Reduction of uranium by Desulfovibrio desulfuricans.

The possibility that sulfate-reducing microorganisms contribute to U(VI) reduction in sedimentary environments was investigated. U(VI) was reduced to U(IV) when washed cells of sulfate-grown Desulfovibrio desulfuricans were suspended in a bicarbonate buffer with lactate or H2 as the electron donor. There was no U(VI) reduction in the absence of an electron donor or when the cells were killed by heat prior to the incubation. The rates of U(VI) reduction were comparable to those in respiratory Fe(III)-reducing microorganisms. Azide or prior exposure of the cells to air did not affect the ability of D. desulfuricans to reduce U(VI). Attempts to grow D. desulfuricans with U(VI) as the electron acceptor were unsuccessful. U(VI) reduction resulted in the extracellular precipitation of the U(IV) mineral uraninite. The presence of sulfate had no effect on the rate of U(VI) reduction. Sulfate and U(VI) were reduced simultaneously. Enzymatic reduction of U(VI) by D. desulfuricans was much faster than nonenzymatic reduction of U(VI) by sulfide, even when cells of D. desulfuricans were added to provide a potential catalytic surface for the nonenzymatic reaction. The results indicate that enzymatic U(VI) reduction by sulfate-reducing microorganisms may be responsible for the accumulation of U(IV) in sulfidogenic environments. Furthermore, since the reduction of U(VI) to U(IV) precipitates uranium from solution, D. desulfuricans might be a useful organism for recovering uranium from contaminated waters and waste streams.     

Reduction of Hexavalent Uranium from Organic Complexes by Sulfate- and Iron-Reducing Bacteria

The influence of organic-hexavalent-uranium [U(VI)] complexation on U(VI) reduction by a sulfate-reducing bacterium (Desulfovibrio desulfuricans) and an iron-reducing bacterium (Shewanella alga) was evaluated. Four aliphatic ligands (acetate, malonate, oxalate, and citrate) and an aromatic ligand (tiron [4,5-dihydroxy-1,3-benzene disulfonic acid]) were used to study complexed-uranium bioavailability. The trends in uranium reduction varied with the nature and the amount of U(VI)-organic complex formed and the type of bacteria present. D. desulfuricans rapidly reduced uranium from a monodentate aliphatic (acetate) complex. However, reduction from multidentate aliphatic complexes (malonate, oxalate, and citrate) was slower. A decrease in the amount of organic-U(VI) complex in solution significantly increased the rate of reduction. S. alga reduced uranium more rapidly from multidentate aliphatic complexes than from monodentate aliphatic complexes. The rate of reduction decreased with a decrease in the amount of multidentate complexes present. Uranium from an aromatic (tiron) complex was readily available for reduction by D. desulfuricans, while an insignificant level of U(VI) from the tiron complex was reduced by S. alga. These results indicate that selection of bacteria for rapid uranium reduction will depend on the organic composition of waste streams.     

Interaction between uranium and the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>3</Subscript> of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis> strain G20

Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.     

Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c3 mutant

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.     

Removal of U and Mo from water by immobilized Desulfovibrio desulfuricans in column reactors

Intact cells of Desulfovibrio desulfuricans were immobilized in polyacrylamide gel and used to remove soluble U and Mo from water by enzymatically mediated reduction reactions in column reactors. Formate or lactate served as the electron donor and oxidized U(VI) and Mo(VI) species served as electron acceptors. Greater than 99% removal efficiencies were achieved for both metals with initial concentrations of 5 mg/L U and 10 mg/L Mo. Hydraulic residence times in the columns were between 24 and 36 h. Sulfate concentrations as high as 2000 mg/L did not inhibit reduction of U or Mo in the columns. However, nitrate inhibited uranium reduction at concentrations near 50 mg/L and inhibited molybdenum reduction at concentrations near 150 mg/L. The results indicate that enzymatic reduction of U and Mo by immobilized cells of D. desulfuricans may be a practical method for removing these contaminants from solution in continuous-flow reactors.     

Microbial populations stimulated for hexavalent uranium reduction in uranium mine sediment

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 (UO(2)) 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.     

