Geobacter

Previous studies have suggested that the conductive pili of Geobacter sulfurreducens are essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the ...

Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in diVerent cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining diVerent strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15 N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV-visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.

del Agua (2000/60/CE) establece un marco común a nivel europeo para la protección de las aguas y el tratamiento de aguas residuales. En España, el aspecto de tratamiento está regulado mediante el RD 1290/2012, que en parte transpone la normativa comunitaria así como la Directiva 91/271/CE. En él se establece la obligatoriedad de contar con sistemas de tratamiento de aguas residuales para todos los municipios de más de 2000 habitantes, además, el resto de municipios deberán contar con sistemas colectores con un tratamiento adecuado.

Microbial fuel cells (MFCs) can be used to directly generate electricity from the oxidation of dissolved organic matter, but optimization of MFCs will require that we know more about the factors that can increase power output such as the type of proton exchange system which can affect the system internal resistance. Power output in a MFC containing a proton exchange membrane was compared using a pure culture (Geobacter metallireducens) or a mixed culture (wastewater inoculum). Power output with either inoculum was essentially the same, with 4071 mW/m 2 for G. metallireducens and 3871 mW/m 2 for the wastewater inoculum. We also examined power output in a MFC with a salt bridge instead of a membrane system. Power output by the salt bridge MFC (inoculated with G. metallireducens) was 2.2 mW/m 2 . The low power output was directly attributed to the higher internal resistance of the salt bridge system ð19920 AE 50 OÞ compared to that of the membrane system ð1286 AE 1 OÞ based on measurements using impedance spectroscopy. In both systems, it was observed that oxygen diffusion from the cathode chamber into the anode chamber was a factor in power generation. Nitrogen gas sparging, L-cysteine (a chemical oxygen scavenger), or suspended cells (biological oxygen scavenger) were used to limit the effects of gas diffusion into the anode chamber. Nitrogen gas sparging, for example, increased overall Coulombic efficiency (47% or 55%) compared to that obtained without gas sparging (19%). These results show that increasing power densities in MFCs will require reducing the internal resistance of the system, and that methods are needed to control the dissolved oxygen flux into the anode chamber in order to increase overall Coulombic efficiency. r

A hydrogen utilizing exoelectrogenic bacterium (Geobacter sulfurreducens) was compared to both a nonhydrogen oxidizer (Geobacter metallireducens) and a mixed consortium in order to compare the hydrogen production rates and hydrogen recoveries of pure and mixed cultures in microbial electrolysis cells (MECs). At an applied voltage of 0.7 V, both G. sulfurreducens and the mixed culture generated similar current densities (ca. 160 A/m 3 ), resulting in hydrogen production rates of ca. 1.9 m 3 H 2 /m 3 /day, whereas G. metallireducens exhibited lower current densities and production rates of 110 ؎ 7 A/m 3 and 1.3 ؎ 0.1 m 3 H 2 /m 3 /day, respectively. Before methane was detected in the mixed-culture MEC, the mixed consortium achieved the highest overall energy recovery (relative to both electricity and substrate energy inputs) of 82% ؎ 8% compared to G. sulfurreducens (77% ؎ 2%) and G. metallireducens (78% ؎ 5%), due to the higher coulombic efficiency of the mixed consortium. At an applied voltage of 0.4 V, methane production increased in the mixed-culture MEC and, as a result, the hydrogen recovery decreased and the overall energy recovery dropped to 38% ؎ 16% compared to 80% ؎ 5% for G. sulfurreducens and 76% ؎ 0% for G. metallireducens. Internal hydrogen recycling was confirmed since the mixed culture generated a stable current density of 31 ؎ 0 A/m 3 when fed hydrogen gas, whereas G. sulfurreducens exhibited a steady decrease in current production. Community analysis suggested that G. sulfurreducens was predominant in the mixed-culture MEC (72% of clones) despite its relative absence in the mixed-culture inoculum obtained from a microbial fuel cell reactor (2% of clones). These results demonstrate that Geobacter species are capable of obtaining similar hydrogen production rates and energy recoveries as mixed cultures in an MEC and that high coulombic efficiencies in mixed culture MECs can be attributed in part to the recycling of hydrogen into current.

