Extracellular Electron Acceptors Research Articles

Multiple roles of released c-type cytochromes in tuning electron transport and physiological status of Geobacter sulfurreducens.

Dissimilatory metal-reducing bacteria (DMRB) can transfer electrons to extracellular insoluble electron acceptors and play important roles in geochemical cycling, biocorrosion, environmental remediation, and bioenergy generation. c-type cytochromes (c-Cyts) are synthesized by DMRB and usually transported to the cell surface to form modularized electron transport conduits through protein assembly, while some of them are released as extracellularly free-moving electron carriers in growth to promote electron transport. However, the type of these released c-Cyts, the timing of their release, and the functions they perform have not been unrevealed yet. In this work, after characterizing the types of c-Cyts released by Geobacter sulfurreducens under a variety of cultivation conditions, we found that these c-Cyts accumulated up to micromolar concentrations in the surrounding medium and conserved their chemical activities. Further studies demonstrated that the presence of c-Cyts accelerated the process of microbial extracellular electron transfer and mediated long-distance electron transfer. In particular, the presence of c-Cyts promoted the microbial respiration and affected the physiological state of the microbial community. In addition, c-Cyts were observed to be adsorbed on the surface of insoluble electron acceptors and modify electron acceptors. These results reveal the overlooked multiple roles of the released c-Cyts in acting as public goods, delivering electrons, modifying electron acceptors, and even regulating bacterial community structure in natural and artificial environments.

Fe(OH)3 induced the Anammox system to perform extracellular electron transfer for enhancement of NH4+ removal

It is widely found that the ratio of nitrite consumption to ammonia consumption (ΔNO2--N/ΔNH4+-N) in Anammox systems is lower than its stoichiometry of 1.32, but the reason is unclear. In this study, ΔNO2--N/ΔNH4+-N of an Anammox system decreased to 0.80 with the addition of Fe(OH)3, which was significantly lower than a control system without the addition of Fe(OH)3. The lower ratio or more NH4+ removal was likely related to the reduction of Fe(OH)3 that served as extracellular electron acceptor to anaerobically oxidize NH4+, namely Feammox. Chemical or biological oxidation of the produced Fe2+ formed the Fe(III)/Fe(II) cycle to likely cause the continuous Feammox in the Anammox system. Isotopic analysis verified that the Anammox sludge collected from the Fe(OH)3-added reactor produced more 30N2 by Feammox. Raman analysis showed that the outer membrane cytochrome c of Fe(OH)3-added Anammox sludge could participate in the extracellular electron transfer during Feammox. Metagenomic analysis further confirmed that the gene encoding redox enzymes of cytochrome c of Candidatus Brocadia were enriched with adding Fe(OH)3. The results suggested that the Fe(OH)3 induced Anammox bacteria to perform extracellular electron transfer, allowing the Anammox system to effectively remove NH4+ via the Feammox pathway.

Structure of Geobacter cytochrome OmcZ identifies mechanism of nanowire assembly and conductivity.

OmcZ nanowires produced by Geobacter species have high electron conductivity (>30 S cm-1). Of 111 cytochromes present in G. sulfurreducens, OmcZ is the only known nanowire-forming cytochrome essential for the formation of high-current-density biofilms that require long-distance (>10 µm) extracellular electron transport. However, the mechanisms underlying OmcZ nanowire assembly and high conductivity are unknown. Here we report a 3.5-Å-resolution cryogenic electron microscopy structure for OmcZ nanowires. Our structure reveals linear and closely stacked haems that may account for conductivity. Surface-exposed haems and charge interactions explain how OmcZ nanowires bind to diverse extracellular electron acceptors and how organization of nanowire network re-arranges in different biochemical environments. In vitro studies explain how G. sulfurreducens employ a serine protease to control the assembly of OmcZ monomers into nanowires. We find that both OmcZ and serine protease are widespread in environmentally important bacteria and archaea, thus establishing a prevalence of nanowire biogenesis across diverse species and environments.

Genetic Manipulation of Desulfovibrio ferrophilus and Evaluation of Fe(III) Oxide Reduction Mechanisms.

