# Comparative Analysis of Nanosilver Particles Synthesized by Different Approaches and Their Antimicrobial Efficacy

Authors: Hala M. Abdelmigid; Maissa M. Morsi; Nahed Ahmed Hussien; Amal Ahmed Alyamani; Noha Moslah Al Sufyani
Journal: Journal of Nanomaterials
 (2021)

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## Abstract

Silver nanoparticles (AgNPs) were extensively used in different fields worldwide. There is a continued increase in their productions to fulfill various uses. Biological and chemical AgNP syntheses were the most popular mechanisms in this field. Agrowastes are rich in proteins, phenolics, and flavonoids that could act as bioreductant agents in AgNP biological synthesis. The present study was aimed at synthesizing AgNPs via chemical and biological methods using trisodium citrate, pomegranate fruit peel, and coffee ground waste extracts. Moreover, silver nanoparticles were monitored by UV-vis spectroscopy and characterized using zeta potential, size distribution mean, scanning electron microscope (SEM), X-ray diffractometer (XRD), and Fourier transforms infrared spectroscopy (FTIR). Four pathogenic bacterial strains (Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, and MRSA) were used to assess the antimicrobial effect of the synthesized AgNPs (2, 4, and 8 mg/ml). Results report the successful formation of silver nanoparticles chemically (AgNPs_Chem) and biologically by using pomegranate peel extract (AgNPs_PPE) and coffee ground waste extract (AgNPs_CE) due to the change of color to dark brown that is confirmed by UV-vis sharp absorption spectra at specific wavelengths. Characterization using SEM and XRD revealed their crystalline shape with a mean size of AgNPs_Chem=62.75, AgNPs_CE=273.7nm, and AgNPs_PPE=591.9nm. AgNPs_Chem show higher negativity of zeta potential (−46.7 mV) than AgNPs_CE (−12.6 mV), followed by AgNPs_PPE (−7.98 mV), which had the least stability. All the synthesized AgNPs show antimicrobial potential on all selected strains. However, 8 mg/ml shows the most effective concentration and has more efficiency on K. pneumoniae than others. Overall, the results highlight that the use of agrowastes could be an ecofriendly way to synthesize AgNPs biologically that have the same antimicrobial effect as the chemically synthesized AgNPs.


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## Body

## 1. Introduction

Owing to the extensive applications of nanomaterials in various areas of industry, technology, and agriculture along with medicine, the worldwide request for nanomaterials is increasing exponentially. Due to the commercial consumption of silver nanoparticles (AgNPs) in various applications, the annual increase in AgNP production was estimated to be hundreds of tons worldwide [1]. In chemical preparation methods, producing stable colloidal AgNPs of desired sizes within the nanometer scale, apart from being an expensive chemical, can also generate oxidized species during the synthesis process, which cannot be easily separated from the nanomaterial product [2]. Such the undesirable chemical entities can significantly restrict the application opportunities as well as the biological compatibility of the produced nanomaterials.Consequently, the utilization of nanomaterials must be complemented with innovative, low-cost, ecofriendly production processes, which reduce the use of hazardous chemicals and reduce the cohort of unsafe wastes [2, 3]. Green synthesis strategies of metal nanoparticles have been recently growing, as more safe alternatives to conventional chemical methods and low impact on the environment. Such approaches apply mild experimental conditions such as ambient temperature and pressure; require nontoxic, environmentally benign solvents, and reducing agents as well as capping materials [4]. They involve either living organisms, such as bacteria [5] or fungi [6]. Natural extracts [7] obtained from whole plants, extracts of different parts of plants, and fruit waste have been well applied for green nanomaterial synthesis. Plant extracts contain various metabolites (e.g., polysaccharides, proteins, vitamins, or alkaloids), which are generally nontoxic, biodegradable, and can act both as reducing and capping agents, thereby promoting the formation, and preventing the agglomeration of nanoparticles [2, 4, 8]. Application of plant- or fruit waste-based extracts could be more favorable due to a simple, cost-effective, and ecofriendly process relative to other biological paths of NP synthesis [9]. Nanoparticles have been effectively synthesized by a wide range of agrowastes such as Cocos nucifera coir, corn cob, fruit seeds and peels, wheat bran, rice bran, and palm oil mill effluent [10–16]. These agrowastes have been reported to be rich in biomolecules such as phenolics, flavonoids, and proteins, and that can serve as bioreductant agents in the green synthesis of diverse nanoparticles.Pomegranate (Punica granatum) is one of the most important crops cultivated in Saudi Arabia. This fruit is rich in polyphenolic phytochemicals which have been shown to exhibit excellent antioxidant properties and therefore has been widely used in the food industry and in traditional medicine to treat various diseases is Saudi Arabia [17, 18]. The pomegranate peel is an agrowaste, which constitutes about 60% of the weight of pomegranate fruit. The pomegranate peel extract (PE) is also known for its high-nutrient content, consisting of minerals (e.g., potassium), vitamins, phenolics, flavonoids, antioxidants, and anticarcinogenic properties [19–21].Coffee is one of the most popular beverages in the world that are commercially cultivated [22]. They are considered a rich source of biologically active compounds, especially polyphenols [23]. Its seeds (i.e., coffee beans) contain two types of alkaloids, caffeine and trigonelline, as major components. In addition to caffeine, there are adenine, xanthine, hypoxanthine, and guanine [24]. The coffee industry is responsible for the production of massive amounts of wastes [25]. Solid coffee ground (SCG) consists mainly of (hemi) cellulose, melanoidins, proteins, and polyphenols (lignins, tannins, and chlorogenic acids) [26]. Given the presence of organic components, Sunoqrot et al. [27] assumed that the polyphenol-rich aqueous extracts of arabica coffee beans represented by chlorogenic acids and melanoidins may produce nanostructures through oxidative coupling.In this present work, biocompatible silver nanoparticles were synthesized via a variety of approaches using chemical and biological methods. Pomegranate fruit peel and coffee ground waste extracts have been used as capping and stabilizing agents for synthesizing silver nanoparticles from silver ions. The antimicrobial effect against pathogenic bacterial strains was measured, and an opportunity of their chemical versatility to load antimicrobial agents and surface ligands was taken.


