Impact of Ball-Milling and Thermal Hydrolysis on Physicochemical Properties and Anaerobic Digestion Kinetics of Mixed Slaughterhouse and Agricultural Wastes

Slaughterhouse by-products are promising feedstocks for anaerobic digestion due to their high lipid and protein content. However, their complex structures often limit hydrolysis, and excessive pretreatment can induce inhibitory conditions. This study evaluates the effects of ball-milling (BM), ball-milling with water (BM + water), and combined thermal hydrolysis and ball-milling (THP + BM) on the digestion performance of a mixed substrate of slaughterhouse and agricultural wastes. The results demonstrate that all BM-based pretreatments significantly improved digestion kinetics, reducing the lag phase by 26-66% and shortening the <i>T</i>₅₀ values by approximately 40% compared to the untreated substrate. While no statistically significant differences were observed in the ultimate methane yield, the onset of methanogenesis was markedly accelerated in the BM and BM + water treatments. In contrast, despite achieving superior solubilization, the THP + BM treatment failed to provide proportional kinetic enhancements. This was attributed to a severe initial metabolic imbalance-characterized by a pH drop below the inhibitory threshold (6.33)-which induced physiological stress and delayed the functional recovery of methanogens. These findings indicate that while ball-milling effectively facilitates digestion initiation by enhancing physical accessibility, the intensity of combined thermal-mechanical processes must be strategically optimized. For high-strength organic biomass, managing pretreatment severity is crucial to prevent initial acid stress and maximize process efficiency.