Photoassisted Synthesis of Silver Nanoparticles Using Riceberry Rice Extract and Their Antibacterial Application

The green synthesis of silver nanoparticles (AgNPs) has been attractive in biomedical applications due to its nontoxic and eco-friendly approach. This study presents the facile, rapid, and cost-effective synthesis of AgNPs by photoassisted chemical reduction using Riceberry (RB) rice extract as a reducing agent. The effects of reaction parameters including photoirradiation, irradiation time, the volume ratio of silver nitrate (AgNO3) to RB extract, and pH condition on the AgNP formation were also investigated. The characterization of AgNPs was determined by UV–visible spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier-transform infrared (FT-IR) spectroscopy. For antibacterial application, the synthesized AgNPs were studied by disc diffusion method against Escherichia coli and Staphylococcus aureus. The results indicated that light irradiation was an important factor to accelerate the formation of AgNPs. The synthesis parameters including volume of RB extract and pH condition significantly affected the particle size and crystallinity of AgNPs. The volume ratio of AgNO3 to RB extract 1 : 12.5 at pH 2.5 under photoirradiation was the successful condition to form nanometer-sized crystalline particles (average particle size of 
 
 59.48
 ±
 0.37
  
 nm
 
 ) within 30 min with a rate constant of 0.210 min–1. The FT-IR measurement also suggested that the phytochemical constituents in RB extract were served as reducing and stabilizing agents for the synthesis of AgNPs. Additionally, the obtained AgNPs from various conditions demonstrated the antibacterial activity against both strains. Therefore, this study proposes an effective integration technique to synthesize AgNPs within a short time for antibacterial application.


Introduction
In the past decade, nanoparticles (NPs), which are defined as particles having one or more dimensions between 1 and 100 nanometers (nm), have become popular in a wide range of application areas such as engineering [1], food [2], cosmetic [3], and textile [4,5], as well as pharmaceutical field [6]. Among the metal NPs, silver NPs (AgNPs) is commonly used as an active antibacterial agent in medical applications [7], because the nanosize of Ag exhibits unique physicochemical characteristics related to the improvement of antibacterial activity [8]. In addition, AgNPs can be used as a coating material on hospital textile, catheter, and implantable materials to prevent microbial infection [9]. However, these applications depend on the size and shape of AgNPs, and minimizing toxic residue from synthesis methods needs to be concerned [10,11].
To date, synthesis of AgNPs can be achieved by physical [12], chemical [13], biological [14,15], and hybrid methods [16]. The chemical-based synthesis by organic and inorganic reducing agents is the most common approach because it offers the reliability of large-scale production and short time synthesis [9,14,17]. However, a major drawback of chemical reduction is the use of toxic-reducing agents, such as sodium borohydride, trisodium citrate, and ascorbate, which produce hazardous wastes and greatly limit their biomedical applications [18]. Therefore, the biogenic synthesis of AgNPs using bacteria, algae, fungi, and plants has been attractive due to its nontoxic and eco-friendly method. The utilization of plantbased synthesis provides advantages over a microorganismbased approach because it is a simple, easy scale-up, costeffective, and nonaseptic process [19]. In comparison to microbial synthesis, a phytochemical synthesis can also produce AgNPs faster [20], and the formation of the metallic NPs is more stable without organic impurity produced by microorganisms [20,21]. The extracts of edible and nonedible plants from various parts including roots of Zingiber officinale [22], stem bark of Diospyros montana [23], leaves of Ziziphus mauritiana [24], flowers of Cassia angustifolia [25], fruits of Cordia dichotoma [26], and seeds of Psoralea corylifolia [27] have been used for AgNP synthesis. Regarding phytomolecules, the polyol heterocyclic compounds of the plant extract serve as a reducing agent to convert silver ion (Ag + ) to AgNPs, whereas the flavonoid and terpenoid compounds act as a capping agent to stabilize AgNPs [28,29]. Therefore, polyphenol-rich plants are a good alternative source to synthesize AgNPs.
Thai pigmented rice, namely, Riceberry (Oryza sativa L.), has been recently developed from a crossbreed strain of Hom Nin rice and Khoa Dawk Mali 105 for nutritional purposes. The purple rice barn extracts have been reported to have antioxidants, anti-inflammatory, anticancer [30], and antidiabetic activities [31]. The major constituents in a crude extract of RB bran are phenolic compounds [32], which possess hydroxyl (-OH) and carboxyl (-COOH) groups. These functional groups are capable of reducing the Ag + in an aqueous solution to form metallic silver. In addition, RB rice is commonly available in supermarkets all year round, inexpensive, no toxicity, and environment-friendly. Based on these reasons, RB rice extract becomes a potential agent for the production of nanomaterials. However, studies on the synthesis of AgNPs using different parts of nonpigmented and pigmented rice are limited. Utilizing rice bran, rice husk, rice germ, and rice leaf from the nonpigmented cultivar as a reducing agent could produce AgNPs with an approximate size range of 15-346.4 nm [33][34][35]. The differences in size, size distribution, and shape of AgNPs are affected by plant variety, plant parts, and synthesis conditions [36]. In addition, the optimal reaction time for AgNP synthesis using rice extract required at least an hour to complete the reaction at room temperature [35]. Therefore, it is necessary to consider the combination technique for reducing reaction time at room temperature and determine the factors influencing AgNPs synthesis utilizing Thai RB rice.
Recently, photoirradiation from UV light (or sunlight) has been employed to accelerate the formation of nanoparticles [37,38]. However, few studies have reported the synthesis of AgNPs by incorporating light energy and plant-based reducing agent [39,40]. Therefore, the objective of this study was to develop AgNPs by a combination technique using RB rice extract and photoirradiation. The effects of experimental parameters including photoirradiation, irradiation times with the kinetic reaction, volume ratio of AgNO 3 to rice extract, and pH conditions were also determined to identify desirable conditions. The obtained AgNPs have been characterized by using UV-Vis spectroscopy, SEM, XRD, and FT-IR, respectively. For the antibacterial application, the AgNPs were investigated by a disc diffusion method against both Gram-positive and Gram-negative bacteria.

