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Green synthesis and characterization of silver nanoparticles using banana peel extract and their antimicrobial activity against representative microorganisms
Q3 Haytham M.M. Ibrahim*
Department of Radiation Microbiology, National Center for Radiation Research and Technology (NCRRT), Atomic Q1 Energy Authority, P.O. Box 29, Nasr City 11731, Cairo, Egypt
ARTICLE INFO
ABSTRACT
Article history:
Received 25 November 2014 Received in revised form 14 January 2015 Accepted 20 January 2015 Available online xxx
Keywords: Agricultural waste Silver nanoparticles Characterization Antimicrobial activity
The present study reports an eco-friendly, cost efficient, rapid and easy method for synthesis of silver nanoparticles using banana peel extract (BPE) as a reducing and capping agent. The different factor affecting silver reduction was investigated. The optimum conditions were silver nitrate (1.75 mM), BPE (20.4 mg dry weight), pH (4.5) and incubation time (72 h). BPE can reduces silver ions into silver nanoparticles within 5 min after heating the reaction mixture (40—100 °C) as indicated by the developed reddish brown color. The UV —Vis spectrum of silver nanoparticles revealed a characteristic surface plasmon resonance (SPR) peak at 433 nm. Silver nanoparticles were characterized. X ray diffraction revealed their crystalline nature. Scanning electron microscope and field emission scanning electron microscope showed spherical shaped and monodispersed nanoparticles. Transmission electron microscope confirmed the spherical nature and the crystallinity of nanoparticles. The average size of nanoparticles was 23.7 nm as determined by dynamic light scattering. Energy dispersive X-ray spectroscopy analysis showed the peak in silver region confirming presence of elemental silver. Fourier transform infrared spectroscopy affirmed the role of BPE as a reducing and capping agent of silver ions. Silver nanoparticles showed effective antibacterial activity against representative pathogens of bacteria and yeast. The minimum inhibitory concentration and minimum bactericidal concentration were determined. The synthesized nanoparticles showed synergistic effect with levo-floxacin antibiotic, the antimicrobial activity increased by 1.16—1.32 fold. Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
* Tel.: +20 222746791. E-mail address: haythammibrahim@yahoo.com.
Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/jjrras.2015.01.007
1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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1. Introduction
Nanotechnology is an emerging field in the area of interdisciplinary research, especially in biotechnology (Natarajan, Selvaraj, & Ramachandra, 2010). Nanoparticle research is inevitable today not because of only application and also by the way of synthesis (Gopinath et al., 2012).
Silver is a nontoxic, safe inorganic antibacterial agent that is capable of killing about 650 types of diseases causing microorganisms (Jeong, Yeo, & Yi, 2005). There is an increasing interest in silver nanoparticles on account of the antimicrobial properties that they display (Choi et al., 2008). They are even being projected as future generation antimicrobial agents (Rai, Yadav, & Gade, 2009). Silver nanoparticles are important materials that have been studied extensively, such nanoparticles possess unique electrical, optical as well as biological properties and are thus applied in catalysis, bio-sensing, imaging, drug delivery, nanodevice fabrication and in medicine (Jain, Huang, El-Sayed, & El-Sayed, 2008; Nair & Laurencin, 2007).
Synthesis of silver nanoparticles was extensively studied employing chemical and physical methods, but the development of reliable technology to produce nanoparticles is an important aspect of nanotechnology (Natarajan et al., 2010). Synthesis of nanoparticles by physical and chemical methods may have considerable environmental defect, technically laborious and economically expensive (Gopinath et al., 2012). The biological methods, using microorganisms and enzymes, have been suggested as possible eco-friendly alternatives (Mohanpuria, Rana, & Yadav, 2008).
