Green synthesis of biogenic silver particles, process parameter optimization and application as photocatalyst in dye degradation
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A stable dispersion of nano-structured silver particles (AgNPs) was synthesized using an aqueous extract of Citrus sinensis (orange) peel extract. Formation of AgNPs was confirmed using UV–visible spectroscopy. In the process, orange peel was used as both reducing agent and stabilizing agent. In the presence of extract, nanoparticles remained stable even after a time span of 20 days of their synthesis. Different physical parameters such as concentration of reducing agent, temperature, pH and time were being optimized for determination of optimal condition for synthesis of stable dispersion of nanoparticles. The AgNPs were further characterized using various techniques such as X-ray diffraction, dynamic light scattering, field emission scanning electron microscopy and energy-dispersive X-ray spectroscopy for getting information about their formation, composition, hydrodynamic diameter and size. The stability of the colloidal dispersion of nanoparticles over a wide range of temperatures and pHs was evaluated. The AgNPs were found to be stable over a pH range of 2–10 and temperatures ranging 25–75 °C. Later, these synthesized AgNPs were evaluated for their potentiality in antimicrobial activity. It was found that the nanoparticles showed significant bactericidal as well as fungicidal activity depending on the size of nanoparticles. In addition to this, AgNPs were evaluated for photocatalytic degradation of an azo dye, Congo red. Kinetic study of degradation has also been carried out and was found that the reaction followed pseudo-first-order kinetics.
KeywordsSilver nanoparticles Orange peel extract pH Temperature Antimicrobial activity Congo red Photocatalyst
Recently, nanoparticles have gained momentum due to their various beneficial applications in various fields. The unique feature of the nanoparticles is that their properties such as optical, magnetic and electronic vary from their bulk material . The specific properties of nano materials have been attributed to the fact that these particles have characteristic size, shape, area and surface chemistry. Nanoparticles can be synthesized using a variety of methods such as laser ablation, photochemical reduction and sono-chemical method [2, 3, 4, 5]. Though there are several processes available for synthesis of these particles, the processes using chemicals are not eco-friendly and relatively expensive. An alternative way needs to be searched which should be environmental-friendly and cheaper. The most efficient and cost-effective method for synthesis of metal nanoparticles is synthesis using natural resources like plant or fruit or fruit peel extract as stabilizing and reducing agent [6, 7, 8]. The recent literature revealed the enthusiasm of green route to synthesize AgNPs using banana peel extract , Azadirachta indica leaf , Garcinia mangostana leaf , Terminalia arjuna leaf , Murraya koenigii leaf , Punicia granatum peel  and Cinnamomum zeylanicum leaf . Therefore, this green process can be used for production of nanoparticles at large scale also. Biogenic methods of synthesis of nanoparticles have several advantages over physiochemical method. Besides being environmental-friendly and non-toxic in nature, this method leads to production of nanoparticles having well-defined morphology and size . In the proposed work, Citrus sinensis has been employed for the synthesis of nanoparticles. Citrus sinensis (orange) belonging to a family Rutaceae are mainly found in dry and arid regions of tropical and subtropical areas.
Metal nanoparticles have got vital role in various areas such as pharmaceutical, biotechnological and industrial. In particular, silver nanoparticles possess antibacterial, antifungal and larvicidal properties [16, 17, 18]. In fact, silver has the lowest toxicity to animal cells and was found to be most effective against bacterial action. Besides their antimicrobial property, AgNPs also exhibit photocatalytic properties in the field of dye detoxification and its removal [19, 20, 21]. Dyes are used in different industries and are directly discharged into the water bodies without any treatment. These dyes which are non-biodegradable and potentially hazardous can cause serious damage to ecology [22, 23]. Various other methods are also being practiced for treatment of waste water such as UV light degradation, redox treatment, carbon sorption and flocculation; however, these techniques are not that effective and, therefore, need better approach [24, 25, 26, 27].
The objective of this work is to yield relatively stable dispersion of AgNPs in a simple and cheap manner by reducing the silver salt solution using orange peel extract. Peel extract was used as both reducing agent and stabilizing agent. Optimal condition to obtain stable dispersion of AgNPs was obtained by evaluating different parameters such as pH, temperature and concentration of reducing agent. The synthesized nanoparticles were then evaluated for its antimicrobial property, and finally, these AgNPs were also used as photocatalyst for the removal of an azo dye, i.e. Congo red, present in waste water and the kinetic study of degradation has also been reported.
