Synthesis, Characterization, and Optimization of Green Silver Nanoparticles Using Neopestalotiopsis clavispora and Evaluation of Its Antibacterial, Antibiofilm, and Genotoxic Effects
Article Category: Research Article - Pharmaceutical Biotechnology
Publicado en línea: 24 jul 2021
Páginas: 109 - 122
DOI: https://doi.org/10.2478/ebtj-2021-0020
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© 2021 Tuğba Kahraman, Safiye Elif Korcan, Recep Liman, İbrahim Hakkı Ciğerci, Yaser Acikbas, Muhsin Konuk, Gülderen Uysal Akkuş, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
The reduction of silver ions by a variety of chemical or biological agents can yield Ag-NPs. The biological methods which involve nanoparticle synthesis have exhibited higher effectiveness than that of chemical methods on account of slower kinetics; this provides a greater degree of control over crystal growth and a reduction in capital expenses. Such biogenic synthesis methods are easy, safe, renewable, and economical, offering greater biocompatibility through the use of nanoparticles. In particular, fungal AgNPs synthesis is environmentally safe on account of the elimination of dangerous compounds such as hydrazine supports (1,2); such synthesis also does not require elevated temperatures and prolonged synthesis periods. These particles have also been confirmed to be three times more stable than those produced in non-biological methods (3). In addition, fungal biomass can be easily obtained and no additional steps are required for extracting the filtrate. Due to the aforementioned greater stability, fungal biomass can be utilized in the large-scale synthesis of nanoparticles using simple purification methods including filtration, dialysis, and ultracentrifugation due to their higher resistance to agitation and pressure. Furthermore, optimization of the nanoparticles can be achieved by adjusting pH, temperature, agitation, light, amount of biomass and culture medium, etc. (6, 7, 8, 9, 10).
The synthesis of fungal AgNPs which is mediated extracellularly or intracellularly has been used in many industrial and medicinal applications due to efficient antimicrobial, antifungal, antioxidant immunomodulating, and anticancer activities (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) and further on account of their high tolerance of metals and ability to produce large amounts of extracellular protein, which contributes to nanoparticle stability.
The genus Pestalotiopsis contains a wide range of secondary metabolites with several properties; including antitumor, anti-fungal, and antimicrobial (16, 17, 18). The discovery of metal-tolerant endophytic fungi for the synthesis of metal nanoparticles could be of further major advantage (19, 20, 21).
In this study, we report the synthesis of AgNPs using cell-free filtrate (CFF) of
The fungal strain used for the biosynthesis of AgNPs was isolated from decaying wood samples in Usak University Campus, Turkey (38° 40΄ 08˝ N and 29° 19΄ 4˝ E). 1 g of grounded wood sample was mixed with 10 mL of sterile distilled water and then shaking in an incubator at 110 rpm for 15 min at 27±2 °C. 0.1 mL mixture was incubated onto Potato Dextrose Agar (PDA, Merck 110130) for 7 days at 28±2 °C in the dark. After this, the sample was sub-cultured to achieve a pure culture on PDA and kept at 4 °C to use for future studies. Morphological properties of fungus were determined on Czapek-Dox Agar (CDA, Merck 105460) and Malt Extract Agar (MEA, Merck 105398) according to Biju et al. (23).
