Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity

Abstract

Green synthesis method is being increasingly used in the development of safe, stable, and eco-friendly nanostructures with biological resources. In this study, extracellular and intracellular synthesis of gold nanoparticles (AuNPs) was carried out using green algae Chlorella sorokiniana Shihira & R.W. Fresh algae were isolated and identified from Musaözü Pond located in the province of Eskişehir and then extraction process were performed. Optimization studies were studied using pH value, metal salt concentration, and time parameters for extracellular synthesis and using only time parameter for intrasellular synthesis. Since more controlled and optimum conditions can be achieved in the production of AuNPs by extracellular synthesis, these nanoparticles (NPs) were used for characterization and antifungal activity studies. Optical, physical, and chemical properties of synthesized NPs were characterized by UV visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), Zetasizer, X-Ray diffraction (XRD), Fourier transform ınfrared spectroscopy (FTIR), field emission scanning electron microscope (FE-SEM), ınductively coupled plasma mass spectrometer (ICP-MS) and transmission electron microscope (TEM) analysis. The optimum conditions for AuNPs synthesis were determined as 1 mM for HauCl₄ concentration, 6 for pH value, and 60th min for time. AuNPs obtained from extracellular synthesis from C. sorokiniana extract are 5-15 nm in size and spherical shape. TEM images of extracellular synthesis show noticeable cell wall and membrane damages, cytoplasma dissolutions, and irregularities. AuNPs obtained by intracellular synthesis are in 20-40 nm size and localized in the cell wall and cytoplasm. These NPs exhibited significant antifungal activity against C. tropicalis, C. glabrata, and C. albicans isolates. AuNPs obtained by algae-mediated green synthesis have a significant potential for medical and industrial use, and this eco-friendly synthesis method can be easily scaled for future studies.

Keywords: Chlorella; antifungal; gold nanoparticles; green synthesis; transmission electron microscope (TEM).

Figure 1

(a) UV–visible absorption spectrum of AuNPs of intracellularly synthesized using C. sorokiniana after 24 h and 48 h reaction (b) The standard spectrum of AuNPs was calculated based on the study of Yang et al., 2015.

Figure 2

TEM micrographs of intracellular synthesis of AuNPs using C. sorokiniana (a, b) Control Chlorella cells cultured with BG-11 medium only showing regular cellular morphology: cell wall (cw), cell membrane (cm), lipid droplet (ld), thylakoids (thy) and pyrenoid (p); (c, d, e, f) Cells treated with 1 mM Au solution for 24–48 h indicate ultrastructural changes in the cell structure: membrane wall damage (mwd), nanoparticles on cell wall (np), vacuoles (V), and cytoplasm condensation (cc) (Scale bar: 500 nm for a-c-d-e images; 200 nm for b and f images).

Figure 3

UV-visible absorbtion spectra of extracellulary synthesized C-AuNPs recorded for optimization of (a) salt concentration, (b) pH, (c) time parameters.

Figure 4

DLS and Zeta potential diagrams of C-AuNPs (a) To determine size distribution of C-AuNPs (DLS) (b) Zeta potential to determine the stability of C-AuNPs.

Figure 5

SEM and TEM images of C-AuNPs (a) SEM images and morphology of synthesized C-AuNPs. (b, c) TEM images and size values of synthesized C-AuNPs at (100 nm scale); (d) Analysis of energy dispersive X ray (EDX) spectrometer of AuNPs.

Figure 6

FTIR analysis. FTIR spectra of C. sorokiana (a). Synthesized AuNPs (b).

Figure 7

XRD pattern analysis of synthesized AuNPs using C. sorokiana.

Figure 8

Antifungal activity of C-AuNPs on Candida isolates by disc diffusion (a). C. sorokiana extract. (b). Amphotericin B, an antifungal standard. (c). C. albicans ATCC 14053 treated with C-AuNPs. (d). C. tropicalis 1660 treated with C-AuNPs. (e). C. glabrata 1744 treated with C-AuNPs.

Figure 9

TEM micrographs of C. albicans ATCC 14053 treated with C-AuNPs. (a) Untreated control Candida cells; (b, c and d) Cells treated with C-AuNPs. Cells without treatment showed regular and well-conserved features, homogenous cytoplasm and distinctive membrane and wall structure: cell wall (cw), cell membrane (cm), nucleus (nu); After treatment with AuNPs, cytoplasm damage (cd), folded cell shapes (fcs), ghost cells (gc), nanoparticles (np), and membrane invagination (mi) were evident. (Scale bar: 1 μm for a-b-c-d images).

Figure 10

TEM micrographs of C. tropicalis 1660 treated with C-AuNPs. (a) Untreated control Candida cells; (b, c and d) Cells treated with C-AuNPs. Cells without treatment showed well preserved cellular morphology, and entire cell structure: cell wall (cw), cell membrane (cm); After treatment with C-AuNPs, ghost cells (gc), membrane invagination (mi), nanoparticles on the cell wall (np), and membrane ruptures (mr) were apparent. (Scale bars is 200 nm for a–b–d; 1 μm for c images).

Figure 11

TEM micrographs of C. glabrata 1744 treated with C-AuNPs. (a) Untreated control Candida cells; (b, c and d) Cells treated with C-AuNPs. Control Candida cells show homogenous cytoplasm and regular morphological features: cell wall (cw), cell membrane (cm); After treatment with AuNPs, cytoplasm damage (cd), folded cell shapes (fcs), ghost cells (gc), nanoparticles (np), vacuoles (V), and membrane invagination (mi) were observed. (Scale bars is 200 nm for a image; 500 nm for b,c, and d images).