Uranium Biogeochemistry in the Rhizosphere of a Contaminated Wetland.

The objective of this study was to determine if U sediment concentrations in a U-contaminated wetland located within the Savannah River Site, South Carolina, were greater in the rhizosphere than in the nonrhizosphere. U concentrations were as much as 1100% greater in the rhizosphere than in the nonrhizosphere fractions; however and importantly, not all paired samples followed this trend. Iron (but not C, N, or S) concentrations were significantly enriched in the rhizosphere. XAS analyses showed that in both sediment fractions, U existed as UO22+ coordinated with iron(III)-oxides and organic matter. A key difference between the two sediment fractions was that a larger proportion of U was adsorbed to Fe(III)-oxides, not organic matter, in the rhizosphere, where significantly greater total Fe concentrations and greater proportions of ferrihydrite and goethite existed. Based on 16S rRNA analyses, most bacterial sequences in both paired samples were heterotrophs, and population differences were consistent with the generally more oxidizing conditions in the rhizosphere. Finally, U was very strongly bound to the whole (unfractionated) sediments, with an average desorption Kd value (Usediment/Uaqueous) of 3972 ± 1370 (mg-U/kg)/(mg-U/L). Together, these results indicate that the rhizosphere can greatly enrich U especially in wetland areas, where roots promote the formation of reactive Fe(III)-oxides.

Effect of Siderophore DFOB on U(VI) Adsorption to Clay Mineral and Its Subsequent Reduction by an Iron-Reducing Bacterium.

Uranium mining and nuclear fuel production have led to significant U contamination. Past studies have focused on the bioreduction of soluble U(VI) to insoluble U(IV) as a remediation method. However, U(IV) is susceptible to reoxidation and remobilization when conditions change. Here, we demonstrate that a combination of adsorption and bioreduction of U(VI) in the presence of an organic ligand (siderophore desferrioxamine B, DFOB) and the Fe-rich clay mineral nontronite partially alleviated this problem. DFOB greatly facilitated U(VI) adsorption into the interlayer of nontronite as a stable U(VI)-DFOB complex. This complex was likely reduced by bioreduction intermediates such as the Fe(II)-DFOB complex and/or through electron transfer within a ternary Fe(II)-DFOB-U(VI) complex. Bioreduction with DFOB alone resulted in a mobile aqueous U(IV)-DFOB complex, but in the presence of both DFOB and nontronite U(IV) was sequestered into a solid. These results provide novel insights into the mechanisms of U(VI) bioreduction and the stability of U and have important implications for understanding U biogeochemistry in the environment and for developing a sustainable U remediation approach.

Surface biomineralization of uranium onto Shewanella putrefaciens with or without extracellular polymeric substances.

The influence of extracellular polymeric substances (EPS) on the interaction between uranium [U(VI)] and Shewanella putrefaciens (S. putrefaciens), especially the U(VI) biomineralization process occurring on whole cells and cell components of S. putrefaciens was investigated in this study. The removal efficiency of U(VI) by S. putrefaciens was decreased by 22% after extraction of EPS. Proteins were identified as the main components of EPS by EEM analysis and were determined to play a major role in the biosorption of uranium. SEM-EDS results showed that U(VI) was distributed around the whole cell as 500-nanometer schistose structures, which consisted primarily of U and P. However, similar uranium lamellar crystal were wrapped only on the surface of EPS-free S. putrefaciens cells. FTIR and XPS analysis indicated that phosphorus- and nitrogen-containing groups played important roles in complexing U (VI). XRD and U LIII-edge EXAFS analyses demonstrated that the schistose structure consisted of hydrogen uranyl phosphate [H2(UO2)2(PO4)2•8H2O]. Our study provides new insight into the mechanisms of induced uranium crystallization by EPS and cell wall membranes of living bacterial cells under aerobic conditions.

Combined Effects of Fe(III)-Bearing Clay Minerals and Organic Ligands on U(VI) Bioreduction and U(IV) Speciation.