According to the manufacturer’s instructions, total DNA isolation from a single colony of fungus was carried out using the EurX GeneMATRIX Plant&Fungi DNA isolation kit (Poland). 30 μL of Activation Buffer P was added to the spin column without spin and kept at room temperature for 10 min. Fungal tissue was homogenized with a cooled mortar and pestle under liquid nitrogen. 20 mg dry fungal tissue was placed in an empty Eppendorf tube; 400 μL of Lysis Buffer F, 3 μL of RNase A, and 10 μL of Protein kinase K were added. The tube was mixed and incubated at 65 °C for 30 min. After adding 130 μL Buffer AC, the tube was incubated for 5 min on ice. The sample was centrifuged for 10 min at 14000×g. The mixture (400 μL supernatant, 350 μL buffer Sol P and 250 μL 96% ethanol) was centrifuged for 1 min at 12000×g. After 600 μL of lysate was transferred to the DNA binding spin column, the sample was centrifuged for 1 min at 11000×g. Pellet was washed with 500 μL of Wash PX two times at 11000×g for 1 min. Spin columns were placed in a new tube and 50-150 μL of elution buffer was added to elute the bound DNA. Isolated DNA was stored at -20 °C. The universal primer ITS1 (5’- TCC GTA GGT GAA CCT GCG G -3’) and ITS4 (5’- TCC TCC GCT TAT TGA TAT GC -3’) were used to amplify to ITS sequences of nuclear ribosomal DNA) according to Gardes and Bruns (24). The amplification conditions were set as follows: initial denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 45 s (denaturing), annealing temperature at 57 °C for 45 s, followed by extension at 72 °C for 60 s, and finally 72 °C for 5 min (final elongation). PCR reaction was performed with Solis Biodyne (Estonia) FIREPol® DNA Polymerase Taq polymerase enzyme. After PCR, a single band was obtained in agarose gel using 100 bp DNA Ladder Ready to Load (Solis BioDyne) marker, and it was observed that the PCR process was successful. ExoSAP-IT™ PCR Product Cleanup Reagent (ThermoFisher Scientific, USA) was used to purify PCR products. For this purpose mix 5 μL of a post-PCR reaction product with 2 μL of ExoSAPIT™ reagent for a combined 7 μL reaction volume. The ABI 3730XL Sanger sequencing device (Applied Biosystems, Foster City, CA) and the BigDye Terminator v3.1 Cycle sequencing kit were used for the Sanger sequencing. Consensus sequences were used to scan for homologous sequences using the Basic Local Alignment Search Tool (BLAST) program at the National Center for Biotechnology Data (NCBI;
Biosynthesis of silver nanoparticles was done according to Maliszewska et al. (25) with slight modifications. Fungal isolate was grown in 100 mL flask containing medium (0.025 g/ L yeast extract, 0.012 g/L CaCl22H2O, 0.05 g/L MgSO47H2O, 1 g/L NH4H2PO4, and 10 g/L glucose) at 27 °C on a rotary shaker at 110 rpm 7 d and then filtered with Whatman filter paper No. 1 to obtain fungal biomass. The mixture (3.5 g fungal biomass and 150 mL sterile distilled water) was agitated at 27± 2°C in an orbital shaker at 125 rpm for 24 h. After filtration, CFF was mixed with 2.5 mM AgNO3 in a ratio of 1:9, and then the mixture was agitated at 27± 2°C in an orbital shaker (125 rpm) for 72 h. When the color turns brown, the absorbance of fungal synthesized AgNPs was scanned in the range of 200-800 nm on a UV-vis spectrophotometer (Shimadzu, UV-1800) at 1 nm resolution. The CFF and 2.5 mM AgNO3 were used as controls.
Optimization of extracellular fungal AgNPs biosynthesis for AgNPs formation using various parameters including pH (2, 4, 7, 9 and 12; at 25±2 °C ; substrate concentration 0.25 mM AgNO3), temperature (25, 35 and 45 °C; at pH 7; substrate concentration 0.25 mM AgNO3), reaction time ( from 8 to72 hour; at pH 7; substrate concentration 0.25 mM AgNO3) and substrate concentration (substrate concentration, 0.05, 0.1, 0.15, 0.25mM; at pH 7 and 0.25 mM AgNO3; ) were determined according to Sobhy et al. (26) with minor changes until UV measurements were fixed. Each variable was optimized by varying only a single parameter.
Perkin Elmer 1605 FTIR System carried out the characterization of functional groups on the surface of the synthesized Ag-NPs. Spectrum BX spectrophotometer was screened in spectra of 4000-450 cm-1 (27). The size and shape of the synthesized AgNPs were determined by TEM (Hitachi TEM HT 7800) at 100kV voltage. Average nanoparticle sizes were determined by the amount of approximately 100 nanoparticles in various regions of the growing sample. Also, the zeta potential of silver nanoparticles was determined on the MALVERN NANO-ZS device.
