Green Chemistry Approach for the Synthesis of Gold Nanoparticles Using the Fungus Alternaria sp.

Abstract

The synthesis of gold nanoparticles has gained tremendous attention owing to their immense applications in the field of biomedical sciences. Although several chemical procedures are used for the synthesis of nanoparticles, the release of toxic and hazardous by-products restricts their use in biomedical applications. In the present investigation, gold nanoparticles were synthesized biologically using the culture filtrate of the filamentous fungus Alternaria sp. The culture filtrate of the fungus was exposed to three different concentrations of chloroaurate ions. In all cases, the gold ions were reduced to Au(0), leading to the formation of stable gold nanoparticles of variable sizes and shapes. UV-Vis spectroscopy analysis confirmed the formation of nanoparticles by reduction of Au³⁺ to Au⁰. TEM analysis revealed the presence of spherical, rod, square, pentagonal, and hexagonal morphologies for 1 mM chloroaurate solution. However, quasi-spherical and spherical nanoparticles/heart-like morphologies with size range of about 7-13 and 15-18 nm were observed for lower molar concentrations of 0.3 and 0.5 mM gold chloride solution, respectively. The XRD spectrum revealed the face-centered cubic crystals of synthesized gold nanoparticles. FT-IR spectroscopy analysis confirmed the presence of aromatic primary amines, and the additional SPR bands at 290 and 230 nm further suggested that the presence of amino acids such as tryptophan/tyrosine or phenylalanine acts as the capping agent on the synthesized mycogenic gold nanoparticles.

Introduction

The physicochemical and optoelectronic properties of the nanoscale matter depend on the particle size and shape [8]. Their unique and indispensable properties depend on a high surface to volume ratio, which makes them highly superior to any other conventional macroscopic materials. The synthesis of noble metal nanoparticles with varied chemical compositions, sizes, shapes, and controlled monodispersity is one of the challenging aspects in the field of nanotechnology. Metal nanoparticles like silver, gold, and platinum can be synthesized through a wide range of methods. Among the noble metals, gold nanoparticles have received colossal attention in recent years owing to their wide range of potential applications in the field of catalysis [5], biosensors [24], bio-imaging, [25], and drug delivery [3], and also in the treatment of some cancers [15]. Although chemical attempts are the most popular methods for the production of nanoparticles, the usage of unsafe, toxic chemicals and the release of hazardous by-products impede their usage for biomedical applications [17, 36]. Thus, there is an immediate need to develop clean, non-toxic, dependable, biocompatible, and environmentally benign procedures for the synthesis and fabrication of nanoparticles and other high nanostructured materials. This made researchers in the field of material sciences to use microorganisms as possible eco-friendly nanofactories.

It has been well known that microbes such as bacteria [9, 11, 12], fungi [6, 27, 33], diatoms (silica nanomaterials) [18], yeast [13], and actinomycetes [1] are used for the production of a large number of inorganic and heavy metal nanoparticles. However, the production of large biomass, easy handling/bioavailability, high metal tolerance, mineral solubilizing activity, and less time make fungi extremely superior over other microbial resources such as bacteria and yeast [6, 27]. In addition, the studies on fungi can be easily extrapolated to others. The biosynthesis of nanoparticles through microbes primarily fungi, has been carried out both intracellularly and extracellularly [4, 5, 19, 20, 27, 34, 35]. Recently, fungi such as Rhizopus oryzae, Aspergillus oryzae var. viridis, Colletotrichum sp., Penicillium brevicompactum, Aspergillus niger, and Phanerochaete chrysosporium were reported in the synthesis of gold nanoparticles [16, 22]. However, owing to the easy downstream processing and cost-effectiveness, extracellular synthesis finds more extensive applications in industries than the intracellular route. Mukherjee et al. [20] have been able to produce gold nanoparticles of various morphologies through incubation of fungal extract with 10-3 M AuCl4- in the dark. CastroLongoria et al. [4] have demonstrated the synthesis of silver, gold, and bimetallic nanoparticles by the fungus Neurospora crassa strain N150. Thakker et al. [32] synthesized extracellular gold nanoparticles using a plant pathogenic fungus, Fusarium oxysporum f. sp. cubense JT1. Kar et al. [14] also demonstrated the anthelmintic efficacy of gold nanoparticles synthesized using a phytopathogenic fungus, Nigrospora oryzae. In the present study, we report for the first time the use of cellfree filtrate of filamentous fungus Alternaria sp. for the synthesis of isotrophic and anisotrophic gold nanoparticles. The methodology adopted here is a simple, feasible, and single-step process, which does not require the usual usage of toxic and hazardous chemicals.

