Mechanism study of silver nanoparticle production using Neurospora intermedia

Abstract

Elucidation of the molecular mechanism of silver nanoparticle (AgNP) synthesis is necessary to control nanoparticle size, shape, and monodispersity. In this study, the mechanism of AgNP formation by Neurospora intermedia was investigated. The higher production rate of AgNP formation using a culture supernatant heat‐treated at 100° and 121°C relative to that with an un‐treated culture supernatant indicated that the native form of the molecular species is not essential. The effect of the protein molecular weight (MW) on the nanoparticle size distribution and average size was studied by means of ultraviolet–visible spectroscopy and dynamic light scattering. Using un‐treated and concentrated cell‐free filtrate passed through 10 and 20 kDa cut‐off filters led to the production of AgNPs with average sizes of 25, 30, and 34 nm, respectively. Also, using the permeate fraction of cell‐free filtrate passed through a 100 kDa cut‐off filter led to the formation of the smallest nanoparticles with the narrowest size distribution (average size of 16 nm and polydispersity index of 0.18). Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of the fungal extracellular proteins showed two notable bands with the MWs of 15 and 23 kDa that are involved in the reduction and stabilisation of the nanoparticles, respectively.

1 Introduction

Nanoparticles have advantages over bulk materials due to their surface plasmon resonance (SPR), enhanced Rayleigh scattering, and surface enhanced Raman scattering. Therefore, nanoparticles are considered as the building blocks of the various nanostructures and devices used in nanotechnology [1]. Among metal nanoparticles, silver nanoparticles (AgNPs) have become the major focus of many studies due to its several application in non‐linear optics, catalysis, biology (biolabelling and DNA sequencing), and medicine (antimicrobial, antiviral, and anticancer actions) [2, 3, 4, 5, 6]. A number of chemical and physical methods have been developed for the synthesis of AgNPs. However, these methods generally employ expensive and toxic reagents as reducing and stabilising agents, which pose potential risks to human health and the environment [7, 8, 9, 10]. Furthermore, the use of toxic chemicals on the surface of AgNPs in the synthesis procedure limits their applications in clinical fields [1]. Hence, there is an increasing need to develop high yield, low cost, non‐toxic, biocompatible, and eco‐friendly processes for the production of AgNPs [11]. Recently, utilisation of biological resources such as bacteria, fungi, and plants for nanoparticle synthesis has become an alternative process [12, 13, 14, 15, 16, 17, 18]. Fungi are more efficient candidates for fabrication of AgNPs owing to their high protein secretion capacity, higher productivity, and easy handling in large‐scale production. In addition, extracellular biosynthesis using fungi can also make downstream processing much easier than when employing bacteria [19, 20]. Numerous investigations have been published regarding the biosynthesis of AgNPs using various fungi such as Fusarium oxysporium [21, 22, 23, 24], Cladosporium cladosporioides [25], Penicillium sp. [26, 27], Aspergillus flavus [28], and Macrophomina phaseolina [29]. Nonetheless, the precise mechanism of AgNP formation remains poorly understood. However, it has been evidenced that NADH‐dependent nitrate reductase plays a key role in the biosynthesis of AgNPs [30, 31, 32, 33]. Previously, we established the efficacy of employing the Neurospora intermedia as a non‐pathogenic fungus [34]. Also, the characterisation of the synthesised AgNPs was performed in different conditions using X‐ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy (SEM) and dynamic light scattering (DLS) analyses. The main aim of this paper is to investigate the AgNP biosynthesis mechanism inherent within this synthesis method. For this reason, the pattern of the proteins in the culture supernatant and cell‐free filtrate of this fungus before and after mixing with Ag nitrate (AgNO₃) under different conditions was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) analysis. Furthermore, the impact of the molecular weight (MW) of the proteins on the size and size distribution of the synthesised nanoparticles was investigated.

2 Materials and methods

2.1 Microorganism

N. intermedia (PTCC 5291) was prepared from the Persian Type Culture Collection of the Iranian Research Organization for Science and Technology. The maintenance of the stock cultures was performed on a potato dextrose agar supplemented with 0.5% (w/v) yeast extract at 24°C. This strain was stored at 4°C and sub‐cultured periodically to preserve its viability during the period of the present study.

