Geobacter Metallireducens GS-15 Research Articles

Enhancing cadmium immobilization by AQDS-mediated dissimilatory reduction under coexisting conditions of iron and manganese oxides

The serious contamination of cadmium (Cd) in sediments has become an urgent environmental problem. Quinones mediated dissimilatory iron and manganese reduction widely occurs in sediments, this process may enhance Cd immobilization by promoting production of secondary minerals. Nevertheless, the enhancing effect of Cd immobilization is still poorly understood. Here, 9,10-anthraquinone-2,6-disulfonic acid (AQDS) was used to facilitate the microbial reduction of iron and manganese oxides by Geobacter metallireducens GS-15, and its effects on Cd species was investigated. Compared to goethite (FeOOH), microorganisms preferentially reduced birnessite (MnO2) due to more dramatic interfacial electron transfer between birnessite and AQDS. AQDS thus significantly enhanced microbial reduction of iron and manganese coexisted minerals as well as Cd2+ immobilization. After 80 days, an impressive 83% of reducible-Cd appeared in the FeOOH–MnO2-AQDS group, demonstrating the most effective Cd immobilization. Furthermore, XANES results suggested that AQDS promoted the increase of vivianite-Cd in secondary minerals in MnO2-AQDS and FeOOH–MnO2-AQDS groups. Based on the structural equation model, HCl-extracted Fe(II) indirectly affected the increase of reducible-Cd mainly through vivianite-Cd. The AQDS enhancement on microbial reduction of iron and manganese oxides promotes the recrystallization of secondary minerals and the immobilization of Cd, offering a promising strategy for the remediation of Cd-contaminated sediments.

AQDS-mediated dissimilatory reduction of iron (hydr)oxides induces the formation of large grain vivianite: A new insight for phosphorus pollution control in sediment

Quinones plays a crucial role in iron (hydr)oxides dissimilatory reduction and the geochemical cycle of elements. However, the interfacial reactions and the subsequent effect of phosphorus (P) in sediments remain unclear. To clarify it, 9,10-anthraquinone-2,6-disulfonic acid (AQDS) was used to participate in reduction of hematite and goethite by Geobacter metallireducens GS-15, and the interfacial electron transfer between iron (hydr)oxides and AQDS were also explained. Results showed that AQDS had a higher promotion effect on the reduction of goethite than hematite, and more than 41% of goethite was reduced compared to the control check, with an 80.6 mg g−1 phosphorus immobilized. Vivianite was produced in this system, and the size of vivianite in hematite-AQDS and goethite-AQDS groups increased 7.59% and 57.78%, respectively, compared to the corresponding groups without AQDS. The highest weight of vivianite-P was in the Goe-AQDS group by X-ray absorption near edge structure (XANES). Electrochemical characteristics showed that the interfacial electron transfer rates of hematite and goethite with AQDS adding were 7.49 s−1 and 9.03 s−1, respectively. Therefore, high electron transfer efficiency led more microbial iron reductions, which generated high concentrations of Fe(Ⅱ) and facilitated the increasing of production and grain size of vivianite. These findings provide a new understanding of vivianite generation and phosphorus immobilization.

Design of a Microbial Remediation Inoculation Program for Petroleum Hydrocarbon Contaminated Sites Based on Degradation Pathways.

