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PubMed2004

Effect of iron bioavailability on dissolved hydrogen concentrations during microbial iron reduction.

Dissolved hydrogen (H2) concentrations have been shown to correlate with specific terminal electron accepting processes (TEAPs) in aquifers. The research presented herein examined the effect of iron bioavailability on H2 concentrations during iron reduction in flow-through column experiments filled with soil obtained from the uncontaminated background area of the Field Research Center (FRC), Oak Ridge, TN and amended with acetate as the electron donor. The first column experiment measured H2 concentrations over 500 days of column operation that fluctuated within a substantial range around an average of 3.9 nM. Iron reduction was determined to be the dominant electron accepting process. AQDS (9,10-anthraquinone-2,6-disulfonic acid) was then used to determine if H2 concentrations during iron reduction were related to iron bioavailability. For this purpose, a 100-day flow-through column experiment was conducted that compared the effect of AQDS on iron reduction and subsequent H2 concentrations using two columns in parallel. Both columns were packed with FRC soil and inoculated with Geobacter sulfurreducens but only one was supplied with AQDS. The addition of AQDS increased the rate of iron reduction in the flow-through column and slightly decreased the steady-state H2 concentrations from an average of 4.0 nM for the column without AQDS to 2.0 nM for the column with AQDS. The results of this study therefore show that H2 can be used as an indicator to monitor rate and bioavailability changes during microbial iron reduction.

Anthraquinones↗
PubMed2005

Sustained and complete hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation in zero-valent iron simulated barriers under different microbial conditions.

Flow-through columns packed with "aged" zero-valent iron (ZVI) between layers of soil and sand were constructed to mimic a one-dimensional permeable reactive iron barrier (PRB). The columns were continuously fed RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine, ca. 18 mg l(-1)) for over one year. Two columns were bioaugmented with dissimilatory iron reducing bacteria (DIRB) Shewanella algae BrY or Geobacter metallireducens GS-15 to investigate their potential to enhance the reactivity of aged iron by reductive dissolution of passivating iron oxides or via production of biogenic reactive minerals. A third column was not bioaugmented to evaluate colonization by indigenous soil microorganisms. [14C]-RDX was completely removed in all columns at the start of the iron layer, and concentration profiles showed rapid and sustainable RDX removal over one year; however, a phylogenetic profile conducted after one year using DGGE analysis of recovered DNA did not detect S. algae BrY or G. metallireducens in their respective columns. Bacterial DNA was recovered from within the ZVI. Several unidentified 14C-labeled byproducts were present in the effluent of all columns. Dissolved 14C removal and the detection of dissolved inorganic 14C in these columns (but not in the sterile control) suggest microbial-mediated mineralization of RDX and sorption/precipitation of degradation products. Enhanced RDX mineralization in bioaugmented columns was temporary relative to the indigenously colonized column. However, shorter acclimation periods associated with bioaugmented PRBs may be desirable for rapid RDX mineralization, thereby preventing breakthrough of potentially undesirable byproducts. Overall, these results show that high RDX removal efficiency by ZVI-PRBs is achievable and sustainable and that the efficacy and start-up of ZVI-PRBs might be enhanced by bioaugmentation.

Biodegradation, Environmental↗
PubMed2004

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer.

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.

Amino Acid Sequence↗
PubMed2005

Iron-reducing bacteria unravel novel strategies for the anaerobic catabolism of aromatic compounds.

Although the aerobic degradation of aromatic compounds has been extensively studied in many microorganisms, the anaerobic mineralization of the aromatic ring is a more recently discovered microbial capacity on which very little information is available from facultative anaerobic bacteria. In this issue of Molecular Microbiology, Wischgoll and colleagues use proteomic and reverse-transcription polymerase chain reaction (PCR) approaches to identify for the first time the gene clusters involved in the central pathway for the catabolism of aromatic compounds in Geobacter metallireducens, a strictly anaerobic iron-reducing bacterium. This work highlights that the major difference in anaerobic benzoate metabolism of facultative and strictly anaerobic bacteria is the reductive process for dearomatization of benzoyl-CoA. The authors propose that a new type of benzoyl-CoA reductase, comprising molybdenum- and selenocysteine-containing proteins, is present in strictly anaerobic bacteria. This work paves the way to fundamental studies on the biochemistry and regulation of this new reductive process and provides the first genetic clues on the anaerobic catabolism of benzoate by strict anaerobes.

Acyl Coenzyme A↗
PubMed2006

Microbially mediated biodegradation of hexahydro-1,3,5-trinitro-1,3,5- triazine by extracellular electron shuttling compounds.

