Biogenic Synthesis and Characterization of Nano-Hydroxyapatite Using Bacillus Subtilis, Bacillus Licheniformis, and Pseudomonas Fluorescens

This study investigates the biogenic synthesis of nano-hydroxyapatite (nHAP) using Bacillus licheniformis,Bacillus subtilis, and Pseudomonas fluorescens, followed by comprehensive characterization to assess their physicochemicaland structural properties. UV–Vis spectroscopy confirmed nanoparticle formation with absorption peaks at 238 and 280 nm(B. licheniformis), 241 and 278 nm (B. subtilis), and 245 and 281 nm (P. fluorescens), suggesting nucleation and presence oforganic moieties. Spectral shoulders in B. subtilis and P. fluorescens indicated biomolecular interaction, unlike the sharperprofile of B. licheniformis. FTIR analysis showed characteristic phosphate (PO₄³⁻) bands (~1040 and 560–565 cm⁻¹),hydroxyl groups (~3570 cm⁻¹), and minor carbonate (~1450 cm⁻¹) in all samples. Strong amide I (~1640 cm⁻¹) and II (~1540cm⁻¹) bands in B. subtilis and P. fluorescens pointed to proteinaceous capping and biogenic origin, whereas B. licheniformishad sharper phosphate peaks and minimal organic signatures, indicating higher crystallinity. EDX confirmed elementalcomposition with Ca/P ratios near the ideal 1.67. B. licheniformis (1.61) produced the most stoichiometric and pure nHAP,followed by B. subtilis (1.59) and P. fluorescens (1.58), aligning with FTIR observations. DLS and zeta potential resultsshowed B. licheniformis synthesized the smallest (32.4 ± 1.2 nm), most monodisp erse (PDI 0.186), and stable (−34.2 ± 1.7mV) particles. In contrast, P. fluorescens-derived nHAP was larger (39.3 ± 1.9 nm), more polydisperse (PDI 0.264), and lessstable (−26.5 ± 2.2 mV).In conclusion, all three strains sy nthesized nHAP, but only B. subtilis and P. fluorescens exhibitedstrong biogenic characteristics. B. licheniformis yielded highly crystalline, near-stoichiometric nHAP, resembling chemicallysynthesized material, making it ideal for high-purity applications such as agriculture.