Biogenic synthesis, optimisation and antibacterial efficacy of extracellular silver nanoparticles using novel fungal isolate Aspergillus fumigatus MA

Abstract

To eliminate the elaborate processes employed in other non‐biological‐based protocols and low cost production of silver nanoparticles (AgNPs), this study reports biogenic synthesis of AgNPs using silver salt precursor with aqueous extract of Aspergillus fumigates MA. Influence of silver precursor concentrations, concentration ratio of fungal extract and silver nitrate, contact time, reaction temperature and pH are evaluated to find their effects on AgNPs synthesis. Ultraviolet–visible spectra gave surface plasmon resonance at 420 nm for AgNPs. Fourier transform infrared spectroscopy and X‐ray diffraction techniques further confirmed the synthesis and crystalline nature of AgNPs, respectively. Transmission electron microscopy observed spherical shapes of synthesised AgNPs within the range of 3–20 nm. The AgNPs showed potent antimicrobial efficacy against various bacterial strains. Thus, the results of the current study indicate that optimisation process plays a pivotal role in the AgNPs synthesis and biogenic synthesised AgNPs might be used against bacterial pathogens; however, it necessitates clinical studies to find out their potential as antibacterial agents.

1 Introduction

Nanotechnology is a multidisciplinary field that implicates material scientists, mechanical and electronic engineers, medical researchers, biologists, physicists and chemists to share their knowledge to take it into new realms. Synthesis of silver nanoparticles (AgNPs) is one of the most fascinating areas of research. The excellent biocompatibility and antibacterial property of AgNPs has raised considerable interest as nanoparticles for biomedical applications. Nano‐scaled size (1–100 nm) of AgNPs has a great impact on these silver particles’ physicochemical, optical and electronic [1] characteristics. These nanoparticles’ characteristics are considered ideal for applications such as catalysis, plasmonics, optoelectronics, biological sensors, and for surface‐enhanced Raman scattering, DNA sequencing and antimicrobials application [2, 3, 4, 5, 6]. In medical research, researchers have made many efforts to develop antimicrobial agents that could be used in clinical treatments against pathogenic bacteria. In this respect, AgNPs show potent antimicrobial properties and are used in a variety of products for microbial control [7].

The synthesis of metal nanoparticles is usually carried by chemical reduction method, irradiation and thermal decomposition in organic solvent, but all these methods are toxic and expensive [8]. Hence there is a need to evolve procedures for nanoparticle synthesis through environmentally benign routes which are inexpensive and involve use of an eco‐friendly process. Concerning the various drawbacks in the synthesis of AgNPs, it has been emphasised that biologically mediated synthesis would make the nanoparticles more biocompatible. Earlier reports showed that it can be synthesised by bacteria such as Bacillus subtilis and Enterobacteria [9, 10] fungi, such as Penicillium atramentosum KM and Aspergillus clavatus [11, 12], and plants such as Psidium guajava and Mangifera indica [13, 14]. Fungal‐mediated synthesis of AgNPs possess several advantages over bacteria such as tolerance towards metal ions, easy to culture on a large scale, provision of enough biomass for processing and much higher amounts of protein expressions. In addition, the extracellular synthesis of AgNsP using fungi would make the process easier and simpler for downstream processing [15]. To increase the productivity of AgNPs with minimum investment, it is necessary to optimise the various parameters such as concentration of silver nitrate (AgNO₃), ratio of fungal extract and AgNO₃, time, temperature and pH.

Hence, the objectives of this study are to provide the potential of fungal isolate Aspergillus fumigates MA for biogenic synthesis of AgNPs, to optimise the various physiological conditions and provide an evaluation of their antibacterial efficacy.

2 Material and methods

2.1 Preparation of fungal extract

Aspergillus fumigatus MA fungal strain used in the present investigation was isolated from the tannery effluent [16]. Culture was grown and was maintained on potato dextrose agar plates at 30°C and preserved at 4°C until use. The spores of A. fumigatus MA were inoculated into potato dextrose broth and incubated at 25°C for 72 h on rotator shaker at 120 rpm. After 72 h, the fungal biomass was harvested by using Whatman filter paper no.1 and washed twice with sterile double distilled water to remove any medium component. Ten gram of fungal biomass was mixed with 100 ml of double deionised water and then incubated at 25°C for 72 h and agitated at 120 rpm. After incubation, the fungal filtrate (FF) was obtained by passing this through Whatman filter paper no.1.