Materials and Methods
2.1. Materials. The analytical grade of AgNO 3 (99%, purity) was purchased from Merck, Germany. Commercial RB rice (Hong Thong Life Riceberry Rice®) was purchased from a supermarket, Thailand. Hydrochloric acid (HCl) was purchased from J.T. Beker, USA. Sodium hydroxide (NaOH) was purchased from Merck, Germany. Analytical grade solvents including hexane, ethanol, and isopropanol were purchased from Fisher Scientific. Deionized (DI) water was used throughout the experiments.

Preparation and
Standardization of RB Extracts. The rice grains (100 g) were extracted using 1% v/v HCl in DI water and ethanol at the ratio of 1 : 3 w/v for 12 h at 30°C. Then, the grains were extracted thrice with the same procedure. The obtained extract was defatted twice with hexane (1 : 1 v /v). Then, the aqueous extract was separated from the nonpolar part and filtered through a filter paper (Whatman® No.1, Merck, Germany), and subsequently, rotary evaporated under reduced pressure at 45°C. After the alcoholic solvents were evaporated, the remaining aqueous residue was freezedried to generate an RB extract with a yield of 3.62% w/w (on the basis of dry weight). The total phenolic content of the extract was analyzed using the Folin-Ciocalteu colorimetric method [41]. The RB extract was found to contain 12:94 ± 0:19 mg/g of total phenolic compounds expressed as the gallic acid equivalent (GAE, mg/g of extract).

