Two-phase anaerobic digestion with sub-thermophilic hydrolysis: Regulating metabolites to accelerate electron transfer and enhance methanogenesis.

Sub-thermophilic anaerobic digestion accelerates the hydrolysis and acidogenesis of complex substrates to improve methane production, but methanogens may be inhibited under such condition. Two-phase anaerobic digestion (TPAD) system can decouple the hydrolytic-acidogenic and methanogenic phases to optimize microbial activity in each phase. Therefore, in this study, a novel temperature-phased strategy combining a 45°C hydrolytic-acidogenic phase with a 37°C methanogenic phase (TPAD45°C/37°C) was developed to accelerate the degradation of agricultural waste and avoid the sub-thermophilic temperature-induced inhibition of methanogenesis. Results showed that 45°C hydrolytic phase increased ethanol and acetate production, and decreased propionate production compared to the 37°C control. Consequently, at a solid retention time of 20 days, the methane yield in the TPAD45°C/37°C group was 31.8% and 13.0% higher than that in the TPAD37°C/37°C and TPAD45°C/45°C groups, respectively. Mechanistically, the TPAD45°C/37°C group exhibited the highest McrA activity and coenzyme F420 content, indicating superior methanogenic activity. Furthermore, Tafel polarization and temperature-dependent conductivity analyses revealed that the higher levels of ethanol from 45°C hydrolysate provided a stronger thermodynamic driving force to minimize the energy barrier and improve intrinsic electron transfer rates, thereby enhancing methanogenesis. Additionally, the TPAD45°C/37°C group exhibited the highest overall metabolic potential. Microbial community analysis revealed that this system enriched the methanogens, which in turn promoted the degradation of complex substrates and increased methane production. This study provided an economically viable, energy-positive, and highly resilient technological strategy for the sustainable valorization of agricultural waste.