Shuttling happens: soluble flavin mediators of extracellular electron transfer in <Emphasis Type="Italic">Shewanella</Emphasis>

The genus Shewanella contains Gram negative γ-proteobacteria capable of reducing a wide range of substrates, including insoluble metals and carbon electrodes. The utilization of insoluble respiratory substrates by bacteria requires a strategy that is quite different from a traditional respiratory strategy because the cell cannot take up the substrate. Electrons generated by cellular metabolism instead must be transported outside the cell, and perhaps beyond, in order to reduce an insoluble substrate. The primary focus of research in model organisms such as Shewanella has been the mechanisms underlying respiration of insoluble substrates. Electrons travel from the menaquinone pool in the cytoplasmic membrane to the surface of the bacterial cell through a series of proteins collectively described as the Mtr pathway. This review will focus on respiratory electron transfer from the surface of the bacterial cell to extracellular substrates. Shewanella sp. secrete redox-active flavin compounds able to transfer electrons between the cell surface and substrate in a cyclic fashion—a process termed electron shuttling. The production and secretion of flavins as well as the mechanisms of cell-mediated reduction will be discussed with emphasis on the experimental evidence for a shuttle-based mechanism. The ability to reduce extracellular substrates has sparked interest in using Shewanella sp. for applications in bioremediation, bioenergy, and synthetic biology.     

Mercury Methylation by Dissimilatory Iron-Reducing Bacteria

The Hg-methylating ability of dissimilatory iron-reducing bacteria in the genera Geobacter, Desulfuromonas, and Shewanella was examined. All of the Geobacter and Desulfuromonas strains tested methylated mercury while reducing Fe(III), nitrate, or fumarate. In contrast, none of the Shewanella strains produced methylmercury at higher levels than abiotic controls under similar culture conditions. Geobacter and Desulfuromonas are closely related to known Hg-methylating sulfate-reducing bacteria within the Deltaproteobacteria.     

Unsaturated Organic Acids as Terminal Electron Acceptors for Reductase Chains of Anaerobic Bacteria

This paper summarizes the current knowledge of unsaturated organic acids in their role as terminal electron acceptors for reductase chains of anaerobic bacteria. The mechanisms and enzyme systems involved in the reduction of fumarate by Escherichia coli, Wolinella succinogenes, and some species of the genus Shewanella are considered. Particular attention is given to reduction of the double bond of the unnatural compound methacrylate by the δ-proteobacterium Geobacter sulfurreducens AM-1. Soluble periplasmic flavocytochromes c, found in bacteria of the genera Shewanella and Geobacter, are involved in the hydrogenation of fumarate (in Shewanella species) and methacrylate (in G. sulfurreducens AM-1). In E. coli and W. succinogenes, fumarate is reduced in cytosol by membrane-bound fumarate reductases. The prospects for research into organic acid reduction at double bonds in bacteria are discussed.     

Abundance and diversity of iron reducing bacteria communities in the sediments of a heavily polluted freshwater lake

Iron reduction mediated by Fe(III)-reducing bacteria (FeRB) occurs in aqueous environments and plays an essential role in removing contaminates in polluted freshwater lakes. Two model FeRB species, Shewanella and Geobacter, have been intensively studied because of their functions in bioremediation, iron reduction, and bioelectricity production. However, the abundance and community diversity of Shewanella and Geobacter in eutrophic freshwater lakes remain largely unknown. In this work, the distribution, abundance and biodiversity of Shewanella, Geobacter and other FeRB in the sediments of a heavily polluted lake, Chaohu Lake, China, across four successive seasons were investigated. Shewanella, Geobacter, and other FeRB were found to be widely distributed in the sediment of this heavily eutrophic lake. Geobacter was abundant with at least one order of magnitude more than Shewanella in cold seasons. Three Shewanella-related operational taxonomic units were detected and sixty one Geobacter-related operational taxonomic units were grouped into three phylogenetic clades. Thiobacillus, Desulfuromonas and Geobacter were identified as the main members of FeRB in the lake sediments. Interestingly, nutrients like carbon, nitrogen, and phosphorus were found to be the key factors governing the abundance and diversity of FeRB. Total FeRB, as well as Geobacter and Shewanella, were more abundant in the heavily eutrophic zone than those in the lightly eutrophic zone. The abundance and diversity of FeRB in the sediments of freshwater lakes were highly related with the degree of eutrophication, which imply that FeRB might have a great potential in alleviating the eutrophication and contamination in aqueous environments.

     

Initial development and structure of biofilms on microbial fuel cell anodes

Background  

Microbial 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.     