 

Materials and Methods

Chemicals and Fungal Culture

Tetrachloroauric(III) acid and potato dextrose broth (PDB) were purchased from Hi-Media Laboratories Pvt. Ltd. (Mumbai, India). The fungus Alternaria sp. belonging to the family Pleosporaceae was obtained from the Microbial Culture Collection (MCC), Pondicherry University, India. Milli-Q water was used throughout the experiment.

Preparation of Cell-Free Culture Filtrate

The culture, growth, and the processing of fungi for the synthesis of gold nanoparticles were carried out using standard procedures with slight modifications [23]. Briefly, the fungal culture was maintained previously on potato dextrose agar slants at 25℃. For fungus culturing, the fungus was grown under aerobic conditions in a 500 ml Erlenmeyer flask containing 100 ml of PDB at 30℃, pH 4.0 in an orbital shaker at 150 rpm. After 5 days of culturing, the fungal mycelial biomass was filtered using 2-3 layers of sterile Whatman qualitative filter paper No. 1 in aseptic condition, and washed three times with sterile Milli-Q water to remove the media components. Then 10 g of washed fungal biomass was weighed (wet wt.) and transferred into a 500 ml Erlenmeyer flask containing 100 ml of Milli-Q water. The flask containing the fungal biomass was agitated aerobically at 150 rpm for 72 h at 25℃. After 3 days of incubation, the mycelial biomass in Milli-Q water was centrifuged at 7,000 rpm for 20 min at 10℃, and the culture supernatant was filtered using 2-3 layers of sterile Whatman qualitative filter paper No. 1. The obtained cell-free culture filtrate was then used for the synthesis of gold nanoparticles.

Biosynthesis of Gold Nanoparticles Using the Cell-Free Filtrate

In order to compare the kinetics of gold nanoparticle formation and its stability, three different concentrations of chloroauric acid were used. First, 10 ml of the cell-free filtrate was added to 5 ml of gold chloride solution in a 100 ml Erlenmeyer flask, to get a final concentration of 3 × 10-4, 5 × 10-4, and 1 × 10-5M gold chloride. The three conical flasks containing the cell-free filtrate containing the required concentration of auric acid solution were placed in a shaker at 150 rpm, 25℃. The whole reaction was carried out for a period of 72 h. Simultaneously, the cell-free filtrate and the gold chloride solutions were maintained under the same conditions as a positive and a negative control, respectively.

Characterization of the Synthesized Gold Nanoparticles

UV-Vis spectroscopy measurements. The change in color was visually monitored in the conical flask containing gold chloride solution incubated with the cell-free filtrate of Alternaria sp. The biotransformation of Au3+to Au0 was also monitored routinely by periodic sampling of aliquots (1 ml) of the aqueous solution and measuring the UV-vis spectra of the solution. The absorption spectra were recorded as a function of reaction time on a portable dual mode UV-vis spectrophotometer (Thermo Scientific Nano Drop 2000c).

X-ray diffraction (XRD) studies. The colloidal solution of gold nanoparticle was drop-coated onto a glass substrate, and the Xray diffraction patterns were recorded using a PANalytical X’pert PRO X-ray diffractometer (The Netherlands) with Cu Kα1 radiation under the operating voltage and tubing current of about 40 kV and 30 mA, respectively. The diffracted patterns were recorded at 2θ from 10° to 80° with the scanning speed of 0.02°/min.

Fourier transform infra-red (FTIR) spectroscopy. The functional groups present on the surface of the synthesized gold nanoparticles were analyzed using an FTIR (Thermo Nicolet Model 6700) spectrophotometer. For FTIR measurement, the biotransformed gold chloride solution was centrifuged at 7,000 rpm for 20 min at 15℃ and the pellet was washed three times with Milli-Q water to remove any unbound components, which were not capping the gold nanoparticles. The samples were then dried in a hot air oven at 50℃ and the dried samples were pelleted with potassium bromide (KBr). In order to obtain a good signal-to-noise ratio, 512 scans were performed under diffuse transmittance mode with a resolution of 4 cm-1 in the spectral range of 4,000 to 400 cm-1.

Energy dispersive X-ray analysis (EDX). Energy dispersive Xray analysis was done to detect the presence of gold in the synthesized gold nanoparticles. The gold nanoparticle dispersed in deionized water was drop-coated on a microscopic slide and allowed to dry in a hot air oven at 50℃. The thin film formed as a result of drying was sputter-coated on a carbon stub, and the signals were recorded in spot profile mode using a Noran six energy dispersive X-ray microscopic analysis system (Thermo Electron Corporation, USA) coupled with SEM.

Transmission electron microscopy analysis. Transmission electron microscopy (TEM) was performed to examine the size and morphology of the synthesized gold nanoparticles. The sample preparation was carried out by placing 2–4 µl of gold nanoparticle solution on carbon-coated copper grids. The thin film formed was air-dried under ambient conditions and observed using Philips 10 Technai with an accelerating voltage of about 180 keV with a wavelength (λ) of 0.0251 Å.