Silver nitrate (AgNO3, Extra Pure, 99%) and sodium borohydride of Molychem were being used for different purposes. The pH of each solution was adjusted to 8 using 0.1 M sodium hydroxide (Sigma Aldrich) and 0.1 M hydrochloric acid (Fischer Scientific) solution. Citrus sinensis peels were collected from the local market. A total of two bacterial strains, namely Pseudomonas aeruginosa (P.aeruginosa) and Pseudomonas syringae (P. syringae), and a fungal strain, Alternaria brassicicola (A. brassicicola), were used in the study. The bacterial and fungal strains were obtained from CSIR-National Botanical Research Institute, Lucknow, India. For preparation of solution, double distilled water was used.
2.2 Synthesis of AgNPs
Composition of sample solution
2.3 Characterization of AgNPs
At the initial stage, the synthesis of AgNPs was monitored using LAB India UV–Vis 3200 spectrophotometer in a wavelength range of 300–700 nm. A glass cuvette having optical path of 1 cm was used. The stability of colloidal AgNPs was studied by taking the UV–Vis absorption spectra of all samples after 30 min of synthesis and was compared with spectra taken after 7 days and then after 20 days. The UV–Vis absorption spectra for different samples were taken at room temperature. PANalytical X′pert Pro MPD diffractometer was used to perform powder XRD to obtain the diffraction pattern from the sample. The AgNPs were further characterized by DLS study using a Nano Brook 90 plus PALS at 25 °C. The hydrodynamic diameter (Z-average), PDI and width distribution of particles were also determined through DLS study. Morphology and size of the nanoparticles were determined using FESEM. FESEM images of the nanoparticles were taken by using ZEISS microscope (accelerating voltage ranged from 5 to 20 kV). FESEM samples were prepared by placing 2 μL of AgNPs on thin aluminium foil. The chemical composition was verified using elemental analysis on a ZEISS electron microscope equipped with an EDX analyser. To prevent samples from charging, a thin gold coating was sputtered onto the samples prior to the analysis. Coating the sample with a layer of metal resists charging, prevents thermal damage and improves the secondary electron signals, which is required for topographic examination of the sample.
3 Stability of AgNPs at different pHs and temperatures
The colloidal stability of synthesized AgNPs was screened at different pHs and temperatures. The hydrodynamic diameter and PDI of the nanoparticles were measured over a pH range of 2–10 and temperature range of 25–75 °C using NanoBrook 90 plus PALS. 0.1 M NaOH solution and 0.1 M HCl were used to adjust the desired pH environment.
4 Antimicrobial assay of AgNPs
4.1 Antibacterial activity assessment
Antibacterial potential of biogenic AgNPs of different KExt/Ag ratios was tested against Pseudomonas aeruginosa and Pseudomonas syringae (Gram-negative bacteria) by disc diffusion method . To study the effect of AgNPs on pathogens, 100 µl of cultures, which were allowed to grow overnight, was spread on nutrient agar plates in a uniform manner. Pre-sterilized cotton of 1 cm2 was placed on the centre of the plates, and 50 µL of desired nanoparticles was added to it and was dried in air. The plates were incubated at 27 °C for 24 h. After 24 h, zone of inhibition was measured. Peel extract (3000 ppm) was used as control in the process.
4.2 Antifungal activity evaluation
Antifungal assay of the synthesized nanoparticles against Alternaria brassicicola was conducted to evaluate the effect of AgNPs on the mycelial growth of the fungi. Three plates of autoclaved potato dextrose agar (PDA) were prepared in which nanoparticles synthesized using different KExt/Ag ratios were added to each of these plates. The other plate was kept as control containing only Milli-Q Water (without AgNPs). A disc of mycelia was withdrawn from the fungal culture and was placed at the centre of the plate containing silver nanoparticles assimilated in PDA Medium. The plates were then incubated at 25 °C temperature for 7 days. The efficiency of the synthesized nanoparticles was evaluated by measuring the diameter of the fungal colony growth.
The photocatalytic activity of the AgNPs was studied by employing an aqueous solution of an azo dye, i.e. Congo red (20 mg L−1). Thereafter, the photocatalytic reaction was conducted outdoor under the sunlight as main energy source. The experiment was set up by suspending 5 mL of AgNPs solution in 50 mL of dye solution. The mixture was kept in dark under continuous stirring for 30 min to bring the AgNPs to constant equilibrium in the mixture. The mixture was then kept under sunlight for 4–5 h. Degradation of congo red aqueous solution was analysed using UV–Vis spectroscopy by collecting the samples at regular time interval. The kinetics of the reaction has also been studied.
