2000 RESEARCH PROJECTS Program Element 1 Biotransformation and Biodegradation PROJECT: Biodegradation of PuEDTA and Impacts on Pu Mobility PRINCIPAL INVESTIGATOR: Harvey Bolton Jr. PROGRAM ELEMENT 1 Biotransformation and Biodegradation Plutonium (Pu) contamination of sediments and groundwater at many Department of Energy (DOE) sites is a long-term problem. Ethylenediaminetetraacetate (EDTA) was co-disposed with Pu, forming strong PuEDTA complexes, and enhancing Pu transport at many sites. EDTA poses a long-term problem of potentially disseminating Pu and other radionuclides (e.g., 60Co) in the subsurface environment, because it is recalcitrant to biodegradation. Biodegradation of EDTA is a permanent solution to decrease chelate assisted radionuclide transport through destruction of the PuEDTA complex and the precipitation of insoluble PuO2. However, this biodegradation is not well understood because of the lack of information on PuEDTA aqueous species, microbial degradation (e.g., uptake into the cell and enzymology of degrading enzymes), and the effect of physicochemical factors (e.g., Pu:EDTA ratio, CO2 partial pressure, pH, redox, and other metals) on the rate and ability of microorganisms to degrade PuEDTA. We will investigate the aerobic biodegradation of Pu(IV)EDTA and the location and mobility of the Pu, transport of EDTA complexes into the cell, and the genetics and enzymology of aerobic EDTA biodegradation. We will also enrich and isolate an anaerobic EDTA degrading bacterium to determine how the anaerobic biodegradation of PuEDTA may impact the groundwater mobility of Pu. This research will provide the necessary mechanistic understanding of how microbial biodegradation of PuEDTA will affect the groundwater mobility, fate, and transport of Pu in both oxidizing and reducing groundwaters present at DOE sites. PROJECT: Reductive Precipitation and Stabilization of Uranium Complexed with Organic Ligands by Anaerobic Bacteria PRINCIPAL INVESTIGATOR: A. J. Francis PROGRAM ELEMENT 1 Biotransformation and Biodegradation This research addresses the principal mechanisms of microbial alteration of organic- radionuclide complexes and the resultant impacts on radionuclide solubility and stability under anaerobic conditions. This is a collaborative research involving Brookhaven National Laboratory, Colorado School of Mines (B.D. Honeyman), and State University of New York at Stony Brook (G.P. Halada). We propose to (i) elucidate the mechanisms of biotransformation and fate of uranium complexed with organic chelating agents under anaerobic conditions; (ii) identify the factors which regulate the bioreduction of complexed uranium leading to decomplexation and precipitation of reduced uranium, and (iii) enhance the reductive precipitation and stabilization of soluble complexes of uranium under anaerobic conditions in the subsurface. In this study biotransformation of uranium-organic complexes by iron-reducing, fermentative- and sulfate-reducing bacteria, and mixed cultures isolated from NABIR field site will be examined. The influence of soluble and particulate organic matter, pH and ionic strength on the rate and extent of biotransformation and stabilization of reduced uranium will be investigated. Speciation and characterization of uranium complexes in solution and solid phases will be accomplished by using the advanced spectroscopic techniques (XPS, XANES, EXAFS, EDX, SIMS, FTIR, TOF-SIMS and LD-ITMS). Basic information obtained from this study can be used in the development of in situ stabilization of radionuclides by enhancing the biotransformation of organic/inorganic radionuclide complexes in the subsurface environments by anaerobic microorganisms. PROJECT: Impacts of Mineralogy and Competing Microbial Respiration Pathways on the Fate of Uranium in Contaminated Groundwater PRINCIPAL INVESTIGATOR: Joel E. Kostka PROGRAM ELEMENT 1 Biotransformation and Biodegradation The proposed research will elucidate how mineral-bacteria interactions limit the migration of U in contaminated sediments from the NABIR-Field Research Center (FRC). We will focus on the competition between Fe(III)-reducing bacteria (FeRB) and the sulfate-reducing bacteria (SRB), their impact on Fe mineralogy in the subsurface, and how these microbially-mediated mineralogical changes will govern U speciation. The proposed work will 1) comprehensively characterize the dominant Fe and S minerals that are likely to limit U speciation in situ, 2) directly quantify reaction rates and pathways of terminal electron-accepting processes which control subsurface sediment chemistry, and 3) identify and enumerate the organisms mediating U geochemistry using molecular biological anlysis. We will focus on representative subsurface sediments which vary substantially in sediment chemistry such as parent rock mineralogy, groundwater sulfate and nitrate concentrations. For the less studied layer silicate and sulfide mineral groups, we will quantify the impacts of terminal electron-accepting pathways and the resulting reductive dissolution processes mediated by bacteria on the sorption of U. Through determination of reaction rates of important microbial respiration pathways and an in-depth characterization of minerals likely to predominate U sorption, we will provide important inputs for reactive transport models which may be used to predict U flow in subsurface sediments. PROJECT: Sediment Studies of the Biological Factors Controlling the Reduction of U(VI) PRINCIPAL INVESTIGATOR: Derek R. Lovley PROGRAM ELEMENT 1 Biotransformation and Biodegradation Uranium is one of the most prevalent metal contaminants in the subsurface at