The plants or plants extract, which act as reducing and capping agents for nanoparticles synthesis, are more advantageous over other biological processes (Valli & Vaseeharan, 2012), because they eliminate the elaborated process of culturing and maintaining of the cell, and can also be scaled up for large-scale nanoparticle synthesis (Saxena, Tripathi, Zafar, & Singh, 2012). Moreover, plant-mediated nano-particles synthesis is preferred because it is cost-effective, environmentally friendly, a single-step method for biosynthesis process and safe for human therapeutic use (Kumar & Yadav, 2009). Different parts of plant materials such as extracts (MubarakAli, Thajuddin, Jeganathan, & Gunasekaran, 2011), fruit (Prathna, Chandrasekaran, Raichur, & Mukherjee, 2011), bark (Satishkumar et al., 2009), fruit peels (Bankar, Joshi, Kumar, & Zinjarde, 2010), root (Ahmad et al., 2010) and callus (Nabikhan, Kandasamy, Raj, & Alikunhi, 2010) have been studied so far for the synthesis of silver, gold, platinum and titanium nanoparticles in different sizes and shapes (Gopinath et al., 2012).
Bananas are consumed all over the world, after consumption of the pulp, the peels are generally discarded (Bankar et al., 2010). A few applications of banana peels discussed in the literature include (i) exploitation for their medicinal properties (Parmar & Kar, 2008); (ii) in ethanol fermentation (Tewari, Marwaha, & Rupal, 1986); (iii) application as a substrate for generating fungal biomass (Essien, Akpan, & Essien, 2005); (iv) use in the production of laccase (Osma, Toca, Rodriguez Couto, 2007); and (v) utilization as a biosorbent for heavy metal removal (Annadurai, Juang, & Lee, 2003). In
addition, banana peels that are inherently rich in polymers such as lignin, cellulose, hemicellulose and pectins (Emaga, Andrianaivo, Wathelet, Tchango, & Paquot, 2007) could be used in the synthesis of silver nanoparticles.
The present study aims to synthesize silver nanoparticles by a green biological route, using an extract derived from banana peel waste, and characterization of the synthesized nanoparticles utilizing UV-visible spectroscopy, scanning electron microscope (SEM), energy dispersive X-ray spectros-copy (EDX), field emission scanning electron microscope (FESEM), transmission electron microscope (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FT-IR) analysis. Besides, their antimicrobial activity against representatives of humane pathogenic microorganisms was investigated.
Materials and methods
2.1. Microorganisms
Representative microorganisms of Gram-positive bacteria (Bacillus subtilis; local isolate, Staphylococcus aureus; ATCC 6538) and Gram-negative bacteria (Pseudomonas aeruginosa; ATCC 9027, P. aeruginosa; local isolate, Escherichia coli; ATCC 8739) as well as the yeast Candida albicans, ATCC 120231 were used to evaluate the antimicrobial activity of prepared silver nano-particles. Bacterial strains were maintained on nutrient agar slants and the yeast was maintained on potato-dextrose agar slants at 4 °C.
Banana peel extract (BPE) preparation
Banana (Musa paradisiaca) peels were washed and boiled in distilled water for 30 min at 90 °C. The peels (100 g) were crushed in 100 ml distilled water and the extract was filtered through a cheese cloth to remove insoluble fractions and macromolecules. This filtrate was treated with equal volume of chilled acetone and the resultant precipitate was centri-fuged at 1000 rpm for 5 min. This precipitate was resuspended in distilled water and stored in refrigerator 4 ° C for further studies. This extract was used as reducing as well as stabilizing agent.
2.3. Synthesis of silver nanoparticles using BPE
The source of silver was silver nitrate (AgNO3) in distilled water. Typical reaction mixtures contained 1 ml of BPE (equivalent to 6.8 mg dry weight) in 50 ml of silver nitrate solution (1 mM) unless otherwise stated. The reaction mixture was incubated in the dark at 30 °C to avoid the photo activation of silver nitrate under static conditions. Banana peels extract as well as silver nitrate solution (1 mM) were used as control. All experiments were carried out in triplicates and representative data is presented here. The effect of the silver was determined by varying the AgNO3 concentration (0.25,0.5, 1.0,1.25,1.50,1.75, 2.0 mM). The BPE concentration was varied (0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5 or 3.0 ml) while keeping the AgNO3 concentration at a level of 1.75 mM. The effect of pH was studied by adjusting the pH of the reaction mixtures (3 ml
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BPE, 1.75 mM AgNO3) to 2.0, 3.0,3.5,4.0,4.5, 5.0 or 6.5. To study the effect of temperature on nanoparticle synthesis, the reaction mixtures containing 3 ml BPE, and 1.75 mM AgNO3 at pH 4.5 were incubated at 30, 40, 50, 60, 80 or 100 °C for 5 min. The effect of reaction time was evaluated by incubating the reaction mixtures with optimum composition for 3, 5, 10, 15, 20, 25, 30, 45 min, 1, 24, 48, 72 and 96 h.
