2004 RESEARCH PROJECTS
Field Studies

PROJECT: Field-Scale Evaluation Of Biological Uranium Reduction And  Reoxidation In The Near-Source Zone At The Nabir Field Research Center In Oak Ridge, Tn
PRINCIPAL INVESTIGATOR: Craig Criddle
Field Studies

Over the past three years we designed, fabricated, and are now operating a field research station that enables controlled studies of microbial U(VI) reduction in the near-source zone at the NABIR Field Research Center.  The station has both above- and below-ground elements.   The aboveground system removes contaminants that interfere with controlled experiments and U(VI) reduction, such as aluminum, calcium, and nitrate.   The below-ground system consists of two injection and extraction well pairs.  One pair defines an outer flow cell. This cell is connected to the aboveground system, enabling injection of clean, treated water into the outer cell.  The second well pair is nested within the first, and includes provisions for removal of N2 and addition of electron donor and alkalinity. Water quality is monitored at multilevel sampling wells within the inner cell. Breakthrough profiles indicate that water is efficiently transported between the injection and MLS wells, with short breakthrough times. We have obtained evidence of denitrification and biological U(VI) removal within this zone. We propose to advance this work through a series of "single pass" field experiments in which well-defined solutions of uranium and tracer are injected into the inner cell. Reduction experiments will explore hypotheses related to the rate and extent of U(VI) reduction and the role of microbial community composition in stabilization of the process. Single pass re-oxidation experiments will similarly test hypotheses related to the concentration and duration of oxidant and strategies for stabilization of the reduced system to prevent remobilization of uranium when an oxidant enters the system.  These experiments will be accompanied by complementary laboratory studies and modeling. Our overall goal is to develop the operational strategies, tools and understanding needed to enable long-term stabilization of uranium in the near-source zone. Anticipated outcomes include (1) improved knowledge and information on the rates and mechanisms of microbially mediated U reduction and immobilization, (2) information needed to enhance the long-term stability of a remediated site; (3) a validated reactive transport model, (4) trained students, and (5) a published body of work that will inform and enhance future research and remediation activities for immobilization of uranium and other radionuclides.


PROJECT: Field-Integrated Studies of Long-Term Sustainability of Chromium Bioreduction at Hanford 100H Site
PRINCIPAL INVESTIGATOR: Terry Hazen
Field Studies

The project build upons and expands an ongoing NABIR-EM project Field Investigations of Lactate-Stimulated Bioreduction of Cr(VI) to Cr(III) at Hanford 100H.  Pilot field-scale testing is in progress, intermediate key findings using Hanford sediments are: the presence of several types of bacteria, including Bacillus/Arthrobacter and Geobacter species, could contribute to effective Cr(VI) bioreduction; and injection of lactate/polylactate (Hydrogen Release Compound - HRCTM) is an effective method for Cr(VI) bioreduction in ground water.  We also determined that because Cr(VI) reduction in sediments is diffusion rate-limited, a small fraction of Cr(VI) in groundwater could remain unreduced and continue moving with the regional flow.  In addition, the presence of dissolved oxygen within the zone of fluctuations of the water table and the existence manganese oxides could cause a small portion of Cr(III) to reoxidize to Cr(VI).  The main goals of field-integrated studies are to investigate coupled hydraulic, geochemical, and microbial conditions, and to determine critical biogeochemical parameters necessary to maximize the extent of Cr(VI) bioreduction and minimize Cr(III) reoxidation in ground water.  The project will include field monitoring, using water sampling and geophysical methods, of the active zone of Cr(VI) bioreduction and Cr(III) reoxidation in groundwater at the Hanford 100H site.  Using the results of field monitoring, we will design bench-scale flow-through column experiments to obtain diffusion and kinetic parameters characterizing Cr(VI) bioreduction and Cr(III) reoxidation.  To simulate coupled biological and geochemical processes, we plan to develop a new multiphase, multicomponent 3D reactive transport code, TOUGHREACT-BIO, which will be calibrated to bench-scale column experiments and validated with field tests and monitoring.  An improved understanding and ability to predict the biogeochemical-hydrogeological conditions under which Cr-bioreduction is sustainable will facilitate the transfer of our conceptual and numerical models to other analogous Hanford and DOE contaminated sites with a variety of metal contaminants.