6 Results and discussion
6.1 Visual change
The preliminary analysis, which indicates the synthesis of nanoparticles, is the visible change in the colour on addition of peel extract to the silver nitrate solution. On adding different concentrations of peel extract to silver nitrate, a significant change in colour was observed. The colour of the solution turned from colourless to orange when the extract ratio was 0.5, and on increasing the extract ratio to 2.5, brown colour was observed, which turned to dark brown on further increasing the extract ratio to 5.0. The reason for the change in colour is excitation in the surface plasmon resonance (SPR). The SPR is a distinctive optical property, which is exhibited when all the electrons present in conduction band vibrate in resonance, which, in turn, is responsible for the absorption ranging between 380 and 500 nm in UV–Vis spectra for the synthesized silver nanoparticles. Thus, the colour change confirmed the synthesis of silver nanoparticle, which was further confirmed using UV–Vis spectrometer.
6.2 UV–Vis results
UV–Vis absorption spectrum of the synthesized nanoparticles using sodium borohydride as reducing agent and without adding peel extract (stabilizing agent) is depicted in Fig. 2a. The UV–Vis spectrum taken immediately after synthesizing AgNPs showed peak at 390 nm, which indicated the formation of spherical AgNPs, but the spectrum taken after 1 day did not show any peak. The peak at 390 nm disappeared which illustrated the instability of synthesized nanoparticles while spectrum of the nanoparticles obtained in the presence of peel showed no changes even after a week time. The UV–Vis absorption spectrum for nanoparticles having KExt/Ag = 0.5 and 2.5 was stable even after a week, but the stability of nanoparticles obtained from KExt/Ag = 5.0 was even higher (Fig. 2b–d). There are two factors, which determine the stability of nanoparticles. These are (a) changes in the maximum absorption wavelength (b) sharpness of the peak. AgNPs having lowest change in the absorption and also sharpest peak have the highest stability . Based on these results, the solution of nanoparticles with KExt/Ag = 5.0 has the highest stability. This shows that peel extract acts as a stabilizer and the increase in its concentration increased the stability of nanoparticles. Even slight change in the shape, size and distribution of the solution has an influence on the UV–Vis extinction characteristics such as shifting of the peak, intensity and full width half maxima . The results obtained are well in agreement with those reported in Santiago et al.
6.3 XRD analysis
6.4 DLS results
Effect of different concentrations of peel extract on Z-average and zeta potential of AgNPs
Zeta potential (mV)
6.5 FESEM results
6.6 EDX analysis
6.7 Effect of pH on the stability of AgNPs
6.8 Stability of nanoparticles at different temperatures
The stability of nanoparticles was studied over a temperature range 25–75 °C. The particle size and PDI of synthesized nanoparticles using KExt/Ag = 0.5 with varying temperature are shown in Fig. 7b. The size of the nanoparticles increased from 51 to 72 nm on increasing the temperature from 25 to 75 °C. The PDI of synthesized nanoparticles decreased till 45 °C (from 0.415 to 0.204), then increased at 55 °C and then further decreased. The PDI presents the homogeneity of the size distribution in a dispersion of colloidal AgNPs. A lower PDI indicates the presence of more homogeneous particles. Therefore, the most optimum temperature for the stability of nanoparticles was found to be 45 °C at which the particles were smaller in size and homogenous in nature.
6.9 Antibacterial assay result
Zone of inhibition (Mean ± SD) exhibited by AgNPs against Gram-negative bacteria P. syringae and P. aeruginosa
Zone of inhibition (mm)
20.833 ± 0.8498
0.0 ± 0.0
24.833 ± 0.6236
12.166 ± 0.2357
26.33 ± 0.6236
14.33 ± 0.2357
The highest KExt/Ag ratio of 5.0 exhibited maximum antibacterial activity as compared to other counterparts. As observed in Fig. 8, the diameter of zone of inhibition increased with the increase in KExt/Ag ratio, which reflects that synthesized AgNPs using higher concentration of extract exhibited higher bactericidal properties as compared to others.
The increase in the antibacterial activity is because of the fact that with increase in the concentration of peel extract, small-sized nanoparticles were obtained (Table 2) due to which larger surface area became available for the interaction, and hence, bactericidal activities increased as compared to large-sized particles and killed the microorganism [35, 38].
When nanoparticles interacted with the bacterial membrane, certain changes occurred on bacterial surface. The changes produced on the surface of bacterial membrane lead to increase in its permeability, which ultimately affected the transport through plasma membrane. The disturbance in the transportation through plasma membrane consequently caused cell death [39, 40]. The silver nanoparticles, which penetrated the bacterial surface, interacted with compounds containing sulphur and phosphorous  and disturbed the regular cycle leading to ultimate demise of the cell. In case of Psedomonas syringae, an increase in the KExtact/Ag ratio resulted in increase in the killing rate of bacteria. Psedomonas aeruginosa also exhibited the same trend, but the zone of inhibition exhibited by it was comparatively smaller as compared to Pseudomonas syringae (as shown in Table 3). Similar kind of results has been reported when AgNPs were used to kill E. coli and S. aureus .
