Calcium synergy in the enhancing single-stage partial nitritation-anammox process for high-strength ammonium wastewater treatment.

The single-stage partial nitritation-anammox (PNA) process is widely regarded as a sustainable strategy for nitrogen removal from high-strength ammonium wastewater, yet its stability remains challenged under elevated ammonium conditions. In this study, a lab-scale airlift inner-circulation reactor with a working volume of 1.9 L was continuously operated for 247 days to evaluate the synergistic effects of calcium ion addition on process performance, microbial structure, and gene expression. The influent ammonium concentration was progressively increased from 850 mg/L to 1800 mg/L, while calcium ion concentration, added as calcium chloride dihydrate, was raised from 0 mg/L to 100 mg/L. Moderate calcium addition between 40 mg/L and 100 mg/L promoted significant sludge granulation, with mixed liquor volatile suspended solids nearly tripling and sludge volume index decreasing markedly, indicating enhanced biomass retention and sedimentation. Accompanying this, the secretion of extracellular polymeric substances, particularly tightly bound protein and polysaccharides, was substantially stimulated, resulting in a more compact and cohesive matrix. High-throughput 16S rRNA gene sequencing revealed that Ca addition selectively enriched key autotrophic taxa, such as Candidatus Brocadia and Nitrosomonas, while metagenomic analysis showed upregulation of functional genes. The increased abundance of these genes suggests an enrichment of functional microorganisms involved in autotrophic nitrogen metabolism, which may contribute to enhanced nitrogen transformation capacity, thereby supporting stable total inorganic nitrogen removal performance above 85 % despite increasing ammonium stress. These findings deepen the mechanistic understanding of calcium's synergistic role in reinforcing microbial aggregation, regulating EPS biosynthesis, and enhancing nitrogen-converting functionality, providing theoretical guidance for the robust application of PNA systems in treating ammonium-rich industrial wastewaters.