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.

Anthraquinones↗
PubMed2004

In situ expression of nifD in Geobacteraceae in subsurface sediments.

In order to determine whether the metabolic state of Geobacteraceae involved in bioremediation of subsurface sediments might be inferred from levels of mRNA for key genes, in situ expression of nifD, a highly conserved gene involved in nitrogen fixation, was investigated. When Geobacter sulfurreducens was grown without a source of fixed nitrogen in chemostats with acetate provided as the limiting electron donor and Fe(III) as the electron acceptor, levels of nifD transcripts were 4 to 5 orders of magnitude higher than in chemostat cultures provided with ammonium. In contrast, the number of transcripts of recA and the 16S rRNA gene were slightly lower in the absence of ammonium. The addition of acetate to organic- and nitrogen-poor subsurface sediments stimulated the growth of Geobacteraceae and Fe(III) reduction, as well as the expression of nifD in Geobacteraceae. Levels of nifD transcripts in Geobacteraceae decreased more than 100-fold within 2 days after the addition of 100 microM ammonium, while levels of recA and total bacterial 16S rRNA in Geobacteraceae remained relatively constant. Ammonium amendments had no effect on rates of Fe(III) reduction in acetate-amended sediments or toluene degradation in petroleum-contaminated sediments, suggesting that other factors, such as the rate that Geobacteraceae could access Fe(III) oxides, limited Fe(III) reduction. These results demonstrate that it is possible to monitor one aspect of the in situ metabolic state of Geobacteraceae species in subsurface sediments via analysis of mRNA levels, which is the first step toward a more global analysis of in situ gene expression related to nutrient status and stress response during bioremediation by Geobacteraceae.

Acetates↗
PubMed2006

Mercury methylation from unexpected sources: molybdate-inhibited freshwater sediments and an iron-reducing bacterium.

Methylmercury has been thought to be produced predominantly by sulfate-reducing bacteria in anoxic sediments. Here we show that in circumneutral pH sediments (Clear Lake, CA) application of a specific inhibitor of sulfate-reducing bacteria at appropriate concentrations typically inhibited less than one-half of all anaerobic methylation of added divalent mercury. This suggests that one or more additional groups of microbes are active methylators in these sediments impacted by a nearby abandoned mercury mine. From Clear Lake sediments, we isolated the iron-reducing bacterium Geobacter sp. strain CLFeRB, which can methylate mercury at a rate comparable to Desulfobulbus propionicus strain 1pr3, a sulfate-reducing bacterium known to be an active methylator. This is the first time that an iron-reducing bacterium has been shown to methylate mercury at environmentally significant rates. We suggest that mercury methylation by iron-reducing bacteria represents a previously unidentified and potentially significant source of this environmental toxin in iron-rich freshwater sediments.

DNA, Ribosomal↗
PubMed2002

Reduction kinetics of Fe(III), Co(III), U(VI), Cr(VI), and Tc(VII) in cultures of dissimilatory metal-reducing bacteria.

The reduction kinetics of Fe(III)citrate, Fe(III)NTA, Co(III)EDTA-, U(VI)O(2) (2+), Cr(VI)O(4) (2-), and Tc(VII)O(4) (-) were studied in cultures of dissimilatory metal reducing bacteria (DMRB): Shewanella alga strain BrY, Shewanella putrefaciens strain CN32, Shewanella oneidensis strain MR-1, and Geobacter metallireducens strain GS-15. Reduction rates were metal specific with the following rate trend: Fe(III)citrate > or = Fe(III)NTA > Co(III)EDTA- >> UO(2)(2+) > CrO(4)(2-) > TcO(4)(-), except for CrO(4) (2-) when H(2) was used as electron donor. The metal reduction rates were also electron donor dependent with faster rates observed for H(2) than lactate- for all Shewanella species despite higher initial lactate (10 mM) than H2 (0.48 mM). The bioreduction of CrO(4) (2-) was anomalously slower compared to the other metals with H(2) as an electron donor relative to lactate and reduction ceased before all the CrO(4)(2-) had been reduced. Transmission electron microscopic (TEM) and energy-dispersive spectroscopic (EDS) analyses performed on selected solids at experiment termination found precipitates of reduced U and Tc in association with the outer cell membrane and in the periplasm of the bacteria. The kinetic rates of metal reduction were correlated with the precipitation of reduced metal phases and their causal relationship discussed. The experimental rate data were well described by a Monod kinetic expression with respect to the electron acceptor for all metals except CrO(4)(2-), for which the Monod model had to be modified to account for incomplete reduction. However, the Monod models became statistically over-parameterized, resulting in large uncertainties of their parameters. A first-order approximation to the Monod model also effectively described the experimental results, but the rate coefficients exhibited far less uncertainty. The more precise rate coefficients of the first-order model provided a better means than the Monod parameters, to quantitatively compare the reduction rates between metals, electron donors, and DMRB species.

