. The system was flushed with nitrogen gas to generate anaerobic conditions. H2 fermentation was conducted at 37 C with rotary shaking at 150 rpm. After H2 fermentation was exhausted, the non-heat pretreated seed of 4.75 g VS per batch was added, the initial pH was adjusted to 7.0, and the system was reflused with nitrogen gas, and the CH4 fermentation was conducted at 37 C. All experiments were setup in duplicate. During the fermentation experiment, total gas volume and composition were periodically monitored by gas
counters and gas chromatography, respectively. The liquid samples were analyzed for pH and volatile fatty acids (VFAs) every 4-6 h.