Response mechanism of the DAMO-associated denitrification system to oxytetracycline stress

Antibiotics and denitrifying anaerobic methane oxidation (DAMO) processes frequently coexist in natural ecosystems and wastewater treatment systems. This study investigated the performance and microbial ecology of a denitrification system coupled with Nitrite-dependent anaerobic methane oxidation (N-DAMO) under oxytetracycline (OTC) stress. Specifically, 1 mg/L OTC enhanced nitrogen removal efficiency by 15% relative to the control, whereas 10 mg/L OTC exerted a significant inhibition of 58%. The Michaelis-Menten kinetic model predicted that the system could tolerate the maximum OTC concentration of 26.76 mg/L. Mechanistically, the secretion of protein-rich extracellular polymeric substances (EPS) served as a protective barrier against toxicity. The abundance of the DAMO bacterium Candidatus Methylomirabilis correlated negatively with OTC concentration. At 1 mg/L OTC, denitrification was enhanced through the enrichment of Thauera. However, 10 mg/L OTC damaged EPS structure and suppressed microbial activity, and led to a decrease in the abundance of related functional bacteria and an increase in the abundance of antibiotic resistant bacteria such as Hyphomicrobium and Thermomonas. Metagenomic analysis revealed that denitrification genes (e.g., norB, norC) were upregulated with 1 mg/L OTC, whereas high-concentration OTC induced pronounced enrichment of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), with frequently co-localization within the same hosts. This suggests an increased potential for horizontal gene transfer (HGT) occurred within the DAMO community, which may contribute to the dissemination of ARGs. These findings provide new insights into the adaptive mechanisms of N-DAMO systems under antibiotic stress and highlight their potential for nitrogen removal in contaminated environments.