Synthesis of AgNPs.
AgNPs were prepared by a lightassisted chemical reduction method. First, AgNO 3 solution (50 mM) was mixed with 0.1% w/w of RB extract. After sonication for 5 min in an ice bath, the solution was irradiated by simulated sunlight under 1 sun (1.5 AM, 1,000 W/m 2 , Newport 91150 V solar simulator) for 30 min at room temperature. The dispersed AgNPs were centrifuged and washed with DI water at 6,000 rpm for 30 min. The washing step was repeated 5 times to remove unreacted substances. The AgNPs were finally rinsed with isopropanol before drying in a vacuum oven at 40°C overnight. The effects of photoirradiation, irradiation times, volume ratios of AgNO 3 to extracted RB solution, and pH conditions were systematically investigated.
2.3.1. Effect of Photoirradiation, Irradiation Time, and the Kinetic of Reaction. The effect of irradiation light was observed under light irradiation and dark condition for 30 min by repeating the above procedure using the volume ratio of AgNO 3 to extracted RB at 1 : 12.5 and the pH condition of 2.5. The kinetic profile of photocatalytic reaction was investigated for 180 min. Then, the effect of irradiation time on the size and shape of AgNP was also studied.

Effect of Volume Ratio between AgNO 3 and Extracted RB Solution.
To evaluate the effect of volume ratio, six different proportions of AgNO 3 to RB solution (3 : 1, 1 : 1, 1 : 3, 1 : 5, 1 : 7, and 1 : 10) were determined by repeating the above 2 Journal of Nanomaterials synthesis using a fixing pH condition of 2.5 and irradiation time of 30 min.

Effect of pH.
The effect of pH conditions on the synthesis of AgNPs was investigated at pH values of 3, 5, 7, 9, and 11. The volume ratio of AgNO 3 to RB extract was 1 : 12.5, and the irradiation time was 30 min.

Characterization of Synthesized
AgNPs. The optical property of synthesized AgNPs was determined by a UV-Vis spectrophotometer (Thermo Scientific UV-Genesys 10s). UV spectral analysis was studied in the range from 300 to 800 nm. Morphology of synthesized AgNPs was conducted through SEM (FEI Nova Nanosem 450). The average size of AgNPs was obtained from the particle size distribution carefully measured from the FE-SEM image. The particle size of Ag NPs was determined by the image processing in ImageJ software. By using "Analyze Particles," the automatic measurement of the area of each AgNP was obtained. The average diameter of each particle was calculated by using A = πD 2 /4, where A is the area of AgNPs derived from Ima-geJ software and D is the average diameter of AgNPs. The crystal structure of synthesized AgNPs was investigated using an X-ray diffractometer (Bruker D8 ADVANCE diffractometer) operating at 40 kV and 40 mA with CuK α radiation. XRD pattern of AgNPs recorded in the 2θ range from 20°t o 80°. The average crystallite size of synthesized AgNPs was evaluated by using Scherrer's formula (as shown in Equation (1)).
where D: the crystallite size K: the dimensionless shape factor (0.9) λ: the X-ray wavelength (1.5418 Å for CuK α ) β: the line broadening at half the maximum intensity (FWHM) (radian) θ: the Bragg angle (radian). The functional groups present in RB extract and AgNPs derived by RB extract were analyzed by using FT-IR spectrophotometer (Thermo Scientific, Nicolet 6700 spectrophotometer). The spectra were recorded over the range of 400-4000 cm -1 .
2.5. Antibacterial Activities of AgNPs. S. aureus ATCC 25923 (Gram-positive bacteria) and E. coli ATCC 25922 (Gramnegative bacteria) were grown separately on Muller Hilton broth (Becton Dickinson & Co., France) at 37°C for 18-24 h. The bacterial-suspension turbidity was adjusted spectrophotometrically at 600 nm to obtain an optical density (OD) of 0.2, which contained approximately 10 7 colonyforming units (CFU)/mL, before use as inoculum in the antibacterial activity test. The antibacterial activities of AgNPs were evaluated against S. aureus and E. coli by the disc diffusion method [42]. Briefly, each prepared inoculum was swabbed on Mueller-Hinton agar (Becton Dickinson & Co., France) and air-dried at room temperature (25°C). The 6 mm sterile discs were loaded with 10 μL of AgNPs (20 mg/mL) placed on the agar plate. The plates were then incubated at 37°C for 24 h under aerobic conditions. The AgNO 3 solution (50 mM) and DI (10 μL/disc) were used as positive and negative controls, respectively. All disc diffusion tests were performed in triplicate, and the antibacterial activity was expressed as the mean of the inhibition diameter ð mmÞ ± standard deviation ðS:D:Þ.
2.6. Statistical Analysis. All statistical analyses were carried out using IBM SPSS (version 26.0 for Mac). Analysis of variance was performed by ANOVA. Significant differences between the means were determined using Tukey's pairwise comparison test at a significance level of p < 0:05.

