Extracellular synthesis of silver nanoparticles using entomopathogenic fungus: characterization and antibacterial potential
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Present study involves the simple, rapid, non-toxic and in vitro method of extracellular silver nanoparticles synthesis using Entomopathogenic fungus (Beauveria bassiana). The development of silver nanoparticle in fungal supernatant was confirmed by the absorbance peak at 450 nm in UV–Vis spectrophotometer. Further, presence of AgNPs and its crystal lattice was confirmed by EDS and XRD, respectively. TEM micrograph confirmed the presence of differently shaped (triangular, circular, hexagonal) nanoparticles with size ranging from 10 to 50 nm. Variable shape and size of fungal assisted AgNps was also confirmed in SEM study. The optimal pH and temperature for biosynthesis of nanoparticles was found to be 6.0 and 25 °C, respectively. The continuous effects of AgNPs against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus in time dependent manner was confirmed in growth kinetic studies. During 36 h of growth study, maximum reduction in O.D560 was found in E. coli (67.2%) followed by P. aeruginosa (63.3%) and S. aureus (56.8%) at 30 °C. The MIC values of fungal assisted AgNPs against E. coli, P. aeruginosa and S. aureus was found to be 2.5, 3 and 4.5 ppm, respectively. The MIC values of Ciprofloxacin was observed to be 0.5, 0.5 and 0.7 ppm, whereas MICs of AgNPs + Ciprofloxacin showed 0.4, 0.4, 0.5 ppm against E. coli, P. aeruginosa and S. aureus, respectively, clearly highlighting the synergistic effect of AgNPs in combination with Ciprofloxacin. In the view of challenges for developing antimicrobial nanoparticles of variable shape and size by various other methods, tuning nanoparticles synthesis via fungi can be a wonderful approach to resolve existing hurdles.
KeywordsSilver nanoparticles Entomopathogenic fungi Antimicrobial Antibiotic
The term “nano” is derived from a Greek word which means “dwarf” [1, 2]. Synthesis of nanoparticles has gained attention due to their applications in multiple fields such as antifungal agents, antibacterial agents, biosensor, bioremediation, anticancer, drug delivery, etc. [3, 4, 5, 6, 7, 8, 9, 10]. In addition to this, unique properties of nanoparticles such as size, shape, surface to volume ratio, high catalytic activity, etc. make them a unique candidate for multiple applications described above. The synthesis of nanoparticles via biological means has proved to be the better mode as compared to chemical and physical methods . Chemical methods involve toxic chemicals and may generate hazardous or toxic by-product during synthesis, while physical methods have low efficiency and involves high consumption of energy to maintain optimal pressure and temperature during the synthesis process [11, 12]. On the other hand, biological methods for the synthesis of silver nanoparticles provides many advantages i.e., environmental friendly, non-toxic and easy to scale up for large-scale synthesis [13, 14, 15]. Researcher across the world utilized biological entities such as plant (leafs, flower, seeds), bacteria, algae and fungi for the production of different type of metallic nanoparticles such as gold, silver, cadmium, iron, etc. and studied their applications in the field of medical, bioremediation, drug delivery etc. [16, 17, 18, 19].
Among other metal nanoparticles, synthesis of silver nanoparticles (AgNPs) via biological mean gain wide acceleration due to their profound applications in medical application, textile dye removal, wastewater treatment, biological sensors etc. [16, 20, 21, 22]. Synthesis of AgNPs via fungi gain more attention because they produce large amount of biomass in a short period of time under lab conditions and produce large amount of extracellular protein that help in synthesis of nanoparticles [23, 24]. Dhanaraj et al.  observed the AgNPs synthesis potential of fungi Aspergillus niger and observed significant anti-bacterial properties of AgNPs against Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, and Salmonella typhi. In another study, Verma et al.  investigated the antimicrobial potential of AgNPs synthesized through Aspergillu sclavatus against Candida albicans, Pseudomonas fluorescens and Escherichia coli. Results clearly indicated anti-microbial activity with average minimum inhibitory concentration of 5.83 µg mL−1 against above microbial pathogens. However, the literature discussing about synthesis of AgNPs using entomopathogenic fungus and antimicrobial potential of synthesized nanoparticles is lacking [26, 27]. Most of the studies investigates larvicidal and insecticidal properties of these fungi . Synthesis of AgNPs using entomopathogenic fungus provides additional advantages as they are non-pathogenic and insecticidal in nature. Earlier reports have highlighted the insecticidal potential of B. basssiana (entomopathogenic fungi) AgNPs. As per our findings, only one report has studied the antimicrobial efficacy of B. bassiana AgNPs against Staphylococcus aureus and Escherichia coli using well diffusion assay .