Biodegradation, Environmental↗
PubMed2004

Utilization of microbial biofilms as monitors of bioremediation.

A down-well aquifer microbial sampling system was developed using glass wool or Bio-Sep beads as a solid-phase support matrix. Here we describe the use of these devices to monitor the groundwater microbial community dynamics during field bioremediation experiments at the U.S. Department of Energy Natural and Accelerated Bioremediation Research Program's Field Research Center at the Oak Ridge National Laboratory. During the 6-week deployment, microbial biofilms colonized glass wool and bead internal surfaces. Changes in viable biomass, community composition, metabolic status, and respiratory state were reflected in sampler composition, type of donor, and groundwater pH. Biofilms that formed on Bio-Sep beads had 2-13 times greater viable biomass; however, the bead communities were less metabolically active [higher cyclopropane/monoenoic phospholipid fatty acid (PLFA) ratios] and had a lower aerobic respiratory state (lower total respiratory quinone/ PLFA ratio and ubiquinone/menaquinone ratio) than the biofilms formed on glass wool. Anaerobic growth in these systems was characterized by plasmalogen phospholipids and was greater in the wells that received electron donor additions. Partial 16S rDNA sequences indicated that Geobacter and nitrate-reducing organisms were induced by the acetate, ethanol, or glucose additions. DNA and lipid biomarkers were extracted and recovered without the complications that commonly plague sediment samples due to the presence of clay or dissolved organic matter. Although microbial community composition in the groundwater or adjacent sediments may differ from those formed on down-well biofilm samplers, the metabolic activity responses of the biofilms to modifications in groundwater geochemistry record the responses of the microbial community to biostimulation while providing integrative sampling and ease of recovery for biomarker analysis.

Acetates↗
PubMed2003

Cloning and characterization of a novel gene which encodes a protein interacting with the mitosis-associated kinase-like protein NTKL.

NTKL is an evolutionarily conserved kinase-like protein. The cell-cycle-dependent centrosomal localization of NTKL suggested that it was involved in centrosome-related cellular function. The mouse NTKL protein is highly homologous with human NTKL. A novel mouse protein was identified as an NTKL-binding protein (NTKL-BP1) by yeast two-hybrid screening, and the full-length cDNA was amplified based on the result of a sequence data analysis cloning strategy. The full-length cDNA sequence of the NTKL-BP1 gene consists of 2,537 bp, which encode 368 amino acids. A database search revealed that homologues of NTKL-BP1 exist in different organisms, including Arabidopsis thaliana, Drosophila melanogaster, Plasmodium falciparum, Geobacter metallireducens, Anopheles gambiae and human. It suggests that NTKL-BP1 is an evolutionarily conserved protein. The expression of NTKL-BP1 was observed in multiple normal mouse tissues. The interaction of the two proteins was confirmed by co-immunoprecipitation. Moreover, immunofluorescent staining indicated that NTKL and NTKL-BP1 were all localized in the cytoplasm.

Amino Acid Sequence↗
PubMed2026

Abundance-transcription decoupling reveals functional partitioning in bioelectrochemical denitrification biofilms.

Bioelectrochemical denitrification (BED) is often attributed to electroactive microorganisms that access electrode-derived electrons, yet the relative functional contribution of electroactive taxa and denitrifying populations within complex BED biofilms remain unclear. Here, we integrated reactor measurements with genome-resolved metagenomics and metatranscriptomics to examine microbial community structure, functional potential, and gene transcription across contrasting BED operational regimes differing in dissolved oxygen (DO), hydraulic retention time (HRT)/loading, and poised potential. Nitrate removal exceeded 90% across all tested conditions, but nitrogen intermediate accumulation, current generation, and theoretical electron balance differed substantially. Electroactive taxa such as Geobacter dominated (>80% abundance) under longer HRT and stronger poised potential, but contributed minimally to the transcription of canonical denitrification genes. Weaker cathodic potential enriched transcriptionally active denitrifying taxa such as Stutzerimonas, Acidovorax, and MR-S7, while oxygen exposure induced redox-stress responses and reshaped nitrogen metabolism beyond being a competing electron acceptor. Together, these results reveal a decoupling between taxonomic abundance, genomic functional potential, and transcriptional contribution in BED biofilms, indicating that nitrate-removal performance cannot be inferred from current generation or electroactive-taxon abundance alone.