2.4. Characterization of synthesized silver nanoparticles
The UV-Visible spectra of silver nanoparticles were recorded as a function of wavelength using UV—Vis spectrophotometer (Helios Gamma, Thermo Corporation, England) operated at a resolution of 0.5 nm. The shape and size of silver nanoparticles were determined by SEM equipped with EDX, FESEM and TEM. For SEM and elemental analysis the dried reaction mixtures were subjected to JE0L-JSM-5400, Japan, SEM operating at 30 kV. The shape of nanoparticles was further characterized by FESEM (Agilent, 8500, USA). For TEM, a drop of aqueous silver nanoparticles sample was loaded on a carbon-coated copper grid, and it was allowed to dry in room temperature, the micrographs were obtained using TEM (JEOL-JEM-1200 EX, Japan) operating at 80 kV. The electron diffraction pattern for a selected area was also recorded. The average particle size and size distribution were determined by PSS-NICOMP 380-ZLS particle sizing spectrophotometer, St. Barbara, USA. XRD pattern was carried out using X'Pert Pro X-ray diffractometer (PANalytical, Japan). The target was Cu ka radiation and with l = 1.54 A, the generator operated at 40 kV and 40 mA, and the scanning mode was continuous with scanning range (20) from 4° ~ 90°. FT-IR measurements were carried out using Nicolet 6700, USA FT-IR spectrophotometer by employing KBr pellet technique. The FT-IR spectra were collected from 50 scans at a resolution of 4 cm-1 in the transmission mode (4000—400 cm-1).
2.5. Antimicrobial activity of synthesized silver nanoparticles
The antimicrobial activity of silver nanoparticles was investigated utilizing agar well diffusion assay Nanda and Saravanan (2009). The tested microorganisms were swabbed uniformly on nutrient agar- or sabouraud dextrose agar plates using sterile cotton swab, then, five wells of 6-mm diameter were made using sterile well borer. Twenty microliter of silver nanoparticles solutions with various concentrations (0.25, 0.50,1.0,1.50, and 2.0 mM) was poured into the corresponding well. Control sample (BPE) was used to assess the antimicrobial activity of BPE. The plates were incubated at 37 °C for 24 or 48 h for the bacterial and yeast cultures, respectively. The diameter of inhibition zone was measured. To determine the combined effect of silver nanoparticles and the standard antibiotic (levofloxacin), 15 ml of 0.5 mM antibiotic solution was mixed with 15 ml of silver nanoparticles solution (0.5 mM), and placed into the corresponding well of agar plate, inoculated with the tested microorganisms. The plates were incubated at 37 °C for 24 h. Zone of inhibition was measured and compared with that of levofloxacin, AgN03 solution and silver nanoparticles individually.
2.6. Analysis of antimicrobial activity of silver nanoparticles
The antimicrobial activity of silver nanoparticles in terms of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was estimated. For MIC determination, flasks containing sterile 50 ml of nutrient- or sabouraud dextrose broth media, supplemented with various silver nanoparticles concentrations (0.85,1.70, 3.4, 5.1, 6.8 and 10.2 mg/ml), were inoculated with 0.1 ml of the microbial cultures broth (0.8 O.D600). The flasks were incubated in shaking incubator for 24 h at 37 °C and 150 rpm. Silver nanoparticles-free broth media were used as a positive control. The microbial growth was indexed by measuring the optical density (0.D600) using UV—Vis spectrophotometer. For MBC estimation, a loopful of microbial cultures grown in nutrient broth medium supplemented with silver nano-particles were inoculated onto nutrient agar plates or sabo-uraud dextrose plates free from silver nanoparticles and incubated at 37 °C for 24 h. The lowest concentration of nanoparticles that prevent the microbial growth was designated as the MBC.
