1998 RESEARCH PROJECTS
Program Element 1
Biotransformation and Biodegradation

PROJECT: Biodegradation of PuEDTA and Impacts on Pu Mobility
PRINCIPAL INVESTIGATOR: Harvey Bolton
PROGRAM ELEMENT 1 Biotransformation and Biodegradation

OBJECTIVE: 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, formed strong PuEDTA complexes, and enhanced Pu transport at many sites. EDTA poses a long-term problem of potentially disseminating Pu in the subsurface environment, because it is recalcitrant to biodegradation. Biodegradation of EDTA should decrease Pu mobility 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, and other metals) on the rate and ability of microorganisms to degrade PuEDTA. The objectives of this research are to determine the dominant PuEDTA complexes and their stability, the biological and geochemical factors influencing the rate and extent of PuEDTA biodegradation, and the resulting impacts on Pu mobility. Research will focus on determining the dominant PuEDTA aqueous species and their biodegradation, the uptake of Pu- and metal-EDTA complexes into the cell, the distribution and mobility of the Pu during and after EDTA biodegradation (e.g., extracellular, biosorbed, or intracellularly bioaccumulated), and the enzymology and genetics of EDTA biodegradation. This information will provide mechanistic understanding and approaches to bioremediate PuEDTA contaminated sites, determine the influence of PuEDTA biodegradation on Pu immobilization, and provide information necessary for modeling the fate and transport of PuEDTA in the environment.

PROJECT: Microbial Reduction and Immobilization of Uranium in Fe(III)- and Mn(IV)- Containing Sediments
PRINCIPAL INVESTIGATOR: Jim K. Fredrickson
PROGRAM ELEMENT 1 Biotransformation and Biodegradation

OBJECTIVE: Solid and liquid wastes discharged to the ground over a 40-year period constitute a major environmental problem at Department of Energy (DOE) sites nationwide. Metallic and radionuclide contaminants from DOE site wastes that have been found in groundwaters include U, Co, Pu, As, Cd, Cr, Cu, Hg, Ni, and Pb. Uranium is the most frequently-occurring radionuclide in soils, sediments, and groundwater at DOE sites and, therefore, is of particular environmental concern. At many of these sites, co-disposal of metals and radionuclides with organic ligands has led to the formation of metal-organic complexes with enhanced mobility in soils and groundwater.

Dissimilatory iron-reducing bacteria (DIRB) can utilize ferric iron associated with aqueous or solid phases as a terminal electron acceptor coupled to the oxidation of H2 or organic substrates. DIRB are also capable of reducing other metal ions such as Mn(IV), Cr(VI), and U(VI). The reduction of Fe and other metals and radionuclides has a pronounced effect on their solubility and overall geochemical behavior. It is these changes in geochemical behavior that hold promise for using DIRB to alter the mobility of metals and radionuclides in situ for remediation purposes.

The focus of this research will be on coupled microbiological-geochemical investigations of: a) microbial transformations of U-organic (EDTA, citrate, humic acids) complexes in the presence of reactive solid phases (synthetic and naturally-occurring) containing Fe- and Mn-oxides and b) humic acid-accelerated microbial growth, metabolism, and reduction of Fe (III). These processes must be more fully understood before the in situ immobilization of U or mobilization/solubilization of other metals and radionuclides using microbial processes can be fully realized.

This research is focused on three central hypotheses:

  1. Redox Disequilibria Hypothesis. In sediments containing Mn-oxides, U(VI)-carbonate complexes will be reduced by DIRB in preference to Mn(IV)(s), but the resulting U(IV) will be reoxidized by Mn(IV) oxides. Hence, U(IV) will function as an electron shuttle and accelerate the rate and extent of reduction of Mn-oxides.

  2. DIRB U Reduction & Nucleation Hypothesis. The rate of biologic reduction will far exceed the abiotic reduction rate at comparable pe because of the active participation of the cell surface in both the electron transfer reaction and the post reduction nucleation of U(IV) solids. In the absence of strong aqueous complexants, U(IV) will strongly sorb to cell surfaces, eventually nucleating as insoluble uraninite.

