Deciphering the structural and stoichiometric regulation of anaerobic digestion: A cross-scale perspective from molecular thermodynamics to methanogenic pathways

Proteins and polysaccharides are the predominant organic fractions of waste activated sludge (WAS). However, the regulation mechanisms of their distinct molecular structures and compositional ratios on the efficiency of anaerobic digestion (AD) remain unclear. This study comprehensively investigates their impacts on AD performance, focusing on molecular thermodynamics and functional gene regulation involved in electron transfer, energy conversion, and methanogenic pathways. The results demonstrate that molecular structure is a key factor determining substrate bioavailability. The protein with a mainly β-structure (xylanase) and randomly coiled polysaccharide (pullulan) exhibited superior hydrolysis, acidification, and methanogenic efficiency due to increased enzyme binding affinity. Conversely, α-helical protein and triple-helix polysaccharide displayed restricted enzymatic accessibility. Further studies revealed the combination of xylanase and pullulan at the optimal C/N ratio (35) effectively balanced nutrition, thereby achieving the highest cumulative methane yield. Metagenomic and metatranscriptomic analyses revealed that the optimal structures and C/N stoichiometry not only enriched GH13 enzymes, but also shifted the metabolic pathway from acetoclastic to hydrogenotrophic methanogenesis. Moreover, it enhanced interspecies electron transfer and energy conversion efficiency by promoting NADH dehydrogenases, formate dehydrogenase and heterodisulfide reductase, thereby establishing a highly efficient and stable metabolic network in AD system. These findings provide novel insights into the microbial and biochemical regulation driven by substrate structure and stoichiometry from cross-scale perspective, thereby offering a theoretical basis and regulatory strategy for the efficient resource recovery of waste activated sludge. © 2026 Elsevier Ltd.