Dissolved hydrogen concentrations, in conjunction with other geochemical indicators, are becoming an accepted means to determine terminal electron acceptor processes (TEAPs) in groundwater aquifers. Aqueous hydrogen concentrations have been found to fall within specific ranges under methanogenic, sulfate-reducing, iron-reducing, and denitrification conditions. Although hydrogen is gaining in acceptance for determining subsurface TEAPs, there is a dearth of data with regards to the kinetic coefficients for hydrogen utilization in the presence or absence of an additional electron donor under different TEAPs. This study expands the kinetic data for hydrogen utilization through a series of batch experiments, which were conducted to study the utilization of acetate and hydrogen by Geobacter sulfurreducens under iron-reducing conditions. The results of these experiments indicate that the kinetic coefficients (cell yield and first-order degradation rate) describing the rate of hydrogen utilization by G. sulfurreducens under iron-reducing conditions correlate energetically with the coefficients found in previous experiments under methanogenic and sulfate-reducing conditions. In addition, with acetate and hydrogen as simultaneous electron donors, there is slight inhibition between the two electron donors for G. sulfurreducens, and this can be modeled through competitive inhibition terms in the classic Monod formulation. Finally, a key result of this study is that the TEAP-dependent hydrogen concentration in aquifers is not related solely to the microbial kinetics of the hydrogen-consuming organisms as previously suggested but is affected by the multi-substrate kinetics of hydrogen being consumed simultaneously with other electron donors as well as the availability of the electron acceptor.

Aromatic compounds are widely distributed in nature and can only be biomineralized by microorganisms. In anaerobic bacteria, benzoyl-CoA (BCoA) is a central intermediate of aromatic degradation, and serves as substrate for dearomatizing BCoA reductases (BCRs). In facultative anaerobes, the mechanistically difficult reduction of BCoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA (dienoyl-CoA) is driven by a stoichiometric ATP hydrolysis, catalyzed by a soluble, three [4Fe-4S] cluster-containing BCR. In this work, an in vitro assay for BCR from the obligately anaerobic Geobacter metallireducens was established. It followed the reverse reaction, the formation of BCoA from dienoyl-CoA in the presence of various electron acceptors. The benzoate-induced activity was highly specific for dienoyl-CoA (K m ‫؍‬ 24 ؎ 4 M). The corresponding oxygen-sensitive enzyme was purified by several chromatographic steps with a 115-fold enrichment and a yield of 18%. The 185-kDa enzyme comprised 73-and 20-kDa subunits, suggesting an ␣2␤2composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The ␣␤ unit contained 0.9 W, 15 Fe, and 12.5 acid-labile sulfur. Results from EPR spectroscopy suggest the presence of one [3Fe-4S] 0/؉1 and three [4Fe-4S] ؉1/؉2 clusters per ␣␤ unit; oxidized BamBC exhibited an EPR signal typical for a W(V) species. The FeS clusters and the W-cofactor could only be fully reduced by dienoyl-CoA. BamBC represents the prototype of a previously undescribed class of dearomatizing BCRs that differ completely from the ATP-dependent enzymes from facultative anaerobes.

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Rotating bioelectrochemical contactor Nitrification Denitrification 16S rRNA gene pyrosequencing a b s t r a c t Integrating microbial fuel cell (MFC) into rotating biological contactor (RBC) creates an opportunity for enhanced removal of COD and nitrogen coupled with energy generation from wastewater. In this study, a three-stage rotating bioelectrochemical contactor (referred to as RBCeMFC unit) integrating MFC with RBC technology was constructed for simultaneous removal of carbonaceous and nitrogenous compounds and electricity generation from a synthetic medium containing acetate and ammonium. The performance of the RBCeMFC unit was compared to a control reactor (referred to as RBC unit) that was operated under the same conditions but without current generation (i.e. open-circuit mode). The effect of hydraulic loading rate (HLR) and COD/N ratio on the performance of the two units was investigated. At low (3.05 gCOD g À1 N) and high COD/N ratio (6.64 gCOD g À1 N), both units achieved almost similar COD and ammonia-nitrogen removal.