Synthesis of AgNPs Using RB Extract under
Photoirradiation. AgNPs were successfully synthesized by irradiating the simulated sunlight to the solution of AgNO 3 (50 mM) and RB extract (0.1% w/w in DI water) (as illustrated in Figure 1). After light irradiation, the color of the solution was changed from white turbid to dark red within 5 min suggesting that Ag + was reduced to metallic Ag (Ag 0 ). On the other hand, the AgNPs from preliminary experiment were gradually formed within 60 min at pH 7 without sun irradiation. This suggested that photoenergy can accelerate chemical reduction process. 3 Journal of Nanomaterials photoirradiation on AgNPs production, fixing conditions with and without the presence of RB extract (AgNO 3 : RB extract; 1 : 12.5) at pH 2.5 were observed under light irradiation comparing to the dark condition. The UV-vis technique was utilized to investigate the effect of RB extract on the formation of AgNPs. After 30 min of photoirradiation, the AgNO 3 solution with RB extract clearly showed the formation of AgNPs in dark red color solution with high absorption peak (Figure 2(a), black line), whereas the absorption band of AgNO 3 without RB extract showed significantly lower intensity than that of the AgNO 3 : RB extract solution ( Figure 2(a), red line). On the contrary, no absorbance peak was observed in both AgNO 3 solution with and without RB extract under the dark condition (pink and blue lines, respectively), indicating no AgNP formation. These results suggested that direct irradiation of light onto the silver solution was slightly influenced on AgNP formation or crystal growth process of Ag 0 . In contrast, the incorporating light and RB extract acting as a reducing agent in the solution system can strongly induce the formation of AgNPs.
The kinetic of AgNP synthesis was also investigated by using the AgNO 3 : RB extract ratio of 1 : 12.5 and pH 2.5  Figure 2(b) shows the UV-vis spectra of AgNPs suspended in the synthesized solution with different exposure times. As displayed in Figure 2(c), the kinetic graph demonstrated the sigmoid nature, which was characterized as autocatalysis [43]. The reaction demonstrated that the seed particles were formed within the first 10 min of the reaction (nucleation phase), followed by a rapid growth phase to reach the stabilization phase within 30 min. The reaction rate of RB extract for AgNP synthesis was determined by plotting ln ½a/ð1 − aÞ versus time (where a = ½AðtÞ/Að∞Þ; AðtÞ and Að∞Þ are the absorbance at time t and ∞, respectively) [43,44]. The rate constant (k obs ) of the reaction was 0.210 min -1 (inset in Figure 2(c)). Therefore, the results indicated the optimized irradiation time of 30 min.

