INTRODUCTION:
Nanoparticles are particles having one or more dimensions of the order of 100 mm or less, nanoparticles synthesize by a biogenic enzymatic process are far superior, compare to those particles produced by chemical methods. Despite that the latter methods are able to produce large quantities of nanoparticles with a defined size and shape in a relatively short time, they are complicated, outdated, very cost, inefficient and produce harmful toxic wastes that are dangerous, not only to the environment but also to human health.
With an enzymatic method, the use of expensive chemicals is eliminated, and the more useful “green” route is not as energy intensive as the chemical method and is also environment friendly [1]. ‘Green synthesis’ is a procedure of synthesis and assembly of nanoparticles and has been used for a series of special production procedures. This process, defined as the development of clean, non-toxic and environmentally acceptable ways which include organisms ranging from bacteria to fungi and even plants [2]. An important character of metal nanoparticles synthesis is their ability to remain dispersed in liquids without clusters due to the proteins in fungus filtrate play role as stabilizing and capping agents[3]. Microorganisms such as bacteria, actinomycetes, and fungi play an important role in the treatment of toxic metal through reduction of metal ions and are considered as potential Nano factories. Filamentous fungi are ideal candidates for environmental friendly synthesis of AgNPs [4]. A number of researchers have been experimenting on the microbes, especially fungi like Cladosporium tropicum, Aspergillus niger, Pencillium sp. But few experiments have been informed using bacteria like Bacillus thuringiensis, to synthesize silver nanoparticles [5, 6]. The anamorphic entomopathogenic fungi Beauveria bassiana belong to the order Hypocreales (Ascomycota) which have a worldwide distribution as members of the natural soil flora [7]. B. bassiana is a cosmopolitan ascomycete fungus that is able to inhabiting a wide range of environments, including soil, insects, and plants. The fungus is able to live as a saprophyte in the soil, as an endophyte in plants, or as an entomopathogen affecting a wide range of arthropods [8].
In the present study, we have isolated, pure fungal strain for biosynthesis of silver nanoparticles and the synthesized AgNPs were characterized by using UV-Visible spectroscopy spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope, (SEM) hot stage microscopy and Atomic force microscopic analysis (AFM).
MATERIALS AND METHODS:
Chemicals:
PDA was manufacturered in United Kingdom , Silver nitrate was manufacturered in Germany, Hydroxide sodium was manufacturered in Iraq, PDB was manufacturered in India. All the chemicals purchased were of analytical grade.
Preparation of broth and culture of Beauveria bassiana:
The fungal Beauveria bassiana was obtained from the Directorate of Agriculture and Research / Ministry of Science and Technology. It was cultured on potato dextrose agar (PDA). The media was sterilized in the autoclave at 121ºC for 15min. After cooling it was supplemented with chloramphenicol 250mg/L as bacteriostatic agent. The fungal spores were taken from original cultures and inoculated in PDA and incubated for 7 days at 25ºC [9]. PDB broth was prepared in1000 ml Erlenmeyer flask and sterilized in the autoclave. It was allowed to cool broth and provided with chloramphenicol 250 mg/L then inoculated aseptically with a 5Mm actively grown culture disc of the fungus (5 discs per 250ml) and incubated for 2 weeks at 25ºC [10].
Synthesis of silver nanoparticles:
The biomass were harvested after 2 weeks of the growth by sieving through Whatman No.1 filter paper followed by 3 times extensive wash with distilled water to remove any medium components from the biomass. Twenty grams of fresh and clean biomass were taken into an Erlenmeyer flask containing 500 ml of deionized water. The flask was incubated at 25ºC for120h. After incubation, the cell filtrates were obtained by passing it through Whatman No. 1 filter paper and later by Millipore filter 0.2 μm. Then it was brought in contact with 50mM of 100ml (AgNO3) (Fig.1)
Fig (1) Biomass of Beauveria bassiana
Molecular weight of AgNo3:
Ag=107.87, N=14, O=16 = 107.87 + 14 + 16 × 3 = 169.87. So the molar mass of AgNo3 is 169.87, therefor 1000 ml of 50 mM contains 169.87 × 50 /1000 = 8.4935 gm of AgNo3 (Was added to 1000 ml of deionized water, 800 ml of this solution was added 200 ml of cell filters and heated at 60 c for 10 min and was shacked with magnetic hot plate stirrer and adjust PH to 7 by adding drops of NaOH solution, the flasks were covered with aluminium foil to prevent photoreaction of silver nitrate [11]. the flask was incubated at 25°C in a dark condition for 120 h, AgNPs turned brownish yellow color solution [12] .The control without adding AgNO3 was maintained under the same conditions, separately. The protein, enzyme and other compound existing in the fungal liquid work as reducing agents and are responsible for conversion of silver nitrate to silver nanoparticles (Fig 2). The reaction may be written as:
B. bassiana (fungal liquid) + silver nitrate solution
=silver nanoparticles
The culture filterate was efficaciously used as straight bio-reductant to convert Ag+into Ag ° [13].
