2003 RESEARCH PROJECTS Community Dynamics and Microbial Ecology PROJECT: Ecological interactions between metals and microbes that impact bioremediation PRINCIPAL INVESTIGATOR: Allan Konopka Community Dynamics and Microbial Ecology This project’s focus is to understand the structure and function of microbial communities present in subsurface soils contaminated with metals. Although these metals are toxic to many living organisms, some microbes contain genetic traits that make them resistant to these metals. Furthermore, microbes have mechanisms whereby these resistance genes can be transferred to unrelated bacteria. We are specifically interested in chromium, an important pollutant at DOE sites; neither the biochemical basis nor the genetics of Cr resistance is well understood. Our objectives are (1) to determine the effects of environmental factors on community responses to Cr(VI) contamination. These effects may be mediated by physiological responses, species selection, or gene transfer. (2) Determine the role of mobile genetic elements that confer Cr resistance. Microbes with these elements might function as either “bioprotectants” through their physiological activity, or reservoirs of transferable resistance genes. (3) Identify novel physiological and genetic bases for high-level bacterial resistance to Cr(VI). In addition, metals often are unevenly distributed in soils, which can lead to local differences in microbial activity and diversity. We will also determine the distribution of bacterial diversity and metal-resistance genes at these small local scales. The research entails a combination of field-oriented analyses and laboratory simulations of field conditions in microcosms. Both biochemical and molecular techniques are used to determine the dynamics of microbial community structure and function under these conditions, with the long-term objective of identifying microbes and environmental conditions that can are optimal for bioremediation of contaminated sites. PROJECT: Subsurface Bacterial Community Dynamics in the Presence of Plutonium and Uranium PRINCIPAL INVESTIGATOR: Cheryl R. Kuske Community Dynamics and Microbial Ecology The goals of this project are to determine the effects of Pu(VI) on natural bacterial communities in anaerobic subsurface sediments, and to compare bacterial community dynamics in the presence of Pu(VI) or U(VI). Using subsurface sediments from DOE sites, we will conduct laboratory time course experiments where native bacteria are exposed to Pu(VI) or U(VI). We will use a combination of methods to specifically analyze Fe(III)- and sulfate-reducing bacterial groups known to reduce U(VI), and to conduct a broader survey of potentially important soil species that are active in the presence of Pu or U. We will measure bacterial abundance of target bacterial groups using real-time quantitative PCR assays, followed by analysis of species composition in those groups found to be active and/or correlated with actinide reduction. In parallel, we will use terminal restriction fragment profiling to determine broad changes in the total bacterial community as affected by actinide presence. To insure that potentially important species that are not members of our quantitative PCR groups are not missed, we will also assess total bacterial diversity and composition by 16S clone/sequence libraries conducted at certain points in our time course experiments. This study will contribute to the NABIR program by providing information on the dynamics of natural sediment communities in the presence of Pu(VI) and U(VI) and by identifying groups of indigenous bacteria from subsurface sediments that are active in the presence of Pu(VI) or U(VI).   PROJECT: Molecular Analysis of Rates of Metal Reduction and Metabolic State of Geobacter Species During In Situ Uranium Bioremediation PRINCIPAL INVESTIGATOR: Derek Lovley  Community Dynamics and Microbial Ecology Our previously funded NABIR Biotransformations research conclusively demonstrated that microbial U(VI) reduction in uranium-contaminated aquifers can be stimulated with the simple addition of acetate and that stimulating this process can effectively remove uranium from contaminated groundwater. A field experiment confirmed the results of the laboratory sediment incubations, but also revealed that long-term injection of acetate may deplete Fe(III) oxides from the site of acetate injection and promote the growth of acetate-oxidizing sulfate reducers, which appeared to be less effective than Geobacter species in reducing U(VI). These results from the field experiment demonstrate a need for further study of subsurface biotransformations in order to optimize the long-term in situ uranium bioremediation process. Therefore, the objective of this research is to evaluate previously unconsidered aspects of the competition for electron donors near injection wells during in situ bioremediation, the implications of this for U(VI) reduction, and strategies for manipulating biotransformations to promote long-term U(VI) reduction. Studies in which sediments and groundwater from uranium-contaminated sites will be incubated in flow-through columns, as well as field studies, will examine the effect of providing different organic electron donors at various concentrations on long-term U(VI) reduction. It is hypothesized that addition of acetate in concentrations sufficient to consume all of the sulfate near the point of injection will provide enough acetate to maintain an active, downgradient population of Geobacter species, which will continue to remove U(VI) from the groundwater. One alternative strategy is to diminish the competitiveness of sulfate reducers near the point of injection by increasing Fe(III) concentrations in the sediments. Another strategy is to add lactate, instead of acetate, because lactate may stimulate the growth of lactate-oxidizing sulfate-reducing microorganisms which can effectively reduce U(VI) as well as provide acetate for Geobacter species. The potential for dissimilatory metal-reducing microorganisms to reduce U(VI) adsorbed onto sediments will also be examined as this is significant pool of U(VI) at many subsurface sites. Furthermore, the kinetics of U(IV) oxidation with nitrate and oxygen once addition of electron donor is terminated will be evaluated. These studies are expected to expand the basic understanding of biotransformation processes in uranium-contaminated subsurface sediments and help identify successful strategies for long-term in situ bioremediation. PROJECT: Importance of mobile genetic elements and conjugal gene transfer for subsurface microbial community adaptation to biotransformation of metals PRINCIPAL INVESTIGATOR: Soren Sorensen Community Dynamics and Microbial Ecology  Based on the results of past NABIR projects we will expand our studies on the role of horizontal gene