Soluble Electron Acceptors Research Articles

Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella

Many enzymes involved in bioenergetic processes contain chains of redox centers that link the protein surface, where interaction with electron donors or acceptors occurs, to a secluded catalytic site. In numerous cases these redox centers can transfer only single electrons even when they are associated to catalytic sites that perform two-electron chemistry. These chains provide no obvious contribution to enhance chemiosmotic energy conservation, and often have more redox centers than those necessary to hold sufficient electrons to sustain one catalytic turnover of the enzyme. To investigate the role of such a redox chain we analyzed the transient kinetics of fumarate reduction by two flavocytochromes c3 of Shewanella species while these enzymes were being reduced by sodium dithionite. These soluble monomeric proteins contain a chain of four hemes that interact with a flavin adenine dinucleotide (FAD) catalytic center that performs the obligatory two electron–two proton reduction of fumarate to succinate. Our results enabled us to parse the kinetic contribution of each heme towards electron uptake and conduction to the catalytic center, and to determine that the rate of fumarate reduction is modulated by the redox stage of the enzyme, which is defined by the number of reduced centers. In both enzymes the catalytically most competent redox stages are those least prevalent in a quasi-stationary condition of turnover. Furthermore, the electron distribution among the redox centers during turnover suggested how these enzymes can play a role in the switch between respiration of solid and soluble terminal electron acceptors in the anaerobic bioenergetic metabolism of Shewanella.

U(VI) Reduction by Diverse Outer Surface c -Type Cytochromes of Geobacter sulfurreducens

Early studies with Geobacter sulfurreducens suggested that outer-surface c-type cytochromes might play a role in U(VI) reduction, but it has recently been suggested that there is substantial U(VI) reduction at the surface of the electrically conductive pili known as microbial nanowires. This phenomenon was further investigated. A strain of G. sulfurreducens, known as Aro-5, which produces pili with substantially reduced conductivity reduced U(VI) nearly as well as the wild type, as did a strain in which the gene for PilA, the structural pilin protein, was deleted. In order to reduce rates of U(VI) reduction to levels less than 20% of the wild-type rates, it was necessary to delete the genes for the five most abundant outer surface c-type cytochromes of G. sulfurreducens. X-ray absorption near-edge structure spectroscopy demonstrated that whereas 83% ± 10% of the uranium associated with wild-type cells correspond to U(IV) after 4 h of incubation, with the quintuple mutant, 89% ± 10% of uranium was U(VI). Transmission electron microscopy and X-ray energy dispersion spectroscopy revealed that wild-type cells did not precipitate uranium along pili as previously reported, but U(IV) was precipitated at the outer cell surface. These findings are consistent with those of previous studies, which have suggested that G. sulfurreducens requires outer-surface c-type cytochromes but not pili for the reduction of soluble extracellular electron acceptors.

Extracellular Electron Transfer to Fe(III) Oxides by the Hyperthermophilic Archaeon Geoglobus ahangari via a Direct Contact Mechanism

The microbial reduction of Fe(III) plays an important role in the geochemistry of hydrothermal systems, yet it is poorly understood at the mechanistic level. Here we show that the obligate Fe(III)-reducing archaeon Geoglobus ahangari uses a direct-contact mechanism for the reduction of Fe(III) oxides to magnetite at 85°C. Alleviating the need to directly contact the mineral with the addition of a chelator or the electron shuttle anthraquinone-2,6-disulfonate (AQDS) stimulated Fe(III) reduction. In contrast, entrapment of the oxides within alginate beads to prevent cell contact with the electron acceptor prevented Fe(III) reduction and cell growth unless AQDS was provided. Furthermore, filtered culture supernatant fluids had no effect on Fe(III) reduction, ruling out the secretion of an endogenous mediator too large to permeate the alginate beads. Consistent with a direct contact mechanism, electron micrographs showed cells in intimate association with the Fe(III) mineral particles, which once dissolved revealed abundant curled appendages. The cells also produced several heme-containing proteins. Some of them were detected among proteins sheared from the cell's outer surface and were required for the reduction of insoluble Fe(III) oxides but not for the reduction of the soluble electron acceptor Fe(III) citrate. The results thus support a mechanism in which the cells directly attach and transfer electrons to the Fe(III) oxides using redox-active proteins exposed on the cell surface. This strategy confers on G. ahangari a competitive advantage for accessing and reducing Fe(III) oxides under the extreme physical and chemical conditions of hot ecosystems.