The electrically conductive pili (e-pili) of Geobacter sulfurreducens serve as a model for a novel strategy for long-range extracellular electron transfer. e-pili are also a new class of bioelectronic materials. However, the only other Geobacter pili previously studied, which were from G. uraniireducens, were poorly conductive. In order to obtain more information on the range of pili conductivities in Geobacter species, the pili of G. metallireducens were investigated. Heterologously expressing the PilA gene of G. metallireducens in G. sulfurreducens yielded a G. sulfurreducens strain, designated strain MP, that produced abundant pili. Strain MP exhibited phenotypes consistent with the presence of e-pili, such as high rates of Fe(III) oxide reduction and high current densities on graphite anodes. Individual pili prepared at physiologically relevant pH 7 had conductivities of 277 ± 18.9 S/cm (mean ± standard deviation), which is 5,000-fold higher than the conductivity of G. sulfurred...

The transformation of carbon tetrachloride (CT) by biogenic iron species produced from the bioreduction of various Fe(III) oxides in the presence of Geobacter sulfurreducens and electron shuttles were investigated. Cysteine and anthraquinone-2,6-disulfonate (AQDS) at concentrations of 0.5 mM and 10 M, respectively, were added as the electron shuttles. Addition of electron shuttles enhanced the extent of reduction and rate of ferric oxide reduction. The bioreduction extents of ferric oxides by G. sulfurreducens in the presence of electron shuttles were 22.8-48.3% for ferrihydrite, 6.5-17.2% for hematite, and 3.0-11.3% for goethite. After normalization to the surface areas, a higher rate of CT reduction was observed per unit of adsorbed Fe(II) on crystalline oxides. The produced biogenic Fe(II) from crystalline iron oxides was 2.8-7.6 times lower than that obtained from ferrihydrite, while the surface area-normalized rate constant for ironmediated CT transformation in the presence of goethite and hematite were, by factors of 2-21, higher than that obtained using ferrihydrite. These results clearly depict that G. sulfurreducens drove the reduction of CT primarily through the formation of biogenic iron species in the presence of electron shuttle under iron-reducing conditions and that it is a surface area dependent process. (R.-a. Doong).

While electrochemical characterization of enzymes immobilized on electrodes has become common, there is still a need for reliable quantitative methods for study of electron transfer between living cells and conductive surfaces. This work describes growth of thin (<20 m) Geobacter sulfurreducens biofilms on polished glassy carbon electrodes, using stirred three-electrode anaerobic bioreactors controlled by potentiostats and nondestructive voltammetry techniques for characterization of viable biofilms. Routine in vivo analysis of electron transfer between bacterial cells and electrodes was performed, providing insight into the main redox-active species participating in electron transfer to electrodes. At low scan rates, cyclic voltammetry revealed catalytic electron transfer between cells and the electrode, similar to what has been observed for pure enzymes attached to electrodes under continuous turnover conditions. Differential pulse voltammetry and electrochemical impedance spectroscopy also revealed features that were consistent with electron transfer being mediated by an adsorbed catalyst. Multiple redox-active species were detected, revealing complexity at the outer surfaces of this bacterium. These techniques provide the basis for cataloging quantifiable, defined electron transfer phenotypes as a function of potential, electrode material, growth phase, and culture conditions and provide a framework for comparisons with other species or communities.

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uraniumcontaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a preypredator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.