Effect of Irradiation Time on the AgNP Synthesis.
Based on the kinetic reaction, the influence of irradiation time on the size and morphology of AgNP was investigated for 30 min as shown in Figure 3. The formation of AgNPs was initially identified by changing the solution color from light brown to dark brown with increasing sunlight irradiation time from 5 to 30 min (Figure 3(b)). As shown in Figure 3(a), the intensity of UV-vis absorption spectra increased over an increasing irradiation time, indicating the formation of AgNPs. The slight change of absorption peak was consistent with the SEM results that the mean particle size demonstrated a small decrease from 62:34 ± 1:00 to 59:48 ± 0:37 nm with an increase irradiation time from 5 to 30 min. As illustrated in Figure 3(d), the symmetrical narrow range of size distribution could be observed with the synthesized AgNPs after irradiation for 30 min. Moreover, approximately 50% of total particle synthesis for 30 min was distributed in the narrow range between 55 and 65 nm. It has been reported that the particle size and shape of AgNPs synthesized by light-assisted technique could be controlled by wavelength(s) of light and irradiation time [45,46]. However, the irradiation time used in this study obtained a similar shape of AgNPs (Figure 3(c)).
3.1.3. Effect of the Volume Ratio of AgNO 3 to RB Solution on the AgNP Synthesis. The effect of the volume ratio of AgNO 3 to RB solution was performed at six different proportions. An increase of RB volume changed color of AgNP solutions from light red to dark red (Figure 4(b)). As demonstrated in the UV-vis spectra (Figure 4(a)), when the volume ratio of extracted RB solution was reduced from 10 to 1, the absorption peaks showed a decreasing intensity. In addition, the surface plasmon peaks were shifted towards shorter wavelengths from 500 to 400 nm. This result indicated a reduction in the mean diameter of AgNPs [47]. However, it should be noted that the UV-vis extinction spectra including the shifting intensity, and the full width of absorption peaks, were positively related to not only size but also size distribution and shape of AgNPs [48].
SEM images of the synthesized AgNPs are displayed in Figure 4(c). All synthesized AgNPs demonstrated the spherical morphology, and the agglomerated particles could be observed in all conditions. As shown in Figure 4   Journal of Nanomaterials nm, and the particles were distributed in a narrow range when the volume ratio of RB solution was reduced from 10 to 1. This finding was in agreement with the blue-shifted UV-vis spectra. However, when the volume ratio of AgNO 3 to RB extract was increased from 1 : 1 to 3 : 1, the results showed a slight decrease of particle size from 37:98 ± 0:85 to 34:54 ± 0:94 nm, implying the limited capacity of reducing agents in RB extract. This finding was consistent with the previous study of Jain and Mehata [49] that the size of AgNPs could be increased by increasing a volume ratio of leaf extracts of Ocimum sanctum or quercetin, which is a polyphenolic flavonoid.
3.1.4. Effect of pH on the AgNP Synthesis. AgNPs synthesized by using RB extract were performed over a pH range of 3-11. Figure 5(a) demonstrates the UV-visible spectra of AgNP formation. The intensity of absorbance peaks was increased with increasing pH from 3 to 11. The blue-shift wavelength was also observed with an increase of pH condition and indicated the larger size of AgNPs in the acidic medium than in     Journal of Nanomaterials the alkaline medium. In accordance with Figure 5(b), the color of AgNP solutions was also changed from light brown to dark red-brown color with increasing pH from 3 to 11. It should be noted that the color of the AgNP solution provided a convenient spectroscopic signature relating to the effect of pH on nanoparticle formation. In addition, the SEM images confirmed that the particles produced in all pH conditions were in nanometer size in different shapes including hexagonal and spherical shape ( Figure 5(c)). As demonstrated in Figure 5(d), it was clear that the mean particle size of AgNPs was decreased from 98:55 ± 1:22 to 67:93 ± 0:80 nm when the pH of reaction was increased from 3 to 11. Additionally, the particle sizes synthesized in acidic conditions (pH 3 and 5) were distributed in a wider range (20-180 nm) than those in basic conditions (pH 9 and 11; 40-90 nm) (as shown in Figure 5(d)). These results indicated that the alkaline pH medium enhanced the reducing and stabilizing capability of the polyphenolic compounds in RB extract. The reaction pH could influence the pKa and electrical charges of phenolic molecules, which affects the capping and stabilizing abilities and subsequently the growth of the nanoparticles [50]. At alkaline pH, a number of electron-donating groups (-OH) of phenol molecules in RB extract were more deprotonated and facilitated a higher number of Ag + ions to bind and subsequently obtained AgNPs with smaller diameters than those synthesized in an acidic environment. Because polyphenolic compounds including flavonoids and tannins [51] are known to be major secondary metabolites of several plants, the effect pH on the synthesis of metal NPs was similarly reported by using a reducer from Terminalia chebula fruit [52], olive leaf [50], and oak fruit hull extracts [53].
