Anaerobic digestion (AD) is a well-established technology for the sustainable conversion of agricultural organic by-products and waste into bioenergy. Temperature is crucial for optimizing methane production through inocula preservation and reactor start-up in AD. The preservation of inocula induced by temperature has rarely been assessed from an engineering perspective. There has also been limited exploration of the influence of high-to-moderate temperature transition on the initiation of AD. This study employed continuous mesophilic AD reactors with potential engineering applications to conduct revival tests. These tests evaluated the methane production activity of sludge stored at different temperatures and investigated the impact of high-temperature initiation on mesophilic AD. Additionally, we elucidated the correlation between these assessments and microbial diversity as well as composition. The results indicated that bacterial diversity was higher in the inoculum stored at 35 °C compared to 15 °C, ensuring a stable start-up operation of the mesophilic AD. The richness of the bacteria and diversity of the archaea remained stable during the transition from high to mesophilic temperatures. This was conducive to enhancing methanogenic activity of mesophilic AD initiated at 55 °C. The continuously operated AD system showed significant differences in microbial composition compared to its inoculum. Increased abundance of Coriobacteriaceae and Prevotellaceae led to propionate and butyrate accumulation, respectively, reducing AD operational capacity. Methanogenic archaea were less diverse in AD initiated with low-temperature preserved inoculum compared to that with a medium temperature. Streptococcaceae induced by high temperarure could promote AD stability. Hydrogenotrophic methanogens had a competitive advantage in mesophilic AD due to their prior exposure to high-temperature initiation, possibly influenced by Thermotogaceae.
