The preparation of silver nanoparticles (Ag NPs) via an environmentally friendly green synthesis method using both bacterial and plant extracts represents a promising alternative approach from an ecological point of view. Thermophilic bacteria were isolated from the Ilıca hot spring in Erzurum, and the Caldibacillus thermoamylovorans SA1 strain was identified as having the highest lignin peroxidase enzyme activity. Using waste walnut shells in combination with this bacterium, the optimal conditions for lignin peroxidase enzyme production were determined by two different methods (5 g/L shell amount, 96 h incubation time, 140 rpm agitation, 60°C temperature, and pH 8.0). Characterization of the synthesized AgNPs was performed using various analytical techniques, including UV‒Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM), which confirmed the successful production of the nanoparticles. Furthermore, the biological efficacy of the synthesized AgNPs was evaluated by assessing their antibacterial activity against pathogenic bacteria, such as Escherichia coli O157:H7, Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, and Bacillus cereus. The AgNPs showed significant efficacy against all five pathogenic bacteria, with the antibacterial potency ranking as follows: B. cereus > E. coli = S. aureus > K. pneumoniae > S. pyogenes. In particular, the highest activity was observed against the pathogen B. cereus (15 mm). The broad-spectrum antibacterial properties exhibited by the Ag NPs synthesized in this study offer a promising and sustainable solution for diverse applications in various sectors, including the environmental, agricultural, medical and pharmaceutical fields. In addition, the use of walnut shell, an agricultural waste product, as a substrate for enzyme production not only enhances the value of agricultural byproducts but also ensures the continued use of renewable and sustainable resources.
Research Article
Eco-Friendly Synthesis of Silver Nanoparticles Using Thermophilic Bacteria (Caldibacillus thermoamylovorans) and Walnut Shells: Promising Antibacterial Agents for Diverse Applications
https://doi.org/10.21203/rs.3.rs-4161831/v1
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Caldibacillus thermoamylovorans SA1
lignin peroxidase enzyme
walnut shell
silver nanoparticle
antimicrobial activity
Among metal nanoparticles, silver nanoparticles (Ag NPs) are at the forefront of other nanoparticles due to their numerous applications in addition to their unique chemical, physical and biological properties. Ag NPs can be produced by physical, chemical and green synthesis methods (bacteria, fungi, plant extracts, etc.); however, studies involving green synthesis methods have yielded more remarkable results (Rafique et al. 2017; Alabdallah et al. 2021). The green synthesis of Ag NPs using microorganisms is both more environmentally friendly and less costly. Among microorganisms, bacteria are the most widely used. The use of bacteria in the synthesis of Ag NPs has been shown to be effective in producing silver nanoparticles with unique properties (Rafique et al. 2017). In addition to the antimicrobial and antioxidant activities of silver nanoparticles, they are also used in burn dressings, food packaging, water treatment systems, electronics, textiles and medical devices (Devi et al. 2015; Zhang et al. 2016; Alabdallah et al. 2021).
Silver nanoparticles were synthesized by a green synthesis method and were reported to exhibit both anticancer and antibacterial properties. This green synthesis is once again efficient, environmentally friendly, and reproducible (Singh et al. 2023). The application of Ag NPs in the field of nanomedicine has been associated with their small particle size, large surface area and electrochemical properties, thus demonstrating an environmentally friendly approach to creating bioactive materials that may be useful in medical applications (Khatun et al. 2023). Furthermore, the incorporation of silver nanoparticles into nanofibers result in effective antibacterial effects, making them good candidates for biomedical materials (Zhang et al. 2016).
