Influence of Nitrate on Microbial Reduction of Pertechnetate

Feb 8, 2008 - During this transition, the microbial community changed from being ... Azoarcus FRC-B1, and a fermentative Clostridium FRC-C11 were ...
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Environ. Sci. Technol. 2008, 42, 1910–1915

Influence of Nitrate on Microbial Reduction of Pertechnetate XIANGZHEN LI AND LEE R. KRUMHOLZ* Department of Botany and Microbiology and Institute for Energy and the Environment, The University of Oklahoma, Norman, Oklahoma 73019

Received May 18, 2007. Revised manuscript received November 21, 2007. Accepted December 6, 2007.

Factors influencing microbial reduction of Tc(VII) in nitrate and radionuclide contaminated aquifer sediments were investigated using sediment microcosms containing organic electron donor, nitrate and Tc(VII). Microcosms underwent nitrate reduction followed by Tc(VII) reduction. During this transition, the microbial community changed from being dominated by bacteria affiliated with the genus Paenibacillus during the nitrate reduction phase, to those affiliated with genera Agrobacterium, Geothrix, and Desulfosporosinus during the Tc(VII) reduction phase. To investigate the mechanism of Tc(VII) reduction, the nitrate reducing strains Agrobacterium FRC-A2, Azoarcus FRCB1, and a fermentative Clostridium FRC-C11 were isolated from sediment microcosms undergoing Tc(VII) reduction. Nitrate reducing bacteria reduced Tc(VII) effectively only in the presence of Fe(III) and after nitrate was reduced, implying a major role for Fe(II) as an electron shuttle in Tc(VII) reduction. It is likely that accumulation of nitrite blocks Fe(II) production and hence Tc(VII) reduction during the active nitrate reduction phase. The pure culture of Clostridium FRC-C11 is able to reduce Tc(VII) enzymatically with H2 or glucose as electron donor and deposits insoluble Tc compounds within the cells in a manner that is not significantly influenced by the presence of nitrate. These results provided a possible mechanism for Tc(VII) reduction independent of Fe(III) and not influenced by nitrate.

Introduction Technetium is highly mobile in its oxidized form (pertechnetate TcVIIO4-), and with its long-half-life (213000 years), it constitutes one of the most hazardous components of typical radionuclide wastes (1). Under anaerobic conditions, Tc(VII) can be effectively immobilized through chemical or microbial reduction processes, resulting in the formation of low solubility Tc containing solid phases (mainly Tc(IV) and Tc(V)) under neutral conditions (2, 3). The product of Tc(VII) reduction in a range of sediment environments is almost exclusively a hydrous TcIVO2-like mineral (4–6). Chemical reduction of Tc(VII) occurs as Tc(VII) interacts with (a) Fe(II) or reduced S associated with synthetic minerals (6), (b) biogenic reduction products (5, 7, 8), or (c) natural geologic materials (6, 19). Direct enzymatic reduction of Tc(VII) has been shown to occur by the Fe(III)-reducing bacteria, Geobacter metallireducens and Shewanella putrefaciens (2, 11)as well as the sulfate reducing bacteria Desulfovibrio desulfuricans (12) and D. fructosovorans (13). These concepts have been applied to the in situ remediation of Tc(VII) contaminated groundwater. Recent studies * Correspondence author phone: 405-325-0437; fax: 405-325-7619; e-mail: [email protected]. 1910

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have shown that biostimulation of indigenous microbial communities by the addition of an organic electron donor will create reducing conditions that effectively promote Tc(VII) precipitation (14, 15). Unfortunately, radionuclide waste is often cocontaminated with nitrate, derived from use of nitric acid or ammonium in ore processing and isotope separation processes (1). In situ levels of nitrate can reach up to 170 mM in groundwater at the U.S. Department of Energy’s Field Research Center (FRC) in Oak Ridge, TN (14). Nitrate serves as a competing and energetically more favorable electron acceptor for metal reducing bacteria and has been shown to prevent the reduction of Tc(VII) in laboratory studies (16, 17). The mechanism of the inhibition has not been described. An understanding of the mechanism by which nitrate disrupts Tc(VII) reduction is further complicated by reports that, under in situ biostimulation conditions with ethanol, glucose, or acetate, Tc(VII) reduction may occur in the presence of nitrate (14). Previous work has shown that nitrate reduction is carried out by a phylogenetically and metabolically diverse group of bacteria (18) which includes many Fe(III) reducing bacteria (19). Fe(III) reducing bacteria in the family Geobacteraceae have been observed in microcosms undergoing Tc(VII) reduction (4, 7) as well as during in situ Tc(VII) bioreduction experiments at the FRC (14). However Geobacter is not typically observed to be the most abundant group in some environments, such as high-salinity or high-nitrate sediment (20, 21). It remains to be determined which microbial groups are responsible for in situ Tc(VII) reduction in nitrate contaminated environments. Our research aimed to determine the effects of nitrate on bacterial Tc(VII) reduction in laboratory microcosm experiments as well as pure cultures of nitrate reducing and fermentative bacteria isolated after enrichment on Tc(VII). To determine the role of specific functional groups on Tc(VII) reduction, changes in the microbial community as it makes the transition from active nitrate reduction to Tc(VII) reduction were investigated using culture-independent methods.

