Enhancing the participation of water in the anaerobic digestion of sewage sludge for highly efficient methanogenesis

Water is widely present in sewage sludge, in which it constitutes the largest proportion; however, its participation in the methanogenesis of sludge has been overlooked. Here we revealed the mechanisms enhancing the participation of water in methanogenesis of sludge. Through stable isotope tracing experiments, we observed that isoelectric point pretreatment significantly enhanced the participation of water in CO₂-reduction methanogenesis. Experimental results show that solid-liquid non-covalent interactions and interfacial water ordering in sludge were significantly enhanced. The former outcome drove electron transfer, while the latter provided an efficient proton channel. Combined with hydrogen/deuterium kinetic isotope effect (KIE) tests, it demonstrated that the water-mediated proton-coupled electron transfer (PCET) in the sludge were enhanced, accompanied by possible quantum tunnelling effect (KIE >> 10). Variations in the concentrations of key enzymes indicated that enhancing water-mediated PCET promoted both intracellular and extracellular electron–proton flow and accelerated the efficiency of mutual conversion between NADH and NAD⁺, strongly driving ATP synthesis. Further genome-centric metagenomic analysis and reaction thermodynamic calculations revealed that enhancing water-mediated PCET triggered enrichment of CO₂-reduction methanogenic consortia and effectively bypassed the limitation of H₂ partial pressure, providing a thermodynamic advantage to promote collaborative methanogenic metabolisms. These findings provide a theoretical basis for regulating the methanogenesis of perishable organic solid waste by water.