Biosynthesis of silver nanoparticles by thermophilic spore-forming bacilli: screening for high-performance strains and characterization of silver nanoparticles from <i>Anoxybacillus</i> sp. D401a

In this study, thermophilic, spore-forming bacteria from the families <i>Bacillaceae</i> and <i>Anoxybacillaceae</i> were analyzed for their ability to produce silver nanoparticles (AgNPs). <i>Anoxybacillus</i> sp. D401a was selected due to its high AgNP synthesis potential. The culture conditions (NaCl-free nutrient broth, 60 °C, pH 8.5, 48 hr) and synthesis parameters (1.5 mM AgNO₃, 48 hr at 60 °C, pH 8.5) were optimized, resulting in a sevenfold increase in AgNP yield. Scale-up production yielded 139.4 mg of dry AgNPs with strong antimicrobial activity that exhibited significant minimum inhibitory concentration (MIC) values against Gram-positive, Gram-negative and fungal pathogens, outperforming commercial AgNPs. Sub-MIC concentrations of the biosynthesized AgNPs also inhibited biofilm formation and quorum sensing in <i>Chromobacterium violaceum</i> ATCC 12472. Characterization by ultraviolet–visible spectroscopy revealed a surface plasmon resonance peak at 400–450 nm, and Fourier-transform infrared spectroscopy indicated the presence of organic residues that stabilized the particles. Transmission electron microscopy analysis showed predominantly spherical AgNPs (24–57 nm). MTT assays showed a dose-dependent cytotoxicity against human keratinocytes (HaCaT), fibroblasts (HDF, L929), and cancer cells (HT-29, MCF-7), indicating a therapeutic window compared to commercial AgNPs. These results emphasize the potential of <i>Anoxybacillus</i>-derived AgNPs as a sustainable alternative for biomedical applications.