Electroactive bacteria—molecular mechanisms and genetic tools

In nature, different bacteria have evolved strategies to transfer electrons far beyond the cell surface. This electron transfer enables the use of these bacteria in bioelectrochemical systems (BES), such as microbial fuel cells (MFCs) and microbial electrosynthesis (MES). The main feature of electroactive bacteria (EAB) in these applications is the ability to transfer electrons from the microbial cell to an electrode or vice versa instead of the natural redox partner. In general, the application of electroactive organisms in BES offers the opportunity to develop efficient and sustainable processes for the production of energy as well as bulk and fine chemicals, respectively. This review describes and compares key microbiological features of different EAB. Furthermore, it focuses on achievements and future prospects of genetic manipulation for efficient strain development.     

Secretion of Flavins by Shewanella Species and Their Role in Extracellular Electron Transfer

Fe(III)-respiring bacteria such as Shewanella species play an important role in the global cycle of iron, manganese, and trace metals and are useful for many biotechnological applications, including microbial fuel cells and the bioremediation of waters and sediments contaminated with organics, metals, and radionuclides. Several alternative electron transfer pathways have been postulated for the reduction of insoluble extracellular subsurface minerals, such as Fe(III) oxides, by Shewanella species. One such potential mechanism involves the secretion of an electron shuttle. Here we identify for the first time flavin mononucleotide (FMN) and riboflavin as the extracellular electron shuttles produced by a range of Shewanella species. FMN secretion was strongly correlated with growth and exceeded riboflavin secretion, which was not exclusively growth associated but was maximal in the stationary phase of batch cultures. Flavin adenine dinucleotide was the predominant intracellular flavin but was not released by live cells. The flavin yields were similar under both aerobic and anaerobic conditions, with total flavin concentrations of 2.9 and 2.1 μmol per gram of cellular protein, respectively, after 24 h and were similar under dissimilatory Fe(III)-reducing conditions and when fumarate was supplied as the sole electron acceptor. The flavins were shown to act as electron shuttles and to promote anoxic growth coupled to the accelerated reduction of poorly crystalline Fe(III) oxides. The implications of flavin secretion by Shewanella cells living at redox boundaries, where these mineral phases can be significant electron acceptors for growth, are discussed.     

Shewanella oneidensis MR-1 Msh pilin proteins are involved in extracellular electron transfer in microbial fuel cells

Shewanella is a microbial genus that can oxidize lactate for the reduction of insoluble electron acceptors. This reduction is possible by either direct (cell-surface interaction, nanowires) or indirect (soluble redox mediators) mechanisms. However, the actual molecular identification of a nanowire has not been determined. Through mutational studies, Shewanella oneidensis MR-1 was analyzed for its ability to transfer electrons to an electrode after deletion of the structural pilin genes (ΔmshA-D) or the entire biosynthetic expression system (ΔmshH-Q) of one of its pilin complexes (Msh type IV pilus gene locus). The complete removal of the Msh complex (ΔmshH-Q) significantly decreased the current generated from a fuel cell compared to MR-1. However, the mutant with only extracellular Msh structural proteins removed (ΔmshA-D) was able to generate 80% of the current compared to MR-1. Thus, the intracellular and membrane bound Msh biogenesis complex is a pathway for extracellular electron transfer in S. oneidensis MR-1.     

Evidence for the coexistence of direct and riboflavin-mediated interspecies electron transfer in Geobacter co-culture

Geobacter species can secrete free redox-active flavins, but the role of these flavins in the interspecies electron transfer (IET) of Geobacter direct interspecies electron transfer (DIET) co-culture is unknown. Here, we report the presence of a new riboflavin-mediated interspecies electron transfer (RMIET) process in a traditional Geobacter DIET co-culture; in this process, riboflavin contributes to IET by acting as a free-form electron shuttle between free Geobacter species and serving as a bound cofactor of some cytochromes in Geobacter co-culture aggregates. Multiple lines of evidence indicate that RMIET facilitates the primary initiation of syntrophic growth between Geobacter species before establishing the DIET co-culture and provides additional ways alongside the DIET to transfer electrons to achieve electric syntrophy between Geobacter species. Redox kinetic analysis of riboflavin on either Geobacter species demonstrated that the Gmet_2896 cytochrome acts as the key riboflavin reduction site, while riboflavin oxidation by Geobacter sulfurreducens is the rate-limiting step in RMIET, and the RMIET makes only a minor contribution to IET in Geobacter DIET co-culture. The discovery of a new RMIET process in Geobacter DIET co-culture suggests the complexity of IET in syntrophic bacterial communities and provides suggestions for the careful examination of the IET of other syntrophic co-cultures.     