Antibacterial effects of green synthesized AgNPs, AgNO3 and CFF were evaluated on, and Bacillus subtilis, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCCC 25922 using the agar well diffusion method (28). Under aseptic conditions, MHA agar plates (Merck, 1.05437) were inoculated with bacterial (0.5 McFarland) strain. Wells (R=6mm) were filled with 50 μL fungal synthesized of AgNPs (10, 1 and 0.1 ppm), CFF and 0.25 mM AgNO3. After plates were incubated at 24 h for 37 ºC, inhibition zones have been recorded in mm in diameters. Antibiotic sensitivity of bacteria was also determined by the disc diffusion method according to the guideline established by the CLSI were used as control groups. Both experiments have been conducted in triplicate.
Biofilm inhibition assay was performed according to Sand-berg et al. (29) with minor modifications. Fresh, exponentially grown P. aerouginosa ATCC 27853 culture (106 CFU/mL) was used for biofilm inhibition assay. Subsequently, different concentrations of AgNPs (10, 1, and 0.1 ppm) were added (1/1 v/v) to the P. aerouginosa cultures and incubated at 37 °C for 24 h. Following incubation, samples were gently washed two times with sterile distilled water to remove the medium. The biofilms were stained with a 0.5% (w/v) crystal violet at 27±2 °C for 45 min. After washing 3 times with distilled water, 1.5 mL of ethanol: acetic acid (95: 5) mixture was added for 10 min. The antibiofilm activity of the AgNPs was determined at 570 nm by using the following formula.
% Inhibition = (A control – A sample /A control) × 100 A control: Absorbance value not containing AgNPs A sample: Absorbance value with different concentrations of AgNPs
Two Drosophila strains, flr3/TM3, BdS (female), and mwh/ mwh (male) were used for the Drosophila SMART assay (30). The SMART test is focused on the lack of heterozygosity in the wing cells. Transheterozygous larvae were extracted from mating virgin flr3/TM3, BdS females, and mwh males (31). In this study, eggs from virgin flr3 females and mwh males were collected in a healthy nutrient medium for 8 h. After 72±4 h, 3 days old larvae were gathered under tap water using a sieve and transferred to vials containing 4.5 g of Drosophila Instant Medium and 9 mL of test chemicals (0.1, 1, and 10 ppm of AgNO3 and AgNPs). Distilled water and 1 mM EMS were used as a negative and positive control, respectively. Adult individuals were obtained after the application. Then flies were kept at +4 °C in 70% ethanol until wing preparations were prepared. Wings were removed and mounted on microscope slides in Faure’s solution. Prepared wing arrangements were tested with an optical microscope magnification of 40X.
The data obtained in the SMART experiment were evaluated employing a computer program (MICROSTA) prepared for Drosophila wing somatic mutation and recombination tests. Original and alternative hypotheses were calculated by using the binomial conditional test. Kastenbaum and Bowman’s (32) charts were used when original and alternative hypotheses were adopted or rejected.
Fungi were isolated from decaying wood samples in the Usak University Campus, Turkey (Fig. 1A). Colonies were white and cottony with edge undulates and circular growth appearance on CDA (Fig. 1B1-B2) and MEA (Fig. 1C1-C2). During the microscopic examination, sterile hyphae structures were observed, but conidia were not; identification according to its microscopic morphology could therefore not be made. The fungus was identified as
Figure 1
Isolation and identification of
After the addition of 2.5 mM AgNO3 (1:9), the color of the CFF was altered from a light yellow to dark brown due to the reduction of the silver ion. This is the first indication of fungal synthesis AgNPs within 24 h. Figure 2 shows that the reaction had run for 24 h, and the detection of a specific absorption peak at 414 nm, indicating the formation of AgNPs.
Figure 2
UV–Vis spectroscopy of AgNPs synthesized using CFF.
