2004 RESEARCH PROJECTS Biotransformation PROJECT: Anaerobic Biotransformation and Mobility of Pu and of Pu-EDTA PRINCIPAL INVESTIGATOR: Harvey Bolton Biotransformation The complexation of radionuclides (e.g., plutonium (Pu) and 60Co) by co-disposed ethylenediaminetetraacetate (EDTA) has enhanced their transport in sediments at DOE sites.  Our previous NABIR research investigated the aerobic biodegradation and biogeochemistry of Pu(IV)-EDTA.  Plutonium(IV) forms stable complexes with EDTA under aerobic conditions and an aerobic EDTA degrading bacterium can degrade EDTA in the presence of Pu and decrease Pu mobility.  However, our recent studies indicate that while Pu(IV)-EDTA is stable in simple aqueous systems, it is not stable in the presence of relatively soluble Fe(III) compounds (i.e., Fe(OH)3(s) - 2-line ferrihydrite).  Since most DOE sites have Fe(III) containing sediments, Pu(IV) can not be the mobile form of Pu-EDTA in groundwater.  The only other Pu-EDTA complex stable in groundwater relevant to DOE sites would be Pu(III)-EDTA, which only forms under anaerobic conditions.  Research is therefore needed to investigate the biotransformation of Pu and Pu-EDTA under anaerobic conditions.  The biotransformation of Pu and Pu-EDTA under various anaerobic regimes is poorly understood including the reduction kinetics of Pu(IV) to Pu(III) from soluble (Pu(IV)-EDTA) and insoluble Pu(IV) as PuO2(am) by metal reducing bacteria, the redox conditions required for this reduction, the strength of the Pu(III)-EDTA complex, how the Pu(III)-EDTA complex competes with other dominant anoxic soluble metals (e.g., Fe(II)), and the oxidation kinetics of Pu(III)-EDTA.  Finally, the formation of a stable soluble Pu(III)-EDTA complex under anaerobic conditions would require degradation of the EDTA complex to limit Pu(III) transport in geologic environments.  Anaerobic EDTA degrading microorganisms have not been isolated.  These knowledge gaps preclude the development of a mechanistic understanding of how anaerobic conditions will influence Pu and Pu-EDTA fate and transport to assess, model, and design approaches to stop Pu transport in groundwater at DOE sites. PROJECT: Uranium immobilization through Fe(II) biooxidation:  A column study PRINCIPAL INVESTIGATOR: John Coates Biotransformation Current research on the bioremediation of heavy metals and radionuclides is focused on the ability of reducing organisms to use these metals as alternative electron acceptors in the absence of oxygen and thus precipitate them out of solution.  However, many aspects of this proposed scheme need to be resolved, not the least of which is the time frame of the treatment process.  Once treatment is complete and the electron donor addition is halted, the system will ultimately revert back to an oxic state and potentially result in the abiotic reoxidation and remobilization of the immobilized metals.  In addition, the possibility exists that the presence of more electropositive electron acceptors such as nitrate or oxygen will also stimulate the biological oxidation and remobilization of these contaminants.  The selective nitrate-dependent bio-oxidation of added Fe(II) may offer an effective means of “capping off” and completing the attenuation of these contaminants in a reducing environment making the contaminants less accessible to abiotic and biotic reactions and allowing the system to naturally revert to an oxic state. Our previous NABIR funded studies have demonstrated that radionuclides such as uranium and cobalt are rapidly removed from solution during the biogenic formation of Fe(III)-oxides.  In the case of uranium, X-ray spectroscopy analysis indicated that the uranium was in the hexavalent form (normally soluble) and was bound to the precipitated Fe(III)-oxides thus demonstrating the bioremediative potential of this process.  Recently, we demonstrated that nitrate-dependent Fe(II)-oxidizing bacteria are prevalent in the sediment and groundwater samples collected from sites 1 and 2 and the background site of the NABIR FRC in Oakridge, TN.  However, all of our studies to date were performed in batch experiments in the laboratory with pure cultures and although a significant amount was learned about the microbiology of nitrate-dependent bio-oxidation of Fe(II), the effects of complex processes (such as advective flow) present in the natural environment are unknown.  The objective of the current proposal is to address some of these short-comings in an attempt to develop this bioremediative strategy into a robust, field applicable technology.  These studies will be based on continuous flow experiments performed with “two-dimensional” columns using both model sand matrices and natural soils collected from the NABIR Field Research Center (FRC), TN.  They will yield important information on the controlling variables involved and will identify crucial parameters required for the successful application of this technology.  