2004
RESEARCH PROJECTS
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| PROJECT: | Aqueous Complexation Reactions Governing the Rate and Extent of Biogeochemical U(VI) Reduction |
| PRINCIPAL INVESTIGATOR: | Scott Brooks |
| Integrative Studies | |
Laboratory research has shown that dissimilatory metal reducing bacteria (DMRB) can effectively reduce oxidized uranium (U(VI)) to the sparingly soluble U(IV) with the concomitant precipitation of UO2 phases. Despite the promise of bioreduction as a remediation strategy, the factors thatenhance or inhibit the rate and extent of biogeochemical U(VI) reduction under representative environmental conditions are not well defined. Before effective bioimmobilization can be realized, the factors governing contaminant reactivity in multicomponent systems must bebetter understood. Only recently has the quantification of a few key interactions been established. For example, we recently reported the inhibition of bacterial U(VI) reduction by DMRB in the presence of environmentally realistic concentrations of soluble calcium (Ca) (Brookset al., 2003). This finding has significant implications for field applications of bioreduction because Ca2+ is a dominant soluble and cation-exchangeable species in soils and aquifers. We propose to identify and quantify the important biogeochemical reactions that alsoequilibrate with the U-carbonate solution species and may inhibit or enhance U(VI) reduction. Initially, cation exchange resins, with well-defined Ca2+ selectivities, will be employed to establish the distribution of Ca-U-carbonate species in the presence of varying amounts of cation-exchangeable forms of Ca2+; other potentially important competing cations in the exchange equilibria (e.g., Mg2+, Sr2+) will be examined in later phases of the proposed research.Concurrent with the measurement of the competing equilibria among soluble and cation exchangeable phases, the reduction of the major cation-U-carbonate species will be studied using both abiotic and microbial agents. Our state-of-the-art measurement techniques (XAS,XRFS, EDX, TEM, radioisotopes, ICPMS, and KPA) will be applied to quantify these soluble complexes and precipitated phases. By understanding these important key equilibria, more predictable and effective approaches can be established for in situ bioremediation of Uunder realistic field conditions.
| PROJECT: | Reaction-Based Reactive Transport Modeling Of Iron Reduction And Uranium Immobilization At Area 2 Of The NABIR Field Research Center |
| PRINCIPAL INVESTIGATOR: | William Burgos |
| Integrative Studies | |
The proposed research is focused on developing mechanistic, phenomenological descriptions of important reactions and mathematical formulations for modeling reactions for the in situ immobilization of uranium promoted via microbial iron(III) reduction. Our overall goal is to develop and validate a robust mathematical model that can simulate all important chemical reactions and physical processes associated with in situ U(IV) immobilization. Experimental conditions will be designed to match those in saturated zone sediments at Area 2 of the DOE NABIR Field Research Center (FRC) in Oak Ridge, TN. This proposal is submitted to the Integrative Studies element within NABIR as it examines research topics related to both the Biogeochemistry and Biotransformation elements and contains a significant mathematical modeling effort. Our previous research has been funded through the Biotransformation element (DE-FG02-98ER62691 and DE-FG02-01ER63180).
The research will pursue three major objectives: (1) elucidate the mechanisms of reduction of solid-associated U(VI) in Area 2 sediment at the NABIR FRC; (2) evaluate the potential for long-term sustained U(IV) reductive immobilization coupled to dissimilatory metal-reducing bacterial (DMRB) activity in Area 2 sediments; (3) numerically simulate the suite of hydrobiogeochemical processes occurring in experimental systems so as to facilitate modeling of in situ U(IV) immobilization at the field-scale. The proposed research is based on the following five hypotheses: (1) the biological and chemical reduction of sediment-associated U(VI) is fundamentally controlled by its mineralogic and coordination environment; (2) the addition of humic substances can stimulate the reduction of solid-associated U(VI); (3) coupled Fe(III)/U(VI) reduction can be sustained in long-term flow-through reactor experiments with hydrologic residence times (week-to-month) comparable to those expected in pore domains likely to be colonized by DMRB in Area 2 sediments; (4) modest levels of nitrate input will not significantly inhibit coupled Fe(III)/U(VI) reduction in the reworked fill of Area 2; and, (5) the kinetics and thermodynamics of simultaneous biogeochemical reactions can be described by a series of reaction-based kinetic and equilibrium formulations, where rate formulations/parameter estimates derived from batch experiments will be applicable to flow-through (semicontinuous culture and constructed column) reactor experiments.
