After incubation, the fungal biomass was obtained and collected. Fig 1 shows the obtained fungal biomass in the culture medium.
Biomass pretreatments
As it was indicated in the materials and methods, the biomass was divided into four separated flasks and each flask was pretreated separately; two of them were used for active (i.e. one placed at the RT and the other one was placed in the refrigerator) and two were used for passive mechanisms (i.e. one was placed in the autoclave and the other one was placed in the hot air oven) of SNPs production. Fig 2 shows the biomass in the four different tested flasks.
SNPs production
After addition of 1mmol final concentration of silver nitrate solution to each flask and incubation, the color of all the tested four flasks was turned from yellow to brown. Different intensities of the color were obtained which indicated the differences in the amounts, shapes or other characteristics of the SNPs. All the solutions were stable in the environment for at least 6 months. Fig 3, shows the four different tested flasks after SNPs production.
Characterization of the produced SNPs
Spectrophotometer analysis
All the color changed flasks were analyzed using visible spectrophotometer. Before the analysis all the samples were diluted 1:10. Results showed that although all the flasks had maximum absorbance peaks about 400-450 nm but amount of the optical density (OD) and the position of the maximum absorbance peak were different. The maximum absorbance peaks for the SNPs that were pretreated at RT, and refrigerator, autoclave and hot air oven instruments were at 425 nm, 449 nm, 400 nm and 445 nm, respectively with different ODs. The maximum and minimum ODs were for the SNPs which were pretreated using hot air oven and refrigerator instruments, respectively. The negative control flask remained unchanged. Fig 4, shows the obtained spectra for all the four tested samples.
Transmission electron microscopy (TEM)
The sizes and shapes of the produced SNPs for the samples were analyzed using TEM. The average sizes of the SNPs were different which indicated the differences in the production methods. The average sizes of the SNPs that were pretreated at RT and refrigerator, autoclave and hot air oven instruments were about 35 nm, 39 nm, 34 nm and 36 nm, respectively.
Totally, the polygonal, round, oval and triangular shapes of the produced SNPs were seen. Polygonal and triangular shapes of the SNPs were seen in the flask which before SNPs production was incubated in the refrigerator and round and oval shapes of the SNPs were seen in the flask that was incubated at RT. Fig 5 shows the obtained TEM photographs in four tested samples.
Xray difraction analysis (XRD)
XRD results for all the four flasks revealed the presence of four distinct peaks corresponded to the cubical structure of the elemental silver. Fig 6 shows the achieved XRD spectrum for the sample that was pretreated at RT.
Determination the SNPs concentration
Before further experiments, the SNPs concentration for each freeze-dried sample was determined using ICP-OES 730-ES instrument. Table 1 shows the obtained amounts of the samples. Briefly, the highest concentration of the SNPs was calculated in the sample that was pretreated in the hot air oven and the lowest one was for the sample that was pretreated in the refrigerator.
Table1 The obtained SNPs concentration for each sample using ICP-OES 730-ES instrument.
Sample
|
Wavelength (nm): 328.068, Element: Ag (1mg/ml)
|
Concentration (ppm)
|
Blank (ppm)
|
0.00
|
Pretreated in the hot air oven
|
3.20
|
Pretreated in the refrigerator
|
2.63
|
Pretreated in the RT
|
3.52
|
Pretreated in the autoclave
|
3.58
|
Antibacterial activity test
In order to analyze the differences in the antibacterial properties of the four different SNPs samples that were produced by the active and inactive biomass samples, three bacterial strains, S.aureus, P.aeruginosa, and E.coli, were used. The tests performed thrice and the diameter of the obtained inhibition zones measured and data analyzed by the aid of ANOVA program using SPSS software version 22.The p value< 0.05 was considered as significance. Table 2 shows the obtained results.
Table2 The antibacterial activity results of the four test samples and silver nitrate solution as a control. The tests were performed thrice.
The produced SNPs after different pretreatments
|
Inhibition zones (mm) of the SNPs against the tested bacterial strains (Mean±SD)
|
E.coli
|
P.aeruginosa
|
S. aureus
|
Hot air oven
|
8.00±0.47
|
11.00±1.00
|
10.50±0.94
|
Refrigerator
|
10.00±0.75
|
7.00±0.62
|
10.50±0.25
|
RT
|
11.00±0.86
|
11.00±0.20
|
11.00±0.33
|
Autoclave
|
10.00±0.75
|
11.00±0.60
|
11.00±0.62
|
Silver nitrate
|
12.50±0.62
|
12.00±0.64
|
11.00±0.67
|
The ANOVA results showed that in the tested samples that were used against E.coli, there was no significance difference between the SNPs which their flasks before production were placed in the refrigerator and the autoclave (p value > 0.05) but significance differences were seen between the other tested samples (p value < 0.05).
In the tested samples that were used against P. aeruginosa, there was no significance difference between the SNPs which their flasks before production were placed in the hot air oven, autoclave and RT (p value > 0.05) but significance difference was seen between these groups and the sample which was pretreated in the refrigerator (p value < 0.05).
In the tested samples that were used against S. aureus, no significant differences were seen between the tested samples (p value > 0.05).
MTT assay
In order to analyze the toxic effects of the SNPs samples, MTT assay performed and the cell viability percentage determined. IC50 for each sample was obtained and results indicated that all the four tested samples induced dose dependent toxic effects. IC50s for the samples of the flasks which before SNPs production were pretreated with the hot air oven, autoclave and at RT were in the third well (0.125 mg/ml of SNPs), and for the sample of the flask which before SNPs production was pretreated with the refrigerator was in the second well (0.5 mg/ml of SNPs). Hence the amounts of the SNPs in theirIC50 dose based on the ICP obtained results were 0.40, 0.45 and 0.66 and0.44ppm for the samples were pretreated with hot air oven, autoclave, refrigerator and at RT, respectively. Fig 7 indicated the cell viability percentages of the cells based on the MTT assay results.
Apoptosis induction assay
Annexin V-FITC/ propidium iodide assay kit was used for apoptosis induction assay and the cells were incubated with four different types of SNPs at their IC50 doses for 6 h. More than 96% of the cells in the control well were alive and in contrast to the control, the biologically produced SNPs induced more apoptosis and less necrosis in the cells after 6 h of incubation. Fig 8 indicated the obtained results.