Mechanisms of Acetate Concentration and Exposure Time on the Performance and Response of Functional Microbial in CANON Reactor
Anaerobic ammonium oxidation (anammox) biofilms are widely applied in high-ammonia wastewater treatment, yet their resilience to small-molecular acids remains underexplored, which is limiting engineering optimization. This study investigated acetate concentration and exposure duration effects on anammox biofilm systems through four parallel CANON reactors operated under acetate gradients (0, 100, 300, 500 mg COD/L). Integrated macro-micro analyses via 16S rDNA sequencing and Tax4Fun2 functional predictions, elucidating the impacts of acetate stress on system functionality. The results showed that 100 mg COD/L acetate promoted short-term synergistic nitrogen removal by moderately upregulating the denitrifier narG/nirS genes and acetyl-CoA synthetase activity (1.54-fold). This improved TNRE to 90.61% within 15 days without compromising anammox activity. Conversely, prolonged exposure to elevated acetate concentrations resulted in a Thauera-dominated heterotrophic overgrowth (35.9% abundance) characterized by increased amino acid metabolism and fatty acid degradation. This metabolic shift resulted in increased carbon competition and inhibition of free ammonia (113.4 mg/L), which suppressed Candidatus_Kuenenia (3.85%) and Nitrosomonas (3.35%) abundances, ultimately leading to system collapse. These results quantify the dual role of acetate while providing theoretical insights that provide actionable guidelines for industrial CANON systems to balance denitrification with biofilm stability and strengthen resilience to small molecule acid shocks in engineered anammox applications.