Microaerophilic strategy with high sludge concentration enhances removal of nitrogen in a two-stage AO bioreactor treating tetracycline-rich wastewater

Tetracycline (TC) often persists in high-ammonia wastewater and interferes with biological nitrogen removal. This study evaluated a microaerophilic two-stage AO bioreactor (DO = 0.8-1.2 mg/L and MLSS = 10,000 mg/L) under increasing TC concentrations (0-5.0 mg/L). At low TC levels (<1.0 mg/L), the system maintained stable COD and TN removal (>90% and >85%), supported by dense sludge flocs, enhanced EPS secretion, and simultaneous nitrification and denitrification (SND). High TC stress strongly inhibited nitrite-oxidizing bacteria (NOB), caused NO2--N accumulation, and altered nitrogen pathways toward partial nitrification and denitrification (PND). Reactor performance recovered after TC withdrawal, and acclimated sludge exhibited improved tolerance during re-exposure. Microbial analyses showed TC-driven selection, including suppression of NOB (e.g., Nitrospira), enrichment of resistant denitrifying bacteria (DNB) (e.g., Rhizobacter, Comamonas, Ottowia), and TC-degrading genus (e.g., Thauera, Delftia, Xanthomonadaceae). Key enzymes involved in nitrification, denitrification, and TC degradation were identified, illustrating the mechanistic coupling of antibiotic degradation and nitrogen metabolism. These results elucidate how TC reshapes microbial structure and nitrogen pathways, providing guidance for robust treatment of antibiotic-contaminated high-ammonia wastewater.