Lignin peroxidase (LiP) plays an important role in the synthesis of silver nanoparticles due to its unique ability to reduce various phenolic and noncophenolic compounds and catalyze the oxidative cleavage of C-C and ether (C-O-C) bonds, especially in noncophenolic compounds (Singh et al. 2017; Taniwan et al. 2020). Among metal nanoparticles, the biosynthesis of silver nanoparticles via the LiP enzyme has been supported by several studies (Sanghi et al., 2011; Chaurasia et al., 2022). Biologically synthesized silver nanoparticles can be found in various Bacillus species (Kalimuthu et al., 2008; Babu et al., 2009). C. thermoamylovorans, a thermophilic bacterium from the genus Bacillus, is an interesting candidate for the production of LiP enzymes and its ability to synthesize silver nanoparticles. The ability of C. thermoamylovorans to form both thermophilic and facultative anaerobes has made it a potential candidate for the biological synthesis of silver nanoparticles (Combet-Blanc et al., 1995).
This research aimed to investigate the synthesis of nanoparticles from walnut shell waste by LiP enzyme production by C. thermoamylovorans and to determine the biological activity of these nanoparticles. Therefore, our study will provide insight into the biological activities of C. thermoamylovorans and lignin peroxidase enzymes in the synthesis of silver nanoparticles, which will enable us to understand the biological activities of these nanoparticles. To the best of our knowledge, this study is the first to report on LiP enzyme production by C. thermoamylovorans.
2.1. Collecting and preparing walnut shells
Walnut shells used in the study were collected in the Tortum district, Erzurum, Turkey. Walnut shells consist of lignin, cellulose and hemicellulose. To extract the lignin, the walnut shell formed on the outer surface of the walnut plant was removed. Then, the sample was washed several times with distilled water and left to dry at 105°C. The dried walnut shells were first ground and then sieved (0.25 mm). To remove the hemicellulose layer from the walnut shells, 10 g of walnut shell powder was dissolved in 100 mL of distilled water. After treatment with 2% NaOH, the mixture was incubated at 100°C for 4 h. The sample was centrifuged at 6000 rpm for 10 min. Finally, the precipitates were washed once with distilled water and dried at 60°C. The dried samples were stored at + 4°C until use (Fig. 1.) (Zheng et al., 2019).
2.2. Bacterial isolation
Thermophilic bacteria were isolated from water samples collected in the Erzurum Ilica District Hot Spring. After the water samples were collected, they were brought to the laboratory under aseptic conditions and stored at + 4°C until use. Water samples were directly inoculated into Tryptone Soy Broth (TSB) and incubated at 55°C for 24–48 h in a shaking incubator for enrichment. Then, a series of dilutions (10− 1-10− 6) were prepared with 0.85% physiological water, and 100 µL of each dilution was spread on tryptone soy agar (TSA). The TSA medium was supplemented with 15 g/L casein peptone. The Petri dishes were incubated at 55°C for 24–48 h. At the end of the incubation, morphologically distinct colonies were selected on the dilution plates and purified. Pure colonies were stored at -86°C (20% glycerol + TSB) (Adıgüzel et al., 2020).
2.3. Determination of lignin peroxidase (LiP) enzyme content
To determine the LiP enzyme production ability of the strains, agar media containing 4.55 g/L potassium phosphate dibasic (K2HPO4), 0.53 g/L potassium phosphate monobasic (KH2PO4), 0.5 g/L magnesium sulfate (MgSO4), 5 g/L ammonium nitrate (NH4NO3), 0.1 g/L yeast extract, 0.1% guaiacol and 30 g/L TSA were prepared and adjusted to pH 7.0 (Falade et al. 2017; Falade et al., 2019). The isolated strains were then inoculated on the media and incubated at 55°C for 72 h. After incubation, Lugol solution (1%) was added to the Petri plates, and whether the plates hydrolyzed guaiacol was examined by observing zone formation. The bacterial isolate coded with SA1 showed the largest zone, and thus, it was selected as the best enzyme producer.
