Highly stable AgNPs prepared via a novel green approach for catalytic and photocatalytic removal of biological and non-biological pollutants.

Increases in biological and non-biological pollutants pose a significant threat to environmental systems. In an effort to develop an effective means to treat such pollutants, the use of Phaseolus vulgaris (kidney beans) as reducing and capping agents is proposed for the green synthesis of highly stable silver nanoparticles (AgNPs) with a face-centered cubic (fcc) crystalline structure (size range: 10-20 nm). The potent role of the resulting AgNPs was found as triple platforms (photocatalyst, catalyst, and antimicrobial disinfectant). AgNPs were able to photocatalytically degrade approximately 97% of reactive red-141 (RR-141) dye within 150 min of exposure (quantum efficiency of 3.68 × 10-6 molecule.photon-1 and a removal reaction kinetic rate of 1.13 × 10-2 mmol g-1 h-1). The role of specific reactive oxygen species (ROS) in the photocatalytic process and complete mineralization of dye was also explored through scavenger and chemical oxygen demand (COD) experiments, respectively. As an catalyst, AgNPs were also capable of reducing 4-nitrophenol to 4-aminophenol within 15 min. Overall, AgNPs showed excellent stability as catalyst and photocatalyst even after five test cycles. As an antimicrobial agent, the AgNPs are effective against both gram-positive (Bacillus subtilis) and -negative bacteria (Escherichia coli), with the zones of clearance as 15 and 18 mm, respectively. Thus, the results of this study validate the triple role of AgNPs derived via green synthesis as a photocatalyst, catalyst, and antimicrobial agent for effective environmental remediation.


Introduction
The continuous growth of global population coupled with industrialization and urbanization has brought rising demand for chemicals, materials, and energy. The release of highly undesirable wastes from numerous man-made sources into environment was inevitably accompanied to lower the quality of environmental systems, of particular aquatic reservoirs (Ali et al., 2019). For example, azo dyes (e.g., Reactive Red 141 : Table 1), which contain two azo groups and one sulfonic acid group, are identified as a major industrial effluent contaminating freshwater bodies. The fixation rate of reactive dyes in the dying process is highly inefficient, less than 50%; with 10% to 15% of used azo dyes are thus released directly into the environment (Telke et al., 2008). Similarly, chemicals such as 4-nitrophenol (4-NP) that are increasingly used in the production of herbicides, insecticides, and synthetic dyestuffs are regarded as an environmentally harmful substance O'Connor and Young, 1989;Singh et al., 2017;Trapido and Kallas, 2000).
Many conventional methods based on physical/chemical principles are available to treat the contamination of dye. However, the practical use of these methods is generally restricted as their applications are associated with expensive chemicals and/or equipment, complex steps and processes, and production of secondary pollutants (some of which can be more toxic than the primary pollutants) (Nawaz and Ahsan, 2014;Singh and Arora, 2011). Advanced oxidation processes (AOP) are often suggested as a proper means to address some of these limitations.
In particular, nanoparticle (NP)-mediated processes based on generation of reactive oxygen species (ROS) in the presence of light (visible, ultraviolet [UV], or both) are highly effective photocatalysts for dye degradation (Ahmed et al., 2017;Baruah et al., 2018;Deng and Zhao, 2015;Trapido and Kallas, 2000;Vieira et al., 2018). However, many NP-based photocatalysts only utilize a limited portion of the solar spectrum. For example, TiO 2 NPs are effective photocatalysts only under UV irradiation. In contrast, silver nanoparticles (AgNPs) are regarded as a highly effective option for photocatalytic treatment as they can accommodate both the UV and visible regions of the electromagnetic spectrum for actual operation (Leong et al., 2018). The utility of AgNPs has further been recognized as catalytic media to treat 4-NP (Mody et al., 2010;Sreekanth et al., 2016;Suchomel et al., 2018). In addition, the utility of AgNP structures has been identified as effective inhibitors for multiple microbial-resistant pathogens (Rolim et al., 2019;Slavin et al., 2017;Wang et al., 2017). As such, AgNPs can be employed based on diverse principles or multiple platforms (as photocatalyst, catalyst, and antimicrobial agent) to remove different types of pollutants such as RR-141, 4-NP, and pathogenic bacteria from aquatic environments, respectively.
Several physical and chemical methods have been explored for the synthesis of AgNPs by employing organic and inorganic stabilizing/ capping agents (Reverberi et al., 2016;Van Dong et al., 2012;Wani et al., 2011;Zhang et al., 2016). However, these approaches often use noxious and expensive chemicals, involve complex and/or time-consuming processes, and may generate toxic wastes and by-products. Therefore, the development of an environmentally friendly, renewable, easy-to-implement, and cost-effective means of synthesizing AgNPs has become one of the foremost demands in the field of environmental remediation. AgNPs can be produced through a variety of green synthesis routes if one can effectively utilize the biomolecules (e.g., proteins and vitamins) and phytochemicals (e.g., phenols and flavonoids) within plants (biomass) as natural reducing and capping agents (Raveendran et al., 2003;Singh et al., 2018a;Yadi et al., 2018). This eliminates the need for harmful and expensive chemicals in reduction of metal salts and capping of NPs (as is the case in chemical reduction methods).
Herein, a facile and inexpensive green synthesis method is proposed to prepare highly stable AgNPs with the aid of Phaseolus vulgaris seed extract. The produced AgNPs were then employed to investigate their potential as environmental remediation agents (photocatalyst, catalyst and antimicrobial). P. vulgaris seeds contain several essential vitamins, proteins, minerals, flavonoids, phenolic compounds, and antioxidants (e.g., lutein, zeaxanthin, and β-carotene) that play important roles in the synthesis of AgNPs (Udani et al., 2018).
To date, only a few studies have been reported to address the utility of P. vulgaris, in the synthesis of AgNPs (Deb, 2014;Khandelwal et al., 2020;Paul et al., 2015). However, from almost none of them, the catalytic/photocatalytic potential or stability of the synthesized AgNPs has been assessed in detail at the same time. In this study, the potential of AgNP as triple remediation platforms for photocatalyst, catalyst, and disinfectant is assessed against RR-141, 4-NP, and Escherichia coli/Bacillus subtilis bacteria, respectively. Furthermore, studies on the ROS and chemical oxygen demand (COD) were also carried out to support the verification of their remediation potential and recyclability/reusability.

