| Summary: | The development of MFC using electroactive industrial microorganisms has seen a surge of interest because of the co-generation for bioproduct and electricity production. <i>Vibrio natriegens</i> as a promising next-generation industrial microorganism chassis and its application for microbial fuel cells (MFC) was first studied. Mediated electron transfer was found in <i>V. natriegens</i> MFC (VMFC), but <i>V. natriegens</i> cannot secrete sufficient electron mediators to transfer electrons to the anode. All seven electron mediators supplemented are capable of improving the electronic transfer efficiency of VMFC. The media and carbon sources switching study reveals that VMFCs have excellent bioelectricity generation performance with feedstock flexibility and high salt-tolerance. Among them, 1% glycerol as the sole carbon source produced the highest power density of 111.9 ± 6.7 mW/cm<sup>2</sup>. The insight of the endogenous electronic mediators found that phenazine-1-carboxamide, phenazine-1-carboxylic acid, and 1-hydroxyphenazine are synthesized by <i>V. natriegens</i> via the shikimate pathway and the phenazine synthesis and modification pathways. This work provides the first proof for emerging industrial biotechnology chassis <i>V. natriegens</i> as a novel high salt-tolerant and feedstock flexibility electroactive microorganism for MFC, and giving insight into the endogenous electron mediator biosynthesis of VMFC, paving the way for the application of <i>V. natriegens</i> in MFC and even microbial electrofermentation (EF).
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