2.4. Identification of the selected isolate by molecular methods
The genomic DNA of the isolate encoding SA1 was extracted using a Wizard Genomic DNA Purification Kit (A2360). Then, the 16S rRNA gene was PCR amplified using the universal primers 27F (5'-AGAGAGTTGATCCTGGCTCA-3') and 1492R (5'-GGTTACCTTGTACGACTT-3'). The amplified gene region was cloned and inserted the pGEM-T Easy Vector system. After cloning, plasmid isolation was performed, and the 16S rRNA gene sequence (1509 bp) was sequenced at Macrogen Company (Amsterdam, The Netherlands). The 16S rRNA gene sequences available in GenBank and EzBioCloud (http://blast.ncbı.nlm.nih and https://www.ezbiocloud.net) were compared with other bacterial sequences to determine their similarity and to obtain GenBank accession numbers. A phylogenetic tree was constructed with the MEGA11 program based on sequence analysis of 16S rRNA sequences of strains similar to the isolate (Adıgüzel et al., 2020).
2.5. Detection of lignin peroxidase (LiP) enzyme activity
Guaiacol (2-methoxyphenol) and veratryl alcohol (3,4 dimethoxybenzaldehyde), which are lignin monomers, were used to demonstrate the lignin utilization and degradation potential of the isolate (Falade et al. 2017). After the production process, the medium was centrifuged at 12.000–13.000 rpm for 10 min. The pellet was removed, and the supernatant was used as the enzyme source.
Guaiacol (2-methoxyphenol)
The reaction mixture (100 mL) was prepared using 498 µL (15 mM) guaiacol, 228 µL (7.5 mM) hydrogen peroxide (H2O2) and 1.3609 g/L (0.03 M) sodium phosphate buffer (Na2HPO4) (pH 6.0). The reaction was initiated by adding 2 µL of enzyme, and measurements were recorded at 470 nm for 3 min at 60 s intervals. One unit of enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 1 µmol guaiacol produced in 1 min (Angelini et al., 1989; Oztekin et al. 2019).
Veratryl alcohol (3,4-dimethoxybenzaldehyde)
The reaction mixture was prepared with 1 mL (125 mM) sodium potassium tartrate buffer (KNaC4H4O6) (pH 3.0), 500 µL (10 mM) veratryl alcohol, 500 µL (2 mM) hydrogen peroxide solution (H2O2) and 500 µL culture medium. The reaction was initiated by adding hydrogen peroxide, and the absorbance change was observed at 310 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 1 µmol of veratrilaldehyde produced in 1 min (Arora et al., 2001).
2.6. Optimization of culture conditions for lignin peroxidase (LiP) production
For lignin peroxidase production, 100 mL of production medium (Falade et al. 2017; Falade et al., 2019) containing 1.0 g/L walnut shell, 4.55 g/L potassium phosphate dibasic (K2HPO4), 0.53 g/L potassium phosphate monobasic (KH2PO4), 0.5 g/L magnesium sulfate (MgSO4), and 5 g/L ammonium nitrate (NH4NO3) was prepared in 250 mL flasks and autoclaved at 121°C for 15 min. The experimental factors and their associated levels for medium formulation via one-factor-at-a-time OFAT experiments, such as incubation time with 24 h intervals (24–144 h), shell amount with a one gram interval (1.0–9.0 g/L), pH with 0.5 intervals (5–8.5), agitation rate with 10 intervals (130–160 rpm) and temperature with 5 intervals (50–65°C), were optimized. In all optimization steps, a bacterial suspension (OD 600 nm ≈ 1.0) was inoculated into the fermentation medium under aseptic conditions.
2.7. Green hydrothermal synthesis of silver nanoparticles (Ag NPs)
For the synthesis of Ag nanoparticles, AgNO3 (0.1 M) stock solutions were prepared by mixing them in 50 mL of distilled water. The extracellular medium from the bacteria grown in walnut shell medium was then added to the solution and stirred under optimal conditions for 4 h. To this solution, 0.5 M NaOH (25 mL) was added until the pH of the reaction medium was between 8 and 11. Then, citric acid and urea were added, and the mixture was transferred to a Teflon lined, sealed stainless steel autoclave and kept at 180–200°C for 8 h. At the end of the experiment, the pellet was allowed to cool to room temperature. After the reaction was completed, the obtained white solid Ag NPs were washed with ethanol, filtered and dried under vacuum in a laboratory oven at 60°C (Nadaroglu et al., 2020; Nadaroglu et al., 2023).