Whole-genome analysis of gene expression in Geobacter sulfurreducens revealed 474 genes with transcript levels that were significantly different during growth with an electrode as the sole electron acceptor versus growth on Fe(III) citrate. The greatest response was a more than 19-fold increase in transcript levels for omcS , which encodes an outer-membrane cytochrome previously shown to be required for Fe(III) oxide reduction. Quantitative reverse transcription polymerase chain reaction and Northern analyses confirmed the higher levels of omcS transcripts, which increased as power production increased. Deletion of omcS inhibited current production that was restored when omcS was expressed in trans. Transcript expression and genetic analysis suggested that OmcE, another outer-membrane cytochrome, is also involved in electron transfer to electrodes. Surprisingly, genes for other proteins known to be important in Fe(III) reduction such as the outer-membrane c -type cytochrome, OmcB, and the electrically conductive pilin 'nanowires' did not have higher transcript levels on electrodes, and deletion of the relevant genes did not inhibit power production. Changes in the transcriptome suggested that cells growing on electrodes were subjected to less oxidative stress than cells growing on Fe(III) citrate and that a number of genes annotated as encoding metal efflux proteins or proteins of unknown function may be important for growth on electrodes. These results demonstrate for the first time that it is possible to evaluate gene expression, and hence the metabolic state, of microorganisms growing on electrodes on a genome-wide basis and suggest that OmcS, and to a lesser extent OmcE, are important in electron transfer to electrodes. This has important implications for the design of electrode materials and the genetic engineering of microorganisms to improve the function of microbial fuel cells.

Background: Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process. Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites. This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis compared to non-subsurface Geobacter species.

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. Highyield purification of T1 lipase was achieved through twostep affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na + , Ca 2+ , Mn 2+ , K + and Mg 2+ , but inhibited by Cu 2+ , Fe 3+ and Zn 2+ . Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra.

Power densities produced by microbial fuel cells (MFCs) in natural systems are changed by exposure to light through the enrichment of photosynthetic microorganisms. When MFCs with brush anodes were exposed to light (4000 lx), power densities increased by 8-10% for glucose-fed reactors, and 34% for acetate-fed reactors. Denaturing gradient gel electrophoresis (DGGE) profiles based on the 16S rRNA gene showed that exposure to high light levels changed the microbial communities on the anodes. Based on 16S rRNA gene clone libraries of light-exposed systems the anode communities using glucose were also significantly different than those fed acetate. Dominant bacteria that are known exoelectrogens were identified in the anode biofilm, including a purple nonsulfur (PNS) photosynthetic bacterium, Rhodopseudomonas palustris, and a dissimilatory iron-reducing bacterium, Geobacter sulfurreducens. Pure culture tests confirmed that PNS photosynthetic bacteria increased power production when exposed to high light intensities (4000 lx). These results demonstrate that power production and community composition are affected by light conditions as well as electron donors in single-chamber air-cathode MFCs.

Microbial fuel cells (MFCs) are often inoculated from a single wastewater source. The extent that the inoculum affects community development or power production is unknown. The stable anodic microbial communities in MFCs were examined using three inocula: a wastewater treatment plant sample known to produce consistent power densities, a second wastewater treatment plant sample, and an anaerobic bog sediment. The bog-inoculated MFCs initially produced higher power densities than the wastewater-inoculated MFCs, but after 20 cycles all MFCs on average converged to similar voltages (470 ± 20 mV) and maximum power densities (590 ± 170 mW m À 2 ). The power output from replicate bog-inoculated MFCs was not significantly different, but one wastewater-inoculated MFC (UAJA3 (UAJA, University Area Joint Authority Wastewater Treatment Plant)) produced substantially less power. Denaturing gradient gel electrophoresis profiling showed a stable exoelectrogenic biofilm community in all samples after 11 cycles. After 16 cycles the predominance of Geobacter spp. in anode communities was identified using 16S rRNA gene clone libraries (58±10%), fluorescent in-situ hybridization (FISH) (63±6%) and pyrosequencing (81±4%). While the clone library analysis for the underperforming UAJA3 had a significantly lower percentage of Geobacter spp. sequences (36%), suggesting that a predominance of this microbe was needed for convergent power densities, the lower percentage of this species was not verified by FISH or pyrosequencing analyses. These results show that the predominance of Geobacter spp. in acetate-fed systems was consistent with good MFC performance and independent of the inoculum source.

The Fe and N biogeochemical cycles play key roles in freshwater environments. We aimed to determine the spatial positioning and interconnections of the N and Fe cycles in profundal lake sediments. The gradients of O2, NO3(-), NH4(+), pH, Eh, Fe(II) and Fe(III) were determined and the distribution of microorganisms was assessed by most probable numbers and quantitative polymerase chain reaction. The redox zones could be divided into an oxic zone (0-8 mm), where microaerophiles (Gallionellaceae) were most abundant at a depth of 7 mm. This was followed by a denitrification zone (6-12 mm), where NO3(-)-dependent Fe(II) oxidizers and organoheterotrophic denitrifiers both reduce nitrate. Lastly, an iron redox transition zone was identified at 12.5-22.5 mm. Fe(III) was most abundant above this zone while Fe(II) was most abundant beneath. The high abundance of poorly crystalline iron suggested iron cycling. The Fe and N cycles are biologically connected through nitrate-reducing Fe(II) oxidi...

