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
Director, Institute for a Secure & Sustainable Environment
Genome Science and Technology
Center for Environmental Biotechnology
Bredesen Center

The University of Tennessee
Office: 676 Dabney Hall
Office: 507 SERF
Lab: 729 SERF
Knoxville, Tennessee 37996-1605
Cell: 707-631-6763
Phone: 865-974-7709
E-mail: tchazen@utk.edu

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

Presentations and Media

Presentations and Media (1769, 1228 invited, 1326 abstracts or articles published)

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  1. Hazen, T. C.. 2017. Is the Solution to Pollution Dilution i.e. Intrinsic Remediation? Oil Spills, Solvents, Metals, and Radionuclides (OH MY!!!!). University of Tennessee Honors Faculty Lecture Series, Chancellor's Honors Program
  2. Chakraborty, R., X. Wu, T. C. Hazen, Y. Liu, N. Hess, M. W. Fields, P. Zhang, L. Wu, J. Zhou, Q. Li, W. Yang, A. P. Arkin and P. D. Adams. 2017. Microbial Interactions with Natural Organic Matter Extracted from the Oak Ridge FRC. 2017 Genomic Sciences Program Annual PI Meeting. abstract
    Project Goals: Natural organic matter (NOM) is central to microbial food webs and microbially mediated NOM transformations determine much of the carbon (C) flux in subsurface environments. However, little is known about the molecular signature of this pool of C and the microbial activities that regulate NOM turnover are still poorly resolved. The goal of this project was to study the interactions between NOM (extracted from the field site) and native microbial communities present in groundwater at a background site (FW305) at Oak Ridge Field Research Center, TN. Water-soluble NOM was extracted from sediment samples collected from the background uncontaminated site, and the extraction efficiencies were 3.2% for organic carbon and 1.6% for inorganic carbon. Extracted NOM was used as the sole source of carbon in controlled lab incubations, and groundwater from FW305 well served as the microbial inoculum. Subsamples were harvested at several time points during a 50 day incubation for both chemical and microbial analyses. Results indicated a rapid decrease of total organic carbon within the first 1.5 days, concomitant to a rapid burst in CO2, and increased in cell numbers. 16S rRNA gene amplicon sequencing suggested that Massilia spp dominated in the original inoculum, and gradually decreased to below 5% after 50 days, while Azospirillum spp and Cupriavidus spp gradually increased from < 5% to 20-30%. Advanced chemical techniques including FTICR-MS and sXAS were used to characterize the C pool that included NOM metabolites and microbial byproducts produced during the incubation period. The molecular mass of C pool generally ranged from 200-600. Relative abundance of compounds with mass around 400-600 increased with incubation time and were considered to be produced from microbial activities. Also, the C pool shifted during incubation, the proportion of lignin in cultures increased, while proteins decreased. Further, GeoChip was used to identify the changes of microbial communities and expression of functional genes during transformation of the NOM. In conclusion, the data clearly showed that microbial community present at Oak Ridge FRC responded to NOM from the site, and that the community shifted to reveal dominant members in sequence over time in response to transformation of the different functional groups in NOM.
  3. Ling, F., J. Friedman, S. Zhao, M. B. Smith, A. M. Rocha, C. J. Paradis, J. Zhou, T. C. Hazen, E. J. Alm, A. P. Arkin and P. D. Adams. 2017. Microbes at the blurred boundary of natural and built environments. 2017 Genomic Sciences Program Annual PI Meeting. abstract
    Project Goals: This project aims to better understand the assembly of low-diversity communities in a groundwater ecosystem with genomic tools. Humans today spend more time working, living, and recreating in the manmade environment than the natural environment. However, the boundary between built and natural environments isn’t always clear. The Oak Ridge Field Research Center (FRC) presents a case where past anthropogenic contamination to the natural environment caused by uranium enrichment influences the current built environment through the channel of water supply. Microbes living in this environment have been shown to be useful as quantitative biosensors of contamination. In the present study, we found that microbial communities at the FRC sites exhibited an unusual range of diversity, which correlates to the performance of the microbial sensor. We show that the variation in diversity is poorly explained by chemical gradients or cell count, yet well explained by migration when viewed from a dispersal-limited community assembly perspective. Our ongoing work applies shotgun sequencing to infer strain-level diversity that can provide further insights on the community assembly processes. Funding statement. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231. Fangqiong Ling is supported by Alfred P. Sloan Foundation Microbiology of the Built Environment Postdoctoral Fellowship
  4. Ning, D., J. Zhou, Z. He, P. Zhang, J. D. Van Nostrand, L. Wu, R. Tian, E. J. Alm, T. C. Hazen, D. Elias, M. W. Fields, M. W. W. Adams, R. Chakraborty, D. Stahl, J. Wall, A. P. Arkin and P. D. Adams. 2017. Stress mediates relative importance of deterministic and stochastic assembly in groundwater microbial communities. 2017 Genomic Sciences Program Annual PI Meeting. abstract
    Project Goals: Understanding the mechanisms controlling community diversity, distribution and succession is a central, but poorly understood, issue in ecology, particularly in microbial ecology. Although both stochastic and deterministic processes are believed to play roles in shaping community diversity and distribution, their relative importance is hotly debated. The importance of ecological stochasticity in shaping microbial community composition and structure is far less appreciated. Moreover, despite recent intensive studies on ecological community assembly, the factors mediating the relative importance of deterministic vs stochastic processes in shaping community composition and structure remain elusive. Thus, the major goal of this study is to illustrate the relative roles of deterministic and stochastic processes in shaping community structure and the factors controlling their relative importance. To determine whether and how environmental factors mediate community assembly processes, about 100 wells representative of no or low, medium, high and extremely high stress were sampled and more than 200 environmental variables were measured. Null model analysis based on phylogenetic diversity of 16S rRNA gene revealed that the groundwater microbial communities at control or low stress wells without contamination were largely stochastic (~67%). As environmental stresses increased, the communities became less and less stochastic, with 41% of stochasticity at the extremely stressed wells. Also, quantitative analysis showed that variable selection (24~49%) and dispersal limitation (25~57%) played dominant roles while homogeneous selection (7.6~10%), homogeneous dispersal (0~1.6%), and undominated (or drift) (3.9~14%) play minor roles. Environmental stresses had strong positive correlation with variable selection (r=0.96), and negative correlations (r=-0.93) with dispersal limitation. Interestingly, drift (e.g. undominated) were higher at both low (8.6%) and extremely (14%) stressed wells than medium (4.7%) and high (3.9%) stressed wells. The spatial patterns of various processes were consistent with spatial distributions of various contaminants. In addition
  5. Paradis, C. J., T. C. Hazen, A. P. Arkin and P. D. Adams. 2017. Exposure History Dependence of Microbial Mediated Substrate Transformation Rates in Groundwater. 2017 Genomic Sciences Program Annual PI Meeting. abstract
    Project Goals: The goals of this project are to: (1) demonstrate the exposure history dependence of microbial mediated substrate transformation rates in groundwater at the field scale and (2) elucidate the microbial mechanism(s) which control the exposure history dependence of microbial mediated substrate transformation rates The rates at which natural microbial communities can transform a substrate in groundwater have been shown to increase after repeated exposures to the substrate; herein referred to as the “memory effect”. The objectives of this study were to determine: (1) how long the memory effect can last and (2) how the memory effect can alter the structure and function of natural microbial communities. Ethanol substrate was injected into a single groundwater test well for six consecutive weeks in order to establish a memory effect. The groundwater control well, located up-gradient of the test well, was not injected with ethanol during this time. The rate of ethanol removal in the test well was negligible the first week whereas subsequent rates were significant. The test and control wells will be monitored for six additional weeks under ambient conditions. Ethanol substrate will then be injected into both test and control wells in order to determine: (1) if the test well retained its memory effect and (2) if the rate of ethanol removal in the control well is negligible. Here we present the hydrological, geochemical, and microbiological data and analyses in hand from the study site and the experimental well pair. This includes: (1) the direction and magnitude of groundwater velocity, (2) the effective porosity of the groundwater system, (3) diffusive mass transport in the experimental well pair, (4) the rates of ethanol removal in the test well, (5) the extent of nitrate, sulfate, and uranium removal in the test well, (6) the extent of limiting metal nutrient and/or co-factor removal in the test well, (7) microbial community structure (16S rRNA sequencing) at the study site, and (8) microbial community function (GeoChip) at the study site. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231.
  6. Smith, H. J., A. Zelaya, I. Miller, D. Joyner, T. C. Hazen, M. W. Fields, A. P. Arkin and P. D. Adams. 2017. Temporal Variability and Microbial Activity in Groundwater Ecosystems. 2017 Genomic Sciences Program Annual PI Meeting. abstract
    Project Goals: A fundamental goal in the field of microbial ecology is to link the activity and structure of microbial populations and communities to processes occurring within an ecosystem. This project aims to identify the drivers of community structure and succession by identifying the metabolically active fraction of microbial communities from both pristine and contaminated groundwater habitats at the Field Research Center (FRC) at Oak Ridge National Laboratory (ORNL). It is hypothesized that community function is independent of phylogeny and that functionality will be altered as a result of environmental perturbations. The use of geochemically distinct wells in combination with the enumeration and sequencing of translationally-active microorganisms, activity assays, carbon utilization profiles, and geochemical measurements will allow for the elucidation of the mechanisms shaping community structure and function in terms of turnover of natural organic matter and major contaminants (e.g., NO3 -). Multiple assay comparisonswill be used to achieve an accurate characterization of the active fraction of groundwater microbial communities and will ultimately be applied to continuous sediment cores from both contaminated and pristine locations. Abstract: Saturated subsurface environments are estimated to contain approximately 40% of the prokaryotic biomass on Earth, and due to the complexity of these habitats they support highly diverse microbial communities. In addition, it is estimated that over 98% of the Earth’s consumable and available freshwater is in the subsurface as groundwater. However, the factors that determine microbial community assembly, structure, and function in groundwater systems and the impact on water quality and contaminant transport remain poorly understood. Three non-contaminated background wells were sampled for groundwater geochemistry and microbial diversity approximately 3 times a week over a period of three months. Community analysis via ss-rRNA paired-end sequencing and distribution-based clustering revealed temporal differences in richness, diversity, and variability in the groundwater communities. Microbial community composition of a given well was on average >50% dissimilar to any other well at a given time point. Similarities in community structure across wells were observed with respect to the presence of 20 cosmopolitan populations in all samples in all wells; however, wells differed in the relative abundances of these taxa. Similarity percentage (SIMPER) analysis revealed that temporal variability was explained by lowly abundant and transient populations or more highly abundant and frequently present taxa in a sample-dependent manner.
  7. von Netzer, F., D. Gorman-Lewis, E. Shock, S. Turkarslan, C. E. Arens, A. W. Thompson, N. S. Baliga, A. Zhou, J. Zhou, A. Aaring, R. Chakraborty, J. W. Moon, D. Elias, D. C. Joyner, T. C. Hazen, H. Smith, M. Fields, F. Poole, M. W. W. Adams, H. Carlson, A. Deutschbauer, D. Vuono, K. Meinhardt, D. A. Stahl, A. P. Arkin and P. D. Adams. 2017. Understanding the thermodynamic Foundations of microbial Growth Efficiencies in the Lab and Field. 2017 Genomic Sciences Program Annual PI Meeting. abstract
    Project Goals: A key element of microbial growth and therefore microbial community assembly is how microbes partition the available resources between energy required for maintenance and growth. Using microcalorimetry and thermodynamic modelling, we gained a quantitative proxy for microbial growth efficiency under different growth conditions. As the next step, we aim to apply our methods to address the ecological framework guiding the partitioning of denitrification pathways at the Oak Ridge National Laboratory Field Research Site (FRC); through the establishment of microbial activity assays, isotope fractionation analysis as well as establishment of mass balances and stoichiometry for representative nitrate respiring isolates. The assembly of microbial communities is determined by many factors, with the environment setting the stage via availability of electron acceptors, donors and carbon sources as well as with physical/chemical parameters such as temperature and salinity. In this framework microbes have to adapt to either stable or dynamic conditions and to either compete or share resources for survival. Also, bacteria need to balance constantly the division of available energy between maintenance of basic cellular functions and growth. Therefore, microbes with the most favorable ratio between maintenance and growth requirements should be more competitive compared to microbes with higher energy demands for maintenance and growth. Maintenance energy levels as a proxy for microbial competitiveness are usually measured in chemostats near zero growth conditions. Here, we attempt to capture maintenance energies via microcalorimetry and a metabolite-dependent thermodynamic model as a quantitative proxy for microbial growth efficiency. Microcalorimetry offers a direct and highly sensitive method to assess the enthalpy-related terms of microbial growth in relation to the potential energy supplied by growth substrates. This in turn allows for a quantitative description of growth and maintenance in thermodynamic terms under different growth conditions, based on the comparison of the metabolite profile at the start and end of growth. We have examined the influence of temperature stress, simulated environmental dynamics and adaptation to salt stress on the growth efficiencies of different strains of Desulfovibrio vulgaris Hildenborough and D. alaskensis G20. These analyses quantified the cost of maintenance (survival) in relationship to increasingly suboptimal growth conditions, the cost of regulation in a fluctuating environment and the reduction in maintenance costs realized through adaptive evolution. We aim to apply our growth efficiency and maintenance proxy to field conditions by addressing the ecological framework guiding the partitioning of denitrification pathways at the Oak Ridge National Laboratory Field Research Site (FRC). This site has a long contamination history with nitrate among a large variety of other contaminants. Thermodynamic modeling based on mass balances and stoichiometries of site-relevant nitrate respiring isolates will be associated with in situ measurements of nitrate respiration and nitrate stable isotope fractionation analyses to develop a predictive framework for microbial community assembly and activity. As the next step, we are cultivating available nitrate respiring isolates (Rhodanobacter, Acidovorax and Pseudomonas spp.) under different C/N ratios and the application of the thermodynamic model established with metabolite profiles, we aim to record the energy requirements under different biogeochemical conditions. These profiles can then out into context to actual biogeochemical field conditions as characterized by nitrate specific acetylene-block activity assays and the metabolic history as traced by nitrate stable isotope fractionation. ENIGMA (http://enigma.lbl.gov) at LBNL supported by Office of Biological and Environmental Research US Dept of Energy Contract No: DE-AC02-05CH11231
  8. T. C. Hazen. 2017. ENIGMA Project: Lab to Field and Back - Environmental System Microbiome. 2017 DOE TES/SBR Joint Investigators Meeting
  9. Mayes, M. A., Y. Song, Q. Yao, C. Pan, T. C. Hazen, X. Yang, G. Wang, Z. Li, A. Biswas, B. Turner, S. J. Wright, S. G. Tringe and P. Thornton. 2017. Linking Meta-omics with the Microbial ENzyme Decomposition Model. 2017 DOE TES/SBR Joint Investigators Meeting abstract
    Microbes are increasingly included in soil carbon decomposition models, but it is widely recognized that one functional group may be insufficient to represent the diversity of substrates. Microbial metagenomics, metaproteomics, metatranscriptomics, and other related techniques provide important insight into microbial genes and activities, but it remains unclear how to include such detailed information in models of any scale. We provide a solution to this complex problem and demonstrate its application using a pilot study from the Gigante Fertilization Experiment near Barro Colorado Island, Panama. Soils were collected from control and phosphorus (P) addition plots and analyzed for metagenomics, metaproteomics, phosphatase enzyme activities, and CO2 production during incubation experiments. Fertilized soils exhibited around 30% more CO2 release than control soils and had greater microbial biomass than control soils. Control soils exhibited greater monophosphoesterase and diphosphoesterase activities, and had significantly more genes coding for the production of phytase, phospholipase, and exoribonuclease phosphomonoesterase than P addition soils. We also observed differences in genes for carbon decomposition and the reduction of nitrogen and sulfur, and results were consistent between metagenomic and metaproteomic analyses. We incorporated the enzyme functions, the P cycle (Yang et al. 2013, 2016), and a continuum carbon decomposition scheme into the Microbial ENzyme Decomposition model (Wang et al. 2013, 2014, 2015). Model results were able to match the patterns of CO2 evolution in control and P addition soils, indicating the improved model provides a reasonable pathway for including meta-omic information into soil nutrient cycling models. Future work will focus on including anaerobic decomposition pathways.
  10. Campa, M. F., S. M. Techtmann, C. Gibson, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella and T. C. Hazen. . 2017. The Impacts of the Biocide Glutaraldehyde on Community Structures and Degradation Potential in Streams Impacted by Hydraulic Fracturing. American Society for Microbiology Microbe 2017 abstract
    Hydraulic fracturing (HF) involves injecting chemicals, sand, and water into shale formations to create fractures and release hydrocarbons. Large volumes of the hyper-saline fluids used return to the surface as waste. Potential spills of this wastewater raise concerns about the environmental impact these fluids may impose. Biocides are one of the main chemicals of concern used in HF fluids. Biocides are added to prevent biocorrosion of equipment and gas souring. To understand the effect of biocides on the environmental microbial community after a HF surface spill, microcosms were inoculated using stream water impacted and not impacted by Marcellus shale HF operations. Microcosms were incubated aerobically at ambient temperature for 56 days using glutaraldehyde (GA), the most commonly used biocide in HF operations. The microbial community adaptation to biocide was monitored every two weeks using next-generation16S rRNA amplicon sequencing, and abiotic and biotic GA degradation was measured every week using Liquid Chromatography- coupled with Exactive Quadrupole-Orbitrap Mass Spectrometry. 16S rRNA amplicon sequencing showed three methanotrophic taxa enriched in the HF impacted sites after the addition of GA. These taxa were the genera Beijerinckia, Methylobacterium, and Methylosinus. Over time, HF impacted sites kept a higher Simpson alpha diversity than the HF not-impacted sites. Presumably, HF-impacted sites have more organisms capable of tolerating the biocides. The difference of alpha diversity between HF-impacted and HF-not-impacted sites suggests there is a long lasting effect in the microbial population after a HF spill. Furthermore, HF impacted experienced a smaller log reduction of bacterial 16S rRNA gene copy number, that can be attributed to less bacteria dying off. HF not impacted sites show more “enrichment” afterwards, but this could be due to the new carbon sources provided by dead microbes. In 56 days there was not a statistically significant abiotic degradation of GA. However, overtime HF not impacted sites, experienced more biotic degradation of GA as compared to the HF impacted sites. Showing there is a decrease in degradation potential after GA exposure, even though more members of the microbial community are able to tolerate the biocide. These findings show there are lasting effects in microbial community structure and degradation potential in streams impacted by HF operations.
  11. Harik, A., S. M. Techtmann and T. C. Hazen. 2017. Water Swap: Control of Geochemistry Versus Microbial Community Composition on Hydrocarbon Degradation. American Society for Microbiology Microbe 2017 abstract
    Although the ideal for the oil industry is a lack of oil spills and a reliance on preventative measures, the industry must also be prepared for the worst-case scenarios. Current spill response plans are general and can be lacking site and microbial community specific responses. In the water swap experiment the relative control biogeochemistry plays on the microbial degradation of hydrocarbons is looked at in comparison to the role of the pre-spill microbial community composition. Our hypothesis is that the rates of oil biodegradation are primarily controlled by the geochemistry and nutrient limitations. Media simulating near bottom and surface ocean water will be used. Microbial communities will be grown in each type of water then switched to the other, comparing their hydrocarbon degradation rates in both. If microbial community structure is shown to control degradation rates then drilling locations whose communities are not capable of high rates of degradation will need a more extensive spill response plan, where as communities that are better equipped to handle a spill may allow for a smaller response. If geochemistry is found to control oil degradation rates that spill response procedures should include amending with necessary nutrients; further studies will be needed to identify key nutrients.
  12. T. C. Hazen. 2017. Phenotypic Microarray Provides Functional Verification of Genotype. American Society for Microbiology Microbe 2017 abstract
    16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
  13. M. F. Campa and T. C. Hazen. 2017. Phenotypic Microarray for Fracking studies. American Society for Microbiology Microbe 2017
  14. T. C. Hazen. 2017. Environmental Justice and Disposal of Toxic Waste. Integrating Environment and Health, 17th National Conference and Global Forum on Science, Policy and the Environment. http://www.ncseconference.org/
  15. Song, Y., Q. Yao, G. Wang, X. Yang, C. Pan, E. Johnston, M. Kim, K. Konstantinidis, T. C. Hazen and M. Mayes. 2016. Integrating “omics” data into a biogeochemical model: A new model scheme to predict climate feedbacks from microbial function in tropical ecosystems. AGU Fall Meeting abstract
    Soil microorganisms and their activities, which play a significant role in regulating carbon (C) and nutrient biogeochemical cycles, are highly responsive to changes in climate. The diversity of microorganisms, however, complicates the explicit representation of microbial and enzymatic processes in biogeochemical or earth system models. Uncertainties in accounting for microbial diversity therefore limits our ability to incorporate microbial functions into models. However, ‘omics technology provides abundant information to identify the structure and function of the microbial community and strengthens our ability to understand microbially-mediated C and nutrient cycles and their climate feedbacks. We collected soils from control and phosphorus (P) fertilized plots at the Gigante Peninsula long-term fertilization experiment at the Smithsonian Tropical Research Institute in Panama, an ecosystem where P limitation constrains primary productivity and microbial activities. We monitored effects P addition on soil carbon decomposition with respiration measurements and investigated the responsible microbial mechanisms with metagenomics, metatranscriptomics, metaproteomics, and enzyme activity assays. We integrated the P dynamics into the C-N coupled Microbial Enzyme Decomposition (MEND) model. We integrated the ‘omics data with the new microbially-enabled C-N-P model to examine the mechanistic responses of soil microbial activity and heterotrophic respiration to P availability. Our finding indicates that increases in soil P availability can alter both the abundance and activity of enzymes related to soil carbon decomposition and P mineralization in the tropical soil, leading to increased CO2 emissions to the atmosphere. Integrating the ‘omics data into the biogeochemical model enabled scaling of complex ecosystem functions from genes to functional groups to enable predictions of microbial controls on C, N and P cycles.
  16. Wu, X., T. C. Hazen, P. M. Fox, P. S. Nico, Q. Li, W. Yang, Y. Liu, N. J. Hess, P. Zhang, Y. Qin and J. Zhou. 2016. Interactions between Natural Organic Matter and Native Microbes in the Oak Ridge FRC Groundwater. AGU Fall Meeting abstract
    Natural organic matter (NOM) is central to microbial food webs; however, little is known about the interplay between the physical and chemical characteristics of the carbon in NOM and its turnover by microbial communities. Microbial activity changes NOM’s structure and properties, which may further influence the bioavailability of NOM. The change of NOM may reversely affect the microbial community structure as well. To date, our understanding of these interactions is insufficient, and it is critical to identify the role of NOM to carbon turnover, structure of microbial community and to the metabolic potential of that community. In this study, we aimed to study the interactions between NOM and native microbial communities present in groundwater at a background site (FW305 well) at Oak Ridge Field Research Center, TN. The total organic carbon and inorganic carbon in FW305 deep sediment samples were 0.071% and 0.011%, respectively. Water-soluble NOM was extracted from these sediment samples, the extraction efficiencies were 3.2% for organic carbon and 1.6% for inorganic carbon. The extracted NOM was then provided as the sole carbon source to native microbes present in groundwater. Subsamples were harvested several times from these incubations during a 50-day study. 16S rRNA gene amplicon sequencing and Geochip were used to identify the changes of microbial communities and expression of functional genes during transformation of the NOM. Several advanced chemical techniques including FTICR-MS and NEXAFS were used to characterize the C pool (i.e., NOM metabolites and microbial byproducts). Preliminary data clearly showed that microbial community responded to NOM, and shifted as functional groups in NOM transformed. Further detailed metabolite and gene-based analysis to elucidate these changes is currently being conducted.
  17. T. C. Hazen. 2016. Deep Water Horizon Engineering Lessons’ Learned. Engineering Fundamentals College of Engineering
  18. T. C. Hazen. 2016. Oil Biodegradation Potential in Deep Marine Basins Worldwide. 23rd International Petroleum Environmental Conference (IPEC) abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water.  Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels.  Furthermore, this degradation took place without significant oxygen depletion.  Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common.  The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment and microbial community structure on several other potential deep-water drilling sites around the world (Eastern and Central Mediterranean, Caspian Sea, Great Australian Bight, and off the coast of Angola) all of which are quite different then the Gulf of Mexico.
  19. T. C. Hazen and G. S. Sayler. 2016. Environmental Systems Approaches to Bioremediation of Contaminated Sites. The 2016 China-US Joint Annual Symposium “International Nexus of Food, Energy, Water, and Soil”
  20. T. C. Hazen and S. M. Techtmann. 2016. Metagenomic Applications in Environmental Monitoring and Bioremediation. The 2016 China-US Joint Annual Symposium “International Nexus of Food, Energy, Water, and Soil”
  21. T. C. Hazen. 2016. Lessons from Deep Water Horizon?. Microbial Ecology
  22. T. C. Hazen. 2016. Life in the Slow Lane: Limits to Life in the Subsurface. Micro 606 abstract
    A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced using a combination of Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Moorella thermoacetica), as well as other known sulfate reducers and thermophilic organisms. D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea. Additionally, study of the deep subsurface has offered access to the simplest community yet studied by environmental genomics, perhaps consisting of just a single species that is capable of performing all of the tasks necessary for life.
  23. T. C. Hazen. 2016. Life in the Slow Lane: Limits to Life in the Subsurface. Philosophical Society of the Oak Ridge Institute for Continued Learning abstract
    A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced using a combination of Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Moorella thermoacetica), as well as other known sulfate reducers and thermophilic organisms. D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea. Additionally, study of the deep subsurface has offered access to the simplest community yet studied by environmental genomics, perhaps consisting of just a single species that is capable of performing all of the tasks necessary for life.
  24. T. C. Hazen. 2016. Methane: The New Paradigm. UT Science Forum
  25. T. C. Hazen. 2016. Oil bioremediation. Cenovus
  26. T. C. Hazen. 2016. Systems Biology of Oil Biodegradation in 5 Deep Marine Basins, Implications from Deep Water Horizon?. University of Tennessee, Department of Biochemistry & Cellular and Molecular Biology Seminar
  27. T. C. Hazen. 2016. NSF GRFP to Microbiology Graduate Students. University of Tennessee
  28. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral and T. C. Hazen. 2016. Stream water microbial population resistance to biocides used in hydraulic fracturing fluids. International Symposium for Microbial Ecology abstract
    Hydraulic fracturing (HF) has grown very fast in the past several years, with an estimated 700% increase since 2007. During the process of HF a cocktail of chemicals, sand, and water are injected into a shale formation to create fractures, which enable release of gas. In most HF operations, biocides are added to prevent biofouling of equipment and prevent growth of subsurface microbes during gas extraction in order to avoid gas souring. During, extraction some of the injected water returns to the surface carrying with it many of injected chemicals including the remaining biocides. It has been reported that flowback water has been accidentally released into the environment potentially exposing streams to HF chemicals such as biocide. To understand the effect biocides may have in case of HF flowback water surface spills, microcosms were inoculated using stream water impacted by Marcellus shale HF operations (three streams), and as HF not impacted stream water from the same Marcellus shale area (three streams) was used. Two groups of microcosms were set using the two more commonly used biocides: glutaraldehyde (100 ppm concentration) and DBNPA (125 ppm concentration). The microcosms were incubated aerobically at ambient temperature over a period of 8 weeks. The microbial community adaptation to biocide was tracked by sampling every two weeks and performing 16s rRNA amplicon sequencing as compared to a biological control (no biocide added). Chemical degradation of the biocide was tracked every week by HPLC (for DBNPA) and GC-FID (for Glutaraldehyde) as compared to an autoclaved control to track abiotic degradation of the biocides. Preliminary data for the Glutaraldehyde microcosms shows the genus Methylobacterium in enriched in both the HF impacted and not impacted sites, while the biological control shows no enrichment of the genus Methylobacterium. Simpson alpha diversity analysis, shows evenness and similarity of the microbial population decrease drastically in the HF not impacted sites as compared to the HF impacted sites, presumably because the HF affected sites have more members of the microbial community able to tolerate the biocides. Preliminary data for DBNPA, shows an enrichment of an unclassified genus. Simpson alpha diversity analysis shows a decrease in evenness and richness of the HF not impacted sites at week two, but then the alpha diversity increases at the experiment continues. DBNPA quantification shows higher degradation of the biocide in HF not impacted sites as compared to the abiotic control, showing that biodegradation is taking place. However, the HF impacted sites, show less degradation of DBNPA as compared to the abiotic control. This may indicate that HF spill and the amendment of biocide inhibit the microbial population capable of degrading the DBNPA. The aim of this on-going study is to understand the pathways of biocide resistance and the effect they have in the native microbial community in case of a HF fluids spill into the environment.
  29. Curtis, D., P. Zhang, Z. He, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2016. Microbial Populations Influencing Metal and Nitrogen Cycling are Structured Along Contaminant Gradients at a Nuclear Legacy Site. International Symposium for Microbial Ecology abstract
    Microbial communities perform key roles in biogeochemical cycling, which in turn, is an important component of ecosystem function in Earth’s biomes. As such, how anthropogenic contaminants impact microbial community function is a critical question. In this study, the structure and response of subsurface functional microbial populations to uranium and nitrate contamination were evaluated. Groundwater samples were collected along uranium and nitrate gradients and extracted DNA was analyzed with a comprehensive functional gene array. For analysis, samples were categorized as high, moderate and low concentration based on the EPA’s maximum contaminant levels for nitrate (10 mg/L; LN, MN, HN) and uranium (0.03 mg/L; LU, MU, HU). Ordination plots revealed clustering of samples by the level of nitrate or uranium present and genes from known metal and uranium reducing genera were detected in all sample groups. Dissimilatory tests with cytochrome and hydrogenase genes identified significant differences between LU-HU groups and between LU-HU and MU-HU groups, respectively, while nitrate reduction genes showed significant differences among all groups. The abundance of cytochrome, dsrAB and hydrogenase genes decreased from LU to HU, while the abundance of nitrate reduction genes was highest in the MN samples. Canonical correspondence analysis indicated that in addition to NO3- and U, pH and SO42- exerted strong influences on the subsurface community structure, irrespective of whether samples were grouped by nitrate or uranium. These findings point to impacts in biogeochemical cycling within the subsurface with potential downstream effects on ecosystem function.
  30. Justice, N. B., A. Sczesnak, T. C. Hazen and A. P. Arkin. 2016. Unraveling Community Assembly and Organism Interactions with Large Scale Enrichment Culturing. International Symposium for Microbial Ecology abstract
    Bacterial population structures are central to explaining microbial ecosystem function and properties. However, the ecological forces that shape community structures—including species interactions—are myriad and complex, leaving gaps in our ability to understand and predict microbial community structure and functioning. Here we examine microbial community assembly, uncover species interactions, and examine the influence of abiotic factors in microbial community structure by systematically varying the number of organisms founding each of ~1,000 enrichment cultures started from a single groundwater inoculum. We inoculated the groundwater (containing ~37,000 cells mL-1) into both aerobic and anaerobic nitrate-reducing cultures that spanned five dilutions (10-1-10-5) and, following incubation, community structures were evaluated with 16S gene amplicon sequencing. As expected, species richness decreased with increasing inoculum dilution as low abundance individuals were removed. Aerobic and anaerobic communities varied in community composition and taxonomic membership, especially at high inoculum concentrations. Using a most probable number technique, we estimated abundance (as cultivable units/mL of each taxon) of each taxon in the initial sample in aerobic and anaerobic enrichment conditions, and approximated that only ~5-7% of cells from the initial inoculum were cultured. The initial estimated abundances of each OTU were used to develop a null model of community assembly which, compared to measured data, was used to bin organisms as putative strong or weak competitors. Although strong competitors were rare (<5% of cultivated taxa), they drastically shaped community structures when present. Finally, we calculated co-occurrence probabilities for abundant taxa to infer putative positive or negative interspecific interactions amongst organisms. Nearly twice as many interactions were detected in anaerobic samples as aerobic samples, with many of the negative interactions pointing to antagonistic relationships between species of the Bacillaceae with species of Oxalobacteraceae, Paneibacillaceae, and Pseudomonadaceae. Together, this novel approach allows us to show how abiotic and biotic factors interact to structure microbial communities.
  31. Kothari, A., Y.-W. Wu, M. Charrier, L. Rajeev, A. M. Rocha, T. C. Hazen, P. Dehal, D. Chivian, S. Spencer, E. Alm, S. Singer and A. Mukhopadhyay. 2016. Plasmidome studies reveal a variety of horizontally transferred functions within the microbial communities at the Oak Ridge Field Research sites. International Symposium for Microbial Ecology abstract
    The Oak Ridge Field Research Center, in Oak Ridge, TN, has contaminated and uncontaminated areas/wells where research can be conducted on the microbial communities present in the groundwater and sediment. Plasmids host and distribute non-essential genes, independent of the host’s chromosome, thereby benefitting the host bacteria in certain specific environmental conditions. The best-characterized wells at the Oakridge site are now documented to contain several hundred bacterial strains, many of which are likely to contain plasmids. This is the first study to selectively isolate and analyze the plasmid population from these sites. Plasmid isolation was optimized using a model system comprising of three strains containing plasmids of three different sizes (5, 48 and 202 kb). Subsequently, plasmid DNA was isolated from the groundwater of two background wells GW460 and GW456. Genomic DNA was eliminated with the use of a plasmid-safe-DNase, the plasmid DNA was amplified with Phi29 DNA polymerase, and subjected to deep sequencing (Illumina). A total of 42543 (including 67 circular contigs) and 32313 contigs (including 545 circular contigs) greater than 2kb size were assembled from the wells GW456 and GW460, respectively. These encode several known plasmid- associated genes such as those involved in plasmid replication and mobilization. They also encode a variety of other functions, such as genes involved in secondary metabolism, antibiotic resistance, heavy metal resistance, and nitrogen metabolism,. The plasmid-represented functions were comparable in both wells. Interestingly, the most abundant circular contigs (plasmids) from both wells contained genes annotated to be involved in mercury uptake and resistance, along with plasmid mobilization and replication genes. We discuss these and other findings from the plasmidome analysis.
  32. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2016. Microbial Community Changes and Crude Oil Biodegradation in Different Deep Oceans. International Symposium for Microbial Ecology abstract
    Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil and dispersant in various deep-sea basins around the world where oil exploration is anticipated (Eastern and Central Mediterranean Sea, Great Australian Bight and Caspian Sea). In-lab microcosm experiments were set up aerobically to study the microbial respiration, community changes and oil biodegradation. Microbial respiration followed a similar pattern in all of these basins. The treatment of oil and dispersant had the highest CO2 production. The amendment of oil lead to a higher CO2 accumulation compared to control. However, they were all much lower than the Gulf of Mexico (GOM). What’s more, oil biodegradation occurs in all of the sites. The total organic carbon revealed that a big portion of oil was degraded in the first several days, which was consistent with the GC-MS results. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil and dispersant can simplify the community and speed up the oil biodegradation. In particular, the relative abundance of Proteobacteria increased drastically while the relative abundance of archaea decreased. In addition, although oil and dispersant drive the community into a similar endpoint, different bacterial groups were found associating with oil and dispersant.
  33. Ning, D., J. Wang, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, M. B. Smith, A. M. Rocha, S. W. Olesen, C. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, S. M. Techtmann, D. C. Joyner, D. Elias, K. L. Bailey, R. A. Hurt, S. P. Preheim, M. C. Sanders, M. A. Mueller, D. B. Watson, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, E. J. Alm, T. C. Hazen, A. Lancaster, B. J. Vaccaro, F. L. Poole, M. W. Adams and J. Zhou. 2016. Disentangling Ecological Processes and Drivers In Subsurface Microbial Community Assembly In A Nuclear Waste Site. International Symposium for Microbial Ecology
  34. Paradis, C., N. Mahmoudi, S. Jagadamma, D. Driver, K. O’Dell, S. Schaeffer and T. C. Hazen. 2016. Response of soil respiration and microbial community structure to simulated heavy precipitation and drought in a Lexington silt loam. International Symposium for Microbial Ecology abstract
    Mean annual precipitation and consecutive dry day lengths are projected to increase in the Southeastern United States and cause dramatic changes to the natural soil moisture regimes of non-irrigated land. Changes to moisture content can strongly affect the production of soil-respired carbon dioxide (CO2) and the structure of soil microbial communities and are thought to be climate-zone and soil-type specific. The objective of this study was to simulate heavy precipitation and drought stress under highly-controlled laboratory conditions to determine the response of soil-respired CO2 and microbial community structure in a Lexington silt loam. Soil samples were collected from a non-tilled, non-fertilized, non-cover crop plot in West Tennessee during a period of normal weather conditions. Batch microcosms were constructed in triplicate with variable water addition per 6-day drying cycles to simulate ambient, heavy, and drought moisture conditions for 37 days. Simulated heavy precipitation resulted in substantially increased soil-respired CO2 and notable shifts in microbial community structure towards taxa that are structurally resistant to increased osmotic pressure (Firmicutes Clostridia) and capable of oxygen-limited metabolic activity (Delta-proteobacteria). Simulated drought resulted in substantially decreased soil-respired CO2 and a shift in microbial community structure (increase in Actinobacteria and decrease in Acidobacteria) towards those previously observed in arid and semi-arid climate zones. The results of this laboratory study yielded valuable insights as to the directions in which soil-respired CO2 and microbial communities may shift at the ecosystem scale in response to a changing climate.
  35. Ulrich, N., C. McLimans, W. Bernard, J. R. Wright, M. F. Campa, T. C. Hazen and R. Lamendella. 2016. Metagenomics, metatranscriptomics, and single cell sequencing of microbial communities associated with hydraulic fracturing. International Symposium for Microbial Ecology
  36. Paradis, C., N. Mahmoudi, S. Jagadamma, D. Driver, K. O’Dell, S. Schaeffer and T. C. Hazen. 2016. Response of soil respiration and microbial community structure to simulated heavy precipitation and drought in a Lexington silt loam. Oak Ridge Institute for Science and Education (ORISE) Summer 2016 Graduate, Post Graduate, Employee Participant, and Faculty Poster Session
  37. Bailey, R. E., W. A. Henke, C. T. Davis, M. F. Campa, T. C. Hazen, A. W. Johnson, N. O. Hoilett, L. R. McAliley and J. H. Campbell. 2016. Heavy-metal contamination and its effects on microbial community structure in soils near Picher, OK, within the Tar Creek Superfund Site. The Extreme Science and Engineering Discovery Environment (XSEDE16)
  38. D. Ribic, O. G. Brakstad, R. Netzer, T. C. Hazen and F. Drabløs. 2016. From simple to complex degradation of hydrocarbons. A concept of metagenome succession in oil-amended microcosms. MaCuMBA Conference abstract
  39. T. C. Hazen. 2016. Phenotypic Microarray Provides Functional Verification of Genotype. American Society for Microbiology Microbe 2016 abstract
    16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
  40. T. C. Hazen. 2016. Phenotypic Microarray Provides Functional Verification of Genotype. American Society for Microbiology Microbe 2016 abstract
    16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
  41. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Microbial Communities Promoting Metal Reduction are Structured Along the Uranium Gradient at a Nuclear Legacy Site. ASM Microbe Annual Meeting abstract
    Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
  42. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella, C. Grant and T. C. Hazen. 2016. Environmental Microbial Community Tolerance and Adaptation to Biocides Use in Hydraulic Fracturing Operations. ASM Microbe Annual Meeting abstract
    Hydraulic fracturing (HF) in the process of injecting a cocktail of chemicals, sand, and water into an underground rock formation to create fractures and release natural gas. Commonly, biocides are added to prevent biofouling of equipment and prevent microbial growth during gas extraction and gas souring. During extraction some of the injected water and chemicals returns to the surface; this flowback has been accidentally released into the environment, potentially exposing streams to HF chemicals such as biocides. To understand the effect biocides have in steams, microcosms were inoculated using stream water impacted by HF operations (three streams), and as control pristine stream water from the same area were used. Two groups of microcosms were set using the two more commonly used biocides, glutaraldehyde and DBNPA. The microcosms were incubated at ambient temperature over a period of 8 weeks. The microbial community adaptation to biocide was tracked by sampling every two weeks and performing 16s rRNA amplicon sequencing as compared to a biological control (no biocide added). Chemical degradation of the biocide was tracked every week by HPLC, as compared to an autoclaved control to track abiotic degradation. Finally, RNA was sampled at time zero and at the end of the experiment to understand what transcripts were upregulated by the presence of biocide, and hence allow the bacteria to adapt and tolerate the biocide. Our aim is that the results of this experiment will help understand the pathways of biocide resistance and the effect they have in the native microbial community.
  43. Curtis, D., P. Zhang, Z. He, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2016. Microbial Communities Promoting Metal Reduction are Structured Along the Uranium Gradient at a Nuclear Legacy Site. ASM Microbe Annual Meeting abstract
    Prominent pH, nitrate and uranium gradients characterize the Oak Ridge Integrated Field Research Center and provide a setting to understand how the unique subsurface geochemistry influences microbial structure and function. This body of work aims to identify the effect of the uranium gradient on structuring the dissimilatory metal reducing bacteria (DMRB), which influence metal reduction in the subsurface. Samples were grouped into low (LU, <0.03 mg/L, 11 wells), moderate (MU, 0.03-1 mg/L, 15 wells) and high U (HU, >1 mg/L, 15 wells) categories. The community structure and functional potential were discerned through data generated via hybridizing groundwater community DNA to a functional gene array (GeoChip 5.0). Samples did not cluster tightly when relating site characteristics to community assemblage using ordination; however dissimilarity tests did reveal significant differences among groups of samples (p<0.05). Significant differences were observed between HU and LU samples when focusing on cytochrome genes while differences between HU-LU and HU-MU samples were found to be significant when using hydrogenase genes as indicators. DMRB including known uranium reducing genera (Anaromyxobacter, Desulfovibrio, Geobacter) were detected in all groups of samples. Their abundance was found to decrease along the uranium gradient. Sulfate, nitrate and pH were the primary geochemical variables influencing the community structure based on the total repertoire of functional genes detected. These results point to structured functional populations within the subsurface and further our understanding the impact of such gradients on DMRB involved biogeochemical cycling.
  44. Harik, A-M. and T. C. Hazen. 2016. Methanotrophically Mediated Bioaggregation to Control Sand Dust. ASM Microbe Annual Meeting abstract
    Sand and Sand dust events are commonplace throughout many parts of the world and they pose a threat to public health, infrastructure, and daily life. Current techniques to control sand dust are limited, expensive, and not environmentally friendly. Some traditional techniques have been outlawed in the US, among other countries, due to concerns about groundwater contamination. Looking towards safer methods current research has begun to focus on bacteria and their byproducts as a substitute for chemical aggregates. We believe that methanotrophic bacteria - bacteria that can live off of methane - and the sugars they excrete could be applied to the sand surface where they will act as a glue to form a stable layer. Methanotrophic bacteria are known for producing excessive amounts of sugars known as extracellular polysaccharides, these sugars are currently used in the pharmaceutical and food additive industries. While methanotrophic bacteria are not the only type of bacteria that can produce sugars they are unique in a few useful ways; the first being that their targeted growth can be encouraged in the environment with the addition of methane gas, other bacteria will not be able to utilize the methane and will not change in abundance. This is useful when applying the bacteria to sand to encourage the bacteria we want to grow - thus stabilizing the sand - but not others; other bacteria with non-specific sources of carbon would not easily be able to have such targeted growth. A second useful aspect of these bacteria is that they are capable of breaking down many pollutants, this is important because the reactors used to grow the bacteria can have a second purpose of destroying environmental contaminants. The production of sugars is not unique to any one species of methanotrophic bacteria, allowing us the possibility to utilize native bacteria from each location’s sand preventing the introduction of new species to the environment. This project is still in the early lab scale stage, but results so far have been promising.
  45. He, Z., P. Zhang, L. Wu, A. Rocha, Q. Tu, Z. Shi, Y. Qin, J. Wang, D. Curtis, J. Van Nostrand, L. Wu, D. Elias, D. Watson, M. Adams, M. Fields, E. Alm, T. C. Hazen, P. Adams, A. Arkin and J. Zhou. 2016. Microbial Functional Diversity Predicts Groundwater Contamination and Ecosystem Functioning. ASM Microbe Annual Meeting abstract
    Background: Anthropogenic activities have significantly impacted the biosphere of Earth due to contamination of air, water and soil environments, thus decreasing biodiversity and destabilizing ecosystem functions. However, little is known about how environmental contamination affects the biodiversity of groundwater microbial communities and its feedbacks to ecosystem functioning.Methods: We used a comprehensive functional gene array (GeoChip 5.0) to analyze the functional diversity of groundwater microbial communities from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide range of uranium, nitrate and other contaminant concentrations, as well as pH and dissolved gases (e.g., CO2, CH4, N2O).Results: It is hypothesized that the functional diversity would decrease as environmental contamination (e.g., uranium, nitrate) increased, or at low or high pH; however, specific populations capable of utilizing or resistant to those contaminants would increase. Thus, it would be possible to predict groundwater contamination and ecosystem functioning using those key microbial functional genes. Our results indicate that the functional richness/diversity significantly (p < 0.05) decreased as uranium (but not nitrate) increased in groundwater, and about 5.9% of specific key functional populations (e.g., dsrA, cytochrome genes, nirK, norB, nosZ, napA) significantly (p < 0.05) increased as uranium or nitrate increased. Also, we showed that microbial functional diversity could successfully predict uranium and nitrate contamination and ecosystem functioning.Conclusions: This study provides new insights into our understanding of the effects of environmental contamination on groundwater microbial communities and the potential for predicting environmental contamination and ecosystem functioning.
  46. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2016. Microbial Community Response and Crude Oil Biodegradation in Different Deep Oceans. ASM Microbe Annual Meeting abstract
    Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern and Central Mediterranean Sea, Great Australian Bight and Caspian Sea). In this study, microcosms were set up aerobically with three different treatments: seawater, seawater + oil and seawater + oil + oil dispersant. Samples were taken at three time points for the analysis of oil degradation by fluorescence and GC-MS, and microbial community changes by16S rRNA sequencing. CO2 evolution followed a similar pattern among off the basins. The treatment of seawater + oil + dispersant had the highest CO2 production. The amendment of oil lead to a more CO2 accumulation than seawater treatment. However, it’s much higher in the Gulf of Mexico (GOM) than other oceans. What’s more, the dissolved organic mater analysis revealed that application of oil dispersant lead to better oil degradation, which was consistent with the GC-MS results. Oil biodegradation appears to occur rapidly in all of the sites. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil amendment affected how quickly the diversity decreased. The relative abundance of Proteobacteria increased drastically while the relative abundance of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
  47. McBride, K., A. Rossi, H. Woo, J. Wang, N. Labbe and T. C. Hazen. 2016. Xylan-degrading Bacteria Isolated and Characterized from Eastern Mediterranean Sea. ASM Microbe Annual Meeting abstract
    Hemicellulose xylan is an important structural component within plant cell walls that is difficult to degrade. There is relatively little information on the diversity of bacteria that produce xylan-degrading enzymes, or the necessary conditions to maximize enzyme production. Additional knowledge about xylanase producing bacteria could lead to more efficient ways to break down plant material for biofuels. Marine environments were sampled because they are rich in hemicellulosic biomass. This study aims to identify xylan-degrading bacteria by sequencing isolates from Eastern Mediterranean Sea, and measuring their growth rates on xylan media. Isolates were cultivated from a laboratory incubation of xylan-amended seawater. A set of ninety isolates were sub-cultured several times on synthetic seawater agar with xylan as the sole carbon source. All strains were then identified by 16S rRNA gene sequencing. Isolates were closely related to Halomonas, Pseudomonas, Joostella, Glaciecola and Janibacter. None of the species found are well-known xylan degraders. Growth curve data and preliminary metabolic tests with xylan show that several isolates have high xylanase producing ability. Understanding more about xylan degradation could find more cost effective ways to produce biofuels.
  48. Ning, D., J. Wang, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, M. B. Smith, A. M. Rocha, S. W. Olesen, C. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, S. M. Techtmann, D. C. Joyner, D. Elias, K. L. Bailey, R. A. Hurt, S. P. Preheim, M. C. Sanders, M. A. Mueller, D. B. Watson, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, E. J. Alm, T. C. Hazen, A. Lancaster, B. J. Vaccaro, F. L. Poole, M. W. Adams and J. Zhou. 2016. Disentangling Ecological Processes And Drivers In Subsurface Microbial Community Assembly In A Nuclear Waste Site. ASM Microbe Annual Meeting abstract
    Background: A central issue in ecology is understanding the processes shaping biodiversity. The groundwater in Oak Ridge Integrated Field Research Challenge site provides a rare opportunity to examine ecological processes and drivers shaping subsurface microbial diversity.Methods: Groundwater samples from 98 wells were analyzed for 205 environmental variables, as well as 16S rRNA genes by an Illumina MiSeq sequencer. After the phylogenetic signal was determined, the influence of selection was estimated based on beta nearest taxon index (βNTI). Then, turnovers not dominated by selection were analyzed using the Raup-Crick metric (RC) based on Bray-Curtis index to estimate the influence of dispersal limitation (RC>0.95) and homogenizing dispersal (RC<-0.95).Results: Across the entire site, both variable selection (44.6%) and dispersal limitation (46.3%) were important in shaping microbial diversity. Environment variables were used to fit the models with βNTI and RC using distance-based redundancy analysis. The model of βNTI identified 25 significant principal coordinates (PCs) imposing selection, with high loading of heavy metals, pH, nitrate, and DO. The model of RC identified 10 significant PCs imposing dispersal limitation, with high loading of spatial eigenvectors or metals in pellets. The spatial distribution of ecological processes showed the areas with little dispersal limitation for the microbial communities were those areas having low elevation, good diffusion of contaminants or a high level of dissolved oxygen.Conclusions: while contaminants and oxygen availability imposed obvious selection on subsurface microbial communities, the microbial spatial turnover was also largely affected by dispersal limitation, which may reflect actual underground dispersal conditions.
  49. T. C. Hazen. 2016. Paradigm change? Predicting water geochemistry from microbial community structure. American Ecological Engineering Society Annual Meeting abstract
    At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry.
  50. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
  51. Yongquist, E., S. M. Hagan, S. M. Techtmann and T. C. Hazen. 2016. Genomic Diversity of Pseudoalteromonas spp. from Geographically Distant Deep Marine Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    The 32 closely related Pseudoalteromonas spp. we isolated were found in different environments not previously sampled throughout the world’s deep oceans. The isolates’ 16S rRNA gene sequences were more than 99% similar, while the genomic makeup was less than 99% similar. The environmental parameters of these locations vary greatly and little is known about how adaptation to growth under different environmental conditions affects the genomic makeup of very closely related bacteria. To address this, we sought to investigate what physical conditions affect the genomic diversity of 10 closely related oil-degrading Pseudoalteromonas strains from three distant deep marine locations. We wanted to determine how physical and chemical factors of the environment contribute to the genomic differences of the 10 Pseudoalteromonas isolates. This was done by comparing the phenotypes of each strain to one another. We compared the optimal growth temperature, along with the carbon, nitrogen, and osmolyte metabolisms of each isolate in this experiment. We hypothesize that the different physical and chemical environmental factors are influencing the genomic differences of the Pseudoalteromonas species. This work will help better understand how diverse a bacterial species is and how the environment is contributing to driving these differences.
  52. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. National Council Undergraduate Research Annual Meeting abstract
    Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
  53. McBride, K., A. Rossi, H. Woo, J. Wang, N. Labbe and T. C. Hazen. 2016. Xylan-degrading Bacteria Isolated and Characterized from Eastern Mediterranean Sea. National Council Undergraduate Research Annual Meeting abstract
    Hemicellulose xylan is an important structural component within plant cell walls that is difficult to degrade. There is relatively little information on the diversity of bacteria that produce xylan-degrading enzymes, or the necessary conditions to maximize enzyme production. Additional knowledge about xylanase producing bacteria could lead to more efficient ways to break down plant material for biofuels. Marine environments were sampled because they are rich in hemicellulosic biomass. This study aims to identify xylan-degrading bacteria by sequencing isolates from Eastern Mediterranean Sea, and measuring their growth rates on xylan media. Isolates were cultivated from a laboratory incubation of xylan-amended seawater. A set of ninety isolates were sub-cultured several times on synthetic seawater agar with xylan as the sole carbon source. All strains were then identified by 16S rRNA gene sequencing. Isolates were closely related to Halomonas, Pseudomonas, Joostella, Glaciecola and Janibacter. None of the species found are well-known xylan degraders. Growth curve data and preliminary metabolic tests with xylan show that several isolates have high xylanase producing ability. Understanding more about xylan degradation could find more cost effective ways to produce biofuels.
  54. Terry C. Hazen. 2016. Oil Biodegradation in Marine Environments. Environmental Writing Course
  55. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella, C. Grant and T. C. Hazen. 2016. Environmental Microbial Community Tolerance and Adaptation to Biocides Use in Hydraulic Fracturing Operations. ACS Annual Meeting abstract
    Hydraulic fracturing (HF) in the process of injecting a cocktail of chemicals, sand, and water into an underground rock formation to create fractures and release natural gas. Commonly, biocides are added to prevent biofouling of equipment and prevent microbial growth during gas extraction and gas souring. During extraction some of the injected water and chemicals returns to the surface; this flowback has been accidentally released into the environment, potentially exposing streams to HF chemicals such as biocides. To understand the effect biocides have in steams, microcosms were inoculated using stream water impacted by HF operations (three streams), and as control pristine stream water from the same area were used. Two groups of microcosms were set using the two more commonly used biocides, glutaraldehyde and DBNPA. The microcosms were incubated at ambient temperature over a period of 8 weeks. The microbial community adaptation to biocide was tracked by sampling every two weeks and performing 16s rRNA amplicon sequencing as compared to a biological control (no biocide added). Chemical degradation of the biocide was tracked every week by HPLC, as compared to an autoclaved control to track abiotic degradation. Finally, RNA was sampled at time zero and at the end of the experiment to understand what transcripts were upregulated by the presence of biocide, and hence allow the bacteria to adapt and tolerate the biocide. Our aim is that the results of this experiment will help understand the pathways of biocide resistance and the effect they have in the native microbial community.
  56. Hazen, T. C.. 2016. Microbial Community Structure Predicts Groundwater and Marine Geochemistry. . . . Seminar Department of Biological Sciences, Michigan Technological University
  57. Brewer, S. S., M. F. Campa, A. Garcia de Matos Amaral, S. M. Techtmann, K. Fitzgerald, J. L. Fortney and T. C. Hazen. 2016. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. 3rd Annual Southeastern Biogeochemistry Symposium abstract
    Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed and is projected to keep growing. This water intensive drilling process creates toxic wastewater without an efficient disposal method. Because this method involves projecting fluid 1-3 km deep into the Earth, it is likely that microbial communities adapted to the extreme conditions of the subsurface have accumulated in the produced water. The goal of this study is to identify microorganisms that might have bioremediation capabilities for the toxic flowback water and characterize microbes isolated from fracking water samples in anaerobic conditions. Water samples were obtained from hydraulic fracturing locations in the Marcellus shale of Pennsylvania. These water samples include six different collections of flowback water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown in anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. Samples were also grown at ambient temperature and at 37°C. DNA was extracted, and 16S rRNA gene Sanger sequencing was used to identify individually isolated microbes. Illumina sequencing was used to yield genetic information about the overall microbial communities. The Biolog Omnilog, a high-throughput phenotype microarray, was used to determine the genotype-phenotype characteristics of some of the most significant isolates. Early results show presence of numerous anaerobic microbes with metabolic variability and bioremediation potential including sulfate reducers and hydrocarbon degraders. There also have been a considerable number of human pathogens identified with the capability for antibiotic resistance from biocide exposure.
  58. Kim, M., E. R. Johnston, T. C. Hazen, M. A. Mayes, C. Pan, Q. Yao and K. T. Konstantinidis. 2016. Response of Soil Microbial Communities to Phosphorus in Tropical Ecosystems. March 12, 2016. Knoxville, TN. 3rd Annual Southeastern Biogeochemistry Symposium. 3rd Annual Southeastern Biogeochemistry Symposium
  59. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2016. Microbial Community Response and Crude Oil Biodegradation in Different Deep Oceans. 3rd Annual Southeastern Biogeochemistry Symposium abstract
    Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern and Central Mediterranean Sea, Great Australian Bight and Caspian Sea). In this study, microcosms were set up aerobically with three different treatments: seawater, seawater + oil and seawater + oil + oil dispersant. Samples were taken at three time points for the analysis of oil degradation by fluorescence and GC-MS, and microbial community changes by16S rRNA sequencing. CO2 evolution followed a similar pattern among off the basins. The treatment of seawater + oil + dispersant had the highest CO2 production. The amendment of oil lead to a more CO2 accumulation than seawater treatment. However, it’s much higher in the Gulf of Mexico (GOM) than other oceans. What’s more, the dissolved organic mater analysis revealed that application of oil dispersant lead to better oil degradation, which was consistent with the GC-MS results. Oil biodegradation appears to occur rapidly in all of the sites. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil amendment affected how quickly the diversity decreased. The relative abundance of Proteobacteria increased drastically while the relative abundance of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
  60. Woo, H. L. and T. C. Hazen. 2016. Using high throughput sequencing methods to identify keystone bacterial species in recalcitrant terrestrial organic matter transformation. 3rd Annual Southeastern Biogeochemistry Symposium abstract
    The microbial transformation of terrestrial organic matter, particularly recalcitrant lignin and hemicellulose, is a significant but poorly understood phenomenon. 16S rRNA gene amplicon sequencing and metagenomic sequencing are relatively inexpensive molecular tools to quickly assess microbial diversity and functional genes in response to different carbon sources. Using sequencing and bioinformatics, we aim to elucidate the diversity and metabolic potential of bacterial communities subsisting on lignin and hemicellulose in laboratory cultures. Seawater microcosms were incubated with an added concentration of purified lignin or xylan. CO2 respirometry and enzyme assays showed high microbial activity on both substrates. Using an Illumina MiSeq platform, both amended cultures and unamended controls were sequenced for 16S rRNA gene amplicons and metagenomics. Reads were annotated using Qiime and MG-Rast. Annotated data was compared in detail using phyloseq, vegan, and DESeq2 in R. Several species belonging to phylum of Proteobacteria, Flavobacteria, and Firmicutes significantly increased in abundance when lignin or hemicellulose was added. Groups of functional genes related to carbohydrates and aromatic catabolism significantly increased in abundance. Our methods may uncover important species that have been previously overlooked for terrestrial organics degradation.
  61. Hazen, T. C.. 2016. Laboratory Safety. Lunch and Learn University of Tennessee
  62. Chakraborty, R., X. Wu, S. Jagadamma, T. C. Hazen, N. Justice, S. Jenkins, T. R. Northen, M. W. Fields, P. Fox, P. Nico, A. P. Arkin and P. D. Adams. 2016. The Properties of and Microbial Interactions with Natural Organic Matter Extracted from Oak Ridge FRC. DOE Genomic Sciences Contractor Annual Meeting abstract
    Project Goal: Natural organic matter (NOM) availability and transformations determines much of the carbon (C) flux in subsurface environments. However, the molecular signature of this pool of C is largely unknown, and the microbial activities that regulate NOM turnover are still poorly resolved. The goal of this discovery project was to ascertain physical and chemical characteristics of NOM, using minimally destructive techniques that retains the originality of the material, and to use it as a C source in enrichments to study it’s turnover by microbial communities at Oak Ridge FRC. Discovery projects are short term, high impact, investigatory efforts to drive changes in science or technological capability that deeply impact the program in some way. NOM was obtained by extracting FW305 and FW306 sediment samples from different depths using MilliQ-water via shaking and sonication. Using this extraction, total organic C in NOM was 0.4% in the surficial layers and dropped to 0.15% in the deeper layers. The amount of inorganic C in extracted NOM decreased significantly with depth. Results from UV and FTIR analyses showed that extracted NOM mainly contained aromatic and unsaturated compounds in shallower depths and mostly polysaccharides in deeper sediments. HPSEC was used to study the molecular weight distribution of the NOM and a stirred ultrafiltration cell was used to fractionate the extracted NOM based on molecular weight. Extracted NOM was provided as the sole C source to microbes present in background well waters of Oak Ridge FRC. Analysis of the enriched microbial community, and transformed NOM metabolites was carried out. Several fine-scale chemical techniques including FTIR, LC-TOF-MS, and Orbitrap were used to characterize the metabolites, and 16S rRNA sequencing and metatranscriptomics were used to identify the changes in microbial communities in these enrichments. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
  63. Kothari, A., Y.-W. Wu, M. Charrier, L. Rajeev, A. M. Rocha, T. C. Hazen, P. S. Dehal, D. Chivian, S. J. Spencer, E. J. Alm, S. Singer, A. Mukhopadhyay, A. P. Arkin and P. D. Adams. 2016. Extrachromosomal Plasmid DNA Project. DOE Genomic Sciences Contractor Annual Meeting abstract
    Project Goals: The extrachromosomal plasmid DNA project is one of the discovery projects funded by ENIGMA. It is a short term, high impact, investigatory effort to study the plasmid populations of the ENIGMA wells. The goal of this project is to exclusively explore the prevalence of plasmid DNA in these communities. Additionally, the project aims at studying the relevant functional genes that are typically encoded on the plasmids, conferring advantageous traits to the host, in these communities. Plasmids are autonomously replicating extra-chromosomal genetic elements that often act as mediators of horizontal gene transfer in the environment. Plasmids host and distribute non-essential genes, independent of the host’s chromosome, thereby benefitting the host bacteria in certain specific environmental conditions. Native plasmids typically range from 2 kb to 250 kb in size and have been shown to be present in 10-30% of the cultivated isolates from varied environments. The best-characterized wells at the Oakridge site are now documented to contain several hundred bacterial strains, many of which are likely to contain plasmids. This is the first study to selectively isolate and analyze the plasmid population from these sites. To optimize a robust method that isolates a range of plasmid sizes, we developed a model system comprising of three strains containing plasmids of sizes – 5kb, 48kb and 202kb in equal proportions, and tested the potential of various alkaline hydrolysis based methods to isolate plasmids from the serial dilutions of the mixed population. The presence of each plasmid was determined by targeting a unique plasmid borne gene via qPCR. In order to get rid of genomic DNA contamination, the isolated DNA was subjected to Plasmid-Safe-ATP-Dependent DNase and the lack of genomic DNA contamination was confirmed using degenerate primers targeting the 16s rRNA coding sequence. The total plasmid DNA thus obtained was amplified using Phi29 DNA polymerase to generate high-quality template for use in DNA sequencing. To increase the sensitivity of the plasmid isolation procedure, the extraction procedures and Phi29 amplification conditions were optimized. Subsequently, plasmid DNA was isolated from the wells GW460 and GW456 and subjected to deep sequencing using the Illumina paired-end protocol. The reads obtained were trimmed using Trimmomatics, assembled using IDBA-UD (Iterative De Bruijn graph Assembler for reads with Highly Uneven Sequencing Depth) and subjected to MG-RAST to produce gene calls, functional annotations and taxonomic classification. A total of 42543 (including 130 circular contigs) and 32313 contigs (including 760 circular contigs) above 2kb size were detected from the wells GW456 and GW460, respectively. These encode several known plasmid associated genes along with genes involved in secondary metabolism, antibiotic resistance, metal resistance, and nitrogen metabolism, to name a few. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
  64. Rocha, A. M., B. Adams, C. Paradis, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. M. Klingeman, D. B. Watson, D. C. Joyner, S. Jagadamma, J. L. Fortney, J. J. Zhou, J. D. Van Nostrand, M. W. W. Adams, R. Chakraborty, D. Elias, E. J. Alm, T. C. Hazen, A. P. Arkin and P. D. Adams. 2016. Temporal Variation in Groundwater Microbial Community Structure: Implications for Groundwater Monitoring. DOE Genomic Sciences Contractor Annual Meeting abstract
    Project goals: The overarching goal of the Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) field microbiology component is to understand the interactions between environmentally relevant microbial communities and their environment. As part of this, we must understand the mechanisms that may potentially impact microbial community structure, function, and activity. At the DOE Oak Ridge field site, groundwater wells are subject to temporal and seasonal changes in groundwater hydrology and geochemistry, which may result in temporal bias in microbial community monitoring data. Here, we characterize the microbial community response to temporal variability to determine to what extent variation in groundwater geochemistry and hydrology impacts microbial community structure and function. Large-scale groundwater sampling events, such as those associated with biomonitoring, may span the course of several weeks or months. Based on the sampling methods and time-scales involved, microbial community and geochemical data may contain significant temporal bias, as well as, biases attributed to external factors. In this study, we characterized the temporal dynamics of microbial groundwater communities at the background site of the Oak Ridge Field Research Center in Oak Ridge, TN to determine (1) if, and to what extent, temporal and/or seasonal variation of the groundwater geochemistry affects the microbial community structure and (2) to assess the impact of groundwater flow and transport of geochemical constituents on the microbial structure. To determine how resilient microbial communities are to daily and weekly changes in groundwater chemistry, the temporal dynamics of microbial communities from six groundwater wells were monitored and compared to geochemical and hydrological measurements. Of the six wells, we physically and chemically cleaned four wells to remove biofilm and attached particulates from the well casing. The remaining two wells that weren’t cleaned served as controls. All wells were sampled prior to cleaning to establish a baseline microbial community profile. Post-cleaning, each well was sampled a total of twelve times from December 1, 2014 – January 12, 2015. For each well and time-point, groundwater samples were collected for geochemical and microbial community analyses using low flow sampling methods. Nucleic acids were collected by sequentially filtering water through a 10μm pre-filter and 0.2μm-membrane filter and then extracted using a Modified Miller method. A total of 6,959 OTUs were identified across all six wells and size fractions. Of the OTUs Proteobacteria represented a significant portion of the taxa. Analyses of microbial community data indicate overall diversity of the taxa did not vary significantly during time-course sampling. However, significant shifts in the population were observed between cleaning treatments and during sampling time point 01/5/15 for select wells. Additionally, daily and weekly variation in the relative OTU abundance within each well was detected. Throughout the study, groundwater geochemical measurements were relatively stable. However, shallow wells varied in concentrations of Na+, K+, Ca2+, HCO3 -, and CO3 -2 following rain events. The geochemical values for these ions are consistent with distinct differences in water types between deep and shallow wells. The stability of the geochemical measurements may indicate that groundwater chemistry is not a dominant factor in the observed daily and weekly variances, but rather contributes to taxonomic differences observed between well depths. However, further analysis of geochemical shifts at higher resolutions is necessary to understand the full impact of geochemistry on microbial response. Furthermore, analyses of groundwater results indicate that the pumping/sampling of wells did not contribute to sampling bias. Overall, results demonstrate that groundwater microbial community data contain temporal biases. As such caution must be used when designing large-scale sampling events. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231
  65. Spencer, S. J., M. V. Tamminen, A. M. Rocha, T. C. Hazen, E. J. Alm, A. P. Arkin and P. D. Adams. 2016. Assays for spatial structure and transdomain dynamics in environmental communities. DOE Genomic Sciences Contractor Annual Meeting abstract
    Project Goals: Our group focuses on biotechnology development that moves the field of microbial ecology toward complete genomic awareness. Specifically, we use bulk water-in- oil emulsion droplets combined with tailored molecular biology to provide a more comprehensive microscale view of transdomain ecological players and their functional capacity within complex environments. The bulk activity of microbial communities is composed of the additive effect of microscale interactions between bacteria, viruses, and eukaryotes coexisting within a dynamic environment. These microscale competitive or mutualistic exchanges bridge between the foundational principles of ecology and the global activity of microbial communities that we observe in bulk assays. Before we are able to understand, model, or perturb systems at the macroscopic scale, we need improved methods at the resolution of individual cells. Our group recently developed an emulsion-based droplet assay termed epicPCR (emulsion, paired isolation, and concatenation PCR) to physically link functional genes with phylogenetic indicators within single cells. Here we expand upon this platform to map the physical associations of bacteria with each other and with eukaryotic hosts. We are beginning to assay biofilm and host-prey structures by capturing small aggregates of cells in nanoliter droplets, then physically linking segments of the 16S rRNA gene between cells. In biofilms a preliminary untargeted assay, trying to link every cell with every other adjacent cell, highly favored only the most abundant strains present. We’ve now redesigned primer sets that anneal to specific phyla of interest and their physical partners, a semi-targeted version of the assay. With this approach we’ve recovered library contructs enriched for cells as rare as 1 in 10,000 within complex biofilms. In parallel to assays of bacterial proximity, we’ve refined the same droplet methodology to capture eukaryotes with their bacterial symbionts and prey. Sequenced co-aggregations between eukaryotes and bacteria in both wastewater and lake water are enriched for predators, heterotrophs, and known symbionts. We plan to apply this approach to Oak Ridge FRC samples, and in preparation we’ve completed standard eukaryotic and bacterial sequencing from control wells. These structural assays in combination will provide novel microscale data about the complex interchanges connecting bacteria with each other and their broader ecological context. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231
  66. Thorgersen, M. P., B. J. Vaccaro, W. A. Lancaster, F. L. Poole, A. E. Kazakov, L. Rajeev, M. Garber, G. M. Zane, M. N. Price, K. M. Wetmore, A. M. Rocha, T. Mehlhorn, P. S. Novichkov, A. M. Deutschbauer, A. Mukhopadhyay, J. D. Wall, R. Chakraborty, T. C. Hazen, M. W. W. Adams, A. P. Arkin and P. D. Adams. 2016. Microbial responses to toxic metals in the Oak Ridge Reservation environment. DOE Genomic Sciences Contractor Annual Meeting abstract
    Project Goals: The environmentally relevant isolation of several metal-resistant microorganisms is described along with the characterization of a novel uranium-binding complex from a strain isolated from the ORR site. Abstract: The metal resistance campaign is focused on investigating molecular mechanisms to microbial metal resistance. One of the defining characteristics of the Oak Ridge Reservation (ORR) environment is the presence of mixed industrial waste and the effect this waste has on the groundwater microbial community. Metals are a key component of the mixed waste with concentrations of uranium, aluminum, manganese, cadmium and cobalt in contaminated groundwater wells over 1,000 times greater than those in pristine background wells. The metal resistance campaign is exploring the effects of metal toxicity on the ORR groundwater community in several ways. Strains will be described that were isolated from ORR groundwater in media that contain metal concentrations similar to those present in the contaminated environments. The physiology of metal toxicity was studied through measurement of genomewide gene fitness under copper, zinc, chromium and uranium toxicity using the model organism Pseudomonas stutzeri RCH2. In addition, the properties of a uranium-binding protein complex from the ORR isolate Pelosinus fermentans UFO1 will be presented.
  67. T. C. Hazen. 2016. Deepwater Horizon Oil Spill. Seminar to 12th grade science classes at Lincoln International Academy. abstract
    The microbial transformation of terrestrial organic matter, particularly recalcitrant lignin and hemicellulose, is a significant but poorly understood phenomenon. 16S rRNA gene amplicon sequencing and metagenomic sequencing are relatively inexpensive molecular tools to quickly assess microbial diversity and functional genes in response to different carbon sources. Using sequencing and bioinformatics, we aim to elucidate the diversity and metabolic potential of bacterial communities subsisting on lignin and hemicellulose in laboratory cultures. Seawater microcosms were incubated with an added concentration of purified lignin or xylan. CO2 respirometry and enzyme assays showed high microbial activity on both substrates. Using an Illumina MiSeq platform, both amended cultures and unamended controls were sequenced for 16S rRNA gene amplicons and metagenomics. Reads were annotated using Qiime and MG-Rast. Annotated data was compared in detail using phyloseq, vegan, and DESeq2 in R. Several species belonging to phylum of Proteobacteria, Flavobacteria, and Firmicutes significantly increased in abundance when lignin or hemicellulose was added. Groups of functional genes related to carbohydrates and aromatic catabolism significantly increased in abundance. Our methods may uncover important species that have been previously overlooked for terrestrial organics degradation.
  68. 2015. Yeast-Filled Fibers Could Treat Polluted Wastewater.
  69. Hazen, T. C.. 2015. Chair: Genotype/Phenotype. 3rd Conference of Phenotype MicroArrays
  70. Hazen, T. C.. 2015. Phenotypic and Genomic Heterogeneity among Colwellia psychrerythraea Strains from Distant Deep-Sea Basins. 3rd Conference of Phenotype MicroArrays abstract
    16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
  71. Hazen, T. C.. 2015. International Scientific Advisory Committee. 3rd Conference of Phenotype MicroArrays
  72. 2015. Postdoctoral Fellow Honored for Helping Advance Latinas in Tech Field.
  73. Adams, B. G., A. M. Rocha, C. Paradis and T. C. Hazen. 2015. Geochemical Response to Temporal Variations in Groundwater Head. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
  74. Campa, M. F., S. Techtmann, M. L. Patterson, A. Garcia de Matos Amaral, R. Lamendella, C. Grant and T. C. Hazen. 2015. Environmental microbial community tolerance and adaptation to biocides use in hydraulic fracturing operations. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
  75. Chen, C., G. Pan, W. Shi, F. Xu, S. M. Techtmann, S. M. Pfiffner and T. C. Hazen. 2015. How does clay flocculation of harmful algal blooms affect microbial community composition in water and sediment. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
  76. Hazen, T. C., M. B. Smith, A. M. Rocha, C. S. Smillie, S. W. Olesen, C. Paradis, L. Wu, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, D. A. Elias, K. L. Bailey, R. A. Hurt, S. P. Preheim, M. C. Sanders, J. Yang, M. A. Mueller, S. Brooks, D. B. Watson, P. Zhang, Z. He, E. A. Dubinsky, P. D. Adams, A. P. Arkin, M. W. Fields, J. Zhou and E. J. Alm. 2015. Microbial Community Structure Predicts Groundwater Geochemistry. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
  77. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2015. Microbial Community changes and Crude Oil Biodegradation and Microbial Community Changes in Deep Oceans. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
  78. Woo, H. L. and T. C. Hazen. 2015. Using high throughput sequencing methods to identify keystone bacterial species in recalcitrant terrestrial organic matter transformation. Critical Zone Science, Sustainability, and Services in a Changing World. Purdue University
  79. 2015. Department of Energy Honors Rocha as Part of Series.
  80. Campa, M. F., S. Techtmann, S. Brewer, A. Garcia de Matos Amarral, K. Manz, K. Carter, R. Lamandella and T. C. Hazen. 2015. Flowback Water: A Look into the Subsurface Microbial Community and Intrinsic Bioremediation. American Society for Microbiology Annual Meeting abstract
    Hydraulic fracturing is growing exponentially, causing a 702% increase in shale gas production in the U.S. since 2007. This process is water intensive, requiring between 3.3 to 27m3 of water per million cubic meters of gas extracted. As this process keeps growing, and water scarcity issues continue to arise throughout the world, questions keep rising on how to efficiently deal with the flowback water (which is up to 40% of the total injected water). The goal of this investigation is to study the microbial community from six flowback water samples and harness intrinsic bioremediation capabilities to develop an inexpensive and efficient method to treat the flowback water for reuse. Flowback water contains over 700 different chemicals; we will select a couple of contaminants of interest (COI) by Gas Chromatography- Mass Spectrometry (GC-MS). In this study, we collected raw flowback and treated flowback water from a fracking site at the Marcellus Shale. The first aim of the study is to determine the microbial community from the flowback water, and how it changes at different stages of the treatment process. This is being done using 16S rRNA amplicon sequencing using Illumina MiSeq. The second aim is to isolate aerobic and anaerobic bacteria with physiologies of interest. To do this, marine broth plates and ORN7a plus oil plates were inoculated at room temperature and 37°C. The marine broth plates are meant to mimic the high salinity environment in the subsurface and the ORN7a + oil plates were used to isolate hydrocarbon degraders. Preliminary observations showed high growth of halophilic bacteria and the presence of sulfate reducing bacteria. The isolates will be sequenced and compared to the microbial community 16S rRNA amplicon data. The isolates that are most representative of the community will be selected for high-throughput phenotypic microarray, OmniLog, analysis. This will be used to do kinetic and metabolic studies of COI present in the flowback water and the isolates. The study is currently in progress. Characterizing the microbial community structure of the flowback water and understanding its function will help develop and optimize a bioremediation strategy, so that flowback water can be inexpensively treated and reused.
  81. Chen, C., T. C. Hazen, G. Pan, W. Shi and F. Xu. 2015. Impact of Clay Flocculation of Algal Blooms on Pond Microbial Community. American Society for Microbiology Annual Meeting abstract
    16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
  82. Curtis, D., P. Zhang, Z. He, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2015. Changes induced in the subsurface microbial community by the nitrate gradient at a nuclear legacy site. American Society for Microbiology Annual Meeting abstract
    It is important to understand the impact of contamination events on subsurface microbial community-induced biogeochemical cycling. The Oak Ridge Integrated Field Research Center (OR-IFRC) is a nuclear legacy site characterized by a contamination plume emanating from the former S-3 ponds. Wastes within the plume have contributed to significant pH, nitrate and uranium gradients across the site. This work aims to identify the effect of the nitrate gradient on the subsurface microbial community involved in nitrogen cycling. DNA extracted from groundwater was utilized to study the functional composition of microbial communities from low (LN, 1-5mg/L, 13 wells), moderate (MN, 10-100mg/L, 12 wells) and high NO3- (HN, >100mg/L, 13 wells) areas using a functional gene array (GeoChip 5.0). Detrended correspondence analysis revealed that samples were not generally clustered based on the nitrate concentration, though significant differences (p<0.05) between LN-MN, LN-HN and MN-HN groups were observed using dissimilatory tests. Canonical correspondence analysis indicated NO3- strongly influenced the community structure only in HN samples. The abundance of genes contributing to denitrification and nitrate reduction was higher in the MN group relative to the LN and HN groups. A significant decrease in abundance of genes contributing to denitrification (narG, nirS, norB) and dissimilatory nitrate reduction to ammonia (nrfA) was evident between MN-HN groups (p<0.05). The findings presented offer an insight into both the structure of and the factors influencing the subsurface microbial communities in relation to a large nitrate gradient.
  83. Fortney, J. L., J. Liu, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2015. Oil Biodegradation in Oxygen Minimum Zones. American Society for Microbiology Annual Meeting
  84. He, Z., P. Zhang, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, D. Curtis, J. D. Van Nostrand, L. Wu, E. J. Alm, M. W. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. D. Adams and J. Zhou. 2015. Microbial Functional Genes Predict Groundwater Contamination and Ecosystem Functioning. American Society for Microbiology Annual Meeting
  85. Jagadamma, S., C. J. Paradis, S. W. Olesen, A. M. Rocha, D. C. Joyner, J. L. Fortney, D. B. Watson, D. Elias, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, P. Zhang, R. Chakraborty, M. Fields, M. W.W. Adams, J. Zhou, E. J. Alm and T. C. Hazen. 2015. The Memory Effect: Investigating the exposure-history dependence of electron donor biodegradation rates in groundwater. American Society for Microbiology Annual Meeting abstract
    Biodegradation rates of electron donors in groundwater are well known to depend on numerous site-specific physical, chemical and biological parameters. However, emerging scientific evidence suggests that the exposure history of a groundwater zone to an electron donor can have a prolonged and positive effect on its biodegradation rate, referred to here as “memory effect”. The goal of this study was to investigate the long-term memory effect in a groundwater zone previously exposed to an electron donor. The study site is located on the Oak Ridge Reservation in East Tennessee and is contaminated with nitrate and uranium. In 2009, emulsified vegetable oil (EVO) was pulse-injected into a contaminated groundwater zone in order to stimulate in–situ microbial-mediated nitrate and uranium reduction coupled to EVO oxidation. EVO was rapidly biodegraded and concentrations have been below detectable limits since 2010. In this study, we tested whether the planktonic microbial community from groundwater wells with and without previous EVO exposure exhibited a memory effect. We hypothesized that EVO biodegradation rates would be higher in wells with previous exposure versus those without due to sustained enrichment of EVO-degrading functional genes within genomes. This study specifically aimed to answer the following questions: (i) Can a long-term memory effect be detected? (ii) If detectable, is the memory effect significant? (iii) How long can the memory effect last? and (iv) What mechanism(s) are likely responsible for the memory effect? The first phase of this study is in progress and aimed to determine if the memory effect is detectable. We are currently assessing the planktonic microbial community structure in groundwater from wells with and without an EVO exposure history using 16S rRNA sequencing. If the microbial community structure has returned to its pre-EVO state, we will conduct a second EVO injection in wells with and without previous exposure to investigate the in-situ memory effect. The results of the initial EVO exposure in 2009 and the current (2015) state of the planktonic microbial community structure will be presented here. Investigating the magnitude, duration and mechanism(s) of the memory effect may provide more accurate prediction of the fate of contaminants in environments with various electron donor exposure histories.
  86. Johnston, E. R., Z. Li, A. Harris, B. L. Turner, S. J. Wright, C. Pan, K. T. Konstantinidis, T. C. Hazen and M. A. Mayes. 2015. Predicting climate feedbacks: metabolic response of soil microbial communities to phosphorus and oxygen availability in tropical ecosystems. American Society for Microbiology Annual Meeting abstract
    Soil microbial communities exert significant control over release of greenhouse gases (GHGs) through degradation of soil organic matter (SOM). Global environmental change is expected to substantially impact these processes, especially in humid tropical forests, where a predicted warmer and wetter weather pattern will likely lead to depletion of O2 below-ground. Phosphorus (P) limitation, a defining feature of many tropical soil systems, may also constrain plant productivity and SOM turnover. Tropical soils remain understudied, substantially limiting our predictive ability of GHG emissions from such ecosystems. In this study, soils were collected from the Gigante Peninsula fertilization experiment at the Smithsonian Tropical Research Institute in Panama. Soils were sampled from 40x40 m P fertilized and unfertilized plots in lowland and upland zones and incubated at 26°C under aerobic and anaerobic conditions in the laboratory. A Micro-Oxymax respirometer was used to continuously measure several gases (CO2, CH4, H2S, H2, and O2). A second treatment was employed to compare how those fluxes were affected by addition of P to the incubation experiments. Microbial gene expression under different incubation conditions was investigated through RNA-seq on Illumina HiSeq platform. Illumina HiSeq and PacBio scaffold sequencing from metagenomes was used to assess community functional attributes between plots and for the assembly of dominant microbial populations. Early results reveal that P addition significantly increased SOM turnover and CO2 release in unfertilized plots compared to fertilized plots. This suggests that P availability constrained SOM degradation only in the unfertilized plots. DNA and RNA sequencing was used to assess these results by detecting the presence and activity, respectively, of genes related to phosphorus acquisition and organic carbon oxidation. We will also report on our efforts to incorporate omics-derived information to models of GHG emissions from these tropical soils.
  87. Lamendella, R., J. Wright, N. Weit, S. Rummel, T. C. Hazen, M. Fernanda Campa, D. C. Joyner and C. Grant. 2015. Microbial Community Structure of a Passive Abandoned Coal Mine Remediation System In Pennsylvania. American Society for Microbiology Annual Meeting
  88. Liu, J., J. L. Fortney, S. M. Techtmann, D. C. Joyner and T. C. Hazen. 2015. Microbial Activity and Community Changes to Crude Oil in Deep Oceans. American Society for Microbiology Annual Meeting abstract
    Oil spills such as the Deepwater Horizon Oil spill in the Gulf of Mexico have the potential to drastically impact marine systems. However, offshore oil and gas prospecting is expanding with increased demand for petroleum. As a result, the risk of oil contamination in the marine system remains a huge concern to the public and governments around the world. Many studies have shown that microbial communities can play an important role in oil spill clean up. However, very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. Therefore, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern Mediterranean Sea, Central Mediterranean Sea, Great Australian Bight and Southeastern Atlantic Ocean). In this study, microcosms were set up aerobically in three different conditions: seawater, seawater + oil and seawater + oil + oil dispersant (Corexit). Different treatments were set up with 125ml serum bottles and connected to a Micro-OxyMax respirometer, to constantly measure CO2 levels. Samples were taken at three time points for the analysis of oil degradation by fluorescence and GC/MS analysis and microbial community changes by16S rRNA sequencing. CO2 evolution followed a similar pattern in all of the basins sampled. The treatment of seawater + oil + Corexit had the highest CO2 production.. The amendment of oil lead to a higher CO2 accumulation than seawater only treatment. The CO2 accumulation of seawater samples from the Eastern Mediterranean and Great Australian Bight are higher than the Southeastern Atlantic Ocean and Central Mediterranean Sea. However, they were all much lower than the data from the Gulf of Mexico (GOM). What’s more, the fluorescence of dissolved organic mater revealed that most oil in the microcosms was degraded in the first several days, which was consistent with the GC-MS results. Oil biodegradation appears to occur rapidly in all of the sites, but much slower than what was observed in GOM. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. Oil amendment affected how quickly the diversity decreased. The relative abundance of Proteobacteria increased significantly, while the relative abundance of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Southeastern Atlantic Ocean and Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
  89. Rocha, A. M., T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. M. Klingeman, D. B. Watson, D. C. Joyner, J. L. Fortney, S. Jagadamma, B. Detienne, B. Adams, J. J. Zhou, J. D. Van Nostrand, M. W. W. Adams, F. L. Poole, W. A. Lancaster, R. Chakraborty, E. J. Alm and T. C. Hazen. 2015. The Impact of Groundwater Well Disinfection on Microbial Community Response and Regrowth. American Society for Microbiology Annual Meeting abstract
    Hydrogeological studies have shown that groundwater microbial communities and/or geochemical measurements can be biased by a number of external factors, such as, well construction, well maintenance and sampling methodologies. In a recent study at the Oak Ridge Field Research Center’s (ORFRC) background site, the temporal variation of groundwater microbial communities was monitored during November 2013. Findings showed little to no fluctuation in overall geochemistry. However, 16S rRNA sequencing of the microbial community structure varied on a daily basis. Due to the stability of the geochemistry, we hypothesize that the variation in microbial communities is a result of both a pumping effect and from the inclusion of communities associated with biofouling in the well casing and sloughing off during sampling. In this study our goal is to determine if the daily variation in groundwater microbial community profiles are naturally occurring within the aquifer or if (and to what extent) the variation in community structure is a result of factors associated with biofouling or sampling artifacts. To determine the impact of biofouling on the temporal variation of the groundwater microbial community structure we sampled six wells at the ORFRC background site. Of these, we physically and chemically cleaned four wells to remove biofilm and attached particulates from the well casing. The remaining two wells that weren’t cleaned served as controls. All wells were sampled prior to cleaning to establish a baseline microbial community profile. Post-cleaning, each well was sampled a total of twelve times from December 2014 – January 2015. For each well and time-point, groundwater samples were collected for geochemical and microbial community analyses. Nucleic acids were collected by sequentially filtering water through a 10µm pre-filter and 0.2µm-membrane filter. Currently, the microbial and geochemical data are still being analyzed although the preliminary results indicate geochemical variation in response to rain events during the course of the study. Here, we present the temporal variation in microbial response to the well disinfection. Results of this study will further our understanding of the natural temporal variations versus external factors, such biofouling or sampling-related effects on microbial communities.
  90. Techtmann, S., S. Stelling, D. Joyne, S. Uttukar, A. Harris, N. Alshibli, S. Brown and T. C. Hazen. 2015. Phenotypic and Genomic Heterogeneity among Colwellia psychrerythraea Strains from Distant Deep-Sea Basins. American Society for Microbiology Annual Meeting abstract
    16S rRNA gene sequencing is routinely used to identify the taxonomy of prokaryotes. Recent evidence suggests that microbes with nearly identical 16S rRNA genes can have substantial genotypic heterogeneity. To better understand the diversity within a single microbial species, we set out to characterize the phenotypic and genomic diversity of three strains that would be classified as Colwellia psychrerythraea based on 16S rRNA taxonomy. Colwellia are psychrophilic heterotrophic marine bacteria found in many cold ecosystems. Additionally, Colwellia species have been shown to respond to marine oil spills and were important members of the microbial community in the Gulf of Mexico during the Deepwater Horizon oil spill. In this study we compare the carbon source utilization profiles and genomic diversity for three Colwellia psychrerythraea strains isolated from geographically distant deep-sea basins. We have recently isolated two strains of C. psychrerythraea; strain ND2E from the Eastern Mediterranean and strain, GAB14E from the Great Australian Bight. These two recently isolated strains were compared with the type strain C. psychrerythraea 34H, which was isolated from arctic sediments. To understand the phenotypic diversity of these strains, we employed Biolog phenotype microarrays to test the carbon source utilization profiles of these isolates. To investigate the genomic heterogeneity of these three strains we sequenced the genomes of the two recently isolated strains and compared them with the genome of the type strain. These three isolates share greater than 98.2% 16S rRNA identity. However, the carbon source utilization profiles were distinct for each of the strains with less than half of the carbon sources being shared between all three strains. There were also dramatic differences in the genetic makeup of these three strains. The two most closely related strains, 34H and GAB14 (99.3% 16S rRNA identity), are very divergent on the genomic level (79.8% average nucleotide identity). These differences in genomic content are in part due to large insertions and deletions, which, in some cases, correspond to predicted genomic islands. These findings combine to suggest that there can be substantial phenotypic and genomic heterogeneity among a single microbial species in different geographical locations.
  91. Ulrich, N., J. Wright, A. Rosenberger, T. C. Hazen, M. Campa, D. C. Joyner and C. J. Grant. 2015. A Temporal Analysis of Impacts of Unconventional Natural Gas Extraction on Microbial Communities in Headwater Stream Ecosystems in Northwestern Pennsylvania. American Society for Microbiology Annual Meeting
  92. Wang, J., P. Zhang, L. Wu, Z. He, M. B. Smith, A. M. Rocha, C. S. Smillie, S. W. Oleson, C. J. Paradis, J. H. Campbell, J. L. Fortney, T. L. Mehlhorn, K. A. Lowe, J. E. Earles, J. Phillips, S. M. Techtmann, D. C. Joyner, S. P. Preheim, M. S. Sanders, J. Yang, M. A. Mueller, S. Brooks, D. B. Watson, M. W. W. Adams, W. A. Lancaster, F. L. Poole, E. Dubinsky, D. A. Elias, P. D. Adams, A. P. Arkin, M. W. Fields, E. J. Alm, T. C. Hazen and J. Zhou. 2015. Free-living and particle-attached bacterial communities of groundwater along multiple environmental gradients. American Society for Microbiology Annual Meeting abstract
    We characterized the groundwater bacterial communities of 93 wells from the Oak Ridge Integrated Field-scale Research Challenge site with two habitat fractions presenting different species dispersal traits: free-living (0.22-um filter) and particle-associated (10-um filter). Along the multiple environmental gradients (including pH and contaminants), environmental filtering significantly controlled community spatial variations, as indicated by the significant correlations between community variations and environmental changes, and the non-significant distance-decay relationships for both communities. The dominant effect of environmental filtering was further supported by the significantly lower phylogenetic relatedness than null expectations for all communities, and the species partitioning between the two habitats was mainly controlled by nitrate, but not pH, uranium or spatial factors. For the free-living communities, the diversity was more constrained by spatial factors (latitude or longitude) than their counterpart particle-associated communities, while the latter was more affected by environmental variables (e.g., magnesium, dissolved oxygen and nitrate). Unexpectedly, we found there were no significant differences in the diversity between these two habitats, contrasting with our prediction that particles would provide more niches (e.g., microenvironments in nutrients) to support more species than the surrounding groundwater. Furthermore, the particle-associated communities had a significantly higher spatial variation than the free-living ones, indicating the former have a higher retardation rate and lower dispersal rate. Collectively, our results showed that the multiple environmental filters were dominant in controlling the bacterial communities in the studied contaminated aquifer, but the dispersal effects also played important roles in partitioning the species distribution between different habitat fractions.
  93. Woo, H., K. O'Dell, S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2015. What happens to lignin in the ocean? Evidence of Lignin Transformation from Marine Microcosms. American Society for Microbiology Annual Meeting abstract
    The microbial transformation of allochthonous terrestrial organic matter, particularly recalcitrant aromatic lignin, is a significant but poorly understood phenomenon in the marine ecosystem. The genetic basis of microbial lignin degradation is of high interest because of its potential application to lignocellulosic biofuel and lignin valorization. In this study, we aim to use metagenomics to elucidate the metabolic potential of a marine bacterial community subsisting on lignin. Marine seawater from the oxic and ultra oligotrophic Eastern Mediterranean, near the Egyptian Nile Delta outlet, was amended with insoluble Organosolv lignin and incubated for 2 weeks. During the incubation, microbial activity was assessed by CO2 respirometry and oxidative enzyme assays. The community structure and metabolic potential of the microcosms were assessed using selective cultivation of lignin-degrading isolates, 16S rRNA gene amplicon (microbiome) sequencing, and metagenomic sequencing. Lignin amendment caused higher rates of respiration and oxidative enzyme activity in comparison to the control. Cultivated isolates of Halomonas, Idiomarina, Thalassospira spp. from the lignin amended microcosm possessed oxidative activity. The strains represented several of the dominant OTUs in the microbiome. The metagenome of the lignin amended microcosm had higher abundance of the broad COG categorizes, particularly information storage and processing, and genes encoding aromatic catabolism. Conversely, the unamended control metagenome had signs of starvation conditions, such as desiccation stress, spore protection, plasmid function that were not found in the lignin amended metagenome. We believe the positive effect of lignin on the microbial activity and abundance of aromatic catabolism genes supports the notion of marine lignin degradation by microbes.
  94. Wright, J. R., D. Marabello, J. McDermott, W. Wang, T. Macbeth, M. F. Campa, D. C. Joyner, T. C. Hazen and R. Lamendella. 2015. Microbial Community Structure and Function Associated with Dichloromethane Contaminated Groundwater. American Society for Microbiology Annual Meeting
  95. Wu, X., S. Jagadamma, T. C. Hazen, T. Northen, M. Fields and R. Chakraborty. 2015. Microbial Interactions with Native Natural Organic Matter at Contaminated Sites from Oak Ridge FRC. American Society for Microbiology Annual Meeting abstract
    Natural organic matter (NOM) is central to microbial food webs. Physical and chemical characteristics of NOM differ based on their biogenic precursors as well as the redox state and biogeochemistry of the specific environment where they are present. Microbial activity may cause degradation, oxidation, or reduction of NOM, changing the structures and properties that influence their further bioavailability. So far our understanding of these interactions are insufficient, and indigenous microbial activities that regulate NOM turnover are poorly resolved. Therefore, in order to connect NOM to the structure of the microbial community and to the metabolic potential of that community, our goal was to extract indigenous NOM using minimal destructive techniques. The extracted NOM will then be used to feed microbes (as electron donor/acceptor/carbon source). Sediment samples were collected from the background area well FW305 of our field site- Oak Ridge Field Research Center (FRC),TN. We developed methods that using three mild extracting solvents (pH 6.5–7.0), e.g., phosphate buffered saline (PBS), pyrophosphate, and warm de-ionized water with shaking or mild sonication as extraction method were tested. We also tested the efficacy of glass beads as a physical abrasive. The extracted dissolved organic carbon (DOC) was in the range of 8.8–28 mg/L. Sonication with PBS could extract higher levels of DOC and total iron of 1mM when compared with other tested methods. The extracted NOM was fractionated using molecular weight cut-off filters (100 kDa). Groundwater from well FW305 was used as an inoculum to which the fractionated NOM was added to feed native microbes to identify the microbial isolates/community that preferentially grew with these NOM. Analysis of the enriched microbial community, and transformed NOM metabolites was also carried out. Advanced characterization of extracted NOM and transformed NOM will be performed using FTIR and NMR techniques to identify the specific functional groups of NOM that indigenous microbes are interacting with.
  96. Zhang, P., A. Rocha, Z. He, J. Van Nostrand, E. Alm, T. C. Hazen, D. Elias, M. Fields, A. Arkin, P. Adams and J. Zhou. 2015. Impacts of Environmental Contaminants on Diversity of Groundwater Microbial Communities at a U(VI)-contaminated Aquifer. American Society for Microbiology Annual Meeting abstract
    Microbial diversity in groundwater ecosystems has not been well studied but can be impacted by different geochemical conditions such as contaminant type and level and pH. As part of the Global Survey at the US DOE Oak Ridge site, groundwater samples were collected from 69 wells that cover a large scale gradient of contaminant and pH levels at this site, with U(VI) ranging from 0.002 to 55.3 mg/L, nitrate ranging from 0.1 to 11648 mg/L, and pH ranging from 3 to 10.5. Groundwater functional communities were analyzed using a comprehensive functional gene microarray (GeoChip 5.0), and phylogenetic communities were analyzed using Illumina sequencing of 16 rRNA genes. The results indicated that both functional and phylogenetic diversity and structure of groundwater microbial communities were significantly affected by different geochemical characteristics. The community differences were largely correlated with the differences in U(VI), nitrate, pH, dissolved organic carbon and sulfate in the groundwater. The microbial functional and phylogenetic diversity significantly decreased with U(VI) concentration, while high nitrate concentrations showed less inhibition on the microbial diversity. Both functional and phylogenetic diversity was highest at neutral pH and decreased with increased and decreased pH levels. Our results also suggested that although high contaminant levels and pH inhibited functional and phylogenetic diversity, the metabolic potential of indigenous groundwater microbial communities was less impacted than phylogenetic diversity. This study improves our understanding of the diversity of groundwater microbial communities across a large scale of contaminant and pH levels at this site.
  97. 2015. Bacteria the newest tool in detecting environmental damage. ENVASS
  98. 2015. The Promise of Water Security. Southeast Green
  99. 2015. Using Microbial Communities to assess Environmental Contamination. TerraDaily
  100. 2015. Hazen to Lead UT’s Institute for a Secure and Sustainable Environment. Tennessee Today
  101. 2015. Hazen to Lead UT’s Institute for a Secure and Sustainable Environment. Southeast Green
  102. 2015. Using Microbial Communities to assess Environmental Contamination. NZ Health Tec
  103. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. University of Tennessee Alumni Reception
  104. 2015. Bacteria detects presence of Pollutants, Contamination. American Laboratory
  105. 2015. Bacteria the newest tool in detecting environmental damage. Yourwebapps
  106. 2015. Using microbial communities to assess environmental contamination. Lab Manager
  107. 2015. Bacteria the newest tool in detecting environmental damage. AZ News
  108. 2015. Bacteria the newest tool in detecting environmental damage. Science World
  109. 2015. Bacteria the newest tool in detecting environmental damage. ScienceDaily
  110. 2015. Bacteria the newest tool in detecting environmental damage. SciGuru
  111. 2015. Bacteria the newest tool in detecting environmental damage. Tennessee Today
  112. 2015. Bacteria the newest tool in detecting environmental damage. Eurekalert
  113. 2015. Bacterial communities can act as precise biosensors of environmental damage. Kalen2Utech
  114. 2015. Bacterial communities can act as precise biosensors of environmental damage. Biology News Net
  115. 2015. Bacterial communities serve as ready-made biosensors of environmental contaminants. mBioblog
  116. 2015. Bakteri bias jadi alat deteksi polusi lingkungan. Antara News
  117. 2015. Latinas at Tech Giants. Diversity News
  118. 2015. Microbes serve as markers for environmental contamination. AnchorFree
  119. 2015. Microbes serve as markers for environmental contamination. Science News
  120. 2015. Microbes serve as markers for environmental damage. Lastminutestuff
  121. 2015. Microbes serve as markers for environmental damage. Weird4U.com
  122. 2015. Using microbial communities to assess environmental contamination. R&D Magazine
  123. 2015. Using microbial communities to assess environmental contamination. PhysOrg
  124. 2015. Using microbial communities to assess environmental contamination. LBNL Newscenter
  125. Campa, M. F., S. M. Techtmann, S. Brewer, A. Garcia de Matos Amaral, J. Wright, N. Ulrich, R. Lamandella and T. C. Hazen. 2015. Hydraulic Fracturing Flowback Water: A Look into the Subsurface Microbial Community and Intrinsic Bioremediation. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    Treatment and reuse of hydraulic fracturing flowback water is the best alternative to handle the enormous volume of wastewater produced. Intrinsic bioremediation and bioaugmentation are efficient ways to treat the high salinity and chemical content of this water. The microbial community in flowback water can provide an insight into what facilitates bioremediation. Thus, raw and treated flowback waters from the Marcellus Shale were collected. The microbial community from the flowback water, and how it changes at different stages of the treatment process was determined by sequencing of 16S rRNA amplicons. Furthermore, aerobic and anaerobic bacteria with physiologies of interest were isolated. Samples were plated on marine broth and ORN7a supplemented with oil. High growth of halophilic bacteria as well as the presence of sulfate reducing bacteria was observed. The isolates will be sequenced (16S rRNA amplicon) and compared to the microbial community structure data. The isolates that are most representative of the community will be selected for high-throughput phenotypic microarray analysis. The study is currently in progress. Characterizing the microbial community structure of the flowback water and understanding its function will help develop and optimize a bioremediation strategy so that flowback water can be inexpensively treated and reused.