2.8. Characterization of Ag NPs
The characterization of the NPs was carried out at Atatürk University Eastern Anatolia High Technology Application and Research Center (DAYTAM). The instruments used for characterization are listed below:
Scanning electron microscopy (SEM) for SEM analysis: A Zeiss Sigma 300 instrument was used. The particle size and morphology of the sample were analyzed.
X-ray Diffraction (XRD): A Panalytical Empyrean instrument was used. The X-ray crystal structure of biologically prepared Ag NPs was investigated.
Fourier transform infrared (FTIR) spectroscopy: A Bruker Vertex 70 model was used. The surface chemistry of the Ag NPs was investigated.
2.9. Investigation of the antibacterial activity of synthesized Ag NPs against pathogenic bacteria
By using the disk diffusion method, the synthesized Ag NPs (50 mg/mL) were tested against Escherichia coli O157: H7 (ATCC 43884) (Gram-negative), Klebsiella pneumoniae (ATCC 13883) (Gram-negative), Staphylococcus aureus (ATCC 25923) (Gram-positive), Streptococcus pyogenes (ATCC 19615) (Gram-positive) and Bacillus cereus (ATCC 11778) (Gram-positive) (More et al. 2023). Nutrient agar (NA) was prepared for the growth of pathogenic bacteria and autoclaved at 121°C for 15 min. Media and disks were prepared according to the National Committee for Clinical Laboratory Standards (NCCLS). In the presence of positive (kanamycin) or negative control (water), the synthesized silver nanoparticles were impregnated on the disks, and the Petri dishes were grown at 37°C for 18–24 h (Cheon et al. 2019). Three replicates were performed for each bacterium, and the antibacterial effect of Ag NPs was determined by measuring the average diameter (mm) of the zones around each disk.
3.1. Isolation of isolates from thermal springs and determination of LiP enzyme content
A total of 9 bacteria were isolated from the thermal spring in Erzurum. The isolates were cultured on solid media supplemented with guaiacol to determine lignin peroxidase enzyme activity. 4Four strains (coded as SA1, SA2, SA3 and SA4) displayed lignin peroxidase activity, and SA1, which had the largest zone, was selected as the best producer (Table 1).
3.2. Identification of the SA1 strain by conventional and molecular methods
SA1 was identified using conventional and molecular methods. According to conventional analysis, the SA1 strain is Gram-, oxidase- and catalase-positive. The cells of the test strain had a Bacillus morphology. For molecular characterization of the 16S rRNA gene region of the isolate, rRNA sequence analysis was performed. This isolate was identified as Caldibacillus thermoamylovorans (C. thermoamylovorans SA1, 99.7% identity), and its phylogenetic tree structure is shown in Fig. 2.
In the literature, it has been reported that bacterial genera belonging to Pseudomonas sp., Cellulomonas sp., Streptomyces sp., (Woo et al., 2014) Ensifer sp., (Falade et al., 2019) and Bacillus sp. (Falade et al., 2020; Fall et al., 2022) have lignolytic enzyme activity. The fact that the SA1 strain we used in our study is a Bacillus species supported that it is in agreement with the studies conducted for LiP enzyme production. In addition, lignin peroxidase enzyme production using C. thermoamylovorans bacteria has not been previously reported. Therefore, our study is important for revealing the LiP enzyme production capabilities of Bacillus species.
3.3. Optimization studies of lignin peroxidase production from C. thermoamylovorans SA1
The production of microbial enzymes, including lignin peroxidase, is significantly affected by culture parameters (Baltacı et al., 2019). The culture conditions, such as shell amount, temperature, pH, incubation time, and agitation rate, for the production of the lignin peroxidase enzyme by C. thermoamylovorans SA1 were optimized using the OFAT approach. The highest enzyme production was achieved at 60°C, pH 8.0, 5 g/L walnut shell, 140 rpm, and 96 h of incubation. This production process can be regarded as effective and efficient in terms of waste management and sustainability since the bacterium utilizes walnut shells as a carbon source and increases the extracellular production of lignin peroxidase enzymes. (Fig. 3, 4).
