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Synthesis and Characterization of Silver Nanoparticles from Fuzarium Oxysporum and Investigation of Their Antibacterial Activity
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ScienceDirect Materials Today: Proceedings 9 (2019) 506–514
www.materialstoday.com/proceedings
GMSP&NS’18
Synthesis and Characterization of Silver Nanoparticles from Fuzarium Oxysporum and Investigation of Their Antibacterial Activity Shareefraza J. Ukkund,a,b* Momin Ashraf,a Apoorva B. Udupa,a Mayur Gangadharan,b Aswanth Pattiyeri,b Yashawantha K. Marigowda,b Ravika Patila, Prasad Puthiyllama,b a
b
Department of Nano Technology, Srinivas Institute of Technology, Mangalore- India Srinivas Centre for Nano Science and Technology, Srinivas Group of Colleges, Mangalore- India c Department of Mechanical Engineering, Srinivas Institute of Technology, Mangalore- India.
Abstract Nanotechnology is the fastest growing field of 21st century due to this reason that the nanomaterials synthesis finds more importance in field of research. The nanomaterials can be synthesized by physical, chemical and biological methods. The biological methods are more advantageous than the other two methods because of manipulation of the size and morphology of nanostructures by microbes and plants. The Penicillium sps. Fuzarium Oxysporum was used to synthesize silver nanoparticles and the synthesis was extracellular. The nanoparticles were characterized for various studies using UV spectrophotometer, XRD, Zetasizer Nano S90, SEM, EDAX and AFM. The synthesized silver nanoparticles were found to have size in range 30-45 nm confirmed by SEM and AFM analysis. The XRD pattern confirms FCC structure of silver nanoparticles. Further, the antibacterial activity of the silver nanoparticles was tested against several antibiotics by conjugating them with antibiotics, with the help of zone of inhibition. Among several antibiotics used the efficiency of erythromycin increased by 3 fold. © 2018 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Green Methods for Separation, Purification and Nanomaterial Synthesis, GMSP&NS’18, 24–25th April 2018, Centre for Nano and Material Sciences, Jain University, Bangalore 562112, Karnataka, India. Keywords: Biosynthesis; Silver nanoparticles (AgNPs); Antibacterial activity; Fuzarium oxysporum; UV-spectrophotometer.
* Corresponding author. Tel.: +91- 8884975771. E-mail address: [email protected] 2214-7853© 2018 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Green Methods for Separation, Purification and Nanomaterial Synthesis, GMSP&NS’18, 24–25th April 2018, Centre for Nano and Material Sciences, Jain University, Bangalore 562112, Karnataka, India.
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1. Introduction The nanotechnology is revolutionizing the world from past 2-3 decades. The major discoveries like carbon nanotubes by S. Iijima and Scanning Tunneling Microscope by IBM laid a foundation to the nanotechnology. Since then almost all fields ranging from biomedical to the material science making use of the nanoscale materials and devices either to replace or to improve the existing devices [1]. Nanotechnology finds its significant importance in each and every field including electrical, civil, renewable energy, environmental, agriculture, food industry, chemical industry, textile, and medical, automobile, aeronautical and information like quantum computation etc. The nanomaterials of different morphology will have different importance based on the properties which can be used in above mentioned applications. These nanomaterials can be synthesized, fabricated and surface is modified to design nanobased devices or tools which will bring the revolutionary change in future [2]. The nanomaterials can be defined as the materials of size 1-100 nm. Nanomaterials exhibit very strange and different excellent electrical and optical properties as compared to the bulk materials and also the surface to volume ratio of nanomaterials is very high [3]. These strange and important properties make nanomaterials and nanostructures of great interest in research field to understand the properties at nanoscale. These properties are size dependent and vary with tuning of the nanomaterials size [4-5]. This tunability of the nanoparticles can be done during synthesis process. The nanoparticles can be synthesized by chemical, physical and biological methods [6]. The farmer two methods exhibit many drawbacks including poor tunability of size and incorporation of impurities, which play major impact on properties of nanoparticles. The latter method can overcome these drawbacks and can provide the purest form of nanoparticles [7]. The biosynthesis of nanoparticles can be done by using microbes and plants. The microbes and plants will have reducing agents which are responsible for the reduction of metal salt into metal nanoparticles [8-9]. The microbes are extensively used for the biosynthesis of metal nanoparticles for their excellent control over the morphology. Among all nanomaterials, silver finds many applications including antibacterial property. Silver in bulk only exhibits the antibacterial property so many researchers have taken this metal and reduced it to nanoscale to get innovative and excellent properties especially antibacterial activity. Silver exhibits more than hundred times effective in their antibacterial activity when it reduced to nanoscale. Several nanoparticles are produced by many microbes including synthesized by penicillium sps. [8], E. Coli [9], yeast [10], Aspergillus fumigates [11], Bacillus sps. [12] and Aspergillus Niger [13. Silver nanoparticles are nowadays finding significant impact in biomedical devices and implants including heart valves, masks, wound dressing and bandages. The comparative study was carried out of silver nanoparticles with gold nanoparticles where in silver showed much excellent antibacterial activity than gold nanoparticles [14]. The silver nanoparticles were synthesised by fungi fuzarium oxysporum and tested against the antibiotics to improve their antibacterial activity. Several antibiotics are conjugated with silver nanoparticles and the efficiency of antibiotics has been improved by several folds and erythromycin showed 2 fold increments [8]. Most recent years many studies have been carried out on silver nanoparticles for their use in medical fields and their safety issues but there are very fewer studies have been done on silver nanoparticles usage as antiseptic agent in fabrics [15]. Silver has antibacterial property which makes it as very influential substance because of its ability to fight against mammalian tissue where it can be used as antiseptic agent [8, 16]. Silver results cytotoxic effects against microbes thus it can be utilized as antimicrobial agent [8]. The catalytic characteristics of metal nanostructures have received significant importance in biotechnological fields for targeted drug delivery, bionanosensors, antibacterial and antifungal agents [8, 17, 18]. Silver nanoparticles play vital role in medical fields as it is toxic to the microbial cell wall [8, 19, 20]. The present study involves in the biosynthesis of silver nanoparticles from Penicillium sps. Fuzarium Oxysporum. The extracellular synthesis method was utilized for the biosynthesis of silver nanoparticles which were then characterized by UV-visible spectrophotometer for primary confirmation, XRD for structural analysis, SEM and AFM for morphological analysis and EDAX for compositional analysis. Further the synthesized silver nanoparticles were treated with the four different antibiotics and their activities were investigated.