Proteins involved in electron transfer to Fe(III) and Mn(IV) oxides by Geobacter sulfurreducens and Geobacter uraniireducens

Whole-genome microarray analysis of Geobacter sulfurreducens grown on insoluble Fe(III) oxide or Mn(IV) oxide versus soluble Fe(III) citrate revealed significantly different expression patterns. The most upregulated genes, omcS and omcT, encode cell-surface c-type cytochromes, OmcS being required for Fe(III) and Mn(IV) oxide reduction. Other electron transport genes upregulated on both metal oxides included genes encoding putative menaquinol : ferricytochrome c oxidoreductase complexes Cbc4 and Cbc5, periplasmic c-type cytochromes Dhc2 and PccF, outer membrane c-type cytochromes OmcC, OmcG and OmcV, multicopper oxidase OmpB, the structural components of electrically conductive pili, PilA-N and PilA-C, and enzymes that detoxify reactive oxygen/nitrogen species. Genes upregulated on Fe(III) oxide encode putative menaquinol : ferricytochrome c oxidoreductase complexes Cbc3 and Cbc6, periplasmic c-type cytochromes, including PccG and PccJ, and outer membrane c-type cytochromes, including OmcA, OmcE, OmcH, OmcL, OmcN, OmcO and OmcP. Electron transport genes upregulated on Mn(IV) oxide encode periplasmic c-type cytochromes PccR, PgcA, PpcA and PpcD, outer membrane c-type cytochromes OmaB/OmaC, OmcB and OmcZ, multicopper oxidase OmpC and menaquinone-reducing enzymes. Genetic studies indicated that MacA, OmcB, OmcF, OmcG, OmcH, OmcI, OmcJ, OmcM, OmcV and PccH, the putative Cbc5 complex subunit CbcC and the putative Cbc3 complex subunit CbcV are important for reduction of Fe(III) oxide but not essential for Mn(IV) oxide reduction. Gene expression patterns for Geobacter uraniireducens were similar. These results demonstrate that the physiology of Fe(III)-reducing bacteria differs significantly during growth on different insoluble and soluble electron acceptors and emphasize the importance of c-type cytochromes for extracellular electron transfer in G. sulfurreducens.

Photocurrent Generation from Surface Assembled Photosystem I on Alkanethiol Modified Electrodes

Photosystem I (PSI) is a key component of oxygenic photosynthetic electron transport because of its light-induced electron transfer to the soluble electron acceptor ferredoxin. This work demonstrates the incorporation of surface assembled cyanobacterial trimeric PSI complexes into a biohybrid system for light-driven current generation. Specifically, this work demonstrates the improved assembly of PSI via electrophoretic deposition, with controllable surface assembled PSI density, on different self-assembled alkanethiol monolayers. Using artificial electron donors and acceptors (Os(bpy)(2)Cl(2) and methyl viologen) we demonstrate photocurrent generation from a single PSI layer, which remains photoactive for at least three hours of intermittent illumination. Photoelectrochemical comparison of the biohybrid systems assembled from different alkanethiols (hexanethiol, aminohexanethiol, mercaptohexanol, and mercaptohexanoic acid) reveals that the PSI generated photocurrent is enhanced by almost 5 times on negatively charged SAM surfaces as compared to positively charged surfaces. These results are discussed in light of how PSI is oriented upon electrodeposition on a SAM.

Flavin Electron Shuttles Dominate Extracellular Electron Transfer by Shewanella oneidensis

ABSTRACTShewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δbfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria.

Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems

Biological photo-voltaic systems are a type of microbial fuel cell employing photosynthetic microbes at the anode, enabling the direct transduction of light energy to electrical power. Unlike the anaerobic bacteria found in conventional microbial fuel cells that use metals in the environment as terminal electron acceptors, oxygenic photosynthetic organisms are poorly adapted for electron transfer out of the cell. Mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 were created in which all combinations of the three respiratory terminal oxidase complexes had been inactivated. These strains were screened for the ability to reduce the membrane-impermeable soluble electron acceptor ferricyanide, and the results were compared to the performance of the mutants in a biological photo-voltaic system. Deletion of the two thylakoid-localised terminal oxidases, the bd-quinol oxidase and cytochrome c oxidase resulted in a 16-fold increase in ferricyanide reduction rate in the dark compared to the wild-type. A further improvement to a 24-fold increase was seen upon deletion of the remaining "alternative respiratory terminal oxidase". These increases were reflected in the peak power generated in the biological photo-voltaic systems. Inactivation of all three terminal oxidase complexes resulted in a substantial redirection of reducing power; in the dark the equivalent of 10% of the respiratory electron flux was channelled to ferricyanide, compared to less than 0.2% in the wild-type. Only minor improvements in ferricyanide reduction rates over the wild-type were seen in illuminated conditions, where carbon dioxide is preferentially used as an electron sink. This study demonstrates the potential for optimising photosynthetic microbes for direct electrical current production.

Shewanella frigidimarina microbial fuel cells and the influence of divalent cations on current output

The genes involved in the proposed pathway for Shewanella extracellular electron transfer (EET) are highly conserved. While extensive studies involving EET from a fresh water Shewanella microbe (S. oneidensis MR-1) to soluble and insoluble electron acceptors have been published, only a few reports have examined EET from marine strains of Shewanella. Thus, Shewanella frigidimarina (an isolate from Antarctic Sea ice) was used within miniature microbial fuel cells (mini-MFC) to evaluate potential power output. During the course of this study several distinct differences were observed between S. oneidensis MR-1 and S. frigidimarina under comparable conditions. The maximum power density with S. frigidimarina was observed when the anolyte was half-strength marine broth (1/2 MB) (0.28μW/cm2) compared to Luria–Bertani (LB) (0.07μW/cm2) or a defined growth minimal medium (MM) (0.02μW/cm2). The systematic modification of S. frigidimarina cultured in 1/2 MB and LB with divalent cations shows that a maximum current output can be generated independent of internal ionic ohmic losses and the presence of external mediators.

Enhanced nitrate reduction and current generation by Bacillus sp. in the presence of iron oxides

Purpose Fe(III) has been reported as a strictly competitive electron acceptor with respect to other substrate reductions by dissimilatory Fe(III)-reducing bacteria (DIRB). However, the effect of Fe(III) oxides on the substrate reduction by other microorganisms remain unknown. The aims of this study were to investigate the effects of iron oxides on the nitrate reduction and current generation by Bacillus sp., in which the nitrate and carbon anodes served as soluble and insoluble electron acceptors, respectively.

Escherichia coli knock-out mutants with altered electron transfer activity in the Micredox® assay and in microbial fuel cells

Electron transfer from bacteria to external electron acceptors is a biologically important phenomenon that is increasingly being harnessed as useful technology such as in the Micredox® assay and in microbial fuel cells (MFCs). Optimisation of these systems is limited by incomplete knowledge of the underlying genetics of electron transfer. The Keio collection of single gene knock-out Escherichia coli strains is being tested to find genes involved in electron transfer from bacteria to external electron acceptors. Initially, 21 E. coli strains from the Keio collection were selected and tested for altered electro-activity using the Micredox® assay. The Micredox® assay provides a rapid measurement of electron transfer from cells to a soluble electron acceptor (potassium hexacyanoferrate(III)) and was previously developed as a general test for BOD and toxicant measurement. Of the 21 Keio strains, 10 were found to have significantly reduced electron transfer and two were found to have significantly increased electron transfer. The mutant with the lowest electron transfer rate (nuoA) and the highest electron transfer rate (arcA) were then tested for electron transfer in microbial fuel cells (MFCs). The arcA mutant had slightly higher electron transfer rates than the wild type in mediator-less MFC while the nuoA mutant strain had very similar electro-activity to the wild type. However, in a mediated MFC, the mutants were consistently different from the wild type. These results demonstrate that single gene deletion strains of E. coli can have significantly altered electron transfer capabilities, both in the Micredox® assay and in MFCs. Importantly, the Micredox® assay was found to be a rapid and easily scaled-up method to discover genes that are important in electron transfer.