Reduction of U(VI) to U(IV) drastically reduces its solubility and has been proposed as a method for remediation of uranium contamination. However, much is still unknown about the kinetics, mechanisms, and products of U(VI) bioreduction in complex systems. In this study, U(VI) bioreduction experiments were conducted with Shewanella putrefaciens strain CN32 in the presence of clay minerals and two organic ligands: citrate and EDTA. In reactors with U and Fe(III)-clay minerals, the rate of U(VI) bioreduction was enhanced due to the presence of ligands, likely because soluble Fe3+- and Fe2+-ligand complexes served as electron shuttles. In the presence of citrate, bioreduced U(IV) formed a soluble U(IV)-citrate complex in experiments with either Fe-rich or Fe-poor clay mineral. In the presence of EDTA, U(IV) occurred as a soluble U(IV)-EDTA complex in Fe-poor montmorillonite experiments. However, U(IV) remained associated with the solid phase in Fe-rich nontronite experiments through the formation of a ternary U(IV)-EDTA-surface complex, as suggested by the EXAFS analysis. Our study indicates that organic ligands and Fe(III)-bearing clays can significantly affect the microbial reduction of U(VI) and the stability of the resulting U(IV) phase.

Adsorption of Selenite onto Bacillus subtilis: The Overlooked Role of Cell Envelope Sulfhydryl Sites in the Microbial Conversion of Se(IV).

Microbial activities play a central role in the global cycling of selenium. Microorganisms can reduce, methylate, and assimilate Se, controlling the transport and fate of Se in the environment. However, the mechanisms controlling these microbial activities are still poorly understood. In particular, it is unknown how the negatively charged Se(IV) and Se(VI) oxyanions that dominate the aqueous Se speciation in oxidizing environments bind to negatively charged microbial cell surfaces in order to become bioavailable. Here, we show that the adsorption of selenite onto Bacillus subtilis bacterial cells is controlled by cell envelope sulfhydryl sites. Once adsorbed onto the bacteria, selenite is reduced and forms reduced organo-Se compounds (e.g., R1S-Se-SR2). Because sulfhydryl sites are present within cell envelopes of a wide range of bacterial species, sulfhydryl-controlled adsorption of selenite likely represents a general mechanism adopted by bacteria to make selenite bioavailable. Therefore, sulfhydryl binding of selenite likely occurs in a wide range of oxidized Se-bearing environments, and because it is followed by microbial conversion of selenite to other Se species, the process represents a crucial step in the global cycling of Se.

U(VI) Reduction by Biogenic and Abiotic Hydroxycarbonate Green Rusts: Impacts on U(IV) Speciation and Stability Over Time.

Green rusts (GRs) are redox active FeII-FeIII minerals that form in the environment via various biotic and abiotic processes. Although both biogenic (BioGR) and abiotic (ChemGR) GRs have been shown to reduce UVI, the dynamics of the transformations and the speciation and stability of the resulting UIV phases are poorly understood. We used carbonate extraction and XAFS spectroscopy to investigate the products of UVI reduction by BioGR and ChemGR. The results show that both GRs can rapidly remove UVI from synthetic groundwater via reduction to UIV. The initial products in the ChemGR system are solids-associated UIV-carbonate complexes that gradually transform to nanocrystalline uraninite over time, leading to a decrease in the proportion of carbonate-extractable U from ∼95% to ∼10%. In contrast, solid-phase UIV atoms in the BioGR system remain relatively extractable, nonuraninite UIV species over the same reaction period. The presence of calcium and carbonate in groundwater significantly increase the extractability of UIV in the BioGR system. These data provide new insights into the transformations of U under anoxic conditions in groundwater that contains calcium and carbonate, and have major implications for predicting uranium stability within redox dynamic environments and designing approaches for the remediation of uranium-contaminated groundwater.

Bioreduction of hydrogen uranyl phosphate: mechanisms and U(IV) products.