## 2. Materials and Methods

### 2.1. Waste Collection and Preparation of Plant Extract

Fresh pomegranate (Punica granatum L.) fruits and coffee beans were purchased from local shop in Taif governorate, KSA. Pomegranate fruits were washed thoroughly using distilled water, and the seeds were then separated to obtain the peel. The isolated peels were oven dried at low temperature overnight until full dryness and ground into uniform coarse powder using a domestic blender.Coffee beans were ground and used in preparing the coffee drink, and their waste was air dried at room temperature. Steps of pomegranate peel (PP) and coffee ground waste collection are briefly shown in Figure1. For each waste product separately, 50 g powder was mixed with sterile distilled water (500 ml), boiled (10 min), filtered with Whatman filter paper, and cooled down at room temperature. Finally, pomegranate peel extract (PPE) and coffee ground extract (CE) were stored at 4°C for later use.Figure 1
Steps of pomegranate peel (a–c) and coffee ground (d–g) waste collection.
(a)(b)(c)(d)(e)(f)(g)


### 2.2. Green Synthesis of Silver Nanoparticles

Aqueous solution of silver nitrate (AgNO3, 10-3 M, Alfa Aeser, UK) was mixed with each waste plant extract (PPE and CE) with ratio 9 : 1. Mixture was heated at 80°C with continuous stirring until change of extract color from pale yellow (PPE)/pale brown (CE) to dark brown, which refers to Ag nanoparticle (AgNP) formation (Figure 2). To obtain synthesized AgNPs, mixtures were centrifuged at 15000 rpm for10 min, supernatant was discarded, and the pellet was washed with sterile distilled water (3x) and centrifuged to get rid of any adsorbed substances on nanoparticles’ surfaces. Biosynthesized silver nanoparticles (AgNPs_PPE and AgNPs_CE) were oven dried (60°C) overnight in glass petri dishes and then scraped out for further evaluations [28].Figure 2
Change of pomegranate peel extract (PPE) (a) and coffee ground extract (CE) (b) color due to silver nanoparticle AgNP_PPE (c) and AgNP_CE (d) preparations, respectively.
(a)(b)(c)(d)


### 2.3. Chemical Synthesis of Silver Nanoparticles

Silver nanoparticles were prepared through chemical reduction according to Fang et al. [29]. 50 ml of 10-3 M AgNO3 was boiled followed by gentle addition of trisodium citrate (1%, 5 ml, Sigma-Aldrich, Milan, Italy). Solution was heated with vigorous stirring until its color change from colorless to pale brown color (Figure 3). Chemical synthesized silver nanoparticles (AgNPs_Chem) were collected by centrifugation and dried as previously done with biologically prepared AgNPs_PPE and AgNPs_CE.Figure 3
Preparation of silver nanoparticles by chemical reduction (AgNPs_Chem).


### 2.4. Characterization of Silver Nanoparticles

#### 2.4.1. UV-Vis Spectroscopy

The absorption spectra of the synthesized silver nanoparticles (AgNPs_PPE, AgNPs_CE, and AgNPs_Chem) were measured in their aqueous form, before dryness, using a UV-vis-NIR spectrophotometer (UV-1601, Shimadzu, Japan), using wavelength range from 200 to 800 nm. Autoclaved distilled water was used as a reference in measurement.


#### 2.4.2. Size Distribution and Zeta Potential

The average size and surface charge of all synthesized silver nanoparticles were evaluated by using a particle size analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., UK) at 25°C with a detection angle 90°. All powder samples were freshly resuspended in 0.9% saline solution and sonicated for 30 mins at high speed before assessment to prevent nanoparticle aggregations.


#### 2.4.3. Surface Morphology

Silver nanoparticles AgNPs_PPE, AgNPs_CE, and AgNPs_Chem (dried powder) were separately coated with gold using a Cressington Sputter Coater (108 Auto, thickness controller MTM-10, UK) for 10 mins prior to scanning. Scanning electron microscope (SEM, JEOL JSM-639OLA, Analytical Scanning Electron Microscope, Tokyo, Japan, that is present in the Electron Microscope Unit of Taif University) at 20 kV was used with different magnifications of ×500, ×2000, and ×3000 with scale bars of 50μm, 10 μm, and 5 μm, respectively, to determine surface morphology. The X-ray diffractometer (XRD, Pan Analytical, X-pert pro, Netherland) was used to confirm the surface crystalline shape of the synthesized nanoparticles. XRD was used at 30 kV and 100 mA, and the spectrum was recorded by CuKα radiation with a wavelength of 1.5406 Å in the 2θ (from the range of 20°-80°). Patterns of XRD were plotted by the software OriginLab and compared with JCPDS card no. 040783.


#### 2.4.4. Fourier Transforms Infrared Spectroscopy (FTIR)

FTIR spectroscopy (Agilent Technologies, Santa Clara, CA, USA) was used to detect the possible functional groups found in PPE, CE, and sodium citrate (at wavelength range between 450 and 4000 cm-1) that are responsible to silver nanoparticle synthesis and stabilization.


### 2.5. Agar Well Diffusion Assay

The antibacterial potential of silver nanoparticles (AgNPs_PPE, AgNPs_CE, and AgNPs_Chem) was evaluated by well diffusion method. Inhibition zones were recorded in millimeters (mm). Briefly, plates of the Mueller-Hinton agar were inoculated with five different pathogenic bacterial strains, separately:Enterobacter aerogenes, Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA). Wells with a diameter of 7 mm were cut using sterile blue tips. For each plate, five wells were cut for plant extract, antibiotic, and three different concentrations of AgNPs. 100 μl of plant extract (PPE/CE) and 100 μl of the antibiotic ciprofloxacin 750 (1.6 mg/ml) were added as reference and positive control, respectively. Different concentrations (2 mg/ml, 4 mg/ml, and 8 mg/ml) of each silver nanoparticle were suspended in distilled water and were properly sonicated. 100 μl for each treatment and concentration were added per well. Plates were incubated at 37°C overnight. After incubation, diameters of the inhibition zone were measured by using a standard metric ruler.


#### 2.5.1. Statistical Analysis

Data were expressed as themean±standarderror (M±SE). Statistical analysis was conducted using one-way ANOVA to differentiate between the inhibition zone diameter of different bacterial strains groups within the same AgNP treatment using GraphPad software (GraphPad®, 2017), in which, ∗∗∗ indicates P≤0.001, ∗∗ indicates P≤0.01, ∗ indicates P≤0.05, and ns (nonsignificant) means P>0.05.


