2001 RESEARCH PROJECTS Program Element 1 Biotransformation and Biodegradation PROJECT: The Kinetics of Direct Enzymatic Reduction of Uranium(VI): Effects of Ligand Complexation and U(VI) Speciation PRINCIPAL INVESTIGATOR: Calvin Ainsworth PROGRAM ELEMENT 1 Biotransformation and Biodegradation Calculations based on literature values for reduction of the U(VI)- (poly)maleic acid complex (an analog for fulvic acid) suggest that dissolved humic substances will decrease U(VI) reduction rates to an extent that would require an in situ bioreduction zone 32 meters thick. Interdisciplinary laboratory research is proposed to investigate U(VI)-ligand species reduction rates by anthraquinone-2,6-disulfonate (AQDS), hematin (a core structure in cytochrome c3), cytochrome c3 (from D. vulgaris), and intact cells of D. vulgaris (a DMRB organism) as a function of ligand type and structure. The central tenets are: i) reduction of U(VI) follows the electron-transfer (ET) mechanism developed by Marcus; and ii) it is this transfer reaction that dominates the mechanism of U(VI) reduction and is the step that is most affected by complexation, and hence rate-limiting in cell-free systems. Research is based on hypotheses related to i) only those ligands that exhibit complexation and configuration constraints toward the ET process will be significant, ii) the ET step in these reactions will follow Marcus theory, and iii) only those ligands exhibiting both complexation and conformational constraints will decrease the ET rates to such an extent that they will govern whole cell U(VI) reduction. The reduction of complexed U(VI) will be investigated via stopped-flow kinetics with fluorescence, UV/Vis and electron spin resonance spectroscopy detection. Experimental rate data will be collected in a systematic manner, through the selection of specific ligands under specific conditions (pH, pCO2 , temperature, etc.), for AQDS, Hematin, Cytochrome c3, and whole cells to understand the variations in U(VI) reduction observed in the environment. Marcus theory will be used to develop a fundamental basis and understanding of electron-transfer reaction in these systems and to develop a predictive structure-reactivity relationship based on theory and experimental data. PROJECT: Field-Portable Immunoassay Instruments And Reagents to Measure Hexavalent Uranium, Other Metal Contaminants, and Chelators PRINCIPAL INVESTIGATOR: Diane A. Blake PROGRAM ELEMENT 1 Biotransformation and Biodegradation The single most expensive component of any environmental remediation process is the cost of analyzing samples before, during, and following the remediation process. The costs of laboratory analyses continue to climb, and the outlay for the assessment of a single site can frequently reach hundreds of thousands of dollars. The goal of this project is to continue the development and validation of a portable antibody-based sensor that will yield reliable data in real time (i.e., <1 hour), be field-ready (i.e., simple, durable, and accurate), and present low costs (i.e., << $100/test and <$5000 for the initial equipment investment. The goals for the 3-year project period are 1) to test and validate the present uranium sensor and develop protocols for its use at the NABIR Field Research Center; 2) to develop new reagents that will provide superior performance for the present hand-held immunosensor; and 3) to develop new antibodies that will permit this sensor to also measure other environmental contaminants (chromium, mercury, and/or DTPA). A better understanding of in situ bioremediation processes and the development of strategies to enhance bacterial remediation of contaminated sites depend either directly or indirectly upon accurate detection and measurement of organics, metals, and other toxic elements prior to, during, and following the remediation process. This project will provide basic scientific knowledge that could revolutionize clean-up technologies and significantly reduce future costs PROJECT: Iron Reduction and Radionuclide Immobilization: Influence of Natural Organic Matter & Reaction-Based Modeling PRINCIPAL INVESTIGATOR: William D. Burgos PROGRAM ELEMENT 1 Biotransformation and Biodegradation The purposes of the proposed research are to evaluate the potential for humic substance addition to stimulate in situ Fe(III)-reducing activity and U(VI) bioimmobilization, and to construct and validate a comprehensive reaction-based model to simulate these processes. Humic substances are known to stimulate solid-phase metal oxide reduction by their ability to shuttle