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2. Experimental Procedure 2.1 Collection of Sample The sample was collected from the shore of Nethravathi river, Mangalore, Karnataka, India. Aseptic method was used to reduce contamination during collection of the sample. 3 various samples were collected from the in 1 square foot area by method of Random Sampling. 2.2 Preparation of specimen The specimen is prepared by mixing the collected soil with double distilled water. The specimen then subjected to serial dilution up to 10-9 dilution. 2.3 Serial dilution The sterilised plates are tagged. Then 9 mL of buffer is added into 5 test-tubes using sterilised pipettes (20 mL) by using aseptic method. By utilising sterilised pipette (2 mL), 1 mL of water is transferred to 10-1 test-tube. The resultant was vortex for few minutes. The dilution method is continued for sterilised tubes of 10-2 to 10-5. Further, every dilution, the sample is poured into Nutrient agar plate. 2.4 Spread Plate method 5 samples (10-1 to 10-5) from serial dilution are transferred to the petriplates, which contains the nutrient agar media for microbial growth. 2.4.1Media preparation Media used here is Nutrient agar. 200 mL of Nutrient agar is prepared and then the solution is sterilised with autoclave at 1210 C for 15 mins to obtain sterilised media. Procedure: The 1 mL of sample is spread on agar plate by sterilised spreader. Then the culture was kept for incubation for colony growth. In this step it is essential to notice the surface of the plate should be dry for proper soaking of the solution. 2.4.2 Pure culture Varoous kinds of colonies were observed after incubation and one single colony was selected. Further this selected colony was inoculated. Then the plates were incubated at room temperature about 2 days until colonies appear. The colonies of pink colour observed which gave the confirmation of Fuzarium Oxysporum. The single colony pink colour was inoculated into liquid broth. 2.4.3 Culture of Fuzarium Oxysporum A pure culture of obtained sps was then subjected to microbial analysis and the present specific fungi “Fuzarium Oxysporum”, which was utilised for the extracellular biosynthesis of silver nanoparticles. 2.5 Preparation of Biomass The production of biomass for synthesis of silver nanostuctures from Fuzarium Oxysporum was carried out under aerobic culturing of fungi in a nutrient broth prepared of Monopotassium phosphate (7.0 g/L), Dipotassium phosphate (2.0 gLl), Magnesium sulphate (0.1 g/L), Ammonium sulfate (1.0 g/L) yeast extract, (0.6 g/l) and glucose (10 g/L).
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2.5.1 Preparation of liquid broth 250 mL liquid broth was prepared. The pink colony was identified and used for the inoculation to the liquid broth. Further this liquid broth with culture was incubated at room temperature for 7 days for significant growth of fungus. 2.6 Biosynthesis of Ag nanostructure by AgNO3 After the growth of the fungus in the flask containing liquid broth was then filtered using Whattman channel paper No.1, to get homogenous solution. This of pH the solution reported to be 6.5. The 1 mM (0.017 g/100 mL) of silver nitrate is prepared and then 50 mL was added to the 50 mL of filtrate (nutrient broth solution containing suspended microbes). The reaction was conducted under dark. Two flasks were taken and one flask was transferred with only cell filtrate with no Ag NPs (control) and other flask was employed for cell filtrate with Ag NPs (test). The biosynthesis of Ag NPs was traced for various time intervals at 12, 24, 48 and 72 h using UV-visible spectrophotometer and the absorbance was recorded at wave lengths range 200 to 800 nm. 2.7 Optimization of synthesized AgNPs The solution having Ag NPs were tested for their maximum production at different parameters such as concentration of AgNO3, pH and salt concentration and synthesis is optimized. The variable factors: like AgNO3 concentration (0.5, 1.0, 1.5, 2.0, 2.5 mM), pH (5, 6, 7, 8) and salt concentration (0.1, 0.2, 0.3, 0.4, 0.5% NaCl) at varied time interval. 300µl of solution was removed to quantify the absorbance at a wavelength of 430 nm by using UV-visible spectrophotometer. 2.8 Characterization of AgNPs The silver nanoparticles after 72 hr of incubation were then subjected to UV-vis spectrophotometer for primary confirmation of synthesized silver nanoparticles. The solid nanoparticles were obtained by centrifugation of solution. The smear was prepared with the pellet obtained. Further the Ag NPs were characterized by XRD for structural analysis, EDAX compositional analysis, SEM and AFM for morphological studies. 2.9 Antimicrobial Activity of AgNPs An assessment was made to focus the significant impact of antibiotics against the gram +ve and gram -ve microbes with the produced Ag NPs. Standard discs of antibiotics were utilized and poured on Muller-Hinton Agar with the Ag NPs. 20µL of Ag NPs were poured on the standard antibiotics disc. The solution was inoculated and the culture was then kept for incubation at 37 0C overnight. The antibiotics used were erythromycin, streptomycin, fusidic acid, clavulanic acid, daptomycin and spiramycin. 3. Results and Discussion 3.1 Primary confirmation Visual confirmation: The very first characterization of synthesised silver nanostructures was done by observing the colour change from yellow to brown. This can be called as a primary confirmation of formation of silver nanoparticles. After the addition of the 1.5 mM silver nitrate and incubation for 48 h the solution turned to brownish in colour in natural biosynthesis and where in microwave synthesis the brown colour was obtained in 5 minutes. 3.2 UV-Spectrophotometer The synthesized silver nanoparticles can be traced by UV-visible spectrophotometer. The UV absorbance reading was taken at different time intervals of synthesized silver nanoparticles. The synthesis time was traced at 12,
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24, 48, and 72 h and the absorbance measurements with respect to wavelength are given in Figure 1, for a wavelength scan of 200 to 800 nm. The UV absorbance v/s wavelength graph indicates that the absorbance peak was observed in the range of 430 – 460 nm at all the time intervals. The maximum absorbance was observed at 48 h. The absorbance after 48 h found to decrease, the reason might be because of the inactivation or degradation of nitrate reductase enzyme at this point of time. We can conclude that the enzyme activity may reach maximum at 48 h beyond that it may starts losing its activity. The highest peak of absorbance reported was around 2.7 and wavelength at 430 nm.
