Green synthesis of silver nanoparticles (AgNPs) from G. stearothermophilus GF16: stable and versatile nanomaterials with antioxidant, antimicrobial, and catalytic properties

Abstract

Background: Silver nanoparticles (AgNPs) have attracted considerable interest for their distinctive physicochemical properties and wide-ranging applications in nanomedicine, environmental catalysis, and antimicrobial applications. However, sustainable and robust biosynthesis methods remain a challenge.

Results: In this study, we report the biosynthesis of thermostable AgNPs using the secretome of Geobacillus stearothermophilus GF16, a thermophilic and metal-resistant bacterium isolated from the hydrothermal volcanic area of Pisciarelli, Italy. The synthesis was performed without specialized growth media, relying solely on the cell-free bacterial supernatant, and was systematically optimized by varying precursor concentration, temperature, pH, and reaction time. The nanoparticles were characterized by UV-Vis spectroscopy, dynamic light scattering, Fourier-transform infrared spectroscopy, scanning (SEM) and transmission (TEM) electron microscopy. Morphological analysis showed predominantly subspherical nanoparticles with average diameters of 17 ± 5 nm (SEM) and 16 ± 5-7 nm (TEM), depending on precursor concentration. Thermogravimetric analysis demonstrated excellent thermal stability with retention of structural integrity up to 120 °C, an exceptional feature among biogenic AgNPs. The obtained AgNPs exhibited remarkable radical scavenging activity, reaching up to 79% in DPPH and 75% in ABTS assays at 100 µg/mL, highlighting a level of antioxidant performance rarely observed in AgNPs of bacterial origin. In addition to their redox properties, the nanoparticles demonstrated efficient catalytic activity as evidenced by the complete degradation of Congo Red in 20 min and 4-nitrophenol in 35 min. Time-kill assays and minimum inhibitory concentration (MIC) also showed a broad-spectrum antimicrobial potential with complete inhibition of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella Typhimurium at 100 µg/mL. Interestingly, MIC values were significantly lower than those reported for comparable AgNPs. Notably, the nanoparticles also displayed hemocompatibility, validated by hemolysis assays performed on both healthy and β-thalassemic erythrocytes, with hemolysis rates consistently below the 2% safety threshold.

Conclusions: Overall, this study presents the first comprehensive characterization of AgNPs biosynthesized by a thermophilic bacterium, highlighting their multifunctional potential. The use of a thermophilic bacterium as a robust and flexible microbial nanofactory offers a novel eco-friendly and scalable strategy for AgNP production. The resulting nanoparticles exhibit unique thermal stability, broad-spectrum bioactivity, and clinically relevant hemocompatibility, underscoring their promising applicability in nanomedicine, green catalysis, and environmental remediation.

Keywords: Antimicrobial activity; Biocompatibility; Catalytic efficiency; Extremophiles; Nanofactory; Silver nanoparticles.

Fig. 1

Colorimetric evidence of AgNP biosynthesis from bacterial secretome. Cuvettes containing the secretome of G. stearothermophilus GF16, showing the control (left) and AgNP samples with progressive color changes depending on AgNO₃ concentration and incubation time

Fig. 2

Effect of temperature on AgNP biosynthesis and plasmonic response. UV–Vis spectra of AgNPs synthesized at 50 °C (a), 60 °C (b), and 80 °C (c) with varying AgNO₃ concentrations (0.25–2.0 mM) after 24 h of incubation

Fig. 3

Effect of pH on AgNP biosynthesis and plasmonic response. UV–Vis absorption spectra of AgNPs synthesized at pH 5 (a), 8 (b), and 12 (c), with AgNO₃ concentrations ranging from 0.25 to 2.0 mM after 24 h at 60 °C

Fig. 4

Effect of reaction time on AgNP biosynthesis. UV–Vis spectra comparing AgNPs synthesized at 60 °C for 24 and 48 h, using AgNO₃ at 0.25 and 0.5 mM (a), and 1.0 and 2.0 mM (b)

Fig. 5

DLS analysis. Dynamic light scattering (DLS) results showing size distribution by intensity for AgNP0.5 (red) and AgNP2.0 (green)

Fig. 6

FTIR analysis. FTIR spectra of AgNP0.5 (a) and AgNP2.0 (b). Panel (c) overlays both spectra to highlight concentration-dependent differences

Fig. 7

Morphological characterization via TEM and SEM. Electron micrographs of AgNP0.5 and AgNP2.0, showing changes in aggregation and particle morphology

Fig. 8

TGA-based assessment of AgNP thermal stability. Isothermal thermogravimetric analysis showing thermal stability of AgNP0.5 (light pink) and AgNP2.0 (dark purple)

Fig. 9

Antioxidant activity of AgNPs using DPPH and ABTS assays. Antioxidant activity of AgNP0.5 and AgNP2.0 evaluated using (a) DPPH and (b) ABTS assays, compared to secretome and Trolox as reference controls

Fig. 10

Hemocompatibility of AgNPs on normal and thalassemic RBCs. Hemolytic activity of AgNP0.5 and AgNP2.0 at concentrations of 25, 50, 75, and 100 µg/mL on normal and β-thalassemic RBCs. Includes positive and negative controls. Mean ± SD from triplicates

Fig. 11

Antimicrobial activity of AgNPs against bacterial and yeast strains. Effect of AgNP0.5 and AgNP2.0 (100 µg/mL) on pathogens: (a) S. aureus, (b) P. aeruginosa, (c) S. Typhimurium, (d) L. monocytogenes, and (e) C. albicans. Data expressed as mean ± SE, with statistical significance

Fig. 12

Dose-dependent antimicrobial activity of AgNPs. Antimicrobial activity of AgNP0.5 and AgNP2.0 (25, 50, and 100 µg/mL) against pathogens: (a) S. aureus, (b) P. aeruginosa, (c) S. Typhimurium, (d) L. monocytogenes, and (e) C. albicans. Significance levels indicated: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 13

Catalytic degradation of CR and 4-NP by AgNPs. UV–Vis spectra showing CR (a, b, c) or 4-NP (d, e, f) degradation in presence of NaBH₄: (a, d) control, (b, e) AgNP0.5 AgNP2.0, (c, f) AgNP2.0. CR degradation was monitored over 20 min, while 4-NP degradation was monitored over 35 min