The present study investigated the extracellular AgNPs synthesis using Beauveria bassiana (entomopathogenic fungus) along with their antimicrobial potential. The antimicrobial activity of silver nanoparticles was compared with the antibiotic and AgNPs-antibiotic combination. The present report is the first investigation on the kinetic study of B. bassiana synthesized AgNPs against gram positive and negative bacteria at different time intervals with additional studies on AgNPs and antibiotic combination. Further, AgNPs were characterised via Transmission electron microscopy (TEM) equipped Energy dispersive spectrometer (EDS), Scanning electron microscopy (SEM), X-ray diffraction (XRD) and zeta potential.
2 Materials and methods
2.1 Micro-organisms and growth conditions
In the present work, Beauveria bassiana was obtained from Institute of Microbial Technology (Chandigarh, India). The strain was stored at 4 °C on potato dextrose agar slant. Prior to experiments, culture was reactivated on potato dextrose agar at 25 °C for 72 h . Culture of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus stored at 4 °C on nutrient agar slant. Prior to experiments, fresh culture of each bacterial strain was prepared on nutrient broth at 37 °C.
2.2 Extracellular synthesis of silver nanoparticles
Spores from freshly prepared slants of B. bassiana was inoculated to a liquid composite media 100 mL (NH4NO3, 0.5 g L−1; MgSO4·7H2O, 0.1 g L−1; K2HPO4, 0.5 g L−1; NaCl, 1 g L−1; Glucose, 10 g L−1; Yeast extract, 2.5 g L−1) in an Erlenmeyer flask (250 mL) . The flask was incubated at 30 °C at 150 rpm for 72 h (Orbitek, India). The biomass was harvested after 72 h of growth by using Whatman No. 1 filter paper. After harvesting the biomass, extensive washing with distilled water was performed to remove any media components from the biomass. Ten gram of above wet biomass was taken into an Erlenmeyer flask (250 mL) containing 100 mL distilled water and flask was incubated for 48 h at 25 °C and 150 rpm. After 48 h, the filtrate from the flask was obtained by passing flask content through Whatman No. 1 filter paper. The filtrate was mixed with 1 mM silver nitrate (AgNO3) in the ratio of 1:9 (v/v) to obtain the desired reaction mixture. Afterward, reaction mixture was incubated at 25 °C in dark for 30 min at 100 rpm. Control (without AgNO3) was incubated under the same conditions.
Colour change of the above reaction mixture was the first indicator of nanoparticle synthesis. The reaction mixture with silver nanoparticles (AgNPs) turned brown in colour and it was stored under ambient dark conditions for further analysis and experiments.
2.3 Optimization of physio-chemical parameters for AgNPs synthesis
Effect of temperature and pH on the synthesis of AgNPs was investigated by varying the the temperature (15–40 °C) and pH (3–9). To obtained the desired pH value of fungal extract NaOH and HCl was used prior to the addition of silver nitrate. Sample of 1 mL was withdrawn after 30 min and the absorbance was measured at 450 nm.
2.4 Characterization of AgNPs
2.4.1 Ultraviolent–visible spectroscopy
The Ultraviolet–visible spectroscopy analysis of freshly prepared AgNPs was obtained using the Agilent spectrophotometer with water as control. Samples from reaction mixture for UV–Vis spectra were withdrawn at a regular interval of 10 min for overall reaction time of 30 min. The spectra were recorded in a wavelength ranging from 300 to 1000 nm at a resolution of 0.5 nm.
2.4.2 Zeta potential-dynamic light scattering
Zeta potential was used to determine the charge that exists between the AgNPs using Nano ZS90 Zeta sizer (Malvern Instruments) equipped with He–Ne laser (633 nm, 5 Mw).
