Sulfamethoxazole and acetaminophen increase methane yield while exerting different effects on key metabolic pathways and antibiotic resistance gene dynamics during anaerobic digestion

Sulfamethoxazole (SMX, antibiotic) and acetaminophen (ACT, non-antibiotic) are two pharmaceuticals frequently detected in anaerobic digesters. This study evaluated their impact on biogas production, microbial community structure, and antibiotic resistance gene (ARG) dynamics in anaerobic digestion (AD) batch reactors operated at 5 mg/L dosage (SMX-AD and ACT-AD), alongside a control (Con). Compared to Con, cumulative methane yield increased by 62% and 63% in SMX-AD and ACT-AD, respectively, accompanied by accelerated propionate consumption between 5 and 10 d and enrichment of methanogens, which differed between reactors. In SMX-AD, Methanosarcina increased from 0.04% to 3.19%, while in ACT-AD, Methanothrix increased from 3.37% to 6.69%, remaining the dominant methanogen. ARG dynamics also diverged substantially. In SMX-AD, the total abundance of ARGs increased by 23% at 30 d, driven predominantly by the increase in hosts carrying multi-drug, aminoglycoside, and sulfonamide resistance genes, suggesting vertical gene transfer (VGT) as the primary mechanism driving ARG proliferation. Conversely, ACT-AD exhibited a 65% reduction in total ARG abundance, yet retained distinct ARG types within shared host genera. Horizontal gene transfer (HGT) mechanisms also differed: SMX-AD was characterized by upregulation of oxidative stress protein clusters and a transient increase in conjugation, while ACT-AD showed dominance of LexA protein clusters, transcriptional repressors of the SOS response (stress-induced DNA damage response), suggesting a regulated SOS state rather than SOS execution. SMX and ACT induced distinct microbial and genetic responses, both enhancing methane production but driving divergent ARG trajectories via VGT and HGT pathways.