates the bacterial community structure of groundwater samples differentially contaminated with DCM to better understand the biodegradation potential of these autochthonous bacterial communities. Bacterial community analysis was completed using high-throughput sequencing of the 16S rRNA gene of groundwater samples (n = 26) with DCM contamination ranging from 0.89 to 9,800,000 mu g/L. Significant DCM concentration-driven shifts in overall bacterial community structure were identified between samples, including an increase in the abundance of Firmicutes within the most contaminated samples. Across all samples, a total of 6,134 unique operational taxonomic units (OTUs) were identified, with 16 taxa having strong correlations with increased DCM concentration. Putative DCM degraders such as Pseudomonas, Dehalobacterium and Desulfovibrio were present within groundwater across all levels of DCM contamination. Interestingly, each of these taxa dominated specific DCM contamination ranges respectively. Potential DCM degrading lineages yet to be cited specifically as a DCM degrading organisms, such as the Desulfosporosinus, thrived within the most heavily contaminated groundwater samples. Co-occurrence network analysis revealed aerobic and anaerobic bacterial taxa with DCM-degrading potential were present at the study site. Our 16S rRNA gene survey serves as the first in situ bacterial community assessment of contaminated groundwater harboring DCM concentrations ranging over seven orders of magnitude. Diversity analyses revealed known as well as potentially novel DCM degrading taxa within defined DCM concentration ranges, indicating niche-specific responses of these autochthonous populations. Altogether, our findings suggest that monitored natural attenuation is an appropriate remediation strategy for DCM contamination, and that high-throughput sequencing technologies are a robust method for assessing the potential role of biodegrading bacterial assemblages in the apparent reduction of DCM concentrations in environmental scenarios.
  89. Woo, H. L., K. M. DeAngelis, H. Teshima, K. Davenport, H. Daligault, T. Erkkila, L. Goodwin, W. Gu, C.-C. Lo, C. Munk, M. Scholz, Y. Xu, P. Chain, D. Bruce, C. Detter, R. Tapia, C. Han, B. A. Simmons and T. C. Hazen. 2017. High Quality Draft Genome Sequences of Four Lignocellulose-degrading Bacteria from Puerto Rican Forest Soil- Gordonia sp., Paenibacillus sp., Variovorax sp., and Vogesella sp.. Genome Announcement 4. abstract pdf
    Here, we report the high-quality draft genome sequences of four phylogenetically diverse lignocellulose-degrading bacteria isolated from tropical soil (Gordonia sp., Paenibacillus sp., Variovorax sp., and Vogesella sp.) to elucidate the genetic basis of their ability to degrade lignocellulose. These isolates may provide novel enzymes for biofuel production
  90. Techtmann, S. M., M. B. Zhuang, P. Campo, E. Holder, M. Elk, T. C. Hazen, R. Conmy and J. W. S. Domingo. 2017. Corexit 9500 Enhances Oil Biodegradation and Changes Active Bacterial Community Structure of Oil-Enriched Microcosms. Applied and Environmental Microbiology 83:14. abstract pdf
    To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25 degrees C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25 degrees C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25 degrees C consortia were dominated by Vibrio, Idiomarina, Marinobacter, Alcanivorax, and Thalassospira species, while the 5 degrees C consortia were dominated by several species of the genera Flavobacterium, Alcanivorax, and Oleispira. Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5 degrees C consortia, while at 25 degrees C, a decrease in the relative abundance of Marinobacter was observed. At 25 degrees C, Thalassospira, Marinobacter, and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5 degrees C. IMPORTANCE While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups.
  91. Techtmann, S. M., N. Mahmoudi, K. T. Whitt, M. F. Campa, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2017. Comparison of Thaumarchaeotal populations from four deep sea basins. FEMS Microbiology Ecology 93:10. abstract pdf
    The nitrogen cycle in the marine environment is strongly affected by ammonia-oxidizing Thaumarchaeota. In some marine settings, Thaumarchaeotes can comprise a large percentage of the prokaryotic population. To better understand the biogeographic patterns of Thaumarchaeotes, we sought to investigate differences in their abundance and phylogenetic diversity between geographically distinct basins. Samples were collected from four marine basins (The Caspian Sea, the Great Australian Bight, and the Central and Eastern Mediterranean). The concentration of bacterial and archaeal 16S rRNA genes and archaeal amoA genes were assessed using qPCR. Minimum entropy decomposition was used to elucidate the fine-scale diversity of Thaumarchaeotes. We demonstrated that there were significant differences in the abundance and diversity of Thaumarchaeotes between these four basins. The diversity of Thaumarchaeotal oligotypes differed between basins with many oligotypes only present in one of the four basins, which suggests that their distribution showed biogeographic patterning. There were also significant differences in Thaumarchaeotal community structure between these basins. This would suggest that geographically distant, yet geochemically similar basins may house distinct Thaumarchaeaotal populations. These findings suggest that Thaumarchaeota are very diverse and that biogeography in part contributes in determining the diversity and distribution of Thaumarchaeotes.
  92. Liu, J., S. M. Techtmann, H. L. Woo, D. L. Ning, J. L. Fortney and T. C. Hazen. 2017. Rapid Response of Eastern Mediterranean Deep Sea Microbial Communities to Oil. Scientific Reports 7:11. abstract pdf
    Deep marine oil spills like the Deepwater Horizon (DWH) in the Gulf of Mexico have the potential to drastically impact marine systems. Crude oil contamination in marine systems remains a concern, especially for countries around the Mediterranean Sea with off shore oil production. The goal of this study was to investigate the response of indigenous microbial communities to crude oil in the deep Eastern Mediterranean Sea (E. Med.) water column and to minimize potential bias associated with storage and shifts in microbial community structure from sample storage. 16S rRNA amplicon sequencing was combined with GeoChip metagenomic analysis to monitor the microbial community changes to the crude oil and dispersant in on-ship microcosms set up immediately after water collection. After 3 days of incubation at 14 degrees C, the microbial communities from two different water depths: 824 m and 1210 m became dominated by well-known oil degrading bacteria. The archaeal population and the overall microbial community diversity drastically decreased. Similarly, GeoChip metagenomic analysis revealed a tremendous enrichment of genes related to oil biodegradation, which was consistent with the results from the DWH oil spill. These results highlight a rapid microbial adaption to oil contamination in the deep E. Med., and indicate strong oil biodegradation potential.
  93. King, A. J., S. P. Preheim, K. L. Bailey, M. S. Robeson, T. R. Chowdhury, B. R. Crable, R. A. Hurt, T. Mehlhorn, K. A. Lowe, T. J. Phelps, A. V. Palumbo, C. C. Brandt, S. D. Brown, M. Podar, P. Zhang, W. A. Lancaster, F. Poole, D. B. Watson, M. W. Fields, J. M. Chandonia, E. J. Alm, J. Z. Zhou, M. W. W. Adams, T. C. Hazen, A. P. Arkin and D. A. Elias. 2017. Temporal Dynamics of In-Field Bioreactor Populations Reflect the Groundwater System and Respond Predictably to Perturbation. Environmental Science & Technology 51:2879-2889. abstract pdf
    Temporal variability complicates testing the influences of environmental variability on microbial community structure and thus function. An in-field bioreactor system was developed to assess oxic versus anoxic manipulations on in situ groundwater communities. Each sample was sequenced (16S SSU rRNA genes, average 10,000 reads), and biogeochemical parameters are monitored by quantifying 53 metals, 12 organic acids, 14 anions, and 3 sugars. Changes in dissolved oxygen (DO), pH, and other variables were similar across bioreactors. Sequencing revealed a complex community that fluctuated in-step with the groundwater community and responded to DO. This also directly influenced the pH, and so the biotic impacts of DO and pH shifts are correlated. A null model demonstrated that bioreactor communities were driven in part not only by experimental conditions, but also by stochastic variability and did not accurately capture alterations in diversity during perturbations. We identified two groups of abundant OTUs important to this system; one was abundant in high DO and pH and contained heterotrophs and oxidizers of iron, nitrite, and ammonium, whereas the other was abundant in low DO with the capability to reduce nitrate. In-field bioreactors are a powerful tool for capturing natural microbial community responses to alterations in geochemical factors beyond the bulk phase.
  94. Justice, N. B., A. Sczesnak, T. C. Hazen and A. P. Arkin. 2017. Environmental Selection, Dispersal, and Organism Interactions Shape Community Assembly in High-Throughput Enrichment Culturing. Applied and Environmental Microbiology 83:16. abstract pdf
    A central goal of microbial ecology is to identify and quantify the forces that lead to observed population distributions and dynamics. However, these forces, which include environmental selection, dispersal, and organism interactions, are often difficult to assess in natural environments. Here, we present a method that links microbial community structures with selective and stochastic forces through highly replicated subsampling and enrichment of a single environmental inoculum. Specifically, groundwater from a well-studied natural aquifer was serially diluted and inoculated into nearly 1,000 aerobic and anaerobic nitrate-reducing cultures, and the final community structures were evaluated with 16S rRNA gene amplicon sequencing. We analyzed the frequency and abundance of individual operational taxonomic units (OTUs) to understand how probabilistic immigration, relative fitness differences, environmental factors, and organismal interactions contributed to divergent distributions of community structures. We further used a most probable number (MPN) method to estimate the natural condition-dependent cultivable abundance of each of the nearly 400 OTU cultivated in our study and infer the relative fitness of each. Additionally, we infer condition-specific organism interactions and discuss how this high-replicate culturing approach is essential in dissecting the interplay between overlapping ecological forces and taxon-specific attributes that underpin microbial community assembly. IMPORTANCE Through highly replicated culturing, in which inocula are subsampled from a single environmental sample, we empirically determine how selective forces, interspecific interactions, relative fitness, and probabilistic dispersal shape bacterial communities. These methods offer a novel approach to untangle not only interspecific interactions but also taxon-specific fitness differences that manifest across different cultivation conditions and lead to the selection and enrichment of specific organisms. Additionally, we provide a method for estimating the number of cultivable units of each OTU in the original sample through the MPN approach.
  95. Jenkins, S., T. L. Swenson, R. Lau, A. M. Rocha, A. Aaring, T. C. Hazen, R. Chakraborty and T. R. Northen. 2017. Construction of Viable Soil Defined Media Using Quantitative Metabolomics Analysis of Soil Metabolites. Frontiers in Microbiology 8:12. abstract pdf
    Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil bacteria. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil microbial metabolites from a saprolite soil collected from the Oak Ridge Field Research Center (ORFRC). To broadly characterize metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Molecular weight cut-off filtration determined the fraction of carbon accounted for by the quantified metabolites and revealed that these soil metabolites have an uneven quantitative distribution (e.g., trehalose accounted for 9.9% of the <1 kDa fraction). This quantitative information was used to formulate two soil defined media (SUM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate the viability of the SDM, we examined the growth of 30 phylogenetically diverse soil bacterial isolates from the ORFRC field site. The simpler SDM1 supported the growth of 13 isolates while the more complex SDM2 supported 15 isolates. To investigate SDM1 substrate preferences, one isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time -series exometabolomics analysis. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.
  96. Chen, S., H. C. Cheng, J. Liu, T. C. Hazen, V. Huang and Q. He. 2017. Unexpected competitiveness of Methanosaeta populations at elevated acetate concentrations in methanogenic treatment of animal wastewater. Applied Microbiology and Biotechnology 101:1729-1738. abstract pdf
    Acetoclastic methanogenesis is a key metabolic process in anaerobic digestion, a technology with broad applications in biogas production and waste treatment. Acetoclastic methanogenesis is known to be performed by two archaeal genera, Methanosaeta and Methanosarcina. The conventional model posits that Methanosaeta populations are more competitive at low acetate levels (< 1 mM) than Methanosarcina and vice versa at higher acetate concentrations. While this model is supported by an extensive body of studies, reports of inconsistency have grown that Methanosaeta were observed to outnumber Methanosarcina at elevated acetate levels. In this study, monitoring of anaerobic digesters treating animal wastewater unexpectedly identified Methanosaeta as the dominant acetoclastic methanogen population at both low and high acetate levels during organic overloading. The surprising competitiveness of Methanosaeta at elevated acetate was further supported by the enrichment of Methanosaeta with high concentrations of acetate (20 mM). The dominance of Methanosaeta in the methanogen community could be reproduced in anaerobic digesters with the direct addition of acetate to above 20 mM, again supporting the competitiveness of Methanosaeta over Methanosarcina at elevated acetate levels. This study for the first time systematically demonstrated that the dominance of Methanosaeta populations in anaerobic digestion could be linked to the competitiveness of Methanosaeta at elevated acetate concentrations. Given the importance of acetoclastic methanogenesis in biological methane production, findings from this study could have major implications for developing strategies for more effective control of methanogenic treatment processes.
  97. Chakraborty, R., H. Woo, P. Dehal, R. Walker, M. Zemla, M. Auer, L. A. Goodwin, A. Kazakov, P. Novichkov, A. P. Arkin and T. C. Hazen. 2017. Complete genome sequence of Pseudomonas stutzeri strain RCH2 isolated from a Hexavalent Chromium Cr(VI) contaminated site. Standards in Genomic Sciences 12:9. abstract pdf
    Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water in several DOE sites, including Hanford 100 H area. In order to stimulate microbially mediated reduction of Cr(VI) at this site, a poly-lactate hydrogen release compound was injected into the chromium contaminated aquifer. Targeted enrichment of dominant nitrate-reducing bacteria post injection resulted in the isolation of Pseudomonas stutzeri strain RCH2. P. stutzeri strain RCH2 was isolated using acetate as the electron donor and is a complete denitrifier. Experiments with anaerobic washed cell suspension of strain RCH2 revealed it could reduce Cr(VI) and Fe(III). The genome of strain RCH2 was sequenced using a combination of Illumina and 454 sequencing technologies and contained a circular chromosome of 4.6 Mb and three plasmids. Global genome comparisons of strain RCH2 with six other fully sequenced P. stutzeri strains revealed most genomic regions are conserved, however strain RCH2 has an additional 244 genes, some of which are involved in chemotaxis, Flp pilus biogenesis and pyruvate/2-oxogluturate complex formation.
  98. Woo, Hannah L., Kaela B. O'Dell, Sagar Utturkar, Kathryn R. McBride, Marcel Huntemann, Alicia Clum, Manoj Pillay, Krishnaveni Palaniappan, Neha Varghese, Natalia Mikhailova, Dimitrios Stamatis, T. B. K. Reddy, Chew Yee Ngan, Chris Daum, Nicole Shapiro, Victor Markowitz, Natalia Ivanova, Nikos Kyrpides, Tanja Woyke, Steven D. Brown and Terry C. Hazen. 2016. Near-Complete Genome Sequence of Thalassospira sp. Strain KO164 Isolated from a Lignin-Enriched Marine Sediment Microcosm. Microbiology Resource Announcements 4. abstract pdf
    Thalassospira sp. strain KO164 was isolated from eastern Mediterranean seawater and sediment laboratory microcosms enriched on insoluble organosolv lignin under oxic conditions. The near-complete genome sequence presented here will facilitate analyses into this deep-ocean bacterium's ability to degrade recalcitrant organics such as lignin.
  99. Techtmann, S. M. and T. C. Hazen. 2016. Metagenomic applications in environmental monitoring and bioremediation. J Ind Microbiol Biotechnol 43:1345-54. abstract pdf
    With the rapid advances in sequencing technology, the cost of sequencing has dramatically dropped and the scale of sequencing projects has increased accordingly. This has provided the opportunity for the routine use of sequencing techniques in the monitoring of environmental microbes. While metagenomic applications have been routinely applied to better understand the ecology and diversity of microbes, their use in environmental monitoring and bioremediation is increasingly common. In this review we seek to provide an overview of some of the metagenomic techniques used in environmental systems biology, addressing their application and limitation. We will also provide several recent examples of the application of metagenomics to bioremediation. We discuss examples where microbial communities have been used to predict the presence and extent of contamination, examples of how metagenomics can be used to characterize the process of natural attenuation by unculturable microbes, as well as examples detailing the use of metagenomics to understand the impact of biostimulation on microbial communities.
  100. Techtmann, Stephen M., Kathleen S. Fitzgerald, Savannah C. Stelling, Dominique C. Joyner, Sagar M. Uttukar, Austin P. Harris, Noor K. Alshibli, Steven D. Brown and Terry C. Hazen. 2016. Colwellia psychrerythraea Strains from Distant Deep Sea Basins Show Adaptation to Local Conditions. Frontiers in Environmental Science 4:33. abstract pdf
    Many studies have shown that microbes, which share nearly identical 16S rRNA genes, can have highly divergent genomes. Microbes from distinct parts of the ocean also exhibit biogeographic patterning. Here we seek to better understand how certain microbes from the same species have adapted for growth under local conditions. The phenotypic and genomic heterogeneity of three strains of Colwellia psychrerythraea was investigated in order to understand adaptions to local environments. Colwellia are psychrophilic heterotrophic marine bacteria ubiquitous in cold marine ecosystems. We have recently isolated two Colwellia strains: ND2E from the Eastern Mediterranean and GAB14E from the Great Australian Bight. The 16S rRNA sequence of these two strains were greater than 98.2% identical to the well-characterized C. psychrerythraea 34H, which was isolated from arctic sediments. Salt tolerance, and carbon source utilization profiles for these strains were determined using Biolog Phenotype MicoArrays. These strains exhibited distinct salt tolerance, which was not associated with the salinity of sites of isolation. The carbon source utilization profiles were distinct with less than half of the tested carbon sources being metabolized by all three strains. Whole genome sequencing revealed that the genomes of these three strains were quite diverse with some genomes having up to 1600 strain-specific genes. Many genes involved in degrading strain-specific carbon sources were identified. There appears to be a link between carbon source utilization and location of isolation with distinctions observed between the Colwellia isolate recovered from sediment compared to water column isolates.
  101. Shatsky, M., M. Dong, H. Liu, L. L. Yang, M. Choi, M. E. Singer, J. T. Geller, S. J. Fisher, S. C. Hall, T. C. Hazen, S. E. Brenner, G. Butland, J. Jin, H. E. Witkowska, J. M. Chandonia and M. D. Biggin. 2016. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions. Mol Cell Proteomics 15:2186-202. abstract pdf
    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.
  102. Shatsky, M., S. Allen, B. L. Gold, N. L. Liu, T. R. Juba, S. A. Reveco, D. A. Elias, R. Prathapam, J. He, W. Yang, E. D. Szakal, H. Liu, M. E. Singer, J. T. Geller, B. R. Lam, A. Saini, V. V. Trotter, S. C. Hall, S. J. Fisher, S. E. Brenner, S. R. Chhabra, T. C. Hazen, J. D. Wall, H. E. Witkowska, M. D. Biggin, J. M. Chandonia and G. Butland. 2016. Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported. Mol Cell Proteomics 15:1539-55. abstract pdf
    Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.
  103. Rocha, A. M., Q. Yuan, D. M. Close, K. B. O'Dell, J. L. Fortney, J. Wu and T. C. Hazen. 2016. Rapid detection of microbial cell abundance in aquatic systems. Biosens Bioelectron 85:915-23. abstract pdf
    The detection and quantification of naturally occurring microbial cellular densities is an essential component of environmental systems monitoring. While there are a number of commonly utilized approaches for monitoring microbial abundance, capacitance-based biosensors represent a promising approach because of their low-cost and label-free detection of microbial cells, but are not as well characterized as more traditional methods. Here, we investigate the applicability of enhanced alternating current electrokinetics (ACEK) capacitive sensing as a new application for rapidly detecting and quantifying microbial cellular densities in cultured and environmentally sourced aquatic samples. ACEK capacitive sensor performance was evaluated using two distinct and dynamic systems - the Great Australian Bight and groundwater from the Oak Ridge Reservation in Oak Ridge, TN. Results demonstrate that ACEK capacitance-based sensing can accurately determine microbial cell counts throughout cellular concentrations typically encountered in naturally occurring microbial communities (10(3)-10(6) cells/mL). A linear relationship was observed between cellular density and capacitance change correlations, allowing a simple linear curve fitting equation to be used for determining microbial abundances in unknown samples. This work provides a foundation for understanding the limits of capacitance-based sensing in natural environmental samples and supports future efforts focusing on evaluating the robustness ACEK capacitance-based within aquatic environments.
  104. Paradis, C. J., S. Jagadamma, D. B. Watson, L. D. McKay, T. C. Hazen, M. Park and J. D. Istok. 2016. In situ mobility of uranium in the presence of nitrate following sulfate-reducing conditions. J Contam Hydrol 187:55-64. abstract pdf
    Reoxidation and mobilization of previously reduced and immobilized uranium by dissolved-phase oxidants poses a significant challenge for remediating uranium-contaminated groundwater. Preferential oxidation of reduced sulfur-bearing species, as opposed to reduced uranium-bearing species, has been demonstrated to limit the mobility of uranium at the laboratory scale yet field-scale investigations are lacking. In this study, the mobility of uranium in the presence of nitrate oxidant was investigated in a shallow groundwater system after establishing conditions conducive to uranium reduction and the formation of reduced sulfur-bearing species. A series of three injections of groundwater (200 L) containing U(VI) (5 muM) and amended with ethanol (40 mM) and sulfate (20 mM) were conducted in ten test wells in order to stimulate microbial-mediated reduction of uranium and the formation of reduced sulfur-bearing species. Simultaneous push-pull tests were then conducted in triplicate well clusters to investigate the mobility of U(VI) under three conditions: 1) high nitrate (120 mM), 2) high nitrate (120 mM) with ethanol (30 mM), and 3) low nitrate (2 mM) with ethanol (30 mM). Dilution-adjusted breakthrough curves of ethanol, nitrate, nitrite, sulfate, and U(VI) suggested that nitrate reduction was predominantly coupled to the oxidation of reduced-sulfur bearing species, as opposed to the reoxidation of U(IV), under all three conditions for the duration of the 36-day tests. The amount of sulfate, but not U(VI), recovered during the push-pull tests was substantially more than injected, relative to bromide tracer, under all three conditions and further suggested that reduced sulfur-bearing species were preferentially oxidized under nitrate-reducing conditions. However, some reoxidation of U(IV) was observed under nitrate-reducing conditions and in the absence of detectable nitrate and/or nitrite. This suggested that reduced sulfur-bearing species may not be fully effective at limiting the mobility of uranium in the presence of dissolved and/or solid-phase oxidants. The results of this field study confirmed those of previous laboratory studies which suggested that reoxidation of uranium under nitrate-reducing conditions can be substantially limited by preferential oxidation of reduced sulfur-bearing species.
  105. Olesen, S. W., S. Vora, S. M. Techtmann, J. L. Fortney, J. R. Bastidas-Oyanedel, J. Rodriguez, T. C. Hazen and E. J. Alm. 2016. A Novel Analysis Method for Paired-Sample Microbial Ecology Experiments. PLoS One 11:e0154804. abstract pdf
    Many microbial ecology experiments use sequencing data to measure a community's response to an experimental treatment. In a common experimental design, two units, one control and one experimental, are sampled before and after the treatment is applied to the experimental unit. The four resulting samples contain information about the dynamics of organisms that respond to the treatment, but there are no analytical methods designed to extract exactly this type of information from this configuration of samples. Here we present an analytical method specifically designed to visualize and generate hypotheses about microbial community dynamics in experiments that have paired samples and few or no replicates. The method is based on the Poisson lognormal distribution, long studied in macroecology, which we found accurately models the abundance distribution of taxa counts from 16S rRNA surveys. To demonstrate the method's validity and potential, we analyzed an experiment that measured the effect of crude oil on ocean microbial communities in microcosm. Our method identified known oil degraders as well as two clades, Maricurvus and Rhodobacteraceae, that responded to amendment with oil but do not include known oil degraders. Our approach is sensitive to organisms that increased in abundance only in the experimental unit but less sensitive to organisms that increased in both control and experimental units, thus mitigating the role of "bottle effects".
  106. Hemme, C. L., S. J. Green, L. Rishishwar, O. Prakash, A. Pettenato, R. Chakraborty, A. M. Deutschbauer, J. D. Van Nostrand, L. Wu, Z. He, I. K. Jordan, T. C. Hazen, A. P. Arkin, J. E. Kostka and J. Zhou. 2016. Lateral Gene Transfer in a Heavy Metal-Contaminated-Groundwater Microbial Community. MBio 7:e02234-15. abstract pdf
    Unraveling the drivers controlling the response and adaptation of biological communities to environmental change, especially anthropogenic activities, is a central but poorly understood issue in ecology and evolution. Comparative genomics studies suggest that lateral gene transfer (LGT) is a major force driving microbial genome evolution, but its role in the evolution of microbial communities remains elusive. To delineate the importance of LGT in mediating the response of a groundwater microbial community to heavy metal contamination, representative Rhodanobacter reference genomes were sequenced and compared to shotgun metagenome sequences. 16S rRNA gene-based amplicon sequence analysis indicated that Rhodanobacter populations were highly abundant in contaminated wells with low pHs and high levels of nitrate and heavy metals but remained rare in the uncontaminated wells. Sequence comparisons revealed that multiple geochemically important genes, including genes encoding Fe(2+)/Pb(2+) permeases, most denitrification enzymes, and cytochrome c553, were native to Rhodanobacter and not subjected to LGT. In contrast, the Rhodanobacter pangenome contained a recombinational hot spot in which numerous metal resistance genes were subjected to LGT and/or duplication. In particular, Co(2+)/Zn(2+)/Cd(2+) efflux and mercuric resistance operon genes appeared to be highly mobile within Rhodanobacter populations. Evidence of multiple duplications of a mercuric resistance operon common to most Rhodanobacter strains was also observed. Collectively, our analyses indicated the importance of LGT during the evolution of groundwater microbial communities in response to heavy metal contamination, and a conceptual model was developed to display such adaptive evolutionary processes for explaining the extreme dominance of Rhodanobacter populations in the contaminated groundwater microbiome. IMPORTANCE: Lateral gene transfer (LGT), along with positive selection and gene duplication, are the three main mechanisms that drive adaptive evolution of microbial genomes and communities, but their relative importance is unclear. Some recent studies suggested that LGT is a major adaptive mechanism for microbial populations in response to changing environments, and hence, it could also be critical in shaping microbial community structure. However, direct evidence of LGT and its rates in extant natural microbial communities in response to changing environments is still lacking. Our results presented in this study provide explicit evidence that LGT played a crucial role in driving the evolution of a groundwater microbial community in response to extreme heavy metal contamination. It appears that acquisition of genes critical for survival, growth, and reproduction via LGT is the most rapid and effective way to enable microorganisms and associated microbial communities to quickly adapt to abrupt harsh environmental stresses.
  107. Hazen, T. C. and G. S. Sayler. 2016. Environmental Systems Microbiology of Contaminated Environments. Manual of Environmental Microbiology 4th Edition:5.1.6-1-5.1.6-10. abstract pdf
    Environmental Systems Microbiology is well positioned to move forward in dynamic complex system analysis probing new questions and developing new insight into the function, robustness and resilience in response to anthropogenic perturbations. Recent studies have demonstrated that natural bacterial communities can be used as quantitative biosensors in both groundwater and deep ocean water, predicting oil concentration from the Gulf of Mexico Deep Water Horizon spill and from groundwater at nuclear production waste sites (16, 17, 25). Since the first demonstration of catabolic gene expression in soil remediation (34) it has been clear that extension beyond organismal abundance to process and function of microbial communities as a whole using the whole suite of omic tools available to the post genomic era. Metatranscriptomics have been highlighted as a prime vehicle for understanding responses to environmental drivers (35) in complex systems and with rapidly developing metabolomics, full functional understanding of complex community biogeochemical cycling is an achievable goal. Perhaps more exciting is the dynamic nature of these systems and their complex adaptive strategies that may lead to new control paradigms and emergence of new states and function in the course of a changing environment.
  108. Hazen, T. C., R. C. Prince and N. Mahmoudi. 2016. Marine Oil Biodegradation. Environ Sci Technol 50:2121-9. abstract pdf
    Crude oil has been part of the marine environment for millions of years, and microbes that use its rich source of energy and carbon are found in seawater, sediments, and shorelines from the tropics to the polar regions. Catastrophic oil spills stimulate these organisms to "bloom" in a reproducible fashion, and although oil does not provide bioavailable nitrogen, phosphorus or iron, there are enough of these nutrients in the sea that when dispersed oil droplets dilute to low concentrations these low levels are adequate for microbial growth. Most of the hydrocarbons in dispersed oil are degraded in aerobic marine waters with a half-life of days to months. In contrast, oil that reaches shorelines is likely to be too concentrated, have lower levels of nutrients, and have a far longer residence time in the environment. Oil that becomes entrained in anaerobic sediments is also likely to have a long residence time, although it too will eventually be biodegraded. Thus, data that encompass everything from the ecosystem to the molecular level are needed for understanding the complicated process of petroleum biodegradation in marine environments.
  109. Zhou, A., K. L. Hillesland, Z. He, W. Schackwitz, Q. Tu, G. M. Zane, Q. Ma, Y. Qu, D. A. Stahl, J. D. Wall, T. C. Hazen, M. W. Fields, A. P. Arkin and J. Zhou. 2015. Rapid selective sweep of pre-existing polymorphisms and slow fixation of new mutations in experimental evolution of Desulfovibrio vulgaris. ISME J 9:2360-72. abstract pdf
    To investigate the genetic basis of microbial evolutionary adaptation to salt (NaCl) stress, populations of Desulfovibrio vulgaris Hildenborough (DvH), a sulfate-reducing bacterium important for the biogeochemical cycling of sulfur, carbon and nitrogen, and potentially the bioremediation of toxic heavy metals and radionuclides, were propagated under salt stress or non-stress conditions for 1200 generations. Whole-genome sequencing revealed 11 mutations in salt stress-evolved clone ES9-11 and 14 mutations in non-stress-evolved clone EC3-10. Whole-population sequencing data suggested the rapid selective sweep of the pre-existing polymorphisms under salt stress within the first 100 generations and the slow fixation of new mutations. Population genotyping data demonstrated that the rapid selective sweep of pre-existing polymorphisms was common in salt stress-evolved populations. In contrast, the selection of pre-existing polymorphisms was largely random in EC populations. Consistently, at 100 generations, stress-evolved population ES9 showed improved salt tolerance, namely increased growth rate (2.0-fold), higher biomass yield (1.8-fold) and shorter lag phase (0.7-fold) under higher salinity conditions. The beneficial nature of several mutations was confirmed by site-directed mutagenesis. All four tested mutations contributed to the shortened lag phases under higher salinity condition. In particular, compared with the salt tolerance improvement in ES9-11, a mutation in a histidine kinase protein gene lytS contributed 27% of the growth rate increase and 23% of the biomass yield increase while a mutation in hypothetical gene DVU2472 contributed 24% of the biomass yield increase. Our results suggested that a few beneficial mutations could lead to dramatic improvements in salt tolerance.
  110. Zhang, P., J. D. Van Nostrand, Z. He, R. Chakraborty, Y. Deng, D. Curtis, M. W. Fields, T. C. Hazen, A. P. Arkin and J. Zhou. 2015. A Slow-Release Substrate Stimulates Groundwater Microbial Communities for Long-Term in Situ Cr(VI) Reduction. Environ Sci Technol 49:12922-31. abstract pdf
    Cr(VI) is a widespread environmental contaminant that is highly toxic and soluble. Previous work indicated that a one-time amendment of polylactate hydrogen-release compound (HRC) reduced groundwater Cr(VI) concentrations for >3.5 years at a contaminated aquifer; however, microbial communities responsible for Cr(VI) reduction are poorly understood. In this study, we hypothesized that HRC amendment would significantly change the composition and structure of groundwater microbial communities, and that the abundance of key functional genes involved in HRC degradation and electron acceptor reduction would increase long-term in response to this slowly degrading, complex substrate. To test these hypotheses, groundwater microbial communities were monitored after HRC amendment for >1 year using a comprehensive functional gene microarray. The results showed that the overall functional composition and structure of groundwater microbial communities underwent sequential shifts after HRC amendment. Particularly, the abundance of functional genes involved in acetate oxidation, denitrification, dissimilatory nitrate reduction, metal reduction, and sulfate reduction significantly increased. The overall community dynamics was significantly correlated with changes in groundwater concentrations of microbial biomass, acetate, NO3-, Cr(VI), Fe(II) and SO4(2-). Our results suggest that HRC amendment primarily stimulated key functional processes associated with HRC degradation and reduction of multiple electron acceptors in the aquifer toward long-term Cr(VI) reduction.
  111. Thorgersen, M. P., W. A. Lancaster, B. J. Vaccaro, F. L. Poole, A. M. Rocha, T. Mehlhorn, A. Pettenato, J. Ray, R. J. Waters, R. A. Melnyk, R. Chakraborty, T. C. Hazen, A. M. Deutschbauer, A. P. Arkin and M. W. Adams. 2015. Molybdenum Availability Is Key to Nitrate Removal in Contaminated Groundwater Environments. Appl Environ Microbiol 81:4976-83. abstract pdf
    The concentrations of molybdenum (Mo) and 25 other metals were measured in groundwater samples from 80 wells on the Oak Ridge Reservation (ORR) (Oak Ridge, TN), many of which are contaminated with nitrate, as well as uranium and various other metals. The concentrations of nitrate and uranium were in the ranges of 0.1 muM to 230 mM and <0.2 nM to 580 muM, respectively. Almost all metals examined had significantly greater median concentrations in a subset of wells that were highly contaminated with uranium (>/=126 nM). They included cadmium, manganese, and cobalt, which were 1,300- to 2,700-fold higher. A notable exception, however, was Mo, which had a lower median concentration in the uranium-contaminated wells. This is significant, because Mo is essential in the dissimilatory nitrate reduction branch of the global nitrogen cycle. It is required at the catalytic site of nitrate reductase, the enzyme that reduces nitrate to nitrite. Moreover, more than 85% of the groundwater samples contained less than 10 nM Mo, whereas concentrations of 10 to 100 nM Mo were required for efficient growth by nitrate reduction for two Pseudomonas strains isolated from ORR wells and by a model denitrifier, Pseudomonas stutzeri RCH2. Higher concentrations of Mo tended to inhibit the growth of these strains due to the accumulation of toxic concentrations of nitrite, and this effect was exacerbated at high nitrate concentrations. The relevance of these results to a Mo-based nitrate removal strategy and the potential community-driving role that Mo plays in contaminated environments are discussed.
  112. Techtmann, S. M., J. L. Fortney, K. A. Ayers, D. C. Joyner, T. D. Linley, S. M. Pfiffner and T. C. Hazen. 2015. The unique chemistry of Eastern Mediterranean water masses selects for distinct microbial communities by depth. PLoS One 10:e0120605. abstract pdf
    The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.
  113. Smith, M. B., 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., Jr. 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, E. J. Alm and T. C. Hazen. 2015. Natural bacterial communities serve as quantitative geochemical biosensors. MBio 6:e00326-15. abstract pdf
    Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. IMPORTANCE: Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.
  114. Sitte, J., S. Loffler, E. M. Burkhardt, K. C. Goldfarb, G. Buchel, T. C. Hazen and K. Kusel. 2015. Metals other than uranium affected microbial community composition in a historical uranium-mining site. Environ Sci Pollut Res Int 22:19326-41. abstract pdf
    To understand the links between the long-term impact of uranium and other metals on microbial community composition, ground- and surface water-influenced soils varying greatly in uranium and metal concentrations were investigated at the former uranium-mining district in Ronneburg, Germany. A soil-based 16S PhyloChip approach revealed 2358 bacterial and 35 archaeal operational taxonomic units (OTU) within diverse phylogenetic groups with higher OTU numbers than at other uranium-contaminated sites, e.g., at Oak Ridge. Iron- and sulfate-reducing bacteria (FeRB and SRB), which have the potential to attenuate uranium and other metals by the enzymatic and/or abiotic reduction of metal ions, were found at all sites. Although soil concentrations of solid-phase uranium were high, ranging from 5 to 1569 mug.g (dry weight) soil(-1), redundancy analysis (RDA) and forward selection indicated that neither total nor bio-available uranium concentrations contributed significantly to the observed OTU distribution. Instead, microbial community composition appeared to be influenced more by redox potential. Bacterial communities were also influenced by bio-available manganese and total cobalt and cadmium concentrations. Bio-available cadmium impacted FeRB distribution while bio-available manganese and copper as well as solid-phase zinc concentrations in the soil affected SRB composition. Archaeal communities were influenced by the bio-available lead as well as total zinc and cobalt concentrations. These results suggest that (i) microbial richness was not impacted by heavy metals and radionuclides and that (ii) redox potential and secondary metal contaminants had the strongest effect on microbial community composition, as opposed to uranium, the primary source of contamination.
  115. Ramsay, B. D., C. Hwang, H. L. Woo, S. L. Carroll, S. Lucas, J. Han, A. L. Lapidus, J. F. Cheng, L. A. Goodwin, S. Pitluck, L. Peters, O. Chertkov, B. Held, J. C. Detter, C. S. Han, R. Tapia, M. L. Land, L. J. Hauser, N. C. Kyrpides, N. N. Ivanova, N. Mikhailova, I. Pagani, T. Woyke, A. P. Arkin, P. Dehal, D. Chivian, C. S. Criddle, W. M. Wu, R. Chakraborty, T. C. Hazen and M. W. Fields. 2015. High-Quality Draft Genome Sequence of Desulfovibrio carbinoliphilus FW-101-2B, an Organic Acid-Oxidizing Sulfate-Reducing Bacterium Isolated from Uranium(VI)-Contaminated Groundwater. Microbiology Resource Announcements 3:2. abstract pdf
    Desulfovibrio carbinoliphilus subsp. oakridgensis FW-101-2B is an anaerobic, organic acid/alcohol-oxidizing, sulfate-reducing beta-proteobacterium. FW-101-2B was isolated from contaminated groundwater at The Field Research Center at Oak Ridge National Lab after in situ stimulation for heavy metal-reducing conditions. The genome will help elucidate the metabolic potential of sulfate-reducing bacteria during uranium reduction.
  116. O'Dell, K. B., H. L. Woo, S. Utturkar, D. Klingeman, S. D. Brown and T. C. Hazen. 2015. Genome Sequence of Halomonas sp. Strain KO116, an Ionic Liquid-Tolerant Marine Bacterium Isolated from a Lignin-Enriched Seawater Microcosm. Microbiology Resource Announcements 3. abstract pdf
    Halomonas sp. strain KO116 was isolated from Nile Delta Mediterranean Sea surface water enriched with insoluble organosolv lignin. It was further screened for growth on alkali lignin minimal salts medium agar. The strain tolerates the ionic liquid 1-ethyl-3-methylimidazolium acetate. Its complete genome sequence is presented in this report.