Materials and methodology
2.1. Materials P. Vulgaris seeds were purchased from a local market in Fatehgarh Sahib, Punjab, India, and thoroughly rinsed with distilled water to remove dirt and debris. The bacteria E. coli (DH-5α) and B. subtilis were procured from the Department of Biotechnology, SGGSWU, Punjab, India. Silver nitrate (AgNO 3 ), sodium hydroxide (NaOH), sodium borohydride (NaBH 4 ), and 4-NP were purchased from Merck, Germany. RR-141 was purchased from Parswanath Dye Stuff Industries, Ahmedabad, India. All glassware was washed with aqua regia (1:3 vol of concentrated HNO 3 /HCl) and then rinsed with deionized (DI) water before use.

Synthesis of AgNPs
A mixture of P. Vulgaris seeds (10 g) and distilled water (100 mL) was heated at 80°C for 1 h, followed by filtration. AgNPs were synthesized by adding 1, 2, 3, and 4 mL of the resulting filtrate (extract) into the AgNO 3 solution (0.01 M, 50 mL) and stirred at a constant speed (300 rpm) for 30 min. (The pH of the extract was maintained at 10 using buffer solution to ensure optimal outcomes (Singh et al., 2019)). With time, the color of the solution changed from colorless to light yellow and dark brown, indicative of the reduction of silver ions into AgNPs.