  126. Chen, C., T. C. Hazen, G. Pan, W. Shi and F. Xu. 2015. Impact of Clay Flocculation of Algal Blooms on Pond Microbial Community. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    One of the major environmental concerns of clay flocculation in harmful algal blooms control is the ecological impact of the clay and modified clay. Here we investigated the impact of the clay flocculation technique on the microbial composition in water samples collected in June 2014 at eutrophic ponds in Datong, China. Successional changes in the microbial community structure due to the clay flocculation for algal cells removal were determined by deep sequencing of 16S rRNA genes. Quantitative PCR was used to quantify the biomass in water samples. In addition, total phosphate, soluble reactive phosphate, total nitrate, NH4-N, and NO3-N were measured. The 16S data revealed that microbial community structure significantly changed after clay flocculation in pond water. Water samples were dominated by Actinobacteria, Spartobacteria, Betaproteobacteria, Acidimicrobiia, and Synechococcophycideae before clay flocculation, whereas Actinobacteria, Sphingobacteriia and Betaproteobacteria were dominant during the first 10 days after flocculation, and Actinobacteria decreased dramatically in the following 5 days. Deltaproteobacteria as well as Gammaproteobacteria and Bacilli were dominant in water samples on the 11th day and 15th day. In addition, the total coliform test results indicated a decrease of indicator bacteria after clay flocculation. This study provides an ecological impact assessment of clay flocculation for harmful algae blooms in pond water.
  127. Hagen, S., N. Mahmoudi, A. Steen and T. C. Hazen. 2015. Quantifying Extracellular Enzyme Activity in Deep-Sea Sediment from the Mediterranean Sea through the use of Fluorometric Assays. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    The Mediterranean Sea is home to a diverse community of heterotrophic microbes responsible for cycling much of the organic carbon that enters its waters. The subset of those organisms that utilize macromolecules produce extracellular enzymes as a means of carbon degradation. However, this hydrolytic community is poorly characterized at water column depths greater than a few hundred meters where physical properties, such as pressure and temperature, create a unique environment for influencing enzyme behavior. Here we describe enzyme activities of a suite of hydrolases in surface sediment collected at four sampling stations in the Mediterranean Sea at water depths ranging from 800-2200m. In total, nine enzymes were studied- three peptidases, one esterase, and five glycosidases. Fluorometric assays revealed alkaline phosphatase and aminopeptidase to be active on the magnitude of 100x the other enzymes studied here. Furthermore, assays revealed a significant correlation between activity and depth for a majority of the studied community, indicating adaptation to environmental conditions. A qualitative assessment of the data also showed difference in the activity of enzymes from cores taken from the same site, suggesting variability in hydrolytic potential over a short (approximately 1 m) spatial scale.
  128. Harik, A.-M., S. Techtmann, J. Fortney and T. C. Hazen. 2015. Water Swap. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    To determine change in microbial community structure and hydrocarbon degradation rates when placed in foreign waters, as compared to ambient waters. Microbial directed biodegradation has a large impact on hydrocarbon degradation; from naturally occurring oil seeps and from spills. The microbial communities from two separate Angola ocean waters, one surface and one deep water, were removed via 0.22 μm filter then either placed back in their original water source or placed into the other depth’s water by flipping the filter and filtering the water back through it. 16S rRNA sequencing and GC analysis are being performed, we expect there to be differences in microbial community structure and hydrocarbon degradation rates of the communities in their ambient waters versus their swapped waters. If it is found that microbial community structure plays a much larger role than nutrients on biodegradation than oil drilling locations whose microbial communities are not capable of high rates of oil biodegradation will need to have a more extensive spill response than those locations whose microbial communities are well equipped. However, if nutrients are found to be the major factor then spill response procedures can include supplementing the waters with necessary nutrients.
  129. Liu, J., J. Fortney, S. Techtmann, D. Joyner and T. C. Hazen. 2015. Crude Oil Biodegradation and Microbial Community Changes in Deep Oceans. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    Crude oil contamination in the ocean from oil spills becomes a huge concern to the public and governments. Very limited information is available on the oil degradation potential and microbial community response to crude oil contamination in deep oceans. In this study, we investigated the response of microbial communities to crude oil in various deep-sea basins from around the world where oil exploration is anticipated (Eastern Mediterranean Sea, Central Mediterranean Sea and Great Australian Bight). Microcosms were set up aerobically in three different conditions: seawater, seawater + oil and seawater + oil + oil dispersant (Corexit). CO2 evolution followed a similar pattern in all of the basins sampled. The treatment of seawater + oil + Corexit had the highest CO2 production. In addition, there was a clear succession of microbial communities during degradation of oil. The microbial diversity decreased in all of the microcosms over time. The relative abundance of Proteobacteria increased drastically while the population of archaea decreased. In particular, the percentage of Betaproteobacteria increased in samples from the Central Mediterranean Sea. However, Gammaproteobacteria increased in abundance in the microcosms from the Eastern Mediterranean Sea and Great Australian Bight, which was very similar to GOM.
  130. Paradis, C., N. Mahmoudi, D. Driver, K. O’Dell, J. Fortney, S. Jagadamma, S. Schaeffer and T. C. Hazen. 2015. Soil Microbial Respiration and Community Structure in Response to Severe Drought and Precipitation Events. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    There is strong evidence of an increasing trend of severe drought and precipitation events in the Unites States (US) due to greenhouse gas emissions. However, there is a considerable knowledge gap between these severe weather events and soil microbial respiration. The objective of this study was to quantify soil microbial respiration and assess microbial community structure under ambient and heavy weekly precipitation drying/wetting cycles and continuous drought conditions. Laboratory microcosms were constructed using soil native to the southeastern US. Soil moisture and CO2 were measured periodically during the 6-week experiment. Soil microbial DNA (16S rRNA) was extracted and analyzed before and after incubation. The rate of CO2 production in the drought treatment was substantially less compared the ambient and heavy moisture treatments after 2 weeks. The cumulative CO2 production in the ambient and heavy moisture treatments were similar. The rate of CO2 production was substantially higher immediately after wetting of the drying soil (wetting effect) in the ambient treatment compared to the heavy moisture treatment. The microbial community taxa was similar among the treatments before and after incubation. These results suggested that extended drought may result in decreased respiration and the wetting effect becomes negligible during frequent heavy precipitation. Further, our study suggested that severe drought or precipitation events may simply decrease or increase respiration, respectively, of taxa already present rather than select for specific microbial communities.
  131. Techtmann, S., S. Stelling, D. Joyner, S. Uttukar, A. Harris, N. Alshibli, S. Brown and T. C. Hazen. 2015. Phenotypic and Genomic Heterogeneity among Colwellia psychrerythraea Strains from Distant Deep-Sea Basins. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    16S rRNA sequencing is routinely used to investigate the diversity of prokaryotes in environmental settings. Evidence suggests that microbes with nearly identical 16S rRNA genes can have genotypic heterogeneity. To better understand the diversity within a single microbial species, we characterized the phenotypic and genomic heterogeneity of three strains of Colwellia psychrerythraea. Colwellia are psychrophilic heterotrophic marine bacteria ubiquitous in cold ecosystems. We have recently isolated two Colwellia strains: ND2E from the Eastern Mediterranean and GAB14E from the Great Australian Bight. The 16S rRNA of these two strains are greater than 98.2% identical to the type strain C. psychrerythraea 34H, which was isolated from Arctic sediments. Carbon source utilization profiles for these strains were determined using the Biolog phenotype microarrays. The carbon source utilization profiles were distinct with less than half of the carbon sources being shared between all three strains. Whole genome sequencing revealed that the genomes of these three strains were quite diverse with some genomes having up to 1600 unique genes. These findings suggest that a single microbial species can exhibit substantial phenotypic and genomic heterogeneity. This diversity must be taken into account when trying to interpret 16S rRNA sequencing data from complex environmental microbial communities.
  132. Woo, H., K. O'Dell, S. Techtmann and T. C. Hazen. 2015. What Happens to Lignin in the Ocean? Evidence of Bacterial Lignin Degradation in Marine Microcosms. 2nd Annual Southeastern Biogeochemistry Symposium abstract
    The microbial transformation of allochthonous terrestrial organic matter, particularly recalcitrant aromatic lignin, is a significant but poorly understood phenomenon in the marine ecosystem. We aim to elucidate the diversity and metabolic potential of a marine bacterial community subsisting on lignin. Seawater from the oxic and ultra-oligotrophic Mediterranean Sea, near the Egyptian Nile Delta, was amended with lignin and incubated for 2 weeks. Microbial activity was assessed by CO2 respirometry and enzyme assays. Community structure and metabolic potential of the microcosms were assessed using 16S rRNA gene amplicon sequencing, and metagenomic sequencing. Lignin amendment caused higher respiration rates and oxidative enzyme activity in comparison to the control. Halomonas, Idiomarina, Thalassospira spp. were several of the dominant OTUs in the microbiome. The metagenome of the lignin-amended microcosm had higher abundance of genes encoding information storage and processing, and aromatic catabolism. Conversely, the unamended control metagenome had signs of starvation conditions, such as higher abundance of genes encoding desiccation stress, spore protection, and plasmid function that were not found in the lignin-amended metagenome. We believe the positive effect of lignin on microbial activity and abundance of aromatic catabolism genes supports the notion of lignin degradation by marine microbes
  133. T. C. Hazen. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. Georgia Tech, School of Earth and Atmospheric Sciences & Biology Seminar abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
  134. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. College of Engineering Distinguished Lecture abstract
    Good: The US is expected by many to achieve energy independence by 2016 and to surpass Saudi Arabia as the top producer of oil by 2020. These changes have been largely due to Shale Fracking for gas and oil. We have seen a rapid decrease in the price of oil and gasoline at the pump in the last few months reaching 5 year lows. This has in turn stimulated the economy. The combustion of CH4 produces about half the amount of CO2 relative to coal and a third as much as oil, suggesting reduced overall CO2 emissions. Methane has other attendant environmental benefits including less SO2, NOx, and volatile organic carbon emissions produced during combustion relative to other forms of carbon. Methane can also be a cheap feedstock for valuable products, including remediation of other toxic organics. Bad: Methane is 24 times more potent as a greenhouse gas then carbon dioxide. The recent Intergovernmental Panel on Climate Change report (IPCC 2014) suggests that greenhouse gas emissions must be substantially curbed over the next 35 years with overall fuel switching from coal to gas and greater reliance on nuclear and renewable energy sources. However the US EPA predicts methane emissions from oil and gas activities are projected to grow 4.5% from 2011 to 2018. Ugly: Aging infrastructure in cities and oil/gas production/distribution facilities is a major source of fugitive methane emissions and it is increasing dramatically with more reliance on cheap natural gas. The public in places has become furious about fracking activities with some cities, counties and states banning all or some of fracking activities. Informed decisions are sorely missing.
  135. Terry C. Hazen. 2015. "OMICS" the Fantasy is Over: We need multiple lines of evidence. RemTEC
  136. T. C. Hazen. 2015. Oil biodegradation in five deepwater basins around the world. SINTEF Sea Lab
  137. T. C. Hazen. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. Seminar Nanjing University abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
  138. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. Seminar Shenyang China Agriculture Institute
  139. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. Seminar Beijing China Agriculture Institute
  140. T. C. Hazen. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. European Bioremediation Conference VI abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
  141. Savannah Gillman. 2015. UT, ORNL: partners in science. The Daily Beacon
  142. Brown, Randall. 2015. Hazen Encourages UT-ORNL Internships. @COE e-newsletter abstract
    Dr. Terry Hazen, UT-Oak Ridge National Laboratory (ORNL) Governor's Chair Professor in the Department of Civil Engineering, discussed the advantages of UT-ORNL internships in a recent article in the Daily Beacon.
  143. Hazen, T. C.. 2015. Deepwater Horizon Oil Spill: Do Microbial Communities at other Deep Water Drilling Sites around the World Respond the Same?. Biology Department Seminar Series, Central Michigan University
  144. Hazen, T. C.. 2015. Methane: the Good, the Bad, and the Ugly. University of Tennessee Sigma Xi
  145. 2015. Terry Hazen Discusses Gulf Recovery on WBIR. WBIR TV News
  146. Adams, B. G., A. M. Rocha, S. Jagadamma, C. Paradis and T. C. Hazen. 2015. Impact of Temporal Variations of Hydrology on Groundwater Geochemistry. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    The Department of Energy’s (DOE) Oak Ridge field research uncontaminated site is located within the Bear Creek Valley (BCV) in Oak Ridge, TN. At the site there are 6 groundwater monitoring wells of multiple depths ranging from 20-71ft deep. Previous hydrologic and geochemical studies have identified the presence of two geochemically distinct groundwater zones, as well as, fluctuations in the gradients between flow in shallow and deep wells. Although we know that fluctuations occur in the height of the hydraulic head, it is unclear how these variations affect the geochemistry in shallow versus deep wells. The goals of this study are to (1) determine the direction and magnitude of the horizontal and vertical hydraulic gradients, and (2) determine how the stability (or instability) of the hydraulic head impacts groundwater geochemistry. Use of In-Situ Aqua TROLL® 200 CTD Logger allowed for continual monitoring of the following physical parameters; conductivity, dissolved oxygen, and water level/pressure. Using water level/pressure, we determined direction and magnitude of the horizontal and vertical hydraulic gradients. By combining geochemical data and time-series hydrologic data from the 6 wells, we hope to better understand the geochemical response to variations in hydraulic gradient.
  147. Brewer, S. S., M. F. Campa, S. M. Techtmann, J. L. Fortney and T. C. Hazen. 2015. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Hydrocarbon production from hydraulic fracturing of gas shale in the US has skyrocketed recently and is projected to keep growing. With the increase of this unconventional drilling method, concerns have been proposed about environmental safety and dangers to human health. Because this method involves injecting fluid between 1-3 km deep into the Earth and the fluid produced from the well after drilling is often reused in other hydraulic fracturing operations. The goal of this study is to identify novel organisms that might have bioremediation capabilities for the toxic flowback water and compare microbial communities isolated from fracking water samples in anaerobic conditions. Water samples from Pennsylvania include six different collections of produced (flowback) water, a flowback mix tank, and three different treatment tanks. Inoculations from the water samples were grown under anaerobic conditions in high salinity marine media and halotolerant hydrocarbon degradation dependent media. DNA was extracted, and 16S rRNA gene sequences were used to identify the isolated microbes, and the microbial communities were characterized by 16S rRNA gene amplicon Illuminia sequencing. The physiological conditions of some significant isolated microbes were further characterized by the Omnilog phenotypic microarray system. Early results show presence of numerous anaerobic microbes including sulfate reducers.
  148. Detienne, B. L., A. M. Rocha, S. Pfiffner, S. Jagadamma and T. C. Hazen. 2015. Microbial Community Structure and Abundance in Uranium and Nitrate Contaminated Groundwater. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Based on geochemical and geographical differences between groundwater wells, the distribution and abundance of microbial communities in each well could vary drastically. At the Department of Energy’s Oak Ridge field site, 243-acres of contaminated area exist. Contaminants in this site include nitrate and uranium with concentrations ranging from <0.05 -14400ppm and <0.005 - 55ppm, respectively. In order to understand relationship of microbial communities to different concentrations of uranium, groundwater wells were sampled for geochemical and microbial analyses. The goal of this study is to characterize microbial community structure and diversity of uranium and nitrate contaminated wells versus non-contaminated wells. Microbial diversity and biomass were determined using phospholipid fatty acid (PLFA). To measure PLFAs, groundwater was filtered through 0.2 μm pore size Sterivex filter, immediately frozen, and stored at -80°C. Lipids were extracted using the modified Bligh-Dyer method. Currently, PLFA results are being analyzed. However, preliminary PLFA data indicates community diversity in uranium contaminated wells have greater microbial diversity compared to non-contaminated wells. Additionally stress indicators for Gram-negative bacteria were identified in a small fraction of the wells. Results from this study indicate that groundwater microbial communities can vary in respect to the geochemistry and environmental stress factors at the site.
  149. Fitzgerald, K., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2015. Diversity and Distribution of Archaeal amoA Genes in Geochemically Distinct Marine Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Microbes are essential in the cycling of nutrients within the environment. Since the discovery of ammonia-oxidizing archaea (AOA), it is now understood that Thaumarchaeota may play an important role in the nitrogen cycle within marine environments. AOA are mainly responsible for the nitrogen cycle's first step, which is oxidation of ammonia to nitrite. However, investigation of AOA's diversity and distribution has only recently begun. In this study, we aimed to understand the diversity and distribution of the Archaeal amoA gene, which is required for ammonia oxidation. We used high-throughput next-generation sequencing to investigate the amoA diversity in 59 samples taken from three marine basins at varying depths. Our preliminary data suggest that diversity of Archaeal amoA is low and the relative abundance varies by basin. We will investigate if there are distinct populations of amoA genes within each basin and the phylogenetic diversity of amoA in our samples. These basins are geochemically distinct and grant the opportunity to understand how geochemical variables affect the diversity and distribution of amoA. The study will lead to a greater understanding of AOA in distinct marine basins.
  150. Garcia de Matos Amaral, A., M. F. Campa and T. C. Hazen. 2015. Community Structure of Fracking Flowback water from Marcellus shale of Pennsylvania. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Hydraulic fracking is an important process for shale gas extraction. It has caused an exponential growth of gas extraction in the United States, and is helping the country get a step closer to energy independence. However, this process has also raised questions about its impact on the environment. Millions of gallons of water are produced as waste from the process, increasing water stress in many areas that are already experiencing drought. Bioremediation is a possible way to treat and reuse the produced wastewater. Particularly, intrinsic bioremediation may be aided through the characterization and understanding of microorganisms present into the flowback water. The aim of this study is to investigate raw and treated flowback from the Marcellus shale of Pennsylvania, and identifying all the microorganisms present and understanding their physiology. To do this DNA was extracted from the samples, and 16S rRNA gene amplicons sequencing was performed. The molecular and metagenomics techniques used will aid to explore and help to understand the physiology and metabolism of them the microbes present This knowledge will be used to suggest intrinsic bioremediation capabilities of the microorganisms and treat the wastewater generated through the fracking process.
  151. S. Hagen, S. Techtmann and T. C. Hazen. 2015. Quantifying Extracellular Enzyme Activity In Deep-Sea Sediment From the Mediterranean Sea Through The Use Of Fluorometric Assays. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    The Mediterranean Sea is home to a diverse community of heterotrophic microbes responsible for cycling much of the organic carbon that enters its waters. The subset of those organisms that utilize macromolecules produce extracellular enzymes as a means of carbon degradation. However, this hydrolytic community is poorly characterized at water column depths greater than a few hundred meters where physical properties, such as pressure and temperature, create a unique environment for influencing enzyme behavior. Here we describe enzyme activities of a suite of hydrolases in surface sediment collected at four sampling stations in the Mediterranean Sea at water depths ranging from 800-2200m. In total, nine enzymes were studied- three peptidases, one esterase, and five glycosidases. Fluorometric assays revealed alkaline phosphatase and aminopeptidase to be active on the magnitude of 100x the other enzymes studied here. Furthermore, assays revealed a significant correlation between activity and depth for a majority of the studied community, indicating adaptation to environmental conditions. A qualitative assessment of the data also showed difference in the activity of enzymes from cores taken from the same site, suggesting variability in hydrolytic potential over a short (approximately 1 m) spatial scale.
  152. McBride, K. R., C. Chen and T. C. Hazen. 2015. Assessing Ecological Impact of Clay Flocculation Techniques by Measuring Microbial Community Structure. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Clay flocculation is a recently developed technique that restores water systems experiencing harmful algal blooms; but the ecological impact of flocculation on the quality of these water systems has not been thoroughly studied. This project aims to assess the ecological impact of different flocculation treatments by measuring microbial community structure. In order to do this, sedimentary columns treated with three different types of flocculation were tested to observe microbial community composition. For this, one of the methods used was 16s RNA sequencing, which gave a general profile of microbial diversity; the other method was Phospholipid Fatty Acid (PLFA) analysis, which measures biomass in relation to the community structure. The results of the 16s RNA sequencing showed no significant variation in microbial composition between the different flocculation treatments and control. Preliminary PLFA results suggest small changes in the biomass between different techniques. Results of this project entail that flocculation may still have an impact on microbial community structure, which could lead to alteration of nutrient levels in sediment and affect overall water quality. This study can help broaden our understanding of potential ecological impact of clay flocculation on water and in sedimentary environments.
  153. Whitt, K., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2015. Abundance and Diversity of Thaumarcheaota in Four Ocean Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Microbes are the most prevalent living organisms in the world. They are found in almost every environment on earth, and are essential for the cycling of nutrients, such as carbon and nitrogen, in the environment. The nitrogen cycle is essential to life as it converts nitrogen into forms that can be used as nutrients for life. While much is known about the nitrogen cycle in soils, less is known about the marine nitrogen cycle. Recently, Thaumarchaeota, members of the Domain Archaea have been found to be major contributors to the nitrogen cycle in marine environments. Very little is known about the diversity and distribution of Thaumarchaeota in the world’s oceans. This study aims to determine the abundance and diversity of Thaumarcheaota in four ocean basins. We performed qPCR to determine the copy number of genes from Thaumaracheaotes and used statistical analyses to determine differences in gene abundance between basins. Additionally, we investigated the correlations between gene abundance and different environmental factors. 16S rRNA sequences of Thaumarchaeotes was also used to investigate the diversity of Thaumarcheaotes in different environments. Preliminary data shows certain Thaumaracheaotes are specific to particular marine locations and their abundance is related to different environmental factors.
  154. Yongquist, E., S. Techtmann and T. C. Hazen. 2015. Genomic Diversity of Pseudoaltermonas species from Geographically Distant Marine Basins. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    Microbes are extremely diverse and capable of catalyzing many functions. Currently, microbial species are defined though characterization of the microbe’s metabolism and through sequencing of 16S rRNA. Recent studies suggest that microbes classified as the same species through 16S rRNA sequencing may demonstrate high genetic diversity. The goal of the study is to identify how much genomic diversity exists within a single species from different marine locations. Differences in the physical and chemical parameters of these locations may select for particular populations of microbes capable of growing in these environments. We isolated 99 microbial strains from the coasts of Australia, Angola, and Bermuda. Out of those isolates, we chose 32 Pseudoalteromonas spp. that have greater than 99% 16S rRNA identity. We will sequence the entire genomes of the 32 strains and compare their genomes to better understand the genomic heterogeneity within this species. This work will result in a better understanding of biogeographic patterns in marine microbial species. We hope that this work will demonstrate the utility of performing whole genome sequencing to differentiate between closely related taxa.
  155. Wu, X., S. Jagadamma, A. Lancaster, M. Adams, T. C. Hazen, N. Justice and R. Chakraborty. 2015. Microbial Interactions with Natural Organic Matter Extracted from the Oak Ridge FRC. American Geophysical Union Annual Meeting abstract
    Natural organic matter (NOM) is central to microbial food webs; however, little is known about the interplay between the physical and chemical characteristics of NOM and its turnover by microbial communities based upon biotic and abiotic parameters (e.g., biogenic precursors, redox state, bioavailability). Microbial activity changes the structures and properties that influence further bioavailability of NOM. To date, our understanding of these interactions is insufficient, and indigenous microbial activities that regulate NOM turnover are poorly resolved. It is critical to identify NOM characteristics to the structure and composition of microbial communities and to the metabolic potential of that community. Towards that end, sediment samples collected from the background area well FW305 (Oak Ridge Field Research Center, Oak Ridge, TN) were tested for NOM extraction methods that used three mild solvents, e.g., phosphate buffered saline (PBS), pyrophosphate, and MilliQ-water. MilliQ-water was finally chosen for extracting sediment samples via shaking and sonication. Groundwater from well FW301 was used as an inoculum to which the extracted NOM was added as carbon sources to feed native microbes. To identify the specific functional groups of extracted NOM that are bioavailable to indigenous microbes, several techniques, including FTIR, LC-MS, EEM, were applied to characterize the extracted NOM as well as the transformed NOM metabolites. 16S rDNA amplicon sequencing was also performed to identify the specific microbial diversity that was enriched and microbial isolates that preferentially grew with these NOM was also cultivated in the lab for future detailed studies.
  156. 2015. Hazen Addresses Wastewater Treatment Idea.
  157. Chakraborty, R., A. Pettenato, X. Wu, S. Jagadamma, T. C. Hazen, M. Fields, T. Northen, S. Jenkins, W. A. Lancaster, M. W. W. Adams, A. P. Arkin and P. D. Adams . 2015. Microbial Interactions with Native Natural Organic Matter in Groundwater and Sediment from the Oak Ridge FRC. Genomics Sciences Program Contractor-Grantee Meeting abstract
    Project Goal: Natural organic matter (NOM) is central to microbial food webs; however, little is known about the interplay between physical and chemical characteristics of NOM and it’s turnover by microbial communities in groundwater and sediments of Oak Ridge Field Research Center (FRC). Microbe mediated molecular and geochemical mechanisms control the flow of carbon that support evolution and maintenance of a community within a given environment. To gain insight into the cycling of carbon, and how the turnover of NOM regulates the development of indigenous microbial community at the FRC, it is critical to extract NOM, identify its characteristics, and correlate this to the structure and composition of microbial communities and to the metabolic potential of that community. In this FY15 Discovery project, we focused on NOM from the background well, FW305. Preliminary experiment initiated with FW305 sediment enriched with 10ppm fulvic acid (as part of the Microparticles Campaign), showed rapid evolution of CO2. Isolations from this fulvic acid enriched samples yielded strains belong to Sphingomonas, Pseudomonas, Undibacterium, Rugamonas, Rhodococcus genera. The same sediment sample (FW305) was tested for NOM extraction methods using three mild solvents, e.g., phosphate buffered saline (PBS), pyrophosphate, and warm de-ionized water with shaking or mild agitation. The pH was kept close to in-situ conditions, 6.5–7.0. We also tested the efficacy of glass beads as a physical abrasive to aid in extraction. The dissolved organic carbon (DOC) in the extracted NOM was in the range of 8.8–28 mg/L. Sonication with PBS extracted higher levels of DOC, while shaking with pyrophosphate extracted higher levels of metals such as iron, zinc, cobalt and manganese when compared with other tested methods. A spectrophotometric scan of the sample with highest DOC content showed high absorbance between 300-380nm, indicative of presence of functional groups with high aromaticity such as carboxylic and phenolic groups. The extracted NOM was fractionated using molecular weight cut-off filters (100 kDa). In ongoing experiments, we are testing existing isolates from FW 305 for their ability to transform the NOM, and identify the transformation product. In addition, we will add this fractionated NOM to feed native microbes present in the groundwater of well FW305 to identify the microbial isolates/community that preferentially grew with this NOM. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02-05CH11231
  158. Elias, D. A., A. J. King, K. L. Bailey, S. P. Preheim, M. S. Robeson II, T. R. Chowdhury, B. R. Crable, R. A. Hurt Jr., A. C. Somenahally, S. Techtmann, T. Mehlhorn, K. A. Lowe, A. M. Rocha, A. Zelaya, M. W. Fields, A. P. Arkin, J.-M. Chandonia, T. C. Hazen, E. J. Alm, J. Zhou, T. J. Phelps, C. C. Brandt, S. D. Brown, M. Podar, M. W. W. Adams, D. B. Watson and P. D. Adams. 2015. Groundwater-fed Bioreactors Show Distinct Colonization and Community-wide Response Dynamics to Perturbations. Genomics Sciences Program Contractor-Grantee Meeting abstract
    Laboratory bioreactors have long been used for investigating the characteristics of a microorganism or simple synthetic community. However, for studying natural or in-situ microbial communities, discontinuous “snapshot in time” sampling has mainly occurred. In this study an in-field bioreactor system was developed to temporally monitor and manipulate the in-situ microbial community while maintaining the in-situ community structure. Three above ground, in-field reactors were continuously fed microaerobic (0.2% O2) groundwater directly from an existing well at the Oak Ridge Field Research Center, Oak Ridge, TN, for 12 weeks. Each bioreactor contained 800 ml of groundwater and 8 replicate biofilm coupons filled with sterilized site sediment to monitor both the planktonic and biofilm communities. Gas influx was varied from aerobic (weeks 1-7, & 9) to anaerobic (weeks 8 & 10) to confirm that manipulation of bioreactor microorganisms was tractable. Samples from the incoming groundwater and from each bioreactor were taken every two days to match the dilution rate of the reactors. Each sample was analyzed by 16S rRNA sequencing at an average of 10,000 reads and key biogeochemical properties were measured including pH, dissolved oxygen, ORP, conductivity, 12 organic acids 14 anions and 3 sugars. At every third time point 53 different metals were also measured. Community structure and diversity was highly similar across all three bioreactors according to 16S rRNA sequencing, representing 30-65% of the groundwater OTUs overall and 50-85% of high abundance groundwater OTUs. Biofilm coupons captured a unique subset of the groundwater OTUs but on average were only 27% similar to groundwater and 48% similar to the bioreactor planktonic samples. Community beta-diversity patterns indicated bioreactors were more different to the groundwater than expected if no growth was occurring, thereby suggesting growth in the reactors. Correlations between organic acid profiles and bacterial clades revealed that the metabolic function was conserved across all three bioreactors and the in-situ groundwater community. Transitions to anoxic conditions and subsequent lowering of the pH at weeks 7 and 10 resulted in strong, repeatable bacterial community and individual clade shifts toward the groundwater composition. However, not all bacterial groups in the bioreactors mirrored those in the groundwater. In fact, known metal- and organic acid-metabolizing clades increased in abundance in the bioreactors when the incoming groundwater increased in solute concentration despite no change in the incoming clade abundance. Similarly, co-occurrence relationships of OTUs known for syntrophic and predatorprey interactions were observed over time in the bioreactors. This type of in-field bioreactor system allows for discreet temporal monitoring of microbial community structure and function simultaneously while allowing community responses to be determined during the testing of new strategies for environmental amendment or restoration on a small and affordable scale. Keywords. Bioreactor, Microbial Community, Groundwater, Diversity, Anaerobic, Time Course Funding Statement: The work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
  159. Rocha, A. M., T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. M. Klingeman, D. B. Watson, D. C. Joyner, J. L. Fortney, S. Jagadamma, J. J. Zhou, J. D. Van Nostrand, M. W. W. Adams, F. L. Poole, W. A. Lancaster, R. Chakraborty, D. Elias, P. D. Adams, A. P. Arkin, E. J. Alm and T. C. Hazen. 2015. Temporal Variation in Groundwater Geochemistry and Microbial Community Structure at Oak Ridge Field Site. Genomics Sciences Program Contractor-Grantee Meeting abstract
    Project goals: The goal of the ENIGMA field microbiology component is to identify key microbial populations and determine the community events and mechanisms of these populations that impact and control environmental activities of interest, ultimately predicting how perturbations of the environment may affect community structure and function. From these results, we can develop models that can be applied to microbial populations overlaid with geochemical parameters and engineering controls. An example of such a model is described in the 100-well survey where we demonstrated the ability to utilize natural bacterial communities as in situ environmental sensors that capture environmental perturbations and elucidate key systems biology features (Smith et al., submitted). Here, we expand upon the survey to capture the microbial community response to temporal changes in the groundwater geochemistry to (1) provide for temporal tuning of predictive models, and (2) to determine if and to what extent geochemical variation in groundwater affects microbial community, activity, and genetic diversity along different well depths. Across aquatic and terrestrial environments, numerous studies have sought to characterize key microbial communities and to identify factors that drive changes in microbial community structure and activity. While these studies enable us to further understand and potentially uncover key correlations between the composition of microbial communities and their environment, information regarding temporal community dynamics is often limited or in many cases lacking. One such example is the Oak Ridge Field Research Center (ORFRC) where there has been a large focus to characterize the spatial distribution of groundwater and soil microbial communities across different geochemical transects (e.g. Uranium-Nitrate-pH). In this timeseries study, we aim to bridge this gap by capturing the spatio- and temporal variation of geochemistry on the overall structure, function, and genetic diversity of the groundwater microbial communities in the groundwater wells at the ORFRC. Here, we present our findings from two pilot studies conducted during November 2013 and from November 2014 - January 2015, respectively, at the ORFRC background field site. One of our main objectives of the temporal study is to determine how resilient (or volatile) microbial communities are to daily and weekly changes in groundwater geochemistry. To capture changes in microbial community structure and geochemical constituents, we initially sampled two deep and two shallow groundwater wells over the course of three weeks during November 2013. For each well and time-point, groundwater samples were collected for geochemical and microbial communities analyses. Nucleic acids were collected by filtering water through a 10.0μm pre-filter and 0.2μm membrane filter and then extracted using a Modified Miller method. Results from the study showed that geochemical measurements across all the wells remained fairly stable over the course of the study. However, a decrease in pH and increase in conductivity measurements was observed in the shallow wells during small rain events. Unlike the geochemistry, the 16S rRNA sequencing of the microbial community structure within each well varied on a daily basis in both the 0.2 μm and 10.0 μm size fraction. Statistical analysis of the 16S data using Adonis indicated that there were statistically significant differences (p-value = 0.0001) in the community structure between wells throughout the study. Nonmetric multidimensional scaling analysis of the community structure did not show distinct differences between communities present in shallow versus deep well depth. However, analysis indicated that communities present in wells FW-300 (shallow) and GW-460 (deep) were much more variable throughout the time course. Due to the stability of the geochemistry in the 2013 pilot study, we hypothesize that the variation in microbial communities is a result of both a sampling effect and from the inclusion of communities associated with biofouling in the well casing and sloughing off during sampling. To determine if the daily variation in groundwater microbial community profiles in the first study were naturally occurring within the aquifer or if (and to what extent) the variation in community structure is a result of factors associated with biofouling or sampling artifacts, we sampled six wells from the background site from November 2014 – January 2015. Of the six wells, we physically and chemically cleaned four wells to remove biofilm and attached particulates from the well casing. The remaining two wells that weren’t cleaned, served as controls. Prior to cleaning, all wells were sampled to establish a baseline microbial community profile. Postcleaning, each well was sampled a total of twelve times. For each well and time-point, groundwater samples were collected for geochemical and microbial community analyses. Currently, the microbial and geochemical data are still being analyzed although the preliminary results indicate geochemical variation in response to rain events during the course of the study. Overall, results from both pilot studies suggest evidence of geochemical and microbial response within select wells in response to rain events. Findings from these will enable ENIGMA campaigns for more specialized questions on microbial community structure, provide for temporal tuning on environmental models, and further our understanding of the natural temporal variations versus external factors, such as biofouling or sampling-related effects on microbial communities. This material by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological & Environmental Research under contract number DE-AC02- 05CH11231
  160. Brewer, S., S. M. Techtmann, N. Mahmoudi, D. Niang, S. Pfiffner and T. C. Hazen. 2015. Co-extraction of DNA and PLFA from Soil Samples using Bligh and Dyer PLFA Extraction and Modified Miller DNA Extraction. Posters at the Tennessee Capitol. http://ugresearch.utk.edu/activities/posters-at-the-capitol/posters-at-the-capitol-2015/
  161. Brewer, S. S., M. F. Campa, A. G. Amaral, S. M. Techtmann, J. L. Fortney, K. Fitzgerald and T. C. Hazen. 2015. Isolation and Characterization of Anaerobic Microbial Communities from Hydraulic Fracturing Fluids. Tennessee Experimental Learning Symposium (TELS)
  162. Terry C. Hazen. 2014. "OMICS" the Fantasy is Over: We need multiple lines of evidence. Microbial Insights Webinar
  163. Driver, D. A., K. O’Dell, C. J. Paradis, N. Mahmoudi, J. L. Fortney, S. M. Schaeffer and T. C. Hazen. 2014. Soil Microbial Respiration and Biomass as a Function of Soil Moisture Content in a Lexington Silt Loam from West Tennessee. Geological Society of America Annual Metting
  164. Paradis, C. J., S. Jagadamma, J. L. Fortney, T. Mehlhorn, J. C. Parker, D. B. Watson, L. D. McKay and T. C. Hazen. 2014. The Memory Effect: In situ electron donor biodegradation rates as a function of exposure history in a shallow groundwater system. Geological Society of America Annual Metting abstract
    Microbial-mediated redox reactions in groundwater systems can result in significant degradation or immobilization of contaminants that are harmful to the environment and human health. Groundwater conditions can be perturbed to stimulate native microbial communities via the addition of electron donors, electron acceptors, and/or nutrients in order to increase the rates of contaminant transformation. The degradation rate of an electron donor are well known to vary widely between study sites depending on several site-specific factors including the physical characteristics of the solid subsurface media, the chemical species and concentrations of electron acceptors, and the structure and function of the microbial community. Recent scientific studies have suggested that the electron donor degradation rate can vary significantly within a single monitoring well based on the short-term exposure history of the groundwater system. Moreover, groundwater remediation practitioners suggest that the electron donor degradation rate can increase as a function of exposure history even after the physical and chemical conditions of the groundwater system have returned to their initial, pre-electron donor addition state; we refer to this as the “memory effect”. The objective of this study is to adequately characterize and control the physical and chemical groundwater system conditions before, during and after repeated electron donor exposures in order to scientifically describe the memory effect. The experimental field site is located at the Oak Ridge Field Research Center in Oak Ridge, Tennessee where previous scientific studies focused on electron donor additions for reduction and immobilization of radionuclides. The most recent electron donor addition was in 2005 and the site has since been under natural conditions. This study will use the single well push-pull method to repeatedly expose a shallow and unconfined aquifer to ethanol (electron donor) and nitrate (electron acceptor). The biodegradation rate of ethanol under nitrate-reducing conditions will be compared between groundwater wells with and without a short-term exposure history. The memory effect described here may have broader implications on other electron donors that are contaminants such as crude oil and its refined products.
  165. Hazen, T. C., A. M. Rocha, M. Smith, C. Smillie, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, C. Paradis, K. Bailey, D. Joyner, J. L. Fortney, S. Pfiffner, J. J. Zhou, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, D. Curtis, D. Xu, D. Elias, M. Adams, F. Poole, R. Chakraborty, A. P. Arkin and E. Alm. 2014. Microbial Community Structure Predicts Groundwater Geochemistry. International Symposium of Subsurface Microbiology 2014 (ISSM 14). abstract
    At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry.
  166. Woo, H. L., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Lignin-Degrading Microbes of the Eastern Mediterranean Sediment and Seawater. International Symposium of Subsurface Microbiology 2014 (ISSM 14).
  167. Hazen, T. C.. 2014. Harnessing metagenomics in oil-spill cleanup: lessons from the Deepwater Horizon spill. Genome Canada “Genomics: the Power and the Promise” abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. This has also enabled comparative data for risk assessment on several other potential deep-water drilling site around the world.
  168. Curtis, D., P. Zhang, J. D. Van Nostrand, A. M. Rocha, T. C. Hazen and J. Zhou. 2014. Reduction in U(VI) Concentration Influences the Subsurface Microbial Community during the Titration of Highly Acidic Sediments. American Society for Microbiology Annual Meeting
  169. Handley, K. M., O. U. Mason, T. C. Hazen, J. Gilbert and J. Jansson. 2014. Genomic Insights into Uncultivated Microbial Communities Associated with Spatially Distinct Oil Polluted Marine Sediments. American Society for Microbiology Annual Meeting
  170. Hemme, C. L., S. J. Green, L. Rishishwar, O. Prakash, R. Chakraborty, A. M. Deutschbauer, J. D. Van Nostrand, L. Wu, Z. He, I. Jordan, T. C. Hazen, A. P. Arkin, J. E. Kostka and J. Zhou. 2014. Lateral Gene Transfer and Gene Duplication Contribute to Overabundance of Geochemical Resistance Genes in Uranium-Contaminated Groundwater Communities. American Society for Microbiology Annual Meeting
  171. Liu, J., S. Techtmann, J. Fortney, D. Joyner and T. C. Hazen. 2014. Oil-Induced Changes in the Structure and Function of the Eastern Mediterranean Sea Microbial Community. American Society for Microbiology Annual Meeting abstract
    The 2010 oil spill in the Gulf of Mexico brought intense interest in oil contamination and microbial biodegradation in the deep ocean. We have been researching the co-relationship among geochemical characteristics, microbial community structure and oil degradation in deep basins. Eastern Mediterranean sea has some potential for oil production. It is deeper than Gulf of Mexico and considered as one of the most oligotrophic regions on the earth, in which the primary productivity is phosphorus limited. What’s more, the bottom temperature in this region is between 12 to 14℃, which is much warmer than many other deep basins. Very limited research has been done on the microbial community and its changes according to oil in this area. Eastern Mediterranean sea waters from different sites and various depths were collected and studied using a systems biology approach. The indigenous microbial communities can be grouped according to different depths rather than locations and archaea accounted for more than 20% of the bacterial community in deep water samples. On-ship perturbation tests were carried out to examine the changes of microbial community when exposed to crude oil and dispersant. In general, oil played a role that decreased the diversity of the microbial community. The abundance of archaea decreased during the incubation and it disappeared faster when there was more crude oil, but the population of Oceanspirillales increased in the appearance of oil and dispersant. In addition, microrespirometry experiments were used to study the CO2 accumulation from microorganisms exposed to oil, dispersant and different nutrient conditions. The addition of dispersant and phosphate increased CO2 accumulation, which was much higher than the addition of iron. However, compared with Gulf of Mexico, CO2 accumulation was very low. What’s more, many molecular and systems biology tools, like geochip, are applying to target functional genes and microbial activity from communities and isolates. These results will broaden our knowledge on the microbial community in Eastern Mediterranean sea and make us better bioremediation method for oil contamination in this area.
  172. Mahmoudi, N., M. S. Robeson, S. M. Techtmann, S. M. Pfiffner, S. C. Stelling, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Diversity and Function of Microbial Communities in the Caspian Sea. American Society for Microbiology Annual Meeting abstract
    The Caspian Sea is the world’s largest enclosed body of water and accounts for 40% of the total lacustrine waters of the world. Approximately 130 rivers drain into the Caspian Sea making riverine inputs the primary source of water and nutrients into this system. As a result, the salinity of the Caspian Sea differs significantly from other bodies of water and is approximately a third the salinity of seawater. Furthermore, excessive inputs of organic matter and nutrients via runoff over the last few decades have led to permanent hypoxic conditions in bottom waters with oxygen concentrations of 5% to 7%. The Caspian Sea has been the subject of extensive long-term studies into the geochemical and hydrological context; however, little work has been done to characterize the microbial communities in this unique marine system using the latest molecular techniques. The low salinity levels combined with anoxic conditions could provide niches for distinct microbial communities in seafloor sediments. We investigated the diversity and function of microbial communities in sediments using a combination of high throughput DNA sequencing, Geochip, and PLFA analysis. Sediment cores were collected at bottom depths of 141m, 205m and 600m from the southern Caspian Sea in July 2013. Gammaproteobacteria and Deltaproteobacteria were found to be dominant bacterial groups across all sediment samples. Within Delaproteobacteria, Desulfobacterales was a commonly observed group indicating that sulfate reduction may be a primary metabolism in these sediments. Crenarchaeota, specifically Marine Benthic Group B and Cenarchaeales, were found to be the dominant Archaeal groups across sediment samples. Cell densities ranged from 9.1x106 to 9.1x107 and were found to decrease with core depth. Concurrently, the metabolic function of these microbial communities is being investigated using Geochip functional microarray analysis. Our study is one of the first to provide a comprehensive characterization of microbial communities in the Caspian Sea using both DNA and lipid-based approaches.
  173. Pettenato, A., M. Schicklberger, J. Ray, A. M. Deutschbauer, T. C. Hazen, A. P. Arkin and R. Chakraborty. 2014. Nitrate-Contaminated Groundwater at Oakridge FRC. American Society for Microbiology Annual Meeting abstract
    The groundwater at U.S. Department of Energy’s Field Research Center (FRC) in Oak Ridge contains high plumes of uranium, technetium, nitrate, volatile organic compounds and has a pH gradient from 3-10. Nitrate concentrations in the groundwater ranges from 0 to 14,000mg/L, cell counts varied from 103 cells/ml to 2 X 106 cells/ml. We investigated the diversity and metabolism of nitrate-reducing bacteria isolated from several groundwater wells across these geochemical gradients. A wide range of media was used for the isolations and lactate, acetate, glycerol, simple sugars or simple fatty acids were used as carbon and electron donors. More than 200 diverse clonal isolates were obtained and identified by 16S-rDNA sequencing with representatives from Pseudomonas, Castellaniella, Rhodanobacter, Intrasporangium, Aquaspirillum, Variovorax, Duganella, Delftia, Chromobacter, Cupriavidus genera among others. Isolated strains utilized different pathways for nitrate reduction (nitrogen or ammonia as end products) as demonstrated using physiological and molecular methods, and some of the strains such as Intrasporangium strain GW247B1, grew robustly in very high concentrations of nitrate (300mM). Several nitrate-reducers exhibited chemolithotrophic metabolisms and oxidized Iron and dissolved humics. More than a dozen different denitrifying Pseudomonas species were obtained. These Pseudomonas strains significantly differed in their ability to detoxify nitrate. Preliminary comparative analysis of their genomes revealed that each strain contained circa 1000 genes distinct from each other, while sharing approximately 3000 ‘core’ genes. We present results from our ongoing detailed genotypic and phenotypic characterization of several of such nitrate-reducing strains from FRC.
  174. Rajan, S. S., N. Flournoy, M. J. Beazley, R. J. Martinez, T. C. Hazen and P. A. Sobecky. 2014. Application on Marine Bacterial Populations. American Society for Microbiology Annual Meeting
  175. Rocha, A. M., C. Smillie, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, C. Paradis, K. Bailey, D. Joyner, J. L. Fortney, S. Pfiffner, J. J. Zhou, J. D. Van Nostrand, L. Wu, P. Zhang, D. Curtis, D. Xu, D. Elias, M. W. Adams, A. Lancaster, R. Chakraborty, A. P. Arkin, E. J. Alm and T. C. Hazen. 2014. Temporal Variation in Groundwater Geochemistry Has a Dominant Effect on Microbial Community Structure at the Oak Ridge Field Research Site. American Society for Microbiology Annual Meeting abstract
    Across the DOE Oak Ridge Reservation (ORR), there have been a number of studies aimed at characterizing groundwater microbial community assemblages, activity, and functional diversity at along various geochemical gradients. Results of these studies have led towards further understanding of geochemical-microbial associations, as well as, elucidating microbial response to environmental stress factors. Often these studies focus on the spatial distribution of microbial communities rather than the temporal variability of both microbial assemblages and groundwater chemistry. If variability in groundwater chemistry plays a dominant effect on the assembly and activity of groundwater microbial communities, then key geochemical-microbial associations may be missed if the temporal dynamics of a system is not considered. In this study, our overall goal is to determine to what extent, temporal variation of the groundwater geochemistry effects microbial community structure, function, and genetic diversity in groundwater wells along different depths and geochemical transects. Here, we present the findings from our initial time-series study. To determine how resilient microbial communities are to daily and weekly changes in groundwater chemistry, we sampled 2 shallow and 2 deep wells at the ORR background site three times per week for 3 weeks. For each well and time-point, groundwater samples were collected for geochemical and microbial communities analyses. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Cell abundance in the deep wells was an order of magnitude higher than in the shallow wells. Initial PLFA community structure indicates a predominately dominant Gram-negative community. Conductivity and pH field measurements within the deep wells remained stable over time. However, a decrease in pH was indicated in the shallow wells after a rain event. Similarly, dissolved CO2 estimates were higher in the shallow wells compared to the deep wells. Overall, results from this study suggest evidence of geochemical and microbial response within shallow wells response to rain events.
  176. Song, R., A. Zhou, Z. He, J. D. Wall, A. P. Arkin, T. C. Hazen and J. Zhou. 2014. Evolution and Adaptation of Desulfovibrio vulgaris Hildenborough to Elevated Temperature: Fitness and Trade-Offs. American Society for Microbiology Annual Meeting
  177. Techtmann, S. M., K. Ayers, J. L. Fortney, D. C. Joyner, S. M. Pfiffner and T. C. Hazen. 2014. The Eastern Mediterranean Microbial Community is Strongly Stratified by Water Mass. American Society for Microbiology Annual Meeting abstract
    The Mediterranean is a semi-enclosed sea at the intersection of the European, Asian, and African continents. The waters of the Eastern Mediterranean are characterized by high salinity, elevated deep-water temperatures and ultra oligotrophic conditions. Several studies have sought to characterize the microbial community of the Eastern Mediterranean. However, few studies have applied next-generation sequencing to understanding the relationship between community structure and geochemistry of these waters. Here we characterize the geochemistry and microbial community at various depths throughout the water column at five stations in the Eastern Mediterranean. The geochemistry of the water column is stratified by depth, corresponding to three prominent water masses. The carbon and nutrient levels significantly vary between these water masses. The microbial community was characterized using a combination of high-throughput DNA sequencing and PLFA analysis. These analyses demonstrate that the microbial community in these waters is also highly stratified by depth with the largest differences occurring between the surface water and the deeper water masses. However more subtle differences exist between the intermediate water mass and the deep water mass. The diversity of the microbial community is lowest in the surface waters and increases with depth. The dominant bacterial phyla in the surface waters are Cyanobacteria, Proteobacteria (Gamma- and Alpha-) and Bacteriodetes. In the deeper water masses, in addition to Alpha- and Gammaproteobacteria, the Deltaproteobacteria, Chlorflexi, and Planctomycetes are also significant members of population. Archaea make up a much smaller proportion of the microbial community in the surface waters (<1% of recovered reads) whereas they are more dominant in the deeper waters (28 - 50% of recovered reads). Thaumarchaeaota related to Nitrosopumilis dominate the Archaeal community in the deeper water masses. Indicator species analysis revealed the presence of distinct taxa in the different water masses. These indicator taxa may be microbial signatures for these important water masses. The distinct geochemistry of the water masses in the Eastern Mediterranean may act to structure the microbial community allowing for unique populations to thrive in these adjacent water masses.
  178. Trexler, R., C. Solomon, C. Brislawn, E. McClure, A. Grube, T. C. Hazen, M. Keddache, C. Grant and R. Lamendella. 2014. The Response of Freshwater Aquatic Microbial Communities to Marcellus Shale Natural Gas Extraction. American Society for Microbiology Annual Meeting abstract
    At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
  179. Voordeckers, J. M., P. Zhang, Z. Shi, Y. Deng, J. D. Van Nostrand, L. Wu, Z. He, T. C. Hazen, D. A. Elias, M. M. Fields, A. P. Arkin, P. D. Adams and J. Zhou. 2014. Effects of Uranium Contamination on the Metal Homeostasis Genes of Groundwater Microbial Communities. American Society for Microbiology Annual Meeting
  180. Woo, H. L., S. M. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Investigating Lignin Degradation Potential in the Hypersaline Eastern Mediterranean Deep-Sea Basin. American Society for Microbiology Annual Meeting abstract
    The fate of terrestrial organic carbon (terrOC) in the ocean is unclear as only a fraction of the estimated input is found buried in the sediments, thereby suggesting that the rest is degraded. Since terrOC consists of lignin among other recalcitrant organics, the largely unknown microbial diversity of deep-sea saline environments should be able to degrade lignin even in the presence of ionic liquid, a solvent with salt-like properties used by the lignocellulosic biofuel industry. A better understanding of these microbes can aid the discovery of enzymes that can turn lignin waste into a higher value feedstock as well as inform models of terrOC cycling in the ocean. To obtain a simplified lignolytic microbial community, hypersaline oxic Eastern Mediterranean surface water and sediment were collected from a deep basin near the Nile Delta and incubated aerobically in bottles with insoluble lignin for 2 weeks, during which carbon dioxide was monitored. Ionic liquid, 1-ethyl-3-methylimidazolium acetate, was also added to some treatments as a stressor. The taxonomic diversity was then assessed using 16S rRNA gene amplicon sequencing and the lignolytic enzyme activity was tested using the model lignin L-3,4-dihydroxyphenylalanine. Microcosms with lignin had 10-fold higher respiration rates within 1 day and higher lignolytic activity than the unamended controls. Adding 0.5% ionic liquid did not negatively impact respiration rates. Sequencing revealed that ionic liquid did not alter the community structure either, as Halomonas remained the dominant at 30% relative abundance. Sequencing also revealed that the water and sediment microcosms have different microbial communities with lignin despite having similar respiration rates; Novosphingobium dominated the sediment while Idiomarina dominated the water community. Ionic liquid in combination with lignin caused the Crenarchaeota to increase 10-fold; Archaea have not been well-studied for lignin degradation or ionic liquid tolerance before. Our findings show that the deep-sea has a novel microbial diversity with lignin degradation potential that is unhindered by ionic liquid.
  181. Hazen, T. C.. 2014. Omics and Geochemistry: the ENIGMA 100-Well Survey. TES/SBR Joint Investigators Meeting abstract
    At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
  182. Hazen, T. C.. 2014. How many punches can Mother Nature take in the Gulf of Mexico: Focus on Deepwater Horizon Oil Spill & Science and the Media. Mic/Nite University of Tennessee abstract
    At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
  183. Hazen, T. C.. 2014. ENIGMA at ORNL. Fourth Biennial Southeastern In Situ Soil and Groundwater Remediation Conference 2014.
  184. Bailey, K., R. A. Hurt, T. R. Chowdhury, M. S. Robeson II, S. Techtmann, T. Mehlhorn, A. Zelaya, M. W. Fields, A. P. Arkin, S. D. Brown, M. Podar, D. A. Stahl, T. C. Hazen, J. Zhou, T. J. Phelps, M. W. W. Adams, D. B. Watson and D. A. Elias. 2014. Reproducibility of a Groundwater Microbial Community in Replicate Bioreactors. June, 2014. Goldschmidt, Sacramento CA. Goldschmidt Conference
  185. Hazen, T. C.. 2014. Deepwater Horizon Oil Spill: Deepwater oil-degrading bacterial communities. Seminar Norwegian University of Science and Techonology (NTNU) abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  186. Hazen, T. C.. 2014. Deepwater Horizon Oil Spill: Deepwater oil-degrading bacterial communities. Seminar SINTEFF abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  187. Hazen, T. C. . 2014. Deepwater Horizon Oil Spill: How resilient is the Gulf of Mexico?. Scripps Institution of Oceanography abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently live in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  188. Hazen, T. C.. 2014. Comparing and Contrasting Petroleum Degrading Microbial Communities in Deep-Sea Environments.. MedRem Conference abstract
    Very large reservoirs of oil and gas have recently been discovered in the Eastern Mediterranean, and other deep-sea environments around the world, making this important developing interest for oil and gas prospecting. Using a systems biology approach we have been comparing and contrasting these sites at the molecular level all the way up to the ecosystem level because “the whole is greater then the sum of it’s parts”. These studies are also bridging our ‘response phase” research on the Deep Water Horizon oil spill in the Gulf of Mexico. These studies will shed light on the microbial community structure at different deep-sea sites and will help to clarify how unique physicochemical parameters could affect the microbial response to an oil spill. Practical information will be gained regarding the conditions needed to elicit robust oil biodegradation. This work will expand our understanding of the native microbial communities and their hydrocarbon-degrading potential and how they function at a systems biology level.
  189. Elias, D. A., M. W. W. Adams, R. Chakraborty, M. W. Fields, T. C. Hazen, J. Zhou, T. Northern, N. Baliga, J.-M. Chandonia, A. P. Arkin and P. D. Adams. 2014. Natural and Synthetic Ecology in ENIGMA: Determining the links between Microbial Community Structure and Function. Genomics Sciences Program Contractor-Grantee Meeting abstract
    Project Goals: The overarching goal of the Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA) is to understand environmentally-relevant microbial community structure and function through a series of integrated field-to- laboratory campaigns. The Natural and Synthetic Ecology campaign is designed as an interdisciplinary platform to elucidate the fundamental ties between structure and function, as well as determine the environmental influences on these ties. Our initial focus has been the development of in-field bioreactors as a method of capturing temporal fluctuations in the in-situ community due to either natural or induced influences. Going forward we will determine the influence of naturally-occurring carbon sources, invasive species and selected pressures on microbial activities such as nitrate- and metal- reduction. One of the most difficult aspects of studying microbial ecology is determining and understanding the fundamental ties between microbial community structure (the organism biodiversity and their relative abundances that comprise a given microbial community) and the observed functions (the detectable biochemical activities that support survival of the observed species). Although microorganisms are important in controlling the fate of contaminants in the subsurface, information on the basis of how why microbial communities respond to contaminants is lacking. Hence, it is important to characterize microbial communities, establish linkages between biodiversity and function, and study interactions between different species. As a part of the overall ENIGMA goal to link genotypes to phenotypes, the overall objective of this campaign is to obtain deep understanding of the composition, structure, function, activity and interaction of subsurface microbial communities at DOE contaminated sites (i.e. Oak Ridge Integrated Field Research Center). We have developed a bioreactor system for manipulating and temporally monitoring the in-situ microbial community in the field so as to maintain the in-situ community structure. Community structure was measured through sequencing, PCR and qPCR for selected genes, cell counts and total protein before and after the cells entered the bioreactor system. Temporal community function was qualified by alterations in the concentration of 53 metals, 12 organic acids, 14 anions and 4 sugars, pre- and post- bioreactor exposure. Near future experimental plans include determining an adequate naturally-occurring carbon source that will allow for an increase in total biomass with minimal alteration to the relative abundances of the major phylogenetic groups within the community. Finally, in order to establish the environmental relevance of new field isolates from this site, a pilot study using the bioreactor system will be conducted in collaboration with the Metals Metabolism campaign of ENIGMA. The bioreactors will use synthetic groundwater mimicking the geochemistry of the ORNL wells and will be inoculated with groundwater supplemented with various Mo concentrations since a current hypothesis is that a lack of Mo may inhibit nitrate-reduction. The experimental duration is expected to be 30 days with temporal measurements of metals (53 elements), metabolites, 16S rRNA to determine changes in community structure, and by qPCR of key denitrification genes. End-point samples will be used for the isolation and characterization of new denitrifying strains. This work is highly collaborative, involving several ENIGMA campaigns including the 100 Well Survey, Microparticle Mesogenomics, Microbial Isolations and Characterizations and, Metals with down the line benefits to the Predictive Biology and Printable Worlds campaigns. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02- 05CH11231.
  190. Jansson, J. K., J. Kimbrel, N. Ballor, H. Woo, T. Ruegg, T. C. Hazen, M. P. Thelen, B. A. Simmons and S. W. Singer. 2014. Halophilic Communities as a Source for Novel Lignocellulolytic Enzymes. Genomics Sciences Program Contractor-Grantee Meeting abstract
    Project Goals: Characterize halophilic communities from saline environments as sources of novel halophilic microorganisms, genes, and enzymes for biofuel feedstock deconstruction. Selectively enrich microbial populations from complex microbial communities from saline environments on biofuel feedstocks (Miscanthus, Pine and Eucalyptus) to obtain candidates potentially capable of deconstruction of feedstocks under high salinity conditions. Describe the metabolic potential and gene expression patterns in both natural saline communities and feedstock enrichments by sequencing and screening of metagenomes, metatranscriptomes and metaproteomes. Use functional metagenomics to express a library of genes that potentially represent novel mechanisms for deconstruction of biomass that are currently underrepresented in gene catalogues. Formulate (by synthesis, cloning and expression of genes characterized above) and verify activity of a cocktail of halophilic enzymes for deconstruction of biomass in the presence of ionic liquids. Lignocellulose presents a challenge to next generation biorefineries due to its recalcitrance to microbial degradation. Ionic liquid (IL)-based pretreatment has been successful in preparing biomass for enzyme saccharification, but the most common ILs used for pretreatment inhibit many downstream enzymatic and microbial processes mediated by mesophilic enzymes. Halophiles, by definition, are adapted to high-salt environments and are thus a potential source for IL tolerant enzymes. Here we sought to discover & recover novel lignocellulolytic enzymes from environmental and feedstock-enriched halophilic bacterial communities. We collected both liquid and sediment samples from different saline environments in Puerto Rico and San Francisco including salt flats, saltern ponds and turtle grass beds. For each of the environmental samples we obtained 16S rRNA gene sequences, metagenomes and metaproteomes. The data revealed an increase in relative abundance of haloarchaea and genes and proteins implicated in a hypersaline lifestyle with increasing salinity. In addition, a fosmid library was constructed in an expression vector for high throughput functional metagenomics screening using the robotics platform at JBEI. Samples from a turtle grass bed (3.5% salinity) and a high salinity saltern pond (33.2% salinity) were selected for enrichment on the potential biofuels feedstocks: miscanthus (M), eucalyptus (E) or pine (P) under aerobic and anaerobic conditions and followed through three 2-‐week passages. At the end of each passage cells were harvested, specific enzyme activities were measured and DNA, RNA and proteins were extracted. Data collected include enzyme activities for B glucosidase, cellobiohydrolase and xylanase, 16S rRNA gene sequences, metagenomes metatranscriptomes and metaproteomes. We found that enzyme activity was typically highest after the first passage, with the aerobic turtle grass enrichments having consistent activity on each feedstock. After the third passage, metagenomes were constructed and binned using MaxBin, a binning algorithm developed at JBEI. Bins were subsequently searched against the CAZY/dbCAN HMMs. In addition, expressed transcripts from 11 metatranscriptomes were identified by either alignment to the reference metagenomes or de novo assembled. To date we have identified over 1000 expressed candidate carbohydrate active enzymes from the enrichments and obtained reconstructed genomes for >100 feedstock-enriched archaea/bacteria. Heterologous expression of a diverse collection of 29 putative glycoside hydrolases is ongoing. The next step will be to validate and incorporate these candidate enzymes into a halophilic deconstruction enzyme mixture with high activity and IL tolerance. This work conducted by the Joint Bio Energy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DEAC02-05CH11231.
  191. Rocha, A. M., M. Smith, C. Smillie, J. L. Fortney, S. M. Techtmann, D. C. Joyner, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, J. H. Campbell, S. Pfiffner, K. Ayers, C. Paradis, J. D. Van Nostrand, L. Wu, P. Zhang, Z. He, J. Zhou, M. W. W. Adams, A. Lancaster, P. D. Adams, A. P. Arkin, E. J. Alm and T. C. Hazen. 2014. Microbial Community Structure Predicts Groundwater Geochemistry. Genomics Sciences Program Contractor-Grantee Meeting abstract
    Project goals: ENIGMA’s Field Microbiology component works with microbial communities in the environment to investigate how biological structure and function relate to critical biochemical conditions/changes, such as the immobilization of toxic metals and the flow of carbon and nitrogen. The goal is to identify key microbial populations and determine the community events and mechanisms of these populations that impact and control environmental activities of interest, ultimately predicting how perturbations of the environment may affect community structure. From these results, we can develop models that can be applied to microbial populations overlaid with geochemical parameters and engineering controls. Development of such a model and key findings from a 100-well survey at the Department of Energy’s Oak Ridge Field site is described below. Specifically, we demonstrate the ability to utilize microbial community assembly from independent groundwater environments to accurately predict the geochemistry and elucidate key systems biology features. One of the primary objectives of the ENIGMA Environmental Core Field Microbiology Component is to design an efficient field sampling study that will maximize the geochemical diversity of the study site and provide for enhanced resolution of microbial communities and geochemical associations. At the Department of Energy’s Oak Ridge field research site, 243-‐acres of contaminated area is located within the Y-‐12 plant area of responsibility of the Oak Ridge Reservation (ORR). Here, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 100 groundwater wells in order to (1) characterize key microbial populations at geochemically distinct locations and (2) identify associations between environmental gradients and microbial communities. We hypothesize that differences in geochemistry underlie differences in microbial community structure across the groundwater wells. In order to identify microbe-environment associations, a statistically informed experimental design was developed by ENIGMA’s computational core. To optimize geochemical diversity and identify wells where environmental factors are uncorrelated, k-medians clustering algorithm was used to group 818 wells into 100 clusters using 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering 4-L of water through a 10.0μm pre-filter and 0.2μm-membrane filter and then extracted using a Modified Miller method. Community genomic DNA yields from the 0.2μm and the 10.0μm filters range from 0.096- 8.5μg and 0.096-22μg, respectively. Cell counts for the field samples ranged from 103-106 cells/mL and were consistent with biomass estimates from phospholipid fatty acid analysis (PLFA). Initial analysis of PLFA data suggests stress indicators for Gram Negative populations, such as those associated with pollutants and nutrient limitation, are present in 15% of the wells sampled. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. However, initial analysis indicates there are some organisms unique to the 10.0μm size fraction. A total of 27 wells, which can be classified into 7 groups based on pH and contaminant concentrations, were analyzed with the newest GeoChip 5.0_180K to evaluate functional differences and potentials of microbial communities under different environmental conditions. Our results indicated that the groundwater microbial community functional structure was significantly different among seven different groups of wells, and such community differences were largely correlated with the differences in pH, U(VI), nitrate, dissolved organic carbon (DOC) and sulfate in the groundwater. The microbial functional diversity significantly decreased with Ur concentrations. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. Using the synthetic learning in microbial ecology (SLiME) algorithm and the large independent dataset, we are able to predict the geochemistry from the 16S rRNA. Additionally, using the relationship between the geochemistry and microbiology, the critical OTUs that geochemistry can be identified. Overall, results from this study indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the geochemistry. Such predictions may provide the ability of microbial monitoring for natural attenuation at legacy sites and be enabling for ENIGMA for more specialized questions on microbial community and network structure and function. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02- 05CH11231.
  192. Yilmaz, S., M. Smith, E. J. Alm, D. A. Elias, T. C. Hazen, A. P. Arkin, A. K. Singh and P. D. Adams. 2014. Single Cell Genomics Applications in ENIGMA. Genomics Sciences Program Contractor-Grantee Meeting abstract
    Project Goals: We are developing a versatile single cell genomics pipeline that can respond to the needs of a multi-institutional program like ENIGMA. Our pipeline utilizes FISH (fluorescence in situ hybridization) for targeting species of interest, FACS (fluorescence activated cell sorting) for high throughput isolation of single cells, and MDA (multiple displacement amplification) for production of sufficient DNA for genome sequencing. At present, this pipeline is being used for a number of collaborative projects in ENIGMA. Single cell sequencing is a powerful tool for the analysis of uncultivated microorganisms. Current culture-independent, population based techniques (i.e., metagenomics) relying on pooled nucleic acids from communities of microorganisms can independently measure metabolic activity and the species present, but cannot link the activity deterministically to the species. In an attempt to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities, we developed a high-throughput approach to study uncultivable microorganisms one cell at a time. Our approach includes isolation of individual cells by cell sorting, followed by whole genome amplification and sequencing. This pipeline is being utilized to analyze groundwater samples from DOE bioremediation sites (e.g., Hanford 100H, Oak Ridge FRC) to identify keystone organisms and link their functions to species as well as to estimate the level of horizontal gene transfer within the community; to isolate and identify viruses in deep subsurface groundwater, and investigate their role in microbial community structure and function; to assess the composition of bioaggregates in environmental samples with the ultimate goal of verifying the stereotypical configurations of microorganisms. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
  193. Chen, C., T. C. Hazen and S. Techtmann. 2014. The response of microbial community structure to clay flocculation of harmful algae blooms. International Symposium of Microbial Ecology (ISME 15)
  194. Liu, J., S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen*. 2014. Microbial respiration and community changes to crude oil in deep Eastern Mediterranean and Great Australian Bight. International Symposium of Microbial Ecology (ISME 15)
  195. Mahmoudi, M., M. Robeson, T. Porter, S. Pfiffner, S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Sedimentary microbiomes in the Caspian Sea. International Symposium of Microbial Ecology (ISME 15)
  196. Rocha, A., S. Pfiffner, K. Ayers, J. L. Fortney, S. Techtmann, D. C. Joyner, J. Van Nostrand, P. Zhang, A. Lancaster, J. Zhou, D. Watson, M. Adams, R. Chakraborty, A. Arkin, E. Alm and T. C. Hazen. 2014. Phospholipid fatty acid analysis for characterization of a microbial community structure across a geochemically diverse watershed. International Symposium of Microbial Ecology (ISME 15)
  197. Techtmann, S., K. Ayers, J. L. Fortney, D. C. Joyner, S. Pfiffner and T. C. Hazen. 2014. The structure and function of the eastern Mediterranean microbial community is strongly stratified by water mass. International Symposium of Microbial Ecology (ISME 15)
  198. Woo, H., S. Techtmann, J. L. Fortney, D. C. Joyner and T. C. Hazen. 2014. Investigating lignin degradation potential by microbial communities in the deep-ocean. International Symposium of Microbial Ecology (ISME 15) abstract
    The fate of terrestrial organic carbon (terrOC) in the ocean is unclear as only a fraction of the estimated input is found buried in the sediments, thereby suggesting that the rest is degraded. Since terrOC consists of lignin among other recalcitrant organics, the largely unknown microbial diversity of deep-sea saline environments should be able to degrade lignin even in the presence of ionic liquid, a solvent with salt-like properties used by the lignocellulosic biofuel industry. A better understanding of these microbes can aid the discovery of enzymes that can turn lignin waste into a higher value feedstock as well as inform models of terrOC cycling in the ocean. To obtain a simplified lignolytic microbial community, hypersaline oxic Eastern Mediterranean surface water and sediment were collected from a deep basin near the Nile Delta and incubated aerobically in bottles with insoluble lignin for 2 weeks, during which carbon dioxide was monitored. Ionic liquid, 1-ethyl-3-methylimidazolium acetate, was also added to some treatments as a stressor. The taxonomic diversity was then assessed using 16S rRNA gene amplicon sequencing and the lignolytic enzyme activity was tested using the model lignin L-3,4-dihydroxyphenylalanine. Microcosms with lignin had 10-fold higher respiration rates within 1 day and higher lignolytic activity than the unamended controls. Adding 0.5% ionic liquid did not negatively impact respiration rates. Sequencing revealed that ionic liquid did not alter the community structure either, as Halomonas remained the dominant at 30% relative abundance. Sequencing also revealed that the water and sediment microcosms have different microbial communities with lignin despite having similar respiration rates; Novosphingobium dominated the sediment while Idiomarina dominated the water community. Ionic liquid in combination with lignin caused the Crenarchaeota to increase 10-fold; Archaea have not been well-studied for lignin degradation or ionic liquid tolerance before. Our findings show that the deep-sea has a novel microbial diversity with lignin degradation potential that is unhindered by ionic liquid.
  199. Zhang, P., Z. He, J. D. Van Nostrand, L. Wu, D. Curtis, T. C. Hazen, D. E. Elias, M. W. Fields, A. P. Arkin, P. D. Adams and J. Zhou. 2014. Impacts of Environmental Contaminants on Functional Diversity of Groundwater Microbial Communities at a U(VI)-Contaminated Aquifer. International Symposium of Microbial Ecology (ISME 15) abstract
    Abstract: Microbial diversity in groundwater ecosystems has not been well studied yet. The functional diversity of groundwater microbial communities at heavy metal contaminated sites would be impacted by different geochemical characteristics such as contaminants and pH. As part of the Global 100 Well Survey at the Oak Ridge site, groundwater samples were collected from seven groups of wells with different geochemical characteristics at this site. The first four groups primarily concern the effects of contaminants (U(VI) and nitrate) on microbial diversity: (1) low contaminants (nitrate<2 mg/L and U(VI)<0.01 mg/L) and neutral pH (6.5-7.2), (2) light contaminants (nitrate 4-36 mg/L and U(VI) 0.1-0.2 mg/L) and also neutral pH, (3) moderate contaminants (nitrate 5.5-1471 mg/L and U(VI) 0.1-1.5 mg/L) and neutral pH, and (4) high contaminants (nitrate 2692-11648 mg/L and U(VI) 3.8-55 mg/L) and low pH (3-5.2). The remaining three groups with low contaminants address possible effects of other geochemical characteristics such as groundwater pH: (5) acidic pH (5.2-6.7), (6) high pH (8.9-9.8), and (7) extremely high pH (9.2-10.5). Groundwater microbial communities were analyzed using a comprehensive functional gene microarray (GeoChip 5.0). The results indicated that the groundwater microbial community functional diversity and structure was significantly different among these seven different groups of wells. Such community differences were largely correlated with the differences in U(VI), nitrate, pH, dissolved organic carbon and sulfate in the groundwater. The microbial functional diversity significantly decreased with U(VI) concentrations. Overall, our results suggested that high contaminants and low pH appeared to inhibit the biodiversity and metabolic potential of indigenous groundwater microbial communities. This study improves our understanding of the functional diversity and metabolic potential of groundwater microbial communities across a large scale of contaminant and pH levels at this site.
  200. Bailey, K. L., B. R. Crable, R. A. Hurt, II M. S. Robeson, S. Techtmann, D. A. Stahl, T. C. Hazen, A. P. Arkin, J. Chandonia, T. Northen, M. W. Fields, E. J. Alm, J. Zhou, M. W. W. Adams and D. A. Elias. 2014. Reproducibility of a Groundwater Microbial Community in Replicate Bioreactors. ENIGMA SFA Annual Retreat abstract
    Project Goals: To develop a well-head bioreactor system for temporally monitoring microbial community structure without impacting the microbial community in-situ. Abstract: One of the most difficult aspects of studying microbial ecology is determining and understanding the fundamental ties between[1] microbial community structure and observed functions. Although microorganisms are important in controlling contaminant fate in the subsurface, knowledge of how/why microbial communities respond to contaminants is lacking[2]. Hence, it is important to characterize microbial communities, establish linkages between biodiversity and function, and study interactions between different species. We have developed a bioreactor system for manipulating and temporally monitoring the microbial community without disturbing the community structure in-situ. Three above ground, in-field reactors were fed groundwater from well FW305 at the Oak Ridge Field Research Center, Oak Ridge, TN for 11 weeks. Each bioreactor contained 8 replicate biofilm coupons filled with sterilized sediment obtained from the FRC. The microbial communities from the planktonic and biofilm portions of the reactors were compared to the groundwater community for structure and function. Community structure was measured through sequencing and total protein before and after the cells entered the bioreactor system. Temporal community function was qualified by alterations in the concentration of 53 metals, 9 organic acids, 7 anions and 3 sugars, pre- and post- bioreactor exposure. The findings of this initial experiment will directly impact the number of biological replicates that will be required going forward with future reactor studies at this site.
  201. Deng, Y., P. Zhang, Z. He, Z. Shi, Y. Qin, J. D. Van Nostrand, L. Wu, M. Fields, C. W. Schadt, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin and J. Zhou. 2014. Network dynamics of groundwater microbial community succession during uranium bioremediation and new network analysis approaches. ENIGMA SFA Annual Retreat abstract
    In recent years, the inference of species/population interactions and their relevant network analyses have evoked substantial interests in microbial ecology, but few studies have focused on the dynamic changes of microbial community networks in response to substrate amendments or environmental perturbations. In this study, an improved functional molecular ecological network (fMEN) approach was used to explore the interaction of groundwater microbial community succession during uranium bioremediation with emulsified vegetable oil (EVO) amendment. Samples were collected at 0, 4, 17, 31, 80, 140 and 269 days after a onetime EVO injection and examined with a functional gene array (GeoChip 4.0). Our results showed the species/population interaction was dynamically altered after EVO amendment. Competition among microbial species was notable in groundwater microbial communities after EVO injection, especially through EVO-stimulated microorganisms (e.g., sulfate-reducing bacteria) that restrained the growth of other species, resulting in the decline of microbial community richness and diversity. Also, microbial interactions were dynamically switched during the microbial community succession but the changes appeared to be time-lagging. In addition, seven keystone microorganisms were identified, which may play important roles in uranium bioremediation. This study provides new insights into our understanding of the dynamics of microbial interactions during the groundwater microbial community succession in response to EVO amendment.
  202. He, Z., P. Zhang, A. M. Rocha, L. Wu, Q. Tu, Y. Qin, D. Curtis, J. D. Van Nostrand, L. Wu, E. Alm, M. Fields, D. A. Elias, D. A. Stahl, T. C. Hazen, A. P. Arkin, P. Adams and J. Zhou. 2014. Functional genes of groundwater microbial communities predict contamination and ecosystem functioning. ENIGMA SFA Annual Retreat abstract
    Project Goals: This project aims to understand the biodiversity, composition, structure, dynamics, and assembly mechanisms of groundwater microbiomes and their relationships with environmental factors, especially across a large range of concentrations of uranium, nitrate and pH. Advanced metagenomic approaches are used to survey groundwater microbiomes from 100 wells located at Oak Ridge Field research Center. This study is focused on how environmental contamination affects the biodiversity of groundwater microbial communities and their feedbacks to ecosystem functioning using functional gene arrays and other technologies. The results provide insights into our understanding of dimensions of biodiversity of groundwater microbiomes and their relationships with environmental contamination, and the potential to predict contaminants in the environment towards bioremediation of contaminated sites. Abstract Various contaminants in groundwater, such as heavy metals (e.g., uranium), nitrate, and extreme pH, present numerous threats to the environment and human health. Although studies have shown these contaminants affect groundwater microbial communities, how the overall microbial community functional diversity, composition, structure and function vary and change across a large contaminant gradient is not well understood. It would be interesting to be able to predict environmental contamination and ecosystem functioning bases on the microbial community diversity, composition and structure, or vice versa. It is hypothesized that the functional diversity would decrease as environmental contamination (e.g., increased uranium and nitrate) increases, or as pH is too low or too high; however, specific populations capable of utilizing the available contaminants would increase. In this study, groundwater microbial communities were collected from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN) representing a large range of uranium, nitrate and other contaminant concentrations as well as pH and analyzed using a new version of GeoChip 5.0. The results indicated that the functional diversity significantly decreased as uranium and nitrate increased in the groundwater, particularly at high concentrations, or pH was low or high. About 5% of specific key functional populations significantly (p < 0.05) increased as uranium and/or nitrate increased in the groundwater, including dsrA for sulfur (S) reduction, cytochrome and hydrogenase genes for metal reduction, napA and nrfA for dissimilatory nitrogen (N) reduction, nasA and nirA/B for assimilatory N reduction, nirK, nirS and nosZ for denitrification. These genes may indicate the potential of bioremediation of the contaminated groundwater. Additionally, we used a machine learning method, random forests, to predict uranium and nitrate contamination in groundwater using these key microbial functional genes. Our results showed a high specificity and sensitivity and a low error rate when out-of-bagging (OOB) estimation was used, suggesting it is possible to predict environmental contamination and ecosystem functioning using key microbial functional genes. Overall, this study provides new insights into our understanding of the effects of environmental contamination on groundwater microbial communities and their feedbacks to ecosystem functioning.
  203. Hemme, C. L., Q. Tu, Z. Shi, Y. Qin, W. Gao, Y. Deng, J. D. Van Nostrand, L. Wu, Z. He, S. J. Green, L. Rishishwar, O. Prakash, P. S. G. Chain, S. Tringe, M. W. Fields, R. Chakraborty, A. M. Deutchbauer, I. K. Jordan, J. E. Kostka, E. M. Rubin, J. M. Tiedje, A. P. Arkin, T. C. Hazen and J. Zhou. 2014. Metagenomic Analysis of Pristine and Stressed Groundwater Communities. ENIGMA SFA Annual Retreat abstract
    Metagenomes were sequenced from two sites at the Oak Ridge Integrated Field Research Center (OR-IFRC). The first site, FW301, is from the IFRC background area and represents a pristine groundwater system. The second site, FW106, is from Area 3 at the base of a contaminant plume originating from the S3 waste disposal ponds (see Hemme et al. 2010 ISME J. 4 660-672). The FW106 community experiences constant exposure to high levels of nitric acid, uranium, organic compounds, etc. Previous analyses of the FW106 metagenome show a low-diversity community dominated by denitrifying γ-proteobacteria species. Analysis also suggested the possibility of lateral transfer of genes related to geochemical resistance. To complement these analyses and to address outstanding questions regarding lateral gene transfer and nutrient cycling, the FW106 metagenome was compared to the pristine FW301 metagenome, other environmental metagenomes, and the genomes of Rhodanobacter isolates from IFRC sites.
  204. Paradis, C. J., S. Jagadamma, J. L. Fortney, T. Mehlhorn, D. B. Watson, L. D. McKay and T. C. Hazen. 2014. Hydrogeologic Characterization of a Groundwater System for Investigating Ethanol Biodegradation Rates as a Function Exposure History. ENIGMA SFA Annual Retreat abstract
    Project Goals: The goals of this project are to (1) characterize electron donor biodegradation rates as a function of groundwater system exposure history at the field scale, (2) elucidate the responsible mechanism(s) at the laboratory scale and (3) numerically model both data sets in a groundwater system using a relatively rapid and simple microbial-mediated redox reaction. We hypothesize that the degradation rate of a pulse injection of ethanol is significantly greater when the reaction zone has been previously exposed to ethanol, even after the physical and chemical conditions of the groundwater reaction zone have returned to their initial conditions between exposures. We further hypothesize that the mechanism responsible for the memory effect is either that (1) microbes capable of degrading ethanol are enriched following pulse exposures and remain enriched (although possibly dormant) or (2) pathways for ethanol metabolism are upregulated more rapidly following repeated pulse exposures of ethanol. Abstract: Experimental field studies are critical to elucidating in situ physical, chemical and biological processes. Rigorous characterization of such processes under ambient conditions and prior to controlled perturbation(s) is of utmost importance. However, characterization of environmental systems can be incredibly challenging, time consuming and costly due to considerable temporal variability and spatially heterogeneity. The objective of this study was to rapidly and rigorously characterize the fundamental physical and chemical conditions of a groundwater system using manual and real-time digital in situ monitoring equipment prior to push-pull experimentation. Future push-pull experiments will be focused on characterizing ethanol biodegradation rates and mechanisms as a function of the groundwater system exposure history to the ethanol. The groundwater system is shallow, unconfined and consists of 3 to 4 meters of unconsolidated fill followed by 3 meters of intact saprolite; monitoring wells are screened across the water table and the fill/saprolite contact. The direction, magnitude and temporal variability of the horizontal hydraulic gradient was determined by manual depth to water measurements and geospatial analysis in ArcMap. The magnitude and spatial variability of the hydraulic conductivity was determined by manual flow and drawdown measurements during low-flow steady-state pumping and was visualized in ArcMap. Groundwater quality parameters (temperature, pH, dissolved oxygen, oxidation-reduction potential, and electrical conductivity) were monitored in real-time during low-flow pumping though a standard multimeter with a flow through cell. These results suggested that the horizontal hydraulic gradient (dh/dl) was temporally stable (220±4o at 3.5±0.5%) and may indicate stable groundwater flow direction and magnitude. These results also suggested that wells FW003, FW218, FW219, FW226 and FW230 may not be sufficiently permeable (K≤0.08 m/day) to facilitate push-pull experimentation. Lastly, the groundwater quality results suggested that temperature (24 to 30oC) and pH (cicrumneutral) were relatively stable compared to dissolved oxygen, oxidation-reduction potential and electrical conductivity; variability in these latter parameters may be due to heterogeneity of the groundwater system. This study demonstrated that rapid and rigorous characterization of the fundamental physical and chemical conditions of groundwater systems is feasible using only in situ monitoring equipment combined with manual measurements of groundwater head and flow rates during low-flow pumping. This study also established a robust pre-experimental data set to facilitate optimal push-pull test design, application and data interpretation.
  205. Rocha, A. M., C. Smillie, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, J. Phillips, D. B. Watson, C. Paradis, K. Bailey, D. Joyner, J. L. Fortney, S. Pfiffner, J. J. Zhou, J. D. Van Nostrand, L. Wu, P. Zhang, D. Curtis, D. Xu, D. Elias, M. W. Adams, A. Lancaster, R. Chakraborty, A. P. Arkin, E. J. Alm and T. C. Hazen. 2014. Temporal Variation in Groundwater Geochemistry Has a Dominant Effect on Microbial Community Structure at the Oak Ridge Field Research Site. ENIGMA SFA Annual Retreat abstract
    Across the DOE Oak Ridge Reservation (ORR), there have been a number of studies aimed at characterizing groundwater microbial community assemblages, activity, and functional diversity at along various geochemical gradients. Results of these studies have led towards further understanding of geochemical-microbial associations, as well as, elucidating microbial response to environmental stress factors. Often these studies focus on the spatial distribution of microbial communities rather than the temporal variability of both microbial assemblages and groundwater chemistry. If variability in groundwater chemistry plays a dominant effect on the assembly and activity of groundwater microbial communities, then key geochemical-microbial associations may be missed if the temporal dynamics of a system is not considered. In this study, our overall goal is to determine to what extent, temporal variation of the groundwater geochemistry effects microbial community structure, function, and genetic diversity in groundwater wells along different depths and geochemical transects. Here, we present the findings from our initial time-series study. To determine how resilient microbial communities are to daily and weekly changes in groundwater chemistry, we sampled 2 shallow and 2 deep wells at the ORR background site three times per week for 3 weeks. For each well and time-point, groundwater samples were collected for geochemical and microbial communities analyses. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Cell abundance in the deep wells was an order of magnitude higher than in the shallow wells. Initial PLFA community structure indicates a predominately dominant Gram-negative community. Conductivity and pH field measurements within the deep wells remained stable over time. However, a decrease in pH was indicated in the shallow wells after a rain event. Similarly, dissolved CO2 estimates were higher in the shallow wells compared to the deep wells. Overall, results from this study suggest evidence of geochemical and microbial response within shallow wells response to rain events.
  206. Zeng, L., R. Csencsits, C. Petzold, T. C. Hazen, F. Poole, W. A. Lancaster, M. Adams, M. Fields, D. Stahl, H. Nikaido, B. Jap and P. Walian. 2014. Membrane-based Mechanisms in Stress Response and Community Interactions. ENIGMA SFA Annual Retreat abstract
    Project Goals: In support of our long-standing interest in understanding mechanisms central to stress response and community organization at the molecular level, a primary aim of our project has been the identification and analysis of proteins key to these processes. Particular emphasis is placed on the study of membrane-based systems. Core objectives stemming from this aim involve the isolation and characterization of protein complexes using biochemical and biophysical methods, elucidation of corresponding functional mechanisms, and the development of molecular models and testable hypotheses. Additional goals include supporting objectives of the ENIGMA campaigns, particularly those relevant to microbial communities and interaction mechanisms, metalloprotein function, and the development of technologies for functional screening. Abstract: A central theme of our studies has been the identification and characterization of membranebased molecular mechanisms utilized in the response to environmental conditions, and the establishment and maintenance of communities. In addition to the characterization of targeted protein function, we have been studying growth condition-associated changes in protein abundance, protein-protein interactions, post-translational modifications, and mutations leading to altered function. Recent work has focused on developing antibodies useful for studying the transition from planktonic to biofilm community organization, chromatographic approaches for separating lipidated from non-lipidated proteins, characterization of outer membrane-based metal-binding proteins (where metals can be essential for stabilizing protein conformation, protein-protein interactions, and may play a role in environmental sensing), and investigating the composition and functional properties of vesicles (found to facilitate the protected extracellular transfer of a range of molecules including proteins and nucleic acids; evidence is mounting that this process can support cellular housekeeping, offensive and defensive scenarios, horizontal gene transfer, and biofilm formation). Through these studies we are developing models of how certain membrane proteins (e.g. DVU1422, 3104, 1012 and 0799) factor in community interactions, membrane stability, stress response and outer membrane vesicle production. Sequence homologies suggest that many of the findings derived from these efforts may be applicable across a range of bacteria.
  207. Zhang, P., Z. He, J. D. Van Nostrand, L. Wu, D. Curtis, T. C. Hazen, D. E. Elias, M. W. Fields, A. P. Arkin, P. D. Adams and J. Zhou. 2014. Impacts of Environmental Contaminants on Functional Diversity of Groundwater Microbial Communities at a U(VI)-Contaminated Aquifer. ENIGMA SFA Annual Retreat abstract
    Abstract: Microbial diversity in groundwater ecosystems has not been well studied yet. The functional diversity of groundwater microbial communities at heavy metal contaminated sites would be impacted by different geochemical characteristics such as contaminants and pH. As part of the Global 100 Well Survey at the Oak Ridge site, groundwater samples were collected from seven groups of wells with different geochemical characteristics at this site. The first four groups primarily concern the effects of contaminants (U(VI) and nitrate) on microbial diversity: (1) low contaminants (nitrate<2 mg/L and U(VI)<0.01 mg/L) and neutral pH (6.5-7.2), (2) light contaminants (nitrate 4-36 mg/L and U(VI) 0.1-0.2 mg/L) and also neutral pH, (3) moderate contaminants (nitrate 5.5-1471 mg/L and U(VI) 0.1-1.5 mg/L) and neutral pH, and (4) high contaminants (nitrate 2692-11648 mg/L and U(VI) 3.8-55 mg/L) and low pH (3-5.2). The remaining three groups with low contaminants address possible effects of other geochemical characteristics such as groundwater pH: (5) acidic pH (5.2-6.7), (6) high pH (8.9-9.8), and (7) extremely high pH (9.2-10.5). Groundwater microbial communities were analyzed using a comprehensive functional gene microarray (GeoChip 5.0). The results indicated that the groundwater microbial community functional diversity and structure was significantly different among these seven different groups of wells. Such community differences were largely correlated with the differences in U(VI), nitrate, pH, dissolved organic carbon and sulfate in the groundwater. The microbial functional diversity significantly decreased with U(VI) concentrations. Overall, our results suggested that high contaminants and low pH appeared to inhibit the biodiversity and metabolic potential of indigenous groundwater microbial communities. This study improves our understanding of the functional diversity and metabolic potential of groundwater microbial communities across a large scale of contaminant and pH levels at this site.
  208. Alshbli, N., S. M. Techtmann, Y. M. Piceno, L. M. Tom, G. L. Andersen and T. C. Hazen. 2014. 16S rRNA Microarray Analysis of Microbial Communities in Hydrocarbon-Containing Deep-Sea Environments. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
  209. Stelling, S., S. Techtmann and T. C. Hazen. 2014. Comparison of oil degrading Bacteria in the Gulf of Mexico and Eastern Mediterranean Sea. University of Tennessee Exhibition of Undergraduate Research and Creative Achievement (EUReCA) abstract
    At the Department of Energy’s Oak Ridge field site, over 20 years of historical and published data for more than 800 groundwater wells is available in a computer queryable database. In this study, we conducted a survey of 99 groundwater well clusters in order to (1) characterize key microbial populations at geochemically distinct locations, and (2) identify associations between environmental gradients and microbial communities. To optimize geochemical diversity, wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. At each well, in situ groundwater measurements were recorded and unfiltered and filtered groundwater samples were collected for both geochemical measurements and analysis of microbial communities. Nucleic acids were collected by filtering water through a 10.0µm pre-filter and 0.2µm-membrane filter and then extracted using a Modified Miller method. Evaluation of divergence of microbial communities across all the wells indicates the microbial communities are fairly distinct. Comparison of microbial communities within each well shows taxa are not as divergent compared to across all wells. Metadata correlations of all the wells show many of the geochemical parameters are independent of each other. To evaluate potential microbial-geochemical associations, a random forest classification system was used and trained on the OTU abundances to predict continuous values for each geochemical parameter. Results indicate that with careful design and a large dataset, the groundwater microbial community structure can be used to accurately predict the water geochemistry. This project is part of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) Scientific Focus Area at LBNL (http://enigma.lbl.gov).
  210. 2014. 2013 Research Annual Report. “Mitigating Oil Spills”. 2013 Research Annual Report
  211. Hazen, T. C.. 2013. Systems Biology Approach to an Ecological Disaster Deepwater Horizon Oil Spill. UTK Micro310 abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  212. Hazen, T. C.. 2013. Systems Biology Approach to an Ecological Disaster Deepwater Horizon Oil Spill. Clean Gulf 2013 abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  213. Hazen, T. C.. 2013. A Systems Biology Approach to Biotransformation of Heavy Metals and Radionuclides in Groundwater. China‐US Ecopartnership for Environmental Sustainability abstract
    Environmental biotechnology encompasses a wide range of characterization, monitoring and control or remediation technologies that are based on biological processes. Recent breakthroughs in our understanding of biogeochemical processes and genomics are leading to exciting new and cost effective ways to monitor and manipulate the environment. Indeed, our ability to sequence an entire microbial genome in just a few hours is leading to similar breakthroughs in characterizing proteomes, metabolomes, phenotypes, and fluxes for organisms, populations, and communities. Understanding and modeling functional microbial community structure and stress responses in subsurface environments has tremendous implications for our fundamental understanding of biogeochemistry and the potential for natural attenuation or bioremediation of contaminated sites. Monitoring techniques that inventory and monitor terminal electron acceptors and electron donors, enzyme probes that measure functional activity in the environment, functional genomic microarrays, phylogenetic microarrays, metabolomics, proteomics, and quantitative PCR are also being rapidly adapted for studies in environmental biotechnology. Integration of all of these new high throughput techniques using the latest advances in bioinformatics and modeling will enable break-through science in environmental biotechnology. A review of these techniques with examples from field studies and lab simulations for biotransformation of heavy metals and radionuclides in groundwater will be discussed.
  214. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to Oil Contaminated Beaches, Marshes, and Sediment. China‐US Ecopartnership for Environmental Sustainability abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite significant efforts to protect hundreds of miles of beaches, wetlands and estuaries from the Deepwater Horizon oil spill, oil began washing up on the Gulf Coast in early May 2010. We determined the temporal response of the autochthonous microbial communities to the oil on a heavily-impacted beach on Elmer’s Island, Louisiana, Mobile Bay, Alabama, and on Pensacola Beach, Florida. We also studied sediment cores from around the Macondo Well. Analysis of deep 16S rRNA gene pyrotag sequence data revealed that the oil-contaminated samples were dominated by members of the Alpha- and Gammaproteobacteria and that there was a succession in the microbial community over time. Our combined 16S rRNA and metatranscriptome sequence data revealed a rapid response of the natural microbial community to oil contaminants, including prevalence of bacteria with the capacity to degrade oil. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  215. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. China‐US Ecopartnership for Environmental Sustainability abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  216. K. L. Bailey, J. G. Moberly, T. J. Phelps, M. Podar, S. D. Brown, Z. K. Yang, M. M. Drake, T. C. Hazen, A. P. Arkin, A. V. Palumbo and D. A. Elias. 2013. Transcriptomic and Proteomic Analysis of Geobacter sulfurreducens PCA and Desulfovibrio vulgaris Hildenborough Co-cultures. American Society for Microbiology Annual Meeting abstract
    Synthetic community systems consisting of model organisms are essential to elucidating the mechanisms that mediate microbial community structure and function. These microbial communities form ecosystem foundations, drive biogeochemical processes, and are relevant for biotechnology and bioremediation. A model, metal-reducing community was constructed to study microbial cell to cell interactions, cell signaling and competition for resources. Co-cultures of Geobacter sulfurreducens PCA and Desulfovibrio vulgaris Hildenborough grown in the presence and absence of sulfate were grown in 5 L chemostats and physiological and metabolic analyses (GC, HPLC, species specific fluorescent antibody staining) were performed as well as specially designed multispecies microarrays to follow gene expression changes in the various cultivation conditions. Additionally, comprehensive AMT tag proteomics was used for a protein level assessment of the model community. Lactate oxidation by D. vulgaris was sufficient to support G. sulfurreducens via the excretion of H2 and acetate. Fumarate was utilized by G. sulfurreducens and reduced to succinate and malate. Both transcriptomic and proteomic data reveal significant differences amongst/between the syntrophic and competitive growth of these bacteria. Steady state community cultivation coupled to state-of-the-science analyses such as described herein will allow for a comprehensive, system biology level assessment of a metal-reducing microbial community and may further our understanding interspecies communication for syntrophy or direct competition.
  217. C. L. Hemme, Q. Tu, Z. Shi, Y. Qin, J. D. Van Nostrand, L. Wu, Z. He, M. W. Fields, T. C. Hazen, J. M. Tiedje and J. Zhou. 2013. Metagenomic Analysis of Pristine Groundwater Suggests Robust Community Capable of Efficient Geochemical Cycling. American Society for Microbiology Annual Meeting abstract
    To better understand the patterns of nutrient flow in healthy versus stressed groundwater ecosystems, the metagenome of a pristine groundwater community (well FW301) was sequenced and compared to multiple metagenomes including a previously characterized stressed groundwater metagenome FW106 which is chronically exposed to high concentrations of nitric acid and heavy metals. The phylogenetic and metabolic diversity of the pristine groundwater sample was comparable to analyzed temperate soil and surface freshwater metagenomes and was much higher and more diverse than that of the stressed groundwater metagenome. The FW301 community is dominated by proteobacterial species with Pseudomonadales, Burkholdariales and other Betaproteobacterial lineages representing the major phylotypes. FW301 shows robust nitrogen and sulfur cycles complete with ammonia oxidation, nitrification, denitrification, sulfate reduction and sulfur oxidation activities and a capacity for cycling of carbon monoxide and metabolism of aromatic compounds. The FW301 community displays a robust community metabolism that allows for efficient cycling of key elements such as carbon, nitrogen and sulfur. In contrast, the loss of metabolic diversity coupled with inhibitory concentrations of nitrate and sulfate at FW106 has resulted in truncated geochemical cycles in which carbon, nitrogen and sulfur are utilized inefficiently and significant pools of these elements may be lost to the community. The results suggest that the diverse pristine community encodes multiple metabolic and phylogenetic redundancies that allow for rapid adaptation to changing ecological conditions, while the stressed community is highly adapted to the contaminated groundwater environment but likely has a lower capacity to adapt to changing ecological conditions.
  218. J. Huang, A. Pettenato, M. Schicklberger, A. M. Deutschbauer, A. M. Rocha, D. B. Watson, T. C. Hazen, A. P. Arkin and R. Chakraborty. 2013. Physiology Of Nitrate-reducing Anaerobes Isolated From Background And Nitrate-contaminated Groundwater At Oakridge FRC. American Society for Microbiology Annual Meeting abstract
    The U.S. Department of Energy’s Field Research Center (FRC) in Oak Ridge includes 243-acres of contaminated area, the groundwater contains high plumes of uranium, technetium, nitrate, volatile organic compounds and has a pH gradient from 3-10. The goal of this project was to investigate the diversity of nitrate-reducers present across these geochemical gradients. Groundwater samples collected from several wells were incubated at 25°C in the dark under anaerobic conditions with nitrate as electron acceptor. Several compounds including lactate, acetate, glycerol, simple sugars or simple fatty acids were used as carbon and electron donors. More than 50 colonies were picked and identified by 16S-rDNA sequencing. Clonal isolates obtained from the background sites mostly belonged to Pseudomonadales (Gamma-Proteobacteria) or Burkholderiales (Beta-Proteobacteria). Bacterial isolates obtained from the high nitrate-contaminated site mostly belonged to Actinomycetales (Actinobacteria), Neisseriales and Burkholderiales (Beta-Proteobacteria). Few Pseudomonas strains from the background site preferred nitrite over nitrate as the electron acceptor. Phenotypic studies with these isolates show a differential response in their growth rates and ability to reduce nitrate. Intrasporangium calvum strain GW247B1 grew robustly at nitrate concentration of 200mM with lactate as sole carbon and electron source, Sinobacter strain FW507F5 could only grow with 10mM nitrate under the same conditions. Chromobacterium strain FW507F1 also showed growth at nitrate concentrations beyond 180mM and was most versatile in its ability to utilize different carbon compounds as electron donors. Whole-genome sequence of several of these isolates is underway which will provide a better understanding of the predominant nitrate removal mechanisms in these diverse environmental strains.