  117. Mahmoudi, N., M. S., 2nd Robeson, H. F. Castro, J. L. Fortney, S. M. Techtmann, D. C. Joyner, C. J. Paradis, S. M. Pfiffner and T. C. Hazen. 2015. Microbial community composition and diversity in Caspian Sea sediments. FEMS Microbiol Ecol 91:1-11. abstract pdf
    The Caspian Sea is heavily polluted due to industrial and agricultural effluents as well as extraction of oil and gas reserves. Microbial communities can influence the fate of contaminants and nutrients. However, insight into the microbial ecology of the Caspian Sea significantly lags behind other marine systems. Here we describe microbial biomass, diversity and composition in sediments collected from three sampling stations in the Caspian Sea. Illumina sequencing of 16S rRNA genes revealed the presence of a number of known bacterial and archaeal heterotrophs suggesting that organic carbon is a primary factor shaping microbial communities. Surface sediments collected from bottom waters with low oxygen levels were dominated by Gammaproteobacteria while surface sediments collected from bottom waters under hypoxic conditions were dominated by Deltaproteobacteria, specifically sulfate-reducing bacteria. Thaumarchaeota was dominant across all surface sediments indicating that nitrogen cycling in this system is strongly influenced by ammonia-oxidizing archaea. This study provides a baseline assessment that may serve as a point of reference as this system changes or as the efficacy of new remediation efforts are implemented.
  118. King, G. M., J. E. Kostka, T. C. Hazen and P. A. Sobecky. 2015. Microbial Responses to the Deepwater Horizon Oil Spill: From Coastal Wetlands to the Deep Sea. Annual Review of Marine Science, Vol 7 7:377-401. abstract pdf
    The Deepwater Horizon oil spill in the northern Gulf of Mexico represents the largest marine accidental oil spill in history. It is distinguished from past spills in that it occurred at the greatest depth (1,500 m), the amount of hydrocarbon gas (mostly methane) lost was equivalent to the mass of crude oil released, and dispersants were used for the first time in the deep sea in an attempt to remediate the spill. The spill is also unique in that it has been characterized with an unprecedented level of resolution using next-generation sequencing technologies, especially for the ubiquitous hydrocarbon-degrading microbial communities that appeared largely to consume the gases and to degrade a significant fraction of the petroleum. Results have shown an unexpectedly rapid response of deep-sea Gammaproteobacteria to oil and gas and documented a distinct succession correlated with the control of the oil flow and well shut-in. Similar successional events, also involving Gammaproteobacteria, have been observed in nearshore systems as well.
  119. Hemme, C. L., Q. Tu, Z. Shi, Y. Qin, W. Gao, Y. Deng, J. D. Nostrand, L. Wu, Z. He, P. S. Chain, S. G. Tringe, M. W. Fields, E. M. Rubin, J. M. Tiedje, T. C. Hazen, A. P. Arkin and J. Zhou. 2015. Comparative metagenomics reveals impact of contaminants on groundwater microbiomes. Front Microbiol 6:1205. abstract pdf
    To understand patterns of geochemical cycling in pristine versus contaminated groundwater ecosystems, pristine shallow groundwater (FW301) and contaminated groundwater (FW106) samples from the Oak Ridge Integrated Field Research Center (OR-IFRC) were sequenced and compared to each other to determine phylogenetic and metabolic difference between the communities. Proteobacteria (e.g., Burkholderia, Pseudomonas) are the most abundant lineages in the pristine community, though a significant proportion ( >55%) of the community is composed of poorly characterized low abundance (individually <1%) lineages. The phylogenetic diversity of the pristine community contributed to a broader diversity of metabolic networks than the contaminated community. In addition, the pristine community encodes redundant and mostly complete geochemical cycles distributed over multiple lineages and appears capable of a wide range of metabolic activities. In contrast, many geochemical cycles in the contaminated community appear truncated or minimized due to decreased biodiversity and dominance by Rhodanobacter populations capable of surviving the combination of stresses at the site. These results indicate that the pristine site contains more robust and encodes more functional redundancy than the stressed community, which contributes to more efficient nutrient cycling and adaptability than the stressed community.
  120. 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. J Microbiol Methods 115:64-6. abstract pdf
    Lipid/DNA co-extraction from one sample is attractive in limiting biases associated with microbial community analysis from separate extractions. We sought to enhance established co-extraction methods and use high-throughput 16S rRNA sequencing to identify preferentially extracted taxa from co-extracted DNA. Co-extraction results in low DNA yields and distinct community structure changes.
  121. Billings, A. F., J. L. Fortney, T. C. Hazen, B. Simmons, K. W. Davenport, L. Goodwin, N. Ivanova, N. C. Kyrpides, K. Mavromatis, T. Woyke and K. M. DeAngelis. 2015. Genome sequence and description of the anaerobic lignin-degrading bacterium Tolumonas lignolytica sp. nov. Stand Genomic Sci 10:106. abstract pdf
    Tolumonas lignolytica BRL6-1(T) sp. nov. is the type strain of T. lignolytica sp. nov., a proposed novel species of the Tolumonas genus. This strain was isolated from tropical rainforest soils based on its ability to utilize lignin as a sole carbon source. Cells of Tolumonas lignolytica BRL6-1(T) are mesophilic, non-spore forming, Gram-negative rods that are oxidase and catalase negative. The genome for this isolate was sequenced and returned in seven unique contigs totaling 3.6Mbp, enabling the characterization of several putative pathways for lignin breakdown. Particularly, we found an extracellular peroxidase involved in lignin depolymerization, as well as several enzymes involved in beta-aryl ether bond cleavage, which is the most abundant linkage between lignin monomers. We also found genes for enzymes involved in ferulic acid metabolism, which is a common product of lignin breakdown. By characterizing pathways and enzymes employed in the bacterial breakdown of lignin in anaerobic environments, this work should assist in the efficient engineering of biofuel production from lignocellulosic material.
  122. Zhou, J. Z., Y. Deng, P. Zhang, K. Xue, Y. T. Liang, J. D. Van Nostrand, Y. F. Yang, Z. L. He, L. Y. Wu, D. A. Stahl, T. C. Hazen, J. M. Tiedje and A. P. Arkin. 2014. Stochasticity, succession, and environmental perturbations in a fluidic ecosystem. Proceedings of the National Academy of Sciences of the United States of America 111:E836-E845. abstract pdf
    Unraveling the drivers of community structure and succession in response to environmental change is a central goal in ecology. Although the mechanisms shaping community structure have been intensively examined, those controlling ecological succession remain elusive. To understand the relative importance of stochastic and deterministic processes in mediating microbial community succession, a unique framework composed of four different cases was developed for fluidic and nonfluidic ecosystems. The framework was then tested for one fluidic ecosystem: a groundwater system perturbed by adding emulsified vegetable oil (EVO) for uranium immobilization. Our results revealed that groundwater microbial community diverged substantially away from the initial community after EVO amendment and eventually converged to a new community state, which was closely clustered with its initial state. However, their composition and structure were significantly different from each other. Null model analysis indicated that both deterministic and stochastic processes played important roles in controlling the assembly and succession of the groundwater microbial community, but their relative importance was time dependent. Additionally, consistent with the proposed conceptual framework but contradictory to conventional wisdom, the community succession responding to EVO amendment was primarily controlled by stochastic rather than deterministic processes. During the middle phase of the succession, the roles of stochastic processes in controlling community composition increased substantially, ranging from 81.3% to 92.0%. Finally, there are limited successional studies available to support different cases in the conceptual framework, but further well-replicated explicit time-series experiments are needed to understand the relative importance of deterministic and stochastic processes in controlling community succession.
  123. Woo, H. L., S. Utturkar, D. Klingeman, B. A. Simmons, K. M. DeAngelis, S. D. Brown and T. C. Hazen. 2014. Draft Genome Sequence of the Lignin-Degrading Burkholderia sp. Strain LIG30, Isolated from Wet Tropical Forest Soil. Genome Announc 2. abstract pdf
    Burkholderia species are common soil Betaproteobacteria capable of degrading recalcitrant aromatic compounds and xenobiotics. Burkholderia sp. strain LIG30 was isolated from wet tropical forest soil and is capable of utilizing lignin as a sole carbon source. Here we report the draft genome sequence of Burkholderia sp. strain LIG30.
  124. Woo, H. L., T. C. Hazen, B. A. Simmons and K. M. DeAngelis. 2014. Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils. Systematic and Applied Microbiology 37:60-67. abstract pdf
    Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, beta-D-glucosidase, cellobiohydrolase, beta-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated beta-D-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal-Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts. (C) 2013 Elsevier GmbH. All rights reserved.
  125. Woo, Hannah, Nicholas Ballor, Terry Hazen, Julian Fortney, Blake Simmons, Karen Davenport, Lynne Goodwin, Natalia Ivanova, Nikos Kyrpides, Konstantinos Mavromatis, Tanja Woyke, Janet Jansson, Jeff Kimbrel and Kristen DeAngelis. 2014. Complete genome sequence of the lignin-degrading bacterium Klebsiella sp. strain BRL6-2. Standards in Genomic Sciences 9:19. abstract pdf
    In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated Klebsiella sp. strain BRL6-2 on minimal media with alkali lignin as the sole carbon source. This organism was isolated anaerobically from tropical forest soils collected from the Bisley watershed at the Ridge site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are characterized by cycles of iron oxidation and reduction. Genome sequencing was targeted because of its ability to grow on lignin anaerobically and lignocellulolytic activity via in vitro enzyme assays. The genome of Klebsiella sp. strain BRL6-2 is 5.80 Mbp with no detected plasmids, and includes a relatively small arsenal of genes encoding lignocellulolytic carbohydrate active enzymes. The genome revealed four putative peroxidases including glutathione and DyP-type peroxidases, and a complete protocatechuate pathway encoded in a single gene cluster. Physiological studies revealed Klebsiella sp. strain BRL6-2 to be relatively stress tolerant to high ionic strength conditions. It grows in increasing concentrations of ionic liquid (1-ethyl-3-methyl-imidazolium acetate) up to 73.44mM and NaCl up to 1.5M.
  126. 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. 2014. Microbial biogeography across a full-scale wastewater treatment plant transect: evidence for immigration between coupled processes. Applied Microbiology and Biotechnology 98:4723-4736. abstract pdf
    Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO2 (-) entering the reactor from an upstream trickling filter. Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased > 30-fold between influent and trickling filter effluent concomitant with NO2 (-) production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct "Nitrosomonas-like" lineage dominated in activated sludge. Prior time series indicated that this "Nitrosomonas-like" lineage was dominant when NO2 (-) levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO2 (-) levels were high. This is consistent with the hypothesis that NO2 (-) production may cooccur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors.
  127. Trexler, R., C. Solomon, C. J. Brislawn, J. R. Wright, A. Rosenberger, E. E. McClure, A. M. Grube, M. P. Peterson, M. Keddache, O. U. Mason, T. C. Hazen, C. J. Grant and R. Lamendella. 2014. Assessing impacts of unconventional natural gas extraction on microbial communities in headwater stream ecosystems in Northwestern Pennsylvania. Front Microbiol 5:522. abstract pdf
    Hydraulic fracturing and horizontal drilling have increased dramatically in Pennsylvania Marcellus shale formations, however the potential for major environmental impacts are still incompletely understood. High-throughput sequencing of the 16S rRNA gene was performed to characterize the microbial community structure of water, sediment, bryophyte, and biofilm samples from 26 headwater stream sites in northwestern Pennsylvania with different histories of fracking activity within Marcellus shale formations. Further, we describe the relationship between microbial community structure and environmental parameters measured. Approximately 3.2 million 16S rRNA gene sequences were retrieved from a total of 58 samples. Microbial community analyses showed significant reductions in species richness as well as evenness in sites with Marcellus shale activity. Beta diversity analyses revealed distinct microbial community structure between sites with and without Marcellus shale activity. For example, operational taxonomic units (OTUs) within the Acetobacteracea, Methylocystaceae, Acidobacteriaceae, and Phenylobacterium were greater than three log-fold more abundant in MSA+ sites as compared to MSA- sites. Further, several of these OTUs were strongly negatively correlated with pH and positively correlated with the number of wellpads in a watershed. It should be noted that many of the OTUs enriched in MSA+ sites are putative acidophilic and/or methanotrophic populations. This study revealed apparent shifts in the autochthonous microbial communities and highlighted potential members that could be responding to changing stream conditions as a result of nascent industrial activity in these aquatic ecosystems.
  128. Stelling, S. C., S. M. Techtmann, S. M. Utturkar, N. K. Alshibli, S. D. Brown and T. C. Hazen. 2014. Draft Genome Sequence of Thalassotalea sp. strain ND16A Isolated from Eastern Mediterranean Sea water collected from a depth of 1055 m. Genome Announcement 2:e01231-14. abstract pdf
    Thalassotalea sp. strain ND16A belongs to the family Colwelliaceae and was isolated from eastern Mediterranean Sea water at a depth of 1,055 m. Members of Colwelliaceae are ubiquitous marine heterotrophs. Here, we report the draft genome sequence of Thalassotalea sp. strain ND16A, a member of the newly described genus Thalassotalea.
  129. Piceno, Y. M., F. C. Reid, L. M. Tom, M. E. Conrad, M. Bill, C. G. Hubbard, B. W. Fouke, C. J. Graff, J. B. Han, W. T. Stringfellow, J. S. Hanlon, P. Hu, T. C. Hazen and G. L. Andersen. 2014. Temperature and injection water source influence microbial community structure in four Alaskan North Slope hydrocarbon reservoirs. Frontiers in Microbiology 5:13. abstract pdf
    A fundamental knowledge of microbial community structure in petroleum reservoirs can improve predictive modeling of these environments. We used hydrocarbon profiles, stable isotopes, and high-density DNA microarray analysis to characterize microbial communities in produced water from four Alaskan North Slope hydrocarbon reservoirs. Produced fluids from Schrader Bluff (24-27 degrees C), Kuparuk (47-70 degrees C), Sag River (80 degrees C), and lvishak (80-83 degrees C) reservoirs were collected, with paired soured/non-soured wells sampled from Kuparuk and lvishak. Chemical and stable isotope data suggested Schrader Bluff had substantial biogenic methane, whereas methane was mostly thermogenic in deeper reservoirs. Acetoclastic methanogens (Methanosaeta) were most prominent in Schrader Bluff samples, and the combined delta D and delta C-13 values of methane also indicated acetoclastic methanogenesis could be a primary route for biogenic methane. Conversely, hydrogenotrophic methanogens (e.g., Methanobacteriaceae) and sulfide-producing Archaeoglobus and Thermococcus were more prominent in Kuparuk samples. Sulfide-producing microbes were detected in all reservoirs, uncoupled from souring status (e.g., the non-soured Kuparuk samples had higher relative abundances of many sulfate-reducers compared to the soured sample, suggesting sulfate-reducers may be living fermentatively/syntrophically when sulfate is limited). Sulfate abundance via long-term seawater injection resulted in greater relative abundances of Desulfonauticus, Desulfomicrobium, and Desulfuromonas in the soured lvishak well compared to the non-soured well. In the non-soured lvishak sample, several taxa affiliated with Thermoanaerobacter and Halomonas predominated. Archaea were not detected in the deepest reservoirs. Functional group taxa differed in relative abundance among reservoirs, likely reflecting differing thermal and/or geochemical influences.
  130. Pedersen, K., A. Bengtsson, J. Edlund, L. Rabe, T. Hazen, R. Chakraborty, L. Goodwin and N. Shapiro. 2014. Complete Genome Sequence of the Subsurface, Mesophilic Sulfate-Reducing Bacterium Desulfovibrio aespoeensis Aspo-2. Genome Announc 2. abstract pdf
    Desulfovibrio aespoeensis Aspo-2, DSM 10631(T), is a mesophilic, hydrogenotrophic sulfate-reducing bacterium sampled from a 600-m-deep subsurface aquifer in hard rock under the island of Aspo in southeastern Sweden. We report the genome sequence of this bacterium, which is a 3,629,109-bp chromosome; plasmids were not found.
  131. Mason, O. U., N. M. Scott, A. Gonzalez, A. Robbins-Pianka, J. Baelum, J. Kimbrel, N. J. Bouskill, E. Prestat, S. Borglin, D. C. Joyner, J. L. Fortney, D. Jurelevicius, W. T. Stringfellow, L. Alvarez-Cohen, T. C. Hazen, R. Knight, J. A. Gilbert and J. K. Jansson. 2014. Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill. Isme Journal 8:1464-1475. abstract pdf
    The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of similar to 4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using C-14-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of C-14-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)'s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.
  132. Martinez, R. J., C. H. Wu, M. J. Beazley, G. L. Andersen, M. E. Conrad, T. C. Hazen, M. Taillefert and P. A. Sobecky. 2014. Microbial Community Responses to Organophosphate Substrate Additions in Contaminated Subsurface Sediments. Plos One 9:11. abstract pdf
    Background: Radionuclide-and heavy metal-contaminated subsurface sediments remain a legacy of Cold War nuclear weapons research and recent nuclear power plant failures. Within such contaminated sediments, remediation activities are necessary to mitigate groundwater contamination. A promising approach makes use of extant microbial communities capable of hydrolyzing organophosphate substrates to promote mineralization of soluble contaminants within deep subsurface environments. Methodology/Principal Findings: Uranium-contaminated sediments from the U. S. Department of Energy Oak Ridge Field Research Center (ORFRC) Area 2 site were used in slurry experiments to identify microbial communities involved in hydrolysis of 10 mM organophosphate amendments [i.e., glycerol-2-phosphate (G2P) or glycerol-3-phosphate (G3P)] in synthetic groundwater at pH 5.5 and pH 6.8. Following 36 day (G2P) and 20 day (G3P) amended treatments, maximum phosphate (PO43-) concentrations of 4.8 mM and 8.9 mM were measured, respectively. Use of the PhyloChip 16S rRNA microarray identified 2,120 archaeal and bacterial taxa representing 46 phyla, 66 classes, 110 orders, and 186 families among all treatments. Measures of archaeal and bacterial richness were lowest under G2P (pH 5.5) treatments and greatest with G3P (pH 6.8) treatments. Members of the phyla Crenarchaeota, Euryarchaeota, Bacteroidetes, and Proteobacteria demonstrated the greatest enrichment in response to organophosphate amendments and the OTUs that increased in relative abundance by 2-fold or greater accounted for 9%-50% and 3%-17% of total detected Archaea and Bacteria, respectively. Conclusions/Significance: This work provided a characterization of the distinct ORFRC subsurface microbial communities that contributed to increased concentrations of extracellular phosphate via hydrolysis of organophosphate substrate amendments. Within subsurface environments that are not ideal for reductive precipitation of uranium, strategies that harness microbial phosphate metabolism to promote uranium phosphate precipitation could offer an alternative approach for in situ sequestration.
  133. Lee, S. W., J. T. Geller, T. Torok, C. H. Wu, M. Singer, F. C. Reid, D. R. Tarjan, T. C. Hazen, A. P. Arkin and N. J. Hillson. 2014. Characterization of Wastewater Treatment Plant Microbial Communities and the Effects of Carbon Sources on Diversity in Laboratory Models. Plos One 9:14. abstract pdf
    We are developing a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S-rRNA-gene targeting microarrays, we compared the compositions of sampled communities to those of inocula propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in both aeration basin and laboratory-cultured communities. Laboratory-cultured communities were enriched in gamma-Proteobacteria. Enterobacteriaceae, and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiacea by high (50 mM) concentrations of chloroacetate. Microbial communities cultured with chloroacetate and D-threonine were more similar to sampled field communities than those cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units (OTUs) was lower for laboratory cultures than for field communities, both flask and reactor systems supported phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios.
  134. Lancaster, W. A., A. L. Menon, I. Scott, F. L. Poole, B. J. Vaccaro, M. P. Thorgersen, J. Geller, T. C. Hazen, R. A. Hurt, S. D. Brown, D. A. Elias and M. W. W. Adams. 2014. Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization. Metallomics 6:1004-1013. abstract pdf
    Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.
  135. Lamendella, R., S. Strutt, S. Borglin, R. Chakraborty, N. Tas, O. U. Mason, J. Hultman, E. Prestat, T. C. Hazen and J. K. Jansson. 2014. Assessments of the Deepwater Horizon oil spill impact on Gulf coast microbial communities. Frontiers in Microbiology 5:13. abstract pdf
    One of the major environmental concerns of the Deepwater Horizon oil spill in the Gulf of Mexico was the ecological impact of the oil that reached shorelines of the Gulf Coast. Here we investigated the impact of the oil on the microbial composition in beach samples collected in June 2010 along a heavily impacted shoreline near Grand Isle, Louisiana. Successional changes in the microbial community structure due to the oil contamination were determined by deep sequencing of 16S rRNA genes. Metatranscriptomics was used to determine expression of functional genes involved in hydrocarbon degradation processes. In addition, potential hydrocarbon-degrading Bacteria were obtained in culture. The 16S data revealed that highly contaminated samples had higher abundances of Alpha- and Gammaproteobacteria sequences. Successional changes in these classes were observed over time, during which the oil was partially degraded. The metatranscriptome data revealed that PAH, n-alkane, and toluene degradation genes were expressed in the contaminated samples, with high homology to genes from Alteromonadales, Rhodobacterales, and Pseudomonales. Notably, Marinobacter (Gammaproteobacteria) had the highest representation of expressed genes in the samples. A Marinobacter isolated from this beach was shown to have potential for transformation of hydrocarbons in incubation experiments with oil obtained from the Mississippi Canyon Block 252 (MC252) well; collected during the Deepwater Horizon spill. The combined data revealed a response of the beach microbial community to oil contaminants, including prevalence of Bacteria endowed with the functional capacity to degrade oil.
  136. Kuehl, J. V., M. N. Price, J. Ray, K. M. Wetmore, Z. Esquivel, A. E. Kazakov, M. Nguyen, R. Kuehn, R. W. Davis, T. C. Hazen, A. P. Arkin and A. Deutschbauer. 2014. Functional genomics with a comprehensive library of transposon mutants for the sulfate-reducing bacterium Desulfovibrio alaskensis G20. MBio 5:e01041-14. abstract pdf
    The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5' RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance. IMPORTANCE: Sulfate-reducing bacteria contribute to global nutrient cycles and are a nuisance for the petroleum industry. Despite their environmental and industrial significance, the genomes of sulfate-reducing bacteria remain poorly characterized. Here, we describe a genetic approach to fill gaps in our knowledge of sulfate-reducing bacteria. We generated a large collection of archived, transposon mutants in Desulfovibrio alaskensis G20 and used the phenotypes of these mutant strains to infer the function of genes involved in gene regulation, methionine biosynthesis, and choline utilization. Our findings and mutant resources will enable systematic investigations into gene function, energy generation, stress response, and metabolism for this important group of bacteria.
  137. Harris, A. P., S. M. Techtmann, S. C. Stelling, S. M. Utturkar, N. K. Alshibli, S. D. Brown and T. C. Hazen. 2014. Draft Genome Sequence of Pseudoalteromonas sp. strain ND6B, an oil degrading isolate from Eastern Mediterranean Sea water collected at a depth of 1210 m. Genome Announcement 2:e01212-14. abstract pdf
    Here, we report the draft genome of Pseudoalteromonas sp. strain ND6B, which is able to grow with crude oil as a carbon source. Strain ND6B was isolated from eastern Mediterranean Sea deep water at a depth of 1,210 m. The genome of strain ND6B provides insight into the oil-degrading ability of the Pseudoalteromonas species.
  138. Zhou, A. F., Z. L. He, Y. J. Qin, Z. M. Lu, Y. Deng, Q. C. Tu, C. L. Hemme, J. D. Van Nostrand, L. Y. Wu, T. C. Hazen, A. P. Arkin and J. Z. Zhou. 2013. StressChip as a High-Throughput Tool for Assessing Microbial Community Responses to Environmental Stresses. Environmental Science & Technology 47:9841-9849. abstract pdf
    Microbial community responses to environmental stresses are critical for microbial growth, survival, and adaptation. To fill major gaps in our ability to discern the influence of environmental changes on microbial communities from engineered and natural environments, a functional gene-based microarray, termed StressChip, has been developed. First, 46 functional genes involved in microbial responses to environmental stresses such as changes to temperature, osmolarity, oxidative status, nutrient limitation, or general stress response were selected and curated. A total of 22,855 probes were designed, covering 79,628 coding sequences from 985 bacterial, 76 archaeal, and 59 eukaryotic species/strains. Probe specificity was computationally verified. Second, the usefulness of functional genes as indicators of stress response was examined by surveying their distribution in metagenome data sets. The abundance of individual stress response genes is consistent with expected distributions based on respective habitats. Third, the StressChip was used to analyze marine microbial communities from the Deepwater Horizon oil spill. That functional stress response genes were detected in higher abundance (p < 0.05) in oil plume compared to nonplume samples indicated shifts in community composition and structure, consistent with previous results. In summary, StressChip provides a new tool for accessing microbial community functional structure and responses to environmental changes.
  139. Zhou, A. F., E. Baidoo, Z. L. He, A. Mukhopadhyay, J. K. Baumohl, P. Benke, M. P. Joachimiak, M. Xie, R. Song, A. P. Arkin, T. C. Hazen, J. D. Keasling, J. D. Wall, D. A. Stahl and J. Z. Zhou. 2013. Characterization of NaCl tolerance in Desulfovibrio vulgaris Hildenborough through experimental evolution. Isme Journal 7:1790-1802. abstract pdf
    Desulfovibrio vulgaris Hildenborough strains with significantly increased tolerance to NaCl were obtained via experimental evolution. A NaCl-evolved strain, ES9-11, isolated from a population cultured for 1200 generations in medium amended with 100 mM NaCl, showed better tolerance to NaCl than a control strain, EC3-10, cultured for 1200 generations in parallel but without NaCl amendment in medium. To understand the NaCl adaptation mechanism in ES9-11, we analyzed the transcriptional, metabolite and phospholipid fatty acid (PLFA) profiles of strain ES9-11 with 0, 100- or 250 mM-added NaCl in medium compared with the ancestral strain and EC3-10 as controls. In all the culture conditions, increased expressions of genes involved in amino-acid synthesis and transport, energy production, cation efflux and decreased expression of flagellar assembly genes were detected in ES9-11. Consistently, increased abundances of organic solutes and decreased cell motility were observed in ES9-11. Glutamate appears to be the most important osmoprotectant in D. vulgaris under NaCl stress, whereas, other organic solutes such as glutamine, glycine and glycine betaine might contribute to NaCl tolerance under low NaCl concentration only. Unsaturation indices of PLFA significantly increased in ES9-11. Branched unsaturated PLFAs vertical bar 17: 1 omega 9c, a17: 1 omega 9c and branched saturated i15: 0 might have important roles in maintaining proper membrane fluidity under NaCl stress. Taken together, these data suggest that the accumulation of osmolytes, increased membrane fluidity, decreased cell motility and possibly an increased exclusion of Na+ contribute to increased NaCl tolerance in NaCl-evolved D. vulgaris.
  140. Spier, C., W. T. Stringfellow, T. C. Hazen and M. Conrad. 2013. Distribution of hydrocarbons released during the 2010 MC252 oil spill in deep offshore waters. Environmental Pollution 173:224-230. abstract pdf
    The explosion of the Deepwater Horizon oil platform on April 20th, 2010 resulted in the second largest oil spill in history. The distribution and chemical composition of hydrocarbons within a 45 km radius of the blowout was investigated. All available certified hydrocarbon data were acquired from NOAA and BP. The distribution of hydrocarbons was found to be dispersed over a wider area in subsurface waters than previously predicted or reported. A deepwater hydrocarbon plume predicted by models was verified and additional plumes were identified. Because the samples were not collected systematically, there is still some question about the presence and persistence of an 865 m depth plume predicted by models. Water soluble compounds were extracted from the rising oil in deepwater, and were found at potentially toxic levels outside of areas previously reported to contain hydrocarbons. Application of subsurface dispersants was found to increase hydrocarbon concentration in subsurface waters. (C) 2012 Elsevier Ltd. All rights reserved.
  141. Somenahally, A. C., J. J. Mosher, T. Yuan, M. Podar, T. J. Phelps, S. D. Brown, Z. K. Yang, T. C. Hazen, A. P. Arkin, A. V. Palumbo, J. D. Van Nostrand, J. Z. Zhou and D. A. Elias. 2013. Hexavalent Chromium Reduction under Fermentative Conditions with Lactate Stimulated Native Microbial Communities. Plos One 8:11. abstract pdf
    Microbial reduction of toxic hexavalent chromium (Cr(VI)) in-situ is a plausible bioremediation strategy in electron-acceptor limited environments. However, higher [Cr(VI)] may impose stress on syntrophic communities and impact community structure and function. The study objectives were to understand the impacts of Cr(VI) concentrations on community structure and on the Cr(VI)-reduction potential of groundwater communities at Hanford, WA. Steady state continuous flow bioreactors were used to grow native communities enriched with lactate (30 mM) and continuously amended with Cr(VI) at 0.0 (No-Cr), 0.1 (Low-Cr) and 3.0 (High-Cr) mg/L. Microbial growth, metabolites, Cr(VI), 16S rRNA gene sequences and GeoChip based functional gene composition were monitored for 15 weeks. Temporal trends and differences in growth, metabolite profiles, and community composition were observed, largely between Low-Cr and High-Cr bioreactors. In both High-Cr and Low-Cr bioreactors, Cr(VI) levels were below detection from week 1 until week 15. With lactate enrichment, native bacterial diversity substantially decreased as Pelosinus spp., and Sporotalea spp., became the dominant groups, but did not significantly differ between Cr concentrations. The Archaea diversity also substantially decreased after lactate enrichment from Methanosaeta (35%), Methanosarcina (17%) and others, to mostly Methanosarcina spp. (95%). Methane production was lower in High-Cr reactors suggesting some inhibition of methanogens. Several key functional genes were distinct in Low-Cr bioreactors compared to High-Cr. Among the Cr resistant microbes, Burkholderia vietnamiensis, Comamonas testosterone and Ralstonia pickettii proliferated in Cr amended bioreactors. In-situ fermentative conditions facilitated Cr(VI) reduction, and as a result 3.0 mg/L Cr(VI) did not impact the overall bacterial community structure.
  142. Skerker, J. M., D. Leon, M. N. Price, J. S. Mar, D. R. Tarjan, K. M. Wetmore, A. M. Deutschbauer, J. K. Baumohl, S. Bauer, A. B. Ibanez, V. D. Mitchell, C. H. Wu, P. Hu, T. Hazen and A. P. Arkin. 2013. Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates. Molecular Systems Biology 9:21. abstract pdf
    The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance.
  143. Kimes, N. E., A. V. Callaghan, D. F. Aktas, W. L. Smith, J. Sunner, B. T. Golding, M. Drozdowska, T. C. Hazen, J. M. Suflita and P. J. Morris. 2013. Metagenomic analysis and metabolite profiling of deep-sea sediments from the Gulf of Mexico following the Deepwater Horizon oil spill. Frontiers in Microbiology 4:17. abstract pdf
    Marine subsurface environments such as deep-sea sediments, house abundant and diverse microbial communities that are believed to influence large-scale geochemical processes. These processes include the biotransformation and mineralization of numerous petroleum constituents. Thus, microbial communities in the Gulf of Mexico are thought to be responsible for the intrinsic bioremediation of crude oil released by the Deepwater Horizon (DM) oil spill. While hydrocarbon contamination is known to enrich for aerobic, oil-degrading bacteria in deep-seawater habitats, relatively little is known about the response of communities in deep-sea sediments, where low oxygen levels may hinder such a response. Here, we examined the hypothesis that increased hydrocarbon exposure results in an altered sediment microbial community structure that reflects the prospects for oil biodegradation under the prevailing conditions. We explore this hypothesis using metagenomic analysis and metabolite profiling of deep-sea sediment samples following the DWH oil spill. The presence of aerobic microbial communities and associated functional genes was consistent among all samples, whereas, a greater number of Deltaproteobacteria and anaerobic functional genes were found in sediments closest to the DWH blowout site. Metabolite profiling also revealed a greater number of putative metabolites in sediments surrounding the blowout zone relative to a background site located 127 km away. The mass spectral analysis of the putative metabolites revealed that alkylsuccinates remained below detection levels, but a homologous series of benzylsuccinates (with carbon chain lengths from 5 to 10) could be detected. Our findings suggest that increased exposure to hydrocarbons enriches for Deltaproteobacteria, which are known to be capable of anaerobic hydrocarbon metabolism. We also provide evidence for an active microbial community metabolizing aromatic hydrocarbons in deep-sea sediments of the Gulf of Mexico.
  144. Kang, S., J. D. Van Nostrand, H. L. Gough, Z. L. He, T. C. Hazen, D. A. Stahl and J. Z. Zhou. 2013. Functional gene array-based analysis of microbial communities in heavy metals-contaminated lake sediments. Fems Microbiology Ecology 86:200-214. abstract pdf
    Lake DePue (IL, USA) has been contaminated for >80years by an adjacent Zn-smelting facility. Previous work indicated that sulfate reduction increased and biomass declined as pore-water metal concentrations increased, while 16S rRNA gene profiles remained relatively stable. To better understand this phenomenon, the sediment microbial community structure and functional potential were investigated using a functional gene microarray (GeoChip) targeting >10000 functional genes. Nonmetric multidimensional scaling and clustering analyses showed that the overall community structure was similar across all sites based on the relative abundance of all detected genes, but some individual gene categories did show differences. A subset of sulfate reduction genes (dsr) and the most relevant metal resistance genes were more abundant than other categories and were highly correlated with metal contamination. The most significant correlations were between pore-water metal concentrations and dsr, with Zn, Cd, and Mn as the most predictive for the presence of dsr. These results suggest that metal contamination influences sediment microbial community structure and function by increasing the abundance of relevant metal-resistant and sulfate-reducing populations. These populations therefore appear to contribute significantly to the resistance and stability of the microbial communities throughout the gradient of metal contamination in Lake DePue.
  145. Hazen, T. C., A. M. Rocha and S. M. Techtmann. 2013. Advances in monitoring environmental microbes. Current Opinion in Biotechnology 24:526-533. abstract pdf
    Culture-independent approaches, such as next-generation sequencing and microarray-based tools, provide insight into the identity and functional diversity of microbial communities. Although these approaches are potentially powerful tools in understanding microbial structure and function, there are a number of limitations that may bias conclusions. In order to mitigate these biases, an understanding of potential biases within each stage of the experimental process is necessary. This review focuses on the biases associated with sample collection, nucleic acid extraction, processing, sequencing analyses, and Chip technologies used in microbial ecology studies.
  146. Hazen, T. C.. 2013. The SuperChip for microbial community structure, and function from all environments. Microbial Biotechnology 6:450-452. abstract pdf
    We have the technology and capability to develop an all-in-one microarray that can provide complete information on a microbial community, including algae, protozoa, bacteria, archaea, fungi, viruses, antimicrobial resistance, biotoxins and functional activity. With lab-on-a-chip, nanotechnology integrating a variety of the latest methods for a large number of sample types (water, sediment, waste water, food, blood, etc.) it is possible to make a desktop instrument that would have universal applications. There are two major thrusts to this grand challenge that will allow us to take advantage of the latest biotechnological breakthroughs in real time. The first is a bioengineering thrust that will take advantage of the large multidisciplinary laboratories in developing key technologies. Miniaturization will reduce reagent costs and increase sensitivity and reaction kinetics for rapid turnaround time. New and evolving technologies will allow us to port the designs for state-of-the-art microarrays today to completely new nanotechnology inspired platforms as they mature. The second thrust is in bioinformatics to use our existing expertise to take advantage of the rapidly evolving landscape of bioinformatics data. This increasing capacity of the data set will allow us to resolve microbial species to greatly improved levels and identify functional genes beyond the hypothetical protein level. A cheap and portable assay would impact countless areas, including clean water technologies, emerging diseases, bioenergy, infectious disease diagnosis, climate change, food safety, environmental clean-up and bioterrorism. In my opinion it is possible but it will require a very large group of multidiscplenary scientists from multiple institutions crossing many international boundaries and funding over a 5-year period of more than $100 million. Given the impact that this SuperChip could have it is well worth the price!!!
  147. Dubinsky, E. A., M. E. Conrad, R. Chakraborty, M. Bill, S. E. Borglin, J. T. Hollibaugh, O. U. Mason, Y. M. Piceno, F. C. Reid, W. T. Stringfellow, L. M. Tom, T. C. Hazen and G. L. Andersen. 2013. Succession of Hydrocarbon-Degrading Bacteria in the Aftermath of the Deepwater Horizon Oil Spill in the Gulf of Mexico. Environmental Science & Technology 47:10860-10867. abstract pdf
    The Deepwater Horizon oil spill produced large subsurface plumes of dispersed oil and gas in the Gulf of Mexico that stimulated growth of psychrophilic, hydrocarbon degrading bacteria. We tracked succession of plume bacteria before, during and after the 83-day spill to determine the microbial response and biodegradation potential throughout the incident. Dominant bacteria shifted substantially over time and were dependent on relative quantities of different hydrocarbon fractions. Unmitigated flow from the wellhead early in the spill resulted in the highest proportions of n-alkanes and cycloalkanes at depth and corresponded with dominance by Oceanospirillaceae and Pseudomonas. Once partial capture of oil and gas began 43 days into the spill, petroleum hydrocarbons decreased, the fraction of aromatic hydrocarbons increased, and Colwellia, Cycloclasticus, and Pseudoalteromonas increased in dominance. Enrichment of Methylomonas coincided with positive shifts in the delta C-13 values of methane in the plume and indicated significant methane oxidation occurred earlier than previously reported. Anomalous oxygen depressions persisted at plume depths for over six weeks after well shut-in and were likely caused by common marine heterotrophs associated with degradation of high-molecular-weight organic matter, including Methylophaga. Multiple hydrocarbon-degrading bacteria operated simultaneously throughout the spill, but their relative importance was controlled by changes in hydrocarbon supply.