The mobility of uranium (U) in subsurface environments is controlled by interrelated adsorption, redox, and precipitation reactions. Previous work demonstrated the formation of nanometer-sized hydrogen uranyl phosphate (abbreviated as HUP) crystals on the cell walls of Bacillus subtilis, a non-U VI-reducing, Gram-positive bacterium. The current study examined the reduction of this biogenic, cell-associated HUP mineral by three dissimilatory metal-reducing bacteria, Anaeromyxobacter dehalogenans strain K, Geobacter sulf urreducens strain PCA, and Shewanella putrefaciens strain CN-32, and compared it to the bioreduction of abiotically formed and freely suspended HUP of larger particle size. Uranium speciation in the solid phase was followed over a 10-to 20-day reaction period by X-ray absorption fine structure spectroscopy (XANES and EXAFS) and showed varying extents of U VI reduction to U IV. The reduction extent of the same mass of HUP to U IV was consistently greater with the biogenic than with the abiotic material under the same experimental conditions. A greater extent of HUP reduction was observed in the presence of bicarbonate in solution, whereas a decreased extent of HUP reduction was observed with the addition of dissolved phosphate. These results indicate that the extent of U VI reduction is controlled by dissolution of the HUP phase, suggesting that the metal-reducing bacteria transfer electrons to the dissolved or bacterially adsorbed U VI species formed after HUP dissolution, rather than to solid-phase U VI in the HUP mineral. Interestingly, the bioreduced U IV atoms were not immediately coordinated to other U IV atoms (as in uraninite, UO 2) but were similar in structure to the phosphate-complexed U IV species found in ningyoite [CaU(PO 4) 2 •H 2 O]. This indicates a strong control by phosphate on the speciation of bioreduced U IV , expressed as inhibition of the typical formation of uraninite under phosphate-free conditions.

A novel halophilic and metal-reducing bacterium, Orenia metallireducens strain Z6, was isolated from briny groundwater extracted from a 2.02 km-deep borehole in the Illinois Basin, IL. This organism shared 96% 16S rRNA gene similarity with Orenia marismortui, but demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of genus Orenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)] using H2 as the electron donor. Strain Z6 actively reduced ferrihydrite over broad ranges of pH (6-9.6), salinity (0.4-3.5 M NaCl) and temperature (20-60 °C). At pH 6.5, strain Z6 also reduced more crystalline iron oxides such as lepidocrocite (γ-FeOOH), goethite (α-FeOOH) and hematite (α-Fe2O3). Analysis of X-ray absorption fine structure (XAFS) following Fe(III) reduction by strain Z6 revealed spectra from ferrous secondary mineral phases consistent with the precipitation of vivianite [Fe3(PO4)2] a...

biological electrical connections is an alternative to interspecies H2 transfer as a mechanism for electron exchange in syntrophic cultures. However, it has not previously been determined whether electrons received via DIET yield energy to support cell growth. In order to investigate this, co-cultures of Geobacter metallireducens, which can transfer electrons to wildtype G. sulfurreducens via DIET, were established with a citrate synthase-deficient G. sulfurreducens strain that can receive electrons for respiration through DIET only. In a medium with ethanol as the electron donor and fumarate as the electron acceptor, co-cultures with the citrate synthase-deficient G. sulfurreducens strain metabolized ethanol as fast as co-cultures with wild-type, but the acetate that G. metallireducens generated from ethanol oxidation accumulated. The lack of acetate metabolism resulted in less fumarate reduction and lower cell abundance of G. sulfurreducens. RNAseq analysis of transcript abundance was consistent with a lack of acetate metabolism in G. sulfurreducens and revealed gene expression levels for the uptake hydrogenase, formate dehydrogenase, the pilus-associated c-type cytochrome OmcS and pili consistent with electron transfer via DIET. These results suggest that electrons transferred via DIET can serve as the sole energy source to support anaerobic respiration.

