Iron (Fe) minerals and ferrous iron (Fe(II)) play an important role in the several natural elemental cycles, including the carbon cycle, nutrient cycles, and the cycling of metals. In this work we have characterized the reactivity structural Fe(II) in several Fe minerals and in natural soil with uranium. We have studied the reactivity of Fe(II) in solution with the Fe oxide goethite conditions relevant to many natural systems. Green rusts are widely recognized as an intermediate phase in the Fe cycle. Here we investigate the reactivity of green rusts containing different structural anions with uraniumVI (UVI). We have also investigated the effect of aqueous bicarbonate on UVI sorption and reduction by green rusts. Our findings indicate that green rusts reduce UVI to UIV, and that environmentally relevant carbonate concentrations have little effect the rate and extent on this reaction. We have also investigated UVI reduction by structural Fe(II) in magnetite. Magnetite with varying stoichiometry (x = Fe2+/Fe3+) was reacted with UVI. Results from x-ray absorption spectroscopy indicate that the redox properties of magnetite dictate whether magnetite reduces UVI. In addition, magnetite reactivity can be "recharged" by electron transfer from aqueous Fe(II). There is little evidence of the reactivity of structural Fe(II) towards UVI in natural materials. We have characterized a naturally reduced soil and found it contains structural Fe(II) in clay minerals and a possible green rust-like phase. When this soil is exposed to UVI we find that Fe(II) reduces a portion of the U added. Our work highlights the potential for abiotic reduction of UVI by Fe(II) in reduced, Fe-rich environments. We have used 57Fe Mössbauer spectroscopy to study redox reactions of Fe(II) with goethite under biogeochemical conditions relevant to natural systems. When Fe(III) in goethite is substituted with aluminum or anions such as phosphate, silicate, carbonate, and natural organic matter are sorbed onto the surface of goethite, interfacial electron transfer occurs between sorbed Fe(II) and goethite. These results indicate that electron transfer between Fe(II) and Fe oxides occurs under environmentally relevant conditions. Electron transfer was blocked by phospholipids, however, suggesting electron transfer may be inhibited under eutrophic conditions.

Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed.

Naturally occurring bacterial populations are capable of detoxifying chlorinated compounds and immobilizing the radionuclide uranium via reductive processes. This study addressed the following three knowledge gaps in the fields of chlorinated solvent and uranium bioremediation, 1) the risks and benefits of coupling bioremediation with thermal treatment for clean-up of chlorinated ethene-contaminated sites, 2) the accuracy of available techniques for the monitoring of chlorinated solvent bioremediation, and 3) the role of gram positive Desulfitobacterium spp. in uranium immobilization. Experiments demonstrated that thermal treatment increases electron donor availability, but the increased electron donor was not used to fuel reductive dechlorination and was actually consumed for methanogenesis. Two approaches for monitoring chlorinated solvent bioremediation were investigated, molecular techniques and compound-specific isotope analysis (CSIA). Results demonstrated that while Dehalococcoides (Dhc) gene expression was up-regulated under conditions inhibitory to dechlorination, the isotope effects associated with dechlorination reactions catalayzed by Dhc populations in consortia and in pure cultures were similar. U(VI) reduction by multiple Desulfitobacterium isolates was demonstrated. Interestingly, while almost all U(VI)-reducing populations have been reported to produce uraninite (UO2), the product of U(VI) reduction by Desulfitobacterium isolates was a unique form of insoluble mononuclear U(IV).

Uranium has been released into the environment due to improper practices associated with mining and refinement for energy and weapons production. Soluble U(VI) species such as uranyl carbonate can be reduced to form the insoluble U(IV) mineral uraninite (UO2) via microbial respiratory processes. Formation of UO2 diminishes uranium mobility and prevents uranium-laden groundwater from being discharged into surface water; however, oxygen and other oxidants re-solubilize UO2. Many organisms have been shown to reduce uranium, but variations in microbial physiology change the dynamics of microbial uranium reduction in situ and affect uraninite stability. Anaeromyxobacter dehalogenans is a metal-reducing delta-Proteobacterium in the myxobacteria family that displays remarkable respiratory versatility and efficiently reduces U(VI). The approach of this research was to enhance characterization of A. dehalogenans by identifying unique genetic traits, describing variability within the species, and examining the environmental distribution of A. dehalogenans strains. Genome analysis revealed that A. dehalogenans shares many traits with the myxobacteria including type IV pilus-based motility and an aerobic-like electron transport chain. In addition, the genome revealed genes that share sequence similarity with strict anaerobes and other metal-reducing organisms. Physiological examination of microaerophilism in A. dehalogenans strain 2CP-C revealed growth at sub-atmospheric oxygen partial pressure. Physiological characterization of novel isolates demonstrated that strain-level variation in the 16S rRNA gene coincides with metabolic changes that can be linked to the loss of specific gene homologs. Anaeromyxobacter spp. were present at the Oak Ridge Integrated Field-scale Subsurface Research Challenge (IFC) site and multiplex qPCR tools designed using a minor-groove binding probe gave insights into strain and species differences in the community. Finally, 16S rRNA gene sequences were identified which suggest a novel Anaeromyxobacter species that is responsible for uranium reduction at the Oak Ridge IFC site. This research contributes new knowledge of the ecophysiology of a widely distributed, metal-reducing bacterial group capable of uranium immobilization. The characterization of Anaeromyxobacter spp. helps to elucidate the dynamics of biological cycling of metals at oxic-anoxic interfaces, like those at the Oak Ridge IFC, and contributes to the broader study of microbial ecology in groundwater and sediment environments.