Nutrient agar medium (70148 nutrient agar) was provided by Fluka, Spain. Glucose was purchased from Koch-Light Lab., England. Silver nitrate was provided by Sisco Research lab, India. Potassium chloride was obtained from Merck, Germany. Agar–Agar was obtained from Fluka, France. Polyester/cotton blend (PET/C 50/50) fabrics in form of filament woven fabric cloth made from filament yarns was kindly supplied by Misr Polyester Co., Kafr EL-Dwar, Egypt. Acid cellulase enzyme (Cellusoft® L) used for fabric pretreatment was obtained from Novo Nordisk, Denmark.
Aspergillus fumigatus DSM819 was cultured and stored on modified Czapek–Dox’s solid medium slants and refreshed before use. For the purpose of antimicrobial determinations, Escherichia coli, Bacillus mycoides, and Candida albicans were applied as representative microorganisms of Gram-negative bacteria, Gram-positive bacteria, and non-filamentous fungi. They were maintained on nutrient agar medium with the following composition (g/l): peptone, 3; yeast extract, 1.5; meat extract, 1.5; glucose, 0.5; NaCl, 0.25; and agar, 20.0 at pH 7.0. All microbial cultures applied in this study were obtained from the culture collection of Microbial Chemistry Dept., NRC, Egypt.
Preparation of cell-free filtrate and AgNP biosynthesis
A. fumigatus DSM819 was cultivated in 250 ml Erlenmeyer conical flasks which contained 50 ml of the modified Czapek–Dox’s liquid medium (g/l): NaNO3, 2; KH2PO4, 1; MgSO4.7H2O, 0.5; KCl, 0.5; and glucose, 20 for 6 days at 28 °C under static conditions. After that, the culture was filtered via Whatman No. 1 filter paper and the resulted supernatant (cell-free filtrate; CFF) was applied for the mediation of AgNP biosynthesis. The obtained CFF was applied in various reaction mixtures containing silver nitrate aqueous solution (1 mM) as silver source for AgNP biosynthesis process. Then, the mixtures were incubated in the dark (to avoid the photoactivation of silver nitrate) at 30 °C and continues shake at 100 rpm. After incubation, the absorbance of the characteristic reddish brown color of the biosynthesized AgNPs was scanned using UV–visible spectrophotometer (Cary 100 UV-Vis; Agilent Technologies, Germany). Cell-free filtrates as well as silver nitrate solution (1 mM) were used as controls.
Optimization of AgNP biosynthesis
The CFF obtained from A. fumigatus DSM819 culturing was added at different concentrations in ratios between 10 and 60% (v/v), which means adding CFF volumes between 0.5 and 3.0 ml to a total reaction mixture of 5.0 ml, while keeping the AgNO3 concentration at a level of 1.0 mM. The pH value effect was studied through preparing different reaction mixtures adjusted at pH values ranging between 9.0 and 11. The effect of reaction time on the AgNP biosynthesis process was evaluated by incubating the reaction mixtures at optimum conditions for 10, 20, 30, 45, 60, 90, and 120 min. The effect of silver ions on the biosynthesis of AgNPs by A. fumigatus DSM819 was determined by varying the AgNO3 concentration to range from 0.5 to 1.5 mM. All experiments were carried out in triplicates, and the average data was presented.
Characterization of the biosynthesized AgNPs
The UV–visible spectra of AgNPs were recorded as a function of wavelength using UV/vis spectrophotometer (Cary 100 UV-Vis; Agilent Technologies, Germany) operated at data interval of 1.0 nm. The AgNP solution was centrifuged for 20 min at 10000 rpm and was drop coated on a carbon-coated copper grid and dried to be applied for studying both the scanning electron microscopic (SEM) and elemental analysis of the biosynthesized AgNPs using scanning electron microscope (SEM-Quanta FEG250) operated at an accelerating voltage of 20 kV and coupled with energy dispersive X-ray analysis (EDAX) for compositional analysis and the conformation of presence of elemental silver.
The shape and size of AgNPs were determined by transmitting electron microscope (TEM). For TEM, a drop of aqueous AgNP sample was loaded on a carbon-coated copper grid, and it was allowed to dry at room temperature; the micrographs were obtained using TEM (JEOL JEM-1230) at 160 kV. Particle size was measured on a dynamic light scattering (DLS) instrument (PSS, Santa Barbara, CA, USA), using the 632-nm line of a HeNe laser as the incident light with angel 90°.