transfer (HGT) in adaptation to metal stress by using culture independent techniques. Thus, for the first time examination of HGT will encompass the huge diversity of uncultured bacteria in the subsurface environment. The overall goal of the project is to reveal the importance of mobile genetic elements and conjugal gene transfer for subsurface microbial community adaptation to mercury and chromium stress and to biotransformation. Our studies will examine the interaction between the fate of these metals in the subsurface and the microbial community structure and activity. Specifically, we will seek to answer whether intrinsic processes in subsurface communities can be harnessed and stimulated to drive metal biotransformation and immobilization. To maximize the relevance of our findings, we will employ DOE-site derived materials to identify and study relevant metal-biotransforming characters (strains and plasmids), examine the incidence of lateral gene transfer events, and perform lab-scale microcosms studies to biostimulate in situ metal transformation rates. Our ultimate goal is to provide a science-based approach to stimulate in situ Hg and Cr reduction kinetics via a minimally intrusive stimulation and control of subsurface microbial communities. Ultimately, our findings will promote development of effective and mechanistically validated bioremediation technologies for subsurface contaminated with Hg and Cr. These technologies will be of direct value to DOE, but also to other federal agencies and industries that may be confronted with subsurface metal contamination. To accomplish these goals we will: Isolate and characterize hitherto uncultured subsurface bacteria (and their plasmids) of relevance for biotransformation of metals (Hg(II) and Cr(VI)). Qualitatively and quantitatively study horizontal gene transfer among uncultured subsurface bacteria. Study the effect of environmental stress on horizontal gene transfer in subsurface microbial communities. Investigate the significance of mobile genetic elements for microbial adaptation to metal stress Attempt to use horizontal gene transfer as a tool to enhance the reduction of Hg(II) and Cr(VI) by indigenous subsurface soil bacteria.   PROJECT: Towards Understanding Population Dynamics of Metal and Radionuclide Reducers at Field Remediation Sites PRINCIPAL INVESTIGATOR: Jim Tiedje  Community Dynamics and Microbial Ecology Uranium, chromium and technetium, are important mobile pollutants in soils and groundwaters at DOE facilities. They potentially can be reduced to non-mobile forms by microbes in nature and thereby arrest the further spread of these metals. Successful remediation by immobilization requires stimulating effective metal and radionuclide reducing microbial populations in a reliable way over large areas, and maintaining these elements in immobilized form. To reliably achieve this remediation process, one needs to establish cause and effect relationships between the treatment, active populations and the metal reduction in the field. This project is aimed at defining which microbial groups actually carryout the metal reductions in the field so that one can better define the necessary conditions for the most effective populations, and to produce a set of field-ready methods that can be used to determine which populations respond in the field at remediation sites. This will be done under the following objectives: (1) Determine the competitive fitness of known metal reducers under conditions that simulate field site(s), (2) determine the microbial populations important for the reduction of U, Cr and Tc at the NABIR-Field Research Center (FRC) using metagenomics-based approaches, and (3) apply the developed molecular technologies for quantifying which microbial populations grew during field implementation of remediation. To achieve these objectives, we will evaluate the competitiveness of the four best known metal reducing genera, Geobacter, Shewanella, Geothrix and Desulfitobacterium, against the FRC native community in column microcosms, and compare these results to the outcome of the field implemented remediation at the FRC. We will evaluate the competitiveness of the populations using multiple primer set real-time (Q)-PCR, RNA-based stable isotope probing using 13C-labeled substrates that would be practical for biostimulation and several types of DNA microarrays, including community genome arrays (CGAs), functional gene arrays (FGAs) and arrays newly developed using metagenomic approaches, to characterize the unculturable metal reducers. In the last year, based on the microcosm and field experience, we will select and optimize a subset of these methods that are deployable for support of DOE remediation objectives. This research is being conducted as a collaborative project by scientists at Michigan State University (MSU) and Oak Ridge National Laboratory (ORNL)   PROJECT: Development and Use of Integrated Microarray-based Genomic Technologies for Assessing Microbial Community Composition and Dynamics PRINCIPAL INVESTIGATOR: Jizhong Zhou  Community Dynamics and Microbial Ecology Rapid, parallel, and cost-effective detection tools that can be operated in real time and in heterogeneous field-scale environments are needed for assessing microbial community dynamics. The overall goal of this project is to develop a rapid, parallel, and cost-effective, integrated microarray-based genomic technologies for assessing the structure and dynamics of microbial populations impacted by radioactive and metal contaminants. Towards this goal, the following objectives will be pursued: (1) To further develop and evaluate the 50mer functional gene arrays and SSU rRNA gene-based oligonucleotide microarrays for assessing microbial community composition and dynamics in the NABIR Field Research Center; (2) To develop and optimize novel methods for uniform, sensitive and quantitative amplification of microbial communities; (3) To determine how radioactive and mixed waste contaminants affect the structure, dynamics, and responses of microbial communities using the developed oligonucleotide arrays; and (4) To develop bioinformatics tools for designing oligonucleotide probes for microbial community analysis. We will first focus on developing various microarray-based experimental and computational genomic technologies. Then we will utilize these technologies to understand the dynamics of the microbial communities at NABIR FRC using the samples from different field research projects. In addition, we will integrate our information on microbial community dynamics with existing site geochemistry data to understand how contaminants and remediation treatments affect microbial community diversity, structure, activities and dynamics. Finally we will construct a database to manage the massive microarray data   [Back to Award Recipients Page]