electrons between dissimilatory metal-reducing bacteria (DMRB) and oxide surfaces. In addition, our recent research has demonstrated that humics can also enhance solid-phase ferric oxide reduction through complexation of biogenic Fe(II). The presence of humic substances is therefore likely to increase the abundance and activity of DMRB. The addition of a natural, native material [e.g., FRC humic substances at the DOE Field Research Center (FRC)] may be more acceptable to the general public and key stakeholders compared to other biostimulants such as quinones or complexants. The proposed research is based on a series of hypotheses regarding the impact of free oxide surface site concentration, chemical speciation of surface-associated Fe(II), natural organic matter (NOM) and hydrologic transport on the kinetics and thermodynamics of ferric oxide bioreduction, uranium reduction and DMRB growth. A central hypothesis is that the rate and extent of the many simultaneous reactions can be described by a series of reaction-based rate formulations/parameters and equilibrium constants, and complex systems (e.g., NOM-U(VI)-ferric oxide-DMRB) can be simulated using a reaction-based biogeochemical model by carefully designing separate experiments to independently parameterize important reactions and incrementally adding system complexity. The objectives of our proposed research are to elucidate the factors controlling the rate and extent of biological ferric oxide reduction in subsurface sediments, the mechanisms of U(VI) immobilization under Fe(III)-reducing conditions, and the influence of NOM on these processes. The emphasis of our proposed research is to independently describe important reactions using reaction-based model formulations/parameters. More complex systems will be examined by the incremental addition of other reactive species (e.g., U(VI) and NOM), reactive processes (e.g., DMRB growth) or hydrologic transport. Our approach involves the following experimental and concurrent modeling efforts using synthetic hematite-coated sand, an FRC reference sediment, and the FRC reference humic substances. (1) Kinetic measurements of Fe(III) bioreduction in subsurface sediments and associated DMRB growth. (2) Kinetic measurements of the bioreduction of U(VI) and FRC humics. (3) Kinetic and equilibrium measurements of Fe(II) and U(VI) complexation by solid sediments and dissolved FRC humics. (4) Kinetic and equilibrium measurements of the abiotic reduction of U(VI) by biogenic Fe(II) and bioreduced FRC humics. (5) Experiments examining the above suite of reactions occuring in concert in batch and column reactors. (6) Construction and validation of a series of reaction-based models (incorporating both kinetic and equilibrium speciation formulations) of uranium immobilization under Fe(III)-reducing conditions. PROJECT: Immobilization of Radionuclides through Anaerobic bio-oxidation of Fe(II) PRINCIPAL INVESTIGATOR: John D. Coates PROGRAM ELEMENT 1 Biotransformation and Biodegradation Current research on the bioremediation of heavy metals and radionuclides is focussed 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, 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 exits that the presence of oxygen will also stimulate the biological oxidation and remobilization of these contaminants. The selective anaerobic 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. Adsorption of heavy metals and radionuclides onto iron and manganese oxides has long been recognized as an important reaction for the immobilization of these compounds. We propose to further investigate the applicability of the anaerobic bio-oxidation of Fe(II) added to contaminated systems resulting in the production of Fe(III)-oxides. Our studies will focus on Dechloromonas species which we have previously demonstrated to be a ubiquitous group of Fe(II)-oxidizers in the environment that can couple anaerobic oxidation of Fe(II) to the reduction of either nitrate or (per)chlorate. We have previously demonstrated that bio-oxidation of Fe(II) by these organisms and their close relatives Dechlorosoma species will result in the adsorption and immobilization of uranium and cobalt. This strategy may be applied in two ways: (i) by precipitating Fe(III)-oxides over previously immobilized heavy metals/radionuclide contamination in-situ, forming an insoluble barrier that will crystallize with time, inhibiting future bio-reduction and adsorbing any leached metal contaminants or (ii) by engineering an Fe(III)-oxide wall in-situ, downstream of the immobilized heavy metal/radionuclide