Fig (2) comparison between three types of solution.50mM AgNo3 solution. B-Fungal cell filtrate. C- AgNPs solution
RESULTS:
Fig (3): 3D AFM images of AgNPs:
(Fig.3) shows the 3D image and granulation characterization biosynthesized nanoparticles characterized using atomic absorption spectroscopy (AA‐3000, Angstroms Advanced Inc. USA AFM contact mode) in the Lab of Baghdad University /Iraq [14]. The figure displays the 3D image and distribution chart for AgNP3 which placed on glass the average size is found to be around 89.99 nm and the roughness and the root mean square are 4.08nm, 4.97nm respectively. Also, the figure refers that the shape of this particle is semi rode distribution as matrix on the vertical axis, which means that the thin film is homogenous and uniform.
Fig (4) shows the solution of the developed nanoparticles of silver, which was centrifuged at 4000rpm for 60 min. The solid residues of AgNPs were mixed with deionized water and centrifuged at 4000 rpm for 60 min. After that, the drops of residue deposited on a slide and heated at 60ºC for 30 min. The phase variety and grain size of synthesized Silver nanoparticles was determined by Shimadzu X-Ray diffractometer (XRD 6000).The silver nanoparticles were studied with CUKα radiation at a voltage of 40 KV and current of 30 (MA) with a scan speed in 5.0000 (deg/min). The particle size of the prepared samples was determined by using the Scherrer′s equation as follows [15].
(1)
Where D is the crystal size, λ is the wavelength
of X-ray, 
is the Braggs angle in radians and β is the full
width at half maximum of the peak in radians, the microstrain value ‘’ and
the dislocation density ‘σ’ was evaluated by using the following relations
[16].
e
(2)
Fig (4): XRD pattern of (AgNPs) thin
Table 1: powder X-ray diffraction data of (AgNPs) thin film
|
2Theta (deg) |
β (deg) |
D (nm) |
x10-4 lines-2.m-4 |
σ x 1014 lines/m2 |
|
28.05 |
0.35 |
23.27295 |
14.88853 |
18.46279 |
|
14.03 |
0.33 |
24.19577 |
14.32068 |
17.0813 |
|
32.67 |
0.10 |
82.36097 |
4.20709 |
1.474202 |
The color intensity of synthesized AgNPs increased with duration of incubation, the color of the solution changed to dark brown after 120 h of incubation for the synthesis of nanoparticles. The bioreduction of silver ions in the solution was observed periodically by measuring the UV-Vis spectroscopy of the solutions. The reaction was rapid as the yellowish- brown color appeared within 120 h and the reaction confirmed the formation of AgNPs and there was no color change further. The optimum time required for the completion of reaction from our study was 120 h; It was observed that the reduction of silver ions reaches saturation within 48 h of incubation. (Fig. 5) Shows the UV-Vis spectra using Shimadzu UV-1800 spectrophotometer from 190nm to 1100 nm at regular intervals. Vis spectroscopy is used for the characterization of colloidal particles. noble metal particles possess strong Surface plasmon resonance (SPR) absorption in the visible region and are highly sensitive to the surface modification [17]. The AgNPs solution was exposed to ultra-sonication at room temperature and their surface plasmon resonance was recorded at 234nm.
Fig (5): Transmittance spectrum of (AgNPs) thin film
For using the fundamental relation of photon transmission and absorbance, the absorbance (A) is defended as the logarithm (base 10) of the reciprocal of the transmittance
A=log 10
(3)
T is the transmittance and A is the absorbance of the (Fe3O4). A thin film was prepared by chemical method and deposited by drop casting technique on glass. The reflection of the film has been found by using the relationship: (Fig. 6)
R+T+A=1 (4)
From the reflection R of the thin film, the refraction index can be calculated from the following relationship [18]:
n =
(5)
The maximum value is 2.5. The optical absorption
coefficient α was evaluated by tauc relationαhv= (hv-Eg)n
whenα=2.303
Where t is the film thickness , hv is
the photon energy, Eg=
And no= 0.5 for allowing direct transition.
Plotting the graph between (αhv)2 versus photon energy (hv) provides
the value of the direct band gap. The extrapolation of the straight line to (αhv)
2 = 0, provides the value of the band gap s [19], shown in (fig. 7). The
optical band gab is 4.95 eV.
FTIR analysis was carried out to identify the possible interactions between silver and bioactive molecules, (Fig. 8) Which may play role in the synthesis and steadiness (capping) of silver nanoparticles, FTIR spectroscopy was used to identify the functional groups of the active compounds depend on the peak value in the infrared region [20]. FTIR spectra obviously show that the biomolecules especially proteins prevailing in the filtrate are responsible for the synthesis and stabilization of silver nanoparticles [21].
Fig (6): Reflectance index spectrum of (AgNPs) thin.
Fig (7): (αhv) versus photon energy plot of (AgNPs) thin film