Escherichia coliknock-out mutants with altered electron transfer activity in the Micredox® assay and in microbial fuel cells
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Abstract 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. Keywords: Micredox®microbial fuel cells E. coli electron transferBOD sensor Acknowledgements This work was supported by funding from the New Zealand Foundation for Research, Science and Technology, Contract LVLX0703. The Keio collection strains used in this work were kindly supplied by National BioResource Project (NIG, Japan): E. coli.Keywords:
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Fe(Ⅲ)-reducing bacteria (FRB) are important electrogenic microorganisms in microbial fuel cells (MFCs). There has been no known evolutionary pressure on the microorganisms to produce electricity. Therefore, it is assumed that the ability of electrogenic microorganisms to produce electricity is related to their capacity to transfer electrons onto natural extracellular electron acceptors, such as Fe(Ⅲ) oxides. An electrode of MFC and Fe(Ⅲ)-oxide in nature are both the extracellular insoluble electron acceptors for FRB. The similar metabolisms between electron transfer to an electrode and reducing insoluble Fe(Ⅲ)-oxide by FRB have been analyzed. This article summarizes the information on the recent development of FRB-based MFCs, and points out that the MFCs based on FRB have a good prospect, because of their high efficiency of energy conversion and needlessness of exotic mediator.
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Genus Shewanella is a gram-negative microorganism which has a unique property of utilizing solid state metal-oxide as a terminal electron acceptor. The extracellular electron transfer (ET) occurs via cytochromes located in the outer membrane (outer membrane-cytochrome, OMC). Due to the unique property, Shewanella has attracted a lot of attentions from the view point of biogeochemical cycle and microbial fuel cell. In this review, our recent results on the extracellular ET will be introduced with Shewanella loihica PV-4. We used various electrochemical techniques as well as spectroscopic ones to investigate the ET between the microbes and the (solid) electrode. Based on the various experimental results, we show evidences of the existence of direct ET path from OMC to electrode. We also show that the addition of water soluble Mn Porphyrines, semiconducting iron oxide nano colloids, etc., greatly enhances the ET efficiency. The extracellular ET process can be applied to microbial fuel cell. The power output of thin fuel cell, so far we achieved, is ca. 2 W/l, which is almost equal to one of the most efficient bio-fuel cells using isolated protein catalysts.
Shewanella
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Shewanella oneidensis
Bioelectrochemistry
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Klebsiella oxytoca
Anaerobic respiration
Cellular respiration
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In this investigation on Anaerobic Sulfate Respiration Bacteria (ASRB), it is reported that the obligate anaerobic microorganism, Desulfuromonas acetoxidans are capable of producing nano scale bacterial appendages for facilitating extracellular electron transfer. The nanowires were resistive and electrically conductive (1.88 × 10–8 Ω•m and 7.32 S•m–1). They also permitted the ASRB to colonize the surface of the solid or insoluble electron acceptors, thereby making it possible for extracellular electron transfer to take place to the insoluble electrode in the MFC directly and without the need of mediators for electron shuttling. The maximum power density reached was 7.9 Wm-3, and nanowire production was stimulated whilst insoluble electron acceptors were present for cellular respiration to occur. The results suggest D. acetoxidans initiates the production of conductive nanowires in case of limited availability of a soluble electron acceptor (SO42-) for ASRB as an alternative means for facilitating electron transfer to the insoluble electron acceptors.
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Abstract Extracellular electron transfer (EET) could enable electron uptake into microbial metabolism for the synthesis of complex, energy dense organic molecules from CO 2 and renewable electricity 1–6 . Theoretically EET could do this with an efficiency comparable to H 2 -oxidation 7,8 but without the need for a volatile intermediate and the problems it causes for scale up 9 . However, significant gaps remain in understanding the mechanism and genetics of electron uptake. For example, studies of electron uptake in electroactive microbes have shown a role for the Mtr EET complex in the electroactive microbe Shewanella oneidensis MR-1 10–14 , though there is substantial variation in the magnitude of effect deletion of these genes has depending on the terminal electron acceptor used. This speaks to the potential for previously uncharacterized and/or differentially utilized genes involved in electron uptake. To address this, we screened gene disruption mutants for 3667 genes, representing ≈99% of all nonessential genes, from the S. oneidensis whole genome knockout collection using a redox dye oxidation assay. Confirmation of electron uptake using electrochemical testing allowed us to identify five genes from S. oneidensis that are indispensable for electron uptake from a cathode. Knockout of each gene eliminates extracellular electron uptake, yet in four of the five cases produces no significant defect in electron donation to an anode. This result highlights both distinct electron uptake components and an electronic connection between aerobic and anaerobic electron transport chains that allow electrons from the reversible EET machinery to be coupled to different respiratory processes in S. oneidensis . Homologs to these genes across many different genera suggesting that electron uptake by EET coupled to respiration could be widespread. These gene discoveries provide a foundation for: studying this phenotype in exotic metal-oxidizing microbes, genetic optimization of electron uptake in S. oneidensis ; and genetically engineering electron uptake into a highly tractable host like E. coli to complement recent advances in synthetic CO 2 fixation 15 .
