Pyrite-mediated antibiotic transformation and antibiotic resistance gene metabolism in recirculation stacking hybrid constructed wetlands.

This study elucidates antibiotic removal mechanisms and antibiotic resistance genes (ARG) attenuation pathways in recirculation stacking hybrid constructed wetlands (RSHCWs) amended with pyrite as a functional substrate. Three pilot-scale RSHCWs with varying pyrite contents were operated to treat sewage spiked with sulfapyridine (SPD), ofloxacin (OFX), and oxytetracycline (OTC). All systems achieved high removal efficiencies for antibiotics (>92% for SPD, >85% for OFX, and >95% for OTC) and ARGs (>90%). Pyrite addition significantly enhanced the adsorption of antibiotic and ARG. Mass balance analysis revealed that degradation/transformation was the key process for antibiotic removal. Pyrite enhanced substrate adsorption, promoted the formation of relatively stable complexes, and facilitated abiotic oxidative transformation and formation of smaller molecular weight transformation products. Major transformation pathways included hydroxylation, bond cleavage, decarboxylation, demethylation, and substitution reactions. Integrated microbial network analyses revealed that ARG attenuation was primarily driven by pyrite mediated adsorption, oxidative damage to antibiotic resistant bacteria, and suppression of resistant bacteria and inhibiting horizontal gene transfer via intensified interspecies microbial competition. Pyrite further altered bacterial diversity, enriching stress-resistant taxa and weakening the association between ARGs and potential host bacteria. Overall, this study underscores the dual function of pyrite in enhancing antibiotic degradation and limiting ARG dissemination.