NMR structure note: oxidized microsomal human cytochrome b5

Cytochrome b5 (cytb5) is a small membrane-bound hemoprotein present in all eukaryotic organisms. In most eukaryotic cells, cytb5 is attached to the cytosolic face of the endoplasmic reticulum and is described as the microsomal form (Mc). In vertebrates, two supplementary cytb5 can be found: soluble in the erythrocytes and membrane-bound attached to the internal face of the outer membrane in the mitochondria (OM) (Wang et al. 2007). Cytb5 can be divided into three domains: an N-terminal hydrosoluble globular domain that contains approximately 90 residues, a C-terminal hydrophobic domain about 25 residues long and a linker between the two above-mentioned domains. The hydrophilic domain contains the redox center, an iron protoporphyrin IX, ligated to the apoprotein by two histidyl residues. Mc cytb5 transfers electrons to various acceptors and is consequently involved in many different metabolic pathways. In the mixed function oxidase system, Mc cytb5 is a facultative electron donor to cytochromes P450 (Vergeres and Waskell 1995). Several mechanisms for the recognition between cytb5 and its physiological or artificial acceptors have been hypothesized. The implication of electrostatic interactions has been mostly studied with the cytochromes P450 as acceptors and is now widely accepted as a dominant factor (Schenkman et al. 1994). Nonetheless, several other factors may contribute positively to the recognition mechanism. First, the soluble domain of cytb5 lacking its C-terminal membrane domain cannot transfer electrons to membrane electron acceptors like cytochrome P450 (Dailey and Strittmatter 1978) while the full-length cytb5 can transfer electrons to both soluble and membranebound electron acceptor proteins (Vergeres and Waskell 1995). The linker is also recognized as an important factor controlling cytb5 to P450 interactions (Clarke et al. 2004). The three dimensional structures of the oxidized or reduced soluble domain of wild-type and mutant cytb5 from different species have been determined, either by X-ray or NMR. So far, no three dimensional structure of the full length protein has been reported, although some solid-state NMR data of the full-length rabbit cytb5 inserted in bicelles was Electronic supplementary material The online version of this article (doi:10.1007/s10858-010-9428-6) contains supplementary material, which is available to authorized users.

Initial development and structure of biofilms on microbial fuel cell anodes

BackgroundMicrobial fuel cells (MFCs) rely on electrochemically active bacteria to capture the chemical energy contained in organics and convert it to electrical energy. Bacteria develop biofilms on the MFC electrodes, allowing considerable conversion capacity and opportunities for extracellular electron transfer (EET). The present knowledge on EET is centred around two Gram-negative models, i.e. Shewanella and Geobacter species, as it is believed that Gram-positives cannot perform EET by themselves as the Gram-negatives can. To understand how bacteria form biofilms within MFCs and how their development, structure and viability affects electron transfer, we performed pure and co-culture experiments.ResultsBiofilm viability was maintained highest nearer the anode during closed circuit operation (current flowing), in contrast to when the anode was in open circuit (soluble electron acceptor) where viability was highest on top of the biofilm, furthest from the anode. Closed circuit anode Pseudomonas aeruginosa biofilms were considerably thinner compared to the open circuit anode (30 ± 3 μm and 42 ± 3 μm respectively), which is likely due to the higher energetic gain of soluble electron acceptors used. The two Gram-positive bacteria used only provided a fraction of current produced by the Gram-negative organisms. Power output of co-cultures Gram-positive Enterococcus faecium and either Gram-negative organisms, increased by 30-70% relative to the single cultures. Over time the co-culture biofilms segregated, in particular, Pseudomonas aeruginosa creating towers piercing through a thin, uniform layer of Enterococcus faecium. P. aeruginosa and E. faecium together generated a current of 1.8 ± 0.4 mA while alone they produced 0.9 ± 0.01 and 0.2 ± 0.05 mA respectively.ConclusionWe postulate that this segregation may be an essential difference in strategy for electron transfer and substrate capture between the Gram-negative and the Gram-positive bacteria used here.

Engineered Pyranose 2‐Oxidase: Efficiently Turning Sugars into Electrical Energy

AbstractDue to the recent interest in enzymatic biofuel cells (BFCs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale‐devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi‐rational protein design to improve the catalytic activity of the enzyme pyranose 2‐oxidase (P2Ox) with its alternative soluble electron acceptors 1,4‐benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BFCs. Using a screening assay based on 96‐well plates, we identified the variant H450G, which showed lower KM and higher kcat values for both 1,4‐benzoquinone and ferricenium ion compared to the wild‐type enzyme, when either D‐glucose or D‐galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BFCs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either “wired” to an osmium redox polymer or using soluble 1,4‐benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4‐fold compared to systems employing the wild‐type enzyme.

