Autoclave-assisted green synthesis of silver nanoparticles using A. fumigatus mycelia extract and the evaluation of their physico-chemical properties and antibacterial activity

2.1 Materials

Silver salt (AgNO₃) was purchased from Dr. Mojallali (Dr. Mojallali Chemical Complex Co. Tehran, Iran). Standard solution of AgNPs (with particle size of 10 nm and concentration of 1000 ppm) was obtained from Tecnan-Nanomat (Navarra, Spain).

Aspergillus fumigatus (PTCC 5009) was purchased from microbial Persian type culture collection (PTCC, Tehran, Iran). Potato dextrose agar (PDA) and potato dextrose broth (PDB) as culture media were provided from Oxoid (Oxoid Ltd., Hampshire, England) and Biomark (Biomark Inc. Richmond, Canada). Escherichia coli (PTCC 1395) and Staphylococcus aureus (PTCC 1189) were provided from microbial PTCC (Tehran, Iran). Nutrient agar (NA) was purchased from Biolife (Biolife Co. Milan, Italy). Deionized double-distilled water was used in preparing all aqueous solutions.

2.2 Preparation of A. fumigatus mycelia extract

A. fumigatus was cultured on PDA for 7 days at 25°C. After incubation, the fungal spores were isolated, and fungal spore suspension containing 10⁶ spores/ml was prepared. Spore concentration of spore suspension was evaluated by using a Neubauer’s chamber (haemocytometer), according to the classical procedure. About 1 ml of the provided spore suspension was added into the 100 ml of the PDB and incubated in a shaker incubator (New Brunswick Scientific, Innova 4000, NJ, USA) for 4 days (120 rpm and 25°C). In order to remove any medium traces, the fungal mycelia was separated by filtering through Whatman filter paper No. 1 and then washed thrice with sterile distilled water. Separated wet mycelia was added into 100 ml sterile double distilled water and placed in shaker incubator (120 rpm and 25°C) for 3 days. The mixture was then centrifuged (Sigma 3-18K, Gottingen, Germany) for 5 min at 6500 rpm and filtered through a 0.2 μm micro filter. The filtered A. fumigatus mycelia extract was stored in the refrigerator at 4°C.

2.3 Synthesis of AgNPs using A. fumigatus mycelia extract

According to the literature, silver nitrate solution (1 mm) was prepared by dissolving 0.017 g of its powder in 100 ml of DI water [11], [13], [14]. Different amounts (5–7 ml) of provided mycelia extract with 3 ml of AgNO₃ solution was mixed, after which the mixture solutions were kept in autoclave at 15 psi pressure and 121°C for different times (10–20 min).

2.4 Analysis

2.5 Experimental design and statistical analysis

Central composite design (CCD) with two independent synthesized variables, namely, amount of A. fumigatus mycelia extract (5–7 ml) (X₁) and autoclaved heating time (10–20 min) (X₂), was applied to the design of experiments using the software Minitab v.16 statistical package (Minitab Inc., PA, USA). Response variables were chosen according to the literature [6], [10]. Therefore, 13 experiment runs, including 4 factorial points, 4 star points, and 5 central points, were generated (Table 1) and carried out in 1 day (one block) [16]. In order to estimate the pure error, a central point (X₁: 6 ml and X₂: 15 min) was repeated for five times [17]. Response surface methodology (RSM) was used to evaluate the effects of the independent variables on the response variable, namely, λ_max (Y, nm) of the synthesized AgNPs. The λ_max (broad emission peak) can be correlated to the particle size of the formed NPs as the longer wavelengths correspond to an increase in particle size [18].

A second-order polynomial equation (Eq. 1) was used to correlate the response variables to the studied synthesized variables

where Y is the response variable, A₀ is a constant, A₁ and A₂ correspond to the linear terms, A₁₁ and A₂₂ represent the quadratic terms, and A₁₂ indicates the interaction terms. The suitability of the model was studied while considering the coefficient of determination (R²) and adjusting the coefficient of determination (R²-adj). Analysis of variance (ANOVA) was also carried out to provide the significance determinations of the resulted models in terms of p-value and F ratio. High values of F ratio and small p-value (<0.05) were considered as statistically significant. Based on the fitted polynomial equations, a three-dimensional surface plot was designed to better visualize the independent variable interaction [19]. In order to obtain the optimum levels of independent synthesized variables with the desired response variables, numerical response optimization and graphical optimization using a two dimensional contour plot were applied [20]. For verifying the validity of the statistical experimental approaches, three additional approval tests were performed at the obtained optimum synthesis conditions.

