Cow bedding is one of the dominant livestock wastes among agricultural waste in China. The bedding is a good raw material for anaerobic digestion (AD) and produces biogas of sustainable energy. To improve the AD performance of bedding, a two-step pretreatment of hydrothermal with ammonia (TPHA) was used at 100, 150, and 200 °C holding for 5–30 min. The results revealed that the highest volatile fatty acids concentration was 4720.1 mg/L at a TPHA of 200 °C for 5 min. The highest removal rates of cellulose and hemicellulose were 35.4% and 97.4% at a hydrothermal pretreatment (HP) of 100 °C for 30 min and 200 °C for 10 min, respectively. The highest methane yield of cow bedding was 169.1 mL/g VS using the TPHA at 150 °C for 10 min, which was 54.5% higher than that of the untreated group. Kinetic analysis showed that the modified Gompertz model was more suitable for TPHA for cow bedding. Therefore, TPHA could improve pretreatment characteristics and enhance the methane yield of cow bedding.

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