2001 RESEARCH PROJECTS Program Element 4 Biogeochemical Dynamics PROJECT: Biogeochemical Processes Controlling Microbial Reductive Precipitation of Radionuclides PRINCIPAL INVESTIGATOR: Jim K. Fredrickson PROGRAM ELEMENT 4 Biogeochemical Dynamics Uranium and technetium, common subsurface contaminants on DOE sites, are problematic because they exist as highly soluble and mobile ions, U as U(VI) carbonate complexes and Tc as (Tc(VII)O4-), in oxidized groundwaters. Laboratory research has demonstrated that dissimilatory metal-reducing bacteria (DMRB) can effectively reduce U(VI) and Tc(VII) to insoluble UO2 and TcO2 phases. Bioreductive immobilization of these contaminants offers considerable promise for in situ remediation of contaminated DOE sites. Before effective in situ bioimmobilization of these contaminants can be realized, complex biogeochemical interaction between contaminants, reactive mineral surfaces, and bacteria must be better understood. Previous NABIR research demonstrated that Mn oxides can oxidize biogenic uraninite (UO2) and that the presence of Mn oxides in suspension of DMRB, U(VI) and H2 impede the reduction of U(VI) and, for some oxides, prevent quantitative reductive precipitation. The goal of this research, building on previous results, is to investigate coupled microbiological-geochemical processes controlling: a) the microbial reduction of U and Tc in the presence of Mn oxides; and b) the role of advective transport on the rate and extent of the coupled reactions. The following hypotheses will be evaluated: the reductive precipitation of U and Tc in relation to cell surface; the extent to which precipitation in the cell periplasm may protect the reduced contaminants against oxidation by Mn oxides; the extent to which Mn oxides, including those present in natural materials from the Oak Ridge FRC and Hanford, oxidize reduced U and Tc and impede reductive precipitation; the influence of reactive transport on net reductive precipitation; and the potential for transport of colloidal contaminant precipitates. This research will utilize two DMRB (Shewanella and Geobacter species) in batch and flow experiments with synthetic and natural Mn oxides to probe hypothesis-driven research. A combination of aqueous chemical, spectroscopy, and microscopy analyses will be used to define U, Tc, and Mn speciation and biogeochemical modeling will be used to aid in the design and interpretation of experiments. Research results are expected to have significant implications for the in situ bioreduction and long-term immobilization of U and Tc and will have immediate applicability to proposed field research at the ORNL Field Research Center and, eventually, other DOE sites such as Hanford and Fernald. PROJECT: Biogeochemistry of Aerobic Solubilization of Pu and U by Microorganisms and their Siderophores, Reductants, and Exopolymers PRINCIPAL INVESTIGATOR: Larry Hersman, Ph.D. PROGRAM ELEMENT 4 Biogeochemical Dynamics Radionuclide contaminated environments are often oxic, including the Rocky Flats Environmental Technology Site (RFETS), and the contaminated groundwater at the NABIR Field Research Center (FRC). Radionuclide distribution within such environments is effected by indigenous biogeochemical processes, including the metabolic activities of aerobic microorganisms, key members being the ubiquitous Pseudomonas and Bacillus genera. Because of the chemical similarities between the actinides, uranium (U) and plutonium (Pu), and iron (Fe), the metabolic processes of these microorganisms that effect the biogeochemistry of Fe could also significantly effect Pu and U distribution. We propose to determine the extent to which metabolic processes involved in Fe acquisition and in exopolymer production affects the distribution of Pu and U between the aqueous and solid phases. First, we will determine the equilibrium distribution of Pu and U between these phases, in the presence and absence of microorganisms, and in relation to Fe bioavailability. Second, using transposon mutagenesis, we will determine to what extent microbial processes (including siderophore production and metabolism, and exopolymer and reductant production) directly or indirectly influence aqueous/solid phase distribution of Pu and U. The results of the research program will contribute to NABIR's stated needs to understand both "the principal biogeochemical reactions that govern the concentration, chemical speciation, and distribution of metals and radionuclides between the aqueous and solid phases" and "what alterations to the environment would increase the long term stability of radionuclides in the subsurface." PROJECT: Microbial Stabilization of Plutonium in the Subsurface Environment PRINCIPAL INVESTIGATOR: Bruce Honeyman PROGRAM ELEMENT 4 Biogeochemical Dynamics Pu contamination is widespread in the surface soils and subsurface sediments throughout the DOE complex. Pu is generally considered to be relatively immobile; however, transport of Pu, albeit at very low concentrations, has been observed at many DOE sites. The focus of this research is to elucidate the processes that can lead to the enhanced stabilization (i.e., immobilization) of soluble (organic- and inorganic-Pu complexes) and colloidal forms of Pu by naturally-occurring microbial communities. While several processes responsible for mobilizing and transporting Pu have been hypothesized, the potential for the stabilization of Pu by microorganisms present in the contaminated environment has not been fully evaluated. Results of this basic research should lead to: (i) a better understanding of environmental conditions likely to foster Pu immobility, and (ii) strategies for engineering the long-term in-situ immobilization of Pu in soils and sediments. At most Pu-contaminated sites, removal of contaminated media is financially prohibitive; the development of methods for the in-situ stabilization of Pu is crucial for the long-term, cost-effective stewardship of Pu contaminated sites. This is a multi-disciplinary, integrated research project involving expertise in actinide microbiology (BNL), surface and coordination chemistry/radiochemistry (Colorado School of Mines) and environmental radiochemistry/biogeochemistry and radiocolloids (Texas A&M). PROJECT: Biogeochemistry of Uranium Under Reducing and Re-oxidizing Conditions: An Integrated Laboratory and