2.2 Fungal growth conditions

Erlenmeyer flasks containing 50 ml potato dextrose broth (PDB) supplemented with 0.5% (w/v) yeast extract were inoculated by N. intermedia inoculums (10⁷ spore/ml). The pH of the culture broth before inoculation was adjusted to 5.8 ± 0.2 using 1 N HCl. The inoculated flasks were incubated on an orbital shaker operating at 24°C and 200 rpm for 72 h.

2.3 AgNP formation using culture supernatant

For the biosynthesis of the nanoparticles, N. intermedia fungus was grown aerobically in culture media containing PDB medium supplemented with 0.5% (w/v) yeast extract. About 1 N HCl was used to adjust the final pH of the medium to 5.8. After 72 h of the fermentation, the culture broth was separated using centrifugation operating at 6000 rpm for 20 min. For AgNP production, the resultant supernatant and 2 mM AgNO₃ solution were mixed together with volume ratios of 1:1 (based on previous experiments in our laboratory). Finally, the mixtures were incubated at 28°C and agitated at 200 rpm to reach the maximum absorbance at λ _max, corresponding to completion of the nanoparticle formation. All reactions were performed in the presence of light.

2.4 AgNP formation using cell‐free filtrate

The fungal mycelia were separated from cultures using filtration and the resultant mycelia were washed thrice with sterilised distilled water. Then, 10 g of harvested mycelia was submerged in 100 ml sterilised distilled water and incubated on an orbital shaker operating at 200 rpm and 4°C for 72 h. This suspension was filtered through Whatman filter paper No. 1. Finally, the obtained filtrate (50 ml) was mixed with 50 ml of a 2 mM aqueous AgNO₃. The reaction mixtures were incubated at 200 rpm, 28°C until the maximum absorbance at λ _max was attained. These experiments were conducted in triplicate under light conditions.

2.5 AgNP formation using cell biomass

After the cultivation period, the mycelial mass was separated from the culture broth by sterile filter paper, and the mycelia were washed three times with sterile distilled water. Subsequently, 10 g of harvested mycelia was submerged in 100 ml of a 1 mM aqueous AgNO₃. Finally, the reaction mixtures were incubated until reaching a maximum absorbance at λ _max in a shaker operated at 200 rpm and 28°C.

2.6 Characterisation of AgNPs

2.6.1 Ultraviolet–visible (UV–vis) spectroscopy

Colour change of the reaction mixtures are an evidence of nanoparticle formation. The absorbance of the colloidal AgNPs was measured using a double beam UV–vis spectrophotometer (Cary 100, Varian). All spectra of the samples were recorded at wavelengths of 200–800 nm.

2.6.2 DLS measurement

To determine the size distribution of the produced nanoparticles, particle size distributions were carried out for size ranges of 0.3–10⁴ nm using DLS (DLS; Malvern Instruments Ltd., UK). Polydispersity indexes (PDIs) obtained from DLS measurements were criteria of the particle agglomerations. Values of the PDI range from 0.01 for monodispersed particles up to values of 0.5–0.7 for aggregated particles.

2.6.3 Scanning electron microscopy

The morphological characterisation of the produced AgNPs was performed by SEM (KYKY‐EM3200, China) at 25.00 kV. The sample was prepared by locating the purified synthesised AgNPs on the SEM holder, followed by gold coating using sputter coater (KYKY‐SBC12, China). SEM images were recorded at 30,000× magnification.

2.7 AgNP biosynthesis under various conditions

2.7.1 Role of extracellular proteins and their structure

To evaluate the role of proteins in AgNP biosynthesis, proteins in the cell‐free filtrate and culture supernatant of N. intermedia were precipitated using ammonium sulphate powder. At the first stage, the cell‐free filtrate and culture supernatant were concentrated to 1% of their initial volumes using ultrafiltration (Sartorius, Germany) with a 5 kDa cellulose tri acetate membrane in the presence of N₂. The extracellular proteins of the cell‐free filtrate and culture supernatant of N. intermedia fungus were precipitated by adding ammonium sulphate powder at a final concentration of 80% (w/v). Then, the reaction mixtures were stirred slowly overnight at 4°C. The precipitated proteins were separated via centrifugation at 14,000 rpm, 4°C for 15 min. After washing extensively with phosphate buffer or cold acetone, the precipitated proteins were resuspended in a phosphate buffer (pH 7.4) and used for AgNP biosynthesis. The protein‐free supernatant was dialysed using a cellulose acetate membrane with MW cut‐off of 12 kDa. At first, the dialysis bag was pre‐treated according to the manufacturer's instructions. Then, dialysis bag containing protein‐free supernatant was submerged in a 50 mM phosphate buffer solution (pH 7.2) and stirred gently. Finally, the dialysed solution was used for the synthesis of AgNPs.