Materials and Methods Sediment Collection, Culture Media, and Artificial Groundwater Preparation. Sediment samples were collected near well GW-836 from the aquifer underlying area 2 of the U.S. Department of Energy, FRC, located in the Y-12 complex at Oak Ridge National Laboratory in Tennessee (For site description and sampling methods, see Supporting Information SI-A1). For all microcosm experiments in which sediments were used, GW-835 artificial groundwater (AGW) was added. The solution was formulated on the basis of the water composition from well GW-835 (close to GW-836). It contained (mM) CaCl2 (1.85), CaSO4 (1.56), Ca(NO3)2 (0.65), MgCl2 (1.07), NaNO3 (0.76), KCl (0.16), and MnCl2 (0.02). A mineral medium (MM) was used for pure culture experiments. The medium contained (mM) NaCl (1.84), NH4Cl (1.87), KCl (0.13), KH2PO4 (0.02), MgCl2 · 6H2O (0.2), and CaCl2 · 2H2O (0.27). AGW and MM were flushed with N2/CO2 (80/20, v/v). AGW was adjusted to pH 6.4 with HCl. NaHCO3 was added to the MM to 30 mM and to obtain a pH of 7.0-7.2. Nitrate concentrations were adjusted through addition of Ca(nitrate)2 from a stock solution. Microcosm Experiments. Microcosms were prepared in 30 mL serum bottles and contained 2 g of homogenized sediment slurry (ca. 50% moisture) and 10 mL of AGW. Bottles were flushed with N2/CO2 (80/20, v/v) and sealed with rubber 10.1021/es071164j CCC: $40.75

 2008 American Chemical Society

Published on Web 02/08/2008

Bacterial Isolation. MM + nitrate (10 mM) agar plates (1.5%) were prepared with either (a) yeast extract (0.1%), (b) glucose (10 mM), or (c) ethanol (50 mM). Media were innoculated by directly plating dilutions from microcosms, and plates were incubated anaerobically in sealed ammo boxes under N2/CO2 (80/20, v/v) for 1 week at 23 °C. Ammo boxes had been modified by drilling a hole in the lid for a stopper used for flushing with gas. Single colonies were picked and inoculated into liquid MM + nitrate with 10 mM glucose. Pure Culture Microcosms. Pure culture microcosms were established with 10 mL of artificial groundwater along with 1 g of sediment in 20 mL glass serum tubes in the same manner as sediment microcosms. Microcosms were then autoclaved for 1 h at 121 °C and cooled, and Tc(VII) was added to a final concentration of 20 µM. Acetate, glucose, ethanol, and nitrate were added from stock solutions. Duplicate microcosms for each isolate were inoculated with 0.2 mL of an early stationary phase culture (OD600 ) 0.3–0.4 for each species). No Tc(VII) reduction activity was detected in uninoculated controls. Resting Cell Experiments. To generate resting cells, log phase cultures were harvested by centrifugation. Cells were washed and resuspended in anaerobic 30 mM NaHCO3 buffer (pH 7.2) to about 25 µg/mL protein. Aliquots (3 mL) of the washed cell suspension were transferred into serum tubes with a headspace of N2/CO2 (80/20, v/v). Tc(VII) was added from a stock solution to a final concentration of 130 µM. Glucose (10 mM), ethanol (50 mM), formate (20 mM), lactate (20 mM), acetate (20 mM), or H2:CO2 (80/20, v/v, as headspace) was added as electron donor. Cells were incubated for 8 days with periodic sampling (0.2 mL) for Tc(VII) measurements. Chemical Analytical Methods. Ferrihydrite was chemically synthesized (22). nitrate, SO42-, and NO2– were measured by ion chromatography, ethanol by gas chromatography, and Fe(II) with the ferrozine assay (23). Soluble Tc was assayed with a liquid scintillation counter.

