Synthesis of zinc oxide nanoparticles using Chrysopogonzizanioides grass extract, its applications in photodegradation and antimicrobial activity

Zinc oxide nanoparticles (ZnONPs) were synthesized using zinc nitrate hexahydrate as an oxidizer and Chrysopogonzizanioides (Vetiver) grass as a novel fuel using a green approach. The plausible mechanism of synthesis of ZnONPs is explained in detail. The X-ray diffraction pattern as well as the Rietveld refinement showed a single-phase wurtzite structure. The average crystallite size and the lattice strain were estimated using Williamson–Hall plot. The stability of the crystal lattice was confirmed from the extremely small value of lattice strain. The presence of various functional groups in the plant extract and the zinc–oxygen bonding in the ZnONPs were confirmed by FTIR. The surface morphology was investigated using SEM, and it showed a nanorod-like nanostructure. The elemental mapping was carried out using EDS. The EDS spectrum suggests the formation of ZnO nanorods along with the high proportion of carbon and low proportion of Si as well as K. These might have resulted from the rich organic profile of Chrysopogonzizanioides grass extract. Within the UV–visible spectrum at 300 nm, the highly blue-shifted strong absorption band was observed due to the strong quantum confinement effect with the band gap of 3.628 eV. The photodegradation of RB2 dye was studied over ZnONPs catalyst, and it showed excellent photocatalytic activity. The catalyst was active for up to five cycles without losing much of its efficiency. The detailed degradation mechanism of RB2 dye was explained using LCMS technique. Further antimicrobial activity was tested against a broad range of microorganisms, namely Staphylococcus aureus, Escherichia coli and much prevalent human fungal pathogen Candida albicans. The minimum inhibitory concentration (MIC) for each micro-organism was determined using broth micro-dilution assay, and the values were found to be for E. coli (1.0 mg/mL IC100), S. aureus (0.5 mg/mL IC 70) and the fungal strain in planktonic growth state (0.5 mg mL-1 IC 100).


Introduction
Dye contaminants obtained from the textile, printing and many other industries have assumed a pivotal role in damaging the environment. In the textile industries during fabric dying, a large quantity of unbind dye is released into the wastewater stream. Thus, the dying process precipitates highly coloured effluents in water resources, which is aesthetically unpleasant and causes harmful effects on the marine ecosystem [1,2]. Further, the discharge of the unreacted chemicals into the water bodies has caused carcinogenic and mutagenic effects to the biosphere; this can be treated using metal nanoparticles and microbial desalination cells [3,4]. Various physical and chemical techniques have been used for the treatments of dyes along with their effluent; however, these methods are not practically feasible as they are either harmful or highly expensive [5,6].
Mahmoud et al. [7] had synthesized the nanocomposite with various Montmorillonite clays using xanthan gum along with polyvinyl imidazole (XG-g-PVI). These nanocomposites work as an excellent adsorbent to adsorbed malachite green dye. Further, they are also used to inhibit the growth of E. coli and S. aureus the commonly observed pollutant in water. Messih et al. [8] had reported ZnO@SiO 2 nanomaterials using sol-gel method, in the presence of ammonium hydroxide as surfactant and CTAB as the capping agent. These catalysts were found to be very effective for the photodegradation of MB as well as Eosin dye. Sanad et al. [9] had synthesized pure ZnO, ZnS nanoparticles and ZnO@ZnS composites, using the sol-gel method in the presence of CTAB as the capping agent. These catalysts were applied for the degradation of MB as well as EOSIN dye. Further, the ZnO@ZnS nanocomposites were found to be the best catalyst as compared to Pure ZnO and ZnS nanoparticles.