  219. D. B. Bowen De Leon, B. D. Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen and M. W. Fieldss. 2013. Injection of Nitrate as a Competing Electron Acceptor during Stimulation for Cr(VI) Reduction Alters the Microbial Population in Groundwater and Surrogate Sediment. American Society for Microbiology Annual Meeting abstract
    The Hanford 100-H site is a chromium-contaminated site in which stimulation for Cr(VI) reduction using a polylactate compound resulted in depletion of terminal electron acceptors such oxygen, nitrate, and sulfate, and a significant decrease in soluble Cr(VI). Nitrate, common in DOE metal and radionuclide waste sites, results in denitrification, changes in redox conditions, and inhibition of sulfate-reducing organisms, targeted during stimulation for Cr(VI) reduction. The purpose of this study was to study the microbial population dynamics during a simulated episodic nitrate event to better understand the resilience and sustainability of Cr(VI) reduction. Five days following a lactate injection to stimulate Cr(VI) reduction, 55 gallons of potassium nitrate (5,000 ppm) was injected. The lactate injection resulted in reduction of Cr(VI) to below detection; however, following the nitrate injection, Cr(VI) rebounded to concentrations higher than before lactate injection. Nitrate was consumed or dispersed within 13 days. The planktonic and biofilm microbial populations were monitored by groundwater filtration and surrogate sediment sampling in wells upstream and downstream of the lactate and nitrate injections. Pyrosequencing results demonstrated a drastic shift in the injection well of a shift from metal-reducing organism to denitrifiers in both the planktonic and biofilm communities (namely Thauera and Thiobacillus, respectively). This shift was not observed downstream of the injection, indicating that nitrate was consumed before reaching downstream wells. In the injection well, with the exception of the Thiobacillus increasing to ~60% relative abundance, the sediment community was stable compared to the groundwater community where many of the pre-nitrate dominant members became rare or absent post-nitrate injection. SparCC correlation analyses resulted in highly significant (p < 0.001) and strong (cor ≤ -0.8) negative correlations between sulfate- and metal-reducers and denitrifiers in sediment. This was not observed in groundwater. These data indicate that an episodic nitrate event would result in an increase of Cr(VI) in groundwater and an alteration in the microbial population at or near the point-source.
  220. A. M. Rocha, J. L. Fortney, S. M. Techtmann, D. C. Joyner, T. L. Mehlhorn, J. Earles, K. A. Lowe, D. B. Watson, J. H. Campbell, E. Alm, M. Smith, A. P. Arkin and T. C. Hazen. 2013. Geochemical diversity and microbial-environmental associations of uranium-contaminated groundwater at Oak Ridge field research sites. American Society for Microbiology Annual Meeting abstract
    The physical and geochemical nature of the groundwater environment is important towards understanding the taxonomic, genetic and functional diversity of the microbial communities. Within various groundwater environments, key geochemical transects may provide constraints on microbial activities and community composition. At the Department of Energy’s Oak Ridge field research site, 243-acres of contaminated area is located within the Y-12 plant area of responsibility of the Oak Ridge Reservation. Here, over 800 groundwater wells each containing different geochemical properties are present. The goal of this study is to identify key geochemical transects where microbial communities and activities can be assessed. To maximize the geochemical diversity and to enhance the resolution of microbial-geochemical associations, 100 contaminated groundwater wells containing key geochemical features from were surveyed. Wells were selected using k-medians clustering to group 818 wells into 100 clusters by 14 geochemically similar measurements. Within each cluster, sites for sampling were chosen randomly or by accessibility to the well. For each well in situ groundwater parameters, including temperature, pH, dissolved oxygen, conductivity, and oxidation-reduction potential were measured. Additionally, unfiltered and filtered groundwater samples were collected for geochemical and microbial analysis. Initial analysis of groundwater samples shows a pH and nitrate range of 2.9-10 and 0.09-14,000ppm, respectively, with the lowest nitrate concentrations corresponding to higher pH values (>7). Comparison of background sites show that pH values were similar, ranging 6.5-7.16. However, there are notable differences in nitrate concentrations, with FW301 being ten-fold higher (36.3 ppm) than FW300 (3.65 ppm) and FW303 (3.96 ppm). Analysis of nucleic acids collected from 4L of filtered groundwater, indicated low DNA yields (237-423 ng gDNA) at the background sites. Further analysis of the geochemical and microbial communities across the contaminated wells will provide a better understanding of the geochemical transects present across the field site. These transects will allow for elucidation of relationships between the environment and the genetic and functional diversity of the community.
  221. A. C. Somenahally, J. J. Mosher, Jr. R. A. Hurt, T. J. Phelps, S. D. Brown, M. Podar, A. V. Palumbo, T. C. Hazen, A. P. Arkin and D. A. Elias . 2013. Chromium as a Geochemical Determinant of Microbial Community Structure and Function. American Society for Microbiology Annual Meeting abstract
    Background: As part of a continuing effort to facilitate metal-reduction/remediation with in-situ microbial communities, this study focused on influence of the contaminating metal Chromium on microbial community structure and function. Methods: Metal contaminated groundwater was stimulated with lactate for 15 weeks in duplicate continuous-flow anaerobic reactors with 0, 0.1, or 3 mg/L Cr(VI) simultaneously to give a stable, enriched microbial communities. Temporal analyses included GC, HPLC, 16S pyrosequencing, metal-reduction assays, metal uptake, small metabolites, metagenomics and metatranscriptomics. At the experiment conclusion, the consortia were used to attempt to obtain isolates by traditional serial dilutions or fluorescence-activated cell sorting (FACS). Results: Previous experiments using no Cr(VI) revealed a dominance of Pelosinus spp. within the final community and subsequent isolates were capable of reducing U(VI), CrVI) and Fe(III). Here, temporal metal-reduction showed increased Cr(VI)-reduction rates in chemostats with higher Cr(VI). There were no obvious differences in trends of H2 or CH4 and genomic analyses are ongoing. Conclusions: Cultivation and metabolic characterization of organisms from a stable, enriched community allows for a deeper understanding of the community metabolism as a whole. Comprehensive investigations such as these allow the evaluation of remediation strategies and identify which community members are important for bioremediation.
  222. S. M. Techtmann, J. L. Fortney, D. C. Joyner, A. M. Rocha, T. D. Linley and T. C. Hazen. 2013. Hydrocarbon Degrading Bacteria in the Warm Oligotrophic Deep Eastern Mediterranean. American Society for Microbiology Annual Meeting abstract
    Very large reservoirs of oil and gas have recently been discovered in the Eastern Mediterranean, making this area an important developing region for oil and gas prospecting. Many of the physical properties of the Eastern Mediterranean are unique compared to other deep-sea hydrocarbon basins. The Mediterranean is very deep with an average depth of 2500 m. Additionally, the water temperature in the deepest parts of the Mediterranean is very warm (between 12.5 and 14.5°C). In contrast, the temperature of Atlantic waters at similar latitudes and comparable depths are typically below 4°C. Furthermore, the Eastern Mediterranean has been reported to be extremely oligotrophic with a N:P ratio of 27.4 (typically N:P ratios are <16). All of these factors will affect the microbial community structure and may potentially select for a unique complement of oil-degrading microbes in these deep warm waters. The oil-degrading capacity of the microbial community in the deep Eastern Mediterranean is unknown. If a spill of crude oil were to occur in the Eastern Mediterranean, the proper approach for using bioremediation to assist in spill clean up is unclear due to the unique characteristics of this environment. To that end, the hydrocarbon degrading potential of the microbial community from various sites in the Eastern Mediterranean was examined. Lab microrespirometry experiments were performed to examine the effect of oil, dispersant, and various nutrient amendments on oil biodegradation. The addition of oil and oil plus COREXIT 9500 both stimulated respiration. Microbial cell numbers increased in the oil and oil plus dispersant treatments. The effect of these treatments on the microbial community structure is currently being examined using molecular techniques. A variety of hydrocarbon degrading isolates were also obtained from the Eastern Mediterranean waters sampled in this study. Furthermore, molecular techniques are being employed to probe both the phylogenetic diversity and functional diversity of the native microbial community and the hydrocarbon degraders present. This study will shed light on the natural microbial community structure of the Eastern Mediterranean and will help to clarify how the unique physicochemical parameters of the Eastern Mediterranean could affect the microbial response to an oil spill.
  223. S. Yilmaz, Y. K. Light, R. J. Meagher, T. C. Hazen, A. P. Arkin and A. K. Singh. 2013. Single-cell Analysis Platforms for Uncultivable Microorganisms. American Society for Microbiology Annual Meeting abstract
    Single cell sequencing is emerging as a powerful tool for the analysis of uncultivated environmental microorganisms. Current culture-independent, population based techniques (i.e., metagenomics) relying on pooled nucleic acids from lysed bacteria can independently measure metabolic activity and the species present, but cannot link the activity deterministically to species. We are developing high-throughput tools for studying microorganisms one cell at a time, allowing us to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities. Our approach includes FISH-based identification of desired species, enrichment by cell sorting, followed by single-cell encapsulation, whole genome amplification and sequencing. We are utilizing this pipeline to analyze water samples from DOE bioremediation sites (e.g., Oak Ridge FRC) to identify keystone organisms and link their functions to species; to analyze the oral microbiome for targeted sequencing of rare species; to analyze human gut communities to estimate the level of horizontal gene transfer within the community; and to analyze an evolving coculture to determine the genetic, metabolic, and structural basis of the evolution of new social networks that are more efficient, stable, and productive than ancestral networks.
  224. P. Zhang, R. Chakraborty, J. Van Nostrand, Z. He, D. Curtis, Y. Deng, T. C. Hazen, A. Arkin and J. Zhou. 2013. Diversity of Microbial Functional Communities during Long-term Cr(VI) Immobilization Stimulated with a Slow-release Substrate in the Hanford Aquifer. American Society for Microbiology Annual Meeting abstract
    The sustainability of reduction has been one of the greatest interests during design of in-situ subsurface strategies for bioremediation of toxic metals. A one-time amendment of slow-release glycerol polylactate (HRCa) stimulated long-term bioreduction of Cr(VI) at the DOE Hanford site. To understand the microbial functional communities that mediated this dramatic Cr(VI) immobilization, a comprehensive functional gene array was used to analyze the diversity and changes of groundwater microbial communities after HRCa. The results showed that the abundance of a wide diversity of functional genes significantly increased after HRCa. HRCa first stimulated various genes for HRC acetogenesis, bacteriophage replication and lysis, and degradation of microbial biomass. Also, HRCa stimulated most genes involved in reduction pathways of nitrate, Fe(III), Cr(VI), and sulfate. Additionally, many genes for retaining of C, N, S, P nutrients (e.g. N2 and CO2 fixation) in the ecosystem, Cr(VI) resistance, and transformations of organic and metal contaminants were highly enriched. These genes increased immediately after HRCa and were further enriched after one year, resulting in a dramatically altered community functional structure, which exhibited enhanced physiological ecological potential of better utilizing C, N, P, S nutrients, Cr(VI) reduction and resistance, and adaption to the contaminated environment. These community composition and structure changes were closely correlated with levels of electron donors, Cr(VI), Fe(II), sulfate, and nitrate in the groundwater. Cr(VI) reduction appeared mediated enzymatically and indirectly through Fe(II) and H2S production, and Fe(III)-, sulfate-, and nitrate-reducing bacteria could play important roles. This study provides insights into the diversity of microbial functional communities and geochemical factors important to long-term Cr(VI) immobilization in-situ.
  225. J. Zhou, Y. Deng, P. Zhang, K. Xue, J. D. Van Nostrand, Y. Yang, Z. He, D. A. Stahl, T. C. Hazen, J. M. Tiedje and A. P. Arkin . 2013. Stochasticity, Succession and Environmental Perturbations in Fluidic Ecosystems. American Society for Microbiology Annual Meeting abstract
    Unraveling the drivers of community structure, succession, and resilience in response to environmental change is a central issue, but poorly understood in ecology. Although the relative importance of ecological processes (deterministic vs stochastic) in shaping community structure is intensively examined, very little is known about the mechanisms controlling ecological succession, a phenomenon that a community undergoes more or less orderly and predictable changes following disturbance. To understand the relative importance of stochastic and deterministic processes in mediating microbial community succession, here, we examined the responses of a groundwater microbial community to a large change in carbon resource, the addition of slow-release emulsified vegetable oil (EVO) for sustained uranium immobilization at a contaminated site. A novel general theoretical framework was developed to conceptualize the relationships between stochasticity, succession and environmental perturbations. Our results revealed that the groundwater microbial communities diverged substantially after EVO amendment, but subsequently converged to new community structures very similar to, but distinct from, the initial states, indicating the resilience and adaptation of the groundwater microbial communities. Consistent with the proposed conceptual framework but contradictory to the conventional wisdom, null model analyses revealed that the community succession responding to EVO amendment was primarily controlled by stochastic rather than deterministic processes. To our knowledge, this is the first study to explicitly demonstrate the resilience of microbial communities at a field scale and the importance of stochastic processes in mediating ecological succession. Elucidating the mechanisms controlling community structure, succession and resilience is fundamental to biodiversity preservation, ecosystem restoration and environmental management.
  226. Terry C. Hazen. 2013. Big Idea Professor Develops ‘SuperChip’ to Speed Up Lab Results. Tennessee Today
  227. Techtmann, S. and T. C. Hazen. 2013. Can Mother Nature Take a Punch? The Science of the Big Gulf Oil Spill. Harden Valley Academy
  228. Hazen, T. C.. 2013. A Systems Biology Approach to Understanding Metal/Radionuclide Contaminated Sites. RemTEC annual meeting
  229. S. M. Techtmann, J. L. Fortney, D. C. Joyner, A. M. Rocha, T. D. Linley and T. C. Hazen. 2013. Hydrocarbon Degrading Bacteria in the Warm Oligotrophic Deep Eastern Mediterranean. Second International Symposium on Bioremediation and Sustainable Environmental Technologies abstract
    Very large reservoirs of oil and gas have recently been discovered in the Eastern Mediterranean, making this area an important developing region for oil and gas prospecting. Many of the physical properties of the Eastern Mediterranean are unique compared to other deep-sea hydrocarbon basins. The Mediterranean is very deep with an average depth of 2500 m. Additionally, the water temperature in the deepest parts of the Mediterranean is very warm (between 12.5 and 14.5°C). In contrast, the temperature of Atlantic waters at similar latitudes and comparable depths are typically below 4°C. Furthermore, the Eastern Mediterranean has been reported to be extremely oligotrophic with a N:P ratio of 27.4 (typically N:P ratios are <16). All of these factors will affect the microbial community structure and may potentially select for a unique complement of oil-degrading microbes in these deep warm waters. The oil-degrading capacity of the microbial community in the deep Eastern Mediterranean is unknown. If a spill of crude oil were to occur in the Eastern Mediterranean, the proper approach for using bioremediation to assist in spill clean up is unclear due to the unique characteristics of this environment. To that end, the hydrocarbon degrading potential of the microbial community from various sites in the Eastern Mediterranean was examined. Lab microrespirometry experiments were performed to examine the effect of oil, dispersant, and various nutrient amendments on oil biodegradation. The addition of oil and oil plus COREXIT 9500 both stimulated respiration. Microbial cell numbers increased in the oil and oil plus dispersant treatments. The effect of these treatments on the microbial community structure is currently being examined using molecular techniques. A variety of hydrocarbon degrading isolates were also obtained from the Eastern Mediterranean waters sampled in this study. Furthermore, molecular techniques are being employed to probe both the phylogenetic diversity and functional diversity of the native microbial community and the hydrocarbon degraders present. This study will shed light on the natural microbial community structure of the Eastern Mediterranean and will help to clarify how the unique physicochemical parameters of the Eastern Mediterranean could affect the microbial response to an oil spill.
  230. Hazen, T. C. and R. Prince. 2013. Marine Oil Spills. Second International Symposium on Bioremediation and Sustainable Environmental Technologies
  231. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. CSIRO Seminar abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  232. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. CSIRO Seminar abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  233. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Harbin Institute of Technology Seminar abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite spanning more than 200m in the water column and extending more than 10 km from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  234. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to Oil Contaminated Beaches, Marshes, and Sediment. Institute of Soil Science, Chinese Academy of Sciences, China‐US Ecopartnership “Frontiers in Environmental Research” abstract
    The explosion on April 20, 2010 at Deepwater Horizon drilling rig in the Gulf of Mexico resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, and in the dispersment of an oil plume 1,500m below the surface of the water. Despite significant efforts to protect hundreds of miles of beaches, wetlands and estuaries from the Deepwater Horizon oil spill, oil began washing up on the Gulf Coast in early May 2010. We determined the temporal response of the autochthonous microbial communities to the oil on a heavily-impacted beach on Elmer’s Island, Louisiana, Mobile Bay, Alabama, and on Pensacola Beach, Florida. We also studied sediment cores from around the Macondo Well. Analysis of deep 16S rRNA gene pyrotag sequence data revealed that the oil-contaminated samples were dominated by members of the Alpha- and Gammaproteobacteria and that there was a succession in the microbial community over time. Our combined 16S rRNA and metatranscriptome sequence data revealed a rapid response of the natural microbial community to oil contaminants, including prevalence of bacteria with the capacity to degrade oil. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico.
  235. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Gulf of Mexico Oil Spill and Ecosystem Science Conference http://gulfofmexicoconference.org
  236. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Clemson University
  237. 2013. UT experts: BP oil spill gone from deep ocean, but remains in marshes . Knoxville Sentinel
  238. Hazen, T. C.. 2013. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. ACS annual meeting
  239. 2013. Gulf of Mexico Has Greater-Than-Believed Ability to Self-Cleanse Oil Spills. VibesForum
  240. K. L. Bailey, J. G. Moberly, T. J. Phelps, A. M. Rocha, H. Woo, M. Podar, S. D. Brown, Z. K. Yang, M. M. Drake, T. C. Hazen, A. P. Arkin, A. V. Palumbo and D. A. Elias. . 2013. Transcriptomic and Proteomic Analysis of Geobacter sulfurreducens PCA and Desulfovibrio vulgaris Hildenborough Co-cultures. 1st Annual Postdoc Research Symposium, Oak Ridge National Laboratory
  241. A. M. Rocha, M. Smith, C. Smillie, J. L. Fortney, S. M. Techtmann, D. C. Joyner, T. L. Mehlhorn, J. E. Earles, K. A. Lowe, D. B. Watson, J. H. Campbell, E. Alm, A. P. Arkin and T. C. Hazen. 2013. Global Survey of the Department of Energy’s Oak Ridge Field Research Site. 1st Annual Postdoc Research Symposium, Oak Ridge National Laboratory
  242. 2013. Deepwater Horizon Gulf of Mexico 'deep-cleaned' itself . GlasgowWired
  243. 2013. Deepwater Horizon Gulf of Mexico 'deep-cleaned' itself. LiverpoolWired
  244. 2013. Deepwater Horizon Gulf of Mexico 'deep-cleaned' itself. BBC News
  245. 2013. Deepwater Horizon: Gulf of Mexico 'deep-cleaned' itself. EdinburghWired
  246. 2013. Deepwater Horizon: Gulf of Mexico ‘deep-cleaned” itself. Random News
  247. 2013. DID THE GULF OF MEXICO SELF-CLEAN AFTER DEEPWATER DISASTER?. FastCompany
  248. 2013. Environment Is the Gulf of Mexico resilient to oil spills? . Summit County Citizens Voice
  249. 2013. An explosion in oil-munching bacteria made fast work of BP oil spill, scientist says. DemocraticUnderground
  250. 2013. An explosion in oil-munching bacteria made fast work of BP oil spill, scientist says. The Times Picayune
  251. 2013. Gulf Of Mexico Bacteria Found To Break Down And Consume Crude Oil. RedOrbit
  252. 2013. Gulf of Mexico can ‘self-deep-clean’. AfroEdge
  253. 2013. Gulf of Mexico Can Self-Cleanse Oil Spills. Laboratory Equipment
  254. 2013. Gulf of Mexico cleans itself naturally from Deepwater Horizon blowout. Ottawa Citizen
  255. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. e! Science News
  256. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. Bartle Doo
  257. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. Science Codex
  258. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. Phys.Org.
  259. 2013. Gulf of Mexico Has Greater-Than-Believed Ability to Self-Cleanse Oil Spills. ScienceDaily
  260. 2013. Gulf of Mexico has greater-than-believed ability to self-cleanse oil spills. American Chemical Society
  261. 2013. Gulf of Mexico Has Surprising “Innate” Ability to Cleanse Oil Spills. Softpedia
  262. 2013. Microbes ate Macondo oil in Gulf of Mexico . UPI
  263. 2013. Nature’s Key Gulf Cleanup Role After Oil Spill Touted by Researchers. NewsMax
  264. 2013. Oceans clean themselves. Leader-Post
  265. 2013. Oil From Deepwater Horizon Spill Broken Down By Hungry Ocean Bacteria, Researcher Says . Nature World News
  266. 2013. Oil-Eating Bacteria Fixed The Deepwater Horizon Disaster, But They May Not Help Next Time. FastCoexist
  267. 2013. ORNL’s Keller, Babu, Hazen elected AAAS fellows. Oak Ridge Today
  268. 2013. Radio Interview. Deepwater Horizon: Gulf of Mexico 'deep-cleaned' itself. BBC Radio Live 5
  269. 2013. Research shows Gulf of Mexico resilient after spill. R&D Magazine
  270. 2013. Self-Healing. The k2p blog
  271. 2013. Study Oil-Eating Bacteria Mitigated Deepwater Horizon Oil Spill. BrothersJudd
  272. 2013. Study Oil-Eating Bacteria Mitigated Deepwater Horizon Oil Spill. US News and World Report
  273. 2013. Top Thirteen “In the News” Stories for 2013. Tennessee Today
  274. 2013. TV Interview. Gulf of Mexico Has Surprising “Innate” Ability to Cleanse Oil Spills. CNN TV
  275. 2013. UT Professor's Research Shows Gulf of Mexico Resilient After Spill. Tennessee Today
  276. Terry C. Hazen. 2013. Gulf of Mexico Has Surprising “Innate” Ability to Cleanse Oil Spills. CBS Radio
  277. Hienz, W.. 2013. Four Faculty Named AAAS Fellows. Tennessee Today
  278. Kemsley, J. 2013. After The Deepwater Horizon Disaster. Chemical and Engineering News
  279. Hazen, T. C.. 2012. A Systems Biology Approach to the Deepwater Horizon Oil Spill, the 2nd largest marine oil spill in the world. South Central China University abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
  280. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Key Lab on Pollution Ecology & Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
  281. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. The 2012 China-US Joint Symposium “Land Use, Ecosystem Services, and Sustainable Development” abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
  282. Hazen, T. C.. 2012. Deepwater Horizon Oil Spill Ecological Disaster, Engineering Disaster, Science of Opportunity!!!. University of Tennessee, College of Engineering, Board of Advisors
  283. Hazen, T. C.. 2012. Cover The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Environmental Microbiology 14.
  284. Hazen, T. C.. 2012. Dispersants and Oil, What We Learned from the Deepwater Horizon Disaster. University of Tennessee, Earth and Planetary Sciences Seminar
  285. Hazen, T. C.. 2012. Dispersants and Oil, What We Learned from the Deepwater Horizon Disaster.. Rice University abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
  286. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. Wake Forest University, Perspectives in Biology Symposium
  287. Hazen, T. C.. 2012. Science and the Media: perspectives for the Scientist. Wake Forest University, Perspectives in Biology Symposium
  288. Hazen, T. C.. 2012. The Deepwater Horizon Oil Spill A Systems Biology Approach to an Ecological Disaster. AGU
  289. DeAngelis, K., C. Dylan, S. Blake, T. C. Hazen and S. Whendee. 2012. Shifting dynamics of bacteria and fungi during litter decomposition in wet tropical forest soils. International Symposium of Microbial Ecology (ISME 14) abstract
    The rates of decomposition in wet tropical forest soils are extremely fast compared to other biomes, with rapid turnover of labile carbon pools. Fast rates of decomposition are curious considering the low and fluctuating redox potentials. Oxygen limitation coupled to high rates of decomposition suggests that anaerobic or facultative decomposing bacteria are prevalent and active decomposers in tropical forest soils, but the communities involved are poorly understood. This study was intended to test the hypothesis that litter decomposition rates and communities would vary across a rainfall gradient in the Luquillo Experimental Forest LTER in Puerto Rico USA. We employed large-scale microbial community sequencing in combination with measurements of enzyme rates and chemistry of decomposing switchgrass litter and adjacent soil samples over a two-year period at 1, 4, 13, 30, 60 and 96 weeks. The experiment was designed to capture both short- and long-term temporal trends in decomposition, as well as evaluate the roles of rainfall (low and high elevation) and soil oxygen availability (low and high redox) among four sites in the Luquillo Experimental Forest. The low elevation sites had the lowest C:N ratios after two years and the most mass lost. Though the high elevation, low redox site had the least mass lost, it also had the highest litter enzyme activity. Partial small subunit ribosomal RNA genes were sequenced with universal primers for bacteria, archaea and eukaryotes, yielding 40,850 unique taxa after quality filtering, dereplication and clustering. Community profiles based on phylogenetic (UniFrac) distance showed that during early decomposition (weeks 1-13), bacterial litter communities were distinct from soil, while fungal soil and litter communities were similar; during later decomposition (weeks 30-96), bacterial soil and litter communities were indistinguishable while fungal litter communities were distinct from soil. A relatively small set of dominant taxa was highly enriched on litter (>1% relative abundance) during early-stage decomposition, including Beta- and Gammaproteobacteria, Firmicutes and Stramenopiles. Fungi appeared among the dominant taxa after 60 weeks decomposition. Richness estimates revealed that in the high elevation sites, litter selected for a much less diverse community compared to soil. Measures of phylogenetic structure showed that bacteria were more clustered in soil than litter, while the opposite was true for fungi. This suggests that net negative interactions (like competition or predation) are involved to bacterial litter colonization, while net positive interactions (like phenotypic attraction or spatial isolation) are involved in fungal litter colonization. Taken together, these data reveal a succession of fungi, bacteria, and their likely eukaryotic predators that conspire to accomplish decomposition under low and fluctuating redox conditions, as well as strategies for microbial decomposition in low redox potential tropical forest soils.
  290. Piceno, Y., L. Tom, F. Reid, S. Borglin, J. Fortney, D. Joyner, A. Pettenato, T. C. Hazen, C. Spier, W. Stringfellow, J. Wong and G. Andersen. 2012. Microbial community structure differences associated with elevated hydrocarbon concentrations in sediment near and far from the Deepwater Horizon MC-252 wellhead. International Symposium of Microbial Ecology (ISME 14) abstract
    The Deepwater Horizon oil spill in the Gulf of Mexico released large amounts of oil into the water column and may have affected sediments nearby. Sediment cores were collected at distances from less than 1 km to over 250 km from the MC-252 wellhead during September-October 2010. Cores were sectioned at 0-1 cm and 4-5 cm. DNA was extracted, amplified for 16S rRNA genes, and analyzed by PhyloChip. Hydrocarbon concentrations were measured in adjacent cores. Microbial communities in the 0-1 cm horizon in cores with elevated hydrocarbon concentrations were hypothesized to have greater relative abundances of microbes associated with hydrocarbon degradation. Such differences were not predicted for the 4-5 cm horizon communities. For 0-1 cm sediment horizons, results suggested moderate differences in PhyloChip profiles between sample groups. Families with increased relative abundances in the samples with the highest hydrocarbon concentrations were Rhodobacteraceae, Flavobacteraceae, Desulfobulbaceae, Helicobacteraceae, Lachnospiraceae, and Campylobacteraceae. Members of some ofthese families (e.g., Sulfitobacter, Polaribacter, and Arcobacter) have been found in seawater amended with crude oil, while others (e.g., Sulfurovum and members of the Desulfobulbaceae) have been proposed to grow syntrophically under benzene-degrading conditions. The families with higher relative abundances in samples with low or no hydrocarbons were Acidobacteriaceae, Rhodospirillaceae, various Deltaproteobacteria (e.g., Syntrophobacteraceae), Sinobacteraceae, and Chromatiaceae. As hypothesized, there was no evidence of differences in the 4-5 cm horizon. These results suggest the effects of elevated hydrocarbon concentrations on the microbial community were primarily restricted to the upper few cm of the sediment.
  291. Terry C. Hazen. 2012. A Systems Biology Approach to the Deepwater Horizon Oil Spill. University of Aberdeen, OceanLab
  292. De Leon, K. B., B. D. Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen and M. W. Fields. 2012. Microbial population dynamics in groundwater and surrogate sediments during HRC® biostimulation for Cr(VI)-reduction. International Symposium of Microbial Ecology (ISME 14) abstract
    The Hanford 100-H site is a chromium-contaminated site that has been designated by the Department of Energy Environmental Management as a field study site for in situ chromium reduction. In August 2004, the first injection of hydrogen release compound (HRC®) resulted in an increase of microorganisms and a reduction of soluble chromium(VI) to insoluble chromium(III). Little is understood about the microbial community composition and dynamics during stimulation. The aim of this study is to compare microbial communities of groundwater and soil samples across time and space during a second injection of HRC®. A second injection occurred November 2008 and geochemical data collected throughout the study showed an overall decrease in nitrate, sulfate, and chromium(VI). Spatial and temporal water and soil samples (n=34) were collected pre-and post-injection from four wells at the field site. Soil columns constructed from stainless steel mesh were lined with nylon mesh and filled with Hanford soils from the 100-H site. The soil columns were used to represent not only the microbes flowing through the soil via groundwater, but the microbes that require a matrix in order to grow. DNA was extracted from each of the samples and the V1V2 region of the 16S rRNA gene was sequenced via multiplex pyrosequencing. Soil sample populations differed from the corresponding groundwater (even at the phyla level) and were more diverse (p=0.001). While many of the populations were observed in both groundwater and surrogate sediments, the respective matrices appeared to enrich for particular OTUs. Of 667 total genera, 141 and 69 were unique to groundwater and soil, respectively. Genera observed only in the sediment included Marinomonas while genera observed only in the groundwater included Desulfonauticus, Desulfomicrobium, and Syntrophobacter. Pseudomonas, Acidovorax, Clostridium, and Herbaspirillum were dominant regardless of sample type. Results do not indicate a large shift in dominant organisms in soil from pre- to post- injection, and this may be due to the organisms remaining dominant from the first stimulation. Correlation analyses of genera were done for each sample type using SparCC. Metal-reducing organisms such as Geobacter, Desulfovibrio, and Geothrix were correlated in soil while possible fermenting bacteria such as Clostridium, Pelotomaculum, and Pelosinus were correlated in groundwater. For each well, HRC® injection resulted in increased diversity, but the greatest changes during stimulation occurred in the populations of mid-dominance either between wells or across time. These organisms could be important to consider as possible indicator species in future work that includes targeted isolations to better understand the mechanisms of microbial interactions.
  293. Goodheart, D., T. C. Hazen and M. K. Firestone. 2012. Deconstructing the Microbial Community Degrading Plant Material in a Wet, Tropical Forest. International Symposium of Microbial Ecology (ISME 14) abstract
    The lower montane tropical forest in the Luquillo Experimental Forest in Puerto Rico has the highest litter decomposition rate measured to date. This soil undergoes frequent oxic/anoxic fluctuations lasting from days to weeks, caused by rain events. The persistent fluctuating environmental condition in the Luquillo Experimental Forest creates two disparate conditions (oxic and anoxic) under which distinct metabolic processes function. The persistent fluctuation in the environmental conditions could enable the rapid litter decomposition by coupling the oxic and anoxic decomposition processes. The goals of this research are to: i) determine the abundance and identity of the bacterial and fungal communities degrading different components of plant material (whole lignocellulose, cellulose and lignin) and ii) assess the metabolic potential of the microbial community under each environmental condition and substrate in a wet, tropical forest soil. To address these goals, soil from the Luquillo Experimental Forest was used in microcosm studies with 13C-labeled plant material added (either Avena barbata -whole lignocellulose analog, cellulose, or vanillin-lignin analog) under either continuous oxic, continuous anoxic or four day oxic/anoxic fluctuation. Gas samples were taken and triplicate Sample (with substrate) and Control (no substrate) soil jars were destructively sampled over time. DNA was extracted and used for qPCR of the bacterial 16S and the fungal ITS, 16S pyrotags and GeoChip 4 analyses. As expected, cumulative CO2 and CH4 were greatest in the oxic and anoxic conditions, respectively, for each substrate. The cumulative CO2 and CH4 from the fluctuating condition of each substrate were similar to the cumulative CO2 and CH4 of the oxic condition. Fungal abundance increased under oxic conditions and dramatically decreased under anoxic conditions with cellulose as the substrate, suggesting fungi are the main agents of decomposition under oxic conditions for cellulose in this soil. The abundance of bacteria does not change in response to environmental condition. However, 16Spyrotag data shows that the structure of the bacterial community altered in response to the environmental condition and substrate. We used the GeoChip to assess the functional capacity of the microbial community under each environmental condition and substrate. The relative abundances of carbon degradation genes altered in response to environmental condition and substrate. In particular, cellulases and hemicellulases showed the greatest changes in relative abundance due to the presence of substrate and environmental conditions, respectively. The abundance, structure and metabolic capacity of the microbial community altered in response to both the environmental conditions and the substrate. This dynamic response could drive the observed rapid decomposition rate in this wet, tropical forest soil.
  294. Terry C. Hazen. 2012. Metagenomics and the Environment. Society of Industrial Microbiology Annual Meeting
  295. Lamendella, R., S. E. Borglin, R. Chakraborty, T. C. Hazen, J. K. Jansson. 2012. Microbial Community Dynamics on an oil contaminated beach following the Deepwater Horizon Oil Spill. Society of Industrial Microbiology Annual Meeting abstract
    Despite significant efforts to protect hundreds of miles of beaches, wetlands and estuaries from the Deepwater Horizon oil spill, oil began washing up on the Gulf Coast in early May 2010. In this study, we aimed to determine the temporal response of the autochthonous microbial communities to the oil on a heavily-impacted beach on Elmer’s Island, Louisiana. Analysis of deep 16S rRNA gene pyrotag sequence data revealed that the oil-contaminated samples were dominated by members of the Alpha- and Gammaproteobacteria and that there was a succession in the microbial community over time. We also sequenced RNA extracted from the samples to determine which microbes and functions were active. Our combined 16S rRNA and metatranscriptome sequence data revealed a rapid response of the natural beach microbial community to oil contaminants, including prevalence of bacteria endowed with the functional capacity to degrade oil. By correlation to hydrocarbon data from the same samples, we determined that the oil originating from the Deepwater Horizon oil spill underwent further degradation by indigenous microbial consortia on the beach.
  296. Gilbert, D.. 2012. Waves of Berkeley Lab responders deploy omics to track Deepwater Horizon cleanup microbes. EurekAlert http://www.eurekalert.org/pub_releases/2012-06/dgi-wob061512.php
  297. 2012. Business Bulletins. KnoxvileBiz.com
  298. Terry C. Hazen. 2012. Can Mother Nature Take a Punch The Deepwater Horizon Incident. Technical Society of Knoxville
  299. Hazen, T. C. 2012. Fate and distribution of Deepwater Horizon oil. University of Wisconsin at Milwaukee
  300. Zhang, P., W-M. Wu, J. Van Nostrand, Y. Deng, Z. He, D. Curtis, T. Gihring, G. Zhang, C. Schadt, D. Watson, P. Jardine, C. Criddle, S. Brooks, T. Marsh, J. Tiedje, T. C. Hazen, J. Zhou. 2012. Dynamic Changes of Microbial Communities in Response to Stimulation with Emulsified Vegetable Oil for U(VI) Reduction at a Contaminated Aquifer. Annual Meeting of the American Society for Microbiology abstract
    To examine the microbial functional communities stimulated with a slow-biodegrading electron (e) donor for sustainable U(VI) reduction, a one-time injection of emulsified vegetable oil (EVO) was conducted at a U(VI)-contaminated aquifer. Groundwater samples were collected from an up-gradient control well and seven down-gradient wells (W1-7) to monitor functional gene changes of indigenous microbial communities over a 269-day period using GeoChip-based metagenomics technology. Acetate was detected as one of the intermediates from EVO biodegradation that stimulated bioreduction of NO3-, Mn(IV), Fe(III), SO42-, and U(VI) for at least 140 days in W1-7. During this period, the functional gene diversity increased, with significant enrichments of genes involved in acetogenesis, methanogenesis, methane oxidation, denitrification, dissimilatory nitrate reduction, metal reduction, and sulfate reduction, while the community in the control well did not show a significant change. Fe(III)- and sulfate-reducing bacteria, including Geobacter, Anaeromyxobacter, Desulfovibrio and Desulfotomaculum, could play key roles in the sustainable U(VI) reduction, whereas acetogens, denitrifiers and methanotrophs could be important for the production of e-donors and maintaining reducing conditions favorable for U(VI) reduction. After EVO was depleted, the functional gene diversity declined and became more similar to the pre-injection levels by day 269. Dynamic changes of the functional microbial communities were highly correlated with changes of acetate, NO3-, Mn, Fe, SO42-, and U(VI). This study provides the first in-situ evidence that a one-time injection of a slow-biodegrading e-donor effectively stimulated functional communities and sustainable U(VI) reduction. The dynamic changes of the communities were primarily associated with the availability of the e-donor and e-acceptors in the aquifer.
  301. Baidoo, E. E., S. Yilmaz, J. Geller, T. C. Hazen, A. K. Singh, J. D. Keasling. 2012. Differential Analysis of Metabolic Intermediates from Desulfovibrio vulgaris Hildenborough and Methanococcus maripaludis under Syntrophic Growth Conditions. Annual Meeting of the American Society for Microbiology abstract
    Metabolite profiling experiments are normally conducted on the monoculture of microorganisms. However, microorganisms in their natural habitats rarely live alone as they are part of ecosystems that are largely made up of microbial communities forming a network that is responsible for processing and exchanging material. Initial steps towards characterizing the metabolisms of individual microorganisms from their respective communities are being performed at the most basic level in the form of laboratory co-culture experiments. In general, metabolite concentrations from the co-culture are compared to the concentrations of the monoculture. However, monoculture conditions are not a true reflection of the natural environment; and although co-culture studies facilitate accurate characterization of the metabolisms of syntrophic partners, metabolite profiling still has some major challenges. The greatest being the determination of the origin of each metabolite. One way of achieving this is to physically separate the co-culture prior to metabolite analysis. For this purpose, we have designed a dual chamber bioreactor in which the chambers containing monocultures of Desulfovibrio vulgaris Hildenborough (D. vulgaris) and Methanococcus maripaludis (M. maripaludis) are separated by a membrane filter that allows the free passage of nutrients from one population to another. In the absence of sulfate, sulfate reducing bacteria (SRB) such as D. vulgaris species ferment organic acids and alcohols, producing hydrogen, acetate and carbon dioxide. Under these conditions they are reliant upon hydrogen and acetate-scavenging methanogens to convert the aforementioned compounds to methane. This syntrophy increases the chances of survival of SRBs in environments that are limited in electron acceptors. The metabolic versatility of D. vulgaris is highlighted by the complexity of energy metabolism in this SRB and, hence, requires further study. To this end, a combination of LC-MS and CE-MS technologies are being used to provide metabolite data for the accurate characterization of the central metabolisms of D. vulgaris and M. maripaludis under syntrophic growth conditions. Further, this approach is being used to determine metabolite exchange between the organisms.
  302. Butland, G. P., S. R. Chhabra, B. Gold, N. L. Liu, S. Reveco, T. R. Juba, J. D. Wall, B. R. Lam, J. T. Geller, T. C. Hazen, M. Choi, M. D. Biggin, E. D. Szakal, S. Allen, H. Liu, H. E. Witkowska, J‐M. Chandonia. 2012. High Throughput Identification of Protein Complexes from Desulfovibrio vulgaris by a Tandem Affinity Purification Pipeline. Annual Meeting of the American Society for Microbiology abstract
    Desulfuvibrio vulgaris is a sulfate reducing bacterium (SRB) found in DOE sites contaminated with heavy metals or radionuclides and can reduce many of the contaminants to an insoluble form. Environmental change or human intervention can alter the chemical environment in the subsoil, which in turn affects which species predominate as well as microbial physiology. It is therefore critical to learn how such changes affect SRBs and their interaction with other members of the community. We have chosen Desulfovibrio vulgaris to address these questions in molecular detail as it is one of the dominant SRBs found at DOE sites. Most cellular processes in D. vulgaris are mediated by multiple proteins interacting with each other in the form of multi-protein complexes and not by individual proteins acting in isolation. In order to accurately model cellular processes in this organism and its response to stress, our goal is to develop a comprehensive knowledgebase of protein complexes and protein-protein interactions using high throughput tandem affinity purification (TAP). Currently, we have generated 1588 unique TAP-tagged strains, of which 866 with correctly integrated TAP-tagged chromosomal fusions have been subject to TAP analysis and have had their composition analyzed by mass spectrometry. In ~80% of these analyses, the bait was verified to be present by gel-free mass spectroscopy. Despite many bottlenecks including larger culture volumes and strict anaerobiosis associated with working with D. vulgaris, we have obtained throughput statistics, data quality and success rates similar to those previously reported for E. coli. Detected high-confidence interactions cover a range of biological processes including energy conservation, protein secretion, protein folding, and cofactor biosynthesis with both novel and previously predicted interactions. We are now in a position to conduct a system-wide analysis of all stable protein-protein interactions in D. vulgaris and to target how these change in response to stresses typically occurring in the subsoil of contaminated sites for a select set of stress response genes.
  303. Chakraborty, R., Y. M. Piceno, F. C. Reid, S. E. Borglin, E. A. Dubinsky, L. M. Tom, T. C. Hazen, G. L. Andersen. . 2012. Microbial Community Structure and Hydrocarbon Degradation by Isolates Obtained from Different Depths in the Aftermath of the Deepwater Horizon Spill in the Gulf of Mexico. Annual Meeting of the American Society for Microbiology abstract
    The deepwater horizon spill resulting from the exploded drilling rig released over 3 million barrels of crude oil into the Gulf of Mexico. Bio-Sep beads amended with volatile hydrocarbons from MC-252 oil were used to create hydrocarbon-amended traps for attracting oil-degrading microbes in situ. Traps were placed on a drilling rig about 600-m from the original MC-252 oil spill site at four depths: 46-m, 205-m, 1,096-m and 1,509-m. These traps were harvested at different time points to study hydrocarbon degradation, microbial community structure, and to isolate oil-degrading bacteria. While the extractable organics did not show a significant over all decrease, there was a loss of short chain alkanes (
  304. Dubinsky, E. A., L. Tom, F. Reid, S. Borglin, K. Chavarria, J. Fortney, D. Joyner, J. Kuehl, R. Lamendella, H. Lim, O. Mason, Y. Piceno, K. Wetmore, C. Wu, T. C. Hazen, G. L. Andersen. 2012. Succession and persistence of hydrocarbon-degrading microbial communities following the Deepwater Horizon Oil Spill. Annual Meeting of the American Society for Microbiology abstract
    The Deepwater Horizon oil spill created large plumes of dispersed oil deep in the Gulf of Mexico and stimulated growth of indigenous bacteria that can degrade hydrocarbons at cold temperatures. We tracked microbial community composition during and after the spill to determine relationships between microbial dynamics, and hydrocarbon and dissolved-oxygen concentrations. Oil plumes were dominated by a few species of Gammaproteobacteria, but community structure varied over time and was dependent on hydrocarbon concentration and composition. Pelagibacter were the most negatively impacted group of bacteria in oil plumes. Dissolved oxygen anomalies persisted over six weeks after well containment and remained dominated by plume bacteria despite the absence of petroleum hydrocarbons. Methanotrophs were also enriched in lingering oxygen anomalies, consistent with reports that methane consumption lagged behind the biodegradation of other hydrocarbon inputs.
  305. Hemme, C. L., Y. Deng, T. J. Gentry, M. W. Fields, L. Wu, S. Green-Tringe, D. B. Watson, Z. He, P. Chain, T. C. Hazen, J. M. Tiedje, E. M. Rubin, J. Zhou. 2012. Metagenomic Insights into Evolution of a Heavy Metal-Contaminated Groundwater Microbial Community. Annual Meeting of the American Society for Microbiology abstract
    The 30% of global freshwater reserves located in subsurface streams and aquifers represent a critical source of freshwater for human consumption and irrigation. Significant portions of natural groundwater reserves are currently contaminated by natural or anthropogenic means. The introduction of contaminants limits the use of groundwater without extensive processing, contributes to human disease, and has a significant negative effect on local ecosystems. Comprehensive characterization of microbial communities in natural systems remains a challenge due to their extremely high diversity and the as-yet uncultivated status of the vast majority of environmental microorganisms. Metagenomics and associated technologies have revolutionized the study of microbial diversity, adaptation and evolution. Low-complexity microbial communities from extreme environments such as acidic geothermal hot springs and contaminated sites are ideal for high-resolution, in-depth metagenomics studies. In this study, a microbial community from highly uranium-contaminated groundwater was sequenced using a random shotgun sequencing-based strategy and compared to a related pristine groundwater metagenome with the goal of addressing the following questions: (i) How does anthropogenic environmental change such as contamination affect groundwater microbial community diversity and structure? (ii) How do microbial communities adapt to severe environmental changes such as heavy metal contamination? (iii) What ecological trends are expected to manifest in stressed microbial ecosystems? Results indicate an overabundance of contaminant resistance genes (e.g. mercuric resistance, heavy metal ion efflux, etc.) in the stressed metagenome as well as an order of magnitude loss of species, metabolic and allelic diversity. Analysis further shows that carbon monoxide metabolism may be a key metabolic process in pristine groundwater systems that has been almost entirely lost in the stressed system. The cumulative results suggest that expected ecological trends for stressed ecosystems (reduced nutrient turnover, lower diversity, etc.) are consistent for stressed groundwater systems.
  306. Lamendella, R., S. E. Borglin, R. Chakraborty, T. C. Hazen, J. K. Jansson. 2012. Metatranscriptomics of an oil contaminated beach following the Deepwater Horizon Oil Spil. Annual Meeting of the American Society for Microbiology abstract
    The Deepwater Horizon oil spill represents one of the largest environmental catastrophes in the history of the oil industry. There are few molecular surveys describing the response of active indigenous microbial populations in oil-polluted coastal ecosystems. In this study, we aimed to determine the impact of the oil spill on the microbial community dynamics and gene expression over time on a heavily oil-polluted beach in Louisiana. Additionally, we aimed to determine which members of the microbial community and which functional genes were responsible for hydrocarbon degradation. We took samples (n=162) from Elmer’s Island, LA, over three time periods in June 2010, and determined the bacterial community structure by 16S rRNA gene pyrotag sequencing. The pyrotag data was analyzed using the QIIME annotation pipeline. Metadata collected from each sample included hydrocarbon composition, cell counts, sample depth, and location. RNA was extracted and sequenced from one sample from each collection date using the Illumina HiSeq platform. Metatranscriptomic data was assembled using the CLC Genomics Assembly tool, contigs were submitted to MG-RAST for annotation, and statistical analyses were performed in STAMP v2.0. The pyrotag data revealed a higher relative abundance of specific proteobacterial groups and reduced amounts of several other bacterial taxa, including actinobacteria in heavily oiled samples. Community structure was highly correlated to hydrocarbon concentration, extent of oil biodegradation, and bacterial diversity. The metatranscriptomic data revealed that the Rhodobacterales and other alphaproteobacteria were more active at the two earlier sampling points, whereas members of the Firmicutes and Actinobacteria were more active at the later sampling period. Additionally, an increased abundance of transcripts coding for proteins involved in carbon metabolism, respiration, and cell signaling, were identified in the more heavily oiled samples from the first two timepoints. By contrast, expressed genes for nitrogen metabolism were more elevated in the lesser contaminated sample. These findings illustrate successional and functional changes occurred in the beach microbial communities during biodegradation of the oil contaminants following the Deepwater Horizon oil spill.
  307. Lee, S., D. Tarjan, J. T. Geller, M. E. Singer, C. Wu, T. Torok, T. C. Hazen, N. J. Hillson, A. P. Arkin. 2012. Assessing and Mitigating the Biological Risks of Genetically Modified Bacteria in the Environment. Annual Meeting of the American Society for Microbiology abstract
    We are developing a means for understanding and assessing the biological risks and fates of genetically modified bacteria and their genetic elements in the environment. We are using as our model waste effluent such as might be contaminated by the failure of standard safety protocols for industrial bioreactors. This study seeks to estimate the risks associated with this model failure scenario by investigating the feasibility of horizontal gene transfer (HGT) of genetically engineered genes in model sewage reactors that simulate the conditions and microbial communities found in wastewater treatment plants. Here, we present results on the transmissibility and stability of fitness conferring genetic material between engineered strains of E. coli and an activated sludge microbial community sampled from the EBMUD wastewater treatment plant in Oakland, CA. To confer fitness in a controlled environment, we are identifying an exotic carbon source whose catabolism in E. coli is only possible through exogenous genetic material. The stabilities of broad-host range plasmids containing carbon catabolic genes, co-expressed with fluorescent protein markers, are being evaluated in E. coli grown in an artificial sewage minimal medium supplemented with the exotic carbon source. Further, we are characterizing the rate of enrichment of plasmid-bearing strains co-cultured with a wild-type E. coli strain. The transmissibility of these plasmids to the microbial community is being studied using a linked pair of bioreactors that allowed for the exchange of nucleic acids but not cells. One bioreactor cultures the engineered E. coli while the other cultures the activated sludge microbial community. Plasmid prevalence is quantified over time in both chambers using qPCR and FACS. Together this work establishes methodologies for assessing and mitigating the risks of future large-scale microbial metabolic engineering projects, including those extending beyond the bioreactor.
  308. Meyer, B., J. Kuehl, A. Deutschbauer, M. Price, A. Arkin, T. C. Hazen, D. Stah. 2012. Alternative Electron Transfer Systems in Desulfovibrio‐Methanogen Assemblies: Case Study of Desulfovibrio alaskensis str. G20‐Methanogen Cocultures Grown on Lactate. Annual Meeting of the American Society for Microbiology abstract
    In the absence of inorganic electron acceptors, the complete degradation of organic matter relies on the cooperative activities between phylogenetically and metabolically distinct microbial groups, e.g. fermenting, acetogenic bacteria and hydrogenotrophic, methanogenic archaea that are obligately linked through metabolite exchange in syntrophic association. To extend understanding of the genetic and metabolic basis of this common mutualism, we investigated two-member communities composed of alternative acetogenic Desulfovibrio (Dv.) species and methanogen pairings. These studies demonstrated that (1) different pairings vary significantly in their growth characteristics (e.g. growth rate, biomass yield and population dynamics) reflecting differences in the syntrophy-associated enzyme systems of Dv. spp. and (2) certain Dv. species interact differently with varying methanogenic partners in coculture. To investigate these observations in greater detail, we grew Dv. alaskenis str. G20 in combination with either Methanococcus maripaludis or Methanospirillum hungatei in chemostats under various dilution rates. Whole genome microarray-based transcriptional analysis of Dv. alaskensis str. G20 grown at high dilution rates in coculture with Methanococcus maripaludis showed a significant up-regulation of genes encoding periplasmic formate dehydrogenases, Ni-Fe hydrogenases, and membrane-bound, quinone-interacting complexes Qrc and Qmo relative to monoculture growth. The transcriptional patterns were distinct from previously studied Dv. vulgaris str. Hildenborough indicating that Dv. alaskensis str. G20 uses an alternative electron transfer system for syntrophic growth. In addition, the elevated levels of formate measured in the chemostats pointed to its importance as a mediator of electron exchange in Dv. alaskensis str. G20 syntrophic cocultures in contrast to the solely hydrogen exchange-based system of Dv. vulgaris str. Hildenborough. Further analyses revealed that gene expression of syntrophically grown Dv. alaskensis str. G20 varied with both growth rate and the methanogenic partner. As opposed to the existence of a conserved core of genes common to Dv. spp., the study points to considerable plasticity in the genetic and metabolic basis of the syntrophic lifestyle.
  309. Mircea Podar, Jennifer J. Mosher, Steven D. Brown, Dominique C. Joyner, Roseann Csencsits, Tommy J. Phelps, Kenneth H. Downing, Terry C. Hazen, Adam P. Arkin, Anthony V. Palumbo and Dwayne A. Elias. 2012. A Functional Genomic Characterization of Metal-reducing Pelosinus spp. isolated from Cr(VI) Contaminated Groundwater. Annual Meeting of the American Society for Microbiology abstract
    Background As part of a continuing effort to stimulate metal-reduction/remediation with natural microbial communities at sites contaminated by human activities, this study focused on physiological and functional genomics characterization of metal-reducing bacterial isolates from U(VI) and Cr(VI) contaminated groundwater at Hanford, WA. Four strains of Pelosinus spp. were obtained from chemostatic lactate-enrichments of the indigenous microbial community. Methods Each strain was isolated from a stable, enriched microbial community using fluorescence-activated cell sorting (FACS). Inter-strain characterization included metal-reduction assays (HFO, Fe(III), U(VI) and Cr(VI)), growth on >180 carbon sources via Omnilog, morphological TEM characterization, and metal uptake. Additionally, the genomes of all four strains as well as the type strain Pelosinus fermentans R7 have been sequenced. Results While all strains are capable of Fe(III)-reduction, only some were able to reduce Cr(VI) and one could reduce U(VI). Growth characteristics showed that two strains are highly similar to the type strain while the other two were similar to each other but physiologically different from the type strain and also accumulate polyphosphates. Each species appears morphologically similar overall with multiple flagella and all have identical SSU rRNA genes. At the genomic level, while there are large regions with identical sequence, islands where the sequences diverged and with different gene contents were identified. Conclusion Cultivation and metabolic characterization of isolated organisms from a stable, enriched community allows for a deeper understanding of the community metabolism as a whole. Comprehensive investigations such as these allow the evaluation of remediation strategies and identify which community members are important for bioremediation. The closely related genomic sequences suggest these strains have diverged relatively recently. Identification of genes responsible for the physiological differences should shed light into mechanisms of adaptation to changing environments and niche selectivity. This work conducted by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
  310. Walian, P., S. Allen, M. Shatsky, L. Zeng, E. Szakal, H. Liu, B. Lam, J. Geller, K. Hillesland, S. Hall, S. Fisher, M. Fields, D. Stahl, T. C. Hazen, S. Brenner, J‐M. Chandonia, E. Witkowska, M. Biggin, B. Jap. 2012. Membrane Protein Complexes of Desulfovibrio vulgaris‐ Changes in Response to Stress and the Establishment of Communities. Annual Meeting of the American Society for Microbiology abstract
    Background: Cell membranes represent the “front-line” of cellular defense and the interface between a cell and its environment. Significant changes in response to environmental conditions are expected to take place through the proteins situated within these membranes. Membrane protein-associated changes may occur in the form of abundance level, protein-protein interactions, post-translational modifications and even mutations. To characterize these changes we have developed a processing pipeline for membrane proteins and have chosen as our initial subject the Gram-negative sulfate-reducing bacterium, D. vulgaris. Methods: We are utilizing a high-throughput process for the isolation and identification of untagged membrane protein complexes that features mild, but effective, detergent solubilization, liquid chromatography and native electrophoresis methods. Protein identification is accomplished by mass spectrometry analysis of in-gel samples. Bioinformatics methods are used to assess complex stoichiometry and prepare interactome maps. Results: Processing of D. vulgaris membrane proteins from cultures grown under standard and stressed conditions (including growth to stationary phase, and growth under elevated levels of nitrate or NaCl) has been completed. An analysis of stress-associated changes detected in the outer-membrane proteins of D. vulgaris will be presented. In addition to supporting our studies on large-volume planktonic monocultures, we have refined the methods employed in the pipeline so that they would be more effective in processing samples derived from smaller, more native-like, sources. Preliminary results obtained from biofilm and other small-volume samples will be discussed. Conclusions: The catalog of D. vulgaris outer-membrane protein complexes prepared from these studies will serve as an essential reference for the detection and characterization of environment-driven changes in these proteins. Recent improvements in pipeline sensitivity and resolution are helping us to work successfully with relatively small cell samples such as biofilms which in turn will permit characterization of the roles of membrane proteins in the establishment and maintenance of communities.
  311. Yilmaz, S., P. Liu, R. J. Meagher, Y. K. Light, A. P. Arkin, T. C. Hazen, A. K. Singh. 2012. Single‐cell Analysis Platforms for Genomic Analysis of Uncultivable Environmental Microbes. Annual Meeting of the American Society for Microbiology abstract
    Current metagenomic techniques (e.g., microarray or 16S rRNA sequencing) relying on pooled nucleic acids from lysed bacteria can independently measure metabolic activity and the species present, but cannot link the activity deterministically to species. We are developing high-throughput tools for studying bacteria one cell at a time, allowing us to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities. Our approach includes FISH-based identification of desired species, enrichment by cell sorting, followed by single-cell encapsulation, whole genome amplification and sequencing. Encapsulation of bacteria in nanoliter plugs in particular allows us to scale down conventional (microliter-volume) assays, such as WGA, into much smaller reaction volumes better suited to the size of an individual microbe. We are using this pipeline to analyze water samples from DOE bioremediation sites (e.g., Hanford) to identify keystone organisms and link their functions to species. Furthermore, we are also using our single-cell genomics pipeline to complement the metagenomic sequencing efforts in ENIGMA. Metagenomic sequencing typically fails to achieve complete assembly and metabolic reconstruction of individual genomes in a complex community. Single-cell sequencing, together with metagenomics, makes it possible to assemble genomes of novel uncultivated organisms.
  312. Zhou, A., Z. He, E. Baidoo, K. Hillesland, M. P. Joachimiak, J. K. Baumoh, P. Benke, A. Mukhopadhyay, G. M. Zane, P. S. Deha, J. D. Wall, A. P. Arkin, D. Stahl, T. C. Hazen, J. Zhou. 2012. Molecular basis to adaption to salt in Desulfovibrio vulgaris in an evolutionary context. Annual Meeting of the American Society for Microbiology abstract
    High salinity is a key environmental stressor Desulfovibriovulagris often faces in its natural habitats. The molecular basis of its adaptation to elevated salinity in an evolutionary context is unknown. A long-term evolution experiment of 12 replicatepopulations under control or salt stress conditions has been set up. Salt resistance/tolerance was evaluated by growth on media containing higher concentrations of NaCl. The fitness change of evolved D. vulgarispopulations was determined by competition with the ancestor. Molecular basis of adaptation to elevated NaClwas further revealed by whole genome sequencing, global transcriptional profiling and metabolite assays. A rapid phenotypic adaptation to elevated NaCl could be observed as early as 100 generations and then gradual increases of salt resistance/tolerance. SNPs and insertion/deletions were identified in evolved D. vulgarisThe contribution of these mutations to salt resistance/tolerance wasnot only supported by the linkage between phenotype and genotype, but also by phenotype of the site-directed mutants.Parallelism of adaptive evolution was observed at about 5000 generations. Consistent data from transcriptomics and the metabolite assay demonstrated many changes in gene transcription and accumulation of metabolites in evolved clones under non-stress conditions. Increased transcription of genes involved in amino acid synthesis and transport, exclusion of Na+ and energy metabolism was detected in the evolved D. vulgaris. Interestingly, different sets of organic solutes may be responsible for releasing low or high salt stress. In summary, D. vulgaris quickly adapted to salt stress through genetic changes, gene expression changes and accumulation of organic solutes. Glu, Ala and Asp might be major players for coping with NaCl stress; Gln, GB and Gly might play important role at low NaCl stress.
  313. James G. Moberly, Tommy J. Phelps, Mircea Podar, Steven D. Brown, Zamin K. Yang, Meghan M. Drake, Terry C. Hazen, Adam P. Arkin, Anthony V. Palumbo and Dwayne A. Elias. 2012. Development of a Model Microbial Community for a Systems Biology Level Assessment of Metal-reduction. Annual Meeting of the American Society for Microbiology abstract
    BACKGROUND. One of the largest experimental gaps is between the simplicity of pure cultures and the complexity of open environmental systems, particularly in metal-contaminated areas. These microbial communities form ecosystem foundations, drive biogeochemical processes, and are relevant for biotechnology and bioremediation. METHODS. A model, metal-reducing community was constructed to study microbial cell to cell interactions, cell signaling and competition for resources. The microbial community was comprised of the metal-reducing Desulfovibrio vulgaris Hildenborough and Geobacter sulfurreducens PCA along with Methanococcus maripaludis S2 so as to study complete carbon reduction and maintain a low hydrogen partial pressure. Cultivation used 5L chemostats and analysis included physiological and metabolic analyses (GC, HPLC, species specific fluorescent antibody staining) as well as specially designed multispecies microarrays to follow gene expression changes in the various cultivation conditions. State-of -the-science comprehensive AMT tag proteomics was also used for a protein level assessment of the model community. RESULTS. Preliminary data revealed that lactate oxidation by D. vulgaris was sufficient to support both G. sulfurreducens and M. maripaludis via the excretion of H2 and acetate. Fumarate was utilized by G. sulfurreducens and reduced to succinate and malate. Methane was quantified, suggesting acetate and H2 concentrations were sufficient for M. maripaludis. CONCLUSIONS. Steady state community cultivation coupled to state-of-the-science analyses such as described herein will allow for a comprehensive, system biology level assessment of a metal-reducing microbial community and may further our understanding interspecies communication for syntrophy or direct competition.
  314. Jennifer J. Mosher, Tommy J. Phelps, Mircea Podar, Steven D. Brown, Terry C. Hazen, Adam P. Arkin, Anthony V. Palumbo, Boris A. Faybishenko and Dwayne A. Elias. 2012. Determination of the Influence of Chromium on Microbial Community Structure and Function. Annual Meeting of the American Society for Microbiology abstract
    Background: As part of a continuing effort to facilitate metal-reduction/remediation with in-situ microbial communities, this study focused on influence of the contaminating metal Chromium on microbial community structure and function. Methods: Metal contaminated groundwater was stimulated with lactate for 15 weeks in duplicate continuous-flow anaerobic reactors with 0, 0.1, or 3 mg/L Cr(VI) simultaneously to give a stable, enriched microbial communities. Temporal analyses included GC, HPLC, 16S pyrosequencing, metal-reduction assays, metal uptake, small metabolites, metagenomics and metatranscriptomics. At the experiment conclusion, the consortia were used to attempt to obtain isolates by traditional serial dilutions or fluorescence-activated cell sorting (FACS). Results: Previous experiments using no Cr(VI) revealed a dominance of Pelosinus spp. within the final community and subsequent isolates were capable of reducing U(VI), CrVI) and Fe(III). Here, temporal metal-reduction showed increased Cr(VI)-reduction rates in chemostats with higher Cr(VI). There were no obvious differences in trends of H2 or CH4 and genomic analyses are ongoing. Conclusion: Cultivation and metabolic characterization of organisms from a stable, enriched community allows for a deeper understanding of the community metabolism as a whole. Comprehensive investigations such as these allow the evaluation of remediation strategies and identify which community members are important for bioremediation.
  315. Hazen, T. C. 2012. Fate and distribution of Deepwater Horizon oil. Gordon Research Conference on Oceans and Human Health
  316. Spier, C. L., W. T. Stringfellow, T. C. Hazen and M. Conrad. 2012. An investigation of hydrocarbons sampling distribution in subsurface sediment and water samples after the 2010 deepwater horizon oil spill and the relationship between contamination in sediments and the water column. 8th National Monitoring Conference abstract
    The explosion of the Deepwater Horizon oil platform on April 20, 2010 resulted in the third largest oil spill in history. We investigated the distribution and chemical composition of hydrocarbons surrounding the spill site. A complete set of hydrocarbon data were acquired from the NOAA and BP, including data from 16 research missions. Several hydrocarbon plumes were identified including near-surface plumes (0.5 to 200m), a small mid-depth plume (850-880m), and a large deepwater plume between approximately 1000 and 1400m below surface. The vertical, lateral, and temporal distribution of hydrocarbons within the water column was investigated, and we found significant differences in the chemical composition of the plumes. The distribution of hydrocarbons remaining in sediments between August and October, 2010 was investigated. All sediment samples with total polycyclic aromatic hydrocarbons (PAHs) concentrations exceeding chronic toxicity limits were located less than 3.2km from the wellhead. All sediment samples with concentrations above the mean pre-spill PAH levels (>600µg/kg), based on 2006 and 2009 survey’s by the Minerals Management Service in the Deep Gulf of Mexico, were found within 12km of the wellhead.
  317. Hazen, T. C. 2012. Ecogenomics of the Deepwater Horizon Spill. Univiversity of California, Davis abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  318. Hazen, T. C. 2012. Ecogenomics of the Deepwater Horizon Spill. Univiversity of Tennessee, Genomics Sciences Program abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  319. Andersen, G. L., Y. M. Piceno, F. C. Reid, R. Chakraborty, S. E. Borglin, E. A. Dubinsky, L. M. Tom, H.-Y. N. Holman, T. C. Hazen. 2012. Microbial community structure and in situ MC-252 oil degradation at different depths in the Gulf of Mexico. ACS Spring Meeting.
  320. Dubinsky, E. A., L. M. Tom, F. Reid, S. Borglin, K. Chavarria, J. Fortney, D. Joyner, J. Kuehl, R. Lamendella, H.- C. Lim, R. Mackelprang, O. U. Mason, Y. Piceno, K. Wetmore, C. Wu, T. C. Hazen, G. L. Andersen. 2012. Microbial community composition as a highly sensitive biosensor for oil spills in the deep ocean. ACS Spring Meeting.
  321. Hazen, T. C. 2012. Deepwater Horizon Oil Spill Ecogenomics. ACS Spring Meeting.
  322. Lamendella, R., S. E. Borglin, R. Chakraborty, T. C. Hazen, and J. K. Jansson. 2012. Microbial Community Dynamics on an oil contaminated beach following the Deepwater Horizon Oil Spill. ACS Spring Meeting.
  323. Mason, O. U., Terry C. Hazen, T. Woyke and J. K. Jansson. 2012. OMics analyses of the deep-sea microbial community response to the Deepwater horizon oil spill. ACS Spring Meeting.
  324. Schmidt, C.. 2012. Exxon Valdez Vs. Deepwater Horizon ES&T’s Top Feature Article 2011. Envrionmental Science & Technology 46:3603-3604. pdf
  325. Hazen, Terry C. 2012. Can Mother Nature Take a Punch: the Gulf Oil Spill. University of Tennessee, Science Forum. abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill. This team-science approach suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
  326. Cheng, X., W. Reindl, K. Deng, B. Bowen, B. Lai, J M. Gladden, S. W. Singer, A. Wong, T. C. Hazen, B. Fox, K. Sale, B. A. Simmons, A. K. Singh, J. Keasling, P. D. Adams, and T. R. Northen . 2012. Nanostructure-Initiator Mass Spectrometry (NIMS): High Throughput Enzyme Activity Assays for Biofuel Development.. DOE Genomic Science Meeting abstract
    Project Goals: This project meets an urgent need for a highly specific activity screening approach and offers tremendous potential for the high-throughput identification and optimization of industrial enzymes and enabling application of biological approaches utilizing large libraries. The efficient deconstruction of lignocellulosic biomass into biofuels represents a critical and formidable challenge. JBEI is addressing this challenge using a multifaceted approach that is highly dependent on enzyme discovery, optimization and synthetic biology. The optimization of deconstruction processes requires technologies for the high throughput screening and identification of glycoside hydrolase activities. The high sensitivity, specificity, and resolution of mass spectrometry make it well suited for the analysis of sugar molecules. However, the low throughput of conventional GC/MS and LC/MS precludes implementation for screening purposes. Here we present a multiplexed approach based on nanostructure-initiator mass spectrometry (NIMS) that allows for the rapid analysis of several glycolytic activities in parallel under diverse assay conditions. By forming colloids, it was possible to perform aqueous reactions in microwell plates despite the substrate analogs’ hydrophobic perfluorinated tags. Our assay can be used both for the characterization of known enzymes (pH and temperature profiles, kinetic studies, ionic liquid tolerance), and the identification of yet unknown activities, even from complex biological samples (environmental and enrichment cultures). We are now integrating this assay with acoustic printing resulting in a 100-fold increase in throughput.
  327. Dhaeseleer, P. D., J. Gladden, J. Park, A. Redding, C. Petzold, M. Allgaier, D. Chivian, S. Singer, T. C. Hazen, and B. Simmons. 2012. Metagenomics, Metabolic Reconstruction, and High-Resolution Proteomics of Biomass Degradation in a Thermophilic Bacterial Community. DOE Genomic Science Meeting. abstract
    Project Goals: The Microbial Communities group at JBEI aims to develop a fundamental understanding of how microbial communities degrade targeted biomass feedstocks, and to utilize a targeted, function-based screening approach to genomics and proteomics to identify, isolate, and characterize new enzymes that are capable of efficiently degrading lignocellulosic feedstocks. Focusing on a thermophilic switchgrass-adapted enrichment community yields an order of magnitude more useful enzyme sequences compared to our previous work on a more complex community, and the resulting enzymes are more likely to be well suited to our targeted feedstock, pretreatment, and processing conditions. Combining enzymatic assays, metagenomics, zymography, MS proteomics, and metabolic modeling provides a multidimensional view of the internal functioning of this highly active biomass degrading bacterial community. A microbial enrichment culture with high biomass degrading activity was selected for metagenomic sequencing, annotated using JGI’s IMG/M system, and binned into phylogenetic groups. Metabolic reconstructions were generated using Pathway Tools, allowing us to assign metabolic roles to the different members of the bacterial community. High resolution MS metaproteomics by EMSL was mapped to the community members to analyze differential expression of their metabolic pathways and identify highly expressed biomass degrading enzymes.
  328. Auer, M. A. Gorur, P. Arbeleaz, N. Baliga, D. A. Ball, M. Biggin, J. M. Chandonia, S. Chhabra, R. Csencsits, K. H. Downing, M. W. Fields, J. T. Geller, R. Glaeser, T. C. Hazen, G. L. Hura, T. Juba, B. Lam, C. M. Leung, J. Liphardt, J. Malik, J. P. Remis, S. Reveco, J. Tainer, A. Tauscher, J. Wall, A. Deutschbauer, T. Northen, A. Arkin, and P. D. Adams. 2012. ENIGMA Biotechnology: Biofilm Imaging: From Protein Complexes to Intact Microbial Communities. DOE Genomic Science Meeting abstract
    Project Goals: The ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) project aims to elucidate the mechanisms and key processes that enable microorganisms and their communities to function in metal-contaminated soil sites. The ENIGMA Biotechnology Component focuses its efforts on providing crosscutting technologies that will support all other ENIGMA components with a particular focus on biological imaging at different scales of bacteria and microbial communities. These data will help to develop models of microbial community activity and principles of community organization in an effort to predict the role that microbial species and their interactions play in the dynamics of geochemical transformations in a changing environment. Microbial physiology is inherently a multiscale biological process that coordinates complex processes such as extracellular metal reduction and response to environmental stresses and competing species. Bacteria often assemble into sustainable communities that allow individual bacteria to coordinate their respective behavior and thus optimizing the efficiency of biological processes, which may enhance the chances for species survival. ENIGMA is addressing the complexity of multiscale spatiotemporal biofilm organization through a combination of expertise in traditional structural biology and modern multimodal imaging. SAXS (Rambo et al. 2010) and single particle cryo-EM are proven technologies to determine protein complex stoichiometry and shape, allowing the fitting of high-resolution structures into the intermediate resolution density envelope (Han et al. 2009). Cryo-electron tomography of bacterial whole mount samples can detect intra- and extracelluar specializations e.g. those important for metal reduction. Cryo-EM analysis is complemented by widefield 2D section TEM and advanced 3D SEM imaging approaches (FIB/SEM and SBF/SEM) of cryopreserved, freeze substituted and resin-embedded samples. With these novel EM imaging approaches, we have begun to examine large areas and volumes of biofilms in DvH and other soil bacteria. We have found outer membrane vesicles, vesicle chains and cell-cell connections (Palsdottir et al. 2009, Remis et al. 2010, Remis et al. submitted), as well as compartmentalization of metal precipitation (Auer, unpublished observation). These observations suggest a an intricate set of interactions and possibly coordination of function between community members. X-ray and EMbased imaging approaches are complemented by tag-based labeling of proteins both at the light and electron microscopy level, and allow the study of cell-to-cell variations in protein abundance and protein localization (Chabra et al. 2010). Advanced optical super-resolution imaging methods (including PALM and STORM) allow high precision localization and counting (Betzig et al 2006). Further integration of small molecule mass spectrometry imaging, while at a somewhat larger size scale, promises to link structural observation and protein localization with metabolic activity of biofilm regions. Through the integrated application of these imaging modalities ENIGMA is deconstructing a mechanistic understanding of biofilm function. Acknowledgement: This work conducted by ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. References 1. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution. Science 2006 313 (5793): 1642–1645 2. Chhabra SR, Butland G, Elias DA, Chandonia JM, Fok OY, Juba TR, Gorur A, Allen S, Leung CM, Keller KL, Reveco S, Zane GM, Semkiw E, Prathapam R, Gold B, Singer M, Ouellet M, Szakal ED, Jorgens D, Price MN, Witkowska HE, Beller HR, Arkin AP, Hazen TC, Biggin MD, Auer M, Wall JD, Keasling JD. Generalized schemes for highthroughput manipulation of the Desulfovibrio vulgaris genome. Appl Environ Microbiol. 2011 Nov;77(21):7595- 604 3. Han BG, Dong M, Liu H, Camp L, Geller J, Singer M, Hazen TC, Choi M, Witkowska HE, Ball DA, Typke D, Downing KH, Shatsky M, Brenner SE, Chandonia JM, Biggin MD, Glaeser RM. Survey of large protein complexes in D. vulgaris reveals great structural diversity. Proc Natl Acad Sci U S A. 2009 Sep 29;106(39):16580-5. 4. Palsdottir H, Remis JP, Schaudinn C, O’Toole E, Lux R, Shi W, McDonald KL,Costerton JW, Auer M. Three-dimensional macromolecular organization of cryofixed Myxococcus xanthus biofilms as revealed by electron microscopic tomography. J Bacteriol. 2009 Apr;191(7):2077-82 5. Rambo RP, Tainer JA. Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. Curr Opin Struct Biol. 2010 Feb;20(1):128-37. 6. Remis JP, Costerton JW, Auer M. Biofilms: structures that may facilitate cell-cell interactions. ISME J. 2010 Sep;4(9):1085-7 7. Remis JP, Wei W, Gorur A, Allen S, Witkowski HE, Costerton JW, Auer M Bacterial Social Networks: Targeted Delivery of Outer Membrane Proteins via Membrane Vesicle Chains, submitted
  329. Chakraborty, R., S. E Borglin, B. Faybishenko, P. Dehal, A. P. Arkin, T. C. Hazen, M. W. Fields, J. Geller, J. Fortney, D. Joyner, M. Conrad, and P. D. Adams. 2012. Effect of Nitrate Stress on Metal-reducing Microbes and Results of Nitrate push/pull Field Tests at Hanford 100H. DOE Genomic Science Meeting abstract
    Project Goal: The ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) project seeks to elucidate the mechanisms and key processes that enable microorganisms and their communities to function in metal-contaminated soil sites. One goal is to understand the effect of environmental stressors that enable ENIGMA-relevant microorganisms to thrive in such environments. Abstract: As part of the ongoing investigation of sustainable bioremediation of Cr(VI) in groundwater at the Hanford 100H area, we performed groundwater biostimulation tests by injecting Hydrogen Release Compound (HRC ) and three lactate (17mM) injection experiments. To investigate the response of resident microbes to nitrate stress, a push-pull test was then conducted by injecting 55 gals of groundwater (collected from the background well) with KNO3 (nitrate concentration 5,000 ppm) in October, 2010. After one day, pumping began from the same well, and lasted for 16 days. As a result of nitrate injection, total biomass decreased and sulfate concentration increased, but the sulfide and iron concentrations dropped. During pumping, the nitrate concentration decreased about 3 orders of magnitude. PLFA data showed biomass on the order of 107 cells/ml prior to push pull, and dropping off to 105 cells/ml during the test, but recovering toward the end back to 107 cells/ml by the end. Biomarker lipids indicate a shift toward monoenoics indicating an increase in gram negative bacteria and decrease in branched lipids (gram postive) and branched monoenoic (sulfate reducers). We discuss the field test results in details and elucidate the effect of nitrate stress on environmentally relevant microbes Geobacter metallireducens and Desulfovibrio vulgaris as observed through controlled lab experiments. The lab studies and the field study together will help in understanding the overall fate of microbes under changing environmental conditions in the field and the key cellular mechanisms impacted by such stress conditions. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
  330. Chandonia, J.-M., Maxim Shatsky, Ming Dong, Haichuan Liu, Lee Yang, Jil T. Geller, Megan Choi, Barbara Gold, Nancy L. Liu, Marjon Khairy, Sonia Reveco, Tom R. Juba, Bonita R. Lam, Evelin D. Szakal, Simon Allen, Sunil Kumar, Farris L. Poole, Steven E. Brenner, Steven C. Hall, Susan J. Fisher, Michael Adams, T. C. Hazen, Judy D. Wall, Swapnil Chhabra, Jian Jin, H. Ewa Witkowska, Adam P. Arkin, Gareth P. Butland, Mark D. Biggin, and Paul D. Adams. 2012. ENIGMA Microbiology Physiology: Accurate, High-Throughput Identification of Stable Protein Complexes in Desulfovibrio vulgaris. DOE Genomic Science Meeting abstract
    Project Goals: Desulfovibrio vulgaris has been selected as a model bacterium for intensive study by ENIGMA because it can reduce heavy metals and radionuclide contaminants present in the soil at many DOE sites, rendering the contaminants insoluble. ENIGMA seeks to model, at a molecular systems level, how this and similar bacteria respond to natural and human induced changes in their environment and how this alters their ability to stabilize contaminants in the soil. A component of our strategy is to develop and use high throughput pipelines to purify and characterize soluble protein complexes. We expect that these interaction data will improve our ability to produce accurate metabolic and regulatory models of key members of microbial communities. The group led by Mark Biggin has developed a novel method for identification of stable, soluble protein complexes in microbes. In a small-scale pilot study, we showed that many protein complexes survive intact through a series of orthogonal chromatographic methods, with complex components having correlated elution profiles. These profiles were measured with the aid of mass spectrometry (MS) and iTRAQ reagents (Dong et al., 2008). We developed statistical and machine learning methods to analyze a full-scale data set, which were required in order to obtain biologically meaningful results due to the high potential for false positives (FP) caused by co-elution of proteins that are not part of a complex. Our methods were tuned using a manually curated gold standard (GS) set. As a first high-throughput study, we demonstrated this technique in identifying a highprecision subset of stable protein complexes in Desulfovibrio vulgaris. We have shown that our predicted network of interactions is significantly enriched in pairs with similar functional annotations. The quantitative information from elution profiles allowed us to develop a statistical model to estimate the false discovery rate in our predictions; because this varies according to how “crowded” the eluted fractions are, we are able to identify a subset of hundreds of highly reliable (i.e., with very low false discovery rate) interactions, as well as a much larger set of interactions that can be predicted with known false discovery rates. Advantages of the tagless approach include not requiring a mutant library (needed for alternative tag-based approaches such as TAP), and a false discovery rate comparable to TAP. The group led by Gareth Butland has identified a number of protein complexes using TAP. We have developed an automated pipeline for synthesis of tagged gene constructs in collaboration with Swapnil Chhabra (Chhabra et al., 2011). To date, over 700 pulldowns (comprising more than 600 unique D. vulgaris strains) have been subject to analysis. In these experiments, more than 10,000 interactions were detected with over 1,000 distinct prey proteins. Using curated GS datasets (as in the tagless analysis), we filtered out ubiquitous proteins and other likely FP, resulting in a set of high-confidence interactions. A number of these interactions have been reciprocally confirmed, using strains in which the original prey protein was tagged and used as bait. Preliminary analysis of the data have identified several novel complexes, including multiple paralogous versions of the DnaJK-GrpE chaperone complex, each of which is bound to a small protein that may act as an allosteric regulator. References 1. Chhabra SR, Butland GP, Elias D, Chandonia JM, Fok OY, Juba T, Gorur A, Allen S, Leung CM, Keller K, Reveco SA, Zane GM, Semkiw ES, Prathapam R, Gold B, Singer M, Ouellet M, Szakal ED, Jorgens D, Price MN, Witkowska HE, Beller HR, Arkin AP, Hazen TC, Biggin MD, Auer M, Wall JD, Keasling JD. 2011. Generalized Schemes for High Throughput Manipulation of the Desulfovibrio vulgaris Hildenborough Genome. Applied and Environmental Microbiology 77:7595-7604. 2. Dong M, Yang LL, Williams K, Fisher SJ, Hall SC, Biggin MD, Jin J, Witkowska HE. 2008. A “tagless” strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking. J Proteome Res 7:1836-1849. ENIGMA is a Lawrence Berkeley National Laboratory Scientific Focus Area Program supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics:GTL Foundational Science through contract DE-AC02- 05CH11231.
  331. De Leon, K. B., B. D Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen, and M. W. Fields, and Paul D. Adams. 2012. ENIGMA Microbial Community Dynamics in Groundwater and Surrogate Sediments During HRC¿ Biostimulation of Cr(VI)-Reduction. DOE Genomic Science Meeting abstract
    Project Goals: The elucidation of bacterial community dynamics for both groundwater and sediment-associated communities over time and space during biostimulation for chromate reduction in relation to geochemical variables. The Hanford 100-H site is a chromium-contaminated site that has been designated by the Department of Energy Environmental Management as a field study site for in situ chromium reduction. In August 2004, the first injection of hydrogen release compound (HRC®) resulted in an increase of microorganisms and a reduction of soluble chromium(VI) to insoluble chromium(III). Little is understood about the microbial community composition and dynamics during stimulation. The aim of this study is to compare microbial communities of groundwater and soil samples across time and space during a second injection of HRC®. A second injection occurred November 2008 and geochemical data collected throughout the study showed an overall decrease in nitrate, sulfate, and chromium(VI). Spatial and temporal water and soil samples (n=34) were collected pre-and post-injection from four wells at the field site. Soil columns constructed from stainless steel mesh were lined with nylon mesh and filled with Hanford soils from the 100-H site. The soil columns were used to represent not only the microbes flowing through the soil via groundwater, but the microbes that require a matrix in order to grow. DNA was extracted from each of the samples and SSU rDNA gene fragments was sequenced via multiplex pyrosequencing. Sequences were refined by length, primer errors, and Ns, and sequences with a high percentage of low Phred quality score values were removed. Python scripts were developed to filter the pyrotag data with respect to quality scores, and the filtering technique was validated with environmental samples. Soil samples differed from the corresponding groundwater (even at the phyla level) and were more diverse (p=0.001). While many of the populations were observed in both groundwater and surrogate sediments, the respective matrices appeared to enrich for particular OTUs. Predominant populations for the sediments were Psuedomonas, Acidovorax, Clostridium, Aquaspirillum, Methylibium, Anaeromyxobacter while predominant populations for groundwater were Psuedomonas, Pleomorphomonas, Ramlibacter, Arthrobacter, and Herbaspirillum. Genera observed only in the sediment included Marinomonas while genera observed only in the groundwater included Desulfonauticus, Desulfomicrobium, and Syntrophobacter. Results do not indicate a large shift in dominant organisms in soil from pre- to post- injection, and this may be due to the organisms remaining dominant from the first stimulation. However, a prevalence of core genera and rare genera were observed across 34 samples while urban and rural genera were less abundant. There was a shift from Acidovorax to Aquaspirillum from upstream (non-stimulated) to downstream soil both pre- and post-injection. Surrogate soil samples indicated similar changes in the soil community in the injection (Well 45) and downstream (Well 41) wells across time, while water samples seem to indicate more of a pre- and post-injection grouping instead of gradual changes across time. Furthermore, while post-injection soil samples indicate a continuing dominance of Aquaspirillum, corresponding water samples indicate Pseudomonas as a dominant genus. For each well, HRC® injection resulted in increased diversity, but the greatest changes during stimulation occurred in the populations of mid-dominance either between wells or across time. These organisms could be important to consider as possible indicator species in future work. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
  332. Elias, D. A., J. J. Mosher, T. J. Phelps, M. Podar, R. A. Hurt, J. H. Campbell, M. M. Drake, J. G. Moberly, C. W. Schadt, S. D. Brown, T. C. Hazen, A. P. Arkin, A. V. Palumbo, B. A. Faybishenko, and P. D. Adams. 2012. ENIGMA Environmental: Succession of Hanford Groundwater Microbial Communities During Lactate Amendment and Electron-acceptor Limitation. DOE Genomic Science Meeting abstract
    Project Goals Bioremediation strategies involving in-situ microbial stimulation for Cr(VI) - reduction/immobilization are ongoing, but determining their relative success is complex. By using controlled laboratory conditions, the influence of individual variables on the successful community structure, dynamics and the metal-reducing potential can be studied. The goal of the current work was to determine the impact of lactate stimulation during sulfate limitation on the succession of a native microbial community. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI) contaminated groundwater from the Hanford, Washington 100-H area and incubated for 95 days to obtain stable, enriched communities. The microbial community structure shifted with a significant loss of diversity. Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp. with small levels of other organisms including methanogens. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Isolation efforts attained four new strains of Pelosinus spp. Three of the 4 Pelosinus strains were capable of Cr(VI)- reduction and one also reduced U(VI). Under the tested conditions of limited sulfate, it appears that the sulfate-reducers, including Desulfovibrio spp., were outcompeted even though they are capable of fermentative growth. These results suggest that lesser-known organisms, such as Pelosinus spp., may play a more important role in metal-reduction than currently suspected. Currently, a similar bioreactor experiment is underway and builds from the above findings. Hanford groundwater was enriched in duplicate bioreactors with lactate and; 1) no Cr(VI) to emulate the earlier work and establish a baseline, 2) 0.1 mg/L Cr(VI) to reflect the low plume concentrations nearing the Columbia river and 3) 3.0 mg/L Cr(VI) to reflect the source metal concentration. Multiple levels of analysis are now underway after a 105 day experiment. These include temporal measurements of; 16S rRNA gene pyrosequencing, gas and organic acid quantification by GC and HPLC, Geochip, Phylochip, metagenomics, metatranscriptomics, metaproteomics, soluble Fe(III), HFO, Cr(VI) and U(VI) reduction assays, metal uptake characteristics for 36 metals, small metabolite quantification, and fluorescent antibody cell counts for species of Desulfovibrio, Geobacter, Methanococcus, and Pelosinus. These results will help to determine if and how the added complexity of Cr(IV) will influence the microbial community structure and metabolism as well as revealing the any acclimation of the community to Cr and quantifying the relative reduction potential. Acknowledgement This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02- 05CH11231. Oak Ridge National Laboratory is managed by University of Tennessee UTBattelle LLC for the Department of Energy under Contract No. DE-AC05-00OR22725
  333. Geller, J., S. Lee, D. Tarjan, C. Wu, T. Torok, T. C. Hazen, A. P. Arkin, and N. J. Hillson. 2012. ELSI Pilot Assessing and Mitigating the Risks of Large-Scale Metabolic Engineering. DOE Genomic Science Meeting abstract
    Project Goals: Establish methodologies for assessing and mitigating the risks of future large-scale metabolic engineering microbial projects, including those extending beyond the bioreactor. The DOE EERE-funded Advanced Biofuel Process Development Unit (ABPDU) houses two 300 liter microbial fermentation tanks. In the extremely unlikely event that the ABPDU’s post-fermentation microbicidal protocol (e.g. base treatment and neutralization) should catastrophically fail, broth harboring viable genetically engineered micro-organisms could be purged directly to downstream waste-water treatment processes. This Ethical, Legal and Social Implications (ELSI) pilot study seeks to quantitate the risks associated with this scenario, by measuring the viability of the engineered microbes (and perhaps more importantly, their embedded genes) in mock sewage reactors that mimic the conditions and microbial communities found in real-world waste water treatment plants. Furthermore, investigation of how differential genetic backgrounds (e.g. gene deletions) impact survival and gene transmission to sewage sludge communities will guide subsequent forwardengineering efforts to further reduce risk. This pilot study establishes methodologies (leveraging only recently available technologies) for assessing and mitigating the risks of future large-scale metabolic engineering microbial projects, including those extending beyond the bioreactor.