contamination which will “catch” and adsorb any heavy metals and radionuclides that may be solubilized and re-mobilized as a result of environmental fluxes such as re-oxidation (biotically or abiotically) or ligation. Our proposed studies will further investigate several aspects of Fe(II) oxidation including the biochemistry and genetic systems involved in the oxidation of Fe(II) by Dechloromonas species, the crystalline iron mineral phases produced by these organisms and the environmental conditions required to produce them, the stability of the crystalline phases biologically produced, and the binding capacity and stability of binding for uranium of the various Fe(III)-oxides produced. These studies will lend new insight into the potential effects and applicability of Fe(II) oxidation to the permanent immobilization and remediation of radionuclides in the environment. PROJECT: Fe(II)-Induced Inhibition of Dissimilatory Bacterial Reduction of Metals and Radionuclides: The Role and Reactivity of Cell-Surface Precipitates PRINCIPAL INVESTIGATOR: Yuri A. Gorby PROGRAM ELEMENT 1 Biotransformation and Biodegradation Dissimilatory metal reducing bacteria (DMRB) help control the biogeochemistry of anoxic, non-sulfidogenic subsurface and sedimentary environments, principally through the enzymatic reduction of iron and manganese oxide minerals. Biogenic Fe(II) resulting from the metabolic activity of DMRB is chemically reactive and can influence a variety of geochemical processes controlling the fate and transport of heavy metals and radionuclides. Certain forms of Fe(II) serve as electron donors for the reduction and precipitation of U(VI), Cr(VI), and Tc(VII). Hence, conditions that retard or inhibit iron reduction decrease the efficiency by which DMRB ameliorate contaminant transport. Biogenic Fe(II) phases formed at the cell-mineral interface may profoundly influence metal reduction by DMRB and can significantly contribute to redox reactions involving multivalent heavy metals and radionuclides important to DOE environmental restoration efforts. Recent results in our laboratory demonstrate that biogenic Fe(II) accumulates as precipitates on cell surfaces and these precipitates effectively slow or arrest anaerobic respiration. Preliminary computational and experimental results indicate that mixed Fe(II)-Fe(III) layered double hydroxyl-salt green rusts are the dominant mineral phases that form on cell surfaces. Green rusts are redox reactive (i.e., they can chemically reduce multivalent heavy metals and radionuclides) and are common to anaerobic, iron-reducing environments. Circumstantial evidence suggests that DMRB overcome inhibitory effects of these products of iron respiration by forming and releasing small outer membrane vesicles that are encrusted by Fe(II)-Fe(III) minerals. Collectively, these phenomena have important implications for controlling the rate, extent, and products of metal reduction in anoxic, subsurface environments, and the viability of bioremediation schemes based on this process. This project will (1) characterize the composition and evaluate the redox-reactivity of biogenic Fe(II) minerals formed on the surface of DMRB, (2) investigate the nature of cell-surface associated Fe(II) inhibition of metal reduction, (3) identify environmental and physiological conditions, (such as pH, carbonate concentration, ionic strength, and enzymatic reduction rate), that could minimize inhibitory effects of Fe(II). PROJECT: Effect of Microbial Exopolymers on the Spatial Distributions and Transformations of Cr and U at the Bacteria-Geosurface Interface PRINCIPAL INVESTIGATOR: Dr. Ken Kemner PROGRAM ELEMENT 1 Biotransformation and Biodegradation The microenvironment at and adjacent to actively metabolizing cell surfaces can be significantly different from the bulk environment. Cell surface polymers, metabolic products, etc. can set up steep chemical gradients over very short distances. Furthermore, characteristics of the polymers produced and the chemical gradients they create might differ under different biogeochemical conditions. Predicting the behavior of contaminant radionuclides and metals in such microenvironments is currently difficult because the chemistry of these environments has been difficult or impossible to define. The behavior of contaminants in such microenvironments can ultimately affect their macroscopic fates. Information about biogeochemical interactions at the microbe-geosurface microenvironment is paramount for predicting the fate of contaminants and effectively designing bioremediation