  148. DeAngelis, K. M., P. D'Haeseleer, D. Chivian, B. Simmons, A. P. Arkin, K. Mavromatis, S. Malfatti, S. Tringe and T. C. Hazen. 2013. Metagenomes of tropical soil-derived anaerobic switchgrass-adapted consortia with and without iron. Standards in Genomic Sciences 7:382-398. abstract pdf
    Tropical forest soils decompose litter rapidly with frequent episodes of anoxia, making it likely that bacteria using alternate terminal electron acceptors (TEAs) such as iron play a large role in supporting decomposition under these conditions. The prevalence of many types of metabolism in litter deconstruction makes these soils useful templates for improving biofuel production. To investigate how iron availability affects decomposition, we cultivated feedstock-adapted consortia (FACs) derived from iron-rich tropical forest soils accustomed to experiencing frequent episodes of anaerobic conditions and frequently fluctuating redox. One consortium was propagated under fermenting conditions, with switchgrass as the sole carbon source in minimal media (SG only FACs), and the other consortium was treated the same way but received poorly crystalline iron as an additional terminal electron acceptor (SG + Fe FACs). We sequenced the metagenomes of both consortia to a depth of about 150 Mb each, resulting in a coverage of 26x for the more diverse SG + Fe FACs, and 81 x for the relatively less diverse SG only FACs. Both consortia were able to quickly grow on switchgrass, and the iron-amended consortium exhibited significantly higher microbial diversity than the unamended consortium. We found evidence of higher stress in the unamended FACs and increased sugar transport and utilization in the iron-amended FACs. This work provides metagenomic evidence that supplementation of alternative TEAs may improve feedstock deconstruction in biofuel production.
  149. DeAngelis, K. M., D. Chivian, J. L. Fortney, A. P. Arkin, B. Simmons, T. C. Hazen and W. L. Silver. 2013. Changes in microbial dynamics during long-term decomposition in tropical forests. Soil Biology & Biochemistry 66:60-68. abstract pdf
    Humid tropical forest soils are characterized by low and fluctuating redox, conditions which are thought to inhibit organic matter degradation by microbes. However, evidence suggests that soil microbial communities are adapted to the redox conditions in these ecosystems. In this study we tested the hypothesis that soil oxygen (02) availability as an index of redox conditions structures patterns in litter decomposition and associated microbial community dynamics over space and time in humid tropical forests. We conducted a two year decomposition experiment on a common litter substrate in four sites along an elevational gradient with well described climate and redox dynamics. Microbial community sequencing, potential enzyme activities, and litter chemistry measurements were made on litter and soil to determine the relationship between soil and litter communities and biogeochemistry. Decomposition was slowest in the upper elevation site, which was the wettest and had the lowest average soil 02 availability. However, soil hydrolytic and litter phenol oxidase activities were greatest at this site. Small subunit ribosomal RNA genes were sequenced with universal primers for bacteria, archaea and eukaryotes, yielding 40,850 unique taxa after quality filtering and clustering. Across all sites, microbial succession was observed as increasing litter richness, converging bacterial community profiles, and diverging fungal community profiles. Initial decomposers (1-4 weeks) included many r-selected bacteria, including Alpha-, Beta- and Gammaproteobacteria, Clostridia, Bacteroidetes. We also found evidence of anaerobic fungi such as Cryptococcus, as well as the plant-associated Phialocephala and Phyllachora species, suggesting that anaerobic and plant-associated fungi are prevalent later in decomposition in soils with low and fluctuating redox conditions. Because of the striking similarities between sites in functional potential despite differences in wet tropical soil decomposing communities and litter chemistry, we suggest that future climate-driven disruptions to redox fluctuations could significantly alter the terrestrial carbon (C) cycle in tropical forests. (C) 2013 Elsevier Ltd. All rights reserved.
  150. Zhou, A. F., Y. Y. I. Chen, G. M. Zane, Z. L. He, C. L. Hemme, M. P. Joachimiak, J. K. Baumohl, Q. He, M. W. Fields, A. P. Arkin, J. D. Wall, T. C. Hazen and J. Z. Zhou. 2012. Functional Characterization of Crp/Fnr-Type Global Transcriptional Regulators in Desulfovibrio vulgaris Hildenborough. Applied and Environmental Microbiology 78:1168-1177. abstract pdf
    Crp/Fnr-type global transcriptional regulators regulate various metabolic pathways in bacteria and typically function in response to environmental changes. However, little is known about the function of four annotated Crp/Fnr homologs (DVU0379, DVU2097, DVU2547, and DVU3111) in Desulfovibrio vulgaris Hildenborough. A systematic study using bioinformatic, transcriptomic, genetic, and physiological approaches was conducted to characterize their roles in stress responses. Similar growth phenotypes were observed for the crp/fnr deletion mutants under multiple stress conditions. Nevertheless, the idea of distinct functions of Crp/Fnr-type regulators in stress responses was supported by phylogeny, gene transcription changes, fitness changes, and physiological differences. The four D. vulgaris Crp/Fnr homologs are localized in three subfamilies (HcpR, CooA, and cc). The crp/fnr knockout mutants were well separated by transcriptional profiling using detrended correspondence analysis (DCA), and more genes significantly changed in expression in a Delta DVU3111 mutant (JW9013) than in the other three paralogs. In fitness studies, strain fW9013 showed the lowest fitness under standard growth conditions (i.e., sulfate reduction) and the highest fitness under NaCl or chromate stress conditions; better fitness was observed for a Delta DVU2547 mutant (JW9011) under nitrite stress conditions and a Delta DVU2097 mutant (JW9009) under air stress conditions. A higher Cr(VI) reduction rate was observed for strain JW9013 in experiments with washed cells. These results suggested that the four Crp/Fnr-type global regulators play distinct roles in stress responses of D. vulgaris. DVU3111 is implicated in responses to NaCl and chromate stresses, DVU2547 in nitrite stress responses, and DVU2097 in air stress responses.
  151. Walian, P. J., S. Allen, M. Shatsky, L. Zeng, E. D. Szakal, H. C. Liu, S. C. Hall, S. J. Fisher, B. R. Lam, M. E. Singer, J. T. Geller, S. E. Brenner, J. M. Chandonia, T. C. Hazen, H. E. Witkowska, M. D. Biggin and B. K. Jap. 2012. High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris. Journal of Proteome Research 11:5720-5735. abstract pdf
    Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane-associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.
  152. Mosher, J. J., 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 D. A. Elias. 2012. Microbial Community Succession during Lactate Amendment and Electron Acceptor Limitation Reveals a Predominance of Metal-Reducing Pelosinus spp. Applied and Environmental Microbiology 78:2082-2091. abstract pdf
    The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N-2 gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.
  153. Mason, O. U., T. C. Hazen, S. Borglin, P. S. G. Chain, E. A. Dubinsky, J. L. Fortney, J. Han, H. Y. N. Holman, J. Hultman, R. Lamendella, R. Mackelprang, S. Malfatti, L. M. Tom, S. G. Tringe, T. Woyke, J. H. Zhou, E. M. Rubin and J. K. Jansson. 2012. Metagenome, metatranscriptome and single-cell sequencing reveal microbial response to Deepwater Horizon oil spill. Isme Journal 6:1715-1727. abstract pdf
    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea. The ISME Journal (2012) 6, 1715-1727; doi:10.1038/ismej.2012.59; published online 21 June 2012 Subject Category: integrated genomics and post-genomics approaches in microbial ecology
  154. Lu, Z. M., Y. Deng, J. D. Van Nostrand, Z. L. He, J. Voordeckers, A. F. Zhou, Y. J. Lee, O. U. Mason, E. A. Dubinsky, K. L. Chavarria, L. M. Tom, J. L. Fortney, R. Lamendella, J. K. Jansson, P. D'Haeseleer, T. C. Hazen and J. Z. Zhou. 2012. Microbial gene functions enriched in the Deepwater Horizon deep-sea oil plume. Isme Journal 6:451-460. abstract pdf
    The Deepwater Horizon oil spill in the Gulf of Mexico is the deepest and largest offshore spill in the United State history and its impacts on marine ecosystems are largely unknown. Here, we showed that the microbial community functional composition and structure were dramatically altered in a deep-sea oil plume resulting from the spill. A variety of metabolic genes involved in both aerobic and anaerobic hydrocarbon degradation were highly enriched in the plume compared with outside the plume, indicating a great potential for intrinsic bioremediation or natural attenuation in the deep sea. Various other microbial functional genes that are relevant to carbon, nitrogen, phosphorus, sulfur and iron cycling, metal resistance and bacteriophage replication were also enriched in the plume. Together, these results suggest that the indigenous marine microbial communities could have a significant role in biodegradation of oil spills in deep-sea environments. The ISME Journal (2012) 6, 451-460; doi: 10.1038/ismej.2011.91; published online 4 August 2011
  155. Liang, Y. T., J. D. Van Nostrand, L. A. N'Guessan, A. D. Peacock, Y. Deng, P. E. Long, C. T. Resch, L. Y. Wu, Z. L. He, G. H. Li, T. C. Hazen, D. R. Lovley and J. Z. Zhou. 2012. Microbial Functional Gene Diversity with a Shift of Subsurface Redox Conditions during In Situ Uranium Reduction. Applied and Environmental Microbiology 78:2966-2972. abstract pdf
    To better understand the microbial functional diversity changes with subsurface redox conditions during in situ uranium bioremediation, key functional genes were studied with GeoChip, a comprehensive functional gene microarray, in field experiments at a uranium mill tailings remedial action (UMTRA) site (Rifle, CO). The results indicated that functional microbial communities altered with a shift in the dominant metabolic process, as documented by hierarchical cluster and ordination analyses of all detected functional genes. The abundance of dsrAB genes (dissimilatory sulfite reductase genes) and methane generation-related mcr genes (methyl coenzyme M reductase coding genes) increased when redox conditions shifted from Fe-reducing to sulfate-reducing conditions. The cytochrome genes detected were primarily from Geobacter sp. and decreased with lower subsurface redox conditions. Statistical analysis of environmental parameters and functional genes indicated that acetate, U(VI), and redox potential (Eh) were the most significant geochemical variables linked to microbial functional gene structures, and changes in microbial functional diversity were strongly related to the dominant terminal electron-accepting process following acetate addition. The study indicates that the microbial functional genes clearly reflect the in situ redox conditions and the dominant microbial processes, which in turn influence uranium bioreduction. Microbial functional genes thus could be very useful for tracking microbial community structure and dynamics during bioremediation.
  156. Kinner, N.. 2012. The Future of Dispersant Use in Spill Response. abstract pdf
    The National Oceanic and Atmospheric Administration (NOAA) is the primary scientific adviser to the U.S. Coast Guard (USCG), and a trustee for the Nation’s marine natural resources. In support of these responsibilities, NOAA conducted an evaluation of the observations and science conducted during the Deepwater Horizon (DWH) spill to build a foundation for planning and decision making in future spills, identify key information gaps and develop a research plan for closing the gaps. This initiative included academic and agency scientists and stakeholders to collect and evaluate the available scientific evidence concerning both surface and subsurface application of dispersants during the DWH emergency response. To broaden this initiative it is being organized and coordinated under the Interagency Coordinating Committee for Oil Pollution Research (ICCOPR), established under the Oil Pollution Act of 1990. The Coastal Response Research Center (CRRC), a partnership between the University of New Hampshire and NOAA’s Office of Response and Restoration (ORR), and Research Planning, Incorporated (RPI) are partnering with NOAA to coordinate and carry out this project. Contained in this report are: an overview of the information synthesis; expert white papers on dispersant efficacy and effectiveness, degradation of dispersants and dispersed oil, physical transport and chemical behavior of dispersed oil, biological effects of dispersants and dispersed oil on surface and deep ocean species, dispersants and seafood safety, dispersants and human health, and dispersants and risk communication; an independent chemical analysis on the COREXIT dispersants used during the DWH spill; and the outcomes of the workshop. If you have any comments about the initiative or this report, please contact us. This effort is part of NOAA ORR’s on-going activities to improve oil spill response and restoration in the aftermath of Deepwater Horizon and support preparedness for future spills.
  157. Khudyakov, J. I., P. D'Haeseleer, S. E. Borglin, K. M. DeAngelis, H. Woo, E. A. Lindquist, T. C. Hazen, B. A. Simmons and M. P. Thelen. 2012. Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium, Enterobacter lignolyticus. Proceedings of the National Academy of Sciences of the United States of America 109:E2173-E2182. abstract pdf
    To process plant-based renewable biofuels, pretreatment of plant feedstock with ionic liquids has significant advantages over current methods for deconstruction of lignocellulosic feedstocks. However, ionic liquids are often toxic to the microorganisms used subsequently for biomass saccharification and fermentation. We previously isolated Enterobacter lignolyticus strain SCF1, a lignocel-lulolytic bacterium from tropical rain forest soil, and report here that it can grow in the presence of 0.5 M 1-ethyl-3-methylimidazolium chloride, a commonly used ionic liquid. We investigated molecular mechanisms of SCF1 ionic liquid tolerance using a combination of phenotypic growth assays, phospholipid fatty acid analysis, and RNA sequencing technologies. Potential modes of resistance to 1-ethyl-3-methylimidazolium chloride include an increase in cyclopropane fatty acids in the cell membrane, scavenging of compatible solutes, up-regulation of osmoprotectant transporters and drug efflux pumps, and down-regulation of membrane porins. These findings represent an important first step in understanding mechanisms of ionic liquid resistance in bacteria and provide a basis for engineering microbial tolerance.
  158. Hazen, T. C. and S. Wuertz. 2012. Environmental biotechnology Editorial overview. Current Opinion in Biotechnology 23:414-414. abstract pdf
    The tools of metagenomics, transcriptomics, proteomics and metabolomics, which are commonly summarized under the heading omics, have begun to redefine how we approach research questions in environmental biotechnol- ogy. This issue contains a majority of articles that either describe the tools of omics or invoke systems biology — a nonreductionist approach intent on observing and integrating the information generated from omics analyses to model emerging properties of the systems studied — to chart the path ahead toward a better understanding and management of a variety of environ- ments. These applications vary from the human gut microbiome all the way to bioremediation of chemically contaminated sites using chip-based tech- nology. And while these new approaches will continue to provide invaluable insights, especially into the vast and still mostly untapped reservoir of genes and functions in hitherto uncultivable microbes, it is equally important to acknowledge the contributions dealing with physiological traits and bio- technological technologies that study and interpret the functions of microbial communities and isolated strains using more traditional genetic, molecular and engineering-based methods. Examples in this issue relate to enrichment and isolation of metabolically diverse micoorganisms — still the ultimate goal for physiological characterization and many biotechnological applications — low temperature anaerobic digestion, and solvent tolerance in Gram-negative bacteria. In conclusion, we believe that in their entirety the articles summarize many important research areas and a range of state-of-the-art methodologies, which we hope will guide the reader in the rapidly emerging field of environmental biotechnology.
  159. Gladden, J. M., S. A. Eichorst, T. C. Hazen, B. A. Simmons and S. W. Singer. 2012. Substrate perturbation alters the glycoside hydrolase activities and community composition of switchgrass-adapted bacterial consortia. Biotechnology and Bioengineering 109:1140-1145. abstract pdf
    Bacteria modulate glycoside hydrolase expression in response to the changes in the composition of lignocellulosic biomass. The response of switchgrass-adapted thermophilic bacterial consortia to perturbation with a variety of biomass substrates was characterized to determine if bacterial consortia also responded to changes in biomass composition. Incubation of the switchgrass-adapted consortia with these alternative substrates produced shifts in glycoside hydrolase activities and bacterial community composition. Substantially increased endoglucanase activity was observed upon incubation with microcrystalline cellulose and trifluororacetic acid-pretreated switchgrass. In contrast, culturing the microbial consortia with ionic liquid-pretreated switchgrass increased xylanase activity dramatically. Microbial community analyses of these cultures indicated that the increased endoglucanase activity correlated with an increase in bacteria related to Rhodothermus marinus. Inclusion of simple organic substrates in the culture medium abrogated glycoside hydrolase activity and enriched for bacteria related to Thermus thermophilus. These results demonstrate that the composition of biomass substrates influences the glycoside hydrolase activities and community composition of biomass-deconstructing bacterial consortia. Biotechnol. Bioeng. 2012; 109:11401145. (C) 2011 Wiley Periodicals, Inc.
  160. Genome Alberta and the Public Policy Forum. 2012. Metagenomics and Bioremediation. Enhancing Energy Production and Environmental Outcomes through Genomics: The case for innovation abstract pdf
    Bioremediation By identifying the microorganisms and genetic pathways capable of detoxifying or eliminating toxic compounds, metagenomics can be used to develop biological solutions to clean up impacted sites in a less expensive and more environmentally friendly manner compared to alternative technologies.
  161. Dougherty, M. J., P. D'Haeseleer, T. C. Hazen, B. A. Simmons, P. D. Adams and M. Z. Hadi. 2012. Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization. Bmc Biotechnology 12:9. abstract pdf
    Background: Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. Results: Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to degrees 60-70 degrees C. Conclusions: Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.
  162. DeAngelis, K. M., J. L. Fortney, S. Borglin, W. L. Silver, B. A. Simmons and T. C. Hazen. 2012. Anaerobic Decomposition of Switchgrass by Tropical Soil-Derived Feedstock-Adapted Consortia. Mbio 3:9. abstract pdf
    Tropical forest soils decompose litter rapidly with frequent episodes of anoxic conditions, making it likely that bacteria using alternate terminal electron acceptors (TEAs) play a large role in decomposition. This makes these soils useful templates for improving biofuel production. To investigate how TEAs affect decomposition, we cultivated feedstock-adapted consortia (FACs) derived from two tropical forest soils collected from the ends of a rainfall gradient: organic matter-rich tropical cloud forest (CF) soils, which experience sustained low redox, and iron-rich tropical rain forest (RF) soils, which experience rapidly fluctuating redox. Communities were anaerobically passed through three transfers of 10 weeks each with switchgrass as a sole carbon (C) source; FACs were then amended with nitrate, sulfate, or iron oxide. C mineralization and cellulase activities were higher in CF-FACs than in RF-FACs. Pyrosequencing of the small-subunit rRNA revealed members of the Firmicutes, Bacteroidetes, and Alphaproteobacteria as dominant. RF- and CF-FAC communities were not different in microbial diversity or biomass. The RF-FACs, derived from fluctuating redox soils, were the most responsive to the addition of TEAs, while the CF-FACs were overall more efficient and productive, both on a per-gram switchgrass and a per-cell biomass basis. These results suggest that decomposing microbial communities in fluctuating redox environments are adapted to the presence of a diversity of TEAs and ready to take advantage of them. More importantly, these data highlight the role of local environmental conditions in shaping microbial community function that may be separate from phylogenetic structure. IMPORTANCE After multiple transfers, we established microbial consortia derived from two tropical forest soils with different native redox conditions. Communities derived from the rapidly fluctuating redox environment maintained a capacity to use added terminal electron acceptors (TEAs) after multiple transfers, though they were not present during the enrichment. Communities derived from lower-redox soils were not responsive to TEA addition but were much more efficient at switchgrass decomposition. Though the communities were different, diversity was not, and both were dominated by many of the same species of clostridia. This reflects the inadequacy of rRNA for determining the function of microbial communities, in this case the retained ability to utilize TEAs that were not part of the selective growth conditions. More importantly, this suggests that microbial community function is shaped by life history, where environmental factors produce heritable traits through natural selection over time, creating variation in the community, a phenomenon not well documented for microbes.
  163. Chakraborty, R., C. H. Wu and T. C. Hazen. 2012. Systems biology approach to bioremediation. Current Opinion in Biotechnology 23:483-490. abstract pdf
    Bioremediation has historically been approached as a 'black box' in terms of our fundamental understanding. Thus it succeeds and fails, seldom without a complete understanding of why. Systems biology is an integrated research approach to study complex biological systems, by investigating interactions and networks at the molecular, cellular, community, and ecosystem level. The knowledge of these interactions within individual components is fundamental to understanding the dynamics of the ecosystem under investigation. Understanding and modeling functional microbial community structure and stress responses in environments at all levels have tremendous implications for our fundamental understanding of hydrobiogeochemical processes and the potential for making bioremediation breakthroughs and illuminating the 'black box'.
  164. Chakraborty, R., S. E. Borglin, E. A. Dubinsky, G. L. Andersen and T. C. Hazen. 2012. Microbial response to the MC-252 oil and Corexit 9500 in the Gulf of Mexico. Frontiers in Microbiology 3:6. abstract pdf
    The Deepwater Horizon spill released over 4.1 million barrels of crude oil into the Gulf of Mexico. In an effort to mitigate large oil slicks, the dispersant Corexit 9500 was sprayed onto surface slicks and injected directly at the wellhead at water depth of 1,500 m. Several research groups were involved in investigating the fate of the MC-252 oil using newly advanced molecular tools to elucidate microbial interactions with oil, gases, and dispersant. Microbial community analysis by different research groups revealed that hydrocarbon degrading bacteria belonging to Oceanospirillales, Colwellia, Cycloclasticus, Rhodobacterales, Pseudoalteromonas, and methylotrophs were found enriched in the contaminated water column. Presented here is a comprehensive overview of the ecogenomics of microbial degradation of MC-252 oil and gases in the water column and shorelines. We also present some insight into the fate of the dispersant Corexit 9500 that was added to aid in oil dispersion process. Our results show the dispersant was not toxic to the indigenous microbes at concentrations added, and different bacterial species isolated in the aftermath of the spill were able to degrade the various components of Corexit 9500 that included hydrocarbons, glycols, and dioctyl sulfosuccinate.
  165. Brown, S. D., M. Podar, D. M. Klingeman, C. M. Johnson, Z. K. Yang, S. M. Utturkar, M. L. Land, J. J. Mosher, R. A. Hurt, T. J. Phelps, A. V. Palumbo, A. P. Arkin, T. C. Hazen and D. A. Elias. 2012. Draft Genome Sequences for Two Metal-Reducing Pelosinus fermentans Strains Isolated from a Cr(VI)-Contaminated Site and for Type Strain R7. Journal of Bacteriology 194:5147-5148. abstract pdf
    Pelosinus fermentans 16S rRNA gene sequences have been reported from diverse geographical sites since the recent isolation of the type strain. We present the genome sequence of the P. fermentans type strain R7 (DSM 17108) and genome sequences for two new strains with different abilities to reduce iron, chromate, and uranium.
  166. Borglin, S., D. Joyner, K. M. DeAngelis, J. Khudyakov, P. D'Haeseleer, M. P. Joachimiak and T. Hazen. 2012. Application of phenotypic microarrays to environmental microbiology. Current Opinion in Biotechnology 23:41-48. abstract pdf
    Environmental organisms are extremely diverse and only a small fraction has been successfully cultured in the laboratory. Culture in micro wells provides a method for rapid screening of a wide variety of growth conditions and commercially available plates contain a large number of substrates, nutrient sources, and inhibitors, which can provide an assessment of the phenotype of an organism. This review describes applications of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in development of culture media for newly discovered strains, and for assessment of phenotype of environmental communities. Also discussed are considerations and challenges in data interpretation and visualization, including data normalization, statistics, and curve fitting.
  167. Beazley, M. J., R. J. Martinez, S. Rajan, J. Powell, Y. M. Piceno, L. M. Tom, G. L. Andersen, T. C. Hazen, J. D. Van Nostrand, J. Z. Zhou, B. Mortazavi and P. A. Sobecky. 2012. Microbial Community Analysis of a Coastal Salt Marsh Affected by the Deepwater Horizon Oil Spill. Plos One 7:13. abstract pdf
    Coastal salt marshes are highly sensitive wetland ecosystems that can sustain long-term impacts from anthropogenic events such as oil spills. In this study, we examined the microbial communities of a Gulf of Mexico coastal salt marsh during and after the influx of petroleum hydrocarbons following the Deepwater Horizon oil spill. Total hydrocarbon concentrations in salt marsh sediments were highest in June and July 2010 and decreased in September 2010. Coupled PhyloChip and GeoChip microarray analyses demonstrated that the microbial community structure and function of the extant salt marsh hydrocarbon-degrading microbial populations changed significantly during the study. The relative richness and abundance of phyla containing previously described hydrocarbon-degrading bacteria (Proteobacteria, Bacteroidetes, and Actinobacteria) increased in hydrocarbon-contaminated sediments and then decreased once hydrocarbons were below detection. Firmicutes, however, continued to increase in relative richness and abundance after hydrocarbon concentrations were below detection. Functional genes involved in hydrocarbon degradation were enriched in hydrocarbon-contaminated sediments then declined significantly (p<0.05) once hydrocarbon concentrations decreased. A greater decrease in hydrocarbon concentrations among marsh grass sediments compared to inlet sediments (lacking marsh grass) suggests that the marsh rhizosphere microbial communities could also be contributing to hydrocarbon degradation. The results of this study provide a comprehensive view of microbial community structural and functional dynamics within perturbed salt marsh ecosystems.
  168. Baelum, J., S. Borglin, R. Chakraborty, J. L. Fortney, R. Lamendella, O. U. Mason, M. Auer, M. Zemla, M. Bill, M. E. Conrad, S. A. Malfatti, S. G. Tringe, H. Y. Holman, T. C. Hazen and J. K. Jansson. 2012. Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill. Environmental Microbiology 14:2405-2416. abstract pdf
    The Deepwater Horizon oil spill resulted in a massive influx of hydrocarbons into the Gulf of Mexico (the Gulf). To better understand the fate of the oil, we enriched and isolated indigenous hydrocarbon-degrading bacteria from deep, uncontaminated waters from the Gulf with oil (Macondo MC252) and dispersant used during the spill (COREXIT 9500). During 20 days of incubation at 5 degrees C, CO2 evolution, hydrocarbon concentrations and the microbial community composition were determined. Approximately 60% to 25% of the dissolved oil with or without COREXIT, respectively, was degraded, in addition to some hydrocarbons in the COREXIT. FeCl2 addition initially increased respiration rates, but not the total amount of hydrocarbons degraded. 16S rRNA gene sequencing revealed a succession in the microbial community over time, with an increase in abundance of Colwellia and Oceanospirillales during the incubations. Flocs formed during incubations with oil and/or COREXIT in the absence of FeCl2. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy revealed that the flocs were comprised of oil, carbohydrates and biomass. Colwellia were the dominant bacteria in the flocs. Colwellia sp. strain RC25 was isolated from one of the enrichments and confirmed to rapidly degrade high amounts (approximately 75%) of the MC252 oil at 5 degrees C. Together these data highlight several features that provide Colwellia with the capacity to degrade oil in cold, deep marine habitats, including aggregation together with oil droplets into flocs and hydrocarbon degradation ability.
  169. Zhou, J. Z., Q. He, C. L. Hemme, A. Mukhopadhyay, K. Hillesland, A. F. Zhou, Z. L. He, J. D. Van Nostrand, Terry C. Hazen, D. A. Stahl, J. D. Wall and A. P. Arkin. 2011. How sulphate-reducing microorganisms cope with stress: lessons from systems biology. Nature Reviews Microbiology 9:452-466. abstract pdf
    Sulphate-reducing microorganisms (SRMs) are a phylogenetically diverse group of anaerobes encompassing distinct physiologies with a broad ecological distribution. As SRMs have important roles in the biogeochemical cycling of carbon, nitrogen, sulphur and various metals, an understanding of how these organisms respond to environmental stresses is of fundamental and practical importance. In this Review, we highlight recent applications of systems biology tools in studying the stress responses of SRMs, particularly Desulfovibrio spp., at the cell, population, community and ecosystem levels. The syntrophic lifestyle of SRMs is also discussed, with a focus on system-level analyses of adaptive mechanisms. Such information is important for understanding the microbiology of the global sulphur cycle and for developing biotechnological applications of SRMs for environmental remediation, energy production, biocorrosion control, wastewater treatment and mineral recovery.
  170. Van Nostrand, J. D., L. Y. Wu, W. M. Wu, Z. J. Huang, T. J. Gentry, Y. Deng, J. Carley, S. Carroll, Z. L. He, B. H. Gu, J. Luo, C. S. Criddle, D. B. Watson, P. M. Jardine, T. L. Marsh, J. M. Tiedje, T. C. Hazen and J. Z. Zhou. 2011. Dynamics of Microbial Community Composition and Function during In Situ Bioremediation of a Uranium-Contaminated Aquifer. Applied and Environmental Microbiology 77:3860-3869. abstract pdf
    A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U. S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter(-1)). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.
  171. Singer, S. W., A. P. Reddy, J. M. Gladden, H. Guo, T. C. Hazen, B. A. Simmons and J. S. Vandergheynst. 2011. Enrichment, Isolation and Characterization of Fungi Tolerant to 1-Ethyl-3-Methylimadazoium Acetate. Journal of applied microbiology abstract pdf
    Aims: This work aimed to characterize microbial tolerance to 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. Methods and Results: Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populations dominated by Aspergilli. Ionic liquid-tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]-tolerant A. fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. Conclusions: The results of the study suggest that tolerance to ionic liquids is a general property of the Aspergilli. Significance and Impact of Study: Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction.
  172. Reindl, W., K. Deng, J. M. Gladden, G. Cheng, A. Wong, S. W. Singer, S. Singh, J. C. Lee, C. H. Yao, T. C. Hazen, A. K. Singh, B. A. Simmons, P. D. Adams and T. R. Northen. 2011. Colloid-based multiplexed screening for plant biomass-degrading glycoside hydrolase activities in microbial communities. Energy & Environmental Science 4:2884-2893. abstract pdf
    The enzymatic hydrolysis of polysaccharides into fermentable sugars is a crucial step in the conversion of biomass to lignocellulosic biofuels. An efficient hydrolysis is highly dependent on the identification and characterization of optimal glycoside hydrolases. However, existing techniques for characterizing activity are limited by the range of reaction conditions that can be used, sample complexity, and throughput. The method we present is 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 tubes and microwell plates despite the substrate analogs' hydrophobic perfluorinated tags. This method was validated by analyzing standard enzymatic parameters (temperature, pH, and kinetics) of beta-glucosidase and beta-xylosidase in separate setups. The multiplexity of this assay system was demonstrated by the simultaneous analysis of beta-glucosidase and beta-xylosidase activities, which was then used to profile environmental samples. Enzymes secreted by microbial communities within these samples were extracted by washing the sample with buffer. Enzymatic activities were directly detected within this crude extract without any further sample pretreatment steps. The multiplexed analysis of beta-glucosidase, exo-/endoglucanase, and xylanase activities was applied for a detailed characterization of previously unknownglycoside hydrolase activities. The results show the suitability of the described method for the rapid screening of crude environmental samples under a wide range of conditions to determine enzyme activities from the microbial communities present within these samples.
  173. Reddy, A. P., M. Allgaier, S. W. Singer, T. C. Hazen, B. A. Simmons, P. Hugenholtz and J. S. VanderGheynst. 2011. Bioenergy Feedstock-Specific Enrichment of Microbial Populations During High-Solids Thermophilic Deconstruction. Biotechnology and Bioengineering 108:2088-2098. abstract pdf
    Thermophilic microbial communities that are active in a high-solids environment offer great potential for the discovery of industrially relevant enzymes that efficiently deconstruct bioenergy feedstocks. In this study, finished green waste compost was used as an inoculum source to enrich microbial communities and associated enzymes that hydrolyze cellulose and hemicellulose during thermophilic high-solids fermentation of the bioenergy feedstocks switch-grass and corn stover. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase and endoglucanase activity from deconstructed solids. Xylanase and endoglucanase activity increased by more than a factor of 5, upon four successive enrichments on switchgrass. Overall, the changes for switchgrass were more pronounced than for corn stover; solids reduction between the first and second enrichments increased by a factor of four for switchgrass while solids reduction remained relatively constant for corn stover. Amplicon pyrosequencing analysis of small-subunit ribosomal RNA genes recovered from enriched samples indicated rapid changes in the microbial communities between the first and second enrichment with the simplified communities achieved by the third enrichment. The results demonstrate a successful approach for enrichment of unique microbial communities and enzymes active in a thermophilic high-solids environment. Biotechnol. Bioeng. 2011; 108: 2088-2098. (C) 2011 Wiley Periodicals, Inc.
  174. Mohanty, S. R., B. Kollah, E. L. Brodie, T. C. Hazen and E. E. Roden. 2011. 16S rRNA Gene Microarray Analysis of Microbial Communities in Ethanol-Stimulated Subsurface Sediment. Microbes and Environments 26:261-265. abstract pdf
    A high-density 16S rRNA gene microarray was used to analyze microbial communities in a slurry of ethanolamended, uranium-contaminated subsurface sediment. Of specific interest was the extent to which the microarray could detect temporal patterns in the relative abundance of major metabolic groups (nitrate-reducing, metal-reducing, sulfate-reducing, and methanogenic taxa) that were stimulated by ethanol addition. The results show that the microarray, when used in conjunction with geochemical data and knowledge of the physiological properties of relevant taxa, provided accurate assessment of the response of key functional groups to biostimulation.
  175. American Academy of Microbiology. 2011. Microbes and Oil Spills (FAQ). American Society for Microbiology abstract pdf
    Let’s begin by defi ning some terms – microbes, clean-up, and oil. First, what do we mean by microbes? When we talk about microbes that are able to clean up oil, we’re talking primarily about bacteria and fungi. Bacteria can break down oil to carbon dioxide and water. However, no single organism can break down all the components of crude oil or refi ned fuels spilled into the environment. The tens of thousands of different compounds that make up oil can only be biodegraded by communities of microorganisms acting in concert. Some bacteria can degrade several hydrocarbons or a class of hydrocarbons. The combined action of the community can degrade almost all of the components
  176. Olivia U. Mason and Terry C. Hazen. 2011. New insights into microbial responses to oil Spills from the Deepwater Horizon incident. SIM News 2011. abstract pdf
    Taken together, these studies clearly demonstrated that there was a profound and significant response by certain members of the in situ microbial community in the deep-sea in the Gulf of Mexico. In particular putative hydrocarbon degrading Bacteria appeared to bloom in response to the Deepwater Horizon oil spill, even though the temperature at these depths is never >5°C. As the plume aged the shifts in the microbial community on a temporal scale suggested that different, yet metabolically important members of the community were able to respond to a myriad of plume constituents, e.g. shifting from propane/ethane to alkanes and finally to methane. Thus, the biodegradation of hydrocarbons in the plume by Bacteria was a highly significant process in the natural attenuation of many compounds released during the Deepwater Horizon oil spill.
  177. Liu, P., R. J. Meagher, Y. K. Light, S. Yilmaz, R. Chakraborty, A. P. Arkin, T. C. Hazen and A. K. Singh. 2011. Microfluidic fluorescence in situ hybridization and flow cytometry (mu FlowFISH). Lab on a Chip 11:2673-2679. abstract pdf
    We describe an integrated microfluidic device (mu FlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The mu FlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of mu FlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The mu FlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome.
  178. Kostka, J. E., O. Prakash, W. A. Overholt, S. J. Green, G. Freyer, A. Canion, J. Delgardio, N. Norton, T. C. Hazen and M. Huettel. 2011. Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spill. Applied and Environmental Microbiology 77:7962-7974. abstract pdf
    A significant portion of oil from the recent Deepwater Horizon (DH) oil spill in the Gulf of Mexico was transported to the shoreline, where it may have severe ecological and economic consequences. The objectives of this study were (i) to identify and characterize predominant oil-degrading taxa that may be used as model hydrocarbon degraders or as microbial indicators of contamination and (ii) to characterize the in situ response of indigenous bacterial communities to oil contamination in beach ecosystems. This study was conducted at municipal Pensacola Beach, FL, where chemical analysis revealed weathered oil petroleum hydrocarbon (C(8) to C(40)) concentrations ranging from 3.1 to 4,500 mg kg(-1) in beach sands. A total of 24 bacterial strains from 14 genera were isolated from oiled beach sands and confirmed as oil-degrading microorganisms. Isolated bacterial strains were primarily Gammaproteobacteria, including representatives of genera with known oil degraders (Alcanivorax, Marinobacter, Pseudomonas, and Acinetobacter). Sequence libraries generated from oiled sands revealed phylotypes that showed high sequence identity (up to 99%) to rRNA gene sequences from the oil-degrading bacterial isolates. The abundance of bacterial SSU rRNA gene sequences was similar to 10-fold higher in oiled (0.44 x 10(7) to 10.2 x 10(7) copies g(-1)) versus clean (0.024 x 10(7) to 1.4 x 10(7) copies g(-1)) sand. Community analysis revealed a distinct response to oil contamination, and SSU rRNA gene abundance derived from the genus Alcanivorax showed the largest increase in relative abundance in contaminated samples. We conclude that oil contamination from the DH spill had a profound impact on the abundance and community composition of indigenous bacteria in Gulf beach sands, and our evidence points to members of the Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria (Rhodobacteraceae) as key players in oil degradation there.
  179. Hazen, Terry C.. 2011. Subsurface Biogeochemical Research (SBR) Contractor-Gantee Workshop Abstracts. Subsurface Biogeochemical Research (SBR) Contractor-Gantee Workshop Abstracts pdf
  180. Gladden, J. M., M. Allgaier, C. S. Miller, T. C. Hazen, J. S. VanderGheynst, P. Hugenholtz, B. A. Simmons and S. W. Singer. 2011. Glycoside Hydrolase Activities of Thermophilic Bacterial Consortia Adapted to Switchgrass. Applied and Environmental Microbiology 77:5804-5812. abstract pdf
    Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60 degrees C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80 degrees C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.
  181. DeAngelis, K. M., C. H. Wu, H. R. Beller, E. L. Brodie, R. Chakraborty, T. Z. DeSantis, J. L. Fortney, T. C. Hazen, S. R. Osman, M. E. Singer, L. M. Tom and G. L. Andersen. 2011. PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip. Applied and Environmental Microbiology 77:6313-6322. abstract pdf
    Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.
  182. DeAngelis, K. M., P. D'Haeseleer, D. Chivian, J. L. Fortney, J. Khudyakov, B. Simmons, H. Woo, A. P. Arkin, K. W. Davenport, L. Goodwin, A. Chen, N. Ivanova, N. C. Kyrpides, K. Mavromatis, T. Woyke and T. C. Hazen. 2011. Complete genome sequence of Enterobacter lignolyticus SCF1. Standards in Genomic Sciences 5:69-85. abstract pdf
    In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated "Enterobacter lignolyticus" SCF1 on minimal media with alkali lignin as the sole source of carbon. This organism was isolated anaerobically from tropical forest soils collected from the Short Cloud Forest site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are net methane producers. Because of its ability to grow on lignin anaerobically, we sequenced the genome. The genome of "E. lignolyticus" SCF1 is 4.81 Mbp with no detected plasmids, and includes a relatively small arsenal of lignocellulolytic carbohydrate active enzymes. Lignin degradation was observed in culture, and the genome revealed two putative laccases, a putative peroxidase, and a complete 4-hydroxyphenylacetate degradation pathway encoded in a single gene cluster.