Fig. 1. UV-visible spectrophotometer recordings at 12, 24, 48 and 72 h.
The above data imply that the enzyme nitrate reductase liberated into the cell filtrate solution after the adding of AgNO3. Then the Ag ions are reduced to Ag nanostructures by the act of enzymatic reduction thus bringing extracellular synthesis of silver nanoparticles. 3.3 Optimization of Ag nanoparticles 3.3.1 Influence of AgNO3 concentration The synthesized silver nanoparticles were examined with 0.5, 1.0, 1.5, 2.0, 2.5 mM of silver nitrate and incubated. The highest yield was sobserved at 1 mM concentration as given in Figure 2.
Fig. 2. Influence of silver nitrate on silver nanoparticles.
3.3.2: Influence of pH Figure 3(a) shows the influence of pH during the biosynthesis of silver nanoparticle and maximum yield was obtained at pH 6.5 compared to other conditions (pH 5, 7, 8). It was also reported the significant formation of silver
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nanoparticles was found at around pH 6.5 as the Ag nanostructures tend to absorb maximum light. Shareef et al. [8] studied on the same conditions, where they reported that the maximum production was found at pH 6 which was acidic for applications related to clinical studies. As adapted from their study, the reason for the reduction in peak as it moves for neutral pH might be because of deactivation of enzymes, which are essential for the reduction of AgNO3 to Ag nanoparticles. 3.3.3: Influence of salt concentration The formation of nanoparticles are tuned by varying salt concentration (0.1, 0.2, 0.3, 0.4, 0.5% NaCl) (Figure 3(b)). The significant formation of nanoparticles was found at NaCl concentration of 0.2%. It was also examined that the concentration of salt above 0.2% was unfavourable for the synthesis of nanoparticle. The absorbance value has reduced because of the deactivation of enzyme at higher salt concentration. The salt concentration at 0.2% results the stable and excellent formation of silver nanoparticles.
Fig. 3(a) Influence of pH on Ag NPs and (b) Influence of salinity on Ag NPs
3.4 FTIR analysis FTIR peaks for the silver nanoparticles specimen fit in to the series of the microbial enzymes and proteins present in the specimen which acts as a reducing mediator (Figure 4(a)). FTIR examination on the synthesized silver nanoparticles in the range of 4500-500 cm-1 wave number to observe the interactions between the silver ions and enzymes in microbial solution that used to stabilize the silver nanoparticles. Figure 4(a) distinct the presence of microbial proteins observable because of the twisting shaped by amide bonds. Silver nanoparticles exhibit well-known peaks at 3332.9, 1645.3 and 1025.7 cm-1, signifying the involvement of NH elongating vibrations, N-H bending vibrations and C=O elongating vibrations that refers to the Microbial-Silver nanoparticles aggregates. The C-N and C-O-C stretching vibration recommended the existence of nitrate reductase enzymes on the surface of the Ag NPs.
Fig. 4(a) FTIR spectrum and (b) XRD pattern of the synthesized silver nanoparticles
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3.5 XRD analysis The XRD patterns (Figure 4(b)) reveal that the synthesized silver nanoparticles were of size range 50-60 nm by using Debye-Scherrer’s formula. The obtained XRD data were matching with standard data for silver. The miller indices (111), (200) and (220) confirm the FCC structure of synthesized silver nanoparticles and these planes are indexed as per the (JCPDS- 40-0783). The crystalline silver nanoparticles size was calculated from XRD analysis and found to be 16.98 nm, 15.77 nm and 19.94 nm. 3.6 SEM Analysis The biosynthesized Ag NPs are then subjected to centrifuge to obtain a pellet to obtain SEM results image clearly shows silver nanoparticles of size range 25-40 nm (Figure 5(a)). 3.7 AFM analysis The Atomic Force Microscope image reveals the surface texture of Ag NPs. The obtained image clearly shows Ag NPs of size range 30-45 nm with the spherical shapes from the AFM image (Figure 5(b)). 3.8 EDAX analysis The biosynthesized Ag NPs were characterized by EDAX and the presence of silver nanoparticles was confirmed by the EDAX pattern (Figure 5(c)).
Fig. 5(a) SEM image, (b) AFM image and EDAX spectrum of the synthesized silver nanoparticles.