It should be noted that the irradiation time has small effect on the particle size of AgNPs, compared with the effect of volume ratio and pH. Therefore, the effect of volume ratio and pH is plot against the average particle size of AgNPs (as illustrated in Figure 6). The AgNPs in the synthesis condition at pH 2.5 showed the smallest size, although the trend of particle size was increased with a decreasing pH solution from 11 to 3. The nonlinear relationship between pH and the particle size could be similarly observed in the synthesis of AgNPs using herbal extract [54,55]. This could be related to different charged surface on NPs in different pH [55,56]. By fixing the solution pH at 2.5, a varied volume ratio of RB extract obtained the smaller size of AgNPs in comparison with other pH conditions ( Figure 6). However, the ratio of 1 : 12.5 (AgNO 3 : RB extract) obtained a slightly decreasing particle size (approximately 60 nm), comparing with the lower ratios of 1 : 7 and 1 : 10 (about 63 and 69 nm, respectively). This result is consistent with the synthesized AgNPs by tannic acid in polyol reaction [57]. The average size of AgNPs was decreased from 23 to17 nm with increasing concentration of tannic acid of from 10 to 100 μM due to the accelerated reduction of Ag + by tannic acid [57][58][59]. However, tannic acid at a very high concentration of 1000 μM obtained a large particle size of 85 nm because the reduction of Ag + was decelerated by the chelation ability of tannic acid [57][58][59]. Moreover, the effect of plant extraction ratios on the AgNP synthesis resulted from the overall chemical reaction. One phytomolecule can perform dual functions as reducing and capping agents to contribute AgNPs, and some of them can induce the opposite effects on the synthesis reaction depending on the concentration [57]. Therefore, the desired size of particles was appropriately adjusted by varying RB extract ratios.
The comparison with the previous methods using rice extract for AgNP synthesis is shown in Table 1.This study presents a rapid and effective combination approach to synthesize AgNPs with sub-100-nm-sized spherical and hexagonal particles within 30 min at room temperature by using a ratio of AgNO 3 to RB extract 1 : 12.5 at pH 2.5, whereas the condition of previous reports required a high temperature (75 and 121°C) or a long reaction time (60 min) for synthesis.
3.2. XRD. The selected AgNPs, which were obtained from the conditions providing the high UV-visible peak intensity results, were investigated the crystallinity by XRD. The crystal phase structure of AgNPs prepared with different synthesis conditions was investigated by XRD (Figure 7). The characteristic peaks of metallic silver constructed with facecentered (fcc) cubic structure JCPDS (Card no. 04-0783) were observed in the sample prepared with AgNO 3 : RB extract volume ratio of 1 : 12.  [60]. This clearly confirms that a pure crystalline structure of AgNPs was successfully prepared by using RB extract. In addition, the XRD pattern of AgNPs synthesized using high pH conditions exhibited amorphous structures. We assume that the growth rate of AgNPs is very fast, leading to the formation of a nonordered crystal structure inside the lattice of Ag particles. On the other hand, the growth rate of Ag crystal in acidic condition (low pH) is well-controlled to form crystal structure. The average crystallite size of synthesized AgNPs was evaluated  3.3. FT-IR. FT-IR has been conducted to investigate functional groups of RB extract and formation mechanism of AgNPs. From Figure 8, IR spectrum of RB extract exhibited broad absorption band at 3300-3200 cm −1 that correlates to hydroxyl (OH) stretching of phenolic component and amide (NH) stretching of protein component. The major peaks at 1722, 1637, 1435, 1217, and 1022 cm −1 are corresponded to C=O stretching of carbonyl group, conjugated C=C or amide I, CH 2 bending vibrations, C-O-C, and C-OH vibration, respectively [61][62][63][64]. For FT-IR spectrum of synthesized AgNPs, it was clearly seen that the OH absorption band at 3300-3200 cm −1 region was decreased, while the C=O signal was enhanced. This indicated that the OH of phenolic compounds in RB extract were involved in reducing process to form metallic Ag capped with carbonyl functionality. In addition, new absorption peaks at 1545, 1377, 1288, and 1072 cm −1 were observed. These peaks could be assigned to amide II stretching, C-N stretching, and C-O-C vibration characteristic, suggesting that ether linkage and protein in RB extract could adsorb on the surface of AgNPs [65][66][67].