  3. Humic Acid Electron Shuttle Hypothesis. Environmentally relevant concentrations of humic acids (e.g., 5-20 mg C L-1) and quinones will support rates of metabolism and cell growth significantly higher than in their absence and will result in a more rapid and extensive reduction of Fe- and Mn-oxides. The acceleration of oxide reduction will be especially enhanced in porous media where cells can not readily access solid phase Fe and Mn due to pore size or other physical restrictions.

APPROACH: The overall goal of this research project is to develop a fundamental understanding of coupled microbiological-geochemical processes including: a) microbial transformations of U-carbonate and -organic (EDTA, citrate) complexes in the presence of reactive solid phases (synthetic and naturally-occurring) containing Fe- and Mn-oxides and b) humic acid-accelerated microbial growth, metabolism, and reduction of Fe (III). These processes must be more fully understood before the in situ immobilization of U or mobilization/solubilization of other metals and radionuclides using microbial processes can be fully realized. The proposed research is hypothesis-driven and will utilize a series of synthetic and natural subsurface materials and organic co-contaminants of relevance to DOE sites in experiments to address these hypotheses. Combined geochemical and spectroscopic methods will be used to probe the distribution of the metals and characteristics of the solids during microbial metal reduction. A coupled geochemical-microbial reaction model will be used to assess our understanding of these processes and to develop a predictive capability. Although the research is laboratory based, it is expected that the information from this project will be linked to related projects in NABIR that address microbially-driven oxidation-reduction processes. Ultimately, we expect this information and project to contribute to NABIR field experiment(s) focusing on inorganic contaminant mobilization/immobilization processes.

PROJECT: Bioremediation of Actinide and Transition Metal Contamination: Mechanistic Studies
PRINCIPAL INVESTIGATOR: Kenneth H. Nealson
PROGRAM ELEMENT 1 Biotransformation and Biodegradation

OBJECTIVE: The long term objectives of this project are to study various facets of uranium (U6+) and chromium (Cr6+) chemistry and biochemistry, with the goal of understanding the mechanisms of removal (biotic and abiotic) under both oxic and anoxic conditions. If these mechanisms can be understood, it should be possible to plan strategies for bioremediation that are both efficient and predictable. The immediate objectives (for years 1 and 2) include:

  1. Characterization of abiotic (indirect) binding of U and Cr to metal oxides.

    1. Characterization of U6+ and Cr6+ binding to manganese and iron oxides as a function of T, pH, Eh, mineral type, and concentration.

    2. Characterization of release of U or Cr from metal oxides under various conditions (see above).

  2. Characterization of U and Cr binding to biologically produced metal oxides.

    1. Binding of U and Cr to biologically formed metal oxides, using bacterial spores to synthesize Mn oxides in situ.

  3. Characterization of cell-catalyzed reduction of U and Cr, using S. putrefaciens isolates.

    1. Definition of the conditions favoring reduction of Mn, Fe, U, and Cr.

    2. Biochemical characterization of the cell free reduction system.

    3. Fate of released U and Cr under various conditions.

APPROACH: This work will be done using a three faceted approach. The first is wet microbiology and microbial physiology and biochemistry. We will examine both whole cells and cell extracts of oxidizing and reducing bacteria to characterize both U and Cr uptake, release, and reduction. We will use a series of mutants that are defective in metal reduction as controls to separate specific cell mediated effects from general anaerobic processes not specifically connected with metal reducing abilities. The second approach is that of ESEM (environmental scanning electron microscopy) and CLSM (confocal laser scanning microscopy) in which the cells and cell products will be directly examined under high resolution without the need for cell fixation or cell coating. Finally, we will utilize X-ray spectroscopy and X-ray microscopy, the latter being a new technique under development by our group. These approaches allow high resolution redox state determination of Mn, Fe, U and Cr, as well as high resolution imaging of bacteria inside solid structures, such as metal oxides.


