Effects of Alternating Polarity Frequency on the Assembly of Electro-Methanogenic Microbial Communities

Electro-methanogenesis can enhance methane production and has attracted extensive interest, but the current strategies for building electro-methanogenic microbial communities suffer from several limitations. Alternating polarity has been shown as a new strategy to build electro-methanogenic communities, but the impact of alternation frequency on community assembly remains to be understood. In this study, the impact was evaluated using bioelectrochemical systems amended with granular activated carbon, and the working-electrode potential was alternated between +0.4 and -0.4 V vs. SHE at six frequencies ranging from 0.5 to 12 days. Among the tested conditions, 4-d alternation led to the best overall performance, producing approximately 250 L/L/kg cumulative biogas, 96% methane content, and a 45% methane yield, while preserving a stable and reproducible community structure. The community contained two Methanobacterium populations, two putatively electroactive Geobacter populations, and one putatively electroactive Pseudomonas population, together with diverse fermentative and syntrophic bacteria. Transcript-based analyses revealed clear frequency- and potential-dependent shifts in the activity of these functional groups. Redundancy analysis associated the Methanobacterium formicicum-related population with cathodic current and methane production, whereas the Geobacter sulfurreducens-related population was linked to anodic current and methane production. Bayesian and local-similarity networks further connected methanogen dynamics with specific electroactive, fermentative, and syntrophic partners. Collectively, these results establish alternating polarity frequency as a critical parameter for building robust communities capable of high-rate, high-quality methane production.