2.2 Biosynthesis of AgNPs

AgNO₃ used in this study was of analytical grade and procured from Hi Media Laboratories Pvt. Ltd., Mumbai, India. The aqueous solution of AgNO₃ was prepared in double deionised water in a sterile amber colour bottle and kept at room temperature for 24 h. For the synthesis of AgNPs, fungal extract was mixed with an aqueous solution of AgNO₃ in 250 ml Erlenmeyer flask and incubated in the dark. A control set‐up was also maintained without A. fumigates MA extract.

2.3 Optimisation of process parameter for AgNPs synthesis

Different parameters such as concentration of silver precursor, concentration ratio of fungal extract and AgNO₃, time, temperature and pH were optimised to influence the rate of synthesis of AgNPs.

2.3.1 Effect of silver precursor concentration

AgNO₃ was used as silver precursor and it acts as substrate for the reaction. The effect of the silver salt on nanoparticle synthesis was determined by varying concentration of AgNO₃, i.e. 1, 2, 3, 4 and 5 mM.

2.3.2 Effect of concentration ratio of fungal extract and AgNO₃

The concentration ratio of fungal extract and AgNO₃ varied for the maximum production of AgNPs; the reaction was carried out by using different ratios of AgNO₃ and fungal extract (1:9, 2:8, 3:7, 4:6 and 5:5).

2.3.3 Effect of time

The optimum time for bio‐reduction of pure silver ions was monitored by measuring the UV–visible spectrum (UV–vis) of the reaction medium at 0, 24, 48, 72 and 96 h.

2.3.4 Effect of temperature

The effect of temperature on the synthesis of AgNPs was carried out at 5, 15, 25, 35 and 45°C. The reaction temperature was maintained using water bath.

2.3.5 Effect of pH

Ideal pH selected by incubating the reaction mixture at different pH varied from 2, 5, 7, 9 and 11. The pH was maintained with the help of 0.1N HCl and 0.1N NaOH. Various parameters for optimisation of AgNPs synthesis was monitored by using UV–vis spectrophotometer (Systronic‐115, India) with a resolution of 1.0 nm between 300 and 600 nm.

2.4 Characterisation of synthesised AgNPs

AgNPs were synthesised under optimised conditions in suspension purified twice by centrifugation at 10,000 rpm for 30 min and redispersed the pellet in sterile distilled water and was further analysed using Fourier‐transform infrared spectroscopy (FTIR), X‐ray diffraction (XRD) and transmission electron microscopy (TEM).

2.4.1 FTIR analysis ()

The FTIR spectrum of aqueous solution of AgNPs was recorded using Thermo Scientific Nicolet iS50‐India with resolution at 4.000 from 400 to 4000 nm.

2.4.2 XRD measurements

After drying the purified silver particles, the structure and composition were studied by X‐ray diffractometer. XRD measurement was recorded by using X'Pert Pro PANalytical X‐ray diffractometer instrument. The instrument was operated at a voltage of 40 kV and a current of 40 mA with CuKα radiation. The crystallite domain size was calculated from the width of the XRD peaks using the Scherrer formula

$$D=0.94\lambda /\beta \cos \theta$$

where D is the average crystallite size perpendicular to the reflecting planes, λ is the wavelength, β is the full width at half maximum and θ is the diffraction angle.

2.4.3 Transmission electron microscopy

TEM technique was employed to evaluate the size and shape of synthesised AgNPs using a H‐7500 electron microscope (Hitachi, Japan) at an accelerating voltage of 120 kV. For TEM measurements, a drop of diluted sample of AgNPs was placed on the carbon‐coated copper grids and water was allowed to evaporate.

2.4.4 Sodium dodecyl sulphate (SDS) electrophoresis

SDS‐polyacrylamide gel electrophoresis(PAGE) electrophoresis was carried out to observe the involvement of proteins in the synthesis and stability of AgNPs. Both purified AgNPs suspension and FF were precipitated by adding solid ammonium sulphate (75%w/v). For desalting, the concentrated proteins were washed with sodium phosphate buffer (0.05 M, pH 7.0) at least three times. Afterwards, the samples were dialysed overnight against the sodium phosphate buffer (0.05 M, pH 7.0). Then the protein was investigated by running the samples in SDS‐PAGE as per standard procedure. The sample was dissolved in sample buffer and then heated at 80°C for 10 min. After cooling, the sample was mixed with bromophenol blue and was loaded in the gel. After running the protein sample in the gel, the gel was stained with Coomassie brilliant blue and then observed.

