Co-occurrence of direct and indirect extracellular electron transfer mechanisms during electroactive respiration in a dissimilatory sulfate reducing bacterium.

Understanding the extracellular electron transfer mechanisms of electroactive bacteria could help determine their potential in microbial fuel cells (MFCs) and their microbial syntrophy with redox-active minerals in natural environments. However, the mechanisms of extracellular electron transfer to electrodes by sulfate-reducing bacteria (SRB) remain underexplored. Here, we utilized double-chamber MFCs with carbon cloth electrodes to investigate the extracellular electron transfer mechanisms of Hildenborough (H), a model SRB, under varying lactate and sulfate concentrations using different H mutants. Our MFC setup indicated that H can harvest electrons from lactate at the anode and transfer them to cathode, where H could further utilize these electrons. Patterns in current production compared with variations of electron donor/acceptor ratios in the anode and cathode suggested that attachment of H to the electrode and biofilm density were critical for effective electricity generation. Electron microscopy analysis of H biofilms indicated H utilized filaments that resemble pili to attach to electrodes and facilitate extracellular electron transfer from cell to cell and to the electrode. Proteomics profiling indicated that H adapted to electroactive respiration by presenting more pili- and flagellar-related proteins. The mutant with a deletion of the major pilus-producing gene yielded less voltage and far less attachment to both anodic and catholic electrodes, suggesting the importance of pili in extracellular electron transfer. The mutant with a deficiency in biofilm formation, however, did not eliminate current production indicating the existence of indirect extracellular electron transfer. Untargeted metabolomics profiling showed flavin-based metabolites, potential electron shuttles.IMPORTANCEWe explored the application of Hildenborough in microbial fuel cells (MFCs) and investigated its potential extracellular electron transfer (EET) mechanism. We also conducted untargeted proteomics and metabolomics profiling, offering insights into how DvH adapts metabolically to different electron donors and acceptors. An understanding of the EET mechanism and metabolic flexibility of H holds promise for future uses including bioremediation or enhancing efficacy in MFCs for wastewater treatment applications.