| Summary: | 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. <i>Anaerolineales, Thermotogales</i>, and <i>Lactobacillales</i> were strongly influenced by temperature. <i>Synergistales</i> had a synergistic relationship with <i>Desulfovibrionales</i>. <i>Clostridiales</i> was responsible for acetate and butyrate production and closely related to <i>Lactobacillales</i>. Acetoclastic <i>Methanosaetaceae</i> was the predominant methanogen. Methanogens could survive and maintain their population even though methanogenesis was limited under high OLRs and low temperatures.
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