Abstract
Robust interspecies interactions are essential for efficient methanogenesis in anaerobic digestion. This study investigated the impact of quorum sensing (QS) enhancement on the succession of methanogenic communities during anaerobic digestion. The QS stimulation via exogenous N-acyl-homoserine lactones enhanced methane production by 18.8%-22.1%. Moreover, QS shaped microbial community succession toward a more deterministic assembly, selectively enriching key syntrophs (Pelotomaculum, Smithella), and methanogens (Methanobacterium, Methanothrix). Metagenomic analysis revealed that QS induced genes related to transcription, transport, and cofactor biosynthesis instead of directly regulating carbon metabolism. In this context, interspecies electron transfer emerges as a critical factor regulating interspecies interactions under QS regulation. Specifically, QS enhancement boosted redox mediator secretion, and the concentration of 2-amino-3-carboxy-1,4-naphthoquinone and phenazine increased by 7.8- and 4.8-fold, respectively. QS enhancement also induced higher abundance of c-type cytochromes. Moreover, the higher electron transfer coefficients were detected with 40.2%-89.9% increase. Further, QS also enhanced relative abundance of genes involved in Complex I/III and ferredoxin-dependent hydrogenases, promoting electron flow from syntrophs to methanogens. These effects induced higher relative abundance of genes associated with syntrophic propionate/butyrate oxidation and hydrogenotrophic/acetotrophic methanogenesis. Collectively, given that the similar regulation pathway is widely distributed in anaerobes, these findings identify QS as a critical ecological signal that drives functional microbial succession.