2.5 Determination of antibacterial efficacy

The antimicrobial activities of synthesised AgNPs were determined using the agar well diffusion method. All the test cultures were procured from the Microbial Type Culture Collection Centre (MTCC), Chandigarh, India. Antibacterial efficacy of AgNPs was tested against Bacillus cereus (MTCC‐1305), Staphylococcus aureus (MTCC‐3160) Salmonella typhimurium (MTCC‐1253), Aeromonas hydrophila (MTCC‐1739), Enterobacter aerogenes (MTCC‐2823) and Micrococcus luteus (MTCC‐1809). Cultures were maintained at 4°C on nutrient agar (Hi‐Media, India) and inoculated into the Muller–Hinton Agar plates for antibacterial assay. Then the 20 μl nanoparticles suspension was poured into the well with the help of a micropipette. The plates were then incubated at 37°C for 24 h and zone of inhibition was measured.

2.6 Statistical analysis

Each experiment was carried out in triplicate and results are presented as the mean ± standard deviation in the respective figures.

3 Results and discussion

As the A. fumigatus MA extract is mixed with aqueous solution of the AgNO₃, it changes from colourless to a yellowish brown due to reduction of silver ion, indicating the formation of AgNPs (Fig. 1). The colour changes are due to the surface plasmon resonance exhibited by the AgNPs [17]. It is evident that the shape and size of metal nanoparticles produced from metallic precursor in solution depends on various reaction conditions such as concentration of metal precursor, ratio of metallic precursor/reducing agent, time, temperature and pH [18]. The fungus‐mediated reduction may be due to the presence of biomolecules such as amino acids, enzymes/proteins, polysaccharides and vitamins found in fungal extract. The mechanism of fungal‐mediated synthesis of AgNPs has not as yet been fully understood; however, a few earlier reports by Ahmad et al. (2003) and Duran et al. (2007) [19, 20] suggested that plausible proteins such as nitrate reductase and nicotinamide adenine dinucleotide hydride‐dependent reductase are responsible for the reduction of silver ions into AgNPs by using fungal extract of Fusarium oxysporum. UV–vis absorption spectrum of AgNPs formation exhibited absorbance peak at 420 nm. Similarly Rai et al. (2010) and Jain et al. (2010) [21, 22] reported peaks in the range of 400–450 nm by using fungal extract of Phoma spp. and A spergillus flavus NJP08. The studies of Sastry et al. (2003) [23] demonstrated that the peak at 420 nm indicated the sizes of AgNPs in the range of 2–100 nm.

Fig. 1.

Erlenmeyer flask containing cell‐free filtrate of A. fumigatus MA without (A) and with (B) AgNO₃ solution (3 mM) after 72 h of reaction

3.1 Effect of silver precursor concentration

3 mM concentration of AgNO₃ gave a sharp and characteristic absorption peak at 420 nm in UV–vis spectrum; however, there is a shift in peak positions when using 1, 2, 4 and 5 mM concentrations (Fig. 2). Thus the optimum concentration of AgNO₃ was found to be 3 mM.

Fig. 2.

Effect of concentration of AgNO₃ on production of AgNPs

Similar results are reported by El‐rafie et al. (2012) and Wei et al. (2011) [24, 25] for synthesis of AgNPs by using Fusarium solani and Bacillus amyloliquefaciens, respectively. The dependence of conversion of silver ions to AgNPs on the concentration of AgNO₃ could be associated with the amount of proteins that exist in the fungal extract. By increasing the concentration of AgNO₃ up to 3 mM, there are enough proteins and enzymes in the extract to enable the reduction of all AgNO₃ to AgNPs. Further increase in concentration might have some toxic effects on A. fumigatus MA extract.

3.2 Effect of concentration ratio of fungal extracts and AgNO₃ solution

The ratio of 5 ml of fungal extract along with 5ml of AgNO₃ showed characteristic trough at 420 nm; on the other hand, the spectra got shifted at concentration ratio of 1:9, 2:8, 3:7 and 4:6 (Fig. 3). This indicated that equal concentration of fungal extracts and AgNO₃ (5:5) was optimum for AgNPs synthesis.

Fig. 3.