Bioelectrochemical system↗
PubMed2006

Identification and characterization of the gene encoding the Acidobacterium capsulatum major sigma factor.

Acidobacterium capsulatum is an acid-tolerant, encapsulated, Gram-negative member of the ubiquitous, but poorly understood Acidobacteria phylum. Little is known about the genetics and regulatory mechanisms of A. capsulatum. To begin to address this gap, we identified the gene encoding the A. capsulatum major sigma factor, rpoD, which encodes a 597-amino acid protein with a predicted sequence highly similar to the major sigma factors of Solibacter usitatus Ellin6076 and Geobacter sulfurreducens PCA. Purified hexahistidine-tagged RpoD migrates at approximately 70 kDa under SDS-PAGE conditions, which is consistent with the predicted MW of 69.2 kDa, and the gene product is immunoreactive with monoclonal antibodies specific for either bacterial RpoD proteins or the N-terminal histidine tag. A. capsulatum RpoD restored normal growth to E. coli strain CAG20153 under conditions that prevent expression of the endogenous rpoD. These results indicate we have cloned the gene encoding the A. capsulatum major sigma factor and the gene product is active in E. coli.

Amino Acid Sequence↗
PubMed1999

Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria.

The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) for evaluating the metabolic activity of aerobic bacteria has gained wide application in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the factors contributing to abiotic reduction of CTC were also examined. CTC was used in conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF), that targets bacterial cell wall proteins, to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bacteria, including Escherichia coli grown fermentatively, and Pseudomonas chlorophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudoalcalegenes grown under nitrate-reducing conditions, actively reduced CTC during all phases of growth. Greater than 95% of these cells accumulated intracellular CTC-formazan crystals during the exponential phase. Obligate anaerobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desulfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. The optimal CTC concentration for all organisms examined was 5 mM. Anaerobic CTC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing ferric citrate as electron acceptor following growth of either G. metallireducens or G. sulfurreducens. Abiotic reduction of CTC was observed in the presence of ascorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)Cl2, sodium thioglycolic acid and sodium sulfide. These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacteria, care must be taken to avoid abiotic reduction of CTC.

Bacteria, Anaerobic↗
PubMed2003

Evaluation of electron-shuttling compounds in microbial ferric iron reduction.

Iron-reducing bacteria can transfer electrons to ferric iron oxides which are barely soluble at neutral pH, and electron-shuttling compounds or chelators are discussed to be involved in this process. Experiments using semipermeable membranes for separation of ferric iron-reducing bacteria from ferric iron oxides do not provide conclusive results in this respect. Here, we used ferrihydrite embedded in 1% agar to check for electron-shuttling compounds in pure and in enrichment cultures. Geobacter sulfurreducens reduced spatially distant ferrihydrite only in the presence of anthraquinone-2,6-disulfonate, a small molecule known to shuttle electrons between the bacterial cell and ferrihydrite. However, indications for the production and excretion of electron-shuttling compounds or chelators were found in ferrihydrite-containing agar dilution cultures that were inoculated with ferric iron-reducing enrichment cultures.

Anthraquinones↗
PubMed2002

Carbon tetrachloride transformation in a model iron-reducing culture: relative kinetics of biotic and abiotic reactions.

Contributions of biotic (cell-mediated) and abiotic (mineral-mediated) reactions to carbon tetrachloride (CT) transformation were studied in a model iron-reducing system that used hydrous ferric oxide (HFO) as the electron acceptor, acetate as the substrate, and Geobacter metallireducens as a representative dissimilative iron-reducing bacteria (DIRB). Over a period of 2-3 weeks, nanoscale magnetite particles, Fe3O4, were consistently formed as a product of iron respiration in this system. CT transformation rates were measured independently in resting cell suspensions of G. metallireducens or in suspensions of washed magnetite particles recovered from spent cultures. Protein and surface area-normalized expressions were derived for the biotic and abiotic reaction rates, respectively. Using the yield of total protein and magnetite surface area formed during growth in the model system as a basis for comparison, the mineral-mediated (abiotic) reaction was estimated to be 60-260-fold faster than the biotic reaction throughout the incubation period. We conclude that G. metallireducens induces CT transformation in this system primarily through the formation of reactive mineral surfaces rather than via co-metabolic mechanisms. The findings indicate that reactive biogenic minerals could play a significant role in the natural attenuation of chlorinated solvents in iron-reducing environments. A novel approach for stimulating reductive transformation of contaminants may be to enhance the formation of reactive biogenic minerals in situ.

Biotransformation↗
PubMed2003

Inhibition of bacterial U(VI) reduction by calcium.