The bands typical for AgNPs were observed in the wavelength range of 4010-419 nm under changing conditions. The peak was obtained at 414 nm in every tested condition, and accordingly, the bio-reduction of Ag ions in the CFF was monitored periodically by measuring UV-Vis spectroscopy at 414 nm. Different parameters including pH, temperature, substrate concentration, and reaction time are shown in Fig 3. A pH range of 2–12 was selected for this study. Absorbance is shown to increase with pH, suggesting that an alkaline environment is more suitable for AgNP biosynthesis (Figure3A). The optimal temperature found for AgNP biosynthesis was 35°C and 45°C (Figure 3C). Absorbance values also increased with time, up to 64 h, but did not increase thereafter (Figure 3D). The effect of AgNO3 concentrations on AgNPs formation is shown in Fig. 3C. Increasing levels of AgNPs formation were observed with an increasing AgNO3 concentration. The highest AgNPs were obtained from 0.25 mM AgNO3 concentration.
Figure 3
Effect of pH (A), temperature (B), substrate concentration (C), and exposure periods (D) on the substrate on the stability of AgNPs synthesis.
FTIR analysis of the synthesized AgNPs revealed visible bands at 3425.58, ~2920, 2858.51, 2355.08, 1647.21, 1556.55, 1456.26, 1382.96, and 1068.56 cm-1, as shown in Fig. 4. The band seen at 3425.58 cm-1 can be attributed to O–H (alcohol) stretching. Two peaks are attributed to C–H (alkane) stretching which is observed at ~2920 and 2858.51 cm-1. A further band observed at 2355.08 cm-1 represents the H-C=O (aldehyde hydrogen) stretching. When analyzing the FTIR spectra, it was seen that the stretching vibrations of the amide I and amide II bands of the proteins were at 1647.2 cm-1. The last band, which was seen at 1068.56 cm-1, is accounted for by C–O (alcohol/ether) stretching. FTIR data exhibit that the biological compounds or molecules can be preferred for both stabilization and synthesis of AgNPs.
Figure 4
FTIR spectrum of green synthesized AgNPs
The TEM image of synthesized AgNPs displayed small particles with clear morphology. Figure 5a (scale bar of 50 nm) and Figure 5b (scale bar of 100 nm) present TEM images of AgNPs for two different scale bars. TEM analysis demonstrated the spherical shape of synthesized silver nanoparticles; these ranged in size from 4.77 nm to 20.68 nm with an average length of 11.88±5.06 nm.
Figure 5
TEM images of AgNPs for scale bar of a) 50 nm b) 100 nm.
The average diameter obtained for AgNPs in ultrahigh-purity water suspension is in the size distribution of between 32.5 and 75.6 nm with a mean diameter of 55.15±14.83 nm for silver nanoparticles. The obtained PdI value of AgNPs was 0.185. Zeta potential and electrophoretic mobility of AgNPs were defined as –19.38±0.73 mV and -1.51±0.06 μm cm/sV, respectively. This zeta potential and electrophoretic mobility results are reported as an average value of three measurements.
The fungal AgNPs have been tested for antibacterial activity against E.coli (ATCC 25922), P. aerouginosa ATCC 27853 (Gram-negative), and B. subtilis, S. aureus ATCC 25923 (Gram-positive). Testing for antimicrobial activity showed that the synthesized AgNPs had antimicrobial activity on all the tested bacteria except E. coli in a dose-dependent manner (Table 1). CFF did not show antimicrobial effects on gram-positive and negative bacteria. 0.25 mM AgNO3 showed antimicrobial effects on all tested bacteria. 10 ppm AgNPs showed more antibacterial effects (12.33±1.53) than Vancomycin (8.67±0.58) and Erythromycin (9±1) on
Antimicrobial activity of CFF, AgNO3, and AgNPs.