Results and Discussion FIGURE 1. Tc(VII) reduction during microcosm incubation with varying initial nitrate and ethanol concentration. Microcosms were incubated with GW-835 groundwater. ), nitrate; (, nitrite; O, Tc(VII); 2, Fe2+; *, SO42-. Arrow indicates sampling time for clone library analysis. Values represent the means of triplicate with standard deviation. For clarity, error bars are shown only when they are larger than the symbols (same in other figures). stoppers. Technetium-99 was amended to a final concentration of 5.6 µM as ammonium pertechnetate. Ethanol and nitrate were added to 50 or 100 mM each, and yeast extract was added where indicated to 0.1%. Sediments were incubated at 23 °C. Experiments were carried out in triplicate. Periodic sampling was carried out in an anaerobic glovebox (95% N2, 5% H2). Microcosms were first shaken, and the sediment slurry samples were removed with a syringe, dispensed into microcentrifuge tubes, and centrifuged at 20000g for 5 min, or filtered (0.2 µm). The aqueous phase was analyzed for nitrate, nitrite, SO42-, Fe2+, and soluble Tc as described below. For the measurement of sorbed Fe(II), sediment samples were extracted with 0.5 N HCl for 30 min prior to centrifugation. For the analysis of microbial community composition, unamended microcosms or microcosms amended with 100 mM ethanol and 100 mM nitrate were prepared (Figure 1). Amended bottles were sacrificed at zero time, 20 days, and 40 days. Unamended microcosms were sacrificed at 14 days. Contents of microcosms were centrifuged in a 50 mL centrifuge tube at 5000g for 10 min. DNA was then extracted from the pellet, and 16s rRNA clone libraries were constructed as described in Supporting Information SI-A2.

Tc(VII) Reduction in the Presence of Other Electron Acceptors. Microcosm experiments incubated at different nitrate concentrations showed that the reduction cascade of terminal-electron-accepting processes occurred (Figure 1). No instantaneous Tc removal was observed at the initial time points, indicating that neither rapid sorption nor rapid chemical reduction occurred. In microcosms without addition of nitrite and ethanol, endogenous nitrate was reduced within 3 days. The majority of the Tc was removed from solution between 6 and 10 days, during which time the sediment bound (0.5 M HCl extractable) Fe(II) also increased (Figure 1). When nitrate and ethanol (50 or 100 mM each) were added to sediment slurries, nitrite accumulated early in the incubations (Figure 1). During the period when nitrite was present, Fe(III) and Tc(VII) concentrations did not change, but after nitrite was completely reduced, Tc(VII) reduction and Fe(II) production occurred simultaneously. In the controls with nitrate but without ethanol addition, neither nitrate nor Tc(VII) reduction occurred (data not shown), indicating that the ethanol was being used as electron donor for Tc(VII) reduction. Microbial Community Shift during Tc Reduction. Microcosms containing 100 mM each of ethanol and nitrate were sampled for microbial community analysis at three time points, representing the initial community (0 day), active nitrate reduction (20 days), and at 40 days toward the end of the Tc(VII) and Fe(III) reduction phase (Figure 1). A reference microcosm (low nitrate) without addition of nitrate or ethanol was also prepared and sampled at day 14. Comparative sequence analysis revealed shifts in the composition of the microbial community during the reducVOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Class level distribution of clones from microcosm sediments sampled over the incubation period with n being the total number of clones from each sample. tion processes. Initially, sediments were highly enriched in microorganisms with β-proteobacterial 16S rRNA gene sequences (Figure 2 and Figure S4) with the majority (72.2%) affiliated with the genus Aquaspirillum. The β-proteobacteria remained important at later time points but decreased to less than 20% of clones at 20 and 40 days. While nitrate reduction occurred, the majority of clones (72.5%) were related to the genus Paenibacillus, suggesting its important role in nitrate reduction. Members of the genus Paenibacillus are facultative organisms capable of polysaccharide degradation, fermentation, and nitrate reduction (24). Paenibacillus related clones have been previously detected in iron reducing enrichment cultures from FRC sediment incubated with glucose or glycerol (25). During active nitrate reduction, less than 4% of the clones were related to the genus Agrobacterium. However when the final sample was removed after nitrate reduction was complete, the fraction of Paenibacillus clones had dropped dramatically to less than 2% with a concomitant increase in clones related to other nitrate reducing bacteria, metal reducing bacteria, and sulfate reducing bacteria. The dominant clones were related to Agrobacterium (41.5%), Geothrix (13.2%), and Desulfosporosinus (13.2%). Geothrix fermentans, the only named species of Geothrix, was isolated on acetate/ Fe(III) medium from a petroleum-contaminated aquifer (26). This organism is within the Phylum Acidobacteria. Desulfosporosinus species are gram-positive sulfate reducing bacteria that are known to grow on ethanol and short-chain fatty acids. They have been detected in uranium contaminated subsurface sediments (20) and gasoline-contaminated groundwater (27). In microcosms without ethanol or nitrate additions, the most abundant clones were affiliated with the genus Herbaspirillum (71.8%), with clones related to Desulfosporosinus only accounting for 2.9% of the library. Tc(VII) Reduction by Nitrate Reducing Bacteria. Sediment dilutions from day 20 and 40 microcosms were directly plated on agar containing 50 mM ethanol or 10 mM glucose along with 10 mM nitrate, and 10 strains were isolated. Surprisingly, nine of them had identical 16s rRNA sequences with a similarity of 98.6% to Agrobacterium tumefaciens (AF508099)and98.4%toSinorhizobiumsp.DAO10(DQ336178), and also with a similarity of >97% to the Agrobacterium 1912