 

PROJECT: Stability of U(VI) and Tc(VII) Reducing Microbial Communities to Environmental Perturbation: Development and Testing of a Thermodynamic Network Model
PRINCIPAL INVESTIGATOR: Jonathan Istok
 Field Studies

Rationale:  In situ field experiments at the NABIR Field Research Center have shown that cooperative metabolism of denitrifiers and Fe(III)/sulfate reducers is essential for creating subsurface conditions favorable for U(VI) and Tc(VII) bioreduction (Istok et al., 2004).  Although much has been learned about the physiology and metabolic potential of specific microorganisms with these capabilities that have been isolated from the FRC and other sites using pure cultures of microorganisms, major gaps exist in our understanding of the functioning of these organisms when they are present in intact microbial communities.  For example, although ongoing NABIR studies have demonstrated the large genetic diversity of subsurface microorganisms at the FRC, many of these have never been isolated in pure culture.  Yet it is the collective metabolic capability of these largely uncharacterized microorganisms that must be relied on for effective U(VI) and Tc(VII) bioimmobilization. 

Research Goal and Hypotheses:The overall goal of this project is to develop and test a thermodynamic network model for predicting the effects of substrate additions and environmental perturbations on the composition and functional stability of subsurface microbial communities.  The overall scientific hypothesis is that a thermodynamic analysis of the energy-yielding reactions performed by broadly defined groups of microorganisms can be used to make quantitative and testable predictions of the change in microbial community composition that will occur when a substrate is added to the subsurface or when environmental conditions change.

Approach:  The proposed research will be conducted at the FRC using four intermediate-scale (~ 2 m) bioreactor models currently deployed in Areas 1 and 2. The network model will be used to predict the effects of substrate additions on the microbial community composition in the bioreactors by predicting the growth of major metabolic groups of organisms (aerobes, fermenters, denitrifiers, Fe(III)/sulfate/metal reducers).  Model predictions will be tested by quantifying changes in the abundance of each of these groups using a combination of functional genes and lipid analysis.   The network model will also be used to examine the stability of the U(VI) and Tc(VII) reducing microbial communities to changing environmental conditions.  These predictions will be tested by challenging the microbial community in the bioreactors with a series of perturbations representative of those likely to occur (due to heterogeneity in groundwater geochemistry) in a full-scale bioreactor at the FRC.  These will include variations in pH, nitrate, and sulfate concentrations in the bioreactor influent. 

Project Significance:  As will be shown below, the ability to predict the effects of donor addition on change inmicrobial community composition is essential for creating conditions that favor the long-term stability of bioreduced U and Tc.  Moreover, the ability of a microbial community to maintain functional stability (i.e. maintain high rates of U(VI) and Tc(VII) reduction) when subjected to various environmental perturbations (e.g., fluctuating pH and concentrations of electron acceptors) is of critical importance for the ultimate use of bioimmobilization at DOE legacy waste sites.  The principal immediate significance of this project will be in establishing and testing a theoretical framework for designing and interpreting complex field experiments and to aid in “bridging-the-gap” between basic NABIR laboratory and field research.

PROJECT: Factors Controlling In Situ Uranium and Technetium Bio-Reduction and Reoxidation at the NABIR Field Research Center
PRINCIPAL INVESTIGATOR: Jonathan Istok
 Field Studies

Rationale:  Ongoing laboratory and field studies by our group have demonstrated that it is possible to stimulate denitrification and microbially-mediated U reduction at FRC Area 1.  Microcosm studies showed that (1) NO3- removal is necessary for U reduction, (2) rates of U reduction are limited by the availability of suitable electron donors, (3) rates of U reduction are greatly reduced below pH 5, and (4) NO3- and the products of denitrification can rapidly reoxidize (and therefore remobilize) microbially-reduced U.  Although denitrification has been successfully stimulated  in field tests (as measured by NO2- production) through the addition of various electron donors (e.g. acetate, glucose, and ethanol), unequivocal evidence for U reduction has yet to be obtained, in part because NO3- levels, although reduced, have remained high following donor addition.  Similar processes are believed to control the reduction pertechnetate (Tc(VII)O4-) to Tc(IV)-containing mineral phases, although the limited scope of our current project precludes their examination.  Note that although Tc concentrations are monitored during our ongoing field tests, unequivocal evidence for Tc reduction following electron donor addition has not been obtained. 

