Co-occurrence of Direct and Indirect Extracellular Electron Transfer Mechanisms during Electroactive Respiration in a Dissimilatory Sulfate Reducing Bacterium

Extracellular electron transfer (EET) propels microbial fuel cell (MFC) technology and contributes to the mobility of redox active minerals and microbial syntrophy in nature. Sulfate-reducing bacteria (SRB), especially the genus Desulfovibrio corrode metal electrodes but are of interest for sulfate-containing MFCs providing wastewater treatment. Although extensive studies on SRB-mediated metal electrode corrosion have been done, there remain knowledge gaps on SRB EET to electrodes. We aimed to determine SRB EET mechanisms towards improving SRB performance in MFC wastewater treatment. Our MFCs with Desulfovibrio vulgaris Hildenborough ( Dv H), a model SRB, indicated that Dv H can harvest and send electrons to the carbon cloth electrode. Electricity production with a maximum power density of ∼0.074 W/m ² was observed when the ratio of lactate (electron and carbon donor) to sulfate (electron acceptor) was 60:20 and 0:10 in the anodic and cathodic chamber, respectively. Patterns in current production compared to variations of electron donor/acceptor ratios in the anode and cathode suggested that attachment of Dv H to the electrode and biofilm density were critical for effective electricity generation. Analysis of Dv H biofilms at different conditions (planktonic dissimilatory sulfate reduction respiration vs. electroactive respiration) by electron microscopy indicated Dv H utilized filaments that resemble nano-pili to attach on electrodes and facilitate EET from cell-to-cell and to the electrode. Proteomics profiling of electroactive respiration proteins indicated Dv H adapted to electroactive respiration by presenting more pili-, flagellar-related proteins and histidine kinases on electrodes. To investigate the role of pili and biofilm, we grew two Dv H mutants in MFCs under the same conditions. The mutant with a deletion of the major pilus-producing gene yielded less voltage and far less attachment to the electrode, suggesting the importance of pili in EET. The mutant with a deficiency in biofilm formation, however, did not eliminate current production indicating the existence of indirect EET. Untargeted metabolomics profiling showed flavin-based metabolites, potential electron shuttles, were dysregulated between respiration modes. This work revealed the metabolic flexibility of Dv H to thrive in less than ideal conditions with solid surfaces as both an electron acceptor (growth on anode) and donor (growth on cathode) by using a combination of direct and indirect EET mechanisms. Understanding Dv H EET mechanism could enhance the application of Dv H in MFCs treating wastewater.