The sulfate-reducing microbe Desulfovibrio ferrophilus is of interest due to its relatively rare ability to also grow with Fe(III) oxide as an electron acceptor and its rapid corrosion of metallic iron. Previous studies have suggested multiple agents for D. ferrophilus extracellular electron exchange including a soluble electron shuttle, electrically conductive pili, and outer surface multiheme c-type cytochromes. However, the previous lack of a strategy for genetic manipulation of D. ferrophilus limited mechanistic investigations. We developed an electroporation-mediated transformation method that enabled replacement of D. ferrophilus genes of interest with an antibiotic resistance gene via double-crossover homologous recombination. Genes were identified that are essential for flagellum-based motility and the expression of the two types of D. ferrophilus pili. Disrupting flagellum-based motility or expression of either of the two pili did not inhibit Fe(III) oxide reduction, nor did deleting genes for multiheme c-type cytochromes predicted to be associated with the outer membrane. Although redundancies in cytochrome or pilus function might explain some of these phenotypes, overall, the results are consistent with D. ferrophilus primarily reducing Fe(III) oxide via an electron shuttle. The finding that D. ferrophilus is genetically tractable not only will aid in elucidating further details of its mechanisms for Fe(III) oxide reduction but also provides a new experimental approach for developing a better understanding of some of its other unique features, such as the ability to corrode metallic iron at high rates and accept electrons from negatively poised electrodes. IMPORTANCE Desulfovibrio ferrophilus is an important pure culture model for Fe(III) oxide reduction and the corrosion of iron-containing metals in anaerobic marine environments. This study demonstrates that D. ferrophilus is genetically tractable, an important advance for elucidating the mechanisms by which it interacts with extracellular electron acceptors and donors. The results demonstrate that there is not one specific outer surface multiheme D. ferrophilus c-type cytochrome that is essential for Fe(III) oxide reduction. This finding, coupled with the lack of apparent porin-cytochrome conduits encoded in the D. ferrophilus genome and the finding that deleting genes for pilus and flagellum expression did not inhibit Fe(III) oxide reduction, suggests that D. ferrophilus has adopted strategies for extracellular electron exchange that are different from those of intensively studied electroactive microbes like Shewanella and Geobacter species. Thus, the ability to genetically manipulate D. ferrophilus is likely to lead to new mechanistic concepts in electromicrobiology.

Fluid-like electrodes and Purple Phototrophic Bacteria: bridging the gap in wastewater biorefineries

Wastewater biorefineries aim to generate value-added products in an economically viable process while removing pollutants. In this scenario, Purple phototrophic bacteria (PPB), the most versatile microorganisms on earth, are highly effective for sustainable wastewater treatment and nutrient recovery as cell protein. One of the most innovative approaches for applying PPB in the wastewater sector is their capacity for interchanging electrons with electroconductive materials. In contrast with classical biofilm-based techniques, we have demonstrated that a fluid-like electrode can accept electrons from planktonically grown PPB. We anticipate that such findings will impact in wastewater electrobioremediating capacity of PPB. Moreover, controlling the electrochemical nature of the extracellular electron acceptor (fluid-like electrode) allows for fine-tuning the metabolism of a planktonic PPB-dominated community to enhance their biodegradation rate (2-fold) while growing on brewery wastewater. For this purpose, a twin set of microbial electrochemical fluidized bed reactors (ME-FBR) were operated in identical conditions, except for illumination conditions (dark vs. infrared), to promote the development of PPB. Illumina sequencing revealed that both infrared radiation and polarization led to changes in the microbial population while producing an electrical current of 7 A·m-3. Indeed, the Geobacter genus was the electroactive bacteria outcompeting under dark conditions. In contrast, electroactive PPB genera like the Rhodopseudomonas and Rhodobacter outcompeted others under infrared illumination and electrostimulation. In this work, we have demonstrated how microbial selection can contribute to the sustainability of an electrobioremediation wastewater treatment by avoiding emissions of greenhouse gases such as methane. In addition, fluid-like bed bioreactors have shown their usefulness in recovering nutrients as PPB biomass, favoring planktonic growth and thus facilitating the recovery of a valuable product: the biomass of PPB.

