Bacterial interfaces and surface chemistry shape hydroxyapatite mineral architecture during microbially induced calcium phosphate precipitation

Calcium phosphate biomineralization plays a crucial role in the formation of geological deposits, biological materials, and environmental remediation. While microorganisms are known to influence calcium phosphate precipitation, the relative contributions of bacterial cell surfaces and extracellular polymeric substances (EPS) remain poorly defined. This study presents the first microscale investigation of the distinct roles of Gram-positive and Gram-negative bacterial cells and EPS in regulating hydroxyapatite (HAp) mineralization, employing abiotic, EPS-mediated, and bacterial precipitation systems. Zeta potential measurements and Fourier Transform Infrared Spectroscopy (FTIR) revealed that both bacterial cells and isolated EPS provide negatively charged, chemically distinct interfaces capable of interacting with dissolved Ca 2+ ions. Solution chemistry demonstrated greater phosphate and calcium ion depletion in biotic systems, consistent with enhanced crystal precipitation compared to abiotic controls. Field emission scanning electron microscopy (FESEM) and Brunauer–Emmett–Teller (BET) analyses showed that biological interfaces produced distinct crystal morphologies and surface characteristics. X-ray diffraction (XRD) revealed significant differences in phase composition: abiotic precipitation produced mixed calcium phosphate phases, whereas EPS-mediated precipitation promoted nucleation but remained dominated by precursor phases. In contrast, whole-cell systems favored HAp formation, with Sporosarcina pasteurii producing phase-pure crystalline HAp and Pseudomonas fluorescens producing HAp-dominant precipitates. Acid dissolution experiments further confirmed the increased stability of biogenic precipitates. These findings demonstrate that the spatial organization of bacterial surface functional groups, rather than surface charge alone, governs hydroxyapatite phase evolution, crystal growth, and mineral stability. While localized metabolic activity may contribute to microenvironmental conditions, the results highlight the importance of bacterial cell surfaces and EPS as key determinants of hydroxyapatite biomineralization, providing a mechanistic basis for the design of biologically mediated mineral materials.