## 2.1. Waste Collection and Preparation of Plant Extract

Fresh pomegranate (Punica granatum L.) fruits and coffee beans were purchased from local shop in Taif governorate, KSA. Pomegranate fruits were washed thoroughly using distilled water, and the seeds were then separated to obtain the peel. The isolated peels were oven dried at low temperature overnight until full dryness and ground into uniform coarse powder using a domestic blender.Coffee beans were ground and used in preparing the coffee drink, and their waste was air dried at room temperature. Steps of pomegranate peel (PP) and coffee ground waste collection are briefly shown in Figure1. For each waste product separately, 50 g powder was mixed with sterile distilled water (500 ml), boiled (10 min), filtered with Whatman filter paper, and cooled down at room temperature. Finally, pomegranate peel extract (PPE) and coffee ground extract (CE) were stored at 4°C for later use.Figure 1
Steps of pomegranate peel (a–c) and coffee ground (d–g) waste collection.
(a)(b)(c)(d)(e)(f)(g)


## 2.2. Green Synthesis of Silver Nanoparticles

Aqueous solution of silver nitrate (AgNO3, 10-3 M, Alfa Aeser, UK) was mixed with each waste plant extract (PPE and CE) with ratio 9 : 1. Mixture was heated at 80°C with continuous stirring until change of extract color from pale yellow (PPE)/pale brown (CE) to dark brown, which refers to Ag nanoparticle (AgNP) formation (Figure 2). To obtain synthesized AgNPs, mixtures were centrifuged at 15000 rpm for10 min, supernatant was discarded, and the pellet was washed with sterile distilled water (3x) and centrifuged to get rid of any adsorbed substances on nanoparticles’ surfaces. Biosynthesized silver nanoparticles (AgNPs_PPE and AgNPs_CE) were oven dried (60°C) overnight in glass petri dishes and then scraped out for further evaluations [28].Figure 2
Change of pomegranate peel extract (PPE) (a) and coffee ground extract (CE) (b) color due to silver nanoparticle AgNP_PPE (c) and AgNP_CE (d) preparations, respectively.
(a)(b)(c)(d)


## 2.3. Chemical Synthesis of Silver Nanoparticles

Silver nanoparticles were prepared through chemical reduction according to Fang et al. [29]. 50 ml of 10-3 M AgNO3 was boiled followed by gentle addition of trisodium citrate (1%, 5 ml, Sigma-Aldrich, Milan, Italy). Solution was heated with vigorous stirring until its color change from colorless to pale brown color (Figure 3). Chemical synthesized silver nanoparticles (AgNPs_Chem) were collected by centrifugation and dried as previously done with biologically prepared AgNPs_PPE and AgNPs_CE.Figure 3
Preparation of silver nanoparticles by chemical reduction (AgNPs_Chem).


## 2.4. Characterization of Silver Nanoparticles

### 2.4.1. UV-Vis Spectroscopy

The absorption spectra of the synthesized silver nanoparticles (AgNPs_PPE, AgNPs_CE, and AgNPs_Chem) were measured in their aqueous form, before dryness, using a UV-vis-NIR spectrophotometer (UV-1601, Shimadzu, Japan), using wavelength range from 200 to 800 nm. Autoclaved distilled water was used as a reference in measurement.


### 2.4.2. Size Distribution and Zeta Potential

The average size and surface charge of all synthesized silver nanoparticles were evaluated by using a particle size analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., UK) at 25°C with a detection angle 90°. All powder samples were freshly resuspended in 0.9% saline solution and sonicated for 30 mins at high speed before assessment to prevent nanoparticle aggregations.


### 2.4.3. Surface Morphology

Silver nanoparticles AgNPs_PPE, AgNPs_CE, and AgNPs_Chem (dried powder) were separately coated with gold using a Cressington Sputter Coater (108 Auto, thickness controller MTM-10, UK) for 10 mins prior to scanning. Scanning electron microscope (SEM, JEOL JSM-639OLA, Analytical Scanning Electron Microscope, Tokyo, Japan, that is present in the Electron Microscope Unit of Taif University) at 20 kV was used with different magnifications of ×500, ×2000, and ×3000 with scale bars of 50μm, 10 μm, and 5 μm, respectively, to determine surface morphology. The X-ray diffractometer (XRD, Pan Analytical, X-pert pro, Netherland) was used to confirm the surface crystalline shape of the synthesized nanoparticles. XRD was used at 30 kV and 100 mA, and the spectrum was recorded by CuKα radiation with a wavelength of 1.5406 Å in the 2θ (from the range of 20°-80°). Patterns of XRD were plotted by the software OriginLab and compared with JCPDS card no. 040783.


### 2.4.4. Fourier Transforms Infrared Spectroscopy (FTIR)

FTIR spectroscopy (Agilent Technologies, Santa Clara, CA, USA) was used to detect the possible functional groups found in PPE, CE, and sodium citrate (at wavelength range between 450 and 4000 cm-1) that are responsible to silver nanoparticle synthesis and stabilization.


## 2.4.1. UV-Vis Spectroscopy

The absorption spectra of the synthesized silver nanoparticles (AgNPs_PPE, AgNPs_CE, and AgNPs_Chem) were measured in their aqueous form, before dryness, using a UV-vis-NIR spectrophotometer (UV-1601, Shimadzu, Japan), using wavelength range from 200 to 800 nm. Autoclaved distilled water was used as a reference in measurement.


## 2.4.2. Size Distribution and Zeta Potential

The average size and surface charge of all synthesized silver nanoparticles were evaluated by using a particle size analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd., UK) at 25°C with a detection angle 90°. All powder samples were freshly resuspended in 0.9% saline solution and sonicated for 30 mins at high speed before assessment to prevent nanoparticle aggregations.


## 2.4.3. Surface Morphology

Silver nanoparticles AgNPs_PPE, AgNPs_CE, and AgNPs_Chem (dried powder) were separately coated with gold using a Cressington Sputter Coater (108 Auto, thickness controller MTM-10, UK) for 10 mins prior to scanning. Scanning electron microscope (SEM, JEOL JSM-639OLA, Analytical Scanning Electron Microscope, Tokyo, Japan, that is present in the Electron Microscope Unit of Taif University) at 20 kV was used with different magnifications of ×500, ×2000, and ×3000 with scale bars of 50μm, 10 μm, and 5 μm, respectively, to determine surface morphology. The X-ray diffractometer (XRD, Pan Analytical, X-pert pro, Netherland) was used to confirm the surface crystalline shape of the synthesized nanoparticles. XRD was used at 30 kV and 100 mA, and the spectrum was recorded by CuKα radiation with a wavelength of 1.5406 Å in the 2θ (from the range of 20°-80°). Patterns of XRD were plotted by the software OriginLab and compared with JCPDS card no. 040783.


