Size-associated biomineralization and mineral speciation differences in anammox granules under low-phosphate...

Biomineralization influences the structural stability, biomass retention, and mass transfer of anaerobic ammonium oxidation (anammox) granules, yet size-associated mineral differences under low-phosphate conditions remain insufficiently resolved. Mature granules from a long-term upflow anaerobic sludge blanket reactor operated at 2.7 mg P L^-1 were separated into five size fractions (<0.6, 0.6-1.18, 1.18-2.36, 2.36-4.0, and >4.0 mm) and characterized using integrated mineralogical, spectroscopic, and microbial analyses. Ash contents ranged from 65.29 to 74.43%, while specific anammox activity increased with granule size. Short-term assays showed apparent decreases in dissolved Ca²⁺ and phosphate, with net Ca/P removal ratios above 4, indicating that stoichiometric hydroxyapatite (HAP) precipitation alone could not account for the observed aqueous changes. Calcite-rich particles were recovered from fine or peripheral material, whereas internally recovered particles contained HAP with features consistent with carbonate substitution. Tightly bound extracellular polymeric substances (EPS) were more abundant and protein-rich than loosely bound EPS and exhibited greater Ca²⁺-binding capacity. Larger granules showed higher relative abundances of several heterotrophic taxa, while the dominant anammox genus remained comparatively stable, suggesting size-associated differentiation of microbial partners potentially involved in organics and nitrogen turnover. These findings connect size-associated mineral differences with EPS binding properties, microbial community composition, and anammox activity, informing the evaluation of granule management under low-phosphate conditions.