Brewery spent grain (BSG) is a widely generated agro-industrial residue with considerable potential for energy recovery through anaerobic digestion (AD). However, its complex lignocellulosic structure limits hydrolysis and access to organic matter, constraining methane recovery under conventional AD conditions. Thermo-alkaline pretreatment is a promising strategy to overcome these limitations by disrupting biomass structure and increasing organic solubilization. This study evaluated the effect of thermo-alkaline pretreatment (3% NaOH, 121 °C, 30 min) on methane production from brewery spent grain (BSG). Batch assays were performed at 37 °C for 70 days, using mixtures of raw and hydrolyzed BSG in varying proportions. Results showed that a moderate addition of hydrolysate (25%) significantly improved performance, increasing methane yield by 57.5% and the maximum methane production rate by 95.2% compared with untreated BSG. Conversely, hydrolysate alone reduced methane production by 81.9%, extended the lag phase, and caused microbial inhibition, largely due to the accumulation of p-cresol and volatile fatty acids. Microbial analysis revealed a shift in community structure under stress, with increased relative abundance of Synergistota. Functional predictions indicated that methanogenesis and energy metabolism pathways were stimulated under balanced substrate conditions, while fermentative pathways predominated under inhibitory scenarios. These findings highlight the importance of hydrolysate proportion in optimizing AD performance and suggest that controlled application of thermo-alkaline pretreatment can substantially improve methane recovery from BSG.