  183. DeAngelis, K. M., M. Allgaier, Y. Chavarria, J. L. Fortney, P. Hugenholtz, B. Simmons, K. Sublette, W. L. Silver and T. C. Hazen. 2011. Characterization of Trapped Lignin-Degrading Microbes in Tropical Forest Soil. Plos One 6:9. abstract pdf
    Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition.
  184. Chhabra, S. R., M. P. Joachimiak, C. J. Petzold, G. M. Zane, M. N. Price, S. A. Reveco, V. Fok, A. R. Johanson, T. S. Batth, M. Singer, J. M. Chandonia, D. Joyner, T. C. Hazen, A. P. Arkin, J. D. Wall, A. K. Singh and J. D. Keasling. 2011. Towards a Rigorous Network of Protein-Protein Interactions of the Model Sulfate Reducer Desulfovibrio vulgaris Hildenborough. Plos One 6:16. abstract pdf
    Protein-protein interactions offer an insight into cellular processes beyond what may be obtained by the quantitative functional genomics tools of proteomics and transcriptomics. The aforementioned tools have been extensively applied to study Escherichia coli and other aerobes and more recently to study the stress response behavior of Desulfovibrio vulgaris Hildenborough, a model obligate anaerobe and sulfate reducer and the subject of this study. Here we carried out affinity purification followed by mass spectrometry to reconstruct an interaction network among 12 chromosomally encoded bait and 90 prey proteins based on 134 bait-prey interactions identified to be of high confidence. Protein-protein interaction data are often plagued by the lack of adequate controls and replication analyses necessary to assess confidence in the results, including identification of potential false positives. We addressed these issues through the use of biological replication, exponentially modified protein abundance indices, results from an experimental negative control, and a statistical test to assign confidence to each putative interacting pair applicable to small interaction data studies. We discuss the biological significance of metabolic features of D. vulgaris revealed by these protein-protein interaction data and the observed protein modifications. These include the distinct role of the putative carbon monoxide-induced hydrogenase, unique electron transfer routes associated with different oxidoreductases, and the possible role of methylation in regulating sulfate reduction.
  185. Chhabra, S. R., G. Butland, D. A. Elias, J. M. Chandonia, O. Y. Fok, T. R. Juba, A. Gorur, S. Allen, C. M. Leung, K. L. Keller, S. Reveco, G. M. Zane, E. Semkiw, R. Prathapam, B. Gold, M. Singer, M. Ouellet, E. D. Szakal, D. Jorgens, M. N. Price, H. E. Witkowska, H. R. Beller, A. P. Arkin, T. C. Hazen, M. D. Biggin, M. Auer, J. D. Wall and J. D. Keasling. 2011. Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome. Applied and Environmental Microbiology 77:7595-7604. abstract pdf
    The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
  186. Chakraborty, R., T. C. Hazen, D. C. Joyner, K. Kusel, M. E. Singer, J. Sitte and T. Torok. 2011. Use of immunomagnetic separation for the detection of Desulfovibrio vulgaris from environmental samples. Journal Of Microbiological Methods 86:204-209. abstract pdf
    Immunomagnetic separation (IMS) has proved highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted the separation of viable cells of the sulfate-reducing bacterium, Desulfovibrio vulgaris. Streptavidin-coupled paramagnetic beads and biotin labeled antibodies raised against surface antigens of this microorganism were used to capture D. vulgaris cells in both bioreactor grown laboratory samples and from extremely low-biomass environmental soil and subsurface drilling samples. Initial studies on detection, recovery efficiency and viability for IMS were performed with laboratory grown D. vulgaris cells using various cell densities. Efficiency of cell isolation and recovery (i.e., release of the microbial cells from the beads following separation) was followed by microscopic imaging and acridine orange direct counts (AODC). Excellent recovery efficiency encouraged the use of IMS to capture Desulfovibrio spp. cells from low-biomass environmental samples. The environmental samples were obtained from a radionuclide-contaminated site in Germany and the chromium (VI)-contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Field deployable IMS technology may greatly facilitate environmental sampling and bioremediation process monitoring and enable transcriptomics and proteomics/metabolomics-based studies directly on cells collected from the field. (C) 2011 Elsevier B.V. All rights reserved.
  187. Brodie, E. L., D. C. Joyner, B. Faybishenko, M. E. Conrad, C. Rios-Velazquez, J. Malave, R. Martinez, B. Mork, A. Willett, S. Koenigsberg, D. J. Herman, M. K. Firestone and T. C. Hazen. 2011. Microbial community response to addition of polylactate compounds to stimulate hexavalent chromium reduction in groundwater. Chemosphere 85:660-665. abstract pdf
    To evaluate the efficacy of bioimmobilization of Cr(VI) in groundwater at the Department of Energy Hanford site, we conducted a series of microcosm experiments using a range of commercial electron donors with varying degrees of lactate polymerization (polylactate). These experiments were conducted using Hanford Formation sediments (coarse sand and gravel) immersed in Hanford groundwater, which were amended with Cr(VI) and several types of lactate-based electron donors (Hydrogen Release Compound, HRC: primer-HRC, pHRC; extended release HRC) and the polylactate-cysteine form (Metal Remediation Compound, MRC). The results showed that polylactate compounds stimulated an increase in bacterial biomass and activity to a greater extent than sodium lactate when applied at equivalent carbon concentrations. At the same time, concentrations of headspace hydrogen and methane increased and correlated with changes in the microbial community structure. Enrichment of Pseudomonas spp. occurred with all lactate additions, and enrichment of sulfate-reducing Desulfosporosinus spp. occurred with almost complete sulfate reduction. The results of these experiments demonstrate that amendment with the pHRC and MRC forms result in effective removal of Cr(VI) from solution most likely by both direct (enzymatic) and indirect (microbially generated reductant) mechanisms. (C) 2011 Elsevier Ltd. All rights reserved.
  188. Atlas, R. M. and T. C. Hazen. 2011. Oil Biodegradation and Bioremediation: A Tale of the Two Worst Spills in US History. Environmental Science & Technology 45:6709-6715. abstract pdf
    The devastating environmental impacts of the Exxon Valdez spill in 1989 and its media notoriety made it a frequent comparison to the BP Deepwater Horizon spill in the popular press in 2010, even though the nature of the two spills and the environments impacted were vastly different. Fortunately, unlike higher organisms that are adversely impacted by oil spills, microorganisms are able to consume petroleum hydrocarbons. These oil degrading indigenous microorganisms played a significant role in reducing the overall environmental impact of both the Exxon Valdez and BP Deepwater Horizon oil spills.
  189. Zhou, A. F., Z. L. He, A. M. Redding-Johanson, A. Mukhopadhyay, C. L. Hemme, M. P. Joachimiak, F. Luo, Y. Deng, K. S. Bender, Q. A. He, J. D. Keasling, D. A. Stahl, M. W. Fields, T. C. Hazen, A. P. Arkin, J. D. Wall and J. Z. Zhou. 2010. Hydrogen peroxide-induced oxidative stress responses in Desulfovibrio vulgaris Hildenborough. Environmental Microbiology 12:2645-2657. abstract pdf
    P>To understand how sulphate-reducing bacteria respond to oxidative stresses, the responses of Desulfovibrio vulgaris Hildenborough to H2O2-induced stresses were investigated with transcriptomic, proteomic and genetic approaches. H2O2 and induced chemical species (e.g. polysulfide, ROS) and redox potential shift increased the expressions of the genes involved in detoxification, thioredoxin-dependent reduction system, protein and DNA repair, and decreased those involved in sulfate reduction, lactate oxidation and protein synthesis. A gene coexpression network analysis revealed complicated network interactions among differentially expressed genes, and suggested possible importance of several hypothetical genes in H2O2 stress. Also, most of the genes in PerR and Fur regulons were highly induced, and the abundance of a Fur regulon protein increased. Mutant analysis suggested that PerR and Fur are functionally overlapped in response to stresses induced by H2O2 and reaction products, and the upregulation of thioredoxin-dependent reduction genes was independent of PerR or Fur. It appears that induction of those stress response genes could contribute to the increased resistance of deletion mutants to H2O2-induced stresses. In addition, a conceptual cellular model of D. vulgaris responses to H2O2 stress was constructed to illustrate that this bacterium may employ a complicated molecular mechanism to defend against the H2O2-induced stresses.
  190. Xu, M. Y., W. M. Wu, L. Y. Wu, Z. L. He, J. D. Van Nostrand, Y. Deng, J. Luo, J. Carley, M. Ginder-Vogel, T. J. Gentry, B. H. Gu, D. Watson, P. M. Jardine, T. L. Marsh, J. M. Tiedje, T. Hazen, C. S. Criddle and J. Z. Zhou. 2010. Responses of microbial community functional structures to pilot-scale uranium in situ bioremediation. Isme Journal 4:1060-1070. abstract pdf
    A pilot-scale field test system with an inner loop nested within an outer loop was constructed for in situ U(VI) bioremediation at a US Department of Energy site, Oak Ridge, TN. The outer loop was used for hydrological protection of the inner loop where ethanol was injected for biostimulation of microorganisms for U(VI) reduction/immobilization. After 2 years of biostimulation with ethanol, U(VI) levels were reduced to below drinking water standard (<30 mu g l(-1)) in the inner loop monitoring wells. To elucidate the microbial community structure and functions under in situ uranium bioremediation conditions, we used a comprehensive functional gene array (GeoChip) to examine the microbial functional gene composition of the sediment samples collected from both inner and outer loop wells. Our study results showed that distinct microbial communities were established in the inner loop wells. Also, higher microbial functional gene number, diversity and abundance were observed in the inner loop wells than the outer loop wells. In addition, metal-reducing bacteria, such as Desulfovibrio, Geobacter, Anaeromyxobacter and Shewanella, and other bacteria, for example, Rhodopseudomonas and Pseudomonas, are highly abundant in the inner loop wells. Finally, the richness and abundance of microbial functional genes were highly correlated with the mean travel time of groundwater from the inner loop injection well, pH and sulfate concentration in groundwater. These results suggest that the indigenous microbial communities can be successfully stimulated for U bioremediation in the groundwater ecosystem, and their structure and performance can be manipulated or optimized by adjusting geochemical and hydrological conditions. The ISME Journal (2010) 4, 1060-1070; doi:10.1038/ismej.2010.31; published online 18 March 2010
  191. Wu, C. H., B. Sercu, L. C. Van de Werfhorst, J. Wong, T. Z. DeSantis, E. L. Brodie, T. C. Hazen, P. A. Holden and G. L. Andersen. 2010. Characterization of Coastal Urban Watershed Bacterial Communities Leads to Alternative Community-Based Indicators. Plos One 5:11. abstract pdf
    Background: Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. Methodology/Principal Findings: Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and alpha-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC: A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip-analyzed studies. Conclusions/Significance: This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.
  192. Jose Malave-Orengo, Sharon E. Borglin, Terry C. Hazen and Carlos Rios-Velazquez. 2010. A modified cell extraction method to access microbial community structure in soil samples by phospholipid fatty acid analysis. Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology 1:1562-1568. abstract pdf
    The aim of this chapter is to present a modified cell extraction method that is effective in increasing biomass recovery, especially when biochemical techniques like Phospholipid Fatty Acid Analysis (PLFA) are use to determine biomarkers for soil community structure. Extremely low biomass is a limiting factor for obtaining accurate and representative prokaryotic and eukaryotic community structure and composition in soil matrices. In this study, a combination of an inorganic cell releasing buffer and surfactants along with a density gradient separation provides an effective method for cell elution from sediment over other approaches from low biomass soil samples by avoiding traditional problems with direct biochemical extraction methods, e.g. ionic adsorption, pH, and humic acid interferences. Loam soil (106 cells/g of soil) and experimentally determined low biomass (102 cells/g of soil) samples were treated with modified cell extraction method to compare numbers of signature phospholipids biomarkers, their response (concentration), and microbial community structure in each tested soil. A higher biomass yield, measured in pico moles (pm) total lipid, and higher microbial diversity were obtained in both high and low biomass soils when the enhanced extraction method was used. Bacteria were a dominant community component in both high and low biomass soil samples, but several important groups such as methane oxidizers, sulfate reducers, fungi and iron reducers, were only detected with the new method. Genomic DNA extractions were also successfully extracted from the PLFA aqueous phase, showing high molecular weight recovery suitable for further DNA finger-printing techniques.
  193. Hemme, C. L., Y. Deng, T. J. Gentry, M. W. Fields, L. Y. Wu, S. Barua, K. Barry, S. G. Tringe, D. B. Watson, Z. L. He, T. C. Hazen, J. M. Tiedje, E. M. Rubin and J. Z. Zhou. 2010. Metagenomic insights into evolution of a heavy metal-contaminated groundwater microbial community. Isme Journal 4:660-672. abstract pdf
    Understanding adaptation of biological communities to environmental change is a central issue in ecology and evolution. Metagenomic analysis of a stressed groundwater microbial community reveals that prolonged exposure to high concentrations of heavy metals, nitric acid and organic solvents (similar to 50 years) has resulted in a massive decrease in species and allelic diversity as well as a significant loss of metabolic diversity. Although the surviving microbial community possesses all metabolic pathways necessary for survival and growth in such an extreme environment, its structure is very simple, primarily composed of clonal denitrifying gamma-and beta-proteobacterial populations. The resulting community is overabundant in key genes conferring resistance to specific stresses including nitrate, heavy metals and acetone. Evolutionary analysis indicates that lateral gene transfer could have a key function in rapid response and adaptation to environmental contamination. The results presented in this study have important implications in understanding, assessing and predicting the impacts of human-induced activities on microbial communities ranging from human health to agriculture to environmental management, and their responses to environmental changes. The ISME Journal (2010) 4, 660-672; doi:10.1038/ismej.2009.154; published online 25 February 2010
  194. He, Z. L., A. Zhou, E. Baidoo, Q. He, M. P. Joachimiak, P. Benke, R. Phan, A. Mukhopadhyay, C. L. Hemme, K. Huang, E. J. Alm, M. W. Fields, J. Wall, D. Stahl, T. C. Hazen, J. D. Keasling, A. P. Arkin and J. Zhou. 2010. Global Transcriptional, Physiological, and Metabolite Analyses of the Responses of Desulfovibrio vulgaris Hildenborough to Salt Adaptation. Applied and Environmental Microbiology 76:1574-1586. abstract pdf
    The response of Desulfovibrio vulgaris Hildenborough to salt adaptation (long-term NaCl exposure) was examined by performing physiological, global transcriptional, and metabolite analyses. Salt adaptation was reflected by increased expression of genes involved in amino acid biosynthesis and transport, electron transfer, hydrogen oxidation, and general stress responses (e.g., heat shock proteins, phage shock proteins, and oxidative stress response proteins). The expression of genes involved in carbon metabolism, cell growth, and phage structures was decreased. Transcriptome profiles of D. vulgaris responses to salt adaptation were compared with transcriptome profiles of D. vulgaris responses to salt shock (short-term NaCl exposure). Metabolite assays showed that glutamate and alanine accumulated under salt adaptation conditions, suggesting that these amino acids may be used as osmoprotectants in D. vulgaris. Addition of amino acids (glutamate, alanine, and tryptophan) or yeast extract to the growth medium relieved salt-related growth inhibition. A conceptual model that links the observed results to currently available knowledge is proposed to increase our understanding of the mechanisms of D. vulgaris adaptation to elevated NaCl levels.
  195. He, Z. L., Y. Deng, J. D. Van Nostrand, Q. C. Tu, M. Y. Xu, C. L. Hemme, X. Y. Li, L. Y. Wu, T. J. Gentry, Y. F. Yin, J. Liebich, T. C. Hazen and J. Z. Zhou. 2010. GeoChip 3.0 as a high-throughput tool for analyzing microbial community composition, structure and functional activity. Isme Journal 4:1167-1179. abstract pdf
    A new generation of functional gene arrays (FGAs; GeoChip 3.0) has been developed, with similar to 28 000 probes covering approximately 57 000 gene variants from 292 functional gene families involved in carbon, nitrogen, phosphorus and sulfur cycles, energy metabolism, antibiotic resistance, metal resistance and organic contaminant degradation. GeoChip 3.0 also has several other distinct features, such as a common oligo reference standard (CORS) for data normalization and comparison, a software package for data management and future updating and the gyrB gene for phylogenetic analysis. Computational evaluation of probe specificity indicated that all designed probes would have a high specificity to their corresponding targets. Experimental analysis with synthesized oligonucleotides and genomic DNAs showed that only 0.0036-0.025% false-positive rates were observed, suggesting that the designed probes are highly specific under the experimental conditions examined. In addition, GeoChip 3.0 was applied to analyze soil microbial communities in a multifactor grassland ecosystem in Minnesota, USA, which showed that the structure, composition and potential activity of soil microbial communities significantly changed with the plant species diversity. As expected, GeoChip 3.0 is a high-throughput powerful tool for studying microbial community functional structure, and linking microbial communities to ecosystem processes and functioning. The ISME Journal (2010) 4, 1167-1179; doi: 10.1038/ismej.2010.46; published online 29 April 2010
  196. He, Q. A., Z. L. He, D. C. Joyner, M. Joachimiak, M. N. Price, Z. K. Yang, H. C. B. Yen, C. L. Hemme, W. Q. Chen, M. W. Fields, D. A. Stahl, J. D. Keasling, M. Keller, A. P. Arkin, T. C. Hazen, J. D. Wall and J. Z. Zhou. 2010. Impact of elevated nitrate on sulfate-reducing bacteria: a comparative Study of Desulfovibrio vulgaris. Isme Journal 4:1386-1397. abstract pdf
    Sulfate-reducing bacteria have been extensively studied for their potential in heavy-metal bioremediation. However, the occurrence of elevated nitrate in contaminated environments has been shown to inhibit sulfate reduction activity. Although the inhibition has been suggested to result from the competition with nitrate-reducing bacteria, the possibility of direct inhibition of sulfate reducers by elevated nitrate needs to be explored. Using Desulfovibrio vulgaris as a model sulfate-reducing bacterium, functional genomics analysis reveals that osmotic stress contributed to growth inhibition by nitrate as shown by the upregulation of the glycine/betaine transporter genes and the relief of nitrate inhibition by osmoprotectants. The observation that significant growth inhibition was effected by 70mM NaNO3 but not by 70mM NaCl suggests the presence of inhibitory mechanisms in addition to osmotic stress. The differential expression of genes characteristic of nitrite stress responses, such as the hybrid cluster protein gene, under nitrate stress condition further indicates that nitrate stress response by D. vulgaris was linked to components of both osmotic and nitrite stress responses. The involvement of the oxidative stress response pathway, however, might be the result of a more general stress response. Given the low similarities between the response profiles to nitrate and other stresses, less-defined stress response pathways could also be important in nitrate stress, which might involve the shift in energy metabolism. The involvement of nitrite stress response upon exposure to nitrate may provide detoxification mechanisms for nitrite, which is inhibitory to sulfate-reducing bacteria, produced by microbial nitrate reduction as a metabolic intermediate and may enhance the survival of sulfate-reducing bacteria in environments with elevated nitrate level. The ISME Journal (2010) 4, 1386-1397; doi: 10.1038/ismej.2010.59; published online 6 May 2010
  197. Hazen, T. C., E. A. Dubinsky, T. Z. DeSantis, G. L. Andersen, Y. M. Piceno, N. Singh, J. K. Jansson, A. Probst, S. E. Borglin, J. L. Fortney, W. T. Stringfellow, M. Bill, M. E. Conrad, L. M. Tom, K. L. Chavarria, T. R. Alusi, R. Lamendella, D. C. Joyner, C. Spier, J. Baelum, M. Auer, M. L. Zemla, R. Chakraborty, E. L. Sonnenthal, P. D'Haeseleer, H. Y. N. Holman, S. Osman, Z. M. Lu, J. D. Van Nostrand, Y. Deng, J. Z. Zhou and O. U. Mason. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. Science 330:204-208. abstract pdf
    The biological effects and expected fate of the vast amount of oil in the Gulf of Mexico from the Deepwater Horizon blowout are unknown owing to the depth and magnitude of this event. Here, we report that the dispersed hydrocarbon plume stimulated deep-sea indigenous gamma-Proteobacteria that are closely related to known petroleum degraders. Hydrocarbon-degrading genes coincided with the concentration of various oil contaminants. Changes in hydrocarbon composition with distance from the source and incubation experiments with environmental isolates demonstrated faster-than-expected hydrocarbon biodegradation rates at 5 degrees C. Based on these results, the potential exists for intrinsic bioremediation of the oil plume in the deep-water column without substantial oxygen drawdown.
  198. Hazen, Terry C.. 2010. Cometabolic Bioremediation. Handbook of Hydrocarbon Microbiology: Microbial interactions with hydrocarbons, oils, fats and related hydrophobic substrates and products pdf
  199. Hazen, Terry C.. 2010. In Situ Groundwater Bioremediation. Handbook of Hydrocarbon Microbiology: Microbial interactions with hydrocarbons, oils, fats and related hydrophobic substrates and products pdf
  200. Hazen, Terry C.. 2010. Biostimulation. Handbook of Hydrocarbon Microbiology: Microbial interactions with hydrocarbons, oils, fats and related hydrophobic substrates and products pdf
  201. Terry C. Hazen. 2010. Subsurface Biogeochemical Research (SBR) Contractor-Gantee Workshop Abstracts. Subsurface Biogeochemical Research (SBR) Contractor-Gantee Workshop Abstracts pdf
  202. Gorur, A., C. M. Leung, D. Jorgens, A. Tauscher, J. P. Remis, D. A. Ball, S. Chhabra, V. Fok, J. T. Geller, M. Singer, T. C. Hazen, T. Juba, D. Elias, J. Wall, M. Biggin, K. H. Downing, M. Auer. 2010. Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion. Microscopy & Microanalysis 16:864-865. abstract pdf
  203. DeAngelis, K. M., J. M. Gladden, M. Allgaier, P. D'Haeseleer, J. L. Fortney, A. Reddy, P. Hugenholtz, S. W. Singer, J. S. Vander Gheynst, W. L. Silver, B. A. Simmons and T. C. Hazen. 2010. Strategies for Enhancing the Effectiveness of Metagenomic-based Enzyme Discovery in Lignocellulolytic Microbial Communities. Bioenergy Research 3:146-158. abstract pdf
    Producing cellulosic biofuels from plant material has recenSSU rRNA gene amplicon pyrosequencing or phylogenetic microarray analysis revealed that the adapted communities were significantly simplified compared to the natural communities from which they were derived. Several members of the lignin-adapted and switchgrass-adapted consortia are related to organisms previously characterized as biomass degraders, while others were from less well-characterized phyla. The decrease in complexity of these communities make them good candidates for metagenomic sequencing and will likely enable the reconstruction of a greater number of full-length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to feedstocks and conditions of interest.tly emerged as a key US Department of Energy goal. For this technology to be commercially viable on a large scale, it is critical to make production cost efficient by streamlining both the deconstruction of lignocellulosic biomass and fuel production. Many natural ecosystems efficiently degrade lignocellulosic biomass and harbor enzymes that, when identified, could be used to increase the efficiency of commercial biomass deconstruction. However, ecosystems most likely to yield relevant enzymes, such as tropical rain forest soil in Puerto Rico, are often too complex for enzyme discovery using current metagenomic sequencing technologies. One potential strategy to overcome this problem is to selectively cultivate the microbial communities from these complex ecosystems on biomass under defined conditions, generating less complex biomass-degrading microbial populations. To test this premise, we cultivated microbes from Puerto Rican soil or green waste compost under precisely defined conditions in the presence dried ground switchgrass (Panicum virgatum L.) or lignin, respectively, as the sole carbon source. Phylogenetic profiling of the two feedstock-adapted communities using SSU rRNA gene amplicon pyrosequencing or phylogenetic microarray analysis revealed that the adapted communities were significantly simplified compared to the natural communities from which they were derived. Several members of the lignin-adapted and switchgrass-adapted consortia are related to organisms previously characterized as biomass degraders, while others were from less well-characterized phyla. The decrease in complexity of these communities make them good candidates for metagenomic sequencing and will likely enable the reconstruction of a greater number of full-length genes, leading to the discovery of novel lignocellulose-degrading enzymes adapted to feedstocks and conditions of interest.
  204. Byrne-Bailey, K. G., K. C. Wrighton, R. A. Melnyk, P. Agbo, T. C. Hazen and J. D. Coates. 2010. Complete Genome Sequence of the Electricity-Producing "Thermincola potens" Strain JR. Journal of Bacteriology 192:4078-4079. abstract pdf
    "Thermincola potens" strain JR is one of the first Gram-positive dissimilatory metal-reducing bacteria (DMRB) for which there is a complete genome sequence. Consistent with the physiology of this organism, preliminary annotation revealed an abundance of multiheme c-type cytochromes that are putatively associated with the periplasm and cell surface in a Gram-positive bacterium. Here we report the complete genome sequence of strain JR.
  205. 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 Community. Plos One 5:9. abstract pdf
    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, similar to 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 degrees C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.
  206. Walker, Christopher B., Sergey Stolyar, Dylan Chivian, Nicolas Pinel, Jeffrey A. Gabster, Paramvir S. Dehal, Zhili He, Zamin Koo Yang, Huei-Che B. Yen, Jizhong Zhou, Judy D. Wall, Terry C. Hazen, Adam P. Arkin and David A. Stahl. 2009. Contribution of mobile genetic elements to Desulfovibrio vulgaris genome plasticity. Environmental Microbiology 11:2244-2252. abstract pdf
    P>The genome of Desulfovibrio vulgaris strain DePue, a sulfate-reducing Deltaproteobacterium isolated from heavy metal-impacted lake sediment, was completely sequenced and compared with the type strain D. vulgaris Hildenborough. The two genomes share a high degree of relatedness and synteny, but harbour distinct prophage and signatures of past phage encounters. In addition to a highly variable phage contribution, the genome of strain DePue contains a cluster of open-reading frames not found in strain Hildenborough coding for the production and export of a capsule exopolysaccharide, possibly of relevance to heavy metal resistance. Comparative whole-genome microarray analysis on four additional D. vulgaris strains established greater interstrain variation within regions associated with phage insertion and exopolysaccharide biosynthesis.
  207. Walker, Christopher B., Zhili He, Zamin K. Yang, Joseph A., Jr. Ringbauer, Qiang He, Jizhong Zhou, Gerrit Voordouw, Judy D. Wall, Adam P. Arkin, Terry C. Hazen, Sergey Stolyar and David A. Stahl. 2009. The Electron Transfer System of Syntrophically Grown Desulfovibrio vulgaris. Journal of Bacteriology 191:5793-5801. abstract pdf
    Interspecies hydrogen transfer between organisms producing and consuming hydrogen promotes the decomposition of organic matter in most anoxic environments. Although syntrophic coupling between hydrogen producers and consumers is a major feature of the carbon cycle, mechanisms for energy recovery at the extremely low free energies of reactions typical of these anaerobic communities have not been established. In this study, comparative transcriptional analysis of a model sulfate-reducing microbe, Desulfovibrio vulgaris Hildenborough, suggested the use of alternative electron transfer systems dependent on growth modality. During syntrophic growth on lactate with a hydrogenotrophic methanogen, numerous genes involved in electron transfer and energy generation were upregulated in D. vulgaris compared with their expression in sulfate-limited monocultures. In particular, genes coding for the putative membrane-bound Coo hydrogenase, two periplasmic hydrogenases (Hyd and Hyn), and the well-characterized high-molecular-weight cytochrome (Hmc) were among the most highly expressed and upregulated genes. Additionally, a predicted operon containing genes involved in lactate transport and oxidation exhibited upregulation, further suggesting an alternative pathway for electrons derived from lactate oxidation during syntrophic growth. Mutations in a subset of genes coding for Coo, Hmc, Hyd, and Hyn impaired or severely limited syntrophic growth but had little effect on growth via sulfate respiration. These results demonstrate that syntrophic growth and sulfate respiration use largely independent energy generation pathways and imply that to understand microbial processes that sustain nutrient cycling, lifestyles not captured in pure culture must be considered.
  208. Waldron, Patricia J., Liyou Wu, Joy D. Van Nostrand, Chris W. Schadt, Zhili He, David B. Watson, Philip M. Jardine, Anthony V. Palumbo, Terry C. Hazen and Jizhong Zhou. 2009. Functional Gene Array-Based Analysis of Microbial Community Structure in Groundwaters with a Gradient of Contaminant Levels. Environmental Science & Technology 43:3529-3534. abstract pdf
    To understand how contaminants affect microbial community diversity, heterogeneity, and functional structure, six groundwater monitoring wells from the Field Research Center of the U.S. Department of Energy Environmental Remediation Science Program (ERSP; Oak Ridge, TN), with a wide range of pH, nitrate, and heavy metal contamination were investigated. DNA from the groundwater community was analyzed with a functional gene array containing 2006 probes to detect genes involved in metal resistance, sulfate reduction, organic contaminant degradation, and carbon and nitrogen cycling. Microbial diversity decreased in relation to the contamination levels of the wells. Highly contaminated wells had lower gene diversity but greater signal intensity than the pristine well. The microbial composition was heterogeneous, with 17-70% overlap between different wells. Metal-resistant and metal-reducing microorganisms were detected in both contaminated and pristine wells, suggesting the potential for successful bioremediation of metal-contaminated groundwaters. In addition, results of Mantel tests and canonical correspondence analysis indicate that nitrate, sulfate, pH, uranium, and technetium have a significant (p < 0.05) effect on microbial community structure. This study provides an overall picture of microbial community structure in contaminated environments with functional gene arrays by showing that diversity and heterogeneity can vary greatly in relation to contamination.
  209. Van Nostrand, Joy D., Wei-Min Wu, Liyou Wu, Ye Deng, Jack Carley, Sue Carroll, Zhili He, Baohua Gu, Jian Luo, Craig S. Criddle, David B. Watson, Philip M. Jardine, Terence L. Marsh, James M. Tiedje, Terry C. Hazen and Jizhong Zhou. 2009. GeoChip-based analysis of functional microbial communities during the reoxidation of a bioreduced uranium-contaminated aquifer. Environmental Microbiology 11:2611-2626. abstract pdf
    P>A pilot-scale system was established for in situ biostimulation of U(VI) reduction by ethanol addition at the US Department of Energy's (DOE's) Field Research Center (Oak Ridge, TN). After achieving U(VI) reduction, stability of the bioreduced U(IV) was evaluated under conditions of (i) resting (no ethanol injection), (ii) reoxidation by introducing dissolved oxygen (DO), and (iii) reinjection of ethanol. GeoChip, a functional gene array with probes for N, S and C cycling, metal resistance and contaminant degradation genes, was used for monitoring groundwater microbial communities. High diversity of all major functional groups was observed during all experimental phases. The microbial community was extremely responsive to ethanol, showing a substantial change in community structure with increased gene number and diversity after ethanol injections resumed. While gene numbers showed considerable variations, the relative abundance (i.e. percentage of each gene category) of most gene groups changed little. During the reoxidation period, U(VI) increased, suggesting reoxidation of reduced U(IV). However, when introduction of DO was stopped, U(VI) reduction resumed and returned to pre-reoxidation levels. These findings suggest that the community in this system can be stimulated and that the ability to reduce U(VI) can be maintained by the addition of electron donors. This biostimulation approach may potentially offer an effective means for the bioremediation of U(VI)-contaminated sites.
  210. Tang, Yinjie J., Rajat Sapra, Dominique Joyner, Terry C. Hazen, Samuel Myers, David Reichmuth, Harvey Blanch and Jay D. Keasling. 2009. Analysis of Metabolic Pathways and Fluxes in a Newly Discovered Thermophilic and Ethanol-Tolerant Geobacillus Strain. Biotechnology and Bioengineering 102:1377-1386. abstract pdf
    A recently discovered thermophilic bacterium, Geobacillus thermoglucosidasius M10EXG, ferments a range of C5 (e.g., xylose) and C6 sugars (e.g., glucose) and is tolerant to high ethanol concentrations (10%, v/v). We have investigated the central metabolism of this bacterium using both in vitro enzyme assays and C-13-based flux analysis to provide insights into the physiological properties of this extremophile and explore its metabolism for bio-ethanol or other bioprocess applications, Our findings show that glucose metabolism in G. thermoglucosidasius M10EXG proceeds via glycolysis, the pentose phosphate pathway, and the TCA cycle; the Entner-Doudoroff pathway and transhydrogenase activity were not detected. Anaplerotic reactions ( including the glyoxylate shunt, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase) were active, but fluxes through those pathways could not he accurately determined using amino acid labeling. When growth conditions were switched from aerobic to micro-aerobic conditions, fluxes (based on it normalized glucose uptake rate of 100 units (gDCW) (1)h (1)) through the TCA cycle and oxidative pentose phosphate pathway were reduced front 64 +/- 3 to 25 +/- 2 and from 30 +/- 2 to 19 +/- 2, respectively. The carbon flux under micro-aerobic growth was directed to ethanol, L-lactate (>99% optical purity), acetate, and formate. Under fully anerobic conditions, G. thermoglucosidasius M10EXG Used a mixed acid fermentation process and exhibited a maximum ethanol yield of 0.38 +/- 0.07 mol mol(-1) glucose. In silico flux balance modeling demonstrates that lactate and acetate production from G. thermoglucosidasius M10EXG, reduces the maximum ethanol yield by approximately threefold, thus indicating that both pathways should be modified to maximize ethanol production.
  211. Holman, Hoi-Ying N., Eleanor Wozei, Zhang Lin, Luis R. Comolli, David A. Ball, Sharon Borglin, Matthew W. Fields, Terry C. Hazen and Kenneth H. Downing. 2009. Real-time molecular monitoring of chemical environment in obligate anaerobes during oxygen adaptive response. Proceedings of the National Academy of Sciences of the United States of America 106:12599-12604. abstract pdf
    Determining the transient chemical properties of the intracellular environment can elucidate the paths through which a biological system adapts to changes in its environment, for example, the mechanisms that enable some obligate anaerobic bacteria to survive a sudden exposure to oxygen. Here we used high-resolution Fourier transform infrared (FTIR) spectromicroscopy to continuously follow cellular chemistry within living obligate anaerobes by monitoring hydrogen bond structures in their cellular water. We observed a sequence of well orchestrated molecular events that correspond to changes in cellular processes in those cells that survive, but only accumulation of radicals in those that do not. We thereby can interpret the adaptive response in terms of transient intracellular chemistry and link it to oxygen stress and survival. This ability to monitor chemical changes at the molecular level can yield important insights into a wide range of adaptive responses.
  212. Hazen, Terry C., Romy Chakraborty, James M. Fleming, Ingrid R. Gregory, John P. Bowman, Luis Jimenez, Dai Zhang, Susan M. Pfiffner, Fred J. Brockman and Gary S. Sayler. 2009. Use of gene probes to assess the impact and effectiveness of aerobic in situ bioremediation of TCE. Archives of Microbiology 191:221-232. abstract pdf
    Gene probe hybridization was used to determine distribution and expression of co-metabolic genes at a contaminated site as it underwent in situ methanotrophic bioremediation of trichloroethylene (TCE). The bioremediation strategies tested included a series of air, air:methane, and air:methane:nutrient pulses of the test plot using horizontal injection wells. During the test period, the levels of TCE reduced drastically in almost all test samples. Sediment core samples (n = 367) taken from 0 m (surface)-43 m depth were probed for gene coding for methanotrophic soluble methane monooxygenase (sMMO) and heterotrophic toluene dioxygenase (TOD), which are known to co-metabolize TCE. The same sediment samples were also probed for genes coding for methanol dehydrogenase (MDH) (catalyzing the oxidation of methanol to formaldehyde) to assess specifically changes in methylotrophic bacterial populations in the site. Gene hybridization results showed that the frequency of detection of sMMO genes were stimulated approximately 250% following 1% methane:air (v/v) injection. Subsequent injection of 4% methane:air (v/v) resulted in an 85% decline probably due to nutrient limitations, since addition of nutrients (gaseous nitrogen and phosphorus) thereafter caused an increase in the frequency of detection of sMMO genes. Detection of TOD genes declined during the process, and eventually they were non-detectable by the final treatment, suggesting that methanotrophs displaced the TOD gene containing heterotrophs. Active transcription of sMMO and TOD was evidenced by hybridization to mRNA. These analyses combined with results showing the concomitant decline in TCE concentrations, increases in chloride concentration and increases in methanotroph viable counts, provide multiple lines of evidence that TCE remediation was caused specifically by methanotrophs. Our results suggest that sMMO genes are responsible for most, if not all, of the observed biodegradation of TCE. This study demonstrates that the use of nucleic acid analytical methods provided a gene specific assessment of the effects of in situ treatment technologies.
  213. Terry C. Hazen. 2009. 4th Annual DOE-ERSP PI Meeting: Abstracts. 4th Annual DOE-ERSP PI Meeting: Abstracts pdf
  214. Han, Bong-Gyoon, Ming Dong, Haichuan Liu, Lauren Camp, Jil Geller, Mary Singer, Terry C. Hazen, Megan Choi, H. Ewa Witkowska, David A. Ball, Dieter Typke, Kenneth H. Downing, Maxim Shatsky, Steven E. Brenner, John-Marc Chandonia, Mark D. Biggin and Robert M. Glaeser. 2009. Survey of large protein complexes in Desulfovibrio vulgaris reveals great structural diversity. Proceedings of the National Academy of Sciences of the United States of America 106:16580-16585. abstract pdf
    An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr approximate to 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate approximate to 10 times greater than that of previous "proteomic'' screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.
  215. Elias, Dwayne A., Aindrila Mukhopadhyay, Marcin P. Joachimiak, Elliott C. Drury, Alyssa M. Redding, Huei-Che B. Yen, Matthew W. Fields, Terry C. Hazen, Adam P. Arkin, Jay D. Keasling and Judy D. Wall. 2009. Expression profiling of hypothetical genes in Desulfovibrio vulgaris leads to improved functional annotation. Nucleic Acids Research 37:2926-2939. abstract pdf
    Hypothetical (HyP) and conserved HyP genes account for >30% of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 347 of the 3634 genes were annotated as conserved HyP (9.5%) along with 887 HyP genes (24.4%). Given the large fraction of the genome, it is plausible that some of these genes serve critical cellular roles. The study goals were to determine which genes were expressed and provide a more functionally based annotation. To accomplish this, expression profiles of 1234 HyP and conserved genes were used from transcriptomic datasets of 11 environmental stresses, complemented with shotgun LC-MS/MS and AMT tag proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. One thousand two hundred and twelve of these genes were transcribed with 786 producing detectable proteins. There was no evidence for expression of 17 predicted genes. Except for the latter, monocistronic gene annotation was expanded using the above criteria along with matching Clusters of Orthologous Groups. Polycistronic genes were annotated in the same manner with inferences from their proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the relevance of the inferred functional annotations.
