Excessive hydroxyapatite crystallization drives architectural heterogeneity and functional differentiation in...

Hydroxyapatite (HAP) crystallization promotes anaerobic ammonium oxidation (anammox) granulation, but excessive crystallization may suppress anammox activity. In this study, we investigated the architectural and functional differentiation of HAP-anammox granules cultivated in an expanded granular sludge bed reactor using physicochemical characterization, metagenomic sequencing, and batch assays. During long-term operation, inorganic solids accumulated continuously, while the ratio of volatile suspended solids to suspended solids (VSS/SS) decreased from 0.78 to 0.40 and total nitrogen removal efficiency (TNRE) declined to below 50%. Reducing influent Ca and P concentrations increased VSS/SS to 0.63 and restored TNRE to above 87%, indicating that performance deterioration was associated with excessive hydroxyapatite crystallization. Size- and color-based fractionation further revealed granule heterogeneity. Red-brown granules retained a higher proportion of active biomass and were the main contributors to nitrogen removal. Notably, the 2.0-2.8 mm red-brown granules exhibited the highest anaerobic ammonium-oxidizing bacteria (AnAOB) abundance (44.77%) and specific anammox activity (SAA) (402.92 mg N⋅gVSS^-1⋅d^-1). White granules contained more inorganic solids, had higher Ca and P contents and stronger HAP-associated mineral signals but exhibited lower anammox activity. Excessive HAP crystallization reduced the active biomass fraction and drove functional differentiation between nitrogen removal and phosphorus retention. These findings provide a basis for Ca-P regulation, granule separation, and performance recovery in HAP-anammox systems.