Quantitation of AgNPs
The concentration of AgNPs was estimated from the concentration of AgNO 3 solution (0.01 M, 50 mL) using a procedure described by Kalishwaralal et al. (2010) as follows: Step 1: Average number of atoms per nanoparticle (N): (It was assumed that 100% of the silver atoms were converted into silver NPs.) where π = 3.14, ρ is the density of the face-centered cubic crystalline (fcc) structure of silver (i.e., 10.5 g/cm 3 ), D is the average diameter of AgNPs (16 nm), M is the atomic mass of silver (107.86 g), and N A is the number of atoms per mole (Avogadro's number = 6.023 × 10 23 ). Therefore,  (1) = N 125683 Step 2: Molar concentration of AgNPs solution (C): where N T is the total number of silver atoms, which is equal to the molarity of silver nitrate (0.01 M) × atoms present in one mole (6.023 × 10 23 ), N is number of atoms per nanoparticle (calculated in step 1), and V is the volume of solution (L). By putting the value of N obtained from Step 1 into Eq. (2), 0.01 6.023 10 125682.98 0.05 6.023 10 23 23 (3) C = 1.59 × 10 -6 M.L -1 = 1590 nM

Photocatalytic activity: Degradation of RR-141
An aqueous solution of RR-141 (50 mL, 20 ppm) was allowed to equilibrate at room temperature (in dark condition over 30 min). Thereafter, the as synthesised AgNP (5 mg) was added and stirred (magnetic stirrer, 200 rpm) for 20 min. The solution was then directly exposed to sunlight. Following exposure, the AgNPs were separated from the dye solution through centrifugation (at 10,000 rpm for 15 min), and the UV-Vis spectrum of the supernatant (2 mL) was recorded. The degradation efficiency (R) was estimated by equations (4) (Gautam et al., 2016): where C 0 /C and A 0 /A represent the concentration and absorbance of dyes, respectively, at time t = 0/t. The quantum efficiency (QE) of the removal process was also calculated by:

Complete mineralization evaluation and reactive oxygen species (ROS) assessment
The complete mineralization of the dye was determined by monitoring the reduction in COD. For this purpose, 2 mL of test solution was pipetted into the standard amount of potassium dichromate and kept at 150°C for 2 h. Then, COD was estimated on COD meter (Bansal and Sud, 2012).
A scavenger experiment was undertaken to investigate the ROS in photocatalysis. In this regard, different quenchers (with concentration 0.5 mM) were used such as: (i) methanol for hydroxy radical (OH • ), (ii) p-benzoquinone (p-BQ) for superoxide radicals (  Optical study of AgNPs: (a) UV-visible spectra of AgNPs prepared using 1-4mLP. vulgaris seed extracts, (b) FTIR spectrum of finally prepared AgNPs, (c) and (d) the UV-visible absorbance and Zetasizer graph, respectively, of freshly prepared AgNPs and 3-month-old AgNPs. P. Rani, et al. E n v ir o n m e n t I n t e r n a t io n a l 1 4 3 ( 2 0 2 0 ) 1 0 5 9 2 4 2019). After adding these scavengers with AgNPs, photoacatalytic activity was monitored under the same reaction conditions described above.

Assessment of antimicrobial activity
A conventional well-plate diffusion technique was used to evaluate the antibacterial potential of the as-synthesized AgNPs. First, bacterial cultures were grown in Luria broth (LB) overnight and then incubated in LB at 37°C. Subsequently, wells were punched in the LB plates. The prepared AgNPs were then added into the wells at different volumes (20, 40, 60, and 80 µL) and placed at 37°C for one day.

Characterization of AgNPs
An optical study of synthesized AgNPs was undertaken using a Shimadzu-UV 2600 spectrophotometer at wavelengths from 200 to 800 nm. The role of biomolecules in the extract and their interaction with AgNPs were investigated by an Alpha Fourier-transform infrared (FTIR) spectrophotometer (Bruker Corp.). The average particle size was estimated by a particle-size analyzer (Malvern-ZEN-1690). Morphological characteristics and accurate particle sizes were measured from high-resolution transmission electron microscope (HR-TEM; Jeol JEM-2100) images. Similarly, the crystalline structure and interplanar d-spacing between lattice planes were estimated using selected area electron diffraction (SAED). The quantitative stoichiometric configuration of the prepared sample was determined by energy-dispersive X-ray spectroscopy on an Oxford Instruments X-Max 51 -XMX0004.