The proposed research addresses the long-term BER program goal of developing science-based solutions for cleanup of DOE contaminated sites by creating a two-dimensional model system designed to test the remediative potential of Fe(II) biooxidation on uranium contamination in-situ. This project is a collaborative effort between the laboratories of Drs. John D. Coates at University of California Berkeley, Laurie A. Achenbach at Southern Illinois University, and Michelle Scherer at the University of Iowa.  Dr. Coates will responsible for the oversight of the entire project, the construction and operation of the columns, and basic microbial enumeration studies and geochemical analysis.  Dr. Achenbach will be responsible for all molecular aspects of this project, while Dr. Scherer will be responsible for iron mineral characterization and identification.   PROJECT: Composition, Reactivity, and Regulation of Extracellular Metal-Reducing Structures (Bacterial Nanowires) Produced by Dissimilatory Metal Reducing Bacteria PRINCIPAL INVESTIGATOR: Yuri Gorby Biotransformation Microbial growth and metabolism are typically limited by low concentration of available carbon and electrons in oligotrophic subsurface environments.  Stimulation of indigenous microbial populations following injection of compounds such as acetate and lactate confirm this condition.  However, the burgeoning microbial populations quickly deplete dissolved electron acceptors, such as oxygen, and thereby produce a condition of electron acceptor limitation.  This transition from one limitation to another significantly influences metabolic status of bacteria that experience it. We recently discovered that the dissimilatory metal reducing bacterium Shewanella oneidensis strain MR-1 displays extracellular appendages composed of a structural pilin under conditions of electron acceptor limitation.  We have compelling evidence that decaheme cytochromes are associated with and probably adorn the surface of these appendages and that heme iron in these cytochromes is in the reduced (ferrous) state under conditions of electron acceptor limitation.  When electron acceptor limitation is relieved by the addition of either dissolved or solid phase electron acceptor (including nitrate, fumarate, hydrous ferric oxide, ferric-NTA, uranium, etc.) heme iron is instantaneously oxidized and the appendages are rapidly (within seconds) consumed and sequestered in the periplasmic region of the cell. This response can also facilitate the transport of mineral precipitates from the cell exterior to the periplasmic region through pores in the outer membrane.  This strategy for disposing of electrons under electron acceptor limited conditions is not unique to Shewanella.  Searches of complete genomic sequences reveal that Geobacter sulfurreducens, among others, contain homologous gene sequences for components of the respiratory appendages.  Moreover, we have demonstrated that Geobacter displays morphologically similar appendages in response to electron acceptor limitation. This discovery fundamentally changes our perception of the biological mechanisms involved in dissimilatory metal reduction.  Understanding this complex response and its biogeochemical implications will significantly advance NABIR science.  Furthermore our emphasis on membrane surface structures and extracellular appendages has potential to provide totally new insights and concepts on how bacteria engage with mineral surfaces to transfer electrons across the complex solid-liquid interface, and the mechanisms of iron and trace metal/radionuclide mineral biosynthesis.  These scientific issues are basic in concept and have implications well beyond the NABIR program such as to global biogeochemical cycles, fundamental biogeochemical controls on contaminant concentrations in vadose zone porewaters and groundwater, and the bacterial access and utilization of energy stored in mineral solids.  However, this proposal is limited to scientific investigations that address the objectives of the NABIR program. This proposal seeks to study metabolic, physiologic, and morphologic response of metal reducing bacteria to electron acceptor limited conditions expected within subsurface sediments.  Particular emphasis is given to evaluating the structure and reactivity of “bacterial nanowires” and their role in reducing and precipitating heavy metals and radionuclides.   PROJECT: Investigating In Situ Bioremediation Approaches for Sustained Uranium Immobilization Independent of Nitrate Reduction PRINCIPAL INVESTIGATOR: Tom Phelps  Biotransformation Uranium and radionuclide contamination at DOE facilities frequently occurs with nitrate. Current bioremediation strategies reduce nitrate/nitrite before reducing U and metals. While consistent with traditional thinking, this approach is problematic within the geochemical environment of DOE wastes where nitrate may persist for centuries.  