## 2.4.4. Fourier Transforms Infrared Spectroscopy (FTIR)

FTIR spectroscopy (Agilent Technologies, Santa Clara, CA, USA) was used to detect the possible functional groups found in PPE, CE, and sodium citrate (at wavelength range between 450 and 4000 cm-1) that are responsible to silver nanoparticle synthesis and stabilization.


## 2.5. Agar Well Diffusion Assay

The antibacterial potential of silver nanoparticles (AgNPs_PPE, AgNPs_CE, and AgNPs_Chem) was evaluated by well diffusion method. Inhibition zones were recorded in millimeters (mm). Briefly, plates of the Mueller-Hinton agar were inoculated with five different pathogenic bacterial strains, separately:Enterobacter aerogenes, Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA). Wells with a diameter of 7 mm were cut using sterile blue tips. For each plate, five wells were cut for plant extract, antibiotic, and three different concentrations of AgNPs. 100 μl of plant extract (PPE/CE) and 100 μl of the antibiotic ciprofloxacin 750 (1.6 mg/ml) were added as reference and positive control, respectively. Different concentrations (2 mg/ml, 4 mg/ml, and 8 mg/ml) of each silver nanoparticle were suspended in distilled water and were properly sonicated. 100 μl for each treatment and concentration were added per well. Plates were incubated at 37°C overnight. After incubation, diameters of the inhibition zone were measured by using a standard metric ruler.


### 2.5.1. Statistical Analysis

Data were expressed as themean±standarderror (M±SE). Statistical analysis was conducted using one-way ANOVA to differentiate between the inhibition zone diameter of different bacterial strains groups within the same AgNP treatment using GraphPad software (GraphPad®, 2017), in which, ∗∗∗ indicates P≤0.001, ∗∗ indicates P≤0.01, ∗ indicates P≤0.05, and ns (nonsignificant) means P>0.05.


## 2.5.1. Statistical Analysis

Data were expressed as themean±standarderror (M±SE). Statistical analysis was conducted using one-way ANOVA to differentiate between the inhibition zone diameter of different bacterial strains groups within the same AgNP treatment using GraphPad software (GraphPad®, 2017), in which, ∗∗∗ indicates P≤0.001, ∗∗ indicates P≤0.01, ∗ indicates P≤0.05, and ns (nonsignificant) means P>0.05.


## 3. Results and Discussion

### 3.1. Characterization of Silver Nanoparticles

#### 3.1.1. UV-Vis Spectroscopy

The change of PPE, CE, and trisodium citrate colors from pale yellow, brown, and colorless, respectively, to dark brown color after AgNO3 addition indicates successful formation of silver nanoparticles (Figures 2 and 3). The present results are in consistent with Jabir et al. [15]; they have reported color change of Annona muricata peel extract from deep yellow to dark brown due to the green synthesis of silver nanoparticles. AgNP_PPE, AgNP_CE, and AgNP_Chem syntheses were confirmed by UV absorbance because it is the simplest way to confirm colloidal metal nanoparticle formation due to their high surface plasmon resonances (SPR) [30, 31]. Figure 4 shows UV-vis sharp absorption spectra of AgNPs_PPE (350 nm), AgNPs_CE (2 sharp peaks at 320 nm and 440 nm), and AgNPs_Chem (430 nm) that typically detected previously for colloidal silver nanoparticles within the range from 350 to 600 nm [16, 32, 33]. The formed AgNPs have different UV spectra, due to different physical characteristics of formed silver nanoparticles, including size and shape, that affect their SPR [34].Figure 4
UV-visible spectral analysis of green synthesized AgNPs_PPE and AgNPS_CE and chemically synthesized AgNPs_Chem.The present study was consistent with previous reports, which recorded AgNP synthesis due to reduction of Ag+ to Ag0 in the presence of UV-vis spectroscopy using PPE, spent coffee ground extracts, and trisodium citrate at SPR 421 nm, 405–430 nm, and 420 nm, respectively [35–37]. It was known that SPR peak shifting towards a higher wavelength is accompanied by a decrease in the prepared AgNP size that is reported in the present study [38].


#### 3.1.2. Size Distribution and Zeta Potential

Zeta potential was used to estimate the stability of the synthesized AgNPs, in which an increase in positivity/negativity of zeta potential means a more stable nanoparticle. It was recorded that chemically synthesized AgNPs (−46.7 mV) were more stable than AgNPs_CE (−12.6 mV) followed by AgNPs_PPE (−7.98 mV) which had the least stability. Previously, it was reported that a high negative potential value increases nanoparticles’ repulsion force that in turn helps in their dispersal, stability, and good colloidal nature [39]. AgNP stability affects their size, in which less stability initiates particle aggregation and in turn increased their size as reported in the present study with AgNPs_PPE=591.9nm, AgNPs_CE=273.7nm, and AgNPs_Chem=62.75nm (Figure 5). It was clear that chemically synthesized silver nanoparticles were more stable than biologically synthesized, and AgNPs synthesized from CE were more stable than those formed from PPE extracts. Therefore, AgNPs_Chem revealed the smallest size followed by AgNPs_CE and AgNPs_PPE.Figure 5
Average size of green synthesized (a) AgNPs_PPE (591 nm), (b) AgNPs_CE (273.7 nm), and chemically synthesized (C) AgNPs_Chem (62.68 nm).
(a)(b)(c)The present results were incompatible with previous studies, in which AgNPs synthesized from PPE (31.6 nm; 118.6–231.7 nm), CE (21–255 nm), and trisodium citrate (5–10 nm) showed smaller size [35, 37, 40, 41]. It was known that change of AgNO3 concentration and/or pH affects the size of synthesized silver nanoparticles. pH is inversely proportional to AgNP size, where at alkaline pH, the reaction rate increases with successive crystallization into smaller particles. Increasing pH reduces the aggregation tendency of nanoparticles due to complete surface charging and in turn increases their repulsion. In addition, increasing silver nitrate concentrations (1 mM to 5 mM) decreased the size of formed AgNPs as previously reported [38].