ZnO nanoparticles had been synthesized by various methods; each method has its own advantages and disadvantages. However, green approach has an edge over all the other methods of synthesis. Green methods are eco-friendly as they avoid the usage of toxic chemicals and reagents, and the process of synthesis is also very easy. In this method of synthesis, the fruit juices, plant extract, plant latex acts as fuelling, reducing and capping agents. Yadav et al. had used the juice of watermelon and sugarcane, as fuel and reported excellent photocatalytic activity of ZnONPs [10,11]. Alamelu et al. reported the synthesis of ZnONPs using tapioca starch in the photodegradation of methylene blue dye [12]. Photocatalytic properties of ZnONPs were studied using the plant extract of Cassia fistula and Garcinia xanthochymus [13,14]. Additionally, along with photocatalytic activity, ZnONPs exhibit antimicrobial properties also. In recent years, the development of antimicrobial agents and external coatings has received significant attention. The strong UV-adsorption capacity of ZnONPs makes them a very good antibacterial agent [15]. Nowadays due to continuous discharge of antibiotics into the environment it has created very serious problems. It leads to an increase in resistance of microorganisms many folds as the antibiotics rarely metabolized and remain active after their removal [16,17]. Hence, there is a need to work out on this issue very seriously and one of the solutions to meet this challenge is the synthesis of metal nanoparticles and their applications in antimicrobial studies. The recent approach of metal nanoparticles has gain importance in the development of potent antimicrobial agents [18]. ZnO material synthesized by spin coating method has shown antimicrobial activity against gram-positive as well as gram-negative bacteria (such as S. pnemoniae, S. aureus, E. coli and E. hermannii) at concentrations ranging from 100 to 600 lg/mL by agar well diffusion method [19]. The MIC value of ZnONPs synthesized using neem extract was found to be 10.42 lg/mL against Escherichia coli and Salmonella typhi [20]. The antifungal activity of ZnO biosynthesized using Ziziphus nummularia leaf extract showed an effective MIC of 1.25 mg/mL for C. albicans and C. glabrate [21]. However, despite all the above advantages many of the ZnONPs also suffered from some evident disadvantages, namely weak crystallinity [22], very restricted spectrum reaction range [22], less photocatalytic activity [23], weak antibacterial activity [24]. Therefore, an easy and schematic approach to assembling ZnO nanostructures is extremely desirable for effective solar energy conversion as well as for effective antimicrobial activity.
In continuation of our earlier research in photodegradation of industrial dyes [25,26], herein, we report the green synthesis of ZnONPs using Chrysopogonzizanioides grass extract and its application in the photodegradation of dyes. The green approach is planned to make the process cost-effective as well as environment-friendly. Chrysopogonzizanioides grass extract is a plant of Indian origin; its oil (extracted from the roots) has been traditionally used as the medicine, an aroma ingredient. However, the aerial part of the plant is discarded and is considered worthless; we have used this aerial part to synthesize ZnONPs. The as-synthesized ZnONPs were characterized by using various physicochemical techniques such as XRD, FTIR, SEM-EDS, UV-visible and LCMS. To the best of our knowledge, this is the first study in which the photodegradation of RB2 dye was studied using ZnONPs. The kinetic study and mechanism of RB2 dye degradation over ZnONPs were discussed in detail. Further, the antimicrobial activity of ZnONPs against Staphylococcus aureus (gram-positive), Escherichia coli (gram-negative) and Candida albicans (Fungus) was discussed in detail.

Preparation of plant extract
Chrysopogonzizanioides grass was collected and thoroughly washed with distilled water to eliminate impurities. The grass was dried in the shade for 15 days, and the dried leaves were powdered using mortar and pestle. 20 g of the prepared powder was boiled at 60°C in 300 mL of distilled water. It was heated until the colour of the aqueous solution turned brown. The plant extract was cooled at room temperature and filtered using Whatman 41, and the filtrate was used for the synthesis of ZnONPs. The plant extract is stored in the glass bottle and kept in the refrigerator to avoid fungal growth and was used as and when required.

Synthesis of ZnONPs
Ten millilitre of Chrysopogonzizanioides grass extract was heated till 60°C, followed by the addition of 2.0 g of zinc nitrate hexahydrate. The temperature of the solution was maintained to 80°C under constant stirring till a paste of yellowish-white colour was obtained. The paste was sintered at 400°C for 2 h in the muffle furnace. The yellowish-white colour powder obtained was finely pulverized using mortar and pestle. The resulting sample was stored in a glass vial at room temperature. Many researchers had synthesized ZnONPs using chemical reagents such as NaOH or hydrazine or any other chemicals as a stabilizing or capping agent. As down the line, these reagents change the pH of water bodies causing threats to aquatic flora and fauna, and thus, it is of immense ecological concern. Few researchers have avoided the use of such chemicals in the synthesis of ZnONPs. By inspiring their work, we also did the same; the only chemical used in the synthesis of ZnONPs was zinc nitrate salt. Thus, green chemistry principles were followed in the synthesis of ZnONPs by avoiding the usage of chemical reagents.