Effect of ratio of concentration of A. fumigatus MA extract and AgNO₃ on production of AgNPs

Our results found accordance with that of Vaidyanathan et al. (2010) [17] where they optimised the synthesis of AgNPs by using Bacillus licheniformis. Optimisation of extract led to the enhanced synthesis of AgNPs. The synthesis was found to be dependent on the enzymes and proteins activity. This has revealed that increases in the amount of extract enhance the enzymes and proteins that accelerate the reduction rate of silver ions.

3.3 Effect of time period

UV–vis spectra showed strong and characteristic absorption peak at 420 nm after 72 h; however, after 72 h no change in the peak was observed. Therefore, an optimum 72 h are required for obtaining the desired metal nanoparticles (Fig. 4).

Fig. 4.

Effect of time on production of AgNPs

The results agreed with previous work carried out by Jain et al. (2010) and Mukherjee et al. (2001) [22, 26] using A. flavus NJP08 and Verticillium showed that 72 h gave enough time for maximum productivity of AgNPs. Previously some authors had reported some variations in the fungal extract incubation periods and their UV spectra recorded for the biosynthesis of AgNPs by using different fungi. For example, Ingle et al. (2009) [27] and Ranjan and Nilotpala. (2011) [28] reported that the synthesis of AgNPs by the fungus F. solani and P. purpurogenum NPMF occurred at an incubation time ranging between 2 and 3 h and 24 h, respectively. Such variations in biosynthesis of AgNPs might be due to the selected fungal species and the culture conditions applied. The reduction of silver ions to AgNPs dependence on time indicates that the increase in incubation time will enhance the rate of reduction.

3.4 Effect of temperature

UV–vis spectra recorded at different temperature showed a sharp peak at 25°C. This indicated that the enzyme present in extract of A. fumigatus MA showed maximum activity at 25°C (Fig. 5). These findings are in similarity with other previous studies using different fungal species such as A. flavus NJP08, Verticillium and Penicillium sp. for the synthesis of AgNPs [22, 26, 29]. The authors found that temperature of 25°C showed maximum productivity of AgNPs. This could be associated with the enzyme stability that exists in the fungal extract. Result suggested that increase and decrease of temperature could decrease the reduction process. This might be due to the inactivation of the degradation of proteins present in the extract that are responsible for the reduction of silver.

Fig. 5.

Effect of temperature on production of AgNPs

3.5 Effect of pH

A sharp peak was observed at pH 7. However, there was no sharp peak at acidic pH (2 and 5) as well as basic pH (9 and 11). This indicated that pH 7 was optimum for the completion of the reaction (Fig. 6). Similar change in absorption spectra with pH reported by Singh et al. (2014) and Krishanraj et al. (2012) – by using endophytic fungi Penicillium spp. and Acalypha indica leaf extract – indicated that the maximum productivity of AgNPs occur at pH 7 [29, 30]. This is not surprising as each enzyme has an optimum pH for its maximum activity. The enzymes secreted by the fungus A. fumigatus MA is stable at neutral pH; at higher pH and lower pH, the enzymes did not show any functional activity for synthesis of AgNPs.

Fig. 6.

Effect of pH on production of AgNPs

3.6 FTIR analysis

FTIR measurements of the purified AgNPs suspension were carried out to identify the possible interactions between silver and bioactive molecules that may be responsible for synthesis of AgNPs. The FTIR spectrum of AgNPs showed three distinct peaks – at 462.56, 1643.42 and 3360.52 cm⁻¹ (Fig. 7). The FTIR spectrum of AgNPs showed a trough in the region 3360.52 cm⁻¹ that refers to the stretching vibrations of primary amines while at 1643.56 cm⁻¹ is due to the carbonyl stretch vibrations in the amide linkages of proteins and 462.56 cm⁻¹ is the fingerprint. The carbonyl groups of amino acid residues and peptides have a strong ability to bind to silver. The broad trough indicated the presence of amino groups that are plausible proteins. Similar observations are also reported by Huang et al. and Sastry et al. [23, 31]. These observations indicated that the presence and binding of proteins with AgNPs can lead to their plausible stabilisation. FTIR results are evidence that secondary structures of proteins have not been affected by the reaction with silver ions or binding with AgNPs.

Fig. 7.

