Green synthesis of gold nanoparticles (AuNPs) using microorganisms has been generally studied aiming for high-yield production and morphologies appropriated for various applications, such as bioremediation, (bio)sensors, and (bio)catalysis. Numerous approaches showed the individual effect of factors influencing the synthesis of AuNPs with limited analysis of the governing factors enhancing the production and desired quality of the precipitates. This study proposes a fractional-factorial design to investigate the simultaneous influence of seven environmental factors (cell concentration, temperature, anoxic/oxic conditions, pH, gold concentration, electron donor type, and bacterial species) on the recovery yield and synthesis of targeted AuNPs. Various sizes and morphologies of the AuNPs were obtained by varying the environmental factors studied. The factors with significant effects (i.e., 0.2 mM Au and pH 5) were selected according to statistical analysis for optimal removal of 88.2 ± 3.5% of gold and with the production of valuable 50 nm AuNPs, which are known for their enhanced sensitivity. Implications of the cytochrome-C on the bacterial mechanisms and the provision of electron donors via an electrochemical system are further discussed. This study helps develop gold recovery and nanoparticle synthesis methods, focusing on the determining factor(s) for efficient, low-cost, green synthesis of valuable materials.

Green synthesis of metal nanoparticles (NPs) using biological systems provides a sustainable alternative to conventional chemical fabrication methods. This protocol presents reproducible experimental and analytical procedures for the synthesis of gold (Au) and silver (Ag) NPs using bacterial cultures, fungal filtrates, and plant leaf extracts as reducing and stabilizing agents. AgNPs were synthesized using extracts from Psidium guayaquilensis, Acanthophora spicifera, and Earliella sp., exhibiting characteristic ultraviolet-visible (UV-Vis) absorption bands between 405 and 425 nm that confirmed NP formation. A design-of-experiments approach was implemented to evaluate the influence of environmental factors, including oxygen conditions, pH, Au concentration, cell concentration, electron donor type, and temperature, on the synthesis of AuNPs by Shewanella oneidensis and Cupriavidus metallidurans. Under ideal conditions (0.2 mM Au and pH 5), S. oneidensis produced predominantly spherical AuNPs with an average size of 43.6 ± 11.0 nm and a characteristic absorption peak at 520 nm. NP formation and morphology were confirmed using UV-Vis spectroscopy and transmission electron microscopy. This workflow provides a reproducible platform for biogenic NP synthesis and supports applications in biosensing, bioremediation, and antimicrobial technologies.