Microbial adaptation mechanism of the syntrophic propionate-oxidizing methanogenic system from 36 °C to 20 °C

Operating anaerobic digestion (AD) at 20 °C offers substantial economic advantages; however, propionate accumulation at low temperatures remains a major bottleneck. Developing a robust propionate-degrading methanogenic consortium suitable for 20 °C is therefore essential. In this study, a stepwise temperature decrease from 37 to 20 °C in CSTRs yielded a stable methane production of 212 mL gVS^-1, representing 91 % of the mesophilic level. The enriched propionate-oxidizing bacteria consisted primarily of Thermovirga (13 %), Cryptanaerobacter (11 %), and Smithella (3 %). Compared with the mesophilic community, the cold-acclimated consortium exhibited more diverse propionate oxidation pathways, including the MMC pathway, propionate-dependent sulfate reduction, and the dismutation pathway. The acetoclastic methanogen Methanothrix remained dominant. Furthermore, the upregulation of genes associated with temperature regulation and membrane fluidity supported microbial adaptation to the temperature downshift from 36 °C to 20 °C. Overall, this study demonstrates the feasibility of efficient propionate conversion to methane at 20 °C and provides possibility to improve underperforming low-temperature AD systems.