6.10 Antifungal assay result
Fungal growth (colony diameter, mm) in the presence of AgNPs synthesized using variable KExt/Ag
Diameter of fungal (A. brassicicola) growth (mm)
55.5 ± 1.0
30.0 ± 1.0
27.7 ± 0.7
7 Mechanism of silver nanoparticles synthesis using orange peel
On exposing the Congo red solution to the solar radiation, the peak observed at 507 nm suppressed significantly, which depicts the mineralization of azo bonds by the attack of hydroxyl radicals. On the other hand, the peak centred at 355 nm, which was attributed to naphthalene ring, exhibited a lesser suppression. Thus, it can be concluded that azo bonds are more susceptible to degradation by hydroxyl radicals as compared to the aromatic rings.
8.1 Mechanism of degradation of dye
8.2 Kinetics of degradation of dye
The rate of reaction was determined by plotting a graph between ln (A0/At) versus reduction, and slope of the graph depicts the rate of reaction. The order of the reaction was found to be of pseudo-first order, and the rate constant calculated from the slope of the graph was found to be 0.2028 h−1 (Fig. 12c).
In the study, AgNPs were prepared using green route and stabilized in the presence of orange peel extract. The concentration of peel extract influenced not only the size, PDI and FWHM but also had an effect on colloidal stability of nanoparticles. According to the UV–Vis data obtained, KExt/Ag = 5.0 is the most optimum concentration for obtaining nanoparticles of higher stability. A considerable increase in the intensity of the UV–Vis spectrum peak was observed in the presence of extract, which indicates synthesis of large number of nanoparticles. FESEM images confirmed the role of peel extract in the synthesis of silver nanoparticles. The morphology and composition of AgNPs are in good agreement with those reported by Gonzalez et al. . The hydrodynamic diameter of the AgNPs was found to be smallest at pH 10 and 45 °C temperature, and therefore, it was concluded that the optimum condition for the maximum stability of nanoparticles was found to be at pH 10 and 45 °C temperature. Orange peel is not only cost-effective but is also readily available and hence provides a cheap and easy method to synthesize stable silver nanoparticles, which can be further used for various applications. Synthesized and stabilized AgNPs exhibited good antimicrobial activity against bacteria Pseudomonas syringae and Psedomonas aeruginosa and fungi Alternaria brassicicola. The AgNPs were also capable of degrading Congo red dye efficiently and exhibited higher catalytic activity. Thus, greener route for synthesis of nanoparticles could be used for making comparatively better antimicrobial and catalytic agent for various purposes.
The authors acknowledge Department of Civil Engineering, IIT-Kanpur, for DLS measurement and Advanced Centre for Materials Science, IIT-Kanpur, for FESEM and XRD analysis.
The authors declare no competing financial interests. This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 9.Gopi D, Kanimozhi K, Bhuvaneshwari N, Indira J, Kavitha L (2014) Novel banana peel pectin mediated green route for the synthesis of hydroxyapatite nanoparticles and their spectral characterization. Spectrochim Acta Part A Mol Biomol Spectrosc 118:589–597. https://doi.org/10.1016/j.saa.2013.09.034 CrossRefGoogle Scholar
- 13.Sajeshkumar NK, Vazhacharickal PJ, Mathew JJ, Sebastin A (2015) Synthesis of silver nano particles from curry leaf (Murraya koenigii) extract and its antibacterial activity. J Pharm Sci 4:15–25Google Scholar
- 19.Varadavenkatesan T, Vinayagam R, Selvaraj R (2019) Green synthesis and structural characterization of silver nanoparticles synthesized using the pod extract of Clitoria ternatea and its application towards dye degradation. Mater Today. http://doi.org/10.1016/j.matpr.2019.04.216
- 35.Kaviya S, Santhanalakshmi J, Viswanathan B, Muthumary J, Srinivasan K (2011) Biosynthesis of silver nanoparticles using Citrus sinensis peel extract and its antibacterial activity. Spectrochim Acta Part A: Mol Biomol Spectrosc 79:594–598. https://doi.org/10.1016/j.saa.2011.03.040 CrossRefGoogle Scholar
- 41.Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO (2000) A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 52:662–668. https://doi.org/10.1002/1097-4636(20001215)52:4%3C662:aid-jbm10%3E3.0.co;2-3 CrossRefGoogle Scholar