3.1 Characterization of the A. fumigatus mycelia extract

As clearly observed in the FT-IR spectra of the A. fumigatus mycelia (Figure 1), three main absorption peaks were centered at 1638.99, 3438.19, and 3487.81 cm⁻¹ which were in the region range of 400–4000 cm⁻¹. The most wide spectra absorption peaks were observed at 3487.81 and 3438.19 cm⁻¹, which can be attributed to the stretching vibrations of the OH (hydroxyl groups) of saccharides in the fungal cell wall [21]. Furthermore, the peak at 3438.19 cm⁻¹ indicated N-H stretching vibrations in amide linkages of proteins. The band at 1638.99 cm⁻¹, referring to the carbonyl stretch, was assigned to the amide I bond of protein. The results indicated that the presence of the proteins and enzymes can reduce the Ag⁺ ions to atoms and form AgNPs. It seems that the main possible mechanism for the formation of AgNPs by the A. fumigatus mycelia extract is through the nitrate reductases. The obtained results are in agreement with findings of Phanjom and Ahmed [22], who reported that nitrate reductase of A. oryzai is the main reducing agent in the synthesis of AgNPs. Meanwhile, the presence of proteins in A. fumigatus mycelia extract may have been responsible for stabilizing the synthesized NPs [10].

Figure 1:

FT-IR spectra of A. fumigatus mycelia extract.

3.2 Fitting the response surface models

According to the design of experiments, second-order polynomial models were fitted using the response variables obtained from the experimental runs (Table 1). The predictable regression coefficients and the corresponding significance of regressions for the model are given in Table 2. The F ratio and p-values of the all terms in obtained model, which are used to evaluate their effectiveness, are also shown in Table 3.

Given that the overall model performance could be manifested in coefficients of determinations, the resulting quite high values for R² (0.938) and R²-adj (0.862) verified the fitness of the proposed model. Moreover, the achieved non-significant lack of fit for the proposed final model (p>0.05) confirmed its sufficient fitness to the synthesis parameter effects (Table 2). As clearly observed in Table 3, the main and quadratic effects of heating time had non-significant (p>0.05) effects on the λ_max (particle size) of the synthesized AgNPs. However, its interaction with the amount of the A. fumigatus mycelia extract had a significant effect on the λ_max of the synthesized AgNPs.

3.2.1 λ_max of the synthesized AgNPs

The λ_max of the obtained AgNPs ranged from 436 to 482 nm (Table 1). In fact, the particle size of synthesized AgNPs can be manifested in the λ_max of the AgNPs. Generally, the longer wavelengths in the absorption spectra of metal NPs are correlated to their bigger size [23]. The λ_max of the synthesized AgNPs as a function of A. fumigatus mycelia extract and autoclaved heating time is shown in Figure 2. As clearly observed in the figure, at constant heating time, the λ_max of the synthesized AgNPs decreased when the amount of mycelia extract increased. The obtained result can be explained by the fact that increasing the amount of fungal extract increased the concentration of its reducing molecule (nitrate reductase) and stabilizing agents (saccharides and proteins), which in turn, stabilized the molecules, thus creating a layer around the synthesized AgNPs and decreasing AgNP agglomeration [1], [2]. The results also indicated that at higher amount of fungal extract, the λ_max of the synthesized AgNPs decreased when the heating time increased. However, at a lower amount of mycelia extract, heating time did not show a significant (p<0.05) effect on the λ_max of the formed AgNPs. It seems that by increasing the hearing time, the moving speed of the formed AuNPs in the solution was enhanced, which in turn, increased the collision frequency between NPs increased and resulted in their decreased particle size [2]. The presence of a slim curvature in the λ_max curve (Figure 2) confirmed the significant effects of the amount of A. fumigatus mycelia extract and autoclaved heating time.

Figure 2:

Surface plot for λ_max of the synthesized AgNP solution as a function of the A. fumigatus mycelia extract and heating time.