The goal of this research is to investigate bioremediation strategies involving the addition of specific energy sources and nutrients, such as propane, hydrogen and triethylphosphate, that can effectively immobilize metals and radionuclides without requiring complete nitrate removal.  Such strategies would be compatible with the current FRC Area 1 environmental constraints of high nitrate, low permeability, and low pH. We hypothesize that if even a fraction of the microbial reducing potential can be directed towards U and metals, the reduced species can be stable in anaerobic waters making nitrate/nitrite removal unnecessary. Our initial objective is to demonstrate that the stringent removal of oxygen from groundwater is a key for U(IV) stability rather than removal of nitrate/nitrite.  Accordingly, transient influxes of nitrate would effect little change if oxygen was rapidly eliminated and metal reducing conditions reestablished.  A second objective is to stimulate extant microorganisms to reduce metals in microcosm experiments with high nitrate and low pH.  Our final objective is to develop supporting approaches minimizing impacts of nitrate reduction that might facilitate U(IV) re-oxidation and mobilization. Evidence that a small percentage of the potential bioreduction capacity within microcosms can be directed toward U reduction and its sustained immobilization would constitute success.  Through this microcosm-based laboratory research, we will investigate innovative biotransformation strategies providing long-term stability of immobilized metals and radionuclides without requiring nitrate removal.  This research will also enhance our understanding of interrelationships between biological nitrate and metal reduction in low pH environments.   PROJECT: Promoting uranium immobilization by the activities of microbial phosphatases PRINCIPAL INVESTIGATOR: Patricia Sobecky  Biotransformation The goal of this proposal is to examine the role of nonspecific phosphohydrolases in naturally occurring subsurface microorganisms for the purpose of promoting the immobilization of radionuclides through the production of phosphate precipitates. This study will focus on uranium (U), a radionuclide that poses significant risk to human health and the environment.  Nonspecific acid phosphohydrolases are a broad group of secreted microbial phosphatases that function in acidic-to-neutral pH ranges and utilize a wide variety of organophosphoester substrates. We hypothesize that subsurface microorganisms that exhibit (acid) phosphatase activity and are resistant to heavy metals have the potential to immobilize U via a biomineralization process. Biomineralization, defined as the immobilization of an element by non-redox microbial precipitation, could prove to be a feasible alternative or complementing remediation approach to U bioreduction and adsorption processes.  As a proof-of-principle for the proposed strategy, genetically modified subsurface microbes with enhanced phosphatase activity were previously tested.  During aerobic growth on a model organophosphorus compound (i.e., 10 mM glycerol-3-phosphate) in sterile soil incubations, the phosphatase activity of the modified microbes resulted in the accumulation of dissolved inorganic phosphate [(PO43-); >200 µM].  In cell-free supernatants the liberated reactive PO43- was shown to remove as much as 69% of U from aqueous solution. In this proposal the primary objective is to demonstrate that the intrinsic phosphatase activities of indigenous subsurface microbes result in the release/accumulation of sufficient PO43- to cause the formation and precipitation of low solubility U-phosphate minerals in oxygenated groundwater and soil. We will examine three critical hypotheses (1) acid phosphatases of subsurface microbes provide resistance to heavy metals and this phosphatase-mediated resistance trait has been disseminated in subsurface populations by lateral gene transfer; (2) phosphatase activity of these subsurface microbes will promote U immobilization by formation of insoluble U-phosphate precipitates; and (3) subsurface geochemical parameters (pH, nitrate) will affect phosphate mineral formation by altering microbial phosphatase activity and/or affecting the stability of the metal phosphate precipitates.  The proposed biotransformation, with its emphasis on an aerobic process, can be considered to serve as a secondary biobarrier strategy for U immobilization should the metal precipitates formed by dissimilatory mechanisms remobilize due to a change in redox state. The research hypotheses and experimental approaches are thus designed to gain insight and knowledge into the microbial metabolic process and environmental factors that promote the immobilization of U via the formation of U-phosphate mineral.     [Back to Award Recipients Page]