Geobacter sulfurreducens can protect 304L stainless steel against pitting in conditions of low electron acceptor concentrations

The effect of Geobacter sulfurreducens cells was studied on the electrochemical behaviour of 304L stainless steel, emphasizing the role of the soluble electron acceptor (fumarate). In fumarate-lacking media, the presence of G. sulfurreducens induced free potential ennoblement in a few hours. This ennoblement has already been observed in standard media that contained fumarate. Our previous studies have shown that G. sulfurreducens shifted the pitting potential toward the positive values. The pits induced by the presence of the bacteria were wider and deeper than in the absence of bacteria. Here, in fumarate-lacking media, similar shift in pitting potential was observed, but the repassivation phase was strongly improved. AFM analysis showed that pits were identical with those observed in the absence of bacteria at lower potential. In contrast with all the previous work where G. sulfurreducens enhanced corrosion, here at a low concentration of electron acceptor, the presence of the bacteria protected the steel against pitting.

Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells

Geobacter sulfurreducens produces current densities in microbial fuel cells that are among the highest known for pure cultures. The possibility of adapting this organism to produce even higher current densities was evaluated. A system in which a graphite anode was poised at −400 mV (versus Ag/AgCl) was inoculated with the wild-type strain of G. sulfurreducens, strain DL-1. An isolate, designated strain KN400, was recovered from the biofilm after 5 months of growth on the electrode. KN400 was much more effective in current production than strain DL-1. This was apparent with anodes poised at −400 mV, as well as in systems run in true fuel cell mode. KN400 had current (7.6 A/m 2) and power (3.9 W/m 2) densities that respectively were substantially higher than those of DL1 (1.4 A/m 2 and 0.5 W/m 2). On a per cell basis KN400 was more effective in current production than DL1, requiring thinner biofilms to make equivalent current. The enhanced capacity for current production in KN400 was associated with a greater abundance of electrically conductive microbial nanowires than DL1 and lower internal resistance (0.015 versus 0.130 Ω/m 2) and mass transfer limitation in KN400 fuel cells. KN400 produced flagella, whereas DL1 does not. Surprisingly, KN400 had much less outer-surface c-type cytochromes than DL1. KN400 also had a greater propensity to form biofilms on glass or graphite than DL1, even when growing with the soluble electron acceptor, fumarate. These results demonstrate that it is possible to enhance the ability of microorganisms to electrochemically interact with electrodes with the appropriate selective pressure and that improved current production is associated with clear differences in the properties of the outer surface of the cell that may provide insights into the mechanisms for microbe–electrode interactions.

The PsaE subunit of photosystem I prevents light-induced formation of reduced oxygen species in the cyanobacterium Synechocystis sp. PCC 6803

The PsaE protein is located at the reducing side of photosystem I (PSI) and is involved in docking the soluble electron acceptors, particularly ferredoxin. However, deletion of the psaE gene in the cyanobacterium Synechocystis sp. strain PCC 6803 inhibited neither photoautotrophic growth, nor in vivo linear and cyclic electron flows. Using photoacoustic spectroscopy, we detected an oxygen-dependent, PSI-mediated energy storage activity in the ΔpsaE null mutant, which was not present in the wild type (WT). The expression of the genes encoding catalase ( katG) and iron superoxide dismutase ( sodB) was upregulated in the ΔpsaE mutant, and the increase in katG expression was correlated with an increase in catalase activity of the cells. When catalases were inhibited by sodium azide, the production of reactive oxygen species was enhanced in ΔpsaE relative to WT. Moreover, sodium azide strongly impaired photoautotrophic growth of the ΔpsaE mutant cells while WT was much less sensitive to this inhibitor. The katG gene was deleted in the ΔpsaE mutant, and the resulting double mutant was more photosensitive than the single mutants, showing cell bleaching and lipid peroxidation in high light. Our results show that the presence of the PsaE polypeptide at the reducing side of PSI has a function in avoidance of electron leakage to oxygen in the light (Mehler reaction) and the resulting formation of toxic oxygen species. PsaE-deficient Synechocystis cells can counteract the chronic photoreduction of oxygen by increasing their capacity to detoxify reactive oxygen species.