3.9 Zone of inhibition The synergistic antibacterial movement of silver nanoparticles with distinctive anti-infection agents was studied. The zone of inhibition of silver nanoparticles is studied against gram +ve and gram -ve were examined independently. The results are organized in Table1. Table 1: Zone of inhibition of various antibiotics against the test organisms Fusidic acid (10 µg/disc) Erythromycin (10 µg/disc) Antibiotics Fus (zone in mm) Fus+AgNPs Fold increase Ery (zone in mm) Ery+Ag NPs Fold increase (zone in mm) (in %) (zone in mm) (in %) S.aureas 6 9 50 0 0 0 E coli 0 0 0 2 6 300 Antibiotic Clavulanic acid (10µg/disc) Daptomycin (10µg/disc) Cla (zone in mm) Cla +AgNPs Fold increase Dap (zone in mm) Dap+AgNPs Fold increase (zone in mm) (in %) (zone in mm) (in %) S.aureas 13 16 63.07 18 36 100 E coli 0 0 0 0 0 0 Streptomycin (10µg/disc) Spiramycin (10µg/disc) Stp (zone in mm) Stp+AgNPs Fold increase Spi (zone in mm) Spi+AgNPs Fold increase (zone in mm) (in %) (zone in mm) (in %) S.aureas 0 0 0 0 0 0 E coli 4 6 50 8 12 50
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The results showed that there was exactly 3 fold increment in the efficiency of antibiotics. The antibiotic Fusidic acid and Streptomycin showed half (50%) fold improvements in the efficiency whereas, the Daptomycin showed 2 (100%) fold improvement and Erythromycin showed 3 (300%) fold improvements, which showed the significant effective antimicrobial property of silver nanoparticles. Other antibiotics such as Clavulanic acid and Spiramycin also showed excellent improvement in their efficiency by 63.07 % and 50% respectively. These results can be compared with other literature results. As compared to the study done by Guangquan et. al. [21] which showed an increase of 70-80% efficiency in the zone of inhibition test, our study showed around 300% increase (threefold increase) for the antibiotic Erythromycin. Also, we can compare the results of Shareef et. al. [8], which showed an increase of 100% efficiency in the zone of inhibition test and the present study showed 300% improvement in efficiency of Erythromycin. This difference might be because of the size of the silver nanoparticles, as the size goes on reducing, the antibacterial activity of the silver nanoparticles is increasing. With such observations, one can interpret that the smaller the Ag NPs tend to show better antibacterial activity. 4. Conclusion Fuzarium Oxysporum was used for the biosynthesis of silver nanoparticles. The biological method always finds a better approach as compare to the chemical and physical methods as it is eco-friendly and economical. The Fuzarium Oxysporum was observed to have a maximum production of nanoparticles in 48 h by biosynthesis method. The Fuzarium Oxysporum was examined to have a maximum yield of Ag NPs at pH 6.5, AgNO3 concentration of 1 mM and at a salt concentration of 0.2%. The average size of the Ag NPs was found to be 30-45 nm by SEM and AFM analysis and XRD peaks showed that the structure of nanoparticles was FCC in nature. FTIR pattern revealed the presence of silver nanoparticles with nitrate reductase enzymes on the surface of silver nanoparticles. EDAX results confirmed the presence of silver nanoparticles. Zone of inhibition test proved that there was an increase in stability of nanoparticles. Antibacterial activity of silver nanoparticles was checked against six antibiotics, wherein the Fusidic acid, Spiramycin and Streptomycin showed 50% improvement in efficiency, Clavulanic acid showed 63.07% improvement, Daptomycin showed 100% improvement and Erythromycin showed highest fold increase 300% improvement. Acknowledgements We acknowledge the support from the Dept. of Nanobiotechnology, Srinivas centre for Nano Science and Technology, Srinivas University, Dept. of Nano Technology Srinivas Institute of Technology, Dept. of Nanotechnology VTU, CPGC, Bangalore, for providing facilities for the characterization tools XRD, EDAX and SEM. We also acknowledge VIT, Vellore, NITK Surathkal and Mangalore University, Mangalore for providing opportunity to use AFM and FTIR. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
Bhushan B., “Handbook of Nanotechnology” Springer Handbooks. Springer, Berlin, Heidelberg, 2017. Baldev Raj and U. Kamachi Mudali, “Nanotechnologies in Sodium‐Cooled Fast Spectrum Reactor and Closed Fuel Cycle Sustainable Nuclear Energy System”, Nanotechnology for Energy Sustainability, 2017, pp. 271-294. https://doi.org/10.1002/9783527696109.ch12 Tarakanova A., Chang SW., Buehler M.J. “Computational Materials Science of Bionanomaterials: Structure, Mechanical Properties and Applications of Elastin and Collagen Proteins”. In: Bhushan B., Luo D., Schricker S., Sigmund W., Zauscher S. (eds) Handbook of Nanomaterials Properties. Springer, Berlin, Heidelberg, 2014. A.H. Rajasab, “Biosynthesis of Silver Nanoparticles from Fungi”, (PhD thesis), Shodhganga, 2017, http://hdl.handle.net/10603/138900 Ninganagouda S., Rathod V. and Singh D., “Characterization and biosynthesis of silver nanoparticles using a fungus Aspergillus niger” International Letters of Natural Sciences, 10, 2014, pp.49-57 K. J. P. Anthony, M. Murugan, M. Jeyaraja, N. K.Rathinam and G.Sangiliyandi, “Synthesis of silver nanoparticles using pine mushroom extract: A potential antimicrobial agent against E. coli and B. subtilis” Ind. Eng. Chem. Res., 20, 2014, pp. 2325-2331. G. Cao and Ying wang “Nanostructures & nanomaterials: synthesis, properties & applications”, 2nd edition, world scientific publishing, Singapore, 2011. JU Shareef, M Navya Rani, S Anand, Dinesh Rangappa,Synthesis and characterization of silver nanoparticles from penicillium sps., Materials Today: Proceedings, 4, 2017, pp.11923-11932 S. Gurunathan, K. Kalishwarlal, R. Vaidhyanathan, V. Deepak, S.R.K. Pandian, J. Muniyandi, N. Hariharan and S.H. Eom, “Biosynthesis, purification and characterization of silver nanoparticles using Escherichia coli,” Colloids and Surfaces B: Biointerfaces, 74, 2009, pp. 328335.