RpoS, the sigma S subunit of RNA polymerase, is vital during the growth and survival of Geobacter sulfurreducens under conditions typically encountered in its native subsurface environments. We investigated the conservation of sites that may be important for RpoS function in G. sulfurreducens. We also employed sequence information and expression microarray data to predict G. sulfurreducens genome sites that may be related to RpoS regulation. Hierarchical clustering identified three clusters of significantly downregulated genes in the rpoS deletion mutant. The search for conserved overrepresented motifs in co-regulated operons identified likely −35 and −10 promoter elements upstream of a number of functionally important G. sulfurreducens operons that were downregulated in the rpoS deletion mutant. Putative − 35/− 10 promoter elements were also identified in the G. sulfurreducens genome using sequence similarity searches to matrices of − 35/−10 promoter elements found in G. sulfurreducens and in Escherichia coli. Due to a sufficient degree of sequence similarity between −35/−10 promoter elements for RpoS, RpoD, and other sigma factors, both the sequence similarity searches and the search for conserved overrepresented motifs using microarray data may identify promoter elements for both RpoS and other sigma factors. Gene 384 (2006) 73 -95 www.elsevier.com/locate/gene

In this survey, a total of 80 787 reads and 28 171 unique NifH protein sequences were retrieved from soil RNA. This dataset extends our knowledge about the structure and diversity of the functional diazotrophic communities in agricultural soils from Argentinean Pampas. Operational taxonomic unit (OTU)-based analyses showed that nifH phylotypes related to Geobacter and Anaeromyxobacter (44.8%), Rhizobiales (29%), Cyanobacteria (16.7%), and Verrucomicrobiales (8%) are key microbial components of N2 fixation in soils associated with no-till management and soil depth. In addition, quantification of nifH gene copies related to Geobacter and Cyanobacteria revealed that these groups are abundant in soils under maize-soybean rotation and soybean monoculture, respectively. The correlation of physicochemical soil parameters with the diazotrophic diversity and composition showed that soil stability and organic carbon might contribute to the functional signatures of particular nifH phylotypes i...

Butyrate is an important product of anaerobic fermentation; however, it is not directly used by characterized strains of the highly efficient anode respiring bacteria (ARB) Geobacter sulfurreducens in microbial electrochemical cells. By combining a butyrate-oxidizing community with a Geobacter rich culture, we generated a microbial community which outperformed many naturally derived communities found in the literature for current production from butyrate and rivaled the highest performing natural cultures in terms of current density (∼ 11A/m(2)) and Coulombic efficiency (∼ 70%). Microbial community analyses support the shift in the microbial community from one lacking efficient ARB in the marine hydrothermal vent community to a community consisting of ∼ 80% Geobacter in the anode biofilm. This demonstrates the successful production and adaptation of a novel microbial culture for generating electrical current from butyrate with high current density and high Coulombic efficiency, by c...

The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.

Analysis of the genome of Geobacter sulfurreducens revealed four genes encoding putative symporters with homology to ActP, an acetate transporter in Escherichia coli. Three of these genes, aplA, aplB and aplC, are highly similar (over 90 % identical) and fell within a tight phylogenetic cluster (Group I) consisting entirely of Geobacter homologues. Transcript levels for all three genes increased in response to acetate limitation. The fourth gene, aplD, is phylogenetically distinct (Group II) and its expression was not influenced by acetate availability. Deletion of any one of the three genes in Group I did not significantly affect acetate-dependent growth, suggesting functional redundancy. Attempts to recover mutants in which various combinations of two of these genes were deleted were unsuccessful, suggesting that at least two of these three transporter genes are required to support growth. Closely related Group I apl genes were found in the genomes of other Geobacter species whose genome sequences are available. Furthermore, related genes could be detected in genomic DNA extracted from a subsurface environment undergoing in situ uranium bioremediation. The transporter genes recovered from the subsurface were most closely related to Group I apl genes found in the genomes of cultured Geobacter species that were isolated from contaminated subsurface environments. The increased expression of these genes in response to acetate limitation, their high degree of conservation among Geobacter species and the ease with which they can be detected in environmental samples suggest that Group I apl genes of the Geobacteraceae may be suitable biomarkers for acetate limitation. Monitoring the expression of these genes could aid in the design of strategies for acetate-mediated in situ bioremediation of uranium-contaminated groundwater.