In another set of experiments, both the cell‐free filtrate and the culture supernatant, were boiled at 100°C for 30 min, and after cooling were added to aqueous AgNO₃ to produce AgNPs. The cell‐free filtrate was also mixed with the AgNO₃ solution after exposure to a temperature of 121°C and pressure of 15 psi for 15 min in an autoclave, and the production of AgNPs was evaluated.

2.7.2 AgNP biosynthesis using active and inactive cell‐free filtrates

In these experiments, active and inactive cell‐free filtrates of the fungus were used for AgNP synthesis. The active cell‐free filtrate was prepared as described in Section 2.4.

The inactive cell‐free filtrate was prepared by autoclaving of the fungal mycelia suspension in distilled water at a temperature of 121°C and pressure of 15 psi for 15 min. Then, 10 g of the inactive mycelia was separated by filtration, resuspended in 100 ml of distilled water, and incubated at 28°C under shaking at 200 rpm for 72 h. The inactive biomass was separated by passage via Whatman filter paper No. 1, and 50 ml of inactive cell‐free filtrate was mixed with 50 ml of a 2 mM aqueous AgNO₃ for AgNP synthesis. Then, the reaction mixtures were incubated at 200 rpm, 28°C for 72 h. Finally, the AgNPs produced by both active and inactive cell‐free filtrates were evaluated by UV–vis and DLS analyses.

2.7.3 Effect of protein MW on AgNP biosynthesis

The cell‐free filtrate fractions were prepared using cellulose tri acetate membranes (Sartorius, Germany) with 100, 20, and 10 kDa cut‐off filters. The retentive and permeated fractions were mixed separately with 2 mM AgNO₃ solutions (1:1 volume ratio) and incubated at 200 rpm, 28°C for 72 h.

2.7.4 SDS‐PAGE analysis

The secreted proteins present in the cell‐free filtrate and culture supernatant of the fungus were concentrated using filtration with a 5 kDa cut‐off membrane and dialysed thoroughly against a phosphate buffer (pH 7.4) by a 10 kDa cut‐off dialysis bag. The resulting protein containing extracts were assessed using 12% SDS‐PAGE analysis.

For determination of the specific proteins acting as a reducing agent, the fungal biomass after the cultivation period was harvested, extensively washed, and divided into three equal portions. These portions were suspended in distilled water, 1 mM potassium nitrate, and 1 mM AgNO₃ solutions (10% w/v), respectively, and incubated on an orbital shaker operating at 24°C, and 200 rpm for 24 h. The cell‐free filtrates resulting from these three media were concentrated via filtration through a 5 kDa cut‐off filter, and, finally, analysed by SDS‐PAGE.

To investigate the profile of the proteins that attached to the surfaces of AgNPs, the as‐synthesised nanoparticles were concentrated via centrifugation at 10,000 rpm for 20 min, washed with acetone, and then boiled with 1% SDS solution for 10 min. Finally, SDS‐treated solution subjected to centrifugation at 8000 rpm for 10 min to release proteins from the AgNP surfaces. The SDS‐treated and un‐treated AgNPs were analysed using 12% SDS‐PAGE.

3 Results and discussion

This is the first study elucidating the mechanism of AgNP synthesis by extracellular protein secretion of the novel fungal strain, N. intermedia. The possible involvement of protein components in nanoparticle synthesis was confirmed using SDS‐PAGE analysis. The effects of protein structure and MW on AgNP characteristics were also investigated.