This paper analyzed the degradation pathways of petroleum hydrocarbon degradation bacteria, screened the main degradation pathways, and found the petroleum hydrocarbon degradation enzymes corresponding to each step of the degradation pathway. Through the Copeland method, the best inoculation program of petroleum hydrocarbon degradation bacteria in a polluted site was selected as follows: single oxygenation path was dominated by Streptomyces avermitilis, hydroxylation path was dominated by Methylosinus trichosporium OB3b, secondary oxygenation path was dominated by Pseudomonas aeruginosa, secondary hydroxylation path was dominated by Methylococcus capsulatus, double oxygenation path was dominated by Acinetobacter baylyi ADP1, hydrolysis path was dominated by Rhodococcus erythropolis, and CoA path was dominated by Geobacter metallireducens GS-15 to repair petroleum hydrocarbon contaminated sites. The Copeland method score for this solution is 22, which is the highest among the 375 solutions designed in this paper, indicating that it has the best degradation effect. Meanwhile, we verified its effect by the Cdocker method, and the Cdocker energy of this solution is −285.811 kcal/mol, which has the highest absolute value. Among the inoculation programs of the top 13 petroleum hydrocarbon degradation bacteria, the effect of the best inoculation program of petroleum hydrocarbon degradation bacteria was 18% higher than that of the 13th group, verifying that this solution has the best overall degradation effect. The inoculation program of petroleum hydrocarbon degradation bacteria designed in this paper considered the main pathways of petroleum hydrocarbon pollutant degradation, especially highlighting the degradability of petroleum hydrocarbon intermediate degradation products, and enriching the theoretical program of microbial remediation of petroleum hydrocarbon contaminated sites.

Inhibition effect of polyvinyl chloride on ferrihydrite reduction and electrochemical activities of Geobacter metallireducens.

Geobacter metallireducens GS15, a model of dissimilatory iron-reducing bacteria, is the key regulator in biogeochemical iron cycling. How the emerging contaminant microplastics involved in the iron cycling are driven by microbes on the microscale remains unknown. Hence, the influences of two typical microplastics, polybutylene terephthalate-hexane acid (PBAT) and polyvinyl chloride (PVC), were explored on the activity of G. metallireducens GS15 with ferrihydrite or ferric citrate as the respective electron acceptors. The results showed that the iron (II) contents in PBAT- and PVC-treatment groups were 16.79 and 6.81 mM, respectively, at the end of the experiment. Compared with the PBAT-treatment group, scanning electron microscopy and energy dispersive spectrometery revealed that merely a small amount of iron-containing products covered the surface of PVC. Moreover, PBAT and PVC could both retard the electroactivity of G. metallireducens GS15 at the beginning of microbial fuel cell operation. On the basis of the results above, microplastic PVC might exhibit potential inhibition of the iron cycling process driven by G. metallireducens GS15 with ferrihydrite as the terminal electron acceptor. This study extended our understanding of the influence of the microplastics PBAT and PVC on microbially mediated biogeochemical iron cycling. The findings might have an important implication on the biogeochemical elements cycling in the ecosystem with the involvement of emerging contaminant microplastics.

Mercury methylation by Geobacter metallireducens GS-15 in the presence of Skeletonema costatum

In this study, bacterial mercury (Hg) methylation was investigated under the influence of red-tide algae of Skeletonema costatum (S. costatum). The distribution and speciation of total mercury (THg) and methylmercury (MeHg) were profiled by employing Geobacter metallireducens (G. metallireducens GS-15) as the methylating bacteria. G. metallireducens GS-15 showed different capabilities in methylating different inorganic forms of Hg(II) (HgCl2) and Hg(II)-Algae (HgCl2 captured by S. costatum) to MeHg. In the absence of S. costatum, a maximum methylation efficiency of 4.31 ± 0.47% was achieved with Hg(II) of 1–100 μg L−1, while accelerated MeHg formation rate was detected at a higher initial Hg(II) concentration. In the presence of S. costatum, there were distinct changes in the distribution of THg and MeHg by altering the bioavailability of Hg(II) and Hg(II)-Algae. A larger proportion of THg tended to be retained by a higher algal biomass, resulting in decreased methylation efficiencies. The methylation efficiency of Hg(II) decreased from 3.01 ± 0.10% to 1.01 ± 0.01% with 10-mL and 250-mL algal media, and that of Hg(II)-Algae decreased from 0.83 ± 0.13% to 0.22 ± 0.01% with 10-mL and 250-mL Hg(II)-Algae media. Fourier transform infrared spectrometry, surface charge properties and elemental compositions of S. costatum were used to infer that amine, carboxyl and sulfonate functional groups were most likely to interact with Hg(II) through complexation and/or electrostatic attraction. These results suggest that red-tide algae may be an influencing factor on bacterial Hg methylation in eutrophic water bodies.

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens.

Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET_RS10855 was heterologously expressed in Escherichia coli cells. The His6-tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K4[Fe(CN)6]. Temperature optimum was 75°C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions.

Electron Transfer Strategies Regulate Carbonate Mineral and Micropore Formation.

Some microbial carbonates are robust biosignatures due to their distinct morphologies and compositions. However, whether carbonates induced by microbial iron reduction have such features is unknown. Iron-reducing bacteria use various strategies to transfer electrons to iron oxide minerals (e.g., membrane-bound enzymes, soluble electron shuttles, nanowires, as well as different mechanisms for moving over or attaching to mineral surfaces). This diversity has the potential to create mineral biosignatures through manipulating the microenvironments in which carbonate precipitation occurs. We used Shewanella oneidensis MR-1, Geothrix fermentans, and Geobacter metallireducens GS-15, representing three different strategies, to reduce solid ferric hydroxide in order to evaluate their influence on carbonate and micropore formation (micro-size porosity in mineral rocks). Our results indicate that electron transfer strategies determined the morphology (rhombohedral, spherical, or long-chained) of precipitated calcium-rich siderite by controlling the level of carbonate saturation and the location of carbonate formation. Remarkably, electron transfer strategies also produced distinctive cell-shaped micropores in both carbonate and hydroxide minerals, thus producing suites of features that could potentially serve as biosignatures recording information about the sizes, shapes, and physiologies of iron-reducing organisms. Key Words: Microbial iron reduction-Micropore-Electron transfer strategies-Microbial carbonate. Astrobiology 18, 28-36.

Concentration of sheep manure bacteria in the immobilization of arsenic from groundwater using zero-valent

Permeable reactive barrier column tests were performed to investigate whether anaerobic bacteria in sheep manure could help remove As from groundwater. One column served as a control and was packed with zero-valent iron (ZVI), compost leaf, wood chips, glass beads, and gravel, after which it was sterilized. The other (‘inoculated column’) was packed with the same ingredients, with the addition of sheep manure as a source of anaerobic bacteria. Simulated As-contaminated groundwater was prepared based on groundwater samples from Sumbawa Island, Indonesia, but with the arsenic concentration adjusted to 50 mg/L. The inoculated column was found to remove As more effectively from the simulated groundwater than the sterilized one. A gradual decrease in sulfate concentration was observed in the inoculated column at the rate of 0.26 mmol of sulfate/L/day, suggesting that there was sulfate-reducing activity in the microbial population. In addition, the sulfur isotope ratio showed -4.3 (‰) and 0.2 (‰) in influent and effluent, respectively, indicating that sulfate-reducing bacteria (SRB) consumed δ32S preferentially. Using population size estimates from the most probable number (MPN) method, the population of SRB was found to increase with distance traveled in the column. Profiling the community composition of the bacteria in different fractions of the inoculated column using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) on 16S rRNA sequences suggested that a majority of bacteria were various Clostridium species and one species of Proteobacteria, Geobacter metallireducens GS-15. Some of them may contribute to the removal of arsenic.Keywords: Sheep manure, zero valence iron, arsenic, immobilization, sulfate-reducing bacteria

Geobacter sp. SD-1 with enhanced electrochemical activity in high-salt concentration solutions.

An isolate, designated strain SD-1, was obtained from a biofilm dominated by Geobacter sulfurreducens in a microbial fuel cell. The electrochemical activity of strain SD-1 was compared with type strains, G. sulfurreducens PCA and Geobacter metallireducens GS-15, and a mixed culture in microbial electrolysis cells. SD-1 produced a maximum current density of 290 ± 29 A m−3 in a high-concentration phosphate buffer solution (PBS-H, 200 mM). This current density was significantly higher than that produced by the mixed culture (189 ± 44 A m−3) or the type strains (< 70 A m−3). In a highly saline water (SW; 50 mM PBS and 650 mM NaCl), current by SD-1 (158 ± 4 A m−3) was reduced by 28% compared with 50 mM PBS (220 ± 4 A m−3), but it was still higher than that of the mixed culture (147 ± 19 A m−3), and strains PCA and GS-15 did not produce any current. Electrochemical tests showed that the improved performance of SD-1 was due to its lower charge transfer resistance and more negative potentials produced at higher current densities. These results show that the electrochemical activity of SD-1 was significantly different than other Geobacter strains and mixed cultures in terms of its salt tolerance.

