Biosynthesis of Silver Nanoparticles Using Escherichia coli: Materials, Characterization, and Biosafety Guidelines.

Abstract

Silver nanoparticles (AgNPs) have applications in antimicrobial materials, sensing, catalysis, and biomedicine, but conventional synthesis methods may require hazardous reducing agents, high energy inputs, or specialized equipment. This article describes the extracellular biosynthesis of AgNPs using cell-free supernatant from Escherichia coli K-12 DH5α. Basic Protocol 1 covers cultivation of the bacterial biomass, preparation of cell-free supernatant, reduction of silver ions, recovery and washing of the nanoparticle fraction, storage, and waste management. Basic Protocol 2 describes characterization using ultraviolet-visible spectroscopy, scanning electron microscopy, and X-ray diffraction. The accompanying Commentary addresses the effects of silver-ion concentration, pH, medium composition, and sample purification, with particular attention to AgCl formation and contamination by residual biological material. Troubleshooting and interpretation guidance help users evaluate nanoparticle formation, aggregation, morphology, phase composition, and batch-to-batch reproducibility. The protocols provide an accessible framework for microbial AgNP synthesis while emphasizing appropriate interpretation criteria, biosafety practices, and management of silver-containing waste. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Biosynthesis and purification of AgNPs using cell-free E. coli supernatant Basic Protocol 2: Characterization of E. coli-derived AgNPs.

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