| Agents | Diameter zone (mm) ± Standard Deviation | ||||
|---|---|---|---|---|---|
| S. aureus ATCC 25923 | E. coli ATCCC 25922 | B. subtilis | |||
| Vancomycin (30 μg) | 21±2 | 8.67±0.58 | 25±1 | 2±1.73 | |
| Penicillin (10 μg) | 41±3 | Not determined | 33.67±0.58 | 30.67±2.08 | |
| Chloramphenicol (30 μg) | 26.67±0.58 | 16±1.73 | 30.33±1.53 | 36±1.73 | |
| Erythromycin (15 μg) | 30.33±2.08 | 9±1 | 26.67±1.53 | 29±1 | |
| Tetracycline (30 μg) | 30.33±2.52 | 14.67±0.58 | 11.67±0.58 | 16±2 | |
| Cell-Free Filtrate | 0 | 0 | 0 | 0 | |
| Ag NO3 2,5 mM | 18.33±1.53 | 13±1 | 14.33±1.15 | 16.33±1.53 | |
| AgNPs | 10 ppm | 10±1.73 | 12.33±1.53 | 12.33±1.53 | 17± 1 |
| 1 ppm | 9±1 | 1±1.73 | 0 | 16.33± 2.08 | |
| 0.1 ppm | 0 | 0 | 0 | 8.33±0.58 | |
That fungal synthesized AgNPs can effectively inhibit biofilms formed by
Biofilm inhibition (%) of AgNPs against
| AgNPs | |||
|---|---|---|---|
| Inhibition % (n=3) | 10 ppm | 1 ppm | 0.1 ppm |
| 81.13 | 67.63 | 40.28 | |
| 76.25 | 65.45 | 45.11 | |
| 80.62 | 63.32 | 40.43 | |
| Average biofilm inhibition (%) ± Standard Deviation | 79.33±2.68 | 65.47±2.16 | 41.94±2.75 |
AgNO3 and AgNPs (0.1, 1, and 10 ppm) whose genotoxic properties are evaluated with SMART, as shown in Table 3. 72±4 h 3rd stage trans-heterozygous larvae were exposed to 3 different concentrations (0.1, 1, 10 ppm) of AgNO3 and AgNPs, distilled water (negative control) and 1mM of Ethyl methanesulfonate (positive control). In the evaluating genotoxic effects of AgNO3 and AgNPs, wing preparations were prepared from normal poultry individuals for each concentration. In 72±4 h distilled water application, a total of 14 clones were determined, including 12 small uniform clones and 2 large uniform clones in 80 wings. Additionally, the total number of mwh clones found was 13. In distilled water application, clone induction frequency was calculated to be 0.76. Comparing the results obtained from the application of different concentrations of AgNO3 and AgNPs and those obtained from the control group application of distilled water, it was observed that there was no statistically significant difference between any of the clone types. The results for EMS applications used as positive control were compared with those for distilled water applications, and in all clone types, a positive reaction was observed. Neither increase nor decrease was seen in the total clone induction frequency at a specific rate depending on the dose increase. The obtained application results for all concentrations showed that the differences were not statistically significant compared with those obtained from the distilled water, which is the wing preparations, the control group.
