Molecular gatekeeping in sludge EPS: pH-thermal forcing reshapes biopolymer interactions and carbon release pathways

The complex structure of extracellular polymeric substances (EPS) in waste activated sludge severely limits the recovery of endogenous carbon from sludge via thermal hydrolysis. In this study, a three-dimensional pH-time-layer framework was established to elucidate how pH and thermal effects jointly regulate EPS disintegration, molecular transformation, and carbon release pathways. Bulk measurements were combined with LC-MS/MS-based metabolomics analysis to reveal both macroscopic redistribution and molecular-level dynamic changes. The results showed that alkaline condition (pH 12) significantly promoted carbon release and generated a clear outward gradient (S-EPS > LB-EPS > TB-EPS). The Layer Distribution Index (LDI) indicated that the molecular gate was opened and the resistance to interlayer migration was reduced. In contrast, under acidic (pH 2) and neutral (pH 7) conditions, the EPS structure remained more compact, resulting in limited carbon release. The LDI further revealed that approximately 60 min was a critical transition point, corresponding to the initial loosening of the gatekeeping structure and the onset of outward migration. At the molecular level, proteins underwent sequential transformation from peptides to amino acids and amines, accompanied by rapid outward migration under alkaline conditions. In contrast, carbohydrates followed a staged pathway of "depolymerization-transient accumulation (60-90 min)-delayed migration," reflecting stronger structural hysteresis. Humic-like substances exhibited relatively limited molecular-level variation (LDI ≈ 1.6-2.0) but substantial bulk release, indicating that their behavior was primarily driven by structural collapse rather than fragmentation-controlled migration. Under alkaline conditions, these components were transformed into more polar species, facilitating the co-solubilization of proteins and carbohydrates. Overall, these findings demonstrate that pH governs the opening of EPS gatekeeping pathways through distinct structural and molecular mechanisms. Under alkaline conditions, an optimal operational window (60-120 min) was identified to maximize the release of biodegradable carbon while limiting secondary reactions. This framework provides a mechanistic basis for achieving controllable and predictable carbon recovery from sludge.