1999 RESEARCH PROJECTS Program Element 1 Biotransformation and Biodegradation PROJECT: Impact of Iron-Reducing Bacteria on Metals and Radionuclides Adsorbed to Humic-Coated Iron (III) Oxides PRINCIPAL INVESTIGATOR: William Burgos PROGRAM ELEMENT 1 Biotransformation and Biodegradation A chemical mixture fundamental to the understanding of contaminant mobilization at U.S. Department of Energy sites is cationic metals and radionuclides in combination with natural organic matter such as humic acids. The overall goal of this proposed research is to provide an improved understanding of the mechanisms that control the sorption of cationic metals and radionuclides to humic-coated Fe(III) oxides, the subsequent biotransformations of the humic-coated oxides and solubilization of sorbed contaminants by dissimilatory iron reducing bacteria (DIRB), and the conditions that promote maximum sustained DIRB activity for the bioremediation of contaminated subsurface environments. In addition to this basic science focus, we plan to couple a mechanistic geochemical-biochemical reaction model with our experimental results for future upscaling applications. Little information on metal ion-humic acid-Fe(III) oxide systems is available, yet humic-coated Fe(III) oxides are a critical subsurface component controlling the speciation and distribution of metals and radionuclides. Similarly, many aspects of humic acid-Fe(III) oxide-DIRB interactions are not well characterized yet vital to accurately predicting the field-scale performance of a DIRB bioremediation system. This proposed research is based on four multidisciplinary hypotheses: (1) hydrous metal oxides and oxide-bound humic substances control the speciation and distribution of metals and radionuclides; (2) the interaction of solubilized Fe(II) with "clean" or humic-coated Fe(III) oxides will control DIRB activity and contaminant solubilization; (3) adsorbate-humic acid-Fe(III) oxide surface speciation, DIRB growth rates, and contaminant solubilization can be described by a series of formation constants and rate equations; and (4) upscaling to field conditions can be achieved by coupling our BIOKEMOD model describing the evaluated batch reactions with the HYDROBIOGEOCHEM flow model. The first three hypotheses will be tested by this research. Our experimental approach involves: (1) kinetic and equilibrium measurements of the cationic metal Zn(II) and the cationic radionuclide Sr(II) with an International Humic Substance Society reference humic acid that will be partitioned between solution and adsorbed phases; (2) measurement of the reductive dissolution of the humic-coated Fe(III) oxides and solubilization of associated contaminants with a DIRB-pure culture and -natural consortia recovered from an Fe(III)-reducing subsurface environment; (3) construction of a biogeochemical model for this system, based on experiments and quality-controlled using the mechanistic BIOKEMOD model; and (4) predictively upscaling the suite of processes to field-scale using the reactive transport HYDROBIOGEOCHEM model. This research does not include validation of the model using field data, which will be the subject of subsequent long-term research. Our results will provide fundamental information for the future use of DIRB for the bioremediation of contaminated subsurface environments and support on-going research within DOE's NABIR Program. PROJECT: Immobilization of Heavy Metals and Radionuclides Through Bioxidation of Reducing Environments PRINCIPAL INVESTIGATOR: John D. Coates PROGRAM ELEMENT 1 Biotransformation and Biodegradation Radionuclides and other contaminating heavy metals are readily bound and adsorbed to manganese and ferric oxides present in the environment. In this way, low level contamination of these compounds are immobilized and pose little threat to groundwater supplies. However, if the environment becomes reducing due to co-contamination with organic compounds, microbial activity will rapidly deplete dissolved oxygen. As the next most abundant microbial electron acceptors in many of these environments, Mn(IV)- and Fe(III)-oxides are readily reduced to Mn(II) or Fe(II) with a resulting release of the bound metals. We propose to microbially reoxidize Fe(II) to insoluble Fe(III)-oxides in the anaerobic contaminated environment by taking advantage of the unique metabolism of chlorate-reducing bacteria (ClRB) that can use Fe(II) as an electron donor with chlorate as the sole electron acceptor in anaerobic environments. The Fe(III) produced will abiotically reoxidize any Mn(II) and the Mn(IV)- and Fe(III)-oxides formed will adsorb and immobilize any contaminating heavy metals/radionuclides. We will also take advantage of the fact that many ClRB can be induced to produce extracellular O2 through the dismutation of chlorite into Cl- and O2 in anoxic environments. The proposed process will function at a number of levels (i) the stimulation of the indigenous chlorate-reducing population will reoxidize the Fe(II) and Mn(II) in the environment to immobilize heavy metals and radionuclides (ii) once established, the ClRB will be induced to dismutate chlorite and the biologically produced O2 will stimulate the rapid aerobic