As common pollutants in wastewater, heavy metals and antibiotics have negative effects on the performance, microbial community and antibiotic resistance genes (ARGs) propagation of biological treatment processes. This study investigated the differential responses of heterotrophic denitrification (HD) and sulfur autotrophic denitrification (SAD) process to Cu(II) and sulfamethazine (SMZ) stress. Exposure to 12 mg/L Cu(II) and 25 mg/L SMZ, either alone or combined, reduced nitrogen removal efficiency by 4.22 % to 32.6 % for HD and 38.7 % to 63.2 % for SAD, respectively. HD exhibited greater robustness and microbial cooperation compared to SAD under stress. Cu(II) and SMZ intensified bacterial reactive oxygen species (ROS) responses, inhibited cell activity, and reduced extracellular polymeric substance secretion. Interestingly, microbially reduced sulfides (e.g., H2S and S2−) in SAD alleviated the cellular oxidative stress induced by Cu(II) and SMZ through ROS neutralization and CuS precipitation. Under stress, the relative abundance of ARGs increased significantly more in HD than in SAD compared to their respective control groups. Network analysis revealed more potential ARGs hosts in HD than in SAD. This study underscored the potential of the microbially reduced sulfides generated in the SAD process to effectively mitigate the spread of ARGs under selective pressure from heavy metals and antibiotics.
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