#### 3.1.3. Scanning Electron Microscope (SEM) and XRD Analysis

Figure6 shows the crystalline shape of biologically synthesized AgNPs_PPE and AgNPs_CE and chemically synthesized AgNPs_Chem by using SEM. This was confirmed by XRD evaluation, in which four main standard peaks were reported according to Bragg’s reflection of silver nanoparticles that indicates the face-centered cubic (FCC) crystalline shape. Those four main peaks were present at 2θ=38.3°, 44.49°, 64.6°, and 77.5° that corresponds to Ag planes 111, 200, 220, and 311, respectively (Figure 7). FCC crystalline shape was previously reported in silver nanoparticle synthesis using various plant extracts as well as trisodium citrate as the chemical approach [19, 42–44]. The presence of other unpredicted diffraction peaks of AgNPs_PPE at 2θ of 27.90°, 32.38°, 46.43°, 55.00°, 57.00°, and 67.00° may be related to phytochemicals present in PPE. However, unassigned crystalline peaks at 2θ of 32.38° and 46.43° were also reported in previous studies [45, 46].Figure 6
Scanning electron microscope photos showing the crystalline shape of AgNPs_PPE (a), AgNPS_CE (b), and AgNPs_Chem (c) with different magnifications.
(a)(b)(c)Figure 7
XRD patterns of AgNPs_PPE, AgNPs_CE, and AgNPs_Chem that confirm their crystalline shapes.


#### 3.1.4. FTIR Evaluation of AgNPs

FTIR analysis was conducted to identify the possible biomolecules present in PPE, and the CE extract that are responsible for capping of silver nanoparticles, reduction, and stabilization. Figure8 shows differences between each synthesized AgNP with its reference. Any change in sharpness or position of any peak is referred to group contribution in silver nanoparticles’ synthesis. As reported earlier, AgNPs_PPE different peaks showed higher sharpness compared to PPE only. The recorded sharp peak at 2927 cm−1 was referred to C–H stretching; 1641 cm−1 was referred to C–O stretching vibration in quinine; 1351 cm−1 to C=C aromatic ring; 1228 cm−1 to C–O stretching in ether, ester, or phenol; and 1055 cm−1 to C–N stretching of the aliphatic primary amine [47]. Regions at 1315–1037 and 1456–1600 cm−1 were referred to phenolic groups participating in ion replacement response of flavanones’ plant extract [48]. Therefore, flavonoids present in PPE are responsible for Ag+ to Ag0 reduction, capping, and stability of AgNPs [49]. Shankar et al. [50] had suggested that flavanones of plant extract might be adsorbed on the metal nanoparticle surface and determined by strong bands at 1074 cm−1. PPE has different constituents including flavonols, gallotannins, quercetin, ellagic acid derivatives, catechins, procyanidins, and kaempferol that aid in its efficacy [51].Figure 8
FTIR spectra of synthesized silver nanoparticles in comparison with their standard, in which (a) represents difference between AgNPs_PPE and PPE extract and (b) represents difference between AgNPs_CE and CE extract, while (c) shows difference between chemically synthesized AgNPs_Chem and sodium citrate.
(a)(b)(c)FTIR analysis of AgNPs_CE shows different sharp peaks in comparison to its extract CE. The AgNP_CE FTIR pattern shows a broad band at 3600–3000 cm−1, two sharp peaks at 2922 cm−1 and 2852 cm−1, and band at 1741 cm−1 due to –N-H and –O-H stretching vibrations, C–H bond asymmetric stretching of the methyl (–CH3) group, and C=O vibration, respectively. Peaks at the range from 1700 cm−1 to 1500 cm−1 may be assigned to C–C and C–N stretching vibration. Vibrations of different bonds such as -C-H, -C-O, and -C-N were determined in wavelength from 1400 cm−1 to 900 cm−1. All assigned bands or peaks were previously reported in literature data that confirmed the presence of CO, OH, and NH2 groups in CE [52, 53]. Those different groups of CE that are found in its constituents (hemicellulose, melanoidins, proteins, and polyphenols) might share in AgNP synthesis, capping, and stabilization [26].In AgNP_Chem synthesis, trisodium citrate acts as a reducing agent. As previously reported, different peaks were assigned that might be referred to C–H stretching deformation (1282 cm−1), nitro compound (NO) as NO2 (1402 cm−1), and C=O stretching asymmetric in COO– (1582 cm−1), while an amine stretching appeared at higher wavelength at 3312 cm−1 [54, 55].


### 3.2. Antibacterial Activity Assay

Plant extracts (PPE and CE) and green and chemical synthesized AgNPs (AgNPs_PPE, AgNPs_CE, and AgNPs_Chem, respectively) were examined for their antimicrobial activity by using a well diffusion method. After 24 h incubation at 37°C, PPE and CE extracts did not show any inhibition on different bacterial strains. However, different concentrations of AgNPs (2 mg/ml, 4 mg/ml, and 8 mg/ml) suppress bacterial growth that appears as an inhibitory zone (Figure9). Growth of the inhibitory zone increased by increasing AgNP concentration; 8 mg/ml shows the highest zone. In addition, Gram-negative bacteria K. pneumoniae show a higher zone inhibition in comparison to other Gram-negative bacteria (Enterobacter aerogenes and Pseudomonas aeruginosa) and the Gram-positive bacteria MRSA (Figure 10). There is no significant difference between the antibacterial potentials of the three different AgNP treatments on the selected bacterial strains.Figure 9
Representative photographs of silver nanoparticles (AgNPs) (2, 4, and 8 mg/ml) antimicrobial potential on different bacterial cells that show inhibition zones;Enterobacter aerogenes (a), Pseudomonas aeruginosa (b), and MRSA (c).
(a)(b)(c)Figure 10
Antibacterial effect of biologically synthesized AgNPs_PPE, AgNPs_CE, and chemically synthesized AgNPs_Chem on different pathogenic bacterial strainsEnterobacter aerogenes, K. pneumoniae, Pseudomonas aeruginosa, and MRSA. All groups within the same AgNP treatment were statistically nonsignificant (ns) in comparison with each other with means P>0.05.Our results are consistent with Rautela et al. [56], in which they have reported antimicrobial effect of green synthesized AgNPs using Tectona grandis seed extracts on Staphylococcus aureus, Bacillus cereus, and E. coli. Also, they have recorded a higher inhibition zone on Gram-negative bacteria (E. coli) in comparison to Gram-positive bacteria (B. cereus and S. aureus) at the same concentration of AgNPs. Sensitivity of Gram-negative bacteria towards AgNPs compared to Gram-positive bacteria could be explained by variation in their cell wall composition. The cell wall of Gram-positive bacteria consists of a thicker peptidoglycan layer than that of Gram-negative bacteria. This leads to a more rigid structure that in turn increases difficulties towards AgNP penetration [57]. It is still unclear about the action of the mechanism of AgNPs on the bacteria. Rautela et al. [56] suggested that AgNPs could enhance the permeability of the membrane, by increasing sugar and protein leakage from the cytoplasm.