Results and discussion
3.1. Visual observation and UV—visible spectroscopy
Noble metals are known to exhibit unique optical properties due to the property of surface plasmon resonance (SPR) (Bindhu & Umadevi, 2013). The formation of silver nano-particles was monitored with color change and UV—Vis spectroscopy. The color of the reaction mixture started changing to yellowish brown within 10 min and to reddish brown after 1 h, indicating the generation of silver nano-particles, due to the reduction of silver metal ions Ag+ into silver nanoparticles Ag° via the active molecules present in the BPE (Ahmad et al., 2003). This color is attributed to the excitation of SPR. As shown in Fig. 1a, a characteristic and well-defined SPR band for silver nanoparticles is obtained at around l 433 nm (Mulvaney, 1996). Control silver nitrate solution neither developed the reddish brown color nor did they display the characteristic band, indicating that abiotic reduction of silver nitrate did not occur under the used conditions.
3.2. Effect of silver nitrate concentration
Yellowish brown and light reddish brown colors were observed at salt concentrations of 0.25 and 0.5 mM, and darker shades of reddish brown color were observed at silver nitrate concentrations ranged from 1.0 to 2.0 mM. The SPR peak of silver nanoparticles became distinct with increasing the concentration of silver nitrate, the maximum peak intensity was obtained at 1.75 mM of AgN03 (Fig. 1b). A variation in the biological material and metal salt concentration is known to influence nanoparticle synthesis (Pimprikar, Joshi, Kumar, Zinjarde, & Kulkarni, 2009).
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Fig. 1 — UV—Visible absorption spectra of synthesized silver nanoparticles, showing the surface plasmon resonance peak of silver nanoparticles at 433 nm (a); inset showing the color change upon formation of silver nanoparticles, at different AgNO3 concentrations (b), at variable concentrations of BPE (c), at different reaction temperatures (d), at different pH of reaction mixture (e), and at different incubation time (f). Q2
3.3. Effect of BPE
The reaction mixtures containing 0.25, 0.5, and 0.75 ml of BPE developed a light reddish brown color, while those containing 1.0 up to 3.0 ml of BPE developed darker reddish brown color. The SPR peaks were proportionally more intense and the maximum peak intensity was observed at BPE content of 3.0 ml (Fig. 1c). When the concentration of the biological material mediating nanoparticle synthesis is increased, higher contents of the biomolecules involved in the metal reductive
process are available resulting in a more intense color. Such an effect has been reported with the bark extract of Cinnamon zeylanicum (Satishkumar et al., 2009) and with the leaves of Cinnamon camphora (Huang et al., 2007).
3.4. Effect of pH
The color of reaction mixture and the intensity of the SPR peaks were pH dependent; when the reaction was conducted at pH 2.0 neither color change nor characteristic SPR peak of
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silver nanoparticles were observed (Fig. 1d). At pH values 2.0-4.0 a white precipitate was obtained and no color change was observed also. Varying shades of reddish brown color were observed at pH values 4.5-6.5. The highest color intensity was obtained at pH 4.5. In agreement, Roopan et al. (2013) reported that at pH 2.0 no reaction occurred while at pH 11 highly monodispersed nanoparticles were obtained with an average size of 23 ± 2 nm. A variety of biomolecules are postulated to be involved in biological nanoparticle synthesis, such biomolecules are likely to be inactivated under the extremely acidic conditions (pH 2.0). The differences in the colors obtained over the range of pH could be ascribed to a variation in the dissociation constants (pKa) of functional groups on the biomass that are involved (Pimprikar et al., 2009).