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related clone sequences that made up 41.5% of the clone library in the day 40 sample. Both A. tumefaciens and Sinorhizobium DAO10 are denitrifying strains that are often isolated from contaminated sites (28, 29). From this group of nine strains, one designated Agrobacterium FRC-A2 was chosen for further study. The last colony isolated from the day 40 sample was related to Azoarcus and designated Azoarcus FRC-B1. This latter strain uses ethanol, lactate, acetate, formate, and glucose as electron donors for nitrate reduction. The 16s rRNA sequence was 99.8% similar to that of Azoarcus toluclasticus (AF123077), a denitrifying bacterium that degrades aromatic compounds (30). Previous investigations at the FRC with nitrate- and uranium-contaminated groundwater have yielded nirS and nirK clones from organisms related to Agrobacterium and Rhizobium (31), indicating the more general role for this genus in nitrate and possibly Tc(VII) reduction at the FRC. Both strains were used to investigate the relationship between nitrate and Tc(VII) reduction. At a concentration of 10 mM nitrate, Agrobacterium FRC-A2 reduced Tc(VII) with glucose, acetate, lactate, or ethanol as electron donors in the presence of ferrihydrite (data not shown). At low nitrite concentration, nitrate was reduced within 3 days followed by Tc(VII) reduction (Figure 3). Over 99% of added Tc(VII) was removed during 15 days. The HCl-extractable Fe2+ concentration was more than 150 µM during the incubation. Controls lacking ferrihydrite did not reduce Tc(VII), indicating a role of Fe(II) in Tc(VII) reduction. Tc(VII) was not reduced with or without ferrihydrite with formate as electron donor, and nitrite was detected throughout the experiment. Similarly, in the presence of 100 mM nitrate and 40 mM glucose, Tc(VII) reduction did not occur and nitrite was present throughout the 20 day incubation (Figure 3). When the nitrite concentration increased at day 6, HCl-extractable Fe(II) decreased, perhaps as a result of nitrite-dependent oxidation of Fe(II). The above experiments were repeated with Azoarcus FRC-B1, and reduction behavior was similar to that of Agrobacterium FRC-A2. Inoculation of Agrobacterium FRC-A2 into sterilized sediment along with AGW containing 2 mM nitrate and 10 mM glucose (Figure S1) induced nitrate and Tc(VII) reduction, and significant amounts of Fe(II) were detected. In the incubations with 50 or 100 mM nitrate (glucose 40 mM),