The mobility of uranium (U) in subsurface environments is controlled by interrelated adsorption, redox, and precipitation reactions. Previous work demonstrated the formation of nanometer-sized hydrogen uranyl phosphate (abbreviated as HUP) crystals on the cell walls of Bacillus subtilis, a non-U(VI)-reducing, Gram-positive bacterium. The current study examined the reduction of this biogenic, cell-associated HUP mineral by three dissimilatory metal-reducing bacteria, Anaeromyxobacter dehalogenans strain K, Geobacter sulfurreducens strain PCA, and Shewanella putrefaciens strain CN-32, and compared it to the bioreduction of abiotically formed and freely suspended HUP of larger particle size. Uranium speciation in the solid phase was followed over a 10- to 20-day reaction period by X-ray absorption fine structure spectroscopy (XANES and EXAFS) and showed varying extents of U(VI) reduction to U(IV). The reduction extent of the same mass of HUP to U(IV) was consistently greater with the biogenic than with the abiotic material under the same experimental conditions. A greater extent of HUP reduction was observed in the presence of bicarbonate in solution, whereas a decreased extent of HUP reduction was observed with the addition of dissolved phosphate. These results indicate that the extent of U(VI) reduction is controlled by dissolution of the HUP phase, suggesting that the metal-reducing bacteria transfer electrons to the dissolved or bacterially adsorbed U(VI) species formed after HUP dissolution, rather than to solid-phase U(VI) in the HUP mineral. Interestingly, the bioreduced U(IV) atoms were not immediately coordinated to other U(IV) atoms (as in uraninite, UO2) but were similar in structure to the phosphate-complexed U(IV) species found in ningyoite [CaU(PO4)2·H2O]. This indicates a strong control by phosphate on the speciation of bioreduced U(IV), expressed as inhibition of the typical formation of uraninite under phosphate-free conditions.

In situ bioremediation of uranium with emulsified vegetable oil as the electron donor.

A field test with a one-time emulsified vegetable oil (EVO) injection was conducted to assess the capacity of EVO to sustain uranium bioreduction in a high-permeability gravel layer with groundwater concentrations of (mM) U, 0.0055; Ca, 2.98; NO3(-), 0.11; HCO3(-), 5.07; and SO4(2-), 1.23. Comparison of bromide and EVO migration and distribution indicated that a majority of the injected EVO was retained in the subsurface from the injection wells to 50 m downgradient. Nitrate, uranium, and sulfate were sequentially removed from the groundwater within 1-2 weeks, accompanied by an increase in acetate, Mn, Fe, and methane concentrations. Due to the slow release and degradation of EVO with time, reducing conditions were sustained for approximately one year, and daily U discharge to a creek, located approximately 50 m from the injection wells, decreased by 80% within 100 days. Total U discharge was reduced by 50% over the one-year period. Reduction of U(VI) to U(IV) was confirmed by synchrotron analysis of recovered aquifer solids. Oxidants (e.g., dissolved oxygen, nitrate) flowing in from upgradient appeared to reoxidize and remobilize uranium after the EVO was exhausted as evidenced by a transient increase of U concentration above ambient values. Occasional (e.g., annual) EVO injection into a permeable Ca and bicarbonate-containing aquifer can sustain uranium bioreduction/immobilization and decrease U migration/discharge.

Reaction of U(VI) with titanium-substituted magnetite: influence of Ti on U(IV) speciation.

Reduction of hexavalent uranium (U(VI)) to less soluble tetravalent uranium (U(IV)) through enzymatic or abiotic redox reactions has the potential to alter U mobility in subsurface environments. As a ubiquitous natural mineral, magnetite (Fe3O4) is of interest because of its ability to act as a rechargeable reductant for U(VI). Natural magnetites are often impure with titanium, and structural Fe(3+) replacement by Ti(IV) yields a proportional increase in the relative Fe(2+) content in the metal sublattice to maintain bulk charge neutrality. In the absence of oxidation, the Ti content sets the initial bulk Fe(2+)/Fe(3+) ratio (R). Here, we demonstrate that Ti-doped magnetites (Fe3 - xTixO4) reduce U(VI) to U(IV). The U(VI)-Fe(2+) redox reactivity was found to be controlled directly by R but was otherwise independent of Ti content (xTi). However, in contrast to previous studies with pure magnetite where U(VI) was reduced to nanocrystalline uraninite (UO2), the presence of structural Ti (xTi = 0.25-0.53) results in the formation of U(IV) species that lack the bidentate U-O2-U bridges of uraninite. Extended X-ray absorption fine structure spectroscopic analysis indicated that the titanomagnetite-bound U(IV) phase has a novel U(IV)-Ti binding geometry different from the coordination of U(IV) in the mineral brannerite (U(IV)Ti2O6). The observed U(IV)-Ti coordination at a distance of 3.43 Å suggests a binuclear corner-sharing adsorption/incorporation U(IV) complex with the solid phase. Furthermore, we explored the effect of oxidation (decreasing R) and solids-to-solution ratio on the reduced U(IV) phase. The formation of the non-uraninite U(IV)-Ti phase appears to be controlled by availability of surface Ti sites rather than R. Our work highlights a previously unrecognized role of Ti in the environmental chemistry of U(IV) and suggests that further work to characterize the long-term stability of U(IV) phases formed in the presence of Ti is warranted.

