1. INTRODUCTION:

Nanotechnology involves engineering the materials at the molecular and sub-molecular level. Nanotechnology is a friendly manufacturing process ultimately leading to atomically precise molecular manufacturing with zero waste. It mainly focuses on developing natural as well as synthetic systems for the production of structures and materials at nano-scale [1]. Unique physical properties such as good electrical conductivity, optical band gap, refractive index and magnetic properties, and superior mechanical properties such as hardness of nanomaterials and chemical properties has led to the extensive studies on the nano-sized materials. These unique properties arise due to the small size and large specific surface area. Nanomaterials are useful in developing diagnostic tool, drug delivery system, sunscreens formulation, antimicrobial bandages, disinfectants, nanobiosensors, as catalyst for greater efficiency in current manufacturing process by minimizing the use of toxic materials and an alternative energy production [2].

Nanoparticles and nanomaterials are synthesized by physical, chemical, mechanical and biological methods. Synthesis of nanomaterial is done based on three strategies (i) liquid phase synthesis (ii) gas phase synthesis (iii) vapor phase synthesis. Methods such as co-precipitation [3], sol-gel processing [4], micro-emulsions processing, hydrothermal/ solvo-thermal processing [5], microwave processing, sono-chemical processing, and template processing, high temperature solid state reaction, high energy ball milling [6-8], liquid mix process [9], rapid quenching process [10], thermal plasma [11], UV irradiation and lithography are used for synthesizing nanoparticles. These methods are expensive, toxic and involve the use of harmful chemicals apart from other complexities like low stability of the produced nanoparticles and aggregation of the particles [12]. Hence, there is an increasing need to develop high-yield, low cost, nontoxic, and environmentally benign procedures for synthesis of metallic nanoparticles. The simplest method used for producing the nanoparticle is the reduction of their respective salts [13]. Therefore, the biological approach for synthesis of nanoparticles became important. A vast array of biological resources available in nature including plants and plant products, algae, fungi, yeast, bacteria, and viruses are employed for synthesis of nanoparticles. Both unicellular and multicellular organisms have been used to produce intracellular or extracellular inorganic nanomaterials [14]. Mostly fungi are chosen instead of bacteria because of their tolerance and better metal bioaccumulation ability [15]. Other advantages include the ease in the scale up process, economic viability, ability to secrete large amount of enzymes and ease in handling the biomass [16].

Zinc oxide nanoparticles synthesis is mainly concentrated because of its wide band gap and large excitation energy. It is used to create various nanostructures, nanowires, nanotubes, nanorods, nanoribbons, nanoneedles, nanocables etc. Zinc oxide nanoparticles are used in many devices such as solar cells, batteries, photodetectors, nanolasers, varistors and biosensors [2]. They are used in making transparent UV protection film and it is used as electrostatic dissipative coating [17]. Indiscriminate use of antibiotics has led to the biogenesis of mutant strains of microbes that are resistant to antibiotics and antimicrobial drugs. This has triggered and increased the need for studying the antimicrobial activity of nanoparticles and to prove it as an efficient antimicrobial agent. Zinc is an essential element, levels above threshold level inhibits bacterial enzymes such as glutathione reductase, thiol peroxidase, dehydrogenase etc there by acts as an antibacterial agent [18]. The synthesis of zinc oxide nanoparticles using culture filtrate of Aspergillus terreus, characterization of the synthesized nanoparticles and antifungal activity towards Aspergillus niger, Aspergillus fumigatus and Aspergillus aculeatus using well diffusion method were reported here.

2. MATERIALS AND METHODS:

2.1. Fungi used and growth conditions:

The fungus Aspergillus terreus was obtained from Institute of Microbial Technology, Microbial Type Culture Collection and Gene Bank, Chandigarh, India. The fungus was subcultured by growing on Czepak agar slants for 96 h at 32°C and refrigerated at 4°C.

2.2. Materials used:

Luria Bertani broth, mycological peptone, glucose and agar were purchased from Himedia, Mumbai, India. A. niger, A. fumigatus and A. aculeatus were obtained from Institute of Microbial Technology, Microbial Type Culture Collection and Gene Bank, Chandigarh, India..

2.3. Mycological synthesis of zinc oxide nanoparticles using Aspergillus terreus:

The fungus Aspergillus terreus was grown aerobically in 500 ml Erlenmeyer flasks containing 200 ml Capek-Dox liquid medium. The culture was agitated in an orbital shaker at 160 rpm at 32°C for 4 days. The fungal culture was filtered under vacuum through Whatman #2 filter paper. 1 mM ZnSO₄salt was added to the filtrate and kept in a shaking incubator at 150 rpm at 32°C for 2 days. White precipitate deposition started to occur at the bottom of the flask which indicated the transformation process. The white aggregate formed in the bottom of the flask was separated from the filtrate by centrifugation at 10,000 rpm for 10 min and lyophilized.