Genotoxic evaluation of AgNO3 and AgNPs using SMART assay
| Treatments | Number of Wings (N) | Small uniform clones (1-2 cells) (m=2) | Large uniform clones (> 2 cells) (m=5) | Twin clones (m=5) | Total mwh clones (m=2) | Total clones (m=2) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| No. | Fr. | D. | No. | Fr. | D. | No. | Fr. | D. | No. | Fr. | D. | No. | Fr. | D. | ||
| Normal Wing | ||||||||||||||||
| Distilled Water | 80 | 12 | (0.16) | 2 | (0.02) | 0 | (0.00) | 13 | (0.19) | 14 | (0.19) | 0.76 | ||||
| 1 EMS mM | 31 | 69 | (2.21) | + | 27 | (0.83) | + | 9 | (0.27) | + | 105 | (3.31) | + | 104 | (3.40) | + 5.38 |
| 0.1 | 80 | 15 | (0.16) | i | 5 | (0.06) | i | 0 | (0.00) | i | 20 | (0.25) | i | 20 | (0.25) | i 1.02 |
| 1 | 80 | 9 | (0.07) | - | 2 | (0.02) | i | 0 | (0.00) | - | 8 | (0.10) | - | 1 | (0.10) | - 0.41 |
| 10 | 80 | 5 | (0.01) | - | 2 | (0.02) | i | 0 | (0.00) | - | 3 | (0.03) | - | 7 | (0.03) | - 0.15 |
| 0.1 | 80 | 15 | (0.16) | i | 5 | (0.06) | i | 0 | (0.00) | i | 20 | (0.25) | i | 20 | (0.25) | i 1.02 |
| 1 | 80 | 6 | (0.05) | - | 2 | (0.02) | i | 0 | (0.00) | - | 8 | (0.8) | - | 8 | (0.8) | - 0.39 |
| 10 | 80 | 4 | (0.01) | - | 2 | (0.02) | i | 0 | (0.00) | - | 3 | (0.03) | - | 6 | (0.03) | - 0.15 |
EMS.. Ethyl methanesulfonate; Fr.. frequency; D.. display of statistics results; +.. positive; -.. negative; i.. trivial difference; m..multiplication factor; probability level = 0.05
Extracellular biosynthesis of AgNPs is a method that uses a cell-free fungi filtrate as the reducing agent (3) and is both environmentally friendly and energy-efficient. In this study, the extracellular synthesis of AgNPs using
In the stabilization and accumulation of the particles, the optimization phase plays a crucial role. By regulating parameters such as pH, temperature, the concentration of the substrate, and exposure period, the size of nanoparticles is controllable (47). Different fungi and conditions can be utilized to generate various AgNPs (48). The stability of AgNPs, on the other hand, remains a challenge to control; biosynthesis efficiency is additionally reportedly very limited (49). The methods making use of biomass extracted from the reaction medium produced more AgNPs, but also a narrower AgNPs size range (1.5–20 nm) in comparison to those solutions that used biomass and biomass filtrates (50). Researchers have attempted to optimize the conditions to control the yield, scale, and properties of fungal AgNPs (51). Fig. 3A shows that the synthesis of fungal Ag-NPs increased with an increase in pH; high absorption values were observed via spectrophotometer at pH 12. Nanoparticle synthesis increase is possible at alkaline pH values, due to competition for the creation of negatively charged bonds between proton and metal ions (52). With the use of citrate-capped Ag-NPs, there has been observed a linear correlation between pH and the concentration of generated AgNPs (53). Our FTIR data show that the biological compounds can be capped fungal Ag-NPs. Maximum AgNPs production was observed between pH 9 and 11 for Fusarium oxysporum (54), at pH 12 for Sclerotinia sclerotiorum MTCC 8785 (5), and Penicillium oxalicum (6).
High absorption values in the spectrophotometer for Ag-NPs were observed at 35°C and 45 °C (Fig. 3B). Similar to our results, the optimum temperature for fungus mediated AgNPs was observed at 40 °C for Trichoderma harzianum (56) and Rhizopopus stonolifer (57), at 50 °C for Fusarium oxysporium (54), and at 30 °C for Guignardia mangifera (58). The temperature degrees used in the synthesis of fungal AgNPs can affect the synthesis rate, the size, and the stability of the NPs (59). AgNPs formation increased by increasing AgNO3 concentration (Fig. 3C.). The highest AgNPs were obtained from a 0.25 mM concentration of AgNO3. 0.25 mM AgNO3 was selected for AgNPs production to obtain well-dispersed small nanoparticles because high AgNO3 concentrations can produce large nanoparticles with irregular morphological features (60). In addition, the toxicity of the AgNPs may increase with increasing AgNO3 concentration (57). Absorbance values also increased with time, up to 64 h, but did not increase thereafter (Figure 3D). Ottoni et al. (61) reported that the maximum AgNPs absorption values were determined at 72 h.