Nanoscale magnetite can facilitate microbial extracellular electron transfer that plays an important role in biogeochemical cycles, bioremediation and several bioenergy strategies, but the mechanisms for the stimulation of extracellular electron transfer are poorly understood. Further investigation revealed that magnetite attached to the electrically conductive pili of Geobacter species in a manner reminiscent of the association of the multi-heme c-type cytochrome OmcS with the pili of Geobacter sulfurreducens. Magnetite conferred extracellular electron capabilities on an OmcS-deficient strain unable to participate in interspecies electron transfer or Fe(III) oxide reduction. In the presence of magnetite wild-type cells repressed expression of the OmcS gene, suggesting that cells might need to produce less OmcS when magnetite was available. The finding that magnetite can compensate for the lack of the electron transfer functions of a multi-heme c-type cytochrome has implications not only for the function of modern microbes, but also for the early evolution of microbial electron transport mechanisms.

The proteome of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high-pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under six different growth conditions in order to enhance the potential that a wide range of genes would be expressed. The AMT tag approach was able to identify a much greater number of proteins than could be detected with the 2-D PAGE approach. With the AMT approach over 3,000 gene products were identified, representing about 90% of the total predicted gene products in the genome. A high proportion of predicted proteins in most protein role categories were detected; the highest number of proteins was identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT tag approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.

The activity of microorganisms often plays an important role in dynamic natural attenuation or engineered bioremediation of subsurface contaminants, such as chlorinated solvents, metals, and radionuclides. To evaluate and/or design bioremediated systems, quantitative reactive transport models are needed. State-of-the-art reactive transport models often ignore the microbial effects or simulate the microbial effects with static growth yield and constant reaction rate parameters over simulated conditions, while in reality microorganisms can dynamically modify their functionality (such as utilization of alternative respiratory pathways) in response to spatial and temporal variations in environmental conditions. Constraint-based genome-scale microbial in silico models, using genomic data and multiple-pathway reaction networks, have been shown to be able to simulate transient metabolism of some well studied microorganisms and identify growth rate, substrate uptake rates, and byproduct rates under different growth conditions. These rates can be identified and used to replace specific microbially-mediated reaction rates in a reactive transport model using local geochemical conditions as constraints. We previously demonstrated the potential utility of integrating a constraint-based microbial metabolism model with a reactive transport simulator as applied to bioremediation of uranium in groundwater. However, that work relied on an indirect coupling approach that was effective for initial demonstration but may not be extensible to more complex problems that are of significant interest (e.g., communities of microbial species and multiple constraining variables). Here, we extend that work by presenting and demonstrating a method of directly integrating a reactive transport model (FORTRAN code) with constraint-based in silico models solved with IBM ILOG CPLEX linear optimizer base system (C library). The models were integrated with BABEL, a language interoperability tool. The modeling system is designed in such a way that constraint-based models targeting different microorganisms or competing organism communities can be easily plugged into the system. Constraint-based modeling is very costly given the size of a genome-scale reaction network. To save computation time, a binary tree is traversed to examine the concentration and solution pool generated during the simulation in order to decide whether the constraint-based model should be called. We also show preliminary results from the integrated model including a comparison of the direct and indirect coupling approaches and evaluated the ability of the approach to simulate field experiment.