 

Results and Discussion

The reducing components produced by the microorganisms play a significant role in the extracellular synthesis of nanoparticles. The mycosynthesis of gold nanoparticles can be monitored visually through a gradual change in the color of the reaction solution, and hence the reaction has been monitored through UV-Vis spectroscopy. Nevertheless, spectroscopic analysis forms a basic and ideal technique to understand the optical properties of metal nanoparticles. The three flasks, namely the mycelia-free filtrate and chloroauric acid solutions (controls) and the mycelia-free filtrate incubated with chloroauric acid (0.3, 0.5, 1 mM), were routinely monitored for the change of color. No significant color change was observed in both the controls. However, the change of color from dark yellow to crystal violet after 24 h indicated the formation of gold nanoparticles in the flasks containing the fungal filtrate with the concentrations of 0.3 and 0.5 mM HAuCl4. However, the flask containing the fungal filtrate with 1 mM HAuCl4 solution turned pinkish-red in color. Thus, there iwas a gradual change in the color from crystal violet to pink-red with the increase in the concentration of gold chloride. We believe the reducing enzymes secreted by the fungus into the surrounding medium might be responsible for the reduction of chloroaurate ions. It is well known that gold nanoparticles produce a wide range of colors, due to the excitation of the surface plasmon resonance [21]. After 24 h, a single intense SPR band was centered at 535 nm in the flask containing 0.3 mM HAuCl4, which is a clear indication of nanoparticle formation (Fig. 1A). The intensity increased with the increase in the reaction time, and it was saturated after 72 h, illustrating the complete reduction of Au3+ions. In most cases, the SPR band of gold nanoparticles centered between 500 and 600 nm, depending on the dielectric constant of the medium, and size and shape of the nanoparticles [30, 31]. With the increase in the concentration from 0.3 to 0.5 mM HAuCl4, there was a red-shift in the SPR band from 545 to 550 nm, which was recorded after 48 h of the reaction time (Fig. 1B). This slight increase in SPR and the red-shift indicates the increase in the particle size of the synthesized gold nanoparticles with the increase in the concentration of HAuCl4 [7, 19]. Interestingly, the solution containing 1 mM HAuCl4 produced two additional bands at 290 and 250 nm apart from the standard peak at 535 nm (Fig. 1C). The peak at 290 nm finds the presence of aromatic amino acids such as tryptophan/tyrosine or phenylalanine. On the other hand, the band at 230 nm is attributed to the presence of peptide linkage, thus stabilizing the nanoparticles in aqueous solution [2]. Nevertheless, the production of one or more SPR bands indicates the anisotropic nature of the synthesized gold nanoparticles [23]. After 72 h, no further additional plasmon resonance peaks were observed, indicating the completion of nanoparticle synthesis. The synthesized nanoparticles exhibited no change in color, and the intensity remained the same even after 45 days, suggesting that the synthesized gold nanoparticles were more stable at room temperature.

Fig. 1.UV-Vis spectra of gold nanoparticles measured as a function of time by exposing the cell-free filtrate of the filamentous fungus Alternaria sp. to three different concentrations of chloroaurate ions: (A) 0.3 mM, (B) 0.5 mM, and (C) 1 mM HAuCl4.

X-ray diffraction studies help in understanding the crystalline nature of the mycogenic gold nanoparticles. Fig. 2 represents the typical XRD profile of the gold nanoparticle films deposited on a thick glass substrate. The gold nanoparticles were crystalline in nature, with four distinct intense diffraction peaks at 38.2°, 44.3°, 64.5°, and 77.6° corresponding to the crystal planes (111), (200), (220), and (311), respectively (International Centre for Diffraction Data, ICDD No. 4-0783). The high intense Bragg’s reflection at 38.2° was a very good indication that the developed gold nanoparticles were oriented (111) in a flat position on the planar surface. On the other hand, the reflections corresponding to (200), (220), and (311) were weak and widened, which indicate the nanoparticles are of smaller size [30]. To confirm the presence of gold in the synthesized nanoparticles, EDX analysis was done in the spot profile mode (Fig. 3). The presence of sharp signals at 1.8, 2.1, and 2.5 keV was typical for the absorption of metallic gold nanocrystallites (~25% (w/w)). In addition, the weak peak of element oxygen might have arisen from the proteins/ enzymes of the fungal filtrate, which capped the gold nanoparticles, preventing it from aggregation and contributing to its stability [29]. Fig. 4 shows the FTIR spectrum of the gold nanoparticles. The sharp bands at 3,400 and 2,916 cm-1 suggest the presence of stretching vibrations (N–H) of primary and secondary amines, and aldehydic C-H stretching, respectively [28]. Moreover, this also indicates the possibility of hydroxyl functional groups, which might have arisen from alcohols or phenols present in the fungal filtrate. On the other hand, their corresponding N–H bending vibration was seen at 1,650 and 1,532 cm-1, respectively. In addition, the weak bands at 1,392 and 1,070 cm-1 were assigned to C–N stretching vibrations of aromatic and aliphatic amines. Hence, the presence of amide linkages to hold the amino acid residues of the protein biomolecules, such as tryptophan/tyrosine secreted extracellularly by the fungal cells, are responsible for the possible stabilization of the synthesized gold nanoparticles [10, 34].