FIGURE 3. Tc(VII), NO3–, and Fe(III) reduction by Agrobacterium FRC-A2. Cultures were grown in a mineral medium supplied with 10 mM nitrate with 10 mM glucose or 130 mM NO3– with 40 mM glucose with or without 50 mM Fe(III): ), nitrate; (, nitrite; O, Tc(VII); 2, Fe2+. Averages of two measurements with mean deviation are given. nitrite accumulated, Fe(II) concentrations were low, and no Tc(VII) was reduced within 30 days. Tc(VII) Reduction with Clostridium FRC-C11. Microcosms were set up with yeast extract (0.1%) as the only organic supplement along with 10 mM nitrate in artificial groundwater. After Tc(VII) reduction occurred, sediments were diluted and plated onto MM with either glucose or yeast extract as substrate (10 mM nitrate). The most abundant organism isolated was a Clostridium sp. designated as strain FRC-C11. The 16s rRNA sequence of this strain had a 99.2% similarity to that of Clostridium favosporum (DSM 5907). Strain FRC-C11 grows with glucose or yeast extract, but not ethanol, acetate, lactate, or formate. It does not reduce nitrate (Figure 4 and Figure S2).

FIGURE 4. Tc(VII), nitrate, and Fe(III) reduction by Clostridium FRC-Cl1 in microcosms with sterilized sediment at an initial nitrate concentration of 2 ((), 50 (0), and 100 mM (O). The GW-835 groundwater was supplied with 10, 40, and 40 mM glucose for 2, 50, and 100 mM nitrate treatments, respectively. Averages of two measurements with mean deviation are given. When pure cultures were incubated with glucose and 20 µM Tc(VII), 58% of the Tc(VII) was reduced within 20 days (Figure S2). The addition of 10% H2 to the headspace had no significant effect on Tc reduction. Addition of 100 mM nitrate briefly delayed Tc reduction, but 68% of the Tc(VII) was still reduced by day 20. The delay indicates that nitrate may be transformed at low levels. When ferrihydrite was added to cultures with 2 mM nitrate, 96% of the Tc(VII) was removed from solution by 20 days. These latter cultures produced low levels of Fe(II) (up to 104 µM), which is presumably also involved in Tc(VII) reduction. When Clostridium FRC-Cl1 was inoculated into sterilized sediments, in the presence of nitrate (2, 50, and 100 mM) (Figure 4), the majority of the Tc(VII) (96, 87, and 89%, respectively) was reduced within 20 days. Nitrite did not accumulate, but the addition of 50 or 100 mM nitrate appeared to suppress the accumulation of Fe(II) presumably impacting the extent of Tc(VII) reduction. These and the VOL. 42, NO. 6, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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above pure culture results indicate that both a direct and a Fe(II) mediated reduction of Tc(VII) are likely in this system. Clostridium FRC-C11 sequences were not retrieved from ethanol microcosms, most likely because it does not use ethanol. However, in situ biostimulation with glucose at several FRC sites enriched for sequences related to Clostridium beijerinckii (32), a fermentative bacterium whose SS rRNA sequence is 98% similar to FRC-C11. Reduction of Tc(VII) by Resting Cells of Clostridium FRCC11. Resting cells of Clostridium FRC-C11 were prepared to determine their ability to directly reduce Tc(VII). During an 8 day incubation with a 80% H2 headspace, 89% of the total Tc(VII) (130 µM) was removed from solution. In the controls without cells or with only N2 in the headspace, no Tc(VII) reduction occurred. Glucose was a relatively poor electron donor for Tc(VII) reduction by resting cells. After the experiments were terminated, the suspension was separated by paper chromatography and shown to contain a Tc(V) compound (Figure S3A). Transmission electron microscopy (TEM) showed an electron-dense deposit in the cytoplasm (Figure S3B) and EDAX demonstrated that it contained Tc (Figure S3C), indicating cytoplasmic Tc(VII) reduction. Role of Fe(II) on Tc Reduction. Through pure culture studies with Agrobacterium FRC-A2, we have shown that the coupled biotic reduction of Fe(III) with the abiotic reduction of Tc(VII) occurs in sediments. Previous work has investigated mechanisms for in situ Tc(VII) reduction and suggested that the active Fe(II) production in sediments as a result of Fe(III) reduction is the most likely controlling mechanism for Tc(VII) reduction (4, 5, 8). Our pure culture work is in agreement with this conclusion. When the ratio of sediment associated Fe(II) to Tc(VII) was higher than 4.3, Tc(VII) was shown to be completely reduced (5). Microcosm experiments reported here had HCl-extractable Fe2+ reaching several millimolar with Tc(VII) at 5.6 µM. Most of Fe2+ is solid associated, reinforcing the importance of surface reactions in Tc(VII) reduction (8). In our microcosm experiments in which an excess of electron donor was present, simultaneous reduction of Fe(III), sulfate, and Tc(VII) occurred. These processes were accompanied by an increase in dissolved Fe(II) and the presence of 16s rRNAs from sulfate reducing (SRB) and Fe(III) reducing bacteria. This simultaneous increase would only happen when electron donor was in excess. As SRB are known to be capable of reducing Fe(III) (3), both the SRB and denitrifying bacteria may be providing Fe(II) needed for Tc(VII) reduction. Clostridium FRC-C11 has been shown to carry out Tc(VII) reduction through Fe(II) and also through enzymatic processes with either sugars or H2 as the electron donor. Similarly, other Clostridia (33) have been shown to be capable of Tc(VII) reduction. The fact that Tc(IV) accumulates in the cytoplasm of the cell provides additional evidence for direct ezymatic reduction by this species. This process would be independent of the Fe(II) and nitrate concentration. It seems likely, however, that due to the natural abundance of iron minerals and typical low concentrations of degradable organic compounds and H2, indirect Tc(VII) reduction may dominate during in situ Tc(VII) transformation. These results and those obtained in previous laboratory investigations of Tc(VII) reduction, clearly point to a mechanism involving indirect (Fe coupled) reduction. However Tc(VII) reduction has been observed in laboratory experiments with nitrate present (34) and field experiments with nitrate (14). In these situations, it is possible that bacteria like Clostridia are also involved in a direct reduction process. Nitrate Effects on Tc Reduction. As nitrate reduction occurs in laboratory incubated sediments and pure cultures of nitrate reducing bacteria, no Tc(VII) reduction was observed. Nitrate likely competes with Tc(VII) as an electron acceptor (1), or it may inhibit Tc(VII) reduction through the 1914