Characterization
Purity and crystalline structure were characterized at room temperature on Philips (Xpert) X-ray diffractometer (XRD) with Cu ka radiation having wavelength 1.540 A 0 . FTIR spectra of the samples were recorded on 3000 Hyperion Microscope with vertex 8 FTIR using KBr pellets in the range of 400-4000/cm. The microstructure and the sample morphology of particles were characterized by FESEM ULTRA PLUS manufacture Carl Zeiss Germany. The surface area was analysed using micromeritics BET surface area analyser. Photodegradation of RB2 was studied on UV-visible spectrophotometer 1800 of Shimadzu make. Photocatalytic oxidative degradation of RB2 dye was studied using LCMS of Agilent technologies, USA. The model is 1290 infinity UHPLC, 1260 infinity Nano-HPLC with Chipcube, 6550 iFunnel Q-TOFs.

Photocatalytic degradation of dye
Degradation of RB2 dye over ZnONPs was evaluated under solar irradiation. The photoreactor used was 250-mL borosilicate beaker. 50 mL, 20 ppm aqueous solution of RB2 dye-containing 0.025 g of catalyst was stirred in dark for 30 min until sunlight irradiation to maintain adsorption equilibria. This was followed by the addition of oxidizing agent H 2 O 2 . The mixture was then exposed to irradiation with constant stirring. Aliquots of the irradiating mixture were taken at a constant interval of 30 min and were analysed on a UV-visible spectrophotometer (Shimadzu-1800). The reaction was performed by controlling various parameters, viz. the amount of catalyst, pH of the dye solution and the amount of H 2 O 2 .

Antimicrobial activity
MIC of ZnONPs of S. aureus ATCC 6538, E. coli ATCC 8739 and C. albicans was performed using the broth micro-dilution assay in 96-well microtitre plates. Briefly, overnight grown bacterial and fungal cultures were re-suspended in nutrient broth and Sabouraud broth, respectively. The OD of all three cultures was adjusted to 0.1 at 600 nm to give the count of 8 9 10 7 cells/mL. To determine the MIC of each microbial culture, 100 lL of respective broth and 10-lL cultures were added into the 96-well plate which gave maximum growth (positive or growth control). The stock solution was prepared by adding ZnONPs in broth and sonicated for 2 min to obtain a uniform suspension. A twofold dilution was then made to obtain different concentrations of ZnONPs ranging from 1000 to 0.48 lg/mL. 100 lL of varying concentrations ZnONPs solution was added to each well with and without 10lL of the bacterial or fungal cell suspension to give test and colour blank readings, respectively. The microtitre plate was then incubated at 37°C for 24 h in case of bacteria and at room temperature for 48 h in case of fungus. The least ZnONPs concentration in the well where no microbial growth was observed was the MIC. A negative control containing only broth was used in the study. MIC was reported by reading the microtitre plate by microtitre plate reader at 600 nm [27,28].

Results and discussion
The Rietveld refined XRD pattern of ZnONPs is depicted in Fig. 1. The structure was refined with the space group P63mc using the Full Prof program. The XRD analyses designated that ZnO nanorods have a hexagonal unit cell, single-phase wurtzite structure. The crystal data, the observed, calculated and difference XRD profiles for ZnONPs after the final cycle of refinement, and the refinement factors of ZnONPs obtained from X-ray powder diffraction data are depicted in Table 1. The observed profile and the calculated profile are perfectly matching with each other (Fig. 1). The value of the goodness factor (v2) is equal to 2.56, which is attributed as an excellent value for the assessment. The profile fitting is excellent if the v2 value is low; hence, the procedure adopted for profile fitting was by minimizing the v2 function [29]. The crystal lattice parameters of ZnONPs are in good agreement with the literature report (JCPDS No. #36-1451). Williamson-Hall plot for ZnONPs is illustrated in the inset of Fig. 1. The values of average crystallite size and the lattice strain are 42 nm and 0.0027, respectively, obtained from a linear least square fitting to g cosh-sinh. The lattice strain value is low, indicating the stable structure of ZnONPs; this may be due to the green method of synthesis. The crystal structure of ZnONPs from the Rietveld refinement is depicted in Fig. 2.