Extracellularly formed nanoparticles have been stabilized with proteins and reducing agents secreted by the fungus. In total, four high molecular weight proteins produced from fungal biomass have been reported in combination with nanoparticles. Furthermore, removing metal ions and the surface attachment of proteins to nanoparticles did not weaken the tertiary structure of proteins (62). The compounds of aliphatic and aromatic hydrocarbons comprise a broad variety of functional groups, (–CN, –SH, –COOH, –NH2); these are reported to have a strong propensity for the functionalization of noble metal nanoparticles, rendering them valuable as surface-protective functional groups (63, 64, 65, 66, 67, 68). Carbonyl bound in peptide-protein residues and amino acid has been confirmed to have a larger capacity to bind metals, to the extent that proteins form a coating covering the metal nanoparticles to prevent and stabilize the agglomeration of particles. From this, we can surmise that proteins can be attached to nanoparticle surfaces via free amine groups or cysteine residues which will act as encapsulating agents and stabilize particles. FTIR data demonstrate that the biological compounds or molecules can be preferred for both stabilization and synthesis of AgNPs (Figure 4).
TEM images of synthesized AgNPs displayed the tiny particles with clear morphology. TEM analysis demonstrated a spherical shape of synthesized silver nanoparticles. The average nanoparticle size was determined as 11.88±5.06 nm (Figure 5). The obtained average diameter for AgNPs in ultrahigh-purity water suspension presents a distribution of values ranging between 32.5 and 75.6 nm with a mean diameter of 55.15±14.83 nm for silver nanoparticles. Furthermore, the PdI value of AgNPs in ultrahigh-purity water suspension was obtained as 0.185. Zeta potential (ζ) and electrophoretic mobility of Ag-NPs in ultrahigh-purity water were found as –19.38±0.73 mV and -1.51±0.06 μm cm/sV. Electrophoretic mobility for AgNPs was calculated by Henry’s equation (69). Similar studies have been carried out using the extracellular CFF of Punctularia atropurpurascens, Penicillium expansum, and Phanerochaete chrysosporium fungi to produce SNPs. The size distribution and average sizes of PaNPs, PeNPs, and PchNPs were observed between 12 nm-30 nm, and between 11 nm-38 nm via DLS and TEM, respectively. When the average sizes of these three nanoparticles were compared with the data obtained from our current study, the comparison showed consistency of results; the average diameter of AgNP synthesized using
The antimicrobial action of Ag+ is thought to be exhibited through the absorption of ions by the microorganism cell, along with an accumulation within the cell leading to a shrinkage of the cytoplasm membrane or the attraction of the cytoplasm to itself by the cell wall. It is reported that in this way DNA molecules are damaged, and cells lose their ability to replicate due to the infiltration of Ag+. Furthermore, Ag+ has been shown to act upon the -SH bonds of proteins and cause them to be inactivated (73). Cho et al. (74) reported that silver clumps act as a catalyst for the oxidation of microorganisms in oxygen-added solutions. Antimicrobial activity results showed that synthesized AgNPs possessed antimicrobial activity on all the tested bacteria except E. coli, in a dose-dependent manner (Table 1). The antibacterial mechanism of AgNPs has only tentative explanations. According to some studies, gram-negative bacteria show more sensitivity to AgNPs compared to gram-positive bacteria (9,75), while other research reports the reverse (76,77). However, it is known that the antimicrobial activity depends on the type of microbial species and the concentration of AgNPs (78, 79, 80, 81). It has been further observed elsewhere that AgNPs resistance in the E. coli K-12 MG1655 strain can develop rapidly, with relatively few mutational steps. This does not bode well for the prospect of continuous use of AgNPs as antimicrobial agents (82). After repeated exposure to silver nanoparticles, Gram-negative bacteria as Escherichia coli 013, Pseudomonas aeruginosa CCM 3955, and E. coli CCM 3954 were all able to develop resistance to the antimicrobial effect. This resistance is caused by the production of the adhesive flagellum protein flagellin, which in turn causes the nanoparticles to clump together. The resistance develops without requiring any genetic changes; what is required is only a phenotypic modification, to reduce the colloidal stability of the nanoparticles and thus eradicate their antibacterial activity (83).
We have demonstrated that fungal synthesized AgNPs can be effective for the inhibition of biofilms formed by
AgNPs were synthesized for the first time using CFF of