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oxidation of Fe(II). Nitrate will not directly oxidize Tc(IV) sulfide (35) and does not prevent Tc(VII) from being reduced by Clostridium FRC-C11, which is unable to produce nitrite (this study). However, nitrite is often accumulated during denitrification at high initial nitrate concentrations (14, 19). In the incubation experiments reported here in which accumulation of nitrite occurred, Tc(VII) reduction did not occur. Anaerobic incubation of 10 mM nitrite with reduced Tc(IV) in sterilized sediment did not solubilize Tc(IV) (data not shown); the effect of nitrite must therefore be in preventing Tc(VII) reduction. Although nitrite is a poor oxidant of Fe(II) at low nitrite:Fe(II) molar ratios (36), nitrite will rapidly oxidize Fe(II) when the nitrite:Fe(II) molar ratio increases (19), as in the systems described here where nitrite: Fe(II) molar ratios reached more than 200:1 (Figure 1). Experiments in which 15 mM nitrite was added to sediment microcosms containing 5 mM Fe(II) showed that the Fe(II) was rapidly oxidized (Figure S4) and Tc(VII) reduction did not occur within 20 days. In the same microcosms which had no nitrite added, but were supplemented with 100 mM nitrate and ethanol, Fe(II) was quickly oxidized between 6 and 10 days, during the time that active nitrate reduction occurred and nitrite accumulated (Figure 1). Fe(II) oxidation was much weaker in the microcosms without ethanol, in which microbial nitrite reduction activity was low due to lack of organic substrate, and less than 1 mM nitrite was detected. These data indicate that chemical reoxidation of Fe(II) by nitrite occurred as nitrate reduction was occurring. This observation is similar to that in previous work in which active nitrate reduction resulted in accumulation of Fe3+ (19, 37) which in turn results in U(IV) oxidation in U containing microcosms (19). This process plus the competition for electron acceptor in the nitrate reducing conditions prevents Tc(VII) from being reduced.

Acknowledgments This research was supported by the Environmental Remediation Science Program from the U.S. Department of Energy. We thank David B. Watson for conducting sediment sampling and Anne Spain for technical assistance.

Supporting Information Available Detailed description of experimental methods and Tc(VII) reduction by isolated strains. This material is available free of charge via the Internet at http://pubs.acs.org.

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