Fig. 2.XRD analysis of the gold nanoparticles obtained from drop-coating of the aqueous solution of gold nanoparticles on a silica substrate. The gold colloid showed strong diffraction peaks at 38.2°, 44.3°, 64.5°, and 77.6°, respectively.

Fig. 3.Spot profile mode of energy dispersive X-ray analysis of synthesized gold nanoparticles.

Fig. 4.FTIR spectrum of the gold nanoparticles obtained using a cell-free filtrate of the filamentous fungus Alternaria sp.

Transmission electron microscopy gives complete information regarding the size and morphology of the synthesized gold nanoparticles. Fig. 5 represents the TEM images of gold nanoparticles synthesized using three different concentrations (viz., 0.3, 0.5, and 1 mM) of gold chloride with the cell-free filtrate of the filamentous fungus Alternaria sp. Monodispersed isotropic quasi-spherical nanoparticles with a size range of 7–13 nm were obtained in the case of low concentration of HAuCl4 (0.3 mM) (Fig. 5A). The clear separation of nanoparticles indicates the presence of amino acids/ enzymes, which may behave as the capping material, and thus preventing the agglomeration. The selected area diffraction pattern with bright circular rings corresponding to (111), (200), (220), and (311) planes show the face-centered cubic crystalline nature of the synthesized gold nanoparticles (Fig. 5A). Similarly, the reaction mixture containing 0.5 mM of gold salt resulted in the production of spherical nanoparticles with a much larger size distribution of 15– 18 nm (Fig. 5B). Besides this, spherical nanoparticles with heart-like morphologies were observed for the first time with the mycogenic filtrate of Alternaria sp. The reaction mixture containing a higher concentration of gold chloride (1 mM) resulted in the synthesis of a wide range of anisotropic nanoparticles such as rod, spherical, square, pentagonal, and hexagonal shaped with size ranging from 69 to 93 nm (Figs. 5C and 5D). Such a wide variety of nanoparticles has already been reported with the fungus Fusarium oxysporum f. sp. lycopersici [26]. This tremendous increase in size and the clustered nature of the nanoparticles are well attributed from the spectroscopic measurements of the synthesized gold nanoparticle (broad nature of SPR at 535 nm). Thus, the increase in the concentration of gold chloride has resulted in increased particle size with different morphologies. This result exactly coincides with the report previously established by Mishra et al. [19] with the fungal filtrate of Penicillium brevicompactum. This ability to tune the optical properties of nanoparticles, which results in various morphologies, finds extensive applications in the fields of biomedicine and biotechnology.

Fig. 5.Representative TEM images of gold nanoparticles synthesized by exposing different concentrations of chloroaurate ions to fungal filtrate. (A) Quasi-spherical nanoparticles in the size range of 7–13 nm obtained with 0.3 mM HAuCl4. Inset shows the corresponding SAED pattern of the monodispersed quasi-spherical nanoparticles; (B) spherical nanoparticles along with heart-like morphologies in the size range of 15–18 nm obtained with 0.5 mM HAuCl4 ; (C) and (D) spherical, square, rod, pentagonal, and hexagonal shaped nanoparticles in the size range of 69–93 nm obtained with 1 mM HAuCl4.

In conclusion, a bio-inspired, simple, low-cost, and green chemistry approach has been adopted for the synthesis of gold nanoparticles under ambient conditions. The reduction of chloroaurate ions on exposure to the cultural filtrate of the fungus Alternaria sp. has resulted in a wide range of nanoparticles, such as quasi-spherical for 0.3 mM, spherical for 0.5 mM, and spherical, rod, square, hexagonal, and pentagonal-like morphologies for 1 mM auric acid solutions. The reduction of gold ions and stabilization of gold nanoparticles take place with the help of proteins present in the fungal filtrate. The combination of cell-free filtrate with chloroaurate ions at ambient conditions makes it clear that a biological system can be used to modulate the size and shape of gold nanoparticles. Thus, this remarkably new and rapid technology identifies immense contribution to the field of green chemistry and nanomaterial sciences.