FTIR spectrum of synthesised AgNPs

3.7 XRD analysis

The XRD pattern shows four peaks in the spectrum of 2θ values, ranging from 20 to 80. XRD spectra of pure crystalline silver structures matched with that by the Joint Committee on Powder Diffraction Standards (file nos. 04‐0783 and 84‐0713). This was evident from the peaks at 2θ values of 36.45°, 59.47°, 62.58° and 67.53° corresponding to 111, 200, 220 and 311 planes for silver, respectively (Fig. 8).

Fig. 8.

XRD pattern of synthesised AgNPs

Our result clearly indicated the crystalline nature of the synthesised AgNPs. These results are similar to previous work where the crystalline nature of synthesised AgNPs have been reported by using different fungi [19, 32, 33].

3.8 Transmission electron microscopy

A TEM micrograph captured for the AgNPs film spread over a carbon‐coated copper grid is presented in Fig. 9. From the micrographs, the synthesised AgNPs appeared to be spherical with a size range of 3–20 nm. TEM is being preferably used to determine the morphology and size of nanostructures. AgNPs size ranges of 5–60, 5–25, 5–105 and 5–40 nm have been reported for Fusarium spp. [19, 34, 35], Aspergillus spp. [21, 33, 36], Penicillium spp. [28, 29, 37] and Trichoderma viride [32], respectively.

Fig. 9.

TEM micrograph showing AgNPs

3.9 SDS electrophoresis

Protein concentration was in the same range for both the FF and synthesised AgNPs suspension (AgNPs). The proteins of 98, 80, 66, 43, 30, 28, 20 and 6 kDa were observed in both FF and AgNPs suspension during electrophoretic runs (Fig. 10).

Fig. 10.

SDS‐PAGE analysis of proteins secreted from A. fumigatus MA. Lane 1 (molecular size marker 97.4 kDa phosphorylase b; 66 kDa bovine serum albumin; 43 kDa, ovalbumin; 29 kDa carbonic anhydrase; 18.4 kDa b‐lactoglobulin; 6.5 kDa aprotinin). Lane 2, purified extracellular proteins from FF. Lane 3, purified AgNPs with bound proteins

Similar protein bands in both A. fumigatus MA and AgNPs suspension indicated that proteins present in FF remained unchanged during the formation of the nanoparticles. Furthermore, it is possible to state that these proteins stabilise the synthesised AgNPs by capping to their surfaces [38].

3.10 Determination of antibacterial efficacy

The synthesised AgNPs show significant antimicrobial activity against bacterial pathogens B. cereus, S. aureus, S. typhimurium, A. hydrophila, E. aerogenes and M. luteus (Fig. 11). Synthesised AgNPs showed highest antimicrobial activity against B. cereus, and A. hydrophila showed a zone of inhibition of 20 nm and 22 mm. Our study revealed the broad spectrum antibacterial efficacy of the AgNPs against Gram‐negative and Gram‐positive pathogenic bacteria. The AgNPs exposure to six different test bacteria resulted in formation of the zones of inhibition, whereas no visible zones were observed with the use of only deionised Milli‐Q water.

Fig. 11.

Antimicrobial activities of AgNPs against bacteria. All values represented in the table are average of results of three separately conducted experiments

Silver ions exhibit an antibacterial effect by denaturing the cellular proteins, inhibition of DNA replication and by the alteration in cell membrane permeability. The efficient antibacterial property of AgNPs compared with the silver precursors is due to their large surface area that provides better contact with the bacteria. Fayaz et al. (2007) [32] investigated antimicrobial activity of AgNPs against Salmonella typhi, Escherichia coli, S. aureus and M. luteus. Our findings of antimicrobial activity have synergistic effects that corroborate with the earlier reported study. The AgNPs release silver ions into the bacterial cells and enhance their bactericidal activity [5, 6, 7].

4 Conclusion

An eco‐friendly and low‐cost procedure for biosynthesis of AgNPs using water filtrate of A. fumigatus MA is demonstrated. UV–vis absorbance spectral analysis confirmed the biosynthesis of AgNPs. FTIR and XRD provided additional evidence of the presence and crystalline nature of AgNPs. TEM was used to validate the morphology and size of AgNPs. Furthermore, biogenic synthesised AgNPs displayed a pronounced antimicrobial activity against different pathogenic bacteria. Thus, the present investigation showed a simple, rapid and environment‐friendly route for synthesising AgNPs. The results from this study clearly indicated that the optimisation process played a crucial role in the silver precursor reduction and nanoparticles synthesis.