## 3.1. Characterization of Silver Nanoparticles

### 3.1.1. UV-Vis Spectroscopy

The change of PPE, CE, and trisodium citrate colors from pale yellow, brown, and colorless, respectively, to dark brown color after AgNO3 addition indicates successful formation of silver nanoparticles (Figures 2 and 3). The present results are in consistent with Jabir et al. [15]; they have reported color change of Annona muricata peel extract from deep yellow to dark brown due to the green synthesis of silver nanoparticles. AgNP_PPE, AgNP_CE, and AgNP_Chem syntheses were confirmed by UV absorbance because it is the simplest way to confirm colloidal metal nanoparticle formation due to their high surface plasmon resonances (SPR) [30, 31]. Figure 4 shows UV-vis sharp absorption spectra of AgNPs_PPE (350 nm), AgNPs_CE (2 sharp peaks at 320 nm and 440 nm), and AgNPs_Chem (430 nm) that typically detected previously for colloidal silver nanoparticles within the range from 350 to 600 nm [16, 32, 33]. The formed AgNPs have different UV spectra, due to different physical characteristics of formed silver nanoparticles, including size and shape, that affect their SPR [34].Figure 4
UV-visible spectral analysis of green synthesized AgNPs_PPE and AgNPS_CE and chemically synthesized AgNPs_Chem.The present study was consistent with previous reports, which recorded AgNP synthesis due to reduction of Ag+ to Ag0 in the presence of UV-vis spectroscopy using PPE, spent coffee ground extracts, and trisodium citrate at SPR 421 nm, 405–430 nm, and 420 nm, respectively [35–37]. It was known that SPR peak shifting towards a higher wavelength is accompanied by a decrease in the prepared AgNP size that is reported in the present study [38].


### 3.1.2. Size Distribution and Zeta Potential

Zeta potential was used to estimate the stability of the synthesized AgNPs, in which an increase in positivity/negativity of zeta potential means a more stable nanoparticle. It was recorded that chemically synthesized AgNPs (−46.7 mV) were more stable than AgNPs_CE (−12.6 mV) followed by AgNPs_PPE (−7.98 mV) which had the least stability. Previously, it was reported that a high negative potential value increases nanoparticles’ repulsion force that in turn helps in their dispersal, stability, and good colloidal nature [39]. AgNP stability affects their size, in which less stability initiates particle aggregation and in turn increased their size as reported in the present study with AgNPs_PPE=591.9nm, AgNPs_CE=273.7nm, and AgNPs_Chem=62.75nm (Figure 5). It was clear that chemically synthesized silver nanoparticles were more stable than biologically synthesized, and AgNPs synthesized from CE were more stable than those formed from PPE extracts. Therefore, AgNPs_Chem revealed the smallest size followed by AgNPs_CE and AgNPs_PPE.Figure 5
Average size of green synthesized (a) AgNPs_PPE (591 nm), (b) AgNPs_CE (273.7 nm), and chemically synthesized (C) AgNPs_Chem (62.68 nm).
(a)(b)(c)The present results were incompatible with previous studies, in which AgNPs synthesized from PPE (31.6 nm; 118.6–231.7 nm), CE (21–255 nm), and trisodium citrate (5–10 nm) showed smaller size [35, 37, 40, 41]. It was known that change of AgNO3 concentration and/or pH affects the size of synthesized silver nanoparticles. pH is inversely proportional to AgNP size, where at alkaline pH, the reaction rate increases with successive crystallization into smaller particles. Increasing pH reduces the aggregation tendency of nanoparticles due to complete surface charging and in turn increases their repulsion. In addition, increasing silver nitrate concentrations (1 mM to 5 mM) decreased the size of formed AgNPs as previously reported [38].


### 3.1.3. Scanning Electron Microscope (SEM) and XRD Analysis

Figure6 shows the crystalline shape of biologically synthesized AgNPs_PPE and AgNPs_CE and chemically synthesized AgNPs_Chem by using SEM. This was confirmed by XRD evaluation, in which four main standard peaks were reported according to Bragg’s reflection of silver nanoparticles that indicates the face-centered cubic (FCC) crystalline shape. Those four main peaks were present at 2θ=38.3°, 44.49°, 64.6°, and 77.5° that corresponds to Ag planes 111, 200, 220, and 311, respectively (Figure 7). FCC crystalline shape was previously reported in silver nanoparticle synthesis using various plant extracts as well as trisodium citrate as the chemical approach [19, 42–44]. The presence of other unpredicted diffraction peaks of AgNPs_PPE at 2θ of 27.90°, 32.38°, 46.43°, 55.00°, 57.00°, and 67.00° may be related to phytochemicals present in PPE. However, unassigned crystalline peaks at 2θ of 32.38° and 46.43° were also reported in previous studies [45, 46].Figure 6
Scanning electron microscope photos showing the crystalline shape of AgNPs_PPE (a), AgNPS_CE (b), and AgNPs_Chem (c) with different magnifications.
(a)(b)(c)Figure 7
XRD patterns of AgNPs_PPE, AgNPs_CE, and AgNPs_Chem that confirm their crystalline shapes.


### 3.1.4. FTIR Evaluation of AgNPs

FTIR analysis was conducted to identify the possible biomolecules present in PPE, and the CE extract that are responsible for capping of silver nanoparticles, reduction, and stabilization. Figure8 shows differences between each synthesized AgNP with its reference. Any change in sharpness or position of any peak is referred to group contribution in silver nanoparticles’ synthesis. As reported earlier, AgNPs_PPE different peaks showed higher sharpness compared to PPE only. The recorded sharp peak at 2927 cm−1 was referred to C–H stretching; 1641 cm−1 was referred to C–O stretching vibration in quinine; 1351 cm−1 to C=C aromatic ring; 1228 cm−1 to C–O stretching in ether, ester, or phenol; and 1055 cm−1 to C–N stretching of the aliphatic primary amine [47]. Regions at 1315–1037 and 1456–1600 cm−1 were referred to phenolic groups participating in ion replacement response of flavanones’ plant extract [48]. Therefore, flavonoids present in PPE are responsible for Ag+ to Ag0 reduction, capping, and stability of AgNPs [49]. Shankar et al. [50] had suggested that flavanones of plant extract might be adsorbed on the metal nanoparticle surface and determined by strong bands at 1074 cm−1. PPE has different constituents including flavonols, gallotannins, quercetin, ellagic acid derivatives, catechins, procyanidins, and kaempferol that aid in its efficacy [51].Figure 8
FTIR spectra of synthesized silver nanoparticles in comparison with their standard, in which (a) represents difference between AgNPs_PPE and PPE extract and (b) represents difference between AgNPs_CE and CE extract, while (c) shows difference between chemically synthesized AgNPs_Chem and sodium citrate.
(a)(b)(c)FTIR analysis of AgNPs_CE shows different sharp peaks in comparison to its extract CE. The AgNP_CE FTIR pattern shows a broad band at 3600–3000 cm−1, two sharp peaks at 2922 cm−1 and 2852 cm−1, and band at 1741 cm−1 due to –N-H and –O-H stretching vibrations, C–H bond asymmetric stretching of the methyl (–CH3) group, and C=O vibration, respectively. Peaks at the range from 1700 cm−1 to 1500 cm−1 may be assigned to C–C and C–N stretching vibration. Vibrations of different bonds such as -C-H, -C-O, and -C-N were determined in wavelength from 1400 cm−1 to 900 cm−1. All assigned bands or peaks were previously reported in literature data that confirmed the presence of CO, OH, and NH2 groups in CE [52, 53]. Those different groups of CE that are found in its constituents (hemicellulose, melanoidins, proteins, and polyphenols) might share in AgNP synthesis, capping, and stabilization [26].In AgNP_Chem synthesis, trisodium citrate acts as a reducing agent. As previously reported, different peaks were assigned that might be referred to C–H stretching deformation (1282 cm−1), nitro compound (NO) as NO2 (1402 cm−1), and C=O stretching asymmetric in COO– (1582 cm−1), while an amine stretching appeared at higher wavelength at 3312 cm−1 [54, 55].