3.1 Comparison of AgNP biosynthesis

The appearance of yellowish‐brown colour in reaction mixtures confirmed the formation of AgNPs, and is related to excitation of SPR [35]. The UV–vis spectra of the produced AgNPs using culture supernatant, cell‐free filtrate, and biomass of N. intermedia fungus at different periods of time are shown in Fig. 1. As indicated in Fig. 1, the absorbance intensities of all spectra (AgNP concentration) increased with increasing of the reaction time. The lower region of the UV–vis spectrum verified the formation of smaller nanoparticles [36]. As presented in Fig. 1, a strong SPR is centred near 403, 420, and 405 nm for AgNPs produced using cell‐free filtrate, culture supernatant, and biomass, respectively. As indicated, synthesis of AgNPs using the culture supernatant resulted in the formation of the smallest nanoparticles. The absorbance intensity presented insight into the bioreduction of Ag ions (Ag⁺) and the amount of AgNPs produced [37]. As shown in Figs. 1 a and c, the highest and lowest plasmon SPR peaks were obtained from the AgNPs produced using the culture supernatant and biomass, respectively. The higher production rate of AgNPs synthesised using the culture supernatant may be due to the presented metabolites in the culture supernatant in addition to the greater amounts of proteins and enzymes compared with the other media. One reason for the lowest production rate of AgNPs synthesised by the biomass may be the absence of reducing agents at the beginning of the reaction and secretion of these factors after mixing the biomass with Ag⁺.

Fig. 1.

UV–vis spectra of colloidal AgNPs in reaction solutions containing

(a) 1:1 Volume ratio of N. intermedia culture supernatant and AgNO₃, (b) 1:1 Volume ratio of N. intermedia cell‐free filtrate and AgNO₃, (c) N. intermedia biomass and AgNO₃ (1:10 v/w); at 12 h (I), 24 h (II), 36 h (III), and 72 h (IV)

The absorption band at 265 nm (data not shown) is attributed to the aromatic amino acids such as tyrosine and tryptophan residues of the proteins [38]. This may be attributed to the secretion of extracellular proteins by N. intermedia in both cell‐free filtrate and culture supernatant and offers a probable mechanism for the bioreduction of Ag⁺ present in the reaction mixtures.

3.2 AgNP biosynthesis using the treated and un‐treated culture supernatant

Mixing of the protein‐free supernatant with AgNO₃ solution resulted in no production of AgNPs. This result establishes the importance of proteins in AgNP biosynthesis, which has verified the UV–vis analysis data. To achieve further insight into the mechanism of AgNP formation, the culture supernatants were heat‐treated initially for 30 min at 100°C or autoclaved for 15 min at 121°C and 15 psi, and then added to the AgNO₃ solutions. UV–vis spectra of the colloidal AgNPs formed using the un‐treated and heat‐treated culture supernatants at 100 and 121°C are compared, as shown in Fig. 2. While all the samples considered demonstrate the capability of synthesising AgNPs, the intensity and λ _max for the samples vary. It is of interest that the absorbance intensity of the autoclaved supernatant sample is highest. This could be a consequence of amino acid release owing to protein hydrolysis, which provides greater availability of some reducing agents for Ag⁺ reduction. Previous studies have also reported that interactions between amino acids and metal ions are responsible for synthesis of metal nanoparticles [39]. Heating of protein molecules results in the breakdown of hydrogen bonds and dismantling the hydrophobic core, which, in turn, enhances the interactions between amino acids and Ag⁺. Thus, the higher rate of AgNP synthesis in the case of denatured (heat‐treated) proteins reveals the important role of amino acids in reduction of Ag⁺ ions.

Fig. 2.

Comparison of UV–vis spectra of colloidal AgNPs in reaction solutions containing AgNO₃

(a) Cell‐free filtrate, (b) Heat‐treated cell‐free filtrate at 121°C, (c) Heat‐treated cell‐free filtrate at 100°C

3.3 AgNP biosynthesis using active and inactive cell‐free filtrates

Fig. 3 shows the UV–vis spectra of the nanoparticles generated using active and inactive cell‐free filtrates. The absorption intensity of the spectrum obtained from AgNPs generated using the inactive cell‐free filtrate is considerably higher than that obtained using the active cell‐free filtrate, which confirms the higher productivity of AgNP biosynthesis using the inactive cell‐free filtrate medium. This may be attributable to the higher organic matter content, acting as reducing agents, present in the inactive cells, as reported by other researchers [40]. It appears that autoclaving leads to the rupture of cell walls, and, consequently, a greater release of organic materials.

Fig. 3.