Immobilization of U(VI) from oxic groundwater by Hanford 300 Area sediments and effects of Columbia River water.

Regions within the U.S. Department of Energy Hanford 300 Area (300 A) site experience periodic hydrologic influences from the nearby Columbia River as a result of changing river stage, which causes changes in groundwater elevation, flow direction and water chemistry. An important question is the extent to which the mixing of Columbia River water and groundwater impacts the speciation and mobility of uranium (U). In this study, we designed experiments to mimic interactions among U, oxic groundwater or Columbia River water, and 300 A sediments in the subsurface environment of Hanford 300 A. The goals were to investigate mechanisms of: 1) U immobilization in 300 A sediments under bulk oxic conditions and 2) U remobilization from U-immobilized 300 A sediments exposed to oxic Columbia River water. Initially, 300 A sediments in column reactors were fed with U(VI)-containing oxic 1) synthetic groundwater (SGW), 2) organic-amended SGW (OA-SGW), and 3) de-ionized (DI) water to investigate U immobilization processes. After that, the sediments were exposed to oxic Columbia River water for U remobilization studies. The results reveal that U was immobilized by 300 A sediments predominantly through reduction (80-85%) when the column reactor was fed with oxic OA-SGW. However, U was immobilized by 300 A sediments through adsorption (100%) when the column reactors were fed with oxic SGW or DI water. The reduced U in the 300 A sediments fed with OA-SGW was relatively resistant to remobilization by oxic Columbia River water. Oxic Columbia River water resulted in U remobilization (∼7%) through desorption, and most of the U that remained in the 300 A sediments fed with OA-SGW (∼93%) was in the form of uraninite nanoparticles. These results reveal that: 1) the reductive immobilization of U through OA-SGW stimulation of indigenous 300 A sediment microorganisms may be viable in the relatively oxic Hanford 300 A subsurface environments and 2) with the intrusion of Columbia River water, desorption may be the primary process resulting in U remobilization from OA-SGW-stimulated 300 A sediments at the subsurface of the Hanford 300 A site.

Redox behavior of uranium at the nanoporous aluminum oxide-water interface: implications for uranium remediation.

Sorption-desorption experiments show that the majority (ca. 80-90%) of U(VI) presorbed to mesoporous and nanoporous alumina could not be released by extended (2 week) extraction with 50 mM NaHCO(3) in contrast with non-nanoporous α alumina. The extent of reduction of U(VI) presorbed to aluminum oxides was semiquantitatively estimated by comparing the percentages of uranium desorbed by anoxic sodium bicarbonate between AH(2)DS-reacted and unreacted control samples. X-ray absorption spectroscopy confirmed that U(VI) presorbed to non-nanoporous alumina was rapidly and completely reduced to nanoparticulate uraninite by AH(2)DS, whereas reduction of U(VI) presorbed to nanoporous alumina was slow and incomplete (<5% reduction after 1 week). The observed nanopore size-dependent redox behavior of U has important implications in developing efficient remediation techniques for the subsurface uranium contamination because the efficiency of in situ bioremediation depends on how effectively and rapidly U(VI) bound to sediment or soil can be converted to an immobile phase.

Microbial reductive transformation of phyllosilicate Fe(III) and U(VI) in fluvial subsurface sediments.