  216. Borglin, Sharon, Dominique Joyner, Janet Jacobsen, Aindrila Mukhopadhyay and Terry C. Hazen. 2009. Overcoming the anaerobic hurdle in phenotypic microarrays: Generation and visualization of growth curve data for Desulfovibrio vulgaris Hildenborough. Journal of Microbiological Methods 76:159-168. abstract pdf
    Growing anaerobic microorganisms in phenotypic microarrays (PM) and 96-well microtiter plates is an emerging technology that allows high throughput survey of the growth and physiology and/or phenotype of cultivable microorganisms. For non-model bacteria, a method for phenotypic analysis is invaluable, not only to serve as a starting point for further evaluation, but also to provide a broad understanding of the physiology of an uncharacterized wild-type organism or the physiology/phenotype of a newly created mutant of that organism. Given recent advances in genetic characterization and targeted mutations to elucidate genetic networks and metabolic pathways, high-throughput methods for determining phenotypic differences are essential. Here we outline challenges presented in studying the physiology and phenotype of a sulfate-reducing anaerobic delta proteobacterium. Desulfovibrio vulgaris Hildenborough. Modifications of the commercially available OmniLog (TM) system (Hayward, CA) for experimental setup, and configuration, as well as considerations in PM data analysis are presented. Also highlighted here is data viewing software that enables users to view and compare multiple PM data sets. The PM method promises to be a valuable strategy in our systems biology approach to D. vulgaris studies and is readily applicable to other anaerobic and aerobic bacteria. Published by Elsevier B.V.
  217. Wu, Liyou, Xueduan Liu, Matthew W. Fields, Dorothea K. Thompson, Christopher E. Bagwell, James M. Tiedje, Terry C. Hazen and Jizhong Zhou. 2008. Microarray-based whole-genome hybridization as a tool for determining procaryotic species relatedness. ISME Journal 2:642-655. abstract pdf
    The definition and delineation of microbial species are of great importance and challenge due to the extent of evolution and diversity. Whole-genome DNA-DNA hybridization is the cornerstone for defining procaryotic species relatedness, but obtaining pairwise DNA-DNA reassociation values for a comprehensive phylogenetic analysis of procaryotes is tedious and time consuming. A previously described microarray format containing whole-genomic DNA (the community genome array or CGA) was rigorously evaluated as a high-throughput alternative to the traditional DNA-DNA reassociation approach for delineating procaryotic species relationships. DNA similarities for multiple bacterial strains obtained with the CGA-based hybridization were comparable to those obtained with various traditional whole-genome hybridization methods (r = 0.87, P<0.01). Significant linear relationships were also observed between the CGA-based genome similarities and those derived from small subunit (SSU) rRNA gene sequences (r = 0.79, P<0.0001), gyrB sequences (r = 0.95, P<0.0001) or REP- and BOX-PCR fingerprinting profiles (r = 0.82, P<0.0001). The CGA hybridization-revealed species relationships in several representative genera, including Pseudomonas, Azoarcus and Shewanella, were largely congruent with previous classifications based on various conventional whole-genome DNA-DNA reassociation, SSU rRNA and/or gyrB analyses. These results suggest that CGA-based DNA-DNA hybridization could serve as a powerful, high-throughput format for determining species relatedness among microorganisms.
  218. Wan, Jiamin, Tetsu K. Tokunaga, Yongman Kim, Eoin Brodie, Rebecca Daly, Terry C. Hazen and Mary K. Firestone. 2008. Effects of Organic Carbon Supply Rates on Uranium Mobility in a Previously Bioreduced Contaminated Sediment. Environmental Science & Technology 42:7573-7579. abstract pdf
    Bioreduction-based strategies for remediating uranium (U)-contaminated sediments face the challenge of maintaining the reduced status of U for long times. Because groundwater influxes continuously bring in oxidizing terminal electron acceptors (O-2, NO3-), it is necessary to continue supplying organic carbon (OC) to maintain the reducing environment after U bioreduction is achieved. We tested the influence of OC supply rates on mobility of previously microbial reduced uranium U(IV) in contaminated sediments. We found that high degrees of U mobilization occurred when OC supply rates were high, and when the sediment still contained abundant Fe(Ill). Although 900 days with low levels of OC supply minimized U mobilization, the sediment redox potential increased with time as did extractable U(VI) fractions. Molecular analyses of total microbial activity demonstrated a positive correlation with OC supply and analyses of Geobacteraceae activity (RT-qPCR of 16S rRNA) indicated continued activity even when the effluent Fe(II) became undetectable. These data support our hypothesis on the mechanisms responsible for remobilization of U under reducing conditions; that microbial respiration caused increased (bi)carbonate concentration and formation of stable uranyl carbonate complexes, thereby shifted U(IV)/U(VI) equilibrium to more reducing potentials. The data also suggested that low OC concentrations could not sustain the reducing condition of the sediment for much longer time. Bioreduced U(IV) is not sustainable in an oxidizing environment for a very long time.
  219. Tokunaga, Tetsu K., Jiamin Wan, Yongman Kim, Rebecca A. Daly, Eoin L. Brodie, Terry C. Hazen, Don Herman and Mary K. Firestone. 2008. Influences of Organic Carbon Supply Rate on Uranium Bioreduction in Initially Oxidizing, Contaminated Sediment. Environmental Science & Technology 42:8901-8907. abstract pdf
    Remediation of uranium-contaminated sediments through in situ stimulation of bioreduction to insoluble UO2 is a potential treatment strategy under active investigation. Previously, we found that newly reduced U(IV) can be reoxidized under reducing conditions sustained by a continuous supply of organic carbon (OC) because of residual reactive Fe(III) and enhanced U(VI) solubility through complexation with carbonate generated through OC oxidation. That finding motivated this investigation directed at identifying a range of OC supply rates that is optimal for establishing U bioreduction and immobilization in initially oxidizing sediments. The effects of OC supply rate, from 0 to 580 mmol of OC (kg of sediment)(-1) year(-1), and OC form (lactate and acetate) on U bioreduction were tested in flow-through columns containing U-contaminated sediments. An intermediate supply rate on the order of 150 mmol of OC (kg of sediment)(-1) year(-1) was determined to be most effective at immobilizing U. At lower OC supply rates, U bioreduction was not achieved, and U(VI) solubility was enhanced by complexation with carbonate (from OC oxidation). At the highest OC supply rate, the resulting highly carbonate-enriched solutions also supported elevated levels of U(VI), even though strongly reducing conditions were established. Lactate and acetate were found to have very similar geochemical impacts on effluent U concentrations (and other measured chemical species), when compared at equivalent OC supply rates. While the catalysts of U(VI) reduction to U(IV) are presumably bacteria, the composition of the bacterial community, the Fe-reducing community, and the sulfate-reducing community had no direct relationship with effluent U concentrations. The OC supply rate has competing effects of driving reduction of U(VI) to low-solubility U(IV) solids, as well as causing formation of highly soluble U(VI)-carbonato complexes. These offsetting influences will require careful control of OC supply rates in order to optimize bioreduction-based U stabilization.
  220. Hubbard, Susan S., Ken Williams, Mark E. Conrad, Boris Faybishenko, John Peterson, Jinsong Chen, Phil Long and Terry Hazen. 2008. Geophysical monitoring of hydrological and biogeochemical transformations associated with Cr(VI) bioremediation. Environmental Science & Technology 42:3757-3765. abstract pdf
    Understanding how hydrological and biogeochemical properties change over space and time in response to remedial treatments is hindered by our ability to monitor these processes with sufficient resolution and over field relevant scales. Here, we explored the use of geophysical approaches for monitoring the spatiotemporal distribution of hydrological and biogeochemical transformations associated with a Cr(VI) bioremediation experiment performed at Hanford, WA. We first integrated hydrological wellbore and geophysical tomographic data sets to estimate hydrological zonation at the study site. Using results from laboratory biogeophysical experiments and constraints provided by field geochemical data sets, we then interpreted time-lapse seismic and radar tomographic data sets, collected during thirteen acquisition campaigns over a three year experimental period, in terms of hydrological and biogeochemical transformations. The geophysical monitoring data sets were used to infer: the spatial distribution of injected electron donor; the evolution of gas bubbles; variations in total dissolved solids (nitrate and sulfate) as a function of pumping activity; the formation of precipitates and dissolution of calcites; and concomitant changes in porosity. Although qualitative in nature, the integrated interpretation illustrates how geophysical techniques have the potential to provide a wealth of information about coupled hydrobiogeochemical responses to remedial treatments in high spatial resolution and in a minimally invasive manner. Particularly novel aspects of our study include the use of multiple lines of evidence to constrain the interpretation of a long-term, field-scale geophysical monitoring data set and the interpretation of the transformations as a function of hydrological heterogeneity and pumping activity.
  221. Terry C. Hazen, Boris Faybishenko and Preston Jordan. 2008. Complexity of Groundwater Contaminants at DOE Sites. pdf
  222. Faybishenko, Boris, Terry C. Hazen, Philip E. Long, Eoin L. Brodie, Mark E. Conrad, Susan S. Hubbard, John N. Christensen, Dominique Joyner, Sharon E. Borglin, Romy Chakraborty, Kenneth H. Williams, John E. Peterson, Jinsong Chen, Shaun T. Brown, Tetsu K. Tokunaga, Jiamin Wan, Mary Firestone, Darrell R. Newcomer, Charles T. Resch, Kirk J. Cantrell, Anna Willett and Stephen Koenigsberg. 2008. In Situ Long-Term Reductive Bioimmobilization of Cr(VI) in Groundwater Using Hydrogen Release Compound. Environmental Science & Technology 42:8478-8485. abstract pdf
    The results of a field experiment designed to test the effectiveness of a novel approach for long-term, in situ bioimmobilization of toxic and soluble Cr(VI) in groundwater using a hydrogen release compound (HRC)-a slow release glycerol polylactate-are described. The field experiment was conducted at the Hanford Site (Washington), a U.S. Department of Energy nuclear production facility, using a combination of hydrogeological, geophysical, geochemical, and microbiological measurements and analyses of water samples and sediments. The results of this experiment show that a single HRC injection into groundwater stimulates an increase in biomass, a depletion of terminal electron acceptors O-2, NO3-, and So(4)(2-,) and an increase in Fe2+, resulting in a significant decrease in soluble Cr(VI). The Cr(VI) concentration has remained belowthe background concentration in the downgradient pumping/ monitoring well, and below the detection limit in the injection well for more than 3 years after the HRC injection. The degree of sustainability of Cr(VI) reductive biclimmobilization under different redox conditions at this and other contaminated sites is currently under study.
  223. Chivian, Dylan, Eoin L. Brodie, Eric J. Alm, David E. Culley, Paramvir S. Dehal, Todd Z. DeSantis, Thomas M. Gihring, Alla Lapidus, Li-Hung Lin, Stephen R. Lowry, Duane P. Moser, Paul M. Richardson, Gordon Southam, Greg Wanger, Lisa M. Pratt, Gary L. Andersen, Terry C. Hazen, Fred J. Brockman, Adam P. Arkin and Tullis C. Onstott. 2008. Environmental genomics reveals a single-species ecosystem deep within earth. Science 322:275-278. abstract pdf
    DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, composes > 99.9% of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style well suited to long-term isolation from the photosphere deep within Earth's crust and offers an example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.
  224. Tang, Yinjie J., Romy Chakraborty, Hector Garcia Martin, Jeannie Chu, Terry C. Hazen and Jay D. Keasling. 2007. Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-nitrilotriacetic acid. Applied and Environmental Microbiology 73:3859-3864. abstract pdf
    We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13c isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO, via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.
  225. Tang, Yinjie, Francesco Pingitore, Aindrila Mukhopadhyay, Richard Phan, Terry C. Hazen and Jay D. Keasling. 2007. Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using gas chromatography-mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry. Journal of Bacteriology 189:940-949. abstract pdf
    Flux distribution in central metabolic pathways of Desuffiovibrio vulgaris Hildenborough was examined using C-13 tracer experiments. Consistent with the current genome annotation and independent evidence from enzyme activity assays, the isotopomer results from both gas chromatography-mass spectrometry (GC-MS) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) indicate the lack of an oxidatively functional tricarboxylic acid (TCA) cycle and an incomplete pentose phosphate pathway. Results from this study suggest that fluxes through both pathways are limited to biosynthesis. The data also indicate that > 80% of the lactate was converted to acetate and that the reactions involved are the primary route of energy production [NAD(P)H and ATP production]. Independently of the TCA cycle, direct cleavage of acetyl coenzyme A to CO and 5,10-methyl tetrahydrofuran also leads to production of NADH and ATP. Although the genome annotation implicates a ferredoxin-dependent oxoglutarate synthase, isotopic evidence does not support flux through this reaction in either the oxidative or the reductive mode; therefore, the TCA cycle is incomplete. FT-ICR MS was used to locate the labeled carbon distribution in aspartate and glutamate and confirmed the presence of an atypical enzyme for citrate formation suggested in previous reports [the citrate synthesized by this enzyme is the isotopic antipode of the citrate synthesized by the (S)-citrate synthase]. These findings enable a better understanding of the relation between genome annotation and actual metabolic pathways in D. vulgaris and also demonstrate that FT-ICR MS is a powerful tool for isotopomer analysis, overcoming the problems with both GC-MS and nuclear magnetic resonance spectroscopy.
  226. Stolyar, Sergey, Qiang He, Marcin P. Joachimiak, Zhili He, Zamin Koo Yang, Sharon E. Borglin, Dominique C. Joyner, Katherine Huang, Eric Alm, Terry C. Hazen, Jizhong Zhou, Judy D. Wall, Adam P. Arkin and David A. Stahl. 2007. Response of Desulfovibrio vulgaris to alkaline stress. Journal of Bacteriology 189:8944-8952. abstract pdf
    The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580).
  227. Mukhopadhyay, Aindrila, Alyssa M. Redding, Marcin P. Joachimiak, Adam P. Arkin, Sharon E. Borglin, Paramvir S. Dehal, Romy Chakraborty, Jil T. Geller, Terry C. Hazen, Qiang He, Dominique C. Joyner, Vincent J. J. Martin, Judy D. Wall, Zamin Koo Yang, Jizhong Zhou and Jay D. Keasling. 2007. Cell-wide responses to low-oxygen exposure in Desulfovibrio vulgaris Hildenborough. Journal of Bacteriology 189:5996-6010. abstract pdf
    The responses of the anaerobic, sulfate-reducing organism Desulfovibrio vulgaris Hildenborough to low-oxygen exposure (0.1% O-2) were monitored via transcriptomics and proteomics. Exposure to 0.1% O-2 caused a decrease in the growth rate without affecting viability. Concerted upregulation of the predicted peroxide stress response regulon (PerR) genes was observed in response to the 0.1% O-2 exposure. Several of the candidates also showed increases in protein abundance. Among the remaining small number of transcript changes was the upregulation of the predicted transmembrane tetraheme cytochrome c(3) complex. Other known oxidative stress response candidates remained unchanged during the low-O-2 exposure. To fully understand the results of the 0.1% O-2 exposure, transcriptomics and proteomics data were collected for exposure to air using a similar experimental protocol. In contrast to the 0.1% O-2 exposure, air exposure was detrimental to both the growth rate and viability and caused dramatic changes at both the transcriptome and proteome levels. Interestingly, the transcripts of the predicted PerR regulon genes were downregulated during air exposure. Our results highlight the differences in the cell-wide responses to low and high O-2 levels in D. vulgaris and suggest that while exposure to air is highly detrimental to D. vulgaris, this bacterium can successfully cope with periodic exposure to low O-2 levels in its environment.
  228. MacLean, L. C. W., T. J. Pray, T. C. Onstott, E. L. Brodie, T. C. Hazen and G. Southam. 2007. Mineralogical, chemical and biological characterization of an anaerobic biofilm collected from a borehole in a deep gold mine in South Africa. Geomicrobiology Journal 24:491-504. abstract pdf
    A biofilm sample was collected from an anaerobic water and gas-flowing borehole, 1.474 km below land surface in the Evander Au mine, Republic of South Africa. The biofilm was 27 wt% ZnS, which was similar to 2 x 10(7) times more concentrated than the dissolved Zn measured in the borehole water. X-Ray diffraction indicated that the Zn was present in the form of fine grained, 4.7 +/- 0.9 nm particles with smaller amounts of pyrite (FeS2). Scanning electron microscopy, coupled with energy-dispersive X-ray spectroscopy confirmed the identity of these minerals in the biofilm. Using transmission electron microscopy, the fine-grained ZnS minerals were found to coat the 1 mu m-diameter rod-shaped bacteria that made up the primary sub-structure of the biofilm. The FeS2 was present as framboids (spherical aggregates of 0.5-1 mu m FeS2 crystals) up to 10 mu m in diameter and as large, 2-3 mu m euhedral crystals that were not nucleated on the bacterial surfaces, but were found within the biofilm. Analyses of 16S rDNA utilizing clone libraries and a phylochip indicates that the ZnS rich biofilm is dominated by methanogens with a significant sulfate-reducing bacterial population and minor sulfide and CH4-oxidizing chemolithotrophs. This biofilm community is sustained by sulfate, bicarbonate and H-2-bearing paleometeoric water.
  229. Jacobsen, J. S., D. C. Joyner, S. E. Borglin, T. C. Hazen, A. P. Arkin and E. W. Bethel. 2007. Visualization of growth curve data from phenotype microarray experiments. 11th International Conference Information Visualization 535-544. abstract pdf
    Phenotype microarrays provide a technology to simultaneously survey the response of an organism to nearly 2,000 substrates, including carbon, nitrogen and potassium sources; varying pH; varying salt concentrations; and antibiotics. In order to more quickly and easily view and compare the large number of growth curves produced by phenotype microarray experiments, we have developed software to produce and display color images, each of which corresponds to a set of 96 growth curves. Using color images to represent growth curves data has proven to be a valuable way to assess experiment quality, compare replicates, facilitate comparison of the responses of different organisms, and identify significant phenotypes. The color images are linked to traditional plots of growth versus time, as well as to information about the experiment, organism, and substrate. In order to share and view information and data project-wide, all information, plots, and data are accessible using only a Web browser.
  230. Hazen, Terry C.. 2007. 2nd Annual DOE-ERSP PI Meeting:Abstracts. 2nd Annual DOE-ERSP Meeting pdf
  231. Garczarek, Florian, Ming Dong, Dieter Typke, H. Ewa Witkowska, Terry C. Hazen, Eva Nogales, Mark D. Biggin and Robert M. Glaeser. 2007. Octomeric pyruvate-ferredoxin oxidoreductase from Desulfovibrio vulgaris. Journal of Structural Biology 159:9-18. abstract pdf
    Pyruvate-ferredoxin oxidoreductatse (PFOR) carries out the central step in oxidative decarboxylation of pyruvate to acetyl-CoA. We have purified this enzyme from Desulfovibrio vulgaris Hildenborough (DvH) as part of a systematic characterization of as many multiprotein complexes as possible for this organism, and the three-dimensional structure of this enzyme has been determined by a combination of electron microscopy (EM), single particle image analysis, homology modeling and computational molecular docking. Our results show that the 1 MDa DvH PFOR complex is a homo-octomer, or more precisely, a tetramer of the dimeric form of the related enzyme found in DesuUbvibrio africanus (Da), with which it shares a sequence identity of 69%. Our homology model of the DVH PFOR dimer is based on the Da PFOR X-ray structure. Docking of this model into our 17 angstrom resolution EM-reconstruction of negatively stained DvH PFOR octomers strongly suggests that the difference in oligomerization state for the two species is due to the insertion of a single valine residue (Val383) within a surface loop of the DvH enzyme. This study demonstrates that the strategy of intermediate resolution EM reconstruction coupled to homology modeling and docking can be powerful enough to infer the functionality of single amino acid residues. (c) 2007 Elsevier Inc. All rights reserved.
  232. Bender, Kelly S., Huei-Che Bill Yen, Christopher L. Hemme, Zamin Yang, Zhili He, Qiang He, Jizhong Zhou, Katherine H. Huang, Eric J. Alm, Terry C. Hazen, Adam P. Arkin and Judy D. Wall. 2007. Analysis of a ferric uptake regulator (Fur) mutant of Desulfovibfio vulgaris Hildenborough. Applied and Environmental Microbiology 73:5389-5400. abstract pdf
    Previous experiments examining the transcriptional profile of the anaerobe Desulfovibrio vulgaris demonstrated up-regulation of the Fur regulon in response to various environmental stressors. To test the involvement of Fur in the growth response and transcriptional regulation of D. vulgaris, a targeted mutagenesis procedure was used for deleting the fur gene. Growth of the resulting Delta fur mutant (JW707) was not affected by iron availability, but the mutant did exhibit increased sensitivity to nitrite and osmotic stresses compared to the mild type. Transcriptional profiling of JW707 indicated that iron-bound Fur acts as a traditional repressor for ferrous iron uptake genes (feoAB) and other genes containing a predicted Fur binding site within their promoter. Despite the apparent lack of siderophore biosynthesis genes within the D. vulgaris genome, a large 12-gene operon encoding orthologs to TonB and TolQR also appeared to be repressed by iron-bound Fur. While other genes predicted to be involved in iron homeostasis were unaffected by the presence or absence of Fur, alternative expression patterns that could be interpreted as repression or activation by iron-free Fur were observed. Both the physiological and transcriptional data implicate a global regulatory role for Fur in the sulfate-reducing bacterium D. vulgaris.
  233. Redding, A. M., A. Mukhopadhyay, D. C. Joyner, Terry C. Hazen and J. D. Keasling. 2006. Study of nitrate stress in Desulfovibrio vulgaris Hildenborough using iTRAQ proteomics. Brief Funct Genomic Proteomic 5:133-43. abstract pdf
    The response of Desulfovibrio vulgaris Hildenborough (DvH), a sulphate-reducing bacterium, to nitrate stress was examined using quantitative proteomic analysis. DvH was stressed with 105 mM sodium nitrate (NaNO(3)), a level that caused a 50% inhibition in growth. The protein profile of stressed cells was compared with that of cells grown in the absence of nitrate using the iTRAQ peptide labelling strategy and tandem liquid chromatography separation coupled with mass spectrometry (quadrupole time-of-flight) detection. A total of 737 unique proteins were identified by two or more peptides, representing 22% of the total DvH proteome and spanning every functional category. The results indicate that this was a mild stress, as proteins involved in central metabolism and the sulphate reduction pathway were unperturbed. Proteins involved in the nitrate reduction pathway increased. Increases seen in transport systems for proline, glycine-betaine and glutamate indicate that the NaNO(3) exposure led to both salt stress and nitrate stress. Up-regulation observed in oxidative stress response proteins (Rbr, RbO, etc.) and a large number of ABC transport systems as well as in iron-sulphur-cluster-containing proteins, however, appear to be specific to nitrate exposure. Finally, a number of hypothetical proteins were among the most significant changers, indicating that there may be unknown mechanisms initiated upon nitrate stress in DvH.
  234. Mukhopadhyay, A., Z. L. He, E. J. Alm, A. P. Arkin, E. E. Baidoo, S. C. Borglin, W. Q. Chen, Terry C. Hazen, Q. He, H. Y. Holman, K. Huang, R. Huang, D. C. Joyner, N. Katz, M. Keller, P. Oeller, A. Redding, J. Sun, J. Wall, J. Wei, Z. M. Yang, H. C. Yen, J. Z. Zhou and J. D. Keasling. 2006. Salt stress in Desulfovibrio vulgaris Hildenborough: An integrated genomics a pproach. Journal of Bacteriology 188:4068-4078. abstract pdf
    The ability of Desullfovibrio vulgaris Hildenborough to reduce, and therefore contain, toxic and radioactive metal waste has made all factors that affect the physiology of this organism of great interest. Increased salinity is an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in striking elongation of D. vulgaris cells. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we used a systems approach to explore the effects of excess NaCl on D. vulgaris. In this study we demonstrated that import of osmoprotectants, such as glycine betaine and ectoine, is the primary mechanism used by D. vulgaris to counter hyperionic stress. Several efflux systems were also highly up-regulated, as was the ATP synthesis pathway. Increases in the levels of both RNA and DNA hellicases suggested that salt stress affected the stability of nucleic acid base pairing. An overall increase in the level of branched fatty acids indicated that there were changes in cell wall fluidity. The immediate response to salt stress included up-regulation of chemotaxis genes, although flagellar biosynthesis was down-regulated. Other down-regulated systems included lactate uptake permeases and ABC transport systems. The results of an extensive NaCl stress analysis were compared with microarray data from a KCl stress analysis, and unlike many other bacteria, D. vulgaris responded similarly to the two stresses. Integration of data from multiple methods allowed us to develop a conceptual model for the salt stress response in D. vulgaris that can be compared to those in other microorganisms.
  235. Lin, Li-Hung, Pei-Ling Wang, Douglas Rumble, Johanna Lippmann-Pipke, Erik Boice, Lisa M. Pratt, Barbara Sherwood Lollar, Eoin L. Brodie, Terry C. Hazen, Gary L. Andersen, Todd Z. DeSantis, Duane P. Moser, Dave Kershaw and T. C. Onstott. 2006. Long-term sustainability of a high-energy, low-diversity crustal biome. Science 314:479-482. abstract pdf
    Geochemical, microbiological, and molecular analyses of alkaline saline groundwater at 2.8 kilometers depth in Archaean metabasalt revealed a microbial biome dominated by a single phylotype affiliated with thermophilic sulfate reducers belonging to Firmicutes. These sulfate reducers were sustained by geologically produced sulfate and hydrogen at concentrations sufficient to maintain activities for millions of years with no apparent reliance on photosynthetically derived substrates.
  236. He, Q., K. H. Huang, Z. L. He, E. J. Alm, M. W. Fields, Terry C. Hazen, A. P. Arkin, J. D. Wall and J. Z. Zhou. 2006. Energetic consequences of nitrite stress in Desulfovibrio vulgaris Hildenborough, inferred from global transcriptional analysis. Applied and Environmental Microbiology 72:4370-4381. abstract pdf
    Many of the proteins that are candidates for bioenergetic pathways involved with sulfate respiration in Desulfovibrio spp. have been studied, but complete pathways and overall cell physiology remain to be resolved for many environmentally relevant conditions. In order to understand the metabolism of these microorganisms under adverse environmental conditions for improved bioremediation efforts, Desuffiovibrio vulgaris Hildenborough was used as a model organism to study stress response to nitrite, an important intermediate in the nitrogen cycle. Previous physiological studies demonstrated that growth was inhibited by nitrite and that nitrite reduction was observed to be the primary mechanism of detoxification. Global transcriptional profiling with whole-genome microarrays revealed coordinated cascades of responses to nitrite in pathways of energy metabolism, nitrogen metabolism, oxidative stress response, and iron homeostasis. In agreement with previous observations, nitrite-stressed cells showed a decrease in the expression of genes encoding sulfate reduction functions in addition to respiratory oxidative phosphorylation and ATP synthase activity. Consequently, the stressed cells had decreased expression of the genes encoding ATP-dependent amino acid transporters and proteins involved in translation. Other genes up-regulated in response to nitrite include the genes in the Fur regulon, which is suggested to be involved in iron homeostasis, and genes in the Per regulon, which is predicted to be responsible for oxidative stress response.
  237. Hazen, Terry C., Ken Timmis, Dave Stahl, Ed DeLong and Michael Wagner. 2006. This issue of environmental microbiology is dedicated to the memory of David. C. White. Environmental Microbiology 8:2059-2061. pdf
  238. Hazen, Terry C. and D. A. Stahl. 2006. Using the stress response to monitor process control: pathways to more effective bioremediation. Current Opinion in Biotechnology 17:285-290. abstract pdf
    Environmental contamination with a variety of pollutants has prompted the development of effective bioremediation strategies. But how can these processes be best monitored and controlled? One avenue under investigation is the development of stress response systems as tools for effective and general process control. Although the microbial stress response has been the subject of intensive laboratory investigation, the environmental reflection of the laboratory response to specific stresses has been little explored. However, it is only within an environmental context, in which microorganisms are constantly exposed to multiple changing environmental stresses, that there will be full understanding of microbial adaptive resiliency. Knowledge of the stress response in the environment will facilitate the control of bioremediation and other processes mediated by complex microbial communities.
  239. Terry C. Hazen and Romy Chakraborty. 2006. Book Review: Environmental Biotechnology by alan Scragg Oxford Press. The Quarterly Review of Biology 81:304. pdf
  240. Hazen, Terry C.. 2006. DOE-NABIR PI Workshop: Abstracts. DOE-NABIR PI Workshop: Abstracts pdf
  241. Fields, M. W., C. E. Bagwell, S. L. Carroll, T. Yan, X. Liu, D. B. Watson, P. M. Jardine, C. S. Criddle, Terry C. Hazen and J. Zhou. 2006. Phylogenetic and functional biomakers as indicators of bacterial community responses to mixed-waste contamination. Environmental Science & Technology 40:2601-2607. abstract pdf
    Few studies have demonstrated changes in community structure along a contaminant plume in terms of phylogenetic, functional, and geochemical changes, and such studies are essential to understand how a microbial ecosystem responds to perturbations. Clonal libraries of multiple genes (SSU rDNA, nirK, nirS, amoA, pmoA, and dsrAB) were analyzed from groundwater samples (n = 6) that varied in contaminant levels, and 107 geochemical parameters were measured. Principal components analyses (PCA) were used to compare the relationships among the sites with respect to the biomarker (n = 785 for all sequences) distributions and the geochemical variables. A major portion of the geochemical variance measured among the samples could be accounted for by tetra chloroethene, Tc-99, NO3, SO4, Al, and Th. The PCA based on the distribution of unique biomarkers resulted in different groupings compared to the geochemical analysis, but when the SSU rRNA gene libraries were directly compared (Delta C-xy values) the sites were clustered in a similar fashion compared to geochemical measures. The PCA based upon functional gene distributions each predicted different relationships among the sites, and comparisons of Euclidean distances based upon diversity indices for all functional genes (n = 432) grouped the sites by extreme or intermediate contaminant levels. The data suggested that the sites with low and high perturbations were functionally more similar than sites with intermediate conditions, and perhaps captured the overall community structure better than a single phylogenetic biomarker. Moreover, even though the background site was phylogenetically and geochemically distinct from the acidic sites, the extreme conditions of the acidic samples might be more analogous to the limiting nutrient conditions of the background site. An understanding of microbial community-level responses within an ecological framework would provide better insight for restoration strategies at contaminated field sites.
  242. Clark, M. E., Q. He, Z. He, K. H. Huang, E. J. Alm, X. F. Wan, Terry C. Hazen, A. P. Arkin, J. D. Wall, J. Z. Zhou and M. W. Fields. 2006. Temporal transcriptomic analysis as Desulfovibrio vulgaris hildenborough transitions into stationary phase during electron donor depletion. Applied and Environmental Microbiology 72:5578-5588. abstract pdf
    Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.
  243. Chhabra, S. R., Q. He, K. H. Huang, S. P. Gaucher, E. J. Alm, Z. He, M. Z. Hadi, Terry C. Hazen, J. D. Wall, J. Zhou, A. P. Arkin and A. K. Singh. 2006. Global analysis of heat shock response in Desulfovibrio vulgaris Hildenborough. Journal of Bacteriology 188:1817-1828. abstract pdf
    Desulfovibrio vulgaris Hildenborough belongs to a class of sulfate-reducing bacteria (SRB) and is found ubiquitously in nature. Given the importance of SRB-mediated reduction for bioremediation of metal ion contaminants, ongoing research on D. vulgaris has been in the direction of elucidating regulatory mechanisms for this organism under a variety of stress conditions. This work presents a global view of this organism's response to elevated growth temperature using whole-cell transcriptomics and proteomics tools. Transcriptional response (1.7-fold change or greater; Z >= 1.5) ranged from 1,135 genes at 15 min to 1,463 genes at 120 min for a temperature up-shift of 13 degrees C from a growth temperature of 37 degrees C for this organism and suggested both direct and indirect modes of heat sensing. Clusters of orthologous group categories that were significantly affected included posttranslational modifications; protein turnover and chaperones (up-regulated); energy production and conversion (down-regulated), nucleotide transport, metabolism (down-regulated), and translation; ribosomal structure; and biogenesis (down-regulated). Analysis of the genome sequence revealed the presence of features of both negative and positive regulation which included the CIRCE element and promoter sequences corresponding to the alternate sigma factors sigma(32) and sigma(54). While mechanisms of heat shock control for some genes appeared to coincide with those established for Escherichia coli and Bacillus subtilis, the presence of unique control schemes for several other genes was also evident. Analysis of protein expression levels using differential in-get electrophoresis suggested good agreement with transcriptional profiles of several heat shock proteins, including DnaK (DVU0811), HtpG (DVU2643), HtrA (DVU1468), and AhpC (DVU2247). The proteomics study also suggested the possibility of posttranslational modifications in the chaperones DnaK, AhpC, GroES (DVU1977), and GroEL (DVU1976) and also several periplasmic ABC transporters.
  244. Brodie, Eoin L., Todd Z. DeSantis, Dominique C. Joyner, Seung M. Baek, Joern T. Larsen, Gary L. Andersen, Terry C. Hazen, Paul M. Richardson, Donald J. Herman, Tetsu K. Tokunaga, Jiamin M. Wan and Mary K. Firestone. 2006. Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation. Applied and Environmental Microbiology 72:6288-6298. abstract pdf
    Reduction of soluble uranium U(VI) to less-soluble uranium U(M is a promising approach to minimize migration from contaminated aquifers. It is generally assumed. that, under constant reducing conditions, U(M is stable and immobile; however, in a previous study, we documented reoxidation of U(IV) under continuous reducing conditions (Wan et al., Environ. Sci. Technol. 2005, 39:6162-6169). To determine if changes in microbial community composition were a factor in U(IV) reoxidation, we employed a high-density phylogenetic DNA microarray (16S microarray) containing 500,000 probes to monitor changes in bacterial populations during this remediation process. Comparison of the 16S microarray with clone libraries demonstrated successful detection and classification of most clone groups. Analysis of the most dynamic groups of 16S rRNA gene amplicons detected by the 16S microarray identified five clusters of bacterial subfamilies responding in a similar manner. This approach demonstrated that amplicons of known metal-reducing bacteria such as Geothrix fermentans (confirmed by quantitative PCR) and those within the Geobacteraceae were abundant during U(VI) reduction and did not decline during the U(IV) reoxidation phase. Significantly, it appears that the observed reoxidation of uranium under reducing conditions occurred despite elevated microbial activity and the consistent presence of metal-reducing bacteria. High-density phylogenetic microarrays constitute a powerful tool, enabling the detection and monitoring of a substantial portion of the microbial population in a routine, accurate, and reproducible manner.
  245. Abulencia, Carl B., Denise L. Wyborski, Joseph A. Garcia, Mircea Podar, Wenqiong Chen, Sherman H. Chang, Hwai W. Chang, David Watson, Eoln L. Brodie, Terry C. Hazen and Martin Keller. 2006. Environmental whole-genome amplification to access microbial populations in contaminated sediments. Applied and Environmental Microbiology 72:3291-3301. abstract pdf
    Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi 29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.
  246. Wan, J. M., T. K. Tokunaga, E. Brodie, Z. M. Wang, Z. P. Zheng, D. Herman, Terry C. Hazen, M. K. Firestone and S. R. Sutton. 2005. Reoxidation of bioreduced uranium under reducing conditions. Environmental Science & Technology 39:6162-6169. abstract pdf
    Nuclear weapons and fuel production have left many soils and sediments contaminated with toxic levels of uranium (U). Although previous short-term experiments on microbially mediated U(VI) reduction have supported the prospect of immobilizing the toxic metal through formation of insoluble U(IV) minerals, our longer-term (17 months) laboratory study showed that microbial reduction of U can be transient, even under sustained reducing conditions. Uranium was reduced during the first 80 days, but later(100-500 days) reoxidized and solubilized, even though a microbial community capable of reducing U(Vl) was sustained. Microbial respiration caused increases in (bi) carbonate concentrations and formation of very stable uranyl carbonate complexes, thereby increasing the thermodynamic favorability of U(IV) oxidation. We propose that kinetic limitations including restricted mass transfer allowed Fe(111) and possibly Mn(IV) to persist as terminal electron acceptors (TEAS) for U reoxidation. These results show that in-situ U remediation by organic carbon-based reductive precipitation can be problematic in sediments and groundwaters with neutral to alkaline pH, where uranyl carbonates are most stable.
  247. Tokunaga, T. K., J. M. Wan, J. Pena, E. L. Brodie, M. K. Firestone, Terry C. Hazen, S. R. Sutton, A. Lanzirotti and M. Newville. 2005. Uranium reduction in sediments under diffusion-limited transport of organic carbon. Environmental Science & Technology 39:7077-7083. abstract pdf
    Costly disposal of uranium (U) contaminated sediments is motivating research on in situ U(VI) reduction to insoluble U(IV) via directly or indirectly microbially mediated pathways. Delivery of organic carbon (OC) into sediments for stimulating U bioreduction is diffusion-limited in less permeable regions of the subsurface. To study OC-based U reduction in diffusion-limited regions, one slightly acidic and another calcareous sediment were treated with uranyl nitrate, packed into columns, then hydrostatically contacted with tryptic soy broth solutions. Redox potentials, U oxidation state, and microbial communities were well correlated. At average supply rates of 0.9 mu mol OC (g sediment)(-1) day(-1), the U reduction zone extended to only about 35-45 mm into sediments. The underlying unreduced U(VI) zone persisted over 600 days because the supply of OC was diffusion-limited and metabolized within a short distance. These results also suggest that low U concentrations in groundwater samples from OC-treated sediments are not necessarily indicative of pervasive U reduction because interior and exterior regions of such sediment blocks can contain primarily U(VI) and U(IV) respectively.
  248. Koenigsberg, S. S., Terry C. Hazen and A. D. Peacock. 2005. Environmental Biotechnology: a Bioremediation Perspective. Remediation Journal 15:5-25. abstract pdf
    The term environmental biotechnology has a certain air of modernity when in fact it has a long history of use, if one considers the underlying principles and not the appellation. However, as part of its complex meaning, there is a dynamic new definition and purpose in this discipline with regard to bioremediation. The ability to probe the environment at the molecular level with exquisite methods, to create a new awareness of fundamental biological processes therein, has created an important new paradigm in remediation engineering design and management. Further, biological lines of evidence made extremely robust through the merger of biotechnology and environmental science are poised to be incorporated into the very fabric of site evaluation and disposition at the regulatory level. At the operational level, the field of environmental biotechnology is driven by the “omics,” the common suffix for disciplines like genomics, proteomics, and metabolomics. An introduction to these elements of the process is followed by a review of how they are being used right now in a commercial framework, with the understanding that the entire process is still in the formative stages of its vast potential.