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[10] A. B. Moghaddam, Mona, Moniri, S. Azizi, R. A. Rahim, A. B. Ariff, W. Z. Saad, F. Namvar, M. Navaderi, R. Mohamad, “Biosynthesis of ZnO nanoparticles by a new Pichia kudriavzevi yeast starin and evaluation of their antimicrobial and antioxidant activities” Molecules, 22, 2017, 872. [11] K.C. Bhainsa and S.F. D’Souza, “Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigates,” Colloids and Surfaces B: Biointerfaces, 47, 2006, pp. 160-164. [12] M. Saravanan, S. K. Barik, D. M. Ali, P. Prakash, A. Pugazhendhi, “Synthesis of silver nanoparticles from Bacillus brevis (NCIM 2533) and their antibacterial activity against pathogenic bacteria” Microbial Pathogenesis, 116, 2018, pp. 221-226. [13] W. Chengzheng, W. Jiazhi, C. Shuangjiang, M. K. Swamy, U. R. Sinniah, M. Akhtar, A. Umar, “Biogenic synthesis, characterization and evaluation of silver nanoparticles from Aspergillus Niger JX556221 against human colon cancer cell line HT-29” Journal of Nanoscience and Nanotechnology, 18, 2018, pp. 3673- 3681. [14] Shirin Nourbakhsh and Ali Ashjaran, “Laser treatment of cotton fabric for durable antibacterial properties of silver nanoparticles” Materials (Basel), 5,2012, pp. 1247–1257. [15] Wilkinson L. J., White R. J. ,Chipman J. K. “Silver and nanoparticles of silver in wound dressings: a review of efficacy and safety” Journal of wound care, 20, 2011, (https://doi.org/10.12968/jowc.2011.20.11.543) [16] C. Sundaramoorth, M. Kalaivani, D.M. Mathews, S. Palanisamy, A. Kalaiselvan and Rajasekaran A, “Biosynthesis of silver nanoparticles from Aspergillis niger and evaluation of its wound healing activity in experimental rat model,” International Journal of PharmTech Research, 1, 2009, pp. 1523- 1529. [17] A. Ahmad, P. Mukherjee, S. Senapati, D. Mandal, M.I. Khan, R. Kumar and M. Sastry, “Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum,” Colloids and Surfaces B: Biointerfaces, 28, 2003, pp. 313-318. [18] K.C. Bhainsa and S.F. D’Souza, “Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigates,” Colloids and Surfaces B: Biointerfaces, 47, 2006, pp. 160-164. [19] N. Duran, P.D. Marcato, O.I. Alves, G.I. DeSouza and E. Esposito, “Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporium strains,” Journal of Nanobiotechnology, 3, 2005, pp. 8. [20] K. Vahabi, G.A. Mansoori and S. Karimi, “Biosynthesis of silver nanoparticles by fungus Trichoderma reesei,” Insciences Journal, 1, 2011, pp. 65-79. [21] Guangquan Li 1, Dan He 1, Yongqing Qian 1, Buyuan Guan, Song Gao, Yan Cui, Koji Yokoyama and Li Wang. (2012). Fungus-Mediated Green Synthesis of Silver Nanoparticles Using Aspergillus terreus International Journal of Molecular Science, 13, 2012, pp.466-476.
ScienceDirect Materials Today: Proceedings 9 (2019) 506–514
www.materialstoday.com/proceedings
GMSP&NS’18
Synthesis and Characterization of Silver Nanoparticles from Fuzarium Oxysporum and Investigation of Their Antibacterial Activity Shareefraza J. Ukkund,a,b* Momin Ashraf,a Apoorva B. Udupa,a Mayur Gangadharan,b Aswanth Pattiyeri,b Yashawantha K. Marigowda,b Ravika Patila, Prasad Puthiyllama,b a
b
Department of Nano Technology, Srinivas Institute of Technology, Mangalore- India Srinivas Centre for Nano Science and Technology, Srinivas Group of Colleges, Mangalore- India c Department of Mechanical Engineering, Srinivas Institute of Technology, Mangalore- India.
Abstract Nanotechnology is the fastest growing field of 21st century due to this reason that the nanomaterials synthesis finds more importance in field of research. The nanomaterials can be synthesized by physical, chemical and biological methods. The biological methods are more advantageous than the other two methods because of manipulation of the size and morphology of nanostructures by microbes and plants. The Penicillium sps. Fuzarium Oxysporum was used to synthesize silver nanoparticles and the synthesis was extracellular. The nanoparticles were characterized for various studies using UV spectrophotometer, XRD, Zetasizer Nano S90, SEM, EDAX and AFM. The synthesized silver nanoparticles were found to have size in range 30-45 nm confirmed by SEM and AFM analysis. The XRD pattern confirms FCC structure of silver nanoparticles. Further, the antibacterial activity of the silver nanoparticles was tested against several antibiotics by conjugating them with antibiotics, with the help of zone of inhibition. Among several antibiotics used the efficiency of erythromycin increased by 3 fold. © 2018 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Green Methods for Separation, Purification and Nanomaterial Synthesis, GMSP&NS’18, 24–25th April 2018, Centre for Nano and Material Sciences, Jain University, Bangalore 562112, Karnataka, India. Keywords: Biosynthesis; Silver nanoparticles (AgNPs); Antibacterial activity; Fuzarium oxysporum; UV-spectrophotometer.
* Corresponding author. Tel.: +91- 8884975771. E-mail address: [email protected] 2214-7853© 2018 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on Green Methods for Separation, Purification and Nanomaterial Synthesis, GMSP&NS’18, 24–25th April 2018, Centre for Nano and Material Sciences, Jain University, Bangalore 562112, Karnataka, India.