The microbial reduction of Fe(III) and U(VI) was investigated in shallow aquifer sediments collected from subsurface flood deposits near the Hanford Reach of the Columbia River in Washington State. Increases in 0.5 N HCl-extractable Fe(II) were observed in incubated sediments and (57)Fe Mössbauer spectroscopy revealed that Fe(III) associated with phyllosilicates and pyroxene was reduced to Fe(II). Aqueous uranium(VI) concentrations decreased in subsurface sediments incubated in sulfate-containing synthetic groundwater with the rate and extent being greater in sediment amended with organic carbon. X-ray absorption spectroscopy of bioreduced sediments indicated that 67-77% of the U signal was U(VI), probably as an adsorbed species associated with a new or modified reactive mineral phase. Phylotypes within the Deltaproteobacteria were more common in Hanford sediments incubated with U(VI) than without, and in U(VI)-free incubations, members of the Clostridiales were dominant with sulfate-reducing phylotypes more common in the sulfate-amended sediments. These results demonstrate the potential for anaerobic reduction of phyllosilicate Fe(III) and sulfate in Hanford unconfined aquifer sediments and biotransformations involving reduction and adsorption leading to decreased aqueous U concentrations.

Microscale geochemical gradients in Hanford 300 Area sediment biofilms and influence of uranium.

The presence and importance of microenvironments in the subsurface at contaminated sites were suggested by previous geochemical studies. However, no direct quantitative characterization of the geochemical microenvironments had been reported. We quantitatively characterized microscale geochemical gradients (dissolved oxygen (DO), H(2), pH, and redox potential) in Hanford 300A subsurface sediment biofilms. Our results revealed significant differences in geochemical parameters across the sediment biofilm/water interface in the presence and absence of U(VI) under oxic and anoxic conditions. While the pH was relatively constant within the sediment biofilm, the redox potential and the DO and H(2) concentrations were heterogeneous at the microscale (<500-1000 µm). We found microenvironments with high DO levels (DO hotspots) when the sediment biofilm was exposed to U(VI). On the other hand, we found hotspots (high concentrations) of H(2) under anoxic conditions both in the presence and in the absence of U(VI). The presence of anoxic microenvironments inside the sediment biofilms suggests that U(VI) reduction proceeds under bulk oxic conditions. To test this, we operated our biofilm reactor under air-saturated conditions in the presence of U(VI) and characterized U speciation in the sediment biofilm. U L(III)-edge X-ray absorption spectroscopy (XANES and EXAFS) showed that 80-85% of the U was in the U(IV) valence state.

Influence of magnetite stoichiometry on U(VI) reduction.

Hexavalent uranium (U(VI)) can be reduced enzymatically by various microbes and abiotically by Fe(2+)-bearing minerals, including magnetite, of interest because of its formation from Fe(3+) (oxy)hydroxides via dissimilatory iron reduction. Magnetite is also a corrosion product of iron metal in suboxic and anoxic conditions and is likely to form during corrosion of steel waste containers holding uranium-containing spent nuclear fuel. Previous work indicated discrepancies in the extent of U(VI) reduction by magnetite. Here, we demonstrate that the stoichiometry (the bulk Fe(2+)/Fe(3+) ratio, x) of magnetite can, in part, explain the observed discrepancies. In our studies, magnetite stoichiometry significantly influenced the extent of U(VI) reduction by magnetite. Stoichiometric and partially oxidized magnetites with x ≥ 0.38 reduced U(VI) to U(IV) in UO(2) (uraninite) nanoparticles, whereas with more oxidized magnetites (x < 0.38) and maghemite (x = 0), sorbed U(VI) was the dominant phase observed. Furthermore, as with our chemically synthesized magnetites (x ≥ 0.38), nanoparticulate UO(2) was formed from reduction of U(VI) in a heat-killed suspension of biogenic magnetite (x = 0.43). X-ray absorption and Mössbauer spectroscopy results indicate that reduction of U(VI) to U(IV) is coupled to oxidation of Fe(2+) in magnetite. The addition of aqueous Fe(2+) to suspensions of oxidized magnetite resulted in reduction of U(VI) to UO(2), consistent with our previous finding that Fe(2+) taken up from solution increased the magnetite stoichiometry. Our results suggest that magnetite stoichiometry and the ability of aqueous Fe(2+) to recharge magnetite are important factors in reduction of U(VI) in the subsurface.