  249. Terry C. Hazen and H. H. Tabak. 2005. Developments in bioremediation of soils and sediments polluted with metals and radionuclides: 2. Field research on bioremediation of metals and radionuclides. Reviews in Environmental Science and Bio/Technology 4:157-183. abstract pdf
    Bioremediation of metals and radionuclides has had many field tests, demonstrations, and full-scale implementations in recent years. Field research in this area has occurred for many different metals and radionuclides using a wide array of strategies. These strategies can be generally characterized in six major categories: biotransformation, bioaccumulation/bisorption, biodegradation of chelators, volatilization, treatment trains, and natural attenuation. For all field applications there are a number of critical biogeochemical issues that most be addressed for the successful field application. Monitoring and characterization parameters that are enabling to bioremediation of metals and radionuclides are presented here. For each of the strategies a case study is presented to demonstrate a field application that uses this strategy.
  250. Hazen, Terry C.. 2005. SERDP and ESTCP Expert Panel Workshop on Research and Development Needs for the Environmental Remediation Application of Molecular Biological Tools. pdf
  251. Terry C. Hazen. 2005. DOE-NABIR PI Workshop: Abstracts. DOE-NABIR PI Workshop: Abstracts pdf
  252. Borglin, Sharon E, Terry C Hazen, Curtis M Oldenburg, Peter T Zawislanski and Comparative Study. 2004. Comparison of aerobic and anaerobic biotreatment of municipal solid waste. Journal of the Air & Waste Management Association 54:815-822. abstract pdf
    To increase the operating lifetime of landfills and to lower leachate treatment costs, an increasing number of municipal solid waste (MSW) landfills are being managed as either aerobic or anaerobic bioreactors. Landfill gas composition, respiration rates, and subsidence were measured for 400 days in 200-L tanks filled with fresh waste materials to compare the relative effectiveness of the two treatments. Tanks were prepared to provide the following conditions: (1) air injection and leachate recirculation (aerobic), (2) leachate recirculation (anaerobic), and (3) no treatment (anaerobic). Respiration tests on the aerobic wet tank showed a steady decrease in oxygen consumption rates from 1.3 mol/day at 20 days to 0.1 mol/day at 400 days. Aerobic wet tanks produced, on average, 6 mol of carbon dioxide (CO2)/kg of MSW as compared with anaerobic wet tanks, which produced 2.2 mol methane/kg of MSW and 2.0 mol CO2/kg methane. Over the test period, the aerobic tanks settled on average 35%, anaerobic tanks settled 21.7%, and the no-treatment tank settled 7.5%, equivalent to overall mass loss in the corresponding reactors. Aerobic tanks reduced stabilization time and produced negligible odor compared with anaerobic tanks, possibly because of the 2 orders of magnitude lower leachate ammonia levels in the aerobic tank. Both treatment regimes provide the opportunity for disposal and remediation of liquid waste.
  253. Tokunaga, T. K., J. M. Wan, Terry C. Hazen, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti and W. Rao. 2003. Distribution of chromium contamination and microbial activity in soil aggregates. Journal of Environmental Quality 32:541-549. abstract pdf
    Biogeochemical transformations of redox-sensitive chemicals in soils can be strongly transport-controlled and localized. This was tested through experiments on chromium diffusion and reduction in soil aggregates that were exposed to chromate solutions. Reduction of soluble Cr(VI) to insoluble Cr(III) occurred only within the surface layer of aggregates with higher available organic carbon and higher microbial respiration. Sharply terminated Cr diffusion fronts develop when the reduction rate increases rapidly with depth. The final state of such aggregates consists of a Cr-contaminated exterior, and an uncontaminated core, each having different microbial community compositions and activity. Microbial activity was significantly higher in the more reducing soils, while total microbial biomass was similar in all of the soils. The small fraction of Cr(VI) remaining unreduced resides along external surfaces of aggregates, leaving it potentially available to future transport down the soil profile. Using the Thiele modulus, Cr(VI) reduction in soil aggregates is shown to be diffusion rate- and reaction rate-limited in anaerobic and aerobic aggregates, respectively. Thus, spatially resolved chemical and microbiological measurements are necessary within anaerobic soil aggregates to characterize and predict the fate of Cr contamination. Typical methods of soil sampling and analyses that average over redox gradients within aggregates can erase important biogeochemical spatial relations necessary for understanding these environments.
  254. Tokunaga, Tetsu K, Jiamin Wan, Mary K Firestone, Terry C Hazen, Keith R Olson, Donald J Herman, Stephen R Sutton and Antonio Lanzirotti. 2003. In situ reduction of chromium(VI) in heavily contaminated soils through organic carbon amendment. Journal of Environmental Quality 32:1641-1649. abstract pdf
    Chromium has become an important soil contaminant at many sites, and facilitating in situ reduction of toxic Cr(VI) to nontoxic Cr(III) is becoming an attractive remediation strategy. Acceleration of Cr(VI) reduction in soils by addition of organic carbon was tested in columns pretreated with solutions containing 1000 and 10 000 mg L-1 Cr(VI) to evaluate potential in situ remediation of highly contaminated soils. Solutions containing 0, 800, or 4000 mg L-1 organic carbon in the form of tryptic soy broth or lactate were diffused into the Cr(VI)contaminated soils. Changes in Cr oxidation state were monitored through periodic micro-XANES analyses of soil columns. Effective first-order reduction rate constants ranged from 1.4 x 10(-8) to 1.5 x 10(-7) s(-1), with higher values obtained for lower levels of initial Cr(VI) and higher levels of organic carbon. Comparisons with sterile soils showed that microbially dependent processes were largely responsible for Cr(VI) reduction, except in the soils initially exposed to 10 000 mg L-1 Cr(VI) solutions that receive little (800 mg L-1) or no organic carbon. However, the microbial populations (less than or equal to2.1 x 10(5) g(-1)) in the viable soils are probably too low for direct enzymatic Cr(VI) reduction to be important. Thus, synergistic effects sustained in whole soil systems may have accounted for most of the observed reduction. These results show that acceleration of in situ Cr(VI) reduction with addition of organic carbon is possible in even heavily contaminated soils and suggest that microbially dependent reduction pathways can be dominant.
  255. McCullough, J. and Terry C. Hazen. 2003. Bioremediation of Metals and Radionuclides: What It Is and How It Works (2nd Edition). pdf
  256. Looney, B. B., Terry C. Hazen, D. L. Eaton and E. Charoglu-Boemer. 2003. Technical Assistance to Kansas City Plant: Mitigation of Polychlorinated Biphyenyl Discharges. pdf
  257. Hazen, Terry C., A. Tien, A. Worsztynowicz, D. J. Altman, K. Ulfig and T. Manko. 2003. Biopiles For Remediation Of Petroleum-Contaminated Soils: A Polish Case Study. Proceedings of the NATO Advanced Research Workshop on The Utilization of Bioremediation to Reduce soil Contamination: Problems and Solutions 19:229-246. abstract pdf
    The US Department of Energy and the Institute for Ecology of Industrial Areas of Poland demonstrated bioremediation techniques for the clean up of acidic petroleum sludge impacted soils at an oil refinery in southern Poland. The waste was composed of high molecular weight paraffinic and polynuclear aromatic hydrocarbons. Benzo(a)pyrene and BTEX compounds were identified as the contaminants of concern. Approximately 3,300 m3 of contaminated soil (TPH ~ 30,000 ppm) was targeted for treatment. A biopile design which employed a combination of passive and active aeration in conjunction with nutrient and surfactant application was used to increase the biodegradation of the contaminants of concern. Over the 20 month project, more than 81% (120 metric tons) of petroleum hydrocarbons were biodegraded. Despite the fact the material treated was highly weathered and very acidic, biodegradation rates of 121 mg/kg soil/day in the actively aerated side (82 mg/kg soil/day in the passive side) were achieved in this biopile. Microbial counts and dehydrogenase measurements gave the best correlation with the biodegradation rates. Costs were competitive or significantly lower when compared with other ex situ treatment processes.
  258. Curtis M. Oldenburg, Sharon E. Borglin and Terry C. Hazen. 2002. Multiphase modeling of flow, Transport, and biodegradation in a mesoscale landfill bioreactor. pdf
  259. Hazen, T. C.. 2002. Basic Research Needs for Countering Terrorism. pdf
  260. Hazen, Terry. 2002. Technical assistance to Ohio closure sites; Technologies to address leachate from the on-site disposal facility at Fernald Environmental Management Project, Ohio. pdf
  261. Hazen, Terry. 2002. Technical assistance to Ohio closure sites; Recommendations to address contaminated soils, concrete, and corrective action management unit/groundwater contamination at Ashtabula, Ohio. pdf
  262. Hazen, Terry. 2002. SCFA lead lab technical assistance at Lawrence Berkeley National Laboratory: Baseline review of three groundwater plumes. pdf
  263. Hazen, T.. 2002. SCFA lead lab technical assistance at Oak Ridge Y-12 national security complex: Evaluation of treatment and characterization alternatives of mixed waste soil and debris at disposal area remedial action (DARA) solids storage facility (SSF). pdf
  264. Tokunaga, Tetsu K., Jiamin Wan, Terry C. Hazen, Egbert Schwartz, Mary K. Firestone, Stephen R. Sutton, Matthew Newville, Keith R. Olson, Antonio Lanzirotti and William Rao. 2001. Distribution of Chromium Contamination and Microbial Activity in Soil Aggregates. Journal of Environmental Quality 32. pdf
  265. Tokunaga, T. K., J. Wan, M. K. Firestone, T. C. Hazen, E. Schwartz, S. R. Sutton and M. Newville. 2001. Chromium diffusion and reduction in soil aggregates. Environmental Science & Technology 35:3169-74. abstract pdf
    The distribution of metal contaminants such as chromium in soils can be strongly localized by transport limitations and redox gradients within soil aggregates. Measurements of Cr(VI) diffusion and reduction to Cr(III) were obtained in soil columns representing transects into soil aggregates in order to quantify influences of organic carbon (OC) and redox potentials on Cr transport distances and microbial community composition. Shifts in characteristic redox potentials, and the extent of Cr(VI) reduction to Cr(III) were related to OC availability. Depth profiles of Cr(VI, III) obtained with micro X-ray absorption near edge structure (micro-XANES) spectroscopy reflected interdependent effects of diffusion and spatially dependent redox potentials on reduction kinetics and microbial community composition. Shallow diffusion depths (2-10 mm) and very sharply terminated diffusion fronts in columns amended with OC (80 and 800 ppm) reflected rapid increases in Cr reduction kinetics over very short (mm) distances. These results suggest that Cr contamination in soils can be restricted to the outsides of soil aggregates due to localized transport and rapid reduction and that bulk sample characterization is inadequate for understanding the controlling biogeochemical processes.
  266. Plaza, Grazyna, Krzysztof Ulfig, Terry C Hazen and Robin L Brigmon. 2001. Use of molecular techniques in bioremediation. Acta Microbiologica Polonica 50:205-218. abstract pdf
    In a practical sense, biotechnology is concerned with the production of commercial products generated by biological processes. More formally, biotechnology may be defined as "the application of scientific and engineering principles to the processing of material by biological agents to provide goods and services" (Cantor, 2000). From a historical perspective, biotechnology dates back to the time when yeast was first used for beer or wine fermentation, and bacteria were used to make yogurt. In 1972, the birth of recombinant DNA technology moved biotechnology to new heights and led to the establishment of a new industry. Progress in biotechnology has been truly remarkable. Within four years of the discovery of recombinant DNA technology, genetically modified organisms (GMOs) were making human insulin, interferon, and human growth hormone. Now, recombinant DNA technology and its products - GMOs are widely used in environmental biotechnology (Glick and Pasternak, 1988; Cowan, 2000). Bioremediation is one of the most rapidly growing areas of environmental biotechnology. Use of bioremediation for environmental clean up is popular due to low costs and its public acceptability. Indeed, bioremediation stands to benefit greatly and advance even more rapidly with the adoption of molecular techniques developed originally for other areas of biotechnology. The 1990s was the decade of molecular microbial ecology (time of using molecular techniques in environmental biotechnology). Adoption of these molecular techniques made scientists realize that microbial populations in the natural environments are much more diverse than previously thought using traditional culture methods. Using molecular ecological methods, such as direct DNA isolation from environmental samples, denaturing gradient gel electrophoresis (DGGE), PCR methods, nucleic acid hybridization etc., we can now study microbial consortia relevant to pollutant degradation in the environment. These techniques promise to provide a better understanding and better control of environmental biotechnology processes, thus enabling more cost effective and efficient bioremediation of our toxic waste and contaminated environments. Microbiology, Institute for Ecology of Industrial Areas, ul. Kossutha, 40-833, Katowice, Poland
  267. Eaton, D., D. Janeday, D. Woodward, J. Imrich, J. Evans, M. Morris, P. Reimus and T. Hazen. 2001. SCFA lead lab technical assistance review of the Pit 7 Complex source containment. pdf
  268. Murdoch, L., J. S. Girke, J. Rossabi, J. Reed, D. Conley, J. Phelan, R. W. Falta, W. Heath, Terry C. Hazen, R. L. Sieqrist, O. R. West, M. A. Urynowicz, W. W. Slack, P. Bishop, V. Hebatpuria, L. E. Erickson, L. C. Davis and P. A. Kulakow. 2000. Remediation of Organic Chemicals in the Vadose Zone. Vadose Zone Science and Technology Solutions 2:949-1156. abstract pdf
    The current status, applications, and future developments of vadose-zone characterization and monitoring technologies are described using case-study data from practicing scientists and engineers. The basic principles, advantages, and limitations of existing vadose-zone characterization and monitoring methods are considered, and evidence is presented that the central problem of the vadose-zone investigation is the preferential fast-flow phenomenon and accelerated deep-contaminant transport toward groundwater. Included are water flow and chemical transport processes in deep and shallow vadose zones, field vadose-zone characterization and monitoring, and determination of unsaturated hydraulic properties of variably saturated soils and rocks. A number of case studies are appended that illustrate the concepts and technologies addressed in the chapter.
  269. Hazen, T. C., A. J. Tien, A. Worsztynowicz, D. J. Altman, K. Ulfig and T. Manko. 2000. Biopiles for remediation of petroleum-contaminated soils: a Polish case study. Polish refinery biopile. NATO Advanced Research Workshop on Bioremediation pdf
  270. Hazen, Terry C.. 2000. Bioremediation Education Science and Technology (BEST) Program Annual Report 1999. abstract pdf
    The Bioremediation, Education, Science and Technology (BEST) partnership provides a sustainable and contemporary approach to developing new bioremedial technologies for US Department of Defense (DoD) priority contaminants while increasing the representation of underrepresented minorities and women in an exciting new biotechnical field. This comprehensive and innovative bioremediation education program provides under-represented groups with a cross-disciplinary bioremediation cirruculum and financial support, coupled with relevant training experiences at advanced research laboratories and field sites. These programs are designed to provide a stream of highly trained minority and women professionals to meet national environmental needs.
  271. Faybishenko, B., M. Bandurraga, M. Conrad, P. Cook, C. Eddy-Dilek, L. Everett, Terry C. Hazen, S. Hubbard, A. R. Hutter, P. Jordan, C. Keller, F. J. Leij, N. Loaiciga, E. L. Majer, L. Murdoch, S. Renehan, B. Riha, J. Rossabi, Y. Rubin, A. Simmons, S. Weeks and C. V. Williams. 2000. Vadose Zone Characterization and Monitoring: Current Technologies. Vadose Zone Science and Technology Solutions 1:133-395. abstract pdf
    The current status, applications, and future developments of vadose-zone characterization and monitoring technologies are described using case-study data from practicing scientists and engineers. The basic principles, advantages, and limitations of existing vadose-zone characterization and monitoring methods are considered, and evidence is presented that the central problem of the vadose-zone investigation is the preferential fast-flow phenomenon and accelerated deep-contaminant transport toward groundwater. Included are water flow and chemical transport processes in deep and shallow vadose zones, field vadose-zone characterization and monitoring, and determination of unsaturated hydraulic properties of variably saturated soils and rocks. A number of case studies are appended that illustrate the concepts and technologies addressed in the chapter.
  272. Worsztynowicz, A., A. Tien, K. Ulfig, K. Zacharz, M. Adamski, D. Rzychon, Terry C. Hazen and D. Altman. 1999. Soil Cleaning at Czechowice Refinery. Proceedings Fourth International Symposium and Exhibition on Envirnomental Contamination in Central and Eastern Europe (Warsaw '98) pdf
  273. Ulfig, K, G. Plaza, K. Lukasik, T. Manko, A. Worsztynowicz, A. Tien and Terry C. Hazen. 1999. Microbiological Changes in Petroleum-Contaminated Soil During Bioremediation at a Polish Petroleum Refinery.. Proceedings Fourth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe pdf
  274. Tien, A.J., D.J. Altman, A. Worsztynowicz, K. Zacharz, K. Ulfig, T. Manko and T.C. Hazen. 1999. Bioremediation of a process waste lagoon at a southern Polish oil refinery - DoE's first demonstration project in Poland. Proceedings Fourth International Symposium and Exhibition on Envirnomental Contamination in Central and Eastern Europe (Warsaw '98) abstract pdf
    Nearly a century of continued use of a sulfuric acid-based oil refining technology by the Czechowice Oil Refinery, located in southern Poland, has produced an estimated 120 thousand tons of acidic, highly weathered, petroleum sludges. This waste has been deposited into three unlined process waste lagoons, three meters deep, now covering 3.8 hectares. Initial analysis indicated that the sludges were composed mainly of high molecular weight paraffinic and polycyclic aromatic hydrocarbons (PAHs). The overall objective of this project is to provide a cost-effective bioremediation demonstration with petroleum-sludge contaminated soil for Poland. The specific goal of the remediation is to reduce the environmental risk from PAH compounds in soil and provide a green zone (grassy area) adjacent to the site boundary. A 0.3 hectare site, the smallest of the waste lagoons, was selected for remediation using a technology known as biopiling. The original lagoon sludge was amended and sold as a fuel to a local cement company thereby eliminating the contaminants while providing the refinery an additional revenue source. Approximately 3300 m3 of contaminated soil with a mean total petroleum hydrocarbon (TPH) concentration of 27,000 ppm was targeted for treatment. The biopile was divided into two sections; an area of approximately 1610 m2 passively aerated using BaroballsTM and an area of approximately 1390 m2 actively aerated via air injection. Use of both passive and active aeration methods allows for an accurate assessment of cost and efficiency, with the most appropriate design to be deployed for future lagoon remediation at the refinery. Since the fall of 1997, approximately 80 tons of TPH have been removed from the biopile as a whole. The remediation strategies that have been applied at the Czechowice Oil Refinery waste lagoon in Czechowice-Dziedzice, Poland were designed, managed and implemented under direction of the Westinghouse Savannah River Company (WSRC) for the United States Department of Energy (DOE), the Institute for Ecology of Industrial Area (IETU), Ames Laboratory and Florida State University (FSU). FSU has the overall lead for the DOE EM-50 Polish Initiative Program. This collaboration between IETU, DOE and its partners, provides the basis for international technology transfer of new and innovative remediation technologies which can be applied in Poland and Eastern Europe as well.
  275. Peters, N. E.. 1999. Water Quality Degradation and Freshwater Availability - Need for a Global Initiative. Proceedings UNESCO Colloquium entitled "Water - A Looming Crisis" pdf
  276. McCullough, J., Terry Hazen and Sally Benson. 1999. Bioremediation of metals and radionuclides: What it is and How it Works. pdf
  277. Hazen, Terry C.. 1999. Critical Biogeochemical Parameters Used for In Situ Bioremediation of Solvents in Fractured Rock.. Proceedings Dynamics of Fluids in Fractured Rocks pdf
  278. Radway, J. C., J. W. Santo Domingo, Terry C. Hazen and E. W. Wilde. 1998. Evaluation of biodegradation potential of foam embedded Burkholderia cepacia G4. Biotechnology Letters 20:663-666. abstract pdf
    Foam embedded Burkholderia cepacia G4 removed up to 80% and 60% of a 3 mg/l solution of trichloroethylene (TCE) and a 2 mg/l solution of benzene, respectively. Removal of TCE and benzene decreased more than 50% when readily metabolizable carbon sources were present. TCE degradative activity was observed with G4 cells induced with phenol or benzene prior or after immobilization of cells.
  279. Legrand, R., A. J. Morecraft, J. A. Harju, T. D. Hayes and T. C. Hazen. 1998. Field application of in situ methanotrophic treatment for TCE remediation. First International Conference on Remediation of Chlorinated and Recalcitrant Compounds, Vol 4 - Bioremediation and Phytoremediation 193-198. abstract pdf
    In situ methanotrophic treatment technology (MTT) was evaluated at a site contaminated with chlorinated ethenes and hydrocarbons near a natural gas pipeline compressor station. The formation is characterized by approximately 50 ft (15 m) of saprolitic overburden above bedrock, and a depth to groundwater of 8 to 10 ft (2.4 to 3 m). The MTT system was automated and normally unattended. Air and methane were injected, along with nitrous oxide (N2O) and triethylphosphate (TEP), as nutrient sources; TEP delivery was intermittent because of design issues. Trichloroethene (TCE) levels dropped from 2130 to 150 mu g/L in the well initially exhibiting the highest concentration. The radius of influence of the air injection was approximately 30 ft (9 m). Methanotrophic bacteria increased over six orders of magnitude and eventually dominated the subsurface microbiota. The results indicate that, as long as nitrogen and phosphorus were reliably supplied, rapid (two to four weeks) growth of methanotrophs and associated oxidation of TCE followed. This pilot system was expanded to bioremediate the entire plume above bedrock; three additional injection wells were installed, along with observation wells, and a new TEP diffusion system was developed.
  280. Fuentes, F. A., J. W. Santo Domingo and Terry C. Hazen. 1998. Survival of Candida albicans and Pseudomonas aeruginosa in oil polluted tropical coastal waters. Water Research 32:2154-2170. abstract pdf
    The effect of environmental abiotic factors on survival and activity of oil degrading isolates, Pseudomonas aeruginosa OD-1 and Candida albicans OD-2, was determined in situ using membrane diffusion chambers. The study sites were located in a tropical bay on the Atlantic Ocean with history of petroleum contamination by oil tankers. Microbial densities were measured by direct counts. H-3-thymidine uptake, microautoradiography, INT-reduction, adenosine triphosphate (ATP), and frequency of dividing cells were used to assess microbial activity. Both organisms showed a significant decrease in cell density over a three day period, although, temporal increases in densities were observed. Significant decreases in total activity vr;ere observed ibr both populations during the study; however, the respiration potential and ATP content per organism remained constant or even increased by the end of the study suggesting that a significant fraction of these populations were capable of withstanding in situ environmental conditions. Results suggest that increases in ambient phosphorus concentrations played a role in the prolonged ill situ survival of these petroleum degrading isolates at some sites. The traffic of oil tankers might have played a significant impact on microbial survival in this bay, as violent agitation of sediments increased phosphorus concentrations available to microorganisms. Published by Elsevier Science Ltd.
  281. Young, J. D., D. J. Altman, K. H. Lombard, A. W. Bourquin, D. C. Mosteller, T. C. Hazen and Inst Battele Mem. 1997. Sanitary landfill optimization test for remediation of chlorinated solvents. In Situ and on-Site Bioremediation, Vol 5 4(5):315-316. pdf
  282. Ulfig, K., G. Plaza, Terry C. Hazen, Carl B. Fliermans, M. M. Franck and K. H. Lombard. 1997. Bioremediation treatability and feasibility studies at a Polish petroleum refinery. Proceedings Warsaw 96 pdf
  283. Santo Domingo, J. W., J. C. Radway, E. W. Wilde, P. Hermann and Terry C. Hazen. 1997. Immobilization of Burkholderia cepacia in polyurethane-based foams: Embedding efficiency and effect on bacterial activity. Journal of Industrial Microbiology & Biotechnology 18:389-395. abstract pdf
    Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam, All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O-2 consumption and CO2 evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds.
  284. Santo Domingo, J. W., J. C. Radway, Terry C. Hazen and E. W. Wilde. 1997. Use of microrespirometry to determine viability of immobilized Burkholderia cepacia G4. Biotechnology Techniques 11:571-575. abstract pdf
    Embedding of Burkholderia cepacia G4 cells in a polyurethane-based foam decreased their culturability by more than four orders of magnitude. However, respiration rates of immobilized cells were at least 33-41% of unimmobilized cells. Embedded cells also degraded trichloroethylene. Therefore, respirometry is a more reliable indicator of viability of polyurethane immobilized bacteria than culturing methods.
  285. Santo Domingo, J. W., B. E. Bumgarner, D. J. Altman, C. J. Berry, T. C. Hazen and Inst Battele Mem. 1997. Physiological response of subsurface microbial communities to nutrient additions. In Situ and on-Site Bioremediation, Vol 5 4(5):307-312. abstract pdf
    Studies were conducted to evaluate the physiological response of subsurface microbial communities to nutrient addition campaigns implemented as part of an landfill in situ bioremediation project. Different injection campaigns were performed throughout this study involving the addition of air, with triethylphosphate (TEP), nitrous oxide (N2O), and methane. Samples were taken from saturated zone piezometer wells and analyzed for the following parameters: direct and viable counts, frequency of dividing cells, and percent of cells hybridizing to eubacterial rRNA targeting probes. Less than 2% of the microscopical counts from the saturated zone hybridized to an eubacterial probe before any of the injection campaigns. However, the number of hybridizing cells increased to nearly 42% after air injections, reaching a maximum of 63% a few weeks later. A steady decrease in the number of hybridizing cells was observed after maximum activity was reached, despite additional nutrient campaigns. In contrast, direct and viable counts, and the frequency of dividing cells remained relatively unchanged after the initial microbial biomass stimulation. Interestingly, the ratio of beta and gamma Proteobacteria changed during this period suggesting that nutrient campaigns had an effect in the in situ microbial community structure.
  286. Santo Domingo, Jorge W., Christopher J. Berry and Terry C. Hazen. 1997. Use of conventional methods and whole cell hybridization to monitor the microbial response to triethylphosphate. Journal of Microbiological Methods 29:145-151. abstract pdf
    The effect of triethylphosphate (TEP) on the activity of a landfill aquifer microbial community was evaluated using standard techniques and in situ hybridizations with phylogenetic probes. Benzene was used as an external carbon source to monitor degradation of an aromatic compound in TEP amended microcosms. Microscopic and viable counts were higher in TEP containing microcosms when compared to unamended controls. A significant increase in ribosomal activity was also observed for TEP amended samples as determined by the number of cells hybridizing to an eubacterial probe. In addition, the number of beta and gamma Proteobacteria increased from undetectable levels prior to the study to 15-29% of the total bacteria in microcosms containing TEP and benzene. In these microcosms, nearly 40% of the benzene was degraded during the incubation period compared to less than 5% in unamended microcosms. While TEP has previously been used as an alternate phosphate source in the bioremediation of chlorinated aliphatics, this study shows that it can also stimulate the microbial degradation of aromatics in phosphate limited aquifers. (C) 1997 Published by Elsevier Science B.V.; copyright held by the U.S. government.
  287. Radway, Joann c., Jorge W. Santo Domingo, Christopher J. Berry, Ed W. Wilde and Terry C. Hazen. 1997. Degradation of Trichloroethylene and Benzene by Embedded Burkholderia cepacia. In Situ and On Site Bioremediation 1:85. pdf
  288. Pfiffner, S. M., A. V. Palumbo, T. J. Phelps and Terry C. Hazen. 1997. Effects of nutrient dosing on subsurface methanotrophic populations and trichloroethylene degradation. Journal of Industrial Microbiology & Biotechnology 18:204-212. abstract pdf
    In in situ bioremediation demonstration at the Savannah River Site in Aiken, South Carolina, trichloroethylene-degrading microorganisms were stimulated by delivering nutrients to the TCE-contaminated subsurface via horizontal injection wells, Microbial and chemical monitoring of groundwater from 12 vertical wells was used to examine the effects of methane and nutrient (nitrogen and phosphorus) dosing on the methanotrophic populations and on the potential of the subsurface microbial communities to degrade TCE. Densities of methanotrophs increased 3-5 orders of magnitude during the methane- and nutrient-injection phases; this increase coincided with the higher methane levels observed in the monitoring wells, TCE degradation capacity, although not directly tied to methane concentration, responded to the methane injection, and responded more dramatically to the multiple-nutrient injection. These results support the crucial role of methane, nitrogen, and phosphorus as amended nutrients in TCE bioremediation. The enhancing effects of nutrient dosing on microbial abundance and degradative potentials, coupled with increased chloride concentrations, provided multiple lines of evidence substantiating the effectiveness of this integrated in situ bioremediation process.
  289. Lombard, K. H., T. C. Hazen, A. Worsztynowicz, B. Jagosz and Inst Battele Mem. 1997. Bioremediation techniques for the cleanup of a petroleum waste lagoon. In Situ and on-Site Bioremediation, Vol 5 4(5):467-467. pdf
  290. Kastner, J. R., K. H. Lombard, J. A. Radway, J. Santo Domingo, G. L. Burbage and Terry C. Hazen. 1997. Characterization using laser induced fluorescence and Bioremediation. In Situ and On-Situ Bioremediation 1:385-932. pdf
  291. Hazen, Terry C., A. Tien, K. H. Lombard, D. J. Altman and A. Worsztynowicz. 1997. Czechoqice Oil Refinery Bioremediation Demonstration Test Plan. pdf
  292. Hazen, Terry C., K. H. Lombard, B. B. Looney, M. V. Enzien, J. M. Dougherty, Carl B. Fliermans, J. Wear and C. A. Eddy-Dilek. 1997. Full Scale Demonstration of In Situ Bioremediation of Chlorinated Solvents in the Deep Subsurface Using Gaseous Nutrient Biostimulation. Progress in Microbial Ecology 597-604. pdf
  293. Hazen, Terry C.. 1997. Bioremediation. Microbiology of the Terrestrial Subsurface 247-266. pdf
  294. Hazen, Terry C.. 1997. Controlled Phosphate-Enhanced Bioremediation Tested. EPA Tech Trends 25:3-5. pdf
  295. Fliermans, C. B., M. M. Franck, T. C. Hazen and R. W. Gorden. 1997. Ecofunctional enzymes of microbial communities in ground water. Fems Microbiology Reviews 20:379-389. abstract pdf
    Biolog(TM) technology was initially developed as a rapid, broad spectrum method for the biochemical identification of clinical microorganisms. Demand and creative application of this technology has resulted in the development of Biolog(TM) plates for Gram-negative and Gram-positive bacteria, for yeast and Lactobacillus sp. Microbial ecologists have extended the use of these plates from the identification of pure culture isolates to a tool for quantifying the metabolic patterns of mixed cultures, consortia and entire microbial communities. Patterns that develop on Biolog(TM) microplates are a result of the oxidation of the substrates by microorganisms in the inoculum and the subsequent reduction of the tetrazolium dye to form a color in response to detectable reactions. Depending upon the functional enzymes present in the isolate or community one of a possible 4 x 10(28) patterns can be expressed. The patterns were used to distinguish the physiological ecology of various microbial communities present in remediated groundwater. The data indicate that one can observe differences in the microbial community among treatments of bioventing, 1% and 4% methane injection, and pulse injection of air, methane and nutrients both between and among wells. The investigation indicates that Biolog(TM) technology is a useful parameter to measure the physiological response of the microbial community to perturbation and allows one to design enhancement techniques to further the degradation of selected recalcitrant and toxic chemicals. Further it allows one to evaluate the recovery of the microbial subsurface ecosystem after the perturbations have ceased. We propose the term 'ecofunctional enzymes' (EFE) as the most descriptive and useful term for the Biolog(TM) plate patterns generated by microbial communities. We offer this designation and provide ecological application in an attempt to standardize the terminology for this relatively new and unique technology.
  296. Carrasco, C. E., H. J. Alvarez, N. Ortiz, M. Bishal, W. Arias, J. W. Santo Domingo and Terry C. Hazen. 1997. Multiple antibiotic resistant Escherichia coli from a tropical rain forest stream in Puerto Rico. Caribbean Journal of Science 33:191-197. abstract pdf
    The resistance to antibiotics and presence of plasmids in fecal coliforms isolated from the tropical rain forest of El Yunque, Puerto Rico, was studied. Densities of fecal coliforms obtained from a pristine site and a sewage contaminated site in this forest's watershed were higher than maximum levels allowed for recreational waters. Approximately 70% of the fecal coliform isolates were identified as Escherichia coli. Multiple antibiotic resistance (MAR) was common for isolates at both sites; however, the site receiving sewage effluent had a greater proportion of MAR isolates. Antibiotic resistance (R) plasmids were recovered from MAR isolates of each site. All recovered plasmids were approximately 1 kilobase. The recovered plasmids seemed capable of transforming E. coli HB101 in vitro. The high concentrations of enteric bacteria, R plasmid mobility, and documented long term survival of fecal bacteria in tropical freshwater environments give increasing importance to adequate sewage treatment and to better methods to monitor bacterial indicators of fecal contamination for tropical areas.
