Temperature transition strategy shapes methanogenic adaptation and process resilience during increasing organic loading in anaerobic digestion

Temperature transition and organic loading rate (OLR) are major operational factors governing anaerobic digestion performance; however, the influence of temperature-transition strategy on subsequent reactor resilience during increasing substrate loading remains insufficiently understood. Six continuous stirred tank reactors were operated under distinct strategies: constant mesophilic operation (37°C); gradual transitions to 42, 46, or 55°C; rapid stepwise transition to 46°C; and abrupt transition to 46°C. After thermal adaptation, OLR was progressively increased from 3 to 10 kg VS m −3 d −1 while hydraulic retention time (HRT) simultaneously decreased. Reactor performance and methanogenic community responses were assessed using methane yield, biogas composition, volatile fatty acids, total ammoniacal nitrogen, calculated free ammonia, and quantitative PCR (qPCR). Moderate temperature increases to 42–46°C transiently improved methane yield but produced no sustained long-term gains. Transition to 55°C caused pronounced short-term inhibition, followed by recovery after extended acclimation and stable methane production at the highest loading conditions. Segmented regression identified a methane-yield transition region centered at 6.75 kg VS m −3 d −1 (95% confidence interval: 4.92–8.57), while generalized least-squares modelling and block-bootstrap analyses supported the robustness of the reactor-response patterns. Greater loading tolerance was associated with increased abundances of Methanosarcinaceae and hydrogenotrophic methanogens, indicating restructuring of the quantified methanogenic community. Overall, reactor resilience was related not only to final operating temperature but also to the transition pathway and acclimation time. These findings provide an engineering basis for adaptive temperature management and loading optimization in continuously operated agricultural anaerobic digesters.