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.     

Bio-electrochemical synthesis of commodity chemicals by autotrophic acetogens utilizing CO<Subscript>2</Subscript> for environmental remediation

Bio-electrochemical synthesis (BES) is a technique in which electro-autotrophic bacteria such as Clostridium ljungdahlii utilize electric currents as an electron source from the cathode to reduce CO₂ to extracellular, multicarbon, exquisite products through autotrophic conversion. The BES of volatile fatty acids and alcohols directly from CO₂ is a sustainable alternative for non-renewable, petroleum-based polymer production. This conversion of CO₂ implies reduction of greenhouse gas emissions. The synthesis of heptanoic acid, heptanol, hexanoic acid and hexanol, for the first time, by Clostridium ljungdahlii was a remarkable achievement of BES. In our study, these microorganisms were cultivated on the cathode of a bio-electrochemical cell at ?400 mV by a DC power supply at 37°C, pH 6.8, and was studied for both batch and continuous systems. Pre-enrichment of bio-cathode enhanced the electroactivity of cells and resulted in maximizing extracellular products in less time. The main aim of the research was to investigate the impact of low-cost substrate CO₂, and the longer cathode recovery range was due to bacterial reduction of CO₂ to multicarbon chemical commodities with electrons driven from the cathode. Reactor design was simplified for cost-effectiveness and to enhance energy efficiencies. The Columbic recovery of ethanoic acid, ethanol, ethyl butyrate, hexanoic acid, heptanoic acid and hexanol being in excess of 80% proved that BES was a remarkable technology.     

The utility of Shewanella japonica for microbial fuel cells

Shewanella-containing microbial fuel cells (MFCs) typically use the fresh water wild-type strain Shewanella oneidensis MR-1 due to its metabolic diversity and facultative oxidant tolerance. However, S. oneidensis MR-1 is not capable of metabolizing polysaccharides for extracellular electron transfer. The applicability of Shewanella japonica (an agar-lytic Shewanella strain) for power applications was analyzed using a diverse array of carbon sources for current generation from MFCs, cellular physiological responses at an electrode surface, biofilm formation, and the presence of soluble extracellular mediators for electron transfer to carbon electrodes. Critically, air-exposed S. japonica utilizes biosynthesized extracellular mediators for electron transfer to carbon electrodes with sucrose as the sole carbon source.     

Electron transfer mechanisms between microorganisms and electrodes in bioelectrochemical systems

Microbes have been shown to naturally form veritable electric grids in which different species acting as electron donors and others acting as electron acceptors cooperate. The uptake of electrons from cells adjacent to them is a mechanism used by microorganisms to gain energy for cell growth and maintenance. The external discharge of electrons in lieu of a terminal electron acceptor, and the reduction of external substrates to uphold certain metabolic processes, also plays a significant role in a variety of microbial environments. These vital microbial respiration events, viz. extracellular electron transfer to and from microorganisms, have attracted widespread attention in recent decades and have led to the development of fascinating research concerning microbial electrochemical sensors and bioelectrochemical systems for environmental and bioproduction applications involving different fuels and chemicals. In such systems, microorganisms use mainly either (1) indirect routes involving use of small redox-active organic molecules referred to as redox mediators, secreted by cells or added exogenously, (2) primary metabolites or other intermediates, or (3) direct modes involving physical contact in which naturally occurring outer-membrane c-type cytochromes shuttle electrons for the reduction or oxidation of electrodes. Electron transfer mechanisms play a role in maximizing the performance of microbe?Celectrode interaction-based systems and help very much in providing an understanding of how such systems operate. This review summarizes the mechanisms of electron transfer between bacteria and electrodes, at both the anode and the cathode, in bioelectrochemical systems. The use over the years of various electrochemical approaches and techniques, cyclic voltammetry in particular, for obtaining a better understanding of the microbial electrocatalysis and the electron transfer mechanisms involved is also described and exemplified.     

Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System

Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme c-type cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an ∼35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.     

