Water hardness-mediated EBPR-BIPP synergistic phosphorus removal and microbial community response

Water hardness, often overlooked in enhanced biological phosphorus removal (EBPR), plays a decisive role in biologically induced phosphate precipitation (BIPP). While hardness effects on chemical phosphorus removal are documented, hardness-mediated BIPP impacts on EBPR performance remain poorly understood. This study operated four sequencing batch reactors at 0, 150, 300, and 600 mg L^-1 hardness (as CaCO₃). Results demonstrated that hardness simultaneously promoted biological uptake and chemical precipitation, maintaining stable high performance (88% TP removal, SVI 89 mL g^-1) even at 600 mg L^-1, whereas the control failed due to sludge disintegration. Sequential extraction and speciation analysis (³¹P MAS-NMR, XRD, XPS) revealed phosphorus transformation from soluble to stable calcium- and magnesium-bound forms, predominantly hydroxyapatite (HAP) and struvite (MAP), with the MAP proportion increasing at higher hardness. Under high hardness, PPK and PPX activities increased by 109.1% and 80.7%, respectively, and the relative abundance ratio of PAO/DPAO-associated taxa to GAO/DGAO-associated taxa increased from 0.24 to 2.5. A 190-day operation confirmed the synergistic interactions between BIPP and EBPR. Microbial analysis revealed that the relative abundance of GAO/DGAO-associated taxa decreased from 5.0% to 2.0%, while the phoR gene showed its highest relative abundance (0.45%) under high-hardness conditions, suggesting a potential association between extracellular chemical fixation and intracellular storage. This study elucidates the hardness-driven synergistic mechanisms, providing a theoretical basis for EBPR optimization in high-hardness regions.