  334. Hazen, T. C., M. W. Fields, J. Zhou, J. Van Nostrand, D. A. Elias, J. J. Mosher, S. D. Brown, T. J. Phelps, M. Podar, A. V. Palumbo, R. A. Hurt, A. Singh, E. Alm, M. B. Smith, D. C. Joyner, R. Chakraborty, B. Faybishenko, K. DeLeon, J. Geller, B. Lam, T. Torok, J. Fortney, S. E. Borglin, D. Stahl, J. Wall, A. P. Arkin, and P. D. Adams. 2012. ENIGMA Environmental Overview: Field to Lab to Field. DOE Genomic Science Meeting abstract
    Project Goals: ENIGMA working hypotheses: 1) Key transects in the environment provide constraints on community composition and activity that are discernable at multiple scales. The uranium/nitrate/pH gradient may provide one such transect and we will examine the communities and activities in different in situ (and laboratory simulated) uranium/chromium contaminated environments. 2) These environmental constraints change in time due in part to the structure and function of these communities and causal relationships may be discovered and quantified. 3) Community structure is established and maintained by varying factors that include transient populations, niche diversification, optimized interactions, and resource competition. We will be attempting to dissect these by field perturbation and time series studies and laboratory simulations of the environment. 4) There are important stable communities that achieve the above, which we can dissect at the level of molecular interactions, and we will drive toward isolation of key organisms, discovery of key activities and interactions, and dissect cellular networks that controls when and how these activities are expressed. The implications of the above hypotheses suggest: 1) There is a core set of metabolic factors that are the direct effectors of metal-reduction in soil and while exact species may vary, the molecular network will be relatively constant. This effort includes the identification of the biomolecular determinants of metal reduction in key populations that directly and/or indirectly interact with other populations that have programmed responses to important environmental parameters. 2) There are particular variants of these that adapt them for different metals and different concentrations and these are deployed differently in planktonic and attached communities over time during the reductive and reoxidative processes following stimulation. 3) At a particular site there are core, relatively stable sub-communities of microbes whose interspecific interactions are responsible for the stable reduction (and ultimate reoxidation) of metals. There is also a core community structure of necessary functional classes of microbes that form a stable “food web” to near optimally exploit the available energy in the environment. For example at the Hanford 100-H study site, soil samples differed from the corresponding groundwater (even at the phyla level) and were more diverse (p=0.001). While many of the populations were observed in both groundwater and surrogate sediments, the respective matrices appeared to enrich for particular OTUs. Results do not indicate a large shift in dominant organisms in soil from pre- to post- injection, and this may be due to the organisms remaining dominant from the first stimulation. However, a prevalence of core genera and rare genera were observed across 34 samples while urban and rural genera were less abundant. Background and Significance. ENIGMA is planning on shifting the principle field focus from Hanford to Oak Ridge. For the last 12 years ORNL has been characterizing, monitoring, and conducting field experiments at DOE’s BER ORNL Field Research site. The focus has been on elucidating the mechanisms and efficacy of bioreduction and bioimmobilization of U, one of the DOE’s most common waste site contaminant. This has involved a number of field studies including pump tests, hydrological modeling, characterization of sediment and groundwater and amendments of ethanol, bromide, and nitrate, etc. More than 300 wells have been established and characterized and are available for analysis in a searchable database Research and Design • Overarching Driver: microbial community structure and function in both in situ environments and constructed consortia - environment to the laboratory. • Elucidate structure to function during key biogeochemical transformations - immobilization of metals • Determine key succession events and mechanisms - stability in the context of geochemical and thermodynamic constraints • Parameterization at phylogenetic and functional level in conjunction with key biogeochemical variables that together, impact and control activities of interest (e.g., metal-reduction; N flow; C flow) • Identify key populations, directly and/or indirectly related to activities of interest • in situ and laboratory consortia will be used to explicate levels of biological organization from populations to proteins • models will be developed using various bioinformatic tools (e.g., AdaptML, random matrix theory, multivariate statistics) commonly used for genes and proteins but applied to populations overlaid with geochemical parameters and engineering controls. The current tasks for the Environmental Core are as follows: Task 1. Optimize omics protocols for environmental samples. Task 2. Use existing data from the ORNL FRC database to design efficient field sampling studies that maximize the geochemical diversity of study sites. This strategy is expected to enhance the resolution of associations between microbial communities and key geochemical features. Toward this end, we have developed a Monte Carlo search algorithm to optimize site selection for geochemical diversity. To this end we are currently looking for 100 wells that we can do a metagenome analysis in cooperation with the existing a planned SBR IFRC program at ORNL FRC. Overarching principle for Field Studies and Field Linked Resources: Establish as a team with the cooperation of the ORNL IFRC the hypotheses, experiments, sampling, analyses, and schedule as a test plan, including all protocols, amounts, and responsibilities. Prioritize everything so that time, money, shipping, sample limitations, and unforeseen contingencies will not limit delivery of key milestones. This includes 50%, 75%, and 95% evaluations of test plans by team prior to execution, and fatal-flaw analyses at each step. We are currently statistically trying to maximize the geochemical diversity of study sites from the ORNL FRC database. This strategy is expected to enhance the resolution of associations between microbial communities and key geochemical features. Toward this end, we have developed a Monte Carlo search algorithm to optimize site selection for geochemical diversity. We are currently considering a 100 well survey for metagenomics, this is still under planning with the ORNL IFRC. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
  335. Lancaster, W. A., Israel Scott, Brian Vacarro, Angeli Lal Menon, Farris L. Poole, Jil Geller, Jennifer J. Mosher, T. C. Hazen, Dwayne A. Elias, Michael W.W. Adams, Adam P. Arkin and Paul Adams. 2012. ENIGMA Microbial Physiology Assimilatory and Dissimilatory Metallomics of Desulfovibrio vulgaris and Pelosinus Strain A11. DOE Genomic Science Meeting abstract
    Project Goals: The goals of the ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies) project are to understand at a molecular systems biology level the microbial communities at DOE sites contaminated with heavy metals or radionuclides with sufficient detail to predictively model interactions within microbial and community processes that drive complex geochemistry in key environments. We expect to define biological principles governing selection of microbial community function and composition in given environments. While some heavy metals are environmental contaminants, metals in general have a very positive role in biological systems as they afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and greatly impact protein stability. Consequently, metal-containing proteins play key roles in virtually all biological processes. However, the full complement of metalloproteins within a given cell cannot be predicted solely from bioinformatic analyses of a genome sequence since metal coordination sites are diverse and poorly recognized. Hence it is not possible to predict the number and types of metals that a microorganism utilizes and how these might vary with the growth conditions. Determining the metals that are taken up during microbial growth and the metal content of fractionated native biomass can provide insight into these issues and, using coupled MS/MS analyses with extensive fractionations, can reveal completely new aspects of metal metabolism (1). Herein we report comparative metallomic analyses of the model microbe, Desulfovibrio vulgaris strain Hildenborough (DvH), and a newly described microbe, Pelosinus strain A11, which was recently isolated from an enrichment of a groundwater sample from the Hanford site by Elias and coworkers at ORNL. DvH was grown on lactate under sulfate-reducing conditions in a 600-liter metal fermenter and in a glass 5-liter fermenter and the nature of metals assimilated were compared. Pelosinus A11 was also grown at the 600-liter scale on fructose in the absence of sulfate and the assimilated metals were determined using ICP-MS (53 elements). Both organisms assimilated 20 or so metals into their cytoplasmic fractions, but the types and amounts were species specific. For example, DvH assimilated cobalt, cadmium and tungsten into high molecular weight complexes (>3 kDa) but Pelosinus A11 did not. In contrast to DvH, Pelosinus A11 assimilated vanadium, copper and uranium into >3kDa complexes. A number of additional factors affecting metal metabolism were analyzed, including growth with and without added chromium or uranium to study assimilation and dissimilatory reduction of these metals. The results will be presented in terms of the ranges and types of metals assimilated by DvH and Pelosinus A11 and the metals available in the organisms’ natural environments, including Hanford groundwater. Reference 1. Cvetkovic, A., Menon, A. L., Thorgersen, M., Scott, J. W., Poole, F. L., Jenney, F. E., Lancaster, W. A., Praismann, J. L., Shanmukh, S., Vaccaro, B., Trauger, S. A., Kalisiak, E., Apon, J. V., Siuzdak, G., Yannone, S. M., Tainer, J. A. and Adams, M. W. W. (2010) Microbial metalloproteomes are largely uncharacterized. Nature 466, 779-782 This work was conducted as part of the ENIGMA project and was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
  336. Meyer, B., K. L Hillesland, J. Flowers, N. Pinel, N. Elliott, J. D. Wall, M. Joachimiak, A. Zhou, J. Zhou, J. Kuehl, A. Deutschbauer, M. Price, Z. He, A. Mukhopadhyay, E. Baidoo, T. Northern, N. Baliga, M. Biggin, M. Dong, P. Walian, A. Singh, S. Yilmaz, D. Elias, M. Fields, H. Garcia-Martin, T. C. Hazen, A. P. Arkin, and D. A. Stahl. 2012. ENIGMA Laboratory Evolutionary and Ecological Origins of Community Assembly, Stability, and Efficiency. DOE Genomic Science Meeting abstract
    Project Goals: A goal of DOE and ENIGMA is to understand and ultimately predict microbial community assembly and the adaptive response of communities to environmental change. To address these questions, we are examining assemblies of simple two-member communities composed of a secondary fermenter like Desulfovibrio species and hydrogenotrophic methanogens. This type of association (syntrophy) is representative of a trophic interaction sustaining both pollutant transformation and organic matter mineralization in many anoxic environments typical of the subsurface. To study the metabolic versatility and specificity of these assemblies, we first investigated the association of twelve different pairings of Dv. species and two methanogenic species (Methanococcus maripaludis and Methanospirillum hungatei). The results demonstrated that different Desulfovibrio-methanogen pairings vary significantly in their growth characteristics, most notably in their ability to ferment lactate at elevated hydrogen levels, presumably reflecting differences in their syntrophy-associated enzyme systems (e.g., hydrogenases and electron transfer complexes). Those studies now serve to direct a systems-level approach to the study of common and divergent features of community interaction: focusing on the genetic and metabolic signatures of efficient species interaction, major determinants of community stability, and the capacity for these communities to improve through adaptive evolution. Abstract: Comparative studies have so far shown that both the electron transfer system and the mediator for electron transfer differ among Desulfovibrio species. For example, comparison of different assemblies grown in chemostats under various dilution rates demonstrated the importance of formate as major mediator of electron exchange in Dv. alaskensis strain G20 syntrophic cocultures in contrast to the hydrogen exchange-based system of Dv. vulgaris strain Hildenborough. Notably, the transcript analyses revealed that gene expression during syntrophic growth of Dv. alaskensis str. G20 also varies with both growth rate and the methanogenic partner. These conclusions were subsequently confirmed using a tagged-transposon Dv. alaskenis G20 mutant mini-library (1200 strains) to examine the relative fitness of different insertion mutants grown syntrophically in chemostats. Complementary studies are examining the adaptive evolutionary response of the two species to syntrophic growth. Ongoing laboratory evolution experiments of 24 replicated lines have so far documented a remarkable capacity for rapid improvement in the stability and efficiency of this mutualism after only 1000 generations of cooperative growth. The genetic basis of improved cooperation is now being examined by genome resequencing, initially of twelve of the pairings at 1000 generations using both Illumina and SOLiD next generation sequencing platforms and microarrays. Since some lines have evolved to obligate syntrophy, the history of their adaptive evolution will be reconstructed using single cell genome sequencing of earlier generations. The first mutations in Dv. vulgaris to become fixed in multiple lines were in an outer membrane porin (DVU0799), suggesting that amino acid replacements near the outer face of this porin alters the flux of metabolites and/or substrates. Other notable and frequent mutations were in genes implicated in EPS synthesis and regulation of lactate metabolism. Collaborative biophysical studies with ENIGMA members are now exploring altered function of the different porin mutants and mutants implicated in EPS synthesis. Of particular note was the replacement in all evolved lines of six amino acids in the sensory PAS domain of a histidine kinase (DVU3022) implicated in the regulation of lactate metabolism. The high frequency replacement with an identical short amino acid sequence having predicted sensory function suggests a novel phase variation-like mechanism of adaptive response. Together these ongoing studies point to both common and divergent mechanisms of interspecies interaction, and offer a framework to better resolve genetic, structural, and metabolic features contributing to stability and efficiency of community assembly. Funding This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231
  337. Walian, P. J., S. Allen, Max Shatsky, Lucy Zeng, Evelin D. Szakal, Haichuan Liu, Bonita Lam, Jil T. Geller, Kristina L. Hillesland, Steven C. Hall, Susan J. Fisher, Matthew W. Fields, David A. Stahl, T. C. Hazen, Steven E. Brenner, Adam M. Deutschbauer, Trent R. Northen, John-Marc Chandonia, H. Ewa Witkowska, Mark D. Biggin, Bing K. Jap, and Paul D. Adams. 2012. ENIGMA Biotechnology: Membrane Protein Complexes—Their Roles in Desulfovibrio vulgaris Stress Response and in the Establishment and Maintenance of Communities. DOE Genomic Science Meeting abstract
    Project Goals: Key aims of this project are—to develop a system for the high-throughput isolation and identification of membrane protein complexes, optimizing this process for effectiveness across a range of sample types including planktonic cultures and biofilms; to apply this system in the study of DOE relevant microbes such as Desulfovibrio vulgaris in order to detect and characterize changes in their membrane protein complexes brought about by environmental stressors, and through the role of these proteins in the establishment and maintenance of communities. A central goal of the ENIGMA consortium is to develop robust molecular-level models capable of predicting how target microbes respond to a range of environmental conditions. In support of this goal, our interests within ENIGMA have centered on the dynamic role of membrane protein complexes in this process. Cell membranes represent the “front-line” of cellular defense and the interface between a cell and its environment. Significant changes in response to environmental conditions are expected to take place through the proteins situated within these membranes. Membrane protein-associated changes may occur in the form of abundance level, protein-protein interactions, post-translational modifications and even mutations. To understand some of the earliest and perhaps most critical responses to stress, characterization of these changes on a molecular level is needed. The study of membrane proteins presents a major challenge in protein biochemistry; to address this we have developed a unique high-throughput process for the isolation and identification of untagged membrane protein complexes that features mild, but effective, detergent solubilization, liquid chromatography and native electrophoresis methods. We have been applying this system in two main areas of investigation, one of which has been our work on developing a D. vulgaris membrane protein complex database covering standard and stressed growth conditions, and the second to characterize the roles of selected membrane proteins in the establishment and maintenance of communities. Our study of membrane protein complexes in the outermembrane of D. vulgaris grown under standard conditions is complete and we are at an advanced stage with the innermembrane component. An interactome of proteins identified in D. vulgaris outer-membrane preparations is in the final stages of refinement. These preparations have yielded 69 outer-membrane protein identifications (which is over 80% of the number expected); 90% of these proteins were found to be in complexes. The most prevalent categories of proteins detected were the lipoproteins, and proteins with non-specific annotations (hypothetical and conserved hypothetical). This compendium of D. vulgaris outer-membrane protein complexes will serve as an essential reference for the detection and characterization of environment-driven changes in these proteins. Processing of outer-membrane proteins from stressed D. vulgaris cultures (including growth to stationary phase, and growth under elevated levels of nitrate or NaCl) has recently been completed. Initial analysis of stress-associated changes in outer-membrane protein abundance suggests that for many proteins there are significant differences between these changes and the changes in expression level inferred from mRNA experiments. Efforts on the preparation of the inner-membrane protein interactome and completion of the analysis of stress-induced changes occurring in the outer-membrane proteins of D. vulgaris are on-going. In addition to our studies on large-volume planktonic monocultures, we have refined the methods employed in the pipeline so that they may be used to process samples derived from more native-like sources. Recent improvements made in pipeline sensitivity and resolution are now allowing us to work successfully with relatively small cell samples such as biofilms. We anticipate that through additional optimization of the system, we will be able to process yet smaller samples, not only cultured in the lab but obtained directly from field sites. To assess the potential for discovery from such sources, we have begun pilot studies on biofilm samples. Early results contain evidence of protein changes occurring during the transition from stationary phase to biofilms, suggesting that this will be a productive direction for future studies of microbial communities. Recent work by the Stahl group on adaptive evolution experiments with co-cultures of Methanococcus maripaludis and D. vulgaris, has identified mutations of soluble and membrane protein genes likely to be important in establishing the syntrophic mutualism between these species. These results suggest a large influence of acquired membrane protein mutations leading to improved growth rates within this co-culture community. We have begun to process membranes from clonal isolates to characterize changes in the D. vulgaris membrane protein population. Interestingly, the most abundant protein of the D. vulgaris outer-membrane (DVU_0799) is also the most consistently mutated protein in these experiments. Therefore, a key goal of ours will be to purify and functionally characterize those mutated membrane proteins found to play a role in facilitating improved rates of growth. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
  338. Yilmaz, S., P. Liu, R. J. Meagher, Y. Light, A. P. Arkin2 T. C. Hazen, A. K. Singh, and P. D. Adams. 2012. Single-cell Analysis Platforms for Genomic Analysis of Uncultivable Environmental Microbes. DOE Genomic Science Meeting abstract
    Project Goals: We are developing a pipeline for single cell genomics that utilizes FISH (fluorescence in situ hybridization) for targeting species of interest, FACS (fluorescence activated cell sorting) for high throughput isolation of single cells, and MDA (multiple displacement amplification) for production of sufficient DNA from single cells for genome sequencing. This pipeline is being used for a number of collaborative projects in ENIGMA. Current metagenomic techniques (e.g., microarray or 16S rRNA sequencing) relying on pooled nucleic acids from lysed bacteria can independently measure metabolic activity and the species present, but cannot link the activity deterministically to species. We are developing high-throughput tools for studying bacteria one cell at a time, allowing us to unravel the complex dynamics of population, gene expression, and metabolic function in mixed microbial communities. Our approach includes FISH-based identification of desired species, enrichment by cell sorting, followed by single-cell encapsulation, whole genome amplification and sequencing. Encapsulation of bacteria in nanoliter plugs in particular allows us to scale down conventional (microlitervolume) assays, such as WGA, into much smaller reaction volumes better suited to the size of an individual microbe. We are using this pipeline to analyze water samples from DOE bioremediation sites (e.g., Hanford) to identify keystone organisms and link their functions to species. Furthermore, we are also using our single-cell genomics pipeline to complement the metagenomic sequencing efforts in ENIGMA. Metagenomic sequencing typically fails to achieve complete assembly and metabolic reconstruction of individual genomes in a complex community. Single-cell sequencing, together with metagenomics, makes it possible to assemble genomes of novel uncultivated organisms. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
  339. Zhou, J., Z. He, Y. Deng, A. Zhou, P. Zhang, Q. Tu, J. Van Nostrand, H. Yu, Z. Shi, J. Voordeckers, Y. Lee, R. Song, L. Wu, M. W. Fields, D. A. Stahl, J. D. Wall, T. C. Hazen, A. P. Arkin and P. D. Adams. 2012. ENIGMA Environmental Metagenomics-Enabled Understanding Of Microbial Communities At DOE Contaminated Sites. DOE Genomic Science Meeting abstract
    Project Goals: Although high throughput sequencing and associated (meta)genomic technologies provide an avenue to determine genetic and organismal diversity of an ecosystem, linking the genetic/population diversity to phenotypic diversity across different organizational levels (e.g., molecular, cellular, populations, communities and ecosystems) is extremely challenging. As a part of the overall ENIGMA goal, the ultimate aim of this project is to utilize (meta)genomic technologies to better understand the mechanistic connections between molecular-level interactions/processes and community-level processes/functions. The following three specific objectives have been pursued: (i) To determine adaptation and molecular mechanisms of Desulfovibrio vulgaris Hildenborough (DvH) in response to multiple environmental stresses; (ii) To understand microbial community functional diversity at U/Cr-contaminated sites and develop high-throughput functional gene arrays (FGAs) for microbial community analysis; and (iii) To determine the responses, interactions, mechanisms and dynamics of groundwater/sediment microbial communities to U/Cr contamination and bioremediation treatments. Long-term experimental evolution of DvH. To better understand the mechanistic connections between molecular-level functions and community-level processes, experimental evolution has been carried out to determine molecular mechanisms of DvH in response to high salinity. Significantly increased salt resistance was observed in evolved DvH (eDvH) with increased biomass, higher growth rate and shorter lag phases. Whole genome sequencing of eDvH at 1200 generations revealed specific point mutations and deletions. Their contribution to increased salt resistance has been proven by mutagenesis and phenotype analyses. Glu and Ala significantly increased in eDvH. After 5000 generations, the final biomass and growth rate of eDvH in the medium with high salinity was similar to that in the medium without extra salt. To further investigate the dynamics of evolution, repeatability of the evolution, whole genome sequencing, fitness assay and site-directed mutagenesis are in progress. GeoChip-based metagenomic technology development. We have developed the GeoChip 4.0 series (4.0-4.2) for characterizing microbial communities. The GeoChip 4.0 series are manufactured based on the NimbleGen microarray format. For example, GeoChip 4.0 contains 120,054 distinct probes, and covers 200,393 coding sequences for 539 gene families in different microbial functional processes. The StressChip subset contains 22,855 probes covering 79,628 gene sequences for 46 genes involved in microbial responses to environmental stresses (e.g., temperature, osmolarity, oxidative status, nutrient limitation). The specificity, sensitivity and quantification of the developed GeoChip 4.0 series were evaluated computationally and experimentally. High specificity was observed for both synthesized oligonucleotides and genomic DNA from pure strains; the sensitivity was estimated to be 0.5 µg of DNA; the log(signal intensity) vs. log(DNA concentration) was highly correlated (R = 0.925). All the results showed that the GeoChip 4.0 series are specific, sensitive, and quantitative tools for characterizing microbial communities. GeoChip applications. GeoChips have been used to study groundwater microbial communities to examine sustained reduction of contaminants using slow-degrading/slow-hydrolysis e-donors. At the Oak Ridge site, a one-time injection of emulsified vegetable oil (EVO) was used to examine U(VI) bioreduction and immobilization. Samples collected from the control and treatment wells (W1-7) were analyzed using GeoChip 3.0. Acetate, from EVO biodegradation, stimulated NO3-, Mn(IV), Fe(III), SO42-, and U(VI) bioreduction in W1-7 and increased functional gene diversity. After EVO depletion, functional gene diversity declined. Fe(III)- and sulfate-reducing bacteria could play key roles in U(VI) reduction, whereas acetogens, denitrifiers and methanotrophs could be important for e-donor production and maintaining favorable reducing conditions. At the Hanford site, a one-time injection of poly-lactate was used to test Cr(VI) bioreduction. The groundwater microbial communities were monitored for 390 days using GeoChip 4.0. Cr(VI) was effectively reduced and functional gene diversity increased. Fe(III)- and sulfate-reducing bacteria could play key roles in Cr(VI) reduction, whereas denitrifiers could be important for maintaining reducing conditions. Metagenomic sequencing. We have sequenced or resequenced metagenomes and dominant isolates from Oak Ridge FRC wells FW106 (contaminated with uranium, nitric acid, organics, and mercury) and FW301 (pristine). The FW106 metagenome was previously sequenced, and the results suggest extensive lateral transfer of metal resistance and organic compound metabolism genes. To extend these analyses, the metagenomes of FW106 and FW301, and genomes of multiple isolates of the dominant Rhodanobacter strain found in FW106 were sequenced or resequenced using Illumina sequencing technology. We are currently conducting comparative analyses of FW106 to FW301 to identify ecological trends observed between pristine and highly stressed groundwater communities. Furthermore, we are comparing the FW106 metagenome to Rhodanobacter isolates to confirm predicted lateral transfer events. Molecular ecological network analysis. A novel random matrix theory-based approach has been developed to construct molecular ecological networks (MENs) based on GeoChip hybridization or high-throughput sequence data. Various mathematic and statistical tools and methods have been integrated into a comprehensive MEN analysis pipeline (MENAP). We have applied this approach to construct and analyze MENs from the Oak Ridge EVO experiment described above. Functional MENs were constructed from three GeoChip datasets: (i) Early EVO injection (≤31 days), (ii) Days 80 to 140, and (iii) Control well (7 time points) and pre- injection (0 day). All three constructed functional MENs posed general network characteristics (scale-free, small world and modularity), and the topology of these functional MENs was distinctly different, suggesting that the interactions among different microbial functional groups/populations in each community were dynamically altered during uranium bioremediation. Additionally, the changes in network structure were significantly correlated with environmental geochemical dynamics and EVO concentrations. Acknowledgements. This work conducted by ENIGMA was supported by the Office of Science, Office of Biological and Environmental Research, of the U. S. Department of Energy under Contract No. DE-AC02-05CH11231.
  340. Dubinsky, E. A., Y. M. Piceno, F. C. Reid, L. M. Tom, Terry C. Hazen and G. L. Andersen. 2012. Microbial Community Composition in a Deep Water Oil Plume and Dissolved Oxygen Anomalies. 2012 Ocean Sciences Meeting. abstract
    In the aftermath of the Deepwater Horizon blowout, a dilute hydrocarbon plume was observed between 1,100 and 1,300 M below the surface. We collected water samples at plume depth on three ships from May 25 to August 26, 2010. Samples from transects starting near the source followed the prevailing current at this depth. No detectable hydrocarbons were observed at plume depth by the end of July, however, multiple samples taken in August had small reductions in DO suggesting microbial respiration and oxygen consumption. Microbial communities in plume samples were distinctive from non-plume with increased relative abundance of potential hydrocarbon-degrading gammaproteobacteria and Vibrio. Community compositions in August samples with reduced DO were similar to plume communities with a few notable differences. Taxa from the Methylococcaceae were more abundant in the late season, reduced DO samples. This supports the hypothesis that methanotrophs bloom late in plume succession in response to methane in water column. These results demonstrate that microbial communities at depth respond to trace amounts (<1 ppb) of hydrocarbon and microbial communities can be effective biosensors for hydrocarbon
  341. Hazen, T. C. 2012. Ecogenomics of the Deepwater Horizon Spill. 2012 Ocean Sciences Meeting abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  342. Spier, C. L., W. T. Stringfellow, T. C. Hazen and M. Conrad. 2012. An investigation into the distribution of hydrocarbons in sediments and the subsurface water column after the 2010 explosion of the Macondo 252 Deepwater Oil Rig. 2012 Ocean Sciences Meeting. abstract
    The explosion of the Deepwater Horizon oil platform on April 20, 2010 resulted in the third largest oil spill in history. We investigated the distribution and chemical composition of hydrocarbons surrounding the spill site. A complete set of hydrocarbon data were acquired from the NOAA and BP, including data from 16 research missions. Several hydrocarbon plumes were identified including near-surface plumes (0.5 to 200m), a small mid-depth plume (850-880m), and a large deepwater plume between approximately 1000 and 1400m below surface. The vertical, lateral, and temporal distribution of hydrocarbons within the water column was investigated, and we found significant differences in the chemical composition of the plumes. The distribution of hydrocarbons remaining in sediments between August and October, 2010 was investigated. All sediment samples with total polycyclic aromatic hydrocarbons (PAHs) concentrations exceeding chronic toxicity limits were located less than 3.2km from the wellhead. All sediment samples with concentrations above the mean pre-spill PAH levels (>600µg/kg), based on 2006 and 2009 survey’s by the Minerals Management Service in the Deep Gulf of Mexico, were found within 12km of the wellhead.
  343. Hazen, Terry C. 2012. Ecogenomics enables a new systems biology understanding of the Deepwater Horizon oil spill. Genome Alberta Hydrocarbon Metagenomics Workshop. abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  344. Hazen, Terry C. 2012. Horizon Oil Spill Degradation of oil by indigenous deep water microbes. Manitoba Environmental Industries Association annual workshop abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  345. Hazen, Terry C.. 2012. Ecogenomics enables a new systems biology understanding of the Deepwater Horizon oil spill. University of Alaska, Fairbanks. abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  346. Hazen, Terry C. 2012. Can Mother Nature Take a Punch Systems Biology of the Gulf Oil Spill. University of Tennessee, Civil & Environmental Engineering. abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  347. Hazen, T. C. 2012. What happened to the Gulf Oil Spill: A Systems Biology Approach. Biosciences Division, Oak Ridge National Lab. abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in an immense oil plume 4,000 ft deep. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis, phenotypic microarrays, metagenomes, metatranscriptomes, single cell sequencing, stable isotope analysis in combination with a variety of hydrocarbon and micronutrient analyses we were able to characterize the deep-sea microbial ecosystem and the effect of the oil spill.
  348. Boxall, B. 2012. Gulf currents aided breakdown of oil after BP spill, study says. http://www.miamiherald.com/2012/01/09/2581760/gulf-currents-aided-breakdown.html
  349. Hazen, T. C. 2011. NOAA Marine Microbes Workshop. NOAA.
  350. Spier, C.L., W.T. Stringfellow, E. Sonnenthal, M. Conrad, and T. C. Hazen. 2011. The distribution of hydrocarbons in surface and deepwater plumes during the MC252 oil spill in the Gulf of Mexico. American Geophysical Union Fall Meeting. abstract
    The explosion of the Deepwater Horizon oil platform on April 20, 2010 resulted in the third largest oil spill in history. We investigated the distribution and chemical composition of hydrocarbons surrounding the spill site. A complete set of hydrocarbon data were acquired from the NOAA and BP, including data from 16 research missions. Several hydrocarbon plumes were identified including near-surface plumes (0.5 to 200m), a small mid-depth plume (850-880m), and a large deepwater plume between approximately 1000 and 1400m below surface. The vertical, lateral, and temporal distribution of hydrocarbons within the water column was investigated, and we found significant differences in the chemical composition of the plumes. The distribution of hydrocarbons remaining in sediments between August and October, 2010 was investigated. All sediment samples with total polycyclic aromatic hydrocarbons (PAHs) concentrations exceeding chronic toxicity limits were located less than 3.2km from the wellhead. All sediment samples with concentrations above the mean pre-spill PAH levels (>600µg/kg), based on 2006 and 2009 survey’s by the Minerals Management Service in the Deep Gulf of Mexico, were found within 12km of the wellhead.
  351. Conrad, M. E., M. Bill, W.T. Stringfellow, S. E. Borglin, O. U. Mason, E. A. Dubinsky, Y. M. Piceno, J. L. Fortney, L. M. Tom,? K. L. Chavarria, R. Lamendella, D. C. Joyner, K. Wetmore, J. Kuehl, R. Mackelprang, C. Wu, J. Lim, F. Reid, and T. C. Hazen. 2011. Isotopic evidence for microbial oxidation of dissolved methane in the Gulf of Mexico oil spill deep plume. Goldschmidt Conference.
  352. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume. Goldschmidt Conference. abstract
    The explosion on April 20, 2010 at the BP-leased Deepwater Horizon drilling rig in the Gulf of Mexico off the coast of Louisiana, resulted in oil and gas rising to the surface and the oil coming ashore in many parts of the Gulf, it also resulted in the dispersment of an immense oil plume 4,000 feet below the surface of the water. Despite spanning more than 600 feet in the water column and extending more than 10 miles from the wellhead, the dispersed oil plume was gone within weeks after the wellhead was capped – degraded and diluted to undetectable levels. Furthermore, this degradation took place without significant oxygen depletion. Ecogenomics enabled discovery of new and unclassified species of oil-eating bacteria that apparently lives in the deep Gulf where oil seeps are common. Using 16s microarrays, functional gene arrays, clone libraries, lipid analysis and a variety of hydrocarbon and micronutrient analyses we were able to characterize the oil degraders. Metagenomic sequence data was obtained for the deep-water samples using the Illumina platform. In addition, single cells were sorted and sequenced for the some of the most dominant bacteria that were represented in the oil plume; namely uncultivated representatives of Colwellia and Oceanospirillum. In addition, we performed laboratory microcosm experiments using uncontaminated water collected from The Gulf at the depth of the oil plume to which we added oil and COREXIT. These samples were characterized by 454 pyrotag. The results provide information about the key players and processes involved in degradation of oil, with and without COREXIT, in different impacted environments in The Gulf of Mexico. We are also extending these studies to explore dozens of deep sediment samples that were also collected after the oil spill around the wellhead. This data suggests that a great potential for intrinsic bioremediation of oil plumes exists in the deep-sea and other environs in the Gulf of Mexico.
  353. Butland, G. P., S. R. Chhabra, B. Gold, N. L. Liu, S. Reveco, T. R. Juba, J. D. Wall, B. R. Lam, J. T. Geller, T. C. Hazen, M. Choi, M. D. Biggin, E. D. Szakal, S. Allen, H. Liu, H. E. Witkowska, and J.-M. Chandonia. 2011. High Throughput Identification of Protein Complexes from Desulfovibrio vulgaris by a Tandem Affinity Purification Pipeline. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  354. Chakraborty, R., D. Joyner, B. A. Faybishenko, M. Fields, T. Torok, G. L. Andersen, and T. C. Hazen. 2011. Integrated Microbiological Approaches to Characterize Cr(VI)-Reducing Microbial Community at the DOE Hanford 100H Site. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  355. Chandonia, J.-M., M. Dong, M. Shatsky, H. Liu, L. Yang, T. C. Hazen, J. T. Geller, M. Choi, E. D. Szakal, S. Allen, S. E. Brenner, S. C. Hall, S. J. Fisher, S. Kumar, F. L. Poole, M. Adams, J. Jin, H. E. Witkowska, A. P. Arkin, and M. D. Biggin. 2011. Accurate, High-Throughput Identification of Stable Protein Complexes in Desulfovibrio vulgaris using a Tagless Strategy. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  356. Chhabra,, S., M. Auer, G. Butland, J.-M. Chandonia, T. C. Hazen, J. D. Wall, E. Witkowska, D. Elias, M. Adams, M. Fields, J. Liphardt, G. Hura, and D. Stahl. 2011. High Throughput Production and Analysis of Genetically Engineered Desulfovibrio vulgaris Strains via Homologous Recombination. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  357. De Leon, K. B., B. D. Ramsay, D. R. Newcomer, B. Faybishenko, T. C. Hazen, J. Zhou, and M. W. Fields. 2011. Microbial Community Dynamics from Groundwater and Surrogate Sediments During HRC¿ Biostimulation for Cr(VI)-Reduction. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  358. DeAngelis, K. M., J. Fortney, S. Borglin, W. Silver, and T. C. Hazen. 2011. Feedstock-Adapted Anaerobic Consortia Derived from Tropical Forest Soils. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy
  359. DHaeseleer, P. and A. M. Redding-Johanson, C. J. Petzold, P. I. Benke, M. Allgaier, D. C. Chivian, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, and S. W. Singer J. M. Gladden. 2011. Metagenomics, Proteomics, and Metabolic Reconstruction of a Thermophilic Feedstock-Adapted Bacterial Community. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy
  360. Gorur, A., C. M. Leung, S. Chhabra, T. Juba, A. Tauscher, S. Reveco, J. P. Remis, B. Lam, J. T. Geller, T. C. Hazen, M. Biggin, J. M. Chandonia, K. H. Downing, J. Wall, and M. Auer. 2011. Subcellular Localization of Proteins in the Anaerobic Sulfate Reducer Desulfovibrio vulgaris via SNAP-Tag Labeling and Photoconversion. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  361. Hillesland,, K. L., B. Meyer, N. Pinel, N. Elliott, M. Joachimiak, J. Kuehl, A. Deutschbauer, A. Zhou, Z. He, J. Zhou, D. Elias, T. C. Hazen, A. P. Arkin, and D. A. Stahl. 2011. Adaptive Evolution and Physiology of Nascent Microbial Mutualisms. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  362. Liu, P., R. J. Meagher, Y. K. Light, S. Yilmaz, R. Chakraborty, A. P. Arkin, T. C. Hazen, and A. K. Singh. 2011. Microfluidic Tools for Single-Cell Genomic Analysis of Environmental Bacteria. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  363. Mosher, J. J., J. G. Moberly, C. W. Schadt, T. J. Phelps, M. Podar, S. D. Brown, A. V. Palumbo, M. W.W. Adams, D. A. Stahl, K. L. Hillesland, J. D. Wall, M. W. Fields, T. C. Hazen, and D. A. Elias. 2011. Characterization of Naturally Occurring and Model Microbial Communities. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy
  364. Tu, Q., Y. Deng, Z. He, H. Yu, Y. Qin, A. Zhou, J. Xie, Z. Lu, J. Voordeckers, Y. Lee, K. Xue, J. Van Nostrand, L. Wu, Y. Jiang, T. C. Hazen, P. Adams, and J. Zhou. 2011. Development of Metagenomic Technologies for Analyzing Microbial Communities. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  365. Walian, P. J., S. Allen, L. Zeng, E. D. Szakal, H. Liu, S. C. Hall, S. J. Fisher, R. Santos, B. Lam, J. T. Geller, T. C. Hazen, J.-M. Chandonia, H. E. Witkowska, M. D. Biggin, and B. K. Jap. 2011. High-Throughput Pipeline for the Purification and Identification of Desulfovibrio vulgaris Membrane Protein Complexes. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  366. Yoon, S. H., D. J. Reiss, J. C. Bare, D. Tenenbaum, M. Pan, J. Slagel, S. Lim, M. Hackett, A.-L. Menon, M. W.W. Adams, A. Barnebey, S. M. Yannone, J. A. Leigh, and N. S. Baliga. PI: N. S. Baliga, Co-PIs: J. A. Leigh, M. Hackett, W. Whitman, P. Adams, A. P. Arkin, T. C. Hazen, M. W.W. Adams, G. Hura, S. M. Yannone, S. Holbrook, G. Siuzdak, and J. A. Tainer. 2011. Parallel Evolution of Transcriptome Structure During Genome Reorganization. Joint Meeting 2011 Genomic Science Awardee Meeting IX and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Meeting, U. S. Department of Energy.
  367. Chakraborty, R., T. C. Hazen, S. E. Borglin, and D. C. Joyner. 2011. Degradation and oil and dispersant by microbes isolated from the Gulf of Mexico in the aftermath of MC252 oil spill. SIM annual meeting.
  368. Grimes, D. J., T. C. Hazen, S. McClellan, M. Sogin, and J. B. Paul. 2011. Vibrios and petroleum biodegradation ? capable but contributory ?. SIM annual meeting.
  369. Hazen, T. C. 2011. Ecogenomics enables a new systems biology understanding of the Deepwater Horizon oil spill. UC-Berkeley, Department of Plant and Microbial Biology.
  370. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. West Kazakhstan Technical University
  371. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. West Kazakhstan State University
  372. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. American Club, US State Department Visit to Kazakhstan.
  373. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Atyrau Institute of Oil and Gas, US State Department Visit to Kazakhstan.
  374. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Atyrau Region Akimat, US State Department Visit to Kazakhstan.
  375. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Aktau Region Akimat, US State Department Visit to Kazakhstan.
  376. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Caspian State University of Technologies and Engineering, US State Department Visit to Kazakhstan.
  377. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Kazakhstan National Center for Biotechnology, US State Department Visit to Kazakhstan.
  378. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. The Euroasian National University, US State Department Visit to Kazakhstan.
  379. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. Kazakhstan National Library, US State Department Visit to Kazakhstan.
  380. Hazen, T. C. 2011. Deepwater Horizon Oil Spill - popular media vs. science lessons learned. University of North Carolina Wilmington Planet Ocean Seminar Series.
  381. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. IPEC Conference.
  382. Hazen, T. C. 2011. The Deepwater Horizon Oil Spill: Ecogenomics and biodegradation of the deep-sea plume---Remediation of Hydrocarbon Spills. University of North Carolina Wilmington Planet Ocean Seminar Series.
  383. Baelum, J., S. Borglin, J. L. Fortney, R. Lamendella, O. U. Mason, M. Bill, M. E. Conrad, H-Y. Holman, S. A. Malfatti, S. Tringe, T. C. Hazen, and J. K. Jansson. 2011. Microcosm Simulations of the Microbial Community Response to the Deepwater Horizon Oil Spill in the Gulf of Mexico. ASM annual Meeting.
  384. Baelum, J., S. E. Borglin, J. L. Fortney, R. Lamendella, O. U. Mason, M. Bill, M. E. Conrad, S. Malfatti, S. Tringe, H.-Y. Holman, T. C. Hazen, and J. K. Jansson. 2011. Simulations of the microbial community response to the Deepwater Horizon Oil spill in the Gulf of Mexico using a microcosm approach. BAGECO2011 Meeting.
  385. Bohan, S. 2011. Lawrence Berkeley Lab scientists tinker with microbes to battle climate change. Contra Costa Times
  386. Borglin, S. E., Y. Piceno, D. C. Joyner, J. Fortney, and T. C. Hazen. 2011. Analysis of microbial community structure and alkane composition in Mississippi Canyon oil spill using phospholipid fatty acid analysis. ASM annual Meeting.
  387. Chakraborty, R., S. E. Borglin, D. H. Long, D. C. Joyner, and T. C. Hazen. 2011. Interaction of MC252 oil and COREXIT with isolates and enrichments from Gulf of Mexico. ASM annual Meeting.
  388. Chandonia, J-M., M. Dong, M. Shatsky, H. Liu, S. E. Brenner, L. Yang, T. C. Hazen, J. T. Geller, M. Choi, E. D. Szakal, J. Jin, H. E. Witkowska, A. P. Arkin, and M. D. Biggin. 2011. Accurate, High-Throughput Identification of Stable Protein Complexes Using a Tagless Strategy. ASM annual Meeting.
  389. Chhabra, S., B. Gold, N. L. Liu, S. Reveco, T. R. Juba, J. D. Wall, B. R. Lam, J. T. Geller, T. C. Hazen, M. Choi, M. D. Biggin, E. D. Szakal, S. Allen, H. Witkowska, J-M. Chandonia, and G. P. Butland. 2011. Engineering Desulfovibrio vulgaris Hildenborough for High Throughput Tandem Affinity Purification of Protein Complexes. ASM annual Meeting.
  390. DHaeseleer, P. and A. M. Redding-Johanson, C. J. Petzold, P. I. Benke, M. Allgaier, D. C. Chivian, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, and S. W. Singer J. M. Gladden. 2011. Metagenomics, Proteomics, and Metabolic Reconstruction of a Thermophilic Feedstock-adapted Bacterial Community. ASM annual Meeting.
  391. Dickey Zakaib, G.. 2011. Role of bacteria in Gulf oil spill under the microscope.
  392. Fancher, L. 2011. Mother Nature Cleans up Gulf Oil Spill. Walnut Creek Patch
  393. Geller, J. T., H. Woo, D. C. Joyner, S. Kendall, and T. C. Hazen. 2011. Microfluidic Studies of Nitrate Stress on Shewanella oneidensis Biofilms. ASM annual Meeting.
  394. Gorur, A., C. M. Leung, A. Tauscher, D. Jorgens, S. Reveco, J. Remis, B. Lam, J. T. Geller, T. C. Hazen, T. Juba, S. Chhabra, J. Wall, M. Biggin, K. H. Downing, and M. Auer. 2011. High Throughput Subcellular Protein Expression and Localization Studies in the Anaerobic Sulfate Reducer Desulfovibrio vulgaris. ASM annual Meeting.
  395. Grossman, E. 2011. Murky Waters.
  396. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. REMTEC.
  397. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Tulane University
  398. Hazen, T. C. 2011. Impact Of BP Spill May Not Be Catastrophic. KTVU http://www.ktvu.com/news/27986393/detail.html
  399. Hazen, T. C. 2011. Microbes Cope with Calamity: Gulf Oil Spill. JGI@Lesher: The Deal with Carbon: How Earth?s Mighty Microbes Respond
  400. Hazen, T. C. 2011. Microbial Ecogenomic Response to the Deepwater Horizon Oil Spill in the Gulf of Mexico - Deepwater Horizon & Beyond. ASM annual Meeting.
  401. Hazen, T. C. 2011. Microbial Response to the Deepwater Horizon Oil Spill in the Gulf of Mexico. BAGECO2011 Meeting.
  402. Hu, P., C. H. Wu, T. Desantis, P. Jasrotia, H. Woo, K. Kearcher, S. Meiss, T. Torok, L. D. Taylor, W. Overholt, S. Green, G. L. Andersen, J. E. Kostka, and T. C. Hazen. 2011. Validation of MycoChip ? A Microarray for Fungal Community Studies. ASM annual Meeting.
  403. Joyner, D. C., R. Chakraborty, S. E. Borglin, D. H. Long, and T. C. Hazen. 2011. High Throughput Metabolic Phenotype Profiling of Oil and Dispersant Degrading Consortia from the MC252 Oil Spill in the Gulf of Mexico. ASM annual Meeting.
  404. Lamendella, R., S. Borglin, J. Hultman, O. U. Mason, F. Reid, J. Fortney, K. Wetmore, J. Kuehl, H.- C. Lim, T. C. Hazen, and J. Jansson. 2011. Impact of the Deepwater Horizon Oil Spill on beach microbial community dynamics. ASM annual Meeting.
  405. Liu, P., R. J. Meagher, Y. Light, S. Yilmaz, R. Chakraborty, A. P. Arkin, T. C. Hazen, and A. K. Singh. 2011. Microfluidic Fluorescence in situ Hybridization and Flow Cytometry Microdevice for Environmental Microbial Detection. ASM annual Meeting.
  406. Martinez, R. J., C. H. Wu, M. J. Beazley, G. L. Andersen, T. C. Hazen, M. Taillefert, and P. A. Sobecky. 2011. Microbial Phosphatase Activity Involved in Subsurface Uranium Sequestration. ASM annual Meeting.
  407. Mason, O. U., R. Lamendella, J. Hultman, R. Mackelprang, S. Borglin, L. M. Tom, E. A. Dubinsky, J. Fortney, T. C. Hazen, and J. K. Jansson. 2011. Metagenomic analysis of the deep-sea microbial community response to the Deepwater Horizon oil spill. ASM annual Meeting.
  408. Moberly, J. G., T. J. Phelps, C. W. Schadt, M. Podar, S. D. Brown, Z. K. Yang, M. M. Drake, T. C. Hazen, A. P. Arkin, A. V. Palumbo, and D. A. Elias. 2011. Development of a Model Microbial Community for a Systems Biology Level Assessment of Metal-reduction. ASM annual Meeting.
  409. Mosher, J. J., T. J. Phelps, S. L. Carroll, M. M. Drake, C. W. Schadt, M. Podar, S. D. Brown, T. C. Hazen, A. P. Arkin, A. V. Palumbo, B. A. Faybishenko, and D. A. Elias. 2011. Isolation of metal reducing organisms from lactate-enriched contaminated groundwater. ASM annual Meeting.
  410. Rajan, S., R. J. Martinez, M. J. Beazley, Y. Piceno, G. L. Andersen, T. C. Hazen, P. A. Sobecky, and B. Mortazavi. 2011. Coastal Alabama Microbial Responses to the Deepwater Horizon Oil Spill. ASM annual Meeting.
  411. Walian, P. J., S. Allen, L. Zeng, E. Szakal, S. C. Hall, S. J. Fisher, R. Santos, B. Lam, J. T. Geller, T. C. Hazen, J. M. Chandonia, H. E. Witkowska, M. D. Biggin, and B. K. Jap. 2011. High-throughput Pipeline for the Purification and Identification of Desulfovibrio vulgaris Membrane Protein Complexes. ASM annual Meeting.
  412. Zhang, P., W-M. Wu, J. Van Nostrand, Y. Deng, Z. He, T. Gihring, G. Zhang, C. Schadt, D. Watson, P. Jardine, C. Criddle, S. Brooks, T. Marsh, J. Tiedje, T. C. Hazen, and J. Zhou. 2011. Diverse Functional Genes of Microbial Community Stimulated with Emulsified Vegetable Oil for in situ U(VI) Reduction. ASM annual Meeting.
  413. Zhou, A., Z. He, E. Baidoo, K. Hillesland, M. P. Joachimiak, J. Baumohl, P. Benke, A. Mukhopadhyay, P. S. Dehal, A. P. Arkin, D. Stahl, T. C. Hazen, and J. Zhou. 2011. Transcriptomics and Metabolites Assay of Salt-Adapted Desulfovibrio vulagris Hildenborough in Experimental Evolution. ASM annual Meeting.
  414. DeAngelis, K. M., W. L. Silver, and T. C. Hazen. 2011. Anaerobic deconstruction of switchgrass by tropical soil-derived feedstock adapted consortia. DOE Joint Genome Institute annual user meeting.
  415. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Science Night for Orinda Intermediate School.
  416. Hazen, T. C. 2011. The Gulf Oil Spill ? Ecogenomics and Ecoresilience Genomics of Energy & Environment. DOE Joint Genome Institute annual user meeting
  417. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. Presentation arranged by the Center for Safe Energy: for the Environment and Energy ministers of the Republic of Kazakhstan.
  418. Mason, O. U., T. C. Hazen, P. Chain, E. A. Dubinsky, J. Fortney, J. Han, J. Hultman, R. Lamendella, R. Mackelprang, L. M. Tom, S. G. Tringe, T. Woyke, E. M. Rubin, and J. K. Jansson. 2011. Omics analyses of the deep-sea microbial community response to the Deepwater Horizon Oil Spill. DOE Joint Genome Institute annual user meeting.
  419. 2011. Berkeley Lab Scientist Wins PNNL?s Outstanding Lecture Award. Today at Berkeley Lab, http://today.lbl.gov/2011/06/17/berkeley-lab-scientist-wins-pnnl?s-outstanding-lecture-award/
  420. Hazen, T. C., B. Faybishenko, H. Beller, E. Brodie, E. Sonnenthal, C. Steefel, J. Larsen, M. Conrad, M. Bell, J. Christensen, S. Brown, D. Joyner, S. Borglin, J. Geller, R. Chakraborty, P. Nico, P. Long, D. Newcomer, and E. Arntzen. 2011. Comparison of Field Groundwater Biostimulation Experiments Using Polylactate and Lactate Solutions at the Chromium Contaminated Hanford 100-H Site. The International Symposium on Bioremediation and Sustainable Environmental Technologies.
  421. Hazen, T. C. 2011. The Gulf Oil Spill. JBEI Seminar Program.
  422. Hazen, T. C. 2011. The Gulf Oil Spill. The International Symposium on Bioremediation and Sustainable Environmental Technologies.
  423. Hazen, T. C. 2011. A Systems Biology Approach to the Deepwater Horizon Oil Spill ? an example of Team Science for ecological disasters. Pacific Northwest National Laboratory Frontiers Lecture Series.
  424. Andersen, G. L., E. Dubinsky, Y. M. Piceno, L. Tom, K. Sublette, T. Z. DeSantis, T. C. Hazen, and S. E. Borglin. 2011. Response of petroleum-degrading microbial communities at different depths to the Deepwater Horizon oil spill. SIM annual meeting.
  425. DeAngelis, K. M., P. D'Haeseleer, J. Fortney, S. Borglin, W. L. Silver, and T. C. Hazen. 2011. Metagenomics of anaerobic lignocellulolytic feedstock-adapted consortia derived from tropical forest soils. SIM annual meeting
  426. Hazen, T. C. 2011. The Gulf Oil Spill. SIM annual meeting.
  427. Hazen, T. C. 2011. Metagenome and single cell sequencing. SIM annual meeting.
  428. Hazen, T. C. 2011. A Systems Biology Approach?to the Deepwater Horizon Oil Spill ??an?example of Team Science for ecological disasters.. Grimes Distinguished Lecturer Program, Gulf Coast Research Lab.
  429. Mason, O. U., T. C. Hazen, and J. R. Jansson. 2011. Metagenomic, Metatranscriptomics and single cell genomic analysis of the deep-sea microbial community response the to Deep Water Horizon oil Spill. SIM annual meeting
  430. Sobecky, P. A., M. Beazley, R. J. Martinez, S. S. Rajan, J. Powell, Y. Piceno, G. L. Andersen, L. Tom, T. C. Hazen, J. D. Nostrand, J. Zhou, and B. Mortazav. 2011. Coastal Alabama Bacterial Community Responses to the Deepwater Horizon Oil Spill. SIM annual meeting
  431. 2011. Bacteria. The Current, Canadian Broadcast Company
  432. 2011. Bacteria devoured methane gas from gulf oil spill, scientists say. http://berkeleylabreport.blogspot.com/2011/01/bacteria-devoured-methane-gas-from-gulf.html
  433. Forrester, M. 2011. Study: Undersea bugs ate natural gas released in oil spill. http://video-game-schools.freeawx.com/study-undersea-bugs-ate-natural-gas-released-in-oil-spill/
  434. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Regional Science Council Seminar Series, EPA region 9.
  435. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. Stanford University.
  436. Hotz, R. L. 2011. Microbes Devoured Methane From BP Spill, Study Says. http://online.wsj.com/article/SB10001424052748703730704576065942040672186.html?mod=googlenews_wsj
  437. Morrison, J. 2011. Dirty Work. American Way Magazine.
  438. Raloff, J. 2011. Methane from BP spill goes missing. http://www.sciencenews.org/view/generic/id/68461/title/Methane_from_BP_spill_goes_missing
  439. Vastag, B. 2011. Bacteria devoured methane gas from gulf oil spill, scientists say. http://www.washingtonpost.com/wp-dyn/content/article/2011/01/06/AR2011010603570.html
  440. Vergano, D. 2011. Study: Undersea bugs ate natural gas released in oil spill. http://www.usatoday.com/tech/science/environment/2011-01-06-gulf-oil-spill-methane-natural-gas-bacteria_N.htm
  441. Yong, E. 2011. Bacteria ate up all the methane that spilled from the Deepwater Horizon well. http://blogs.discovermagazine.com/notrocketscience/2011/01/06/bacteria-ate-up-all-the-methane-that-spilled-from-the-deepwater-horizon-well/
  442. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Luncheon Seminar to Ex-LBNL employees.
  443. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. AAAS annual meeting.
  444. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. Purdue University Sigma Xi meeting.
  445. Hazen, T. C. 2011. Open ocean studies on the Deepwater Horizon oil incident. University of Wisconsin.
  446. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. BP Headquarters.
  447. Yarris, L. 2011. Lessons learned from the two worst oils spills in U.S. history. http://www.physorg.com/news/2011-08-lessons-worst-oils-history.html
  448. Anthony, L. 2011. UC researcher reflects on Gulf oil spill.
  449. Cassidy, G. 2011. One Year Later: Did Bacteria Save Oil and Gas Stocks?. http://www.benzinga.com/trading-ideas/long-ideas/11/04/1011228/one-year-later-did-bacteria-save-oil-and-gas-stocks
  450. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Department of Microbiology, University of Georgia.
  451. Hazen, T. C. 2011. Can Mother Nature Take a Punch? - Microbes and the BP Oil Spill in the Gulf of Mexico. Darden Lecture, Department of Biology, University of Alabama.
  452. Hazen, T. C. 2011. Panel 1. The Interpretation of Scientific Data for Disaster. Response After the Spill: A Dialogue Between Students and Policymakers. Roosevelt Institute, University of Georgia.
  453. Lovett, R. A. 2011. Why Did Huge Oil Plumes Form After the Gulf Spill?. http://news.nationalgeographic.com/news/2011/04/110420-gulf-oil-spill-anniversary-plumes-dispersants-science-nation/
  454. Hulick, K. 2011. Learning from Disasters Exxon-Valdez vs. Deepwater Horizon. Odyssey January 2011 pg 24-26.
  455. Voigt, E. 2011. Invasion of the Oil-Eating Microbes. Odyssey January 2011 pg 16-18.
  456. Hazen, T. C., B. Faybishenko, H. R. Beller, E. Brodie, E. L. Sonnenthal, C. I. Steefel, J. Larsen, M. E. Conrad, J. N. Christensen, S. T. Brown, D. C. Joyner. S. E. Borglin, J. Geller, R. Chakraborty, P. Nico, P. Long, D. Newcomer, and E. Armtzen. 2011. Comparison of field groundwater biostimulation experiments using polylactate and lactate solutions at the Chromium-contaminated Hanford 100-H Site. American Geophysical Union Annual Meeting.
  457. 2011. Lessons learned from the two worst oils spills in US history: Microbes matter. http://www.bayoubuzz.com/louisiana-local-news/bp-oil-spill/323275-lessons-learned-from-the-two-worst-oils-spills-in-us-history-microbes-matter
  458. 2011. Microbes? role in oil spills investigated. http://www.istockanalyst.com/business/news/5370015/microbes-role-in-oil-spills-investigated
  459. Berwyn, B. 2011. Microbial cleanups touted for major oil spills. http://summitcountyvoice.com/2011/08/20/microbial-cleanups-touted-for-major-oil-spills/
  460. Raloff, J. 2011. Bacteria binged on BP oil but didn?t grow. http://www.sciencenews.org/view/generic/id/333086/title/Bacteria_binged_on_BP_oil_but_didnt_grow__
  461. Kaufman, L. 2011. Nearly a year after spill, Gulf studies yield more than damage. http://www.bendbulletin.com/article/20110412/NEWS0107/104120407/
  462. Goldenberg, S. 2011. Has BP really cleaned up the Gulf oil spill? April 14. http://www.guardian.co.uk/environment/2011/apr/13/deepwater-horizon-gulf-mexico-oil-spill
  463. Hazen, T. C. 2010. The Gulf of Mexico Oil Spill. Florence Conference on Phenotype Microarray Analysis of Microorganisms.
  464. Hazen, T. C. 2010. Phenotypic MicroArray for bioenergy applications. Florence Conference on Phenotype Microarray Analysis of Microorganisms.
  465. Joyner, D. C., Hazen, T. C. 2010. Phenotypic MicroArray for bioenergy applications. Florence Conference on Phenotype Microarray Analysis of Microorganisms.
  466. Chakraborty, R., E. L. Brodie, B. Faybishenko, Y. M. Piceno, L. Tom, S. Choudhuri, H. R. Beller, J. Liu, T. Torok, D. C. Joyner , P. E. Long, D. R. Newcomer, G. L. Andersen, and T. C. Hazen. 2010. Microbial community changes during sustained Cr(VI) reduction at the 100H site in Hanford, WA. Annual meeting of the American Society for Microbiology Meeting.
  467. DeAngelis, K. M., M. Allgaier, Y. Chavarria, J. Fortney, P. Hugenholtz, B. Simmons, K. Sublette and and T. C. Hazen W. L. Silver. 2010. Trapping Lignin Degrading Microbes in Tropical Forest Soil. Annual meeting of the American Society for Microbiology Meeting.
  468. Fortney, J. L., K. M. DeAngelis, Y. Chavarria, W. Silver, and T. C. Hazen. 2010. Anaerobic feedstock-adapted consortia and isolates from Puerto Rico tropical forest soils. Annual meeting of the American Society for Microbiology Meeting.
  469. Geller, J. T., S. E. Borglin, J. L. Fortney, B. R. Lam, T. C. Hazen, and M. D. Biggin. 2010. Large-Scale, Continuous-Flow Production of Stressed Biomass (Desulfovibrio vulgaris Hildenborough). Annual meeting of the American Society for Microbiology Meeting.
  470. Joachimiak, M., R. Chakraborty, A. Zhou, J. L. Fortney, Z. He, P. Dehal, M. R. Price, J. Wall, J. Zhou, A. P. Arkin, T. C. Hazen, J. D. Keasling, and S. R. Chhabra. 2010. A revised bioenergetic model of Desulfovibrio vulgaris strain Hildenborough. Annual meeting of the American Society for Microbiology Meeting.
  471. Joachimiak, M. P., R. Chakraborty, A. Zhou, J. L. Fortney, J. T. Geller, Z. He, J. Wall, J. Zhou, A. P. Arkin, T. C. Hazen, J. D. Keasling and S. R. Chhabra. 2010. Revisiting modes of energy generation in sulfate reducing bacteria. Annual meeting of the American Society for Microbiology Meeting.
  472. Joyner, D. C., J. L. Fortney, R. Chakraborty, and T. C. Hazen. 2010. Adaptation the Biolog OmniLog Phenotype MicroArray plate technology to profile the strict metal reducing anaerobe Geobacter metallireducens. Annual meeting of the American Society for Microbiology Meeting.
  473. Liu, H., M. Dong, L. L Yang, E. D Szakal, S. Allen, S. C. Hall, S. J. Fisher, T. C. Hazen, J. T. Geller, M. E Singer, J. Jin, M. D. Biggin, and H. E. Witkowska. 2010. Development and Refinement of an iTRAQ-Based Tagless Strategy for High-Throughput Purification and Identification of Soluble Protein Complexes. 58th ASMS Conference on Mass Spectrometry.
  474. Mosher, J. J., M. M. Drake, S. L. Carroll, Z. K. Yang, C. W. Schadt, S. D. Brown, M. Podar, T. C. Hazen, A. P. Arkin, T. J. Phelps, A. V. Palumbo, B. A. Faybishenko, and D. A. Elias. 2010. Microbial community dynamics of lactate enriched Hanford groundwaters. Annual meeting of the American Society for Microbiology Meeting.
  475. Piceno, Y. M., D. Venkateswaran, L. Tom, S. Chaudhuri, M. Vu, T. C. Hazen, R. Chakraborty, G. L. Andersen. 2010. Optimization of extraction techniques for microbial community analysis of MEOR samples. Annual meeting of the American Society for Microbiology Meeting.
  476. Timberlake, S. C., M. P. Joachimiak, D. Joyner, R. Chakraborty, J. K. Baumohl, P. S. Dehal, A. P. Arkin, T. C. Hazen, and E. J. Alm. 2010. Conservation of Modules but not Phenotype in Bacterial Stress Responses. Annual meeting of the American Society for Microbiology Meeting.
  477. Woo, H. L., K. M. DeAngelis, T. C. Hazen, and B. A. Simmons. 2010. Isolation of Aerobic Lignin- and Cellulose- degrading Bacteria from Tropical Soils from Biofuel Feedstock Deconstruction. Annual meeting of the American Society for Microbiology Meeting.
  478. Christensen, J. N., E. Sonnenthal, S. T. Brown, M. E. Conrad, L. Yang, S. Mukhopadhyay, C. I. Steefel, B. Faybishenko, and T. C. Hazen. 2010. Using Cr Isotopic Measurements Together with Reactive Transport Modeling to Monitor Stimulated Bio-containment at the 100H Test Site, Hanford, Washington. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
  479. Hazen, T. C. 2010. Ecogenomics with MicrobesOnline and KnowledgeBase. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
  480. Hazen, T. C. and P. Bayer. 2010. Systems Environmental Microbiology: Innovative Approaches to Understand Cellular and Microbial Community Activity and Function. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
  481. Hubbard, S. S., J. Ajo-Franklin, H. Beller, E. Brodie, J. Chen, J. Christensen, M. Conrad, D. DePaolo, B. Faybishenko, S. Finsterle, T. C. Hazen, M. Kowalsky, E. Sonnenthal, N. Spycher, C. Steefel, T. Tokunaga, J. Wan, K. Williams, Y. Wu, M. Denham, Y. Fujita, L. Li, and P. Long. 2010. Overview of the LBNL Sustainable Systems SFA. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
  482. Zhang, P., W. Wu, J. D. Van Nostrand, Y. Deng, Z. He, T. Gihring, G. Zhang, C. W. Schadt, D. Watson, P. Jardine, S. Brooks, T. L. Marsh, J. M. Tiedje, T. C. Hazen, and J. Zhou. 2010. Geochip-based analysis of metabolic diversity of microbial communities during in situ biostimulation at a uranium-contaminated aquifer. Subsurface Biogeochemical Research (SBR) Contractor-Grantee Workshop.
  483. Reddy, A. P., M. Allgaier, J. M. Gladden, S. Singer, P. Hugenholtz, B. Simmons, T. C. Hazen, and J. S. VanderGheynst. 2010. Enrichment of highly efficient thermophilic microbial communities active on switchgrass and corn stover in a high-solids environment. 239th ACS National Meeting & Exposition.
  484. Hazen, T. C. 2010. Systems Biology Approach to Bioremediation: Omics and Hydrobiogeochemical Processes. Goldschmidt 2010: Earth, Energy and the Environment.
  485. Allgaier, M., A. Reddy, J. I. Park, N. Ivanova, P. D?haeseleer, S. Lowry, R. Sapra, T. C. Hazen, B. A. Simmons, J. S. VanderGheynst, and P. Hugenholtz. 2010. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost microbial community. ISME 13 ? 13th International Symposium on Microbial Ecology.
  486. Chakraborty, C., J. Fortney, A. Zhou, M. Joachimiak, A. Mukhopadhyay, S. Borglin, Z. He, A. P. Arkin, J. Zhou, and T. C. Hazen. 2010. Investigation of osmotic stress response in the anaerobic metal-reducing microbe Geobacter metallireducens, strain GS -15. ISME 13 ? 13th International Symposium on Microbial Ecology.
  487. De Leon, K., D. Newcomer, B. Faybishenko, B. Ramsay, T. C. Hazen, and M. Fields. 2010. Microbial community dynamics from groundwater and surrogate sediments during HRC¿ stimulation at a chromium contaminated field site. ISME 13 ? 13th International Symposium on Microbial Ecology.
  488. DeAngelis, K. M., M. Allgaier, P. D?haeseleer, J. L. Fortney, P. Hugenholtz, B. Simmons, and T. C. Hazen. 2010. Analysis of anaerobic lignocellulose decomposing consortia from Puerto Rico tropical forest soils. ISME 13 ? 13th International Symposium on Microbial Ecology.
  489. Dubinsky, E., T. DeSantis, Y. Piceno, O. Mason, N. Singh, A. Probst, D. Joyner, R. Chakraborty, T. C. Hazen, G. Andersen. 2010. Phylochip assay finds deepwater oil plume enrichment of psychrophilic oil-degrading bacteria. ISME 13 ? 13th International Symposium on Microbial Ecology.
  490. Goodheart, D. B., W. L. Silver, T. C. Hazen, and M. K. Firestone. 2010. The Diversity and Activity of Methanogens in a Wet, Tropical Forest during Plant Decomposition. ISME 13 ? 13th International Symposium on Microbial Ecology.
  491. Hazen, T. C. 2010. Microbial ecology of the 2010 Deepwater Horizon Oil Spill. ISME 13 ? 13th International Symposium on Microbial Ecology.
  492. Hazen, T. C. 2010. The Spill and Ecogenomics. ISME 13 ? 13th International Symposium on Microbial Ecology.
  493. Martinez, R., M. Beazley, C. Wu, G. Andersen, T. C. Hazen, M. Taillefert, and P. Sobecky. 2010. Microbial phosphatase activity involved in subsurface uranium sequestration. ISME 13 ? 13th International Symposium on Microbial Ecology.
  494. Mason, O., A. Iavarone, L. Tom, S. Borglin, R. Chakraborty, T. C. Hazen, and J. Jansson. 2010. Pressure assisted omics analyses of resident microbes in petroleum reservoirs. ISME 13 ? 13th International Symposium on Microbial Ecology.
  495. Mason, O. U., A. Probst, E. Dubinsky, Y. Piceno, L. Tom, J. Fortney, R. Lamendella, T. C. Hazen, and J. Jansson. 2010. The microbial community of a dispersed, deep-sea oil plume. ISME 13 ? 13th International Symposium on Microbial Ecology.
  496. Mosher, J. J., M. M. Drake, S. L. Carroll, Z. K. Yang, C. W. Schadt, S. D. Brown, M. Podar, T. C. Hazen, A. P. Arkin, T. J. Phelps, A. V. Palumbo, B. A. Faybishenko, and D. A. Elias. 2010. Lactate enrichment of uranium and chromium contaminated Hanford groundwater samples. ISME 13 ? 13th International Symposium on Microbial Ecology.
  497. Singer, S., J. Gladden, M. Allgaier, A. Reddy, J. VanderGheynst, T. C. Hazen, P. Hugenholtz, P. D'haeseleer, B. Simmons. 2010. Targeted enzyme discovery in thermophilic feedstock-adapted microbial communities using proteogenomic and biochemical techniques. ISME 13 ? 13th International Symposium on Microbial Ecology.
  498. Van Nostrand, J., P. Waldron, D. Watson, Z. He, L. Wu, P. Jardine, T. C. Hazen, and J. Zhou. 2010. Geochip-based analysis of groundwater microbial communities across a gradient of pH, heavy metal, and nitrate contamination. ISME 13 ? 13th International Symposium on Microbial Ecology.
  499. Wells, G. F., C. H. Wu, Y. M. Piceno, B. Eggleston, E. L. Brodie, T. Z. DeSantis, G. L. Andersen, T. C. Hazen, C. A. Francis, and C. S. Criddle. 2010. Immigration as a driver of microbial community structure: sloughing from a biofilm bioreactor is linked to downstream-activated sludge community dynamics. ISME 13 ? 13th International Symposium on Microbial Ecology.
  500. Wu, C. H., E. A. Dubinsky, J. Hulls, S. R. Osman, T. C. Hazen, and G. L. Andersen. 2010. Temporal dynamics of cattle and human fecal microbial communities in fresh and marine waters. ISME 13 ? 13th International Symposium on Microbial Ecology.
  501. Zhang, P., W. Wu, J. Van Nostrand, Y. Deng, Z. He, T. Gihring, G. Zhang, C. Schadt, D. Watson, P. Jardine, S. Brooks, T. Marsh, J. Tiedje, T. C. Hazen, and J. Zhou. 2010. Microarray-based characterization of microbial community functional structure during in situ biostimulation at a uranium-contaminated aquifer. ISME 13 ? 13th International Symposium on Microbial Ecology>
  502. Borglin, S. E., O. U. Mason, E. Dubinsky, J. Fortney, R. Lamendella, D. Joyner, Y. Piceno, and T. C. Hazen. 2010. Analysis of microbial community structure in crude oil and oil spill samples using phospholipid fatty acid analysis. Annual Meeting of the Society for Industrial Microbiology.
  503. Chakraborty, R., and T. C. Hazen. 2010. Microbial community changes during sustained Cr(VI) reduction at the 100H site in Hanford, WA. Annual Meeting of the Society for Industrial Microbiology.
  504. Dubinsky, E. A., G. L. Andersen, M. E. Conrad, O. U. Mason, Y. M. Piceno, W. T. Stringfellow, J. Zhou, and T. C. Hazen. 2010. Response of petroleum-degrading microbial communities to the Deepwater Horizon oil spill at the surface and in the deep. Annual Meeting of the Society for Industrial Microbiology.
  505. Hazen, T. C. 2010. Environmental Microbiology - In Situ Bioremediation and Ecogenomics. Annual Meeting of the Society for Industrial Microbiology.
  506. Hazen, T. C. 2010. Environmental Microbiology ? Deconstruction Ecogenomics for Biofuels. Annual Meeting of the Society for Industrial Microbiology.
  507. Khudyakov, J. I., K. M. DeAngelis, H. Woo, S. Borglin, T. C. Hazen, and M. Thelen. 2010. Ionic liquid tolerance in Enterobacter cloacae, a lignocellulolytic bacterium isolated from tropical rain forest soil. Annual Meeting of the Society for Industrial Microbiology.
  508. Mason, O. U., E. A. Dubinsky, Y. M. Piceno, A. T. Iavarone, L. M. Tom, T. C. Hazen, and J. K. Jansson. 2010. Omics analyses of resident microbes in extreme environments such as petroleum reservoirs and deep sea oil plumes. Annual Meeting of the Society for Industrial Microbiology.
  509. Woo, H. L., K. M. DeAngelis, T. C. Hazen, and B. A. Simmons. 2010. Isolation of lignin- and cellulose- degrading bacteria from tropical soils for biofuel feedstock deconstruction. Annual Meeting of the Society for Industrial Microbiology.
  510. Reddy, A. P., M. Allgaier, J. M. Gladden, S. W. Singer, P. Hugenholtz, B. A. Simmons, T. C. Hazen, and J. S. VanderGheynst. 2010. Characterization of the activity of thermophilic microbial communities on bioenergy feedstocks. 32nd Symposium on Biotechnology for Fuels and Chemicals.
  511. Singer, S. W., J. M. Gladden, M. Allgaier, A. P. Reddy, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, and P. Hugenholtz. 2010. Targeted Enzyme Discovery in Thermophilic Feedstock-adapted Microbial Communities. 32nd Symposium on Biotechnology for Fuels and Chemicals.
  512. Goldenberg, S. 2010. Gulf oil spill: White House accused of spinning report. Guardian. http://www.guardian.co.uk/environment/2010/aug/05/oil-spill-white-house-accused-spin
  513. The Medical Muckraker. 2010. Did oil-gobbling bacteria rid the Gulf of BP?s submarine oil plumes?.
  514. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. National Commission on the BP Deepwater Horizon Oil Spill and Offshore Drilling.
  515. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. BP Deep Sea Oil Release Water Column Meeting.
  516. Hazen, T. C. 2010. Ecogenomics and biogeochemistry enables understanding of the Deepwater Horizon disaster in the Gulf of Mexico. University of Granada.
  517. Grimes, D. J., T. C. Hazen, and S. McLellan. 2010. Marine Bioremediation: The Microbial Response to the Deepwater Horizon Incident. International Marine Biotechnology Convention 2010.
  518. Hazen, T. C. 2010. Deep Water Horizon Oil Spill - Intrinsic Bioremediation or Mother Natures? Abilities to Cleanup Our Messes. City College of San Francisco.
  519. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. LBNL Open House.
  520. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. Northern California Science Writers Association Dinner.
  521. Hazen, T. C. 2010. Deep-Sea Oil Plume Enriches Indigenous Oil-Degrading Bacteria. Civil and Environmental Engineering, University of California at Berkeley.
  522. Hazen, T. C. 2010. MEHR Program Ecogenomics Core Review. Energy Biosciences Institute, University of California at Berkeley.
  523. Hazen, T. C., R. M. Atlas, D. J. Grimes, J. Spain, and J. M. Suflita. 2010. Microbes and Oil Spills Mini-Colloquium. American Academy of Microbiology.
  524. Borglin, S., O. U. Mason, E. Dubinsky, J. Fortney, R. Lamendella, D. Joyner, Y. Piceno, and T. C. Hazen. 2010. Analysis of Microbial Community Structure in Crude Oil and Oil Spill Samples Using Phospholipid Fatty Acid Analysis. Sustainable Approaches to Remediation of Contaminated Land (SARCL-2010) and Contaminated Site Management (CSM-2010).
  525. Dubinsky, E., G. L. Andersen, M. E. Concord, O. U. Mason, Y. Piceno, W. T. Stringfellow, J. Zhou, and T. C. Hazen. 2010. Response of Petroleum-Degrading Microbial Communities to the Deepwater Horizon Oil Spill at the Surface and in the Deep. Sustainable Approaches to Remediation of Contaminated Land (SARCL-2010) and Contaminated Site Management (CSM-2010).
  526. Hazen, T. C. 2010. Biosciences in Earth Sciences Division. LBNL Scientific Advisory Committee.
  527. Hazen, T. C. 2010. The Gulf of Mexico Oil Spill - Rescue from Microbiology?. Danish Society for Microbiology annual meeting.
  528. Hazen, T. C. 2010. LBNL Oil Spill Research. Town Hall Office of Chief Financial Officer LBNL.
  529. Hazen, T. C. 2010. Oil Spill Aftermath. Discover Cal Lecture Series.
  530. Hazen, T. C. 2010. Oil Spill Aftermath. Discover Cal Lecture Series.
  531. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. Vibrios in the Environment 2010.
  532. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. Contra Costa Watershed Forum.
  533. Northen, T., W. Reindl, K. Deng, J. Gladden, S. Singer, A. Singh, T. C. Hazen, B. Simmons, P. Adams, and J. Keasling. 2010. High Throughput Multiplexed GlycoChip Enzymatic Assays for Biofuels Development. American Institute of Chemical Engineers annual meeting.
  534. Singer, S. W., J. M. Gladden, P. D'haeseleer, M. Allgaier, D. C. Chivian, T. C. Hazen, J. S. VanderGheynst, P. Hugenholtz, and B. A. Simmons. 2010. Targeted Discovery of Enzymes From Enriched Microbial Consortia for High Temperature Saccharification of Ionic-Liquid Pre-Treated Biomass. American Institute of Chemical Engineers annual meeting.
  535. -----. 2010. 3 Future Oil-Spill Fighters: Sponges, Superbugs, and Herders. Berkeley Lab Media Report.
  536. -----. 2010. Caution Required for Cleanup of Oil Spill. Today at Berkeley Lab.
  537. -----. 2010. Caution Required for Gulf Oil Spill Clean-Up, Bioremediation Expert Says. www.sciencedaily.com.
  538. -----. 2010. Caution urged in oil spill cleanup. www.upi.com/Science_News.
  539. -----. 2010. Chemical Detergents May Make Gulf Oil Disaster Worse, Say Experts. www.foxnews.com.
  540. -----. 2010. Deepwater Horizon Spill Detergents Could Make Bad Situation Worse. www.outlookseries.com.
  541. -----. 2010. Disperse and Conquer. www.mnn.com.
  542. -----. 2010. Extreme caution required in Gulf Oil-spill clean up: Berkeley expert. www.thaindian.com.
  543. -----. 2010. Oil spill. KCBS all news radio.
  544. -----. 2010. Plan B in the Gulf. www. Almendron.com.
  545. -------. 2010. Lab Scientist Pens New York Times Op-Ed on Gulf Spill. Today at Berkeley Lab.
  546. Adame, J. 2010. Clean-up role for microbes is sought. Abilene Reporter News.
  547. Fowler, P. 2010. Oil Cleanup Could Be Worse For Environment Than Actual Spill, Expert Says. www.newsroomamerica.com.
  548. Graffis, M. 2010. Caution Required for Gulf Oil Spill Clean-up. misc.activism.progressive.
  549. Harrell, A. 2010. Your Hair Is Not Enough to Combat Major Oil Spill. SFweekly, blogs.sfweekly.com.
  550. Hazen, T. C. 2010. Extreme Caution Required for Gulf Oil Spill Clean-up. Axis of Logic.
  551. Kaufman, R. 2010. 3 Future Oil-Spill Fighters: Sponges, Superbugs, and Herders. National Geographic Daily News.
  552. Lowe, M. 2010. Tactics and Techniques for Tackling the Oil Spill. Magblog.audubon.org.
  553. Ott, R., K. Arnold, J. Hofmeister, T. Hazen, and K. M. Yeager. 2010. Plan B in the Gulf. The New York Times (www.nytimes.com)
  554. Outzen, R. 2010. Doing nothing is better than dispersants. http://ricksblog.biz
  555. Siegel, R. P. 2010. How to Clean Up the Oil. www.triplepundit.com.
  556. Tunnicliffe, H. 2010. BP plugs one leak, containment dome ready. www.tcetoday.com.
  557. Vieru, T. 2010. Berkeley Lab Scientists on Hot to Clean Gulf Oil Spill. Sci Pry.
  558. Yarris, L. 2010. Caution Required for Gulf Oil Spill Clean-up. Berkeley Lab News Center.
  559. Yarris, L. 2010. Caution Required for Gulf Oil Spill Clean-up. www.articleant.com
  560. Hazen, T. C., E. Dubinsky, and O. U. Mason. 2010. Deepwater Horizon Oil Spill. LBNL chapter of Institute for Nuclear and Particle Astrophysics (INPA)
  561. Hazen, T. C. 2010. Forum on BP Oil Disaster. Mt. Diablo Peace & Justice Center.
  562. Hazen, T. C. 2010. Gulf Oil Spill, Update. LBNL Earth Sciences Division Town Hall meeting.
  563. Paine, A. 2010. Tennesseans donate hair, pantyhose to sop up oil spill.
  564. Smith, M. 2010. Bugs in the Water.
  565. Tasker, F. 2010. BP's Gulf leak boosts interests in oil-eating microbes.
  566. Tierney, J. 2010. BP's Detergents Might Cause More Harm.
  567. Hazen, T. C. 2010. Gulf Oil Spill, Bioremediation the Hope and the Hype. LBNL Center for Science and Engineering Education.
  568. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. LBNL invited press conference for AGU annual meeting.
  569. Hazen, T. C. 2010. Open ocean studies on the Deepwater Horizon oil incident. Science@Cal.
  570. Vilcaez, J., L. Li, S. S. Hubbard, and T. C. Hazen. 2010. Biodegradation of Deep-Sea Oil Spill at the Gulf of Mexico: an Estimate of Half Life Time. AGU annual meeting.
  571. .... 2010. BP Watch: Hiding the oil.
  572. ……. 2010. Gulf oil spill: White House accused of spinning report.
  573. Hazen, T. C. 2010. Gulf spill biodegradation rates and ecogenomics. National Incident Response JAG.
  574. Hazen, T. C. 2010. Gulf spill biodegradation rates and ecogenomics. NOAA National Science Response Team.
  575. Hazen, T. C. 2010. Systems Biology: The New Frontier for Environmental Biotechnology. Army Sponsored Microbial Data Integration Workshop.
  576. -------. 2010. Cleaning Hazardous Waste with Green Microbes. Today at Berkeley Lab.
  577. Trevedi, B. 2010. Feature Story ? The Right Bugs. www.miller-mccune.com.
  578. -----. 2010. Plan B in the Gulf. Bloomberg Surveillance.
  579. Byrne-Bailey, K. G., K. C. Wrighton, R. A. Melnyk, T. C. Hazen, and J. D. Coates. 2010. The First Genome Sequence of a Gram-Positive Bacterium Isolated from a Microbial Fuel Cell: Thermincola potens strain JR. Joint Genome Institute Users Meeting.
  580. Hazen, T. C. 2010. A Systems Biology Approach to the Environment Using Ecogenomics. Earth Sciences Division Review.
  581. Metzner, J. 2010. Pulse of the Planet.
  582. ……. 2010. Despite BP efforts to clean Gulf, nature will do most of it.
  583. Morgan, C. 2010. When will spill be cleaned up? Maybe never. The Republic. http://www.therepublic.com/view/story/OILSPILL-CLEANUP_2910483/OILSPILL-CLEANUP_2910483/
  584. Morgan, C. 2010. When will spill be cleaned up? Maybe never. Kansas City Star. http://www.kansascity.com/2010/07/31/2119839/when-will-spill-be-cleaned-up.html
  585. Raval, A. 2010. The right spill response: cautious optimism about the Gulf?s future.
  586. Schoof, R. 2010. Despite Gulf cleanup efforts, nature will have to do most it. http://www.mcclatchydc.com/2010/07/16/97702/despite-gulf-cleanup-efforts-nature.html
  587. Hazen, T. C. 2010. Life in the Slow Lane: Ecogenomics in Extreme Environments - Implications for Enhanced Oil Recovery. Harbin Institute of Technology
  588. Hazen, T. C. 2010. Systems Biology (Integration of the Omics, Bioinformatics, and Biogeochemistry): The New Frontier for Environmental Biotechnology. Harbin Institute of Technology
  589. Hazen, T. C. 2010. Systems Biology (Integration of the Omics, Bioinformatics, and Biogeochemistry): The New Frontier for Environmental Biotechnology. Central South China University
  590. Hazen, T. C. 2010. Targeted Enzyme Discovery using Metagenomics. Harbin Institute of Technology
  591. Arkin, A. P., E. Baidoo, P. Dehal, D. Elias, M. Fields, J. Geller, T. C. Hazen, Z. He, K. Hillesland, J. Keasling, K. Keller, M. Keller, L. Krumholz, B. Meyer, L. Miller, J. Mosher, A. Mukhopadhyay, A. Palumbo, T. Phelps, M. Podar, L. Rajeev, A. Redding, C. Schadt, D. Stahl, S. Stolyer, A. Venkateswaren, C. Walker, J. Wall, Z. Yang, G. Zane, A. Zhou, and J. Zhou. 2010. Laboratory models for the study of community interaction, functional stability, and survival. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
  592. Arkin, A. P., E. Baidoo, K. Bender, P. I. Benke, A. Deutschbauer, M. Fields, T. C. Hazen, Z. He, D. C. Joyner, J. Keasling, K. Keller, E. G. Luning, A. Mukhopadhyay, L. Rajeev, J. Ray, J. D. Wall, G. Zane, A. Zhou, and J. Zhou. 2010. Laboratory models for the study of community interaction, functional stability, and survival. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
  593. Ball, D. A., S. Chhabra, D. Elias, V. Fok, J. T. Geller, A. Gorur, T. C. Hazen, D. Jorgens, T. Juba, A. Leung, J. Remis, m. E. Singer A. Tauscher, J. Wall, M. Auer, and K. H. Downing. 2010. Towards localization of functionality in Desulfovibrio vulgaris by electron microscopy. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
  594. Chhabra, S., G. Butland, D. Elias, S. Reveco, V. Fok, B. Gold, T. Juba, J.-M. Chandonia1, E. Witkowska, T. C. Hazen, J. Wall, and J. Keasling. 2010. Protein Complex Analysis Project (PCAP): Large-scale identification of protein-protein interactions in Desulfovibrio vulgaris using tandem-affinity purification. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
  595. DeAngelis, K. M., M. Allgaier, W. L. Silver, Y. Chavarria, J. Fortney. P. Hugenholtz, B. Simmons, K. Sublette, and T. C. Hazen. 2010. Trapping lignin-degrading microbes in tropical forest soil. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
  596. Gladden, J. M., A. M. Reddy, J. S. VanderGheynst, T. C. Hazen, B. A. Simmons, P. Hugenholtz, and S. W. Singer. 2010. Targeted enzyme discovery in feedstock-adapted microbial communities. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
  597. Han, B. G., H. Liu, M. Dong, M. Shatsky, S. E. Brenner, P. Arbelaez, J. Malik, D. Typke, T. C. Hazen, J. T. Geller, H. J. Sterling, L. Yang, M. Choi, E. D. Szakal, S. Allen, S. C. Hall, Susan J. Fisher, E. R. Williams, J.-M. Chandonia, J. Jin, H. E. Witkowska, R. M. Glaeser, M. D. Biggin. 2010. High Throughput Identification, Purification and Structural Characterization of Soluble Protein Complexes in Desulfovibrio vulgaris. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
  598. Hazen, T. C., G. Anderson, S. Borglin, E. Brodie, S. van Dien, M. Fields, J. Fortney, J. Geller, E. Hendrickson, K. L Hillesland, H.-Y. Holman, J. Leigh, T. Lie, D. Joyner, R. Chakraborty, D. Elias, A. Mukhopadhyay, C. Schadt, D. Stahl, S. Stolyar, C. Walker, J. Wall, Z. Yang, H.-C. Yen, G. Zane, J. Zhou. 2010. Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers ENIGMA:VIMSS:ESPP. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
  599. Holman, H.-Y., E. Wozei, L. R. Comolli, S. A. Ball, S. E. Borglin, M. W. Fields, T. C. Hazen, and K. H. Downing. 2010. Real-Time monitoring of Chemical Environment in Cells during Stress-Adaptive Response. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
  600. Van Nostrand, J. D., L. Wu, P. Waldron, P. Zhang, Y. Deng, Z. He, W. Wu, S. Carroll, C. Schadt, A. Palumbo, D. Watson, C. Criddle, P. Jardine, T. C. Hazen, and J. Zhou. 2010. Applications of GeoChip for analysis of different microbial communities. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010.
  601. Walian, P. J., S. Allen, L. Zeng, E. Szakal, E. Johansen, H. Liu, S. C. Hall, S. J. Fisher, M. E. Singer, J. T. Geller, S. Lin, T. C. Hazen, H. E. Witkowska, M. D. Biggin, and B. K. Jap. 2010. Pipeline for Large-scale Purification and Identification of Desulfovibrio vulgaris Membrane Protein Complexes. Genomics:GTL Contractor-Grantee Workshop VIII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2010
  602. ---------. 2010. Hazen et. al. Deep-sea oil plume enriches psychrophilic oil-degrading bacteria.
  603. Gupta, S. 2010. Deep oil in Gulf appears to have vanished.
  604. Gupta, S. 2010. Gulf spill: Is the oil lurking underwater?.
  605. Hazen, T. C. 2010. Undersea oil-eating bacteria. http://www.sciencefriday.com/program/archives/201008275
  606. Morgan, C. 2010. When will spill be cleaned up? Maybe never. Brandenton. http://www.bradenton.com/2010/08/02/2474305/recovery-test-of-technology-nature.html
  607. Wurth, J. 2010. Professor's expertise might help with cleanup of future oil spills. The News Gazette. http://www.news-gazette.com/news/environment/2010-08-08/professors-expertise-might-help-cleanup-future-oil-spills.html
  608. -----. 2010. 5-May. www.thaindian.com.
  609. -----. 2010. Gulf Oil Spill Solution ? Donated Hair? May 6. Today at Berkeley Lab.
  610. Waters, K. 2010. Oil Spill Eaten By RidX? June 22.
  611. Dubinsky, E., C. Wu, J. Hulls, Terry C. Hazen and G. L. Andersen. 2009. A complete microbial community approach to tracking fecal pollution in coastal waters. 9th Biennial State of the San Francisco Estuary Conference
  612. Hazen, Terry C. and G. L. Andersen. 2009. LBNL SuperChip Grand Challenge. Science and Technology, U. S. Department of Homeland Security
  613. Hazen, Terry C. 2009. Life in the Slow Lane: Ecogenomics in Extreme Environments - Implications for Enhanced Oil Recovery. University of California at Berkeley, Energy Biosciences Institute Seminar
  614. Martinez, R. J. and C. W., Terry C. Hazen, G. L. Andersen, M. Taillfert, P. A. Sobecky, Patricia K. Salome. 2009. Uranium Immobilization by the Activities of Microbial Phosphatases. Geological Society of America Annual Meeting
  615. Chakraborty, R., T. C. Hazen, D. C. Joyner, M. E. Singer and T. Torok. 2009. Use of Immunomagnetic Separation to Detect Microorganisms in Environmental Samples. Annual meeting of the American Society for Microbiology abstract
    Background: Immunomagnetic separation (IMS) has been shown highly efficient for recovering microorganisms from heterogeneous samples. Current investigation targeted extremely low-biomass environmental samples. Methods: Paramagnetic beads coated with antibodies to surface antigens of bacteria were used to capture Desulfovibrio vulgaris cells in both bioreactor grown laboratory samples and real-world subsurface drilling samples. First, detection and recovery efficiency for IMS were studied with laboratory grown D. vulgaris cells using various cell densities. Field samples were obtained from the chromium (VI)- contaminated Hanford site, an ongoing bioremediation project of the U.S. Department of Energy. Results: Anti-Desulfovibrio vulgaris antibodies were raised in rabbit, collected, and purified by Pacific Immunology Corporation (Ramona, CA). Molecular Probes (Eugene, OR) DSB-X Biotin Protein Labeling Kit was used to label antibodies with the unique biotin ligand. After antigenantibody reaction, Dynabeads® FlowComp™ Flexi Kit (Dynal Invitrogen, Oslo, Norway) captured the targeted D. vulgaris cells. 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). Conclusion: Excellent recovery efficiency encouraged us to use IMS to capture Desulfovibrio spp. cells from low-biomass real-world environmental samples. Field deployable adaptation of IMS may change environmental sampling and bioremediation process monitoring by enabling transcriptomic and proteomic based studies directly on cells collected from the field.
  616. Chhabra, S. R. and D. Elias, V. Fok, R. Prathapam, T. Juba, J-M. Chandonia, E. Witkowska, Terry C. Hazen, J. Wall, J. Keasling G. Butland. 2009. A High Throughput Genetics Pipeline for Identifying Protein-Protein Interactions in Desulfovibrio vulgaris Using Tandem-affinity Purification. Annual meeting of the American Society for Microbiology abstract
    Most cellular processes are mediated by multiple proteins interacting with each other in the form of multi-protein complexes and not by individual proteins acting in isolation. In order to extend our functional genomics analyses of stress responsepathways in Desulfovibrio vulgaris, we want to study the role of protein complexes in this sulfate reducing bacterium which has been found to exist in several DOE waste sites. Here we report the development of a technological platform for rapid identification of protein-protein interactions from a library of D. vulgaris mutant strains expressing tagged proteins. Our existing platform is based on the single cross-over chromosomal integration of tagged constructs generated in E. coli and we demonstrate the successful implementation of tagged strain generation, verification and identification of interacting protein partners. We demonstrate the strain generation process using automated software and hardware tools such as LIMS for automated sequence alignments and strain tracking, liquid handling systems for processing nucleic acids. We generated 363 tagged (STF/SPA) clones using the two-step TOPO-Gateway® cloning approach (Invitrogen) of which 278 were electroporated into competent D. vulgaris cells. We confirmed the single cross-over integration of 76 strains and the expression of affinity tagged fusion proteins using anti-FLAG IP-western blots and the verified strains were then subjected to TAP purification and MS based identification of interacting proteins. The TOPO-Gateway® mediated single-crossover approach works best for genes located at terminal ends of operons. In order to overcome these limitations, we have further developed the use of a double cross-over approach mediated through Sequence and Ligation Independent Cloning (SLIC). This approach may be applied for genome wide manipulation of gene insertions or deletions. We will report our efforts towards adapting this method into a high throughput affinity tagged strain generation platform applicable to non-model organisms. Acknowledgments/References: This work was part of the Virtual Institute for Microbial Stress and Survival (http://VIMSS.lbl.gov) supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomics:GTL Program through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. This document was prepared as an account of work sponsored by the United States Government.
  617. Elias, D. A. and M. D. Biggin, G. Butland, S. Chhabra, A. Fagorala, Terry C. Hazen, D. Jorgans, D. C. Joyner, T. R. Juba, M. Perez, J. P. Remis, A. Tauscher, J. D. Wall M. Auer. 2009. Protein Complex Analysis Project (PCAP): Localization of Multi-Protein Complexes through SNAP-Tag Labeling. Annual meeting of the American Society for Microbiology abstract
    The broad goal of this subproject of PCAP is to develop tag-based labeling approaches for highthroughput subcellular localization of proteins in microorganisms of interest to DOE. Our goal is to determine the abundance, the spatial organization and relative locations of proteins within Desulfovibrio vulgaris Hildenborough (DvH) cells in pure cultures, as well as in DvH in microbial communities under both routine laboratory and environmentally relevant stress conditions. The key to an effective high-throughput approach has been the development of a functional genetic tagging approach. We chose to introduce tags onto single copies of the genes encoding the target proteins that were regulated by gene-specific wild-type promoter sequences. We have concentrated our efforts on SNAP-labeling (Covalys Technologies) which fuses the 20 kDa DNA repair enzyme O6-alkylguanine-DNA alkyltransferase to the target protein. This enzyme reacts specifically and rapidly with fluorescently labeled benzylguanine derivatives, leading to the covalent labeling of the SNAP-tag with the fluorescent probe. Both the commercially available Gateway vectors and Sequence and Ligation Independent Cloning (SLiC) plasmids and methodologies were used to ligate the SNAP tag onto the carboxy terminal end of the gene of interest. To date, it appears that the SLiC methodology for tagged gene plasmid production is cheaper, faster and has the advantage of ease of introduction of a promoter for expression of downstream genes in an operon. As a proof of principle for our fluorescent tag visualization, we have extended our approach to a small number of E.coli constructs where the localization of the proteins are better established. Once the labeling and photoconversion protocol are sufficiently robust, they can be applied to a growing number of DvH tagged strains under a variety of environmental conditions, including stress conditions.
  618. Brileya, K. A. and S. Stolyar, D. A. Stahl, A. P. Arkin, Terry C. Hazen, M. W. Fields C. B. Walker. 2009. Temporal and Spatial Organization within a Syntrophic Bacterial-Archaeal Biofilm. Annual meeting of the American Society for Microbiology abstract
    A syntrophic co-culture of the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and the methanogenic archaeon, Methanococcus maripaludis, was selected as a basal community that can directly and indirectly interact as a biofilm. It was hypothesized that hydrogen transfer would dictate co-culture biofilm formation in the absence of sulfate as terminal electron acceptor for D. vulgaris and without addition of hydrogen as electron donor for the methanogen. M. maripaludis did not form significant biofilms on a glass surface in batch mono-culture experiments, but D. vulgaris did. However, M. maripaludis did form a pellicle-like structure in batch, static cultures. A biofilm reactor was developed to co-culture D. vulgaris and M. maripaludis during syntrophic growth, and spatial and temporal organization was characterized using qPCR, epifluorescent microscopy, field emission electron microscopy, methane production and protein and carbohydrate analysis. During early development, the biofilm initiated as a monolayer of D. vulgaris cells, and the mainly D. vulgaris biofilm contained extracellular filaments that have been previously described. Soon after the development of the D. vulgaris biofilm, M. maripaludis cells were observed, and the number of planktonic phase cells declined as the number of biofilm cells increased for both populations. Over time, the methanogenic biofilm stabilized, and the ratio of D. vulgaris to M. maripaludis cells was approximately 2.5 and this is a similar ratio observed for cultures entirely populated by planktonic cells. However, at later time points, the planktonic populations had a ratio of approximately 0.2, and this ratio was significantly lower compared to biofilm. Both populations had 1 to 2 log more cells in the biofilm than the planktonic phase. As the methanogenic biofilm developed, extracellular structures continued to be observed. The results suggested that D. vulgaris initiated and established a biofilm that then recruited M. maripaludis, and the biofilm grew and changed over time as the numbers of both populations increased.
  619. Chakraborty, R. and A. Zhou, M. Joachimiak, S. Borglin, A. Mukhopadhyay, Z. He, A. P Arkin, J. Zhou, T. C Hazen J. Fortney. 2009. Investigation of salt stress responses in the metal reducing organism Geobacter metallireducens. Annual meeting of the American Society for Microbiology abstract
    In an effort to investigate effects of environmental stressors on bacteria that are found to co-exist in several DOE-contaminated sites, we studied the effect of salt stress on Geobacter metallireducens strain GS15. A comparison of these results with salt stress in Desulfovibrio vulgaris and Shewanella oneidensis will provide a better understanding of bioremediation by these bacteria under hypersaline conditions. Strain GS15 was grown in the defined LS4D medium at 30°C containing 10mM acetate and 10mM Fe-NTA. In these growth conditions, the minimum inhibitory concentration of NaCl on this organism was determined to be 100mM. To examine salt stress response of GS15 in detail, 100mM NaCl was added to a midlog phase culture and cells were harvested after 1, 2 and 4 hours for different analyses including Fe(III) reduction, transcriptomics and phospholipids fatty acid analysis (PLFA). In stressed cultures, after 4 hours of exposure to salt, cell numbers reached 5.1 X 10^7/ ml compared to 9.4 X 10^7/ml in non-stressed controls. Transcriptomic analysis revealed that after 4 hrs, the most up-regulated genes observed were those that encode heat shock proteins, such as Hsp90, Hsp 20, GrpE, DnaK, and DnaJ. These heat shock proteins perform protein repair functions which can be in demand during stress. Also up-regulated were genes encoding proteins in the large and small subunits of the ribosome The most down-regulated genes included those encoding the, sodium ion symporter, some genes of cytochrome C family and flagellar proteins (flagellin, FlaG, and FlgJ). It was also observed that several proteins involved in the process of chemotaxis like CheA, CheR, CheY, CheX along with several histidine kinases and response regulators were down-regulated. PLFA analysis of stressed cells was performed and compared to non-stressed cells, showing small shifts in relative levels of saturated and unsaturated fatty acids in response to salt stress. The results of this study suggests that presence of 100mM NaCl causes some perturbation in the cell machinery of strain GS15 and that cells are able to overcome this stress without fatal consequences. Acknowledgments/References: U.S.
  620. DeAngelis, K. M, W. L. Silver and Terry C. Hazen. 2009. Identification and Characterization of Lignocellulolytic Microbial Communities in Puerto Rican Wet Tropical Forest Soils. Annual meeting of the American Society for Microbiology abstract
    Soils in humid tropical forests have extremely fast decomposition rates, with most mass lost within a year. Fluctuating redox and Fe-oxide mineralogy likely contribute to high activity, and frequent episodes of anoxia are likely brought on both by limitation of oxygen diffusion through water as well as biological oxygen demand induced by imported organic carbon. These conditions suggest that decomposing consortia are primarily bacteria, not fungi as are usually observed, and that makes this system attractive for investigating their capability for deconstructing plant material to basic biofuel components. The objective of this research is to define field conditions and microbial communities responsible for high decomposition and methane production in Puerto Rico forest soils. Towards this end, we designed a field experiment in which litterbags filled with switchgrass were buried in June 2008 in four different forest types in the Luquillo LTER (El Yunque National Forest, Puerto Rico, USA). The four forest types range from more aerobic soils, warmer temperatures and annual precipitation of ~1,000 mm, to fluctuating redox soils, to mostly anaerobic, cooler soils and annual precipitation exceeding 4,000 mm. The most aerobic site was instrumented with oxygen sensors to measure trace gasses at sampling and soil oxygen levels on an hourly basis over the course of this year-long incubation. At each of 6 time points over a year, litter bags and soil are collected from the field and assayed for microbial community composition by 16S ribosomal DNA PhyloChip as well as quantitative PCR of target populations, potential enzyme activity (b-glucosidase, endoglucanase xylosidase, chitinase, phenol oxidase and peroxidase), and mass loss as an indication of decomposition. We also buried biosep beads as bug traps baited with lignin to identify microbes specifically able to decompose lignin. Results suggest that a subset of the soil community is responsible for switchgrass decomposition; that most enzyme activities are independent of oxygen availability; and that belowground decomposition is accelerated in warmer, more aerated sites.
  621. Faybishenko, B., Terry C. Hazen and S. S. Hubbard. 2009. Application of Innovative Bioremediation Technologies for Metals and Radionuclides in Soils and Groundwater. IAEA International Conference on Remediation of Land Contaminated by Radioactive Material Residues abstract
    Bioremediation of metals and radionuclides is a relatively new approach for cleaning up metal and radioactively soils and groundwater at contaminated sites. The presentation will include: (1) a review of the main types of bioremediation technologies—biotransformation, bioaccumulation/bisorption, biodegradation using chelation, biovolatilization, and natural attenuation—as well as an application of multiple treatment methods; (2) a discussion of the promising results of ongoing research and case studies from field tests, demonstrations, and full-scale implementation of different bioremediation technologies at several contaminated sites, along with a summary of biogeochemical criteria needed to implement these technologies for cleaning up metals and radionuclides in soils and groundwater; and (3) a series of innovative in situ site characterization and long-term monitoring methods associated with these technologies. We will also emphasize the need for implementing a combination of both in situ and ex situ remediation strategies.
  622. He, Z., Y. Deng, J. D. Van Nostrand, L. Wu*, C. L. Hemme, T. J. Gentry, J. Liebich, Q. Tu, A. P. Arkin, Terry C. Hazen, J. Zhou. 2009. Development and Applications of GeoChip 3.0 for Analysis of Microbial Community Structures, Compositions, Potential Functions. Annual meeting of the American Society for Microbiology abstract
    Functional gene arrays (FGAs), or GeoChip constructed with key genes involved in various biological and geochemical processes have been widely used to analyze microbial communities. Based on GeoChip 2.0, a new generation of Geo- Chip (GeoChip 3.0) has been developed. GeoChip 3.0 has several new features in terms of coverage, design, and data analysis. GeoChip 3.0 covers approximately 50,000 gene sequences for 306 gene families, including functional genes involved in antibiotic resistance and energy metabolism. The phylogenic marker GyrB has been added, which allows us to obtain additional phylogenetic information and to analyze more diverse environmental samples. Sequence-specific, exclusive group-specific, and inclusive group-specific probes with different specificities were selected for probe design. These various types of probes greatly increase gene coverage.Three strategies have been implemented for data analysis. First, a universal standard has been implemented so that data normalization and comparison of samples from different sites, time points, or laboratories can be conducted. Second, a genomic standard is used to quantitatively analyze gene abundance. Third, a software package (including databases) has been developed for sequence retrieval, probe design, information storage, and especially, data analysis and automatic updates, which greatly facilitates the management and analysis of complicated GeoChip data sets. GeoChip 3.0 has been used for analyses of responses of microbial communities to elevated CO2 and increased temperature. The results showed that autotrophic CO2 and N2 fixation by microorganisms increased significantly in response to elevated CO2. This also demonstrates that GeoChip can provide insights into biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, global climate change, ecosystem management, and environmental cleanup and restoration. It is also particularly useful for providing direct linkages of microbial genes/populations to ecosystem processes and functions. Acknowledgments/References: This work was supported by the US Department of Energy under the Genomics: GTL Program through the Virtual Institute of Microbial Stress and Survival (VIMSS, http://vimss.lbl.gov), by Environmental Remediation Science Program (ERSP), Office of Biological and Environmental Research, Office of Science, and by the Oklahoma Center for Advancement of Science and Technology (OCAST) under Oklahoma Applied Research Support (OARS) Project AR062-034.
  623. Hemme, C. L. and T. J. Gentry, M. W. Fields, L. Wu, K. Barry, S. Green-Tringe, D. B. Watson, Z. He, Terry C. Hazen, J. M. Tiedje, E. M. Rubin, J. Zhou Y. Deng. 2009. Analysis of a Microbial Metagenome from a Pristine Groundwater Ecosystem. Annual meeting of the American Society for Microbiology abstract
    Previous analyses of a microbial metagenome from uranium and nitric-acid contaminated groundwater (FW106) showed significant environmental effects resulting from the rapid introduction of multiple contaminants. Effects include a massive loss of species and strain biodiversity, accumulation of toxinresistant genes in the metagenome and lateral transfer of toxin resistance genes between community members. To better understand these results in an ecological context, a second metagenome from a pristine groundwater system located along the same geological strike was sequenced and analyzed (FW301). It is hypothesized that FW301 approximates the ancestral FW106 community based on phylogenetic profiles and common geological parameters; however, even if is not the case, the datasets still permit comparisons between healthy and stressed groundwater ecosystems. Complex carbohydrate metabolism has been almost entirely lost in the stressed ecosystem. In contrast, the pristine system encodes a wide diversity of complex carbohydrate metabolism systems, suggesting that carbon turnover is very rapid in the healthy groundwater system. FW301 encodes many (~160+) carbon monoxide dehydrogenase genes while FW106 encodes none. This result suggests that the community is frequently exposed to oxygen from aerated rainwater percolating into the subsurface, with a resulting high rate of carbon metabolism and CO production. When oxygen levels fall, the CO then serves as a major carbon source for the community. FW301 appears to fix CO2 via the reductive carboxylase (reverse TCA) cycle and possibly by acetogenesis, activities; these activities are lacking in the heterotrophic FW106 system which relies exclusively on respiration of nitrate and/or oxygen for energy production. Overall comparative analysis suggests that the introduction of contaminants is accompanied by a decrease in biodiversity, loss of nutrient cycling, less metabolic diversity, increased respiration and dominance by r-strategists. These results are consistent with trends predicted for stressed ecosystems. Acknowledgments/References: The authors would like to thank Dr. Fares Najar and Dr. Bruce Roe for providing sequencing services, and Dr. Tommy Phelps and Dr. Christopher W. Schadt assisted in groundwater sampling. This research was supported by The United States Department of Energy under the Environmental Remediation Science Program (ERSP), and Genomics: GTL program through the Virtual Institute of Microbial Stress and Survival (VIMSS; http://vimss.lbl.gov), Office of Biological and Environmental Research, Office of Science, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52- 07NA27344, and Los Alamos National Laboratory under contract No. DE- AC02-06NA25396. Oak Ridge National Laboratory is managed by University of Tennessee UT-Battelle LLC for the Department of Energy under contract DE-AC05-00OR22725
  624. Kang, S. and J. Van Nostrand, Z. He, L. Wu, D. A. Stahl, Terry C. Hazen, J. Zhou H. L. Gough. 2009. Controlling Factors of Sediment Microbial Communities at the Metal Contaminated Freshwater Lake (Lake DePue). Annual meeting of the American Society for Microbiology abstract
    Microbes are known for their versatility under different and even certain extreme environmental conditions. The effect of extreme environments on microbial distribution is of special interest. Lake DePue (Illinois, USA) was subjected to metal contamination for about 80 years from adjacent zinc smelting activities. Sediments were collected in triplicate from five areas of the lake along a transect from near a creek inlet to the main body of the lake. GeoChip II, with over 10,000 microbial functional genes, was used to investigate metal impact on the microbial communities and their distributions. In addition to comparison of the overall microbial community, three microbial communities of functional subgroups, defined by genes relevant to metal contamination (S cycling, metal resistance & reduction, and C cycling genes) were examined. While most environmental variables were spatially structured, the pore water metal concentration, total and three subgroup microbial communities were not spatially structured except for a very significant single patch at ~250 m in microbial communities. Polyphasic approaches identified 2-4 metals (e.g., zinc, arsenic, lead etc) were the likely controlling factors for predicting different microbial communities. While geographic distance between areas was held constant, selected pore water metals (Zn, As and Pb) showed fairly significant correlations with microbial communities (P < 0.099) except for the S cycling community. Canonical correspondence analysis (CCA) models with the same sets of pore water metals were also very significant based on Monte Carlo permutation (P < 0.013) except for the S cycling community. Canonical variance partitioning analysis (VPA) results indicated that selected pore water metals explained more variances than geographic distance, and the significance of the pore water metals effect was much higher in all microbial communities. In conclusion, microbial communities in sediment from Lake DePue varied significantly with the level of metal contamination.
  625. Knierim, B. and S. Singh, D. Jorgens, M. Zemla, K. DeAngelis, A. P. Reddy, J. VanderGheynst, Terry C. Hazen, B. M. Holmes, R. Sapra, B. A. Simmons, P. D. Adams, M. Auer L. Prak. 2009. Electron Microscopic Imaging at JBEI. 31st Symposium on Biotechnology for Fuels and Chemicals, Society for Industrial Microbiology
  626. Kozina, C. L. and D. Joyner, K. L. Sale, D. S. Reichmuth, Terry C. Hazen, R. Sapra A. S. Pawate. 2009. Metabolic engineering of a novel thermophilic ethanologen Geobacillus thermoglucosidasius M10EXG for enhanced ethanol production. 31st Symposium on Biotechnology for Fuels and Chemicals, Society for Industrial Microbiology abstract
    The thermophilic bacterium Geobacillus thermoglucosidasius (Gth) M10EXG is a facultative anaerobe that has an optimal growth temperature of 60 oC. It can metabolize both C5 (xylose) and C6 (glucose) sugars and is tolerant to 10% ethanol, making it an attractive candidate for industrial bioethanol production from lignocellulosic biomass. However, in order to maximize the production of ethanol from the fermentative pathway, it is essential to understand the fermentative metabolism, operational pathways, and the flux through the different fermentative pathways. We have completed a metabolic analysis of the growth of Gth M10EXG using both xylose and glucose under varying concentrations of oxygen. As expected, ethanol production is detected only under anaerobic conditions using either xylose or glucose as the sole carbon source. Furthermore, metabolic flux analysis of the anaerobic and aerobic growth using glucose as the sole carbon source shows that 0.6 mol lactate, 0.9 mol acetate, 0.4 mol ethanol, and 1.0 mol formate are produced per mole of glucose metabolized. With recent genome sequencing and metabolic flux analysis completed, we have targeted both lactate and formate production pathways for modification to increase ethanol production. We present results from the metabolic engineering of the aforementioned pathways and the effect on ethanol production.
  627. Martinez, R. J. and C. H. Wu, G. L. Andersen, Terry C. Hazen, M. Taillefert, P. A. Sobecky M. J. Beazley. 2009. Microbial Phosphatase Activity Involved in Subsurface Uranium Sequestration. Annual meeting of the American Society for Microbiology abstract
    Soils and groundwater contaminated with heavy metals and radionuclides remain a legacy of Cold War nuclear weapons development. Due to the widespread contamination of soils and groundwater, in situ sequestration of heavy metals and radionuclides has proven to be a cost-effective strategy for remediation. We are currently investigating a remediation approach that utilizes the phosphatase activity of bacterial strains extant within the contaminated soils of the DOE Field Research Center (ORFRC) in Oak Ridge, TN, for in situ contaminant sequestration. We have previously demonstrated the accumulation of PO4 3- and concomitant mineralization of U(VI) using pure cultures grown under both oxic and anoxic conditions with glycerol phosphate as the sole C and P source and NO3 -, the sole N source and terminal electron acceptor in the absence of oxygen. To determine the microbial diversity of U(VI) and NO3 - contaminated ORFRC Area 2 soils as well as the microbial community response to exogenous organophosphate additions under oxic and anoxic growth conditions, soil slurry experiments were conducted at pH 5.5. Incubations were conducted for 36 days at 25ÅãC with 10 mM G2P and 15mM NO3 - as the sole C, P and N sources, respectively. Under oxic growth conditions, greater than 4 mM soluble PO4 3- was measured at the end of the slurry incubations and NO2 - was not detected. Preliminary data obtained for anoxic soil slurry incubations indicated an accumulation of greater than 1mM PO4 3- as well as the accumulation and subsequent removal of NO2 -. Following triplicate incubations, total DNA was extracted from the slurries and 16S diversity analyzed with a high-density oligonucleotide microarray (PhyloChip). Our soil slurry studies demonstrate the efficacy of organophosphate-mediated sequestration of U(VI) driven by the diverse microbial community extant within ORFRC contaminated subsurface soils. Thus, a strategy which employs microbial hydrolysis of organophosphates could complement current remediation approaches.
