Ergothioneine improves methane recovery and reduces indicators of oxidative stress in ammonia-stressed food waste anaerobic digestion.

Anaerobic digestion is a promising route for food waste valorization, but ammonia inhibition remains a major constraint on methane recovery. Nine semi-continuous digesters were operated under stepwise increases in total ammonia nitrogen (TAN; 2.0-4.0 g·L-1). Three treatments were evaluated: the control (C; 0 mg·L-1·d-1), lower-dose ergothioneine (EGT) treatment (R1; 0.67 mg·L-1·d-1), and higher-dose EGT treatment (R2; 6.67 mg·L-1·d-1). C deteriorated after TAN increased to 2.7 g·L-1, whereas R2 maintained a mean daily specific methane production of 369 mL CH4·g-1 VS and 77% total chemical oxygen demand removal under 4.0 g·L-1 TAN. Fluorescence derived from 2',7'-dichlorodihydrofluorescein diacetate was 22.6% and 56.8% lower in R1 and R2, respectively, than in C. The EGT treatments also showed higher antioxidant-enzyme activity indicators derived from enzyme-linked immunosorbent assays and higher methanogenesis-associated indicators. Gene-based taxonomic profiles revealed distinct bacterial community configurations, while metaproteomic analysis showed greater representation of proteins associated with antioxidant responses, energy conservation, and hydrogenotrophic methanogenesis in R2. Together, the reactor, physiological, microbial, and protein-level patterns were consistent with an EGT-associated attenuation of oxidative-stress-related responses and better maintenance of methane-forming functions during progressive ammonia exposure. These findings identify EGT as a promising metabolic additive for improving methane recovery from nitrogen-rich organic wastes, while further work is needed to refine dosing and assess long-term economic feasibility.