Shelly D. Kelly

X-ray absorption fine-structure determination of pH-dependent U-bacterial cell wall interactions.

X-ray absorption fine structure (XAFS) measurements was used at the U L3-edge to directly determine the pH dependence of the cell wall functional groups responsible for the absorption of aqueous UO22+ to Bacillus subtilis from pH 1.67 to 4.80. Surface complexation modeling can be used to predict metal distributions in water–rock systems, and it has been used to quantify bacterial adsorption of metal cations. However, successful application of these models requires a detailed knowledge not only of the type of bacterial surface site involved in metal adsorption/desorption, but also of the binding geometry. Previous acid-base titrations of B. subtilis cells suggested that three surface functional group types are important on the cell wall; these groups have been postulated to correspond to carboxyl, phosphoryl, and hydroxyl sites. When the U(VI) adsorption to B. subtilis is measured, observed is a significant pH-independent absorption at low pH values (<3.0), ascribed to an interaction between the uranyl cation and a neutrally charged phosphoryl group on the cell wall. The present study provides independent quantitative constraints on the types of sites involved in uranyl binding to B. subtilis from pH 1.67 to 4.80. The XAFS results indicate that at extremely low pH (pH 1.67) UO22+ binds exclusively to phosphoryl functional groups on the cell wall, with an average distance between the U atom and the P atom of 3.64 ± 0.01 A. This U-P distance indicates an inner-sphere complex with an oxygen atom shared between the UO22+ and the phosphoryl ligand. The P signal at extremely low pH value is consistent with the UO22+ binding to a protonated phosphoryl group, as previously ascribed. With increasing pH (3.22 and 4.80), UO22+ binds increasingly to bacterial surface carboxyl functional groups, with an average distance between the U atom and the C atom of 2.89 ± 0.02 A. This U-C distance indicates an inner-sphere complex with two oxygen atoms shared between the UO22+ and the carboxyl ligand. The results of this XAFS study confirm the uranyl-bacterial surface speciation model.

Adsorption of cadmium to Bacillus subtilis bacterial cell walls: A pH-dependent X-ray absorption fine structure spectroscopy study

The local atomic environment of Cd bound to the cell wall of the gram-positive bacterium Bacillus subtilis was determined by X-ray absorption fine structure (XAFS) spectroscopy. Samples were prepared at six pH values in the range 3.4 to 7.8, and the bacterial functional groups responsible for the adsorption were identified under each condition. Under the experimental Cd and bacterial concentrations, the spectroscopy results indicate that Cd binds predominantly to phosphoryl ligands below pH 4.4, whereas at higher pH, adsorption to carboxyl groups becomes increasingly important. At pH 7.8, we observe the activation of an additional binding site, which we tentatively ascribe to a phosphoryl site with smaller Cd-P distance than the one that is active at lower pH conditions. XAFS spectra of several cadmium acetate, phosphate, and perchlorate solutions were measured and used as standards for fingerprinting, as well as to assess the ability of FEFF8 and FEFFIT to model carboxyl, phosphoryl, and hydration environments, respectively. The results of this XAFS study in general corroborate existing surface complexation models; however, some binding mechanism details could only be detected with the XAFS technique.

Microbial Populations Stimulated for Hexavalent Uranium Reduction in Uranium Mine Sediment

ABSTRACT Uranium-contaminated sediment and water collected from an inactive uranium mine were incubated anaerobically with organic substrates. Stimulated microbial populations removed U almost entirely from solution within 1 month. X-ray absorption near-edge structure analysis showed that U(VI) was reduced to U(IV) during the incubation. Observations by transmission electron microscopy, selected area diffraction pattern analysis, and energy-dispersive X-ray spectroscopic analysis showed two distinct types of prokaryotic cells that precipitated only a U(IV) mineral uraninite (UO2) or both uraninite and metal sulfides. Prokaryotic cells associated with uraninite and metal sulfides were inferred to be sulfate-reducing bacteria. Phylogenetic analysis of 16S ribosomal DNA obtained from the original and incubated sediments revealed that microbial populations were changed from microaerophilic Proteobacteria to anaerobic low-G+C gram-positive sporeforming bacteria by the incubation. Forty-two out of 94 clones from the incubated sediment were related to sulfate-reducing Desulfosporosinus spp., and 23 were related to fermentative Clostridium spp. The results suggest that, if in situ bioremediation were attempted in the uranium mine ponds, Desulfosporosinus spp. would be a major contributor to U(VI) and sulfate reduction and Clostridium spp. to U(VI) reduction.

Direct microbial reduction and subsequent preservation of uranium in natural near-surface sediment.

