Establishing endogenous partial denitrification coupled with anammox as a stable nitrogen removal pathway in an AOA system: Co-enrichment and synergistic interactions between GAOs and AnAOB.

The mainstream application of anammox in municipal wastewater treatment is constrained by unstable nitrite supply and poor retention of anammox bacteria (AnAOB). In this study, a continuous-flow anaerobic/oxic/anoxic (AOA) system treating real low-C/N municipal wastewater was operated for 265 d to establish endogenous partial denitrification coupled with anammox (EnPDA). Sidestream phosphorus release was applied to suppress phosphorus-accumulating organism (PAO) metabolism and enhance glycogen-accumulating organisms (GAOs), while mature anammox biofilms and blank carriers were jointly introduced into the anoxic zone to promote AnAOB retention and in situ enrichment. Anaerobic carbon allocation gradually shifted from the coexistence of phosphate-associated metabolism (PAM) and glycogen-associated metabolism (GAM) to GAM dominance, strengthening endogenous denitrification in the anoxic zone. Accordingly, the nitrogen removal efficiency increased from 60.41 ± 3.87% in Phase I to 82.42 ± 1.70% in Phase IV, while the effluent total nitrogen concentration decreased from 25.12 ± 3.07 to 11.28 ± 1.32 mg/L. The anammox contribution increased from 4.14 ± 1.99% to 29.55 ± 2.85%. Meanwhile, Ca. Competibacter increased from 6.70% to 11.68% in flocculent sludge, whereas Ca. Brocadia increased from 0.27% to 2.08% on the initially blank biofilm carriers. Preferential transcription of nitrate reduction genes over nitrite reduction genes further supported nitrite retention for AnAOB utilization. These results indicate that coordinating GAO-mediated nitrite supply with biofilm-based AnAOB retention enabled the stable establishment of EnPDA, providing a carbon-efficient strategy for nitrogen removal from low-C/N municipal wastewater.