In this study, the optimum incubation time for the metabolic activities of the bacterium C. thermoamylovorans SA1 was determined. The optimum LiP enzyme production was tested over a wide range of incubation times (24–144 h). The extracellularly produced enzyme source was determined by spectrophotometric measurements with substrates of both guaiacole and veratryl alcohol. The effect of incubation time on LiP enzyme activity was investigated, and the results are shown in Fig. 3A. The activity increased exponentially at a certain rate from 24 h to 96 h. However, due to the hardness of the walnut shell structure, the highest activity was observed after 96 h, indicating that the activity of veratryl alcohol was 126.88 EU/mL and that of guaiacole was 3.13 EU/mL. In a study performed by Falade et al. (2019), optimum peroxidase production in Bacillus sp. was reported to occur after 48 h of addition of 0.1% w/v pure lignin to the medium (Falade et al. 2019). In another study, manganese peroxidase was produced from Fusarium sp. by adding rice straw or wood shavings to the medium. The highest enzyme activity values were reported at the end of the 9th and 12th days (Huy et al. 2017). Reddy et al. (2003) used P. ostreatus and P. pulmonarius fungi, and the maximum specific laccase activity was determined on day 10, while lignin peroxidase and xylanase activities were determined on day 20 (Reddy et al. 2003). In studies using fungi, enzyme production was not time-effective compared to that of bacteria (Reddy et al. 2003; Huy et al. 2017). It is known that obtaining long-term enzyme activity is not preferred in biotechnology. Considering the short fermentation time of bacteria, the use of bacteria was preferred in our study. In this way, the production of LiP enzymes by bacteria will offer a better alternative in industrial fields.
There are studies on the depolymerization of lignin-containing wastes generated in the environment as a result of agricultural and industrial applications by microorganisms (Reddy et al. 2003; Huy et al. 2017; Falade et al. 2020). Both industrial byproducts and organic and agricultural wastes can be utilized as eco-friendly and cost-effective alternatives. The nutrients present in wastes support enzyme production and microbial growth. These waste materials have been recycled biotechnologically to eliminate their environmental side effects and to develop eco-friendly bioprocessing methods. Therefore, walnut shells were used to obtain the LiP enzyme. To determine the optimum amount of shell, walnut shell was added to the medium in the range of 1–9 g, and the LiP enzyme activity was monitored for 96 h. The results are shown in Fig. 3B. The highest LiP enzyme activity was obtained from 5 g (veratryl alcohol: 161.65 EU/mL; guaiacol: 5.26 EU/mL) of walnut shell. Banana peel substrate was used as an alternative to agricultural and/or industrial wastes such as sawdust, straw and bagasse. The production and specific activities of various lignolytic and cellulolytic enzymes in P. ostreatus and P. pulmonarius fungal strains were investigated. Laccase, xylanase, carboxy methyl cellulase and lignin peroxidase enzymes were also analyzed (Reddy et al. 2003). In the studies performed by Falade et al. (2019) and Falade et al. (2020), different nitrogen sources and lignin monomers were used for quantification (Falade et al. 2019; Falade et al. 2020). In our study, only a certain amount of salt and waste walnut shells were added to the media. The production of lignin peroxidase from waste walnut shells showed that we can produce this enzyme without the need for other lignin monomers or nitrogen sources. In this study, the conversion of environmental waste into LiP using C. thermoamylovorans SA1 bacteria was performed for the first time.