UV–vis spectra of the colloidal AgNPs generated in reaction solutions containing AgNO₃

(a) Active cell‐free filtrate, (b) Inactive cell‐free filtrate after: 24 h (I), 48 h (II), and 72 h (III)

3.4 AgNP biosynthesis using retentate and permeate fractions of cell‐free filtrate

The permeate and retentate fractions of the cell‐free filtrate passed through filters with 10, 20, and 100 kDa cut‐offs were added to AgNO₃ solutions. The UV–vis spectra of the colloidal nanoparticles biosynthesised using the retentate and permeate fractions of the cell‐free filtrate are shown in Figs. 4 a and b, respectively.

Fig. 4.

UV–vis spectra of the colloidal AgNPs formed in reaction solutions containing AgNO₃

(a) Fractions remained after passing cell‐free filtrates through cellulose filters with (a) 10 kDa, (b) 20 kDa, and (c) 100 kDa cut‐offs [Inset shows the UV–vis spectra of produced AgNPs using cell‐free filtrate concentrated via a 100 kDa cut‐off filter paper: after reaction completion (I), 72 h after reaction completion (II)], (b) Permeate cell‐free filtrates passed through filters with (a) 10 kDa, (b) 20 kDa, and (c) 100 kDa cut‐offs after 72 h [Inset shows the spectra of the AgNPs synthesised using diluted cell‐free filtrate passed through a 10 kDa cut‐off filter paper after: 48 h (I), 72 h (II)]

As presented in Fig. 4 a, a strong SPR band is centred at 412, 418, and 427 nm for AgNPs synthesised using the retentate fractions of cell‐free filtrate passed through filters with 10, 20, and 100 kDa cut‐offs, respectively. These results indicate that higher MW proteins tend to produce AgNPs of larger size (concentrated from a 100 kDa cut‐off filter). Higher MW proteins allow for larger AgNP growth because more space is available around the Ag nucleus. Also, a mixture of high and low MW proteins (concentrated from a 10 kDa cut‐off filter) results in smaller AgNPs because low MW proteins cap the AgNPs and inhibit their growth. The inset of Fig. 4 a shows that the SPR band of the AgNPs produced using the cell‐free filtrate concentrated with a 100 kDa cut‐off filter shifted to a higher wavelength 72 h after completion of the reaction, which could be due to the aggregation of nanoparticles due to instability. Owing to the presence of smaller MW macromolecules that cap the nanoparticles, the AgNPs produced by the retentate fraction of the 10 kDa cut‐off filter are more stable than those synthesised by the retentate fraction of the 100 kDa cut‐off filter (Fig. 4 a). This explanation is also confirmed by comparing the results obtained for the retentate fractions of the 10 and 20 kDa cut‐off filters (Fig. 4 a). These results reveal that proteins with an MW between 10 and 20 kDa can affect the size and production rate of AgNPs.

The UV–vis spectra of the nanoparticles produced using the permeate fractions of cell‐free filtrates passed through 10, 20, and 100 kDa cut‐off filters are presented in Fig. 4 b. The SPR band of colloidal AgNPs synthesised using the permeate fraction of cell‐free filtrate passed through the 100 kDa cut‐off filter is centred at a shorter wavelength region relative to the SPR band of AgNPs resulting from the permeate fraction of filtrate passed through the 20 kDa cut‐off filter. This illustrates the formation of smaller nanoparticles using the permeate fraction passed through the 100 kDa cut‐off filter. As shown in the inset of Fig. 4 b, the SPR band of AgNPs produced by the permeate fraction of filtrate passed through the 10 kDa cut‐off filter, disappeared after 72 h indicating the instability of synthesised AgNPs using biomolecules with MWs lower than 10 kDa. These observations confirm the presence of important biomolecules with MWs between 10 and 100 kDa that operate as reducing and stabilising agents in AgNP formation, respectively.

