This study investigated the performance of thermophilic-mesophilic (T-M) and mesophilic-thermophilic (M-T) two-phase sludge anaerobic digestion at different inoculation proportions after a change in digestion temperature. After temperature change, the pH, total ammonia nitrogen (TAN), free ammonia nitrogen (FAN), solubility chemical oxygen demand (SCOD), and total alkalinity (TA) levels of two-phase digesters were between thermophilic control digesters and mesophilic control digesters. However, the volatile fatty acid (VFA) levels of two-phase digesters were higher than those of thermophilic or mesophilic control digesters. The bacteria communities of M-T two-phase digesters were more diverse than those of T-M. After a change in digestion temperature, the bacterial community was dominated by Coprothermobacter. After a change of digestion temperature, the relative abundance (RA) of Methanobacterium, Methanosaeta, and Methanospirillum of M-T two-phase digesters was higher than that of T-M two-phase digesters. In comparison, the RA of Methanosarcina of T-M two-phase digesters was higher than that of M-T two-phase digesters. The ultimate methane yields of thermophilic control digesters were greater than those of mesophilic control digesters. Nevertheless, the ultimate methane yield levels of M-T two-phase digesters were greater than those of T-M two-phase digesters. The ultimate methane yields of all two-phase digesters presented an earlier increase and later decrease trend with the increasing inoculation proportion. Optimal methane production condition was achieved when 15% of sludge (T-M15) was inoculated under mesophilic-thermophilic conditions, which promoted 123.6% (based on mesophilic control) or 27.4% (based on thermophilic control). An optimal inoculation proportion (about 15%) balanced the number and activity of methanogens of high-solid sludge anaerobic digestion.
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