Extracellular polymeric substances Govern granule size differentiation and microbial assembly in...

The Complete Ammonium and Nitrate Removal via Denitratation-Anammox over Nitrite (CANDAN) is a promising low-carbon strategy for sustainable nitrogen removal; however, the mechanistic linkage between extracellular polymeric substances (EPS), aggregation behavior, granule size differentiation, and microbial assembly remains unclear. Here, size-fractionated granules from a CANDAN reactor were systematically investigated to establish a multi-scale framework linking EPS structural properties to aggregation-driven granule organization and microbiome assembly. Medium-sized granules (0.5-1.0 mm) exhibited the highest aggregation capacity (74.5%) and fastest aggregation kinetics, indicating a cohesive and dynamically stable matrix. This aggregation advantage was closely associated with optimized functional performance, including high specific anammox activity (8.76 ± 1.36mg N g^-1 VSS h^-1), the highest nitrate reduction and nitrite production rates, and a high nitrite transformation ratio (90.1%), reflecting efficient pathway coupling in CANDAN systems. EPS analyses revealed that aggregation and stability were governed primarily by structural organization rather than bulk EPS content. Medium-sized granules were characterized by a lower protein-to-polysaccharide ratio, enriched hydrophobic functional groups, and β-sheet-dominated protein conformations, which collectively enhanced intercellular cohesion and stabilized the EPS matrix. This structurally optimized aggregation regime further shaped microbial assembly, promoting the enrichment and coordination of Candidatus Brocadia and Thauera. Overall, this study identifies aggregation capacity as a key mechanistic driver linking EPS structural properties to granule size differentiation and microbial assembly, highlighting granule size as a mesoscale regulator bridging physicochemical structure and ecological function.