The SEM images and EDS spectra of as-synthesized ZnONPs are revealed in Fig. 3. The SEM images manifest nanorod-like structures. The low magnification images (Fig. 3a, b) show that these nanorodlike nanostructures are grown by gathering of small distorted hexagonal shape-like structures. The diameter of nanorod-like structures is between 20 and 40 nm. The high magnification image (Fig. 3c) depicts that; the sample consists of agglomerated nanostructures. The corresponding EDS spectra are depicted in Fig. 3d; it demonstrates the atomic percentage of Zn and O in nanorod-like structure and is observed to be 45.3 and 38.92. The further atomic percentage of C, Si and K were found to be 15.07, 0.33 and 0.37, respectively. The high proportion of carbon along with a low proportion of Si and K might have resulted from the rich organic profile of Chrysopogonzizanioides grass extract.
The FTIR spectrum of Chrysopogonzizanioides grass extract and the biosynthesized ZnONPs is shown in Fig. 4. The FTIR spectrum of Chrysopogonzizanioides grass extract (Fig. 4a) exhibited several peaks at 3400, 2900, 2800, 1600, 1400, 1125, 900, 800,650, 490/cm. The peaks at 3400 (O-H), 1600 (N-H), and 1125 (C-O) or 900/cm (RCOO) are related to alkaloids, flavonoids and phenolic compounds, respectively [28][29][30][31], whereas the broad stretching band at 3400/cm stipulates the existence of hydrogen-bonded groups. These results signify the presence of flavonoid derivatives in the plant extract. There is a shift in the position as well as the intensity of the band of the spectrum of ZnONPs (Fig. 4b). This is due to the interrelation of the functional groups of the flavonoids as well as phenols with the ZnONPs. The prominent and very sharp band was observed at 488/cm because of the stretching vibration of Zn-O bond in tetrahedral coordination. A very weak band at 654/cm was allocated to the stretching vibrations of Zn-O bonds in octahedral coordination.
, where y i is the observed intensity and y ic is the calculated intensity at the ith step R wp 12.40 R wp weighted profile factor  mechanism of conversion of zinc nitrate to ZnONPs is depicted in Scheme 1. In the first step of mechanism, Zn 2? makes a complexation with CO-4 carbonyl oxygen and deprotonated C-5 OH group of luteolin and forms zinc luteolin complex. In the next step after reduction, zinc luteolin complex is converted into zinc hydroxide. Finally, after heating zinc hydroxide is converted into ZnONPs [32].
The optical properties of as-prepared Chrysopogonzizanioides grass extract and ZnONPs were examined using UV-visible spectrophotometer in the range of 200-800 nm. The light brown-coloured extract showed a small hump in the near UV region (297.2 nm) as shown in Fig. 5a. The observed hump was probably due to the plant biomolecules (polyphenols, flavonoids, etc.), which have a crucial role in the reduction of metal ions [33,35]. To study the optical properties of ZnONPs, the particles were dispersed in deionized water followed by ultrasonication for about 15 min. The resultant solution showed a very strong band at 300 nm as shown in Fig. 5a; this band is very much blue-shifted when they are compared with their bulk counterpart (360 nm). The strong blue-shifted absorption edge confirms that ZnONPs showed a very strong quantum confinement effect [36]. The change in the spectrum provided the first confirmation for the formation of ZnONPs. The optical band gap was calculated using UV-visible spectra by Tauc plot method (Fig. 5b). Extrapolating of the straight line in the plot of (ahm) 2 vs Energy (hm) gives the value of optical band gap of ZnONPs [37]. The optical band gap was found to be 3.628 eV which shows a slight increase (* 0.4 eV) than the ideal value (at room temperature). The increase in the band gap also supports that ZnONPs exhibit quantum confinement effect. The photocatalytic activity was carried out using ZnONPs as a catalyst to study the photodegradation of RB2 dye at various reaction conditions. The UV-visible spectrum showed an absorption band at 463 nm and a small hump at 280 nm assigned to the visible and UV region, respectively.