2.4.3 Transmission electron microscopy and energy dispersive spectrometer
To determine the size and the shape of the AgNPs, Transmission electron microscopy was performed. The sample for TEM was prepared by dropping the solution of AgNPs on the carbon side of carbon-copper grid. After drying for 30 min, grid was placed on the sample holder and micro-graphs were obtained for size and shape analysis. For TEM images, sample was analysed via EOL JEM-1400. On the other hand, Energy dispersive micrograph was obtained by Bruker-ASX (Model QuanTax 200) was used.
2.4.4 Scanning electron microscopy
The nanoparticle sample was lyophilized and characterized further to study the morphological features using scanning electron microscopy (ZEISS EVO 50).
2.4.5 X-ray diffraction (XRD) measurement
XRD (X-ray diffraction) was conducted for the nanoparticles using Cuka radiation (k = 1.5406 Å) via X’ Pert PRO Philips Analytical-PW 3040/60. The sample was examined at 40 kV with 30 mA at a 2 h angle pattern and was scanned in the region of 25°–80°. The micro-graph obtained after scanning was compared with the Joint committee on Power Diffraction Standards Library to establish the crystalline structure of nanoparticles.
2.4.6 Antimicrobial activity of AgNPs and antibiotic
Three different species of bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) were tested for their susceptibility to the antimicrobial properties of fungal derived AgNPs in term of growth kinetic and Minimum inhibitory concentration (MIC). Effect of AgNPs on growth kinetic of bacterial strain was evaluated in 100 mL sterilized nutrient broth containing 1 ppm of AgNPs and inoculated with one mL bacterial strain (106 CFU/mL). The media was incubated at 30 °C at 150 rpm. Control without AgNPs was incubated under the same conditions. The flask content (0.2 mL) was withdrawn at a regular interval of 2 h for 36 h and optical density of the sample was measured at 560 nm to obtained growth curve. To determine the minimum inhibitory concentrations (MICs) for all three bacteria against AgNPs, antibiotic (Ciprofloxacin) and AgNPs-Antibiotic combination, nutrient broth was added with different concentration of AgNPs (0.5–10 ppm) and antibiotic(0.5–10 ppm), followed by bacterial inoculation. To determine the combined effects AgNPs-Ciprofloxacin different concentration of AgNPs and antibiotic ranging from 0.5 to 10 ppm was used in ratio of 1:1. All the experiment was performed on 96-well titre plate which was incubated at 30 °C after inoculation. The plate was evaluated visually for biofilm formation after 36 h. Minimum inhibitory concentration was expressed as the lowest AgNPs concentration at which no visible growth was observed.
3 Results and discussion
3.1 Extracellular synthesis of sliver nanoparticles
3.2 Characterization of AgNPs
3.3 Zeta potential of AgNPs
3.4 Optimization of physio-chemical parameters for AgNPs synthesis
3.5 Antimicrobial activity of AgNPs (MIC and growth kinetic)
Minimum inhibitory concentrations (MICs; ppm) of AgNPs, Ciprofloxacin and AgNPs-Ciprofloxacin
Minimum inhibitory concentration (ppm)
The present study clearly demonstrated the potential of selected Entomopathogenic fungus, B. bassiana for development of extracellular silver nanoparticles (AgNPs). Various analytical tools were used for characterisation of synthesised AgNPs i.e., UV–vis spectrophotometer, Zeta-DLS, TEM, SEM, XRD etc. The techniques used for characterisation showed synthesis of AgNPs in fungal supernatant with variable shapes (triangular, spherical, hexagonal) and size range of 10–50 nm, the synthesized nanoparticles were found to be stable under fungal solution (Zeta: − 22 mV). The fungal assisted AgNPs showed time dependent increase in antimicrobial efficacy against S. aureus, P. aeruginosa and E. Coli, moreover synergistic effect was also observed with antibiotic, Ciprofloxacin against all the bacterial strains. Further, as development of AgNPs was performed with eco-friendly approach, it can be utilized in various sectors where safety is the primary concern i.e., environment and healthcare.
Authors gratefully acknowledge CST, Uttar Pradesh [Grant no: CST/8276 (young scientist scheme) and CST/1586] for funds. We are thankful to the Department of Textile Engineering (IIT Delhi) and CRF facility (IIT Delhi) for their technical support in EDS and TEM analysis. Guidance received by Prof. Anushree Malik, CRDT, IIT Delhi is acknowledgeable.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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