Co-fitness analysis identifies a diversity of signal proteins involved in the utilization of specific c-type cytochromes

Purposec-Type cytochromes are essential for extracellular electron transfer (EET) in electroactive microorganisms. The expression of appropriate c-type cytochromes is an important feature of these microorganisms in response to different extracellular electron acceptors. However, how these diverse c-type cytochromes are tightly regulated is still poorly understood.MethodsIn this study, we identified the high co-fitness genes that potentially work with different c-type cytochromes by using genome-wide co-fitness analysis. We also constructed and studied the co-fitness networks that composed of c-type cytochromes and the top 20 high co-fitness genes of them.ResultsWe found that high co-fitness genes of c-type cytochromes were enriched in signal transduction processes in Shewanella oneidensis MR-1 cells. We then checked the top 20 co-fitness proteins for each of the 41 c-type cytochromes and identified the corresponding signal proteins for different c-type cytochromes. In particular, through the analysis of the high co-fitness signal protein for CymA, we further confirmed the cooperation between signal proteins and c-type cytochromes and identified a novel signal protein that is putatively involved in the regulation of CymA. In addition, we showed that these signal proteins form two signal transduction modules.ConclusionTaken together, these findings provide novel insights into the coordinated utilization of different c-type cytochromes under diverse conditions.

Dynamic roles of inner membrane electron-transfer hub of Shewanella oneidensis MR-1 in response to extracellular reduction kinetics

The unique extracellular electron transfer (EET) network of exoelectrogens endow them with extraordinary extracellular respiration ability to facilitate environmental remediation and biogeochemical processes. Shewanella oneidensis MR-1 is an environmentally ubiquitous exoelectrogen possessing multiple EET pathways. While the inner membrane CymA protein is widely believed to serve as an essential electron transfer hub in all these pathways, here our experimental evidences suggest another possibility: its EET role is highly dependent on extracellular reduction kinetics. Comparison of the bacterial EET and extracellular reduction performances in response to different species and concentrations of extracellular electron acceptors shows that the fraction of electron efflux contributed by the CymA pathway is positively correlated to the EET rate. This means some unknown EET pathways independent of CymA are activated under slow EET condition, likely as a strategy of self-adaption to environmental changes by feedback regulating the electron transport pathways. Our findings provide a new perspective to facilitate better understanding of the dynamic bacteria-environment interactions, and may inspire the discovery of new EET proteins and the development of more environmentally-robust biotechnologies for environmental remediation applications.

Real-time detection of response regulator phosphorylation dynamics in live bacteria

Bacteria utilize two-component system (TCS) signal transduction pathways to sense and adapt to changing environments. In a typical TCS, a stimulus induces a sensor histidine kinase (SHK) to phosphorylate a response regulator (RR), which then dimerizes and activates a transcriptional response. Here, we demonstrate that oligomerization-dependent depolarization of excitation light by fused mNeonGreen fluorescent protein probes enables real-time monitoring of RR dimerization dynamics in live bacteria. Using inducible promoters to independently express SHKs and RRs, we detect RR dimerization within seconds of stimulus addition in several model pathways. We go on to combine experiments with mathematical modeling to reveal that TCS phosphosignaling accelerates with SHK expression but decelerates with RR expression and SHK phosphatase activity. We further observe pulsatile activation of the SHK NarX in response to addition and depletion of the extracellular electron acceptor nitrate when the corresponding TCS is expressed from both inducible systems and the native chromosomal operon. Finally, we combine our method with polarized light microscopy to enable single-cell measurements of RR dimerization under changing stimulus conditions. Direct invivo characterization of RR oligomerization dynamics should enable insights into the regulation of bacterial physiology.

Self-Assembled Biohybrid: A Living Material To Bridge the Functions between Electronics and Multilevel Biological Modules/Systems.

Exoelectrogens are known to be specialized in reducing various extracellular electron acceptors to form conductive nanomaterials that are integrated with their cell bodies both structurally and functionally. Utilizing this unique capacity, we created a strategy toward the design and fabrication of a biohybrid electronic material by exploiting bioreduced graphene oxide (B-rGO) as the structural and functional linker to facilitate the interaction between the exoelectrogen community and external electronics. The metabolic functions of exoelectrogens encoded in this living hybrid can therefore be effectively translated toward corresponding microbial fuel cell applications. Furthermore, this material can serve as a fundamental building block to be integrated with other microorganisms for constructing various electronic components. Toward a broad impact of this biohybridization strategy, photosynthetic organelles and cells were explored to replace exoelectrogens as the active bioreducing components and as formed materials exhibited 4- and 8-fold improvements in photocurrent intensities as compared with native bioelectrode interfaces. Overall, a biologically driven strategy for the fabrication and assembly of electronic materials is demonstrated, which provides a unique opportunity to precisely probe and modulate desired biofunctions through deterministic electronic inputs/outputs and revolutionize the design and manufacturing of next-generation (bio)electronics.

