Unveiling the Role of Intermittent Electrostimulation: Enhancing Microbial Metabolism and Electron Transfer in Electroactive Biofilms to Optimize V(V) Reduction and Immobilization

Intermittent electrostimulation plays a crucial role in the microbial processing of toxic vanadate [V(V)], influencing microbial metabolism, extracellular polymeric substances (EPS) secretion, and population dynamics. However, its influence on these aspects, particularly regarding microbial metabolism pathways and electron transfer mechanisms mediated by microbial metabolic activities, remains inadequately elucidated. Herein, electric-stimulated reactors incorporating different electrostimulation modes (continuous, intermittent, and none) were constructed and operated for over 150 days, with or without electroactive bacteria (EAB), to evaluate the V(V) reduction and immobilization. The bioreactor employing intermittent electrostimulation with an EAB-enriched inoculum (IP/AS-EB) showed rapid and efficient startup, achieving stable performance with significantly higher V(V) reduction efficiency (RE: 95.8%) and immobilization efficiency (IE: 93.1%) compared to those with continuous electrostimulation (CP/AS-EB: RE, 89.4%; IE, 68.9%) and control groups (AS-EB Ctrl: RE, 65.1%; IE, 52.5%). The reduction product included amorphous VO(OH)₂ and CaV₂(PO₄)₂(OH)₄·3H₂O, both occurring either intracellularly or extracellularly. Increased activity in the IP/AS-EB reactor results from enhanced targeted microbial and EPS, significantly enriching species like Geobacter, Petrimonas, Rhodococcus, Sedimentibacter, and Christensenellaceae_R-7_group. Total EPS, particularly humic-like substances and proteins, was significantly higher in IP/AS and IP/AS-EB reactors than in CP/AS, CP/AS-EB, and control groups. Besides, proteomic analysis further indicated that intermittent electrostimulation enriched bacteria capable of producing significant amounts of humic substances, redox-active proteins, and electron transfer proteins. These components, along with endogenous electron mediators identified via metabolomic analyses, effectively facilitated electron transfer within respiratory chains and interspecies. This study elucidates how intermittent electrostimulation boosts specific microbial communities and their metabolic pathways, offering insights into optimizing V(V) reduction processes and broadening their potential applications in heavy metal bioremediation.