Granule size influences anammox performance through functional differentiation and microbial specialization in a UASB system

Anammox has emerged as an energy-efficient and sustainable technology for nitrogen removal in wastewater treatment. However, the performance of anammox reactors is highly dependent on the structural and functional characteristics of granular sludge. The specific influence of granule size on microbial activity and community structure remains insufficiently understood. This study investigates the anammox performance, amino acid metabolism and microbial community dynamics in relation to granule size in a lab-scale upflow anaerobic sludge blanket (UASB) reactor. Granular sludge was classified into three size categories based on granule diameter: small (< 1 mm), medium (1–2 mm), and large (> 2 mm). Batch tests showed a positive correlation between granule size and specific anammox activity (SAA). Large granules achieved the highest SAA of 21.43 ± 2.83 mg NH 4 + -N/g VSS/day, attributed to enhanced metabolic efficiency and structural robustness. Extracellular polymeric substances (EPS) content in large granules was 5-fold higher than in smaller granules and dominated by polysaccharides, contributing to the maintenance granule integrity. Microbial analysis demonstrated size-dependent community specialization: large granules exhibited greater microbial diversity, enriched anammox bacteria Ca. Brocadia and Ca. Kenenia, and elevated expression of nitrogen metabolism genes. Amino acid profiling identified hydrophobic compounds as key mediators of microbial aggregation. Additionally, cross-feeding interactions were identified between amino acid-synthesizing and anammox bacteria which possibly contribute to enhanced community-level metabolic resilience. This study highlights granule size as a critical factor shaping microbial function and reactor efficiency, providing a foundation for targeted granule management in the development of high-performance anammox-based wastewater treatment systems. • Granule size critically affects anammox performance and microbial community structure. • Large granules (>2 mm) exhibited highest specific anammox activity and EPS levels. • Metabolic cross-feeding and EPS-mediated aggregation enhanced microbial specialization in large granules. • Size-based granule management is essential for efficient nitrogen removal in wastewater systems. • Future work should explore granule-size control strategies in full-scale or hybrid anammox systems.