Based on the Folin-Ciocalteu assay, RB extract in this study contained phenolic compounds approximately 13 mg GAE/g. It has been reported that the top three phenolic compounds found in Thai RB extract included 2-methoxy-vinyl phenol, catechol, and guaiacol [32]. Besides, the documented anthocyanidins in RB extract were cyanidin-3-glucoside and peonidin-3-O-glucoside [68]. Together with the result of FT-IR spectra, the proposed mechanism of biosynthesized AgNPs by phytochemical constituents in RB extract is illustrated in Figure 9. The oxidation of these compounds causes the formation of phenoxy radicals, which their electrons can be delocalized by resonance along with reducing the Ag + ion to form AgNPs [69,70]. Additionally, this resonance did not only contribute to the stabilized phenoxy radicals of phenolic acid but also could possibly prevent the coagulation of prepared metal silver nuclei [44,69]. Apart from the phenolic constituents, RB bran extract was reported to have protein content [71]; therefore, it could be notified that the amino acid may also serve as reducing and stabilizing agents similar to the phenolic compounds.
3.4. Antibacterial Activity of the Synthesized AgNPs. The antibacterial activity of the synthesized AgNPs using different conditions was assessed through the disc diffusion method against Gram-positive S. aureus and Gram-negative E. coli. The antibacterial activity was clearly observed in the AgNO 3 solution (a positive control) and AgNP samples. Conversely, the sterile DI water, which was used as a negative control, showed no antibacterial activity as shown by no inhibition zone ( Figure 10). As presented in Figures 10(a)-10(c), the inhibition zones of the synthesized AgNPs at different conditions against S. aureus (9:17 ± 0:58 to 10:38 ± 0:25 mm) were significantly higher (p < 0:05) than those against E. coli.  Figure 9: Schematic illustration of proposed mechanism of biosynthesized AgNPs by phytochemical constituents in RB extract [44,72]. 13 Journal of Nanomaterials indicated that the synthesized AgNPs using RB extract exhibited more effective antibacterial activity against Grampositive bacteria than against Gram-negative bacteria, which were in agreement with the previous studies of Khalil et al. [50], Huong and Nguyen [73], and Jemilugba et al. [74]. In addition, it could be noted that the smaller size of AgNPs provided the larger inhibition zone as obviously shown in Figure 10(b). Because AgNPs would attach to the bacterial cell membrane and release silver ions to penetrate and interact with biomolecule and DNA, the smaller size of NPs, the higher surface area to volume ratio of NPs could lead to more attachment and stronger binding to the cell membrane, resulting in the higher efficacy [75,76]. On the other hand, several studies have reported that AgNPs were more susceptible to Gram-negative bacteria than Gram-positive bacteria [77,78]. This phenomenon may be caused by the different membrane structure of the microorganism. Due to the thick layer of peptidoglycan in Gram-positive bacteria, it makes them more rigid and makes them less permeable for the silver ions to get inside the cells [79]. However, the opposed antibacterial susceptibility of AgNPs on Gram-negative and Gram-positive bacteria could be affected by the different physicochemical properties of AgNPs, which played a critical effect on their antibacterial potential including shape, size, surface charge, and concentrations [36,80]. Mandal et al. [81] reported that Gram-positive Enterococcus faecalis cells accumulated more Ag + ions than Gram-negative Proteus vulgaris as shown by the lower zeta potential of −15 and −26 mV, respectively, which indicated the preferentially eradicated Gram-positive bacteria and the chargedependent mechanism of AgNP efficacy. In addition, AgNPs in the spherical shape were effective against both Grampositive and Gram-negative bacterial than the rod shape as shown by the lower minimum inhibitory concentrations [82]. Because AgNPs in the spherical shape has larger effective specific contact area as compared to the rod shape, they could achieve closer contact with bacterial cell and causes more damages [83]. So, the antibacterial efficacy of lightassisted AgNPs using RB extracts was based on the bacteria strain and the size of AgNPs. Apart from effective inhibition of susceptible bacteria, AgNPs could inhibit the formation of bacterial biofilm, which is one of the virulence factors of resistant bacteria [84]. Therefore, their antibacterial activity of AgNPs on multidrug resistant bacteria in both standard and clinical isolates, and antibiofilm formation are deserved for further investigation.