Characterization of synthesized AgNPs
Addition of the 1, 2, 3, and 4 mL of P. vulgaris seed extract to AgNO 3 solution at room temperature changed the appearance of the solution from colorless to pale yellow and finally to a dark brownish-red color. The latter is indicative of the formation of AgNPs through the reduction of Ag + ions to Ag 0  as also evidenced by UV-visible spectroscopy (Fig. 1a). A sharp band between 420 and 430 nm was observed to commensurate with surface plasmon excitations. At 2 mL of extract, observations of a blue shift (at 422 nm) confirmed the narrow size distribution of AgNPs (Jain and Mehata, 2017). It was suspected that large amount of reductive phytoconstituents led to a rapid formation of Ag NPs followed by their subsequent growth via Ostwald reopening. Therefore, the size of NPs will increase over time to cause red shift in UV spectrum (Shaik et al., 2018). In the present case, the  P. Rani, et al. E n v ir o n m e n t I n t e r n a t io n a l 1 4 3 ( 2 0 2 0 ) 1 0 5 9 2 4 2 mL volume of extract was treated as an optimal volume for the preparation of AgNPs. Hence, AgNPs prepared through this route were used for further characterization and degradation studies. The functionality present on AgNP surfaces was investigated by FTIR spectroscopy. Fig. 1(b) shows the FTIR spectrum of the as-synthesised AgNPs. Strong absorption bands at 3590 (O-H str.), and 3070 cm −1 (C-H str.), 1670 cm −1 (C = O str.), and 1525 cm −1 (N-H bend, Amide I) are indicative of organic or proteinaceous matter on the surface of AgNPs. The proteinaceous matter acts to cap the AgNPs while preventing agglomeration. Huang et al., 2007). The absorption bands at 816 and 630 cm −1 are attributed to the stretching vibrations of AgNPs (Khan et al., 2013). The stability and longevity of the AgNPs were assessed by recording their UV-vis spectrum after three months storage (Fig. 1 d). A very minor decline in absorbance (without any shift in the absorption band) of synthesized AgNPs was noted, signifying the exceptional stability of AgNPs over extended periods. Likewise, the average particle size (Fig. 1.d) showed a very minor increase, i.e., Z-direction from 31.5 to 35.7 nm).
The size and morphology of the as-prepared AgNPs by P. vulgaris seeds extract were determined by HR-TEM micrographs (Fig. 2). The synthesized AgNPs exhibited a spherical morphology, ranging in size from 10 to 20 nm (average size~16 nm). As shown in Fig. 2(b), the interplanar d-spacing between the lattice fringes was 0.235 nm, corresponding to the (1 1 1) lattice plane. The identification of a crystalline structure was investigated by SAED analysis, as shown in Fig. 2(c). The bright circles confirmed the polycrystalline nature of the AgNPs. From the SAED pattern, the interplanar d-spacings were estimated to be 2.44, 2.09, 1.47, and 1.26 Å, corresponding to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) lattice planes, respectively, as summarized in Table 2. These results resemble the standard data of a crystal structure (JCPDS file no. 00-004-0783) to support the (fcc) crystal structure of our as-synthesized AgNPs. The surface elemental composition of AgNPs ( Fig. 2(d)) showed the predominance of Ag (74%) and O (26%) on mass basis. (Note that only % elemental values of Ag and O was considered in present case.) The presence of oxygen correlates well with FTIR data (O-H str.) and our premise that organic molecules, e.g. proteinaceous matter should be involved in capping of the AgNPs .