## 3.1.1. UV-Vis Spectroscopy

The change of PPE, CE, and trisodium citrate colors from pale yellow, brown, and colorless, respectively, to dark brown color after AgNO3 addition indicates successful formation of silver nanoparticles (Figures 2 and 3). The present results are in consistent with Jabir et al. [15]; they have reported color change of Annona muricata peel extract from deep yellow to dark brown due to the green synthesis of silver nanoparticles. AgNP_PPE, AgNP_CE, and AgNP_Chem syntheses were confirmed by UV absorbance because it is the simplest way to confirm colloidal metal nanoparticle formation due to their high surface plasmon resonances (SPR) [30, 31]. Figure 4 shows UV-vis sharp absorption spectra of AgNPs_PPE (350 nm), AgNPs_CE (2 sharp peaks at 320 nm and 440 nm), and AgNPs_Chem (430 nm) that typically detected previously for colloidal silver nanoparticles within the range from 350 to 600 nm [16, 32, 33]. The formed AgNPs have different UV spectra, due to different physical characteristics of formed silver nanoparticles, including size and shape, that affect their SPR [34].Figure 4
UV-visible spectral analysis of green synthesized AgNPs_PPE and AgNPS_CE and chemically synthesized AgNPs_Chem.The present study was consistent with previous reports, which recorded AgNP synthesis due to reduction of Ag+ to Ag0 in the presence of UV-vis spectroscopy using PPE, spent coffee ground extracts, and trisodium citrate at SPR 421 nm, 405–430 nm, and 420 nm, respectively [35–37]. It was known that SPR peak shifting towards a higher wavelength is accompanied by a decrease in the prepared AgNP size that is reported in the present study [38].


## 3.1.2. Size Distribution and Zeta Potential

Zeta potential was used to estimate the stability of the synthesized AgNPs, in which an increase in positivity/negativity of zeta potential means a more stable nanoparticle. It was recorded that chemically synthesized AgNPs (−46.7 mV) were more stable than AgNPs_CE (−12.6 mV) followed by AgNPs_PPE (−7.98 mV) which had the least stability. Previously, it was reported that a high negative potential value increases nanoparticles’ repulsion force that in turn helps in their dispersal, stability, and good colloidal nature [39]. AgNP stability affects their size, in which less stability initiates particle aggregation and in turn increased their size as reported in the present study with AgNPs_PPE=591.9nm, AgNPs_CE=273.7nm, and AgNPs_Chem=62.75nm (Figure 5). It was clear that chemically synthesized silver nanoparticles were more stable than biologically synthesized, and AgNPs synthesized from CE were more stable than those formed from PPE extracts. Therefore, AgNPs_Chem revealed the smallest size followed by AgNPs_CE and AgNPs_PPE.Figure 5
Average size of green synthesized (a) AgNPs_PPE (591 nm), (b) AgNPs_CE (273.7 nm), and chemically synthesized (C) AgNPs_Chem (62.68 nm).
(a)(b)(c)The present results were incompatible with previous studies, in which AgNPs synthesized from PPE (31.6 nm; 118.6–231.7 nm), CE (21–255 nm), and trisodium citrate (5–10 nm) showed smaller size [35, 37, 40, 41]. It was known that change of AgNO3 concentration and/or pH affects the size of synthesized silver nanoparticles. pH is inversely proportional to AgNP size, where at alkaline pH, the reaction rate increases with successive crystallization into smaller particles. Increasing pH reduces the aggregation tendency of nanoparticles due to complete surface charging and in turn increases their repulsion. In addition, increasing silver nitrate concentrations (1 mM to 5 mM) decreased the size of formed AgNPs as previously reported [38].


## 3.1.3. Scanning Electron Microscope (SEM) and XRD Analysis

Figure6 shows the crystalline shape of biologically synthesized AgNPs_PPE and AgNPs_CE and chemically synthesized AgNPs_Chem by using SEM. This was confirmed by XRD evaluation, in which four main standard peaks were reported according to Bragg’s reflection of silver nanoparticles that indicates the face-centered cubic (FCC) crystalline shape. Those four main peaks were present at 2θ=38.3°, 44.49°, 64.6°, and 77.5° that corresponds to Ag planes 111, 200, 220, and 311, respectively (Figure 7). FCC crystalline shape was previously reported in silver nanoparticle synthesis using various plant extracts as well as trisodium citrate as the chemical approach [19, 42–44]. The presence of other unpredicted diffraction peaks of AgNPs_PPE at 2θ of 27.90°, 32.38°, 46.43°, 55.00°, 57.00°, and 67.00° may be related to phytochemicals present in PPE. However, unassigned crystalline peaks at 2θ of 32.38° and 46.43° were also reported in previous studies [45, 46].Figure 6
Scanning electron microscope photos showing the crystalline shape of AgNPs_PPE (a), AgNPS_CE (b), and AgNPs_Chem (c) with different magnifications.
(a)(b)(c)Figure 7
XRD patterns of AgNPs_PPE, AgNPs_CE, and AgNPs_Chem that confirm their crystalline shapes.