2.4. Characterization of zinc oxide nanoparticles synthesized by Aspergillus terreus:

Various properties of the synthesized zinc oxide nanoparticles were investigated. Optical properties of the nanoparticles were analyzed using from UV-Visible spectroscopy. UV-Visible spectrum was recorded on SYSTRONICS Double Beam UV-Visible spectrophotometer 2201. The spectrum values were obtained between the wavelength range 200 to 900 nm. The characteristic functional groups present in the molecules of synthesized nanoparticles were analyzed using Fourier Transform-Infra Red (FT-IR) spectroscopy. FT-IR spectroscopy was measured on BRUKER α-T FT-IR Spectrometer. The samples were mixed with KBr (binding agent) and were made into discs at high pressure using hydraulic press. These discs were scanned in the range of 500 to 4000 cm^-¹to obtain FT-IR spectra. Structural characterization was analyzed in order to obtain information about particle size, crystal structure and surface morphology using X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM). XRD patterns were recorded on a XPERT-PRO diffractometer. This diffractometer uses Cu-K as an anode, acts as a X-Ray source (wavelength = 1.54060 Å), operating with Cu- tube radiation at 40 Kv and 30 mA. The scan step for 2θ was 0.0170° with a scan step time of 38.1s. Size of the zinc oxide nanoparticles were examined under QUANTA 200 SEM, magnification range 35 to 30,000.

2.5. Preparation of inoculum culture for antimicrobial activity:

Mycological peptone dextrose agar (MPDA) was sterilized and test tube slants were prepared. These test tubes were inoculated with A. niger, A. fumigatus and A. aculeatus and incubated at 37°C for 3 days then used as inoculum culture to study the antifungal activity of zinc oxide nanoparticles.

2.6. Antimicrobial activity of zinc oxide nanoparticles:

Antifungal activity of zinc oxide nanoparticles towards A. niger, A. fumigatus and A. aculeatus was studied using well diffusion method. MPDA plates were inoculated with all these three fungal cultures by streak plate method. Fungal agar plates were inoculated with fungus grown on the slants using sterilized inoculation loops. A well of 5 mm diameter was made in the middle of the plate using gel puncher. 100 µl of control (lacking zinc oxide nanoparticles) and sample (containing zinc oxide nanoparticles) was added to the inoculated petriplates. The inoculated fungal plates were kept at 37°C for 2 days. The zone of clearance was checked in order to find the antifungal properties of zinc oxide nanoparticles.

3. RESULTS AND DISCUSSION:

The zinc oxide nanoparticles synthesized using A. terreus culture filtrate was characterized by UV-Visible spectrophotometer, FT-IR, SEM and XRD and the results are discussed.

3.1. Visual observation of zinc nanoparticles synthesized by A. terreus:

On mixing the A. terreus culture filtrate with the aqueous solution of zinc sulphate, the color of the culture filtrate was changed rom colourless solution to turbid liquid after 48 hrs. This color change is an indication of reduction of the zinc sulphate ions by the proteins present in fungal culture filtrate which resulted in the formation of white aggregates of zinc oxide nanoparticles as shown in Fig. 1.

Fig. 1. Visual confirmation of zinc oxide nanoparticles (B-Blank, S-Zince oxide nanoparticle sample)

3.2. UV spectrum of zinc oxide nanoparticles synthesized by A. terreus:

The findings from the UV-Visible absorption spectrum were considered as a novel technique for preparation of the monodispersed nanoparticles, in this case it is was used to confirm the presence of zinc oxide nanoparticles. An absorption peak obtained at 340 nm as shown in Fig. 2 confirmed the presence of zinc oxide nanoparticles in culture filtrate.