Field Study PRINCIPAL INVESTIGATOR: Brent Peyton PROGRAM ELEMENT 4 Biogeochemical Dynamics Previous work in our laboratories shows that Desulfovibrio desulfuricans G20 can reduce U(VI) to U(IV) while associated with hematite, goethite, ferrihydrite, and quartz surfaces, and that the composition of the secondary mineral phase precipitates was significantly different than previous work where no Fe-mineral phase was initially present. In addition, using ion specific microelectrodes, we have shown that the aqueous phase chemistry near a hematite surface, and in accumulations of hematite -associated D. desulfuricans, was significantly different than near a quartz surface. Our results indicate that the composition of the mineral substratum also significantly affects rates of metal precipitation and immobilization, and that the underlying mineral phase affects the rate and extent of U(IV) reoxidation and subsequent mobilization. Finally, our use of open flow reactors that are more representative of in situ conditions indicates that, in the field, groundwater hydrodynamics and a continual influx of substrate and contaminants can yield significantly different results than obtained with closed serum bottles. The combined laboratory and field research proposed here will extend these fundamental results to examine U(VI) reduction and immobilization with mixtures of sulfate reducing bacteria (SRB) and dissimilatory iron reducing bacteria (DIRB) on hematite which is found in the fractured Nolichucky shale at the DOE NABIR Field Research Center (FRC) test site. Using techniques developed to specifically probe biogeochemical processes at both the micro- and meso-scale, the proposed research will expand our current understanding of the roles played by mineral surfaces, bacterial competition, and local hydrodynamics on the reduction and immobilization of uranium by SRB and DIRB. To this end, the following overall experimental hypothesis is proposed: On a hematite mineral surface under anaerobic conditions, accumulations of secondary inorganic precipitates are controlled by a) the bacteria associated with the mineral surface, b) the electron acceptors available for anaerobic bacterial respiration, and c) the local hydrodynamics governing the micro- and meso-scale transport of electron donors and acceptors, nutrients, and other chemical species between the surface and the bulk solution. At the start of the project, chemical, mineralogical, and physical properties of FRC core samples will be characterized to help focus the laboratory studies on field conditions. We will install in-well sampling devices containing slow-release nutrient sources and inserts for flat mineral surfaces to provide primary information about field processes. Upon removal from the field, these surfaces will be analyzed by the same suite of surface and microscopic techniques proposed for the laboratory studies, thus rendering a high degree of overlap between the laboratory and field data. Laboratory studies using channel flow reactors (CFR) optimized for biogeochemical experiments will simulate the interactions of mineral surfaces with mineral-associated SRB and DIRB in the presence of electron acceptors available for anaerobic bacterial respiration at the FRC site. Microelectrodes will profile aqueous phase chemistry from the bulk liquid, through surface-associated accumulations of bacteria/mineral precipitates, to the mineral surface. These profiles are needed to understand the local chemical flux near the mineral surface and how the nature of the mineral and associated bacteria influences near-surface aqueous chemistry. In the field, surface-associated bacteria and secondary mineral precipitation also have the potential to physically plug fractures that are pervasive at the FRC site. This may yield significant and unexpected changes in local groundwater chemistry, microbial ecology, and rates of radionuclide immobilization. A more complex CFR will be used to simulate fracture-flow to examine the impact of hydrodynamics and substrate addition strategies on meso-scale contaminant bio-immobilization processes. PROJECT: Influence of Reactive Transport on the Reduction of U(VI) in the Presence of Fe(III) and Nitrate: Implications for U(VI) Immobilization by Bioremediation/Biobarriers PRINCIPAL INVESTIGATOR: Brian D. Wood PROGRAM ELEMENT 4 Biogeochemical Dynamics The coupling of biogeochemical and transport processes is important to the bioremediation of metals and radionuclides in the field, but experimental research systems with transport and biologically-mediated redox reactions is severely lacking. We propose to examine the reduction of U(VI) in the presence of nitrate and Fe(III)-containing minerals under conditions representative of biostimulation. This research will establish: (1) mechanisms by which the fluxes of electron acceptors, electron donors, and other species can be controlled to maximize the transfer of reductive equivalents to the aqueous and solid phases, and (2) associated process models that describe the transport and reaction of U(VI) and iron species under conditions relevant to bioremediation. This research will utilize DOE subsurface sediments collected from the Hanford site and the Oak Ridge FRC, and synthetic porous media designed to have specific bioavailable iron mineral phases and contents. The facultative dissimilatory metal reducing bacterium Shewanella putrefaciens (strain CN32) will be adopted as a test organism. Experimental research will be conduced using sediment-packed column systems, and will be focused on three main areas: (1) the importance of the abiotic reduction of U(VI) by biogenic Fe(II); (2) the influence of the transport process on Fe(III) reduction and U(VI) immobilization, with an emphasis on methods for controlling the fluxes of aqueous species to maximize uranium reduction; and (3) the reductive capacity of biologically-reduced sediments (with respect to re-oxidation by convective fluxes of O2 and NO3-) and the long-term stability of immobilized uranium mineral phases after bioremediation processes are complete. The proposed research is unique in the NABIR portfolio, and it will provide scientifically-based information that will be useful in the design and assessment of bioremediation strategies for U(VI) as well as other metals and radionuclides. [Back to Award Recipients Page]