Determinants of Multiheme Cytochrome Extracellular Electron Transfer Uncovered by Systematic Peptide Insertion.

The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate the binding of MtrA to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). The EET efficiency was found to be inversely correlated with the proximity of the insertion to the heme prosthetic groups. Mutants with decreased EET efficiencies also arose from insertions in a subset of the regions that make residue-residue contacts with the porin MtrB, while all sites contacting the extracellular cytochrome MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET efficiencies. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III) respiration conditions correlated with anode reduction. The peptide insertion profile, which represents the first comprehensive sequence-structure-function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for the regulation of EET.

Methane-Dependent Extracellular Electron Transfer at the Bioanode by the Anaerobic Archaeal Methanotroph "Candidatus Methanoperedens".

Anaerobic methanotrophic (ANME) archaea have recently been reported to be capable of using insoluble extracellular electron acceptors via extracellular electron transfer (EET). In this study, we investigated EET by a microbial community dominated by “Candidatus Methanoperedens” archaea at the anode of a bioelectrochemical system (BES) poised at 0 V vs. standard hydrogen electrode (SHE), in this way measuring current as a direct proxy of EET by this community. After inoculation of the BES, the maximum current density was 274 mA m–2 (stable current up to 39 mA m–2). Concomitant conversion of 13CH4 into 13CO2 demonstrated that current production was methane-dependent, with 38% of the current attributed directly to methane supply. Based on the current production and methane uptake in a closed system, the Coulombic efficiency was about 17%. Polarization curves demonstrated that the current was limited by microbial activity at potentials above 0 V. The metatranscriptome of the inoculum was mined for the expression of c-type cytochromes potentially used for EET, which led to the identification of several multiheme c-type cytochrome-encoding genes among the most abundant transcripts in “Ca. Methanoperedens.” Our study provides strong indications of EET in ANME archaea and describes a system in which ANME-mediated EET can be investigated under laboratory conditions, which provides new research opportunities for mechanistic studies and possibly the generation of axenic ANME cultures.

Unexpected role of electron-transfer hub in direct degradation of pollutants by exoelectrogenic bacteria.

Exoelectrogenic bacteria (EEB) are capable of anaerobic respiration with diverse extracellular electron acceptors including insoluble minerals, electrodes and flavins, but the detailed electron transfer pathways and reaction mechanisms remain elusive. Here, we discover that CymA, which is usually considered to solely serve as an inner-membrane electron transfer hub in Shewanella oneidensis MR-1 (a model EEB), might also function as a reductase for direct reducing diverse nitroaromatic compounds (e.g. 2,4-dichloronitrobenzene) and azo dyes. Such a process can be accelerated by dosing anthraquinone-2,6-disulfonate. The CymA-based reduction pathways in S. oneidensis MR-1 for different contaminants could be functionally reconstructed and strengthened in Escherichia coli. The direct reduction of lowly polar contaminants by quinol oxidases like CymA homologues might be universal in diverse microbes. This work offers new insights into the pollutant reduction mechanisms of EEB and unveils a new function of CymA to act as a terminal reductase.

Evidence for Horizontal and Vertical Transmission of Mtr-Mediated Extracellular Electron Transfer among the Bacteria.

ABSTRACTSome bacteria and archaea have evolved the means to use extracellular electron donors and acceptors for energy metabolism, a phenomenon broadly known as extracellular electron transfer (EET). One such EET mechanism is the transmembrane electron conduit MtrCAB, which has been shown to transfer electrons derived from metabolic substrates to electron acceptors, like Fe(III) and Mn(IV) oxides, outside the cell. Although most studies of MtrCAB-mediated EET have been conducted in Shewanella oneidensis MR-1, recent investigations in Vibrio and Aeromonas species have revealed that the electron-donating proteins that support MtrCAB in Shewanella are not as representative as previously thought. This begs the question of how widespread the capacity for MtrCAB-mediated EET is, the changes it has accrued in different lineages, and where these lineages persist today. Here, we employed a phylogenetic and comparative genomics approach to identify the MtrCAB system across all domains of life. We found mtrCAB in the genomes of numerous diverse Bacteria from a wide range of environments, and the patterns therein strongly suggest that mtrCAB was distributed through both horizontal and subsequent vertical transmission, and with some cases indicating downstream modular diversification of both its core and accessory components. Our data point to an emerging evolutionary story about metal-oxidizing and -reducing metabolism, demonstrates that this capacity for EET has broad relevance to a diversity of taxa and the biogeochemical cycles they drive, and lays the foundation for further studies to shed light on how this mechanism may have coevolved with Earth’s redox landscape.