Shewanella oneidensis
Electron acceptor
Anaerobic respiration
Shewanella
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Shewanella spp. are remarkably versatile respirers. These gram-negative facultative anaerobes can not only switch from aerobic to anaerobic respiration of soluble non-metallic species, but can also reduce metal ions, both soluble and insoluble. By performing extracellular electron transfer (EET) across their outer membranes, they can reduce insoluble minerals, such as iron and magnesium oxides, as well as positively poised electrodes. The latter allows them to serve as living catalysts for organic matter oxidation in microbial fuel cells (MFC) [Watson and Logan 2010], an application designed to purify wastewater and produce electricity simultaneously. In such applications, Shewanella form electrically conductive biofilms at the solid electrode. To perform EET Shewanella utilizes three tools: (i) Outer membrane cytochromes (OMC), a general name for several different multiheme cytochromes displayed on its outer membrane, which receive terminal respiratory electrons from the inner membrane via two other multiheme cytochromes [Bretschger et al. 2007; Shi et al. 2012]. The OMC can transfer electrons directly to insoluble electron acceptors, such as electrodes, but also across the biofilm; (ii) Electrically conductive appendages [El Naggar et al. 2010], dubbed bacterial nanowires, which extend from bacterium to bacterium, throughout the biofilm and connect directly to the solid electron acceptor surface. The structure and function of these appendages are still under investigation and have been the center of considerable controversy. A recent study suggests that they are periplasmic extensions [Pirbadian et al. 2014]. (iii) Secreted flavins, which serve either or both as soluble electron transfer mediators [Marsili et al. 2008] and/or OMC cofactors [Paquete et al. 2014; Okamoto et al. 2014]. Because of its respiratory versatility, Shewanella is of great importance and interest, both for biogeochemical processes such as metal cycling and biomineralization [Fredrickson et al. 2008] and for sustainable technology applications, such as bioremediation, wastewater treatment and resource reclamation [Marshall et al. 2006; Watson and Logan 2010]. These bacteria have only been investigated over the last three decades, meaning that there are still considerable gaps in our understanding of the way they function and their possible applications. However, much knowledge has already been accumulated, and it is the goal of this collection to offer the reader a taste of the different relevant aspects, to serve as a first step for further literature exploration. The open access papers you will find here touch upon respiration, taxis, biofilms, environmental aspects and applied research. However, the collection does not pretend to supply exhaustive information or include all the most prominent papers on the subject. Many important papers, which are not available for open access, had to be omitted. It is my hope that this collection helps readers gain preliminary orientation in the wondrous landscape of Shewanella research, and that in the future more open access publications will join it to make it fuller.
Shewanella
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Shewanella oneidensis
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Abstract Electricity production in microbial fuel cells (MFCs) is an emerging green alternative to the use of fossil fuels. Shewanella oneidensis MR‐1 (SOMR‐1) is a Gram‐negative bacterium, adapted to MFCs due to its ability to link its bioenergetic metabolism through the periplasm to reduce extracellular electron acceptors. OmcA is a highly abundant outer‐membrane cytochrome of SOMR‐1 cells and is involved in the extracellular electron transfer to solid acceptors and electron shuttles. To investigate electron transfer performed by OmcA towards final acceptors, site directed mutagenesis was used to disturb the axial coordination of hemes. Interactions between OmcA and redox partners such as iron and graphene oxides, and electron shuttles were characterized using nuclear magnetic resonance and stopped‐flow experiments. Results showed that solid electron acceptors do not come into close proximity to the hemes, in agreement with experimentally observed slow electron transfer. In contrast, mutation of the distal axial ligand of heme VII changes the driving force of OmcA towards electron shuttles and reduces the affinity of the FMN:OmcA complex. Overall, these results reveal a functional specificity of particular hemes of OmcA and provide guidance for the rational design of mutated SOMR‐1 strains optimized for operating in different microbial electrochemical devices.
Shewanella oneidensis
Shewanella
Electron acceptor
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Shewanella oneidensis
Shewanella
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