Calcium chloride modulates the biosynthesis capability and properties of silver nanoparticles synthesized from in vitro-grown lemon Balm.

Plant-mediated synthesis is a well-established green route for silver nanoparticles (AgNPs) production, but a significant challenge remains in controlling the properties of the nanoparticles. This study aims to explore whether altering the nutrient composition of the plant growth medium can influence nanoparticle properties. Lemon balm plants were grown in vitro under three concentrations of CaCl₂ (220, 440 (standard MS condition), and 880 mg/L) in MS medium. Aqueous extracts from plants were used to synthesize AgNPs, and the nanoparticles were characterized by UV-Vis spectroscopy, XRD, FESEM, DLS, FTIR, and antibacterial disc diffusion assays. The results showed that the 220 mg/L CaCl₂ treatment produced the highest yield of AgNPs, with faster reduction kinetics, smaller particle size (12.48 nm), narrower size distribution (PdI = 0.245), and diverse morphologies (spherical, triangular, cubic). The 440 mg/L treatment also produced AgNPs but with larger size (34.64 nm) and predominantly spherical morphology. In contrast, the 880 mg/L treatment largely failed to produce AgNPs; instead, XRD revealed dominant AgCl peaks. FTIR analysis confirmed the participation of plant functional groups in the biosynthesis and capping of the AgNPs. Antibacterial assay showed bactericidal activity of the produced AgNPs against Gram-positive and Gram-negative bacteria. Results indicate, excess CaCl₂ is detrimental, causing immediate precipitation of AgCl, outcompeting the bio-reduction process. The precise mechanisms underlying the differences between 220 and 440 mg/L CaCl₂ treatments are attributed to gene expression changes, secondary metabolites content, residual Cl⁻ content, etc. This study provides a foundation for further investigations into nutrient-mediated control of green nanoparticle synthesis.