Hydroxyl-enriched biochar enhances methanogenesis by shaping microbial functional responses in anaerobic digestion

Biochar is effective in improving methanogenesis during anaerobic digestion (AD), but its underlying mechanisms remain poorly understood. Here, we investigated the role of hydroxyl groups by synthesizing ball-milled biochars (BMBs) with different hydroxyl contents through alkali modification and evaluating their effects on methane production and microbial gene abundance patterns. Hydroxyl content increased from 0.14 mmol/g in BMB₀ to 0.70 and 0.60 mmol/g in BMB₅ and BMB₁₀, respectively. BMB₅ achieved the highest final methane yield of 385.54 mL CH₄/g volatile solids (VS), 20.9% above BMB₀. The hydroxyl content was positively correlated with final methane yield (P < 0.01). Planktonic and sessile communities also showed distinct compositions, with Methanobacterium preferentially enriched on biochar surfaces. Metagenomic analysis showed higher abundances of pili- and Fpo-related genes in BMB-associated communities than in the Blank, whereas variation in the hydrogenotrophic methanogenesis (HM) gene module was significantly associated with hydroxyl content (P = 0.025). These results link hydroxyl enrichment to differences in methanogenesis-related genetic potential and methane production. Overall, this study identifies surface hydroxyl content as a material property closely associated with the methanogenic performance of biochar and provides a basis for rational design of biochar to enhance methane recovery from AD systems.