3.5. Effect of temperature
The temperature also affected the process of silver reduction. Reaction mixtures incubated at 30, 40 and 50 °C showed light reddish brown color and less pronounced SPR peaks. At higher incubation temperature (60,80 and 100 °C) dark reddish brown color and more intense SPR peaks were revealed (Fig. 1e). At room temperature (30 °C) the color change took 10 min to develop, while by heating the reaction mixtures at 40-100 °C the reduction process was faster and the reddish brown color was developed within 5 min. The maximum SPR peak intensity was detected at 100 °C. The increase in reaction temperature, UV spectra show sharp narrow peaks at lower wavelength region (412 nm at 100 °C), which indicate the formation of smaller nanoparticles, whereas, at lower reaction temperature, the peaks observed at higher wavelength regions (440 nm at 30 °C) which clearly indicates increase in silver nanoparticles size. It is a well-known fact that when the temperature is increased, the reactants are consumed rapidly leading to the formation of smaller nanoparticles (Park, Joo, Kwon, Jang, & Hyeon, 2007). Similarly, the size of silver nanoparticles was decreased with an increase in incubation temperature when the fungus Trichoderma viride was employed (Fayaz, Balaji, Kalaichelvan, & Venkatesan, 2009).
1.6 ■
0.4 ■
3.6. Effect of incubation period
The intensity of the reddish brown color was directly proportional to the incubation time of reaction mixture (Fig. 1f). The rate of silver ions reduction was going slowly during the first 45 min, as indicated by the low absorbance values at wavelength 433 nm (Fig. 2a) and color intensity (Fig. 2b). Tangible increase in the absorbance together with color intensity was revealed after longer time periods up to 97 h as shown in Fig. 2(a, b). The maximum reduction of silver ions was obtained after 72 h. This increase in absorbance along with color intensity could be ascribed to an increase in the number of silver nanoparticles with time (Bhainsa & D'Souza, 2006). It has been reported that the time required for complete reduction of the metal ions during biosynthesis of metal nanoparticles using bacteria and fungi range from 24 to 124 h (Korbekandi, Iravani, & Abbasi, 2009). The rapid generation of nanoparticles was owing to the excellent reducing potential of the active components of banana peel extract and their polymeric stabilization within a narrow size spectrum.
3.7. Characterization of silver nanoparticles
The shape of the synthesized silver nanoparticles was analyzed by SEM, representative SEM micrographs of control and treated BPE magnified at 750 x and 1500 x are shown in Fig. 3a and (b, c), respectively. Monodispersed spherical silver nanoparticles were formed on the surface of BPE derived biological materials as indicated in Fig. 3(b, c). The image obtained by the FESEM also showed spherical nanoparticles (Fig. 3d), confirming the result obtained by SEM.
EDX analysis gives qualitative as well as quantitative status of elements that may be involved in formation of nano-particles, 2001 EDX analysis gives qualitative as well as quantitative status of elements that may be involved in formation of nanoparticles. The elemental profile of synthesized nanoparticles using BPE shows higher counts at 3 keV due to silver, confirms the formation of silver nanoparticles (Fig. 3e). Generally metallic silver nanocrystals show typical optical absorption peak approximately at 3 keV due to their surface
10 20 30 40 50 Reaction time (minute)
24 48 72 Reaction time (h)
45 min 1 h 24 h 48 h 72 h 96 h
Fig. 2 - Effect of reaction time on absorbance intensity of synthesized silver nanoparticles at 433 nm (a); inset showing the absorbance during the first 45 min, and color intensity of the reaction mixture (b).
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plasmon resonance (Magudapatty, Gangopadhyayrans, Panigrahi, Nair, & Dhara, 2001). The elemental analysis of the silver nanoparticles shown in Fig. 3e revealed highest proportion of silver followed by Cl and P
The TEM image showed monodispersed silver nano-particles with spherical shape (Fig. 4a), confirming the results obtained by SEM and FESEM. Crystalline nature of the nano-particles is evidenced by the selected area electron diffraction patterns with bright circular spots (Fig. 4a). The average particle size was determined by DLS method, and it was found to be 23.7 nm as revealed in the size distribution graph (Fig. 4b).
The crystalline nature of silver nanoparticles was confirmed by the analysis of XRD pattern as shown in Fig. 5. The four distinct diffraction peaks at 20 values of 38.15°, 44.30°, 64.53° and 76.96° can be indexed to the (111), (2 0 0), (2 2 0) and (311) reflection planes of face centred cubic structure of silver. In addition to the Bragg peaks representative of silver nanocrystals, additional peaks were also observed at 27.89°, 32.24°, 46.26°, and 54.79°. These peaks are due to the organic compounds which are present the extract and responsible for silver ions reduction and stabilization of resultant nano-particles (Roopan et al., 2013). The XRD pattern obtained is
Fig. 3 — SEM micrograph of control BPE (a), BPE treated with AgNO3 (1 mM) magnified 750 x (b) and magnified 1500 x (c), FESEM micrograph of silver nanoparticles (d), and EDX profile of the synthesized silver nanoparticles (e).