In another optimization step, pH optimization, the enzyme activity was examined in the range of pH 5-8.5. It increased from pH 5.0 to pH 8.0. However, in both measurements, the highest enzyme activity was observed at pH 8.0 (veratryl alcohol: 270.25 EU/mL; guaiacol: 6.34 EU/mL) (Fig. 3.C). The graph shows that the bacteria exhibited activity in both acidic and basic environments. It was reported that the pH range of the bacterium was 5.4 to 8.5, and the optimum pH range was 7.0 (Combet-Blanc et al. 1995). Falade et al. (2020) reported that the highest peroxidase enzyme activity value for Bacillus sp. occurred at pH 5.0 (Falade et al. 2020). In a different study by Falade, the highest enzyme activity was reported at pH 8.0 (Falade et al. 2019). As a result, our study is compatible with the findings of Falade et al. (2020).
Shaking speeds of 130, 140, 150 and 160 were tested, and the highest enzyme activity was observed at 140 rpm (273.12 EU/mL for veratryl alcohol and 7.70 EU/mL for guaiacol) (Fig. 4A). In a study conducted by Falade et al. 2019, the enzyme activity was reported at 150 rpm (Falade et al. 2019; Falade et al. 2020). In another study, manganese peroxidase was produced by Fusrium sp. and showed the best activity at 180 rpm (Huy et al., 2017). In our study, the enzyme activity was high even at low rpm.
Finally, a temperature range of 50–65°C was tested. Since it is a thermophilic bacterium, the temperature experiment was started at 50°C. Figure 4. B shows that the highest enzyme activity value in LiP enzyme production was at 60°C (veratryl alcohol: 280.38 EU/mL; guaiacol: 8.1 EU/mL). In both measurements, a large increase in the LiP enzyme activity of the C. thermoamylovorans SA1 strain was observed between 55 and 60°C, while a decrease was observed at 65°C. The maximum growth temperature of the bacterium was previously reported to be 58°C (Combet-Blanc et al. 1995). Nour El-Dein et al. (2014) studied the thermophilic bacterium Streptomyces sp. in a temperature test between 30 and 60°C, which showed the highest peroxidase enzyme activity at 40 and 50°C (Nour El-Dein et al., 2014). In another study, a temperature range of 30–70°C was tested using T. fusca bacteria for extracellular lignocellulolytic enzyme production, and the highest enzyme activity was obtained at 50°C (Tuncer et al. 1999). In the study suggested by Alam, temperature optimization was performed for LiP enzyme production by P. chrysosporium, and the highest enzyme activity was observed at 55°C (Alam et al., 2009). In our study, the fact that the C. thermoamylovorans SA1 strain showed the greatest activity at a high temperature of 60°C is of biotechnological importance.
3.4. Synthesis and characterization of Ag NPs using LiP enzyme homogenates
An enzyme homogenate showing high LiP activity was produced from the bacterium B. thermoamylovorans SA1. This enzyme homogenate was used to synthesize AgNPs continuously. Silver ions were first reduced at 60°C for 4 h, and then, hydrothermal synthesis of Ag NPs (200°C and 8 h) was carried out. The nanoparticles changed from light yellow to dark brown during stirring, indicating that the nanoparticles could be produced. Our study shows the feasibility of silver nanoparticle production at high temperatures, such as 55°C instead of the 25°C used by Nadaroğlu and the 28°C used by Wang (Wang et al. 2016; Nadaroglu et al. 2020).
3.5. Characterization of Ag NPs
UV visible spectroscopy
After hydrothermal synthesis, the Ag NPs were precipitated to the bottom of the thermal cup. The synthesis of Ag NPs was confirmed using UV spectrometry. UV‒visible spectroscopy was used to study the colloidal reaction medium obtained during the synthesis of Ag NPs catalyzed by lignin peroxidase enzymes obtained extracellularly from B. thermoamylovorans. The LiP enzyme assisted in the synthesis of Ag NPs. UV absorbance plots showing the optical properties of the synthesized Ag NPs are shown in Fig. 5. In the given spectrum, the maximum absorbance for the synthesized NPs was 410 nm. The presence of a peak in the 300–500 nm range indicates the synthesis of silver NPs. The LiP enzyme in the oxidoreductase group clearly synthesized Ag NPs, which was confirmed using UV‒visible spectroscopy at wavelengths between 300 nm and 700 nm. UV‒visible spectroscopy is a well-established technique for the analysis of shape- and size-controlled NPs in aqueous solutions. Finally, this spectroscopy allows the measurement of the wavelengths of light absorbed by the NPs.