The particle size distribution and PDI of the produced AgNPs using retentate fractions (passed through 10 and 20 kDa cut‐off filters), permeate fraction (passed through the 100 kDa cut‐off filter), and un‐treated cell‐free filtrates were determined by DLS analysis, as shown in Fig. 5. As presented in Fig. 5, the average sizes of the nanoparticles synthesised using macromolecules with MWs higher than 10 and 20 kDa are 30 and 34 nm, and also their PDI are 0.30 and 0.45, respectively. The average size and PDI of the nanoparticles synthesised using the permeate fraction of cell‐free filtrate with MWs lower than 100 kDa are 16 nm and 0.18, respectively. Using un‐treated cell‐free filtrate led to the production of AgNPs with an average size of 25 nm and PDI of 0.27. These results illustrate that un‐treated cell‐free filtrate produced AgNPs of smaller size and narrower size distribution in comparison with the retentate fraction of cell‐free filtrate passed through 10 and 20 kDa cut‐off filters. This observation indicates the importance of low MW biomolecules in controlling the size and size distribution of nanoparticles. As shown in Fig. 5, the smallest AgNPs with the narrowest size distribution were generated using the permeate fraction of cell‐free filtrate passed through the 100 kDa cut‐off filter. Therefore, a wide range of molecules (<100 kDa) of different capabilities are involved in the AgNPs formation with the desired properties. The average size and PDI of the synthesised nanoparticles using cell‐free filtrates in selected cases are presented in Table 1. Fig. 6 shows the SEM image of produced AgNPs using permeate fraction of cell‐free filtrate passed through the 100 kDa cut‐off filter. As shown in Fig. 6, the shape of AgNPs in this case is predominantly spherical. Also, there is no sign of aggregation, indicating the stabilisation of the nanoparticles by secreted proteins.

Fig. 5.

DLS analysis of colloidal AgNPs generated by mixing of AgNO₃

(a) Cell‐free filtrate, (b) Permeate fraction of cell‐free filtrate passed through a 100 kDa cut‐off filter paper, (c) Retentate fraction of cell‐free filtrate passed through a 10 kDa cut‐off filter paper, (d) Retentate fraction of cell‐free filtrate passed through a 20 kDa cut‐off filter paper

Table 1.

Comparison of the AgNP characteristics synthesised by retentate and permeate fractions of cell‐free filtrate of the N. intermedia fungus

Protein's MW, kDa	Average particle size, nm	PDI
non‐treated	25	0.27
higher than 10 kDa	30	0.30
higher than 20 kDa	34	0.45
lower than 100 kDa	16	0.18

Fig. 6.

SEM image of synthesised AgNPs in reaction mixtures containing: AgNO₃ solution and permeate fraction of cell‐free filtrate passed through a 100 kDa cut‐off filter paper

The results obtained in the present paper are compared with the results obtained by our research group, and also data from the literature (Table 2). As presented in this table, using permeate fraction of N. intermedia cell‐free filtrate (passed through a 100 kDa cut‐off filter) in nanoparticle synthesis led to the formation of the smaller nanoparticles with the higher monodispersity, compared with using polysaccharide, modified polysaccharide, and Tollens methods, which makes it as method of choice for further development. The results of this paper are also comparable with the data obtained from the literature, with additional advantages of operating at ambient temperature without adding stabiliser and chemicals.

Table 2.

Comparison of the results obtained in this study with data from the literature

Method	Reducer	Stabiliser	Condition	Temperature, °C	Size range, nm	Average size, nm	PDIa	Reference
biological (using cell‐free filtrate of N. intermedia)	secreted proteins	secreted proteins	non‐treated	ambient	—	25	0.27	present paper
			MW>10 kDa	ambient	—	30	0.30
			MW>20 kDa	ambient	—	34	0.45
			MW<100 kDa	ambient	—	16	0.18
polysaccharide	β ‐D‐glucose	starch	—	80	—	30	0.35	[22, 24]
modified polysaccharide	β ‐D‐glucose	starch	—	121	—	20	0.24
Tollens	β ‐D‐glucose	starch	adding sodium hydroxide and ammonium	ambient	—	42	0.42
irradiation	starch	starch	in microwave	—	<10	NDb	ND	[41]
solid‐state reduction	starch	starch	adding sodium hydroxide	70	—	30	ND	[42]
chemical reduction	ascorbic acid (vitamin C)	starch	adding sodium hydroxide	30	17–30	ND	ND	[43]

^a PDI: polydispersity index.

^b ND: not determined.