Anaerobic oxidation of methane mediated by microbial extracellular respiration.

Anaerobic oxidation of methane (AOM) can be microbially mediated by the reduction of different terminal electron acceptors. AOM coupled to reduction of sulfate, manganese/iron oxides, humic substances, selenate, arsenic and other artificial extracellular electron acceptors are recognized as processes associated with microbial extracellular respiration. In these processes, methane-oxidizing archaea transfer electrons to external electron acceptors or to interdependent microbial species, which are mechanistically dependent on versatile extracellular electron transfer (EET) pathways. This review compiles recent progress in the research of electromicrobiology of AOM based on the catalogue of different electron acceptors. Naturally distributed and artificially constructed EET-mediated AOM is summarized, with the discussion of their environmental importance and application potentials. The diversity of responsible microorganisms involved in EET-mediated AOM is discussed with both methane-oxidizing archaea and their putative bacterial partners. More importantly, the review highlights progress and deficiencies in our understanding of EET pathways in EET-mediated AOM, raising open research questions for future research.

Geobacter sulfurreducens inner membrane cytochrome CbcBA controls electron transfer and growth yield near the energetic limit of respiration.

Geobacter sulfurreducens utilizes extracellular electron acceptors such as Mn(IV), Fe(III), syntrophic partners, and electrodes that vary from +0.4 to -0.3V versus standard hydrogen electrode (SHE), representing a potential energy span that should require a highly branched electron transfer chain. Here we describe CbcBA, abc-type cytochrome essential near the thermodynamic limit of respiration when acetate is the electron donor. Mutants-lacking cbcBA ceased Fe(III) reduction at -0.21V versus SHE, could not transfer electrons to electrodes between -0.21 and -0.28V, and could not reduce the final 10%-35% of Fe(III) minerals. As redox potential decreased during Fe(III) reduction, cbcBA was induced with the aid of the regulator BccR to become one of the most highly expressed genes in G. sulfurreducens. Growth yield (CFU/mM Fe(II)) was 112% of WT in ∆cbcBA, and deletion of cbcL (an unrelated bc-cytochrome essential near -0.15V) in ΔcbcBA increased yield to 220%. Together with ImcH, which is required at high redox potentials, CbcBA represents a third cytoplasmic membrane oxidoreductase in G. sulfurreducens. This expanding list shows how metal-reducing bacteria may constantly sense redox potential to adjust growth efficiency in changing environments.

Extracellular riboflavin induces anaerobic biofilm formation in Shewanella oneidensis

BackgroundSome microorganisms can respire with extracellular electron acceptors using an extended electron transport chain to the cell surface. This process can be applied in bioelectrochemical systems in which the organisms produce an electrical current by respiring with an anode as electron acceptor. These organisms apply flavin molecules as cofactors to facilitate one-electron transfer catalyzed by the terminal reductases and in some cases as endogenous electron shuttles.ResultsIn the model organism Shewanella oneidensis, riboflavin production and excretion trigger a specific biofilm formation response that is initiated at a specific threshold concentration, similar to canonical quorum-sensing molecules. Riboflavin-mediated messaging is based on the overexpression of the gene encoding the putrescine decarboxylase speC which leads to posttranscriptional overproduction of proteins involved in biofilm formation. Using a model of growth-dependent riboflavin production under batch and biofilm growth conditions, the number of cells necessary to produce the threshold concentration per time was deduced. Furthermore, our results indicate that specific retention of riboflavin in the biofilm matrix leads to localized concentrations, which by far exceed the necessary threshold value.ConclusionThis study describes a new quorum-sensing mechanism in S. oneidensis. Biofilm formation of S. oneidensis is induced by low concentrations of riboflavin resulting in an upregulation of the ornithine-decarboxylase speC. The results can be applied for the development of strains catalyzing increased current densities in bioelectrochemical systems.

