Biomethane is produced by acetate cleavage, not direct interspecies electron transfer: genome-centric view and carbon isotope.

Understanding the source of methane (CH) is of great significance for improving the anaerobic fermentation efficiency in bioengineering, and for mitigating the emission potential of natural ecosystems. Microbes involved in the process named direct interspecies electron transfer coupling with CO reduction, i.e., electrons released from electroactive bacteria to reduce CO into CH, have attracted considerable attention for wastewater treatment in the past decade. However, how the synergistic effect of microbiota contributes to this anaerobic carbon metabolism accompanied by CH production still remains poorly understood, especial for wastewater with antibiotic exposure. Results show that enhancing lower-abundant acetoclastic methanogens and acetogenic bacteria, rather than electroactive bacteria, contributed to CH production, based on a metagenome-assembled genomes network analysis. Natural and artificial isotope tracing of CH further confirmed that CH mainly originated from acetoclastic methanogenesis. These findings reveal the contribution of direct acetate cleavage (acetoclastic methanogenesis) and provide insightsfor further regulation of methanogenic strategies.