degradation of the co-contaminating organics, and (iii) the biologically produced O2 will inhibit any further microbial Mn(IV)- and Fe(III)-reduction The overall result of this process will be the biological removal of the organic contaminants, the reoxidation of the soluble Fe(II), and the readsorption and immobilization of the heavy metals and radionuclides by the insoluble Mn(IV)- and Fe(III)-oxides produced. PROJECT: Biodegradation & Biotransformation of Mixed Wastes Containing Metals & Chlorinated Xenobiotic Compounds by Microbial Consortia Enriched Under Different Physiological Conditions PRINCIPAL INVESTIGATOR: Don L. Crawford PROGRAM ELEMENT 1 Biotransformation and Biodegradation One approach for bioremediation of water contaminated with heavy metals or radionuclides is the use of sulfate-reducing bacteria (SRBs) to precipitate the metals by biogenic sulfide precipitation. Soils can also be bioremediated in this way after acid-leaching the metals from the soil. Bioremediation of acid mine drainage using SRB reactors has been demonstrated full scale. Waters and soils contaminated with organic compounds, including chlorinated aromatic and aliphatic chemicals, can be bioremediated under fermentative, methanogenic, or sulfate-reducing conditions, where biodegradation is carried out by adapted anaerobic bacterial consortia. On the other hand, little research has been devoted to examining how anaerobic bacterial consortia respond to and transform mixed wastes containing both metals and xenobiotics, even though mixed contaminants are found at DOE sites. Incomplete knowledge limits our ability to develop effective bioremediation processes, since we lack an understanding of how microbial populations respond "in situ" to the presence of organics in combination with metals under different physiological conditions. In my laboratory, we have shown that enrichment cultures of fermentative bacteria and SRBs can bioremediate water containing the pentachlorophenol (PCP) in the presence of high concentrations of cadmium (Cd). Our preliminary results are summarized below. There is only a limited amount of other information on anaerobic bioremediation of such mixed wastes. We know little about the specific microbes that are active in degradative consortia that bioremediate xenobiotic-heavy metal wastewater mixtures. We do not well understand how xenobiotic degrading microbial populations respond physiologically to heavy metals or radionuclides as co-contaminants, or the effects that organic contaminants have on heavy metal biotransformations under different physiological conditions. In sulfate-reducing environments, where precipitated metal sulfides are generally insoluble and less toxic, we hypothesize that it will be possible to use adapted anaerobic bacterial consortia containing SRBs for the bioremediation of many heavy metal/xenobiotic-containing waters, provided that the initially soluble forms of the metals, or organic compound metal complexes, do not unacceptably interfere with initial activity of SRBs in the consortia. As compared to sulfate-reducing conditions, we hypothesize that responses to toxicity will differ markedly under fermentative or methanogenic conditions. In the proposed research, we will study biodegradation and biotransformation of heavy metal and chlorinated xenobiotic mixtures in groundwater. We will examine how anaerobic bacterial consortia adapt to, metabolize, and biotransform these mixtures. In phase 1, populations established under aerobic, fermentative, methanogenic and sulfate-reducing conditions will be compared for their metabolism and biotransformation of the chlorinated compounds and metals, initially using a model flask scale system of water contaminated with a mixture such as trichloroethelene (TCE) and cadmium. In phase II, laboratory experiments will be continued in a simulated flowing aquifer by employing core materials obtained from DOE sites. Phase III will involve moving to in situ studies within an aquifer. The 36 month project will be led by Dr. Don L. Crawford, Professor of Microbiology at the University of Idaho. PROJECT: Formation and Reactivity of Biogenic Iron Microminerals PRINCIPAL INVESTIGATOR: Yuri A. Gorby PROGRAM ELEMENT 1 Biotransformation and Biodegradation Radionuclides and heavy metals (e.g., U, Cr, and Ni) pose significant environmental toxicity and health hazards in the subsurface at many of the DOE sites involved in the processing of nuclear materials. The fate and transport of these contaminants are controlled to a large extent by redox chemistry of saturated subsurface sediments and by the nature of the mineral phases that are present. Dissimilatory iron reducing bacteria catalyze many of the reduction reactions in anoxic, non-sulfidogenic environments and are recognized as important agents impacting the migration of metal contaminants in groundwater. Research is proposed to investigate the formation of chemically reactive biogenic ferrous iron minerals formed during the reduction of ferric oxides that are common components of subsurface sediments. The central tenet is that the composition and micromorphology of these minerals are influenced by the microenvironment near the