This study evaluated the influence of hyper-thermophilic pre-hydrolysis stage (70
C) on methane recovery of sewage sludge at 35
C. In this configuration, the process performance in both temperatures were estimated and the microbial communities were characterized by full-length16S rRNA genes and/or microbial activities. In addition, the appropriate solubilization reaction time was assessed. The results revealed that the higher hydrolysis and acidogenesis activities were achieved with longer reaction time of pretreatment (5 days) and thus higher organic nitrogen conversion and alkalinity were attained. Under appropriate pretreatment reaction time, pretreated sludge was characterized by 65% higher organic matters solubilization and 1.4-fold higher volatile fatty acids (VFAs) concentration compared to raw sludge. The overall methane yield produced under this scenario was 179 L CH4. KgVSin, with 15% of the absolute yield was produced in hydrolysis reactor. 50% reduction in bacteria belong to Firmicurtes was observed at mesophilic reactor and meanwhile the relative abundance of Bacteroidetes and Cloacimonetes were enhanced. The predominant methanogens in both stages did not change implying adaptation of Methanothermobacter (>62%) to mesophilic condition. However, increasing acetoclastic methanogens up to 30% in mesophilic reactor indicating methane was produced from pretreated sludge mainly through H2– mediated CO2 reduction and partially from acetate cleavage. The results highlight the key role of