The surface area of ZnONPs was estimated using (BET) surface analyser at the temperature of -196°C . The samples were prepared with the flow of N 2 gas at the temperature of 150°C for 2 h. The Langumir theory was extended by Brunauer, Emmett and Teller, and they gave following equation [38].
The terms involved in the equations are P-equilibrium pressure, P 0 -saturation pressure, Qamount of gas adsorbed on the adsorbate, Q mmonolayer adsorbed, and C is the BET constant. The BET plot {1/[Q(P 0 /P)] vs P/P 0 } of the ZnONPs is depicted in Fig. 6a. It is clear from the figure that the plot is linear, and the values obtained from the slope (A) and intercept (I) gave unique values of Q m and C, respectively. From the BET plots, different variables were calculated such as slope-3.708996 g/cm 3 , Intercept-0.110422 g/cm 3 , Q m -0.2618 cm 3 /g, C-34.589221, S total and S BET -1.1398m 2 /g, and pore width 77.3224 Å . Various empirical and computational values required for plotting the curve are given in Table 2. The values of 'Q m ' and 'C', total surface area (S total ) and specific surface area (S BET ) were calculated according to Eqs. (2)(3)(4)(5).
where Avogadro's number is 'N', the molecular cross-sectional area is 's' (0.1620 nm 2 ), the molar volume of the adsorbate gas is 'V' and the mass of the adsorbent sample is 'M' (0.1304 g). The S total and S BET were found to be 1.5839 m 2 /g and 1.138m 2 /g, Scheme 1 Plausible mechanism of synthesis of ZnONPs through Chrysopogonzizanioides  respectively. The t-plot and BJH adsorption methods were utilized to calculate pore volume and pore size and were found to be 0.00203 cm 3 /g and 77.3224 nm, respectively. The value of BET surface area of ZnONPs was 1.5839 m 2 /g. The nitrogen adsorption and desorption isotherms of ZnONPs were recorded and are depicted in Fig. 6b. According to the IUPAC classification, the recorded isotherms are of the type IV [39]. The adsorption-desorption isotherm of ZnONPs forms type IV hysteresis loop indicating self-assembly of small nanoparticles which exist as complex porous structure. Also, it is to be noted that the type IV isotherms are typical isotherms for mesoporous materials [40]. Mesoporous materials are good adsorbent, and hence, RB2 dye was well adsorbed at the surface of ZnONPs, which leads to the rapid photodegradation of RB2 dye. The degradation of RB2 dye was monitored through the variation observed in the intensity of the absorption peak of the RB2. In the beginning (t = 0), the absorption band was observed at 463 nm in the visible region and a small hump at 280 nm in the UV region. To demonstrate the activity of ZnONPs catalyst on RB2 solution, the experiment was carried out in solar irradiation. To the 50 mL of 20 ppm RB2 solution, 25 mg of the catalyst was mixed, and the solution was kept on the magnetic stirrer for adsorption and desorption in the dark for 30 min. Followed by the addition of 0.2 mL H 2 O 2 and at a maintained pH of the solution 2.5, the beaker was kept in the sunlight to record the absorbance of the dye solution at the interval of 30 min using a UVvisible spectrophotometer. The absorption peak intensity of the RB2 at 463 nm gradually decreased in intensity, and there is a new band observed with the increase in the irradiation time as shown in Fig. 7a. These observations confirmed the degradation of chromophores responsible for the colour of RB2 dye. Further, the hump due to aromatic rings vanished which indicates degradation of aromatic rings. The results indicate that during 30 min of adsorptiondesorption in the dark, the dye degradation was 2.32%, after 30, 60, 90 and 120 min; the degradation was 77.27%, 87.27%, 91.81% and 99.98%. The synthesized catalyst ZnONPs are very effective in the photodegradation of RB2 with almost 100% degradation in just 120 min. The coefficient of determination (R 2 ) was found to be 0.998 which is very close to unity. Thus, the reaction follows pseudo-first-order kinetics [41].