  628. Ray, J. and A. Deutschbauer, K. Keller, J. Robertson, G. Zane, M. Price, S. Chhabra, J. Wall, A. Arkin, Terry C. Hazen, J. Keasling, A. Mukhopadhyay E. Luning. 2009. Study of Two-component Signal Transduction Systems in Desulfovibrio vulgaris Hildenborough. Annual meeting of the American Society for Microbiology abstract
    Two-component systems, comprised of histidine kinase (HK) and response regulator (RR) proteins, represent the primary and ubiquitous mechanism in bacteria for initiating cellular response towards a wide variety of environmental conditions. In D. vulgaris, more than 60 such systems have been predicted, but remain mostly uncharacterized. The ability of D. vulgaris to survive in its environment is undoubtedly linked with the activity of genes modulated by these two-component signal transduction systems. Using recently developed methods for in vitro confirmation of specific phosphotransfer from HKs to their cognate RRs a high throughput mapping of two-component systems is possible. This is aided by in silico methods to predict candidate partners for HKs or RRs that are either ORFans or have no proximal genes that may serve as their cognate partners. Additional corroboration of these predictions may be found from microarray data in our VIMSS database. The requirement of highly pure, active proteins for these high throughput phosphotransfer assays were fulfilled by generating expression vectors for all RRs and the soluble portions of HKs using the Gateway® Cloning system. His-tagged HKs and the corresponding cognate RRs have been purified under native conditions and used successfully in in vitro phosphotransfer mapping for selected two-component systems in D. vulgaris. The immediate applications of this research are for two-component systems discovered to be involved in specific stress studies. Availability of a library of RRs and HKs also allows us to test a variety of broader questions experimentally. For example, do the many highly homologous HKs in D. vulgaris, still maintain specific interactions with one or few RRs? Beyond testing such hypothesis, this library of purified proteins can also be used to map entire two-component signal transduction cascades. In addition, the availability of a library of HK knockout mutants allowed us to follow-up on conclusions from the in vitro phosphotransfer studies. Results from the in vitro assays and follow up studies are presented in this poster.
  629. Reddy, A. P. and P. Hugenholtz, B. A. Simmons, Terry C. Hazen, J. VanderGheynst M. Allgaier. 2009. Tracking Microbial Community Changes during Decomposition of Switchgrass. 31st Symposium on Biotechnology for Fuels and Chemicals, Society for Industrial Microbiology
  630. Van Nostrand, J. D., P. Waldron, D. B. Watson, Z. He, L. Wu, P. Jardine, Terry C. Hazen, J. Zhou. 2009. GeoChip Analysis of Groundwater Microbial Communities across a Gradient of pH, Heavy Metal, Nitrate Contamination. Annual meeting of the American Society for Microbiology abstract
    The Field Research Center (FRC) site of the U.S. DOE ERSP (Environmental Remediation Science Program) at Oak Ridge, TN, is contaminated with radionuclides, heavy metals, and other contaminants due to historical U enrichment activities. To examine the impact of contaminant level on microbial communities, five contaminated and one uncontaminated well, providing a gradient of groundwater nitrate, pH and U concentrations, were sampled. DNA from these samples was analyzed with a comprehensive functional gene microarray (GeoChip 2.0) containing probes for >10,000 genes involved in carbon, sulfur, and nitrogen cycling, contaminant degradation and metal resistance and reduction. Results of principle component analysis of functional genes showed that the most contaminated wells clustered together and had the greatest gene overlap. Gene abundance correlated with contaminant levels. Higher percentages of nitrogen fixation genes were detected in wells with lower nitrate concentrations, while the percentage of nitrate reduction genes generally decreased with decreasing nitrate. Wells with elevated sulfate concentrations had a greater percentage of genes dedicated to sulfate reduction, and higher signal intensities for dsrAB genes than the background, indicating a greater abundance of those genes. The total signal intensity of metal resistance and reduction genes in contaminated wells was greater than the background, indicating that metal-related genes were more prevalent in the contaminated wells. CCA indentified pH, Sr, U, Cs, and NO3 as the most important environmental variables in determining community structure. This study provides an overall view of the functional genes present in a highly contaminated environment, and shows that the contaminant level has significant effects on bacterial community structure.
  631. Van Nostrand, J. D., P. J. Waldron*, W. Wu, L. Wu, Y. Deng, J. Carley, Z. He, C. S. Criddle, P. Jardine, Terry C. Hazen, J. Zhou. 2009. GeoChip-based Analysis of Functional Microbial Communities in a Bioreduced Uranium-contaminated Aquifer during Nitrate Exposure. Annual meeting of the American Society for Microbiology abstract
    A pilot-scale system at the U.S. DOE’s Field Research Center in Oak Ridge, TN was established for biostimulation of subsurface U(VI) reduction by injection of ethanol. The system was able to reduce U(VI) to a level below EPA drinking water standards. In this study, stability of the bioreduced area during oxidation by nitrate was examined. Introduction of nitrate to the reduced area resulted in U(IV) oxidation but subsequent removal of the nitrate and injection of ethanol resulted in reduction of the U(VI) back to the low level. Geochip 2.0, a comprehensive 50mer microarray containing probes for genes involved in the geochemical cycling of N, S, and C, metal resistance and contaminant degradation, was used to monitor the dynamics of the groundwater microbial community structure and function before, during, and after reoxidation of U(IV) by nitrate. The numbers of functional genes detected decreased 10- to 20- fold following the increase in nitrate concentration and were slow to recover after nitrate levels decreased. The relative abundance of each functional gene group was similar prior to the increase in nitrate level, then the relative proportion of nitrogen fixation genes increased from 1-2% to over 20%. By the end of the study period, the relative abundance of all gene groups returned to the original values. Detrended correspondence analysis indicated that communities before and immediately following introduction of nitrate were similar. A shift in community structure occurred during the period of nitrate elevation and for several months afterwards; however, by day 1607, approximately 200 days after nitrate exposure and then injection of ethanol, the communities had begun to recover and were more similar to the initial community. Canonical correspondence analysis indicated that Fe(II), pH, U, and nitrate were the most important environmental variables in controlling community structure (p=0.04). These results demonstrate that introduction of elevated nitrate levels dramatically affected the microbial community structure; however, the community was able to recover.
  632. Wu, C. H. and L. C. Van De Werfhorst, T. Z. DeSantis, E. L. Brodie, Terry C. Hazen, G. L. Andersen, P. A. Holden B. Sercu. 2009. Bacterial Biogeography of an Urban Creek Impacted with Fecal Pollution. Annual meeting of the American Society for Microbiology abstract
    High levels of fecal bacteriological contamination in coastal waters are often attributed to nearby human development. Tracking inland sources of fecal bacteria can be a significant challenge. To address this in Santa Barbara, CA, a multi-phase study has been conducted in an urban coastal creek that discharges into an impacted beach frequently posted with warnings. TRFLP and high-density microarray (PhyloChip) were used to characterize bacterial community composition during dry weather at several locations during three consecutive days. For comparison, human fecal and two sewage samples were analyzed as well. Spatial and temporal variations in bacterial community composition were characterized. The Mantel test was used to compare the TRFLP and the PhyloChip data, and a significant correlation (r=0.5881) was obtained. There is a positive significant correlation (r=0.6188) between the environmental variables similarity matrix and the community similarity matrix. In addition, a strong positive significant correlation (r=0.8132) was determined between the geographical distance of the sites and day 3 of the community composition, but not for day one or two. PhyloChip analysis enabled identification of transient and resident bacterial communities over the three-day sampling period. Bacterial community profiles comparing different habitat types (ocean, lagoon, creek and fecal) indicated that more than 50% of the community composition in ocean, lagoon and creek samples consists of Proteobacteria. Fecal samples had higher percentages of Clostridia and Bacilli than all water samples. When only fecal-associated OTUs (taxa common to all three sewage/fecal samples) were considered, grouping according to location was stronger, and relatedness to sewage remained similar. The microarray metagenomics approach enabled the characterization of bacterial communities in a sewageimpacted urban creek. The results from this study enhance our understanding of bacterial community fluxes over short period of time and small spatial scale, and will guide further development of fecal contamination detection with the PhyloChip.
  633. Wu, L. Y., J. D. Van Nostrand, T. J. Gentry, Z. J. Huang, C. W. Schadt, W. M. Wu, D. Watson, M. W. Fields, C. S. Criddle, J. Tiedje, Terry C. Hazen, J. J. Zhou. 2009. Microbial Community Dynamics and the Effect of Geochemistry in Uranium Bioremediation Revealed by Functional Gene Array Analysis. Annual meeting of the American Society for Microbiology abstract
    Composition and dynamics of the functional microbial communities were examined in a pilot-scale system of in situ bioremediation and immobilization of U(VI) at a highly contaminated aquifer at the U. S. DOE’s Field Research Center, Oak Ridge, TN. Samples were collected from three monitoring wells (FW101-2, 102-2, and 102-3) during a period of active U(VI) reduction (days 161-719) and were studied using GeoChip 2, a functional gene array containing >24,000 probes covering 10,000 functional genes. About 20% of functional genes on the GeoChip 2 were detected. Higher percentages of cytochrome C (25-31%), metal resistant (20-25%), and methane oxidation genes (29% in FW101-2) were detected. Detrended correspondence analysis showed that the microbial communities were more similar withinwells while changed along the process of the U(VI) bioremediation. Microbial populations, as indicated by gene numbers, increased slowly during the early phase of operation due to ethanol injections, reached a peak late around 100 days and then decreased and stabilized. The microbial community structure continued to changeover the course of these experiments. Cytochrome genes in FW102-2 increased from day163 (9.4%) to 184 (12.0%) then decreased and stabilized after day248. Nitrogen cycling genes showed a similar trend, while sulfate reducing genes changed little. The populations of six metal reducing microorganisms (e.g., Desulfovibrio desulfuricanse G20 and Anaeromyxobacter dehalogenans 2CP-C) shifted during the process as U(VI) decreased and followed similar patterns in all three wells. The results of canonical correspondence analyses indicate that the microbial communities were significantly affected by geochemistry including COD, nitrate, sulfate, and pH. Variance portioning analysis revealed that the effect of COD on the microbial communities was independent from other geochemical factors. Metal test results showed that the effects of geochemistry on microbial communities changed at different stages of U(VI) bioremediation. Acknowledgments/References: DOE
  634. Wu, L. Y. and Y. Q. Luo, J. D. Van Nostrand, R. A. Sherry, Z. L. He, Terry C. Hazen, J. Z. Zhou J. P. Xie. 2009. The Responses of the Underground Microbial Communities of Grassland to the Global Warming and Different Land-use Practices Revealed by GeoChip and Pyrosequencing Analyses. Annual meeting of the American Society for Microbiology abstract
    Responses of microbial communities to warming (2oC) and clipping of grasses in grassland soils at the Kessler Farm Field Laboratory in McClain County, Oklahoma, were revealed by GeoChip and pyrosequencing analyses. Samples were collected from a long-term (eight years) experiment involving four treatments (replicated on 6 plots): warmed-unclipped (WU), warmed-clipped (WC), unwarmed-clipped (UC), and unwarmedunclipped( UU). DNA was extracted using a freezegrind method. 16S rDNA was PCR amplified from each sample using tagged primer pairs, and mixed equally for pyrosequencing. 240,000 16S rRNA gene sequences (70,000,000 bp total) were obtained and 12,000 OTUs were identified based on a 97% similarity cutoff. OTUs were identified from each treatment condition: WU (3926), WC (4399), UC (2931), and UU (3328). 23 phyla or phylum equivalents were identified, although 98% of the OTUs were from only 10 phyla. Comparisons were made between treatments using response ratios based on the OTUs identified. The treatments UC and UU had the highest percentage of OTUs in common (44.3%), while WU and UC had the least (36.2%). Of the shared OTUs, 23% had significantly different quantities between WC and UU, while only 21.5% did between WU and WC. When compared to UU, UC has more OTUs that were significantly more abundant; while warmed samples have more OTUs that are less abundant. However, the warmed samples have more unique OTUs than the unwarmed samples. These results indicate that both warming and clipping altered the microbial communities in the grassland soils. Clipping seems to have increased the population of dominant species while warming decreased the abundance of the dominant species. To study the functional diversity and dynamics of the microbial communities, communities were analyzed with GeoChip 3. Cluster analysis of the GeoChip data showed that replicate samples clustered based on warming and then on clipping, indicating obvious differences in the microbial communities under warming and clipping treatments. Results of DCA showed similar relationships. More statistical analyses of the pyrosequencing and GeoChip data are underway. Acknowledgments/References: OBC
  635. Zhou, A., Z. He, G. M. Zane, C. Hemme, Y. Chen*, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou. 2009. Study of Global Gene Regulation by CRP/FNR in Desulfovibrio vulgaris Hildenborough. Annual meeting of the American Society for Microbiology abstract
    Characterization of transcriptional regulatory networks is essential for understanding cellular processes. CRP/FNR regulators are DNA binding proteins which function as positive transcription factors. There are four (DVU2547, DVU0379, DVU3111 and DVU2097) CRP/FNR homologues in the Desulfovibrio vulgaris Hildenborough genome. D. vulgaris is an obligate anaerobe and has been used as a model organism of sulfate-reducing bacterium (SRB) for studying the energy metabolism, sulfur cycling, and biocorrosion. Knowledge about the gene regulation by these regulators is limited. Evidence from other bacteria demonstrates that CRP/FNR regulators function in response to a broad spectrum of intracellular and exogenous signals such as oxidative and nitrosative stress, nitric oxide, carbon monoxide or temperature. Microarray data from D. vulgaris showed that the transcript levels of Crp/Fnr were altered in response to nitrate, nitrite, heat shock, and oxygen stresses. In order to understand the function of D. vulgaris CRP/FNR, knockout mutants for all four CRP/ FNR proteins were generated. The physiological studies of the mutants included the utilization of various electron donors and acceptors, response to different stressors, etc. The global gene regulation by these regulators was investigated by transcriptomic analysis with mutants and wildtype grown under regular and stressed conditions. Recombinant proteins for all four CRP/FNR were obtained and polyclonal antibodies were generated. The transcription data will be confirmed by in vivo evidence of the gene regulation via chromatin immunoprecipitation-chip (ChIP-chip). Then the CRP/FNR binding motif can be predicted by computational analysis. Future studies will focus on the understanding how those homologues respond to different environmental conditions.
  636. Zhou, A. and M. J. Joachimiak, P. S. Dehal, A. Pl Arkin, K. Hillesland, D. Stahl, J. Wall, Terry C. Hazen, J. Zhou Z. He. 2009. Genetic Adaptation to Salt Stress during the Long-Term Evolution of Desulfovibrio vulgaris Hildenborough. Annual meeting of the American Society for Microbiology abstract
    In order to understand the molecular mechanism of salt adaptation, a long-term evolution experiment was carried out under controlled laboratory conditions with Desulfovibrio vugaris Hildenborough, a sulfate reducing bacteria which has been recognized as a model environmental organism. With the recent advances in genome sequencing and highthroughput genomic technologies, it is possible to now link sub-cellular molecular/metabolic processes with population-level processes. Control and stressed lines (6 lines each, from a single colony-based pure culture) grown in the LS4D medium (control) and LS4D with 100 mM NaCl (stressed) were transferred every 48 hrs using one to one hundred dilution. The phenotypic response of salt tolerance was tested periodically by monitoring the growth of all cell lines in LS4D supplemented with 250 mM NaCl. The results demonstrated that the adaptation to salt stress is a dynamic process. Enhanced salt tolerance of stressed lines was observed at 300 generations and became more obvious with the increasing number of generations. De-adaptation of cell lines (500, 1000 and 1200 generations) by removal of salt stress did not affect the increased salt tolerance, indicating that the observed phenotype changes were due to genetic changes instead of physiological adaptation. Further, results of the de-adaptation experiment suggested the dynamic trend of genetic adaptation and that the genetic mutation may have become stable by 1000 generations. Gene expression profiles of the 500 and 1000 generation samples were examined using the D. vulagris whole genome microarray and the microarray data confirms the phenotypic test results. Genes involved in energy production and conversion and signal transduction mechanisms were among the gene categories with the most genes up-regulated. Whole genome sequencing of selected colonies is underway to identify beneficial genetic mutations in the D. vulgaris genome.
  637. Zhou, J. and Terry C. Hazen. 2009. High Throughput Genomic Technologies for Complex Microbial Community Analysis. Annual meeting of the American Society for Microbiology
  638. Hazen, Terry C.. 2009. Environmental Biotechnology – a Tour through the ‘Omics’. AEHS annual west coast meeting abstract
    Environmental biotechnology encompasses a wide range of characterization, monitoring and control or remediation technologies that are based on biological processes. Recent breakthroughs in our understanding of biogeochemical processes and genomics are leading to exciting new and cost effective ways to monitor and manipulate the environment. Indeed, our ability to sequence an entire microbial genome in just a few hours is leading to similar breakthroughs in characterizing proteomes, metabolomes, phenotypes, and fluxes for organisms, populations, and communities. Understanding and modeling functional microbial community structure and stress responses in subsurface environments has tremendous implications for our fundamental understanding of biogeochemistry and the potential for natural attenuation or bioremediation of contaminated sites. Monitoring techniques that inventory and monitor terminal electron acceptors and electron donors, enzyme probes that measure functional activity in the environment, functional genomic microarrays, phylogenetic microarrays, metabolomics, proteomics, and quantitative PCR are also being rapidly adapted for studies in environmental biotechnology. Integration of all of these new high throughput techniques using the latest advances in bioinformatics and modeling will enable break-through science in environmental biotechnology. A review of these techniques with examples from field studies and lab simulations will be discussed.
  639. Hazen, Terry C. 2009. Systems Biology for Bioenergy. DOE Bioenergy Research Centers Biweekly Conference Call
  640. Hazen, Terry C.. 2009. Systems Biology: The New Frontier for Bioenergy. NanoFocus and Bioenergy Oklahoma EPSCoR Annual State Conference 2009
  641. Skerker, J., A. Deutschbauer, P. Novichkov, A. Gerasimova, J. Mar, K. Wetmore, J. Baumohl, M. Price, P. Dehal, J. Kuehl, C. Wu, I. Dubchak, Terry C. Hazen, A. P. Arkin. 2009. An idiosyncratic view of Fuel Synthesis Challenges. Energy Biosciences Institute Annual Retreat
  642. Skerker, J., A. Deutschbauer, J. Mar, K. Wetmore, M. Price, P. Dehal, J. Baumohl, I. Dubchak, Terry C. Hazen, A. P. Arkin. 2009. A systems biology approach for optimizing biofuel production in Zymomonas mobilis. Energy Biosciences Institute Annual Retreat
  643. Hazen, Terry C.. 2009. EBI Microbial Enhanced Hydrocarbon Recovery. Energy Biosciences Institute Annual Retreat
  644. Hazen, Terry C., B. K. Fouke, J. D. Coates. 2009. EBI Microbial Enhanced Hydrocarbon Recovery. Energy Biosciences Institute Annual Retreat
  645. Hazen, Terry C.. 2009. Life in the Slow Lane: Ecogenomics of an Extreme Environment. BAGECON 10 International Conference abstract
    A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced using a combination of Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Moorella thermoacetica), as well as other known sulfate reducers and thermophilic organisms. D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea. Additionally, study of the deep subsurface has offered access to the simplest community yet studied by environmental genomics, perhaps consisting of just a single species that is capable of performing all of the tasks necessary for life.
  646. Hazen, Terry C. 2009. Metagenomics of Soil Rain Forest. Terragenome Workshop
  647. DeAngelis, K., W. Silver, P. D’haseleer, J. Fortney, M. K. Firestone, Terry C. Hazen. 2009. Puerto Rico tropical forest soils as reservoirs for novel lignocellulolytic enzymes and organisms. Society for Industrial Microbiology Annual Meeting
  648. Hazen, Terry C. 2009. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. Master Universitario in Biotecnologia, University of Granada
  649. Hazen, Terry C. and B. A. Simmons. 2009. Systems Biology Approaches to Environmental Microbial Adaptations for Bioenergy Products. Society for Industrial Microbiology Annual Meeting abstract
    Environmental biotechnology encompasses a wide range of characterization, monitoring and control or bioenergy technologies that are based on biological processes. Recent breakthroughs in our understanding of biogeochemical processes and genomics are leading to exciting new and cost effective ways to monitor and manipulate the environment and potentially produce bioenergy fuels. Indeed, our ability to sequence an entire microbial genome in just a few hours is leading to similar breakthroughs in characterizing proteomes, metabolomes, phenotypes, and fluxes for organisms, populations, and communities. Understanding and modeling functional microbial community structure and stress responses in subsurface environments has tremendous implications for our fundamental understanding of biogeochemistry and the potential for making biofuel breakthroughs. Monitoring techniques that inventory and monitor terminal electron acceptors and electron donors, enzyme probes that measure functional activity in the environment, functional genomic microarrays, phylogenetic microarrays, metabolomics, proteomics, and quantitative PCR are also being rapidly adapted for studies in environmental biotechnology. Integration of all of these new high throughput techniques using the latest advances in bioinformatics and modeling will enable break-through science in environmental biotechnology. A review of these techniques with examples from field studies and lab simulations will be discussed.
  650. Hazen, Terry C. 2009. A Systems Biology Approach to Environmental Biotechnology using Ecogenomics. International Society for Microbial Ecology and International Water Association Special Colloquia
  651. Hazen, Terry C.. 2009. Systems Biology the New Frontier for Bioenergy. National University of Singapore
  652. Hazen, Terry C. and G. L. Andersen. 2009. A Systems Biology Approach to Environmental Biotechnology using Ecogenomics. National University of Singapore
  653. Auer, M., M. D. Biggin, G. Butland, S. Chhabra, D. A. Elias*, A. Fagorala, Terry C. Hazen, D. Jorgens, D. C. Joyner, T. R. Juba, M. Perez, J. P. Remis, A. Tauscher, J. D. Wall. 2009. Protein Complex Analysis Project (PCAP): Localization of Multi-Protein Complexes through SNAP-Tag Labeling. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  654. Brileya, K. and S. Stolyar, D. A. Stahl, A. P. Arkin, Terry C. Hazen, M. W. Fields C. Walker. 2009. Temporal and Spatial Organization within a Syntrophic Bacterial-Archaeal Biofilm. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  655. Chhabra, S., G. Butland*, D. Elias, V. Fok, R. Prathapam, T. Juba, J.-M. Chandonia, E. Witkowska, M. Biggin, Terry C. Hazen, J. D. Wall, J. D. Keasling. 2009. A high throughput pipeline for identifying protein-protein interactions in Desulfovibrio vulgaris using tandem-affinity purification. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  656. Chivian, D. and E. L. Brodie, D. E. Culley, P. S. Dehal, T. Z. DeSantis, T. M. Gihring, A. Lapidus, L.-H. Lin, S. R. Lowry, D. P. Moser, P. Richardson, G. Southam, G. Wanger, L. M. Pratt, G. L. Andersen, Terry C. Hazen, F. J. Brockman, A. P. Arkin, T. C. Onstott E. J. Alm. 2009. Environmental Genomics Reveals a Single-Species Ecosystem Deep Earth. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  657. DeAngelis, K. M., W. L. Silver, J. Fortney and Terry C. Hazen. 2009. Discovery and Optimization of Lignocellulolytic Bacteria from Puerto Rican Rainforest Soils. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009 abstract
    Tropical soils in Puerto Rican rain forests have some of the highest decomposition rates recorded in the world, with almost total mass lost in decomposing plant material within one year. These soils are capable of deconstructing biofuel plant materials to basic components, like ethanol, methane or methanol. Rapidly fluctuating redox conditions are characteristic of the highly weathered soils of upland humid tropical forests, which are dominated by Fe-oxide mineralogy and have relatively low sulfate availability. The frequent episodes of anoxic conditions make it likely that these decomposing consortia are primarily bacteria, not fungi as are usually observed in temperate systems. Previous lab incubation under fluctuating redox conditions permitted simultaneous methanogenesis, N2O production, and iron reduction, all accompanied by steady CO2 production. The objective of this research is to define field conditions that result in characteristic high methane production in Puerto Rico forest soils from decomposing plant materials, and determine whether different microbial communities break down different plant materials. Towards this end, we designed a field experiment and accompanying laboratory incubations that would allow us to investigate the rates, controls and mechanisms of switchgrass decomposition in tropical rainforest soils. In June of 2008, we buried litterbags filled with switchgrass in four different forest types in the Luquillo LTER, located at the El Yunque National Forest in Puerto Rico, USA. The four forest types vary from more aerobic soils, warmer temperatures and annual precipitation on the order of 1,000 mm, to fluctuating redox soils, to mostly anaerobic soils, cooler soil temperatures and annual precipitation that can exceed 4,000 mm. The experimental design included 4 field sites, 6 time points, and bags buried in pairs, one for. At each of 6 time point, litter bags and soil are collected from the field and assayed for microbial community analysis using 16S ribosomal DNA PhyloChip, potential enzyme activity (β-glucosidase, endoglucanase xylosidase, chitinase, phenol oxidase and peroxidase), and mass loss as indicators of decomposition. In the driest site, which we expect to also have the highest rates of decomposition, we also buried biosep beads baited with lignin (using unbaited beads as controls) as bug traps to identify and isolate microbes specifically able to decompose lignin. This site was also instrumented with oxygen sensors to measure oxygen levels in soil on an hourly basis over the course of this year-long incubation, and ultimately to correlate decomposition, enzyme activity and microbial community composition with oxygen availability at the end of the experiment. Concomitantly with the field experiment, we are using fresh soil to inoculate mini-reactors with dried ground switchgrass and incubate anaerobically to enrich for lignocellulose-degrading organisms. The initial inoculation of rain forest soil with switchgrass resulted in significant CO2, CH4 and H2S production compared to uninoculated, anaerobic soil incubations, as well as a substantial change in microbial community composition. Switchgrass amendment resulted in significant change in 147 taxa compared to the 1847 detected in the soils. With switchgrass addition to soil, Archaea, methanogens, enteric bacteria, Bacilli and Clostridia were significantly increased, while Acidobacteria, Burkholderia and Verrucomicrobial were significantly reduced in the microbial community. Further passages of the soil microbial community with switchgrass as the sole carbon source has resulted in a low-richness, anaerobic microbial community capable of efficiently converting switchgrass to methane and carbon dioxide as well as depolymerizing cellulose, hemi-cellulose, and lignin in the process.
  658. Dehal, P. S. and D. Chivian, K. H. Huang, M. P. Joachimiak, K. Keller, M. N. Price, R Chakraborty, M. W. Fields, J. Zhou, D. A. Stahl, J. D. Wall, A. P. Arkin, Terry C. Hazen E. J. Alm. 2009. Comparative Sequencing and Analysis of Multiple Desulfovibrio and Other Sulfate Reducing Species. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  659. Deutschbauer, A., J. Kuehl*, M. Price, P. Dehal, Terry C. Hazen, A. P. Arkin. 2009. The development and application of an integrated functional genomics platform in Desulfovibrio desulfuricans G20. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  660. Dong, M., M. Daly, H. Liu, S. Allen, E. Szakal, S. C. Hall, S. J. Fisher, Terry C. Hazen, J. T. Geller, M. E. Singer, L. L. Yang, J. Jin, H. E. Witkowska*, M. D. Biggin. 2009. Analysis of an Intact Dissimilatory Sulfite Reductase Protein Complex from Desulfovibrio vulgaris using an Ion Mobility QTOF Analyzer. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  661. Dong, M. and L. Yang, M. Choi, E. D. Szakal, S. Allen, S. C. Hall, S. J. Fisher, G. Butland, Terry C. Hazen, J. T. Geller, M. E. Singer, P. Walian, B. Jap, J. Jin, J.-M. Chandonia, H. E. Witkowska, M. D. Biggin H. Liu. 2009. Protein Complex Analysis Project (PCAP): Protein Complex Purification and Identification by ”Tagless” Strategy. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  662. Elias, D. A. and A. M. Redding, A. Mukhopadhyay, M. Joachimiak, H.-C. B. Yen, M. W. Fields, Terry C. Hazen, A. P. Arkin, J. D. Keasling, J. D. Wall Elliott C. Drury. 2009. Expression profiling of hypothetical genes in Desulfovibrio vulgaris leads to improved functional annotation. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  663. Hazen, T. C. and S. Borglin, E. Brodie, S. van Dien, M. Fields, J. Fortney, J. Geller, E. Hendrickson, K. L Hillesland, H.-Y. Holman, J. Leigh, T. Lie, J. Jacobsen, D. Joyner, R Chakraborty, M. Keller, A. Mukhopadhyay, C. Schadt, D. Stahl, S. Stolyar, C. Walker, J. Wall, Z. Yang, H.-C. B. Yen, G. Zane, J. Zhou G. Anderson. 2009. Applied Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers VIMSS:ESPP. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  664. Hazen, T. C. and J. Keasling, A. Mukhopadhyay, S. Chhabra, J. T. Geller, M. Singer, D. Joyner, L. Camp, T. Torok, J. Wall, D. Elias, M. D. Biggin H.-Y. Holman. 2009. Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes during Stress Response in Desulfovibrio vulgaris: Microbiology Subproject. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  665. Hazen, T. C. and S. Singer, J. VanderGheynst, P. M. D’haeseleer, M. P. Thelen, K. DeAngelis, A. Reddy, M. Allgaier, J. Fortney, G. Andersen, T. DeSantis, E. Brodie, C. Wu, D. Goodheart, M. Firestone, W. Silver, B. Simmons P. Hugenholtz. 2009. JBEI Microbial Communities Deconstruction Research Activities. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  666. He, Q. and D. C. Joyner, M. Joachimiak, M. N. Price, Z. K. Yang, H.-C. B. Yen, C. L. Hemme, R Chakraborty, W. Chen, M. M. Fields, D. A. Stahl, J. D. Keasling, M. Keller, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou Z. He. 2009. Impact of Elevated Nitrate on Sulfate-Reducing Bacteria: Implications of inhibitory mechanisms in addition to osmotic stress. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  667. He, Z., A. Zhou*, Q. He, A. Mukhopadhyay, E. Baidoo, M. Joachimiak, C. L. Hemme, P. Benke, A. M. Redding, M. M. Fields, D. A. Stahl, J. D. Keasling, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou. 2009. Desulfovibrio vulgaris Hildenborough responses to salt and H2O2 stresses. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  668. Knierim, B. and S. Singh, D. Jorgens, M. Zemla, K. DeAngelis, A. Reddy, J. VanderGheynst, Terry C. Hazen, B. Holmes, R. Sapra, B. Simmons, P. Adams, M. Auer L. Prak. 2009. Electron Microscopic Imaging a JBEI. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  669. Liu, H. and N. Khainovski, M. Dong, E. D. Szakal, M. Choi, S. Allen, Terry C. Hazen, J. T. Geller, M. E. Singer, P. Walian, B. Jap, S. C. Hall, S. J. Fisher, H. E. Witkowska, J. Jin, M. D. Biggin L. Yang. 2009. Protein Complex Analysis Project (PCAP): Introduction of Iterative MS/MS Acquisition (IMMA) to the MALDI LC MS/MS Workflow To Enable High Throughput Protein Complex Identification using Tagless Strategy. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  670. Meagher, R. J. and Y. K. Light, P. Dehal, Terry C. Hazen, A. P. Arkin, A. K. Singh M. Z. Hadi. 2009. Microfluidic tools for single-cell genomic analysis of environmental bacteria. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  671. Miller, L. D. and J. Mosher, M. Drake, Z.K. Yang, M. Rodriguez, S.D. Brown, T. J. Phelps, M. Podar, A. V. Palumbo, C. W. Schadt, M. Keller, D. C. Joyner, Terry C. Hazen, S. Stolyar, K. Hillesland, D.A. Stahl A. Venkateswaran. 2009. Development and Analysis of Multispecies Consortia to Study Microbial Community Stress and Survival. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  672. Mukhopadhyay, A. and J. Ray, A. Deutschbauer, K. Keller, J. Robertson, G. Zane, M. Price, S. Chhabra, J. Wall, A. P. Arkin, Terry C. Hazen, J. Keasling E. Luning. 2009. ESPP2: Study of Two component signal transduction systems in Desulfovibrio vulgaris Hildenborough. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  673. Ramsay, B. and S. Carroll, A. Lapidus, J. C. Detter, C. Han, M. Land, L. Hauser, Terry C. Hazen, A. Arkin, A. Beliaev, R. Sanford, F. Löeffler, M. W. Fields C. Hwang. 2009. Characterization of Metal-Reducing Communities and Isolates from Uranium-Contaminated Groundwater and Sediments. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  674. Van Nostrand, J. D. and P. Waldron, Ye Deng, Z. He, W. Wu, S. Carroll, C. Schadt, A. Palumbo, D. Watson, C. Criddle, P. Jardine, Terry C. Hazen, J. Zhou L. Wu. 2009. Applications of GeoChip for analysis of different microbial communities. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  675. Walian, P. J. and L. Zeng, E. Szakal, E. Johansen, S. C. Hall, S. J. Fisher, M. E. Singer, C. Park, Terry C. Hazen, H. E. Witkowska, M. D. Biggin, B. K. Jap S. Allen. 2009. Protein Complex Analysis Project (PCAP): Isolation and Identification of Membrane Protein Complexes from D. vulgaris. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  676. Hazen, Terry C. 2009. Bioremediation: the Hope and the Hype. Manitoba Environmental Industry Association Meeting
  677. Hazen, Terry C. 2009. Hanford 100H Cr Bioremediation. DOE EM RO conference on 100 area Cr
  678. Hazen, Terry C. 2009. A Systems Biology Approach to Bioremediation. Manitoba Environmental Industry Association Meeting
  679. Hazen, Terry C.. 2009. Systems Biology: The New Frontier for Bioenergy. Department of Microbiology, University of Oklahoma
  680. Dubinsky, E.A., C. Wu, S. Osman, J. Hulls, Terry C. Hazen, G. L. Andersen. 2009. A complete microbial community approach to monitoring and source tracking coastal-zone pollution. U.S. EPA National Beach Conference
  681. Conrad, M. E. and C. H. Wu, M. Bill, K. E. Wright, S. D. Chamberlain, B. R. Lam, E. L. Brodie, P. S. Nico, N. Spycher, Terry C. Hazen Y. Fujita. 2009. Enhanced Immobilization of Metals and Radionuclides in the Vadose Zone. 4th Annual DOE-ERSP PI Meeting
  682. Hazen, T. C. and H. Beller, E. Brodie, S. S. Hubbard, J. Peterson, E. Sonnenthal, C. Steefel, L. Yang, J. Larsen, M. Conrad, J. Christensen, S. Brown, D. Joyner, S. Borglin, J. Geller, R. Chakraborty, P. Nico, T. Tokunaga, J. Wan, M. Firestone, P. Long, D. Newcomer, L. N’Guessan B. Faybishenko. 2009. Field-Scale Investigations of Cryptic Growth and Memory Response Hypotheses at the Chromium Contaminated Hanford 100-H Site. 4th Annual DOE-ERSP PI Meeting
  683. Hazen, T. C. and B. Faybishenko. 2009. Update on Groundwater Issues across the DOE Complex. 4th Annual DOE-ERSP PI Meeting
  684. Hubbard, S. S. and H. Beller, E. Brodie, J. Chen, J. Christensen, M. Conrad, M. Denham, D. DePaolo, B. Faybishenko, S. Finsterle, Y. Fujita, Terry C. Hazen, M. Kowalsky, L. Li, P. Long, P. Nico, E. Sonnethal, N. Spycher, C. Steefel, T. Tokunaga, J. Wan, K. H. Williams, Y. Wu J. Aho-Franklin. 2009. LBNL Sustainable Systems SFA. 4th Annual DOE-ERSP PI Meeting
  685. Sobecky, P. A., R. J. Martinez, M. J. Beazley, K. Salome, C. Wu, Terry C. Hazen, G. L. Andersen, S. M. Webb, M. Taillefert. 2009. Uranium Immobilization by the Activities of Microbial Phosphatases. 4th Annual DOE-ERSP PI Meeting
  686. Van Nostrand, J. D. and M. Xu, W. Wu, L. Wu. Z. He, Y. Deng, C. Hemme, D. Watson, C. Criddle, P. Jardine, Terry C. Hazen, J. Zhou P. J. Waldron. 2009. Improvements to GeoChip 3.0 and Application for Microbial Community Analysis. 4th Annual DOE-ERSP PI Meeting
  687. Chakraborty, R., E. L Brodie, D. C Joyner, T. Torok, J. L Fortney, S. E Borglin, P. E Long, D. R Newcomer, S. Choudhuri, H. R Beller, Y. M Piceno, L. Tom, G. L Andersen, B. Faybishenko, Terry C. Hazen. 2009. Microbial community changes during sustained Cr(VI) reduction at the 100H site in Hanford, WA. American Geophysical Union Annual Meeting
  688. Faybishenko, B., Terry C. Hazen. 2009. Multiple Factor Analysis and k-Means Clustering-Based Classification of the DOE Groundwater Contaminant Database. American Geophysical Union Annual Meeting abstract
    A proper classification of the plume characteristics is critical for selecting the most suitable characterization, monitoring, and remediation technologies. To perform a statistical analysis of the different groundwater plume characteristics, we used the DOE Groundwater Database, including 221 groundwater plumes located at 60 DOE sites. To classify the plume characteristics, we used a multiple factor analysis (MFA), including a principal component analysis (PCA) of quantitative plume characteristics and a multiple correspondence analysis (MCA) of qualitative plume characteristics. The input parameters used for the statistical analysis are: the presence of eight types of contaminant groups—chlorinated hydrocarbons, fuels, explosives, sulfates, nitrates, metals, tritium, and radioisotopes; a number and associations of contaminant groups; a contamination severity index (based on the association of contaminant groups and complexity of remediation); contaminant mass and plume volumes; groundwater depth and velocities; and climatic conditions. The input variables are also partitioned into the active and supplementary plume characteristics. Statistical results include the evaluation of the correlation matrix between the groups of variables and individual plume characteristics. From the results of the MFA, the first four factors can be used to describe the variability of the basic plume characteristics. The contaminant severity index and the number of contaminant groups provide a major contribution to the 1st factor; the types of contaminant groups and carbon tetrachloride concentrations provide the major contribution to the 2nd factor. The contribution of the supplementary data (climate and plume depth and velocity) is insignificant. The presence of radioactive contaminants is mostly related to the 1st factor; the presence of sulfates, and to a lesser degree the presence of nitrates and metals, is related to the 2nd factor. The strongest relationship is, as expected, between the types of contaminant groups and the contamination severity. The relationships between contaminant groups and the plume depth and velocity, and contaminant groups and climate are weak, and there is no a significant relationship with the plume volumes. To visualize the contribution of different factors, the results of MFA calculations are presented using two- and three-dimensional maps. Using the first four factors for the basic plume characteristics, a k-means cluster analysis was applied to classify the plumes into respective clusters. These results can be used to plan characterization, monitoring, and modeling of contaminant behavior at contaminated sites, and to design appropriate remediation technologies.
  689. Hazen, Terry C., B. Faybishenko, P. Jordan. 2009. Characterization of a Contaminant Inventory at DOE Sites, as a Tool for Selecting Monitoring and Remediation Technologies. American Geophysical Union Annual Meeting abstract
    The U.S. Department of Energy (DOE) is responsible for the remediation and long-term stewardship of one of the world’s largest groundwater contamination portfolios, with a significant number of plumes containing various contaminants, and considerable total mass and activity. The frequency of occurrence and ranking of contaminants in groundwater plumes is one of the main criteria needed for decision-making related to planning and prioritizing the types of basic research and the development of site characterization, monitoring, and remedial approaches. Using the data from 60 DOE sites, including 221 groundwater plumes, collected in the DOE Groundwater Database (GWD), we evaluated the frequency of occurrence of specific contaminants and their associations, plume volumes, contaminant maximum concentrations, masses, and isotope activities. Contaminants detected in groundwater at 60 DOE sites and facilities can be categorized into the following eight generic contaminant groups: chlorinated hydrocarbons (chlorinated ethenes), fuels and fuel components (i.e., petroleum/fuel hydrocarbons), explosives, metals, radioactive isotopes (excluding tritium), tritium, sulfates, and nitrates. The most common are plumes containing two (29.4% of all plumes in the GWD) and three (29%) contaminant groups. The most frequent binary combinations of contaminant groups are those of mixed waste, including chlorinated hydrocarbons and tritium—35% and metals and isotopes—28% of all plumes. Our results were compared to the data from 18 DOE sites and 91 plumes, collected in 1992, to illustrate the progress in site characterization and remediation over the past decade. The analysis of contaminant inventory and plume characteristics should be helpful in establishing priorities for basic research needs, which will enable cost-effective and efficient application of new characterization, monitoring, modeling, and remediation technologies.
  690. Hazen, Terry C., E. Sonnenthal, S. Mukhopadhyay, C. Steefel, P. Long, B. Faybishenko. 2009. Field and Numerical Modeling Study of Reductive Bioimmobilization of Cr (VI) in Groundwater at Hanford 100H Site. American Geophysical Union Annual Meeting abstract
    The 2004-2008 field experiment at the Hanford 100-H Site showed that a single injection of the hydrogen release compound (HRC)—a mixture of slow release glycerol polylactate, fast disassociating lactic acid, and glycerol into groundwater— stimulated an increase in biomass and a depletion of terminal electron acceptors, resulting in a significant decrease in soluble Cr (VI). The Cr (VI) concentration remained below the background concentration in the downgradient pumping/monitoring well, and below the detection limit in the injection well, for more than 3 years after the initial HRC injection in 2004. Reaction-transport modeling of the field experiment was performed to elucidate reaction pathways and rates of biogeochemical processes governing Cr (VI) bioimmobilization. Field observation data were used to develop a 3-D hydraulic and reaction-transport-isotopic model, simulated using the TOUGHREACT code. This model was used to assess the degradation kinetics of the HRC, the effects of lactate and acetate-induced bioreduction of Cr and Fe, and the effects on Cr and Sr isotopic compositions in the fluids and mineral phases (calcite dissolution in the acidic plume and precipitation at the fringes). The 2008 experiment involved reinjection of HRC into the same zones as the earlier injection and likely resulted in dissolution of previously precipitated Cr (Fe) hydroxides and additional calcite dissolution in sediments outside of the wellbore. Detailed results of the integrated field and modeling study will be presented.
  691. Wu, C. H. and J. Chou, M. Bill, J. Henriksen, K. E. Wright, E. L. Brodie, G. L. Andersen, Terry C. Hazen, Y. Fujita, M. E. Conrad B. R. Lam. 2009. Microbial metabolism of triethylphosphate, a potential phosphate source for radionuclide mineralization. American Geophysical Union Annual Meeting abstract
    Significant quantities of metals and radionuclides contaminate unsaturated zones at several sites in the western U.S. In many cases, this contamination has migrated to groundwater, sometimes decades after being released into the subsurface. A potentially useful approach for immobilizing radionuclides such as uranium and strontium in the vadose zone is precipitation with microbially-generated phosphate. Triethylphosphate (TEP) is a low-toxicity organophosphate that can be vaporized and delivered to the vadose zone. Microbes can catalyze TEP degradation, leading to the release of inorganic phosphate that can then lead to the precipitation of phosphate minerals. These minerals are typically highly stable and poorly soluble under environmental conditions. Sequestration in phosphate minerals is a promising strategy for mitigating radionuclide transport in the environment. To examine the feasibility of this strategy, we set up lab-scale incubation experiments with TEP-amended synthetic groundwater inoculated with vadose zone-derived mixed cultures from the Idaho National Laboratory (INL), and sediment slurries using solids from the Hanford Reservation in Washington (U.S. Department of Energy facilities with significant radionuclide contamination in the vadose zone). The amount of phosphate released in the cultures was monitored, and the microbial communities were characterized with a high-density microarray (PhyloChip). Significant biodegradation of TEP was observed in the experiments with the synthetic groundwater amended with 5 mM TEP. Phosphate concentrations in live cultures steadily increased to >0.25 mM after 13 months with no phosphate accumulated in killed controls. Surprisingly, no evidence for phosphate mineral precipitation was observed, contrary to expectations based on equilibrium considerations. Studies are underway to investigate potential kinetic inhibition of precipitation under these conditions. Cell counts increased by approximately one order of magnitude during that period. Significant decreases in the d13C values of dissolved inorganic carbon in the live cultures were observed, indicating the microbial community was respiring the carbon in the TEP. In contrast, no significant accumulation of phosphate was observed in the sediment slurries with 5 mM TEP, most likely due to phosphate adsorption to the solids. Microbial community identification indicated that organisms in the families of Xanthomonadaceae, Crenotrichaceae and Comamonadaceae were enriched by the addition of TEP. Further characterization of radionuclide-biota interactions would lead to enhanced understanding of the fate and transport of these contaminants in the subsurface.
  692. Zhou, A., Z. He, M. P. Joachimiak, P. S Dehal, A. P. Arkin, K. Hillesland, D. Stahl, J. Wall, Terry C. Hazen and J. Zhou. 2009. The molecular mechanism of adaptation to salt stress revealed by the long-term evolution of Desulfovibrio vulgaris Hildenborough. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009
  693. Allgaier, M., A. Reddy, J. VanderGheynst, A. Copeland, V. Kunin, P. D’haeseleer, K. DeAngelis, J. Fortney, D. Chivian, P. S. Dehal, B. Simmons, Terry C. Hazen and P. Hugenholtz. 2009. Metagenomic Characterization of Compost and Rain Forest Soil Microbial Communities. Genomics:GTL Contractor-Grantee Workshop VII, USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2009 abstract
    Microorganisms are a promising source of novel carbohydrate-active enzymes (cellulases, hemicellulases, and lignases) since they are primarily responsible for plant biomass degradation in nature. However, most carbohydrate-active enzymes used by industry come from only a few model organisms. To identify new lignocellulolytic enzymes we shotgun sequenced genomic DNA from two sources: a sample of pristine Puerto Rican rain forest soil and a sample obtained from a solid state fermentation experiment in which switchgrass was incubated 30 days in a lab under mesophilic and thermophilic conditions after inoculating with conventional green waste compost. Both ecosystems display high rates of plant biomass degradation and are therefore prime targets for novel carbohydrate-active enzyme discovery. 454-Titanium pyrosequencing was used to generate metagenome data sets from the two samples resulting in a total of 1,412,492 reads (rain forest: 863,759; compost: 548,733) with an average read length of 424 bases. Reads were quality filtered and trimmed in preparation for comparative analyses with the metagenome analysis tools IMG/M and MicrobesOnline. The complexity of the rain forest soil metagenome precluded assembly, so sequence data were analyzed unassembled. However, for the compost sample, significant assembly occurred resulting in contigs up to 50 kb in length. Preliminary comparative analysis of a fraction of the rain forest soil sequence data revealed more than 2,700 cellulases, hemicellulases, and ligninases including glycoside hydrolases as well as glycosyl transferases, representing ~1% of all predicted protein-coding genes (e.g. compared to 1.2% or 0.03% identified in metagenomic data sets from termite guts or silage surface soil, respectively). The enzyme repertoires present in the two metagenome data sets will be further analyzed to identify new deconstruction enzymes and compared to assess differences in activity and community composition between compost and rain forest soil.
  694. Hazen, Terry C.. 2008. Systems Biology the New Frontier for Bioenergy. University of California at Santa Barbara, Bren School of Engineering
  695. Hazen, Terry C.. 2008. Bioremediation: the Hope and the Hype of Environmental Cleanup. Guangdong Institute of Microbiology, Chinese Academy of Sciences - Guangzhou
  696. Hazen, Terry C.. 2008. Life in the Slow Lane: Ecogenomics of an Extreme Environment. Guangdong Institute of Microbiology, Chinese Academy of Sciences - Guangzhou
  697. Hazen, Terry C.. 2008. Life in the Slow Lane: Ecogenomics of an Extreme Environment. Annual Meeting of Ecological Society of China, Microbial Ecology Branch
  698. Hazen, Terry C.. 2008. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. Guangdong Institute of Microbiology, Chinese Academy of Sciences - Guangzhou
  699. Hazen, Terry C.. 2008. Systems Biology the New Frontier for Bioenergy. Guangdong Institute of Microbiology, Chinese Academy of Sciences - Guangzhou
  700. Hazen, Terry C.. 2008. Systems Biology the New Frontier for Bioenergy. Chinese Society for Microbiology and American Society for Microbiology Joint Seminar on Environmental Microbiology and Bioenergy
  701. Hazen, Terry C.. 2008. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. EPA Biotechnology Webnar
  702. Hazen, Terry C.. 2008. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. University of California Berkeley, College wide BioE* Seminar
  703. Hazen, Terry C. 2008. Demonstration Workshop on PM visualization software and Microbesonline. Phenotypic Microarray Conference
  704. Hazen, Terry C. 2008. The Frontiers of Environmental Biotechnology. AICHE Soils Conference
  705. Hazen, Terry C.. 2008. Life in the Slow Lane: Ecogenomics of an Extreme Environment. JGI Users Meeting
  706. Hazen, T. C. and D. C. Joyner, J. Jacobsen S. E. Borglin. 2008. Anaerobic Phenotypic Microarray. Phenotypic Microarray Conference
  707. Joyner, D. and A. Mukhopadhyay, Terry C. Hazen J. Jacobsen. 2008. Assessment of Nitrogen utilization in Desulfovibrio vulgaris using phenotype microarray. Phenotypic Microarray Conference
  708. Chakraborty, R., E. L. Brodie, J. Van Nostrand, J. Zhou and Terry C. Hazen. 2008. Investigation of Cr(VI) Tolerant Bacteria from Cr(VI)-Contaminated 100H Site at Hanford, WA. 108th General Meeting of the American-Society-for-Microbiology 108:465. abstract
    Hexavalent Chromium is a widespread contaminant found in soil, sediment, and groundwater. In order to stimulate microbially mediated reduction of Cr(VI), a poly-lactate compound HRC was injected into the chromium-contaminated aquifers at site 100H at Hanford, WA. Based on the results of the bacterial community composition using high-density DNA microarray analysis of 16S rRNA gene products, we investigated the diversity of the dominant anaerobic culturable microbial populations present at this site and their role in Cr(VI) reduction. Also, functional gene array (GeoChip) analysis of DNA extracted from monitoring well at the site indicated that genes involved in nitrate reduction, sulfate reduction, iron reduction, methanogenesis, as well as many chromium tolerance/reduction genes were highly abundant relative to the injection well. In addition, positive enrichments set up at 30°C using defined anaerobic media resulted in the isolation of an iron-reducing isolate strain HAF, a sulfate-reducing isolate strain HBLS and a nitrate-reducing isolate, strain HLN among several others. Preliminary 16S rDNA sequence analysis identified strain HAF as Geobacter metallireducens, strain HLN as Pseudomonas stutzeri and strain HBLS as Desulfovibrio species. Strain HAF utilized propionate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Growth was optimal at 37°C and pH of 6.5. Strain HLN utilized acetate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Optimal growth was observed at 37°C, at a pH of 7.5. Anaerobic washed cell suspension of strain HLN reduced almost 95μM Cr(VI) within 4 h relative to controls. Further, with 100μM Cr(VI) as sole electron acceptor, cells of strain HLN grew to cell numbers of 4.05 x 10^7/ml over a period of 24 h after an initial lag, demonstrating direct enzymatic Cr(VI) reduction by this species. These results demonstrate that Cr(VI) immobilization at the Hanford 100H site could be mediated by direct microbial metabolism apart from indirect chemical reduction of Cr(VI) by end products of microbial activity.
  709. Chakraborty, R., N. Ramos-Hernandez, E. X. Perez, Y. Katsuura, A. Massol-Deya and Terry C. Hazen. 2008. Characterization of Novel Marine Sulfate-Reducing Bacteria Resistant to RDX and Other Explosives. 108th General Meeting of the American-Society-for-Microbiology 108:420. abstract
    Exposure to nitro-compounds like RDX, TNT contained in unexploded ordinance pose significant risks for people and the environment due to their toxicity and explosive nature. Bombing sites used for military training activities contain considerable amounts of these contaminants. Until 2003 and for more than 60 years, the US Navy used the eastern part of Vieques, Puerto Rico as a bombing range. Since then, leaching of explosive compounds like RDX from unexploded ordinance represents a serious threat to the marine ecosystem of the area. The contribution of sulfate-reducing bacteria (SRB) to natural attenuation of explosives has been demonstrated in freshwater environments; however, little is known about their contribution in marine environments. From the contaminated sediments, we isolated 5 novel SRBs using lactate as the sole electron donor. Preliminary 16S rDNA analyses identify all 5 isolates to be delta proteobacteria most closely related to Desulfovibrio, with 16S rDNA sequences less than 94% of similarity with known sequences, thus representing putative new species. In general, the isolates grew best at 37°C, at a pH of 7.5 and optimal growth was obtained with 1.5-3% of salt added in the media. While all isolates grew with pyruvate as the alternative electron donor, 3 of the isolates grew with 20mM glycerol, one of them showed growth on 50mM fumarate. Electron acceptors sulfate, sulfite and thiosulfate were reduced by all isolates. Preliminary results indicate two of the isolates could also reduce Mn(IV). Some of the bacterial isolates grew better in explosive-amended media. The growth and survival in the presence of TNT increased with added salinity of the media. Based on this result and the fact that the isolates have had prior exposure to explosives, investigations are underway to study the potential of RDX degradation by the novel isolates.
  710. Chakraborty, R., Y. Tang, F. Pingitore, J. D. Keasling and Terry C. Hazen. 2008. Metabolic Pathways in the Pleomorphic Metal-Reducing Organism Desulfovibrio africanus Strain PCS. 108th General Meeting of the American-Society-for-Microbiology 108:361-362. abstract
    Desulfovibrio africanus is a strict anaerobic sulfate-reducing organism that can also reduce toxic metals like Chromium (VI) to non-toxic Chromium (IV). While a lot of researchers have focused on studying the biochemistry, isolation and structure of metalloproteins in this organism, not much is known about the physiology of this organism. Previously, Desulfovibrio africanus strain PCS (99% similar to the type strain) was isolated from sediments in San Diego as a lactate oxidizing sulfate reducer. Using lactate as the sole electron donor, this organism could reduce almost 200μM Chromium(VI) even when subjected to stress by 50mM nitrate. Strain PCS appears as thin spiral shaped bacterium in mid log phase but morphs into spherical form later in the growth phase. It was observed that the spiral morphotype actively utilizes lactate while the spherical morphotype does not. Using 13C-labeled lactate as a single carbon source, we investigated the metabolic pathways of lactate utilization in mid-log cells of strain PCS with sulfate as the terminal electron acceptor. The isotopomer analysis of proteinogenic amino acids was performed using both gas chromatography-mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry. Based on the labeling pattern of 8 key metabolites alanine, histidine, serine, isoleucine, leucine, aspartate, succinate and glutamate, we observed several unique metabolic pathways in strain PCS. In this organism, a branched tricarboxylic acid cycle exists due to no activity of ketoglutarate dehydrogenase. Also, the lack of an oxidative functional pentose phosphate pathway was observed. The results predict presence of a Re-type citrate synthase, similar to the recently characterized citrate synthase of Clostridia, while isoleucine synthesis seems to be completely via citramalate pathway rather than via L-threonine dehydratase. The isotope labeling pattern of amino acids allowed a preliminary prediction of the in vivo metabolic pathways through central pathways especially useful for microbes whose genetic fingerprint have yet to be completely deciphered.
  711. Deutschbauer, A., J. Oh, M. Price, P. Dehal, D. Bruno, J. Kuehl, R. Chakraborty, Terry C. Hazen, C. Nislow, G. Giaever, R. W. Davis and A. P. Arkin. 2008. Phenotypic Characterization of Microorganisms by Barcoded Transposon Mutagenesis. 108th General Meeting of the American-Society-for-Microbiology 108:377. abstract
    Systems-level analyses of less studied bacteria are limited by the presence of numerous uncharacterized genes and an over reliance on annotations from well studied bacteria such as E. coli. To meet this challenge, we are developing a mutagenesis and phenotyping strategy that is comprehensive across the genome and applicable to any microorganism amenable to transposon mutagenesis. We have cloned and sequence-verified ~3000 barcode modules into a Gateway entry vector. Each module is a 175 base pair element containing two unique 20 base pair sequences, the UPTAG and DOWNTAG, flanked by common PCR priming sites. Each module can then be rapidly transferred in vitro to any DNA element, such as a transposon, that is made Gateway compatible. Transposon mutants marked by the modules will be sequenced to determine which of the ~3000 barcode modules was used and which gene was disrupted. Transposon mutants can be rapidly re-arrayed into a single pool containing ~3000 uniquely tagged, sequence-verified mutant strains. By sequencing saturating numbers of transposon mutants, we can identify and assay mutants in most nonessential genes in a given genome. The fitness of each mutant in the pool will be monitored in parallel by the hybridization of the barcodes to an Affymetrix microarray containing the barcode complements in a system identical to that used for the yeast deletion collection. Compared to other approaches for the parallel analysis of transposon mutants such as signature tagged mutagenesis, genetic footprinting, and transposon site hybridization, our approach offers higher throughput, a single microarray design is universal for any organism, single mutational events are assayed, and mutant strains are archived for verification, further analysis, and distribution. The completion of this project will enable the quantitative phenotypic analysis of thousands of mutants across a wide range of conditions. In addition, our genetic resources provide a framework for the systematic genetic interrogation of individual pathways. Here we describe the application of this approach to Shewanella oneidensis MR1 and Desulfovibrio desulfuricans G20.
  712. Dong, M., H. Liu, M. Choi, Terry C. Hazen, J. Geller, M. Singer, L. Camp, S. Allen, S. J. Fisher, S. C. Hall, E. D. Szakal, H. Witkowska, L. Yang, J. Jin and M. D. Biggin. 2008. Tagless Protein Complex Identification: A Novel High Throughput Strategy to Purify Protein Complexes and Identify Them by Mass Spectrometry. 108th General Meeting of the American-Society-for-Microbiology 108:546-547. abstract
    We are developing a high throughput pipeline to purify and identify water soluble protein complexes from a microbe. Our strategy uses a novel "tagless" method that chromatographically separates the water soluble protein contents of a bacterium into a large number of fractions by an optimized four-step fractionation scheme, and then identifies polypeptides and monitors their elution patterns by quantitative iTRAQ mass spectrometry. Co migrating polypeptides are automatically detected as putative protein complexes by mathematical algorithms. We have fractionated 10 g of protein extract from a 400 L culture of Desulfovibrio vulgaris into 6 parts by ammonium sulfate precipitation. Each part has then been separated by ion exchange chromatography followed by hydrophobic interaction chromatography (HIC). The HIC fractions are further separated either by size exclusion chromatography or by multi-channel, native gel electrophoresis using a novel instrument we have developed. About 48,000 fractions will need to be generated and analyzed by iTRAQ. To date we have analyzed 0.2% of this fraction space by assaying 76 sizing column fractions from 4 HIC fractions. In toto, 160 polypeptides were detected by MS/MS that matched to 2 or more peptides and an additional 70 polypeptides were detected that matched to a single peptide. 7 putative heteromeric complexes were found and a further 9 for which only one partner of known E.coli homologs was identified. At least 82 putative homomeric complexes were also detected, based on inconsistencies between their theoretical molecular weight and their migration in sizing columns or native PAGE. The large number of putative complexes detected within a very small portion of the overall fraction space indicates that the tagless strategy holds high potential in characterizing bacterial interactomes. This method will then be used as part of a larger effort by the Protein Complex Analysis Project to model stress responses relevant to the detoxification of metal and radionuclide contaminated sites.
  713. Elias, D. A., A. M. Kucken, S. D. Brown, M. M. Drake, L. A. Fagan, R. Chakraborty, C. C. Brandt, M. Podar, Terry C. Hazen, J. D. Wall and A. V. Palumbo. 2008. Environmental Parameters and the Genes Involved in Mercury Methylation in the Pleomorphic Desulfovibrio africanus. 108th General Meeting of the American-Society-for-Microbiology 108:522. abstract
    The sulfate-reducing bacterium Desulfovibrio africanus ATCC 19997 has been shown to methylate mercury (Hg), a process that increases Hg toxicity. The goals of this work are to determine optimal growth conditions for this strain, assess the rate and extent of Hg methylation, and develop a genetic system for the identification of the genes involved in methylation. Initial Hg methylation analysis indicated that cultures spiked with mercuric nitrate generated 1 ng/L of methyl-Hg after several days. For optimal growth, media, NaCl concentrations, and pHs have been tested. A complex medium with yeast extract, 0.5% (w/v) NaCl, lactate as the carbon and electron donor, and sulfate as the electron acceptor yields 1x109 cells/ml in 48 hours. Microscopic observations revealed that the organism changed from a “lemon-shaped” cell in the lag phase to long, slender multi-flagellated rods in the exponential phase, and back to “lemon-shaped” cells in the transition to stationary phase. This phenomenon has been seen in the fresh-water strain Desulfovibrio africanus ATCC 19996 as well as the newly described Desulfovibrio strain PCS. Metabolic capacity and genetic accessibility of the different morphological types is being explored in strain 19997. Preliminary growth studies toward a genetic system have thus far shown antibiotic resistance to only kanamycin and ampicillin of seven antibiotics tested. We are currently determining transformation parameters. Proof-of-principle testing has targeted three candidate loci in D. africanus ATCC19997 for potential roles in Hg methylation of which orthologs were either not found in non-methylating Desulfovibrio spp. or did not share synteny with non-methlyators. These are a hypothetical gene, a SAM dependent methyltransferase, and a cobalamin-dependent methionine synthase I gene. Hence, heterologous expression of each of these genes in the non Hg-methlyating D. vulgaris Hildenborough will allow for a functional analysis of these proteins, and combined with their deletion in D. africanus may reveal the mechanisms by which methyl Hg is generated in the environment.
  714. Fortney, J. L., R. Chakraborty, A. Zhou, M. Joachimiak, S. Borglin, Z. He, A. P. Arkin, J. Zhou and Terry C. Hazen. 2008. Investigation of Stress Response in Metal-Reducing Organism Geobacter metallireducens Strain GS15. 108th General Meeting of the American-Society-for-Microbiology 108:383. abstract
    As part of the ongoing project on determination of stress response pathways in metal and radionuclide reducing bacteria, it is imperative to compare and contrast effects of environmental stressors on bacteria that are found to co-exist in several DOE-contaminated sites. In this study we have optimized growth kinetics for Geobacter metallireducens strain GS15 (GM) to monitor their responses to stressors NaCl and NO3 through several analyses including Fe(III) reduction, total cell protein production, transcriptomics, direct cell counts, and phospholipid fatty acid analysis (PLFA). Investigating responses of GS15 to these stressors will provide a good comparison for similar studies with Desulfovibrio vulgaris and Shewanella oneidensis, and provide a greater understanding of bioremediation under stress conditions. Growth of GM was optimized in defined LS4D medium at 30°C containing 10mM acetate and 10mM Fe-NTA in place of lactate and sulfate. In this medium, cell numbers of 4.3x107 are achieved in midlog phase of growth. The minimal inhibitory concentration of NaCl on this organism in this media was determined to be 100mM, and that of NO3 was determined to be less than 25mM. The MICs were determined by following Fe(II) concentration and cell counts. To examine salt stress response of GM in detail, 100mM NaCl was amended to a midlog phase culture. After 4 hours of salt stress, the stressed cultures had reduced 7.1 mM Fe(III) and contained 5.23x107 cells/ml, which is significantly less than that of control cultures where 9.0mM Fe(III) was reduced and 9.53x107 cells/ml were present. PLFA analysis of stressed cells was performed and compared to non-stressed cells, which showed shifts in relative amounts of saturated and unsaturated fatty acids in response to stress. Transcriptomic analysis of the cells under different stress conditions is in progress.
  715. Geller, J. T., S. E. Borglin, L. E. Camp, R. Chakraborty, J. L. Fortney, M. E. Singer, M. L. Shelby, T. Torok and Terry C. Hazen. 2008. Application of Custom-Designed Fermentors for Extremophilic Microorganisms. 108th General Meeting of the American-Society-for-Microbiology 108:465. abstract
    BACKGROUND: Extremophilic microorganisms may play key roles in remediation of groundwater contaminants and biofuels development. Standard fermentors are not equipped for anaerobic growth conditions, nor can stainless steel withstand the reactivity of metal-reducing organisms and their metabolic by-products. We have developed methods to grow the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough (DvH), under various, batch and continuous growth conditions for detailed physiological and molecular analyses, using custom-built fermentors. METHODS: Five-liter-volume fermentors have PEEK headplates and agitators. During growth, temperature, pH, OD, and redox potential are continuously controlled. Samples are taken for ion chromatography, phospholipid fatty acid (PLFA) analysis, direct cell counts, RT-PCR, and total cell protein. DvH is grown in batch and turbidostat modes using a defined, lactate-sulfate medium at 30ºC. Batch cultures have also been grown under various stress conditions. RESULTS: The maximum specific growth rate for DvH in the 5-liter fermentor is 0.11 1/h. At the onset of deceleration phase, all lactate (60 mM) is depleted, and 30 mM of the initial 50 mM sulfate is metabolized. The dilution rate to maintain the culture at mid-log phase in the turbidostat is 0.15 1/h. Cell densities and total proteins range from 5-10x10^8 cells/ml, and 80-120 µg/ml, respectively. PLFA profiles for DvH are sensitive to the growth conditions and growth phase, while preliminary results show consistent whole-cell protein patterns when visualized using SDS-PAGE. Melt curves generated by real-time PCR proved to be an excellent quality control tool to monitor culture purity. CONCLUSION: Detailed characterization of DvH during growth in custom-designed pilot-scale fermentors provides insights into physiological changes during different growth phases and stress conditions, and has resulted in the development of protocols for controlled and reproducible production of high quality biomass. The experiments have also proven the utility of custom-designed fermentors for growth of extremophiles.
  716. Hadi, M., R. Chakraborty, Y. Light, J. L. Fortney, R. Meagher, A. P. Arkin, Terry C. Hazen and A. K. Singh. 2008. Multigene Analysis to Elucidate Organisms Involved in Cr Bioreduction. 108th General Meeting of the American-Society-for-Microbiology 108:558. abstract
    In any given microbial ecosystem, 99% of bacteria resist cultivation and can’t be analyzed with traditional high throughput techniques. Metagenomic analysis and dynamics of microbial communities can be gleamed using digital PCR techniques and single cell comparisons. In situ bioremediation using indigenous microorganisms is one of the many techniques that has shown promising results in recent years. Although indigenous bacteria at the site can degrade/sequester a wide range of hazardous compounds however, the bacterial population and decontamination efficiency can be affected by high concentrations of toxic compounds (e.g. heavy metals etc). Furthermore, current molecular techniques do not exist to identify the predominant contributor specie(s) and the mechanism of detoxification/sequestration. Recently a hydrogen release compound (HRC) stimulation field test was preformed to investigate bioreduction of Cr(VI) to Cr (III) at Hanford (site 100H). Post injection of the HRC, the Cr(VI) levels drastically decreased. Microarray analysis identified four key bacterial species that could be the major contributors to this bioreduction process. Based on the microarray results, several anaerobic enrichments were initiated in defined media, and 3 strains were isolated that were very closely related to Desulfovibrio vulgaris, Geobacter metallireducens and Pseudomonas stutzeri spp. When individually tested in the lab, all the isolates were capable of Cr(VI) reduction. However to elucidate the contribution of these organisms to the Cr(VI) reduction process in-situ, we attempted a mesocosm study and monitored the stoichiometry of bacterial population and the expression of several key enzymes reported to be involved in Cr(VI) reduction using digital PCR as a function of the Cr(IV) depletion. This is a novel technique not commonly used for bioremediation studies. We discuss our results and present a preliminary model for Cr(IV) bioreduction in this complex environment. We also outline some future detection strategies for similar studies.
  717. Han, B. G., D. Typke, M. Dong, R. Walton, Terry C. Hazen, J. Geller, M. Singer, M. D. Biggin and R. M. Glaeser. 2008. Initial Structural Survey of Multi-Protein Complexes of Desulfovibrio vulgaris by Electron Microscopy. 108th General Meeting of the American-Society-for-Microbiology 108:350. abstract
    The broad aim of our Protein Complex Analysis Project (PCAP) within DOE’s Genomics:GTL program is to analyze the overall stoichiometry and arrangement of subunits within large, biochemically purified multi-protein complexes of Desulfovibrio vulgaris Hildenborough (DvH). The goals of determining the quaternary structures include (1) determining whether structural changes occur in some molecular machines under markedly different physiological conditions, such as encountered in the field during bioremediation, (2) providing 3-D models of their structures that can be used as templates in order to image the same multi-protein complexes within whole cells by EM tomography, and (3) using both types of information to model the biochemical networks and circuits of micro-organisms in order to better utilize them for applications in bioremediation. Single-particle EM technique has been used on soluble-protein complexes with Mr in the range 400 k to over 1000 k to provide three-dimensional (3-D) reconstructions at a resolution of ~2 nm. Somewhat over half of the 15 complexes studied have been stable enough to produce high-quality 3-D reconstructions. This preliminary phase of characterization has shown surprising differences in the quaternary structures of complexes isolated from DvH and homologous proteins from other microbes. These differences occur frequently enough to make it clear that structures determined for other micro-organisms are inadequate as templates for modeling the biochemical networks within a given microbe of interest. By extension it is clear that the same types of comparisons are essential for the quaternary structures of multi-protein complexes that exist under different physiological conditions. Work that is aimed towards increasing the throughput currently includes the implementation of automated data collection and analysis, and the engineering of new support-film technologies for EM sample preparation.
  718. He, Z., E. Baidoo, A. Zhou, Q. He, P. Benke, R. Phan, M. Joachimiak, M. W. Fields, A. Mukhopadhyay, E. J. Alm, K. Huang, J. D. Wall, Terry C. Hazen, J. D. Keasling, A. P. Arkin and J. Zhou. 2008. Global Transcriptional and Metabolite Analysis of Desulfovibrio vulgaris Hildenborough Responses to Long-Term Exposure to Elevated NaCl. 108th General Meeting of the American-Society-for-Microbiology 108:577. abstract
    The mechanisms of salt adaptation were studied in Desulfovibrio vulgaris Hildenborough using global transcriptional and metabolite analyses. The growth of D. vulgaris was inhibited by high salinity, and salt inhibition could be relieved by an addition of amino acids (e.g., tryptophan) or yeast extract. Salt shock (sudden increase in salt concentration) and salt adaptation (inoculating cells in the medium containing high concentrations of salt) showed a marked difference in respective transcriptomes. Salt adaptation induced expression of genes encoding proteins related to amino acid biosynthesis, formate dehydrogenases, histidine kinases, response regulators, specific transporters, heat-shock proteins, and peptidases. Genes involved in ribosomal protein synthesis, and energy metabolism were repressed. Genes involved in glycine/betaine/proline ABC transport, Na+/H+ transport, K+ uptake and transport, proline biosynthesis and transport, and glycerol biosynthesis and transport were not significantly affected. Metabolite assays suggested that some amino acids (e.g. glutamate) may accumulate as osmoprotectants in D. vulgaris. A conceptual model is proposed to link our observed results to currently available knowledge for further understanding the mechanisms of adaptation of D. vulgaris to sodium chloride.
  719. He, Z., Y. Deng, J. D. Van Nostrand, L. Wu, C. L. Hemme, T. J. Gentry, J. Liebich, A. P. Arkin, Terry C. Hazen and J. Zhou. 2008. Development of GeoChip 3.0 for Microbial Community Analysis. 108th General Meeting of the American-Society-for-Microbiology 108:547-548. abstract
    Microarrays constructed with the genes encoding key enzymes involved in various biological and geochemical processes are referred to as functional gene arrays (FGAs), or GeoChip. On the basis of GeoChip 2.0 (He et al. 2007), which contains 24,243 oligonucleotide (50mer) probes and covers > 10,000 gene sequences in >150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation, a new generation of GeoChip (GeoChip 3.0) has been developed. GeoChip 3.0 has several new features compared to GeoChip 2.0. First, GeoChip 3.0 is expected to cover >38,000 gene sequences for more than 290 gene families, and such a coverage allows us to obtain more information about microbial communities and analyze more diverse environmental samples. Second, the homology of automatically retrieved sequences by key words is verified by HUMMER using seed sequences so that any potential unrelated sequences are removed. Third, GeoChip 3.0 includes phylogenic markers, such as gyrB. Fourth, a software package (including databases) has been developed for sequence retrieval, probe and array design, probe verification, array construction, array data analysis, information storage, and automatic update, which greatly facilitate the management of such a complicated array, especially for future update. Fifth, a universal standard has been implemented in GeoChip 3.0 so that data normalization and comparison of different samples can be conducted. Finally, a genomic standard is also used to quantitatively analyze gene abundance. GeoChip 3.0 will provide more capability for studying biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, global climate change, ecosystem management, and environmental cleanup and restoration. It is also particularly useful for providing direct linkages of microbial genes/populations to ecosystem processes and functions.
  720. Hemme, C., Y. Deng, T. J. Gentry, L. Wu, M. W. Fields, S. Green-Tringe, J. C. Detter, K. Barry, N. Kyrpides, D. Watson, P. Richardson, Terry C. Hazen, J. Tiedje and J. Zhou. 2008. Comparative Metagenomics of Microbial Communities from Pristine and Contaminated Groundwater. 108th General Meeting of the American-Society-for-Microbiology 108:460. abstract
    Microbial community DNA isolated from contaminated groundwater located at the US Dept. of Energy Field Research Center (FRC) in Oak Ridge, TN, was analyzed to determine the effects of chronic exposure to contaminants on microbial community structure. The sample was obtained from a site (FW106) experiencing long-term exposure to high levels of uranium and other heavy metals, nitric acid and organic solvents. Analysis indicates very low species diversity community (~13 OTU) dominated by denitrifying γ- and β-proteobacteria. Metabolic reconstruction of the dominant γ-proteobacterial species revealed adaptations for specific geochemical parameters including the following: denitrification pathways; pathways for degradation of organic compounds such as1,2-dichloroethene, acetone, butanol, methanol and formaldehyde; accumulation of multiple heavy metal efflux systems (czcABC, czcD, cadA-family, mer operon genes, etc.). Analysis indicates that lateral gene transfer is the predominant mechanism of introducing genetic variation in the community, resulting in the lateral acquisition of, for example, an acetone carboxylation pathway and heavy metal efflux systems. The sample was compared to a second groundwater metagenome from a pristine FRC site (FW301) to determine differences between the two communities. In contrast to the low species diversity of FW106, the FW301 is represented by multiple phyla including all 5 classes of proteobacteria, Planctomycetes, Chloriflexi, Actinobacteria, Acidobacteria, Bacteroidetes and Firmicutes. In contrast to the FW106 sample which resulted in significant read assembly, the FW301 sample is composed largely of single reads that do not assemble into contigs (95%). Abundance profiling of geochemical and cytochrome genes between FW106 and FW301 and between FW106 and the acid mine drainage (AMD) metagenome show distinct environmental signatures between the samples. This analysis verified the previous observation of accumulation of heavy metal other toxin efflux mechanisms in FW106 as well as an accumulation of specific c-type cytochromes in FW106 that may be important in heavy metal resistance.
  721. Hubbard, S. S. and M. Conrad, John E. Peterson, Boris Faybishenko, Jonathan Ajo-Franklin, Terry C. Hazen, Phil Long K. Williams. 2008. Geophysical Monitoring of Hydrological and Biogeochemical Transformations associated with Bioremediation. Battelle International Symposium on Recalcitrant Substances
  722. Joyner, D. C., C. B. Walker, R. Chakraborty, J. L. Fortney, J. T. Geller, L. E. Camp, A. Zhou, Z. He, M. P. Joachimiak, S. Stolyar, J. Zhou, D. A. Stahl, A. P. Arkin and Terry C. Hazen. 2008. Characterization of Stress Response in a Sulfate Reducer/Methanogen Coculture. 108th General Meeting of the American-Society-for-Microbiology 108:404. abstract
    Sulfate-reducing bacteria (SRB) and methanogens are found to coexist in a variety of anoxic marine sediments. In these systems they either compete for substrates or engage in successful syntrophic relationships. In our experimental setup, Desulfovibrio vulgaris Hildenborough ferments lactate, producing acetate and hydrogen. Methanococcus maripaludis, a hydrogenotrophic methanogen, then utilizes hydrogen while also incorporating limited amounts of acetate as a carbon source. Mid-log growth phase of this co-culture is achieved in 3 days growing at 37°C at which point, nearly 50% of the initial lactate was depleted. In this study we investigate the stress response of this coculture and compare it to the D. vulgaris monoculture. Minimum Inhibitory Concentration (MIC) determinations of two environmentally relevant stressors (NO3- and NaCl) on the coculture and monoculture suggest nitrate predominantly affects M. maripaludis with a MIC of 25mM while sodium stress affects D. vulgaris with a MIC of 100mM. The response of the coculture to stressors like nitrate, nitrite, salt and peroxide was monitored by several methods. The fate of metabolites was tracked in the cultures and rates of gas evolution/utilization were measured with the Micro-Oxymax. Total biomass was measured over time with direct cell counts (including ratios of SRB: methanogen), cell protein and optical density. Metal reducing capability of log phase co-culture under NO3 stress was investigated and compared to that of under NaCl stress. Phenotype Microarray substrate utilization profiles generated by the Omnilog technology for a variety of metabolic substrates showed differential profiles for the coculture and the monoculture. Whole-genome transcriptional analysis, phenotype microarrays, and growth curves together give us a better understanding of stress response in this coculture.
  723. Kang, S., H. L. Gough, J. Van Nostrand, Z. He, L. Wu, D. A. Stahl, Terry C. Hazen and J. Zhou. 2008. Microbial Communities at the Metal Contaminated Lake Sediment. 108th General Meeting of the American-Society-for-Microbiology 108:428. abstract
    Microbes are known for their versatility under different and even certain extreme environmental conditions. The versatility is of particular interest in many applications including bioremediation. Lake DePue has been subjected to metal contamination over the last 80 years by adjacent zinc smelting activities. Sediments were collected in triplicate from five areas of the lake. These areas were previously identified as having varied metals contamination levels, and are located along a transect from near a creek inlet to the main body of the lake. GeoChip II, with over 10,000 microbial functional genes, was used to investigate metal impact on the microbial diversity and community structure. Three microbial communities of functional subgroups, defined by genes relevant to metal contamination (S cycle, metal resistance and reduction, and C cycle genes) were analyzed. Proximity of sampling sites to the contamination source was important factor in shaping microbial communities. The two sites closest to the contamination source (Sites 1 and 2) clustered together in all three communities, as measured by NMDS, hierarchical clustering and k-means clustering. Interestingly, these two sites also showed the greatest diversity (Shannon’s H’, Simpson’s -lnD and Fisher’s α). Proximity of sampling locations alone could not explain community variability as samples from Site 4, located between Sites 3 and 5, were more similar to samples from Sites 1 and 2. Multivariate correlation analysis by both Mantel test and Procrustes test revealed very significant correlation among three functional categories of communities (P < 0.001). Spatial structures of the three microbial communities were fairly distinctive, in that smaller scale patch size in C cycle community and larger scale patch size in metal-related community were observed while virtually no patchiness was observed in S cycle community by multivariate correlogram analysis. In conclusion, three microbial communities relevant to metal contamination were rather similar in their composition and diversity while their spatial structure was quite distinctive.
  724. Liu, H. and L. L. Yang, S. Allen, P. J. Walian, E. Johansen, S. C. Hall, S. J. Fisher, Terry C. Hazen, J. T. Geller, M. E. Singer, J. Jin, M. D. Biggin, B. Jap, H. E. Witkowska M. Dong. 2008. iTRAQ™ Reagent-Based “Tagless” Strategy of Identification and Purification of Soluble Protein Complexes in Bacteria: Development of High-Throughput Protocols. Annual Meeting ASMS Conference on Mass Spectrometry
  725. Martinez, R. J., M. J. Beazley, C. H. Wu, G. L. Andersen, Terry C. Hazen, M. Taillefert and P. A. Sobecky. 2008. Promoting Uranium Immobilization by the Activities of Microbial Phosphatases. 108th General Meeting of the American-Society-for-Microbiology 108:463. abstract
    Soils and groundwater contaminated with heavy metals and radionuclides remain a legacy of Cold War nuclear weapons development. In situ sequestration of heavy metals radionuclides as insoluble precipitates has proven to be the most cost-effective strategy for remediation. We are currently investigating a remediation approach that utilizes the phosphatase activity of bacterial strains obtained from the contaminated soils of the DOE Field Research Center (ORFRC) in Oak Ridge, TN, for heavy metal and radionuclide sequestration. We have previously demonstrated the accumulation of PO43- and concomitant mineralization of U(VI) by ORFRC Rahnella and Bacillus strains grown aerobically with 10 mM glycerol-3-phosphate (G3P) as the sole C and P source and 15 mM NO3- as the sole N source . We have initiated aerobic soil slurry experiments utilizing U(VI) and NO3- contaminated Area 3 ORFRC soils to determine the changes in microbial community structure following G3P stimulation. Soil slurries were incubated for 10 days at 30°C with 10 mM G3P as the sole C and P source. On average, 4 mM soluble PO43- was measured at the end of the soil slurry experiments. Following incubations (n=3), total DNA was extracted from the slurries and 16S diversity is being analyzed utilizing a high-density oligonucleotide microarray (PhyloChip). Anaerobic phosphate-mediated precipitation of soluble U(VI) have been conducted with Rahnella strain Y9602. Following 5 days of anaerobic growth with previously described concentrations of G3P and NO3-, greater than 1mM soluble PO43- was liberated into the culture media. This concentration of soluble PO43- promoted the mineralization of 90% of the soluble U(VI). Combining our pure culture and soil slurry studies, we plan to demonstrate the efficacy of organophosphate stimulation of microbial communities in heavy metal and radionuclide contaminated soils as a remediation strategy which could complement current approaches.
  726. Schadt, C. W., Z. Yang, A. Venkateswaran, M. Podar, S. Brown, A. Palumbo, T. Hazen and M. Keller. 2008. Developing Methods for Comprehensive Transcriptome Analysis of Environmental Communities. 108th General Meeting of the American-Society-for-Microbiology 108:461. abstract
    We are developing methods for direct cDNA sequencing of environmental samples utilizing high throughput (HT) analysis technologies to study community interactions. Since 80% or more of total RNA from bacteria is represented by the rRNA pool, it is crucial to first remove rRNA without adversely impacting mRNA quality, quantity and composition, prior to HT sequence based methods. We compared three different methods to remove rRNAs and enrich mRNAs. The first utilizes biotin-modified oligos complementary to rRNAs and specific removal by binding to streptavidin-coated beads. The second uses exonuclease that hydrolyzes rRNAs bearing a 5’ phosphate group. The third uses two rounds of reverse transcription, where rRNAs are first reverse transcribed with universal primers for 16 and 23S rRNAs and RNA/DNA hybrids and cDNA are removed by sequential digestion with RNaseH and DNaseI. All three methods significantly enrich mRNA from rRNA without introducing significant biases. Microarray analysis revealed significant differences in mRNA levels in only 0.5% to 5% of genes across the genome as compared to controls of total RNA. However combining the first two methods show much better efficiency for rRNA, removal (up to >65% enrichment compared to less than 50 for individual methods. We have also tested two methods for cDNA amplification based on circularization of the transcripts and phi29 polymerase amplification of either single stranded or double stranded cDNA. Double stranded amplifications resulted in more robust amplifications, greater consistency in gene detection and a greater number of detected genes. Comparisons of microarray results with HT sequencing results from the Solexa platform are currently ongoing. Following this technology development phase, we plan to apply these developed techniques to low biomass contaminated environments to understand the relationships between gene expression patterns and contaminant transformations.
  727. Sercu, B., C. H. Wu, L. C. Van De Werfhorst, E. L. Brodie, Terry C. Hazen, G. L. Andersen and P. A. Holder. 2008. High Density Microarray as an Additional Tool for Microbial Source Tracking in a California Urban Creek. 108th General Meeting of the American-Society-for-Microbiology 108:587. abstract
    High levels of fecal bacteriological contamination in coastal waters are often attributed to nearby human development. Tracking inland sources of fecal bacteria can be a significant challenge. To address this in Santa Barbara, CA, a multi-phase study has been conducted in an urban coastal creek that discharges into an impacted beach frequently posted with warnings. Culture-based IDEXX assays, quantitative PCR specific for human-specific Bacteroides, TRFLP and a high density microarray were used to determine human fecal contamination during dry weather at several locations during 3 consecutive days. Hierarchical clustering using microarray data indicated different degrees of relatedness of the microbial communities of water samples and separately collected sewage samples, and about half of the samples showed relatedness to sewage. When only fecal taxa (taxa most abundant in sewage samples) were considered, grouping according to location was stronger, and relatedness to sewage remained similar. Fecal OTUs belonged to families such as Bacteroidaceae, Lachnospiracae, Enterococcaceae, and Streptococcacea. Quantitative PCR identified a few samples containing elevated concentrations of the human-specific Bacteroides marker. Those samples also contained more fecal taxa, indicating agreement between qPCR and microarray results. However, the microarray results suggested human fecal pollution in additional samples, where no human-specific Bacteroides markers were found. Finally, the absence of important water-related pathogens (e.g. Campylobacter jejuni), and the presence of other pathogens (e.g. Helicobacter pylori) was shown. In conclusion, the microarray metagenomics approach appeared a valuable addition to the DNA toolbox for detection of human fecal waste in urban creek water samples. The results obtained in this study will guide further development of a microarray designed specifically for microbial water quality purposes.
  728. Van Nostrand, J. D., W. M. Wu, L. Wu, Y. Deng, J. Carley, S. Carrol, Z. He, B. Gu, J. Luo, C. Criddle, P. Jardine, Terry C. Hazen and J. Zhou. 2008. Microbial Functional Community Changes in a Bioreduced, Uranium-Contaminated Subsurface During Periods of Resting, Reoxidation, and Recovery. 108th General Meeting of the American-Society-for-Microbiology 108:623-624. abstract
    A pilot-scale system was established for biostimulation of subsurface U(VI) reduction by injection of ethanol at the U.S. DOE’s Field Research Center (FRC) in Oak Ridge, TN. After U(VI) reduction was achieved, the stability of the bioreduced area was examined by suspension of ethanol injections (resting state) and reoxidation of the area by introducing dissolved oxygen (DO) for two months and then ethanol injections were resumed. Geochip 2.0, a comprehensive 50mer microarray containing probes for genes involved in the geochemical cycling of N, S, and C, metal resistance and contaminant degradation, was used to monitor the dynamics of the groundwater microbial community structure and function. The immobilized U was stable during the resting state. After DO was introduced to the reduced area, the monitoring well (FW101-2) located closest to the injection well, had a greater increase in DO (2 mg L-1) than the well located further away (FW102-3; <0.4-0.5 mg L-1). Based on canonical correspondence analysis and Mantel test results, ethanol showed the greatest correlation to community structure, although sulfide did correlate with changes in the functional community. Detrended correspondence analysis showed a shift towards a different community structure after ethanol injections resumed compared to the periods of starvation and exposure to DO. Changes in the functional community structure were similar in the two wells; however, the community in FW101-2 was more affected by DO than in FW102-3. Hierarchical clustering showed that cytochrome c genes grouped based on DO exposure, resting state, or ethanol addition, while dissimilatory sulfite reductase (dsr) genes grouped only by resting state or ethanol addition. However, the relative abundance of dsr genes did decrease when DO levels increased while the relative abundance of cytochrome genes seemed unaffected by changes in DO. Overall, results indicated that ethanol was the main factor affecting community structure, although some changes could be attributed to DO.
  729. Waldron, P. J., J. D. Van Nostrand, D. B. Watson, Z. He, L. Y. Wu, P. M. Jardine, Terry C. Hazen and J. Z. Zhou. 2008. GeoChip Analysis of Subsurface Microbial Communities Impacted by Heavy Metal and Nitrate Contamination. 108th General Meeting of the American-Society-for-Microbiology 108:438-439. abstract
    The objective of this study is to examine the bacterial community structure in wells of varying heavy metal and acid contamination to determine which contaminants have the greatest effect. Five monitoring wells and an uncontaminated background well from the Field Research Center (FRC) site of the U.S. DOE ERSP (Environmental Remediation Science Program) at Oak Ridge, Tennessee, were sampled to provide a gradient of groundwater nitrate, pH and uranium concentrations. DNA from these samples was analyzed with a comprehensive functional gene array containing 24,243 probes for >10,000 genes involved in carbon, sulfur, nitrogen, and metal cycling. Genes with the highest signal intensities from each sample were correlated with the groundwater geochemistry of that well. Wells with similar geochemical profiles had greater gene overlap than dissimilar wells. A higher percentage of nitrogen fixation genes were detected in groundwater with lower nitrate concentrations, while the percentage of nitrate reduction genes generally decreased with decreasing nitrate. Wells with elevated sulfate concentrations had a greater percentage of genes dedicated to sulfate reduction, and higher signal intensities for dsrAB genes than the background, indicating a greater abundance of those genes. Contaminated wells did not have a higher percentage of metal reduction and resistance genes than the background, but the total signal intensity of those genes was 1.4- to 2.3-fold greater than the background, indicating that metal-related genes were more prevalent in the contaminated wells. Uranium, nitrate and sulfate were identified by CCA as important factors in determining community structure. This study provides an overall view of the functional genes present in a highly contaminated environment, and shows the differences in functional populations between wells with varying contamination. As indicated by this work, contaminant level has significant effects on bacterial community structure, the knowledge of which may be important in planning and implementing successful bioremediation strategies in the future.
  730. Waldron, P. J., L. Y. Wu, J. D. Van Nostrand, D. B. Watson, Z. He, C. W. Schadt, Terry C. Hazen, P. M. Jardine and J. Z. Zhou. 2008. Functional Gene Array-Based Analysis of Microbial Community Structure in a Gradient of Nitrate and Heavy Metal Contaminated Groundwaters. 108th General Meeting of the American-Society-for-Microbiology 108:439. abstract
    Six groundwater monitoring wells from the Field Research Center, site of the U.S. DOE Environmental Remediation Science Program (ERSP) at the Oak Ridge Reservation, Oak Ridge, TN, were selected to compose a gradient of pH (3.25 - 7.11), nitrate (1.2 - 41,790 mg/l) and heavy metal contamination (0 - 500 mg/l U; 0 - 39896 mg/l Tc). To determine the functional populations of bacteria present within the gradient, DNA was extracted from groundwater and analyzed with a functional gene array containing 2,006 gene probes for the detection of genes involved in metal-resistance, sulfate-reduction, contaminant degradation and carbon and nitrogen cycling. The signal intensities for each probe were used to measure community diversity and were correlated to the geochemical profile of each well. Diversity decreased in relation to the level of contamination within each well, and each community exhibited a different distribution of genes. Heatmaps of metal resistance genes and nirK and nirS genes indicate that highly contaminated wells had lower gene diversity, but greater signal intensity for detected genes. Wells with the highest sulfate concentrations had the greatest diversity and signal intensity for dsrAB genes. A greater number of carbon fixation genes (cbbL, cbbM) were detected than fermentation genes (FTHFS) in all wells. A variety of organic contaminant degradation genes were detected. Results of Mantel tests and canonical correspondence analysis indicate that nitrate, sulfate, pH, uranium and technetium have a significant (p = 0.05) effect on bacterial community structure. This study provides an overall picture of bacterial community structure in contaminated environments across many different functional genes and shows that diversity can vary widely in relation to the degree of contamination.
  731. Wu, C. H., J. Chou, S. Osman, Y. Fujita, E. L. Brodie, M. Bill, M. S. Conrad, G. L. Andersen and Terry C. Hazen. 2008. Microbial Populations Stimulated by Triethyl Phosphate for in Situ Sequestration of Metals and Radionuclides. 108th General Meeting of the American-Society-for-Microbiology 108:455. abstract
    Significant quantities of metals and radionuclides are contained in thick unsaturated zones at several contaminated sites in the western US. In many cases, this contamination has migrated to underlying groundwater, sometimes decades after being released into the subsurface. Because of the prohibitive costs associated with physically removing the contamination, an attractive remedy to this problem is to develop methods for long-term in situ stabilization of the contamination in the vadose zone. Triethyl phosphate (TEP) gas has been delivered to aquifers and vadose zones to enhance biodegradation of organic pollutants, presumably by alleviating phosphate limitations. We hypothesize that microbially-mediated TEP hydrolysis generating inorganic phosphate could also be used to promote metal and radionuclide sequestration in situ. Phosphate minerals are effective scavengers of contaminants such as Sr-90 and U. However, it is unclear which eubacteria are capable of metabolizing TEP. A high-density microarray (PhyloChip) has been used to characterize the microbial community composition in TEP-enriched subsurface sediments. Results indicate a 30% increase in inorganic phosphate production and 20% lower TEP concentration in sediments without heat sterilization. Microarray analysis reveals that Proteobacteria taxa are enriched by TEP, particulary Methylophilaceae, Comamonadacea, Burkholderiaceae, and Rhizobiales. On-going work includes 1) microarray analysis of metabolically active microbes through extraction and hybridization of rRNA, 2) evaluation of isotopic methods for monitoring microbial utilization of TEP, and 3) demonstration of radionuclide and metal sequestration induced by microbial metabolism of TEP. In conclusion, this research demonstrates the potential of microbial catalysis of TEP in generating inorganic phosphate for the sequestration of metals and radionuclides.
  732. Wu, L., Z. Huang, T. J. Gentry, W. Wu, Z. He, J. D. Van Nostrand, C. W. Schadt, D. Watson, P. Jardine, C. S. Criddle, J. Tiedje, Terry C. Hazen and J. Zhou. 2008. Microbial Community Dynamics and the Effect of Geochemistry in Uranium Bioremediation Revealed by Functional Gene Array Analysis. 108th General Meeting of the American-Society-for-Microbiology 108:436. abstract
    A pilot-scale system was established to examine the feasibility of in situ bioremediation and immobilization of U(VI) at a highly contaminated aquifer at the U. S. DOE’s Field Research Center, Oak Ridge, TN. An above-ground treatment system, including a denitrifying fluidized bed reactor, was used to pre-condition the groundwater to optimize subsurface U immobilization. Treated water was injected into the subsurface along with ethanol to stimulate microbial reduction of soluble U(VI) to insoluble U(IV). Three monitoring wells (FW101-2, 102-2, and 102-3) were analyzed using a functional gene array containing >24,000 probes covering 10,000 genes in >150 gene categories to examine the effect of geochemistry on the functional microbial community dynamics. Microarray results indicate that, during the U (VI) reduction period, both FeRB and SRB populations reached their highest levels on day 212 in FW102-3 and on day 255 in FW102-2 and FW101-2, followed by a gradual decrease over the next 500 days in all three wells and then a rebound on day 719. Mantel tests of functional genes versus the geochemical parameters showed a significant correlation in all three wells between pH and most of the functional gene groups (p-value, <0.01-0.1) detected. The U (IV) concentrations were significantly correlated with the microbial communities in wells FW101-2 and FW102-3 over the entire study period. Once the microbial population peaked, this correlation was also observed for FW102-2, , and even stronger correlations were observed in both FW101-2 and 102-3. Chemical oxygen demand (COD) correlated with the microbial community structure only in well FW101-2. Neither nitrate nor sulfate showed a significant correlation in any of the wells until after the population peak, when significant correlations were observed in FW102-2 and 102-3. Canonical correlation analysis revealed similar correlations between the functional community and the geochemical variables. In addition to correlations with the geochemical parameters, principal components analysis showed that the microbial communities also varied both temporally and spatially.
  733. Wu, L. Y., X. Liu, M. W. Fields, D. K. Thompson, C. E. Bagwell, J. M. Tiedje, Terry C. Hazen and J. Zhou. 2008. Microarray-Based Whole-Genome Hybridization as a Tool for Determining Procaryotic Species Relatedness. 108th General Meeting of the American-Society-for-Microbiology 108:339. abstract
    Due to their small size and diverse metabolic functions, the identification of microorganisms is a great challenge. Whole-genome DNA-DNA hybridization is the cornerstone for defining procaryotic species relatedness, but obtaining pair-wise DNA-DNA reassociation values for a comprehensive phylogenetic analysis of procaryotoes is tedious and time-consuming. A novel format of microarray that contained whole genomic DNA, referred to here as a community genome array (CGA), was developed and 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 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 hybridizations could serve as a powerful, high-throughput format for determining species relatedness of microorganisms.
  734. Zhou, A., Z. He, M. P. Joachimiak, P. S. Dehal, A. P. Arkin, K. Hillesland, D. Stahl, J. D. Walls, Terry C. Hazen and J. Zhou. 2008. The Dynamics and Genetic Adaptation to Salt Stress in Long-Term Experimental Evolution of Desulfovibrio vugaris Hildenborough. 108th General Meeting of the American-Society-for-Microbiology 108:647. abstract
    One of the greatest challenges in biology is to understand the interaction between genotype and environment to determine the fitness of an organism. With the recent advances in genome sequencing and high-throughput genomic technologies, now it is possible to link sub-cellular molecular/metabolic processes with the population-level processes, functions and evolution. Sulfate reducing bacteria Desulfovibrio vugaris Hildenborough (DvH) is an ideal model environmental organism to address such fundamental questions. In this study, we aim to investigate the long-term evolutionary responses, diversifications and adaptation of DvH to salt stress by mimicking the stress condition in the lab culture. LS4D and LS4D + 100 mM NaCl were used as medium for the control lines and stressed lines (6 lines each, from single colony based pure culture) respectively. Cultures were kept at 37oC and transferred every 48 hrs with one to one hundred dilutions. The phenotype of the cell lines on LS4D with higher salt concentration were tested periodically. The results demonstrated that the adaptation to salt stress is a dynamical process. The enhanced salt tolerance to higher salt (LS4D + 250 mM NaCl) of stressed lines was observed at 300 generations; which became more and more obvious with the increase of generations. The de-adaptation experiment on 500, 1000 and 1200 generation cell lines not only provided strong evidence that the phenotype was due to the genetic change instead of physiological adaptation, but also clearly showed the dynamic trend of the genetic adaptation- the genetic mutation became stable at 1000 generation. To further decipher the genetic mystery in behind, the gene expression profile of the 1000 generation were examined by DvH whole genome microarray. Some poly-cistronic operons such as hmcF-E-D-C-B-A, rrf2-rrf1, LysA-2-LysX and DVU3290-3291-3292 (glutamate synthase) were significantly up-regulated in stressed lines. Next, whole genome sequencing on selected colony will be performed to identify the beneficial genetic mutation and more colonies will be checked to confirm whether the mutations are stable or fixed.
