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Sub-moderate temperature (ranging from low to moderate temperature) anaerobic digestion (AD) could balance fermentation efficiency and energy input. We investigated biogas production and the microbial community in wastewater AD at sub-moderate (15 °C, 20 °C, and 25 °C) and moderate (35 °C; control group) temperatures with the organic loading rate (OLR) incrementally increased over 200 days. The impact of temperature on biogas production was found to be minimal at a low OLR but became more significant at a high OLR. Notably, a temperature threshold ranging from 15 °C to 20 °C exerted a strong inhibitory effect on biogas production and disrupted the microbial community. And, SMT-AD is deemed by this study to be the optimal application strategy of wastewater with low temperature and low OLR. Bacterial richness was positively and linearly related to temperature. There is a relevance between methane production and archaeal diversity under the influence of temperature and OLR. Temperature and OLR shaped the ecological function of predominant bacteria. Anaerolineales, Thermotogales, and Lactobacillales were strongly influenced by temperature. Synergistales had a synergistic relationship with Desulfovibrionales. Clostridiales was responsible for acetate and butyrate production and closely related to Lactobacillales. Acetoclastic Methanosaetaceae was the predominant methanogen. Methanogens could survive and maintain their population even though methanogenesis was limited under high OLRs and low temperatures. [ABSTRACT FROM AUTHOR]

[Display omitted] • The effect of temperature rises and falls on anaerobic digestion was assessed. • 45 °C exhibits optimal methanogenic performance during temperature rises and falls. • An effective equilibrium between hydrolysis and methanation is achieved at 45 °C. • The shortest recovery time was 2 days following temperature fluctuation. This study implemented five successive temperature stages (35 °C–45 °C–55 °C–45 °C–35 °C) in a continuous stirred tank reactor, designated as D35-1, D45-1, D55, D45-2, and D35-2, respectively. The objective was to probe the mechanism of temperature transformations on methanogenesis performance, microbial community, and metabolic pathways. The suitability of 45 °C for anaerobic digestion (AD) under varying temperatures was emphatically expounded. Intriguingly, optimal methanogenesis performance was attained at 45 °C, with methane production reaching 238.51 mL/g VS/d in D45-1 and 245.91 mL/g VS/d in D45-2, signifying increases of 17.66% and 21.32% contrasted with D35-2, respectively. Additionally, considerable resistance to temperature shocks at 45 °C was also manifested, as evidenced by a recovery time of 2 days in D45-2. At 45 °C, an effective balance between hydrolysis and methanogenesis was observed, maintaining a dynamic equilibrium between acetoclastic and hydrogenotrophic methanogenesis. The primary drivers in D45-1 were Caldicoprobacter , Bathyarchaeia , and Methanosarcina , while Anaerolinea and Methanosarcina were the key determinants in D45-2. By contrast, methanogenesis was suppressed at other stages. Therefore, 45 °C emerges as a viable alternative to conventional temperatures when confronted with temperature fluctuations to optimize energy efficiency. The results provide novel insights into AD temperature management and pave the way for engineering applications. [ABSTRACT FROM AUTHOR]