الفهرس 
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Abstract
Silver nanoparticles (SNPs) have been proven to be a useful asset in many applications whether industrial, agricultural, medical, or biotechnological owing to its evident antimicrobial activity. Many methods of synthesis of SNPs have been exploited, among which, the biological method of synthesis, in which various living organisms such as plants, algae, bacteria, and fungi are recruited as means of biosynthesis of SNPs has been favored as a more eco-friendly and greener approach of synthesis. Amidst the different organisms which have been used to biosynthesize SNPs, Fungi have gained the most attention as they’re fastidious to grow, easy to handle , their ability to produce large amounts of extracellular bioactive compounds which enable fast, safe , and cost-effective means of synthesis. This study aims to isolate fungi form different sources, screen for their ability to extracellularly biosynthesize silver nanoparticles, select the fungal isolates which have biosynthesized the most stable silver nanoparticles for further optimization, characterize the most stable biosynthesized silver nanoparticles, and to investigate their antimicrobial activity against common human bacterial and fungal pathogens.
(1) Ten samples have been collected. Three soil samples were collected from different highly saline arid regions near Lake Moeris (Qarun Lake) in the northwest area of Faiyum Oasis located southwest of Cairo Governorate, Egypt. Seven plants, three of which are fennel (Foeniculum vulgare), mint (Mentha longifolia) and basil (Ocimum basilicum) were isolated from cultivated high salinity lands alongside Lake Moeris. The other four plants are camelthorn bush (Alhagi graecorum), Phog (Calligomum polygonoids), nitric bush (Nitraria retusa) and Tetraena alba (Zygophyllum album) were isolated from the arid highly saline subtropical region of Wadi El-Rayan Protectorate, Faiyum Governorate, Egypt.
(2) A sum of 60 fungal isolates have been isolated from three soil samples and seven plant specimens, all of which were screened for their ability to support the biosynthesis of SNPs, out of which, 21 fungal isolates (11 endophytic and 10 soil fungal isolates) belonging to 7 genera (Albifimbria, Aspergillus, Candida, chrysosporium, Fennelia, Penicillium, and Scopulariopsis) have successfully supported the biosynthesis of SNPs which was preliminary observed as change in color of reaction mixture to reddish brown followed by UV-Vis spectroscopic confirmation of the biosynthesis.
(3) Among the 21 fungal isolates which have supported the biosynthesis of SNPs, two isolates, Albifimbria verrucaria, an endophyte isolated from roots of basil (Ocimum basilicum), and Aspergillus nidulans, a soil isolate, have synthesized SNPs showing the most long-term stability without tendency for aggregation, which as a result were selected for further optimization of biosynthesis.
(4) Different reaction parameters such as concentration of fungal biomass, concentration of silver nitrate, growth media, pH conditions, and incubation temperatures were investigated using one factor at a time method to synthesize SNPs with the smallest size and highest concentration using both isolates Albifimbria verrucaria and Aspergillus nidulans.
(5) The optimal conditions selected for the synthesis of the highest concentration with the smallest size SNPs using fungal-free filtrate of Albifimbria verrucaria were PDB as growth media, 20 g/100 ml fungal biomass, 1.5 mM silver nitrate, alkaline pH ranging between 9-10, and 60 °C as the incubation temperature of the reaction mixture, whereas PDB as growth media, 25 g/100 ml fungal biomass, 1 mM silver nitrate, alkaline pH 10, and 90 °C as the incubation temperature of the reaction mixture, were the optimum conditions to synthesize silver nanoparticles with the highest concentration and the smallest size SNPs using fungal-free filtrate of Aspergillus nidulans.
(6) UV-Vis absorption spectra of optimally synthesized SNPs using fungal-free filtrate of Albifimbria verrucaria and Aspergillus nidulans were recorded at 410 and 415 nm respectively.
(7) HR-TEM micrographs of optimally synthesized SNPs using fungal-free filtrate of A. verrucaria and A. nidulans confirmed the synthesis of well-dispersed spherical shaped silver nanoparticles with diameters ranging between 1–12 nm and 1.66 and 30.44 nm respectively.
