Green Synthesis of Gold and Silver Nanoparticles Using Plant Extracts, Fungal Filtrates, and Bacterial Cultures

Summary

This protocol describes reproducible green synthesis methods for fabricating gold and silver nanoparticles using plant extracts, fungal filtrates, and bacterial cultures, followed by nanoparticle confirmation through ultraviolet–visible spectroscopy and transmission electron microscopy.

Abstract

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.

Introduction

Metallic nanoparticles (NPs) possess unique physicochemical properties associated with their high surface area, including distinctive electronic, magnetic, and catalytic behaviors, as well as high reactivity. These characteristics have promoted their application across multiple fields, including medicine for disease diagnosis and drug delivery^1,2,3,4,5,6, cosmetics for sunscreen formulations⁷, electronics for transistors and solar cells

Protocol

The research was performed in accordance with the institutional guidelines of the Escuela Superior Politécnica del Litoral (ESPOL, Ecuador). The plant, algal, and fungal biomass used in this study were collected from accessible environments and did not involve protected species or restricted areas. According to institutional and national regulations, no specific permits were required for the collection of freely available biomass under these conditions. Refer to Figure 1 for the integrated experimental workflow used for green synthesis of AgNPs and AuNPs using plant, algal, fungal, and bacterial biological systems.

Results

AgNPs were successfully synthesized through green and biosynthetic routes using different biological reducing agents, and ideal synthesis conditions were identified for each method. In the green synthesis mediated by P. guayaquilensis leaf extract, the optimal conditions consisted of an AgNO₃ concentration of 5 mM, an extract-to-metal solution ratio of 25% (v/v), a reaction temperature of 80°C, and an agitation time of 60 min followed by a resting period of 24 h. Under these conditions, UV–Vis spectro.......

Discussion

When comparing the evaluated synthesis routes in terms of feasibility, NP size control, operational cost, reproducibility, and overall process efficiency, plant-mediated synthesis using P. guayaquilensis leaf extract demonstrated the most favorable balance among the AgNP synthesis methods investigated. This protocol provided improved control over NP size through systematic optimization and direct microscopic characterization while operating under mild conditions without prolonged biological cultivation. In addit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absolute ethanol	Merck	E7023	TEM dehydration series
Aluminum crimp seals	Dwk Wheaton	224178-01	For serum bottles
Amber glass bottles	Duran	2180154	Light-protected storage
ATR-FTIR spectrometer	Thermo Scientific	Nicolet iS10	Spectral range: 300–3600 cm-1
Autoclave	Yamato	SM300	Sterilization at 121 °C
Butyl rubber stoppers	Dwk Kimble	73827-21	Gas-tight sealing
Centrifuge	BIOBASE	BKC-TH20RL	Bench-top centrifuge
Chloroauric acid trihydrate (HAuCl4·3H2O)	Sigma-Aldrich	27988-77-8	Gold precursor
Diamond knife	Aurion	Dyatom 45	Ultrathin TEM sectioning
Distilled or deionized water	Methrom	N/A	Solution preparation
Epoxy resin (EMBED 812/Epon)	Aurion	EMBED 812	TEM embedding medium
Filter paper, qualitative Grade 1	Merck Whatman	WHA1001110	Pore size: 11 µm; diameter: 110 mm
Fourier transform infrared spectrometer (FTIR)	Thermo Scientific	Nicolet iS10	Spectral range: 300–3600 cm-1
Gas manifold system	Custom-built	N/A	For purge cycles
Gas-tight syringe	Hamilton	1001 RN	1 mL
Glutaraldehyde	VWR	219859510	5% Glutaraldehyde, 4% paraformaldehyde in 0.1 M Sodium Cacodylate Buffer
Glycerol	Sigma-Aldrich	G5516	20% stock preservation
Handheld crimper	Biomed	USYGQ-20Z	Crimp sealing of serum bottles
Hand mill/grinder	Corona	L10000	Plant biomass grinding
Hydrochloric acid (HCl)	Merck	320331	pH adjustment
Hydrogen (H2) gas	Linde	Industrial grade	Electron donor gas
ICP-OES system	Varian	Vista-MPX	Metal quantification
Incubator	Memmert	SM 400	Temperature-controlled incubation
Laboratory oven	Barnstead	M100	Drying nanoparticles
Lead citrate	Merck	7398	TEM staining
Nitric acid (HNO3)	Chem-Lab	CL00.0125	Acid reagent
Nitrogen (N2) gas	Linde	Industrial grade	Anoxic conditions
Osmium tetroxide (OsO4)	Merck	201030	TEM fixation
Paraformaldehyde	VWR	101176-014	5% Glutaraldehyde, 4% paraformaldehyde in 0.1 M Sodium Cacodylate Buffer
Potato Dextrose Agar (PDA)	Merck	110130	Fungal cultivation
Potato Dextrose Broth (PDB)	Merck	P6685	Microbiological medium
Rotary shaker	JoanLab	OS-20Pro	Incubation at controlled agitation
Serum bottles (120 mL)	Wheaton	223748	Borosilicate glass
Silver nitrate (AgNO3)	Sigma-Aldrich	209139	ACS reagent, ≥99.0%
Sodium cacodylate buffer	VWR	11650	5% Glutaraldehyde, 4% paraformaldehyde in 0.1 M Sodium Cacodylate Buffer
Sodium chloride (NaCl)	Carl Roth	3957.1	0.9% washing solution
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881	pH adjustment
Sodium lactate	Sigma-Aldrich	L7022	Electron donor/carbon source
Sterile syringes and needles	BD	Various	Sterile liquid handling
Stirring hot plate	Biologix	01-310X	Heating and agitation
Toluidine blue	EMS	DcU-10	Semi-thin section staining
Transmission electron microscope (TEM)	FEI	Tecnai Spirit Twin G20	Equipped with high-resolution digital camera
Tray dryer with forced airflow	Handmade	N/A	Biomass drying at 40 °C
Tryptic Soy Agar (TSA)	Oxoid	CM01301	Microbiological medium
Tryptic Soy Broth (TSB)	Carl Roth	X938.1	Microbiological medium
Uranyl acetate	Sigma-Aldrich	94260 Fluka	TEM staining
UV–Vis spectrophotometer	Thermo Scientific	Genesys 10S	UV–Vis spectral analysis
Vacuum pump	KNF or equivalent	N 820 G	For anoxic conditions
Biological Materials
Biological Material	Source	Identifier	Comments/Description
Acanthophora spicifera	Local collection	N/A	Marine algal biomass
Cupriavidus metallidurans CH34	SCK-CEN	LMG 1195	Bacterial strain
Earliella scabrosa	CIBE-ESPOL	E2	Institutional fungal collection
Psidium guayaquilensis	Local collection	N/A	Wild plant biomass
Shewanella oneidensis MR-1	BCCM/LMG	LMG 19005	Culture collection strain