الفهرس 
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Abstract
5. SUMMARY
Microbial synthesis of nanoparticles has a potential to develop, cost-effective and eco-friendly methods for the production of technology important materials. Silver nanoparticles have a great number of application, e.g. in non-linear optics, spectrally selective coating for solar energy absorption, biolabelling, intercalation materials for electrical batteries, high-sensitivity biomolecular detection and diagnostics, antimicrobials and therapeutics, catalysis and microelectronics. Nanoparticles resulting from some microbial processes have unique chemical and physical properties, different from the properties of conventionally nanoparticles, depending on microorganisms and medium used as well as operational conditions applied. The objective of this study is biosynthesis of the extracellular silver nanoparticles by some local strains through isolation and selection of isolates had the ability for AgNPs synthesis from different sources. Also, optimization of factors affecting silver nanoparticles biosynthesis using classical methods (one variable at a time) and the techniques of interaction between the factors through response surface methodology. Studies were focused on silver nanoparticles stability, characterization and its antimicrobial effects against various pathogens as well as some application.
The obtained results can be summarized as follows:
1) Seventy microbial isolates were isolated from five rhizosphere soil samples and three water samples on specific media. These isolates included different microbial groups being actinomycetes, fungi and bacteria presented in the percentage of 40%, 35.7% and 24.3% of total isolates, respectively. These isolated and 9 microbial strains were tested for their ability to AgNPs synthesis.
2) All tested isolates were found to produce silver nanoparticles by visual observation of microbial filtrates treated with silver nitrate whereas exhibited a gradual change to brown color.
3) The UV-visible spectra of isolates supernatant or filtrates treated with silver nitrate solution was examined at 200-800 nm. The optimal wave length giving the highest optical density (indicating to AgNPs concentration) ranged from 410 to 420 nm.
4) The actinomycetes and fungal isolates were more efficienin for AgNPs synthesis than tested bacterial isolates. Streptomyces aizuneusis produced the highest AgNPs concentration with smallest particle size followed by A14, A29 and A12. Moreover, eleven fungal isolates produced the high AgNPs concentrations ranging from 0.53 to 1.16 with small particle size ranged from 20.56 to 53.52 nm.
5) Streptomyces aizuneusis and Fusarium sp. F17 recorded the highest AgNPs concentration with smallest particle size. So, they were selected as most efficient AgNPs producing organisms. Moreover, their AgNPs solution was extremely stable at room temperature for 2 months without aggregation.
6) The selected fungal isolate (F17) was identified depending on their cultural and morphological properties as a preliminary classification. Then the identity was confirmed by molecular identification using one specific primer for Fusarium genus. Amplified sequences showed 99% similarity to available data of F. oxysporum f. sp. melonis isolate TX388.
7) Optimization of microbial silver nanoparticles production: the production of AgNPs was applied at two stages, the first stage, to produce the fungal or bacterial biomass whereas the second stage; for producing silver nanoparticles by the cell filtrate of biomass. So, the optimization of silver nanoparticles production was carried out on biomass production stage using two stage strategy. In first strategy, screening of carbon and nitrogen sources of AgNPs production by Streptomyces aizuneusis and Fusarium oxysporum F 17 were studied using one factor at -a- time method. In second strategy, interaction among factors was studied subsequently in Plackett-Burman and multi-factorial response surface approach of Face centered Central Composite Design (FCCD).
A. Factor affecting AgNPs production by one variable- at-a-time approach:
• The original carbon sources of MGPY medium was replaced by different carbon sources (8 sources) to study their effect on AgNPs production by tested strains.
• Glycerol and glucose proved to be the best carbon sources for production silver nanoparticles by Streptomyces aizuneusis. Whereas galactose and malt extract were the best carbon sources for silver nanoparticles by Fusarium oxysporum F17. MGPY medium containing galactose as sole carbon recorded the highest AgNPs (1.30) synthesis by Fusarium oxysporum F17 gave the drastic effect for Streptomyces aizuneusis AgNPs production (0.55) whereas the vice versa was true on glycerol medium.
• The organic nitrogen sources (5 sources) gave higher growth and AgNPs synthesis by both tested strains than inorganic sources, expect calcium nitrate medium resulted the same AgNPs concentration of yeast extract medium being 1.65 and lower Streptomyces aizuneusis growth than yeast extract. Whereas yeast extract or beef extract was the most favorable nitrogen source for AgNPs production by Fusarium oxysporum F17.