The rapid kinetics of bacterial U(VI) reduction and low solubility of uraninite (UO2,cr) make this process an attractive option for removing uranium from groundwater. Nevertheless, conditions that may promote or inhibit U(VI) reduction are not well-defined. Recent descriptions of Ca-UO2-CO3 complexes indicate that these species may dominate the aqueous speciation of U(VI) in many environments. We monitored the bacterial reduction of U(VI) in bicarbonate-buffered solution in the presence and absence of Ca. XAFS measurements confirmed the presence of a Ca-U(VI)-C03 complex in the initial solutions containing calcium. Calcium, at millimolar concentrations (0.45-5 mM), caused a significant decrease in the rate and extent of bacterial U(VI) reduction. Both facultative (Shewanella putrefaciens strain CN32) and obligate (Desulfovibrio desulfuricans, Geobacter sulfurreducens) anaerobic bacteria were affected by the presence of calcium. Reduction of U(VI) ceased when the calculated system Eh reached -0.046 +/- 0.001 V, based on the Ca2UO2(CO3)3 --> UO2,cr couple. The results are consistent with the hypothesis that U is a less energetically favorable electron acceptor when the Ca-UO2-CO3 complexes are present. The results do not support Ca inhibition caused by direct interactions with the cells or with the electron donor as the reduction of fumarate or Tc(VII)O4- under identical conditions was unaffected by the presence of Ca.

Bacteria, Anaerobic↗
PubMed2004

Carbon tetrachloride transformation on the surface of nanoscale biogenic magnetite particles.

Iron-reducing conditions in subsurface environments promote dechlorination reactions via both biotic and abiotic pathways, the latter often mediated via biologically activated minerals formed by dissimilatory iron-reducing bacteria (DIRB). Here we report the major products and pathways associated with the abiotic transformation of carbon tetrachloride (CT) by nanoscale biogenic magnetite/maghemite particles produced by the DIRB Geobacter metallireducens. Product formation and free radical/carbene trapping studies indicate that CT transformation occurs via three parallel pathways. The first pathway (hydrogenolysis) results in the formation of chloroform (45-50%) via a trichloromethyl free radical (*CCl3) and possibly a trichloromethyl carbanion (**CCl3-). The second and third pathways involve a dichlorocarbene intermediate (**CCl2), which either hydrolyzes to form CO (approximately 38%) (carbene hydrolysis), or undergoes further reduction to yield methane (8-10%) (carbene reduction). The mechanism of methane formation from **CCl2 is not known, but is speculated to involve a sequence of surface coordinated carbenoid and free radical complexes. The large fraction of relatively benign products formed by the carbene-mediated pathways suggests that magnetite/maghemite particles may have a beneficial application in the remediation of CT contaminated environments.

Carbon Tetrachloride↗
PubMed2004

In situ bioreduction of technetium and uranium in a nitrate-contaminated aquifer.

The potential to stimulate an indigenous microbial community to reduce a mixture of U(VI) and Tc(VII) in the presence of high (120 mM) initial NO3- co-contamination was evaluated in a shallow unconfined aquifer using a series of single-well, push-pull tests. In the absence of added electron donor, NO3-, Tc(VII), and U(VI) reduction was not detectable. However, in the presence of added ethanol, glucose, or acetate to serve as electron donor, rapid NO3- utilization was observed. The accumulation of NO2-, the absence of detectable NH4+ accumulation, and the production of N2O during in situ acetylene-block experiments suggest that NO3- was being consumed via denitrification. Tc(VII) reduction occurred concurrently with NO3- reduction, but U(VI) reduction was not observed until two or more donor additions resulted in iron-reducing conditions, as detected by the production of Fe(II). Reoxidation/remobilization of U(IV) was also observed in tests conducted with high (approximately 120 mM) but not low (approximately 1 mM) initial NO3- concentrations and not during acetylene-block experiments conducted with high initial NO3-. These results suggest that NO3(-)-dependent microbial U(IV) oxidation may inhibit or reverse U(VI) reduction and decrease the stability of U(IV) in this environment. Changes in viable biomass, community composition, metabolic status, and respiratory state of organisms harvested from down-well microbial samplers deployed during these tests were consistent with the conclusions that electron donor additions resulted in microbial growth, the creation of anaerobic conditions, and an increase in activity of metal-reducing organisms (e.g., Geobacter). The results demonstrate that it is possible to stimulate the simultaneous bioreduction of U(VI) and Tc(VII) mixtures commonly found with NO3- co-contamination at radioactive waste sites.

Biodegradation, Environmental↗