Stage-resolved ammonia inhibition mechanisms and resilience in sewage sludge methane fermentation revealed by physicochemical and microbial analyses.

Ammonia inhibition threatens methane recovery from sewage sludge, yet operational nitrogen tolerance boundaries remain poorly defined. By integrating long-term monitoring, kinetic partitioning of four metabolic steps, and microbial dynamics, four functional states were identified under progressive total ammonia nitrogen (TAN) accumulation: stability (0-3 g/L), initial inhibition (3-5.5 g/L), resilience window (5.5-6 g/L, corresponding to an FA concentration of 250-350 mg/L), and syntrophic collapse (>7 g/L). Quantitative partitioning of hydrolysis, acidogenesis, acetogenesis, and methanogenesis under a continuously increasing TAN trajectory revealed distinct inhibition patterns across metabolic stages. Although methanogenesis exhibited a slightly higher initial inhibition threshold, its reaction rate declined most rapidly with increasing TAN, revealing that methanogenesis governed system-level performance deterioration and ultimately determined methane recovery efficiency under ammonia stress. Microbial restructuring supported temporary functional compensation within the resilience window, whereas excessive TAN led to syntrophic breakdown and volatile fatty acid accumulation. These findings define nitrogen risk thresholds and provide a process-microbiome framework linking functional state transitions, metabolic inhibition hierarchy, and microbial adaptation for improving stability and resource efficiency in sludge-based bioenergy systems.