B. Statistical screening of nutritional and physical factors using plackett-Bruman design at first stage.
• Seven variables including culture conditions; (pH, incubation period, agitation speed), and two sources of each carbon and nitrogen source were chosen to perform the optimization process for AgNPs by Streptomyces aizuneusis. The design have 12 runs with two levels for each factor. High production of silver nanoparticles (1.3136 absorbance) was recorded during run number 3 with high levels of glucose, calcium nitrate, yeast extract, agitation speed (150 rpm) and low levels of glycerol, pH adjusted to 8 and incubation periods for 9 days. R2 for this response was 1 indicating that the model terms are significant and high correlation between the experimental and predicted values. All selected independent variables were significantly affected on silver nanoparticles produced by Streptomyces aizuneusis.
• For Fusarium oxysporum F17, a total of four nutritional factors (Galactose, malt extract, yeast extract, beef extract) and four physical factors (pH, temperature, agitation speed and incubation time) were analyzed for their effects on silver nanoparticles production. The maximum silver nanoparticles production (7.594ppm) was achieved by the Run 2 with 14.5 g/l galactose 10g/l malt extract, 12g/l yeast extract, 3g/l beef extract, the pH adjusted 6 and incubated at 28°C for 5days under shacking conditions (150rpm). It was found that galactose, yeast extract and agitation speed had a positive effect on silver nanoparticles production. The coefficient of determination (R2) was 0.95 indicating a satisfactory of the process model and high correlation between the experimental and predicted values.
C. The selected significant factors and their possible interaction effects were also optimized statistically using the full factorial face centered central composite design of response surface methodology (RSM) based on central composite design (CCD) at first stage.
• High value of absorbance for silver nanoparticles production was recorded by Streptomyces aizuneusis at run number 22 (containing 17.25 glucose, 14.8 Glycerol, 13.5 calcium nitrate, 13.5 yeast extract, 8 pH, 9 incubation days, and 150 rpm agitation speed then incubated at 30°C being 1.3428 (O.D). The determination coefficient R2 of the model was 0.83 indicating that 83% of the total variations were explained by the model and revealed good agreement between the experimental results and the predicted values calculated from the model. With respect to production by tested Streptomyces aizuneusis, the coefficients of model term, 3 variables glucose (A), glycerol (B), yeast extract(D) & and interaction between them are significantly influence on the production of silver nanoparticles.
• The highest values of Fusarium oxysporum F17 silver nanoparticles concentrations (81.85 ppm) was achieved at run number 18(containing galactose 15g, yeast extract 15g, and agitation speed 150 rpm). where a total of 20 experiments with 3 different variables with its combination of galactose (A), yeast extract (B), & agitation (C) were performed at three different levels. The determination coefficient R2 of the model was 0.95 indicating that 95% the total variations were explained by the model and revealed good agreement between the experimental results and the predicted values calculated from the model.
D. Statistical screening using central composite design for the variables affecting of silver nanoparticles production at second stage (AgNPs synthesis)
• Twenty experiments with four variables and different combination of concentration of AgNO3 (A), amount of cell filtrate (B), incubation temperature (C), and agitation (rpm)(D) were performed at three levels. The highest value of Streptomyces aizuneusis AgNPs concentration was recorded by run 13 giving actual concentration 84 ppm. Run 13, in presence the 15ml silver nitrate solution (1.5mM), react with 20 ml of cell filtrate at 30°C under shacking condition (150rpm) gave AgNPs concentration was higher than the predicated values (68.31 ppm) by 1.22 fold.
• For Fusarium oxysporum F17 AgNPs synthesis, total 20 experiments for four variables and interactions between them, silver nitrate concentration (A), amount of cell filtrate (ml) (B), temperature (C) and agitation (D) were studied to study their effects at three levels. High value of silver nanoparticles biosynthesis was recorded with run number 17 which have 30ml of cell filtrate react with 15ml of silver nitrate (0.5mM) under agitation speed of 250 rpm at 20°C to give 82.00 ppm AgNPs. The actual value of run 17 was very high comparing to predicted values.
• The coefficient of R2 values of response for Streptomyces aizuneusis and Fusarium oxysporum F17 were 0.95 and 0.99 indicating that 95% and 99% of the total variations were explained by the model and revealed good agreement between the experimental results and the predicted values calculated from the model.
8) characterization of microbial extracellular AgNPs produced
The silver nanoparticles were examined using UV-Visible spectroscopy, Transmition Electron Microscopy (TEM), X-ray Diffractometer (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). Energy dispersive x-ray (EDXA), zeta potential analysis.