PROJECT: Acceptable Endpoints for Metals and Radionuclides: Quantifying the Stability of Uranium and Lead Immobilized Under Sulfate Reducing Conditions
PRINCIPAL INVESTIGATOR: Brent M. Peyton
PROGRAM ELEMENT 1 Biotransformation and Biodegradation

Biologically mediated in-situ immobilization is being considered as a means to significantly decrease transport of metallic contaminants, and hence, produce an acceptable endpoint for treatment of a contaminant plume. The creation of sulfate-reduction conditions to immobilize metals has a high possibility of being selected for use at many contaminated sites because of the large number of metals such as lead (Pb) that form stable sulfide compounds. In addition, effective reduction and subsequent precipitation of uranium (U) and chromium (Cr) under these conditions has been shown in the laboratory. However, as with other possible treatments, the long-term stability of the immobilized metals and factors that affect the immobilization/remobilization process must be quantified to determine whether the treatment can produce an acceptable endpoint.

The goal of this project is to elucidate and quantify the fundamental microbiological and chemical factors that control the formation and subsequent stability of U and Pb precipitates formed under sulfate-reducing conditions. Tests will be performed to assess the effect of redox-sensitive aquifer materials [e.g., amorphous and crystalline iron (Fe) oxides and Fe-bearing clay minerals] and redox-insensitive materials (e.g., quartz) on the immobilization/remobilization processes that control plume stability. To meet this goal, a multi-disciplinary team of microbiologists, geochemists, and engineers has been assembled. In contrast to previous research, we will produce a combined data set from both micro- and meso-scale experiments that quantifies and correlates microbial activity, important aquifer materials, and contaminant properties to yield an accurate determination of whether sulfate-reducing conditions will produce an acceptable endpoint.

PROJECT: Transformation of Heavy Metal Contaminants in Sulfate-Reducing Subsurface Environments: The Role of Thiolated Compounds and Hydrogen Sulfide
PRINCIPAL INVESTIGATOR: Murthy A. Vairavamurthy
PROGRAM ELEMENT 1 Biotransformation and Biodegradation

OBJECTIVE: The overall objective is to obtain a fuller understanding of the role of the sulfur system, comprising mainly thiols and hydrogen sulfide, on the speciation and transformation of toxic heavy metal ions in anaerobic systems undergoing bacterial dissimilatory sulfate reduction. Specific issues to be addressed include (1) the relative importance of thiol complexation versus precipitation as metal sulfides for sequestering metal ions in anoxic environments, (2) the stability and biotransformation of thiol-metal complexes in anaerobic systems, (3) the effect of environmental variables, such as pH, on the anaerobic production of H2S and thiols, and (4) the production of metallothioneins or similar metal-binding proteins by anaerobic bacteria in response to stress by toxic heavy elements.

APPROACH: Although hydrogen sulfide is the primary product of bacterial sulfate reduction, thiols also are generated in sulfate-reducing systems, from either biochemical processes or abiotic reactions involving sulfur nucleophiles and functionalized organic molecules. They occur in a wide range of molecular weights and hydrophilicity. Thiols form their strongest bonds with large, easily polarizable heavy-metal ions such as Cd(II), Hg(II), and Pb(II), and, thus, should play an important role in transforming these metals in anaerobic environments. While low-molecular-weight thiols form soluble complexes of the metals, high-molecular-weight ones, such as humic thiols, can bind the metals to immobilize them. Hence, the kinds of sedimentary thiols involved in metal complexation and the types of metal-thiol complexes formed will be studied. In the initial phase of our research, we will focus on transformations of cadmium in sulfate reducing systems. Time series measurements of sulfur and metal speciation will be conducted with added Cd(II) in model sulfate-reducing systems similar to those of natural sub-surface sediments and soils. The use of model systems is advantageous because the speciation and processes can be more easily controlled and better understood than natural systems which are complex and heterogeneous. We will use a suite of complementary techniques, including x-ray absorption spectroscopy, NMR spectroscopy, and liquid chromatography-mass spectrometry to characterize sulfur and metal speciation in the anaerobic systems. We expect that this multifaceted approach will yield new insights about transformations of heavy metal species in sulfate-reducing systems.