Constraint-based modeling of carbon fixation and the energetics of electron transfer in Geobacter metallireducens.

Geobacter species are of great interest for environmental and biotechnology applications as they can carry out direct electron transfer to insoluble metals or other microorganisms and have the ability to assimilate inorganic carbon. Here, we report on the capability and key enabling metabolic machinery of Geobacter metallireducens GS-15 to carry out CO2 fixation and direct electron transfer to iron. An updated metabolic reconstruction was generated, growth screens on targeted conditions of interest were performed, and constraint-based analysis was utilized to characterize and evaluate critical pathways and reactions in G. metallireducens. The novel capability of G. metallireducens to grow autotrophically with formate and Fe(III) was predicted and subsequently validated in vivo. Additionally, the energetic cost of transferring electrons to an external electron acceptor was determined through analysis of growth experiments carried out using three different electron acceptors (Fe(III), nitrate, and fumarate) by systematically isolating and examining different parts of the electron transport chain. The updated reconstruction will serve as a knowledgebase for understanding and engineering Geobacter and similar species.

Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15.

The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15–20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn2+-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.

A Whole Genome Pairwise Comparative and Functional Analysis of Geobacter sulfurreducens PCA

Geobacter species are involved in electricity production, bioremediations, and various environmental friendly activities. Whole genome comparative analyses of Geobacter sulfurreducens PCA, Geobacter bemidjiensis Bem, Geobacter sp. FRC-32, Geobacter lovleyi SZ, Geobacter sp. M21, Geobacter metallireducens GS-15, Geobacter uraniireducens Rf4 have been made to find out similarities and dissimilarities among them. For whole genome comparison of Geobacter species, an in-house tool, Geobacter Comparative Genomics Tool (GCGT) has been developed using BLASTALL program, and these whole genome analyses yielded conserved genes and they are used for functional prediction. The conserved genes identified are about 2184 genes, and these genes are classified into 14 groups based on the pathway information. Functions for 74 hypothetical proteins have been predicted based on the conserved genes. The predicted functions include pilus type proteins, flagellar proteins, ABC transporters, and other proteins which are involved in electron transfer. A phylogenetic tree from 16S rRNA of seven Geobacter species showed that G. sulfurreducens PCA is closely related to G. metallireducens GS-15 and G. lovleyi SZ. For evolutionary study, acetate kinase protein is used, which showed closeness to Pelobacter propionicus, Pelobacter carbinolicus, and Deferribacteraceae family bacterial species. These results will be useful to enhance electricity production by using biotechnological approaches.

Arsenic Dissolution from Japanese Paddy Soil by a Dissimilatory Arsenate-Reducing Bacterium Geobacter sp. OR-1

Dissimilatory As(V) (arsenate)-reducing bacteria may play an important role in arsenic release from anoxic sediments in the form of As(III) (arsenite). Although respiratory arsenate reductase genes (arrA) closely related to Geobacter species have been frequently detected in arsenic-rich sediments, it is still unclear whether they directly participate in arsenic release, mainly due to lack of pure cultures capable of arsenate reduction. In this study, we isolated a novel dissimilatory arsenate-reducing bacterium, strain OR-1, from Japanese paddy soil, and found that it was phylogenetically closely related to Geobacter pelophilus. OR-1 also utilized soluble Fe(III), ferrihydrite, nitrate, and fumarate as electron acceptors. OR-1 catalyzed dissolution of arsenic from arsenate-adsorbed ferrihydrite, while Geobacter metallireducens GS-15 did not. Furthermore, inoculation of washed cells of OR-1 into sterilized paddy soil successfully restored arsenic release. Arsenic K-edge X-ray absorption near-edge structure analysis revealed that strain OR-1 reduced arsenate directly on the soil solid phase. Analysis of putative ArrA sequences from paddy soils suggested that Geobacter-related bacteria, including those closely related to OR-1, play an important role in arsenic release from paddy soils. Our results provide direct evidence for arsenic dissolution by Geobacter species and support the hypothesis that Geobacter species play a significant role in reduction and mobilization of arsenic in flooded soils and anoxic sediments.