  735. Zhou, A., Z. He, A. Mukhopadhyay, C. Hemme, J. D. Keasling, A. P. Arkin, Terry C. Hazen, J. D. Wall and J. Zhou. 2008. Genome-Wide Transcriptomic and Proteomic Analyses of Desulfovibrio vulgaris Hildenborough Response to Hydrogen Peroxide. 108th General Meeting of the American-Society-for-Microbiology 108:329. abstract
    Oxidative stress is one of the most common environmental stressors. More and more evidences show that the obligate anaerobe Desulfovibrio vulgaris Hildenborough (DvH) is aero-tolerant. However, very little is known about the molecular mechanism of DvH oxidative stress responses. In order to investigate the oxidative stress response of DvH, first, the tolerance of DvH to H2O2 was tested using Bioscreen. Results showed that there was a dosage effect of the H2O2 treatment. Low concentrations (0.1 - 2 mM) of H2O2 did not inhibit the cell growth significantly, while more than 4 mM of H2O2 led to obvious growth retardation. Therefore, in this study, 1 mM of H2O2 was applied to the early mid-log phase DvH cell culture and samples were collected at 6 time-points (0, 30, 60, 120, 240 and 480 min) to study the response at both transcriptional and translational levels. The microarray data demonstrated that the expression profiles among 30, 60 and 120 min were closer; 240 min and 480 min were closer according to PCA analysis and the response peak was at 120 min. The protein content at 120 min after H2O2 shock was analyzed with iTRAQ. A total of 379 proteins were identified with 9 significant increased proteins and 19 significant decreased proteins (Z > 2.0). The comparison between the significant protein changers and the corresponding gene expression changers showed that most of them had the same trend. In gene transcription level, as expected, the predicted regulons such as PerR, FUR were up-regulated; thioredoxin and thioredoxin reductase; transport and binding proteins (copper-translocating, mercuric transport, ferrous iron transport, permease and ABC transporters), protein fate (MsrA, MsrB; DanJ, Daf, heat shock protein etc.), genes involved in DNA replication, recombination, and repair were significantly up-regulated. However, among the known genes involved in oxidative stress, only Rdl, Rbr2 were up-regulated, which indicates that Rdl and Rbr2 may play major roles in the response to H2O2. In addition, the up-regulation of two component system genes such as DVU3269, DVU3381 and DVU3382 imply that they play roles in the oxidative stress response.
  736. Allen, S. and E. Szakal, H. Liu, M. Dong, E. Johansen, L. L. Yang, S. C. Hall, S. J. Fisher, Terry C. Hazen, J. T. Geller, M. E. Singer, J. Jin, M. D. Biggin, B. Jap, H. E. Witkowska P. J. Walian. 2008. Toward the Development of a “Tagless” Method for the Isolation and Identification of Membrane Complexes in Desulfovibrio vulgaris Hildenborough. Annual Meeting ASMS Conference on Mass Spectrometry
  737. Hazen, Terry C.. 2008. Bioremediation: The Hope and the Hype for Environmental Cleanup. National Student Leadership Conference (Engineering)
  738. Hazen, Terry C. 2008. Systems Biology Approaches to Metal/Radionuclide Contaminated Sites in Colloquium on Function and Activity in Microbial Consortia. 108th General Meeting of the American-Society-for-Microbiology
  739. Hazen, Terry C.. 2008. Bioremediation: The Hope and the Hype for Environmental Cleanup. National Student Leadership Conference (Engineering)
  740. Hazen, Terry C.. 2008. A Systems Biology Approach to Environmental Biotechnology: Holes are greater then the parts that plug them!. Oak Ridge National Laboratory
  741. Hazen, Terry C.. 2008. A Systems Biology Approach to Environmental Biotechnology: Holes are greater then the parts that plug them!. University of Tennessee
  742. Hazen, Terry C. 2008. State-of-the-science on microbial processes affecting subsurface contamination. Webnar: Developing a Roadmap to Accelerate Deployment of Field-Scale Models to Simulate Microbially-Mediated Remediation Performance of Contaminated Sites
  743. Arkin, A. P., Terry C. Hazen, C. Abulencia, E. J. Alm, G. Anderson, M. Auer, E. Baidoo, K. S. Bender, P. Benke, S. Borglin, E. Brodie, S. Brown, L. Camp, R. Chakraborty, S. Chhabra, G. Chirica, D. Chivian, M. Cipriano, P. S. Dehal, T. DeSantis, E. Drury, I. Dubchak, D. Elias, M. W. Fields, V. O. Y. Fok, J. Fortney, S. Gaucher, J. Geller, M. Hadi, Z. He, C. Hemme, K. Hillesland, H.-Y. Holman, K. H. Huang, Y. W. Huang, C. Hwang, J. Jacobsen, M. P. Joachimiak, D. Joyner, J. Keasling, K. Keller, M. Keller, Y. Light, E. Luning, R. Meagher, A. Mukhopadhyay, A. Palumbo, R. Phan, T. Phelps, F. Pingitore, M. Podar, M. N. Price, A. Redding, J. Robertson, R. Sapra, C. Schadt, M. Shirley, A. Shutkin, M. Singer, A. Singh, D. A. Stahl, S. Stolyar, A. Sundararajan, Y. Tang, J. V. Nostrand, S. Villa, C. Walker, J. D. Wall, Z. K. Yang, H.-c. Yen, G. Zane, A. Zhou, J. Zhou. 2008. The Virtual Institute of Microbial Stress and Survival - VIMSS:ESPP Overview. Genomics: GTL Awardee Workshop VI
  744. Camp, L., S. Chhabra, D. Elias, J. T. Geller, H.-Y. Holman, D. C. Joyner, J. D. Keasling, A. Mukhopadhyay, M. Singer, T. Torok, J. D. Wall, Terry C. Hazen, S. Allen, G. Butland, M. Choi, M. Dong, S. C. Hall, B. K. Jap, J. Jin, S. J. Fisher , H. Liu, E. Szakal, P. J. Walian, H. E. Witkowska, L. Yang, M. D. Biggin*, P. Arbelaez, M. Auer, D. Ball, F. Garczarek, R. M. Glaeser, D. Jorgens, J. Malik, E. Nogales , H. Palsdottir, J. P. Remis, D. Typke, K. H. Downing, S. S. Andrews, A. P. Arkin, S. E. Brenner, Y. W. Huang, K. Keller, R. Santos, M. Shatsky, J.-M. Chandonia. 2008. Protein Complex Analysis Project (PCAP): Project overview. Genomics: GTL Awardee Workshop VI
  745. Chhabra, S. and D. Elias, V. Fok, B. Gold, J. Jin, A. Mukhopadhyay, R. Prathapam, W. Yang, J.-M. Chandonia, J. D. Wall, Terry C. Hazen, J. D. Keasling G. Butland. 2008. Protein Complex Analysis Project (PCAP): High throughput strategies for tagged-strain generation in Desulfovibrio vulgaris. Genomics: GTL Awardee Workshop VI
  746. Deutschbauer, A., J. Oh, M. Price, P. Dehal, D. Bruno, M. Henriquez, R. Chakraborty, Terry C. Hazen, C. Nislow, G. Giaever, R. W. Davis, A. P. Arkin. 2008. Phenotypic characterization of microorganisms by bar-coded transposon mutagenesis. Genomics: GTL Awardee Workshop VI
  747. Deutschbauer, A., Y. W. Huang, K. H. Huang, E. J. Alm, D. Chivian, P. S. Dehal, M. P. Joachimiak, K. L. Keller, M. N. Price, I. Dubchak, Terry C. Hazen, A. P. Arkin. 2008. A systems Biological Framework for Microbial Pathway Characterization and Engineering. EBI 2008 Research Retreat
  748. Elias, D. A., E. C. Drury, A. M. Redding, A. Mukhopadhyay, H.-C. B. Yen, K. H. Huang, Terry C. Hazen, A. P. Arkin, J. D. Wall. 2008. Expression profiling and gene association of hypothetical and conserved genes in Desulfovibrio vulgaris leads to functional annotation. Genomics: GTL Awardee Workshop VI
  749. Hazen, Terry C., C. Abulencia, G. Anderson, S. Borglin, E. Brodie, S. v. Dien, M. Fields, J. Fortney, J. Geller, E. Hendrickson, H.-Y. Holman, J. Leigh, T. Lie, R. Phan, J. Jacobsen, D. Joyner, R. Chakraborty, M. Keller, A. Mukhopadhyay, C. Schadt, D. Stahl, S. Stolyar, C. Walker, J. D. Wall, E. Wozei, Z. Yang, H.-c. Yen, G. Zane, J. Zhou. 2008. Applied Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers VIMSS:ESPP. Genomics: GTL Awardee Workshop VI
  750. Hazen, Terry C. 2008. JBEI and the implications for Energy Science in the US. University of California Guest Lecture in The Science of Climate Change Mitigation
  751. Hazen, T. C. and J. D. Keasling, A. Mukhopadhyay, S. Chhabra, J. T. Geller, M. Singer, D. C. Joyner, L. Camp, T. Torok, J. D. Wall, D. Elias, M. D. Biggin H.-Y. Holman. 2008. Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes during Stress Response in Desulfovibrio vulgaris: Microbiology Subproject. Genomics: GTL Awardee Workshop VI
  752. He, Z., Y. Deng, J. D. V. Nostrand, L. Wu, C. Hemme, T. J. Gentry, A. P. Arkin, Terry C. Hazen, J. Zhou. 2008. Further development and applications of Geochip 3.0 for microbial community analysis. Genomics: GTL Awardee Workshop VI
  753. Hemme, C. L., Y. Deng, T. J. Gentry, L. Wu, M. W. Fields, S. Green-Tringe, C. Detter, K. Barry, N. Kyrpides, D. Watson, P. Richardson, Terry C. Hazen, J. Tiedje, E. Rubin, J. Zhou. 2008. Comparative Metagenomics of Microbial Communities from Pristine and Contaminated Groundwater. Genomics: GTL Awardee Workshop VI
  754. Holman, H.-Y. N., E. Wozei, Z. Lin, L. Comolli, K. H. Downing, M. Fields, Terry C. Hazen. 2008. Observing Polyglucose Metabolism and Transient Oxygen Stress in Obligate Anaerobes in Vivo. Genomics: GTL Awardee Workshop VI
  755. Mukhopadhyay, A., D. Joyner, E. Luning, K. Keller, J. Robertson, G. Zane, J. Jacobsen, M. Price, S. Chhabra, Terry C. Hazen, A. P. Arkin, J. Wall, J. Keasling. 2008. VIMSS ESPP: Deciphering the roles of two-component systems in Desulfovibrio vulgaris Hildenborough. Genomics: GTL Awardee Workshop VI
  756. Schadt, C., Z. Yang, A. Venkateswaran, M. Drake, S. Carroll, D. Klingeman, M. Podar, T. Phelps, S. Brown, A. Palumbo, S. Stolyar, C. Walker, D. Stahl, Terry C. Hazen, M. Keller. 2008. Applications of Systems Biology Approaches to Understanding Artificial Microbial Consortia and Environmental Communities in the VIMSS Applied Environmental Microbiology Core. Genomics: GTL Awardee Workshop VI
  757. Shutkin, A., A. P. Arkin and Terry C. Hazen. 2008. VIMSS:ESPP2 Scientific Research Project Management. Genomics: GTL Awardee Workshop VI
  758. Van Nostrand, J. D., L. Wu, S. Kang, P. Waldron, Y. Liang, Y. Deng, Z. He, W. Wu, H. Gough, S. Carroll, C. Schadt, A. Palumbo, D. Watson, C. Criddle, P. Jardine, B. Baldwin, A. Peacock, P. Long, D. Stahl, Terry C. Hazen, J. Z. Zhou. 2008. Applications of GeoChip to examine functional microbial communities in metal contaminated environments. Genomics: GTL Awardee Workshop VI
  759. Walker, C. B., Z. He, Z. K. Yang, J. Jacobsen, J. Joseph A. Ringbauer, Q. He, J. Zhou, G. Voordouw, J. D. Wall, A. P. Arkin, Terry C. Hazen, S. Stolyar, D. A. Stahl. 2008. Energy Conservation in a Biogeochemically Significant Microbial Mutualism. Genomics: GTL Awardee Workshop VI
  760. Zhou, A., Z. He, M. P. Joachimiak, P. S. Dehal, A. P. Arkin, K. Hillesland, D. Stahl, J. Wall, Terry C. Hazen, J. Zhou. 2008. The dynamics and genetic adaptation to salt stress in experimental evolution of Desulfovibrio vulgaris Hildenborough. Genomics: GTL Awardee Workshop VI
  761. Hazen, Terry C. 2008. Field-Integrated Studies of Long-Term Sustainability of Chromium Bioreduction at Hanford 100H Site. Annual Meeting American Geophysical Union
  762. Hu, P., R. Chakraborty, E. L. Brodie, G. L. Andersen, Terry C. Hazen. 2008. Reduction of Cr(VI) and survival in Cr-contaminated sites by Caulobacter crescentus. Annual Meeting American Geophysical Union
  763. Zhou, A., A. Mukhopadhyay, Z. He, C. L. Hemme, J. D. Keasling, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou. 2008. Diverse Oxidative Stress Resistance Mechanisms in Sulfate-reducing Bacteria as Revealed by Global Analysis of the Impact of H2O2 Exposure on Desulfovibrio vulgaris Hildenborough. Annual Meeting American Geophysical Union
  764. Chivian, D. and E. Brodie, D. Culley, P. Dehal, T. DeSantis, T. Gihring, A. Lapidus, L.-H. Lin, S. Lowry, D. Moser, P. Richardson, G. Southam, G. Wanger, L. Pratt, G. Andersen, T. Hazen, F. Brockman, A. Arkin, T. Onstott E. Alm. 2008. Environmental Genomics Reveals a Single Species Ecosystem Deep Within the Earth. International Symposium of Microbial Ecology (ISME XII)
  765. Hazen, Terry C. 2008. Symposium: Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology. Society for Industrial Microbiology Annual Meeting
  766. Hazen, Terry C. 2008. The System Biology – Integrated Omics Approach. Society for Industrial Microbiology Annual Meeting
  767. Hazen, T. C. and M. Bailey, P. Hugenholtz, G. L. Andersen, J. Zhou J. F. Banfield. 2008. Roundtable: Ecogenomics, Which Methods are Best?. International Symposium of Microbial Ecology (ISME XII)
  768. He, Z. and J. Van Nostrand, L. Wu, C. Hemme, T. Gentry, J. Liebich, A. Arkin, T. Hazen, J. Zhou Y. Deng. 2008. Development and Application of Geochip 3.0 for Microbial Community Analysis. International Symposium of Microbial Ecology (ISME XII)
  769. Wells, G. and E. Pérez, H.-D. Park, L. Sepúlveda-Torres, B. Eggleston, E. Brodie, T. DeSantis, G. Andersen, T. Hazen, A. Massol-Deyá, J. Zhou, C. Francis, C. Criddle S. Kang. 2008. Investigating the Core and Dispensable Microbiome in Activated Sludge with High-Density Oligonucleotide Phylogenetic and Functional Gene Microarrays. International Symposium of Microbial Ecology (ISME XII)
  770. Wu, C. H. and M. E. Singer, E. L. Brodie, Terry C. Hazen, G. L. Andersen S. R. Oman. 2008. Monitoring sewage microbial community changes in ocean water with the PhyloChip. International Symposium of Microbial Ecology (ISME XII)
  771. Zhou, A., Z. He, M. Joachimiak, P. Dehal, K. Hillesland, A. Arkin, D. Stahl, J. Wall, T. Hazen, J. Zhou*. 2008. The Dynamics and Genetic Adaptation to Salt Stress in Long-Term Laboratory Evolution of Desulfovibrio vulgaris Hildenborough. International Symposium of Microbial Ecology (ISME XII)
  772. Hazen, Terry C.. 2008. Bioremediation: the Hope and the Hype of Environmental Cleanup. Thomas Morton Lecture Series Niagara University
  773. Hazen, Terry C. 2008. Biotechnology approaches to address issues about clean and sustainable energy production and management of waste. 2nd Biotechnology Congress
  774. Hazen, Terry C. 2008. Leveraged programs for site remediation. National Academy of Sciences Committee on Development and Implementation of a Cleanup Technology Roadmap for DOE’s Office of Environmental Management
  775. Hazen, Terry C.. 2008. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. ECSC conference
  776. Hazen, Terry C.. 2008. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. Connecticut Valley Branch, American Society for Microbiology
  777. He, Z. and Y. Deng, Y. Liang, L. Wu, P. Waldron, C. Hemme, T. Gentry, W. Wu, D. Watson, Terry C. Hazen, C. S. Criddle, J. Zhou J. Van Nostrand. 2008. Further Development and Applications of GeoChip for Microbial Community Analysis. DOE Environmental Remediation Sciences Program Annual Review
  778. Hubbard, S. S. and H. Beller, E. Brodie, J. Chen, J. Christensen, M. Conrad, D. DePaolo, B. Faybishenko, S. Finsterle, M. Kowalsky, Terry C. Hazen, P. Nico, S. Pride, E. Sonnenthal, N. Spycher, C. Steefel, T. Tokunaga, J. Wan, K. Williams J. Ajo-Franklin. 2008. Subsurface Science Scientific Focus Area at Lawrence Berkeley National Laboratory. DOE Environmental Remediation Sciences Program Annual Review
  779. Martinez, R. J. and C. Wu, Terry C. Hazen, G. L. Andersen, S. M. Webb, M. Taillefert, P. A. Sobecky M. J. Beazley. 2008. Promoting Uranium Immobilization by the Activities of Microbial Phosphates. DOE Environmental Remediation Sciences Program Annual Review
  780. Hazen, Terry C.. 2007. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. The Second International Conference on Challenges in Site Remediation: Site Characterization & Performance Monitoring
  781. Hazen, Terry C. 2007. Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical and Microbiological Response to Slow Release Electron Donor. The Second International Conference on Challenges in Site Remediation: Site Characterization & Performance Monitoring
  782. Van Nostrand, J. D., Y. Deng, L. Wu, W. Wu, S. Carroll, Z. He, C. Criddle, P. Jardine, Terry C. Hazen and J. Z. Zhou. 2007. Changes in Microbial Community Function during a Period of Reoxidation in a Groundwater Recirculation System. 15th Annual International Conference on Microbial Genomics
  783. Hazen, Terry C.. 2007. Bioremediation: The Hope and the Hype for Environmental Cleanup. Videoconference Seminar for Department of Biology, University of Puerto Rico
  784. Hazen, Terry C.. 2007. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. Kentucky/Tennessee Branch of the American Society for Microbiology
  785. Hazen, Terry C.. 2007. Bioremediation: The Hope and the Hype for Environmental Cleanup. Allegheny Branch American Society for Microbiology
  786. Hazen, Terry C.. 2007. Bioremediation: The Hope and the Hype for Environmental Cleanup. SE Branch American Society for Microbiology
  787. Hazen, Terry C.. 2007. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. Theobald Smith Society, NJ Branch American Society for Microbiology
  788. Biggin, M. D., M. Dong, G. Butland, J. T. Geller, S. C. Hall, Terry C. Hazen, B. K. Jap, J. Jin, S. J. Fisher, M. Singer, P. Walian, H. E. Witkowska and L. Yang. 2007. High throughput purification and identification of water soluble protein complexes in Desulfovibrio vulgaris. 107th General Meeting of the American-Society-for-Microbiology 107:543. abstract
    As part of the Protein Complex Analysis Project (PCAP) we are developing a high throughput pipeline to purify water soluble protein complexes from D. vulgaris, identify their polypeptide constituents by mass spectrometry, determine their stoichiometries, and provide samples suitable for single particle EM characterization. These methods will then be used as part of PCAP's effort to model stress responses relevant to the detoxification of metal and radionuclide contaminated sites. Our strategy uses a novel "tagless" method that fractionates the water soluble protein contents of a bacterium into a large number of fractions, and then identifies the polypeptide composition of a rational sampling of 10,000 - 20,000 of these fractions using MALDI TOF/TOF mass spectrometry. To establish this method to date: We have developed an optimized four-step fractionation scheme. We have built a prototype multi-channel, native gel electrophoresis instrument for high resolution protein separation and automated band collection that elutes a protein band into a 200 micro liter fraction without noticeable loss of sample. We have established an efficient, highly reproducible mass spectrometry sample preparation protocol that uses 96-well PVDF multiscreen plates. We have demonstrated that iTRAQ methodology provides quantitation of the relative abundances of polypeptides in different chromatographic fractions. We have also developed algorithms and graphical display tools for identifying protein complexes from mass spectrometry data, including a method for cluster analysis of iTRAQ data to allow detections of co migrating polypeptides and hence putative protein complexes. Finally, we have refined methods for preparing protein samples suitable for single particle EM analysis. To date, we have identified and purified 15 water soluble protein complexes from D. vulgaris, of which five have been sent for EM structural determination, and one of which has been solved at 17 Angstrom resolution. We are further optimizing and refining our methods to establish a fully functional high throughput pipeline.
  789. Brodie, E. L., Terry C. Hazen, B. A. Faybishenko, J. Van Nostrand, D. C. Joyner, R. Chakraborty, M. S. Conrad, S. E. Borglin, F. E. Long, D. R. Newcomer, T. Z. DeSantis, J. Zhou and G. L. Andersen. 2007. Phylogenetic and functional gene microarray analysis demonstrates direct and indirect mechanisms for sustained chromium bio-immobilization. 107th General Meeting of the American-Society-for-Microbiology 107:444. abstract
    Background: During a field-scale chromium treatability study, a single dose of a slow release electron donor (HRC) was applied to a contaminated aquifer to stimulate microbial reductive precipitation of hexavalent chromium Cr(VI). Here we present analysis of microarray-based prokaryotic population dynamics and correlations with geochemical and geophysical observations following this application over two years. Methods: A high-density 16S rRNA phylogenetic microarray (PhyloChip) and a functional gene array (FGA) were used to analyze groundwater samples from multiple depths in injection and monitoring wells taken at intervals pre- and post-HRC injection. Following filtration, genomic DNA was extracted and PCR amplicons or MDA-amplified community-DNA were analyzed by microarray hybridization. A range of geochemical and geophysical parameters were also monitored. Results: Following HRC injection reducing conditions had rapidly established with a corresponding decline in DO, Eh and nitrate. Cr(VI) concentrations declined steadily over 6 weeks and remained below upgradient concentrations. PhyloChip data demonstrated depth stratified microbial communities with temporal shifts in composition corresponding with observed geochemistry. A sustained enrichment of iron and sulfate reducing bacteria was observed over 2 years, suggesting indirect chromium immobilization through interaction with reactive iron or sulfide by-products. Nitrate reducers such as Pseudomonas also remained elevated over the two years and FGA array data demonstrated a sustained enrichment of Pseudomonas chromate reductase genes suggesting direct reduction of chromate may also be significant in chromium immobilization. Based on this data, organisms representing each of these functional groups have been isolated and characterized. Conclusions: The combination of phylogenetic and functional gene arrays represents a complementary high-throughput approach to elucidating mechanisms responsible for contaminant immobilization in the subsurface.
  790. Chakraborty, R., Y. J. Tang, H. Garcia Martin, J. Chu, J. D. Keasling and Terry C. Hazen. 2007. Flux analysis of central metabolic pathways in the Fe(III)-Reducing organism Geobacter metallireducens via 13-C isotopic labeling. 107th General Meeting of the American-Society-for-Microbiology 107:386. abstract
    Geobacter metallireducens has been known to be one of the dominant microorganisms mediating iron reduction in the environment, the whole genome sequence of which is available. While the genome sequence and proteome are important for understanding a microorganism, they are not necessarily accurate representations of cell physiology and metabolism. Labeling information combined with metabolite balancing and growth kinetics gave an actual in vivo picture of Geobacter metallireducens metabolism under iron reducing conditions. We analyzed the carbon fluxes in central metabolism of strain GS-15 using 13C isotopomer modeling. Acetate labeled in the 1st or 2nd position was a carbon source, and Fe-NTA was the sole terminal electron acceptor. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through central pathways using an isotopomer computation model. The model showed the acetate uptake rate is 21 mmol/gdw/h at the exponential growth phase and over 90% acetate is completely oxidized via TCA cycle. Further, the isotopomer model indicated that the pyruvate carboxylase and phosphoenolpyruvate carboxykinase enzyme are present under these conditions but that the glyoxylate shunt and malic enzyme are absent. Glycolysis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated that the rate-limiting step responsible for slow growth of Geobacter metallireducens on Fe-NTA and acetate is acetyl-CoA transferase. This is based on the surprisingly high reversibility in the reaction between oxoglutarate and succinate . These findings enable a better understanding of the relationship between genome annotation and actual metabolic pathways in G. metallireducens, and provide complementary flux information to the recent in silico model predictions, to further extend our understanding of anaerobic carbon metabolism in this organism.
  791. Chhabra, S. R., A. Mukhopadhyay, G. Zane, C. Hemme, J. Zhou, J. Wall, T. Hazen and J. Keasling. 2007. Functional characterization of the Desulfovibrio vulgaris Hildenborough megaplasmid. 107th General Meeting of the American-Society-for-Microbiology 107:656. abstract
    The D. vulgaris Hildenborough (DvH) genome comprises of a 3.57 Mb chromosome and a 203 kb megaplasmid. From sequence alone it appears that genes for nitrogen fixation, type III secretion, sugar metabolism and metal transport are encoded in the megaplasmid. Interestingly 30% of the genes encoded in the megaplasmid account for hypothetical proteins of unknown function. Using data from functional genomics studies of DvH under a variety of conditions, we summarize the changes in the megaplasmid encoded genes in these growth conditions. Analysis of transcript data also indicates that absolute expression levels of several megaplasmid encoded genes are comparable to the highest expression levels of chromosomally expressed genes. However, cultivation of the wild type strain in ammonium-containing medium typically results in loss of the plasmid from this organism suggesting the importance of the nif genes. Presence or absence of the plasmid also affects the transformation efficiency of DvH. We have compared transformation efficiencies of the wild type strain to those of the megaplasmid lacking strain (MP-). Our results show that control plasmids (pSC27) isolated from E. coli when transformed into DvH display an order of magnitude higher transformation efficiency in the (MP-) strain than the wild type strain. However, control plasmids isolated from wild type DvH transform more efficiently in the wild type strain than the (MP-) strain. In order to systematically characterize how the megaplasmid encoded genes effect cellular responses, we have compared the transcript profiles of the two strains. Results of comparative transcriptome analyses for the wild type and the (MP-) strain and their effect on growth and transformation will be presented.
  792. Chivian, D., E. J. Aim, E. L. Brodie, D. E. Colley, T. Gihring, A. Lapidus, L. H. Lin, S. Lowry, D. P. Moser, P. Richardson, G. Southam, G. Wanger, L. M. Pratt, A. P. Arkin, Terry C. Hazen, F. J. Brockman and T. C. Onstott. 2007. The complete genome of the uncultivated bacterium Desulforudis audaxviator from 2.8 km beneath earth's surface. 107th General Meeting of the American-Society-for-Microbiology 107:470. abstract
    A more complete picture of life on Earth, and even life in the Earth, has recently become possible through the application of environmental genomics. We have obtained the complete genome sequence of a new genus of the Firmicutes, the uncultivated sulfate reducing bacterium Desulforudis audaxviator, by filtering fracture water from a borehole at 2.8 km depth in a South African gold mine. The DNA was sequenced using a combination of Sanger sequencing and 454 pyrosequencing, and assembled into just one genome, indicating the planktonic community is extremely low in diversity. We analyzed the genome of D. audaxviator using the MicrobesOnline annotation pipeline and toolkit (http://www.microbesonline.org), which offers powerful resources for comparative genome analysis, including operon predictions and tree-based comparative genome browsing. MicrobesOnline allowed us to compare the D. audaxviator genome with other sequenced members of the Firmicutes in the same clade (primarily Pelotomaculum thermoproprionicum, Desulfotomaculum reducens, Carboxydothermus hydrogenoformans, and Moorella thermoacetica), as well as other known sulfate reducers and thermophilic organisms. D. audaxviator gives a view to the set of tools necessary for what appears to be a self-contained, independent lifestyle deep in the Earth's crust. The genome is not very streamlined, and indicates a motile, endospore forming sulfate reducer with pili that can fix its own nitrogen and carbon. D. audaxviator is an obligate anaerobe, and lacks obvious homologs of many of the traditional O2 tolerance genes, consistent with the low concentration of O2 in the fracture water and its long-term isolation from the surface. D. audaxviator provides a complete genome representative of the Gram-positive bacteria to further our understanding of dissimilatory sulfate reducing bacteria and archaea. Additionally, study of the deep subsurface has offered access to the simplest community yet studied by environmental genomics, perhaps consisting of just a single species that is capable of performing all of the tasks necessary for life.
  793. Daly, R. A. and Y. Kim, J. M. Wan, T. K. Tokunaga, G. L. Andersen, Terry C. Hazen, M. K. Firestone E. L. Brodie. 2007. Influence of Electron Donor Type and Concentration on Dynamics of Bacterial Populations Associated with Uranium Reduction and Remobilization. 107th General Meeting of the American-Society-for-Microbiology abstract
    Background: Anthropogenic use of uranium has resulted in over 120 contaminated sites in the United States alone. Reductive precipitation of U(VI) by stimulating indigenous microorganisms dramatically decreases uranium solubility and is an attractive, low-cost bioremediation strategy. Our previous long-term sediment studies demonstrated that after an initial period of U(VI) reduction and immobilization significant reoxidation of U(IV) and remobilization of U(VI) occurred. Methods: This long-term column study was designed to determine the effect of differences in organic carbon (OC) substrate type (lactate, acetate), OC concentration and influent pH on the stability of bioreduced U. Microbial community composition and dynamics were analyzed using a custom high-density 16S microarray (16S Phylochip). Results: Analysis of the microbial communities during the reduction and remobilization phases indicated that OC concentration was the primary determinant of the bacterial community composition and that significant shifts in community dynamics occurred between the reduction and remobilization phases. Columns with low OC influent showed decreases in the abundance of α-, β-and γ-proteobacteria, primarily among Caulobacterales, Burkholderiales, Nitrosomonadales, and Rhodocyclales. Columns receiving high OC influent had significantly different populations between the reduction and remobilization phases within the orders Bacteroidales, Flavobacteriales, Clostridiales, and within members of the δ-proteobacteria, including a decrease in abundance of a Geobacter sp. and increases in Desulfobacterales and Syntrophobacterales. Conclusion: The results of this study indicate that the type of organic carbon substrate provided to promote U(VI) reduction has less of an effect on microbial communities compared to OC concentration, and that the microbial communities differ significantly between the uranium reduction and remobilization phases.
  794. Drury, E. C., A. M. Redding, A. Mukhopadhyay, K. H. Huang, Terry C. Hazen, A. P. Arkin, J. D. Wall and D. A. Elias. 2007. A large number of hypothetical proteins are differentially expressed during stress in Desulfovibrio vulgaris. 107th General Meeting of the American-Society-for-Microbiology 107:556. abstract
    Hypothetical and conserved hypothetical proteins make up 30% or more of sequenced bacterial genomes, with few reports confirming expression at either rRNA or protein levels. It is likely that many of these proteins serve significant functions ranging from regulation to unknown steps in carbon or electron flux. Hence, the elucidation of their function(s) is highly relevant to the Virtual Institute Microbial Stress and Survival mission. We have compiled expression profiles for the expected 1167 hypothetical proteins in D. vulgaris from transcriptomic and MS-based iTRAQ proteomic datasets from controlled cultures over 10 stresses. For polycistronic and monocistronic genes respectively, we observed 37 and 46 genes not expressed, 36 and 12 with no stress response but expressed at high rates, 0 and 173 that show no stress response but are expressed at low rates, 445 and 199 with response in two or more stresses, and 104 and 123 that showed differential expression in one stress. While the transcription studies outweigh proteomics, abundance values at the protein level were highly consistent with microarray results. We are presently able to confirm, at the mRNA and protein level, the expression of 253 hypothetical proteins with no evidence for 83 genes encoding a protein. Those expressed should be re-annotated to “expressed protein” while the remainder should be called “non-coding gene”. Of those with no expression, elucidating function without stress-related expression patterns is difficult. Proteins showing differential stress expression are theoretically easier to deduce a putative function, especially those in operons, and these assignments have been completed. Finally, validation of such assignments can only be ascertained by interruption of the gene. We are testing 6 mutants from a transposon library, and will test other hypothetical proteins as the library continues to be sequenced, assigning or confirming putative assignments. By confidently confirming their function in D. vulgaris, there will be a more through understanding of the mechanisms used to survive stresses likely experienced at DOE contaminated sites.
  795. Elias, D. A., G. D. Butland, G. M. Zane, I. B. Hilton, Terry C. Hazen, M. D. Biggin and J. D. Wall. 2007. High-throughput identification of multi-protein complexes via TAP tagging in Desulfovibrio vulgaris. 107th General Meeting of the American-Society-for-Microbiology 107:556. abstract
    The sulfate-reducing bacterium Desulfovibrio vulgaris has repeatedly been shown to have the potential for in-situ metal and radionuclide immobilization. As such, several efforts have focused on the physiology and metabolism this bacterium and the response to stress. The primary goals of this project are to understand the nature of the proteins involved, their function as complexes, and any alterations in complex composition in response to environmental stresses. We are utilizing affinity protein tagging and are comparing the efficiency of three tags; a single and two tandem tags. The tags are “Strep-tag” (Qiagen) that inserts a streptavidin binding peptide; CTF (a.k.a. SPA), a calmodulin binding protein (CBP), a protease (tobacco etch virus) cleavage site, and a 3 x FLAG peptide; and a new “STF” tag that replaces CBP with the streptavidin binding peptide. To test these tags, three proteins (PorB, AtpC, and DsrC) were tagged with all three. Results thus far indicate that the STF tag will be utilized. At issue with DsrC constructs is the C-terminal tag location since the penultimate C-terminal cysteine is involved in protein activity. Mass spectrometry analysis of all three affinity purified DsrC proteins showed no association with other Dsr proteins. This may be valid since all other dsr genes are located in an operon whereas dsrC is monocistronic and interaction for electron delivery may be transient. Proteins are also being tagged with tetracysteine and SNAP (Covalys) for imaging the in-situ location and relative protein density within the cell and as extracellular appendages at different growth states and stresses. We are also constructing a combined STF-SNAP tag, that will allow for the transition to high-throughput. Finally, we are assessing the utility of an ordered plasmid library of D. vulgaris DNA to allow for construction of tagged genes in E. coli in a high-throughput approach. The information from complex identification is expected to lead to a greater understanding of the proteins involved in metal-reduction and their protein-protein interactions, with characterization of the complete pathway(s) for these activities.
  796. Gaucher, S. P., G. S. Chirica, R. Sapra, A. M. Redding, A. Mukhopadhyay, G. M. Buffleben, C. Kozina, R. Phan, D. C. Joyner, J. D. Keasling, Terry C. Hazen, A. P. Arkin and A. K. Singh. 2007. A survey of protein post-translational modifications found in the sulfate-reducing bacterium Desulfovibrio vulgaris hildenborough: Search for stress response mediators. 107th General Meeting of the American-Society-for-Microbiology 107:504-505. abstract
    Post-translational modifications (PTM) play an important role in regulating protein structure (e.g. lipid anchors, disulfide bonds) and function (e.g. phosphorylation, glycosylation). Still others arise through cellular damage such as irreversible oxidation events. Information about these modifications cannot be obtained at the genome level, and so must be characterized at the protein level. We are currently focused on determining the PTM used by the sulfate reducing bacterium, Desulfovibrio vulgaris Hildenborough (DvH). Sulfate reducing bacteria, found widely in nature, have both economic and ecological importance. A goal of the Environmental Stress Pathway Project in the Virtual Institute for Microbial Stress and Survival (VIMSS) is to understand the regulatory networks in DvH for applications to bioremediation. One aspect of this is to determine the types of protein modifications that arise in DvH and how these modifications affect its ability to survive or adapt to its environment. We are using both targeted proteomics and data mining to identify modifications of interest. We are investigating cysteine redox states in DvH proteins using a combination of cysteine labeling chemistries (N-ethylmaleimide and the Applied Biosystems cleavable isotope coded affinity tag) in conjunction with LC/MS/MS. This method allows us to target cysteine residues that undergo reversible oxidation and thus may be candidates for redox mediated activity. To obtain a global survey of PTMs in DvH, we are mining numerous proteomic LC/MS/MS data sets for evidence of modified peptides. Cell lysate for these LC/MS/MS experiments was generated from DvH cultures grown under a variety of stress conditions (nitrate, air, oxygen) as well as in co-culture. The searched-for modifications were determined based on literature precedence and a genome search for the existence of relevant transferases. To date we have found preliminary evidence for cysteine oxidation, lysine acetylation, and methylation of lysine and arginine. Future work will focus on validation of these findings and determining which, if any, of these modifications play a regulatory role in DvH.
  797. Hadi, Masood and J. Kaiser, P. Lane, S. Gaucher, G. Chirica, A. Arkin, T. Hazen, A. Singh Y. Light-Kim. 2007. High-Throughput Identification of Protein Interactions in Electron-Transfer and Stress-Response Pathways in Sulfate-Reducing Bacteria Desulfovibrio vulgaris. 107th General Meeting of the American-Society-for-Microbiology abstract
    Sulfate reducing bacteria (SRB) can derive energy by anaerobic respiration, reducing sulfate or other sulfur compounds to hydrogen sulfide. As energy source Desulfovibrio species can generally use organic or molecular hydrogen for sulfate reduction. The electron chain linking dehydrogenases to the terminal reductases is not well understood in contrast to other modes of respiration. The membrane bound redox complex QMO (quinone-interacting membrane bound oxidoreductase) represents a novel family of respiratory complexes present in the genomes of sulfate reducers and green-sulfur bacterium. Qmo Complex has been hypothesized to be involved in the respiratory electron transfer chain and could be the missing link between the membrane bound menaquinone pool and cytoplasmic reduction of sulfate. We have developed novel high throughput technologies that enable systematic identification, characterization and eventual understanding of molecular machines in bacteria. Our approach relies on expressing ORFs fron DV in exogeneous recombinant systems and using them as baits to pull out complexes from DV cell lysate, followed by Nano-LC and electrospary-MS-MS to identify interacting proteins. Repeating the process for multiple genes allows us to eliminate non-specific binders (false positives) and minimize the probability of missing interacting partners. We have applied this targeted proteomics tool towards the systematic study of molecular machines through the isolation of protein complexes of signature genes. Specifically open reading frames (ORF) of Qmo operon were computationally identified based on homology to other prokaryotes and all the genes cloned. Our approach identified several interacting protein from the "signature" gene set which might be involved in novel complexes related to sulfate/metal reduction. We discuss our results and compare then to known respiratory complexes and the application to these technologies towards proteome wide interaction mapping. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, under contract DE-AC04-94AL85000.
  798. Hazen, Terry C., P. Dehal, A. P. Aikin, M. W. Fields, M. Keller, J. Zhou, G. L. Andersen, F. L. Brodie, D. L. Wyborski, C. B. Abulencia, C. L. Hemme, T. Gentry, D. B. Watson and P. Richardson. 2007. Comparison of multiple ecogenomics methods for determining ecosystem function in uranium-contaminated environments. 107th General Meeting of the American-Society-for-Microbiology 107:444. abstract
    Background: Bioremediation may offer the only feasible strategy for the nearly intractable problem of metal and radionuclide contamination of soil and groundwater. To understand bioremediation in contaminated environments, it is critical to determine the organisms present in these environments, analyze their responses to stress conditions, and elucidate functional position in the environment. Methods: We used multiple molecular techniques on both sediment and groundwater to develop a better understanding of the functional capability and stress level within the microbial community in relationship to over one hundred geochemical parameters. Due to the low pH (3.5-4.5) and high contaminant levels (e.g., uranium) microbial densities and activities were low. We used a phage polymerase amplification system to construct large and small insert DNA libraries, performed metagenome sequencing, constructed clonal libraries of select functional genes (SSU rRNA gene, nirK, nirS, amoA, pmoA, and dsrAB), used a SSU rDNA Phylochip microarray (9,000 taxa), and a functional gene array (23K genes). A complete comparison for community differences and similarities between the different techniques was assessed using several bioinformatics techniques. Results: SSU rDNA analysis revealed the presence of distinct bacterial phyla, including proteobacteria, acidobacteria, and planctomycetes along the contaminant gradient. Metagenome analysis identified many of the same organisms, and diversity was lower in water than sediment. Analysis with functional gene arrays, phylochip, and specific probes for genes and organisms involved in biogeochemical cycling of C, N, and S, metal resistance, stress response, and contaminant degradation suggested that the dominant species could be biostimulated during in situ uranium reduction. Several other findings of difference and similarities between methods are presented. Conclusion: These systems biology field studies could be enabling for strategies to attenuate metal and radionuclide contamination.
  799. He, Q., Z. He, W. Chen, Z. Yang, E. J. Ahn, K. H. Huang, H. C. Yen, D. C. Joyner, M. Keller, A. P. Arkin, Terry C. Hazen, J. D. Wall and J. Zhou. 2007. Understanding the suppression of sulfate reducing bacteria by nitrate: A functional genomics approach. 107th General Meeting of the American-Society-for-Microbiology 107:377. abstract
    Sulfate reducing bacteria (SRB) are of interest for bioremediation with their ability to reduce and immobilize heavy metals. However, field studies show nearly complete suppression of SRB populations by nitrate as a common co-contaminant, which is inconsistent with observations that SRB cultures can readily grow with the presence of over 130 mM nitrate. Transcriptomic profiling of the SRB model Desulfovibrio vulgaris following nitrate treatment found 4, 46, 104, and 142 genes significantly up-regulated and 34, 105, 194, and 146 down-regulated at 30, 60, 120, and 240 min, respectively. Proteomics analysis confirmed the up-regulation of phage shock proteins (PspAC), indicative of a reduced proton motive force, and the repression of multiple ribosomal proteins, which could explain the growth reduction by nitrate. The glycine/betaine transporter genes were also up-regulated, suggesting that NaNO3 also constituted osmotic stress which could be relieved by the transport of osmoprotectants, as validated by the partial relief of growth inhibition by nitrate with the addition of glycine betaine as an osmoprotectant. However, none of these results could explain the complete suppression of SRB by nitrate in the field. It was noted that the hybrid cluster protein (Hcp) was moderately up-regulated in response to nitrate at both the transcript and protein levels, suggesting the likely presence of nitrite, which is known to strongly induce the expression of hcp and is a potent inhibitor for SRB, capable of complete suppression of SRB at milli-molar levels. While insignificant amount of nitrite produced via assimilatory pathways in SRB could induce the expression of hcp and still be readily detoxified, nitrite at toxic levels could be generated from dissimilatory nitrate reduction by other members of the microbial community in the filed; and may subsequently persist in electron donor-limiting environments frequently encountered at contaminated sites. These conditions would diminish the nitrite detoxification capacity of SRB, which requires the supply of reducing equivalents, ultimately leading to the suppression of SRB in these environments.
  800. He, Z., Y. Deng, J. D. Van Nostrand, L. Wu, C. L. Hemme, J. Liebich, T. J. Gentry, Terry C. Hazen, A. P. Arkin and J. Zhou. 2007. GeoChip 3.0: Further development and applications of functional gene Arrays (FGAs) for analysis of microbial communities. 107th General Meeting of the American-Society-for-Microbiology 107:457. abstract
    Microarrays fabricated with the genes encoding key, functional enzymes involved in various biological and geochemical cycling processes are referred to as functional gene arrays (FGAs). Based on GeoChip 2.0, which contains 24,243 oligonucleotide (50mer) probes and covers > 10,000 gene sequences in >150 functional groups involved in nitrogen, carbon, sulfur and phosphorus cycling, metal reduction and resistance, and organic contaminant degradation, a new generation of GeoChip (GeoChip 3.0) is being developed. GeoChip 3.0 has several new features compared to GeoChip 2.0. First, GeoChip 3.0 is expected to contain >30,000 gene sequences of 306 gene families, and such a coverage allows us to obtain more information about microbial communities and analyze more diverse environmental samples. Second, GeoChip 3.0 includes phylogenic markers, such as gyrB. Third, the homology of automatically retrieved sequences by key words is verified by HUMMER using seed sequences so that unrelated sequences are removed. Fourth, a software package (including databases) has been developed for sequence retrieval, probe and array design, probe verification, array construction, array data analysis, information storage, and automatic update, which greatly facilitate the management of such complicated array, especially for future update. Finally, GeoChip 3.0 also includes GeoChip 2.0 probes, and those GeoChip 2.0 probes are checked against new databases. Disqualified probes are flagged. GeoChip 3.0 will provide more capability for studying biogeochemical processes and functional activities of microbial communities important to human health, agriculture, energy, global climate change, ecosystem management, and environmental cleanup and restoration. It is also particularly useful for providing direct linkages of microbial genes/populations to ecosystem processes and functions.
  801. Hemme, C. L., Y. Deng, T. J. Gentry, L. Wu, M. W. Fields, K. Barry, D. B. Watson, N. Krypides, C. Detter, D. C. Bruce, C. W. Schadt, P. Richardson, Terry C. Hazen, J. M. Tiedje, E. Rubin and J. Zhou. 2007. Comparative metagenomics of microbial communities from pristine and contaminated groundwater. 107th General Meeting of the American-Society-for-Microbiology 107:581-582. abstract
    Microbial community DNA from contaminated groundwater from the US Dept. of Energy Field Research Center (FRC) has recently been analyzed to determine the effects of multiple stressors on microbial community structure. The sample was obtained from a site (FW106) experiencing long-term exposure to high levels of uranium, nitric acid and organic solvents. Analysis indicated very low species diversity, with the community dominated by γ- and β-proteobacteria. Furthermore, metabolic reconstruction revealed adaptations for specific geochemical parameters including: denitrification pathways; pathways for degradation of 1,2-dichloroethene, acetone, butanol, methanol and formaldehyde; a large variety of heavy metal resistance systems (czcABC, czcD, cadA, merACPT, etc.). In addition to the resistance systems themselves, the sample shows a significant overabundance of metal and nitrate/nitrite transporters that may serve to increase the baseline transport rate of these compounds. To test hypotheses derived from studies of FW106, a second metagenome was sampled using microbial community DNA from a pristine FRC site (FW301). In contrast to the low species diversity of FW106, the FW301 is represented by multiple phyla including α- and β-proteobacteria, Planctomycetes, Chloriflexi, Actinobacteria and Firmicutes. In contrast to the FW106 sample which yielded large contigs, the FW301 sample is composed largely of single reads that did not assemble into contigs (95%). It was hypothesized that the geochemical resistance genes identified in FW106 also existed in the ancestral pristine sample, thus providing a source of genetic material for adaptation of the community to contamination. A comparison of the geochemical resistance genes of FW106 to FW301 using Blast showed that not only are the genes present in the pristine state but also that they are present in significant numbers. While this does not discount lateral gene transfer and/or gene duplication acting as adaptive mechanisms in FW106, it does suggest that the pool of genetic material necessary for adaptation was greater than previously suspected.
  802. Holman, H. Y. N., E. Wozei, Z. Lin and T. C. Hazen. 2007. Observing molecular-level transient oxygen stress in obligate anaerobes in vivo. 107th General Meeting of the American-Society-for-Microbiology 107:376. abstract
    Aerobic respiration of intracellular polyglucose reserves is postulated to play a central role in oxygen adaptive response in obligatory anaerobes like Desulfovibrio species, but it has been difficult to probe this event at chemical scale in vivo. Here we present a non-invasive synchrotron infrared (SIR) spectromicroscopy approach to reveal time-dependent composition and structure changes at a lateral scale of several individual Desulfovibrio vulgaris cells. The advantage of infrared spectroscopy is that it is non-invasive, and it uses vibration movements of atoms and chemical bonds within functional groups of biomolecules as an intrinsic contrasting mechanism; thus it allows one to immediately detect composition and structure changes within cells. The advantage of using a synchrotron light source is that its high brightness allows us to detect signals ~1000 times weaker than the conventional infrared spectroscopy allows us to. Comparative analysis of SIR spectra of the same individual D. vulgaris exposed to air-level oxygen at different time points reveals chronological information regarding the changes in composition, the level of oxidative stress, and the extent of cellular injury and repair. These results, together with microscopy images, mark a critical step toward the use of SIR spectromicroscopy as an uninterrupted microprobe at a chemical scale level of physiological events in microbiology applications.
  803. Joyner, D. C., J. S. Jacobsen, A. Mukhopadhyay and Terry C. Hazen. 2007. Assesment of nitrogen utilization in Desulfovibrio vulgaris using phenotype microarray. 107th General Meeting of the American-Society-for-Microbiology 107:487. abstract
    Nif genes, found primarily in bacteria, encode the nitrogenase complex and other enzymes involved in nitrogen fixation. Though found in aerobic bacteria, nitrogen fixation occurs optimally in anaerobic conditions and nif genes are present in many obligate and facultative anaerobic bacteria as well as some archaea. The consensus sequences in these genes are highly conserved across nitrogen fixing bacteria, but variability exists in their regulation. In the anaerobic sulfate reducing soil bacterium Desulfovibrio vulgaris Hildenborough the nif genes are encoded on a 200kb megaplasmid which harbors approximately 5% of all of the genes in D. vulgaris. The function of the megaplasmid in the growth and survival of D. vulgaris is not well understood and the megaplasmid is documented to be lost in growth conditions that do not require nitrogen fixation (such as excess ammonium salts). In order determine the growth conditions that play a role in nitrogen utilization a wild type D. vulgaris strain containing the megaplasmid was compared with a D. vulgaris strain lacking the megaplasmid (MP(-)). Nitrogen source utilization is evaluated by direct growth response using Omnilog Phenotype Microarray assays. The selected nitrogen utilization phenotype microarray consists of a single 96-well plate loaded with 95 different nitrogen containing compounds. These assays, were used to track and compare the nitrogen utilization profiles and assess specific metabolic pathways for nitrogen assimilation. Activated megaplasmid nif gene function is seen with the growth of D. vulgaris on N2 in the absence of added inorganic nitrogen compounds. For the megaplasmid containing wild type strain, growth on N2 alone yields a three-fold longer generation time which is not observed with the MP (-) strain. Genes on the megaplasmid may also confer resistance to toxic ions as D. vulgaris growth with NO2 and NO3 reaches higher final yields than the MP (-) strain. Here we demonstrate how phenotypic mapping of the two strains reveal some of the functions of the D. vulgaris megaplasmid .
  804. Mohanty, S. R., E. L. Brodie, B. Kollah, Terry C. Hazen and E. Roden. 2007. Comparative analysis of microbial communities associated with U(VI) bioreduction in ethanol-amended subsurface sediment by 16S rRNA clone libraries and a high density phylogenetic DNA microarray. 107th General Meeting of the American-Society-for-Microbiology 107:572. abstract
    The diversity of metabolically-active microorganisms associated with U(VI) bioreduction and other terminal electron-accepting processes in a slurry of ethanol-amended, U(VI)-contaminated subsurface sediments was assessed by 16S rRNA clone libraries and a high density phylogenetic DNA microarray (16S PhyloChip). Microbial communities were analyzed in samples obtained from the nitrate-reducing, Fe(III)/U(VI)-reducing, sulfate-reducing, and methanogenic phases of the batch incubation experiment, along with a sample from time zero. A total of 88-134 clones of PCR-amplified, reverse-transcribed 16S rRNA were sequenced to construct clone libraries. PhyloChip analyses were performed on PCR-amplified DNA obtained from the same reverse-transcribed rRNA used to construct the clone libraries. The maximum and minimum number of OTUs observed in clone libraries were 54 from the methanogenic and 12 from the sulfate-reducing phases samples. In contrast, the PhyloChip detected a maximum of 1109 and a minimum of 344 OTUs from the methanogenic and nitrate-reducing phase samples, respectively. The two approaches matched each other in detecting the most abundant Clostridiaceae, Desulfotomaculum, Oxalobacteraceae, Dechloromonas, Anaeromyxobacter, and Geobacteraceae sequences. A good correspondence between the 16S rRNA clone libraries and the PhyloChip data was observed in terms of the proliferation of Geobacteraceae during the Fe(III)/U(VI)-reducing phase of the experiment. Collectively our results demonstrate that the 16S PhyloChip provided far greater insight into total microbial diversity than could be achieved with typical clone library techniques, but as the PhyloChip is unable to determine relative abundance of species due to variable probe hybridization efficiencies, a complementary cloning approach is valuable to evaluate the contribution of dominant organisms.
  805. Mukhopadhyay, A., A. M. Redding, A. P. Arkin, S. Borglin, P. Dehal, R. Chakraborty, J. T. Geller, B. Giles, Terry C. Hazen, Q. He, M. Joachimiak, D. C. Joyner, J. D. Wall, Z. Yang, J. Zhou and J. D. Keasling. 2007. Comparison of Desulfovibrio vulgaris Hildenborough response to microaerobic and aerobic exposure. 107th General Meeting of the American-Society-for-Microbiology 107:377. abstract
    Though considered obligate anaerobes for many years after their discovery, sulfate reducing bacteria like Desulfovibrio vulgaris Hildenborough (DvH) are found in environments with very low sulfate and in many environments that are regularly exposed to oxygen or are normally aerobic. The best growth condition for DvH, measured as increase in biomass, remains a completely anaerobic environment. However, DvH is clearly able to tolerate sub-aerobic environments and can survive exposure to air for up to 20 days. Controlled experiments were conducted to expose DvH to aerobic and microaerobic conditions (0.1% O2). Cell-wide responses were monitored via transcriptomics and proteomics measurements. Microaerobic conditions caused an overall decrease in growth without affecting the viability of the bacterium. Cellular responses to microaerobic conditions were mild and primarily included up-regulation of the putative PerR regulon, but other known oxidative stress response candidates remained unchanged. Other transcripts that show an expression profile similar to the PerR regulon genes included the cydA/B operon, encoding putative oxidative phosphorylation proteins. However, comparison with data from prior DvH functional genomics studies suggested that many of these changes could be part of a general stress response in DvH. In contrast, exposure to air produced drastic changes at both the transcriptome and proteome levels and had a detrimental effect on both growth and viability of DvH. During aerobic stress, increases in proteases and chaperones signified air exposure to be a very harsh stress in DvH. However, quantitative proteomics also indicated an accumulation of superoxide-dismutase, catalase as well as ferritins and thioredoxins, and these candidates may be critical for the survival of the small fraction of cells which survive air exposure. Our results indicated that DvH has very different responses towards microaerobic vs. aerobic exposure. Growth of DvH strains under these different O2 exposures and the data from our integrated genomics experiment are presented and have been used to improve the model for O2 stress response in DvH.
  806. Perez, E., N. Ramos-Hernandez, G. Andersen, Y. Piceno, E. Brodie, T. Hazen and A. Massol-Deya. 2007. Bacterial diversity in soil and Sediments from a former bombing range (Vieques, PR). 107th General Meeting of the American-Society-for-Microbiology 107:431. abstract
    The U.S. Navy Atlantic Fleet Weapons Training Area (AFWTA) located in Vieques, Puerto Rico, includes land areas, waters and islets impacted by 63 years of military training operations. High-density universal SSU rRNA gene microarray analysis for Archaea and Bacteria were used to describe the microbial community structure of soil and marine sediment samples from Vieques and two reference locations. Total community DNA was extracted, PCR amplified and hybridized to an array encompassing 16S rRNA for over 8,900 distinguishable taxonomic units. Hierarchical clustering of the 100 most variable sub-families detected by the array demonstrated that variable sequences fell into six (6) primary response groups. Samples from sediments and soils are very similar between each of their kind. Furthermore, samples from closer geographical locations were more similar than distant sites. Higher numbers of OTU’s were observed in soil samples with 193 to 318 sub-families identified in each sample. Bacteroidetes, actinobacterias and acidobacteria were the more common phyla detected in Vieques than reference samples. The presence of diverse groups of bacteria may indicate a great potential for natural or enhanced biological restoration. Understanding the function and community structure of highly disturbed ecosystems could assist environmental restoration strategies.
  807. Phan, R., R. Chakraborty, S. Lam, E. L. Brodie and Terry C. Hazen. 2007. Isolation and characterization of diverse anaerobic Cr(VI) tolerant bacteria from Cr(VI)-contaminated 100H site at Hanford. 107th General Meeting of the American-Society-for-Microbiology 107:575-576. abstract
    Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water. Cr(VI) is more soluble, toxic, carcinogenic, and mutagenic compared to its reduced form Cr(III). In order to stimulate microbially mediated reduction of Cr(VI), a poly-lactate compound HRC was injected into the chromium contaminated aquifers at site 100H at Hanford. Based on the results of the bacterial community composition using high-density DNA microarray analysis of 16S rRNA gene products, we recently investigated the diversity of the dominant anaerobic culturable microbial population present at this site and their role in Cr(VI) reduction. Positive enrichments set up at 30oC in the dark using specific defined anaerobic media resulted in the isolation of an iron reducing isolate strain HAF, a sulfate reducing isolate strain BLS and a nitrate reducing isolate, strain HLN. Preliminary 16S rDNA sequence analysis identifies strain HAF as Geobacter metallireducens, strain HLN as Pseudomonas stutzeri and strain BLS as a member of Geosinus species. Strain HAF isolated with acetate as the electron donor utilized propionate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Growth was optimal at 37oC, pH of 6.5 and 0% salinity. Strain HLN isolated with lactate as electron donor utilized acetate, glycerol and pyruvate as alternative carbon sources, and reduced metals like Mn(IV) and Cr(VI). Optimal growth was observed at 37oC, at a pH of 7.5 and 0.3% salinity. Anaerobic active washed cell suspension of strain HLN reduced almost 95μM Cr(VI) within 4 hours relative to controls. Further, with 100μM Cr(VI) as the sole electron acceptor, cells of strain HLN grew to cell numbers of 4.05X 107/ml over a period of 24hrs after an initial lag, demonstrating direct enzymatic Cr(VI) reduction by this species. 10mM lactate served as the sole electron donor. These results demonstrate that Cr(VI) immobilization at the Hanford 100H site could be mediated by direct microbial metabolism apart from indirect chemical reduction of Cr(VI) by end products of microbial activity.
  808. Ramos-Hernandez, N., R. Chakraborty, D. Joyner, E. Perez, A. Massol-Deya and T. Hazen. 2007. Chemotactic and growth responses to explosives of Desulfovibrio vulgaris H. and sulfate-reducing bacteria isolated from tropical marine sediments. 107th General Meeting of the American-Society-for-Microbiology 107:343. abstract
    Bombing sites used for military training activities can have considerable amounts of contaminants and pose significant risks for people and the environment. Until 2003, the eastern part of Vieques (Puerto Rico) was used by the US Navy as a bombing range. Currently, leaching of explosive compounds from unexploded ordnance represents a serious threat to the marine ecosystem. The contribution of microorganisms in natural attenuation of explosives, including sulfate-reducing bacteria (SRB) has been demonstrated in soils but little is known about their contribution in marine environments. Characterization assays were employed to assess the effects of explosive compounds (TNT, RDX, HMX) on Desulfovibrio vulgaris Hildenborough and five novel SRB isolated from marine sediments in costal waters of Vieques. Pure cultures were combined with media in a covered 96-well micro plate and the opacity was monitored in real time as the bacteria grew in a temperature-controlled plate reader. A dose-response curve was used to estimate minimum inhibitory concentrations (MICs) for TNT, RDX and HMX in 0, 1.5 and 3.0% (w/v) NaCl media. Some of the bacterial isolates grew better in explosive-containing environments than in regular media while at low salt D. vulgaris grew without inhibition at saturated RDX levels. Higher salt concentrations (1.5 and 3.0%) did not support D. vulgaris growth in the presence of RDX. However, this organism did tolerate high levels of TNT at different NaCl concentrations. Elucidating the diversity and behavior of SRBs to explosive compounds in tropical sediments could help us understand the role of these microbial populations in contaminated marine environments.
  809. Singer, M. E., J. T. Geller, R. Chakraborty, Y. Katsuura, M. D. Biggin and Terry C. Hazen. 2007. Reproducible, high quality Desulfovibrio vulgaris Hildenborough biomass production using anaerobic fermentors in batch and continuous flow mode. 107th General Meeting of the American-Society-for-Microbiology 107:609. abstract
    Analysis of multi-protein complexes in a heavy metal reducing organism, Desulfovibrio vulgaris Hildenborough (DvH), is essential in understanding how these complexes control the microbe’s survival in contaminated environments while reducing metals. Investigating microbial multi-protein complexes in an organism requires strictly enforced high quality copious cell production. Our aim was to experimentally test the efficiency of growth and biomass production of DvH in continuous flow mode in an anaerobic bioreactor, and to determine the operational parameters for producing a consistently high volume and high quality output. The 5 L volume bioreactor was custom manufactured with non-metallic wetted parts. After growing the culture to mid-log phase in batch mode, lactate-sulfate media was applied at a dilution rate of 0.15 L/h as a turbidostat to maintain the culture at mid-log phase. It took approximately 3 reactor volumes to achieve equilibrium; a total of over 10 reactor volumes were run. We analyzed total cell counts, total protein concentrations, and concentration of electron donor, acceptor and metabolites of the reactor broth and compared the above parameters within different batch and continuous flow runs. During continuous flow, Acridine orange direct cell counts were found to average 2.8 x 108 cells/ml at mid-log phase, where optical density at 600 nm read 0.6, which is consistent with that observed in batch cultures (3 x 108 cells/ml). Ion chromatography analysis revealed that at the mid-log phase of batch culture, about 40 mM of the lactate and 18mM of the initial sulfate added was utilized. During continuous flow, the lactate utilization was 38 mM, while the sulfate utilization was 22 mM. Total protein patterns at different points during mid-log phase in the continuous flow modes were compared by SDS-PAGE analysis. The results show the pattern to be very consistent. Our findings suggest that cells of DvH can be successfully harvested in continuous flow mode once mid-log phase was reached, to yield reproducible high quality biomass for protein analysis using anaerobic bioreactors.
  810. Van Nostrand, J. D., Y. Liang, L. Wu, W. Wu, S. Carroll, Z. He, C. Criddle, P. Jardine, Terry C. Hazen and J. Z. Zhou. 2007. Changes in microbial community function during a period of reoxidation in a groundwater recirculation system. 107th General Meeting of the American-Society-for-Microbiology 107:570-571. abstract
    A test groundwater recirculation system was constructed at the U.S. DOE’s Field Research Center (FRC) in Oak Ridge, TN to examine the applicability of in situ U(VI) bioremediation of a U-contaminated site. Ethanol was injected into the system to biostimulate the microbial community and a reduction in U concentration has been achieved. To examine the effect of dissolved oxygen (DO) on U(VI) reduction and the stability and re-oxidation of U in this system, air-saturated tap water (9-12 mg L-1 DO) was introduced for a period of 77 d. Geochip, a comprehensive 50mer microarray containing probes for genes involved in the geochemical cycling of N, S, and C as well as genes related to metal reduction and resistance and to organic contaminant degradation, was used to examine changes in the microbial functional community in two monitoring wells before, during, and after the reoxidation period. The well located closest to the injection and extraction wells (101-2), showed a greater increase in DO (2 mg L-1) than the well located further away (102-2; 0.4-0.5 mg L-1). An increase in U(VI) occurred during the reoxidation period and was associated with a decrease in the relative abundance of cycthrome c genes. At 40 d post reoxidation, 101-2 showed an increase in the relative abundance of genes involved in denitrification, nitrate reduction, methane and ammonium oxidation and a decrease for those associated with cytochromes, N fixation and sulfate reduction. By 77 d post reoxidation, the relative abundance of denitrification and ammonia oxidation genes had returned to pre-oxidation levels. At 40 d post reoxidation, 102-2 showed an increase in the relative abundance of genes involved in methane oxidation and nitrate reduction. A decrease was observed with genes associated with ammonification, cytochromes, denitrification, N fixation, and sulfate reduction. The relative abundance of these genes returned to pre-oxidation levels by 77 d post reoxidation. Changes in the functional community were similar in the two wells; however, the community in 101-2 appeared to be affected for a longer period of time, most likely due to the higher levels of DO as compared to 102-2.
  811. Waldron, P. J., J. D. Van Nostrand, D. B. Watson, L. Wu, Z. He, Terry C. Hazen and J. Z. Zhou. 2007. Effects of nitrate, pH and uranium on the subsurface microbial communities revealed by functional gene arrays. 107th General Meeting of the American-Society-for-Microbiology 107:468. abstract
    To understand how contaminants affect microbial community structure, five ground-water samples with varying nitrate, pH and uranium concentrations obtained from the Field Research Center (FRC) site of the U.S. DOE ERSP (Environmental Remediation Science Program) program at Oak Ridge Reservation, Oak Ridge, Tennessee, were analyzed with a comprehensive functional gene arrays containing more than 24,000 gene probes. An uncontaminated background site was also processed in the same manner. Signal intensity was measured for genes involved in the nitrogen cycle, metal reduction, organic carbon degradation and sulfate reduction. Gene diversity and prevalence was correlated to the geochemistry of each well. Wells that were highly contaminated with nitrate had elevated numbers of genes involved in nitrate reduction and denitrification, most likely as a result from the selective pressure exerted by nitrate stress. The well with the highest concentrations of uranium and nitrate and the lowest pH also had the fewest total number of genes reported, indicating that stress from uranium and low pH are likely factors in reducing microbial community diversity. Analysis of the gene diversity from these wells is ongoing.
  812. Walian, P. J., M. Dong, S. J. Fisher, J. T. Geller, S. C. Hall, Terry C. Hazen, D. C. Joyner, M. E. Singer, H. E. Witkowska, M. D. Biggin and B. K. Jap. 2007. Isolation and identification of membrane protein complexes in Desulfovibrio vulgaris hildenborough. 107th General Meeting of the American-Society-for-Microbiology 107:543. abstract
    An important aim of the Genomics GTL Protein Complex Analysis Project (PCAP) is the isolation and identification of membrane protein complexes from D. vulgaris. The cataloging of complex subunit constituents from this organism, grown under normal and stressed conditions, will support the long-range goal of modeling stress responses in D. vulgaris relevant to the detoxification of metal and radionuclide contaminated sites. Isolation of endogenous D. vulgaris membrane protein complexes in quantities sufficient for chromatographic analysis requires substantial amounts of cell membranes. Twenty liter cultures of D. vulgaris typically yielded about 50 milligrams of total membrane proteins. To extract inner and outer membrane protein complexes stably and with maximal yield, we have used a multi-step procedure in which the bacterial membrane is sequentially processed. Membranes were initially treated with a mild detergent to extract proteins primarily from the inner membrane. The residual membrane pellet, enriched with proteins of the outer membrane, was solubilized using a more aggressive detergent. Chromatographic procedures found effective in purifying complexes involved the use of ion exchange, hydroxyapatite and molecular sieve media. SDS-PAGE of the various chromatographic fractions was used to tentatively identify subunits of complexes based on a display of co-elution. Molecular sieve fractions were subjected to native PAGE to further isolate potential complexes. Proteins in the native gel bands, in turn, were extracted and run on SDS-PAGE. These gels revealed whether a putative complex was indeed composed of lower molecular weight subunits, and provided samples well suited for in-gel processing and mass spectrometry analysis. In this manner, a number of homo- and heteromeric complexes, ranging in weight from approximately 70 to 400 kDa, have been identified in this first year. Use of a free-flow electrophoresis device being developed in our group, in combination with larger-scale processing of cell membranes, is expected to significantly improve the isolation and identification of complexes over the next project year.
  813. Wells, G. F., E. X. Perez, H. D. Park, L. C. Sepulveda-Torres, B. Eggleston, E. L. Brodie, T. Z. DeSantis, G. L. Andersen, Terry C. Hazen, A. Francis, C. S. Criddle and A. A. Massol-Deya. 2007. Application of high-density oligonucleotide microarrays to the study of Crenarchaeota community structure and dynamics in an aerated activated sludge wastewater treatment plant. 107th General Meeting of the American-Society-for-Microbiology 107:548. abstract
    Despite the fact that biological wastewater treatment is practiced widely to remove organics, nutrients, toxics, and pathogens from polluted water, little is known about the ecology of microbial communities in bioreactors. In particular, the prevalence, diversity, functional importance, and population dynamics of nonthermophilic Crenarchaeota in aerated activated sludge wastewater treatment systems remain virtually unknown. The aim of this study was to elucidate the diversity and dynamics of nonthermophilic Crenarchaeota in aerated activated sludge systems and to link these dynamics to operational or environmental parameters. To this end, we used high-density 16S rDNA phylogenetic microarrays containing 500,000 probes to monitor bacterial and archaeal population dynamics in monthly samples from the aeration basin of a local municipal activated sludge wastewater treatment plant (WWTP) for a period of one year. Operational data were collected concurrently. Our results revealed a highly diverse prokaryotic community in the WWTP, with an average of 1606 bacterial and archaeal phylotypes detected at each sampling point. Eleven distinct and highly dynamic nonthermophilic Crenarchaeota phylotypes from groups 1.1a and 1.1b were detected in 11 of the 12 months examined. Within each Crenarchaeal phylotype, 16S rRNA gene copy number varied by nearly an order of magnitude during the study period. Seven main covarying archaeal response groups in this time period were identified based on hierarchical clustering. The majority of the identified nonthermophilic Crenarchaeota phylotypes fell within the same response group, indicating significant covariation in population size among these phylotypes during the year of sampling. Our results suggest that nonthermophilic Crenarchaeota may play a previously unrecognized role in aerated activated sludge bioreactors. To our knowledge, this is the first application of high-density phylogenetic DNA microarray methods to the analysis of microbial community structure in activated sludge bioreactors.
  814. Wu, L. Y., T. J. Gentry, Z. J. Huang, C. W. Schadt, W. M. Wu, D. Watson, Z. L. He, C. S. Criddle, J. M. Jiedje, Terry C. Hazen and J. Zhou. 2007. Microarray-based analysis of microbial community composition and dynamics in uranium bioremediation. 107th General Meeting of the American-Society-for-Microbiology 107:570. abstract
    A field-scale system is being used for evaluating in situ biological reduction and immobilization of U(VI) in the DOE ERSP Field Research Center, Oak Ridge, TN. Above-ground treatment of groundwater, including nitrate removal pre-conditions the groundwater for subsurface uranium immobilization. Treated water was then injected into the subsurface with ethanol to stimulate microbial reduction of U(VI) to insoluble U(IV). The microbial community dynamics from one of the 4 frequently sampled monitoring wells (FW 102-3) was intensively analyzed with a functional gene array containing >24,000 probes and covering 10,000 genes in 150 gene categories. The results indicated that during the uranium reduction period, both FeRB and SRB populations reached their highest levels at Day 212, followed by a gradual decrease over 500 days. The uranium concentrations in the groundwater were significantly correlated with the total abundance of c-type cytochrome genes (r=0.73, p<0.05) from Geobacter -type FeRB and Desulfovibrio-type SRB, and with the total abundance of dsrAB (dissimilatory sulfite reductase) genes (r=0.88, p<0.05). The Mantel test of microarray and chemical data also indicated that there was significant correlation between the differences of uranium concentrations and those of total c-cytochrome gene abundance (r=0.75, p <0.001) or dsrAB gene abundance (r=0.72, p<0.01). The changes of more than dozen of individual c-type cytochrome genes from Geobacter sulfurreducens and Desulfovibrio desulfuricans showed significant correlations to the changes of uranium concentrations among different time points. Also the changes of more than 10 dsrAB-containing populations, including both cultured (e.g. D. desulfuricans, D. termitidis, Desulfotomaculum kuznetsovii,) and non-cultured SRB were significantly related to the changes in uranium concentrations, indicating their importance in uranium reduction. Interestingly, as expected, the changes of several dsrAB-containing sulfate-reducing populations previously recovered from this site showed significant correlations to the differences of uranium concentrations.
  815. Yang, Z., C. W. Schadt, Terry C. Hazen and M. Keller. 2007. Towards high-throughput and high sensitivity approaches for uncovering total environmental gene expression patterns. 107th General Meeting of the American-Society-for-Microbiology 107:606. abstract
    Current technologies applied to environmental samples for RNA transcriptional profiling include RT-PCR and functional gene microarrays using total RNAs. However, these methods bear significant limitations that prevent their application in a high throughput manner to de novo communities. To circumvent this limitation, we are developing a method for direct sequencing of cDNA from environmental samples utilizing the HT sequence analysis system Bio454. Since about 80% of total RNA of microorganisms may consist of rRNAs, it is crucial to first remove rRNAs as completely as possible without degrading mRNA quality and quantity. We compared three methods for removing rRNA from the total RNA of Desulfovibrio vulgaris. The first method utilizes biotin modified oligos complementary to conserved regions in 16S & 23S rRNA. The second uses an exonuclease that targets rRNAs bearing a 5’monophosphate. The third method uses first strand transcription with universal primers for 16S & 23S RNAs, and subsequent removal of rRNA and cDNA. All three methods were able to significantly enrich mRNA from rRNA We evaluated the effect of these methods on mRNA quality by microarray analysis. Correlation coefficients between mRNA enriched and control samples ranged from 0.84 to 0.96. Between 0.2 and 2% of genes showed significantly altered expression levels (p=0.05) after rRNA removal with each method, however most showed increased intensity suggesting rRNA removal may increase sensitivity. Currently we are constructing cDNA libraries for HT sequencing with the 454 to further optimize and validate this approach in single species, as well as make comparisons of HT sequence based methods with existing microarrays. The developed tools will then be deployed to understand microbial responses and survival in stressed environmental systems.
  816. Zhou, A., Z. He, C. Hemme, A. Mukhopadhyay, J. Keasling, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou. 2007. Genome-Wide Transcriptomic Analysis of Desulfovibrio vulgaris Hildenborough Response to Hydrogen Peroxide. 107th General Meeting of the American-Society-for-Microbiology abstract
    Oxidative stress is one of the most common environmental stressors. Desulfovibrio vulgaris Hildenborough (DvH), a model organism of sulfate-reducing bacterium (SRB), has been considered an obligate anaerobe. However, more and more evidences show they are aero-tolerant. DvH contains both defense systems which are typical for both aerobic (Sod and Kat) and anaerobic (Rub, Rbr, Rbo etc.) microbes, but little is known about the molecular mechanism of DvH oxidative stress responses. In this study, DvH cells were stressed with H2O2 and the transcript changes were investigated by DvH oligo chip. Results from Bioscreen showed there was a dosage effect upon the H2O2 treatment. Low concentrations (0.1 - 2 mM) did not inhibit growth significantly, and higher concentrations (>4 mM) led to growth retardation. Therefore, two concentrations (1 and 4 mM) and 5 time-points (30, 60, 120, 240 and 480 min) were used to study the transcriptional response. The microarray data demonstrated that the most significant changes in gene expression occurred at 120 min (485 up and 527 down) and 240 min (750 up and 753 down) for 1 mM and 4 mM respectively. The changes in gene number and fold were more significant with 4 mM H2O2 treatment than with 1 mM H2O2, which suggests that more response pathways are triggered with 4 mM of H2O2. Among the known genes involved in oxidative stress, only Rdl, Rbr2 were up-regulated, which indicates that Rdl and Rbr2, rather than Rub-Rbo & Rbr, may play major roles in DvH responses to H2O2 stress. PerR, a key oxidative stress response regulator in organisms that lack OxyR, and genes in the predicted PerR regulon were up-regulated. Two component system genes such as DVU3269, DVU3381 and DVU3382, genes involved in energy metabolism (thioredoxin and thioredoxin reductase, a FMN reductase isf-1), transport and binding proteins (copper-translocating, mercuric transport, ferrous iron transport, permease and ABC transporters), and protein fate (MsrA, MsrB; DanJ, Daf, heat shock protein etc.) were significantly up-regulated. Their functions in oxidative stress response will be confirmed by other approaches such as proteomic analysis and mutagenesis.
  817. Hazen, Terry C. 2007. Life in the slow lane: Deep subsurface extreme environments as analogs to Mars?. UCSF Annual Hamilton Symposium
  818. Hazen, Terry C.. 2007. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. Pioneers in Genomic Biology Lecture Series, University of Illinois
  819. Hazen, Terry C.. 2007. Application of Proteomics to Bioremediation. DOE Workshop Identifying the Future Proteomics Needs for Biological and Environmental Research.
  820. Hazen, Terry C. 2007. ASTAR Advanced Judicial Institute on Nanotechnology, Synthetic Biology and Environmental Biotechnology. ASTAR Platform B Workshop
  821. Hazen, Terry C.. 2007. Bioremediation and Environmental Biotechnology. ASTAR Advanced Judicial Institute on Nanotechnology, Synthetic Biology and Environmental Biotechnology Platform B Workshop
  822. Hazen, Terry C., E. Brodie, D. Favero. 2007. Case Study: Bioremediation (Radian Day). ASTAR Advanced Judicial Institute on Nanotechnology, Synthetic Biology and Environmental Biotechnology Platform B Workshop
  823. Hazen, Terry C. 2007. Systems Biology in Environmental Biotechnology. 50th Anniversary of the Puerto Rican Society of Microbiology
  824. Hazen, Terry C.. 2007. Bioremediation: The Hope and the Hype for Environmental Cleanup. LBNL Summer Lecture Series
  825. Abulencia, C., E. J. Alm, G. Anderson, E. Baidoo, P. Benke, S. Borglin, E. L. Brodie, R. Chakraborty, S. Chhabra, G. Chirica, D. Chivian, M. J. Cipriano, M. E. Clark, P. S. Dehal, E. C. Drury, I. Dubchak, D. A. Elias, M. W. Fields, J. Gabster, S. P. Gaucher, J. Geller, B. Giles, M. Hadi, , Terry C. Hazen and Q. He, Z. He, C. L. Hemme, E. Hendrickson, K. L. Hillesland, H.-Y. Holman, K. H. Huang, Y. W. Huang, C. Hwang, J. Jacobsen, M. P. Joachimiak, D. C. Joyner, J. D. Keasling, K. Keller, M. Keller, J. Leigh, T. Lie, A. Mukhopadhyay, R. Phan, F. Pingitore, M. Price, A. M. Redding, J. Joseph A. Ringbauer, R. Sapra, C. W. Schadt, A. Shutkin, A. K. Singh, D. A. Stahl, S. M. Stolyar, Y. Tang, J. D. Van Nostrand, C. B. Walker, J. D. Wall, E. Wozei, Z. K. Yang, H.-C. Yen, G. Zane, A. Zhou, J. Zhou, A. P. Arkin . 2007. The Virtual Institute of Microbial Stress and Survival: An overview of the Environmental Stress Pathway Project. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  826. Chivian, D., E. J. Alm, E. L. Brodie, D. E. Culley, T. M. Gihring, A. Lapidus, L.-H. Lin, S. Lowry, D. P. Moser, P. Richardson, G. Southam, G. Wanger, L. M. Pratt, A. P. Arkin, , Terry C. Hazen and F. J. Brockman, T. C. Onstott . 2007. The Complete Genome of the Uncultivated Ultra-Deep Subsurface Bacterium Desulforudis audaxviator Obtained by Environmental Genomics. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  827. Drury, E. C., A. M. Redding, A. Mukhopadhyay, K. H. Huang, , Terry C. Hazen and A. P. Arkin, J. D. Wall, D. A. Elias . 2007. A Large Number of Hypothetical Proteins are Differentially Expressed during Stress in Desulfovibrio vulgaris. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  828. Elias, D., S. Chhabra, J. T. Geller, H.-Y. N. Holman, D. Joyner, J. Keasling, A. Mukhopadhyay, M. Singer, T. Torok, J. Wall, , Terry C. Hazen and G. Butland, M. Dong, S. C. Hall, B. K. Jap, J. Jin, S. J. Fisher, P. J. Walian, H. E. Witkowska, L. Yang, M. D. Biggin*, M. Auer, A. Avila-Sakar, F. Garczarek, R. M. Glaeser, J. Malik, E. Nogales, H. Palsdottir, J. P. Remis, D. Typke, K. H. Downing, S. S. Andrews, A. P. Arkin, S. E. Brenner, Y. W. Huang, J. Jacobsen, K. Keller, R. Santos, M. Shatsky, J.-M. Chandonia . 2007. Protein Complex Analysis Project (PCAP): Project Overview. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  829. Gaucher, S. P., A. M. Redding, G. S. Chirica, R. S. G. M. Buffleben, C. Kozina, A. Mukhopadhyay, D. C. Joyner, J. D. Keasling, , Terry C. Hazen and A. P. Arkin, D. A. Stahl, J. D. Wall, A. K. Singh . 2007. A Survey of Protein Post-Translational Modifications Found in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  830. Hazen, Terry C.. 2007. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. School of Biology, Georgia Institute of Technology
  831. Hazen, Terry C. and C. Abulencia, G. Anderson, S. Borglin, E. Brodie, S. v. Dien, M. Fields, J. Geller, H.-Y. Holman, R. Phan, E. Wozei, J. Jacobsen, D. Joyner, R. Chakraborty, M. Keller, A. Mukhopadhyay, D. Stahl, S. Stolyar, J. Wall, H.-C. Yen, G. Zane, J. Zhou, E. Hendrickson, T. Lie, J. Leigh, C. Walker . 2007. VIMSS Applied Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  832. Hazen, T. C. and J. Keasling, A. Mukhopadhyay, S. Chhabra, J. T. Geller, M. Singer, D. Joyner, T. Torok, J. Wall, D. Elias, M. D. Biggin H.-Y. N. Holman. 2007. Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes during Stress Response in Desulfovibrio vulgaris: Microbiology Subproject. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  833. He, Q., Z. He, W. Chen, Z. Yang, E. J. Alm, K. H. Huang, H.-C. Yen, D. C. Joyner, M. Keller, A. P. Arkin, , Terry C. Hazen and J. D. Wall, J. Zhou . 2007. Nitrate stress response in Desulfovibrio vulgaris Hildenborough: Whole-Genome Transcriptomics and proteomics analyses. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  834. He, Z., J. D. Van Nostrand, L. Wu, T. J. Gentry, Y. Deng, C. W. Schadt, W. Wu, J. Liebich, S. C. Chong, B. Gu, P. Jardine, C. Criddle, D. Watson, , Terry C. Hazen and J. Zhou . 2007. Monitoring of Microbial Reduction and Reoxidation Activities in the FRC Sites using a Comprehensive Functional Gene Array. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  835. Hemme, C. L., Y. Deng, T. Gentry, L. Wu, M. W. Fields, D. Bruce, C. Detter, K. Barry, D. Watson, P. Richardson, J. Bristow, , Terry C. Hazen and J. Tiedje, E. Rubin, A. P. Arkin, J. Zhou . 2007. Insights into Stress Ecology and Evolution of Microbial Communities from Uranium-Contaminated Groundwater Revealed by Metagenomics Analyses. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  836. Hwang, C., W.-M. Wu, T. J. Gentry, J. Carley, S. L. Carroll, D. Watson, P. M. Jardine, J. Zhou, , Terry C. Hazen and E. L. Brodie, Y. M. Piceno, G. L. Andersen, E. X. Perez, A. Massol, C. S. Criddle, M. W. Fields . 2007. Changes in Microbial Community Structure during Biostimulation for Uranium Reduction at Different Levels of Resolution. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  837. Klonowska, A., Z. He, Q. He, M. E. Clark, S. B. Thieman, Terry C. Hazen, E. L. Brodie, R. Chakraborty, E. J. Alm, B. Giles, H.-Y. Holman, A. P. Arkin, J. D. Wall, J. Zhou, M. W. Fields. 2007. Desulfovibrio vulgaris Responses to Hexavalent Chromium at the Community, Population, Cellular Levels. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  838. Sapra, R., S. Gaucher, G. Chirica, C. Kozina, G. Buffleben, R. Phan, D. Joyner, , Terry C. Hazen and A. P. Arkin, A. K. Singh . 2007. Redox Proteomics In Desulfovibrio vulgaris Hildenborough: Search for Proteins That Mediate Stress Response via Post-Translational Modification of the Cys Residues. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  839. Walker, C. B., D. Joyner, D. Chivian, S. S. Stolyar, K. Hillesland, J. Gabster, P. Dehal, M. Price, , Terry C. Hazen and A. P. Arkin, P. M. Richardson, D. Bruce, D. A. Stahl . 2007. Genomic Comparisons between a Metal-resistant Strain of Desulfovibrio vulgaris and the Type Strain D. vulgaris Hildenborough. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  840. Yang, Z., C. W. Schadt, , Terry C. Hazen and M. Keller . 2007. Towards High-Throughput and High Sensitivity Approaches for Uncovering Total Environmental Gene Expression Patterns. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  841. Yen, H.-C., , Terry C. Hazen and Z. Yang, J. Zhou, K. H. Huang, E. J. Alm, A. P. Arkin, J. D. Wall . 2007. Response of Desulfovibrio vulgaris Hildenborough to Acid pH. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  842. Zhou, A., Z. He, C. Hemme, A. Mukhopadhyay, J. Keasling, A. P. Arkin, , Terry C. Hazen and J. D. Wall, J. Zhou . 2007. Global Gene Regulation in Desulfovibrio vulgaris Hildenborough. Joint Genomics: GTL Awardee Workshop V and Metabolic Engineering 2007 and USDA-DOE Plant Feedstock Genomics for Bioenergy Awardee Workshop 2007
  843. Chakraborty, R. and Siu Pan Lam, Chin Man Leung, Eoin L. Brodie, Terry C. Hazen Richard Phan. 2007. Diverse anaerobic Cr(VI) tolerant bacteria from Cr(VI)-contaminated 100H site at Hanford 100H site at Hanford. Annual Meeting of the American Geophysical Union
  844. Dong, M., H. Liu, S. Allen, S. Hall, S. Fisher, T. Hazen, J. Geller, M. Singer, L. Yang, J. Jin, M. Biggin and H. E. Witkowska. 2007. Methodological refinements in iTRAQ reagent-based" tagless" strategy of identification and purification of soluble protein complexes in bacteria. 8th International Symposium on Mass Spectrometry in the Health and Life Sciences 6:23. abstract
    Currently, two principle methods are used to identify physical interactions between proteins on a genome-wide basis: two-hybrid screens and tandem affinity purification (TAP). While each method has its undeniable strengths, neither offers an unbiased universal approach for isolating multiple protein complexes from native preparations without prior genetic manipulation. As part of the Genomics: GTL Protein Complex Analysis Project (PCAP) we are developing a high throughput pipeline to purify water soluble protein complexes from Desulfovibrio vulgaris (DvH), identify their polypeptide constituents by mass spectrometry, determine their stoichiometries, and provide samples suitable for single particle EM characterization. These methods will then be used as part of PCAP's effort to model stress responses relevant to the detoxification of metal and radionuclide contaminated sites.Our strategy uses a novel " tagless" method (1) that fractionates the water soluble protein contents of a bacterium into a large number of fractions, and then identifies the polypeptide composition of a rational sampling of these fractions using a LC MALDI TOF/ TOF mass spectrometry (MS) workflow. Protein fractionation employs four orthogonal separation steps optimized to preserve protein complex integrity. Protein elution through the final stage of separation is monitored with the aid of iTRAQ reagent labeling, i. e., iTRAQ reporter ions serve as beacons that signal the presence of the parent protein in each of the examined protein fractions. By definition, a putative complex consists of co-eluting proteins.In preparation for the high-throughput phase of the project, we have examined and refined various aspects of our protocol to improve its robustness and reliability. We have adopted an efficient, highly reproducible MS sample preparation protocol that uses 96well PVDF multiscreen plates (2). We have introduced an internal protein standard to facilitate normalization of the iTRAQ-based quantification of protein levels throughout many protein fractions. We have also developed algorithms and graphical display tools for identifying protein complexes from MS data, including a method for cluster analysis of iTRAQ data to allow detection of co-migrating polypeptides and hence putative protein complexes. Using model systems (standard proteins and Escherichia coli) as well as the target organism (DvH), we have demonstrated that (i) iTRAQ methodology provides a faithful representation of the relative abundances of polypeptides in different chromatographic fractions; (ii) co-elution of components of known complexes can be discerned even under suboptimal conditions of high sample complexity; and (iii) heteromeric and homomeric complexes of DvH are preserved throughout a multistep separation process and detected with the aid of iTRAQ labeling.
  845. Hazen, Terry C.. 2007. Bioremediation: The Hope and the Hype for Environmental Cleanup. National Student Leadership Conference (Engineering)
  846. Hazen, Terry C.. 2007. Systems Microbiology and Exploring Microbial Community Diversity. LBNL Homeland Security S&T Director Walker
  847. Brodie, E. L. and Terry C Hazen. 2007. Molecular Tools in Environmental Microbiology. Advances in Environmental Remediation, Instituto Comunitario de Biodiversidad y Cultura Casa Pueblo de Adjuntas, Programa de Biotecnología Industrial, Universidad de Puerto Rico – Mayaguez
  848. Conrad, M. and N. Spycher, P. Nico, E. L. Brodie, Y. Fujita, A. Ray Terry C. Hazen. 2007. In Situ Sequestration of 90Sr and Uranium in the Vadose Zone through Microbial Precipitation of Phosphate Minerals. DOE Environmental Remediation Sciences Program Annual Review
  849. Daly, R. A. and T. K. Tokunaga, Y. Kim, J. Wan, Terry C. Hazen, M. K. Firestone E. L. Brodie. 2007. Influence of Electron-Donor Form and Supply Rate on Dynamics of Bacterial Populations Associated with Uranium Reduction and Remobilization. DOE Environmental Remediation Sciences Program Annual Review
  850. Hazen, Terry C. 2007. Bioremediation of Metal Contaminated Sites. Advances in Environmental Remediation, Instituto Comunitario de Biodiversidad y Cultura Casa Pueblo de Adjuntas, Programa de Biotecnología Industrial, Universidad de Puerto Rico – Mayaguez
  851. Hazen, Terry C. 2007. Bioremediation of Organic Pollutants under Aerobic and Anaerobic Environmental Conditions. Advances in Environmental Remediation, Instituto Comunitario de Biodiversidad y Cultura Casa Pueblo de Adjuntas, Programa de Biotecnología Industrial, Universidad de Puerto Rico – Mayaguez
  852. Hazen, Terry C. 2007. Ecogenomics for Systems Biology Approaches to Extreme and Contaminated Environments. ASM Branch Foundation Lecture: Ohio ASM Branch
  853. Hazen, Terry C.. 2007. Systems Biology (Integration of the Omics, Bioinformatics, Biogeochemistry): The New Frontier for Environmental Biotechnology. University of Oklahoma
  854. Hazen, T. C. and E. Brodie, D. Joyner, S. Borglin, J. Hanlon, M. Conrad, T. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, R. Chakraborty, P. E. Long, D. R. Newcomer, C. T. Resch, K. Cantrell, A. Willett, S. Koenigsberg B. Faybishenko. 2007. Field-Integrated Studies of Long-Term Sustainability of Chromium Bioreduction at Hanford 100H Site. DOE Environmental Remediation Sciences Program Annual Review
  855. He, Z. and J. Van Nostrand, C. Hemme, T. Gentry, W. Wu, C. Schadt, L. Wu, B. Gu, D. Watson, Terry C. Hazen, P. Jardine, C. S. Criddle, J. Zhou Y. Deng. 2007. GeoChip: Development and Applications for Microbial Community Analysis. DOE Environmental Remediation Sciences Program Annual Review
  856. Tokunaga, T. K. and J. Wan, R. Daly, E. L. Brodie, M. K. Firestone, Terry C. Hazen Y. Kim. 2007. Mesoscale Biotransformation of Uranium. DOE Environmental Remediation Sciences Program Annual Review
  857. Hazen, T. C., A. P. Arkin, M. W. Fields, M. Keller, J. Z. Zhou, G. L. Andersen, E. L. Brodie, D. L. Wyborski, C. L. Abulencia, C. L. Hemme, T. Gentry, D. Watson and P. Richardson. 2006. NUCL 87-Integrated ecogenomics for determining ecosystem function in uranium-contaminated environments. Abstracts Of Papers Of The American Chemical Society 232:1.
  858. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. 3rd Latin American and Caribbean Biotechnology Conference
  859. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. 2da Actividad Regional de Biotechnologia-UPR-Humacao “Biotechnologia en al Dario vivir: mejorando nuestra calidad de vida”
  860. Hazen, Terry C.. 2006. Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical and Microbiological Response to Slow Release Electron Donor. Department of Biology, University of Puerto Rico at Mayaguez
  861. Hazen, T. C. and M. W. Fields, M. Keller, J. Zhou, G. L. Andersen, E. L. Brodie, D. L. Wyborski, C. B. Abulencia, C. L. Hemme, T. Gentry, D. B. Watson, P. Richardson A. P. Arkin. 2006. Ecogenomics for Determining Ecosystem Function in Uranium-Contaminated Environments. Bioremediation in Nuclear Environments, Symposium on Analytical Chemistry in Nuclear Technology, ACS fall meeting
  862. Perez*, E. X., E. M. Rodriguez, N. Ramos, C. Shadt, J. Zhou, Y. Piceno, G. L. Andersen, E. L. Brodie, Terry C. Hazen, A. Massol-Dêya. 2006. Bacteria Diversity in Soil and Sediments from a former Bombing Range (Vieques, PR). 3rd Latin American and Caribbean Biotechnology Conference
  863. Ramos-Hernandez, N. and D. C. Joyner, E. X. Perez, A. Massol-Dêya, Terry C. Hazen R. Chakraborty. 2006. Chemotactic and Growth Responses to Explosives of Desulfovibrio vulgaris H. and Sulfate-Reducing Bacteria Isolated from Tropical Marine Sediments. 3rd Latin American and Caribbean Biotechnology Conference
  864. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. Utah State University
  865. Hazen, T. C. and E. Brodie, D. Joyner, S. Borglin, R. Chakraborty, M. Conrad, T. K. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, A. Willett, S. Koenigsberg B. Faybishenko. 2006. Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical and Microbiological Response to Slow Release Electron Donor. DOE ERSP annual field workshop
  866. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. University of North Carolina, School of Public Health
  867. Alm, E. J., S. C. Borglin, S. C. Chabra, S. P. Gaucher, M. Hadi, Terry C. Hazen, Q. He, H. Y. Holman, K. H. Huang, R. Huang, Z. He, D. C. Joyner, J. D. Keasling, M. Keller, K. Keller, A. Mukhopadhyay, A. Redding, A. Singh, D. D. Stahl, S. Stolyar, Z. Yang, J. Wall, G. Zane, J. Zhou and A. Arkin. 2006. Comparative analysis of bacterial gene expression in response to environmental stress. 106th General Meeting of the American-Society-for-Microbiology 106:579. abstract
    The transcriptional response of bacterial species to environmental stress has been the subject of considerable research, fueled in part by the widespread availability of gene expression microarray technology. Previous studies have established the similarity of gene expression networks across a wide range of organisms, yet in these studies different experiments were performed on different species preventing a direct comparison. We have compiled a core set of stressors including salt, pH, temperature, and oxygen and nitrite/nitrate levels and applied these stressors systematically to a phylogenetically diverse group of metal-reducing bacteria. We compare the expression patterns of orthologous genes and regulons in Desulfovibrio vulgaris, Geobacter metallireducens, and Shewanella oneidensis after exposure to these stressors. We observe that while the overall network may be conserved (genes in the same pathways have high correlations over all conditions), the response of the network the same perturbations can be very different in different species (pathways may respond to the same stressor in different ways). Differences between species can arise from differential behavior of the same regulons and because orthologous regulons may comprise different sets of (non-orthologous) genes, both of which may lead to insights in the ecological factors that shape gene expression.
  868. Borglin, S. E., M. Conrad, T. Hazen, D. Joyner and B. Faybrishenko. 2006. Assessment of bioreduction of Cr(VI) using 13C-PLFA analysis. 106th General Meeting of the American-Society-for-Microbiology 106:501. abstract
    Cr(VI) is a widespread groundwater contaminant. To stimulate bioreduction of Cr(VI) in the groundwater at the Hanford 100H field site, 18 kg of HRC® was injected into the aquifer. HRC® consists of polylactate esterified to a glycerol backbone that slowly releases lactic acid providing a source of carbon. 10 g of 13C-labeled polylactate was added to the HRC® to give it a δ13C value of ~40‰ (versus an unlabeled value of -15‰). Phospholipid fatty acid (PLFA) extracts of biomass in the samples were analyzed to assess the shift in microbial community structure in the extracted groundwater collected from 4 depth intervals in the injection well and in an extraction well located 5 m down-gradient from the injection well. Standard geochemical parameters (dissolved O2, Eh, anion chemistry, etc.) and the δ13C values of dissolved inorganic carbon (DIC), dissolved organic carbon, cell counts, and 16s rDNA analyses are also being monitored. The δ13C values of the primary PLFA peaks (common to many organisms) showed a 13C-enrichment, but reached values (>200‰) much higher the bulk δ13C of the labeled HRC®, reflecting the faster dissolution rate for the 13C-labeled polylactate relative to the HRC®. After the δ13C of the PLFA peaked, it quickly returned to background values (-15‰) indicating rapid turnover of biomass in the system. Several PLFA peaks specific to organisms known to metabolize glycerol (e.g., Flavobacteria) increased significantly but did not show the same increase in δ13C. Biomarkers PLFA associated with Desulfobacter, 10Me16:0, were identified and demonstrated 13C-enrichment. These data are being used to quantify the changes in biological activity resulting from HRC® injection.
  869. Chakraborty, R., E. L. Brodie, R. Phan, D. Joyner, Y. Piceno, G. L. Andersen, M. S. Humphrys, T. H. Hazen, P. Sobecky and Terry C. Hazen. 2006. Diversity of sulfate-reducing bacteria isolated from the Katrina floodwaters in New Orleans. 106th General Meeting of the American-Society-for-Microbiology 106:314. abstract
    Following the hurricane Katrina and subsequent flooding in New Orleans, samples of the floodwater were collected from several isolated sites in and around the city. We investigated the diversity of culturable sulfate reducing bacteria in these unique environmental samples. For isolation, enrichments were set up at 30oC in the dark using lactate (60mM) as the sole electron donor and sulfate (50mM) as the electron acceptor in anaerobic minimal media. Positive enrichments were identified from all site sources within 4 days of incubation and transferred periodically (6 times) before isolation on solid LS4D agar plates. Isolated colonies developed within 4 days and several isolates were obtained from each of the sampling sites. Preliminary analysis of the 16S rRNA genes of these isolates revealed that they belonged primarily to the Desulfovibrio genus and Desulfomicrobium genus of the Proteobacteria. Interestingly, isolates obtained from the water sample taken close to the US Coast Guard Station and the levee fracture site showed dominance of Desulfovibrio species, whereas those obtained from the Aerator site and the University of New Orleans campus showed predominantly Desulfomicrobium species. Representative isolates from each site were chosen for further characterization. Desulfomicrobium strain Nor2a from the Aerator site was highly motile, gram negative, non-sporulating bacterium. However while most Desulfomicrobium species are rod shaped, strain Nor2a is a vibrio, growing optimally at 370C. Apart from lactate, it also utilized alternative electron donors like pyruvate and fumarate. Desulfovibrio strain Nor1a isolated from the Coast Guard station was a coccobacilli, highly motile, gram-negative organism that could also reduce nitrate as an electron acceptor. More detailed characterization of the physiological and biochemical properties of these isolates is ongoing. Comparisons of isolates obtained from the same site as well as those belonging to the same genus obtained from a different site will enhance our understanding of the diversity and biogeography of these sulfate-reducing bacteria.
  870. Chakraborty, R., D. Joyner, E. Wozei, H. Y. N. Holman, S. Lam and Terry C. Hazen. 2006. Desulfovibrio strain PCS, a novel metal reducing pleomorphic sulfate reducing bacterium. 106th General Meeting of the American-Society-for-Microbiology 106:498. abstract
    A sulfate reducing bacterium was isolated from the Paleta Creek site in San Diego Bay with 60mM lactate as the sole carbon source and electron donor, and 50mM sulfate as the electron acceptor. The novel isolate, strain PCS is an anaerobic, non-sporulating, gram-negative organism that is highly motile. The optimum temperature for growth of strain PCS was determined to be 370C. Preliminary 16S rDNA analysis revealed that the closest relative to strain PCS is Desulfovibrio africanus (98 % similar). Light microscopy and SEM images of individual cells reveal sigmoid morphology. Cells of strain PCS appear like slender curved rods during the early log phase and spiral in exponential /stationery phase to approx 5-10μM in length and 0.2μM in width. In this regard, strain PCS is less than half the width of its closest known relative D.africanus. The images also reveal the presence of lemon shaped/spherical structures approx 1μM in diameter especially in early log and stationary phases of growth. Detailed investigations are underway to determine the nature and function of these structures. When grown on LS4D minimal media, strain PCS incompletely oxidizes lactate, accumulating acetate as an end product. Sulfate is reduced to hydrogen sulfide. Apart from lactate, strain PCS also utilizes alternative electron donors like pyruvate, benzoate and dihydroxyacetone. Propionate, butyrate, formate and fumarate were not utilized. Alternative electron acceptors utilized includes Fe-NTA and thiosulfate. Nitrate and chlorate were not reduced. When tested for reduction of toxic metal like Cr(VI), a washed cell suspension of strain PCS could remove almost 150μM of Cr(VI) supplied as potassium chromate with lactate as the electron donor. This high chromium reducing capability of strain PCS is of great significance for the potential utilization of this microbe towards the treatment of Cr(VI) contaminated environments.