Research Goal and Hypotheses: The overall goal of this project is to better understand factors and processes controlling microbially-mediated reduction and reoxidation of U and Tc in the unconsolidated residuum overlying the Nolichucky shale in FRC Areas 1 and 2.  Based on the results of ongoing laboratory and field research conducted by our group in Area 1, we have developed five hypotheses to be tested by the proposed research:

  1. The small rates of denitrification and U bio-reduction observed in laboratory incubations of sediments from Area 1 at low pH (< 5) are due to the presence of high concentrations of toxic metals (especially Al and Ni).  Rates of Tc reduction will also be small at low pH in the presence of high concentrations of toxic metals.
  2. In situ rates of U and perhaps Tc bio-reduction can be increased by increasing system pH and thus precipitating toxic metals from solution.
  3. In situ rates of U and Tc bio-reduction can be increased by the addition of humic substances, which complex toxic metals such as Al and Ni, buffer pH, and serve as electron shuttles to facilitate U and Tc reduction.
  4. Microbially-reduced U and Tc are rapidly oxidized in the presence of high concentrations of NO3- and the denitrification intermediates NO2-, N2O, and NO.
  5. An electron-donor-addition strategy (type and form of donor, with or without pH adjustment and with or without the co-addition of humic substances) can be devised to reduce U and Tc concentrations for an extended period of time in low pH groundwater in the presence of high concentrations of NO3-, Al, and Ni.  This strategy operates by removing or complexing these components of FRC groundwater to allow the subsequent reduction of U(VI) and Tc(VII).

Approach:  Hypotheses will be addressed by an extensive series of field push-pull tests supported by detailed geochemical analyses and laboratory microcosm experiments.  Field tests will be conducted in wells in Areas 1 and 2; laboratory studies will utilize groundwater and sediment from the same locations.  Detailed geochemical analyses will be performed to quantify radionuclide and toxic metal speciation, complexation, precipitation, and co-precipitation as a function of pH, electron donor, and humic substances.  Microcosm studies will be conducted with live and killed microorganisms in sediments (1) to examine effects of toxic metal


concentrations on U and Tc reduction with and without electron donor and humic substances additions and with and without pH adjustment, and (2) to examine the effects of denitrification reactions on the rates of reoxidation of microbially-reduced U and Tc.  Field push-pull tests will be designed to parallel laboratory microcosm studies.  Changes in U and Tc speciation in sediment and groundwater will be determined in laboratory and field samples following electron donor and/or humic substances addition.

PROJECT: In situ microbial community control of the stability of bio-reduced uranium 
PRINCIPAL INVESTIGATOR: David White
 Field Studies

The long-term stability of microbially reduced uranium in the subsurface is a pivotal issue for the eventual application of biostimulation (e.g. electron donor amendment) as a means of bioremediation of uranium contaminated groundwater.  Recent field experiments (Anderson et al. 2003 and unpublished data) demonstrate that U(VI) can be removed from groundwater by electron donor amendment at the field scale.  It was also observed that several months after electron donor (acetate) amendment, some U(VI) reduction was maintained. We hypothesize that, while Geobacter dominates the microbial population during the metal reduction phase, the ongoing U(VI) reduction may be related to metabolically versatile sulfate reducers.  Furthermore, precipitated FeS0.9 is not sufficient, without microbial mediation, to reduce U(VI) under subsurface conditions of flowing groundwater.

However, direct estimation of reoxidation rates is difficult under field conditions.  We propose a series of in situ experiments using in-well sediment incubators that will enable direct measurement of U(IV) removal rates from pre-reduced sediments withspecific microbial and mineralogic amendments.  By comparing U(IV) loss rates with different DIRB and SRB populations we will be able to clearly determine the relative impact of sulfate reducers vs. Fe reducers. The approach we propose also makes it possible to assess actual in situ conditions during the experiment and to directly observe reoxidation (or bioreduction) end points after the field experiment is completed without drilling.  Finally, in-well sediment incubators are relatively inexpensive and could ultimately displace both field-scale electron donor amendment experiments and push-pull tests as the preferred means of assessing site response to bioremediation and long-term stability of both biostimulated and naturally bioattenuated sites.

 

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