many Department of Energy sites. The objective of the studies proposed here is to develop a better understanding of factors which influence the reduction of U(VI) in subsurface environments. The following hypotheses will be tested: 1) direct microbial reduction of U(VI) is a more important process than abiotic U(VI) reduction in uranium-contaminated DOE subsurface sites; 2) microbial reduction of U(VI) can readily be stimulated in contaminated subsurface sediments with the addition of electron donors previously shown to stimulate the reduction of Fe(III) in subsurface sediments; 3) U(VI) is reduced prior to Fe(III) or concurrently with Fe(III) in subsurface environments; 4) nitrate inhibits U(VI) reduction in subsurface sediments, but this is due to competition for electron donors and thus when electron donors are added to accelerate U(VI) reduction, nitrate and U(VI) can be reduced simultaneously; 5) U(VI) is reduced prior to sulfate reduction because sulfate reduction is not competitive for electron donors with U(VI) reduction; 6) biological U(VI) reduction in subsurface sediments follows Michaelis-Menton kinetics with respect to U(VI) at environmentally relevant U(VI) concentrations; and 7) if nitrate is introduced into sediments containing U(IV), the U(IV) will be biologically oxidized to U(VI). These studies will define the parameters controlling biological reduction of U(VI) in uranium-contaminated subsurface sediments. This will provide information that will be useful in predicting intrinsic bioremediation of uranium in the subsurface as well as data that will aid in developing engineered strategies for accelerating subsurface uranium bioremediation in situ. PROJECT: Characterization of Molecular Genetic Events Associated with Colonization of Mineral Surfaces by Geobacter sp. PRINCIPAL INVESTIGATOR: Timothy S. Magnuson, Ph.D. PROGRAM ELEMENT 1 Biotransformation and Biodegradation Recent research has indicated that there is a need for more in-depth investigation into the nature and occurrence of binding to metal surfaces by dissimilatory iron reducing bacteria of the genus Geobacter. It has been demonstrated that these organisms are prevalent in Fe-reducing zones in aquifers and sediments, and likely play an important role in metal cycling, degradation of aromatic hydrocarbons, and detoxification of contaminant metals such as uranium. The objective of the present work involves direct detection of unique RNA transcripts produced by Geobacter grown on different metal oxides and other electron acceptors. An in situ RT-PCR approach, facilitated by the use of primers and molecular beacon probes specific for metal reductase gene sequences, will be used to directly detect the presence of mRNAs that are specifically produced under different growth conditions, including mineral substrate composition and oxygen concentration. We have already demonstrated that primers specific for cytochrome sequences can be used to amplify target DNAs in G. sulfurreducens, G. metallireducens, and G. humireducens. In situ RT-PCR would be particularly useful when using solid phase mineral substrates, because no extraction and purification of RNA from metal oxide containing cultures or samples would be necessary. The results could be used to directly correlate the presence of unique transcripts to colonization of a solid mineral surface. PROJECT: Environmental Actinide Mobilty: Plutonium and Uranium Interactions with Exopolysacharides and Siderophores of Naturally-Occurring Microorganisms PRINCIPAL INVESTIGATOR: Mary P. Neu PROGRAM ELEMENT 1 Biotransformation and Biodegradation The development of actinide and heavy metal bioremediation technologies requires a fundamental understanding of contaminant metal interactions with diverse microbial populations, as well as an understanding of contaminant geochemical behavior. Our goal is to understand how the mobility of radionuclide contaminants is affected by key interactions with aerobic soil microbes: extracellular binding/sorption, siderophore mediated translocation, and metal toxicity. Isolated exopolymer and extracellular binding of U and Pu will be examined using the glutamic acid polymer of B. lichenformis and the polysaccharide exopolymers of R. erythropolis and P. aeruginosa. Siderophore mediated binding and translocation will be studied using S. pilosus, R. rhodochrous strain OFS and P. aeruginosa which produce trihydroxamate, catecholate/carboxylate/hydroxyl, and dihydroxamate/catecholate/carboxylate siderophores, respectively. The stoichiometry and structure of actinide-chelate species and the strength of binding formed will be determined for isolated siderophores and exopolymers. Whole cell studies will focus on determining the quantity of association/translocation, localization, and final speciation of the metal. The effect of U and Pu on microbial viability will be determined which will ensure the validity of our experiments and enhance our knowledge of actinide toxicity. We will perform batch studies in which soil samples from the NABIR field research center at ORNL will be individually combined with actinide species, actinide-siderophore/exopolymer, and actinide-microorganisms. The results of our investigation will fill important gaps in our knowledge of actinide microbial processes for applications as called out by the NABIR Program: 1) fundamental biogeochemistry of actinides 2) effect of chelators on actinide speciation and biotransformation 3) effect of actinide species on common microorganisms 4) effect of microorganisms and their chelators on actinide mobility in soil from a contaminated site. [Back to Award Recipients Page]