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Fig. 4 - TEM micrograph of the silver nanoparticles, the scale bar corresponds to 100 nm (a) (inset: selected area electron diffraction pattern showing the characteristic crystal planes of elemental silver), and their particle size distribution histogram (b).
consistent with earlier reports (Kumar, Palanichamy, & Roopan, 2014).
FT-IR measurements were carried out to identify the major functional groups on the BPE surface and their possible involvement in the synthesis and stabilization of silver nanoparticles, 2007 FT-IR measurements were carried out to identify the major functional groups on the BPE surface and their possible involvement in the synthesis and stabilization of silver nanoparticles. The spectra of BPE before and after reaction with silver nanoparticles are represented in Fig. 6. Control spectrum (BPE untreated with AgNO3) showed several peaks indicating the complex nature of the biological material. The bands appearing at 3411.5, 2932.6, 1749, 1637.6,
40 SO 60
28 (degree)
Fig. 5 - XR diffractograme of silver nanoparticles.
Fig. 6 - FTIR spectra of BPE and silver nanoparticles.
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1386.5, 1146.5, 1077, 829.5 and 642.4 cm-1 were assigned to stretching vibration of O-H of alcohol or N-H of amines, C-H of alkanes, C=O of carboxylic acid or ester, N-C=O amide I bond of proteins, CH2 of alkanes, C-O of carboxylic acid, ester, or ether, C-N of aliphatic amines or alcohol/phenol, N-H deformation of amines, and C-C bending, respectively (Socrates, 1980). After reaction with AgNO3 there was a shift in
the following peaks: 3411.5 to 3420.8, 2932.6 to 2927.7, 1749 to 1742.9,1637.6 to 1626,1386.5 to 1383.3,1146.5 to 1141.1,1077 to 1076.3, 829.5 to 824.5 and 642.4 to 651.3 cm-1 indicating that carboxyl, hydroxyl and amide groups on the surface of BPE may be participating in the process of nanoparticle synthesis (Bankar et al., 2010). Banana peels are mainly composed of pectin, cellulose and hemicelluloses (Emaga et al., 2007) and
P. aeruginosa (ATCC) C albicans
Fig. 7 - Zone of inhibition of silver nanoparticles against various pathogenic microorganisms.
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the functional groups associated with these polymers as well as the proteinaceous matter may thus be involved in reducing the Ag+ to Ag0. Biological components are known to interact with metal salts via these functional groups and mediate their reduction to nanoparticles (Bar et al., 2009).
3.8. Antimicrobial activity of silver nanoparticles
Silver nanoparticles displayed antimicrobial activity against studied pathogenic microorganisms, with varying degrees, as suggested by the diameter of inhibition zone, while BPE didn't show any antimicrobial activity (Fig. 7). The Gram negative bacteria (E. coli and P. aeruginosa) showed larger zones of inhibition, compared with the Gram positive bacteria (B. subtilis and S. aureus) (Fig. 8a), which may due to the variation in cell wall composition. The cell wall of Gram positive bacteria composed of a thick peptidoglycan layer, consisting of linear polysaccharide chains cross linked by short peptides, thus forming more rigid structure leading to difficult penetration of the silver nanoparticles, while in Gram negative bacteria the cell wall possesses thinner peptidoglycan layer (Shrivastava et al., 2007). The antimicrobial activity against C. albicans
>*»>. -«»w c°ti
Microorganisms
0 0.85 1.7 3.4 5.1 6.8 10.2 Silver nanoparticles concentration (ng/ml)
Fig. 8 - Antimicrobial activity of silver nanoparticles against representative pathogenic microorganisms.
was very low as indicated by the small inhibition zone diameter.