(8) DLS technique showed the size distribution and the mean hydrodynamic diameter of SNPs synthesized using Albifimbria verrucaria and Aspergillus nidulans as 14 nm and 28.5 nm respectively, and also showed the zeta potential of SNPs synthesized by both isolates as -24.47 mV and -17.74 mV respectively. The negative charge present on the surface of biosynthesized SNPs is responsible for their stabilization by preventing agglomeration through electrostatic repulsion between particles.
(9) FTIR of silver nanoparticles synthesized by both isolates showed similar absorption spectra peaks corresponding to O-H (hydroxyl) stretching in alcohols, phenols, and flavonoid compounds, C-H (sp3 hybridization) stretching vibration, C=O (carbonyl) stretch in N-C=O amide group of peptide bond and COO- group, as well as, C-N stretch could be observed which is characteristic of proteins demonstrating the role of proteins and silver reducing biomolecules present in fungal-free filtrate of both isolates in synthesizing and stabilizing the silver nanoparticles.
(10) XRD analysis of silver nanoparticles synthesized by both isolates showed four distinct Bragg reflections characteristic of silver nanoparticles with diffraction patterns confirming the crystalline lattice nature of biosynthesized silver nanoparticles.
(11) The antimicrobial activity of silver nanoparticles synthesized by both isolates was evaluated by means of agar well diffusion method. The SNPs synthesized using both isolates Albifimbria verrucaria and Aspergillus nidulans exhibited significant antimicrobial activity against several bacterial and fungal human pathogenic microorganisms including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Bacillus cereus, Candida albicans, Aspergillus flavus, Aspergillus niger, and Aspergillus fumigatus. SNPs synthesized by both isolates showed stronger antifungal than antibacterial activity. Those which were synthesized by Albifimbria verrucaria exhibited stronger antimicrobial activity than those synthesized by Aspergillus nidulans.
(12) Minimum inhibitory concentration of SNPs synthesized by both isolates was evaluated against the previously mentioned bacterial and fungal human pathogens using broth micro-dilution method. SNPs synthesized using Albifimbria verrucaria demonstrated more efficient and consistent antimicrobial activity at lower concentration in comparison with those synthesized using Aspergillus nidulans.
(13) The in vitro antiproliferative activity of SNPs synthesized using both isolates were evaluated against HeLa cell line. The SNPs synthesized by both isolates Albifimbria verrucaria and Aspergillus nidulans showed clearly significant antiproliferative activity against HeLa cells, though silver nanoparticles synthesized by A. verrucaria exhibited more efficient antiproliferative activity than those synthesized by A. nidulans. The IC50 concentration (the concentration of silver nanoparticles that inhibits proliferation of HeLa cells by 50%) of silver nanoparticles derived from A. verrucaria and A. nidulans was equivalent to 2.52 and 16.4 μg/ml respectively after treatment for 48 hours.
(14) Similarly, the cytotoxic activity of SNPs synthesized by A. verrucaria and A. nidulans was evaluated against WI-38 (normal human lung fibroblasts) cell line. SNPs derived from both isolates showed incidence of cytotoxicity against WI-38 cells, thought, those synthesized by A. verrucaria exhibited higher cytotoxic activity against WI-38 cells than those synthesized by A. nidulans, with CC50 (the concentration of SNPs which causes death of 50% of the tested cellular population of WI-38 cells) values equivalent to 10.2 and 39.98 μg/ml respectively after treatment for 48 hours.
(15) The selectivity index values (the ratio between CC50 concentration of SNPs affecting normal WI-38 cells divided by their IC50 against HeLa cancerous cells) of SNPs synthesized by A. verrucaria and A. nidulans were 4 and 2.4 respectively, which are considered within the acceptable range according to various clinical studies.
(16) The data presented in this study show the potential of fungi, particularly endophytic fungal isolate Albifimbria verrucaria and soil fungal isolate Aspergillus nidulans in extracellular biosynthesis of thermostable silver nanoparticles as a possible biofriendly, safe, and efficient antimicrobial agent with promising antiproliferative activity and low cytotoxic effect which can be furtherly exploited and implemented in various biomedical and biotechnological applications.