Fourier transform infrared spectroscopy (FTIR) and XRD analysis
The chemical composition and functional group formation of the synthesized silver nanoparticles were determined by FTIR and are shown in Fig. 6A. Fourier transform infrared spectroscopy (FTIR) of the aqueous silver nanoparticles was used to identify the molecules that might be responsible for the synthesis and stability of the metal nanoparticles via green synthesis. The formation of the bands occurred by the reduction of silver ions by functional groups, and the different bands formed indicated various modes of stretching.
The peak at 2983–3733 cm− 1 in the FTIR spectrum reveals a stretching vibration attributed to the -O-H group. The peaks at 2044 cm− 1 and 2461 cm− 1 are attributed to alkyl groups in the -C-C- structure. The peaks at 1514 cm− 1 and 1223 cm− 1 can be attributed to C = O and amine (NH) groups, respectively. The peak at 680 cm− 1 is attributed to CH groups, while the peak at 501 cm− 1 indicates Ag NPs. The C = O and -OH groups in the structure are effective in the reduction of Ag NPs and are effective in ensuring the stability of Ag NPs after reduction. The findings obtained are compatible with the findings obtained by Khan and his group (Khan et al. 2022) (Fig. 6A).
X-ray powder diffraction analysis is the best and fastest technique used to determine whether nanoscale materials are amorphous or crystalline. XRD analysis is very effective in revealing the crystalline structure of a material. The XRD pattern of the produced Ag NPs is associated with (111), (200), (220) and (311) crystal structures attributed to Ag NPs, with four major peaks positioned at 35.85°, 41.20°, 61.82°, and 71.62°, respectively, corresponding to face-centered cubic (fcc) surfaces (Fig. 6B). The peak at 35.85° in the graph shows the cubic structure of the Ag NPs. In addition, the results are in agreement with those of studies by Elshazly et al., Kanwal et al. and Hassan Afandy et al. (Kanwal et al., 2019; Elshazly et al., 2022; Hassan Afandy et al., 2023).
Scanning electron microscopy (SEM)
The surface morphology of Ag NPs synthesized using the LiP enzyme was investigated by scanning electron microscopy (SEM). The spherical and cubic structures of Ag NPs synthesized by the green synthesis method are shown in Fig. 7A with SEM images magnified to 200 nm. Although Ag NPs have different sizes in the range of 13.33-40 nm, it can be clearly seen in Fig. 7A that they have cubic and spherical structures.
As shown in Fig. 7B, the elemental composition of the Ag NPs was investigated by energy dispersive X-ray (EDX) spectroscopy. EDX analysis was performed to determine which elements were present in the synthesized silver nanoparticles. An area filled with nanoparticles was focused to show the EDX spectrum collected in dot profile mode. The presence of two peaks, one corresponding to silver and one to oxygen, indicated the presence of only oxygen and silver in the EDX spectrum. The EDX spectrum of Ag NPs revealed 38.08% of the main constituent elements. With this result, the enormous purity of Ag NPs has been conclusively demonstrated, and there is no evidence of other contaminants. This consistency in the increase in Ag shown in the spectrum is consistent with findings reported in the literature (Alavi et al., 2018; Magazenkova et al., 2023).