Catalytic activity of synthesized AgNPs
Due to the well-known catalytic properties of AgNPs, they are considered effective catalysts for the degradation of harmful dyes. The catalytic performance of synthesized AgNPs was analyzed by evaluating the reduction and degradation of 4-NP and RR-141, respectively. These performance parameters were evaluated by recording the time-dependent UV-visible spectra of both molecules. In the reduction of 4-NP, a sharp absorption band was evident at 315 nm (Fig. S1). After the addition of sodium borohydride and AgNPs into the reaction medium, 4-NP is initially converted into its nitrophenolate intermediate (evidenced at 400 nm) (Fig. S1), followed reduction to 4-aminophenol. This change is reflected by the appearance of a new band in the UV-visible spectrum at 296 nm (Kästner and Thünemann, 2016). As the reaction proceeds, the intensity of the 400 nm peak decreases, whilst the intensity of the 296 nm peak increase. The effect of amount of AgNPs on the catalytic reaction was also investigated. As shown in Fig. 3(a-c), the reduction of 4-NP was tested using three volumes (10, 20, and 30 µL) of AgNP solution prepared at 535 nM. Accordingly, 20 µL of AgNPs achieved the maximum reduction of 4-NP. With 20 µL of AgNPs, complete degradation of 4-NP took 15 min. The results with 10 and 30 µL took comparatively longer 21 and 24 min, respectively. We therefore concluded that 20 µL of AgNPs was optimal for the 4-NP catalytic performances. This may be due to an increase in amounts of catalyst and capping agents (phytoconstituents), which slowed the reaction rate. As reported previously, the increasing the amount of plant extract and capping agents reduced the reaction rate by slowing restructuration (involving redox reactions between reactants and catalyst) on the surface of the catalyst (Bingwa and Meijboom, 2014;Niu and Li, 2014). In a control sample, the reduction of 4-NP was negligible in the absence of AgNPs (Fig. S2).
The kinetics of the catalytic reaction was determined by plotting a graph of normalized concentration (C t /C 0 ) versus time. Fig. 3(d) shows that the 4-NP reduction using AgNPs was well fit by a pseudo-first-order rate equation. The apparent rate constant (K ' app ) of 4-NP reduction was estimated using a pseudo-first-order rate equation (Thawarkar et al., 2018): where C t and C 0 are concentrations of the reactant (4-NP) at times t = t and t = 0, respectively (Singh et al., 2018b). The K ' app value for 20 µL of AgNPs was calculated to be 0.165 min −1 with correlation coefficient (R 2 ) of 0.935. It can thus be inferred that the catalytic reaction should follow the pseudo first order kinetic rate. The kinetic rate of this reaction was estimated to be 1.59 mmol g -1 h −1 . The high surface-to-volume ratio and active surface sites of AgNPs are speculated to reduce the activation energy barrier between 4-NP and 4-AP. These properties of AgNPs were reported to facilitate the catalytic conversion of 4-NP into 4-AP at ultra-high speed .
A proposed mechanism for 4-NP catalytic reduction is described in Fig. 4. In the first step, the addition of sodium borohydride leads to conversion of 4-NP to nitrophenolate ion. As there is a large potential difference between acceptor (4-NP = 0.76 V) and donor molecules (BH -4 = 1.33 V), a large activation energy barrier is created. However, Fig. 4. Schematic shows the proposed mechanism of 4-NP reduction by Phaseolus vulgaris-mediated AgNPs.  AgNPs efficiently reduce the activation energy barrier (acting as electron reservoir (between donor and acceptor molecules) known as electron relay effect) to convert 4-nitrophenolate ion into 4-aminophenol. Table 3 compares the performance of different catalytic systems for reduction of 4-NP to 4-AP. Previous efforts employed silver-based nanocomposites through complex chemical processes such as precipitation polymerization, hydrolysis, and hydrothermal synthesis. In contrast, in the present study, a simpler and more environmentally friendly system is developed for efficient reduction of 4-NP.