Complete genome, catabolic sub‐proteomes and key‐metabolites of Desulfobacula toluolica Tol2, a marine, aromatic compound‐degrading, sulfate‐reducing bacterium

Among the dominant deltaproteobacterial sulfate-reducing bacteria (SRB), members of the genus Desulfobacula are not only present in (hydrocarbon-rich) marine sediments, but occur also frequently in the anoxic water bodies encountered in marine upwelling areas. Here, we present the 5.2 Mbp genome of Desulfobacula toluolica Tol2, which is the first of an aromatic compound-degrading, marine SRB. The genome has apparently been shaped by viral attacks (e.g. CRISPRs) and its high plasticity is reflected by 163 detected genes related to transposases and integrases, a total of 494 paralogous genes and 24 group II introns. Prediction of the catabolic network of strain Tol2 was refined by differential proteome and metabolite analysis of substrate-adapted cells. Toluene and p-cresol are degraded by separate suites of specific enzymes for initial arylsuccinate formation via addition to fumarate (p-cresol-specific enzyme HbsA represents a new phylogenetic branch) as well as for subsequent modified β-oxidation of arylsuccinates to the central intermediate benzoyl-CoA. Proteogenomic evidence suggests specific electron transfer (EtfAB) and membrane proteins to channel electrons from dehydrogenation of both arylsuccinates directly to the membrane redox pool. In contrast to the known anaerobic degradation pathways in other bacteria, strain Tol2 deaminates phenylalanine non-oxidatively to cinnamate by phenylalanine ammonia-lyase and subsequently forms phenylacetate (both metabolites identified in (13) C-labelling experiments). Benzoate degradation involves CoA activation, reductive dearomatization by a class II benzoyl-CoA reductase and hydrolytic ring cleavage as found in the obligate anaerobe Geobacter metallireducens GS-15. The catabolic sub-proteomes displayed high substrate specificity, reflecting the genomically predicted complex and fine-tuned regulatory network of strain Tol2. Despite the genetic equipment for a TCA cycle, proteomic evidence supports complete oxidation of acetyl-CoA to CO2 via the Wood-Ljungdahl pathway. Strain Tol2 possesses transmembrane redox complexes similar to that of other Desulfobacteraceae members. The multiple heterodisulfide reductase-like proteins (more than described for Desulfobacterium autotrophicum HRM2) may constitute a multifaceted cytoplasmic electron transfer network.

The methylomes of six bacteria

Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and C. jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases, it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTase genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. Two of these proved active. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT® sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active but also revealing their recognition sequences.

Identification of theGeobacter metallireducensBamVW Two-Component System, Involved in Transcriptional Regulation of Aromatic Degradation

Regulation of aromatic degradation in obligate anaerobes was studied in the Fe(III)-respiring model organism Geobacter metallireducens GS-15. A two-component system and a sigma54-dependent promoter were identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, catalyzing the initial step of benzoate degradation.

Distinct Microbial Behavior of Re Compared to Tc: Evidence Against Microbial Re Fixation in Aquatic Sediments