The increasing availability of the genome sequences of microorganisms involved in important bioremediation processes makes it feasible to consider developing genome-scale models that can aid in predicting the likely outcome of potential subsurface bioremediation strategies. Previous studies of the in situ bioremediation of uranium-contaminated groundwater have demonstrated that Geobacter species are often the dominant members of the groundwater community during active bioremediation and the primary organisms catalysing U(VI) reduction. Therefore, a genome-scale, constraint-based model of the metabolism of Geobacter sulfurreducens was coupled with the reactive transport model HYDRO-GEOCHEM in an attempt to model in situ uranium bioremediation. In order to simplify the modelling, the influence of only three growth factors was considered: acetate, the electron donor added to stimulate U(VI) reduction; Fe(III), the electron acceptor primarily supporting growth of Geobacter; and ammonium, a key nutrient. The constraint-based model predicted that growth yields of Geobacter varied significantly based on the availability of these three growth factors and that there are minimum thresholds of acetate and Fe(III) below which growth and activity are not possible. This contrasts with typical, empirical microbial models that assume fixed growth yields and the possibility for complete metabolism of the substrates.

Microbial fuel cells (MFCs) convert biodegradable materials into electricity, potentially contributing to an array of renewable energy production strategies tailored for specific applications. Since there are no known microorganisms that can both metabolize cellulose and transfer electrons to solid extracellular substrates, the conversion of cellulosic biomass to electricity requires a syntrophic microbial community that uses an insoluble electron donor (cellulose) and electron acceptor (anode). Electricity was generated from cellulose in an MFC using a defined coculture of the cellulolytic fermenter Clostridium cellulolyticum and the electrochemically active Geobacter sulfurreducens. In fed-batch tests using twochamber MFCs with ferricyanide as the catholyte, the coculture achieved maximum power densities of 143 mW/ m 2 (anode area) and 59.2 mW/m 2 from 1 g/L carboxymethyl cellulose (CMC) and MN301 cellulose, respectively. Neither pure culture alone produced electricity from these substrates. The coculture increased CMC degradation from 42% to 64% compared to a pure C. cellulolyticum culture. COD removal using CMC and MN301 was 38 and 27%, respectively, with corresponding Coulombic efficiencies of 47 and 39%. Hydrogen, acetate, and ethanol were the main residual metabolites of the binary culture. Cellulose conversion to electricity was also demonstrated using an uncharacterized mixed culture from activated sludge with an aerobic aqueous cathode.

Microbial fuel cells (MFCs) harness the electrochemical activity of certain microbes for the production of electricity from reduced compounds. Characterizations of MFC anode biofilms have collectively shown very diverse microbial communities, raising ecological questions about competition and community succession within these anodereducing communities. Three sets of triplicate, two-chamber MFCs inoculated with anaerobic sludge and differing in energy sources (acetate, lactate, and glucose) were operated to explore these questions. Based on 16S rDNA-targeted denaturing gradient gel electrophoresis (DGGE), all anode communities contained sequences closely affiliated with Geobacter sulfurreducens (>99% similarity) and an uncultured bacterium clone in the Bacteroidetes class (99% similarity). Various other Geobacter-like sequences were also enriched in most of the anode biofilms. While the anode communities in replicate reactors for each substrate generally converged to a reproducible community, there were some variations in the relative distribution of these putative anode-reducing Geobacter-like strains. Firmicutes were found only in glucose-fed MFCs, presumably serving the roles of converting complex carbon into simple molecules and scavenging oxygen. The maximum current density in these systems was negatively correlated with internal resistance variations among replicate reactors and, likely, was only minimally affected by anode community differences in these two-chamber MFCs with high internal resistance.

Extracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronu...

Microbial Fuel cells (MFCs) have been proposed for nutrient removal and energy recovery from different wastes. In this study the anaerobic digestate was used to feed H-type MFC reactors, one with a graphite anode preconditioned withGeobacter sulfurreducensand the other with an unconditioned graphite anode. The data demonstrate that the digestate acts as a carbon source, and even in the absence of anode preconditioning, electroactive bacteria colonise the anodic chamber, producing a maximum power density of 172.2 mW/m2. The carbon content was also reduced by up to 60%, while anaerobic ammonium oxidation (anammox) bacteria, which were found in the anodic compartment of the reactors, contributed to nitrogen removal from the digestate. Overall, these results demonstrate that MFCs can be used to recover anammox bacteria from natural sources, and it may represent a promising bioremediation unit in anaerobic digestor plants for the simultaneous nitrogen removal and electricity generation u...