3.6. Investigation of the Antibacterial Effect of Synthesized Ag NPs
Silver nanoparticles affect both Gram-positive and Gram-negative bacteria (Nadaroğlu et al., 2020; More et al., 2023. In our study, the antibacterial effects of Ag NPs synthesized using the agar disk diffusion method against pathogenic bacteria such as E. coli, K. pneumoniae, S. aureus, S. pyogenes and B. cereus were investigated, and the results are shown in Fig. 8 and Table 2. Silver nanoparticles synthesized with lignin peroxidase (10 mL) showed the highest antimicrobial activity against Bacillus cereus (15.0 mm). In a study by Cicek, silver nanoparticles produced by a green synthesis method were applied to some pathogenic bacteria via the disk diffusion method, and the greatest zone diameter was reported for Klebsiella pneumoniae (19.0 mm). It was also found to be 11.0 mm in diameter in Staphylococcus aureus and 17.0 mm in Streptococcus pyogenes (Cicek et al., 2015). In another study, silver nanoparticles were synthesized from two different bacteria (Ochrobactrum anthropi and Bacillus sp.) and tested for antibacterial activity using the standard well diffusion method. The inhibition zone was reported to be 15.0 mm for Staphylococcus aureus (Thomas et al., 2014). Since silver metal is positively charged, it reacts with proteins in the cell membrane and the main components of DNA bases (such as phosphorus and sulfur) and damages the cell wall morphology of bacteria (More et al. 2023). In addition, due to the small size and large surface area of the silver nanoparticles produced, they easily overcome the cell wall barrier of bacteria. In this way, the high interaction of the surface area increases the antibacterial effect, and our results are in agreement with the literature (Thomas et al. 2014; Cicek et al. 2015; Nadaroğlu et al. 2020; More et al. 2023).
| Pathogenic Microorganisms | Negative Control (water) | Pozitive Control (kanamycin) | Ag NP Zone Diameter (mm) |
|---|---|---|---|
| Bacillus cereus ATCC 11778 | 5.0 mm | 16.0 mm | 15.0 mm |
| Klebsiella pneumoniae ATCC 13883 | 5.0 mm | 21.0 mm | 10.0 mm |
| Escherichia coli O157:H7 ATCC 43894 | 5.0 mm | 15.0 mm | 13.0 mm |
| Streptococcus pyogenes ATCC 19615 | 5.0 mm | 20.0 mm | 8.0 mm |
| Staphylococcus aureus ATCC 25923 | 5.0 mm | 16.0 mm | 13.0 mm |
In this study, we present a new environmentally friendly approach for the green synthesis of silver nanoparticles (Ag NPs) using an enzymatic method that provides control over particle size and shape. B. thermoamylovorans was isolated from Erzurum Ilica Hot Spring, and the optimum LiP enzyme production conditions were determined to be 96 h, 5 g/L shell amount, 140 rpm, pH 8 and 60°C. Silver nanoparticles were synthesized with the lignin peroxidase enzyme homogenate produced after optimization. FTIR, XRD and SEM analyses of the synthesized silver nanoparticles were performed. To determine the biological activity of the synthesized Ag NPs, their antibacterial effects on pathogenic bacteria such as E. coli, K. pneumoniae, S. aureus, S. pyogenes and B. cereus were investigated. As a result, Ag NPs showed activity against all the pathogenic bacteria, and the largest zone was observed for B. cereus (15 mm), while the smallest zone was observed for S. pyogenes (8 mm). This study provides a new approach for the sustainable and innovative synthesis of Ag NPs, and the versatile biological activities of these nanoparticles will allow the establishment of new methods for various applications in agriculture, the environment, medicine and pharmaceuticals. The use of walnut shell waste as a substrate for enzyme production will add value to agricultural and industrial byproducts and support the creation of renewable and sustainable agriculture.
Funding
Not applicable
Declaration of competing interests
The authors declared that there is no conflict of interests.
Author's Contribution
Sefanur Akkaya: Methodology, Conceptualization, Investigation; Mehmet Akif Omeroglu: Conceptualization, Formal analysis, Methodology; Hayrunnisa Nadaroglu: Writing–original draft, Methodology, Conceptualization; Ahmet Adiguzel: Writing–review&editing, Supervision, Conceptualization.
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No competing interests reported.