Ex-situ batch experiments were performed to further investigate whether metformin degradation was mainly induced by biological processes. Anaerobic sludge was obtained from the AnMBR and served as the inoculum source. All batch assays were carried out in 50 mL serum bottles, with each bottle containing 30 mL fresh MSM and 1 mL AnMBR sludge. Prior to incubation, both the liquid phase and headspace were purged with high-purity nitrogen gas for 10 minutes to establish strict anaerobic conditions. The bottles were subsequently sealed with butyl rubber stoppers and incubated at room temperature under static conditions. Two distinct experimental treatments, including a sterilized control group, in which the microbial community was inactivated by autoclaving, and an active untreated consortium. Each treatment was conducted in triplicate with an initial metformin concentration of 20 mg/L.