Both experimental and model designs have been constructed to minimize complexity of the conceptual systems, to build systems that can be validated based on measurements readily made in the field, and to increase conceptual complexity only when absolutely necessary. Based on these principles, we have proposed integrated tasks to resolve our hypotheses and satisfy our objectives. To address the first hypothesis we propose to use surface complexation modeling and spectroscopic techniques (XAS, LIFS) in a complementary fashion. U(VI) sorption experiments will use Area 2 sediments and specimen minerals presumed to control U(VI) sorption and reducibility in these sediments (specifically - illite, goethite, and goethite-coated illite). The second hypothesis will be tested using a series of biotic (Geobacter sulfurreducens) and abiotic [excess Fe(II)] U(VI) reduction experiments with Area 2 sediments and specimen mineral assemblages in the presence/absence of FRC humic substances in batch reactors, semicontinuous culture reactors (SCRs), and constructed column reactors (CCRs). SCR experiments will provide suspension samples over time for solid-phase analyses (XAS, XPS, Mössbauer spectroscopy, ESEM, and wet chemistry). The third hypothesis will be tested in long-term SCR and CCR experiments containing Area 2 sediment inoculated with Geobacter sulfurreducens. Experimental variables will include two hydrologic residence times (10 and 50 days) and the presence/absence of FRC humic substances in the reactor feed solution. The fourth hypothesis will be tested in long-term SCR and CCR experiments with Geobacter metallireducens (capable of both nitrate and metal reduction) and the presence/absence of 0.5 mM nitrate in the reactor feed solution. The fifth hypothesis will be addressed via continued development and calibration/validation of the BIOGEOCHEM – HYDROBIOGEOCHEM series of reaction-based computer models. The proposed research will utilize two national user facilities: the Advance Photon Source (APS) at Argonne National Laboratory, and the W.R. Wiley Environmental Molecular Sciences Laboratory (EMSL) at PNNL. Our graduate students will be hosted at EMSL by PNNL collaborators for 8-week periods of intensive summer research during each year of the project.
| PROJECT: | Integrated Nucleic Acid System for In-Field Monitoring of Microbial Community Dynamics and Metabolic Activity |
| PRINCIPAL INVESTIGATOR: | Darrell Chandler |
| Integrative Studies | |
Molecular analysis of subsurface microbial communities requires some combination of sample collection, concentration, cell lysis, nucleic acid purification, PCR amplification and specific detection in order to address fundamental questions of microbial community dynamics, activity and function in the environment. As a result of our prior NABIR grant, we have now developed a suite of integrated microparticle chemistries, Fe- and SO4-reducer phylogenetic probes and suspension arrays, and combined sample purification/bead array fluidic devices for the automated and direct analysis of 16S rRNA and microbial community dynamics in environmental samples. However, changes in microbial community composition and/or abundance are still insufficient to detect or make conclusions regarding specific microbial activity. Thus, fieldable methods for the direct analysis of functional genes and messenger RNA (mRNA) in the environment are still required. The objective of the proposed work is therefore twofold. First, we seek to verify the 16S rRNA methods, Fe- and SO4-reducer array and instrumentation on sediment and groundwater samples taken from the Schiebe/Roden FRC site, and continue to expand the probe suite for microbial community dynamics as new sequences are obtained from DOE-relevant sites. Second, we propose to address the fundamental molecular biology and analytical chemistry associated with the direct analysis of functional genes and mRNA (hence, microbial activity) in environmental samples. These studies will investigate the behavior of oligonucleotide and cDNA capture and detection probes for direct mRNA purification and detection on microparticle surfaces; “tunable surface” chemistries to increase mRNA capture and detection efficiency; new mRNA reporter and detection chemistries required for the development of in-field monitoring methods and devices; and fluidic strategies for integrating complex biochemistry for direct detection of mRNA in sediments and groundwater, without using the polymerase chain reaction (PCR).
| PROJECT: | Characterizing the Catalytic Potential of Deinococcus, Arthrobacter and other Robust Bacteria in Contaminated Subsurface Environments of the Hanford Site |
| PRINCIPAL INVESTIGATOR: | Michael Daly |
| Integrative Studies | |
Immense volumes of soil and groundwater at numerous U. S. Department of Energy (DOE) sites have low levels of widespread contamination that include mixtures of heavy metals (e.g., Hg & Cr) and radionuclides (e.g., U & Tc), as well as chlorinated hydrocarbons. The remediation of such contaminated sites constitutes an immediate and complex waste management challenge for DOE, particularly in light of the costliness and limited efficacy of current physical and chemical strategies for mixed wastes. In situ bioremediation via natural microbial processes (e.g., metal reduction) remains a potent, potentially cost-effective approach to the reductive immobilization or detoxification of environmental contaminants.