bacterial cell surface and that the chemistry within these microenvironments (e.g., pH, Eh, concentration of cations and anions) is controlled by the rate of iron reduction and physicochemical properties of organic material near the cell surface. Considering that the chemistry of cell-associated microenvironments differ from that of the surrounding medium and that the surfaces of bacterial cells increase the potential for mineral nucleation, the composition and micromorphology of post-reduction minerals may not necessarily be predicted from thermodynamic calculations of the bulk aqueous geochemistry. This research will relate the rate and extent of enzymatic reduction of iron oxide minerals to the incorporation or exclusion of U, Cr, and Ni into post reduction minerals. The results will drive new conceptual models of biomineralization and will expand our knowledge of micro-scale processes that may impact the fate and transport of metal contaminants at pore, core, and field scales. PROJECT: The Role of Natural Organic Matter in Microbial Reduction of Chromate, Pertechnetate, and Uranyl: Linking Chemical Structure to Bioavailability and Redox Reactivity PRINCIPAL INVESTIGATOR: Baohua Gu PROGRAM ELEMENT 1 Biotransformation and Biodegradation The overall goal of the proposed research is to provide a molecular-level understanding of the role that natural organic matter (NOM) plays in facilitating the reductive immobilization of metal and radionuclide contaminants (CrO4-, TcO4-, and UO22+) by anaerobic metal reducing bacteria. The study is motivated by recognition that NOM-mediated electron transfer from microbes to toxic metals (1) may be useful for achieving enhanced immobilization of contaminants, particularly where contaminants are in soil micropores and microbes are excluded due to size or nutrient limitations, but (2) it is still largely a "black box" process due to the complex chemical composition of NOM. Our objectives are to: (1) determine the bioavailability of the major fractions of NOM (i.e., soil humic acid, aquatic humic acid, fulvic acid, and carbohydrate) as electron acceptors for subsurface anaerobic metal-reducing bacteria; (2) quantify the chemical reactivity of redox-active functional groups associated with the NOM fractions that serve to shuttle electrons from microbes to metals and radionuclides; and (3) determine the kinetics of NOM-mediated reductive immobilization of CrO4-, TcO4-, and UO22+ in both batch and column. Advanced spectroscopic and wet-chemical techniques (including NMR, FTIR, and UV molar absorptivity) will be employed to quantify NOM composition in terms of quinone and ketone, aromatic C=C, and carboxyl contents. These functional and structural properties of the NOM fractions will be correlated to observed rates of NOM-mediated electron transfer from microorganisms to metal contaminants. Our findings will directly benefit the application of microbially mediated contaminant stabilization in the field, and contribute to on-going or planned remediation efforts at Hanford's In-Situ Redox Manipulation site, Oak Ridge's Y-12 In-Situ Reactive Barriers site, and Savannah River's Old Burial Ground site.Key Words : Natural organic matter, electron transfer, bioavailability, reductive immobilization, contaminants, metals and radionuclides. PROJECT: Investigation of the Spatial Distributions and Transformations of Cr, Pb, and U Co-contaminant Species at the Bacteria-Geosurface Interface PRINCIPAL INVESTIGATOR: Kenneth M 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 (lipopolysaccharides, extracellular polysaccharides), metabolic products, etc. can set up steep chemical gradients over very short distances. It is currently difficult to predict the behavior of contaminant radionuclides and metals in such microenvironments 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. State-of-the-science X-ray microimaging and spectromicroscopy are powerful techniques for resolving the distribution and speciation of contaminants at the microscopic scale. The objectives of ! this research are (1) to use X-ray microimaging and spectromicroscopy to determine the spatial distribution and chemical speciation of Cr, Pb, and U near the interfaces of Pseudomonas aeruginosa and Shewanella putrefaciens with iron (hydr) oxide and (2) to use this information to identify the interactions among the contaminants, mineral surfaces, and microbial extracellular materials that occur near these interfaces. PROJECT: Bioavailability of Iron (III) in Natural Soils and the Impact on Mobility of Inorganic Contaminants PRINCIPAL INVESTIGATOR: David S. Kosson PROGRAM ELEMENT 1 Biotransformation and Biodegradation Iron oxides, particularly the low crystalline ferrihydrite and amorphous oxyhydroxides, are known to play an important role in the mobility of many inorganic compounds such as heavy metals and radionuclides, as well as serving as an electron acceptor during anoxic biodegradation of many substrates. Our specific objectives are threefold: To determine the potential for increased mobility of heavy metals and radionuclides bound to iron oxides in sediments and floodplain soils at the U.S. DOE Savannah River site (SRS) under conditions supporting