Co-selection of Altered Nitrogen Metabolism and Multidrug Resistance in Wastewater Microbiomes under Long-Term Fluoride Stress

Industrial fluoride (F^-) wastewater threatens the stability of biological treatment systems. However, the adaptive evolution of nitrogen-transforming microorganisms and the spread of antibiotic resistance under long-term F^- stress remain unclear. This study systematically investigated the nitrogen removal performance, microbial community structure, and resistome evolution in a sequencing batch bioreactor under prolonged (120-day) stress from 20 mg/L F^-. The results indicated that long-term F- exposure severely inhibited nitrification, decreasing the ammonia removal efficiency from 99.6% to 74.7% and the specific ammonia oxidation rate (SAOR) by 23.8%. In contrast, a significant enrichment of denitrifying bacteria (e.g., Thauera abundance increased by 274%) enhanced denitrification, boosted the specific nitrate reduction rate (SNRR) by 48.7%, and maintained a stable total inorganic nitrogen (TIN) removal efficiency (64.5%). To counteract F^- toxicity, the microbial community exhibited multi-level adaptations, including enhanced secretion of extracellular polymeric substances (EPS), upregulation of antioxidant and energy metabolism genes, and increased F- efflux capacity. Crucially, F^- stress co-selected for broad-spectrum antibiotic resistance genes (ARGs) and heavy metal resistance genes (MRGs), with total abundances increasing by 19.7% and 31.2%, respectively. Network analysis confirmed that denitrifying bacteria, which gained a competitive advantage under F^- stress, were the primary hosts of these resistance genes. This study reveals the evolutionary mechanisms of nitrogen-converting microbes in the treatment of fluoride-containing wastewater and warns of the potential risks of resistance gene proliferation induced by F^- pollution.