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1. Introduction The nanotechnology is revolutionizing the world from past 2-3 decades. The major discoveries like carbon nanotubes by S. Iijima and Scanning Tunneling Microscope by IBM laid a foundation to the nanotechnology. Since then almost all fields ranging from biomedical to the material science making use of the nanoscale materials and devices either to replace or to improve the existing devices [1]. Nanotechnology finds its significant importance in each and every field including electrical, civil, renewable energy, environmental, agriculture, food industry, chemical industry, textile, and medical, automobile, aeronautical and information like quantum computation etc. The nanomaterials of different morphology will have different importance based on the properties which can be used in above mentioned applications. These nanomaterials can be synthesized, fabricated and surface is modified to design nanobased devices or tools which will bring the revolutionary change in future [2]. The nanomaterials can be defined as the materials of size 1-100 nm. Nanomaterials exhibit very strange and different excellent electrical and optical properties as compared to the bulk materials and also the surface to volume ratio of nanomaterials is very high [3]. These strange and important properties make nanomaterials and nanostructures of great interest in research field to understand the properties at nanoscale. These properties are size dependent and vary with tuning of the nanomaterials size [4-5]. This tunability of the nanoparticles can be done during synthesis process. The nanoparticles can be synthesized by chemical, physical and biological methods [6]. The farmer two methods exhibit many drawbacks including poor tunability of size and incorporation of impurities, which play major impact on properties of nanoparticles. The latter method can overcome these drawbacks and can provide the purest form of nanoparticles [7]. The biosynthesis of nanoparticles can be done by using microbes and plants. The microbes and plants will have reducing agents which are responsible for the reduction of metal salt into metal nanoparticles [8-9]. The microbes are extensively used for the biosynthesis of metal nanoparticles for their excellent control over the morphology. Among all nanomaterials, silver finds many applications including antibacterial property. Silver in bulk only exhibits the antibacterial property so many researchers have taken this metal and reduced it to nanoscale to get innovative and excellent properties especially antibacterial activity. Silver exhibits more than hundred times effective in their antibacterial activity when it reduced to nanoscale. Several nanoparticles are produced by many microbes including synthesized by penicillium sps. [8], E. Coli [9], yeast [10], Aspergillus fumigates [11], Bacillus sps. [12] and Aspergillus Niger [13. Silver nanoparticles are nowadays finding significant impact in biomedical devices and implants including heart valves, masks, wound dressing and bandages. The comparative study was carried out of silver nanoparticles with gold nanoparticles where in silver showed much excellent antibacterial activity than gold nanoparticles [14]. The silver nanoparticles were synthesised by fungi fuzarium oxysporum and tested against the antibiotics to improve their antibacterial activity. Several antibiotics are conjugated with silver nanoparticles and the efficiency of antibiotics has been improved by several folds and erythromycin showed 2 fold increments [8]. Most recent years many studies have been carried out on silver nanoparticles for their use in medical fields and their safety issues but there are very fewer studies have been done on silver nanoparticles usage as antiseptic agent in fabrics [15]. Silver has antibacterial property which makes it as very influential substance because of its ability to fight against mammalian tissue where it can be used as antiseptic agent [8, 16]. Silver results cytotoxic effects against microbes thus it can be utilized as antimicrobial agent [8]. The catalytic characteristics of metal nanostructures have received significant importance in biotechnological fields for targeted drug delivery, bionanosensors, antibacterial and antifungal agents [8, 17, 18]. Silver nanoparticles play vital role in medical fields as it is toxic to the microbial cell wall [8, 19, 20]. The present study involves in the biosynthesis of silver nanoparticles from Penicillium sps. Fuzarium Oxysporum. The extracellular synthesis method was utilized for the biosynthesis of silver nanoparticles which were then characterized by UV-visible spectrophotometer for primary confirmation, XRD for structural analysis, SEM and AFM for morphological analysis and EDAX for compositional analysis. Further the synthesized silver nanoparticles were treated with the four different antibiotics and their activities were investigated.
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2. Experimental Procedure 2.1 Collection of Sample The sample was collected from the shore of Nethravathi river, Mangalore, Karnataka, India. Aseptic method was used to reduce contamination during collection of the sample. 3 various samples were collected from the in 1 square foot area by method of Random Sampling. 2.2 Preparation of specimen The specimen is prepared by mixing the collected soil with double distilled water. The specimen then subjected to serial dilution up to 10-9 dilution. 2.3 Serial dilution The sterilised plates are tagged. Then 9 mL of buffer is added into 5 test-tubes using sterilised pipettes (20 mL) by using aseptic method. By utilising sterilised pipette (2 mL), 1 mL of water is transferred to 10-1 test-tube. The resultant was vortex for few minutes. The dilution method is continued for sterilised tubes of 10-2 to 10-5. Further, every dilution, the sample is poured into Nutrient agar plate. 2.4 Spread Plate method 5 samples (10-1 to 10-5) from serial dilution are transferred to the petriplates, which contains the nutrient agar media for microbial growth. 2.4.1Media preparation Media used here is Nutrient agar. 200 mL of Nutrient agar is prepared and then the solution is sterilised with autoclave at 1210 C for 15 mins to obtain sterilised media. Procedure: The 1 mL of sample is spread on agar plate by sterilised spreader. Then the culture was kept for incubation for colony growth. In this step it is essential to notice the surface of the plate should be dry for proper soaking of the solution. 2.4.2 Pure culture Varoous kinds of colonies were observed after incubation and one single colony was selected. Further this selected colony was inoculated. Then the plates were incubated at room temperature about 2 days until colonies appear. The colonies of pink colour observed which gave the confirmation of Fuzarium Oxysporum. The single colony pink colour was inoculated into liquid broth. 2.4.3 Culture of Fuzarium Oxysporum A pure culture of obtained sps was then subjected to microbial analysis and the present specific fungi “Fuzarium Oxysporum”, which was utilised for the extracellular biosynthesis of silver nanoparticles. 2.5 Preparation of Biomass The production of biomass for synthesis of silver nanostuctures from Fuzarium Oxysporum was carried out under aerobic culturing of fungi in a nutrient broth prepared of Monopotassium phosphate (7.0 g/L), Dipotassium phosphate (2.0 gLl), Magnesium sulphate (0.1 g/L), Ammonium sulfate (1.0 g/L) yeast extract, (0.6 g/l) and glucose (10 g/L).