microbial Fe(III) reduction. To examine the bioavailability of soil Fe(III) as a function of soil pore structure, particularly the fraction of pores smaller than a diameter that physically excludes bacterial transport. To determine if siderophores will increase either the rate or the extent of microbial Fe(III) reduction in natural soils and sediment systems by increasing the solubility, hence improving the transport properties, of extremely insoluble Fe(III) minerals. Fieldwork will include collection of soil and groundwater samples from SRS seepage basins and analysis for Fe(III) and Fe(II) distribution and solubility, iron mineral speciation, heavy metal and radionuclide characterization, and the influence of Fe(III) reduction on radionuclide and heavy metal mobility. The rate and extent of Fe(III) reduction for pure iron minerals and natural soils will be investigated in batch systems and in columns designed to simulate field conditions on a smaller scale. Indigenous soil microorganisms or enrichment cultures of Fe(III)-reducers obtained from sediments at other sites will be used to inoculate these systems. Siderophores will be studied in batch systems only. This project will improve the basic understanding of microbial Fe(III) reduction and Fe(III) bioavailability in natural environments and the possible impacts of microbial Fe(III) reduction on mobility of inorganic compounds. Microbial Fe(III) reduction is one of the many biological processes that can contribute to rates of natural attenuation of organic contaminants at many field sites. This study attempts to further characterize some the fundamental processes involved in Fe(III) reduction. A better understanding of the effects of Fe(III) reduction on the mobility of inorganic compounds such as heavy metals and radionuclides at sites that are contaminated with both organic and inorganic contaminants is essential to proper risk assessment at those sites.   PROJECT: Biotransformation of Mixed Inorganic Ions: Biochemistry, and Contaminant and Species Interactions in Chromate Reducing Consortia PRINCIPAL INVESTIGATOR: James N. Petersen PROGRAM ELEMENT 1 Biotransformation and Biodegradation Mixtures of metallic and radioactive contaminants, including chromate, constitute a major environmental problem at Department of Energy (DOE) facilities. Recent studies have demonstrated that chromate contamination exists at 13 of the 18 DOE installations studied, and that hexavalent chromium constitutes more than 90% of the total chromium present at these sites. Direct microbial reduction of hexavalent chromium to trivalent chromium, a species that forms insoluble precipitates, is one potential treatment technology for such sites. While previous research has shown the potential for this treatment technology, important questions still remain and must be answered before effective field-scale chromate reduction can be accomplished. These questions include: (1) which members of an environmental consortia are actually responsi-ble for reduction of chromate, (2) for these active chromate reducers, what are the biochemical pathways responsible for chromate reduction, (3) what are the effects of carbon source, and perhaps more impor-tantly, of inorganic co-contaminant ions (e.g., SO42-, TcO4-, and UO22+) on the chromate reduction capacity of the system, and 4) can detailed knowledge of active chromate reducers and their biochemistry be used to facilitate preferential growth (in aquifer systems) of subpopulations that have the greatest specific contaminant reduction rates. A hypothesis driven research plan is presented to address these issues. Using a multidisciplinary, multi-pronged approach, for a subsurface environmental consortium previously enriched from the Hanford Site, we will provide insight and answers to the above questions. We will then test our results using fresh soil cores obtained from the Hanford site and from another chromate contaminated DOE site. PROJECT: Determination of Long Term Stability of Metals Immobilized by In Situ Microbial Remediation Processes PRINCIPAL INVESTIGATOR: Bruce M. Thomson PROGRAM ELEMENT 1 Biotransformation and Biodegradation Current alternatives for remediating contamination of soils and ground water by metals, metalloids, and radionuclides are limited and thus present a major challenge to environmental managers. As a result, DOE and other agencies are supporting investigations towards development of in situ stabilization technologies including use of microbial systems to immobilize these contaminants. Much of the work on biological immobilization strategies has focused on the use of microbially mediated reduction. These strategies are based upon the ability of anaerobic organisms to reduce oxidized, and therefore soluble, metals to insoluble precipitates including oxides (e.g. Cr2O3, UO2), sulfides (e.g. FeS, MnS, FeAsS, AsS2, MoS2), and possibly elemental forms (As and Se). These metals are expected to remain insoluble provided that reducing conditions are maintained in the subsurface formation. However, to date no investigation has considered the stability of these phases over long time periods (i.e., decades to centuries) as geochemical conditions in the