Introduction:
Green synthesis has emerged as an environmentally sustainable alternative to conventional chemical approaches for nanoparticle production. Fungal-mediated biosynthesis of silver nanoparticles (AgNPs) is particularly promising due to its efficiency in metal ion reduction and natural secretion of stabilizing biomolecules. This study examined whether stimulating phenol oxidase activity in culture media enhances the rate and yield of AgNP formation using the filamentous fungus Aspergillus niger.

Methods:
Six variants of Czapek–Dox medium were prepared using sucrose, starch, or glucose as carbon sources. Phenol oxidase stimulation was introduced into variants 2, 4, and 6 through copper(II) sulfate, oak sawdust, and hydrogen peroxide. A. niger was cultivated for 72 h in each medium, after which the biomass was transferred to ultrapure water for extracellular metabolite release. Following another 72 h, the biomass was removed, and a 1 mM silver nitrate solution was added to the filtrates. AgNP formation was monitored by UV–Vis spectroscopy over 120 h. Functional groups were identified by FTIR, and crystalline structure was confirmed using XRD.

Results:
A characteristic AgNP surface plasmon resonance peak at ~420 nm appeared in all variants. Media containing phenol oxidase stimulants showed notably higher absorbance and faster nanoparticle formation kinetics. FTIR spectra confirmed the presence of protein, enzyme, and carbohydrate functional groups involved in nanoparticle capping, while XRD verified the crystalline nature of the AgNPs across all samples.

Conclusions:
Phenol oxidase stimulation significantly enhanced the rate of AgNP biosynthesis without altering the nanoparticle structure or capping composition. Medium optimization, therefore, represents a critical step toward scaling fungal-based AgNP production for industrial applications.