Comparative biogas production from Napier grass and sugarcane leaves under varying pH, ammonia stress, and inoculum conditions affecting microbial dynamics and gene expression

Seasonal variations significantly affect the yield of Napier grass (NG), a highly efficient energy crop for biogas production. During low-yield seasons, sugarcane leaves (SCL) offer a promising alternative biomass source. This study compared methane production from NG and SCL using five different inoculum sources under controlled (7.5) and uncontrolled initial pH (8.19–8.44) conditions. Results revealed that uncontrolled pH tended to improve methane yield (MY) and methane production rate (MPR). Under these conditions, the MYs of NG and SCL were comparable across most inocula; however, the rumen fluid (RF) reactors were a notable exception, exhibiting significantly higher performance. Anaerobic sludge (AS) achieved the highest MYs (323.9 and 333.8 mL CH4/g VS) and MPRs (254.6 and 292.8 mL CH4/L·d) for both substrates, despite operating under threshold total ammonia nitrogen (TAN) concentrations. While these high TAN levels strongly extended the acclimation phase (9–11 days) and suppressed methanogen richness, a high proportion of hydrolytic/acidogenic bacteria (53.1%)–primarily Firmicutes and Bacteroidota–played a key role in AS performance. Ammonia-tolerant genes, ackA and acs, were also identified as key contributors to methane production under ammonia stress. Additionally, RF achieved the second-highest MY and MPR, driven by the acetoclastic pathway and supported by a balanced syntrophic relationship between acetogens and Methanosaeta. These findings demonstrate the feasibility of integrating SCL into NG-based biogas reactors during low-yield seasons, supporting Thailand's renewable energy goals and the bio-economy within the sugarcane industry.