formation and ground water change. This project will use a combination of laboratory research and numerical modeling to investigate the long term stability of metals and metalloids immobilized by microbial reduction. The research will focus on arsenic (As), chromium (Cr), selenium (Se) and uranium (U) as these are representative of contaminants found at many DOE sites including U mill tailings sites. Arsenic and Se can also serve as surrogates of high activity radionuclides which are difficult to work with in long duration studies. In addition, iron (Fe) and manganese (Mn) will be included in the experimental program as these are important constituents of most soil and ground water systems, they are biologically active, and their oxidized precipitates are often important scavengers of metal contaminants. Potential release mechanisms include simple dissolution, complexation by organic or inorganic ligands, and oxidation followed by dissolution. The experimental program will involve four sub tasks: (1) generate kg quantities of stabilized metals by techniques established by other investigators, (2) subject these samples to batch leaching tests in the presence and absence of microbial activity, (3) test the leaching of the stabilized metals in one dimensional column tests, and (4) investigate the stability of the stabilized metals in a two dimensional flow cell. The stabilized metals will also be tested for hazardous waste characteristics by the Toxicity Characteristic Leaching Procedure (TCLP) and other tests as appropriate. The theoretical investigation will commence with the use of geochemical modeling to simulate the stability of the immobilized metals in batch tests. This will be extended to incorporate kinetics of abiotic and microbially enhanced leaching. Finally, a coupled geochemical transformation and hydraulic transport code will be used to allow complete simulation of the transformations and transport of metals from an in situ immobilization process. The coupled transformation and transport code will first be calibrated with the experimental results obtained from the two dimenstional flow cell. It will thus permit analysis of the stability of immobilized metals over the very long time periods considered in the performance assessment process. PROJECT: Mesoscale Biotransformation Dynamics as the Basis for Predicting Core Scale Reactive Transport of Chromium and Uranium PRINCIPAL INVESTIGATOR: Jiamin Wan PROGRAM ELEMENT 1 Biotransformation and Biodegradation         Chromium and uranium are among the most common contaminants in subsurface environments at DOE sites. These contaminants have redox-dependent solubilities, mobilities, and toxicities, and biological and chemical reduction can transform these elements to less toxic forms. Although these contaminants have become well understood in batch systems, predictions of their behavior at the core-to-field scales remain an outstanding challenge. Batch studies are inherently deficient for purposes of predicting larger scale subsurface phenomena because they assume well-mixed conditions, whereas most subsurface environments are transport-limited, even at relatively small scales. However, core-scale investigations are equally limited since no direct information is obtained on critical intra-core structure and mechanisms controlling macroscopic observations. Studies based at the heterogeneous core-scale are therefore basically black-box approaches. We propose that studies of realistic heterogeneous model systems and natural systems are needed at the mesoscale (µm-cm), since it is at this unexplored intra-aggregate scale that large gradients in microbial populations, chemical potentials, reaction rates, and transport rates often coexist. The overall objective of this research is to test whether core-scale biotransformations can be predicted based upon understanding mesoscale dynamics. We will follow a process-oriented plan, centered around mesoscale studies of the dynamic nature of contamination events. The processes of waste liquid [containing Cr(VI), U(VI), and organic co-contaminants] infiltration in heterogeneous porous media will be simulated in structured soil mesomodels, in transparent structured pore networks, and in intact soil cores. Changes in microbial communities will be correlated with contaminant distributions and transformations within each experimental system. Chromium and uranium within the soil and glass models will be quantified using the synchrotron X-ray fluorescence microprobe to map elemental distributions, and micro X-ray absorption near-edge structure spectroscopy to map their oxidation state distributions in real time. The in situ spatial distributions of microbes and biofilms will be recorded using confocal and conventional microscopy coupled with activity-dependent visualizing systems. We will summarize results from the mesoscale experiments in terms of quantifiable parameters such as diffusivities, permeabilities, adsorption isotherm coefficients, and effective reduction rates. Then, results of core-scale experiments will be compared with model predictions based upon these independently determined mesoscale-derived parameters. [Back to Award Recipients Page]