  871. Chhabra, S., S. Gaucher, G. Zane, E. Alm, A. Arkin, T. Hazen, J. Wall and A. Singh. 2006. Investigation of protein-protein interactions in the metal-reducing bacterium Desulfovibrio vulgaris. 106th General Meeting of the American-Society-for-Microbiology 106:434. abstract
    Desulfovibrio vulgaris is a sulfate reducing bacteria of interest due to its potential use in bioremediation as well as its economic impact in the petroleum industry (biocorrosion of pumping machinery). This sulfate reducing bacteria has been shown to reduce toxic metals (such as chromium and uranium) to insoluble species making them a good model system for understanding molecular machines involved in bioremediation of contaminated soils and ground water. We have implemented an approach for the isolation of protein complexes from D. vulgaris by generating D. vulgaris cell lines that produce a "bait" protein of interest fused to an affinity tag (strep tag). Lysate from these cells is passed over an avidin column, and the bait protein with its associated proteins is captured and can be selectively eluted from contaminating proteins. One advantage of this approach is that complexes are formed and captured under native conditions. Nanoscale liquid chromatography-tandem mass spectrometry (nLC/MS/MS) is used identify the captured proteins. To determine bait proteins of interest, we computationally identified open reading frames (ORFs) that are involved in stress response (including oxygen, heat, pH and salt) based on homology to known stress related genes from other prokaryotic species. We have also selected proteins that are unique to this sulfate reducer (“signature” genes), expected to yield novel complexes related to sulfate/metal reduction. To validate our methods and address the challenge of non-specific binding, we have included some bait proteins whose interacting partners are well characterized in prokaryotic systems (E. coli, -rpoB and rpoC) and have validated our methods by isolating the binding partners of these targets.
  872. Chivian, D., E. J. Alm, F. J. Brockman, E. L. Brodie, D. E. Culley, T. Gihring, A. Lapidus, L. H. Lin, D. P. Moser, P. Richardson, A. P. Arkin, Terry C. Hazen, T. C. Onstott and E. O. Lawrence. 2006. Environmental genomic characterization of a deep subsurface microorganism. 106th General Meeting of the American-Society-for-Microbiology 106:377. abstract
    The deep subsurface planktonic community found at depths greater than 1.5 km across central South Africa is dominated by a species of Firmicutes that has never been successfully cultivated. A metagenome was assembled from DNA obtained from fracture water emanating from a borehole at 2.8 kilometers depth in a South African Gold mine. The draft assembly is consistent with a single microorganism, and implies that this species is capable of living in isolation in this deep subsurface environment. Genes for dissimilatory sulfate reduction were present in the genome. Based the 16S rRNA gene, the most similar described organism is Desulfotomaculum kuznetsovii (~91% identity), making this one of the first sequenced genomes of a dissimilatory sulfate reducing Gram-positive bacterium. In addition to sulfate reduction, the genome indicates that the organism is capable of formate oxidation. The genome also contains: 1) the acetogenic acetyl-CoA (Wood-Ljungdahl) pathway, 2) a partial TCA cycle, 3) a N2 fixation pathway, 4) genes for sporulation and germination, 5) heat shock proteins, 6) genes for pilus formation, and 7) genes for flagellum formation and chemotaxis. The variety of metabolic pathways and chemotactic capability is not suggestive of a streamlined genome for a sulfate reducer in an energy depleted environment, but rather more consistent with a motile sulfate reducer in an energy rich environment that actively seeks a specific subsurface niche when present and is capable of surviving long periods of time when that niche is absent. The fracture zone geochemistry is consistent with these inferences. The apparent absence of O2 tolerance genes indicates the organism is an obligate anaerobe consistent with an indigenous origin.
  873. Elias, D. A., I. B. Hilton, G. M. Zane, Terry C. Hazen, M. D. Biggin and J. D. Wall. 2006. High-throughput identification of multi-protein complexesvia TAP tagging in Desulfovibrio vulgaris. 106th General Meeting of the American-Society-for-Microbiology 106:284. abstract
    The sulfate-reducing bacterium Desulfovibrio vulgaris has long been of interest to, and has repeatedly been shown to have the potential for, in-situ metal and radionuclide immobilization. As such, several efforts have focused on the physiology and metabolism this bacterium and their response to stress. The primary goals of this project are to understand the nature of the proteins involved, their function as complexes and any alterations in complex composition in response to environmental stresses. Hence, we are currently utilizing tandem affinity tagging of proteins using three distinct tags in order to purify the protein of interest for detailed characterization. The first uses a “Strep-tag” (Qiagen) that inserts a streptavidin binding peptide for easy enrichment, and we have currently constructed 16 such tags. However, in order to attain even higher protein enrichment, we are assessing the proven sequential peptide affinity tag, CTF (a.k.a. SPA), that includes a calmodulin binding protein (CBP), a protease (tobacco etch virus) and a 3 x FLAG site for monoclonal antibody binding versus a new “STF” tag that replaces CBP with a streptavidin binding peptide. At issue is that the latter is only 8 amino acids compared to 125 with CBP, and so should be less likely to interfere with localization/orientation of the protein within the cell. All three approaches are currently being assessed with DsrC (DVU2776), a protein in the dissimilatory sulfite reductase pathway that is essential for cell growth via sulfate respiration. Proteins are also being tagged with a peptide containing a tetracysteine motif for in situ imaging of the location and relative density of the protein within the cell at different growth states and stresses, including redox reactive extracellular appendages. FtsZ (DVU2499), a cell division protein, is the first candidate for testing the efficacy of this procedure. This information is expected lead to a more thorough understanding of not only the proteins involved in metal-reduction but also their protein-protein interactions and characterization of the complete pathway(s) for these activities.
  874. Hadi, M. Z., J. Crawford-Dibble, P. Lane, S. Gaucher, T. Hazen, A. Arkin and A. Singh. 2006. High throughput methods for protein complex isolation and identification. 106th General Meeting of the American-Society-for-Microbiology 106:299. abstract
    Targeted proteomics is the systematic study of protein-protein interactions through the isolation of protein complexes. Several approaches have been developed over the years for purification and identification of protein complexes such as yeast two hybrid methods, co-immunoprecipitaions and endogenous expression of affinity tagged bait proteins (tandem affinity tags -TAPTAG). The TAPTAG approach has been successfully used to build a protein interaction map of the yeast genome. All of these approaches have one thing in common - they are cumbersome, time consuming, require considerable manual handling and are not amenable to automation. We have been developing novel generic high throughput technologies that will enable systematic identification, characterization and eventual understanding of molecular machines that function during stress responses in Desulfovibrio vulgaris [Strain, Hildenborough] from a targeted proteomics perspective. This sulfate reducing bacteria has been shown to reduce toxic metals (such as chromium and uranium) to insoluble species making them a good model system for understanding molecular machines involved in bioremediation of contaminated soils and ground water. We computationally identified open reading frames (ORFs) that are involved in stress response (including oxygen, heat, pH and salt) based on homology to known stress related genes from other prokaryotic species and have used these ORFs as bait proteins to isolate molecular machines. The Exogenous Tagged-Protein method for identification of interacting protein is the most amenable to high throughput and automation. We are now focusing on a set of bait proteins that are unique to Desulfovibrio vulgaris and other sulfate reducer, i.e. "signature" genes which are expected to yield novel complexes related to sulfate/metal reduction. We discuss our results and the application to these technologies towards proteome wide interaction mapping. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy’s NNSA under contract DE-AC04-94AL85000
  875. Hazen, Terry C., B. Faybishenko, E. Brodie, D. Joyner, S. Borglin, R. Chakraborty, M. Conrad, T. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, P. Long, D. Newcomer, A. Willett and S. Koenigsberg. 2006. Long-term chromium Bio-immobilization at the Hanford 100H site: Geochemical and microbiological response to slow release electron donor. 106th General Meeting of the American-Society-for-Microbiology 106:379. abstract
    The focus of these studies is to understand the coupled hydraulic, geochemical, and microbial conditions necessary to maximize Cr(VI) bioreduction and minimize Cr(III) reoxidation in groundwater. Here we present the application of slow release electron donor during a field-scale treatability study over an 18-month period. Samples were taken at intervals pre- and post-injection of a 13C-labeled slow release polylactate compound (HRC) used to stimulate indigenous microbial populations to immobilize hexavalent chromium. Redox potential, pH, dissolved oxygen (DO), nitrate, chromium (VI), and sulfate concentrations in groundwater were monitored. Stable isotope enrichment in dissolved inorganic pools was followed and a fluorescent antibody used to visualize the presence of a sulfate reducer. Following HRC injection (27 days) reducing conditions (-130 mV) had established with a corresponding disappearance of DO and nitrate. Cr(VI) concentrations declined steadily over 6 weeks. Analysis of delta 13C ratios in dissolved inorganic carbon confirmed microbial metabolism of the labeled HRC. Hydrogen sulfide production was first observed after about 20 days post-injection and this corresponded with the enrichment of a Desulfovibrio species identified using fluorescent antibodies. Bacterial densities have remained high (>10e7 cells/ml), Fe(II), and Cr(VI) concentrations in the monitoring and pumping wells have remained below up-gradient concentrations for the first 12 months. Lower parts of the aquifer have maintained sulfate reducing conditions and when pumping was applied to downgradient wells DO declined significantly in the upper parts of the aquifer. A number of sulfate reducers have been isolated from the deeper parts of the aquifer. HRC was still present more than a year after initial injection and was maintaining the overall Cr (VI) levels at non-detectable, suggesting that this may be a long-term bioimmobilization of Cr (VI). Geochemical analysis of groundwater coupled with stable isotope and microbial monitoring allowed for accurate tracking of microbial processes during this field treatability study.
  876. He, Q. and Z. He, Z. Yang, E. J. Alm, K. H. Huang, H.-C. Yen, D. C. Joyner, M. Keller, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou W. Chen. 2006. Nitrate stress response in Desulfovibrio vulgaris Hildenborough: Whole-Genome Transcriptomics and proteomics analyses. 106th General Meeting of the American-Society-for-Microbiology abstract
    Sulfate reducing bacteria (SRB) are of interest for bioremediation with their ability to reduce and immobilize heavy metals. Nitrate, a common co-contaminant in DOE sites, is suggested to inhibit SRB via nitrite. Previous results indicate that nitrite is indeed inhibitory to the growth of Desulfovibrio vulgaris. However, growth inhibition by nitrate alone was also observed. In this study, growth and expression responses to various concentrations of nitrate were investigated using the Omnilog phenotype arrays and whole-genome DNA microarrays. Changes in the proteome were examined with 3D-LC followed by MS-MS analysis. Microarray analysis found 5, 50, 115, and 149 genes significantly up-regulated and 36, 113, 205, and 149 down-regulated at 30, 60, 120, and 240 min, respectively. Many of these genes (~50% at certain time points) were of unknown functions. By comparison to NaCl stress, transcriptional analysis identified changes specific to NaNO3 stress. The hybrid cluster protein was among the highly up-regulated genes, suggesting its role in nitrate stress resistance with its proposed function in nitrogen metabolism. The up-regulation of phage shock protein genes (pspA and pspC) might indicate a reduced proton motive force and the repression of multiple ribosomal protein genes could further explain the growth cessation resulting from nitrate stress. A glycine/betaine transporter gene was also up-regulated, suggesting that NaNO3 also constituted osmotic stress. Osmoprotectant accumulation as the major resistance mechanism was validated by the partial relief of growth inhibition by glycine betaine. Proteomics analyses further confirmed the altered expression of these genes, and in addition, detected increased levels of several enzymes (Sat, DvsB, and AprB) in the sulfate reduction pathway, indicative of the increased energy production during nitrate stress. In conclusion, excess NaNO3 resulted in both osmotic stress and nitrate stress. D. vulgaris shifted nitrogen metabolism and energy production in response to nitrate stress. Resistance to osmotic stress was achieved primarily by the transport of osmoprotectant.
  877. He, Z., Q. He, E. J. Aim, J. D. Wall, M. W. Fields, Terry C. Hazen, A. R. Arkin and J. Zhou. 2006. Exploration of salt adaptation mechanisms in Desuliovibrio vulgaris hildenborough. 106th General Meeting of the American-Society-for-Microbiology 106:355. abstract
    Desulfovibrio vulgaris Hildenborough is a δ-Proteobacterium, a model sulfate-reducing bacterium, and well known for its metabolic versatility and wide distribution. D. vulgaris cells inoculated into medium supplied with NaCl (250-500 mM) survived and grew although at a lower rate and with a longer lag. These cells were used to explore salt adaptation mechanisms combining global transcriptional analysis and physiological studies. This differs from salt shock, in which NaCl was added to cells in mid-log phase and samples were collected after a relatively short exposure (30-240 min). Comparison of D. vulgaris grown in the medium amended with yeast extract plus 500 mM NaCl showed that D. vulgaris growth was inhibited ~ 35% with and ~ 80% without yeast extract. Transcriptomic data revealed that predicted genes encoding leucine biosynthesis proteins, phage- or heat-shock proteins, formate dehydrogenases, sensory box histidine kinases/response regulators, and peptidases were up-expressed in NaCl-adapted cells, and that predicted genes involved in tryptophan biosynthesis, ribosomal protein synthesis, energy metabolism, iron transport, and phage-related proteins were down-expressed. However, genes involved in glycine/betaine/proline ABC transport were not significantly up-regulated, which was different from our previous observations for salt shock. External addition of leucine or/and tryptophan into the medium without yeast extract significantly relieved the inhibition of D. vulgaris growth with 500 mM NaCl, a result consistent with the microarray data since the genes involved in tryptophan biosynthesis are strongly regulated by feedback mechanisms. Addition of other amino acids (e.g. glutamate and serine), precursors of tryptophan, or products of tryptophan could not relieve such an inhibition. The results suggest that the accumulation of metabolites (e.g. leucine and tryptophan) and nutrients may increase in the adaptability of D. vulgaris to high salt conditions. Further studies will focus on the analysis of metabolites and on the elucidation of salt adaptation mechanisms in D. vulgaris.
  878. Hemme, C. L., K. Bender, H. C. Yen, Z. Yang, D. Joyner, J. Jacobsen, Z. He, K. Huang, E. Alm, Terry C. Hazen, A. Arkin, J. Zhou and J. D. Wall. 2006. Characterization of a Desulfovibrio vulgaris hildenborough mutant strain lacking the ferric uptake regulator (fur) gene. 106th General Meeting of the American-Society-for-Microbiology 106:356. abstract
    Analysis of wild-type Desulfovibrio vulgaris Hildenborough under a variety of stress conditions suggests a role for the ferric uptake regulator (FUR) in the general stress response by this organism. A mutant D. vulgaris strain lacking the fur gene has previously been constructed and analyzed using physiological and molecular biology techniques. The mutant strain, designated JW707, was analyzed under iron-replete and iron-depeleted conditions using whole-genome microarray techniques to complement the previous analyses. JW707 cultures grown to mid-log phase with 60 μM Fe showed up-regulation of 51 genes (z ≥ 2). These genes included a ferrous iron transport operon (feoAB), a TonB-dependent ABC transport operon (including tolQR), a putative pepsidase/ABC transporter operon and an uncharacterized hypothetical protein-encoding gene (DVU2681) also shown to be induced under heat shock and nitrite stress conditions. These results are consistent with previously conducted Northern analyses and computational predictions of the D. vulgaris FUR regulon. Similar results were observed for cells grown under iron-depleted conditions (5 μM Fe). In addition, elements of the Trp and Leu biosynthetic operons and the Zur-dependent zinc transport operon were up-regulated and elements of the flagellar apparatus were down-regulated in the mutant. Additional studies are currently underway to distinguish between FUR-dependent and FUR-independent gene regulation in D. vulgaris.
  879. Hemme, C. L., T. Gentry, L. Wu, M. W. Fields, K. Barry, C. Detter, C. Schadt, D. C. Bruce, D. Watson, T. C. Hazen, J. Tiedje, P. Richardson, E. Rubin and J. Zhou. 2006. Metagenomic analysis of uranium-contaminated groundwater. 106th General Meeting of the American-Society-for-Microbiology 106:376. abstract
    One of the challenges inherent in the study of microbial communities is determining how the community responds to stress imposed by changes to the local environment. Community sequence data was isolated from groundwater (FW106) at the DOE NABIR Field Research Center (FRC) in Oak Ridge, TN. The FRC site contains high levels of nitrate, uranium and other heavy metals and low pH (~3.7). The stresses imposed by these conditions have reduced the species diversity of the community, but it is predicted that this loss was compensated for by a period of adaptive evolution of the surviving community members. Sequence analysis of this groundwater sample based on a 16S rDNA library revealed 10 operational taxonomic units (OTUs) at the 99.6% cutoff with >90% of the OTUs represented by an unidentified γ-proteobacterial species similar to Frateuria. The collected DNA was submitted to the DOE Joint Genome Institute (JGI) for sequencing. Three clone libraries (3, 8 and 40 kb) were constructed, and 70+ Mb raw sequences were obtained using a shotgun sequencing approach. The raw sequences were assembled into 2770 contigs totaling ~6 Mb which were further assembled into 224 scaffolds (1.8 kb-2.4 Mb). Preliminary binning of the scaffolds suggests one dominant phylotype consisting of the Frateuria-like γ-proteobacteria and at least three secondary phylotypes encompassing both β- and γ-proteobacteria. Despite the limited species diversity of the sample, analysis of the metagenome suggests the presence of genes necessary for stress responses and survival under the given geochemical conditions, specifically denitrification, metal resistance and pH resistance genes. A significant amount of polymorphism, suggesting strain diversity, was also observed within the predicted phylotypes. Analysis with functional gene arrays based on functional genes involved in biogeochemical cycling of C, N, and S, metal resistance and contaminant degradation suggest that the dominant species could be biostimulated during in situ uranium reduction experiments, suggesting that this species could play a role in uranium bioremediation.
  880. Holman, H. Y. N., E. Wozei and Terry C. Hazen. 2006. Molecular observations of anaerobes in atmospheric oxygen. 106th General Meeting of the American-Society-for-Microbiology 106:297-298. abstract
    A long-standing desire in microbiology is to be able to observe in situ and at a molecular level how anaerobes respond to atmospheric oxygen. Over the past decade, physics, engineering and instrumentation innovations have led to the introduction of synchrotron radiation-based infrared spectromicroscopy. Spatial resolutions of less than ten micrometers and photon energies of less than an electron volt make synchrotron infrared spectromicroscopy non-invasive and useful for following the course of cellular processes. Here we present a comparative study of molecular changes in the obligate anaerobe Desulfovibrio vulgaris and the facultative anaerobe Shewanella oneidensis during their exposure to atmospheric oxygen. Using non-invasive synchrotron radiation-based Fourier transform infrared (SR-based FTIR) spectromicroscopy, we successfully measured directly molecular changes in cellular environments in D. vulgaris and in S. oneidensis during their exposure to air. By comparing measurements, we were able to identify the time-dependent molecular changes in lipids, nucleic acids, proteins, and polyglucose. Images from fluorescence and electron microscopies provide direct visual images of the corresponding morphological changes.
  881. Joyner, D. C., A. Mukhopadhyay, R. Chakraborty, S. Borglin, J. Jacobsen, R. Kim and T. Hazen. 2006. Anaerobic phenotype microarray method for knockout mutant comparison. 106th General Meeting of the American-Society-for-Microbiology 106:550-551. abstract
    Phenotype Microarray (PM) has been developed for the high throughput and rapid assessment of phenotypic responses of microbes to approximately 2,000 metabolites and chemicals under aerobic conditions. Previously in our lab, a method was developed for PM under anaerobic conditions. In the present work we describe a method of inoculum standardization of anaerobes to ensure repeatability of results between replicate runs. Our tests were conducted with the sulfate reducing bacterium Desulfovibrio vulgaris strain Hildenborough in a defined lactate sulfate medium. For optimization of results, several factors were tested that included growth phase of inoculum having the greatest capability for growth after inoculation, optimal centrifugation times at 6000 g for highest retention of cell pellet, optimal inoculum concentration of resuspended cells as determined by AODC which was compared to OD at 600nm and %T. Our results show that standardization was achieved as demonstrated by repeatability of growth data between biological replicates of D. vulgaris in the PM. The application of the anaerobic PM was tested in 2 different studies with a wild type DvH and a single crossover sensor histidine kinase mutant strain of D. vulgaris with a potentially interesting phenotype under salt stress. The differential expression patterns of wt D. vulgaris and the mutant strain of D. vulgaris were compared.Osmotic sensitivity to NaCl and KCl was increased in the mutant strain with inhibition of growth above 3% as compared to 6% and 5% with the wt. No protection of the mutant was conferred by the addition of osmoprotectant. In another test, the mutant strain was used for the novel application of PM technology to investigate phenotypic expression of an organism under stressed conditions. In this study, anaerobic PM of the mutant strain under osmotic stress was generated with 250mM NaCl vs 250mM KCl and compared with the expression pattern of the organism under non stressed conditions. The mutant strain amended with 250mM KCl had greater resistance to osmotic stress up to 10% NaCl and greater resistance to200mM sodium benzoate and 100mM sodium nitrite.
  882. Klonowska, A., Z. He, Q. He, Z. Yang, Terry C. Hazen, S. Thieman, E. J. Alm, A. P. Arkin, J. D. Wall, J. Zhou and M. W. Fields. 2006. Global transcriptomic analysis of chromium(VI) exposure of Desulfovibrio vulgaris hildenborough under sulfate-reducing conditions. 106th General Meeting of the American-Society-for-Microbiology 106:338. abstract
    Desulfovibrio vulgaris is an anaerobic sulfate-reducing bacterium (SRB) able to reduce toxic heavy metals such as chromium and uranium, and D. vulgaris represents a useful SRB model for the bioremediation of heavy metal contamination. Although much work has focused on Cr and U reduction via individual enzymes, less is known about the cellular response to heavy metal stress in Desulfovibrio species. Cells were cultivated in a defined medium with lactate and sulfate, and a sub-lethal concentration of Cr(VI) was added at mid-exponential phase growth. The growth was affected upon addition of Cr(VI), but the treated culture had a similar growth rate to no-treatment culture within approximately 6 h. The major differentially expressed genes included those coding for a presumptive FMN reductase, an hsp20-like protein, a facilitator, an ArsR-like regulator, and a predicted carboxynorspermidine decarboxylase (nspC). A presumptive permease gene was in a predicted operon with the ArsR-like gene, and the permease gene displayed an upward trend of expression during the first 2 h of Cr exposure. At 60 minute post-treatment, genes for a nitroreductase, thioredoxin reductase, and Clp protease adaptor were up-expressed. Presumptive genes for agmatinase, flocculin, Zn-chelator, and a kdpC were also up-expressed. In addition, four predicted metal or drug transporter genes were up-expressed, and included presumptive merP, acrA, and chrA. Interestingly, agmatinase could be an enzymatic activity that could supply a precursor for nspC suggesting a possible role for polyamines. Interestingly, six up-expressed genes were on the megaplasmid, and included hypothetical proteins, a presumptive facilitator, and a predicted chrA (chromate transporter). These results suggested a role of megaplasmid-encoded proteins that may be important to reduce chromate accumulation in the cell. A strain that was missing the megaplasmid was more susceptible to chromium exposure, and this result corroborated the microarray data. Further work is needed to delineate the possible roles of the respective genes.
  883. Mukhopadhyay, A., Z. He, E. Alm, A. Arkin, E. Baidoo, S. Borglin, W. Chen, T. Hazen, Q. He, H. Y. Holman, K. Huang, D. Joyner, M. Keller, P. Oeller, A. Redding, J. Sun, J. Wall, J. Wei, H. C. Yen, J. Zhou and J. Keasling. 2006. Salt stress in Desulfovibrio vulgaris hildenborough: An integrated genomics approach. 106th General Meeting of the American-Society-for-Microbiology 106:298. abstract
    Recent interest in the ability of Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium, to reduce, and therefore contain, toxic and radioactive metal waste, has made all factors that affect its physiology of great interest. Increased salinity constitutes an important and frequent fluctuation faced by D. vulgaris in its natural habitat. In liquid culture, exposure to excess salt resulted in a striking cell elongation in D. vulgaris. Using data from transcriptomics, proteomics, metabolite assays, phospholipid fatty acid profiling, and electron microscopy, we undertook a systems approach to determine the effects of excess NaCl on D. vulgaris. Our data indicates that import of osmoprotectants such as glycine betaine and ectoine constitutes the primary mechanism in D. vulgaris to counter hyper-ionic stress. Many efflux systems were highly upregulated as was the ATP synthesis pathway. Increase in both RNA and DNA helicases suggested that salt stress had affected the stability of nucleic acid base pairing. An overall increase in branched fatty acids indicated changes on cell wall fluidity. An immediate response to salt stress included upregulation of chemotaxis genes though flagellar biosynthesis was down regulated. Other down regulated systems included lactate uptake permeases and ABC transport systems. Unlike many other bacteria, K+ ions did not provide protection from NaCl stress in D.vulgaris. In fact microarray data shows D. vulgaris response to both NaCl and KCl to be very similar. Integration of data from multiple methods allowed us present a conceptual model for the salt stress response in D. vulgaris that can be compared to other microorganisms.
  884. Redding, A., A. Mukhopadhyay, D. Joyner, Terry C. Hazen and J. Keasling. 2006. Quantitative proteomic analysis of nitrate stress in Desulfovibrio vulgaris. 106th General Meeting of the American-Society-for-Microbiology 106:297. abstract
    Desulfovibrio vulgaris Hildenborough (DvH), a sulfate reducing bacterium, has historically been an environmentally important bacterium due its role in bio-corrosion of oil and gas pipelines and is one of the major sources of H2S that cause bio-fouling of petroleum. Another reason for interest in DvH is due to its ability to reduce toxic and radioactive metals to their lower oxidation and insoluble forms, and therefore its potential use in bioremediation. While sulfate typically serves as the electron acceptor in DvH, alternate candidates for electron acceptors such as nitrate also exist. Exposure to excess nitrate occurs frequently since it is a common co-contaminant along with metals such as uranium in many waste sites. Therefore our knowledge of DvH response to nitrate will undoubtedly be critical in developing bioremediation strategies. This poster presents the results from a quantitative proteomic analysis evaluating the response of DvH to nitrate stress. Control proteome was compared with proteome from cells exposed to NaNO3 levels that cause a 50% inhibition in growth. The ITRAQ peptide labeling strategy coupled with tandem liquid chromatography and mass spectrometry (triple-quad time of flight) was used. A total of 1166 unique proteins were identified, representing 34% of the total DvH proteome and spanning every functional category. Our results indicate that this was a mild stress, as confirmed by the lack of change observed in central metabolism or in the sulfate reduction pathway. Increases seen in transport systems for proline, glycine betaine and glutamate indicate that the NaNO3 exposure led to both salt stress and nitrate stress. Up-regulation observed in a large number of ABC transport systems as well as in iron-sulfur cluster containing proteins, however, appear to be specific to the exposure to nitrate. Finally, a number of hypothetical proteins are among the most significant changers, indicating that there may be unknown mechanisms initiated upon nitrate stress. Our poster outlines both our results from the proteomic response to nitrate exposure as well as the methods used to optimize quantitative proteomics for DvH.
  885. Stolyar, S., Q. He, E. Alm, K. Huang, K. L. Hillesland, Terry C. Hazen, S. Borglin, D. Joyner, A. P. Arkin, J. Zhou and D. A. Stahl. 2006. Genomewide gene expression analysis of response of Desulfovibrio vulgaris to high pH. 106th General Meeting of the American-Society-for-Microbiology 106:338. abstract
    High pH can often be encountered in natural environments by microorganisms. As a part of the DOE Genomics:GTL initiative evaluating the use of microbial stress response to monitor the status of environmental systems, we studied the effect of exposure to high alkaline (pH10) media on exponentially growing Desulfovibrio vulgaris Hildenborough (DvH) cells. Any given gram-negative bacterium under high pH stress is challenged by mainly three factors: intracellular alkalinized pH, diminished membrane potential and misfolded degrading proteins. To maintain viability in this stressful state, the cell could transport protons or acids into the cell, synthesize compounds that acidify the cytoplasm, increase protein folding, or increase degradation of denatured proteins. In our experiments, several genes reported to be upregulated in E. coli at high pH were also upregulated in DvH. These include three ATPase genes and a tryptophan synthase gene. As in E. coli, genes involved in flagella synthesis were downregulated during pH 10 stress. DvH also upregulated chaperone and protease genes such as an ATP-dependent Clp protease, an ATP-dependent protease La, and dnaK. Some energy production genes were consistently downregulated at high pH. These include pyruvate carboxylase, desulfoferrodoxin, and ferredoxin II. Finally, the microarray data revealed a potential DvH specific pH homeostasis mechanism. In DvH but not other deltaproteobacteria or E. coli, the antiporter nhaC and a putative L-aspartate oxidase gene are adjacent in the genome and likely to be in the same operon. Both of these genes were upregulated in D. vulgaris during high pH stress. Thus, part of the DvH response to high pH stress appears to involve coupling pumping of protons into the cell with conversion of L-aspartate to oxaloacetate which is likely to increase the acidity of the cell. The effect of several mutations on resistance to high pH will be reported.
  886. Tang, Y. J. and A. Meadows, R. Huang, T. C. Hazen, J. D. Keasling A. Mukhopadhyay. 2006. Investigation of the Central Metabolic Pathways of Desulfovibrio vulgaris Hildenborough Using a Minimal Medium with 13C Labeled Lactate. 106th General Meeting of the American-Society-for-Microbiology abstract
    Desulfovibrio vulgaris Hildenborough, an environmentally important sulfate reducing bacterium, retains research interest due to its ability to reduce toxic and radioactive metals to an insoluble form, and therefore has potential uses in bioremediation. The recent availability of an annotated genomic sequence of D. vulgaris allows us to examine the central metabolic fluxes in this bacterium using 13C isotopomer analysis. By growing D. vulgaris in the LS4D minimal medium with labeled lactate as the sole carbon source, we first developed a Monod model for the kinetics of biomass and waste metabolite production, and lactate/sulfate consumption. Moreover, we determined the cellular quantities of major macromolecules and quantified the carbon labeling patterns of 10 key amino acids in a biomass hydrolysate with GC/MS. Based on the resulting isotopomer distributions and the published genome sequence, a metabolic pathway model was constructed and the central metabolic flux distributions were quantified using a global search algorithm, with the measured growth kinetics, extracellular fluxes, sulfate consumption rate and biomass composition serving as constraints. Our preliminary findings are: 1. Dried biomass composition by weight is 5 % fatty acid, 30% protein, 25% carbohydrate, 10% RNA, 2% DNA, and over 16% ash; 2. D. vulgaris grows in the minimal medium with a specific growth rate of approximately 0.055hr-1 in the middle log phase, and acetate is the major growth associated product, with up to ~73% of consumed lactate being converted stoichometrically to acetate; 3. Although the gene annotation lacks citrate synthase, our isotopomer data strongly indicates that the reaction of oxolacetate + acetylCoA --> citrate may still occur; 4. The flux distribution through central pathways (including TCA cycle, glycolysis and pentose phosphate pathways) was calculated based on our assumed pathway map and measured isotopomer data. These findings and others we will present should enable a better understanding of the basic physiology of D. vulgaris and widen its potential bioremediation applications.
  887. Yen, H. C. B., E. Alm, K. Huang, Terry C. Hazen, A. Arkin, J. Zhou and J. D. Wall. 2006. Response of Desulfovibrio vulgaris to acid medium. 106th General Meeting of the American-Society-for-Microbiology 106:540-541. abstract
    The anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough is currently being explored for environmental bioremediation applications. Among the environmental constraints likely to be encountered in contaminated sites will be acid conditions. Therefore, the effect of low pH on the metabolic capacities of D. vulgaris is being explored. Transcriptional profiling was performed for cells exposed to low pH. According to pH 5.5 microarray data, many clusters of genes in D. vulgaris are up-regulated. Five of these gene clusters were chosen for physiological experiments to determine the relationship between the differentially expressed genes and acid resistance. Cells inoculated into acid pH media had a longer lag phase and lower final protein yields. When cells were pre-grown at pH 6 or 5, they did not show acid adaptation. The acid-grown cells showed greater mobility during microscopic observations and in a soft-agar plate assay, consistent with the increase in the transcription of flagellar genes. However, addition of thiamine, potassium, or individual amino acids showed no significant increase in acid resistance. Addition of casamino acids did have some protective effect. A mutant with a null mutation in pspA, a phage shock gene (JW704) showed an increased sensitivity to acid, having a much longer lag phase before growth in pH 5.5 medium. Finally, the effects of chemicals, such as polyamines which are products of the enzymes encoded in the norspermidine operon which was up-regulated, were investigated. Acid resistance and biofilm formation by adding spermidine, norspermidine, putrescine, or ornithine in growth medium will be reported.
  888. Brodie, E. and Terry C. Hazen. 2006. Integration of the Omics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. The Fifth International Conference on Remediation of Chlorinated and Recalcitrant Compounds
  889. Faybishenko, B., Terry C. Hazen, E. Brodie, D. Joyner, S. Borglin, J. Hanlon, M. Conrad, T. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, C. T. Resch, A. Willett and S. Koenigsberg. 2006. Tracer Tests and Field Monitoring of In situ Cr(VI) Bioreduction at the Hanford 100H Site. Spring meeting of American Geophysical Union
  890. Long, P. E. and C. T. Resch, K. Cantrell, B. Faybishenko, Terry C. Hazen, E. Brodie, D. Joyner, S. Borglin, J. Hanlon, M. Conrad, T. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, A. Willett, S. Koenigsberg D. R. Newcomer. 2006. Evaluation of the Effectiveness of Cr(VI) Biostimulation in Groundwater at Hanford 100H Site. Spring meeting of American Geophysical Union
  891. Tabak, H. H., Terry C. Hazen*. 2006. Advances in Bioremediation of Soils and Sediments Polluted with Metals and Radionuclides: 2. Field Research on Bioremediation of Metals and Radionuclides. The Fifth International Conference on Remediation of Chlorinated and Recalcitrant Compounds
  892. Onstott, T. C., Terry C. Hazen, Adam Arkin, Eric Alm, Eoin Brodie, Dylan Chivian, Paul Richardson, Alla Lapidus, David Culley, Fred Brockman, Li Hung Lin, Thomas Gihring and Duane P. Moser. 2006. Metagenomic characterization of a deep subsurface microorganism. Astrobiology Science Conference (AbSci Con 2006) 6:112-113. abstract
    A metagenome was assembled from DNA obtained from fracture water emanating from a borehole at 2.8 kilometers depth in a South African Au mine. Prior analyses of SSU rRNA and dsrAB gene clone libraries indicated that the planktonic Community was dominated by a species of Firmicutes that has only been detected at depths greater than 1.5 km across central South Africa but never successfully cultivated. The draft assembly is consistent with a single microorgranism. Based on the SSU rRNA gene this organism is most similar to Desulfotomaculum kuznetsovii at 91% identity, which makes it one of the first sequenced genomes of a Sulfate reducing gram positive bacterium. In addition to sulfate reduction it appears to be capable of H-2 and formate oxidation. The assembled genome also contains; 1) an acetyl-CoA pathway; 2) a partial TCA cycle; 3) a N-2 fixation pathway; 4) genes for germination and sporulation; 5) heat shock proteins; 6) genes for pilus formation; and 7) genes for flagellum formation and chemotaxis. The variety of metabolic pathways and inferred chemotactic capability is not suggestive of a streamlined genome for a sulfate reducer in an energy-depleted environment, but rather more consistent with a motile sulfate reducer in an energy rich environment that actively seeks a specific Subsurface niche when present and is capable of surviving long periods of time when that niche is absent. The fracture zone geochemistry is consistent with these inferences. The apparent absence of O-2 tolerance genes indicates the organism is an obligate anaerobe consistent with an indigenous origin.
  893. Chivian, D., E. J. Alm, F. J. Brockman, E. L. Brodie, D. E. Culley, T. Gihring, A. Lapidus, L-H. Lin, D. P. Moser, P. Richardson, A. P. Arkin, Terry C. Hazen and T. C. Onstott. 2006. Environmental Genomic Characterization of a Deep Subsurface Microorganism. JGI Users Meeting
  894. Hazen, Terry C.. 2006. Bioremediation Strategies for Contaminated Sediments. The Sixteenth Annual AEHS Meeting and West Coast Conference on Soils, Sediments and Water
  895. Hazen, Terry C. 2006. Engineered Microbial Systems. Georgia Tech Environmental Systems Microbiological Symposium
  896. Hazen, T. C. and H. H. Tabak. 2006. Field Research on Bioremediation of Metal Contamination in Soils and Sediments. The Sixteenth Annual AEHS Meeting and West Coast Conference on Soils, Sediments and Water
  897. Zhou, J. and C. Hemme, L. Wu, M. W. Fields, C. Detter, K. Barry, D. Watson, C. W. Schadt, P. Richardson, Terry C. Hazen, J. Tiedje, E. Rubin T. Gentry. 2006. Metagenomic Analysis of Microbial Communities in Uranium-contaminated Groundwaters. JGI Users Meeting
  898. Hazen, Terry C. 2006. Stress Response Pathways and Biofilms. Inland Northwest Research Alliance meeting on Bioremediation
  899. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. Wake Forest University
  900. Hazen, Terry C. 2006. Tutorial on Systems Biology and Bioremediation. Wake Forest University
  901. Abulencia, C., E. Alm, G. Anderson, A. P. Arkin*, K. Bender, S. Borglin, E. Brodie, R. Chakraborty, S. Chhabra, S. van Dien, I. Dubchak, M. Fields, S. Gaucher, J. Geller, M. Hadi, Terry C. Hazen, Q. He, Z. He, H.-Y. Holman, K. Huang, R. Huang, J. Jacobsen, D. Joyner, J. Keasling, K. Keller, M. Keller, A. Mukhopadhyay, R. Phan, M. Price, J. A. Ringbauer, Jr., A. Singh, D. Stahl, S. Stolyar, J. Sun, D. Thompson, C. Walker, J. Wall, J. Wei, D. Wolf, D. Wyborski, H.-C. Yen, G. Zane, J. Zhou, B. Zuniga. 2006. The Virtual Institute of Microbial Stress and Survival (VIMSS): Deduction of Stress Response Pathways in Metal/Radionuclide Reducing Microbes. DOE Genomics:GTL Annual Workshop
  902. Alm, E. J., E. E. Baidoo, P. I. Benke, S. E. Borglin, W. Chen, S. Chhabra, M. W. Fields, S. P. Gaucher, A. Gilman, M. Hadi, Terry C. Hazen, Q. He, H.-Y. Holman, K. Huang, R. Huang, Z. He, D. C. Joyner, J. D. Keasling, M. Keller, K. Keller, A. Mukhopadhyay, P. Oeller, F. Pingitore, A. Redding, A. Singh, D. Stahl, S. Stolyar, J. Sun, Z. Yang, J. D. Wall, G. Zane, J. Zhou and A. P. Arkin. 2006. Comparative Analysis of Bacterial Gene Expression in Response to Environmental Stress. DOE Genomics:GTL Annual Workshop
  903. Biggin, M. D., D. Elias, S. Chhabra, H.-Y. Holman, J. Keasling, A. Mukhopadhyay, T. Torok, J. Wall, Terry C. Hazen, Dong M, S. Hall, B. K. Jap, J. Jin, S. Fisher, P. J. Walian, Witkowska H. E, M. Auer, R. M. Glaeser, J. Malik, J. P. Remis, D. Typke, K. H. Downing, A. P. Arkin, S. E. Brenner, J. Jacobsen and J.-M. Chandonia. 2006. Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes during Stress Response in Desulfovibrio vulgaris: Project Overview. DOE Genomics:GTL Annual Workshop
  904. Clark, M. E., Q. He, Z. He, K. H. Huang, E. J. Alm, X. Wan, Terry C. Hazen, A. P. Arkin, J. D. Wall, J. Zhou, J. Kurowski, A. Sundararajan, A. Klonowska, D. Klingeman, T. Yan, M. Duley, M. W. Fields*. 2006. Temporal Transcriptomic Analysis of Desulfovibrio vulgaris Hildenborough Transition into Stationary-Phase Growth during Electron Donor Depletion. DOE Genomics:GTL Annual Workshop
  905. Gaucher, S., M. Hadi, S. Chhabra, E. Alm, G. Zane, D. C. Joyner, A. P. Arkin, Terry C. Hazen, J. D. Wall, A. Singh*. 2006. Investigation of Protein-Protein Interactions in the Metal-Reducing Bacterium Desulfovibrio vulgaris. DOE Genomics:GTL Annual Workshop
  906. Hazen, Terry C. 2006. Microbial Ecology in the deep subsurface. NSF Deep Underground Science and Engineering Lab Workshop
  907. Hazen, T. C. and G. L. Anderson, S. E. Borglin, E. Brodie, S. van Dien, M. Fields, J. Geller, H.-Y. Holman, R. Huang, R. Phan, E. Wozei, J. Jacobsen, D. Joyner, R. Chakraborty, M. Keller, A. Mukhopadhyay, D. Stahl, S. Stolyar, J. D. Wall, D. Wyborski, H.-C. Yen, G. Zane, J. Zhou, E. Hendrickson, T. Lie, J. Leigh, C. Walker C. Abulencia. 2006. VIMSS Applied Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers. DOE Genomics:GTL Annual Workshop
  908. Hazen, T. C. and J. Keasling, A. Mukhopadhyay, S. Chhabra, T. Torok, J. D. Wall, M. D. Biggin H.-Y. N. Holman. 2006. Protein Complex Analysis Project (PCAP): High Throughput Identification and Structural Characterization of Multi-Protein Complexes during Stress Response in Desulfovibrio vulgaris: Microbiology Subproject. DOE Genomics:GTL Annual Workshop
  909. He, Q. and W. Chen, Z. Yang, E. J. Alm, K. H. Huang, H.-C. Yen, D. C. Joyner, M. Keller, J. Keasling, A. P. Arkin, Terry C. Hazen, J. D. Wall, J. Zhou Z. He. 2006. Nitrate stress response in Desulfovibrio vulgaris Hildenborough: Whole-Genome Transcriptomics and proteomics analyses. DOE Genomics:GTL Annual Workshop
  910. Li, T. and D. Klingeman, L. Wu, X. Liu, T. Yan, Y. Xu, A. Beliaev, Z. He, Terry C. Hazen, A. P. Arkin, J. Zhou J. Guo. 2006. H2O2-Induced Stress Responses of Shewanella oneidensis MR-1. DOE Genomics:GTL Annual Workshop
  911. Mukhopadhyay, A., E. J. Alm, A. P. Arkin, E. E. Baidoo, P. I. Benke, S. E. Borglin, W. Chen, S. Chhabra, M. W. Fields, S. P. Gaucher, A. Gilman, M. Hadi, Terry C. Hazen, Q. He, H.-Y. Holman, K. Huang, R. Huang, Z. He, D. C. Joyner, M. Keller, K. Keller, P. Oeller, F. Pingitore, A. Redding, A. Singh, D. Stahl, S. Stolyar, J. Sun, Z. Yang, J. D. Wall, G. Zane, J. Zhou, J. D. Keasling*. 2006. VIMSS Functional Genomics Core Research on Stress Response Pathways in Metal-Reducers. DOE Genomics:GTL Annual Workshop
  912. Wall, J. D. and E. C. Drury, A. Mukhopadhyay, S. Chhabra, Q. He, M. W. Fields, A. Singh, J. Zhou, Terry C. Hazen, A. P. Arkin H.-C. Yen. 2006. Evaluation of stress responses in sulfate-reducing bacteria through genome analysis: identification of universal responses. DOE Genomics:GTL Annual Workshop
  913. Wyborski, D. L., C. B. Abulencia, J. A. Garcia, M. Podar, W. Chen, S. H. Chang, H. W. Chang, Terry C. Hazen, M. Keller*. 2006. Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments. DOE Genomics:GTL Annual Workshop
  914. Zhou, J. and C. Hemme, L. Wu, M. W. Fields, C. Detter, K. Barry, D. Watson, C. W. Schadt, P. Richardson, J. Bristow, Terry C. Hazen, J. Tiedje, E. Rubin T. Gentry. 2006. Metagenomic Analysis of Microbial Communities in Uranium-Contaminated Groundwaters. DOE Genomics:GTL Annual Workshop
  915. Brodie, E. L., Terry C. Hazen, B. Faybishenko, D. Joyner, S. E. Borglin, R. Chakraborty, E. Shapland, M. Conrad, T. Tokunaga, J. Wan, S. Hubbard, K. Williams, M. Firestone, G. L. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer and S. Koenigsberg. 2006. High Density 16S rRNA Microarray Analysis of Long-Term Chromium Bio-immobilization. 11th International Symposium on Microbial Ecology
  916. Chakraborty, R., E. L. Brodie, R. Phan, Y. Piceno, G. L. Andersen, M. S. Humphrys, T. H. Hazen, P. A. Sobecky and Terry C. Hazen. 2006. Diversity of sulfate-reducing bacteria isolated from the Katrina floodwater in New Orleans. 11th International Symposium on Microbial Ecology
  917. Chakraborty, R., D. C. Joyner, E. Wozei, H.-Y. Holman and Terry C. Hazen. 2006. Desulfovibrio strain PCS, a metal reducing pleomorphic sulfate reducing bacterium. 11th International Symposium on Microbial Ecology
  918. Chivian, D., E. J. Alm, F. J. Brockman, E. L. Brodie, D. E. Culley, T. Gihring, A. Lapidus, L-H. Lin, D. P. Moser, P. Richardson, A. P. Arkin, Terry C. Hazen and T. C. Onstott. 2006. Environmental Genomic Characterization of a Deep Subsurface Microorganism. 11th International Symposium on Microbial Ecology
  919. Hazen, Terry C. 2006. Advances in Field Research on Bioremediation of Metals and Radionuclides. Central South University
  920. Hazen, Terry C.. 2006. Bioremediation technologies for petroleum. Department of Environmental Science & Engineering, Tsinghua University
  921. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. Institute of Microbiology, Chinese Academy of Sciences
  922. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. Central South University
  923. Hazen, Terry C.. 2006. Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical and Microbiological Response to Slow Release Electron Donor. Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences
  924. Hazen, T. C. and M. W. Fields, M. Keller, J. Zhou, G. L. Andersen, E. L. Brodie, D. L. Wyborski, C. B. Abulencia, C. L. Hemme, T. Gentry, D. B. Watson, P. Richardson A. P. Arkin. 2006. Integrated Ecogenomics for Determining Ecosystem Function in a Uranium-Contaminated Environment. 11th International Symposium on Microbial Ecology
  925. Hazen, T. C. and A. P. Arkin. 2006. Integration of Omics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. 11th International Symposium on Microbial Ecology
  926. Hazen, T. C. and E. J. Alm, A. P. Arkin, F. J. Brockman, E. L. Brodie, D. Chivan, D. E. Culley, T. Ghring, A. Lapidus, L.-H. Lin, D. P. Moser, P. Richardson T. C. Onstott. 2006. Metagenomic characterization of a deep subsurface microorganism. 11th International Symposium on Microbial Ecology
  927. Perez, E. X., E. M. Rodriguez, N. Ramos, C. Shadt, J. Zhou, Y. Piceno, G. L. Andersen, E. L. Brodie, Terry C. Hazen, A. Massol-Dêya. 2006. Bacteria Diversity in Soil and Sediments from a former Bombing Range (Vieques, PR). LBNL Summer Student Presentations
  928. Ramos-Hernandez, N., R. Chakraborty, D. C. Joyner, E. X. Perez, A. Massol-Dêya, and Terry C. Hazen. 2006. Chemotactic and Growth Responses to Explosives of Desulfovibrio vulgaris H. and Sulfate-Reducing Bacteria Isolated from Tropical Marine Sediments. LBNL Summer Student Presentations
  929. Clark, M. E., Q. He, Z. He, K. H. Huang, E. J. Alm, X. Wan, Terry C. Hazen, A. P. Arkin, J. D. Wall, J. Zhou and M. W. Fields. 2006. Construction of Whole Genome Microarrays, Expression Analysis of Desulfovibrio vulgaris Cells in Metal-Reducing Conditions: Temporal Transcriptomic Analysis of Desulfovibrio vulgaris Hildenborough Transition into Stationary Phase during Electron Donor Depletion. DOE ERSP annual workshop
  930. Hazen, Terry C. 2006. Bioremediation: Cleaning up using Natures Natural Cleansing Capacity. Orton K. Stark General Lecture, Miami University
  931. Hazen, Terry C. 2006. Genomics:GTL and ERSP. DOE ERSP annual workshop
  932. Hazen, Terry C.. 2006. Integrated Omics in Systems Biology: The New Frontier for Environmental Biotechnology, Ecology and Evolution. Orton K. Stark General Lecture, Miami University
  933. Hazen, Terry C. 2006. Omics and Bioremediation Breakout Session. DOE ERSP annual workshop
  934. Hazen, Terry C. 2006. Stimulating the Microbial Reduction of Chromium. DOE ERSP annual workshop
  935. Hazen, T. C. and E. Brodie, D. Joyner, S. Borglin, R. Chakraborty, M. Conrad, T. K. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, A. Willett, S. Koenigsberg B. Faybishenko. 2006. Long-Term Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical and Microbiological Response to Slow Release Electron Donor. DOE ERSP annual workshop
  936. Long, P. E., Terry C. Hazen and B. Faybishenko, E. Brodie, D. Joyner, S. Borglin, R. Chakraborty, M. Conrad, T. K. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, G. Andersen, T. DeSantis, D. R. Newcomer, A. Willett, S. Koenigsberg . 2006. Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Cr(III) at Hanford 100-H Area 100-H Area. Invited presentation to the Hanford Site Unit Manager Meeting
  937. Tokunaga, T. K. and M. K. Firestone, Terry C. Hazen J. Wan. 2006. Mesoscale Biotransformation of Uranium. DOE ERSP annual workshop
  938. Zhou, J. and C. Hemme, L. Wu, M. W. Fields, C. Detter, K. Barry, D. Watson, C. W. Schadt, P. Richardson, Terry C. Hazen, J. Tiedje, E. Rubin T. Gentry. 2006. Metagenomic Analysis of Microbial Communities in Uranium-contaminated Groundwaters. Microbial Genomes Conference
  939. Conrad, M. E., S. E. Borglin, E. Brodie, K. N. Woods, B. Faybishenko, Terry C. Hazen, P. E. Long and A. Willett. 2005. Quantification of enhanced bioreduction of Cr(VI) using 13C-PLFA monitoring coupled with advance microbiological techniques. Annual Meeting Geological Society of America
  940. Hazen, Terry C. 2005. Biological Agents: Threat, Preparedness, Response and Myths. 2005 ASTAR National Boot Camp for Maryland and Ohio ASTA Resource Judges and Guest Jurists, The Language of the Life Sciences at the Airlie Conference Center
  941. Hazen, Terry C. 2005. Environmental Biotechnology and Bioremediation. 2005 ASTAR National Boot Camp for Maryland and Ohio ASTA Resource Judges and Guest Jurists, The Language of the Life Sciences at the Airlie Conference Center
  942. Hazen, Terry C. 2005. Integration of the Omics, Bioinformatics, Biogeochemistry in Groundwater Remediation. International Conference on Safe Water, Exploring Global Demands and Impacts of Natural Disasters, SAFEWATER
  943. Hazen, Terry C. 2005. Intimate Strangers: The Anatomy, Ecology & Engineering of Microorganisms. 2005 ASTAR National Boot Camp for Maryland and Ohio ASTA Resource Judges and Guest Jurists, The Language of the Life Sciences at the Airlie Conference Center
  944. Hazen, Terry C. 2005. Omics and Biogeochemistry: The New Frontier for Environmental Biotechnology (Lab to the Field and back). Department of Plant and Microbial Biology, University of California at Berkeley
  945. Hazen, Terry C. 2005. VIMSS linkage to NABIR FRC. Annual NABIR FRC Workshop
  946. Wyborski, D. L., C. B. Abulencia, B. Buchner, D. Burton, C. Chang, J. Garcia, Terry C. Hazen, K. Obokata, M. Podar, T. Torok, M. Keller. 2005. Analysis of Organisms Surviving in Highly Contaminated Environments. Annual NABIR FRC Workshop
  947. Shepler, C. G., S. C. Hull, T. E. Letain, Terry C. Hazen, H. Nitsche and S. B. Clark. 2005. The interaction of U(VI) with Bacillus sphaericus. 15th Annual V M Goldschmidt Conference 69:A233-A233.
  948. Hazen, Terry C.. 2005. Integration of the Ecogenomics, Proteomics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. University of California at Santa Barbara, Bren School of Engineering
  949. Hazen, Terry C.. 2005. Integration of the Omics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. Stanford University, Department of Civil and Environmental Engineering
  950. Hazen, Terry C.. 2005. Integration of the Omics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. 15th Annual AEHS West Coast Conference on Soils, Sediments, Water
  951. Hazen, Terry C., B. Faybishenko, E. Brodie, D. Joyner, S. Borglin, J. Hanlon, M. Conrad, T. Tokunaga, J. Wan, S. Hubbard, K. Williams, J. Peterson, M. Firestone, P. Long, D. Newcomer, A. Willett and S. Koenigsberg. 2005. Chromium bio-immobilization at the hanford 100H site: Geochemical response to slow release electron donor. 105th General Meeting of the American-Society-for-Microbiology 105:418-419.
  952. He, Z., Q. He, E. J. Alm, J. D. Wall, M. W. Fields, Terry C. Hazen, A. P. Arkin and J. Zhou. 2005. Adaptation of Desulfovibrio vulgaris to Elevated NaCl Conditions. Annual Meeting American Society for Microbiology
  953. Ringbauer, Jr., J. A., R. B. Payne, H. Zhili, H. Qiang, W. Liyou, Z. Jizhong, M. W. Fields, E. Alm, K. Huang, Terry C. Hazen, A. Arkin, J. D. Wall. 2005. Transposon Mutagenesis of Desulfovibrio vulgaris Yields Insight into Sodium and pH Stresses. Annual Meeting American Society for Microbiology
  954. Hazen, Terry C. 2005. Bioremediation technologies for petroleum. LuK Oil Company
  955. Hazen, Terry C.. 2005. Evaluation of sorption/bioremediation product application. Institute of Biology, Komi Republic
  956. Hazen, Terry C.. 2005. Evaluation of sorption/bioremediation product application. International Science & Technology Center
  957. Hazen, Terry C.. 2005. The Impact of Environmental Biotechnology in Bioremediation – The Dawning of the “Age of Omics”. Eighth International Conference on In Situ and On-Site Bioremediation
  958. Hazen, Terry C.. 2005. The Impact of Environmental Biotechnology in Bioremediation – The Dawning of the “Age of Omics”. LBNL Center for Environmental Biotechnology Summer Student Seminar
  959. Hazen, Terry C. 2005. In situ bioremediation and applicability to geobioreactors. Luca Technologies
  960. Hazen, T. C., B. Faybishenko, E. Brodie, D. Joyner, S. E. Borglin, J. Hanlon, M. Conrad, T. Tokunaga, J. M. Wan, S. Hubbard, K. Williams, J. Peterson, M. K. Firestone, G. L. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, A. Willett and S. Koenigsberg. 2005. Chromium Bio-Immobilization at the Hanford 100H Site: Geochemical Response to Slow Release Electron Donor. International Union of Microbiological Societies
  961. Hazen, Terry C. 2005. Bioremediation of Metals and Radionuclides at DOE Sites. Consensus Conference to Characterize Regulatory Concerns Regarding Bioremediation of Radionuclides and Heavy Metals in Mixed Wastes at Doe Sites
  962. Hubbard, S., J. Peterson, J. Chen, K. Williams, M. Conrad, B. Faybishenko, A. Willet, P. Long and Terry C. Hazen. 2005. Geophysical Monitoring of Cr(VI) bioreduction at the Hanford 100H Site. Annual Meeting of the American Geophysical Union
  963. Mukhopadhyay, A., Z. He, E. Alm, Q. He, B. Yen, K. Huang, E. Baidoo, W. Chen, S. Borglin, A. Redding, H. Y. Holman, J. Sun, D. Joyner, N. Katz, M. Keller, J. Zhou, A. P. Arkin, Terry C. Hazen, J. Wall and J. D. Keasling. 2005. The anatomy of salt stress in Desulfovibrio vulgaris hildenborough. 4th Annual World Congress of the Human-Proteome-Organisation (HUPO) 4:S384.
  964. Brodie, E. L.*, T. Z. DeSantis, J. P. Moberg, I. X. Zubieta, Y. M. Piceño, Terry C. Hazen, G. L. Andersen. 2005. A High Density Microarray for Rapid Profiling of 16S rDNA and 16S rRNA of Prokaryotic Communities. International Symposium Subsurface Microbiology
  965. Hazen, Terry C. 2005. Molecular Biology Characterization of Subsurface Microbial Communities. International Symposium Subsurface Microbiology
  966. Hazen, T. C., E. Brodie, F. Brockman, D. Moser, T. Gihring, D. Culley, L-H. Lin, T. Pray, G. Andersen, P. Richardson, L. Pratt and T. C. Onstott. 2005. Comparison of planktonic and biofilm microbial communities in million year old fissure water from a deep subsurface gold mine. International Symposium Subsurface Microbiology
  967. Hazen, Terry C.. 2005. Field-Integrated Studies of Long-Term Sustainability of Chromium Bioreduction at the Hanford 100H. DOE NABIR Annual Investigators Meeting
  968. Hazen, Terry C., B. Faybishenko, E. Brodie, D. Joyner, S. E. Borglin, J. Hanlon, M. Conrad, T. Tokunaga, J. M. Wan, S. Hubbard, K. Williams, J. Peterson, M. K. Firestone, G. L. Andersen, T. DeSantis, P. E. Long, D. R. Newcomer, A. Willett, S. Koenigsberg. 2005. Field-Integrated Studies of Long-Term Sustainability of Chromium Bioreduction at the Hanford 100H. DOE NABIR Annual Investigators Meeting
  969. Hazen, Terry C.. 2005. Integration of the Ecogenomics, Proteomics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. University of New Hampshire
  970. Hazen, Terry C., L. Krumholz. 2005. What changes in microbial community structure can be expected during and after biostimulation. DOE NABIR Annual Investigators Meeting
  971. He, Z., M. E. Clark, Q. He, L. Wu, J. D. Wall, Terry C. Hazen, J. Zhou, M. W. Fields. 2005. Field-Integrated Construction of Whole Genome Microarrays, Expression Analysis of Desulfovibrio vulgaris Cells under Metal-Reducing Conditions. DOE NABIR Annual Investigators Meeting
  972. Tokunaga, J. Wan, Terry C. Hazen, M. Firestone, E. Brodie, Z. Zheng, J. Larsen, D. Herman. 2005. Mesoscale Biotransformation of Uranium. DOE NABIR Annual Investigators Meeting
  973. Brockman, Fred, Duane Moser, Tom Gihring, David Culley, Eoin Brodie, Gary Andersen, Terry Hazen, Paul Richardson, Lisa Pratt and Tullis Onstott. 2005. Inferred bioenergetics of an uncultured bacterium common in fracture fluids ofSouth African deep mines. Biennial Meeting of the NASA-Astrobiology-Institute (NAI) 5:251-252.
  974. Alin, E. J., A. P. Arkin, M. W. Fields, Terry C. Hazen, J. D. Keasling, M. Keller, A. Mukhopadhyay, A. Singh, D. A. Stahl, D. K. Thompson, J. D. Wall and J. Zhou. 2005. A powerful integrated approach to the systems-level response of bacteria to environmental stress. 105th General Meeting of the American-Society-for-Microbiology 105:305.
  975. Barua, S., C. B. McAlvin, C. L. Hemme, S. E. Borglin, Y. Yang, D. K. Thompson, Terry C. Hazen and J. Zhou. 2005. Role of a LysR family transcriptional regulator in response to osmotic stress in Shewanella oneidensis MR1. 105th General Meeting of the American-Society-for-Microbiology 105:318.
  976. Borglin, S. E., Terry C. Hazen, J. Carlson, J. Wall and D. Joyner. 2005. Phenotypic microarray analysis of Desulfovibrio vulgaris. 105th General Meeting of the American-Society-for-Microbiology 105:510.
  977. Borglin, S. E., Terry C. Hazen, D. Joyner, R. Huang, N. Katz, E. Alm and A. Kazakov. 2005. Phospholipid fatty acid analysis as phenotypic indicators of common stress response pathways in Desulfovibrio vulgaris and Shewanella oneidensis. 105th General Meeting of the American-Society-for-Microbiology 105:410.
  978. Brodie, E., Terry C. Hazen, B. Faybishenko, D. Joyner, S. Borglin, M. Conrad, G. Andersen, T. DeSantis, J. Hanlon, P. Long, D. Newcomer, A. Willett and S. Koenigsberg. 2005. Chromium bio-immobilization at the Hanford 100H site: Comprehensive molecular analysis of microbial population dynamics. 105th General Meeting of the American-Society-for-Microbiology 105:420-421.
  979. Chhabra, S. R., Q. He, Z. He, S. Gaucher, E. Alm, M. Hadi, A. Arkin, T. Hazen, J. Zhou and A. K. Singh. 2005. Analysis of the heat shock response in Desulfovibrio vulgaris through global proteomics and transcriptomics studies. 105th General Meeting of the American-Society-for-Microbiology 105:300.
  980. Clark, M. E., Z. He, Q. He, L. Wu, J. D. Wall, Terry C. Hazen, J. Zhou and M. W. Fields. 2005. Whole-genome expression analysis of Desulfovibrio vulgaris cells throughout exponential phase into stationary phase growth in a defined medium. 105th General Meeting of the American-Society-for-Microbiology 105:361.
  981. Fields, M. W., T. Yan, X. Liu, C. E. Bagwell, S. L. Carroll, P. E. Jardine, D. B. Watson, C. S. Criddle, Terry C. Hazen and J. Zhou. 2005. Identification of different relationships between contaminated groundwater samples based upon extensive geochemical data or multiple gene sequences from microbial communities. 105th General Meeting of the American-Society-for-Microbiology 105:399.
  982. Geller, J. T., Terry C. Hazen, R. Huang, D. Joyner and S. E. Borglin. 2005. Characterization of Desulfovibrio vulgaris grown in extremophile turbidostat reactors. 105th General Meeting of the American-Society-for-Microbiology 105:389-390.
  983. He, Q., Z. He, K. H. Huang, E. J. Alm, A. P. Arkin, J. D. Wall, Terry C. Hazen, M. W. Fields and J. Zhou. 2005. Whole-genome transcriptional response of Desulfovibrio vulgaris hildenboroughto nitrite. 105th General Meeting of the American-Society-for-Microbiology 105:362.
  984. He, Q., Z. He, L. Wu, J. D. Wall, A. P. Arkin, Terry C. Hazen, D. A. Stahl, M. W. Fields and J. Zhou. 2005. Transcriptional gene expression analysis of the response to acetone in Desulfovibrio vulgaris using whole-genome oligonucleotide microarrays. 105th General Meeting of the American-Society-for-Microbiology 105:318.
  985. Holman, H. Y. N., S. E. Borglin, Terry C. Hazen, D. Joyner, R. Huang, N. Katz and E. Wozei. 2005. Spectral comparisons reveal general stress response strategies in Desulfovibrio vulgaris. 105th General Meeting of the American-Society-for-Microbiology 105:409.
  986. Holman, H. Y. N., L. R. Comolli, E. Wozei, Terry C. Hazen and K. H. Downing. 2005. Real-time observations of chemical and structural aspects of Desulfovibrio vulgaris and Caluobacter crescentis in atmospheric oxygen. 105th General Meeting of the American-Society-for-Microbiology 105:410.
  987. Huang, R., D. Joyner, S. E. Borglin, Terry C. Hazen and N. Katz. 2005. Large scale biomass production of obligate anaerobes for simultaneous transcriptomics, proteomics, metabolomics, and lipidomics analysis. 105th General Meeting of the American-Society-for-Microbiology 105:407.
  988. Katz, N., Terry C. Hazen, R. Huang, D. Joyner and S. E. Borglin. 2005. High throughput analysis of stress growth response in Shewanella oneidensis MR-1. 105th General Meeting of the American-Society-for-Microbiology 105:410.
  989. Stolyar, S. M., C. B. Walker, H. C. Yen, B. Zuniga, N. Pinel, H. Cough, Z. He, Q. He, J. Zhou, Terry C. Hazen, S. E. Borglin, J. D. Wall and D. A. Stahl. 2005. Genomic and physiological characterization of Desulfovibrio vulgaris strains isolated from a metal contaminated lake. 105th General Meeting of the American-Society-for-Microbiology 105:411.
  990. Wyborski, D. L., C. B. Abulencia, B. Buchner, D. Burton, C. Chang, J. Garcia, Terry C. Hazen, K. Obokata, M. Podar, T. Torok and M. Keller. 2005. Analysis of organisms surviving in highly contaminated environments. 105th General Meeting of the American-Society-for-Microbiology 105:494.
  991. Shepler, C. G., T. Oshiro, B. N. Bottenus, Terry C. Hazen, H. Nitsche and S. B. Clark. 2005. Effects of ionic strength on the transformation of U(VI) oxyhydroxides to U(VI) phosphate solids. American Chemical Society annual meeting 229:019-NUCL.
  992. Hazen, Terry C. 2004. Risk and Environmental Cleanup, some Global Issues. Workshop on Oral Bioavailability of Organic Compounds
  993. Hazen, Terry C. 2004. Biopile Case Studies for landfills and petroleum contaminated soils. On site remediation technologies: engineering of soil washing, land farming and biopile applications
  994. Hazen, Terry C. 2004. Ecogenomics and Phenomics the New Frontier in Bioremediation of Toxic Waste Sites. LBNL Life Sciences & Genomics Seminar Series
  995. Hazen, Terry C.. 2004. Integration of Ecogenomics, Phenomics, Transcriptomics, Proteomics, Lipidomics, Metabolomics, Fluxomics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. NIH/EPA Annual Superfund Research Meeting
  996. Brodie, Eoin, Terry C. Hazen, Jiamin M. Wan, Tetsu K. Tokunaga, Joern Larsen, Keith Olsen, Dominique Joyner, Gary L. Andersen and Mary K. Firestone. 2004. Biogeography of microbial communities associated with diffusion limited reduction of U(VI) and NO3 as co-contaminants in natural sediments and soils. 104th General Meeting of the American-Society-for-Microbiology 104:460.
  997. Borglin, Sharon, Terry Hazen, Dominique Joyner and Rick Huang. 2004. Effects of environmental stressors on signature lipid biomarkers in Desulfovibrio vulgaris. 104th General Meeting of the American-Society-for-Microbiology 104:424.
  998. Hazen, Terry C., Dominique Joyner, Sharon Borglin, Boris Faybishenko, Mark Conrad, Carlos Rios-Velazques, Josue Malave, Ramon E. Martinez, Gary L. Andersen, Mary Firestone, Eoin Brodie, Philip E. Long, Anna Willett and Stephen Koenigsberg. 2004. Functional microbial changes during lactate and HRC-Stimulated bioreduction of Cr(VI) in hanford 100H sediments. 104th General Meeting of the American-Society-for-Microbiology 104:408.
  999. He, Qiang, Zhili He, Liyou Wu, Judy D. Wall, Terry Hazen, Matthew W. Fields and Jizhong Zhou. 2004. Global transcriptional analysis of Desulfovibrio vulgaris in response to nitrite stress using whole-genome oligonucleotide microarrays. 104th General Meeting of the American-Society-for-Microbiology 104:369.
  1000. He, Zhili, Quiang He, Liyou Wu, Judy D. Wall, Terry Hazen, Matthew W. Fields and Jizhong Zhou. 2004. Transcriptional Analysis of Responses of Desulfovibrio vulgaris to NaCl Stress Using Whole-Genome Oligonucleotide Microarrays. American Society for Microbiology 104th Meeting
  1001. Holman, Hoi-Ying N., Zhang Lin, Terry C. Hazen and Dominque Joyner. 2004. A real-time investigation of Desulfovibrio vulgaris response to oxygen stress. 104th General Meeting of the American-Society-for-Microbiology 104:338-339.
  1002. Joyner, D. C., S. E. Borglin, R. Huang, Terry C. Hazen, J. D. Wall, H. Yen and S. M. Stolyar. 2004. Chemically defined medium for Desulfovibrio vulgaris stress studies and biomass production. 104th General Meeting of the American-Society-for-Microbiology 104:595.
  1003. Malave-Orengo, J., S. Borglin, C. Rios-Velazquez and T. Hazen. 2004. Study of microbial community structure using phospholipid fatty acid analysis (PLEA) at a chromium contaminated site. 104th General Meeting of the American-Society-for-Microbiology 104:530.
  1004. Martinez-Santiago, R. E., C. Rios-Velazquez, G. L. Andersen and Terry C. Hazen. 2004. Molecular analysis of the microbial community structure in chromium contaminated sites before and after in situ bioreduction stimulation by lactate injection. 104th General Meeting of the American-Society-for-Microbiology 104:532.
  1005. Hazen, Terry C., D. Joyner, S. Borglin, B. Faybishenko, J. Wan, T. Tokunaga, S. Hubbard, K. Williams, M. Firestone, E. Brodie, P. E. Long, E. Willett, S. Koenigsberg. 2004. Field Investigations of HRC®-Stimulated Bioreduction of Cr(VI) at Hanford 100H. International Conference on Chlorinated and Recalcitrant Compound Remediation
  1006. Hazen, Terry C., D. Joyner, S. Borglin, B. Faybishenko, M. Conrad, C. Rios-Velazquez, J. Malave-Orengo, R. Martinez-Santiago, M. Firestone, E. Brodie, P. E. Long, E. Willett, S. Koenigsberg. 2004. Functional Microbial Changes during Lactate and HRC-Stimulated Bioreduction of Cr(VI) in Hanford 100H Sediments. International Conference on Chlorinated and Recalcitrant Compound Remediation
  1007. Hazen, Terry C. 2004. Biotechnology to Restore Fouled Environments. How may natural and genetically engineered organisms be used?. Global Biotechnology Forum, United Nations Industrial Development Organization
  1008. Hazen, Terry C., D. Joyner, S. Borglin, B. Faybishenko, J. Wan, T. Tokunaga, S. Hubbard, K. Williams, M. Conrad, C. Rios-Velazquez, J. Malave-Orengo, R. Martinez-Santiago, M. Firestone, E. Brodie, P. E. Long, E. Willett, S. Koenigsberg. 2004. Field Investigations of HRC®-Stimulated Bioreduction of Cr(VI) at Hanford 100H. NABIR Program Annual PI Meeting
  1009. Hazen, Terry C. 2004. Genes and Bacteria Why are bacteria important and what do they do? As life forms, how are they structured?. Global Biotechnology Forum, United Nations Industrial Development Organization
  1010. Hazen, Terry C. 2004. An Overview of Environmental Biotechnology. AEHS West Annual Symposium on Contaminated Soils
  1011. Hazen, Terry C. 2004. Plenary Case Scenario and Discussion Chile Pork Growers v. Alpha Food Laboratories* - a biotechnology liability case. Global Biotechnology Forum, United Nations Industrial Development Organization
  1012. Hazen, Terry C. 2004. Plenary Case Scenario and Discussion People v Soderstein, a food bioterrorism case. Global Biotechnology Forum, United Nations Industrial Development Organization
  1013. Hazen, Terry C. 2004. Round Table on Future of Biotechnologies. Global Biotechnology Forum, United Nations Industrial Development Organization
  1014. Hazen, Terry C. 2004. A Scientific Briefing on Bioremediation. Global Biotechnology Forum, United Nations Industrial Development Organization
  1015. Hazen, Terry C. 2004. VIMSS: Rapid Deduction of Stress Response Pathways in Metal and Radionuclide Reducing Bacteria. NABIR Program Annual PI Meeting
  1016. Wan, J., T. K. Tokunaga, Terry C. Hazen, M. K. Firestone, Z. Zhen, E. Brodie, J. Larsen and D. Herman. 2004. Mesoscale Coupled Transport and Biogeochemical Effects on Reduction of U(VI) and NO3– as Co-contaminants in Natural Sediments and Soils. NABIR Program Annual PI Meeting
  1017. Hazen, Terry C.. 2004. Integration of Ecogenomics, Phenomics, Transcriptomics, Proteomics, Lipidomics, Metabolomics, Fluxomics, Bioinformatics, Biogeochemistry: The New Frontier for Environmental Biotechnology. Flinders University
  1018. Hazen, Terry C. 2004. Latest Advances in Environmental Biotechnology. BIO2004
  1019. Hazen, Terry C.. 2004. Recent Advances in Bioremediation. Seventh Biennial Symposium of the International Society of Environmental Biotechnology
  1020. Hazen, Terry C.. 2004. Recent Advances in Bioremediation. CEB Summer Student Lectures
  1021. Hazen, Terry C., A. P. Arkin. 2004. Workshop on Stress in Metal-Reducing Bacteria: Ecology, Functional Genomics, Bioinformatics. Society for Industrial Microbiology Annual Meeting
  1022. Hazen, Terry C., H.-Y. Holman, S. E. Borglin, D. Joyner, R. Huang, Z. Lin, D. Stahl, S. M. Stolyar, M. Fields, D. Thompson, J. Zhou, J. D. Wall, H.-C. Yen, M. Keller5. 2004. VIMSS Applied Environmental Microbiology Core Research on Stress Response Pathways in Metal-Reducers. DOE Genomes to Life Workshop
  1023. Brodie, Eoin, Joern Larsen, Terry C. Hazen, Jiamin M. Wan, Tetsu K. Tokunaga, Dominique Joyner, Gary L. Andersen, Todd DeSantis, Paul Richardson and Mary Firestone. 2004. High density oligonucleotide array monitoring of bacterial community dynamics during carbon stimulated uranium bioremediation. 10th Annual International Symposium on Microbial Ecology ISME-10 Microbial Planet: Sub-Surface to Space
  1024. Fields, M. W., T. Yan, X Liu, C. E. Bagwell, S. L. Carroll, P. M. Jardine, C. S. Criddle, Terry C. Hazen and J. Zhou. 2004. Polyphasic characterization of microbial communities under the stressful conditions of nitrate, heavy metals, radionuclides, and acidic pH in contaminated groundwater. 10th Annual International Symposium on Microbial Ecology ISME-10 Microbial Planet: Sub-Surface to Space
  1025. Hazen, Terry C., Dominique Joyner, Sharon Borglin, Boris Faybishenko, Mark Conrad, Carlos Rios-Valezques, Josue Malave, Ramon E. Martinez, Gary L. Andersen, Mary Firestone, Eoin Brodie, Philip E. Long, Anna Willet and Stephen Koenigsberg. 2004. Functional Microbial Changes During Lactate and HRC-Stimulated Bioreduction of Cr(VI) in Hanford 100H Sediments. 10th Annual International Symposium on Microbial Ecology ISME-10 Microbial Planet: Sub-Surface to Space
  1026. Holman, Hoi-Ying N., Zhang Lin, Terry C. Hazen and Dominique Joyner. 2004. A real-time investigation of Desulfovibrio vulgaris response to oxygen stress. 10th Annual International Symposium on Microbial Ecology ISME-10 Microbial Planet: Sub-Surface to Space
  1027. Hazen, Terry C., A. P. Arkin. 2004. Roundtable on Rapid Deduction of Stress Response Pathways in Metal-Reducing Bacteria: Ecology, Functional Genomics, Bioinformatics. International Symposium on Microbial Ecology -
  1028. Tokunaga, T. K., J. Wan, J. Pena, E. Brodie, M. K. Firestone, Terry C. Hazen, S. R. Sutton. 2004. Uranium bioreduction dynamics in low permeability sediments. International Symposium on Microbial Ecology -
  1029. Wan, J. M., T. K. Tokunaga, J. Larson, Z. Zheng, Terry C. Hazen, D. Herman, E. Brodie and M. K. Firestone. 2004. Oxidation of bioreduced Uranium under reducing conditions. International Symposium on Microbial Ecology -
  1030. Faybishenko, B., Terry C. Hazen, S. Hubbard, M. Conrad and J. Christensen. 2004. Strategy and Techniques for Monitoring Bioremediation of Metals in the Vadose Zone and Groundwater at Contaminated Sites. Workshop Long-Term Performance Monitoring of Metals and Radionuclides in the Subsurface: Strategies, Tools and Case Studies (DOE/USGS)
  1031. Hazen, Terry C. 2004. From Sequence to Integration of Genomics, Proteomics, Metabolomics, Bioinformatics and Ecology: The New Frontier of Science. University of Puerto Rico
  1032. Hazen, Terry C. 2004. Virtual Institute for Microbial Stress and Survival: Rapid Deduction of Stress Response Pathways. University of Puerto Rico
  1033. Borglin, Sharon, Terry C. Hazen, Dominique Joyner and Rick Huang. 2004. Effects of environmental stressors on signature lipid biomarkers in Desulfovibrio vulgaris. 10th Annual International Symposium on Microbial Ecology ISME-10 Microbial Planet: Sub-Surface to Space
  1034. Hazen, Terry C. 2003. Bioremediation and Natural Attenuation: Stewardship through Science. INRA Subsurface Science Conference
  1035. Hazen, Terry C.. 2003. Bioremediation: using biotechnology to cleanup toxic waste sites. Genetics and the International Court
  1036. Hazen, Terry C. 2003. An Overview of Environmental Biotechnology. AEHS Annual Symposium on Contaminated Soils
  1037. Hazen, Terry C.. 2003. Bioremediation: using biotechnology to cleanup toxic waste sites. Genetics and the Court
  1038. Hazen, Terry C.. 2003. From Research to Practical Applications: Gaseous Nutrient Injection for in situ bioremediation of chlorinated solvents. American Society for Microbiology annual meeting abstract
    For the past 10 years, we have been studying, demonstrating, and deploying several gaseous nutrient injection techniques for in situ bioremediation of groundwater and soil contaminated with a variety of hazardous organics. Fundamental research that provided a sound scientific basis for the strategies that were deployed in the field was essential for successful demonstrations and subsequent deployments. By doing a series of treatability tests for several sites, followed by mesoscale (column) studies we were able to develop a convincing set of data that could be presented to the stakeholders, endusers, and regulators. This allowed us to proceed with pilot-scale demonstrations and then full-scale demonstrations. It was critical to the success of the project that the technology developer and researcher stay involved with the initial permitting process and the subsequent field demonstrations. Subsequent commercialization and wide spread use of these technologies hinged on the full-scale demonstrations that convinced site owners, endusers, and regulators that these technologies were appropriate for the sites being considered. Examples of each step in the process will be presented for chlorinated solvent bioremediation. Case studies for full-scale demonstrations and deployment will also be discussed. Spin-off applications for creosote, PAHs, and petroleum sludge will also be presented briefly. The principle sites presented for this discussion are in CA, FL, VA, GA, SC, MI, and Poland.
  1039. Hazen, Terry C. 2003. Marine Pollution. Under Pressure Dive Club
  1040. Hazen, Terry C., B. Faybishenko, J. Wan, T. Tokunaga, S. Hubbard, M. Firestone, P. E. Long, S. Koenigsberg. 2003. NABIR-EM Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Cr(III) at Hanford 100H. NABIR Program Annual PI Meeting
  1041. Letain, T., C. Gillaspie, M. Doublas, S. B. Clark, Terry C. Hazen and H. Nitsche. 2003. The Role of Biogeochemical Dynamics in the Alteration of U Solid Phases under Oxic Conditions. NABIR Program Annual PI Meeting
  1042. Padilla, E. and Terry C. Hazen. 2003. Molecular analysis of the Microbial Community Structure in Aerobic and Anaerobic Landfill Bioreactors. ACS Junior Technical Meeting PRISM
  1043. Wan, J., T. K. Tokunaga, Terry C. Hazen, M. K. Firestone, E. Brodie, Z. Zheng, J. Larsen and J. Pena. 2003. Mesoscale Coupled Transport and Biogeochemical Effects on Reduction of U(VI) and NO3– as Co-contaminants in Natural Sediments and Soils. NABIR Program Annual PI Meeting
  1044. Hazen, Terry C., S. E. Borglin and C. M. Oldenburg. 2003. Aerobic Landfill Bioremediation. In Situ and On Site Bioremediation: The Sixth International Symposium abstract
    Municipal solid waste (MSW) landfills are rapidly becoming a drain on the resources of local municipalities as the requirements for stabilization and containment become increasingly stringent. EPA guidelines for landfills include complete containment of the MSW mass with liners, covers, leachate collection/treatment systems and landfill gas collection/treatment systems. Consequently landfill costs have more than doubled in the last 15 years. To compare the use of aerobic and anaerobic management strategies and their potential advantages, three 200-liter tanks filled with fresh waste materials were used to provide the following conditions: (a) aerobic (air injection with leachate recirculation), (b) anaerobic (leachate recirculation), and (c) a dry-tomb anaerobic landfill (no air injection, no water addition and no leachate recirculation). Leachate from the aerobic tank had significantly lower concentrations of all potential contaminants, both organic and metal. Indeed the overall concentrations in the aerobic tank leachate were an order of magnitude lower after only a few weeks of operation. Respiration tests on the aerobic tank showed a steady decrease in oxygen consumption rates from 1.3 mol/day at 20 days to 0.1 mol/day at 300 days. Over the test period, the aerobic tank settled 35%, the anaerobic tank 21.7% and the dry-tomb tank 7.5%. The aerobic tank produced negligible odor compared to the anaerobic tanks. These results suggest that aerobic management of MSW landfills could increase the rate of stabilization, produce less potent greenhouse gases, eliminate the need for leachate and air emissions treatment systems, reduce odor, and reduce the need for extensive containment strategies.
  1045. Brodie, E. I., M. K. Firestone, J. Pena, J. Larsen, Terry C. Hazen, T. Tokunaga and J. Wan. 2003. Bioreduction of Co-Contaminating Uranium And Nitrate: Carbon Release Rate Effects. In Situ and On Site Bioremediation: The Sixth International Symposium abstract
    Actinide contamination of subsurface soils near former waste disposal ponds at the Field Research Center (FRC) in Oak Ridge Tennessee is a primary topic of DOE funded NABIR investigations. Uranium (238U) is found at concentrations of up to 50 mg/L in groundwater samples, often co-occurring with high concentrations of nitrate (NO3-). Bioreduction of U(VI) to the less soluble U(IV) species has been shown to be an effective approach in reducing migratory U in groundwater, however nitrate removal is a prerequisite for successful U(VI) reduction. Sediment and groundwater samples were obtained from Area 3 adjacent to these former waste disposal ponds. Nitrate concentrations in groundwater from this area were > 800 mM. Initial microbial biomass in these sediments was extremely low, with acridine orange counts in the order of 3 x 104 cells/g. In this series of laboratory simulations, both U(VI) and NO3- reduction in soil solution were monitored with reference to increased carbon substrate availability and rate of carbon release. The carbon substrate utilized is a commercially available polylactate compound (Regenesis HRC“) used previously in field applications for hydrocarbon bioremediation. Bacterial community activity was followed using headspace CO2 and N2O with nucleic acid-based analyses being used to determine changes in bacterial community structure.
  1046. Hazen, Terry C. 2003. Recent Advances in Environmental Biotechnology. BIO
  1047. Hazen, Terry C., A. J. Tien, A. Worsztynowicz, D. J. Altman, K. Ulfig and T. Manko. 2003. Biopiles for remediation of petroleum-contaminated soils: A Polish case study - Polish Refinery biopile. NATO Advanced Research Workshop on the Utilization of Bioremediation to Reduce Soil Contamination 19:229-246. abstract
    The US Department of Energy and the Institute for Ecology of Industrial Areas of Poland demonstrated bioremediation techniques for the cleanup of acidic petroleum sludge impacted soils at an oil refinery in southern Poland. The waste was composed of high-molar mass paraffinic and polynuclear aromatic hydrocarbons. Benzo(a)pyrene and BTEX compounds were identified as the contaminants of concern. Approximately 3300 m3 of contaminated soil (TPH ~30 000 ppm) was targetted 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 tonnes) of petroleum hydrocarbons were biodegraded. Despite the fact that the material treated was highly weathered and very acidic, biodegradation rates of 121 mg per kg soil per day in the actively aerated side (82 mg per kg soil per 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.
  1048. Hazen, Terry C. 2003. Bioremediation Overview. CEB Summer Student Lectures
  1049. Arkin, A., Terry C. Hazen, J. Keasling, A. Singh, F. Olkin, I. Dubchak, H-Y. Holman, D. Stahl, M. Fields, D. Thompson, J. Zhou, J. Wall and M. Keller. 2003. The Virtual Institute of Microbial Stress and Survival: Rapid Deduction of Stress Response Pathways in Metal/Radionuclide Reducing Bacteria: Overview. DOE Genomes to Life Workshop
  1050. Hazen, Terry C., H-Y. Holman, D. Stahl, M. Fields, D. Thompson, J. Zhou, J. Wall, M. Keller. 2003. The Virtual Institute of Microbial Stress and Survival: Rapid Deduction of Stress Response Pathways in Metal/Radionuclide Reducing Bacteria: Applied Environmental Microbiology Core. DOE Genomes to Life Workshop
  1051. Hazen, Terry C. 2002. Direct characterization of microorganisms on mineral surfaces. International Symposium on Subsurface Microbiology
  1052. Hazen, Terry C., T. K. Tokunaga, J. Wan, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti, W. Rao. 2002. Intra-aggregate biogeochemical dynamics of chromium contamination and in-situ bioremediation. International Symposium on Subsurface Microbiology
  1053. Hazen, Terry C., T. K. Tokunaga, J. Wan, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti and W. Rao. 2002. Intra-aggregate biogeochemical dynamics of chromium contamination and in-situ bioremediation. International Symposium on Subsurface Microbiology abstract
    Transport of redox-sensitive contaminants through the vadose zone is typically complex because of the broad range of transport times and reaction rates encountered over short distances. Multi-region flow and transport models are often used to describe fast advective transport through fractures and macropores, and slower diffusion-dominated transport within sediment blocks and soil aggregates. However, transport and reactions occurring within the diffusion-controlled domains that often make up most of the subsurface are commonly only inferred or assumed. Direct measurements within soil aggregates and sediment blocks are needed to understand biogeochemical processes. This is demonstrated through laboratory studies of chromium contamination of soil aggregates, and subsequent in-situ remediation (reduction of Cr(VI) to Cr(III)) by organic carbon infusion. Spatially resolved determination of Cr concentrations and oxidation states using micro-XANES, and spatially-resolved microbial community analyses were done on synthetic and natural soil aggregates. During the diffusion-limited contamination process, more Cr(VI) was transported, but to shorter distances, in more microbially active aggregates. Sharply terminated diffusion fronts, within 2 to 10 mm of the aggregate surface, result from increasing Cr(VI) reduction rates with depth. Infusion of organic carbon into previous Cr(VI)-contaminated aggregates resulted in more rapid reduction to Cr(III) with higher organic carbon concentrations, and lower reduction rates in more highly contaminated sediments. These results show that intra-aggregate Cr dynamics are strongly diffusion-limited in more microbially active systems, and that bulk soil chemical and microbial characterization can obscure relevant biogeochemical processes.
  1054. Hazen, Terry C., T. K. Tokunaga, J. Wan, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti and W. Rao. 2002. Intra-aggregate biogeochemical dynamics of chromium contamination and in-situ bioremediation. NABIR FRC Workshop
  1055. Letain, T., C. Gillaspie, M. Douglas, S. B. Clark, Terry C. Hazen and H. Nitsche. 2002. The Role of Biogeochemical Dynamics in the Alteration of U Solid Phases under Oxic Conditions. International Symposium on Subsurface Microbiology
  1056. Borglin, S. E., Terry C. Hazen, C. M. Oldenburg and P. Zawislanski. 2002. Biotreatment of Municipal Solid Waste in Aerobic and Anaerobic Laboratory Bioreactors. Second International Landfill Research Symposium
  1057. Borglin, S. E., C. M. Oldenburg and Terry C. Hazen. 2002. Smart Storage: Stabilization of Stored and Landfilled Waste using Aerobic and Anaerobic Biotreatment Technology. SERDP Partners Symposium
  1058. Hazen, Terry C. 2002. Bioremediation field applications. First Annual EICE On-Line Conference on Environmental Biotechnology
  1059. Hazen, Terry C.. 2002. In Situ Bioremediation: From Research to Practical Applications. 2002 Subsurface Science Symposium Inland Northwest Research Alliance, Inc abstract
    For the past 10 years, we have been studying, demonstrating, and deploying several gaseous nutrient injection techniques for in situ bioremediation of groundwater and soil contaminated with a variety of hazardous organics. Fundamental research that provided a sound scientific basis for the strategies that were deployed in the field was essential for successful demonstrations and subsequent deployments. By doing a series of treatability tests for several sites, followed by mesoscale (column) studies we were able to develop a convincing set of data that could be presented to the stakeholders, endusers, and regulators. This allowed us to proceed with pilot-scale demonstrations and then full-scale demonstrations. It was critical to the success of the project that the technology developer and researcher stay involved with the initial permitting process and the subsequent field demonstrations. Subsequent commercialization and wide spread use of these technologies hinged on the full-scale demonstrations that convinced site owners, endusers, and regulators that these technologies were appropriate for the sites being considered. Examples of each step in the process will be presented for chlorinated solvent bioremediation. Case studies for full-scale demonstrations and deployment will also be discussed. Spin-off applications for creosote, PAHs, and petroleum sludge will also be presented briefly. The principle sites presented for this discussion are in CA, FL, VA, GA, SC, MI, and Poland.
  1060. Tokunaga, T.K., J. Wan, T.C. Hazen, M.K. Firestone, E. Schwartz, S.R. Sutton and A. Lanzirotti. 2002. Chromium redox transformations in diffusion-limited domains of soils: Linking micro- and macro-scale processes. Soil Science Society of America Annual Meeting
  1061. Hazen, Terry C.. 2002. Bioremediation: the hope and the hype. ENTEC West ’02
  1062. Hazen, Terry C. 2002. Bioremediation: using biotechnology to cleanup toxic waste sites. Genetics and the Court
  1063. Hazen, Terry C. 2002. Microbial Ecology of Diffusion Limited Environments: Scale Implications for Biogeochemistry and Bioremediation. Georgia Institute of Technology, School of Biology
  1064. Hazen, Terry C. and W. Stringfellow. 2002. Critical Parameters for MNA of Chlorinated Solvents. DOE annual Technology Information Exchange abstract
    Project Type: Environmental Restoration Date: November 1, 2002 Document Type: Abstracts Functional Area: Remediation Operations Office: Oakland Summary Tetrachloroethylene (PCE) and trichloroethylene (TCE) were once thought to be recalcitrant in the environment; however, we now know that there are a number of pathways both abiotic and biotic that can completely degrade both of these contaminants in the subsurface. Biodegradation of PCE and TCE is common in nearly all types of environments and can occur in many environments at rates sufficient to satisfy monitored natural attenuation goals. The pathways are quite different, depending on whether the groundwater or soil has become anoxic or not. Anaerobic pathways of PCE and TCE can result in the complete dechlorination or the accumulation of more toxic daughter products like vinyl chloride. Under anaerobic environments, dichloroethylene (DCE) and vinyl chloride (VC) are biodegraded more slowly then PCE or TCE and under some conditions may not be degraded at all. On the other hand, aerobically PCE cannot be biodegraded at all and VC and DCE are degraded very quickly, while TCE is degraded more slowly. Critical to understanding the natural attenuation capacity of any environment is the presence of oxygen, pH, and the redox potential. These parameters will indicate what types of biodegradation are possible. In addition, the presence of total organic carbon and other limiting nutrients will also suggest if sustainable natural attenuation is possible. Measurements of changes in daughter product inventories (DCE & VC) will also be critical as part of a good monitored natural attenuation strategy. As always, these measurements must be balanced with a thorough understanding of the geology and hydrology of the site, including the source term and all potential risk receptors. Examples from several sites will be presented. Lessons Learned Contact: Terry C. Hazen, Lawrence Berkeley National Laboratory, Center for Environmental Biotechnology, University of California, MS 70A-3317, One Cyclotron Road, Berkeley, CA 94720; fax (510) 486-7152, Phone (510) 486-6223; E-mail tchazen@lbl.gov
  1065. Hazen, Terry C. 2002. Bioremediation of a Polish Oil Refinery sludge Lagoon. Glen T. Seaborg Center Seminar
  1066. Hazen, Terry C., T. Torok. 2002. “What do we do first?!!!” Detecting Biothreat Agents, The Latest Protocols and Procedures. California Conference of Local Health Officers, Health Officers Association of California, California Department of Health Services
  1067. Hazen, Terry C. and E. Majer. 2002. Communicating with Program Managers. Earth Sciences Division Workshop, LBNL
  1068. Hazen, Terry C. and T. Torok. 2002. What do we do first?!!! Detecting Biothreat Agents, The Latest Protocols and Procedures. California Conference of Local Health Officers, Health Officers Association of California, California Department of Health Services
  1069. Arkin, A. P. and Terry C. Hazen. 2002. LBNL Genomes to Life Proposal. DOE Office of Biological and Environmental Research Program Managers
  1070. Letain, T., C. Gillaspie, M. Douglas, S. B. Clark, Terry C. Hazen and H. Nitsche. 2002. The Role of Biogeochemical Dynamics in the Alteration of U Solid Phases under Oxic Conditions. NABIR Program Annual PI Meeting
  1071. Wan, J., T. K. Tokunaga, Terry C. Hazen, M. K. Firestone, E. Schwartz, K. R. Olson, S. R. Sutton, M. Newville, A. Lanzirotti and W. Rao. 2002. Bridging the Batch-Field Gap: Chromium Biogeochemistry in Diffusion-Limited Domains. NABIR Program Annual PI Meeting abstract
    Transport of redox-sensitive contaminants through the vadose zone is typically complex because of the broad range of transport times and reaction rates encountered over short distances. Multi-region flow and transport models are often used to describe fast advective transport through fractures and macropores, and slower diffusion-dominated transport within sediment blocks and soil aggregates. However, transport and reactions occurring within the diffusion-controlled domains that often make up most of the subsurface are commonly only inferred or assumed. Direct measurements within soil aggregates and sediment blocks are needed to understand biogeochemical processes. This is demonstrated through laboratory studies of chromium contamination of soil aggregates, and subsequent in-situ remediation (reduction of Cr(VI) to Cr(III)) by organic carbon infusion. Spatially resolved determination of Cr concentrations and oxidation states using micro-XANES, and spatially-resolved microbial community analyses were done on synthetic and natural soil aggregates. During the diffusion-limited contamination process, more Cr(VI) was transported, but to shorter distances, in more microbially active aggregates. Sharply terminated diffusion fronts, within 2 to 10 mm of the aggregate surface, result from increasing Cr(VI) reduction rates with depth. Infusion of organic carbon into previous Cr(VI)-contaminated aggregates resulted in more rapid reduction to Cr(III) with higher organic carbon concentrations, and lower reduction rates in more highly contaminated sediments. These results show that intra-aggregate Cr dynamics are strongly diffusion-limited in more microbially active systems, and that bulk soil chemical and microbial characterization can obscure relevant biogeochemical processes.
  1072. Hazen, Terry C. and T. Torok. 2002. What do we do first?!!! Detecting Biothreat Agents, The Latest Protocols and Procedures. Berkeley Fire Department
  1073. Hazen, Terry C., T. K. Tokunaga, J. Wan, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti and W. Rao. 2002. Intra-aggregate biogeochemical dynamics of chromium contamination and in-situ bioremediation. Bioremediation and Biodegradation: Current Advances in Reducing Toxicity, Exposure and Environmental Consequences
  1074. Arkin, A. P. and Terry C. Hazen. 2002. Genomes to Life Proposal on Virtual Institute for Microbial Stress and Survival. LBNL DOE Office of Science On-Site Review
  1075. Hazen, Terry C., T. K. Tokunaga, J. Wan, E. Schwartz, M. K. Firestone, S. R. Sutton, M. Newville, K. R. Olson, A. Lanzirotti, W. Rao. 2002. Intra-aggregate biogeochemical dynamics of chromium contamination and in-situ bioremediation. Bioremediation and Biodegradation: Current Advances in Reducing Toxicity, Exposure and Environmental Consequences
  1076. Hazen, Terry C., T. Torok. 2002. “What do we do first?!!!” Detecting Biothreat Agents, The Latest Protocols and Procedures. Berkeley Fire Department
  1077. Hazen, Terry C.. 2002. Bioremediation: the hope and the hype. LBNL Center for Biotechnology Summer Student Seminar Series
  1078. Oldenburg, C. M., S. E. Borglin, Terry C. Hazen and P. T. Zawislanski. 2002. Process Modeling of Flow, Transport, and Biodegradation in Landfill Bioreactors. Earth Sciences Division, LBNL Directors Review
  1079. Borglin, S. E., Terry C. Hazen and C. M. Oldenburg. 2002. Comparison of Leachate Quality from Aerobic and Anaerobic Municipal Solid Waste Bioreactors. American Geophysical Union Annual Meeting abstract
    Municipal solid waste landfills are becoming a drain on the resources of local municipalities as the requirements for stabilization and containment become increasingly stringent. Current regulations limit the moisture in the landfill to minimize leachate production and lower the potential for release of leachate to the environment. Recent research has shown that addition and recycling of moisture in the waste optimizes the biodegradation of stabilization and also provides a means for leachate treatment. This study compares the leachate produced from aerobic and anaerobic laboratory bioreactors, and leachate collected from a full-scale anaerobic bioreactor. The laboratory reactors consisted of 200-liter tanks filled with fresh waste materials with the following conditions: (a) aerobic (air injection with leachate recirculation), (b) anaerobic (leachate recirculation). The leachate from the reactors was monitored for metals, nutrients, organic carbon, and microbiological activity for up to 500 days. Leachate from the aerobic tank had significantly lower concentrations of all potential contaminants, both organic and metal, after only a few weeks of operation. Metals leaching was low throughout the test period for the aerobic tanks, and decreased over time for the anaerobic tanks. Organic carbon as measured by BOD, COD, TOC, and COD were an order of magnitude higher in the leachate from the anaerobic system. Microbiological assessment by lipid analysis, enzyme activity assays, and cell counts showed high biomass and diversity in both the aerobic and anaerobic bioreactors, with higher activity in the anaerobic leachate. Results from the full-scale anaerobic bioreactor were not significantly different from those of the laboratory anaerobic bioreactor. The reduction in noxious odors was a significant advantage of the aerobic system. These results suggest that aerobic management of landfills could reduce or eliminate the need for leachate treatment systems, reduce odor, and reduce the need for extensive containment strategies. This work was supported by Laboratory Directed Research and Development Funds at Lawrence Berkeley National Laboratory under Department of Energy Contract No. DE-AC03-76SF00098.