Characterization of extracellular minerals produced during dissimilatory Fe(III) and U(VI) reduction at 100 degrees C by Pyrobaculum islandicum

In order to gain insight into the significance of biotic metal reduction and mineral formation in hyperthermophilic environments, metal mineralization as a result of the dissimilatory reduction of poorly crystalline Fe(III) oxide, and U(VI) reduction at 100 °C by Pyrobaculum islandicum was investigated. When P. islandicum was grown in a medium with poorly crystalline Fe(III) oxide as an electron acceptor and hydrogen as an electron donor, the Fe(III) oxide was reduced to an extracellular, ultrafine-grained magnetite with characteristics similar to that found in some hot environments and that was previously thought to be of abiotic origin. Furthermore, cell suspensions of P. islandicum rapidly reduced the soluble and oxidized form of uranium, U(VI), to extracellular precipitates of the highly insoluble U(IV) mineral, uraninite (UO₂). The reduction of U(VI) was dependent on the presence of hydrogen as the electron donor. These findings suggest that microbes may play a key role in metal deposition in hyperthermophilic environments and provide a plausible explanation for such phenomena as magnetite accumulation and formation of uranium deposits at ca . 100 °C.     

Reduction of uranium by cytochrome c3 of Desulfovibrio vulgaris.

The mechanism for U(VI) reduction by Desulfovibrio vulgaris (Hildenborough) was investigated. The H2-dependent U(VI) reductase activity in the soluble fraction of the cells was lost when the soluble fraction was passed over a cationic exchange column which extracted cytochrome c3. Addition of cytochrome c3 back to the soluble fraction that had been passed over the cationic exchange column restored the U(VI)-reducing capacity. Reduced cytochrome c3 was oxidized by U(VI), as was a c-type cytochrome(s) in whole-cell suspensions. When cytochrome c3 was combined with hydrogenase, its physiological electron donor, U(VI) was reduced in the presence of H2. Hydrogenase alone could not reduce U(VI). Rapid U(VI) reduction was followed by a subsequent slow precipitation of the U(IV) mineral uraninite. Cytochrome c3 reduced U(VI) in a uranium-contaminated surface water and groundwater. Cytochrome c3 provides the first enzyme model for the reduction and biomineralization of uranium in sedimentary environments. Furthermore, the finding that cytochrome c3 can catalyze the reductive precipitation of uranium may aid in the development of fixed-enzyme reactors and/or organisms with enhanced U(VI)-reducing capacity for the bioremediation of uranium-contaminated waters and waste streams.     

Reduction of Fe(III), Mn(IV), and toxic metals at 100 degrees C by Pyrobaculum islandicum

It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When Pyrobaculum islandicum was grown at 100 degrees C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilum grew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100 degrees C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.     

Modeling reduction of uranium U(VI) under variable sulfate concentrations by sulfate-reducing bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 +/- 3 degrees C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V(max), ranged from 2.4 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB. h(-1)) at 0.25 mM sulfate to 5.0 +/- 1.1 micromol of sulfate/mg (dry weight) of SRB. h(-1) at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V(max) was 1.6 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB. h(-1) at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 +/- 0.003 mg (dry weight) of cells/ml. min(-1) for the mixed culture and 0.137 +/- 0.016 mg (dry weight) of cells/ml. min(-1) (U(0) = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Microbial Populations Stimulated for Hexavalent Uranium Reduction in Uranium Mine Sediment

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.     

Uranium (VI) Reduction by Denitrifying Biomass

Groundwater near the S3 ponds at the US Department of Energy's Y-12 site in Oak Ridge, Tennessee, is contaminated by high levels of nitrate (up to 160 mM) and U(VI) (∼0.3 mM). To minimize nitrate inhibition, the authors proposed extraction of contaminated groundwater, nitrate removal in a denitrifying fluidized bed bioreactor (FBR), and return of nitrate-free effluent to the aquifer to stimulate in situ microbial reduction of U(VI). In the presence of carbonate, U(VI) sorption to biomass was negligible, but in its absence, sorption was significant. Biomass reduced U(VI) to U(IV), exhibiting slow first-order removal with respect to U(VI). Addition of electron donor increased rates. Addition of an inhibitor of sulfate reduction (molybdate) slowed the rate and inhibited sulfate reduction. Denitrifying β-Proteobacteria dominated clone libraries of SSU rRNA and dsrA gene sequences. Approximately 10% were low-G+C microorganisms that had 90% to 92% sequence identity with Sporomusa, Acetonema, and Propionispora. The dsrA sequences were dominated by a single clone with ∼80% nucleotide identity to dsrA of Desulfovibrio vulgaris sub sp. oxamicus. The authors conclude that some members of this denitrifyng community reduce uranium, and that sulfate-reducing bacteria likely contribute to this capability.     

