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

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.