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2.5.1 Preparation of liquid broth 250 mL liquid broth was prepared. The pink colony was identified and used for the inoculation to the liquid broth. Further this liquid broth with culture was incubated at room temperature for 7 days for significant growth of fungus. 2.6 Biosynthesis of Ag nanostructure by AgNO3 After the growth of the fungus in the flask containing liquid broth was then filtered using Whattman channel paper No.1, to get homogenous solution. This of pH the solution reported to be 6.5. The 1 mM (0.017 g/100 mL) of silver nitrate is prepared and then 50 mL was added to the 50 mL of filtrate (nutrient broth solution containing suspended microbes). The reaction was conducted under dark. Two flasks were taken and one flask was transferred with only cell filtrate with no Ag NPs (control) and other flask was employed for cell filtrate with Ag NPs (test). The biosynthesis of Ag NPs was traced for various time intervals at 12, 24, 48 and 72 h using UV-visible spectrophotometer and the absorbance was recorded at wave lengths range 200 to 800 nm. 2.7 Optimization of synthesized AgNPs The solution having Ag NPs were tested for their maximum production at different parameters such as concentration of AgNO3, pH and salt concentration and synthesis is optimized. The variable factors: like AgNO3 concentration (0.5, 1.0, 1.5, 2.0, 2.5 mM), pH (5, 6, 7, 8) and salt concentration (0.1, 0.2, 0.3, 0.4, 0.5% NaCl) at varied time interval. 300µl of solution was removed to quantify the absorbance at a wavelength of 430 nm by using UV-visible spectrophotometer. 2.8 Characterization of AgNPs The silver nanoparticles after 72 hr of incubation were then subjected to UV-vis spectrophotometer for primary confirmation of synthesized silver nanoparticles. The solid nanoparticles were obtained by centrifugation of solution. The smear was prepared with the pellet obtained. Further the Ag NPs were characterized by XRD for structural analysis, EDAX compositional analysis, SEM and AFM for morphological studies. 2.9 Antimicrobial Activity of AgNPs An assessment was made to focus the significant impact of antibiotics against the gram +ve and gram -ve microbes with the produced Ag NPs. Standard discs of antibiotics were utilized and poured on Muller-Hinton Agar with the Ag NPs. 20µL of Ag NPs were poured on the standard antibiotics disc. The solution was inoculated and the culture was then kept for incubation at 37 0C overnight. The antibiotics used were erythromycin, streptomycin, fusidic acid, clavulanic acid, daptomycin and spiramycin. 3. Results and Discussion 3.1 Primary confirmation Visual confirmation: The very first characterization of synthesised silver nanostructures was done by observing the colour change from yellow to brown. This can be called as a primary confirmation of formation of silver nanoparticles. After the addition of the 1.5 mM silver nitrate and incubation for 48 h the solution turned to brownish in colour in natural biosynthesis and where in microwave synthesis the brown colour was obtained in 5 minutes. 3.2 UV-Spectrophotometer The synthesized silver nanoparticles can be traced by UV-visible spectrophotometer. The UV absorbance reading was taken at different time intervals of synthesized silver nanoparticles. The synthesis time was traced at 12,
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24, 48, and 72 h and the absorbance measurements with respect to wavelength are given in Figure 1, for a wavelength scan of 200 to 800 nm. The UV absorbance v/s wavelength graph indicates that the absorbance peak was observed in the range of 430 – 460 nm at all the time intervals. The maximum absorbance was observed at 48 h. The absorbance after 48 h found to decrease, the reason might be because of the inactivation or degradation of nitrate reductase enzyme at this point of time. We can conclude that the enzyme activity may reach maximum at 48 h beyond that it may starts losing its activity. The highest peak of absorbance reported was around 2.7 and wavelength at 430 nm.
Fig. 1. UV-visible spectrophotometer recordings at 12, 24, 48 and 72 h.
The above data imply that the enzyme nitrate reductase liberated into the cell filtrate solution after the adding of AgNO3. Then the Ag ions are reduced to Ag nanostructures by the act of enzymatic reduction thus bringing extracellular synthesis of silver nanoparticles. 3.3 Optimization of Ag nanoparticles 3.3.1 Influence of AgNO3 concentration The synthesized silver nanoparticles were examined with 0.5, 1.0, 1.5, 2.0, 2.5 mM of silver nitrate and incubated. The highest yield was sobserved at 1 mM concentration as given in Figure 2.
Fig. 2. Influence of silver nitrate on silver nanoparticles.
3.3.2: Influence of pH Figure 3(a) shows the influence of pH during the biosynthesis of silver nanoparticle and maximum yield was obtained at pH 6.5 compared to other conditions (pH 5, 7, 8). It was also reported the significant formation of silver
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nanoparticles was found at around pH 6.5 as the Ag nanostructures tend to absorb maximum light. Shareef et al. [8] studied on the same conditions, where they reported that the maximum production was found at pH 6 which was acidic for applications related to clinical studies. As adapted from their study, the reason for the reduction in peak as it moves for neutral pH might be because of deactivation of enzymes, which are essential for the reduction of AgNO3 to Ag nanoparticles. 3.3.3: Influence of salt concentration The formation of nanoparticles are tuned by varying salt concentration (0.1, 0.2, 0.3, 0.4, 0.5% NaCl) (Figure 3(b)). The significant formation of nanoparticles was found at NaCl concentration of 0.2%. It was also examined that the concentration of salt above 0.2% was unfavourable for the synthesis of nanoparticle. The absorbance value has reduced because of the deactivation of enzyme at higher salt concentration. The salt concentration at 0.2% results the stable and excellent formation of silver nanoparticles.
Fig. 3(a) Influence of pH on Ag NPs and (b) Influence of salinity on Ag NPs
3.4 FTIR analysis FTIR peaks for the silver nanoparticles specimen fit in to the series of the microbial enzymes and proteins present in the specimen which acts as a reducing mediator (Figure 4(a)). FTIR examination on the synthesized silver nanoparticles in the range of 4500-500 cm-1 wave number to observe the interactions between the silver ions and enzymes in microbial solution that used to stabilize the silver nanoparticles. Figure 4(a) distinct the presence of microbial proteins observable because of the twisting shaped by amide bonds. Silver nanoparticles exhibit well-known peaks at 3332.9, 1645.3 and 1025.7 cm-1, signifying the involvement of NH elongating vibrations, N-H bending vibrations and C=O elongating vibrations that refers to the Microbial-Silver nanoparticles aggregates. The C-N and C-O-C stretching vibration recommended the existence of nitrate reductase enzymes on the surface of the Ag NPs.
