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[Display omitted] • Two-stage anaerobic digestion affects the hydrolysis-methanogenesis process. • Aerobic or micro-aerobic hydrolysis results in carbon loss from the substrate. • Substrate type significantly affected biogas yield and microbial community. • The gradient anaerobic digestion process (GADP) improved the biogas yield. • GADP enhanced carbon recovery of substrate and synergies of microorganisms. The destruction of the synergistic effect of the hydrolysis-methanogenesis process (HMP) of substrate may lead to the serious carbon loss of substrate and low biogas yield in traditional straw biogas engineering. In this study, the influence of substrate composition on anaerobic digestion (AD) was investigated under pilot conditions, and the relationship between HMP was further investigated under full-scale plants. The results showed that straw with a high content of soluble substances had the highest methane yield (264.25 L/kg VS) and degradation rate (59.32 %). At HRT = 20 d, the full-scale plant achieved the highest biogas yield (576.92 L/kg VS, 3.47 L/L/d) and degradation rate (67.50 %). Microbial analysis showed that substrate composition affected microbial community and methanogenic metabolic pathway. The analysis results of PICRUSt2 confirm that gradient AD guarantees the synergistic relationship of HMP. This study provides the theoretical basis and engineering guidance for improving the AD process in traditional straw biogas engineering. [ABSTRACT FROM AUTHOR]