For Fourier transform infrared (FTIR) spectroscopy measurements, reaction mixtures containing AgNO3 at a concentration of 1.5 mM and A. fumigatus DSM819 CFF were prepared and incubated for 90 min at pH 10 to form AgNPs, then centrifuged at 10,000 rpm for 15 min and re-dispersed in sterile distilled water. The process of centrifugation and re-dispersion was repeated four times to ensure good separation of the AgNPs from other contaminants. The obtained pellets were then dried, and the powders were subjected to FTIR spectroscopy measurement. These measurements were carried out on a JASCO FTIR (Japan) instrument in the diffuse reflectance mode at a resolution of 4 cm−1 in KBr pellets.
Cytotoxic effect on human cell lines
Cell viability was assessed by the mitochondrial-dependent reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to purple formazan  in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME, USA). Cells (as a gift from Dr. Stig Linder, Karolinska institutet, Solna, Sweden) were suspended in RPMI 1640 medium, 1% antibiotic–antimycotic mixture (10,000 U/ml potassium penicillin, 10,000 μg/ml streptomycin sulfate, and 25 μg/ml amphotericin B), and 1% l-glutamine at 37 °C under 5% CO2. Cells were batch cultured for 10 days, then seeded at concentration of 10 × 103 cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37 °C for 24 h under 5% CO2 using a water-jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added, and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration (54.0, 27.0, 13.5, 6.75, 3.37, 1.68, 0.84, and 0.40 μg/ml). After 48 h of incubation, medium was aspirated, and 40 μl MTT salt (2.5 μg/ml) was added to each well and incubated for further 4 h at 37 °C under 5% CO2. To stop the reaction and dissolving the formed crystals, 200 μl of 10% sodium dodecyl sulfate (SDS) in deionized water was added to each well and incubated overnight at 37 °C. A positive control was used as a known cytotoxic natural agent who gives 100% lethality under the same conditions. The absorbance was measured using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, CA, USA) at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t test by SPSS 11 program. DMSO is the vehicle used for dissolution with final concentration on the cells which was less than 0.2%. The percentage of change in viability was calculated according to the formula: ((Reading of extract/Reading of negative control) − 1) × 100. A probit analysis was carried for IC50 and IC90 determination using SPSS 11 program.
Pretreatment of PET/C fabrics by cellulase
Using a high-temperature high-pressure laboratory dyeing machine, cellulase (3%) was placed in stainless-steel bowls, then the fabrics were immersed in the solutions (pH 4.5), and the sealed bowls were rotated in a closed bath containing ethylene glycol at 45 °C. The material to liquor ratio was 1:15. The bath temperature increased at a rate of 5 °C/min, and after 40 min, the enzymatic treatment was then terminated by raising the pH to 10 by using Na2CO3. The fabric samples were removed from the bath, rinsed repeatedly with distilled hot and cold water, and then allowed to dry in the open air.
PET/C fabric treatment by the biosynthesized AgNPs
The activated PET/C blend fabric by cellulase and un-hydrolyzed fabrics (blank without enzymatic treatment) were immersed in the biosynthesized AgNP dispersion, and then, the samples were squeezed to a pickup of 60% (wt/wt) of the solution, dried in air at 22 °C for 24 h, and finally cured in an oven at 140 °C for 10 min. In order to evaluate the AgNP adhesion to the PET/C blend fabrics, the treated fabrics were washed five times according to a standard method AATCC Test Method (61-1989). The chemical structure was determined using FTIR spectrometer (model NEXUS 670, NICOLET, USA) in spectral range from 4000 to 400 cm−1.
Assessment of antimicrobial activity
For the assessment of AgNP antimicrobial activity, agar diffusion method was applied. The microbial strains were grown (seeding inoculation technique using 100 μl of re-suspended overnight culture at 37 °C (1 × 107 CFU/100 μl)) at 37 °C on nutrient agar medium (70148 nutrient agar, Fluka, Spain) with the subsequent composition (g/l): peptone, 5.0; NaCl, 5.0; yeast extract, 2.0; meat extract, 1.0; and agar 15.0 (pH 7.0). AgNP samples (108 μg/ml) obtained from the reaction of A. fumigatus DSM819 CFF and AgNO3 at different pH values were applied in a volume of 250 μl into the pre-made holes in Petri dishes of the previously mentioned nutrient agar medium, whereas in case of PET/C fabrics treated by the biosynthesized AgNPs, circular discs of 12 mm from the treated fabrics were applied. Culture plates were left overnight at 4 °C, and then, plates were incubated for 7 h at 37 °C. The developed inhibition zones were measured based on AATCC test method .