Side-stream fermentation drives partial denitrification/anammox for enhanced nitrogen removal and anaerobic ammonium oxidation bacteria enrichment in a flocs-based continuous-flow anaerobic/aerobic/anoxic system.

Anaerobic ammonium oxidation (Anammox) is a promising technology for mainstream nitrogen removal. However, its application is limited by unstable partial nitrification (PN) and inadequate retention of anaerobic ammonium oxidation bacteria (AnAOB) in flocs. To overcome these bottlenecks, this study established a flocs-based continuous-flow anaerobic/aerobic/anoxic (AOA) system coupled with a side-stream anaerobic fermenter. By introducing sludge fermentation liquid (SFL) and an anaerobic transfer line, the system was strategically shifted from unstable partial nitrification-denitrification to a robust partial denitrification/anammox (PD/A) pathway. During stable operation, the effluent concentration of total inorganic nitrogen (TIN) was 6.5 ± 1.8 mgN/L, with anammox in the anoxic zone contributing 31.9 ± 6.3% to TIN removal. The fermenter not only provided short-chain fatty acids to drive the PD process to generate NO2--N but also achieved a sludge reduction ratio of 22.7 ± 2.3%. Batch tests and functional gene quantification demonstrated that SFL effectively stimulated the PD activity in flocs. The anaerobic transfer line provided NH4+-N for AnAOB and intracellular carbon for endogenous PD. Network analysis suggested a potential synergy between AnAOB and heterotrophs (OLB8 and AKYH767), with the latter possibly supporting AnAOB through niche generation and substrate provision. Prolonged sludge retention time (> 90 days) further enriched diverse AnAOB subgroups in flocs, predominantly including Candidatus Brocadia (0.28%) and Candidatus Kuenenia (0.04%). These enriched AnAOB exhibited a specific growth rate of 0.03 d-1 and flexible metabolic adaptations. Overall, this study offers valuable strategies to validate the potential of anammox in the flocs-based AOA system.