• Both the synthesized AgNPs were polydispersed spherical particles ranging from 11 to 42 nm and from 16 to 34 nm for Streptomyces aizuneusis and Fusarium oxysporumF17 nanoparticles, respectively.
• The strong and broad peak was obtained by UV-Visible spectrum between wave length ranged from 410 nm to 430 nm indicating prescence of both AgNPs in Fusarium oxysporum F17 and Streptomyces aizuneusis cell filtrates.
• FTIR spectrum of for Streptomyces aizuneusis silver nanoparticles show peaks at 647.06, 1172.2, 1382.08, 1634.45, 2078.78, 2808.52, 2880.65, and 3452.89 cm-1. The stronger for Streptomyces aizuneusis silver nanoparticles band observed at 647.06 corresponding to C-S stretching of chloro-alkanes, the band appeared at 1382.08 referring to C-C stretching aromatics, the bands observed at 2078.78, and 2880.65 cm-1 were assigned to aromatic-CH stretching and methyne C-H stretch respectively. FTIR measurements of dried sample of Fusarium oxysporum F17 silver nanoparticles showed the representative spectra of obtained nanoparticles manifests absorption peaks located at about 3453.55, 2093.86, 1639.28, 1090.49, 1051.23 and 659.24 in the region 400 – 4000 cm-1. The FTIR spectra revealed the presence of different functional groups.
• The EDXA spectrum showed a strong signal for silver at 3 Kev by two products which is typical for metallic silver nano crystallites indicated to high purity. The others peaks also recorded cupper metal (cu), most likely because of the borosilicate glass on which the sample was coated.
• The data obtained from Dynamic Light Scattering (DLS) measurements of biosynthesis AgNPs indicates that the particles have negative charge -26±0.2 mV- for Streptomyces aizuneusis whereas Fusarium oxysporum F17 have -22 ±0.2 mV- negative charge.
• XRD analysis showed two distinct diffraction peaks at 38.15◦ & 44.51◦, for Streptomyces aizuneusis and at 32.35◦ & 37.85◦, for Fusarium oxysporum F17 for cubic face-centered silver. The lattice constants calculated from this pattern were a = 3.36 ˚A and 2.55 ˚A, respectively.
9) Some application of extracellular biosynthesized AgNPs
a) Antimicrobial activity of biosynthesized AgNPs:
• The AgNPs biosynthesized by Streptomyces aizuneusis and Fusarium oxysporum F17 exhibited excellent antibacterial effects against Bacillus subtilis, Staphylococcus aureus, Listeria monocytogense, Escherichia coli, Pseudomonas aeruginosa and Salmonella paratyphi and antifungal activity against Fusarium solani, Sclerotium rolfsii and Rhizoctonia solani.
• There are significant effects of both AgNPs on all tested organisms.
• Fusarium oxysporum F17 AgNPs was more affecting than Streptomyces aizuneusis AgNPs on the growth of fungal and bacterial strains. Whereas the latter strains were more susceptible for both AgNPs.
b) Toxicity of silver nanoparticles; Against Drosophila melanogaster larvae
• Effect of three concentrations of AgNPs produced by Streptomyces aizuneusis and Fusarium oxysporum F17 (0.01, 0.05&0.07 ppm) on Drosophila melanogaster larvae toxicity was studied. It was noticed that these AgNPs concentrations had no deleterious effect on development and occurrence of distinct phenotypes. Moreover, the percentage of abnormal files being (1.22%) was obtained after 14 feeding period with 0.07 ppm AgNPs.
c) Silver nanoparticles as antitumor
• The efficacy of biologically synthesized AgNPs as an antitumor agent was determined on human lung and liver carcinoma cell line in vitro using NRU colorimetric techniques.
• The AgNPs produced by Streptomyces aizuneusis and Fusarium oxysporum F17 were added on human liver carcinoma (Hep2) cell line using different concentrations (0.01,0.02,0.04,0.06,0.08,0.1 ppm) to determine their cytotoxicity. All treated tumor cells were inhibited with all tested concentrations beginning from 0.01 to 0.1 ppm giving zero percentage of cell viability.
• The human lung carcinoma and normal cells were treated by both silver nanoparticles at different concentrations (from 0.01 to 0.1ppm) leading to different response for cell viability. The viable cell count decreased gradually with increasing Streptomyces aizuneusis AgNPs concentration giving 4.6% viability at 0.2 ppm with cancer cells and the IC50 were 0.06 and 0.0148ppm for normal and cancer cells, respectively.
• The silver nanoparticles of Fusarium oxysporum F17 at 0.018 ppm as IC50 decreased the viability of normal cell to 50% of the initial level.