Enrichment of Geobacter Species in Response to Stimulation of Fe(III) Reduction in Sandy Aquifer Sediments

Abstract Engineered stimulation of Fe(III) has been proposed as a strategy to enhance the immobilization of radioactive and toxic metals in metal-contaminated subsurface environments. Therefore, laboratory and field studies were conducted to determine which microbial populations would respond to stimulation of Fe(III) reduction in the sediments of sandy aquifers. In laboratory studies, the addition of either various organic electron donors or electron shuttle compounds stimulated Fe(III) reduction and resulted in Geobacter sequences becoming important constituents of the Bacterial 16S rDNA sequences that could be detected with PCR amplification and denaturing gradient gel electrophoresis (DGGE). Quantification of Geobacteraceae sequences with a PCR most-probable-number technique indicated that the extent to which numbers of Geobacter increased was related to the degree of stimulation of Fe(III) reduction. Geothrix species were also enriched in some instances, but were orders of magnitude less numerous than Geobacter species. Shewanella species were not detected, even when organic compounds known to be electron donors for Shewanella species were used to stimulate Fe(III) reduction in the sediments. Geobacter species were also enriched in two field experiments in which Fe(III) reduction was stimulated with the addition of benzoate or aromatic hydrocarbons. The apparent growth of Geobacter species concurrent with increased Fe(III) reduction suggests that Geobacter species were responsible for much of the Fe(III) reduction in all of the stimulation approaches evaluated in three geographically distinct aquifers. Therefore, strategies for subsurface remediation that involve enhancing the activity of indigenous Fe(III)-reducing populations in aquifers should consider the physiological properties of Geobacter species in their treatment design. Received: 19 October 1999; Accepted: 28 December 1999; Online Publication: 25 April 2000     

New exoelectrogen Citrobacter sp. SX-1 isolated from a microbial fuel cell

Aims: Isolation, identification and characterization of a new exoelectrogenic bacterium from a microbial fuel cell (MFC). Methods and Results: Exoelectrogenic bacterial strain SX‐1 was isolated from a mediator‐less MFC by conventional plating techniques with ferric citrate as electron acceptor under anaerobic condition. Phylogenetic analysis of the 16S rDNA sequence revealed that it was related to the members of Citrobacter genus with Citrobacter sp. sdy‐48 being the most closely related species. The bacterial strain SX‐1 produced electricity from citrate, acetate, glucose, sucrose, glycerol and lactose in MFCs with the highest current density of 205 mA m^?2 generated from citrate. Cyclic voltammetry analysis indicated that membrane‐associated proteins may play an important role in facilitating the electrons transferring from bacteria to electrode. Conclusions: This is the first study that demonstrates that Citrobacter species can transfer electrons to extracellular electron acceptors. Citrobacter strain SX‐1 is capable of generating electricity from a wide range of substrates in MFCs. Significance and Impact of the Study: This finding increases the known diversity of power generating exoelectrogens and provided a new strain to explore the mechanisms of extracellular electron transfer from bacteria to electrode. The wide range of substrate utilization by SX‐1 increases the application potential of MFCs in renewable energy generation and waste treatment.     

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.     

The blind men and the filament: Understanding structures and functions of microbial nanowires

Extracellular electron transfer via filamentous protein appendages called ‘microbial nanowires’ has long been studied in Geobacter and other bacteria because of their crucial role in globally-important environmental processes and their applications for bioenergy, biofuels, and bioelectronics. Thousands of papers thought these nanowires as pili without direct evidence. Here, we summarize recent discoveries that could help resolve two decades of confounding observations. Using cryo-electron microscopy with multimodal functional imaging and a suite of electrical, biochemical, and physiological studies, we find that rather than pili, nanowires are composed of cytochromes OmcS and OmcZ that transport electrons via seamless stacking of hemes over micrometers. We discuss the physiological need for two different nanowires and their potential applications for sensing, synthesis, and energy production.     

Humic Acid Reduction by Propionibacterium freudenreichii and Other Fermenting Bacteria

Iron-reducing bacteria have been reported to reduce humic acids and low-molecular-weight quinones with electrons from acetate or hydrogen oxidation. Due to the rapid chemical reaction of amorphous ferric iron with the reduced reaction products, humic acids and low-molecular-weight redox mediators may play an important role in biological iron reduction. Since many anaerobic bacteria that are not able to reduce amorphous ferric iron directly are known to transfer electrons to other external acceptors, such as ferricyanide, 2,6-anthraquinone disulfonate (AQDS), or molecular oxygen, we tested several physiologically different species of fermenting bacteria to determine their abilities to reduce humic acids. Propionibacterium freudenreichii, Lactococcus lactis, and Enterococcus cecorum all shifted their fermentation patterns towards more oxidized products when humic acids were present; P. freudenreichii even oxidized propionate to acetate under these conditions. When amorphous ferric iron was added to reoxidize the electron acceptor, humic acids were found to be equally effective when they were added in substoichiometric amounts. These findings indicate that in addition to iron-reducing bacteria, fermenting bacteria are also capable of channeling electrons from anaerobic oxidations via humic acids towards iron reduction. This information needs to be considered in future studies of electron flow in soils and sediments.     

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.