Rapid formation of partial denitrification granular sludge under an alkaline-assisted stepwise SBR strategy: XDLVO insights into interfacial adhesion and calcium-associated reinforcement

Partial denitrification (PDN) granules can retain nitrite producing biomass for downstream Anammox, but their rapid formation and underlying biological and physicochemical mechanisms remain unclear. In this study, PDN granular sludge was cultivated in an SBR operated for 90 d under an alkaline-assisted stepwise strategy. Reactor performance was evaluated together with granule development, extracellular polymeric substances (EPS), sludge surface properties, microbial community composition, XDLVO interactions, and calcium-associated structural characteristics. Visible granules formed within 36 d. After external influent pH adjustment was discontinued, the mature granules maintained nitrite accumulation and nitrate reduction rates of 2.11 ± 0.22 and 2.47 ± 0.41 kgN·m⁻³·d⁻¹, respectively, with approximately 85% of the reduced nitrate-N retained as nitrite-N. Granule maturation was accompanied by enrichment of Thauera from 1.98% to 69.79%, development of a protein rich tightly bound EPS matrix, an increase in PN/PS from 0.11 to 4.62, greater surface hydrophobicity, and a markedly less negative zeta potential. XDLVO analysis indicated a more adhesion-favorable interfacial state in the mature granules, while SEM-EDS and supplementary XRD and EDTA results were consistent with calcium-associated structural reinforcement. Overall, this study links rapid PDN granulation with functional microbial enrichment, EPS-associated interfacial evolution, and calcium-associated structural reinforcement, providing an integrated basis for developing settleable PDN biomass as a potential nitrite source for Anammox applications.