ABSTRACT The fate of uranium in natural systems is of great environmental importance. X-ray absorption near-edge spectroscopy (XANES) revealed that U(VI) was reduced to U(IV) in shallow freshwater sediment at an open pit in an inactive uranium mine. Geochemical characterization of the sediment showed that nitrate, Fe(III), and sulfate had also been reduced in the sediment. Observations of the sediment particles and microbial cells by scanning and transmission electron microscopy, coupled with elemental analysis by energy dispersive spectroscopy, revealed that uranium was concentrated at microbial cell surfaces. U(IV) was not associated with framboidal pyrite or nanometer-scale iron sulfides, which are presumed to be of microbial origin. Uranium concentrations were not detected in association with algal cells. Phylogenetic analyses of microbial populations in the sediment by the use of 16S rRNA and dissimilatory sulfite reductase gene sequences detected organisms belonging to the families Geobacteraceae and Desulfovibrionaceae. Cultivated members of these lineages reduce U(VI) and precipitate iron sulfides. The association of uranium with cells, but not with sulfide surfaces, suggests that U(VI) is reduced by the enzymatic activities of microorganisms. Uranium was highly enriched (760 ppm) in a subsurface black layer in unsaturated sediment sampled from a pit which was exposed to seasonal fluctuations in the pond level. XANES analysis showed that the majority of uranium in this layer was U(IV), indicating that uranium is preserved in its reduced form after burial.

Reduction of AgI, AuIII, CuII, and HgII by FeII/FeIII hydroxysulfate green rust

Green rusts are mixed Fe{sup II}/Fe{sup III} hydroxides that are found in many suboxic environments where they are believed to play a central role in the biogeochemical cycling of iron. X-ray absorption fine structure analysis of hydroxysulfate green rust suspensions spiked with aqueous solutions of AgCH{sub 3}COO, AuCl{sub n}(OH){sub 4-n}, CuCl{sub 2}, or HgCl{sub 2} showed that Ag{sup I}, Au{sup III}, Cu{sup II}, and Hg{sup II} were readily reduced to Ag{sup 0}, Au{sup 0}, Cu{sup 0}, and Hg{sup 0}. Imaging of the resulting solids from the Ag{sup I}-, Au{sup III}-, and Cu{sup II}-amended green rust suspensions by transmission electron microscopy indicated the formation of submicron-sized particles of Ag{sup 0}, Au{sup 0}, and Cu{sup 0}. The facile reduction of Ag{sup I}, Au{sup III}, Cu{sup II}, and Hg{sup II} to Ag{sup 0}, Au{sup 0}, Cu{sup 0}, and Hg{sup 0}, respectively, by green rust suggests that the presence of green rusts in suboxic soils and sediments can have a significant impact on the biogeochemistry of silver, gold, copper, and mercury, particularly with respect to their mobility.

Immobilization of Radionuclides and Heavy Metals through Anaerobic Bio-Oxidation of Fe(II)

ABSTRACT Adsorption of heavy metals and radionuclides (HMR) onto iron and manganese oxides has long been recognized as an important reaction for the immobilization of these compounds. However, in environments containing elevated concentrations of these HMR the adsorptive capacity of the iron and manganese oxides may well be exceeded, and the HMR can migrate as soluble compounds in aqueous systems. Here we demonstrate the potential of a bioremediative strategy for HMR stabilization in reducing environments based on the recently described anaerobic nitrate-dependent Fe(II) oxidation by Dechlorosoma species. Bio-oxidation of 10 mM Fe(II) and precipitation of Fe(III) oxides by these organisms resulted in rapid adsorption and removal of 55 μM uranium and 81 μM cobalt from solution. The adsorptive capacity of the biogenic Fe(III) oxides was lower than that of abiotically produced Fe(III) oxides (100 μM for both metals), which may have been a result of steric hindrance by the microbial cells on the iron oxide surfaces. The binding capacity of the biogenic oxides for different heavy metals was indirectly correlated to the atomic radius of the bound element. X-ray absorption spectroscopy indicated that the uranium was bound to the biogenically produced Fe(III) oxides as U(VI) and that the U(VI) formed bidentate and tridentate inner-sphere complexes with the Fe(III) oxide surfaces. Dechlorosoma suillum oxidation was specific for Fe(II), and the organism did not enzymatically oxidize U(IV) or Co(II). Small amounts (less than 2.5 μM) of Cr(III) were reoxidized by D. suillum; however, this appeared to be inversely dependent on the initial concentration of the Cr(III). The results of this study demonstrate the potential of this novel approach for stabilization and immobilization of HMR in the environment.

Reduction of Ag{sup I}, Au{sup III}, Cu{sup II}, and Hg{sup II} by Fe{sup II}/Fe{sup III} hydroxysulfate green rust.

Green rusts are mixed Fe{sup II}/Fe{sup III} hydroxides that are found in many suboxic environments where they are believed to play a central role in the biogeochemical cycling of iron. X-ray absorption fine structure analysis of hydroxysulfate green rust suspensions spiked with aqueous solutions of AgCH{sub 3}COO, AuCl{sub n}(OH){sub 4-n}, CuCl{sub 2}, or HgCl{sub 2} showed that Ag{sup I}, Au{sup III}, Cu{sup II}, and Hg{sup II} were readily reduced to Ag{sup 0}, Au{sup 0}, Cu{sup 0}, and Hg{sup 0}. Imaging of the resulting solids from the Ag{sup I}-, Au{sup III}-, and Cu{sup II}-amended green rust suspensions by transmission electron microscopy indicated the formation of submicron-sized particles of Ag{sup 0}, Au{sup 0}, and Cu{sup 0}. The facile reduction of Ag{sup I}, Au{sup III}, Cu{sup II}, and Hg{sup II} to Ag{sup 0}, Au{sup 0}, Cu{sup 0}, and Hg{sup 0}, respectively, by green rust suggests that the presence of green rusts in suboxic soils and sediments can have a significant impact on the biogeochemistry of silver, gold, copper, and mercury, particularly with respect to their mobility.