approaches. The scientific goal of this proposal is to identify the interactions among contaminants (Cr and U), mineral surfaces, and microbial extracellular materials occurring near the mineral-microbe interface. The technological goals are (1) to use X-ray absorption fine structure spectroscopy to determine the chemical speciation of contaminants (Cr and U) exposed to bacterial exopolysaccharides, iron oxides, and bioreducing bacteria (Shewanella putrefaciens) and (2) to use X-ray microimaging and spectromicroscopy to determine the spatial distribution and chemical speciation of Cr and U near the interface of actively reducing S. putrefaciens in contact with a hydrated iron (hydr)oxide surface. Results from this work will directly address the Biotransformation component of the NABIR Program by enabling a better understanding of the role of cell surface polymers and metabolic products in the biotransformation of metals and radionuclides at the mineral-microbe interface. PROJECT: New Catalytic DNA Biosensors for Radionuclides and Metal ions PRINCIPAL INVESTIGATOR: Yi Lu PROGRAM ELEMENT 1 Biotransformation and Biodegradation We aim to develop new DNA biosensors for simultaneous detection and quantification of bioavailable radionuclides, such as strontium, uranium, technetium, and plutonium, and metal contaminants, such as lead, chromium, and mercury. The sensors will be highly sensitive and selective, not only for different metal ions, but also for different oxidation states of the same metal ion. They will be applied to the on-site, real-time assessment of concentration, speciation, and stability of the radionuclides and metal contaminants during and after bioremediation. To achieve this goal, we will employ a combinatorial method called “in vitro selection” to search for catalytic DNA molecules that are highly specific for radionuclides or other metal ions. Comprehensive biochemical and biophysical studies will be performed on the selected DNA molecules. The findings from these studies will elucidate the structure/function relationship in catalytic DNA and thus facilitate the design of improved sensors. The DNA will be labeled with fluorescent donor/acceptor pairs to investigate, and to signal, the structural changes upon metal ion binding. Once a collection of individual DNA sensors is identified, each specific for a particular metal ion at a particular concentration range, they will be assembled into a DNA microarray for the simultaneous detection and quantification of all radionuclide and metal contaminants. PROJECT: Mesoscale Coupled Transport and Biogeochemical Effects on Reduction of U(VI) and NO3- as Co-contaminants in Natural Sediments and Soils PRINCIPAL INVESTIGATOR: Jiamin Wan PROGRAM ELEMENT 1 Biotransformation and Biodegradation The primary objective of this research is to bridge the gap between understanding biotransformations of redox-sensitive metals in well-mixed laboratory batch systems and in natural subsurface environments. While the behavior of contaminants has become better understood in batch systems, predicting contaminant behavior in the field remains an outstanding challenge. Subsurface heterogeneity is a main cause of this gap. Subsurface environments are strongly transport-limited, and can be categorized into advective and diffusion-limited domains. Batch studies are inherently deficient for predicting larger scale phenomena because fluids in subsurface environments are not well-mixed, even at relatively small scales (10-4 to 10-1 m). Core-scale investigations are equally limited in applicability since their study yields no direct information on critical intra-core structure and mechanisms controlling macroscopic observations. Information is specifically lacking concerning local, diffusion-limited processes that arise from small-scale variations in permeability, preferential flow paths, and soil/sediment structure. We propose to study the coupled transport and biogeochemistry of U(VI) and NO3-. The redox reactions of these co-occurring contaminants will be investigated in realistically heterogeneous model and natural systems. We will emphasize direct measurements within diffusion-limited domains at the mesoscale (defined here as the typical diffusion-limited scale, ranging from about 10-4 to 10-1 m). It is within these diffusion-limited domains that large gradients in microbial activity, chemical potentials, reaction rates, and transport rates can coexist. Thus, the dynamics at this unexplored mesoscale can control redox-dependent biotransformations in nature. [Back to Award Recipients Page]