Photocatalytic performance of AgNPs
The photocatalytic degradation of RR-141 dye in the presence of AgNPs was monitored by UV-vis spectroscopy (absorption bands at 534 and 332 nm, see Fig. 5(a)), Upon addition of AgNPs and exposure to sunlight, the intensity of these absorption bands started to decrease, as shown in Fig. 5(a). The average sunlight flux was estimated on the pyranometer as 645 W/m 2 .
A control experiment with and without catalyst in dark and sunlight environments was also performed to determine the effects of catalyst and sunlight alone. In the absence of catalyst, a negligible decline in RR-141 absorbance was observed in both the dark and sunlight. Likewise, when the dye degradation experiment was conducted in the dark in the presence of a catalyst, a minor decline in absorbance was observed ( Fig. 5(b and d)). These results suggest that the progress of dye degradation should rely on both AgNPs and sunlight. A kinetic study revealed the photocatalytic reaction to have a pseudo-first-order relationship, with an apparent rate constant (k ' app ) and R 2 of 0.02 min −1 and 0.941, respectively, as shown in Fig. 5(c). Nonetheless, due to the high molecular weight (1774.19 g/mol), low diffusion rate, and complex structure (two azo groups), RR-141 was not as easily degraded compared with the lab dyes (methylene blue: 319.85 g/mol; Congo red: 696.66 g/mol) (Jagadale et al., 2012;Khataee and Kasiri, 2010;Muhd Julkapli et al., 2014). The dye degradation efficiency was approximately 97% within 150 min. Moreover, the QE of the photocatalytic process was 3.68 × 10 -6 molecule.photon -1 , with a removal rate of 3.66 × 10 13 molecule.sec -1 and kinetic rate was estimated to be 1.13 × 10 -2 mmol.g -1 h −1 .
The dye degradation mechanism by AgNPs is based on plasmonderived photocatalysis. One of the advantages of using AgNPs as photocatalysts is that they can utilize both the visible and UV regions of the solar spectrum due to surface plasmon resonance and their inter-band transition properties. The excitation of sp-band electrons of AgNPs involves absorption of light in the visible region. The plasmon effect produces heat energy or hot electrons, which interact with oxygen and generate oxygen free radicals (e.g., O 2 • ). These energetic free radicals are responsible for degradation of dye molecules. The holes generated in the 5 sp band can enhance degradation performance by acquiring electrons from dye molecules. Similarly, under UV radiation exposure (from sunlight), inter-band transitions took place in the 4d orbital of the AgNPs. This process generated highly energetic oxygen and hydroxy radicals (O 2 • ,O H • ), which degraded dye molecules. (Kale et al., 2014;Kumar et al., 2013;Leong et al., 2018;Roy et al., 2015aRoy et al., , 2015bSumi et al., 2017). The dye degradation mechanism is illustrated in Fig. 6. Furthermore, a scavenger experiment was carried out to determine the role of specific ROS species in the photocatalytic process. As seen in Fig. 7(a), the photocatalytic activity was greatly affected and declined relative to the control when p-BQ and methanol were present as scavengers. Also, a minor decline was observed when AO was employed as a scavenger. Therefore, it can be inferred that the superoxide (O 2 • ), and hydroxyl (OH • ) radicals are the dominant ROS species in the photocatalytic activity of AgNPs. In other words, the relative dominance of ROS species in the photocatalytic process can be estimated as O 2 • >OH • >h + . The complete mineralization was also estimated by evaluating the reduction in COD as shown in Fig. 7(b). The complete mineralization of dye took longer (e.g., 60 min) than photocatalytic degradation. This might be due to the formation of some intermediate (uncoloured) moieties that can intervene in the degradation process (Bansal and Sud, 2012).
To validate the efficacy of photocatalytic activity of AgNPs observed in this work relative to the finding of other studies, all related data were surveyed as described in Table 4. We also estimated the QE of all relevant studies. In most of previous studies on AgNPs with a variety of forms (composites, core-shell, etc.), the efficiency of the photocatalytic process was estimated under highly favorable conditions (e.g., use of artificially intense light sources and lab dyes (highly degradable component with low molecular weight and high diffusion rate)). In comparison, the present AgNPs system was demonstrated to have appreciable photocatalytic potency (as seen from QE values) without such adjustment for favorable experimental conditions. It can thus be inferred that the developed system can be employed to effectively destroy hard-to-degrade reactive dyes.
The re-usability and recyclability of the as-synthesised AgNPs were also tested for catalytic (reduction of 4-NP) and photocatalytic applications (degradation of RR-141) as shown in Fig. 8. The as-synthesised AgNPs showed excellent recyclability and efficacy with up to 5 time reuse. Only, a minor decline in catalytic and photocatalytic activity of AgNPs was observed even after five runs.  (continued on next page) P. Rani, et al. E n v ir o n m e n t I n t e r n a t io n a l 1 4 3 ( 2 0 2 0 ) 1 0 5 9 2 4