  297. E. W. Wilde, j. C. Radway, T. C. Hazen and P. Hermann. 1996. Immobilization of degradative bacteria in polyurethane-based foams: embedding efficiency and effect on bacterial activity. DOE Report pdf
  298. Radway, J. C., K. H. Lombard and Terry C. Hazen. 1996. Final Technology Report for D-Area Oil Seepage Basin Bioventing Optimization Test. Environmental Restoration pdf
  299. Hazen, T. C., K. H. Lombard, B. B. Looney, M. V. Enzien, J. M. Dougherty, Carl B. Fliermans, J. Wear and C. A. Eddy-Dilek. 1996. Innovative Site Characteristics of Microbes. IGT Biotech Conference pdf
  300. Terry C. Hazen. 1996. Sanitary Landfill in situ bioremediation optimization test final report. abstract pdf
    The Savannah River Site (SRS) is a 320 square mile facility located in a rural area along the Savannah River, principally in the Aiken and Barnwell counties of South Carolina. The SRS is approximately 25 miles southeast of Augusta, Georgia, and 20 miles south of Aiken, South Carolina. The SRS is owned by the U.S. Department of Energy and operated by Westinghouse Savannah River Company. SRS has been in operation since 1950 with the mission to produce nuclear materials for national defense, medical, research, and space exploration. It has had 5 production nuclear reactors and 1 pilot scale reactor and all of the associated construction, fuel fabrication, processing, and waste handling operations associated with these activities during the last 40+ years. These operations and the people that worked at the site (as many as 50,000 during the early construction phases) generated large amounts of solid sanitary waste. During the first 20 years most of this waste was handled via burning rubble pits near major construction sites at SRS. In the early 1970's, these areas were consolidated into a single sanitary landfill located near the center of SRS, on Road C near Upper Three Runs Creek. SRS Sanitary Landfill began receiving solid waste from site construction areas, offices, shops, and cafeterias in 1974 in its original 32 acre site. In 1987, as the original area reached its capacity, a 16-acre Northern Expansion and a 22-acre Southern Expansion were added. The Southern Expansion was filled and ceased operations in 1993. The Northern Expansion, also known as the Interim Sanitary Landfill (ISL) continued to receive SRS solid waste until October 1994. Though the ISL is still permitted to receive waste, it now only accepts special waste on a case by case basis and is rigorously controlled to ensure that hazardous waste is not accepted. During the course of its operation, the Sanitary Landfill received numerous materials that can leach or generate hazardous compounds, eg. paints, thinners, solvents, batteries, and rags and wipes used with F-listed solvents. The sanitary landfill was operated using the burrow and cover technique. Burrows were dug, waste was placed in the ditch and then covered with soil. Wastes were cataloged but not segregated within the landfill. In 1988, as a result of recurring evidence of hazardous constituents in the groundwater beneath the site, the Sanitary Landfill was designated as a Resource Conservation and Recovery Act (RCRA) Solid Waste Management Unit. In December 1989, the SRS was added to the National Priority List (NPL). At the time, the Sanitary Landfill was included in a combined RCRA/Comprehensive Environmental Response, Compensation and Liability Act (CERCLA) unit list in the Federal Facilities Agreement (FFA). As a result of an ongoing RCRA permit investigation, the U.S. Environmental Protection Agency (EPA) removed the Sanitary Landfill from the combined RCRA/CERCLA unit list on August 29, 1991. The DOE and the South Carolina Department of Health and Environmental Control (SCDHEC) reached a settlement agreement (SW-91-51) in August 1991 outlining the steps that DOE would take to comply with the RCRA regulations. Principally, the DOE would close the portions of the landfill containing the solvent rags in compliance with Subpart G (Closure and Postclosure) of Part 265 (Interim Status Standards for Owners and Operators of Hazardous Waste Treatment Storage and Disposal Facilities) of the South Carolina Hazardous Waste Management Regulation (SCHWMR). The settlement agreement also states that the DOE shall submit a RCRA Postclosure Part B Permit Application on March 31, 1993 (WSRC 1993a), for the portions of the landfill that received the solvent rags. The RCRA Postclosure Part B Permit Application, submitted on March 31, 1993, contained an Alternate Concentration Limit (ACL) Demonstration. On March 31, 1994 a Corrective Action Plan (CAP) based on the assumption that the ACL Demonstration would be approved was submitted to SCDHEC which addressed corrective actions to remediate the groundwater at the Sanitary Landfill. Based on an evaluation of groundwater analytical data for the period of 1984 through 1993 (up to and including 2Q93), as described in the CAP, the GWPS has been exceeded at or downgradient of the Point of Compliance (POC) for vinyl chloride (VC) and trichloroethylene (TCE). As part of the CAP, Westinghouse Savannah River Company Environmental Restoration Department (WSRC-ER) subcontracted Camp Dresser and Mckee Federal to do an Interim Technology Screening Report for evaluating remediation of contaminated groundwater and vadose zone (WSRC-RP-93-892) using EPA guidance (EPA/540/G-89/004). The vadose zone evaluation report determined that "No action" was necessary. The groundwater report evaluated more than 100 process options for groundwater remediation. The initial screening for ease of implementation reduced the options to 40. The second screening evaluating each technology for: 1) overall protection of human health and the environment, 2) compliance with applicable or relevant and appropriate requirements, 3) long-term effectiveness, 4) reduction of toxicity, mobility, or volume, 5) short-term effectiveness, 6) ease of implementation, and 7) cost. Eight alternatives made it through the second screening. Of these eight, aerobic in situ bioremediation was ranked the highest and deemed the most appropriate for the SRS Sanitary Landfill. Previous studies and on-going demonstrations at SRS had shown that normal soil bacteria are capable of degrading chlorinated solvents in situ if they are stimulated with oxygen and additional nutrients. In situ biodegradation is a highly attractive technology for remediation because contaminants are destroyed in place, not simply moved to another location or immobilized, thus decreasing costs, risks, and time, while increasing efficiency and public and regulatory acceptability. Bioremediation has been found to be among the least costly technologies in applications where it is feasible. Full scale demonstrations of this technology have already been completed as part of the SRS Integrated Demonstration at a solvent disposal basin system in M-area (Hazen, 1994). Because the M basin differed from the Sanitary Landfill in having only TCE and tetrachloroethylene (PCE), no other waste disposal, and a groundwater that was only aerobic (> 2 mg/L dissolved oxygen), it was decided that a treatability study was prudent for the Sanitary Landfill. The nine week bench-scale treatability test was done to determine: 1) if the contaminants of concern (COC), (VC, TCE, and chlorobenzene) were biodegradable in the specific soil and groundwater samples. This included determining if pretreatment was necessary to dilute inhibitory compounds, 2) the rates of biodegradation of the COCs, 3) the extent of contaminant biodegradation, and 4) the optimal conditions for biodegradation, including nutrient optimization and choice of inoculum. The treatability study using soil columns to simulate both vadose and groundwater conditions used soil and groundwater from the most contaminated area of the Sanitary Landfill (WSRC-TR-94-0119). These studies showed that all of the COCs were biodegradable by indigenous soil bacteria and that their ability to degrade the COCs to undetectable levels greatly exceeded the highest concentrations found at the Sanitary Landfill. The soil column simulations showed that the biostimulated soil microbes could reduce more than 100,000 ppb of the contaminants in water to undetectable levels in just a few days (the highest concentrations observed at the Landfill has been 100 ppb). The treatability study showed that the COCs were biodegraded in both the saturated and unsaturated soil columns. The major limitation to soil microbes at the SRS Sanitary Landfill was oxygen, supplemental carbon sources (methane), and trace nutrients (phosphorus and nitrogen), in that order. Historical groundwater data and landfill usage information confirmed that there existed two separate plumes of concern. One plume contained TCE as its major contaminant of concern and the other plume contained VC as its major constituent. Because these two plumes were also quite different in terms of dissolved oxygen concentration, total organic, and other trace nutrients a pilot-scale optimization test was deemed necessary to determine the best strategy for both plumes and also to gather critical physical and chemical information as input for the final remediation system for the two parts of the landfill. This pilot-scale optimization test is the focus of this report. The optimization test objectives were to determine the optimum design parameters for full-scale operation including: 1) radial air/methane flow patterns in the saturated and vadose zones, 2) attainable radius of influence (ROI) for the various injection pressures and vacuum pressures in the saturated and vadose zones, 3) the need for hydraulic controls to prevent outward spreading of contaminants from the sparge wells, 4) air injection/extraction flow rates associated various injection/extraction pressures, 5) air/methane injection rates that optimize biodegradation of chlorinated solvents in both the saturated and vadose zones, 6) biodegradation rates for trichloroethylene, vinyl chloride, chlorobenzene, and methane in the vadose and saturated zones under various test conditions, 7) identification of densities of methanotrophs and chlorobenzene degraders present at sites 1 and 2, 8) the rate of trichloroethylene, vinyl chloride, chlorobenzene, and methane loss under test conditions, and 9) the feasibility of using vertical air injection wells (AIW) for full-scale treatment. Two sites were field tested adjacent to the Sanitary Landfill Southern Expansion. Each site was set up with 18 sampling and injection wells. Three air injection wells were positioned to provide overlapping 20 ft radii and a vacuum was applied to a central air sampling well to provide a controlled air sampling ROI. Air, nutrients (nitrous oxide and triethyl phosphate), and methane were introduced sequentially to determine the ability of each nutrient to stimulate bioremediation. The test system’s subsurface component installation, (i.e. injection screen zone and saturated zone piezometer locations) and the local soil structure exhibited a highly sensitive relationship for a pressure vs. flow threshold as defined for the subsurface soil composition of the Sanitary Landfill (DCN; 5112-005-RP-BFBC). The estimated hydraulic conductivity in the saturated zone at site 1 was 2.30E-04 m/s, while site 2 was 9.97E-05 m/s. These estimated hydraulic conductivity values correlate well with the values presented by Freeze and Cherry (1989). In addition, the estimated hydraulic conductivity values also correlate well with the values obtained for the 1994 field permeability testing of the “D” level wells at the Sanitary Landfill (WSRC-TR-94-0263). The lowest recommended hydraulic conductivity for biostimulation using liquid nutrient injection is 10E-6 m/s and for gaseous nutrient injection or extraction 10E-11 m/s (Baker and Herson, 1990). Pressures below the screen zone head pressure threshold were not great enough to force air into the groundwater, but once the threshold was exceeded, (> 10.5 psig at site 1 and > 6 psig at site 2) the pressure/flow characters immediately exhibited biosparging characteristics, as evidenced by air flow out of associated saturated zone piezometers and pressure differentials in the unsaturated zone piezometers. In addition, due to the ease of air injection from limited head pressures and fluid sand conditions in the screen zones the biosparging regime operated in a narrow pressure/flow range, (± 1 psig). Increases above that range created preferential flow paths between the injection wells and associated saturated zone sampling points (i.e. piezometers) thereby short circuiting biosparging. This phenomena for the proposed full-scale remediation system is very unlikely to occur due to its increased depths, lengths and distances to existing monitoring wells and newly designed sampling and monitoring points associated with the proposed system. Site 1 and Site 2 were also significantly different in terms of COCs, dissolved oxygen, chloride, nitrite, and nitrate concentrations, and response to nutrient stimulation, thus each site is considered separately. Overall, both sites were found to have indigenous microorganisms that could be stimulated to degrade chlorobenzenes, trichloroethylene and, its daughter product, vinyl chloride in situ by the addition of oxygen (as compressed air), nutrients, and methane to the contaminated zone. Biostimulation at both sites resulted in undetectable levels of COCs and many other organics in both the groundwater and vadose zone. It was also shown that chloride concentrations in the groundwater at both sites increased significantly as bacteria densities increased. This correlation shows that biodegradation of chlorinated solvents in situ was complete and resulted in production of chloride. Site 1 had lower levels of carbon, lower levels of natural biological activity, higher oxygen levels, and TCE as the COC. This coincides well with the newer refuse source components in this part of the landfill. The groundwater dissolved oxygen (DO) was at or below 20% saturation normally; however, it could be raised to >95% saturation after only 5 h of air injection. Once the air injection was shut off, the DO saturation returned to < 20% in 4 h. TCE concentration did not change when air alone was the stimulus. When gaseous nutrients were added to the air some decrease in TCE concentration was observed; however, when methane was also added to the nutrient air mixture, the TCE concentration in all affected wells declined to non detect levels (<2 ppb). After the air/nutrient/methane injection was ceased the TCE was detectable in 7 days and reached low pre-injection levels within 3-4 weeks. Biodegrader densities increased only slightly during air alone injection, but increased 2-3 orders of magnitude after air/nutrient/methane injection was started. The densities of biodegraders slowly declined over the course of the campaign. After several weeks the densities of biodegraders still had not reached pre-injection levels. Statistical analyses showed that there was a significant positive correlation between DO and biodegrader density, i.e. as the DO increased, the number of bacteria increased. Nitrate was low in shallow wells and high in deeper wells. Conversely nitrite was higher in shallow wells and low in deep wells. In addition, nitrite could not be detected while air was being injected. Since nitrate is required nutrient for biological activity and nitrite is a daughter product of denitrification under anaerobic conditions, this suggests that the shallow wells have higher amounts of total biological activity and that air injection can create bulk aerobic conditions at this site. All of the data from this site demonstrate that oxygen is limiting to the biodegraders at this site, but that air injection alone is insufficient to affect bioremediation of the site. Carbon, nitrogen and phosphate must be supplied to bioremediate the site to non detect concentrations. Biodegrader activity at this site can be maintained at a level effective for groundwater bioremediation by pulsed injection of gaseous nutrients and a carbon source. Monthly groundwater monitoring should be sufficient to maintain an appropriate pulse schedule. Site 2, as compared to site 1, had lower DO (<15% saturation), higher chloride, nitrite, and total carbon concentrations, and VC and chlorobenzenes as COCs. This again reflects the nature of the point source as being refuse that was put in the landfill many years earlier than site 1. This has allowed more leaching and thus more biological activity which created the VC from TCE under anaerobic conditions caused by the higher carbon content. This higher biological oxygen demand was well demonstrated at site 2 by the respiration experiment which showed that air injection could only increase the DO saturation from 15 to 30%. After the air injection was stopped the DO saturation slowly returned to pre-injection levels after 24 h. All of the chlorinated solvents declined significantly with air alone injection reaching non detect very quickly. The chlorobenzenes declined after nitrous oxide and triethyl-phosphate were also added to the gas injection. After air injection stopped COCs increased very slowly not reaching pre-injection concentrations for several weeks. Contaminant-degrader densities increased 2-3 orders of magnitude after air injection was started and declined slowly after air injection was stopped. Nitrite was undetectable when air was on and > 10 ppm when air was off. Chloride concentrations were always higher when air was being injected and increased concomitantly with increases in contaminant degraders and decreases in chlorinated solvents. These studies show that air injection at this site can stimulate contaminant degraders to completely mineralize contaminants to non-detect levels. They also show that enough co-metabolic carbon is present in this environment that methane injection is not necessary. Air injection increases the DO concentration enough to stop anaerobic process as evidenced by the non-detection of nitrite, but not enough to saturate the environment. Monthly monitoring and pulsed injection of air with occasional nitrogen & phosphorous gaseous supplements should be all that is necessary to maintain complete bioremediation of solvents at this site. The final remediation system should incorporate 2 injection zones along the south and west sides of the landfill, respectively. Since groundwater consistently flows parallel to the long axis of the SRS landfill two horizontal wells, one running along the south side of the southern expansion and the other along the west side should be able to bioremediate any solvents coming from the site. Based on the optimization test and probable future leaching changes both injection systems should inject at a depth of 20-30 ft below the water table. This will provide a sparge zone that will biotreat all current and future leachate since the proposed configuration and prevailing groundwater flow would contain any leachate from these areas. Cost analysis will determine if horizontal wells or a series of vertical injection wells are most appropriate. The injection system will consist of a compressor with the ability to add nitrous oxide, triethyl-phosphate, and methane. The south side injection will need to be controlled separately from the west side injection, since different strategies will be necessary for the most cost effective in situ bioremediation. Both wells will, however, need all capabilities since conditions may change as the landfill ages. The results of the Bioremediation Optimization Test have shown that the use of bioremediation via in situ stimulation of indigenous microorganisms is an efficient and cost effective long-term means of obtaining ultimate groundwater restoration at the SRS Sanitary Landfill.
  301. Alvarez, A. J., G. M. Yumet, C. L. Santiago, Terry C. Hazen, R. Chaudhry and G. A. Toranzos. 1996. In situ survival of genetically engineered microorganisms in a tropical aquatic environment. Environmental Toxicology and Water Quality 11:21-25. abstract pdf
    In this study, the survival of genetically engineered microorganisms (GEMs) and their interactions with the environmental microbiota of a tropical river was investigated. Diffusion chambers were used for the in situ survival experiments with a nonplasmid containing Escherichia coli DH1 strain and two model GEMs, E. coli JM103 containing a 2.6 kilobase plasmid (pUCS) and E. coli DH1 with a 4.8 kb plasmid (pWTAla5'). Pure culture survival studies indicated that after a week in the environment a 1.0 log(10) decrease in bacterial numbers occurred for both E. coli DH1, while a 3.0 log(10) reduction was observed for E. coli JM103. However, a reduction of 4.0 log(10) was observed for the E. coli DH1 (pWTAla5') when placed in a chamber conjointly with the resident microbiota. The data suggest that the presence of a plasmid makes no difference on the survival time of GEMs, whereas the presence of competing bacteria is ultimately what limits the survival time of GEMs in the environment. (C) 1996 by John Wiley & Sons, Inc.
  302. Legrand, R., R. R. Baker, T. D. Hayes, R. F. Hickey, Terry C. Hazen and P. J. Petit. 1995. The methanotrophic fluidized bed bioreactor: from laboratory to field demonstration at the Savannah River site.. Proceedings Air & Waste Management Association 88th meeting 95-RA127.02:1-13:1-13. pdf
  303. Hazen, Terry C.. 1995. Savannah River Site a test bed for cleanup technologies. Environmental Protection abstract pdf
    The effort to develop faster and cheaper ways to clean up the environment can be divided into three basic steps. Between the conceptual spark for developing innovative technology to accomplish the task and performing the actual cleanup lies what is arguably the most crucial step: demonstrating, evaluating and fine-tuning the cleanup method. The Savannah River Site (SRS), a U.S. Department of Energy facility near Aiken, S.C., provides an ideal proving ground for fulfilling that second step - testing innovative technologies to clean soil and groundwater contaminated with volatile organic compounds (VOCs).
  304. Brockman, F. J., W. Payne, D. J. Workman, A. Soong, S. Manley and T. C. Hazen. 1995. Effect of Gaseous Nitrogen and Phosphorus Injection on in-Situ Bioremediation of a Trichloroethylene-Contaminated Site. Journal of Hazardous Materials 41:287-298. abstract pdf
    Methane and air were injected through a horizontal well into a trichloroethylene-contaminated site at a depth of 160ft below ground surface to stimulate methanotrophic biodegradation of trichloroethylene (TCE). Sediment samples were analyzed after 35 weeks of methane and air injection, and after 13 weeks of methane and air injection supplemented with injection of the gases nitrous oxide and triethyl phosphate. Methanotroph most-probable-number (MPN) values were very low in most of the samples prior to the addition of nitrogen and phosphorus to the site, and increased several orders of magnitude following the addition, Similarly, the frequency of TCE biodegradative potential in methanotrophic enrichments increased approximately three orders of magnitude after the addition of nitrogen and phosphorus to the site. The MPN and biodegradative potential data indicated that the zone of influence after the addition of nitrogen and phosphorus extended to at least 60 ft from the injection well in both the vertical and horizontal directions.
  305. Morrissey, C. M., S. E. Herbes, O. M. West, A. V. Palumbo, T. J. Phelps and Terry C. Hazen. 1994. Use of Laboratory Soil columns to Optimize in situ Biotransformation of Tetachloroethylene. Applied Biotechnology for Site Remediation 326-331. pdf
  306. McKay, Danny J., J. S. Morse and Terry C. Hazen. 1994. Biodegradation of Trichloroethylene by Alcaligenes eutrophus Jmp134 in a Laboratory-Scale Bioreactor. Hazardous Waste & Hazardous Materials 11:491-499. abstract pdf
    A single stage recirculating bioreactor with a pure culture of Alcaligenes eutrophus JMP134 and a packed gravel bed was operated for a two week period during which a maximum biodegradation of 88.4% of the influent trichloroethylene was observed with average performance of 71.8% at 8.4 hour hydraulic retention time. The reactor was then operated for a seven day period with the gravel bed removed, demonstrating a maximum degradation of 97.4% and an average of 95.6%. Average influent and effuent concentrations for the second case were 5.97 mg/l and 145 mug/l with a mean retention time of 14.1 hours. Phenol, supplied as the sole source of carbon and energy, was degraded below levels of detection (< 1.6 muM) in the effuent.
  307. Lombard, K. H., J. Borthen and Terry C. Hazen. 1994. Design and Configuration Management of System Control Components for in situ Methanotrophic Bioremediation of Groundwater and Sediment Contaminated with Chlorinated Hydrocarbons. Air Sparging for Site Remediation 81-96. pdf
  308. Lombard, K. and T. Hazen. 1994. A petroleum contaminated soil bioremediation facility. abstract pdf
    The amount of petroleum contaminated soil (PCS) at the Savannah River site (SRS) that has been identified, excavated and is currently in storage has increased several fold during the last few years. Several factors have contributed to this problem: (1) South Carolina Department of Health ad Environmental control (SCDHEC) lowered the sanitary landfill maximum concentration for total petroleum hydrocarbons (TPH) in the soil from 500 to 100 parts per million (ppm), (2) removal and replacement of underground storage tanks at several sites, (3) most recently SCDHEC disallowed aeration for treatment of contaminated soil, and (4) discovery of several very large contaminated areas of soil associated with leaking underground storage tanks (LUST), leaking pipes, disposal areas, and spills. Thus, SRS has an urgent need to remediate large quantities of contaminated soil that are currently stockpiled and the anticipated contaminated soils to be generated from accidental spills. As long as we utilize petroleum based compounds at the site, we will continue to generate contaminated soil that will require remediation.
  309. Hazen, T. C., K. H. Lombard, B. B. Looney, M. V. Enzien, J. M. Dougherty, C. B. Fliermans, J. Wear and C. A. Eddydilek. 1994. Summary of Insitu Bioremediation Demonstration (Methane Biostimulation) Via Horizontal Wells at the Savannah River Site Integrated Demonstration Project. In Situ Remediation: Scientific Basis for Current and Future Technologies, Pts 1 and 2 137-150. pdf
  310. Hazen, Terry C.. 1994. Full-Scale Demonstration of In Situ Bioremediation of Chlorinated-Solvents at SRS. South Carolina Engineer pdf
  311. Hazen, Terry C.. 1994. Preliminary Technology Report for In Situ Bioremediation Demonstration (Methane Biostimulation) of the Savannah Rier Site Integrated Demonstration Project. pdf
  312. Fliermans, C. B., J. M. Dougherty, M. M. Franck, P. C. McKinzey, J. E. Wear and T. C. Hazen. 1994. Immunological Techniques as Tools to Characterize the Subsurface Microbial Community at a Trichloroethylene-Contaminated Site. Applied Biotechnology for Site Remediation 186-203. pdf
  313. Enzien, M. V., F. Picardal, Terry C. Hazen, R. G. Arnold and Carl B. Fliermans. 1994. Reductive Dechlorination of Trichloroethylene and Tetrachloroethylene under Aerobic Conditions in a Sediment Column. Applied and Environmental Microbiology 60:2200-2204. abstract pdf
    Biodegradation of trichloroethylene and tetrachloroethylene under aerobic conditions was studied in a sediment column. Cumulative mass balances indicated 87 and 90% removal for trichloroethylene and tetrachloroethylene, respectively. These studies suggest the potential for simultaneous aerobic and anaerobic biotransformation processes under bulk aerobic conditions.
  314. Cadotte, B. and T.C. Hazen. 1994. Nuclear into Environmental: The Transformation of Savannah River. ECON Magazine December 1994:30-31. pdf
  315. Hazen, T. C., B. B. Looney, M. Enzien, M. M. Franck, C. B. Fliermans and C. A. Eddy. 1993. In-Situ Bioremediation Via Horizontal Wells. Engineering Hydrology - Proceedings of the Symposium 862-867. pdf
  316. Hazen, Terry C., B. B. Looney, M. Enzien, J. M. Dougherty, J. Wear, Carl B. Fliermans and C. A. Eddy. 1993. In situ bioremediation via horizontal wells - IECD & ACS Conference. 247-250. abstract pdf
    The test consisted of methane mixed with air into the contaminated aquifer via a horizontal well and extraction from the vadose zone via a parallel horizontal well. This configuration has the advantage of simultaneously stimulating methanotrophic activity in both the groundwater and vadose zone, and inhibiting spread of the contaminant plume. Groundwater was monitored biweekly from 13 wells for a variety of chemical and microbiological parameters. Groundwater from wells in affected areas showed increases in methanotrophs of more than 1 order of magnitude every 2 weeks for several weeks after 1% methane-in-air injection was started. Some wells had increases as much as 7 orders of magnitude. Simultaneous with the increase in methanotrophs was a decrease in water and soil gas concentrations of trichloroethylene (TCE) and tetrachloroethane (PCE). Two wells declined in TCE/PCE concentration in the water by more than 90% to below 2 ppb. All of the wells in the affected zone showed significant decreases in contaminants in less than one month. Chloride concentrations in the water were inversely correlated with TCE/PCE concentration. Four of five vadose zone piezometers declined from concentration as high as 10,000 ppm to less than 5 ppm in less than 6 weeks. The fifth cluster also declined by more than 95%. After only three months on injection, a decline in TCE/PCE in the sediment of more than 30% was also observed, with TCE/PCE being undetectable in most sediments at the end of the 14-month test. Gene probes and direct isolation from the water and sediment revealed that the right types of methanotrophs were being stimulated and that isolates could degrade TCE at a high rate.
  317. Hazen, Terry C.. 1993. Operations Support of Phase 2 Integrated Demonstration In Situ Bioremediation. Vol 1-4. abstract pdf
    This report contains experimental data collected during the demonstration of in situ bioremediation at the Savannah River Site. This project was designed to demonstrate in situ bioremediation of ground water and sediment contaminated with chlorinated solvents. Indigenous microorganisms were stimulated to degrade trichloroethylene, tetrachloroethylene, and their daughter products in situ by addition of nutrients to the contaminated aquifer and adjacent vadose zone. The principle carbon/energy source nutrient used in this demonstration was methane. In situ biodegradation is a highly attractive technology for remediation because contaminants are destroyed, not simply moved to another location or immobilized, thus decreasing costs, risks, and time, while increasing efficiency, safety, and public and regulatory acceptability. This document consists solely of data acquired during phase 2 of the integrated demonstration project concerning in situ bioremediation performed at the Savannah River Site, Aiken, South Carolina. The data is presented in tabular form. This project was designed to demonstrate in situ bioremediation of ground water and sediment contaminated with chlorinated solvents. Indigenous microorganisms were stimulated to degrade trichlorethylene (TCE), tetrachloroethylene (PCE) and their daughter products in situ by addition of nutrients to the contaminated aquifer and adjacent vadose zone. The principle carbon/energy source nutrient used in this demonstration was methane (natural gas). In situ biodegradation is a highly attractive technology for remediation because contaminants are destroyed, not simply moved to another location or immobilized, thus decreasing costs, risks, and time, while increasing efficiency, safety, and public and regulatory acceptability. This report describes the preliminary results of the demonstration and provides conclusions only for those measures that the Bioremediation Technical Support Group felt were so overwhelmingly convincing that they do not require further analyses. Though this report is necessarily superficial it does intend to provide a basis for further evaluating the technology and for practitioners to immediately apply some parts of the technology. This document contains data collected during the demonstration of in situ bioremediation at Savannah River Site. This project was designed to demonstrate in situ bioremediation of ground water and sediment contaminated with chlorinated solvents. Indigenous microorganisms were stimulated to degrade trichloroethylene, tetrachloroethylene and their daughter products in situ by addition of nutrients to the contaminated aquifer and adjacent vadose zone. The principle carbon/energy source nutrient used in this demonstration was methane. In situ biodegradation is a highly attractive technology for remediation because contaminants are destroyed, not simply moved to another location or immobilized, thus decreasing costs, risks, and time, while increasing efficiency, safety, and public and regulatory acceptability.
  318. Gorden, Robert W., Terry C. Hazen and Carl B. Fliermans. 1993. Rapid Screening for Bacteria Capable of Degrading Toxic Organic-Compounds. Journal of Microbiological Methods 18:339-347. abstract pdf
    Routine procedures for isolating and characterizing microorganisms capable of degrading toxic chemicals are time consuming and labor intensive. The objective of this paper is to describe a new method for screening aerobic bacterial isolates and consortia that will rapidly determine metabolic capacity for various toxic chemicals and access the use of various substrates as inducers in the degradation process. This method uses the Biolog(R) multiwell plate technology - a four-step method that includes inoculation followed by incubation of a uniform suspension of cells into a microtiter plate - to test more than 40 bacterial isolates and mixtures against 30 target toxic chemicals. Several bacteria and consortia have been shown to degrade toxic chemicals at concentrations of 10-500 ppm. These results indicate that Biolog(R) GN and MT plates are useful tools for screening bacterial isolates and consortia for their ability to survive metabolize, and potentially degrade selected organic chemicals.
  319. Fliermans, Carl B., Terry C. Hazen and H. Bledsoe. 1993. Characterization and reclamation assessment for the Central Shops Diesel Storage Facility, Savannah River Site, Aiken, South Carolina. abstract pdf
    The contamination of subsurface terrestrial environments by organic contaminants is a global phenomenon. The remediation of such environments requires innovative assessment techniques and strategies for successful clean-ups. Central Shops Diesel Storage Facility at Savannah River Site was characterized to determine the extent of subsurface diesel fuel contamination using innovative approaches and effective bioremediation techniques for clean-up of the contaminant plume have been established.
  320. Eddy Dilek, C. A., B. B. Looney, Terry C. Hazen, R. L. Nichols, Carl B. Fliermans, W. H. Parker, J. M. Dougherty, D. S. Kaback and J. L. Simmons. 1993. Post-test evaluation of the geology, geochemistry, microbiology, and hydrology of the in situ air stripping demonstration site at the Savannah River Site. abstract pdf
    A full-scale demonstration of the use of horizontal wells for in situ air stripping for environment restoration was completed as part of the Savannah River Integrated Demonstration Program. The demonstration of in situ air stripping was the first in a series of demonstrations of innovative remediation technologies for the cleanup of sites contaminated with volatile organic contaminants. The in situ air stripping system consisted of two directionally drilled wells that delivered gases to and extract contamination from the subsurface. The demonstration was designed to remediate soils and sediments in the unsaturated and saturated zones as well as groundwater contaminated with volatile organic compounds. The demonstration successfully removed significant quantities of solvent from the subsurface. The field site and horizontal wells were subsequently used for an in situ bioremediation demonstration during which methane was added to the injected air. The field conditions documented herein represent the baseline status of the site for evaluating the in situ bioremediation as well as the post-test conditions for the in situ air stripping demonstration. Characterization activities focused on documenting the nature and distribution of contamination in the subsurface. The post-test characterization activities discussed herein include results from the analysis of sediment samples, three-dimensional images of the pretest and post-test data, contaminant inventories estimated from pretest and post-test models, a detailed lithologic cross sections of the site, results of aquifer testing, and measurements of geotechnical parameters of undisturbed core sediments.
  321. Bowman, J. P., L. Jimenez, I. Rosario, Terry C. Hazen and G. S. Sayler. 1993. Characterization of the Methanotrophic Bacterial Community Present in a Trichloroethylene-Contaminated Subsurface Groundwater Site. Applied and Environmental Microbiology 59:2380-2387. abstract pdf
    Groundwater, contaminated with trichloroethylene (TCE) and tetrachloroethylene (PCE), was collected from 13 monitoring wells at Area M on the U.S. Department of Energy Savannah River Site near Aiken, S.C. Filtered groundwater samples were enriched with methane, leading to the isolation of 25 methanotrophic isolates. The phospholipid fatty acid profiles of all the isolates were dominated by 18:1omega8c (60 to 80%), a signature lipid for group II methanotrophs. Subsequent phenotypic testing showed that most of the strains were members of the genus Methylosinus and one isolate was a member of the genus Methylocystis. Most of the methanotroph isolates exhibited soluble methane monooxygenase (sMMO) activity. This was presumptively indicated by the naphthalene oxidation assay and confirmed by hybridization with a gene probe encoding the mmoB gene and by cell extract assays. TCE was degraded at various rates by most of the sMMO-producing isolates, whereas PCE was not degraded. Savannah River Area M and other groundwaters, pristine and polluted, were found to support sMMO activity when supplemented with nutrients and then inoculated with Methylosinus trichosporium OB3b. The maximal sMMO-specific activity obtained in the various groundwaters ranged from 41 to 67% compared with maximal rates obtained in copper-free nitrate mineral salts media. This study partially supports the hypothesis that stimulation of indigenous methanotrophic communities can be efficacious for removal of chlorinated aliphatic hydrocarbons from subsurface sites and that the removal can be mediated by sMMO.
  322. Borthen, J., K. Meyer, Lombard and T.C. Hazen. 1993. Catalytic oxidation of trichloroethylene and perchloroethylene mixtures. pdf
  323. McCabe, D. J., A. W. Wiggins, M. R. Poirier and Terry C. Hazen. 1992. Biofouling of microfilters at the Savannah River Site F/H-Area Effluent Treatment Facility. Waste Management '92: Working Towards a Cleaner Environment. Waste Processing, Transportation, Storage and Disposal, Technical Programs and Public Education. Proceedings of the Symposium on Waste Management. Univ. Arizona. 1992, pp.1545-50 vol.2.. abstract pdf
    The F/H-Effluent Treatment Facility uses state-of-the-art water treatment processes to remove contaminants from low-level radioactive wastewater at the Savannah River Site. The plant replaces seepage basins that were closed to comply with the 1984 amendments to the Resource Conservation and Recovery Act (RCRA). The facility removes both radioactive and nonradioactive contaminants from the effluents originating from onsite waste management facilities. The unit processes involve filtration, ion exchange, activated carbon absorption, and reverse osmosis. The filtration step is prone to considerable fouling, reducing the overall throughput of the facility. The filters utilized in the process are Norton Ceraflo ceramic microfilters. It was discovered that bacteria were primarily responsible for the severe filter fouling. Inorganic fouling was also observed, but was not normally as severe as the bacterial fouling. The bacteria densities necessary to induce severe fouling were not significantly higher than those often found in surface water streams. Diversion of waste streams containing the highest quantity of bacteria, and various methods of source reduction were implemented, which dramatically improved the filter performance. Addition of aluminum nitrate at low pH further improved the filter performance. (2 References).
  324. Lombard, K. H., J. W. Borthen and Terry C. Hazen. 1992. The design and management of system components for in situ methanotrophic bioremediation of chlorinated hydrocarbons. abstract pdf
    The successful operation of an in situ bioremediation system is inherent within its design. Well-organized system components enable ease of maintenance, limited down time, and relatively rapid data acquisition. The design effort in this project focused on injection of a low-pressure air/methane mixture into a horizontal well below the water table, a methane-blending system that provided control of the injected mixture, redundant safety interlocks, vapor-phase extraction from a second horizontal well, and an off-gas treatment system that provided efficient thermal catalytic oxidation of the extracted contaminant vapors. The control instrumentation provided sufficient redundancies to allow the system to remain in operation in the event of a component failure, and equally important, the safe shut down of the system should any designed safety parameters be exceeded (i.e., high methane concentration). Final design approval took into consideration the reliability of the equipment and the components specified. Product knowledge and proper application limited the risk of a component or system failure while providing a safe, efficient, and cost-effective remediation system. Microprocessor data acquisition and system control were integrated with an autodialer to provide 24 hr emergency response and operation without on-site supervision. This integrated system also insured accurate data analysis and minimum downtime. Since operations commenced, the system has operated a total of 7,760 hours out of the possible 8,837 hours available. This equates to an operating efficiency of 87.8%.
  325. Kaback, D. S., B. B. Looney, C. A. Eddy and Terry C. Hazen. 1992. Innovative ground water and soil remediation: In situ air stripping using horizontal wells Fifth national outdoor action conference on aquifer restoration, ground water monitoring, and geophysical methods. abstract pdf
    An innovative environmental restoration technology, in situ air stripping, has been demonstrated at the US Department of Energy's (DOE) Savannah River Site (SRS) in South Carolina. This process, using horizontal wells, is designed to concurrently remediate unsaturated-zone soils and ground water containing volatile organic compounds (VOCs). In situ technologies have the potential to substantially reduce costs and time required for remediation as well as improve effectiveness of remediation. Horizontal wells were selected to deliver and extract fluids from the subsurface because their geometry can maximize the efficiency of a remediation system and they have great potential for remediating contaminant sources under existing facilities. The in situ air stripping concept utilizes two parallel horizontal wells: one below the water table and one in the unsaturated zone. The deeper well is used as a delivery system for the air injection. VOCs are stripped from the ground water into the injected vapor phase and are removed from the subsurface by drawing a vacuum on the shallower well in the vadose zone. The first demonstration of this new technology was conducted for a period of twenty weeks. A vacuum was first drawn on the vadose zone well until a steady-state removal of VOCs was obtained. Air was then injected at three different rates and at two different temperatures. An extensive characterization program was conducted at the site and an extensive monitoring network was installed prior to initiation of the test. Significant quantities of VOCs have been removed from the subsurface (equivalent to an eleven-well 500 gpm pump-and-treat system at the same site). Concentrations of VOCs in the ground water have been significantly reduced in a number of the monitoring wells. In addition, the activity of indigenous microorganisms was increased as much as two orders of magnitude during the air injection.
  326. Siler, J. L., M. R. Poirier, D. J. McCabe and Terry C. Hazen. 1991. Fouling of ceramic filters and thin-film composite reverse osmosis membranes by inorganic and bacteriological constituents. abstract pdf
    Two significant problems have been identified during the first three years of operating the Savannah River Site Effluent Treatment Facility. These problems encompass two of the facility's major processing areas: the microfiltration and reverse osmosis steps. The microfilters (crossflow ceramic filters {minus}0.2{mu} nominal pore size) have been prone to pluggage problems. The presence of bacteria and bacteria byproducts in the microfilter feed, along with small quantities of colloidal iron, silica, and aluminum, results in a filter foulant that rapidly deteriorates filter performance and is difficult to remove by chemical cleaning. Processing rates through the filters have dropped from the design flow rate of 300 gpm after cleaning to 60 gpm within minutes. The combination of bacteria (from internal sources) and low concentrations of inorganic species resulted in substantial reductions in the reverse osmosis system performance. The salt rejection has been found to decrease from 99+% to 97%, along with a 50% loss in throughput, within a few hours of cleaning. Experimental work has led to implementation of several changes to plant operation and to planned upgrades of existing equipment. It has been shown that biological control in the influent is necessary to achieve design flowrates. Experiments have also shown that the filter performance can be optimized by the use of efficient filter backpulsing and the addition of aluminum nitrate (15 to 30 mg/L Al{sup 3+}) to the filter feed. The aluminum nitrate assists by controlling adsorption of colloidal inorganic precipitates and biological contaminants. In addition, improved cleaning procedures have been identified for the reverse osmosis units. This paper provides a summary of the plant problems and the experimental work that has been completed to understand and correct these problems.
  327. McCabe, D. J., A. W. Wiggins, M. R. Poirier and Terry C. Hazen. 1991. Biofouling of microfilters at the Savannah River Site F/H-Area Effluent Treatment Facility. abstract pdf
    The F/H-Effluent Treatment Facility uses state-of-the-art water treatment processes to remove contaminants from low-level radioactive wastewater at the Savannah River Site. The plant replaces seepage basins that were closed to comply with the 1984 amendments to the Resource Conservation and Recovery Act (RCRA). The facility removes both radioactive and nonradioactive contaminants from the effluents orginating from onsite waste management facilities. The unit processes involve filtration, ion exchange, activated carbon absorption, and reverse osmosis. The filtration step is prone to considerable fouling, reducing the overall throughput of the facility. The filters utilized in the process are Norton Ceraflo{trademark} ceramic microfilters. It was discovered that bacteria were primarily responsible for the severe filter fouling. Inorganic fouling was also observed, but was not normally as severe as the bacterial fouling. The bacteria densities necessary to induce severe fouling were not significantly higher than those often found in surface water streams. Diversion of waste streams containing the highest quantity of bacteria, and various methods of source reduction were implemented, which dramatically improved the filter performance. Addition of aluminum nitrate at low pH further improved the filter performance.
  328. Jimenez, L., G. Lopez de Victoria, J. Wear, Carl B. Fliermans and T. C Hazen. 1991. Molecular analysis of deep subsurface bacteria. Proceedings of the First International Symposium on Deep Terrestrial Subsurface Microbiology pdf
  329. Hazen, Terry C., L. Jimenez and S. Pfiffner. 1991. Isolation of Microbial DNA from groundwater environments.. Proceedings of the First International Symposium on Deep Terrestrial Subsurface Microbiology pdf
  330. Hazen, Terry C., L. Jimenez, G. L. Devictoria and Carl B. Fliermans. 1991. Comparison of Bacteria from Deep Subsurface Sediment and Adjacent Groundwater. Proceedings of the First International Symposium on Deep Terrestrial Subsurface Microbiology abstract pdf
    Samples of groundwater and the enclosing sediments were compared for densities of bacteria using direct (acridine orange direct staining) and viable (growth on 1% PTYG medium) count methodology. Sediments to a depth of 550 m were collected from boreholes at three sites on the Savannah River Site near Aiken, South Carolina, using techniques to insure a minimum of surface contamination. Clusters of wells screened at discreet intervals were established at each site. Bacterial densities in sediment were higher, by both direct and viable count, than in groundwater samples. Differences between direct and viable counts were much greater for groundwater samples than for sediment samples. Densities of bacteria in sediment ranged from less than 1.00 x 10(6) bacteria/g dry weight (gdw) up to 5.01 x 10(8) bacteria/gdw for direct counts, while viable counts were less than 1.00 x 10(3) CFU/gdw to 4.07 x 10(7) CFU/gdw. Bacterial densities in groundwater were 1.00 x 10(3)-6.31 x 10(4) bacteria/ml and 5.75 - 4.57 x 10(2) CFU/ml for direct and viable counts, respectively. Isolates from sediment were also found to assimilate a wider variety of carbon compounds than groundwater bacteria. The data suggest that oligotrophic aquifer sediments have unique and dense bacterial communities that are attached and not reflected in groundwater found in the strata. Effective in situ bioremediation of contamination in these aquifers may require sampling and characterization of sediment communities.
  331. Hazen, Terry C.. 1991. Test plan for in situ bioremediation demonstration of the Savannah River Integrated Demonstration Project DOE/OTD TTP No.: SR 0566-01. abstract pdf
    This project is designed to demonstrate in situ bioremediation of groundwater and sediment contaminated with chlorinated solvents. Indigenous microorganisms will be simulated to degrade trichloroethylene (TCE), tetrachloroethylene (PCE) and their daughter products in situ by addition of nutrients to the contaminated zone. in situ biodegradation is a highly attractive technology for remediation because contaminants are destroyed, not simply moved to another location or immobilized, thus decreasing costs, risks, and time, while increasing efficiency and public and regulatory acceptability. Bioremediation has been found to be among the least costly technologies in applications where it will work.
