[en] The proposed effort will identify genes and ultimately physiological mechanisms and pathways that are expressed under in situ conditions and are critical to functioning of aquifer dwelling anaerobic bacteria living in contaminated systems. The main objectives are: (1) Determine which Metal-reducer specific genes are important for activities in normal and contaminated subsurface sediment. To achieve these goals, we have generated a library of chromosomal mutants. These are introduced into contaminated sediments, incubated, allowed to grow, and then reisolated. A negative selection process allows us to determine which mutants have been selected against in sediments and thereby identify genes required for survival in subsurface sediments. (2) Delineate the function of these genes through GeneBank and Clusters of Orthologous Groups (COGs) comparisons and analyze other sediment microorganisms to determine if similar genes are present in these populations. After determining the sequence of the genes identified through the previous objectives, we delineate the role of those specific genes in the physiology of G20, MR-1 and perhaps other microorganisms. (3) Determine the loss in function of a select group of mutants. Cells with mutations in known genes with testable functions are assayed for the loss of that function if specific assays are available. Mutants with unknown loss of function and other mutants are run through a series of tests including motility, attachment, and rate of sulfate or iron reduction. These tests allow us to categorize mutants for subsequent more detailed study

[en] The effectiveness and feasibility of bioremediation at the field scale cannot be fully assessed until the mechanisms of immobilization and U speciation in the solid matrix are resolved. However, characterization of the immobilized U and its valence states is extremely difficult, because microbially mediated mineral precipitates are generally nanometer (nm)-sized, poorly crystalline, or amorphous. We are developing combined field emission gun--scanning electron microscopy (FEG-SEM, at Indiana University) and FEG transmission electron microscopy (TEM, at Hopkins) to detect and isolate uranium containing phases; (1) method developments for TEM sample preparations and parallel electron energy loss spectroscopy (EELS) determination of uranium valence; and (2) to determine the speciation, fate, reactivity, valence states of immobilized uranium, using the state-of-the-art 300-kV, FEG-TEM. We have obtained preliminary results on contaminated sediments from Area 3 at the Oak Ridge Field Research Center (FRC). TEM results show that the sediments contain numerous minerals, including quartz, mica/clay (muscovite and/or illite), rutile, ilmenite, zircon, and an Al-Sr-Ce-Ca phosphate mineral, none of which contain uranium above the EDS detection limit. Substantial U (up to ∼2 wt.%) is, however, clearly associated with two materials: (1) the Fe oxyhydroxide and (2) clots of a chemically complex material that is likely a mixture of several nm-scale phases. The Fe oxyhydroxide was identified as goethite from its polycrystalline SAED pattern and EDS analysis showing it to be very Fe-rich; the aggregate also displays one of several morphologies that are common for goethite. U is strongly sorbed to goethite in the FRC sediment, and the ubiquitous association with phosphorous suggests that complexes containing both U and P may play an important role in that sorption. Results from bulk analysis and SEM had previously demonstrated the association of U with Fe and thus suggested that U may be sorbed by Fe oxide or oxyhydroxide (Dr. Roh, image presented by David Watson). However, rigorous identification of the host minerals for U requires TEM results such as these involving imaging, electron diffraction, and spectroscopic analysis. An even higher concentration of U occurs in the chemically complex material noted above. These ''clots'' are high in Fe but also contain C, O, Mg, Al, Si, P, S, Cl, K, Ca, Mn, and U. This chemical complexity strongly suggests that they consist of aggregates of carbonate, silicate, phosphate, and sulfate phases, and TEM images also suggest that they may be intergrowths of numerous exceedingly small nanoparticles. EELS and EFTEM studies should be able to resolve these various components and identify precisely where the uranium is in these complex materials. From the results, it is clear that the FEG-SEM and FEG-TEM can readily detect uranium in the FRC samples. The FEG-SEM allows a wide field of view of the samples and can detect U-rich aggregates as small as 20-30 nm. The FEG-TEM can then focus on these aggregates and use SAED, EDS, EFTEM, and PEELS techniques to determine the valence states, structures, and compositional data for these aggregates. This research will provide a crucial component for a complete understanding of the efficacy of uranium bioremediation.

[en] The overall goal of this project was to better understand factors and processes controlling microbially-mediated reduction and reoxidation of U and Tc in the unconsolidated residuum overlying the Nolichucky shale at the Field Research Center (FRC) at Oak Ridge National Laboratory. Project activities were designed to test the following hypotheses: (1) The small rates of denitrification and U bio-reduction observed in laboratory incubations of sediments from FRC 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)