Heterotrophic integration enhances structural resilience of anammox granules through extracellular polymeric...

Structural resilience of granular sludge is essential for maintaining stable partial denitrification-anammox (PDA) performance under long-term operational disturbances, yet the mechanisms governing resilience in mixed-trophic granules remain poorly understood. Here, heterotroph-integrated PDA granules were systematically compared with independently cultivated partial denitrification (PD) and anammox (AMX) granules to elucidate how heterotrophic integration influences granule stability. The integrated system established stable metabolic coupling, achieving a nitrate-to-nitrite transformation ratio (NTR) of 91.6% with concurrent ammonium removal. More importantly, after 70 operational cycles, the mean diameter of PDA granules decreased by 17.4%, compared with 29.2% for AMX granules, while the 0.5-1.0 mm fraction remained dominant. PDA sludge also displayed the highest aggregation rate constant (8.45 h^-1), indicating rapid structural recovery. Multiscale analyses suggested that these improvements were associated with extracellular polymeric substance (EPS) interface remodeling rather than increased EPS production alone. Heterotrophic integration promoted a tightly bound and compositionally balanced EPS matrix, with the T-EPS fraction reaching 86.1%, together with diversified protein secondary structures and an O/N-rich surface. These features potentially strengthened hydrogen bonding, hydrophobic interactions, polymer bridging, and other non-electrostatic cohesive forces. Concurrently, mixed-trophic community assembly increased microbial richness and established complementary interactions between heterotrophic denitrifiers and anammox bacteria. Collectively, these results support a proposed framework linking microbial community assembly, EPS interface remodeling, and structural resilience. This work identifies extracellular interface regulation as a potential strategy for improving biomass retention and operational robustness in low-carbon anammox-based wastewater treatment.