1998 RESEARCH PROJECTS Program Element 4 Biogeochemical Dynamics PROJECT: Microbiological Controls of Chelated Radionuclides in Multiscale Structured Media PRINCIPAL INVESTIGATOR: Scott C. Brooks PROGRAM ELEMENT 4 Biogeochemical Dynamics OBJECTIVE: The overall goal of this proposed research is to provide an improved understanding and predictive capability of the mechanisms that allow chelate-degrading bacteria to be effective in the bioremediation of subsurface environments contaminated with toxic metals and radionuclides. The study is motivated by the likelihood that vadose zone microbial activity can effectively consume chelating organic ligands thus facilitating the immobilization of released metals and radionuclides via sorption and precipitation reactions. Our objectives are to (1) develop an improved understanding and predictive capability of the mechanisms governing the biodegradation of cobalt (Co)-NTA and uranium (U)-NTA in unsaturated, structured media from the pore scale to the field macrocosm scale (meter-sized pedon); (2) quantify the microbial and hydrologic conditions that influence the biodegradation of metal-chelating ligands, for the purpose of contaminant containment and remediation in heterogeneous, structured media; and (3) provide integrated experimental and theoretical methodologies for the scale-up of biohydrogeochemical processes from the microscopic scale to the macrocosm (pedon) scale. The proposed work consists of four multidisciplinary hypothesis-driven tasks. APPROACH: Our approach involves the use of (1) a variably saturated dynamic flow technique to quantify the biodegradation of Co(II)-NTA and U(VI)-NTA as a function of pore class size in structured media, (2) multiscale experiments that are designed to control the hydrologic conditions and enhance the microbially induced immobilization of Co-NTA and U-NTA, and (3) existing models that couple microbial and hydrogeochemical processes to conduct multiscale process and parameter upscaling studies. The experimental results will provide new insights concerning the relationship between the biodegradation of radionuclide-chelate complexes and the pore structure and hydrologic connectivity in heterogeneous subsurface environments. PROJECT: Microbially Induced Reduction of Toxic Metals and Radionuclides: Competing Geochemical and Enzymatic Processes PRINCIPAL INVESTIGATOR: Scott E. Fendorf PROGRAM ELEMENT 4 Biogeochemical Dynamics OBJECTIVE: This research is part of the Biogeochemical Dynamics Scientific Program Element, DOE/OBER Natural and Accelerated Bioremediation Research Program (NABIR). Focus will be on the influence of direct microbial reduction versus metabolite induced reduction of Cr and Co-EDTA. Specifically, the research will: (1) define under what conditions microbiological or chemical mechanisms dominate the reduction of Co(III)EDTA and Cr(VI); (2) quantify the reduction rates and mechanisms controlling the mobility of Cr and Co; and (3) identify reaction products both in solution and solid phase. APPROACH: Hypothesis-driven research in batch reactors will be used to: (1) evaluate whether Cr(VI) or Co(III)-EDTA are reduced by direct dissimilatory processes or by secondary reaction with anaerobic microbial metabolites, such as sulfide or ferrous iron; (2) determine reduction rates and mechanisms under a range of environmental conditions; and (3) use electrochemical, chromatographic, and spectroscopic methods to define reaction products in solution and solid phases. PROJECT: Influence of Microbial Nitrate Reduction on Subsurface Iron Biogeochemistry, Microbial Chromium Reduction and Chromium Redox Chemistry PRINCIPAL INVESTIGATOR: Flynn W. Picardal PROGRAM ELEMENT 4 Biogeochemical Dynamics OBJECTIVE: This research is part of the Biogeochemical Dynamics Scientific Program Element, DOE/OBER Natural and Accelerated Bioremediation Research Program (NABIR). Nitrate is a ubiquitous groundwater anion (a byproduct of nuclear fuels reprocessing) that occurs in elevated concentrations in groundwater at DOE sites. The investigators seek to answer questions about the presence of nitrate (in association with metals) at DOE sites: to what extent will nitrate inhibit microbial reduction of iron minerals and what is the effect of iron mineral reduction on the mobility of representative toxic metals? The investigators plan to use natural sediments from Hanford and from the NABIR Oyster, Virginia, site and expect to draw on the PNNL sediment collection for well characterized minerals for which data may exist. They will investigate the complex interactions that occur among (1) populations of microorganisms able to reduce or oxidize metals, (2) solid phase Fe(III)-oxides that can be reduced by bacteria and sorb other metals, and (3) nitrate as it affects the rate of reduction of Fe(III) and other metals. Specifically, the project will examine (1) if and to what extent nitrate will inhibit microbial reduction metals in the presence of Fe minerals; (2) the kinetics of nitrate-dependent, microbial oxidation/reduction of Fe and other metals; and (3) the interactions among microbial reduction of nitrate/Fe(III), nitrate-dependent Fe(II) oxidation, and the speciation of representative metals. As described by the principal investigator, representative metals such as Pb, Cr, and Zn that are sorbed to Fe(III) oxides may be released during reductive dissolution as a result of competition for binding sites by microbially-produced Fe(II); nitrate has the potential to inhibit microbial iron reduction and may, therefore, limit the mobility of sorbed metals. APPROACH: Experiments will be conducted using