Solution and microbial controls on the formation of reduced U(IV) species.

Reduction of U(VI) to U(IV) as the result of direct or indirect microbial activity is currently being explored for in situ remediation of subsurface U plumes, under the assumption that U(IV) solubility is controlled by the low-solubility mineral uraninite (U(IV)-dioxide). However, recent characterizations of U in sediments from biostimulated field sites, as well as laboratory U(VI) bioreduction studies, report on the formation of U(IV) species that lack the U═O(2)═U coordination of uraninite, suggesting that phases other than uraninite may be controlling U(IV) solubility in environments with complexing surfaces and ligands. To determine the controls on the formation of such nonuraninite U(IV) species, the current work studied the reduction of carbonate-complexed U(VI) by (1) five Gram-positive Desulfitobacterium strains, (2) the Gram-negative bacteria Anaeromyxobacter dehalogenans 2CP-C and Shewanella putrefaciens CN32, and (3) chemically reduced 9,10-anthrahydroquinone-2,6-disulfonate (AH(2)QDS, a soluble reductant). Further, the effects of 0.3 mM dissolved phosphate on U(IV) species formation were explored. Extended X-ray absorption fine structure (EXAFS) spectroscopy analysis demonstrated that the addition of phosphate causes the formation of a nonuraninite, phosphate-complexed U(IV) species, independent of the biological or abiotic mode of U(VI) reduction. In phosphate-free medium, U(VI) reduction by Desulfitobacterium spp. and by AH(2)QDS resulted in nonuraninite, carbonate-complexed U(IV) species, whereas reduction by Anaeromyxobacter or Shewanella yielded nanoparticulate uraninite. These findings suggest that the Gram-positive Desulfitobacterium strains and the Gram-negative Anaeromyxobacter and Shewanella species use distinct mechanisms to reduce U(VI).

Multiple mechanisms of uranium immobilization by Cellulomonas sp. strain ES6.

Removal of hexavalent uranium (U(VI)) from aqueous solution was studied using a Gram-positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non-growth conditions in bicarbonate and PIPES buffers. Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate-based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X-ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non-uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)-phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U-free controls suggesting simultaneous precipitation of U and PO4³â». In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High-resolution transmission electron microscopy (HR-TEM) and energy dispersive X-ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone-2,6-disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non-uraninite U(IV) phase. Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6.

U(VI) reduction to mononuclear U(IV) by Desulfitobacterium species.

The bioreduction of U(VI) to U(IV) affects uranium mobility and fate in contaminated subsurface environments and is best understood in Gram-negative model organisms such as Geobacter and Shewanella spp. This study demonstrates that U(VI) reduction is a common trait of Gram-positive Desulfitobacterium spp. Five different Desulfitobacterium isolates reduced 100 microM U(VI) to U(IV) in <10 days, whereas U(VI) remained soluble in abiotic and heat-killed controls. U(VI) reduction in live cultures was confirmed using X-ray absorption near-edge structure (XANES) analysis. Interestingly, although bioreduction of U(VI) is almost always reported to yield the uraninite mineral (UO(2)), extended X-ray absorption fine structure (EXAFS) analysis demonstrated that the U(IV) produced in the Desulfitobacterium cultures was not UO(2). The EXAFS data indicated that the U(IV) product was a phase or mineral composed of mononuclear U(IV) atoms closely surrounded by light element shells. This atomic arrangement likely results from inner-sphere bonds between U(IV) and C/N/O- or P/S-containing ligands, such as carbonate or phosphate. The formation of a distinct U(IV) phase warrants further study because the characteristics of the reduced material affect uranium stability and fate in the contaminated subsurface.

c-Type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis.

Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracellular UO(2) nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2) nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO(2)-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2) nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2) nanoparticles. In the environment, such association of UO(2) nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2) or transport in soils and sediments.