Removal of antibiotics and antibiotic resistance genes from sewage sludge via integrated in situ advanced anaerobic digestion: Application of response surface design

An integrated in situ advanced anaerobic digestion (AAD) process was developed at mesophilic temperatures using zero-valent iron (ZVI), papain, and cellulase to enhance digestion efficiency. Using Response Surface Methodology (RSM), the doses of three additives – zero-valent iron (ZVI), papain, and cellulase – were systematically optimized to maximize the removal efficiencies of five antibiotics and twelve antibiotic resistance genes (ARGs). The study analyzed correlations among antibiotics, ARGs, microbial community shifts, and biogas production, showing significant changes in methane production and microbial diversity. Except for OFL, all antibiotics were effectively removed from sludge, with SMZ, SMR, TC, and ROX removal rates reaching 93.59%, 78.93%, 51.38%, and 52.06%, respectively The RSM model identified the optimal experimental conditions as: ZVI 1063 mg/L, cellulase 23 mg/gSS, and papain 24 mg/gSS, which were validated experimentally at the antibiotics added 20 μg/L achieved from the response surface methods. The removal of ARGs varied, with intl1, tetA and sul2 being more persistent, while other ARGs had removal rates of 44.45%–96.91%. Dominant archaeal phyla in combined in situ AAD were Halobacterota and Euryarchaeota, with Methanomethylovorans as the main methanogen. Microbial community changes due to biological enzymes and ZVI enhanced antibiotic removal, while ARG removal depended on the fate of their potential hosts.