Enrichment of members of the family Geobacteraceae associated with stimulation of dissimilatory metal reduction in uranium-contaminated aquifer sediments

Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family Geobacteraceae, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion, Geobacteraceae accounted for ca. 40% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas Geobacteraceae accounted for fewer than 5% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65% of these Geobacteraceae sequences were most similar to sequences from Desulfuromonas species, with the remainder being most closely related to Geobacter species. Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR and TaqMan analyses indicated that the number of Geobacteraceae sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in Geobacteraceae sequences was observed in control sediments. In contrast to the predominance of Geobacteraceae sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that Geobacteraceae are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that Geobacteraceae will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.     

Multiple influences of nitrate on uranium solubility during bioremediation of uranium-contaminated subsurface sediments

Microbiological reduction of soluble U(VI) to insoluble U(IV) has been proposed as a remediation strategy for uranium-contaminated groundwater. Nitrate is a common co-contaminant with uranium. Nitrate inhibited U(VI) reduction in acetate-amended aquifer sediments collected from a uranium-contaminated site in New Mexico. Once nitrate was depleted, both U(VI) and Fe(III) were reduced concurrently. When nitrate was added to sediments in which U(VI) had been reduced, U(VI) reappeared in solution. Parallel studies with the dissimilatory Fe(III)-, U(VI)- and nitrate-reducing microorganism, Geobacter metallireducens, demonstrated that nitrate inhibited reduction of Fe(III) and U(VI) in cell suspensions of cells that had been grown with nitrate as the electron acceptor, but not in Fe(III)-grown cells. Suspensions of nitrate-grown G. metallireducens oxidized Fe(II) and U(IV) with nitrate as the electron acceptor. U(IV) oxidation was accelerated when Fe(II) was also added, presumably due to the Fe(III) being formed abiotically oxidizing U(IV). These studies demonstrate that although the presence of nitrate is not likely to be an impediment to the bioremediation of uranium contamination with microbial U(VI) reduction, it is necessary to reduce nitrate before U(VI) can be reduced. These results also suggest that anaerobic oxidation of U(IV) to U(VI) with nitrate serving as the electron acceptor may provide a novel strategy for solubilizing and extracting microbial U(IV) precipitates from the subsurface.     

Metal reduction by spores of Desulfotomaculum reducens

The bioremediation of uranium‐contaminated sites is designed to stimulate the activity of microorganisms able to catalyze the reduction of soluble U(VI) to the less soluble mineral UO₂. U(VI) reduction does not necessarily support growth in previously studied bacteria, but it typically involves viable vegetative cells and the presence of an appropriate electron donor. We characterized U(VI) reduction by the sulfate‐reducing bacterium Desulfotomaculum reducens strain MI‐1 grown fermentatively on pyruvate and observed that spores were capable of U(VI) reduction. Hydrogen gas – a product of pyruvate fermentation – rather than pyruvate, served as the electron donor. The presence of spent growth medium was required for the process, suggesting that an unknown factor produced by the cells was necessary for reduction. Ultrafiltration of the spent medium followed by U(VI) reduction assays revealed that the factor's molecular size was below 3 kDa. Pre‐reduced spent medium displayed short‐term U(VI) reduction activity, suggesting that the missing factor may be an electron shuttle, but neither anthraquinone‐2,6‐disulfonic acid nor riboflavin rescued spore activity in fresh medium. Spores of D. reducens also reduced Fe(III)‐citrate under experimental conditions similar to those for U(VI) reduction. This is the first report of a bacterium able to reduce metals while in a sporulated state and underscores the novel nature of the mechanism of metal reduction by strain MI‐1.     