Rhenium is enriched in suboxic and anoxic sediments relative to oxic sediments, a characteristic that is being exploited in its use as a paleoredox indicator. Rhenium is fixed at sediment depths where iron reduction and sulfate reduction are the dominant microbial terminal electron-accepting processes. In order to explore mechanisms of its fixation, we investigated perrhenate behavior in pure, batch cultures of two dissimilatory sulfate-reducing strains (Desulfovibrio desulfuricans subsp. desulfuricans and Desulfovibrio desulfuricans ND132) and two iron-reducing strains (Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1). Perrhenate concentrations tested ranged from 0.04 to 12 μM, roughly 4 to 7 orders of magnitude larger than seawater Re concentrations. Within this broad concentration range, none of the organisms tested actively removed Re from solution during one week's growth to stationary phase. Despite these results, the sulfate-reducing cultures appeared to have reached supersaturation relative to ReS2(s), and the iron-reducing cultures may have reached supersaturation relative to ReO2(s). We conclude that neither direct nor short-term indirect microbial processes involving these bacteria are likely to explain Re fixation in sediments. Our results cannot exclude the possibility that microbial metabolites, such as Fe(II) or sulfide, do drive abiotic Re fixation over longer periods of time. The lack of perrhenate reduction by dissimilatory sulfate-reducing bacteria and iron-reducing bacteria contrasts with published reports of pertechnetate behavior. Despite many qualitative similarities between Re and Tc, it is clear that these two elements are quantitatively dissimilar, with Re fixation requiring more intensely reducing conditions.

Identification of the Electron Transfer Flavoprotein as an Upregulated Enzyme in the Benzoate Utilization ofDesulfotignum balticum

Desulfotignum balticum utilizes benzoate coupled to sulfate reduction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was conducted to detect proteins that increased more after growth on benzoate than on butyrate. A comparison of proteins on 2D gels showed that at least six proteins were expressed. The N-terminal sequences of three proteins exhibited significant identities with the alpha and beta subunits of electron transfer flavoprotein (ETF) from anaerobic aromatic-degraders. By sequence analysis of the fosmid clone insert (37,590 bp) containing the genes encoding the ETF subunits, we identified three genes, whose deduced amino acid sequences showed 58%, 74%, and 62% identity with those of Gmet_2267 (Fe-S oxidoreductase), Gmet_2266 (ETF beta subunit), and Gmet_2265 (ETF alpha subunit) respectively, which exist within the 300-kb genomic island of aromatic-degradation genes from Geobacter metallireducens GS-15. The genes encoding ETF subunits found in this study were upregulated in benzoate utilization.

Identification of Sulfate- and Arsenate-Reducing Bacteria in Sheep Manure as Permeable Reactive Materials after Arsenic Immobilization in Groundwater

Permeable reactive barriers (PRBs) column tests were performed to investigate the contribution of anaerobic microbial community in sheep manure on the removal of As from groundwater. The column was packed with zero valent iron (ZVI), sheep manure, compost, wood chips, glass beads and gravels. All materials were sterilized except for sheep manure that contains anaerobic bacteria. Decrease in sulfate concentration was observed at the maximum rate of 0.26 mmol dm−3 day−1. In addition, the sulfur isotopic ratio of δ34S increased from the influent (−4.3 extperthousand) to the effluent (0.2 extperthousand), suggesting that there was sulfate-reducing activity in the microbial community. Arsenate was more effectively immobilized on ZVI than arsenite. Bacterial community analysis using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) on 16S rRNA sequences suggested that majorities of bacteria were several Clostridium species and one species of Proteobacteria, Geobacter metallireducens GS-15, independently of PRBs column heights. Some of them might have contributed to the removal of arsenic.

Novel Reduction of Mercury(II) by Mercury-Sensitive Dissimilatory Metal Reducing Bacteria

The dissimilatory metal reducing bacterium (DMRB) Shewanella oneidensis MR-1 reduces ionic mercury (Hg[II]) to elemental mercury (Hg[0]) by an activity not related to the MerA mercuric reductase. In S. oneidensis, this activity is constitutive and effective at Hg(II) concentrations too low to induce mer operon functions. Reduction of Hg(II) by MR-1 required the presence of electron donors and electron acceptors. Reduction occurred with oxygen or fumarate, but had the highest rate when ferric oxyhydroxide was used as a terminal electron acceptor. Geobacter sulfurreducens PCA and Geobacter metallireducens GS-15 reduced Hg(II) to Hg(0) with activity comparable to MR-1; however, neither the DMRB Anaeromyxobacter dehalogenans 2CP-C nor the nitrate reducer Pseudomonas stutzeri OX-1 reduced Hg(II) during growth. This discovery of constitutive mercury reduction among anaerobes has implications to the mobilization of mercury and production of methylmercury in anoxic environments.