Antibacterial performance of AgNPs
The antibacterial properties of synthesized AgNPs were investigated against two types of bacteria: E. coli (gram-negative) and B. subtilis (gram-positive). Different volumes of AgNPs were poured into cultured E. coli and B. subtilis plates by the disk-diffusion method, as presented in Fig. 9 (a and c). Agar plates b and d were designed as controls for E. coli and B. subtilis, respectively, by adding water, AgNO 3, and P. vulgaris extract to the three wells. The four wells in the plate were filled with 20, 40, 60, and 80 µL suspension of AgNPs and labelled as 1, 2, 3, and 4, respectively, in the a and c agar plates.
Different zones of clearance were observed in all the AgNP-treated wells, while no such zones were observed in control plates. The zone of clearance revealed a linear increment in the volume of AgNPs ( Fig. 9 and Table 5). It was observed that the synthesized AgNPs showed higher antimicrobial activity towards gram-negative bacteria. This difference may be explained from a number of respects. Firstly, differences in cell wall construction should be considered between the two types of bacteria. As gram-positive bacteria have a thicker cell wall to contain peptidoglycan proteins, they can render resistance to AgNPs relative to the gram-negative bacteria with thinner cell wall. Secondly, it should be noted that the cell wall of gram-positive bacteria is negatively charged. As they can attract silver ions to the surface, it is possible to suppress their amount reaching the plasma membrane (Peiris et al., 2018). Antimicrobial screening analysis confirmed that the biogenic AgNPs possess efficient antimicrobial potential against E. coli and B. subtilis bacteria. It is suggested that, due to the small size and large intake of AgNPs by bacteria, the AgNPs demonstrated enhanced toxicity toward bacterial strains (Dakshayani et al., 2019;Pirtarighat et al., 2019). The main reason for high antimicrobial activity of AgNPs is production of ROS, which is suspected to be the main factor in bacterial cell death (Lee et al., 2019), along with formation of Ag + (Al-Sharqi et al., 2019). Earlier studies on AgNP antimicrobial activity suggested that these NPs should attack bacterial cells through oxidative stress, DNA damage, protein denaturation, and rupture of the cell transport membrane (Roy et al., 2019). Multiple antimicrobial mechanisms of AgNPs are described in Fig. 10. (Behravan et al., 2019). AgNPs synthesized in an environmentally friendly manner are associated with more effective antimicrobial activity due to the presence of biomolecules compared with commercially available or chemically synthesized NPs (Bagherzade et al., 2017).

Conclusions
In this research, a highly stable AgNPs were prepared through an This study * If wavelength information was not available or provided in the literature, the following values were assumed: UVC = 250 nm, UV = 365 nm, and VIS = 420 nm. P. Rani, et al. E n v ir o n m e n t I n t e r n a t io n a l 1 4 3 ( 2 0 2 0 ) 1 0 5 9 2 4 environmentally friendly and cost-effective green synthesis approach using P. vulgaris seed extract as capping and reducing agent. The triple role (as catalyst, photocatalyst, and antimicrobial agent) of the produced AgNPs was demonstrated based on their efficacy toward the reduction of each selected target such as 4-NP to 4-AP, RR-141, and both gram-positive/-negative bacteria (Escherichia coli (achieving an 18 mm maximum zone of clearance) and Bacillus subtilis (15 mm maximum zone of clearance)), respectively. Moreover, the as-synthesised AgNPs recorded remarkable stability after various test cycles for catalyst/ photocatalyst. This work fosters the possible development of a three-inone platform for the environmental remediation of waste water systems.   Fig. 10. Schematic shows the multiple antimicrobial mechanism of AgNPs. P. Rani, et al. E n v ir o n m e n t I n t e r n a t io n a l 1 4 3 ( 2 0 2 0 ) 1 0 5 9 2 4 CRediT authorship contribution statement

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.