Overcoming thermodynamic limitations on anaerobic methanogenesis via DSF/c-di-GMP enhanced indirect interspecies electron transfer

While quorum sensing (QS) holds immense potential to unlock the intrinsic thermodynamic constraints of anaerobic methanogenesis, conventional research predominantly skews toward AHLs-mediated direct interspecies electron transfer. The diffusible signal factor (DSF)/cyclic-diguanosine-monophosphate (c-di-GMP) cascade presents a promising avenue to refine the QS regulatory framework. This study constructed distinct QS microenvironments through exogenous addition of DSF and a DSF-degrading consortium to elucidate how this cascade regulates methanogenesis. The DSF/c-di-GMP cascade increased methane production by 33.2%, achieved 99.7% COD removal, and directed 73.2% of influent carbon toward methane. Mechanistically, indirect interspecies electron transfer (IIET) was significantly enhanced, evidenced by upregulation of hydrogenase genes (hox, frh, mvh) and riboflavin-associated genes, a 165.4% increase in electron transport system activity, and 18.8%–75.4% higher sludge conductivity. This cascade also reshaped the microbial community, enriching syntrophic bacteria (e.g., Smithella and Mesotoga) and hydrogenotrophic methanogens, forming a highly efficient modular co-occurrence network. Furthermore, key genes governing syntrophic propionate oxidation (via the methylmalonyl-CoA pathway), butyrate oxidation, and hydrogenotrophic methanogenesis were synergistically upregulated, constructing an effectively coupled carbon–electron metabolic network. The strategy's engineering feasibility was verified using real domestic wastewater, yielding stable COD removal (∼98.0%) and 14.6%-31.4% higher methane production. This study elucidates the role of DSF/c-di-GMP in QS-mediated methanogenesis by strengthening IIET, providing new mechanistic insights for QS-based strategies in wastewater treatment.