batch systems consisting of (1) pure cultures with known metabolic capabilities in the Fe and nitrate reduction and nitrate-dependent Fe oxidation; (2) synthetic Fe(III) oxides (amorphous ferric hydroxide, goethite of various surface areas, hematite, and sands coated with similar oxides) and native sediments from the Hanford and Oyster sites; and (3) known concentrations of nitrate and metals. PROJECT: The Immobilization of Radionuclides and Metals in the Subsurface by Sulfate Reducing Bacteria PRINCIPAL INVESTIGATOR: Joseph M. Suflita PROGRAM ELEMENT 4 Biogeochemical Dynamics OBJECTIVE: 60Co, a radionuclide produced in nuclear reactors is a widely used source of radioactivity. As a result of its widespread use, it is found in groundwater near at least 5 DOE facilities. Sulfate-reducing bacteria carry out the reduction of sulfate, producing sulfide and at the same time obtaining reducing equivalents through the oxidation of organic compounds. These microorganisms are ubiquitously found in subsurface environments but are most abundant in locations rich in their nutrient sources (sulfate and organic carbon). APPROACH: Initially, experiments were carried out to determine the effectiveness of pure cultures of sulfate-reducing bacteria, specifically precipitating cobalt as a Co-S complex from an aqueous microbiological medium at pH 7.0. A colorimetric assay was used to detect Co2+ remaining in solution after the incubation period. The detection limit was approximately 0.1 µM (6 ppb). Cobalt was originally added at 1 mM concentration as Co(II)Cl2 or as Co(II) nitrilotriacetate. Strains tested include Desulfovibrio desulfuricans, Desulfoarcula baarsii, Desulfotomaculum sp. strain groll and "Desulfomicrobium hypogeia." These organisms represent a selection of different sulfate reducing bacteria, each with different characteristics and capabilities. "Desulfomicrobium hypogeia." is a strain isolated from the subsurface. In the presence of the CoCl2 at 1 mM concentration D. desulfuricans and "D. hypogeia." reduced the Co concentration below 0.1 µM (>99.99% removal) whereas 100 µM remained in solution in the inhibited incubations. Sulfide production by D. baarsii and D. groll were inhibited at 1 mM concentration. When cobalt was added with nitrilotriacetate, used to simulate naturally occurring chelating ligands, none of the organisms were inhibited. With this ligand, results varied with 98 to 99.94 % removal. Dmac. sp. strain groll was the most efficient at precipitating Co2+ but both D. baarsii and D. desulfuricans also precipitated greater than 99.4% of the Co. These early results suggest that sulfate reducing bacteria are effective in removing and immobilizing cobalt from an aqueous medium and most likely will also remove it from contaminated groundwater given the appropriate conditions. Ligands such as nitrilotriacetate may reduce the toxicity of metals allowing for higher levels of growth of sulfate reducing bacteria and hence enable these microorganisms to produce sulfide. Chelators also serve to maintain a slightly greater amount of the metal in aqueous solution during the precipitation process. These experiments demonstrate the effectiveness of sulfate reducing bacteria at precipitating Co(II) apparently as a sulfide complex. Near term future experiments will be directed at the mechanism of metal precipitation and characterization of the precipitated complex. We intend to carry out similar and more in depth studies with both Co and uranium as well as a variety of additional chelators to determine their influence and obtain additional support for our hypotheses. PROJECT: Solubilization of Radionuclides and Metals by Iron-Reducing Bacteria PRINCIPAL INVESTIGATOR: John M. Zachara PROGRAM ELEMENT 4 Biogeochemical Dynamics OBJECTIVE: Interdisciplinary laboratory research is to be performed on the biogeochemistry of dissimilatory iron-reducing bacteria (DIRB) under conditions of water advection, and their potential for releasing sorbed metals and radionuclides associated with Fe(III)-oxides in soils and subsurface materials. DIRB are subsurface microorganisms that utilize Fe(III)-oxides as electron acceptors under anoxic conditions, controlling poly-valent metal speciation and the mass content and distribution of Fe(III)-oxides. A central tenet is that water advection is required for long-term sustenance of DIRB and Fe(III) reduction, both in nutrient supply and the removal of passivating metabolic byproducts. APPROACH: Research is based on three field-derived hypotheses related to the use of solid-phase Fe(III)oxides as electron acceptors for microbial respiration, the nature of the microbial-reduction product as a control on reduction rate and extent, and the mobilization of sorbed metal contaminants. Columns and stirred-flow reactors with synthetic and subsurface material will be inoculated with DIRB to investigate how different C-sources and e-donors, interfacial chemical reactions, and water advection meter the activity of DIRB under subsurface-relevant conditions. The reductive solubilization of sorbed metals by DIRB will be investigated using synthetic and subsurface materials spiked and aged with metals (Co, Ni, Hg, Eu), and a contaminated subsurface material. A novel modeling approach is proposed whereby surface and aqueous complexation reactions, and precipitation, are linked with the multiple Monod formulation and water transport. The research considers the need for scale-up to the field, and will provide requisite knowledge and insights to exploit DIRB for in-situ bioremediation on DOE lands and other sites nationally. [Back to Award Recipients Page]