3.3 Optimization of processing parameters for the synthesized AgNPs

The optimum conditions for AgNPs synthesis can be achieved when the process resulted in the formation of the smallest mean particle size (λ_max). Graphical optimization based on a contour plot was used to find the optimum region for the synthesis parameters to produce AgNPs with the minimum particle size (Figure 3). As clearly observed in Figure 3, the minimum λ_max for the synthesized AgNPs was obtained at a higher heating time and higher amount of A. fumigatus mycelia extract.

Figure 3:

Contour plot for λ_max of the synthesized AgNP solution as function of the A. fumigatus mycelia extract and heating time.

Numerical optimization was also used to determine the optimum levels of the studied variables. The results suggested that the synthesis conditions with 7 ml of fungal mycelia solution and 20 min of autoclaved heating time generated the most desirable AgNPs with λ_max of 404.45 nm. Moreover, three AgNPs solutions were prepared according to the recommended optimal levels by numerical optimization and were characterized in terms of the λ_max. The measured experimental value for the λ_max of these three AgNPs solutions was obtained at 407±3 nm. The non-significant differences found between the predicted and experimental values of the synthesized AgNPs at optimum synthesized conditions indicated the adequacy of the obtained and fitted model by RSM.

3.4 Specifications of the synthesized AgNPs at obtained optimum conditions

The appearance of brownish color proved that the AgNPs formed in the reaction mixture solution as a result of the reduction of the Ag⁺ to Ag. Figure 4 indicates the color change of the mixture solution containing AgNO₃ and A. fumigatus mycelia extract at the beginning of the reaction (heating) and after heating and formation of AgNPs. The obtained result is in agreement with the finding of Bhainsa and D’Souza [24], who observed the brownish color of AgNP solution synthesized using A. fumigatus extract. The color change occurred due to the surface plasmon resonance (SPR) of the formed AgNPs (Figure 5). As clearly observed in Figure 4, λ_max of the synthesized AgNPs was obtained at 407 nm, which was in the favorable range for AgNPs (380–450 nm) [15]. The obtained result indicated that the concentration of AgNP synthesized solution obtained at optimum condition was 65 ppm.

Figure 4:

Color and appearance of the mixture solution containing AgNO₃ and A. fumigatus mycelia extract (A) at the beginning of heating and (B) after heating and subsequent formation of AgNPs.

Figure 5:

Surface plasmon resonance spectrum of the synthesized AgNPs at obtained optimum synthesis conditions.

The particle size of the synthesized AgNPs ranged between 18 and 40 nm with an average size of 23 nm. Figure 6 indicates the particle size distribution of the synthesized AgNPs. The PDI and ζ potential values of the synthesized AgNPs using A. fumigatus mycelia extract at obtained optimum synthesized conditions were 0.270 and +35.3 mV, respectively. The obtained results indicated that the more stable AgNPs formed at the optimum synthesized conditions, which were covered with a thin layer of proteins comprising positively charged groups (NH). The obtained results are in agreement with the results of the FT-IR analysis.

Figure 6:

Particle size distribution of the synthesized AgNPs at obtained optimum synthesis conditions.

A typical TEM image of the synthesized AgNPs is shown in Figure 7. As clearly observed, the synthesized NPs were well dispersed with spherical structures; in fact, spherical NPs were more abundant than NPs of other shapes. This spherical shape indicated that the synthesized NPs had minimum surface energy and high thermodynamic stability, thus confirming the high value of the ζ potential of the synthesized AgNPs.

Figure 7:

TEM image of the synthesized AgNPs at obtained optimum conditions.

3.5 Antibacterial activity of the synthesized AgNPs at obtained optimum conditions

The antibacterial activity of synthesized AgNPs on the growth of Gram-positive (S. aureus) and Gram negative (E. coli) bacteria during incubation are shown in Figure 8. As can be seen in the figure, the zones of inhibition were observed with S. aureus (11 mm) and E. coli (9 mm). The obtained results are in agreement with the findings of Ratnasri and Hemalatha [25], and Bala and Arya [26], who found that the synthesized AgNPs using A. fumigatus have strong antibacterial activities against both Gram-positive and Gram-negative bacteria species.

Figure 8:

Created zones of inhibition with (A) S. aureus and (B) E. coli incubated at 37°C for 24 h.