Several key findings for bacteria belonging to the families Deinococcaceae and Arthrobacteriaceae support their suitability as subjects for the Integrative Study element of NABIR, combining the elements Biomolecular Sciences and Engineering, and Community Dynamics and Microbial Ecology. We have recently isolated several distinct species of Deinococcus and Arthrobacter from pristine and contaminated soils of DOE’s Hanford Site in south-central Washington state. Generally, Deinococcus bacteria are exceptionally robust, not only surviving exposure to radiation, but also to other DNA damaging conditions typical of DOE environments. The ability to reduce a variety of toxic metals also appears to be a characteristic shared by these organisms, and we have shown that D. radiodurans and D. geothermalis are proficient at reducing U(VI), Tc(VII), (in the presence of an electron shuttle) and Cr(VI), and have engineered both species for Hg(II) reduction. Recent experimental advances in the genetic management of D. radiodurans and D. geothermalis will facilitate our efforts to characterize the metal-reducing mechanisms of both organisms. These advances include our comprehensive analysis of the D. radiodurans genome, and for D. geothermalis, the development of a system for genetic transformation and ongoing genome sequencing, annotation, and analysis of this organism. Arthrobacter species are capable of free-living growth in many extreme environments, such as under chronic radiation. Arthrobacter spp. have also recently been reported to efficiently resist and reduce high concentrations of hexavalent chromium. These and other cultured isolates from the Hanford Site will be examined for their metal-reducing capabilities as outlined below.
The four specific aims of this proposal are: 1) Characterizing the genetic basis of metal reduction in D. radiodurans and D. geothermalis, and novel deinococcal isolates from the Hanford Site; 2) Characterizing the microbial ecology of contaminated waste sites and metal-reducing potential of these communities; 3) Defining environmental factors that govern in situ growth, environmental robustness, metabolism, and Cr(VI)-reduction of Deinococcus and Arthrobacter spp.; and 4) Characterizing the genetic basis of metal reduction in Arthrobacter aurescens (TC1) and novel Arthrobacter isolates from the Hanford Site.
| PROJECT: | Biogeochemical Mechanisms Controlling Reduced Radionuclide Particle Properties and Stability |
| PRINCIPAL INVESTIGATOR: | James Fredrickson |
| Integrative Studies | |
Uranium and technetium, common subsurface contaminants on DOE sites, are problematic because they exist as highly soluble and mobile ions, U as U(VI) carbonate complexes and Tc as (Tc(VII)O4-), in oxidized groundwaters. Laboratory research has demonstrated that dissimilatory metal-reducing bacteria (DMRB) can effectively reduce U(VI) and Tc(VII) to insoluble UO2 and TcO2 phases. Bioreductive immobilization of these contaminants offers considerable promise for in situ remediation of contaminated DOE sites. A major focus of the NABIR program is on “understanding the role of microorganisms in long-term immobilization of contaminants in place, and the potential for their remobilization”. Considerable emphasis has been placed on defining the specific biological and biogeochemical mechanisms of contaminant reduction within the NABIR program yet relatively little is know regarding the biological factors controlling the form and behavior of the bioreduced contaminants.
Our previous NABIR research findings demonstrated that UO2 nanoparticles are present in the cell periplasm of metal-reducing Shewanella sp. and that localization of UO2 to this compartment protects U(IV) against oxidation by Mn(III,IV) oxides. More recently, we have established that UO2 and TcO2 particles of relatively uniform nm-size are transported from the periplasm via a protein secretion pathway that is common to many Gram-negative bacteria. This is a rather remarkable finding and the first report we are aware of describing the transport of a solid phase across the outer membrane of bacteria. This process has important implications for the long-term immobilization of contaminants and potential for their remobilization. Once nanoparticles are “secreted” from cells they may be subject to a variety of processes including oxidation and transport. This research will focus on defining the mechanisms by which Gram-negative metal-reducing bacteria export contaminant nanoparticles from the periplasm and on the subsequent biogeochemical behavior of the externalized nanoparticles.
| PROJECT: | Biogeochemical Cycling and Environmental Stability of Pu Relevant to Long-Term Stewardship of DOE Sites |
| PRINCIPAL INVESTIGATOR: | Bruce Honeyman |
| Integrative Studies | |
Plutonium contamination is widespread in surface soils and subsurface sediments throughout the DOE complex. Until the last decade or so, Pu was generally considered to be relatively immobile in the terrestrial environment, with the exception of transport via aeolian and erosional mechanisms. More recently, however, the transport of colloidal forms of Pu has been invoked as providing a mobilization pathway in low intensity stream environments and the subsurface.