  332. Fliermans, Carl B. and Terry C. Hazen. 1991. Proceedings of the First International Symposium on Deep Terrestrial Subsurface Microbiology. Proceedings of the First International Symposium on Deep Terrestrial Subsurface Microbiology pdf
  333. Eddy, C. A., B. B. Looney, J. M. Dougherty, Terry C. Hazen and D. S. Kaback. 1991. Characterization of the geology, geochemistry, hydrology and microbiology of the in-situ air stripping demonstration site at the Savannah River Site. abstract pdf
    The Savannah River Site is the location of an Integrated Demonstration Project designed to evaluate innovative remediation technologies for environmental restoration at sites contaminated with volatile organic contaminants. This demonstration utilizes directionally drilled horizontal wells to deliver gases and extract contaminants from the subsurface. Phase I of the Integrated Demonstration focused on the application and development of in-situ air stripping technologies to remediate soils and sediments above and below the water table as well as groundwater contaminated with volatile organic contaminants. The objective of this report is to provide baseline information on the geology, geochemistry, hydrology, and microbiology of the demonstration site prior to the test. The distribution of contaminants in soils and sediments in the saturated zone and groundwater is emphasized. These data will be combined with data collected after the demonstration in order to evaluate the effectiveness of in-situ air stripping. New technologies for environmental characterization that were evaluated include depth discrete groundwater sampling (HydroPunch) and three-dimensional modeling of contaminant data.
  334. Kaback, D. S., B. B. Looney, C. A. Eddy and Terry C. Hazen. 1990. Ground water and soil remediation: In situ air stripping using horizontal wells. abstract pdf
    An innovative environmental restoration technology, in situ air stripping, has been demonstrated at the US Department of Energy (DOE) Savannah River Site (SRS) in South Carolina. This process, using horizontal wells, is designed to concurrently remediate unsaturated-zone soils and ground water containing Volatile Organic Compounds (VOC). In situ technologies have the potential to substantially reduce costs and time required for remediation as well as improve effectiveness of remediation. Horizontal wells were selected to deliver and extract fluids from the subsurface because their geometry can maximize the efficiency of a remediation system and they have great potential for remediating contaminant sources under existing facilities. The first demonstration of this new technology was conducted for a period of twenty weeks. A vacuum was first drawn on the vadose zone well until a steady-state removal of VOCs was obtained. Air was then injected at three different rates and at two different temperatures. An extensive characterization program was conducted at the site and an extensive monitoring network was installed prior to initiation of the test. Significant quantities of VOCs have been removed from the subsurface (equivalent to an eleven-well, 500-gpm, pump-and-treat system at the same site). Concentrations of VOCs in the ground water have been significantly reduced in a number of the monitoring wells.
  335. Hazen, Terry C. and Gary A. Toranzos. 1990. Microbiology of tropical source water. Advances in Drinking Water Microbiology Research Chapter 2:30-51. pdf
  336. Dunifon, R. E. and Terry C. Hazen. 1990. The effect of vacuum pump oil on the chemotactic behavior of soil bacteria. abstract pdf
    The use of biodegradation in the cleanup and transformation of waste materials is an economical and environmentally safe practice. Using chemotaxis, or the movement of bacteria toward or away from compounds, in biodegradation is an area that is being studied at the Savannah River Laboratory. This study investigates the inhibition of vacuum pump oil on the chemotaxis of soil bacteria. It was found that vacuum pump oil does have an inhibitory effect on the movement of bacteria. This inhibition will have to be considered when studying the possibility of using chemotaxis to degrade vacuum pump oil, or any other petroleum products. 5 refs., 5 figs.
  337. Tyndall, R. L., K. S. Ironside, P. L. Metler, E. L. Tan, Terry C. Hazen and Carl B. Fliermans. 1989. Effect of Thermal Additions on the Density and Distribution of Thermophilic Amebas and Pathogenic Naegleria-Fowleri in a Newly Created Cooling Lake. Applied and Environmental Microbiology 55:722-732. abstract pdf
    Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis. The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions. In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri. During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp. increased as much as 5 orders of magnitude, and the concentration of the pathogen N. fowleri increased as much as 2 orders of magnitude. Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions. Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges. Concentrations of thermophilic amoebae and thermophilic Naegleria spp. correlated significantly with temperature and conductivity. Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp. Isoenzyme patterns of the N. fowleri isolated from the cooling lake were identical to patterns of N. fowleri isolated from other sites in the United States and Belgium.
  338. Sharp, J. C. and Terry C. Hazen. 1989. Biodegradation of vacuum pump oil by naturally occurring bacteria. Georgia Journal of Science abstract pdf
    Bacteria are able to degrade any type of hydrocarbon, given the right conditions and enough time. Indeed, many bacteria have been isolated that use toxic hydrocarbons as their only carbon and energy source. This study examines the biodegradation of vacuum pump oil by bacteria. Bacteria that can use vacuum pump oil as their sole carbon and energy source were isolated from soil collected near a waste oil farming site and a fuel oil depot on the Savannah River Plant, near Aiken, South Carolina. Degradation rates of vacuum pump oil were determined by measuring the amount of carbon dioxide produced by the bacteria in a controlled microcosm. Both high concentrations and low concentrations of vacuum pump oil were inhibitory to vacuum pump oil degradation. Phosphorus and nitrogen were found to be significant limiting factors to the rate of vacuum pump oil degradation in the microcosms. Iron, a common co-factor in hydrocarbon degradation, had no measurable effect on the rate of vacuum pump oil degradation. High concentrations of nitrogen and phosphorus combined, were found to have a greater stimulatory effect on vacuum pump oil degradation then either one alone. Hydrogen peroxide, an oxygen source, at very low concentrations had the greatest stimulatory effect on vacuum pump oil degradation of any of the nutrients tested. The degradation of vacuum pump oil by bacteria in microcosms shows great promise for being a controllable and efficient method for eliminating this common laboratory waste.
  339. Santo Domingo, Jorge W., Francisco A. Fuentes and Terry C. Hazen. 1989. Survival and Activity of Streptococcus faecalis and Escherichia coli in Petroleum-Contaminated Tropical Marine Waters. Environmental Pollution 56:263-281. pdf
  340. Rojas, Yazmin A. and Terry C. Hazen. 1989. Survival of Vibrio cholerae in Treated and Untreated Rum Distillery Effluents. Water Research 23:103-113. pdf
  341. Rivera, S., T. Lugo and Terry C. Hazen. 1989. Autecology of Vibrio vulnificus and Vibrio parahaemolyticus in Tropical Waters. Water Research 23:923-931. pdf
  342. Muniz, I., L. Jimenez, G. A. Toranzos and Terry C. Hazen. 1989. Survival and Activity of Streptococcus faecalis and Escherichia coli in Tropical Fresh-Water. Microbial Ecology 18:125-134. pdf
  343. Jimenez, L., I. Muniz, G. A. Toranzos and Terry C. Hazen. 1989. Survival and Activity of Salmonella typhimurium and Escherichia coli in Tropical Fresh-Water. Journal of Applied Bacteriology 67:61-69. abstract pdf
    The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm, typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium, as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.
  344. Elias Montalvo, E. E., A. Calvo and Terry C. Hazen. 1989. Survival and Distribution of Yersinia enterocolitica in a Tropical Rain-Forest Stream. Current Microbiology 18:119-126. pdf
  345. Rivera, S. C., Terry C. Hazen and G. A. Toranzos. 1988. Isolation of Fecal Coliforms from Pristine Sites in a Tropical Rain-Forest. Applied and Environmental Microbiology 54:513-517. abstract pdf
    Samples collected from water accumulated in leaf axilae of bromeliads (epiphytic flora) in a tropical rain forest were found to harbor fecal coliforms. Random identification of fecal coliform-positive isolates demonstrated the presence of Escherichia coli. This bacterium was also isolated from bromeliad leaf surfaces. These data indicate that E. coli may be part of the phyllosphere microflora and not simply a transient bacterium of this habitat. The isolation of fecal coliforms from these sites was unexpected and raises questions as to the validity of using fecal coliforms as indicators of biological water quality in the tropics.
  346. Perez Rosas, N. and Terry C. Hazen. 1988. In situ Survival of Vibrio cholerae and Escherichia coli in Tropical Coral Reefs. Applied and Environmental Microbiology 54:1-9. abstract pdf
    Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.
  347. Negron Alvira, A., I. Perez Suarez and Terry C. Hazen. 1988. Legionella spp. in Puerto Rico Cooling-Towers. Applied and Environmental Microbiology 54:2331-2334. abstract pdf
    Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.
  348. Lopez Torres, A. J., L. Prieto and Terry C. Hazen. 1988. Comparison of the Insitu Survival and Activity of Klebsiella pneumoniae and Escherichia coli in Tropical Marine Environments. Microbial Ecology 15:41-57. pdf
  349. Lopez de Cardona, I., M. Bermudez, E. Billmire and Terry C. Hazen. 1988. Enteric Viruses In a Mangrove Lagoon Survival and Shellfish Incidence. Caribbean Journal of Science 24:102-111. abstract pdf
    Mangrove oysters (Crassostrea rhizophorae) were screened for enteric viruses. For 18 months oysters were collected from Cano Boqueron, a tropical mangrove lagoon on the southwest coast of Puerto Rico. This popular tourist resort has two primary sewage treatment plants which service 158 single family cabanas. In spite of the heavy seasonal input of treated sewage to Cano Boqueron and high densities of fecal coliform bacteria, enteric viruses were not detected in shellfish meat. Because no viruses were detected in the oysters, a virus survival study was performed. Poliovirus type 1 was placed in diffusion chambers in situ at two sites in Cano Boqueron. More than 95% of the poliovirus inactivation occurred within 24 h. Virus inactivation was significantly different by site, indicating different inactivation rates within the lagoon. Chamber studies done simultaneously with Escherichia coli did not reveal differences between sites. It is suggested that the sewage effluent had an antiviral effect in the absence of an antibacterial effect. This study demonstrated the importance for establishing microbial contamination standards for shellfish growing waters in the tropics based upon in situ studies with tropical species, e.g., mangrove oyster.
  350. Hazen, Terry C. and L. Jimenez. 1988. Enumeration and Identification of Bacteria from Environmental-Samples Using Nucleic-Acid Probes. Microbiological Sciences 5:340-343. abstract pdf
    DNA probes are useful for both identification and enumeration of specific bacteria and functional groups of bacteria in environmental samples. Because probes can detect genes, chromosomes, and plasmids, they also promise to be major sources of information about the relatedness of bacteria and groups of bacteria in the environment.
  351. Hazen, Terry C.. 1988. Fecal Coliforms as Indicators in Tropical Waters - a Review. Toxicity Assessment 3:461-477. pdf
  352. Cruz Cruz, N. E., G. A. Toranzos, D. G. Ahearn and Terry C. Hazen. 1988. Insitu Survival of Plasmid-Bearing and Plasmidless Pseudomonas aeruginosa in Pristine Tropical Waters. Applied and Environmental Microbiology 54:2574-2577. pdf
  353. Broadhead, A. N., A. Negron-Alvira, L. A. Baez, Terry C. Hazen and M. J. Canoy. 1988. Occurrence of Legionella Species In Tropical Rain Water Cisterns. Caribbean Journal of Science 24:71-73. abstract pdf
    Direct fluorescent antibody staining of concentrated water samples from ten cisterns in the U.S. Virgin Islands demonstrated the presence of Legionella pneumophila serogroups 1-6, Legionella micdadei and Legionella gormanii. These potential pathogens were found in concentrations high enough to suggest that cistern water being used for drinking and bathing could be a source for Legionealla disease in tropical areas.
  354. Bermudez, M. and Terry C. Hazen. 1988. Phenotypic and Genotypic Comparison of Escherichia coli from Pristine Tropical Waters. Applied and Environmental Microbiology 54:979-983. abstract pdf
    Nine fecal-coliform-positive strains were isolated from pristine sites in a tropical rain forest. These sites included nonpolluted rivers and water from bromeliads (epiphytes) which were 30 ft (ca. 910 cm) above the ground. Phenotypically, all of these isolates were identified as Escherichia coli. Their DNA was isolated and purified, and the base composition (G + C content) was determined and compared with that of E. coli B (ATCC 11303). The DNA from the environmental isolates was also hybridized to radiolabeled DNA from E. coli B. Eight strains had a DNA base composition similar to that of E. coli B and gave more than 75% homology with E. coli B. One strain had a different DNA base composition and a relatively low percentage of homology with the reference strain. The finding of E. coli in pristine tropical waters suggests that this bacterium could be a natural inhabitant in these environments and is not a reliable indicator of recent human fecal contamination in tropical waters. The indicators that are currently used in the tropics to test the biological quality of water should be reevaluated.
  355. Valdes Collazo, L., A. J. Schultz and Terry C. Hazen. 1987. Survival of Candida albicans in Tropical Marine and Fresh Waters. Applied and Environmental Microbiology 53:1762-1767. abstract pdf
    A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.
  356. Santiago Mercado, J. and Terry C. Hazen. 1987. Comparison of 4 Membrane-Filter Methods for Fecal-Coliform Enumeration in Tropical Waters. Applied and Environmental Microbiology 53:2922-2928. abstract pdf
    Four membrane filter methods for the enumeration of fecal coliforms were compared for accuracy, specificity, and recovery. Water samples were taken several times from 13 marine, 1 estuarine, and 4 freshwater sites around Puerto Rico, from pristine waters and waters receiving treated and untreated sewage and effluent from a tuna cannery and a rum distillery. Differences of 1 to 3 orders of magnitude in the levels of fecal coliforms were observed in some samples by different recovery techniques. Marine water samples gave poorer results, in terms of specificity, selectivity, and comparability, than freshwater samples for all four fecal coliform methods used. The method using Difco m-FC agar with a resuscitation step gave the best overall results; however, even this method gave higher false-positive error, higher undetected-target error, lower selectivity, and higher recovery of nontarget organisms than the method using MacConkey membrane broth, the worst method for temperate waters. All methods tested were unacceptable for the enumeration of fecal coliforms in tropical fresh and marine waters. Thus, considering the high densities of fecal coliforms observed at most sites in Puerto Rico by all these methods, it would seem that these density estimates are, in many cases, grossly overestimating the degree of recent fecal contamination. Since Escherichia coli appears to be a normal inhabitant of tropical waters, fecal contamination may be indicated when none is present. Using fecal coliforms as an indicator is grossly inadequate for the detection of recent human fecal contamination and associated pathogens in both marine and fresh tropical waters.
  357. Ortiz-Roque, C. M. and Terry C. Hazen. 1987. Abundance and Distribution of Legionellaceae in Puerto-Rican Waters. Applied and Environmental Microbiology 53:2231-2236. abstract pdf
    Waters in marine and freshwater areas of Puerto Rico were analyzed for the presence of Legionella spp. by direct fluorescent antibody assay with guinea pig confirmation. Several species, including L. bozemanii, L. dumoffii, L. gormanii, L. longbeachae, L. micdadei, and L. pneumophila, were widely distributed among all sites. Legionellaceae, including L. pneumophila, were found in high densities in water collected in the rain forest from epiphytes in trees 30 ft. (about 9.25 m) above the ground. Both interspecific and intersite variations were significant. L. pneumophila was the most abundant species at all sites, with average densities of 10(4) cells ml-1, very close to the range which is potentially pathogenic for humans. Densities of L. pneumophila were highest in sewage-contaminated coastal waters. These are the highest densities of Legionella spp. ever reported for marine habitats. Densities of L. pneumophila were positively correlated with concentrations of sulfates, phosphates, and pH. A survey of 88 fatal atypical pneumonia cases at a Puerto Rico hospital showed that 15% of the patients had L. pneumophila infections. This study establishes L. pneumophila as a relatively common cause of atypical pneumonia in Puerto Rico and suggests natural aquatic habitats as possible sources or reservoirs of pathogenic Legionella spp. in the tropics.
  358. Lopez Torres, A. J., Terry C. Hazen and G. A. Toranzos. 1987. Distribution and Insitu Survival and Activity of Klebsiella pneumoniae and Escherichia coli in a Tropical Rain-Forest Watershed. Current Microbiology 15:213-218. pdf
  359. Hazen, Terry C., Jesus Santiago-Mercado, Gary A. Toranzos and Madeline Bermudez. 1987. What do Water Fecal Coliforms Indicate in Puerto Rico?. Bulletin of the Puerto Rico Medical Association 79:189-193. pdf
  360. Esch, Gerald W., Terry C. Hazen, D. J. Marcogliese, T. M. Goater and A. E. Crews. 1986. A Long-Term Study on the Population Biology of Crepidostomum cooperi (Trematoda, Allocreadidae) in the Burrowing Mayfly, Hexagenia limbata (Ephemeroptera). American Midland Naturalist 116:304-314. pdf
  361. Fuentes, F. A., Terry C. Hazen, A. J. Lopeztorres and P. Rechani. 1985. Klebsiella pneumoniae in Orange Juice Concentrate. Applied and Environmental Microbiology 49:1527-1529. abstract pdf
    Fecal coliform-positive, capsule-forming Klebsiella pneumoniae cells were observed in high densities (10(4) to 10(8) CFU/100 ml) in two commercial batches of frozen orange juice concentrate at a cannery in Puerto Rico. Contamination of both lots was gross and included off colors and odors. Isolates of K. pneumoniae from these concentrates revealed growth at 4, 25, and 34 degrees C with generation times from 0.39 to 1.84 h.
  362. Carrillo, M., E. Estrada and Terry C. Hazen. 1985. Survival and Enumeration of the Fecal Indicators Bifidobacterium adolescentis and Escherichia coli in a Tropical Rain-Forest Watershed. Applied and Environmental Microbiology 50:468-476. abstract pdf
    The density of Bifidobacterium spp., fecal coliforms, Escherichia coli, and total anaerobic bacteria, acridine orange direct counts, percentages of total bacterial community activity and respiration, and 12 physical and chemical parameters were measured simultaneously at six sites for 12 months in the Mameyes River rain forest watershed, Puerto Rico. The densities of all bacteria were higher than those reported for uncontaminated temperate rivers, even though other water quality parameters would indicate that all uncontaminated sites were oligotrophic. The highest densities for all indicator bacteria were at the site receiving sewage effluent; however, the highest elevation site in the watershed had the next highest densities. Correlations between bacterial densities, nitrates, temperature, phosphates, and total phosphorus indicated that all viable counts were related to nutrient levels, regardless of the site sampled. In situ diffusion chamber studies at two different sites indicated that E. coli could survive, remain physiologically active, and regrow at rates that were dependent on nutrient levels of the ambient waters. Bifidobacterium adolescentis did not survive at either site but did show different rates of decline and physiological activity at the two sites. Bifidobacteria show promise as a better indicator of recent fecal contamination in tropical freshwaters than E. coli or fecal coliforms; however, the YN-6 medium did not prove to be effective for enumeration of bifidobacteria. The coliform maximum contaminant levels for assessing water usability for drinking and recreation appear to be unworkable in tropical freshwaters.
  363. Hazen, Terry C., R. V. Dimock, Gerald W. Esch, A. Mansfield and M. L. Raker. 1984. Chemotactic Behavior of Aeromonas hydrophila. Current Microbiology 10:13-17. pdf
  364. Ortiz-Roque, Carmen and Terry C. Hazen. 1983. Legionellosis and Legionella spp. in the waters of Puerto Rico. Bol Asoc Med P R 75:403-7. pdf
  365. Ortiz-Roque, C. and Terry C. Hazen. 1983. Legionella spp. in Puerto Rico Cooling Towers. Applied and Environmental Microbiology 75:403-407. pdf
  366. Hazen, T. C. and G. W. Esch. 1983. Effect of effluent from a nitrogen fertilizer factory and a pulp mill on the distribution and abundance of Aeromonas hydrophila in Albemarle Sound, North Carolina. Applied and Environmental Microbiology 45:31-42. abstract pdf
    The density of Aeromonas hydrophila, standard count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters were measured simultaneously at six sites for 12 months in Albemarle Sound, N.C. One site was above and two sites were below the discharge plume of a Kraft pulping process paper mill. The fourth site was above and the remaining two sites were below the discharge point of a nitrogen fertilizer factory. The impact of the pulp mill on water quality was acute, whereas that of the nitrogen fertilizer factory was chronic and much more subtle. Diffusion chamber studies indicated that A. hydrophila survival is increased by pulp mill effluent and decreased by nitrogen fertilizer factory effluent. From correlation and regression analysis, A. hydrophila was found to be directly affected by phytoplankton density and, thus, indirectly by concentrations of phosphate, nitrate, and total organic carbon. These two point sources are suspect as indirect causes of red-sore disease epizootics, a disease of fish caused by A. hydrophila.
  367. Hazen, Terry C.. 1983. A Model for the Density of Aeromonas hydrophila in Albemarle Sound, North-Carolina. Microbial Ecology 9:137-153. abstract pdf
    The abundance ofAeromonas hydrophila was measured monthly at 29 sites in Albemarle Sound, North Carolina and its tributaries from April 1977 through July 1979. Simultaneous measurements included heterotrophic plate count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters. Using only 6 water quality parameters, multiple correlation and regression analysis of the data produced a best-fit regression which explained 38% of the variation observed in A. hydrophila density. The 6 water quality parameters included dissolved oxygen, temperature, orthophosphate, chlorophyll A trichromatic, total Kjeldahl nitrogen, and ammonia. Heterotrophic plate count bacteria and fecal coliform densities were highly correlated with A. hydrophila density, but made the model very unstable. The model was successfully tested against similar data collected for 2 other North Carolina reservoirs, Lake Norman and Badin Lake. Data from 10 sites in Badin Lake over 18 months and from 7 sites on Lake Norman over 5 months were not significantly different from the Albemarle Sound model. Conditions of water quality that may give rise to "blooms" of A. hydrophila will simultaneously contribute to the probability of increased epizootics in fish in the southeastern United States.
  368. Fuentes, F. A., E. J. Biamon and Terry C. Hazen. 1983. Bacterial Chemotaxis to Effluent from a Rum Distillery in Tropical near-Shore Coastal Waters. Applied and Environmental Microbiology 46:1438-1441. abstract pdf
    Pseudomonas aeruginosa and Vibrio cholerae showed a strong positive chemotactic response towards rum distillery wastewaters (mostos) and a high oxygen uptake rate in the presence of this complex substrate. Rum slops stimulated only motility in Aeromonas hydrophila and Escherichia coli. The A. hydrophila and E. coli isolates were unable to oxidize mostos significantly.
  369. Biamon, E. J. and Terry C. Hazen. 1983. Survival and Distribution of Aeromonas hydrophila in near-Shore Coastal Waters of Puerto Rico Receiving Rum Distillery Effluent. Water Research 17:319-326. pdf
  370. Hazen, Terry C., Gerald W. Esch, R. V. Dimock and A. Mansfield. 1982. Chemotaxis of Aeromonas hydrophila to the Surface Mucus of Fish. Current Microbiology 7:371-375. abstract pdf
    Isolates of Aeromonas hydrophila from various sources show different chemotactic responses to mucus from the surface of freshwater fish. Some isolates were nonchemotactic tofish surface mucus. Isolates of A. hydrophila from fish lesions had a significantly higher chemotactic index than isolates ofA. hydrophila from water. Maximum chemotactic responses occurred more often to diluted fish mucus than to undiluted samples. Fish which were experimentally stressed did not produce mucus that was more or less chemotactic than that of unstressed fish. Fish with red-sore lesions produced surface mucus which was not chemotactic to A. hydrophila. Differences between fish, for any isolate, were also not significant. Thechemotactic substance(s) in fish mucus has a molecular weight of approximately 100,000 and did not appear to be labile when heated to 56°C
  371. Esch, Gerald W. and Terry C. Hazen. 1982. Studies on the Seasonal Dynamics of Crepidostomum cooperi in the Burrowing Mayfly, Hexagenia limbata. Proceedings of the Helminthological Society of Washington 49:1-7. pdf
  372. Hazen, Terry C., Gerald W. Esch and M. L. Raker. 1981. Agglutinating Antibody to Aeromonas hydrophila in Wild Largemouth Bass. Transactions of the American Fisheries Society 110:514-518. pdf
  373. Glassman, A. B., C. E. Bennett and Terry C. Hazen. 1981. Peripheral-Blood Components in Alligator mississippiensis. Transactions of the American Microscopical Society 100:210-215. pdf
  374. Cunningham, D. S., Terry C. Hazen and R. E. Kuhn. 1981. Increased Resistance to Aeromonas hydrophila in Mice Experimentally Infected with Trypanosoma cruzi. Journal of Parasitology 67:468-474. pdf
  375. Fliermans, Carl B. and Terry C. Hazen. 1980. Immunofluorescence of Aeromonas hydrophila as Measured by Fluorescence Photometric Microscopy. Canadian Journal of Microbiology 26:161-168. pdf
  376. Gerald W. Esch and Terry C. Hazen. 1980. The Ecology of Aeromonas hydrophila in Albemarle Sound, North Carolina. Water Resources Reserach Institute of the University of North Carolina 153. abstract pdf
    Densities of Aeromonas hydrophila, f e c a l and t o t a l coliforms were measured i n conjunction with 18 physical and chemical parameters a t 29 s t a t i o n s located i n Albemarle Sound and various t r i b u t a r i e s including tile Chowan, Roanoke, A l l i g a t o r , Pasquotank, North, Scuppernong and Perquimans Rivers. Data were c o l l e c t e d monthly a t most s t a t i o n s beginning April, 1977, and continuing through J u l y , 1979. Densities of -A. h y d r o p h i l a were c o r r e l a t e d wi t h f e c a l and t o t a l c o l i f o rms , d i s s o l v e d oxygen, t u r b i d i t y , chlorophyll a ( t r i c h r o m a t i c ) , pheophytin a, s u l f a t e , ammonia, t o t a l Kjehldahl n i t r o g e n , t o t a l phosphorus, phosphates and t o t a l organic carbon. Strongest c o r r e l a t i o n c o e f f i c i e n t s were between -A . h y d r o p h i l a d e n s i t y and phosphorus and phospha t e s . Data showed t h a t 87;; of the water samples had t o t a l phosphorus c o n c e n t r a t i o n s which exceeded 50 ug 1-1, i n d i c a t i n g eutrophic conditions. During most months, a t most s t a t i o n s , d e n s i t i e s of Aeromonas hydrophila did not exceed 40 cfu ml-l. However, in J u l y , 1979, d e n s i t i e s of 4. hydrophila exceeded 1,000 cfu ml-l a t f i v e of 13 sampling s t a t i o n s used as r e p r e s e n t a t i v e f o r Albemarle Sound and its t r i b u t a r i e s . These high d e n s i t i e s corresponded with a massive outbreak o f red-sore d i s e a s e Liinong s e v e r a l species of comercial and game'fish i n the Sound. The ri.lationsi~ip between red-sore d i s e a s e and dcnsity of A. hydrophila is tllus reminiscei~t of a s i m i l a r s i t u a t i o n observed f o r A. hydrophila density and prcvalence of red-sore d i s e a s e aniong a of largeiriouth bass (Micropterus scllmoides) s t u d i e d in Par Pond, a 1,012 ha cooling r e s e r v o i r located on the Savannah River Plant near Aiken, South Carolina. 3ased on a s e r i e s of henxtological p~rameters measured i n largemouth oass from the Pdr Pond system, evidence suggests t h a t s t r e s s , produced by elsvated terqeratiire i n the r e s e r v o i r , rnay play a s i g n i f i c a n t r o l e i n , rhc red- sore disedsc: syr;droiie. In Aibemar le Sound, i t i s proposed that I a conbination of f a c t o r s , i n c l u d i n g v a r i o u s forms of n i t r o g e n , phosphate, t o t a l phosphords and cotal organic carbon, a r e involved i n c r e a t i n g canditions conducive to i n c r e a s i n g abundance of L. hydrophila. The same water q u a l i t y f a c t o r s a f f e c t i n g A. hydrophila d e n s i t y may simultaneously lead to conditions which promote s t r e s s i n l o c a l f i s h populations. The co~~binatioonf e x c e s s i v e n u t r i e n t l o a d i n g , i n c r e a s e d d e n s i t y of A. hydrophila and ilost s t r e s s would then lead to outbreaks of red-sore d i s e a s e and fish m o r t a l i t y .
  377. Esch, Gerald W. and Terry C. Hazen. 1980. Stress and Body Condition in a Population of Largemouth Bass - Implications for Red-Sore Disease. Transactions of the American Fisheries Society 109:532-536. pdf
  378. Huizinga, H. W., Gerald W. Esch and Terry C. Hazen. 1979. Histo-Pathology of Red-Sore Disease (Aeromonas hydrophila) in Naturally and Experimentally Infected Largemouth Bass Micropterus salmoides (Lacepede). Journal of Fish Diseases 2:263-277. pdf
  379. Hazen, Terry C. and Carl B. Fliermans. 1979. Distribution of Aeromonas hydrophila in Natural and Man-Made Thermal Effluents. Applied and Environmental Microbiology 38:166-168. pdf
  380. Hazen, Terry C.. 1979. Ecology of Aeromonas hydrophila in a South Carolina Cooling Reservoir. Microbial Ecology 5:179-195. pdf
  381. Gorden, R. W., Terry C. Hazen, Gerald W. Esch and Carl B. Fliermans. 1979. Isolation of Aeromonas hydrophila from the American Alligator, Alligator mississippiensis. Journal of Wildlife Diseases 15:239-243. pdf
  382. Quinn, T., Gerald W. Esch, Terry C. Hazen and J. W. Gibbons. 1978. Long-Range Movement and Homing by Largemouth Bass (Micropterus salmoides) in a Thermally Altered Reservoir. Copeia 542-545. pdf
  383. Hazen, Terry C., G. Smith and R. V. Dimock. 1978. Method for Fixing and Staining Peritrich Ciliates. Microscopica Acta 81:15-16. abstract pdf
  384. Hazen, T. C., M. L. Raker, G. W. Esch and C. B. Fliermans. 1978. Ultrastructure of Red-Sore Lesions on Largemouth Bass (Micropterus-Salmoides) - Association of Ciliate Epistylis Sp and Bacterium Aeromonas-Hydrophila. Journal of Protozoology 25:351-355. pdf
  385. Hazen, T. C., C. B. Fliermans, R. P. Hirsch and G. W. Esch. 1978. Prevalence and distribution of Aeromonas hydrophila in the United States. Appl Environ Microbiol 36:731-8. abstract pdf
    The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.
  386. Hazen, Terry C., Gerald W. Esch, A. B. Glassman and J. W. Gibbons. 1978. Relationship of Season, Thermal Loading and Red-Sore Disease with Various Hematological Parameters in Micropterus salmoides. Journal of Fish Biology 12:491-498. abstract pdf
    One hundred and fifty larh'CTTIOuth ba..:;s (Micmplerus salmoides) were caught duting the mid-winter and mjd--summer months in Par Pond, an 1120 ha cooling reservoir located on Savannah River Planl near Aiken, SC. Equal numbers of fi
  387. Hazen, T. C. and G. W. Esch. 1978. Observations on Ecology of Clinostomum-Marginatum in Largemouth Bass (Micropterus-Salmoides). Journal of Fish Biology 12:411-420. pdf
  388. Hazen, T. C., J. M. Aho, T. M. Murphy, G. W. Esch and G. D. Schmidt. 1978. The parasite fauna of the American alligator (Alligator mississippiensis) in South Carolina. Journal of Wildlife Diseases 14:435-9. abstract pdf
    Twelve American alligators (Alligator mississippiensis) were obtained from three different areas of South Carolina. One species of pentastome (Sebekia oxycephala), two species of nematodes (Dujardinascaris waltoni and Multicaecum tenuicolle), four species of trematodes (Polycotyle ornata, Acanthostomum coronarium, Archaeodiplostomum acetabulatum and Pseudocrocodilicola americaniense) and one species of hemogregarine (Haemogregarina crocodilnorum) were recovered. Polycotyle ornata was observed only in alligators from Par Pond while P. americaniense was found in Par Pond and coastal hosts, A. acetabulatum from Kiawah Island and coastal alligators, and A. coronarium only at Kiawah Island. These patterns suggest disjunct distributions for the trematode species in South Carolina alligators. The other parasites were found in alligators from all three locations. The only parasite observed to initiate damage or lesions in the alligator was the pentastome.
  389. Hazen, T. C.. 1978. Ecology of Aeromonas hydrophila in a South Carolina cooling reservoir. Ph. D. Thesis. Wake Forest University Dissertation Abstract Order No: AAAD78-14969:1112. 278 .
  390. Gibbons, J. W., D. H. Bennett, Gerald W. Esch and Terry C. Hazen. 1978. Effects of Thermal Effluent on Body Condition of Largemouth Bass. Nature 274:470-471. pdf
  391. Esch, G. W. and T. C. Hazen. 1978. Impact of thermal loading and other water quality parameters on the epizootiology of red-sore disease in centrarchids Progress report, December 1, 1977--November 30, 1978.
  392. Esch, Gerald W. and Terry C. Hazen. 1978. Thermal ecology and stress: a case history for red-sore disease in largemouth bass (Micropterus salmoides). Energy and Environmental Stress in Aquatic Systems pdf
  393. Hazen, Terry C., W. K. Kellogg and Gerald W. Esch. 1977. Studies on Population Biology of 2 Larval Trematodes in Amphipod, Hyalella-Azteca. American Midland Naturalist 98:213-219. abstract pdf
    The age frequency and density of a population of amphipods, Hyalella azteca, were compared with the occurrence and densities of two trematode parasites, Crepidostomum cooperi and Plagioporus sp. The overall seasonal density of the host and both parasites was greatest at 1 m depth, next at 3 m and least at 2 m. Prior to sexual maturity, amphipods were not infected. Following sexual maturation of the amphipod, the percentage of infected individuals increased with each succeeding age class. H. azteca and both trematode populations were in­dependently heterogeneous in their spatial distributions. Recruitment rates and densities of Plagioporus sp. were lower than for C. cooperi. Infection percentages by both parasites, number of concurrent infections and neotenic forms of C. cooperi increased with increasing water temperatures.
  394. Fliermans, C. B., T. C. Hazen and T. V. Crawford. 1977. Seasonal distribution of Aeromonas hydrophila in Par Pond. Savannah River Laboratory, Environmental Transport and Effects Research. pdf
  395. Fliermans, Carl B., R. W. Gorden, Terry C. Hazen and Gerald W. Esch. 1977. Aeromonas Distribution and Survival in a Thermally Altered Lake. Applied and Environmental Microbiology 33:114-122. abstract pdf
    Par Pond is a thermally enriched monomictic southeastern lake which receives heated effluent from a production nuclear reactor. Fish populations in the lake have lesions of epizooty from which Aeromonas spp. are readily isolated. Distribution and population densities of Aeromonas in the water column were measured along an oxygen and temperature gradient as well as seasonally. Greater population densities of Aeromonas occurred below the oxygen chemocline when the lake was stratified. Survival of Aeromonas hydrophila under in situ conditions in both epilimnetic and hypolimnetic waters was determined through the use of polycarbonate membrane diffusion chambers during two separate reactor operating conditions. Survival levels of pure cultures of A. hydrophila corresponded to the distribution patterns of the naturally occurring Aeromonas-like populations. The greater survival of A. hydrophila during full reactor operation suggests that the fish populations may be exposed to Aeromonas for a longer period of time than when the reactor is not operating.
  396. Esch, Gerald W., Terry C. Hazen and J. M. Aho. 1977. Parasitism and r- and K-selection. Regulation of parasite populations abstract pdf
    A review of the literature on host-parasite relationships and regulation of parasite populations is presented. Some topics discussed are as follows: host factors such as diet, age, sexual maturity, and behavior; inter- and intraspecific interactions; temperature effects; dispersion; mortality; and competition. (HLW)
  397. Esch, G. W. and T. C. Hazen. 1977. Strain specificity of aeromonas hydrophila: an immunofluorescence study.[Effects of thermal pollution]. abstract pdf
    Fluorescent antibodies prepared to Aeromonas hydrophila were species specific when compared to seven other Aeromonas spp. obtained from the American Type Culture Collection (ATCC). The A. hydrophila were isolated from water, largemouth bass, and alligators from a warm monimictic lake thermally altered by production reactor effluents. Over 200 isolates from water, bass, alligators, and human samples were characterized serologically, biochemically, and for drug sensitivity. The isolates were then grouped. The specificity of the antibodies was used to establish a relationship between the infective agents of the largemouth bass and the alligators when compared to those found in water.
  398. Hazen, Terry C., G. Smith and R. V. Dimock. 1976. Method for Fixing and Staining Peritrich Ciliates. Transactions of the American Microscopical Society 95:693-695. abstract pdf
    A method for fixing and staining peritrich ciliates. Trans. Arner. Micros. Soc., 95: 693-695. A simple tech­nique for fixing and staining peritrich ciliates involving fixation in phosphate buffered formalin and staining with Semichun's Acetic-Carmine is presented. This procedure appears to be superior to conventional methods for achieving relaxation of zooids and for differentiation of the macronucleus.
  399. Esch, Gerald W., Terry C. Hazen, Ronald V. Dimock and J. Whitfield Gibbons. 1976. Thermal Effluent and Epizootiology of Ciliate Epistylis and Bacterium Aeromonas in Association with Centrarchid Fish. Transactions of the American Microscopical Society 95:687-693. abstract pdf
    Thermal effluent and the epizootiology of the ciliate Epistylis and the bacterium Aeromonas in association with centrarchid fish. Trans. Amer. Micros. Soc., 95: 687-693. Epistylis sp. ( Ciliophora: Peritricha) is a facultative ectosymbiont of fresh-water fishes in streams and lakes throughout the southeastern U.S. In combination with the gram­negative bacteria Aeromonas hydrophila, epizootic outbreaks of Epistylis have been implicated in the death of many thousands of game fish, primarily striped bass and several species of Centrarchidae ( sunfish), in several reservoirs in North Carolina. While these epizootics have been correlated with high levels of organic loading, recent studies suggest that thermal loading from a nuclear production facility may influence the level of infection by A. hydrophila and Epistylis sp. (A-E complex) on centrarchid sunfish in Par Pond, Savannah River Plant, Aiken, S.C. Utilizing electro-fishing techniques, in excess of 11,500 sunfish, representing six different spe­cies, were collected and examined for external lesions associated with the A-E complex. Largemouth bass ( Micropterus salmoides) were most heavily infected, followed in incidence of infection by bluegill ( Lepomis macrochirus), warmouth ( Lepomis gulosus) and redbreast sunfish ( Lepomis auritus); lesions were not ob­served on the surlace of black crappie (Pomoxis nigromaculatus). The body condi­tions ( or K-factor) of more than 2,000 largemouth bass were calculated; direct correlations were found between thermal loading, body condition, and incidence of infection. These data are discussed in terms of thermal effluent and the epizootiology of Epistylis sp. and Aeromonas hydrophila.