3.5 SDS‐PAGE analysis

SDS‐PAGE analysis was performed to identify the pattern of proteins secreted by N. intermedia in both the culture supernatant and cell‐free filtrate before mixing with AgNO₃. The results are shown in Fig. 7 a. The Ag staining method was also employed to visualise the protein bands in the gel. As shown in Fig. 7 a, SDS‐PAGE analysis revealed the presence of predominant bands of proteins ranging from 15 to 70 kDa in both cases. This may be the reason for the lower stability of the synthesised AgNPs using fractions with an MW >100 kDa (i.e. the retentate fraction of cell‐free filtrate passed through a 100 kDa cut‐off filter).

Fig. 7.

SDS‐PAGE analysis of the

(a) N. intermedia secretion in cell‐free filtrate (lane 1), culture supernatant (lane 2), and protein MW marker (15–100 kDa) (lane 3), (b) Secreted proteins resulted by suspending N. intermedia biomass in: distilled water (lane 1), 1 mM potassium nitrate solution (lane 2) for 72 h, 1 mM AgNO₃ solution for 24 h (lane 3), and protein MW marker (10–100 kDa) (lane 4), (c) Secreted proteins resulted by: SDS‐treated AgNPs (lane 1), un‐treated AgNPs (lane 2) and suspending N. intermedia biomass in: distilled water for 72 h (lane 3), and protein MW marker (10–100 kDa) (lane 4)

To identify the specific proteins acting as reducing agents in AgNP biosynthesis, the secreted proteins from the fungal biomass in distilled water, potassium nitrate, and AgNO₃ solutions were compared using SDS‐PAGE analysis. A comparison of the appearance bands in Fig. 7 b indicates the presence of same bands with an approximate MW of 15 kDa. The band width in lane 3 is larger, which confirms greater protein secretion in the presence of Ag⁺. This could be due to direct contact between the biomass and Ag⁺ that lead to greater secretion of specific proteins that act as reducers. Further experiments are needed to identify the molecular structure and characteristics of this protein.

SDS‐treated and un‐treated samples of colloidal AgNPs were analysed by 12% SDS‐PAGE to investigate the protein bound to the surface of nanoparticles. As shown in Fig. 7 c, the SDS‐treated AgNP sample shows the presence of a single band with an approximate MW of 23 kDa, which is similar to the band, appeared in the cell‐free filtrate (lane 3). Thus, it is clear that a specific protein with an MW of 23 kDa operates as a stabilising agent. This protein band does not appear for the un‐treated nanoparticles (line 2). This observation underscores that the types of proteins that are involved in reduction and stabilisation of nanoparticles are different. This also reveals the interactions of proteins and AgNPs that inhibit the protein permeation into the gel.

The mechanism of AgNP biosynthesis by extracellular proteins of A. flavus was studied using SDS‐PAGE analysis. Results revealed that two main bands having MWs of 32 and 35 kDa cause to reduction of Ag⁺ and stabilisation of AgNPs, respectively [44].

A schematic representation of the possible mechanism for AgNP biosynthesis by N. intermedia is shown in Fig. 8, which is based on the results of the current study. As indicated, the biosynthesis of AgNPs may occur in two stages: (i) reduction of a part of Ag⁺ in reaction mixtures to Ag atoms (Ag⁰) by the existing reducing agents and (ii) capping of the as‐synthesised AgNPs follows. A specific protein with MW of 15 kDa, along with participation of other biomolecules and metabolites, may be responsible for reduction of Ag⁺ into nanoparticles at the first step. The second stage includes binding of a 23 kDa protein to synthesised AgNPs as a stabilising agent.

Fig. 8.

Possible mechanism for AgNP biosynthesis using N. intermedia fungus

4 Conclusion

The present paper involved an investigation of the mechanism of AgNP biosynthesis using N. intermedia. The role of proteins in biosynthesis of AgNPs was confirmed by the mixing of a protein‐free culture supernatant and AgNO₃ solution, which resulted in no AgNP formation. The high reaction rate for AgNP biosynthesis in the case of heat‐treated proteins further verifies that the rate of synthesis depends on the interactions of amino acids with Ag⁺. Using an inactive cell‐free filtrate demonstrated higher productivity in comparison with an active cell‐free filtrate due to the higher organic contents of the inactive cells. SDS‐PAGE analysis indicates that two main proteins with MWs of 15 and 23 kDa involved in the synthesis and stability of AgNPs.