DSA to grow electrochemically active biofilms of Geobacter sulfurreducens

Biofilms of Geobacter sulfurreducens were grown on graphite and on dimensionally stable anodes (DSA) in medium that did not contain any soluble electron acceptor. Several working electrodes were individually addressed and placed in the same reactor to compare their electrochemical behaviour in exactly the same biochemical conditions. Under constant polarization at 0.20 V versus Ag/AgCl, the electrodes were able progressively to oxidize acetate (5 mM), and average current densities around 5 A m −2 and 8 A m −2 were sustained for days on DSA and graphite, respectively. Removing the biofilm from the electrodes led the current to zero, while changing the medium by fresh one did not disturb the current when contact to air was avoided. This confirmed that the biofilm was fully responsible for the electro-catalysis of acetate oxidation and the current was not due to the accumulation of compounds in the bulk. Cyclic voltammetries performed during chronoamperometry indicated that the oxidation started above 0.05 V versus Ag/AgCl. The difference in maximal current values obtained with DSA and graphite was not linked to the biofilm coverage ratios, which were of the same order of magnitude in the range of 62–78%. On the contrary, the difference in maximal current values matched the ratio of the average surface roughness of the materials, 5.6 μm and 3.2 μm for graphite and DSA, respectively.

Changes in Microbial Community Composition and Geochemistry during Uranium and Technetium Bioimmobilization

In a previous column study, we investigated the long-term impact of ethanol additions on U and Tc mobility in groundwater (M. M. Michalsen et al., Environ. Sci. Technol. 40:7048-7053, 2006). Ethanol additions stimulated iron- and sulfate-reducing conditions and significantly enhanced U and Tc removal from groundwater compared to an identical column that received no ethanol additions (control). Here we present the results of a combined signature lipid and nucleic acid-based microbial community characterization in sediments collected from along the ethanol-stimulated and control column flow paths. Phospholipid fatty acid analysis showed both an increase in microbial biomass (approximately 2 orders of magnitude) and decreased ratios of cyclopropane to monoenoic precursor fatty acids in the stimulated column compared to the control, which is consistent with electron donor limitation in the control. Spatial shifts in microbial community composition were identified by PCR-denaturing gradient gel electrophoresis analysis as well as by quantitative PCR, which showed that Geobacteraceae increased significantly near the stimulated-column outlet, where soluble electron acceptors were largely depleted. Clone libraries of 16S rRNA genes from selected flow path locations in the stimulated column showed that Proteobacteria were dominant near the inlet (46 to 52%), while members of candidate division OP11 were dominant near the outlet (67%). Redundancy analysis revealed a highly significant difference (P = 0.0003) between microbial community compositions within stimulated and control sediments, with geochemical variables explaining 68% of the variance in community composition on the first two canonical axes.

Effect of Natural Organic Matter on Zinc Inhibition of Hematite Bioreduction by Shewanella putrefaciens CN32

The effect of zinc on the biological reduction of hematite (alpha-Fe2O3) by the dissimilatory metal-reducing bacterium (DMRB) Shewanella putrefaciens CN32 was studied in the presence of four natural organic materials (NOMs). Experiments were performed under non-growth conditions with H2 as the electron donor and zinc inhibition was quantified as the decrease in the 5 d extent of hematite bioreduction as compared to no-zinc controls. Every NOM was shown to significantly increase zinc inhibition during hematite bioreduction. NOMs were shown to alter the distribution of both biogenic Fe(II) and Zn(II) between partitioned (hematite and cell surfaces) and solution phases. To further evaluate the mechanism(s) of NOM-promoted zinc inhibition, similar bioreduction experiments were conducted with nitrate as a soluble electron acceptor, and hematite bioreduction experiments were conducted with manganese which was essentially non-inhibitory in the absence of NOM. The results suggest that Me(II)-NOM complexes may be specifically inhibitory during solid-phase bioreduction via interference of DMRB attachment to hematite through the formation of ternary Me(II)-NOM-hematite complexes.

Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.