PROJECT: Environmental Actinide Mobility: Plutonium and Uranium Interactions with Exopolysaccharides and Siderophores of Aerobic Soil Microbes
PRINCIPAL INVESTIGATOR: Laura Vanderberg-Twary
PROGRAM ELEMENT 1 Biotransformation and Biodegradation

OBJECTIVE: Our overall goal is to understand fundamental interactions of actinides and aerobic soil microbes which can affect the mobility of these actinides in the environment. To achieve this goal we will :

Determine the binding and immobiliztion of actinides to cellular surfaces of aerobic soil microbes with focus on: 1) the glutamic acid capsule of Bacillus lichenformis with known metal-binding ability and 2) the partially characterized exopolymer of Rhodococcus erythropolis .

Determine the siderophore-mediated uptake of actinides as a result of cellular translocation by 1) using a well-characterized siderophore (dessferrioxamine) produced by the soil microbe, Streptomyces pilosus and 2) by first isolating and characterizing the siderophore produced by the soil isolate, Rhodococcus rhodochrous strain OFS. Determine the oxidation state, speciation and localization of actinides that are translocated by siderophore-mediated processes.

APPROACH: We will study the interactions which are most likely to affect actinide environmental mobility, binding by extracellular polymers and siderophore complexation and uptake, using proven microbiological and biochemical techniques and a multimethod actinide speciation approach. Understanding these interactions will help to lay the basis for the development of successful in situ bioremediation schemes. The environmentally relevant forms of actinides to be used in our experiments are: uranyl carbonate, UO2(CO3)22- as a representative of hexavalent actinides, the hydrated plutonium(V) ion, PuO2+(H2O)5 as a representative of pentavalent actinides, colloidal Pu(IV) hydroxide and Pu(IV)EDTA as representatives of tetravalent actinides. By considering the lower (III and IV) and higher (V and VI) oxidation states we will study actinide ions with different effective charges, redox properties, and hydrolysis and complexation constants, as well as structural and coordination number differences--spherical, bound by 8-10 ligand donors vs. linear, dioxo, bound equatorially by 5-6 ligand donors. We will take a multimethod approach to determine the speciation and location of the actinides associated with biomolecules and obtain detailed structural and mechanistic information.

B. lichenformis and R. erythropolis will be grown under optimal conditions for exopolymer production. The metal binding capacity of the R. erythropolis capsule will be characterized. Whole cell and purified polymer studies using both microbes will be undertaken to determine actinide binding. Specifically, we can use the different components of the multimethod analytical approach to determine total actinide uptake, to determine if, and to what extent the actinides are reduced upon binding by the exopolymer, to determine the coordination geometry about the actinide associated with the capsular material, and to determine detailed structural information about the actinide-polymer interactions. We will also investigate the effect of pH on the reversibility of actinide exopolymer immobilization.

The siderophore of R. rhodochrous strain OFS will be produced, purified and characterized with respect to its structural type (i.e., hydroxamate, catecholate), molecular weight and functionality prior to initiation of actinide binding experiments. Desferrioxamine for siderophore experiments will be purchased commercially. For both siderophores and each actinide oxidation state (IV, V, VI), we will determine if translocation occurs. We determine if the interaction involves metal reduction. If actinides are translocated by siderophore mediated processes, the actinide is essentially immobilized within the cell. If translocation does not occur, or if the actinide-siderophore complex is found partially translocated, the composition and structure of the complex will be determined. In addition, experiments will be performed to determine how the relevant actinides might compete with iron in the environment for siderophore binding.

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