Reduction of Fe(III), Mn(IV), and Toxic Metals at 100°C by Pyrobaculum islandicum

It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When Pyrobaculum islandicum was grown at 100°C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilum grew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100°C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.     

Enrichment of Members of the Family Geobacteraceae Associated with Stimulation of Dissimilatory Metal Reduction in Uranium-Contaminated Aquifer Sediments

Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family Geobacteraceae, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion, Geobacteraceae accounted for ca. 40% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas Geobacteraceae accounted for fewer than 5% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65% of these Geobacteraceae sequences were most similar to sequences from Desulfuromonas species, with the remainder being most closely related to Geobacter species. Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR and TaqMan analyses indicated that the number of Geobacteraceae sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in Geobacteraceae sequences was observed in control sediments. In contrast to the predominance of Geobacteraceae sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that Geobacteraceae are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that Geobacteraceae will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.     

Kinetics of U(VI) reduction by a dissimilatory Fe(III)-reducing bacterium under non-growth conditions

Dissimilatory metal-reducing microorganisms may be useful in processes designed for selective removal of uranium from aqueous streams. These bacteria can use U(VI) as an electron acceptor and thereby reduce soluble U(VI) to insoluble U(IV). While significant research has been devoted to demonstrating and describing the mechanism of dissimilatory metal reduction, the reaction kinetics necessary to apply this for remediation processes have not been adequately defined. In this study, pure culture Shewanella alga strain BrY reduced U(VI) under non-growth conditions in the presence of excess lactate as the electron donor. Initial U(VI) concentrations ranged from 13 to 1680 muM. A maximum specific U(VI) reduction rate of 2.37 mumole-U(VI)/(mg-biomass h) and Monod half-saturation coefficient of 132 muM-U(VI) were calculated from measured U(VI) reduction rates. U(VI) reduction activity was sustained at 60% of this rate for at least 80 h. The initial presence of oxygen at a concentration equal to atmospheric saturation at 22 degrees C delays but does not prevent U(VI) reduction. The rate of U(VI) reduction by BrY is comparable or better than rates reported for other metal reducing species. BrY reduces U(VI) at a rate that is 30% of its Fe(III) reduction rate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 490-496, 1997.     

U(VI) Reduction in Sulfate-Reducing Subsurface Sediments Amended with Ethanol or Acetate

An experiment was conducted with subsurface sediments from Oak Ridge National Laboratory to determine the potential for reduction of U(VI) under sulfate-reducing conditions with either ethanol or acetate as the electron donor. The results showed extensive U(VI) reduction in sediments supplied with either electron donor, where geochemical and microbiological analyses demonstrated active sulfate reduction.     

Dynamics of microbial community composition and function during in situ bioremediation of a uranium-contaminated aquifer

A pilot-scale system was established to examine the feasibility of in situ U(VI) immobilization at a highly contaminated aquifer (U.S. DOE Integrated Field Research Challenge site, Oak Ridge, TN). Ethanol was injected intermittently as an electron donor to stimulate microbial U(VI) reduction, and U(VI) concentrations fell to below the Environmental Protection Agency drinking water standard (0.03 mg liter(-1)). Microbial communities from three monitoring wells were examined during active U(VI) reduction and maintenance phases with GeoChip, a high-density, comprehensive functional gene array. The overall microbial community structure exhibited a considerable shift over the remediation phases examined. GeoChip-based analysis revealed that Fe(III)-reducing bacterial (FeRB), nitrate-reducing bacterial (NRB), and sulfate-reducing bacterial (SRB) functional populations reached their highest levels during the active U(VI) reduction phase (days 137 to 370), in which denitrification and Fe(III) and sulfate reduction occurred sequentially. A gradual decrease in these functional populations occurred when reduction reactions stabilized, suggesting that these functional populations could play an important role in both active U(VI) reduction and maintenance of the stability of reduced U(IV). These results suggest that addition of electron donors stimulated the microbial community to create biogeochemical conditions favorable to U(VI) reduction and prevent the reduced U(IV) from reoxidation and that functional FeRB, SRB, and NRB populations within this system played key roles in this process.