A unique aromatic residue modulates the redox range of a periplasmic multiheme cytochrome from Geobacter metallireducens

Geobacter bacteria are able to transfer electrons to the exterior of the cell and reduce extracellular electron acceptors including toxic/radioactive metals and electrode surfaces, with potential applications in bioremediation or electricity harvesting. The triheme c-type cytochrome PpcA from Geobacter metallireducens plays a crucial role in bridging the electron transfer from the inner to the outer membrane, ensuring an effective extracellular electron transfer. This cytochrome shares 80% identity with PpcA from Geobacter sulfurreducens, but their redox properties are markedly different, thus determining the distinctive working redox potential ranges in the two bacteria. PpcA from G. metallireducens possesses two extra aromatic amino acids (Phe-6 and Trp-45) in its hydrophobic heme core, whereas PpcA from G. sulfurreducens has a leucine and a methionine in the equivalent positions. Given the different nature of these residues in the two cytochromes, we have hypothesized that the extra aromatic amino acids could be partially responsible for the observed functional differences. In this work, we have replaced Phe-6 and Trp-45 residues by their nonaromatic counterparts in PpcA from G. sulfurreducens. Using redox titrations followed by UV–visible and NMR spectroscopy we observed that residue Trp-45 shifted the redox potential range 33% toward that of PpcA from G. sulfurreducens, whereas Phe-6 produced a negligible effect. For the first time, it is shown that the inclusion of an aromatic residue at the heme core can modulate the working redox range in abundant periplasmic proteins, paving the way to engineer bacterial strains for optimal microbial bioelectrochemical applications.

Biology and biotechnology of microbial pilus nanowires.

Type IV pili (T4P) are bacterial appendages used for cell adhesion and surface motility. In metal-reducing bacteria in the genus Geobacter, they have the unique property of being conductive and essential to wire cells to extracellular electron acceptors and other cells within biofilms. Theseelectroactivebacteria use a conserved pathway for biological assembly and disassembly of a short and aromatic dense peptide subunit (pilin). The polymerization of thepilins clusters aromatic residues optimally for charge transport and exposes ligands for metal immobilization and reduction. The simple design yet unique functionalities of conductive T4P afford opportunities for the scaled-up production of recombinant pilins and their in vitro assembly into electronic biomaterials of biotechnological interest. This review summarizes current knowledge of conductive T4P biogenesis and functions critical to actualize applications in bioelectronics, bioremediation, and nanotechnology.

Isolation and Characterization of Electrogenic Bacteria from Tannery Wastewater

Electrogenic bacteria are able to transfer electrons to extracellular electron acceptors as well as can be used in devices like bioelectrochemical systems (BES). This study was focused to produce electricity from wastewater using microbial fuel cell and find out potential electrogenic bacteria from liquid tannery wastes. After power generation study, six isolates were screened as potential electrogenic bacteria. Of them, two potential bacteria were identified based on their morphological and biochemical characteristics and confirmed by phylogenetic analysis based on 16S rRNA gene sequence. We also investigated the effect of anode surface area on electricity generation in the microbial fuel cells and found that the surface area had positive impact on electricity generation.
 Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 23-27

Mechanism of electricigenic respiration mediated by electron transfer mediator of Klebsiella oxytoca d7

Electricigenic respiration is a new microbial metabolism discovered recently in an anaerobic environment lacking common electron receptors such as oxygen and others, in which some bacteria utilize extracellular electron transfer system. One electricigen, Klebsiella oxytoca d7, was isolated and confirmed its ability of electricigenic respiration in previous study, but its process and mechanism from intracellular respiratory chain to extracellular electron acceptors were not clear. In this paper, a microbial fuel cell (MFC) of this pure bacterium was constructed, and the main mechanism of electron transfer via the electron transfer mediators (ETMs) during electricity generation process of the bacterium was explored. The results show the ETMs can only be generated with the circuit on, and its contribution to the total electricity generation was much greater than that when the carbon source was insufficient. The redox potential of the ETMs of the K. oxytoca d7 was about −250 mV. The electrons were intercepted during the transfer from NADH to coenzyme Q and flow to the extracellular electron acceptors. The site of electron interception in the ETMs was only dependent on the redox potential of the ETMs secreted by the electricigens. Electrons were transferred from carriers in intracellular respiratory chain and with slightly higher redox potential than ETMs to ETMs. Normally the contribution of extracellular electron transfer mediated by ETMs to the total electricity generation amounted to more than 60%, indicating the process directly affected the conversion of organic substance in anode chamber and the electricity generation performance of the MFC.