The key mechanism of methanogenesis-driven anaerobic biologically induced phosphate precipitation from pickled mustard wastewater.

Biologically induced phosphate precipitation (BIPP) has emerged as a cost-effective phosphate removal pathway in hypersaline anaerobic bioprocesses, overcoming the drawbacks of high chemical costs and environmental limitations associated with conventional biological phosphorus removal. While microbial metabolism created favorable pH and interfacial conditions for phosphate precipitation, the key microbial metabolism and mechanisms driving BIPP remain unclear. Long-term operation demonstrated that active methanogenesis was crucial for maintaining bulk pH (∼7.4) and achieving efficient phosphorus removal (66.0 ± 2.6%). Inhibiting methanogenesis led to a pH drop (∼5.9) and deteriorated phosphate removal efficiency (17.0%-23.0%). The sludge phosphorus content (10.0 mg P g-1 DS) was 1.6-4.3 times higher than in inhibited systems, with hydroxyapatite identified as the predominant phosphorus mineral. Methanogenesis promoted phosphorus immobilization through multidimensional regulation of extracellular polymeric substances (EPS, the primary interface for BIPP). Proteomic and spectroscopic analyses revealed that methanogenesis up-regulated the expression of metal-ion-binding proteins, enhanced the secretion of low-molecular-weight compounds and conductive humic substances, and maintained a more stable secondary structure of EPS proteins (higher α-helix content), thereby providing abundant sites and stable interfaces for phosphate precipitation. In situ confocal laser scanning microscopy observations showed a tightly cross-linked three-dimensional network of proteins and α-polycaccharides in the EPS. This functionally and structurally tailored EPS ultimately supported the formation of highly mineralized sludge granules. This study elucidated how methanogenesis dominated phosphorus immobilization by elevating pH and functionally and structurally remodeling EPS, thus providing a solid theoretical foundation for optimizing BIPP.