Central to understanding the environmental behavior of Pu in vadose- and saturated-zones, as well as waste streams, is the contribution of microbial communities to Pu speciation. This proposed research would address the principal mechanisms by which naturally occurring microbial communities regulate transformations in Pu chemical speciation; such changes may lead to either enhanced Pu immobilization or its release from immobile phases and subsequent transport.
The overall objective of this proposed research is to understand the biogeochemical cycling of Pu in environments of interest to long-term DOE stewardship issues. Central to Pu cycling (transport initiation —> immobilization) is the role of microorganisms. The hypothesis underlying this proposal is that microbial activity is the causative agent in initiating the mobilization of Pu in near-surface environments: through the transformation of Pu associated with solid phases, production of extracellular polymeric substances (EPS) carrier phases, and the creation of microenvironments. Also, microbial processes are central to the immobilization of Pu species, through the metabolism of organically complexed Pu species and Pu associated with extracellular carrier phases and the creation of environments favorable for Pu transport retardation.
The following figure illustrates the information flow in this multi-institution project.
Experimental approach and information flow. This work is an integrated program between the three participating institutions. The components of study include the use of model compounds, environmental materials and bacterial exudates produced under controlled laboratory conditions.
| PROJECT: | Stabilization of Plutonium in Subsurface Environments via Microbial Reduction and Biofilm Formation |
| PRINCIPAL INVESTIGATOR: | Mary Neu |
| Integrative Studies | |
Subsurface actinide contaminants at DOE sites that have complicated hydrogeology and redox environments are affected by direct and indirect microbially-mediated processes. The influence of these processes is particularly difficult to predict for plutonium, due to its complicated redox behavior and rich chemistry. We propose to investigate how key reactions, which are known to affect major redox-active transition metals, such as Fe and Mn, can affect Pu speciation and environmental mobility. The overarching goal of the proposed research is to understand and optimize mechanisms for in situ immobilization of Pu species by naturally-occurring bacteria, beginning with an investigation of a) bacterial accumulation and immobilization of Pu species by biofilm formation and b) bacterial mineralization and immobilization via direct enzymatic and indirect biogeochemical reduction of Pu species by metal reducing bacteria. A combination of aqueous chemical, radioanalytical, spectroscopic and microscopic analyses will be used to characterize Pu speciation and solution/solid phase distributions. Biotransformation and biogeochemical research results will fill significant gaps in the scientific basis for monitored natural attenuation and in situ stabilization of widespread and problematic Pu contamination, as well as provide immediately useful data to modeling and risk assessment efforts.
| PROJECT: | An Integrated Assessment of Geochemical and Community Structure Determinants of Metal Reduction Rates in Subsurface Sediments |
| PRINCIPAL INVESTIGATOR: | Anthony Palumbo |
| Integrative Studies | |
Significant advances have been made in the ability to measure the composition of indigenous subsurface microbial communities. However, fundamental questions still persist concerning the interactive effects of geochemistry and community structure on metal reduction rates in the subsurface. Many microorganisms can change the subsurface geochemical conditions (e.g., cause a drop in redox) so metal reduction becomes an energetically favored reaction. Some microbes can directly catalyze the necessary reactions so that metal reduction occurs at a more rapid rate than without microbial activity. Many microorganisms can accomplish the first role but many fewer can accomplish the second. Physical and geochemical factors such as mass transport, oxygen level, and nitrate concentration will likely dominate the rate of microbial change in the redox potential. Thus, it is possible that the importance of community structure at this stage of metal reduction may be minimal. However, the effect of community composition on the rate of metal reduction may be important. We will use controlled laboratory experiment with sediments and groundwater from the NABIR Field Research Center (FRC) to compare the effects of manipulations designed to influence community structure (differences in electron donors) to those designed to influence geochemistry (presences of humics as electron shuttles) on uranium reduction. Also, the effect of carbon:phosphate ratios on community structure and uranium reduction rates will be examined in the context of resource-ratio theory to help predict the nutrient supply rates and ratios that maximize uranium reduction at the FRC site. In the final stage we will extend the research to other sites where toxic metals and radionuclides are problematic.