Several main mechanisms underlie the biocidal properties of silver nanoparticles against microorganisms. First, silver nanoparticles attach to the negatively charged cell surface, alter the physical and chemical properties of the cell membranes and the cell wall and disturb important functions such as permeability, osmoregulation, electron transport and respiration (Marambio-Jones & Hoek, 2010; Nel et al., 2009; Sondi & Salopek-Sondi, 2004; Su et al., 2009). Second, silver nanoparticles can cause further damage to bacterial cells by permeating the cell, where they interact with DNA, proteins and other phosphorus- and sulfur-containing cell constituents (AshaRani, Mun, Hande, & Valiyaveettil, 2009; Nel et al., 2009; Nel et al., 2009). Third, silver nanoparticles release silver ions, generating an amplified biocidal effect, which is size-and dose-dependent (Liu, Sonshine, Shervani, & Hurt, 2010; Marambio-Jones & Hoek, 2010).
The antimicrobial properties of silver nanoparticles were analyzed by means of MIC and MBC. Agar dilution and broth dilution are the most commonly used techniques to determine the MIC of antimicrobial agents, in both approaches; the MIC is defined as the lowest concentration of the antimicrobial agent that prevents visible growth of a microorganism under defined conditions (Wiegand, Hilpert, & Hancock, 2008). After 24 h of incubation, no growth of B. subtilis, S. aureus, P. aeruginosa and E. coli, in the flasks supplemented with 6.8, 5.1, 1.70 and 3.4 mg/ml of silver nanoparticles, and the optical density was 0.026, 0.023, 0.011 and 0.012, respectively. Therefore, the MICs were 6.8, 5.1, 1.70 and 3.4 mg/ml, respectively (Fig. 8b). MBC is defined as the lowest concentration of antimicrobial agent that will prevent the growth of microorganism after subculture onto nanoparticles-free media. The MBCs of silver nanoparticles were found to be 10.2, 10.2, 5.1 and 5.1 mg/ml, respectively.
Silver nanoparticles showed more bactericidal activity compared with the silver salt, the inhibition zone diameter were (12-20 mm) and (10-17 mm), respectively. The high bactericidal activity of silver nanoparticles is due to their extremely large surface area, which provides better contact with microorganisms. Moreover, silver nanoparticles act as reservoirs for the Ag+ bactericidal agent. Combination of silver nanoparticles and antibiotic levofloxacin revealed a syn-ergistic effect, the antimicrobial activity against B. subtilis, S. aureus, P. aeruginosa (ATCC), P. aeruginosa (isolate) and E. coli increased by 1.16-, 1.20-, 1.32-, 1.27- and 1.30-fold, respectively (Table 1).
4. Conclusion
Banana peels as agricultural waste material was successfully utilized for the consistent and quick synthesis of silver nanoparticles. The biosynthesized silver nanoparticles using BPE were characterized; they are crystalline, uniform, spherical and monodispersed nanoparticles with average particle size of 23.7 nm. Synthesized silver nanoparticles revealed good antimicrobial activity against the selected pathogenic microorganisms. Moreover, they showed a synergistic effect on the antimicrobial activity of the standard antibiotic
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Table 1 - Antimicrobial activity of silver nitrate, silver nanoparticles and AgNPs combined with levofloxacin antibiotic against representative human pathogenic bacterial strains.
Microorganisms
Diameter of inhibition zone (mm)
BPE AgNO3 AgNPs Levofloxacin Levofloxacin + AgNPs % of enhancement
B. subtilis 0 10 12 25 29 1.16
S.aureus 0 14 16 30 36 1.20
P. aeruginosa (ATCC) 0 17 20 35 40 1.32
P. aeruginosa (isolate) 0 15 18 30 38 1.27
E. coli 0 13 17 30 39 1.30
*All values represented in the table are average of results of three separately conducted experiments.
levofloxacin against Gram-positive and Gram-negative bacteria under investigation. This green synthesis approach appears to be a cost-effective, non-toxic, ecofriendly alternative to the conventional microbiological, physical and chemical methods, and would be suitable for developing a biological process for large-scale production. These silver nanoparticles may be used in effluent treatment process for reducing the microbial load.
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