Fig. 4(a) FTIR spectrum and (b) XRD pattern of the synthesized silver nanoparticles
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3.5 XRD analysis The XRD patterns (Figure 4(b)) reveal that the synthesized silver nanoparticles were of size range 50-60 nm by using Debye-Scherrer’s formula. The obtained XRD data were matching with standard data for silver. The miller indices (111), (200) and (220) confirm the FCC structure of synthesized silver nanoparticles and these planes are indexed as per the (JCPDS- 40-0783). The crystalline silver nanoparticles size was calculated from XRD analysis and found to be 16.98 nm, 15.77 nm and 19.94 nm. 3.6 SEM Analysis The biosynthesized Ag NPs are then subjected to centrifuge to obtain a pellet to obtain SEM results image clearly shows silver nanoparticles of size range 25-40 nm (Figure 5(a)). 3.7 AFM analysis The Atomic Force Microscope image reveals the surface texture of Ag NPs. The obtained image clearly shows Ag NPs of size range 30-45 nm with the spherical shapes from the AFM image (Figure 5(b)). 3.8 EDAX analysis The biosynthesized Ag NPs were characterized by EDAX and the presence of silver nanoparticles was confirmed by the EDAX pattern (Figure 5(c)).
Fig. 5(a) SEM image, (b) AFM image and EDAX spectrum of the synthesized silver nanoparticles.
3.9 Zone of inhibition The synergistic antibacterial movement of silver nanoparticles with distinctive anti-infection agents was studied. The zone of inhibition of silver nanoparticles is studied against gram +ve and gram -ve were examined independently. The results are organized in Table1. Table 1: Zone of inhibition of various antibiotics against the test organisms Fusidic acid (10 µg/disc) Erythromycin (10 µg/disc) Antibiotics Fus (zone in mm) Fus+AgNPs Fold increase Ery (zone in mm) Ery+Ag NPs Fold increase (zone in mm) (in %) (zone in mm) (in %) S.aureas 6 9 50 0 0 0 E coli 0 0 0 2 6 300 Antibiotic Clavulanic acid (10µg/disc) Daptomycin (10µg/disc) Cla (zone in mm) Cla +AgNPs Fold increase Dap (zone in mm) Dap+AgNPs Fold increase (zone in mm) (in %) (zone in mm) (in %) S.aureas 13 16 63.07 18 36 100 E coli 0 0 0 0 0 0 Streptomycin (10µg/disc) Spiramycin (10µg/disc) Stp (zone in mm) Stp+AgNPs Fold increase Spi (zone in mm) Spi+AgNPs Fold increase (zone in mm) (in %) (zone in mm) (in %) S.aureas 0 0 0 0 0 0 E coli 4 6 50 8 12 50
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The results showed that there was exactly 3 fold increment in the efficiency of antibiotics. The antibiotic Fusidic acid and Streptomycin showed half (50%) fold improvements in the efficiency whereas, the Daptomycin showed 2 (100%) fold improvement and Erythromycin showed 3 (300%) fold improvements, which showed the significant effective antimicrobial property of silver nanoparticles. Other antibiotics such as Clavulanic acid and Spiramycin also showed excellent improvement in their efficiency by 63.07 % and 50% respectively. These results can be compared with other literature results. As compared to the study done by Guangquan et. al. [21] which showed an increase of 70-80% efficiency in the zone of inhibition test, our study showed around 300% increase (threefold increase) for the antibiotic Erythromycin. Also, we can compare the results of Shareef et. al. [8], which showed an increase of 100% efficiency in the zone of inhibition test and the present study showed 300% improvement in efficiency of Erythromycin. This difference might be because of the size of the silver nanoparticles, as the size goes on reducing, the antibacterial activity of the silver nanoparticles is increasing. With such observations, one can interpret that the smaller the Ag NPs tend to show better antibacterial activity. 4. Conclusion Fuzarium Oxysporum was used for the biosynthesis of silver nanoparticles. The biological method always finds a better approach as compare to the chemical and physical methods as it is eco-friendly and economical. The Fuzarium Oxysporum was observed to have a maximum production of nanoparticles in 48 h by biosynthesis method. The Fuzarium Oxysporum was examined to have a maximum yield of Ag NPs at pH 6.5, AgNO3 concentration of 1 mM and at a salt concentration of 0.2%. The average size of the Ag NPs was found to be 30-45 nm by SEM and AFM analysis and XRD peaks showed that the structure of nanoparticles was FCC in nature. FTIR pattern revealed the presence of silver nanoparticles with nitrate reductase enzymes on the surface of silver nanoparticles. EDAX results confirmed the presence of silver nanoparticles. Zone of inhibition test proved that there was an increase in stability of nanoparticles. Antibacterial activity of silver nanoparticles was checked against six antibiotics, wherein the Fusidic acid, Spiramycin and Streptomycin showed 50% improvement in efficiency, Clavulanic acid showed 63.07% improvement, Daptomycin showed 100% improvement and Erythromycin showed highest fold increase 300% improvement. Acknowledgements We acknowledge the support from the Dept. of Nanobiotechnology, Srinivas centre for Nano Science and Technology, Srinivas University, Dept. of Nano Technology Srinivas Institute of Technology, Dept. of Nanotechnology VTU, CPGC, Bangalore, for providing facilities for the characterization tools XRD, EDAX and SEM. We also acknowledge VIT, Vellore, NITK Surathkal and Mangalore University, Mangalore for providing opportunity to use AFM and FTIR. References [1] [2] [3] [4] [5] [6] [7] [8] [9]
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