  1080. Brodie, E. L., M. K. Firestone, J. Pena, J. Larsen, Z. Zheng, Terry C. Hazen, T. K. Tokunaga and J. Wan. 2002. Bioreduction of Co-Contaminating Uranium and Nitrate: Effects of Carbon Release Rate. American Geophysical Union Annual Meeting
  1081. Hazen, Terry C.. 2002. Ecology of Pathogens in the Environment Why do good bugs go bad?. LBNL Center for Environmental Biotechnology Summer Student Seminar Series
  1082. Hazen, Terry C.. 2002. Ecology of Pathogens in the Environment “Why do good bugs go bad?”. University of California at Davis, Annual Open House, Division of Biological Sciences
  1083. Hazen, Terry C.. 2002. Ecology of Pathogens in the Environment Why do good bugs go bad?. LBNL DC Office Seminar Series
  1084. Hazen, Terry C.. 2002. Ecology of Pathogens in the Environment Why do good bugs go bad?. University of California at Davis, Annual Open House, Division of Biological Sciences
  1085. Letain, T. E., R. J. Silva, R. Knopp, Terry C. Hazen, W. T. String-fellow and H. Nitsche. 2002. Defining the interactions between microbial cell surfaces and uranium (VI) in aerobic conditions. 102nd General Meeting of the American Society for Microbiology 102:411. abstract
    To assist the U.S. Department of Energy (DOE) with long-term stewardship issues associated with bioremediation of uranium (U), the overall goal of this work is to define the mechanisms by which microorganisms facilitate the formation of U(VI) solid phases. Under anaerobic conditions, microbial reduction of U(VI) to U(IV) can potentially decrease groundwater U contamination by lowering solubility and by slowing migration through the soil. However, such biological alteration must be considered temporary unless long-term anaerobicity can be maintained. When aerobic conditions return, the more soluble, higher oxidation state of uranium, U(VI), will again be thermodynamically favored. Of the U(VI) solids, U(VI)-phosphates are of interest because of their relatively low solubilities. Microorganisms present in soils may play a role in the formation of U(VI)-phosphate solid phases. We are investigating the role of some individual bacterial strains (Bacillus sphaericus and Shewanella putrefaciens) commonly found in soils as well as microbial consortia isolated from the NABIR Field Research Center at Oak Ridge National Laboratory on U solid phase formation. Data will be presented on the relative abilities of these bacteria to sorb U over a wide pH range, with special attention to the sorption behavior at low concentrations of U, such as would be commonly found in the environment. Cell surface functional groups responsible for U interaction are identified using TRLFS (Time-Resolved Laser-induced Fluorescence Spectroscopy). Bacteria are believed to influence actinide geochemistry through various mechanisms which are a part of the dynamic life cycle that defines the biogeochemical cycle of U. This information can then be used to design remediation systems that stimulate biological activity to favor the formation of U(VI)-phosphate phases.
  1086. Oldenburg, Curtis M., Sharon E. Borglin and Terry C. Hazen. 2002. Simulations of flow, transport, and biodegradation in landfills. American Geophysical Union (AGU) Fall Meeting
  1087. Hazen, Terry C.. 2001. Bioremediation: the hope and the hype. First International Conference on Environmental Recovery of Yugoslavia
  1088. Hazen, T.C., A.J. Tien, A. Worsztynowicz, D.J. Altman, K. Ulfig, G. Plaza and T. Manko. 2001. Poland petroleum refinery sludge lagoon cleanup using a biopile. First International Conference on Environmental Recovery of Yugoslavia
  1089. Hazen, Terry C.. 2001. In situ bioremediation: From research to practical applications. 23rd Biennial Groundwater Conference
  1090. Hazen, Terry C.. 2001. Deep Subsurface Microbiology and the Homestake Gold Mine. National Underground Science Laboratory Conference
  1091. Hazen, Terry C. 2001. DOE EM Restoration Needs and Direction. DOE NABIR Biological and Environmental Research Advisory Committee Review
  1092. Hazen, Terry C. 2001. In Situ Bioremediation: Lab to Field. 23rd Biennial Groundwater Conference and
  1093. Hazen, Terry C.. 2001. The Ecology of Pathogens in the Environment. Lawrence Berkeley National Laboratory abstract
    All pathogens have arisen from naturally occurring organisms. This is not surprising given the amazing adaptability of microbes for even the most extreme environments on earth. The strategies that microbial populations use to maintain their populations in natural environments have given rise to mechanisms that enable them to become pathogenic, eg. antibiotic resistance, spore formation, chemotaxis, symbiosis, capsule formation, niche overlap with man, etc. Field studies will be presented that suggest how these adaptions have allowed some microbes to become pathogenic, eg. E. coli, Legionella, and Bacillus anthracis.
  1094. Hazen, Terry C.. 2001. In situ Bioremediation of Solvent Contaminated Sites using Gaseous Nutrient Injection. California Regional Water Quality Board
  1095. Hazen, Terry C. and T. Torok. 2001. The Ecology of Bacillus anthracis. Lawrence Berkeley National Laboratory
  1096. Letain, Tracy E., Robert J. Silva, Roger Knopp, Terry C. Hazen, William T. Stringfellow and Heino Nitsche. 2001. Defining the interactions between microbial cell surfaces and uranium (VI) in aerobic conditions. American Society for Microbiology
  1097. Hazen, Terry C.. 2001. Aerobic Bioremediation of Landfills: Changing the 'Dry Tomb' Paradigm. Gulf Coast Research Laboratory, University of Southern Mississippi
  1098. Hazen, Terry C.. 2001. Aerobic Bioremediation of Landfills: Changing the 'Dry Tomb' Paradigm. University of Hawaii, Water Resources Center
  1099. Hazen, Terry C. 2001. The survival of indicators and pathogens in tropical waters. EPA workshop on Tropical Indicators
  1100. Nitsche, H., S. B. Clark* and Terry C. Hazen. 2001. The role of biogeochemical dynamics in the alteration of U solid phases under oxic conditions. NABIR Annual Investigators Meeting
  1101. Tokunaga, T., J. Wan, Terry C. Hazen, K. Olson, M. Firestone, E. Schwartz, S. Sutton, M. Newville and A. Lanzirotti. 2001. Mesoscale Biotransformation Dynamics Controlling Reactive Transport of Chromium. NABIR Annual Investigators Meeting
  1102. Hazen, Terry C., A. J. Tien, A. Worsztynowicz, D. J. Altman, K. Ulfig, G. Plaza and T. Manko. 2001. Poland Petroleum Refinery Sludge Lagoon biopile Demonstration Project. In Situ and On Site Bioremediation: The Sixth International Symposium
  1103. Ulfig, K., G. Plaza*, A. J. Tien, A. Worsztynowicz, M. A. Heitkamp and Terry C. Hazen. 2001. Microbial Aspects of Bioremediation. 2001 International Containment & Remediation Technology Conference and Exhibition
  1104. S. Borglin, Terry C. Hazen, C. Oldenburg and P. Zawislanski. 2001. Laboratory Investigation of the Biodegradation of Municipal Landfill Materials. In Situ and On Site Bioremediation: The Sixth International Symposium
  1105. S. Chauhan, L. Méndez, J. Montalvo and Terry C. Hazen. 2001. Gaseous In situ bioremediation of benzo(a)pyrene in soil. June. In Situ and On Site Bioremediation: The Sixth International Symposium
  1106. DeAngelis, K., E. Schwartz, M. Firestone, J. Wan, T. Tokunaga, D. Joyner and Terry C. Hazen. 2001. Microbial community composition and chromium transport in a clay sediment. UC Berkeley, 2nd Ann. Microbiology Symposium
  1107. Faybishenko, B. and Terry C. Hazen. 2001. Fuzzy Systems Modeling of In Situ Bioremediation of Chlorinated Solvents. 2001 International Containment & Remediation Technology Conference and Exhibition
  1108. Hazen, Terry C. 2001. Aerated Landfills, Changing the Subtitle D “Dry Tomb” Paradigm. 2001 International Containment & Remediation Technology Conference and Exhibition
  1109. Hazen, Terry C.. 2001. Bioremediation: The Hope and the Hype. 2001 International Containment & Remediation Technology Conference and Exhibition
  1110. Hazen, Terry C. 2001. Tropical Water Quality: Survival of Pathogens and their Indicators Water Quality Issues Workshop. LBNL Special Workshop
  1111. Hazen, Terry C. 2001. In situ bioremediation of solvents and organic contaminants using gaseous nutrient injection. Lawrence Livermore National Laboratory
  1112. Borglin, S. E., Terry C. Hazen, C. M. Oldenburg and P. T. Zawislanski. 2001. Mesoscale Laboratory Models of the Aerobic Biodegradation of Municipal Landfill Materials. American Geophysical Union Annual Meeting
  1113. Faybishenko, B. and Terry C. Hazen. 2001. Fuzzy systems modeling of in situ bioremediation of chlorinated solvents. American Geophysical Union Annual Meeting
  1114. Greenberg, M. R., Terry C. Hazen, S. E. Borglin and C. M. Oldenburg. 2001. Factors controlling metals concentration in aerobic and anaerobic laboratory landfill bioreactors. American Geophysical Union Annual Meeting
  1115. Hazen, Terry C.. 2001. Deep Subsurface Microbiology and the Homestake Gold Mine. National Underground Science Laboratory Microbiology Conference
  1116. Letain, T. E., R. J. Silva, H. Nitsche, Terry C. Hazen, S. B. Clark, M. Douglas, C. Billaspie, R. Knopp and P. J. Panak. 2001. The role of biogeochemical dynamics in the alteration of Uranium solid phases under oxic conditions. American Geophysical Union Annual Meeting
  1117. Borglin, Sharon E., Terry C. Hazen, Curt M. Oldenburg and Peter T. Zawislanski. 2001. Mesoscale laboratory models of the biodegradation of municipal landfill materials. American Geophysical Union (AGU) Fall Meeting 2001
  1118. Greenberg, Michael R., Terry C. Hazen, Sharon E. Borglin and Curt M. Oldenburg. 2001. Factors controlling concentrations of metals in the leachate from aerobic and anaerobic laboratory landfill bioreactors. AGU Fall Meeting 2001 abstract
    Costs and environmental issues associated with operating municipal landfills have motivated laboratory experiments investigating methods to increase biodegradation and decrease fugitive emissions of both liquid and gas. Rates of settling, biodegradation, and emissions were measured in three large laboratory-scale bioreactors filled with 30 kg of typical municipal waste. The bioreactors (200-L clear acrylic tanks) were instrumented to monitor pressure, temperature, moisture, humidity, gas composition, and leachate composition. Three treatments were applied: 1) aerobic (air injection with water addition and recirculation), 2) anaerobic (no air injection, water addition and recirculation), and 3) a control tank (no air or water injection). Preliminary studies showed measurable concentrations of Fe, Ba, Cu, Al, Mn, Ni, and Zn in the leachate. To investigate this further, bi-weekly leachate samples were, and were analyzed for dissolved Fe, Ba, Cu, Al, Mn, Ni, Zn. NO3-, NO2-, NH4+, PO43-, SO42-, Cl-, Fl-, Na+, Ca2, total organic acid, Eh and pH. The aerobic tank leachate had metals concentrations that were an order of magnitude lower than the leachate from the anaerobic tank. Possible explanations include the existence of fewer organic-metal complexes due to the decrease in the dissolved organic matter in the leachate for the aerobic treatment, or the higher pH and redox potential in the aerobic treatment. A sensitivity analysis was performed using MINTEQA2, a U.S. EPA geochemical speciation model that reports on metal speciation based on water composition parameters, for a combination of actual and estimated data. Initial results suggest that the presence of certain organic acids dramatically increases the metal-organic complexation, consistent with observations of greater metal concentrations in the anaerobic treatment leachate. This work was supported by Laboratory Directed Research and Development Funds at Lawrence Berkeley National Laboratory under Department of Energy Contract No. DE-AC03-76SF00098.
  1119. Hazen, Terry C.. 2001. In situ respiration and direct enzymatic assays for assessing bioremediation. American Geophysical Union (AGU) Fall Meeting 2001
  1120. Oldenburg, Curtis M., Sharon E. Borglin and Terry C. Hazen. 2001. Process modeling of flow, transport, and biodegradation in landfill bioreactors. American Geophysical Union Fall Meeting 2001
  1121. Tokunaga, Tetsu K., Jiamin Wan, Terry C. Hazen, Mary K. Firestone, Egbert Schwartz, Keith R. Olson, Stephen R. Sutton, Matthew Newville, Antonio Lanzirotti and William Rao. 2001. Intra-aggregate biogeochemical dynamics of chromium contamination and in-situ remediation. AGU Fall Meeting 2001
  1122. Schwartz, E., T. Tokunaga, J. Wan, Terry C. Hazen and M. Firestone. 2001. Microbial Communities and Hexavalent Chromium Transport in Soil. 9th International Symposium on Microbial Ecology
  1123. 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. 2001. Diffusion-limited chromium reduction in soil aggregates. American Chemical Society annual meeting 222:U490-U490.
  1124. Hazen, Terry C.. 2001. In situ Bioremediation of Solvent Contaminated Sites using Gaseous Nutrient Injection. Department of Civil and Environmental Engineering, University of Washington
  1125. Hazen, Terry C. 2001. Technical Baseline Reassessments. Oakland Site Technology Coordination Group
  1126. Hazen, Terry C.. 2001. Aerated landfills, changing the "dry tomb" paradigm. First International Conference on Environmental Recovery of Yugoslavia
  1127. 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. 2001. Diffusion-limited chromium reduction in soil aggregates Chromium diffusion and reduction in soil aggregates. The American Chemical Society 222:U490-U490. abstract
    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 COO diffusion and reduction to Cr(III) were. obtained in soil columns representing transacts 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.
  1128. Hazen, Terry C. 2000. Aerobic Landfill Bioreactors. Prague 2000, Fifth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe, Environmental Issues in Central and Eastern Europe
  1129. Hazen, Terry C. 2000. Bioremediation and GMO. The Southeast Regional Judicial Conference on Genetics in the Courtroom
  1130. Hazen, Terry C. 2000. Bioremediation: State of the Science. Prague 2000, Fifth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe, Environmental Issues in Central and Eastern Europe
  1131. Terry C. Hazen. 2000. Bioremediation: the hope and the hype. 8th International Conference on Small Genomes
  1132. K. Ulfig, G. Plaza, A. Tien, D. Altman and Terry C. Hazen. 2000. Microbial Overview of Biopile Demonstration. Prague 2000: Fifth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe, Environmental Issues in Central and Eastern Europe.
  1133. A. Worsztynowicz, D. Rzychon, M. Adamski, K. Zacharz, S. Iwaszenko, K. Ulfig, G. Plaza, T. Manko, J. Krajewska, K. Lukasik, A. Tien, D. Altman, T. Hazen, B. Jagosz and J. Mos. 2000. Bioremediation of Soil Contaminated with Petroleum Wastes using a Biopile Technique - A Case Study. Prague 2000: Fifth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe, Environmental Issues in Central and Eastern Europe
  1134. Terry C. Hazen. 2000. Aerobic Bioremediation of Landfills: Changing the 'Dry Tomb' Paradigm. Environmental and Water Resources Engineering Series, University of California at Davis
  1135. Hazen, Terry C. 2000. Aerobic Landfills: Changing the Subtitle D ‘Dry Tomb’ Paradigm. New Mexico Environmental Health Conference
  1136. Hazen, Terry C. 2000. Ecology of Caribbean Coral Reefs. Vacaville SCUBA Club
  1137. Hazen, Terry C. 2000. In situ Bioremediation of Solvent Contaminated Sites using Gaseous Nutrient Injection. Department of Biology, Chico State University
  1138. Hazen, Terry C.. 2000. Bioremediation: the hope and the hype. 6th Annual DOE Technology Information Exchange
  1139. Terry C. Hazen. 2000. Full-scale Biopile Remediation of PAH's at a Polish Oil Refinery Sludge Lagoon. International Petroleum Conference
  1140. J. Montañez, L. Méndez, S. Chauhan and Terry C. Hazen. 2000. Polynuclear aromatic hydrocarbons in situ bioremediation treatability test; focus on contaminant disappearance by HPLC analysis. Annual meeting American Institute of Chemical Engineers
  1141. Hazen, Terry C. 2000. Bioremediation and DNAPLs. International Conference on Chlorinated and Recalcitrant Compound Remediation
  1142. Hazen, Terry C. 2000. Bioremediation at LBNL. LBNL Open House
  1143. Nitsche, Heino, Sue B. Clark and Terry C. Hazen. 2000. The role of biogeochemical dynamics in the alteration of U solid phases under oxic conditions. 4th Annual NABIR PI Workshop
  1144. Hazen, Terry C. 2000. Bioremediation using GMO. UC Berkeley and Biotechnology: Developing Strategic Partnerships, School of Public Health, University of California at Berkeley
  1145. C. Rivera and Terry C. Hazen. 2000. Chemotaxis of Pseudomonas fluorescens to 2,4 and 2,6-dinitrotoluene. American Chemical Society Spring Meeting
  1146. Hazen, Terry C. 2000. Application of Biopiles. NATO Advanced Research Workshop The Utilization of Bioremediation to Reduce Soil Contamination: Problems and Solutions
  1147. Hazen, Terry C. 2000. Bioremediation Studies of UXO. BEST Program Workshop on Bioremediation
  1148. Hazen, Terry C. 2000. Aerated Landfills. Annual Meeting of the Society for Industrial Microbiology
  1149. Hazen, Terry C. 2000. Intimate Strangers: Creators of the Future – Q&A. LBNL Summer Student Program
  1150. Hazen, Terry C. 2000. Full-scale Biopile Remediation of PAH’s at an Oil Refinery Sludge Lagoon. University of Texas at El Paso
  1151. Terry C. Hazen. 2000. Full-scale Biopile Remediation of PAH's at a Polish Oil Refinery Sludge Lagoon. Department of Civil and Environmental Engineering, University of California at Berkeley
  1152. A. Palumbo, S. Pfiffner, Terry C. Hazen and T. Phelps. 2000. Scaling Lab to the Field. NABIR annual investigators meeting
  1153. J. Wan, T. Tokunaga, D. Joyner, Terry C. Hazen, M. Firestone, E. Schwartz, S. Sutton and M. Newville. 2000. Mesoscale biotransformation dynamics controlling reactive transport of chromium. NABIR annual investigators meeting
  1154. Hazen, Terry C.. 2000. Aerobic Bioremediation of Landfills: Changing the 'Dry Tomb' Paradigm. Department of Civil and Environmental Engineering. Massachusetts Institute of Technology
  1155. Hazen, Terry C.. 2000. Bioremediation: the hope and the hype. Lawrence Livermore National Laboratory
  1156. Hazen, Terry C. 2000. Landfill Aerobic Bioremediation/ Gaseous Nutrient Injection for In Situ Bioremediation of Fuels and Chlorinated Solvents. Land Transfer and Long Term Management of Contaminated Federal Facilities
  1157. Hazen, Terry C. 2000. Poland Petroleum Refinery Sludge Lagoon biopile Demonstration Project. Land Transfer and Long Term Management of Contaminated Federal Facilities
  1158. Hazen, Terry C. 2000. Bioremediation research collaborative research for the EPSCOR Program. DOE EPSCOR annual workshop
  1159. Hazen, Terry C. 2000. Bioremediation research sponsored by DOE. DOE EPSCOR annual workshop
  1160. Rodriguez-Martinez, R. A., Terry C. Hazen, T. Torok and M. B. Clark. 2000. Microbial community studies of differently treated explosive contaminated soils. 100th General Meeting of the American Society for Microbiology 100:495.
  1161. Tien, A. J., A. Worsztynowicz, D. J. Altman, Terry C. Hazen, K. Ulfig, G. Plaza and T. Manko. 2000. Poland petroleum refinery sludge lagoon biopile demonstration project. 100th General Meeting of the American Society for Microbiology 100:555-556.
  1162. Velazquez, C. M. R. and Terry C. Hazen. 2000. Chemotaxis pseudomonas fluorecens to 2,4 and 2,6-dinitrotoluene. The American Chemical Society 219:209.
  1163. Terry C. Hazen. 1999. Chemical/Biological Subsurface Barriers. NABIR Workshop on Chemical/Biological Treatment
  1164. Hazen, Terry C.. 1999. Computer assisted presentations. LBNL Macintosh Users Group
  1165. Terry C. Hazen. 1999. Critical Biogeochemical Parameters for Bioremediation of Solvents in Fractured Rock. Fourth USA/CIS Joint Conference on Environmental Hydrology and Hydrogeology
  1166. Terry C. Hazen. 1999. Full-scale Biopile Remediation of PAH's in an Oil Refinery Sludge Lagoon. Jackson State University
  1167. Terry C. Hazen. 1999. Phytoremediation research at LBNL and DOE. BEST Program Phytoremediation Workshop sponsored by Jackson State University
  1168. Hazen, Terry C, A. J. Tien, A. Worsztynowicz, K. Ulfig and D. J. Altman. 1999. Bioremediation Field Demonstration of a Sludge Lagoon at a Polish Petroleum Refinery. Fourth USA/CIS Joint Conference on Environmental Hydrology and Hydrogeology
  1169. Hazen, Terry C. 1999. Bioremediation of Contaminated Subsurface Environments. American Society for Microbiology Annual Meeting
  1170. Terry C. Hazen. 1999. Bioremediation - State of the Science. Universidad Metropolitano
  1171. Hazen, Terry C. 1999. Innovative Bioremediation Demonstrations of Petroleum Contaminated Sites in Poland and US. Petroleum Environmental Research Forum hosted by Chevron
  1172. Terry C. Hazen. 1999. U.S. Landfills: Changing the Subtitle D Paradigm, Dry Tombs to Bioreactors. University of Puerto Rico
  1173. Terry C. Hazen. 1999. U.S. Landfills: Changing the Subtitle D Paradigm, Dry Tombs to Bioreactors. Earth Sciences Division Colloquia, Lawrence Berkeley National Laboratory
  1174. Hazen, Terry C, J. Radway, J. Kastner and M. Franck. 1999. Biosparging for rapid in situ cleanup of solvent contaminated soil and groundwater at the SRS D-area Oil Seepage Basin. ER-TEC'99 Environmental Restoration Technology End User Conference sponsored by DOE/DOD/EPA
  1175. Hazen, Terry C. 1999. Recent Innovations is Bioremediation. ER-TEC’99 Environmental Restoration Technology End User Conference sponsored by DOE/DOD/EPA
  1176. Hazen, Terry C. 1999. Methane injection pilot study for bioremediation of Old Town solvent Plume. California Regional Water Quality Board
  1177. Hazen, Terry C. 1999. The NABIR FRC Web Page. Annual DOE NABIR Investigators Meeting
  1178. Tien, A. J, A. Worsztynowicz, K. Zacharz, D. J. Altman and Terry C. Hazen. 1999. Technology Development and Transfer for Environmental Remediation: Joint American and Polish Cooperation. Sigma Xi annual meeting
  1179. Wan, J, T. Tokunaga, Terry C. Hazen and M. Firestone. 1999. Mesoscale Biotransformation Dynamics as the Basis for Predicting Core Scale Reactive Transport of Chromium and Uranium. Annual DOE NABIR Investigators Meeting
  1180. Hazen, Terry C. 1999. Critical Biogeochemical Parameters Used for In Situ Bioremediation of Solvents in Fractured Rock. Witherspoon Symposium: Dynamics of Fluids in Fractured Rocks
  1181. Hazen, Terry C.. 1999. U.S. Landfills: Changing the Subtitle D Paradigm, “Dry Tombs” to Bioreactors. Wake Forest University, Biology Department, 1999 Distinguished Alumni Lecture
  1182. Terry C. Hazen. 1999. U.S. Landfills: Changing the Subtitle D Paradigm, Dry Tombs to Bioreactors. Biology Department
  1183. Terry C. Hazen. 1999. Critical Biogeochemical Parameters for Bioremediation of Solvents in Fractured Rock. International Symposium on Deep Subsurface Microbiology
  1184. Hazen, Terry C. 1999. Field studies of aerobic bioremediation of groundwater contaminated by a Sanitary Landfill. International Symposium on Deep Subsurface Microbiology
  1185. Brigmon, R. L, D. J. Altman, M. M. Franck, Terry C. Hazen and C. B. Fliermans. 1999. Evaluation of Methanotrophic Bacteria during Injection of Gaseous Nutrients for In situ Trichloroethylene Bioremediation in a Sanitary Landfill. In Situ and On Site Bioremediation Symposium
  1186. A. D. Drinkwine and Terry C. Hazen. 1999. Remediation of TCE Contaminated Ground Water Using Methane Injection to Enhance Cometabolism Demonstration Project at the Hastings Former Naval Ammunition Depot. Gateway to the New Millennium Conference
  1187. Drinkwine, A. D. and Terry C. Hazen. 1999. Remediation of TCE Contaminated Ground Water Using Methane Injection to Enhance Cometabolism Demonstration Project at the Hastings Former Naval Ammunition Depot. In Situ and On Site Bioremediation Symposium
  1188. Hazen, Terry C.. 1999. U.S. Landfills: Changing the Subtitle D Paradigm, “Dry Tombs” to Bioreactors. Department of Environmental Engineering, University of California, Berkeley
  1189. Terry C. Hazen. 1998. Bioremediation Overview, U.S. Department of Energy-Supported Research and Development n Central and Eastern Europe and Russia. Fourth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe (Warsaw'98)
  1190. Terry C. Hazen. 1998. Bioremediation: State-of-the-Science, Special Workshop on DOE projects in Poland. Fourth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe (Warsaw'98)
  1191. Ulfig, K, A. Tien, G. Plaza, A. Worsztynowicz and Terry C. Hazen. 1998. Microbiological changes in petroleum-contaminated soil during bioremediation at a Polish petroleum refinery. Fourth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe (Warsaw'98)
  1192. Worsztynowicz, A, A. Tien, K. Ulfig, K. Zacharz, M. Adamski, D. Rzychon and Terry C. Hazen. 1998. Soil Cleaning at the Czechowice Refinery. Fourth International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe (Warsaw'98)
  1193. Terry C. Hazen. 1998. Bioremediation of chlorinated solvents. Workshop sponsored by the Army Corps of Engineers and the Nebraska Department of Environmental Quality
  1194. Terry C. Hazen. 1998. Methane biostimulation for cleanup of chlorinated solvents at the Naval Ammunition Depot. Award presentation by the Nebraska Department of Environmental Quality to the Army Corps of Engineers
  1195. Terry C. Hazen. 1998. U.S. Landfills: Changing the Subtitle D Paradigm. Stanford University, School of Engineering Seminar Series
  1196. Hazen, Terry C and B. B. Looney. 1998. Bioremediation using SRS Patented Technologies. Southeastern Environmental Resource Alliance sponsored workshop for remediation industry
  1197. Legrand, R., A. J. Morecraft, J. A. Harju, T. D. Hayes, Terry C. Hazen. 1998. Field Application of in situ methanotrophic treatment for TCE remediation.
  1198. Terry C. Hazen. 1998. How do I know if in situ bioremediation is feasible?. Bioremediation for Industry, Society for Industrial Microbiology
  1199. Terry C. Hazen. 1998. In Situ Bioremediation: Natural vs. Accelerated. Bioremediation for Industry, Society for Industrial Microbiology
  1200. Terry C. Hazen. 1998. Field Bioremediation at SRS. DOE Natural and Accelerated Bioremediation Research Program Investigators meeting
  1201. Peters, N. E., M. Bonell, Terry C. Hazen, S. Foster, M. Meybeck, W. Rast, G. Schneider, V. Tsirkunov, J. Williams. 1998. Water Quality Degradation and Freshwater Availability — Need for A Global Initiative. UNESCO colloquium, entitled Water - A Looming Crisis
  1202. Tien, A. J., A. Worsztynowicz, K. Ulfig, D. Altman and Terry C. Hazen. 1998. Comparison of aeration and nutrient ammendment strategies during bioremediation of petroleum sludge contaminated soils from a Polish refinery- batch and soil column studies. 98th General Meeting of the American Society for Microbiology 98:457.
  1203. Terry C. Hazen. 1997. Bioremediation. Risk Abatement Center of Central and Eastern Europe
  1204. Hazen, Terry C. 1997. Full-Scale field remediation Demonstrations: Lessons Learned. Lawrence Berkeley National Laboratory
  1205. Hazen, Terry C. 1997. In Situ Aerobic Co Metabolic Bioremediation of Chlorinated Solvents: Field Demonstrations. US Air Force Expert Panel on In Situ Aerobic Co Metabolic Bioremediation of Chlorinated Solvents
  1206. Hazen, Terry C. 1997. Aerobic bioremediation strategies for landfills. Department of Microbiology, University of Georgia
  1207. Hazen, Terry C. 1997. SRS Biosensor Research. DOE Biotechnology Interlaboratory Council meeting with Army JPO BioDefense
  1208. Hazen, Terry C. 1997. SRS Biotechnology School-to-Work Program. Aiken County Schools Superintendent, Board and Principals
  1209. Hazen, Terry C. 1997. Aerobic bioremediation. 3rd Annual Florida Remediation Conference
  1210. Hazen, Terry C. 1997. Aerobic bioremediation of chlorinated solvents. George Mason University
  1211. Hazen, Terry C. 1997. Field Bioremediation strategies. DOE Natural and Accelerated Bioremediation Research Program
  1212. Terry C. Hazen. 1997. Bioremediation. University of South Carolina, School of the Environment
  1213. Hazen, Terry C.. 1997. Bioremediation.
  1214. Hazen, Terry C. 1997. Aerobic bioremediation of chlorinated solvents: field demonstrations and studies. U. S. Army Corps of Engineers Seventh Annual Combined Innovative Technology and Chemistry Workshop
  1215. Hazen, Terry C. 1997. Bioremediation Technologies. University of South Carolina, School of the Environment
  1216. Hazen, Terry C. 1997. Dry Tomb vs. Bioremediation: are current regulations increasing our long-term liability. SRS Groundwater Update Meeting
  1217. Hazen, Terry C. 1997. Environmental Biotechnology Section Expertise. Bechtel Environmental Restoration Division
  1218. Hazen, Terry C. 1997. Groundwater Issues and Water Quality. Science Education for Public Understanding Workshop for middle and high school teachers in the Central Savannah River Area
  1219. Hazen, Terry C. 1997. In Situ Bioremediation of Chlorinated Solvents. WISTA and BIOPRACT Inc
  1220. Hazen, Terry C. 1997. In Situ Bioremediation using nutrient injection. Second Annual Novel Remediation Technology Symposium: Innovative Remedial Technologies for Cost Effective Site Solution
  1221. Hazen, Terry C. 1997. Microbiological issues for Global Water Quality. United Nations Task Force on Global Water Quality
  1222. Hazen, Terry C. 1997. Roundtable: Passive vs. Accelerated Remediation. Second Annual Novel Remediation Technology Symposium: Innovative Remedial Technologies for Cost Effective Site Solution
  1223. Hazen, Terry C. 1997. Landfill Bioremediation or Why the 'Dry Tomb' approach should be Doomed. CSRA chapter of AIChE
  1224. Hazen, Terry C. 1997. Bioremediation Demonstrations at the SRS. Army Corps of Engineers & Woodward & Clyde
  1225. Hazen, Terry C. 1997. Methane Biostimulation for Chlorinated Solvent Cleanup. Gas Research Institute, Transco, Radian International
  1226. Hazen, Terry C. 1997. Microbiological aspects of wastewater treatment for small communities and small sources. Sponsored by Heinz Endowment
  1227. Hazen, Terry C and B. B. Looney. 1997. Bioremediation using SRS Patented Technologies. Southeastern Environmental Resource Alliance sponsored workshop for remediation industry
  1228. Hazen, Terry C. 1997. Aerobic bioremediation in situ. Bioremediation Symposium Society for Industrial Microbiology
  1229. Kastner, J. R, K. H. Lombard, J. Radway, Terry C. Hazen, G. Burbage, D. J. Altman, M. M. Franck, F. A. Washburn, \C. J. Berry and R. L. Brigmon. 1997. Bioventing vs. prepared beds for remediation of petroleum contaminated sites. In Situ and On-Site Bioreclamation, The Fourth International Symposium
  1230. Lombard, K. H, A. Worsztynowicz, Terry C. Hazen and B. Jagosz. 1997. Bioremediation techniques for the clean up of a petroleum waste lagoon. In Situ and On-Site Bioreclamation, The Fourth International Symposium
  1231. Radway, J. C, J. W. Santo Domingo, C. J. Berry, E. W. Wilde and Terry C. Hazen. 1997. Degradation of trichloroethylene and benzene by embedded Burkholderia cepacia G4. In Situ and On-Site Bioreclamation, The Fourth International Symposium
  1232. Santo Domingo, J. W, B. E. Bumgarner, D. J. Altman, C. J. Berry and Terry C. Hazen. 1997. Physiological response of subsurface microbial communities to nutrient additions. In Situ and On-Site Bioreclamation, The Fourth International Symposium
  1233. K. Ulfig, G. Plaza, Terry C. Hazen, C. B. Fliermans, M. M. Franck and K. H. Lombard. 1997. Bioremediation treatability and feasibility studies at a polish petroleum refinery. In Situ and On-Site Bioreclamation, The Fourth International Symposium abstract
    Bioremediation is one of the most attractive technologies for remediation of environments contaminated with petroleum products. Indigenous microbes can easily degrade most the components of petroleum contamination. Indeed, many investigations have shown that naturally occurring microbes immediately increase after a oil spill to the environment using the petroleum as both a carbon and energy source, often resulting in the complete remediation of the contaminants without human intervention. However, when the petroleum contaminated soil (PCS) presents an immediate health risk and/or carbon concentrations are out of balance other limiting nutrients like oxygen, nitrogen, and phosphorus, than engineered intervention is required, eg. biostimulation. When in situ biostimulation is desirable then physical and chemical environmental conditions can also play a major role in nutrient availability, nutrient delivery, nutrient compatibility with the environment, and ultimately biodegradation rates. Treatability studies are essential to the successful implementation of bioremediation, either in situ or ex situ. As part of the bioremediation demonstration at a petroleum refinery in Poland treatability studies were done in the laboratory and feasibility studies in the field. The laboratory studies examined different soil matrices, and different amendments with appropriate controls. Samples were monitored for changes or differences in contaminant, nutrient, microbe, and end product concentrations. Biodegradation rates were measured by biomass increases, carbon dioxide production, and oxygen consumption. Field studies involved in situ respiration, carbon dioxide production measurements, and comparison of sediment contaminant components, i.e. biodegradable vs. recalcitrant compound.
  1234. Young, J. D, D. J. Altman, K. H. Lombard, A. W. Bourquin, D. C. Mosteller and Terry C. Hazen. 1997. Sanitary landfill optimization test for remediation of chlorinated solvents. In Situ and On-Site Bioreclamation, The Fourth International Symposium
  1235. Altman, D. J, Terry C. Hazen, R. Legrand, R. Hickey, K. H. Lombard, F. A. Washburn and C. J. Berry. 1996. Methanotrophic treatment of contaminated well water using bioreactors. Third International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe
  1236. Hazen, Terry C. 1996. Advances in bioremediation of groundwater and soil. Third International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe
  1237. Hazen, Terry C.. 1996. Aerobic bioremediation of chlorinated solvents: field demonstrations and studies. EAWAG Swiss Institute
  1238. Hazen, Terry C, K. H. Lombard, D. J. Altman, F. A. Washburn, C. J. Berry, J. D. Young, A. Bourquin, M. M. Franck, R. L. Brigmon, E. Becker and J. Santo Domingo. 1996. Bioremediation of groundwater at a sanitary landfill using biosparging and gaseous nutrient injection. Third International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe
  1239. Hazen, Terry C, K. H. Lombard, J. Wear, B. B. Looney, M. V. Enzien, J. M. Dougherty, C. B. Fliermans, M. M. Franck and C. A. Eddy-Dilek. 1996. Full Scale demonstration of in situ bioremediation of chlorinated solvents in the deep subsurface using gaseous nutrient biostimulation. Third International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe
  1240. Kastner, J. R, J. Radway, Terry C. Hazen, K. H. Lombard, G. Burbage, D. J. Altman, M. M. Franck, F. A. Washburn, C. J. Berry and R. L. Brigmon. 1996. Bioventing vs. prepared beds for remediation of petroleum contaminated sites. Third International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe
  1241. Kastner, J. R, J. Radway, Terry C. Hazen, K. H. Lombard, G. Burbage, D. J. Altman, M. M. Franck, F. A. Washburn, C. J. Berry and R. L. Brigmon. 1996. Bioventing vs. prepared beds for remediation of petroleum contaminated sites. Emerging Technologies in Hazardous Waste Management VIII, American Chemical Society
  1242. Lombard, K. H, A. Worsztynowicz, Terry C. Hazen and B. Jagosz. 1996. The Demonstration Of Bioremediation techniques for the clean up of a process waste lagoon at the Czechowice Oil Refinery in Czechowice-Dziedzice, Poland. Third International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe
  1243. Ulfig, K, G. Plaza, Terry C. Hazen, C. B. Fliermans, M. M. Franck and K. H. Lombard.. 1996. Bioremediation Treatability and Feasibility Studies at a Polish Petroleum Refinery. Third International Symposium and Exhibition on Environmental Contamination in Central and Eastern Europe
  1244. Hazen, Terry C.. 1996. Aerobic bioremediation of chlorinated solvents: field demonstrations and studies. Nebraska Department of Environmental Control and Army Core of Engineers
  1245. Hazen, Terry C.. 1996. Aerobic bioremediation of chlorinated solvents: field demonstrations and studies. Missouri Department of Environmental Control, Region 5 EPA, Army Core of Engineers
  1246. Hazen, Terry C. 1996. Bioremediation demonstrations at SRS. Tour of Japanese EPA (20 officials) of SRS sites
  1247. Hazen, Terry C. 1996. Bioremediation field demonstrations: Lessons learned. Michigan Technological University
  1248. Hazen, Terry C. 1996. Landfill leachate biotreatment. Federal Facilities Quarterly Meeting (EPA-R4
  1249. Hazen, Terry C, K. H. Lombard and E. W. Wilde. 1996. Bioremediation Demonstrations by the Biotechnology Group at SRS. Japanese Business and Technology Reps (12)
  1250. Hazen, Terry C. 1996. Biostimulation. IBC Workshop on Bioremediation vs. Bioaugmentation
  1251. Hazen, Terry C. 1996. Biostimulation vs. Bioaugmentation Workshop. IBC Workshop on Bioremediation vs. Bioaugmentation
  1252. Hazen, Terry C. 1996. Groundwater Session. IBC Conference on Innovative Remediation Technologies
  1253. Hazen, Terry C. 1996. Groundwater technologies. IBC Conference on Innovative Remediation Technologies
  1254. Hazen, Terry C. 1996. In Situ Bioremediation Field Demonstration. Northwestern University
  1255. Hazen, Terry C. and K. H. Lombard. 1996. Tours and presentations of bioremediation at the Poland demonstration site. Polish Institute for Ecology of Industrial Areas
  1256. Hazen, Terry C.. 1996. Bioremediation. Minnesota Pollution Control Agency
  1257. Hazen, Terry C.. 1996. Bioremediation. Army Ammunition Plant
  1258. Hazen, Terry C. 1996. Bioremediation Optimization Plans at the D-area Oil Seepage Basin. South Carolina Department of Health and Environmental Control
  1259. Hazen, Terry C. 1996. Biostimulation using methane and PHOSter. Southern Energy Board
  1260. Hazen, Terry C.. 1996. Biotechnology for SRS. Visiting Swiss officials
  1261. Hazen, Terry C.. 1996. Biotechnology for SRS. Visiting Army General
  1262. Hazen, Terry C.. 1996. Biotechnology for SRS. TNX Operations
  1263. Hazen, Terry C.. 1996. Biotechnology for SRS. Visiting Navy Command
  1264. Hazen, Terry C. 1996. SRS Biotechnology. Argonne National Laboratory
  1265. Hazen, Terry C.. 1996. Biotechnology at SRS. U.S. Army Technology Center Representatives
  1266. Hazen, Terry C.. 1996. Biotechnology at SRS. Booz, Allen, Hamilton Inc
  1267. Hazen, Terry C.. 1996. Monitoring Bioremediation: the hope and the hype. University of Turabo, Ana Menendez Foundation
  1268. Hazen, Terry C.. 1996. Monitoring Bioremediation: the hope and the hype. Center for Environmental Biotechnology, Berkeley National Laboratory and University of California, Berkeley
  1269. Hazen, Terry C, B. B. Looney and K. H. Lombard. 1996. PHOSter technology transfer by Southeastern Technology Center. Aiken Municipal Building
  1270. Hazen, Terry C.. 1996. Natural Attenuation. IBC's Second Annual International Symposium on Intrinsic Bioremediation
  1271. Hazen, Terry C. 1996. Bioremediation demonstrations at SRS - tour. Faculty from the University of Tennessee
  1272. Hazen, Terry C. 1996. Biotechnology “work for others” at SRS. SRS Environmental Advisory Committee
  1273. Hazen, Terry C.. 1996. Biotechnology for SRS. DOE, DOD and visiting Poland Diplomats
  1274. Franck, M. M., Terry C. Hazen, K. H. Lombard, D. J. Altman, R. L. Brigmon and C. B. Fliermans. 1996. The use of immunoassay techniques for TPH and BTEX with diesel contaminated soils in a prepared bed bioreactor. 96th General Meeting of the American Society for Microbiology 96:449.
  1275. Hazen, Terry C., Kenneth H. Lombard, James R. Kastner, Dennis J. Altman, Marilyn M. Franck, Fatina A. Washburn, Christopher J. Berry and Robin L. Brigmon. 1996. Bioventing vs. prepared beds for remediation of petroleum contaminated soil. 211th American Chemical Society National Meeting 211:ENVR 68.
  1276. Hazen, Terry C., K. H. Lombard, J. R. Kastner, D. J. Altman, M. M. Franck, F. A. Washburn, C. J. Berry, R. L. Brigmon, A. J. Alvarez, G. M. Yumet, C. L. Santiago, R. Chaudhry and G. A. Toranzos. 1996. Bioventing vs prepared beds for remediation of petroleum-contaminated soil In-situ survival of genetically-engineered microorganisms in a tropical aquatic environment. American Chemical Society 211:68. abstract
    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.
  1277. Arias, W. and Terry C. Hazen. 1995. Ability of an environmental Escherichia coli isolate to survive and multiply in algal exudates of tropical microbial epilithic communities. Seventh International Symposium on Microbial Ecology
  1278. Hazen, Terry C. 1995. Aerobic Bioremediation of Chlorinated Solvents. International Symposium on Advances in Bioremediation sponsored by American Society for Microbiology and European Society of Microbiologists
  1279. Hazen, Terry C. 1995. Bioremediation of toxic waste sites - using the natural cleansing capacity of the environment. Institute for Industrial Ecology
  1280. Hazen, Terry C. 1995. Full-Scale Demonstration of In Situ Bioremediation of Chlorinated Solvents in the Deep Subsurface Using Gaseous Nutrient Biostimulation. Seventh International Symposium on Microbial Ecology
  1281. Hazen, Terry C.. 1995. Bioremediation Demonstrations at DOE's Savannah River Site. Michigan Technological Institute
  1282. Hazen, Terry C. 1995. Field and Soil Column Studies of Reductive Dechlorination in Bulk Aerobic Aquifers. IBC’s International Symposium on Biological Dehalogenation
  1283. Hazen, Terry C. 1995. Field Studies of Intrinsic Bioremediation of Chlorinated Solvents in Anaerobic/Aerobic areas. IBC’s International Symposium on Intrinsic Bioremediation
  1284. Hazen, Terry C.. 1995. Bioremediation Demonstrations at DOE's Savannah River Site. Medical University of South Carolina
  1285. Hazen, Terry C. 1995. Methanotrophic Bioreactor Demonstration for Ground Water Cleanup. Strategic Environmental Research and Development Program, Scientific annual program review
  1286. Hazen, Terry C. 1995. Advances in Bioremediation of Soil and Groundwater at U. S. Department of Energy Sites. Keystone Symposia on Environmental Biotechnology
  1287. Hazen, Terry C. 1995. Monitoring of in situ Biodegradations. Third International Symposium on the Interface between Analytical Chemistry and Microbiology
  1288. Hazen, T. C., D. Altman and K. H. Lombard. 1995. Bioremediation Options for the Columbia County Landfill. Columbia County Board of Commissioners
  1289. Legrand, R., R. Baker and Terry C. Hazen. 1995. Field Demonstration of the Methanotrophic Fluidized Bed Bioreactor at the Savannah River Site. 88Th Air and Waste Management Associations Annual Meeting and Exhibition
  1290. Hazen, Terry C.. 1995. Bioremediation at SRS. Visiting Group from Minnesota Department of Environmental Protection
  1291. Hazen, Terry C.. 1995. Bioremediation at SRS. Visiting Provost University of South Carolina
  1292. Hazen, Terry C.. 1995. Bioremediation at SRS. Visiting Group from Minnesota Department of Environmental Protection
  1293. Hazen, Terry C.. 1995. Bioremediation at SRS. Visiting Group of State, County and Municipal Regulators from around South Carolina
  1294. Hazen, Terry C.. 1995. Bioremediation Demonstrations at the Savannah River Site. Minnesota Pollution Control Agency
  1295. Hazen, Terry C. 1995. Importance of Interdisciplinary Interactions for Large Field Remediation Projects. From the Flask to the Field:
  1296. Hazen, Terry C. 1995. In Situ Bioremediation of Sanitary Landfills. National Research Council
  1297. Hazen, Terry C. 1995. Bioremediation at SRS and Tour. Special seminar and tour for the SRS Environmental Advisory Committee
  1298. Hazen, Terry C.. 1995. Bioremediation at SRS. Special seminar to Board of Directors of Westinghouse Savannah River Company and Westinghouse Electric Company
  1299. Hazen, Terry C, C. J. Berry and R. Brigmon. 1995. Mobile Methanotrophic Bioreactor for Groundwater Cleanup. SRS Engineering Day Symposium at the Augusta Civic Center
  1300. Hazen, Terry C.. 1995. Measuring Bioremediation. Institute for Gas Technologies Eighth International Symposium on Gas, Oil//Environmental Biotechnology
  1301. Hazen, Terry C.. 1995. SRS Biotechnology. Concurrent Technologies Corporation
  1302. Hazen, Terry C.. 1995. SRS Biotechnology. US Air Force Center for Environmental Excellence, Brooks AFB, San Antonio
  1303. Hazen, Terry C.. 1995. SRS Biotechnology. Utilities Industry Consortium
  1304. Hazen, Terry C.. 1995. SRS Biotechnology. Armstrong Laboratory, Tyndall USAF
  1305. Hazen, Terry C.. 1995. Bioremediation Demonstrations at the Savannah River Site. Florida State University
  1306. Hazen, Terry C. 1995. Bioremediation of landfills with options for the Columbia County landfill. Georgia Environmental Protection Department
  1307. Gorden, R. W, Terry C. Hazen*, K. Lombard, T. Hayes, R. Legrand, R. Hickey and F. Sappington. 1995. Methanotrophic Hybrid Bioreactors for on Site Bioremediation. In Situ and On-Site Bioreclamation, The Third International Symposium
  1308. Hazen, Terry C. 1995. Bioremediation of organic vapors. Kodak Corporation
  1309. Hazen, Terry C. 1995. Full Scale In Situ Bioremediation Demonstration of Chlorinated Solvent Contamination in Soil and Groundwater. Department of Environmental Engineering, Georgia Institute of Technology
  1310. Hazen, Terry C. 1995. Methanotrophic Bioreactors for Treatment of Groundwater Contaminated with Chlorinated Solvents. Strategic Environmental Research and Development Program Annual Symposium
  1311. Hazen, Terry C, A. W. Bourquin*, N. O'Halloran, K. Lombard, D. Jackson, C. J. Berry and C. Lockett. 1995. In Situ Optimization Studies for Bioremediation of a Sanitary Landfill Groundwater Plume. In Situ and On-Site Bioreclamation, The Third International Symposium
  1312. Hazen, Terry C and K. H. Lombard. 1995. SRS Sanitary Landfill Bioremediation Optimization Test. South Carolina Department of Health and Environmental Control RCRA Group
  1313. Hazen, T. C., K. H. Lombard, B. B. Looney, M. V. Enzien, J. M. Dougherty, C. B. Fliermans, J. Wear and C. A. Eddy-Dilek. 1995. In Situ Bioremediation Via Horizontal Wells. In Situ and On-Site Bioreclamation, The Third International Symposium
  1314. Lombard, K. H, M. M. Franck* and Terry C. Hazen. 1995. The Savannah River Site sOILS Facility: Immunoassay Techniques for TPH and BTEX Contamination Monitoring in a Soils Bioremediation Facility. In Situ and On-Site Bioreclamation, The Third International Symposium
  1315. Fliermans, C. B., M. M. Franck, Terry C. Hazen and R. W. Gorden. 1995. Ecofunctional Enzymes of Microbial Communities in Groundwater. 95th General Meeting of the American Society for Microbiology 95:347.
  1316. Hazen, Terry C., K. H. Lombard, B. B. Looney, M. Enzien, J. M. Dougherty, C. B. Fliermans and C. A. Eddy-Dilek. 1995. Bioremediation Visualization using 3D Models of Parameters. 95th General Meeting of the American Society for Microbiology 95:403.
  1317. Hazen, Terry C.. 1995. Advances in Bioremediation of Soil and Groundwater at United-States Department-of-Energy Sites. Journal of Cellular Biochemistry 42-42.
  1318. Hazen, Terry C.. 1994. Bioremediation at the Savannah River Site. Special Seminar for State Senator
  1319. Hazen, Terry C.. 1994. Bioremediation at the Savannah River Site. Special seminar and tour for representatives from Spelman College, Atlanta, Georgia.
  1320. Hazen, Terry C.. 1994. Full-Scale Bioremediation Demonstrations at the Savannah River Site. Department of Civil Engineering, University of South Carolina
  1321. Hazen, Terry C.. 1994. In Situ Bioremediation Demonstration (Methane Biostimulation) of the Savannah River Site Integrated Demonstration Project. Department of Environmental Engineering
  1322. Hazen, Terry C., K. H. Lombard, B. B. Looney, M. V. Enzien, J. M. Dougherty, C. B. Fliermans, John Wear, C. A. Eddy-Dilek. 1994. In Situ Bioremediation Demonstration (Methane Biostimulation) of the Savannah River Site Integrated Demonstration Project. DOE Hanford Symposium
  1323. Hazen, Terry C, K. H. Lombard, B. B. Looney, M. V. Enzien, J. M. Dougherty, C. B. Fliermans, John Wear and C. A. Eddy-Dilek. 1994. In Situ Bioremediation Demonstration (Methane Biostimulation) of the Savannah River Site Integrated Demonstration Project. DOE Hanford Symposium
  1324. Fliermans, C. B., J. E. Wear, M. M. Franck, P. C. McKinsey and Terry C. Hazen. 1994. Immunological Techniques As Tools To Characterize The Subsurface Communities In Pristine and Contaminated Sites. Second International Conference on Groundwater Ecology sponsored by the EPA and the American Water Resources Association
  1325. Hazen, Terry C.. 1994. Bioremediation at SRS. AMTEX, American Textiles Industry Consortium
  1326. Hazen, Terry C. 1994. Methanotrophic Bioreactor Demonstration for Ground Water Cleanup. Strategic Environmental Research and Development Program, Scientific Advisory Board
  1327. Hazen, Terry C. 1994. Methanotrophic Bioremediation Demonstrations at SRS. South Carolina Electric and Gas Company at SRS with Tour of SRS
  1328. Hazen, Terry C, B. B. Looney, M. Enzien, J. M. Dougherty, J. Wear, C. B. Fliermans and C. A. Eddy. 1994. In Situ Bioremediation of Chlorinated Solvents Via Horizontal Wells. Second International Conference on Groundwater Ecology sponsored by the EPA and the American Water Resources Association
  1329. Fliermans, C. B., Terry C. Hazen and R. L. Tyndall. 1994. Decade of Monitoring Legionella pneumophila in Southeastern Cooling Towers. ASHRAE Annual Symposium
  1330. Hazen, Terry C.. 1994. Bioremediation at SRS. Meeting with representatives of Institute for Wood Research
  1331. Hazen, Terry C. 1994. Methanotrophic Bioreactor Demonstrations Proposal. Scientific Advisory Board, Strategic Environmental Research and Development Program (DOD-DOE-EPA)
  1332. Hazen, Terry C. 1994. SRS Sanitary Landfill Treatability Study. WSRC Environmental Restoration Department
  1333. Hazen, Terry C, K. Lombard and C. Berry. 1994. Methanotrophic bioreactors for the remediation of solvent contaminated ground water. WSRC/BSRI technical day at the Augusta/Richmond County Civic Center in Augusta, Georgia
  1334. Hazen, Terry C, K. Lombard, J. M. Dougherty, M. Enzien, J. Wear, C. B. Fliermans and C. A. Eddy. 1994. In Situ Bioremediation Via Horizontal Wells. WSRC Environmental Restoration Department. WSRC/BSRI technical day at the Augusta/Richmond County Civic Center in Augusta, Georgia
  1335. Hazen, Terry C. 1994. Advances in Bioremediation of Soil and Groundwater. Pittsburgh Conference annual meeting
  1336. Hazen, Terry C. 1994. Bioremediation of DOE Waste Sites. Arizona State University
  1337. Hazen, Terry C.. 1994. Integrated In Situ Bioremediation Demonstration. Mid-Year Review
  1338. Hazen, Terry C.. 1994. Integrated In Situ Bioremediation Demonstration. Mid-Year Review DOE-OTD-In Situ Remediation Integrated Program
  1339. Hazen, Terry C.. 1994. sOILS Facility for Bioremediation of Petroleum Contaminated Soil. WSRC Environmental Restoration Department
  1340. Hazen, Terry C.. 1994. sOILS Facility for Bioremediation of Petroleum Contaminated Soil. WSRC Environmental Restoration Department
  1341. Hazen, Terry C.. 1994. Bioremediation at the Savannah River Site. Special seminar for the U. S. Department of Energy Biotechnology Interlaboratory Council
  1342. Hazen, Terry C.. 1994. Bioremediation of Groundwater. Westinghouse Waste & Environmental Information Exchange
  1343. Hazen, Terry C, K. H. Lombard, B. B. Looney, M. V. Enzien, J. M. Dougherty, C. B. Fliermans, John Wear and C. A. Eddy-Dilek. 1994. Preliminary Technology Report for In Situ Bioremediation Demonstration (Methane Biostimulation) of the Savannah River Site Integrated Demonstration Project. American Nuclear Society Spectrum94
  1344. Lombard, K. H. and Terry C. Hazen. 1994. A Petroleum Contaminated Soil Bioremediation Facility. American Nuclear Society Spectrum94
  1345. Hazen, Terry C. 1994. Advances in Bioremediation. Annual meeting of the South Carolina Academy of Science
  1346. Hazen, Terry C. 1994. SRS Bioremediation Technologies. Andrews Environmental
  1347. Enzien, M. V., Terry C. Hazen, C. B. Fliermans, M. M. Franck and P. McKenzie. 1994. Microbial community structure in unsaturated sediments during in situ bioremediation of chlorinated solvents. 94th General Meeting of the American Society for Microbiology 94:329.
  1348. Fliermans, C. B., J. E. Wear, M. M. Franck, P. C. McKinsey and Terry C. Hazen. 1994. Use of biolog technology to assess remediation and groundwater perturbations. 94th General Meeting of the American Society for Microbiology 94:329.
  1349. Hazen, Terry C., B. B. Looney, M. Enzien, J. M. Dougherty, J. Wear, C. B. Fliermans and C. A. Eddy. 1994. In situ bioremediation of chlorinated-solvents via horizontal wells. 94th General Meeting of the American Society for Microbiology 94:329.
  1350. Hazen, Terry C. 1994. Earth Day seminar on Pollution. Sixth Grade Science Classes at Tutt Middle School in Augusta
  1351. Fliermans, C. B, J. E. Wear, M. M. Franck, P. C. McKinsey and Terry C. Hazen. 1993. Immunological techniques as tools to characterize the subsurface communities in pristine and contaminated sites. International Symposium on Subsurface Microbiology
  1352. Fliermans, C. B, J. E. Wear, M. M. Franck, P. C. McKinsey and Terry C. Hazen. 1993. Immunological techniques as tools to characterize the subsurface communities in pristine and contaminated sites. 11th International Symposium on Environmental Biogeochemistry
  1353. Fliermans, C. B, J. E. Wear, M. M. Franck, P. C. McKinsey and Terry C. Hazen*. 1993. Use of BIOLOG(r) technology to access remediation and groundwater perturbations. International Symposium on Subsurface Microbiology
  1354. Fliermans, C. B, J. E. Wear, M. M. Franck, P. C. McKinsey and Terry C. Hazen*. 1993. Use of BIOLOG(r) technology to access remediation and groundwater perturbations. 11th International Symposium on Environmental Biogeochemistry
  1355. Hazen, Terry C. 1993. Biodegradation of Chlorinated Solvents and Vegetation Enhancement. EPA/HSRC & Kansas State University Seminar
  1356. Hazen, Terry C. 1993. Deep Subsurface Microbiology. Department of Agronomy, Kansas State University
  1357. Hazen, Terry C. 1993. SRS In Situ Bioremediation General Principals. Graduate Course on Microbiological Site Assessment: Characterization and Monitoring
  1358. Hazen, Terry C.. 1993. SRS In Situ Bioremediation Integrated Demonstration. Graduate Course on Microbiological Site Assessment: Characterization and Monitoring
  1359. Hazen, Terry C.. 1993. SRS In Situ Bioremediation Integrated Demonstration. EPA/HSRC & Kansas State University Seminar
  1360. Hazen, T. C, J. M. Dougherty, M. Enzien, M. M. Franck, C. B. Fliermans and C. A. Eddy. 1993. In Situ Bioremediation Via Horizontal Wells. Emerging Technologies in Hazardous Waste Management V, The Industrial & Engineering Chemistry Division of the American Chemical Society
  1361. Fliermans, C. B., J. M. Dougherty, M. M. Franck, P. C. McKinzey and Terry C. Hazen. 1993. Immunological techniques as tools to characterize the subsurface microbial community at a TCE contaminated site. 93rd General Meeting of the American Society for Microbiology 93:313.
  1362. Hazen, Terry C., M. Enzien, M. M. Franck and C. B. Fliermans. 1993. In situ bioremediation of chlorinated solvents using horizontal wells to inject air and methane. 93rd General Meeting of the American Society for Microbiology 93:312.
  1363. Wear, J. E., P. C. McKinsey, M. M. Franck, H. G. Findley, M. V. Enzien, C. B. Fliermans and Terry C. Hazen. 1993. A most probable number assay using BIOLOG-GN microtiter plates for the study of ground water microbial communities. 93rd General Meeting of the American Society for Microbiology 93:314.
  1364. Hazen, Terry C. 1993. Bioremediation Programs at SRS. EPA/HSRC & SRTC Technology Development Exchange Meeting
  1365. Hazen, Terry C.. 1993. SRS In Situ Bioremediation Integrated Demonstration. EPA/HSRC & SRTC Technology Development Exchange Meeting
  1366. Hazen, T. C, J. M. Dougherty and C. B. Fliermans. 1993. In Situ Bioremediation of Chlorinated Solvents by Using Horizontal Wells to Inject Air and Methane. Annual Meeting of the American Society for Microbiology
  1367. Hazen, Terry C. 1993. Bioreactors and Vegetation Enhancement of Bioremediation of Chlorinated Solvents. DOE/Office of Technology Transfer Investigators Review
  1368. Hazen, Terry C.. 1993. Bioremediation Ecological Risk Assessment. Department of Fisheries and Wildlife Biology, Colorado State University
  1369. Hazen, Terry C.. 1993. Bioremediation Ecological Risk Assessment. Short Course on Ecological Risk Assessment and Management, Department of Fisheries and Wildlife Biology, Colorado State University
  1370. Hazen, T. C.. 1993. Full-Scale Prepared Bed Bioremediation Facility for Petroleum-Contaminated Soil. Savannah River Site Central Environmental Committee
  1371. Hazen, T. C., J. M. Dougherty, M. Enzien, M. M. Franck, C. B. Fliermans and C. A. Eddy. 1993. In Situ Bioremediation Via Horizontal Wells. Annual Meeting of the American Society of Civil Engineers
  1372. Hazen, Terry C. 1993. Bioremediation at SRS. American Academy of Microbiology, Colloquia on Field Research in Bioremediation
  1373. Hazen, Terry C.. 1993. In Situ Bioremediation of Chlorinated Solvents using Injection of air/Methane via Horizontal Wells. Department of Energy, Office of Technology Development
  1374. Hazen, Terry C. 1993. Bioremediation Demonstrations at SRS. Westinghouse Technology Transfer Council
  1375. Hazen, Terry C. 1993. The Environment and Environmental Scientists. Warren Road Elementary School
  1376. Hazen, Terry C.. 1993. Bioremediation Needs and Future. South Carolina University Research and Education Foundation Strategic Planning Committee
  1377. Hazen, Terry C.. 1993. Bioremediation of DOE Waste Sites. Indiana University
  1378. Hazen, Terry C., J. M. Dougherty, M. Enzien, J. Wear, C. B. Fliermans, C. A. Eddy, K. Lombard. 1993. In Situ Bioremediation Via Horizontal Wells. Institute for Gas Technologies Sixth International Symposium on Gas, Oil, Environmental Biotechnology
  1379. Hazen, Terry C. 1993. Integrated In Situ Bioremediation Demonstration. Indiana University
  1380. Hazen, Terry C, J. M. Dougherty, M. Enzien, J. Wear, C. B. Fliermans, C. A. Eddy and K. Lombard. 1993. In Situ Bioremediation Via Horizontal Wells. Institute for Gas Technologies Sixth International Symposium on Gas, Oil//Environmental Biotechnology
  1381. Berry, C. J. and Terry C. Hazen. 1993. Methanotrophic Treatment of Contaminated Well Water Using a Pilot Scale Bioreactor. SRS-DOE Supplier Information Exchange Forum
  1382. Borthen, J, F. Meyer, K. Lombard and Terry C. Hazen. 1993. Catalytic oxidation of trichloroethylene and perchloroethylene mixtures. American Institute of Chemical Engineers
  1383. Edwards, N. T, B. T. Walton, T. A. Anderson, J. J. Beauchamp, L. W. Cooper, R. J. Luxmoore, E. G. O'Neill, G. S. Sayler, D. C. White and Terry C. Hazen. 1993. The Use of Vegetation for Bioremediation of Surface Soils Contaminated with Trichloroethylene. SRS-DOE Supplier Information Exchange Forum
  1384. Hazen, Terry C., K. Lombard. 1993. Full-Scale Prepared Bed Bioremediation Facility for Petroleum-Contaminated Soil. SRS-DOE Supplier Information Exchange Forum
  1385. Hazen, Terry C. and K. Lombard. 1993. Full-Scale Prepared Bed Bioremediation Facility for Petroleum-Contaminated Soil. SRS-DOE Supplier Information Exchange Forum
  1386. Lombard, K. H., J. Borthen and Terry C. Hazen. 1993. Design and Configuration Management of System Control Components for in situ Methanotrophic Bioremediation of Groundwater and Sediment Contaminated with Chlorinated Hydrocarbons. SRS-DOE Supplier Information Exchange Forum
  1387. Berry, C. J, Terry C. Hazen, M. M. Franck and J. Rossabi. 1993. Methanotrophic Treatment of Contaminated Well Water Using a Pilot Scale Bioreactor. In Situ and On-Site Bioreclamation, The Second International Symposium
  1388. Dougherty, J. M, C. J. Berry, M. M. Franck and Terry C. Hazen*. 1993. Characterization of the Subsurface Microbial Community from a Trichloroethylene Contaminated Site. In Situ and On-Site Bioreclamation, The Second International Symposium
  1389. Edwards, N. T, B. T. Walton, T. A. Anderson, J. J. Beauchamp, L. W. Cooper, R. J. Luxmoore, E. G. O'Neill, G. S. Sayler, D. C. White and Terry C. Hazen. 1993. The Use of Vegetation for Bioremediation of Surface Soils Contaminated with Trichloroethylene. In Situ and On-Site Bioreclamation, The Second International Symposium
  1390. Enzien, M. V, F. W. Picardal, Terry C. Hazen and R. G. Arnold. 1993. Biodegradation of Trichloroethylene and Tetrachloroethylene under Aerobic Conditions with Methane Addition in a Sediment Column. In Situ and On-Site Bioreclamation, The Second International Symposium
  1391. Fliermans, C. B, J. M. Dougherty, M. M. Franck, P. C. McKinsey and Terry C. Hazen. 1993. Immunological Techniques as Tools to Characterize the Subsurface Microbial Community at a Trichloroethylene Contaminated Site. In Situ and On-Site Bioreclamation, The Second International Symposium
  1392. Hazen, Terry C. 1993. In Situ Bioremediation of Groundwater. American Society for Microbiology Conference on Water Quality in the Western Hemisphere
  1393. Hazen, Terry C. 1993. Methanotrophic Bioreactor Demonstrations Proposal. Scientific Advisory Board, Strategic Environmental Research and Development Program (DOD-DOE-EPA)
  1394. Hazen, T. C, J. M. Dougherty and C. B. Fliermans. 1993. DOE/SRS Integrated Demonstration: In Situ Bioremediation of Soil and Groundwater at a Chlorinated Solvent Contaminated Site using Horizontal Wells to Inject Air and Methane. In Situ and On-Site Bioreclamation, The Second International Symposium
  1395. Hazen, T. C, J. M. Dougherty, C. B. Fliermans and B. B. Looney. 1993. Bioremediation of Soil and Groundwater at a Chlorinated Solvent Contaminated Site using Horizontal Wells to Inject Air. In Situ and On-Site Bioreclamation, The Second International Symposium
  1396. Lombard, K. H., J. Borthen and Terry C. Hazen. 1993. Design and Configuration Management of System Control Components for in situ Methanotrophic Bioremediation of Groundwater and Sediment Contaminated with Chlorinated Hydrocarbons. In Situ and On-Site Bioreclamation, The Second International Symposium
  1397. Morrissey, C. M, S. E. Herbes, A. V. Palumbo*, T. J. Phelps and Terry C. Hazen. 1993. Use of Laboratory Soil Columns to Optimize in situ Biotransformation of Tetrachloroethylene. In Situ and On-Site Bioreclamation, The Second International Symposium
  1398. Hazen, Terry C.. 1993. In Situ Bioremediation of Chlorinated Solvents using Injection of air/Methane via Horizontal Wells. Technology Transfer Seminar for Industry
  1399. Hazen, Terry C.. 1992. In Situ Bioremediation Demonstrations at SRS. International Symposium on the Implementation of Biotechnology in Industrial Waste Treatment and Bioremediation
  1400. Hazen, Terry C.. 1992. In Situ Bioremediation of Chlorinated Solvents using Injection of air/Methane via Horizontal Wells. Department of Biological Sciences, Clemson University
  1401. Hazen, Terry C.. 1992. Monitoring In Situ Bioremediation. International Symposium on In Situ Bioremediation'92 sponsored by Environment Canada
  1402. Hazen, Terry C. 1992. SRS Bioremediation Technology Licensing Symposium. SRS Technology Transfer Symposium
  1403. Looney, B. B, D. S. Kaback, Terry C. Hazen and J. C. Corey. 1992. Environmental restoration using horizontal wells: A field demonstration. American Chemical Society Symposium on Emerging Technologies for Hazardous Waste Management
  1404. Hazen, Terry C.. 1992. Environmental Research. Richmond County Schools Education Improvement Program
  1405. Hazen, Terry C.. 1992. Science Fair Workshop for Wheeless Road Elementary School. Group Leader
  1406. Hazen, Terry C.. 1992. In Situ Bioremediation of Chlorinated Solvents using Injection of air/Methane via Horizontal Wells. Department of Environmental Engineering, Rice University
  1407. Hazen, Terry C.. 1992. In Situ Bioremediation of Sanitary Landfills. Westinghouse Savannah River Company Environmental Restoration Department
  1408. Hazen, T. C, J. M. Dougherty, M. Enzien, M. M. Franck, C. B. Fliermans, C. A. Eddy and K. H. Lombard. 1992. DOE/SRS Integrated Demonstration: In Situ Bioremediation of Soil and Groundwater. DOE Technology Information Exchange: Remediation
  1409. Dougherty, J. M., M. M. Franck, C. B. Fliermans and Terry C. Hazen. 1992. Characterization of the Subsurface Microbial Community from a Trichloroethylene Contaminated Site. 92ND GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY 92:296.
  1410. Enzien, M. V., F. W. Picardal, Terry C. Hazen and R. G. Arnold. 1992. Effects of Trichloroethylene Tetrachloroethylene and Methane Exposure on Microbial Community Dynamics in a Sediment Column. 92ND GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY 92:296.
  1411. Hazen, Terry C., J. M. Dougherty and B. B. Looney. 1992. Stimulation of Microbial Communities in Groundwater from Horizontal Well in-Situ Air Stripping at a Chlorinated Solvent Contaminated Site. 92ND GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY 92:296.
  1412. Hazen, Terry C. 1992. Emerging Technologies in Bioremediation. American Society for Microbiology Training Workshop
  1413. Hazen, Terry C.. 1992. In Situ Bioremediation Demonstrations at SRS. American Society for Microbiology Annual Meeting
  1414. Hazen, T. C., J. M. Dougherty and B. B. Looney. 1992. Stimulation of Ground Water and Sediment Communities at a Trichloroethylene Contaminated Site. American Society for Microbiology Annual Meeting
  1415. Hazen, Terry C. 1992. Bioremediation of DOE wastes, lessons from oil-contaminated soil. Rocky Flats EG&G Environmental Restoration Department
  1416. Fliermans, C. B, Terry C. Hazen and R. L. Tyndall. 1992. Decade of Monitoring Legionella pneumophila in Southeaster Cooling Towers. Fourth International Symposium on Legionella
  1417. Fliermans, C. B, Terry C. Hazen and R. L. Tyndall. 1992. Modified Direct Fluorescent Antibody Technique as a Monitoring Tool for Legionella. Fourth International Symposium on Legionella
  1418. Hazen, Terry C. 1992. SRS Integrated Demonstration: Bioremediation Tasks. Gas Research Institute Environmental Advisors annual meeting
  1419. Hazen, Terry C. 1992. Deep Subsurface Microbiology - Traveling Lecture. Eckerd College
  1420. Hazen, Terry C.. 1992. In Situ Bioremediation of Chlorinated Solvents using Injection of air/Methane via Horizontal Wells. South Carolina Department of Health and Environmental Control
  1421. Hazen, Terry C.. 1992. In Situ Bioremediation Demonstrations at SRS. Westinghouse Science and Technology Center
  1422. Hazen, T. C., J. M. Dougherty, C. B. Fliermans and B. B. Looney. 1992. Full Scale Underground Injection of Air, Methane, Other Gases via Horizontal Wells for In Situ Bioremediation of Chlorinated Solvent Contaminated Ground Water and Soil. American Institute of Chemical Engineers annual meeting
  1423. Hazen, T. C, J. M. Dougherty, C. B. Fliermans and B. B. Looney. 1992. Full Scale Underground Injection of Air, Methane//Other Gases via Horizontal Wells for In Situ Bioremediation of Chlorinated Solvent Contaminated Ground Water and Soil. American Institute of Chemical Engineers annual meeting
  1424. 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 1545-50 vol.2|2 vol. xv+1891. abstract
    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
  1425. Hazen, Terry C.. 1991. Technology Demonstration at the Miscellaneous Chemical Basin. Monthly SRS-EPA-SCDHEC Federal Facilities Agreement Meeting
  1426. Hazen, Terry C.. 1991. Bioremediation of SRS waste sites. Department of Biology, University of West Florida
  1427. Hazen, Terry C.. 1991. Bioremediation/Biotechnology. Puerto Rico Conference on Advanced Technology/ InterAmerican University
  1428. T. Hall, T. Savage, G. L. Mills and Terry C. Hazen. 1991. Determination of petroleum hydrocarbons in a contaminated shallow aquifer using chromarodiatroscan TLC-FID. Annual meeting of the Society of Environmental Toxicology and Contamination
  1429. Hazen, Terry C.. 1991. Bioremediation at SRS. Annual Meeting of the Southeastern Society for Microbiology
  1430. Hazen, Terry C.. 1991. Ex-Situ Bioremediation of SRS Waste Sites. US Department of Energy, Office of Environmental Restoration Technology Information Exchange Workshop
  1431. Hazen, Terry C.. 1991. In-Situ Bioremediation of SRS Waste Sites. US Department of Energy, Office of Environmental Restoration Technology Information Exchange Workshop
  1432. Hazen, Terry C.. 1991. Integrated Demonstration Project Bioremediation Tasks for FY92. WSRC Environmental Restoration Department Special Seminar
  1433. Hazen, Terry C. and C. Berry*. 1991. Bioreactors. US Department of Energy, Office of Environmental Restoration Technology Information Exchange Workshop
  1434. Mills, G. L, T. Savage, T. Hall and Terry C. Hazen. 1991. Characterization of Diesel Oil Components in a Shallow Aquifer Contaminant Plume. Annual meeting of the Society of Environmental Toxicology and Contamination
  1435. Hazen, Terry C.. 1991. TCE/PCE Bioremediation at SRS. Department of Civil Engineering
  1436. Kaback, D. S, B. B. Looney, C. A. Eddy and Terry C. Hazen. 1991. Horizontal Wells for remediation. National Water Well Association Outdoor Action Conference
  1437. Hazen, Terry C.. 1991. Outdoor Safety. Warren Road Elementary School
  1438. Hazen, Terry C.. 1991. Bioremediation at SRS. DOE-OTD Midyear Program Review
  1439. Hazen, Terry C., C. B. Fliermans. 1991. Bioremediation Workshop. WSRC-DOE Environmental Restoration Division
  1440. Hazen, Terry C.. 1991. Bioremediation at SRS. U. S. Environmental Protection Agency, Office of Research and Development
  1441. Hazen, Terry C. 1991. Bioreactors: a new method for cleaning chemical-contaminated groundwater using soil bacteria. Meeting of South Carolina Research and University Educational Foundation Steering Committee
  1442. Hazen, Terry C.. 1991. Bioremediation of toxic waste sites at the Savannah River Site. Department of Environmental Engineering and Public Health, University of North Carolina at Chapel Hill
  1443. Hazen, Terry C.. 1991. Bioremediation of toxic waste sites at the Savannah River Site. Savannah River Ecology Laboratory, University of Georgia
  1444. Hazen, Terry C.. 1991. Bioremediation of toxic waste sites at the Savannah River Site. Department of Biology, University of North Carolina at Wilmington
  1445. Hazen, Terry C. 1991. Phase 1 biological report and proposed activity of Phase 2 of the DOE Integrated Demonstration of TCE remediation at the Savannah River Site. Technical meeting of the DOE Integrated Demonstration Project at SRS
  1446. Hazen, Terry C.. 1991. Bioremediation of contaminated sites at the Savannah River Site. Augusta College, University of Georgia
  1447. Hazen, Terry C.. 1991. Bioremediation of contaminated waste sites at the Savannah River Site. Department of Chemical Engineering, University of Virginia
  1448. Hazen, Terry C. 1991. Bioremediation: Teaching Bacteria to Destroy Toxic Chemicals in Contaminated Environments or a Natural Solution to Pollution. Fifteenth Congress of Scientific Investigation, sponsored by InterAmerican University
  1449. Hazen, Terry C.. 1991. SRS Integrated Demonstration: Bioremediation Tasks. Fourth International Institute for Gas Technology Symposium on Gas, Oil, Coal and Environmental Biotechnology
  1450. Siler, J. L, D. J. McCabe and Terry C. Hazen. 1991. Fouling of ceramic filters and thin-film composite reverse osmosis membranes by inorganic and bacterial constituents. Annual meeting of American Institute of Chemical Engineers
  1451. Siler, J. L, D. J. McCabe and Terry C. Hazen. 1991. Fouling of ceramic filters and thin-film composite reverse osmosis membranes by inorganics and bacteria. International Congress on Membranes and Membrane Processes
  1452. Hazen, Terry C.. 1990. The potential for bioremediation of PCBs in soil. Workshop of the Westinghouse Electric Bloomington Project
  1453. Hazen, Terry C.. 1990. The SRS sOILs Facility, a petroleum contaminated soil bioremediation facility. Quarterly meeting of the Region 4 EPA/SCDHEC/SRS
  1454. Hazen, Terry C.. 1990. Bioreactors: a new method for cleaning chemical-contaminated groundwater using soil bacteria. Boy Scout Jamboree of the Central Savannah River Council
  1455. Hazen, Terry C.. 1990. Bioremediation of toxic waste sites at the Savannah River Site. Lawrence Livermore National Laboratory
  1456. Hazen, Terry C.. 1990. Phase 1 biological report and proposed activity of Phase 2 of the DOE Integrated Demonstration of TCE remediation at the Savannah River Site. Groundwater Division of the South Carolina Department of Health and Environmental Control
  1457. King, D. L., J. E. Wear and Terry C. Hazen. 1990. Chemotactic Behavior of Soil Bacteria to Benzene Toluene and Xylene. 90TH ANNUAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY 90:255.
  1458. Wear, J. E. and Terry C. Hazen. 1990. Distribution and Abundance of Bacteria from Pristine and Trichloroethylene Contaminated Groundwater. 90TH ANNUAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY 90:262.
  1459. Hazen, Terry C.. 1990. Bioremediation of toxic waste sites at the Savannah River Site. Idaho National Engineering Laboratory, EG&G
  1460. Hazen, Terry C.. 1990. Bioremediation of toxic waste sites at the Savannah River Site. Department of Environmental Engineering, Utah State University
  1461. Hazen, Terry C.. 1990. Bioremediation of toxic waste sites at the Savannah River Site. Department of Environmental Engineering, Vanderbilt University
  1462. Hazen, Terry C.. 1990. Survival of indicators and pathogens in tropical waters. Department of Biological Sciences, Florida Institute of Technology
  1463. Hazen, Terry C.. 1990. Biotechnology as a Career. NSF-Ruth Patrick Science Center Summer High School Scholar Program
  1464. Hazen, Terry C.. 1990. Environmental Biotechnology at SRS. Semi-Annual meeting of the Westinghouse Savannah River Company Board of Directors
  1465. Hazen, Terry C.. 1990. Environmental Biotechnology at SRS. Geotechnical Advisory Committee meeting of the Savannah River Laboratory, Westinghouse Savannah River Company
  1466. Hazen, Terry C.. 1990. Methanotrophic Degradation of Trichloroethylene at SRS. Annual program review Gas Research Institute and Savannah River Laboratory
  1467. Hazen, Terry C.. 1990. Environmental Biotechnology at SRS. Scientific Ecology Group, Division of Westinghouse Electric Company
  1468. Hazen, Terry C.. 1990. Bioremediation at DOE sites, cooperative research programs. U. S. Department of Energy and U. S. Environmental Protection Agency Workshop
  1469. Hazen, Terry C. 1990. Contaminated Environments, Plenary Session 7. International Symposium on Microbiology of the Deep Terrestrial Subsurface
  1470. Hazen, Terry C. 1990. Deep subsurface bacterial responses to contaminants. International Symposium on Microbiology of the Deep Terrestrial Subsurface
  1471. Hazen, Terry C. 1990. International Symposium on Microbiology of the Deep Terrestrial Subsurface. Westinghouse Savannah River Company and U. S. Department of Energy
  1472. Hazen, Terry C, L. Jiménez and S. Pfiffner. 1990. Isolation of microbial DNA from groundwater environments. International Symposium on Microbiology of the Deep Terrestrial Subsurface
  1473. Jiménez, L, G. López de Victoria, J. Wear, C. B. Fliermans and Terry C. Hazen. 1990. Molecular analysis of deep subsurface bacteria. International Symposium on Microbiology of the Deep Terrestrial Subsurface
  1474. Hazen, Terry C.. 1990. Bioremediation of toxic waste sites at the Savannah River Site. U. S. Environmental Protection Agency Headquarters, EPA-DOE committee on environmental restoration
  1475. Hazen, Terry C.. 1990. Bioremediation, a natural solution to pollution. Department of Microbiology, University of Georgia
  1476. Hazen, Terry C.. 1990. HAZWRAP research/demonstration in TCE bioremediation at the Savannah River Site. U. S. Department of Energy (HAZWRAP) and U. S. Air Force Technology Transfer Symposium
  1477. Hazen, Terry C.. 1990. Microbiology of the Deep Terrestrial Subsurface. Department of Microbiology, University of Georgia
  1478. Hazen, Terry C.. 1990. How clean is clean enough? GRI/IGT Workshop on Environmental Biotechnology. Third International IGT Symposium on Gas, Oil, Coal and Environmental Biotechnology
  1479. Hazen, Terry C.. 1990. Proposed sOILs Facility for bioremediation of petroleum contaminated soil at the Savannah River Site. Groundwater Division of the South Carolina Department of Health and Environmental Control
  1480. Hazen, Terry C.. 1990. Bioreactors for the degradation of trichloroethylene in groundwater. DOE Office of Technology Development Integrated Demonstration Meeting for Program Managers
  1481. Hazen, Terry C.. 1990. Bioremediation of toxic waste sites at the Savannah River Site. DOE Review of Environmental Biotechnology
  1482. Hazen, Terry C.. 1990. International Symposium on Environmental Biotechnology. EERO - GBF International Symposium Environmental Biotechnology
  1483. Hazen, Terry C.. 1989. Microbiology of the Deep Terrestrial Subsurface. Department of Biology, University of Dayton
  1484. Hazen, Terry C.. 1989. Microbiology of the Deep Terrestrial Subsurface. Department of Biology, University of North Carolina at Charlotte
  1485. Hazen, Terry C.. 1989. SRL-GRI cooperative R and D for demonstration of a methanotrophic bioreactor for degradation of TCE and PCE in groundwater. Annual meeting of investigators in the Biotechnology Program of the Gas Research Institute
  1486. Jimenez, L., G. Lopez De Victoria, Terry C. Hazen and C. B. Fliermans. 1989. Molecular Characterization of Heterotrophic Bacterial Communities from Deep Terrestrial Subsurface Environments. 89th annual meeting of the american society for microbiology 89:290.
  1487. Lopez De Victoria, G., L. Jimenez, Terry C. Hazen and C. B. Fliermans. 1989. Chemotactic Behavior of Deep Terrestrial Subsurface Bacteria. 89th annual meeting of the american society for microbiology 89:290.
  1488. Soto, J. M. and Terry C. Hazen. 1989. Total Coliforms Fecal Coliforms and Fecal Streptococci as Indicators of Public Water Supply Quality in the Tropics. 89th annual meeting of the american society for microbiology 89:291.
  1489. Hazen, Terry C. 1989. Biodegradation of Trichloroethylene. Annual meeting of the American Society for Microbiology
  1490. Hazen, Terry C. 1989. Indicators and Pathogens in Tropical Waters. Annual meeting of the American Society for Microbiology
  1491. Hazen, Terry C.. 1989. Bioremediation of Toxic Waste Sites. Department of Biology, Davidson College
  1492. Hazen, Terry C.. 1989. Biotechnology as a Career. Ruth Patrick Science Center, Summer Students
  1493. Hazen, Terry C.. 1989. Central Shops Diesel Storage Facility Bioremediation Feasibility Status Report. DOE Groundwater Update
  1494. Hazen, Terry C.. 1989. Central Shops Diesel Storage Facility Bioremediation Feasibility Status Report. Savannah River Laboratory
  1495. Hazen, Terry C. 1989. Ethics of Genetic Engineering. Ruth Patrick Science Center, Summer Students
  1496. Hazen, Terry C.. 1989. Microbiology of the Deep Terrestrial Subsurface. South Carolina Water Resources Commission
  1497. Hazen, Terry C.. 1989. Colloquia: Indicators and Pathogens in Tropical Waters. International Colloquia on Water Quality in the Humid Tropics UNESCO
  1498. Hazen, Terry C.. 1989. Deep Terrestrial Subsurface Microbiology. Department of Microbiology, University of Hawaii
  1499. Hazen, Terry C.. 1989. What do Fecal Coliforms Indicate in Tropical Waters?. Water Resources Institute, University of Hawaii
  1500. Hazen, Terry C. 1989. Deep Probe investigators meeting progress. U. S. Department of Energy and Florida State University
  1501. Hazen, Terry C.. 1989. Bioremediation of Toxic Waste Sites. Sigma Xi of Puerto Rico
  1502. Hazen, Terry C.. 1989. Bioremediation of Toxic Waste Sites. Sigma Xi of Puerto Rico
  1503. Hazen, Terry C.. 1989. Bioremediation to toxic waste sites at Savannah River Site. Second International Symposium on gas, oil//coal biotechnology, Institute of Gas Technology
  1504. Hazen, Terry C.. 1989. Bioremediation, a natural solution to pollution. Central Savannah River Area Sigma Xi Chapter
  1505. Hazen, Terry C.. 1989. Survival and Activity of Indicators and Pathogens in Water. National Marine Fisheries Laboratory of Charleston
  1506. Hazen, Terry C.. 1989. Symposium: Deep Terrestrial Subsurface Microbiology. Annual meeting of the American Society of Industrial Microbiologists
  1507. Hazen, Terry C.. 1989. Bioremediation of Toxic Waste Sites. Department of Biology, Georgia Institute of Technology
  1508. Hazen, Terry C.. 1989. Microbiology of the Deep Terrestrial Subsurface. Department of Biology, Georgia State University
  1509. Hazen, Terry C.. 1988. Survival of indicators and pathogens in natural environments. Northwest regional meeting of the American Society of Microbiology
  1510. Hazen, Terry C.. 1988. Life in the deep terrestrial subsurface. Rennsalaer Polytechnic Institute
  1511. Bermudez, M. and Terry C. Hazen. 1988. DNA Homology between a Reference Strain of Escherichia-Coli and Escherichia-Coli Isolated from Tropical Pristine Waters. annual meeting of the american society for microbiology 88:245.
  1512. Jimenez, L., I. Muniz and Terry C. Hazen. 1988. Survival and Activity of Salmonella-Typhimurium and Escherichia-Coli in Tropical Freshwater. annual meeting of the american society for microbiology 88:246.
  1513. Lopez De Victoria, G., L. Jimenez, Terry C. Hazen and C. B. Fliermans. 1988. Comparison of Bacteria from Water and Sediment Cores in the Terrestrial Subsurface. annual meeting of the american society for microbiology 88:252.
  1514. Bermúdez, M. and Terry C. Hazen. 1988. Phenotypic and genotypic comparison of Escherichia coli from pristine tropical waters. Annual meeting of the American Society for Microbiology
  1515. Hazen, Terry C.. 1988. What do Fecal Coliforms Indicate in Tropical Waters?. Wake Forest University
  1516. Fliermans, C. B, H. W. Bledsoe, L. Jiménez, Terry C. Hazen, T. J. Phelps and F. J. Wobber. 1988. Microbiology and geological comparisons of the terrestrial deep subsurface. Annual meeting of the American Geophysical Union
  1517. Hazen, Terry C, L. Jiménez, G. López de Victoria and C. B. Fliermans. 1988. Comparison of Bacteria from Water and Sediment Cores in the Terrestrial Subsurface. Annual meeting of the Hazardous Materials Control Research Institute
  1518. Hazen, Terry C.. 1988. Indicators in Tropical Waters. First Biennial Symposium on Water Quality
  1519. Domingo, J. S., F. Fuentes and Terry C. Hazen. 1987. Comparison of the in-Situ Survival Rates and Activity of Escherichia-Coli and Streptococcus-Faecalis in Tropical Marine Waters. 87th annual meeting of the american society for microbiology 87:256.
  1520. Jimenez, L., W. E. Arias, T. Lugo and Terry C. Hazen. 1987. Densities and Physiological Activity of Planktonic and Epilithic Bacteria in Tropical Freshwater Environments. 87th annual meeting of the american society for microbiology 87:246.
  1521. Rojas, Y. A., I. Muniz and Terry C. Hazen. 1987. Survival of Vibrio-Cholerae in Treated and Untreated Rum Distillery Effluents. 87th annual meeting of the american society for microbiology 87:246.
  1522. Hazen, Terry C.. 1987. Computer Data Acquisition and Transfer, Modem and Instrument Interfacing. Compu-Campus IV - Turabo University
  1523. Negrón, A, I. Pérez and Terry C. Hazen. 1987. Isolation of Legionella species from cooling towers in the tropics. Annual meeting of the American Society for Microbiology
  1524. Rivera, S, T. Lugo and Terry C. Hazen. 1987. Autecology of Vibrio vulnificus and Vibrio parahaemolyticus in tropical waters. Annual meeting of the American Society for Microbiology
  1525. Santo Domingo, J, F. A. Fuentes and Terry C. Hazen. 1987. Comparison of the in situ survival rates and activity of Escherichia coli and Streptococcus faecalis in tropical marine waters. Annual meeting of the American Society for Microbiology
  1526. Hazen, Terry C.. 1987. Computer usage in the laboratory, a faculty workshop. Sacred Heart University MBS Program
  1527. Hazen, Terry C.. 1987. Microbial indicators of water quality: Reassessing the standards. Savannah River Laboratory, DuPont
  1528. Hazen, Terry C, G. A. Toranzos and L. Jiménez. 1987. Workshop on detecting Salmonella spp. in the environment. Puerto Rico Department of Health
  1529. Hazen, Terry C.. 1987. Life in the deep terrestrial subsurface. Sigma Xi Club of San Juan
  1530. Hazen, Terry C.. 1986. Drinking water microbiology. Puerto Rico Society of Microbiologists
  1531. Hazen, Terry C.. 1986. Computer use in the sciences. Honor Students Symposium, Sacred Heart University
  1532. Hazen, Terry C.. 1986. Water quality in Puerto Rico. BetaBetaBeta, Biology Week, University of Puerto Rico
  1533. Hazen, Terry C.. 1986. Genetically - Engineered Microbes in the Environment. BetaBetaBeta, Biology Week, University of Puerto Rico
  1534. Hazen, Terry C.. 1986. Microbial Ecology of Tropical Waters. Industrial Microbiology Symposium, University of Puerto Rico
  1535. Hazen, Terry C.. 1986. Pathogens and their indicators in the waters and shellfish of Puerto Rico. Natural History Society of Puerto Rico
  1536. Fuentes, F. A., J. Santo Domingo and Terry C. Hazen. 1986. Behavioral Response of Three Marine Microorganisms Towards Petroleum Hydrocarbons. 86th annual meeting of the american society for microbiology 86:258.
  1537. Lopez De Cardona, I., M. Bermudez and Terry C. Hazen. 1986. Survival of Poliovirus in a Tropical Lagoon. 86th annual meeting of the american society for microbiology, 86:252.
  1538. Rojas, Y. A., A. Negron, W. Arias and Terry C. Hazen. 1986. Characterization of Bacterial Communities from Treated and Untreated Rum Distillery Effluents. 86th annual meeting of the american society for microbiology 86:251.
  1539. Hazen, Terry C.. 1986. Survival and distribution of pathogens in tropical marine waters and seafood. Regional AAAS Symposium Marine Industries: Promising Future to Old and New Ventures
  1540. Hazen, Terry C.. 1986. Aquatic Microbial Ecology. School of Medicine, University of Puerto Rico
  1541. Hazen, Terry C.. 1986. Genetically - Engineered Bacteria in the Environment. University Honors Program, University of Puerto Rico
  1542. Arias, W. E. and Terry C. Hazen. 1986. Epilithic communities in a tropical rain forest watershed. Fourth International Symposium on Microbial Ecology
  1543. Hazen, Terry C.. 1986. Human Pathogens in the Environment. Fourth International Symposium on Microbial Ecology
  1544. Hazen, Terry C.. 1986. Microbial Ecology of Tropical Waters. Chautauqua Short Course, NSF - Chautauqua Field Centers
  1545. Gómez, E, Y. A. Rojas and Terry C. Hazen. 1986. Distribution of Vibrio cholerae in rum effluents. Annual Junior Technical meeting of the American Chemical Society of Puerto Rico
  1546. Hazen, Terry C.. 1986. Genetically - Engineered Bacteria in the Environment. Special Topics in Modern Biology, University of Puerto Rico
  1547. Hazen, Terry C.. 1986. Survival and activity of pathogens and their indicators in tropical waters. Marine Science Program, Humacao University
  1548. Rojas, Y. A. and Terry C. Hazen. 1986. Bacterial communities from treated and untreated rum distillery effluents. Annual Junior Technical meeting of the American Chemical Society of Puerto Rico
  1549. Hazen, Terry C.. 1985. Survival and activity of pathogens and their indicators in tropical environments. Savannah River Ecology Laboratory, University of Georgia
  1550. Hazen, Terry C.. 1985. Data Management and Analysis in Microbiology. Puerto Rico Society of Microbiologists
  1551. Hazen, Terry C.. 1985. Survival and activity of coliforms in tropical waters. Department of Biochemistry, University of Puerto Rico, School of Medicine
  1552. Hazen, Terry C.. 1985. Survival and activity of pathogens and their indicators in tropical waters. Department of Physiology, Cayey School of Medicine
  1553. Hazen, Terry C.. 1985. Water Microbiology. Puerto Rico Society of Microbiologists
  1554. Arias, W. E. and Terry C. Hazen. 1985. Epilithic Communities in a Tropical Rain Forest Watershed. 85th annual meeting of the american society for microbiology 85:222.
  1555. Elias-Maldonado, E. and Terry C. Hazen. 1985. Screening of a Fluorescent Antibody for the Presumptive Detection and Identification of Yersinia-Enterocolitica. 85th annual meeting of the american society for microbiology 85:226.
  1556. Fuentes, F. A. and Terry C. Hazen. 1985. Petroleum Biodegradation in Tropical Nearshore Coastal Waters Receiving the Effluents from a Petroleum Refinery. 85th annual meeting of the american society for microbiology 85:232.
  1557. Elías-Maldonado, E. and Terry C. Hazen. 1985. Screening of a fluorescent antibody for the presumptive detection and identification of Yersinia enterocolitica. Annual meeting of the Puerto Rico Society for Microbiology
  1558. Fuentes, F. A. and Terry C. Hazen. 1985. Petroleum biodegradation in tropical near-shore coastal waters receiving the effluents from a petroleum refinery. Annual meeting of the Puerto Rico Society for Microbiology
  1559. Hazen, Terry C.. 1985. I got it, now what do I do with it? - A seminar on statistical decisions. Annual meeting of the Puerto Rico Society for Microbiology
  1560. Hazen, Terry C.. 1985. Aquatic Microbial Ecology. School of Medicine, University of Puerto Rico
  1561. Hazen, Terry C.. 1985. The ecology of Aeromonas hydrophila and disease in fish and alligators. University of Arizona
  1562. Hazen, Terry C.. 1985. Microbiological pollution of the environment. School of Medicine, University of Puerto Rico
  1563. López de Cardona, I, M. Bermúdez* and Terry C. Hazen. 1985. Survival of poliovirus in a tropical lagoon. Semi-annual meeting of the Puerto Rico Society of Microbiologists
  1564. Santiago, J. and Terry C. Hazen. 1985. Comparison of four membrane filter methods and MPN for enumeration of fecal coliforms in tropical waters. Semi-annual meeting of the Puerto Rico Society of Microbiologists
  1565. Santo-Domingo, J, F. Fuentes and Terry C. Hazen. 1985. Survival and activity of Streptococcus faecalis and Escherichia coli in near-shore marine waters receiving petroleum effluent. Semi-annual meeting of the Puerto Rico Society of Microbiologists
  1566. Hazen, Terry C.. 1985. Workshop on Computer-Assisted-Instruction in Biology. InterAmerican University
  1567. Hazen, Terry C. 1984b. Workshop on computer programming and applications for faculty. Resource Center for Science and Engineering
  1568. Hazen, Terry C. 1984a. Workshop on computer programming and applications for faculty. Resource Center for Science and Engineering
  1569. Hazen, Terry C.. 1984. Seminar on how to do biological research (methods and funding). InterAmerican University
  1570. Hazen, Terry C.. 1984. Workshop on Computer-assisted-instruction modules for biology. InterAmerican University
  1571. Hazen, Terry C.. 1984. Workshop on computer programming and applications for faculty. Resource Center for Science and Engineering
  1572. Carrillo, M., E. Estrada and Terry C. Hazen. 1984. Evaluation of Bifidobacteria as a Potential Indicator of Human Fecal Contamination in Tropical Fresh Water. 84th annual meeting of the american society for microbiology 84:ABSTRACT N34.
  1573. Fuentes, F. A. and Terry C. Hazen. 1984. Diauxic growth of Pseudomonas aeruginosa PRG-1 on glucose and benzothiophene. Annual meeting of the American Society for Microbiology
  1574. Hazen, Terry C.. 1984. The ecology of Aeromonas hydrophila and red-sore disease in the southeastern United States. Oakland University
  1575. Hazen, Terry C.. 1984. How to become a scientist and environmental microbiology. Walled Lake Central High School
  1576. López de Cardona, I, M. Bermúdez, L. Almodovar, E. Medina and Terry C. Hazen. 1984. Enteroviruses in shellfish from a tropical lagoon. Annual meeting of the American Society for Microbiology
  1577. Elias-Maldonado, E. and Terry C. Hazen. 1984. Survival and Activity of Yersinia-Enterocolitica in a Tropical Rain Forest Watershed. 84th annual meeting of the american society for microbiology 84:ABSTRACT N16.
  1578. Satiago, J., Terry C. Hazen and N. Perez-Rosas. 1984. Comparison of 4 Membrane Filter Methods and Most Probable Number for Enumeration of Fecal Coliforms in Tropical Waters. 84th annual meeting of the american society for microbiology 84:ABSTRACT N32.
  1579. Hazen, Terry C.. 1984. Microbiological water quality in Puerto Rico. Catholic University of Puerto Rico
  1580. Hazen, Terry C.. 1984. Biogeochemical cycling. School of Medicine, University of Puerto Rico
  1581. Hazen, Terry C.. 1984. Microbiological pollution of the environment. School of Medicine, University of Puerto Rico
  1582. Hazen, Terry C.. 1984. El uso de la microcomputadora. Honors Program, University of Puerto Rico
  1583. Elías de Maldonado, E. E. and Terry C. Hazen. 1984. Sobrevivencia y actividad de Yersinia enterocolitica en la cuenca de un bosque tropical. American Chemical Society of Puerto Rico annual junior technical meeting
  1584. Hazen, Terry C.. 1984. Environmental Microbiology. Millipore Corp. and the Puerto Rico Society for Microbiology
  1585. López, I, M. Bermúdez*, L. Almodovar, E. Medina and Terry C. Hazen. 1984. Enteroviruses from a tropical lagoon. American Chemical Society of Puerto Rico annual junior technical meeting
  1586. Santiago, J. and Terry C. Hazen. 1984. Comparison of four membrane filter methods and MPN for enumeration of fecal coliforms in tropical waters. American Chemical Society of Puerto Rico annual junior technical meeting
  1587. Hazen, Terry C.. 1983. Microcomputers documentation in general: statistical analysis, graphics, budgeting, etc. Annual Chemical Conference of the Puerto Rico Chemists Association
  1588. Hazen, Terry C.. 1983. Non-fermentative gram-negative bacilli. Puerto Rico Society for Microbiology Workshop
  1589. Hazen, Terry C.. 1983. In situ diffusion chamber studies of bacteria and yeast in tropical environments. American Society for Microbiology annual meeting
  1590. Fuentes, F. A. and Terry C. Hazen. 1983. Bacterial Chemo Tactic Response Towards Mostos. 83rd annual meeting of the american society for microbiology 83:N33.
  1591. Lopez-Torres, A. J. and Terry C. Hazen. 1983. Density Distribution and Activity of Klebsiella-Pneumoniae in a Tropical Rain Forest Watershed. 83rd annual meeting of the american society for microbiology 83:N88.
  1592. Valdes-Collazo, L., Terry C. Hazen and A. J. Schultz. 1983. Survival and Distribution of Candida-Albicans in Tropical Waters. 83rd annual meeting of the american society for microbiology 83:N89.
  1593. Carrillo, M. and Terry C. Hazen. 1983. Bifidobacterium as an indicator of human fecal contamination in tropical freshwaters. Puerto Rico Society for Microbiology annual meeting
  1594. Hazen, Terry C.. 1983. Microcomputers in the laboratory. Colegio de Quimicos de Puerto Rico
  1595. Hazen, Terry C.. 1983. Statistics in research investigation. Workshop for High School Science Teachers
  1596. Esch, G. W. and Terry C. Hazen. 1983. Long-term study of Crepidostomum cooperi in the burrowing mayfly, Hexagenia limbata. Annual meeting of the American Society of Parasitologists
  1597. Hazen, Terry C.. 1983. A comparison of in situ survival and activity of four enteric bacteria and one yeast in tropical marine and freshwaters with special attention to the effects of water quality. Third International Symposium on Microbial Ecology
  1598. Hazen, Terry C.. 1983. R