Mechanistic insights into the enhancement of azithromycin biodegradation by extracellular polymeric substances: Roles of metabolic activity, electron transfer, and reactive oxygen species.

White-rot fungi (WRF) have shown potential as an effective alternative for antibiotic removal in wastewater treatment. However, their slow degradation rates remain a major challenge. In this study, we demonstrate that extracellular polymeric substances (EPS) from Brucella intermedia ZL-06 significantly enhance the degradation of azithromycin (AZI) by the WRF Trametes versicolor, reducing the degradation time from 9 to 6 days and achieving > 99.9 % elimination. Mass-balance calculations revealed a 35.33 % increase in the specific biodegradation rate per unit biomass. Mechanistic analysis showed that EPS-primed T. versicolor rapidly generated extracellular reactive oxygen species (ROS). The essential role of ROS in AZI degradation was confirmed by the significant inhibition of degradation efficiency in radical-scavenging assays. Additionally, EPS treatment enhanced electron flux, as evidenced by a 31.12 % increase in cytochrome C content, upregulated oxidoreductase activities, and a reduction in the extracellular electron-transfer resistance between T. versicolor and AZI, from 139.01 Ω to 47.86 Ω, as measured by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). This study proposes a novel mechanism for synergistic degradation, involving both ROS generation and chem-/biodegradation processes. These findings provide a theoretical foundation for developing ROS-enzyme synergistic platforms in antibiotic bioremediation.