Excessive hydroxyapatite crystallization drives architectural heterogeneity and functional differentiation in hydroxyapatite-anammox granules.

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.