Shift of nitrogen metabolism drives the evolution of multidrug resistance in wastewater microbiomes under long-term fluoride stress.

The discharge of fluoride-containing industrial wastewater poses novel challenges to biological wastewater treatment systems. In particular, the long-term impacts of fluoride (F-) stress on key metabolic fluxes (e.g., nitrogen transformation) and the dissemination risk of biological pollutants (e.g., antibiotic resistance genes) in biological treatment systems 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%. Conversely, the significant enrichment of denitrifying bacteria under F- stress enhanced the denitrification process, elevating the specific nitrate reduction rate by 48.4% and thereby maintaining a stable total inorganic nitrogen removal efficiency (∼ 64.5%). To counteract F- toxicity, the microbial community exhibited multi-level adaptations, including enhanced secretion of extracellular polymeric substances, upregulation of antioxidant and energy metabolism genes, and increased F- efflux capacity. Crucially, F- stress co-selected for the proliferation of broad-spectrum antibiotic resistance genes and heavy metal resistance genes in the activated sludge system, with total abundances significantly increasing by 20.3% and 56.9%, respectively, compared to the seed sludge. Network analysis confirmed that denitrifying bacteria, which had 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 during the treatment of fluoride-containing wastewater and highlights the potential risks of resistance gene proliferation induced by F- pollution.