3.3. FT-IR spectrum analysis of zinc nanoparticle synthesized by A. terreus:

The synthesized zinc nanoparticles were subjected to FT-IR analysis to detect the various characteristic functional group associated with the synthesized nanoparticles. The peaks indicate the characteristics functional group present in the synthesized zinc oxide nanoparticles. It is inferred from Fig. 3 that the samples have absorption peaks in the range of 3687.81 cm^-1, 3075.71 cm^-1, 2927.80 cm^-1, 1675.05 cm^-1, 1538.30 cm^-1, 1385.57 cm^-1, 1269.69 cm^-1, 1051.99 cm^-1, 859 cm^-1and 568.77 cm^-1. The absorption peak at 568 cm^-1corresponds to metal-oxygen (ZnO stretching vibrations) vibrational mode. The peak at 1052 cm^-1is ascribed to the stretching vibration of C-N bond of the primary amine or to the stretching vibration of the C-O bond of the primary alcohol. The peak at 1269 cm^-1and 1385 cm^-1 are ascribed to primary, secondary alcohol in-plane bend or vibrational modes of aromatic primary amine and trimethyl, tertiary alcohol, organic sulphate. The peaks at 1538 cm^-1and 1675 cm^-1are ascribed to the vibrational modes of aromatic nitro compounds and alkyl C=C stretch, amide, open chain imino group. The presence of these functional makes the synthesized zinc oxide nanoparticles as effective antimicrobial agent.

Fig. 2. UV Speectrum of synthesized zinc oxide nanoparticles

Fig. 3. The smoothed FT-IR spectrum of synthesized zinc oxide nanoparticles

3.4. XRD analysis of zinc nanoparticles synthesized by A. terreus:

The X-ray diffraction patterns of ZnO nanoparticles is shown in Fig. 4. Sharper and stronger diffraction peaks were observed from Fig. 4. at 29.38°, 31.84°, 38.87°, 42.55° and 47.84°. The shift in the 2θ peak values of ZnO nanoparticles may be due to the presence of preotein molecule from fungal culture filtrate. The average crystallite size was calculated by the Debye Sherrer formula D = K λ / β_1/2cosθ where K is the Sherrer constant (K=0.9 for spherical particle), λ is the X-ray wavelength (λ=1.54060 Å), β_{1/2 is} the width of the XRD peak at half height, θ is the Bragg diffraction angle. Substituting these values D = 0.9 (1.54060)/ (0.05)(0.96) = 29 nm.

Fig. 4. XRD pattern of synthesized zinc oxide nanoparticles

3.5. Structural characterization of zinc nanoparticles synthesized by A. terreus using SEM:

Scanning Electron Microscope was used to deduce the particle size and morphology of the synthesized zinc oxide nanoparticles. It is concluded from Fig. 5. that the particles in the samples were compactly arranged and were almost spherical in shape. The size of the synthesized zinc oxide nanoparticles were found in range between 54.8 - 82.6 nm.

Fig. 5. SEM image of synthesized zinc oxide nanoparticles

3.6. Antifungal activity of zinc nanoparticles synthesized by A. terreus:

The well diffusion experiments were carried out against A. niger, A. fumigatus and A. aculeatus. The effect of zinc oxide nanoparticles on the growth of A. niger, A. fumigatus and A. aculeatus is shown in Fig. 6. Zone of clearance was observed in all the plates from which it is infered that synthesized zinc oxide nanoparticles have antifungal activity. Zone of clearance was significantly large in A. fumigatus inoculated petriplates when compared to A. aculeatus and A. niger inoculated petriplates. The antimicrobial activity of zinc oxide nanoparticles is mainly be generation of highly reactive species like OH¯, H₂O₂, O₂²^¯. H₂O₂penetrates the cell and OH¯ and O₂^2¯damages the cell wall and cell membrane from outside [17].

Fig. 6. Antifungal activity of synthesized zinc oxide nanoparticles

4. CONCLUSION:

Aspergillus terreus can be used for large scale synthesis of zinc oxide nanoparticles. The functional groups present in the zinc oxide nanoparticles was confirmed by FT-IR analysis with the peaks in the range of 560–3690cm^-1. Sharper and stronger diffraction peaks observed in XRD analysis confirmed the synthesized zinc oxide nanoparticles. Synthesized zinc oxide nanoparticles were in the size range of 54.8 to 82.6 nm and were found to be spherical in shape as confirmed from the SEM analysis. The different functional groups associated were found to be C=O, C-N, N-H, O-H, N=O makes the synthesized zinc oxide nanoparticles as effective antimicrobial agent. Synthesized zinc oxide nanoparticles can be used as effective antifungal agent. These nanoparticles can be added to the food to reduce the food poisoning effect by the various Aspergillus sp., which is legally approved.

5. ACKNOWLEDGEMENT:

The authors would like to thank Anna University, Chennai for their SEM analysis and SRM University, Chennai for their XRD and FT-IR Analysis.

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Received on 18.09.2013 Accepted on 02.10.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 4, Pg 142-146

Mycological Synthesis, Characterization and Antifungal Activity of Zinc Oxide Nanoparticles