Process-resolved effects of dibutyl phthalate on sludge anaerobic fermentation: Enzyme inhibition and metabolic disruption underlie the suppression of acidogenesis.
Growing evidence suggests that leaching of plasticizers such as dibutyl phthalate (DBP) from microplastics inhibits methanogenesis in anaerobic digesters treating waste activated sludge. However, how DBP influences upstream anaerobic fermentation (AF) remains unclear. This study evaluated the effects of DBP on overall sludge AF and separately on solubilization, hydrolysis, and acidogenesis processes. Volatile fatty acid (VFA) production showed a V-shaped response to environmentally relevant DBP concentrations. A reduction of 45.3% was observed at 0.5 mg/L DBP, whereas reductions exceeded 95% at 1-2 mg/L. The inhibition then weakened with increasing DBP, with a 60.5% reduction at 200 mg/L, an upper-bound level selected to bracket the maximum concentration reported in sludge. Stage-specific effects intensified with DBP concentration and peaked at 200 mg/L, where solubilization increased by 34.6%, whereas hydrolysis and acidogenesis decreased by 30.8% and 11.2%, respectively. The combined influence of these processes explained the nonmonotonic VFA response. Notably, enzyme assays and molecular simulations indicated that DBP inhibited ACK in a competitive-like manner and reduced its activity. Metagenomic analysis further indicated that DBP reduced genetic potential for downstream pathways converting pyruvate and acetyl-CoA to fermentation products. Collectively, these results reveal a DBP-induced fluctuating AF response and provide mechanistic insights into optimizing anaerobic treatment of plasticizer-laden sludge.