Decoupling the effects of organic acid and EPS in phosphorus activation by phosphate solubilizing bacteria: From screening to morphological transformation analysis

Phosphate-solubilizing bacteria (PSB) are crucial functional microorganisms that enhance phosphorus bioavailability and regulate phosphorus cycling. However, some knowledge gaps remain regarding: the relative effectiveness of PSB-secreted organic acids (individual vs. combined) in dissolving calcium phosphate, and the underlying mechanisms of phosphorus release and mineral transformation mediated by PSB metabolites (organic acids and EPS). This study investigated the dissolution effects of mono-, di-, and tri-carboxylic acids and their mixtures secreted by high-efficiency PSB on calcium phosphate through strain screening and laboratory incubation experiments. Comprehensive characterization techniques including SEM-EDS, FTIR, XRD, and XPS were employed to analyze morphological and mineralogical changes during dissolution. The results showed that from 22 isolated PSBs, four top performers (Burkholderia and Acinetobacter spp.) achieved 538.34 mg/L phosphorus solubilization via oxalic, malic, and gluconic acids secretion. Abiotic experiments showed all individual organic acids increased phosphorus release versus control, with formic acid being most effective. Mixed-acid systems showed intermediate performance between lactic and formic acids. A significantly positive correlation (r = 0.710, p = 0.014) was observed between the mass fraction ratio of carboxyl groups to organic acids and supernatant phosphorus levels, establishing this ratio as a practical predictive indicator for evaluating carboxylic acids' calcium phosphate dissolution capacity. XRD analysis revealed transformation of precipitates into calcium phosphate-hydroxyapatite (Ca-P-HAP) mixtures after 7-day incubation, regardless of PSB presence. Organic acids promote dissolution by acidification and chelation, while EPS hinders this process by adsorbing phosphorus, inducing precipitation, and encapsulating crystals. PSB-mediated dissolution-recrystallization processes, facilitated by organic acid secretion and EPS, generated orthophosphate minerals with distinct particle sizes and morphologies (granular, flaky, and hexagonal prism structures). These findings enhance our understanding of PSB-mediated morphological evolution and mineral transformation in calcium orthophosphates, offering a theoretical foundation for optimizing phosphorus management in agricultural systems.