## 3.1.4. FTIR Evaluation of AgNPs

FTIR analysis was conducted to identify the possible biomolecules present in PPE, and the CE extract that are responsible for capping of silver nanoparticles, reduction, and stabilization. Figure8 shows differences between each synthesized AgNP with its reference. Any change in sharpness or position of any peak is referred to group contribution in silver nanoparticles’ synthesis. As reported earlier, AgNPs_PPE different peaks showed higher sharpness compared to PPE only. The recorded sharp peak at 2927 cm−1 was referred to C–H stretching; 1641 cm−1 was referred to C–O stretching vibration in quinine; 1351 cm−1 to C=C aromatic ring; 1228 cm−1 to C–O stretching in ether, ester, or phenol; and 1055 cm−1 to C–N stretching of the aliphatic primary amine [47]. Regions at 1315–1037 and 1456–1600 cm−1 were referred to phenolic groups participating in ion replacement response of flavanones’ plant extract [48]. Therefore, flavonoids present in PPE are responsible for Ag+ to Ag0 reduction, capping, and stability of AgNPs [49]. Shankar et al. [50] had suggested that flavanones of plant extract might be adsorbed on the metal nanoparticle surface and determined by strong bands at 1074 cm−1. PPE has different constituents including flavonols, gallotannins, quercetin, ellagic acid derivatives, catechins, procyanidins, and kaempferol that aid in its efficacy [51].Figure 8
FTIR spectra of synthesized silver nanoparticles in comparison with their standard, in which (a) represents difference between AgNPs_PPE and PPE extract and (b) represents difference between AgNPs_CE and CE extract, while (c) shows difference between chemically synthesized AgNPs_Chem and sodium citrate.
(a)(b)(c)FTIR analysis of AgNPs_CE shows different sharp peaks in comparison to its extract CE. The AgNP_CE FTIR pattern shows a broad band at 3600–3000 cm−1, two sharp peaks at 2922 cm−1 and 2852 cm−1, and band at 1741 cm−1 due to –N-H and –O-H stretching vibrations, C–H bond asymmetric stretching of the methyl (–CH3) group, and C=O vibration, respectively. Peaks at the range from 1700 cm−1 to 1500 cm−1 may be assigned to C–C and C–N stretching vibration. Vibrations of different bonds such as -C-H, -C-O, and -C-N were determined in wavelength from 1400 cm−1 to 900 cm−1. All assigned bands or peaks were previously reported in literature data that confirmed the presence of CO, OH, and NH2 groups in CE [52, 53]. Those different groups of CE that are found in its constituents (hemicellulose, melanoidins, proteins, and polyphenols) might share in AgNP synthesis, capping, and stabilization [26].In AgNP_Chem synthesis, trisodium citrate acts as a reducing agent. As previously reported, different peaks were assigned that might be referred to C–H stretching deformation (1282 cm−1), nitro compound (NO) as NO2 (1402 cm−1), and C=O stretching asymmetric in COO– (1582 cm−1), while an amine stretching appeared at higher wavelength at 3312 cm−1 [54, 55].


## 3.2. Antibacterial Activity Assay

Plant extracts (PPE and CE) and green and chemical synthesized AgNPs (AgNPs_PPE, AgNPs_CE, and AgNPs_Chem, respectively) were examined for their antimicrobial activity by using a well diffusion method. After 24 h incubation at 37°C, PPE and CE extracts did not show any inhibition on different bacterial strains. However, different concentrations of AgNPs (2 mg/ml, 4 mg/ml, and 8 mg/ml) suppress bacterial growth that appears as an inhibitory zone (Figure9). Growth of the inhibitory zone increased by increasing AgNP concentration; 8 mg/ml shows the highest zone. In addition, Gram-negative bacteria K. pneumoniae show a higher zone inhibition in comparison to other Gram-negative bacteria (Enterobacter aerogenes and Pseudomonas aeruginosa) and the Gram-positive bacteria MRSA (Figure 10). There is no significant difference between the antibacterial potentials of the three different AgNP treatments on the selected bacterial strains.Figure 9
Representative photographs of silver nanoparticles (AgNPs) (2, 4, and 8 mg/ml) antimicrobial potential on different bacterial cells that show inhibition zones;Enterobacter aerogenes (a), Pseudomonas aeruginosa (b), and MRSA (c).
(a)(b)(c)Figure 10
Antibacterial effect of biologically synthesized AgNPs_PPE, AgNPs_CE, and chemically synthesized AgNPs_Chem on different pathogenic bacterial strainsEnterobacter aerogenes, K. pneumoniae, Pseudomonas aeruginosa, and MRSA. All groups within the same AgNP treatment were statistically nonsignificant (ns) in comparison with each other with means P>0.05.Our results are consistent with Rautela et al. [56], in which they have reported antimicrobial effect of green synthesized AgNPs using Tectona grandis seed extracts on Staphylococcus aureus, Bacillus cereus, and E. coli. Also, they have recorded a higher inhibition zone on Gram-negative bacteria (E. coli) in comparison to Gram-positive bacteria (B. cereus and S. aureus) at the same concentration of AgNPs. Sensitivity of Gram-negative bacteria towards AgNPs compared to Gram-positive bacteria could be explained by variation in their cell wall composition. The cell wall of Gram-positive bacteria consists of a thicker peptidoglycan layer than that of Gram-negative bacteria. This leads to a more rigid structure that in turn increases difficulties towards AgNP penetration [57]. It is still unclear about the action of the mechanism of AgNPs on the bacteria. Rautela et al. [56] suggested that AgNPs could enhance the permeability of the membrane, by increasing sugar and protein leakage from the cytoplasm.


## 4. Conclusion

In the present study, we have provided a green, simple, and economic method for silver nanoparticle synthesis with the help of pomegranate peel and coffee ground extracts. In addition, a chemical reduction method was used for AgNP synthesis by the help of trisodium citrate. AgNP_PPE, AgNP_CE, and AgNP_Chem syntheses were confirmed by UV-vis spectroscopy at different absorbance. Zeta potential reports that chemically synthesized AgNPs were more stable than biologically synthesized AgNPs_CE and AgNPs_PPE that in turn affect their nanosize. XRD and SEM elucidated their crystalline shape, while FTIR determined different groups that might participate in capping and stabilization of silver nanoparticles. Green and chemical synthesized silver nanoparticles showed antibacterial activity which was investigated by the agar well diffusion method against different pathogenic strains. Further experiments are required for cytotoxic and genotoxic evaluation of the synthesized AgNPs and if there are any potential human risks for their application. In addition, large scales might be applied especially for the biological methods using different biological waste products.



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*Source: 2204776-2021-10-15.xml*