Anaerobic digestion faces challenges in treating nitrogen-rich food waste. In this study, side-stream hydrogen domestication strategy was employed to improve the ammonia tolerance of AD system. The results revealed that SHD enhanced the methane yield and improved system stability at a high total ammonia nitrogen concentration, while Control stopped producing CH4. Further analysis using enzymes activity assays, flow cytometry, and metagenomics explored the mechanisms underlying ammonia tolerance of R100. Side-stream hydrogen domestication profoundly reshaped the microbial community structure, enriching homoacetogens and Methanosaeta-dominated methanogenic archaea. Key metabolic pathways including homoacetogenesis, butyrate degradation, propionate degradation, and methane production were upregulated, accompanied by increased enzymes activity. The gene abundance related to energy-generating pathways such as glycolysis was significantly enhanced, ensuring sufficient Adenosine Triphosphate production. Additionally, the high gene abundance of ion transport systems contributed to the compensation for H+ and K+ losses under ammonia stress. This study provides important insights and practical guidance for developing novel techniques in the field of AD.
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