XAFS investigation of the interactions of UVI with secondary mineralization products from the bioreduction of FeIII oxides.

Biogenic Fe(II) phases (magnetite, green rust, siderite, vivianite, etc.) provide a reservoir of reducing capacity in many subsurface environments that may contribute to the reduction of contaminants such as U(VI). We have examined the uptake and reduction of U(VI) in the presence of biogenic green rust (BioGR), magnetite (BioMAG), and siderite (BioSID) formed during the reduction of Fe(III) oxides by Shewanella putrefaciens CN32. Within 48 h, total solution-phase U(VI) concentrations decreased from 500 microM to 1.5 microM, 392 microM, and 472 microM in the U-BioGR, U-BioMAG, and U-BioSID systems, respectively. Analysis of the samples by U L(III) extended X-ray absorption fine structure spectroscopy (EXAFS) indicated that despite a stoichiometric excess of Fe(II), no more than 6% of U(VI) was reduced to U(IV) in the U-BioSID system, and no more than 22% of U(VI) was reduced in the U-BioMAG system. For comparison, in the U-BioGR system, >99% of U(VI) was reduced to U(IV). Uptake of U(VI) by BioGR and BioMAG was accompanied by formation of nanoparticulate uraninite. The U EXAFS data for the U-BioSID system were consistent with partial U(VI)/U(IV) substitution for Fe(II) in the surface layer of siderite particles and adsorption of U(IV).

Effects of nitrate on the stability of uranium in a bioreduced region of the subsurface.

The effects of nitrate on the stability of reduced, immobilized uranium were evaluated in field experiments at a U.S. Department of Energy site in Oak Ridge, TN. Nitrate (2.0 mM) was injected into a reduced region of the subsurface containing high levels of previously immobilized U(IV). The nitrate was reduced to nitrite, ammonium, and nitrogen gas; sulfide levels decreased; and Fe(II) levels increased then deceased. Uranium remobilization occurred concomitant with nitrite formation, suggesting nitrate-dependent, iron-accelerated oxidation of U(IV). Bromide tracer results indicated changes in subsurface flowpaths likely due to gas formation and/or precipitate. Desorption-adsorption of uranium by the iron-rich sediment impacted uranium mobilization and sequestration. After rereduction of the subsurface through ethanol additions, background groundwater containing high levels of nitrate was allowed to enter the reduced test zone. Aqueous uranium concentrations increased then decreased. Clone library analyses of sediment samples revealed the presence of denitrifying bacteria that can oxidize elemental sulfur, H(2)S, Fe(II), and U(IV) (e.g., Thiobacillus spp.), and a decrease in relative abundance of bacteria that can reduce Fe(III) and sulfate. XANES analyses of sediment samples confirmed changes in uranium oxidation state. Addition of ethanol restored reduced conditions and triggered a short-term increase in Fe(II) and aqueous uranium, likely due to reductive dissolution of Fe(III) oxides and release of sorbed U(VI). After two months of intermittent ethanol addition, sulfide levels increased, and aqueous uranium concentrations gradually decreased to <0.1 microM.

Uranium in Framboidal Pyrite from a Naturally Bioreduced Alluvial Sediment

Samples of a naturally bioreduced, U-contaminated alluvial sediment were characterized with various microscopic and spectroscopic techniques and wet chemical extraction methods. The objective was to investigate U association and interaction with minerals of the sediment. Bioreduced sediment comprises approximately 10% of an alluvial aquifer adjacent to the Colorado River, in Rifle, CO, that was the site of a former U milling operation. Past and ongoing research has demonstrated that bioreduced sediment is elevated in solid-associated U, total organic carbon, and acid-volatile sulfide, and depleted in bioavailable Fe(III) confirming that sulfate and Fe(III) reduction have occurred naturally in the sediment. SEM/EDS analyses demonstrated that framboidal pyrites (FeS(2)) of different sizes ( approximately 10-20 microm in diameter), and of various microcrystal morphology, degree of surface weathering, and internal porosity were abundant in the <53 microm fraction (silt + clay) of the sediment and absent in adjacent sediments that were not bioreduced. SEM-EMPA, XRF, EXAFS, and XANES measurements showed elevated U was present in framboidal pyrite as both U(VI) and U(IV). This result indicates that U may be sequestered in situ under conditions of microbially driven sulfate reduction and pyrite formation. Conversely, such pyrites in alluvial sediments provide a long-term source of U under conditions of slow oxidation, contributing to the persistence of U of some U plumes. These results may also help in developing remedial measures for U-contaminated aquifers.