Different control experiments were carried out in the presence of ZnONPs using UV-visible spectroscopy, and the data are depicted in Fig. 7b ? ZnONPs ? light (c)]. This is since photo-induced process produces electron-hole pairs, which when migrate throughout the zinc oxide crystal, and eventually responsible for increasing degradation efficiency [42]. However, the rate of the reaction was drastically increased to 100% when the reaction was carried out with [RB2 ? ZnONPs ? light ? H 2 O 2 (d)]; also the rate constant was found to be highest in this case as depicted in Fig. 7b. Additionally, the effect of adsorption and degradation was studied at different pH and is shown in SI in Fig. 1. Catalyst loading plays a very crucial role in the photodegradation process. It is one of the most important parameters in the catalysis process. To make the process economically viable and industrially important, the optimum amount of the catalyst is to be used in the experiment. The catalyst loading results are depicted in Fig. 8a. The results indicate that the rate of dye degradation decreases as the concentration of the catalyst increases. Due to increase in the concentration of the catalyst, the turbidity of the solution increases and there will be a hindrance to the sunlight in the penetration of the light through the dye solution. This leads to decrease in the rate of the photo-phenton process, and the degradation efficiency decreases [43]. Based on observations, the optimized weight of the catalyst is 0.025 g. The rate constant was also highest with this concentration as depicted in Table 3. All the experiments were performed with 0.025 g of the photocatalyst.
The pH of the solution plays a very critical role in the photodegradation of RB2 dye. To make the photodegradation process very effective and economical, the process is to be carried out at the appropriate pH. Hence, experiments were carried out to check the optimum amount of pH to be maintained to carry out the reaction. The role of pH in the photodegradation is depicted in Fig. 8b. The results indicated high photodegradation at lower pH with maximum photodegradation at pH 2.5. Hence, all the experiments were carried out at pH 2.5. The rate constant was also highest with this pH as depicted in Table 3.
To make the process cost-effective, control dosages of H 2 O 2 are essential since H 2 O 2 is a very costly chemical. The effect of H 2 O 2 dosage on the degradation of RB2 is depicted in Fig. 8c. The results indicate that the rate of photodegradation and the rate constant increase as the concentration of H 2 O 2 increases. Based on the above observations, the optimized concentration of H 2 O 2 is 0.2 mL. The rate constant was also highest with 0.2 mL of H 2 O 2 as depicted in Table 3. The H 2 O 2 plays a key part in the degradation process by producing hydroxyl radicals (OHÁ), which break down organic pollutants to form intermediates, which then give rise to the product CO 2 ? H 2 O [44]. The stability and reusability of the ZnONPs photocatalyst were tested over RB2 dye, after recycling to replicate the experiments under identical conditions as shown in Fig. 8d. More specifically, under sunlight irradiation, ZnONPs photocatalyst displayed sustained and consistent behaviour up to the 5th cycle which proves the catalyst's stability and recyclability (see Table 4).
Based on the above results, we proposed a photocatalytic mechanism for ZnONPs under sunlight irradiation (Fig. 9). The sunlight initially falls on the surface of the ZnONPs photocatalyst, the photocatalyst is activated, and e -/h ? pairs are formed. As the holes are left in the valence band, the electrons in the ZnONPs valence band become stimulated. The holes in the valence band are used for the oxidation process, while electrons are used in the conduction band for the reduction process. The reactive species produced decay reactive brown dye in CO 2 and H 2 O [44]. Based on the findings, the following detailed relevant reactions of the ZnONPs system are proposed: The photooxidation pathway must be confirmed to understand how the ZnONPs degrades RB2, and probable intermediates revealed by the LCMS spectra could be used as powerful evidence in this regard. The SI in Fig. 2 depicts different fragments of the RB2 dyes investigated using LCMS. The detailed mechanism of RB2 dye degradation by photocatalytic oxidative degradation is provided based on the LCMS results (Scheme 2). The absence of a molecular ion peak for RB2 dye at m/z = 966 suggests that the dye has been degraded.

Antimicrobial activity
Due to the wide exploitation of antibiotics in the modern era, the rise of antibiotic-resistant bacteria has been increasing exponentially. To put this situation under control, it is necessary to find antibiotics which show broad-spectrum killing. Nanoparticles have a broad range of applications in many fields of life sciences. ZnONPs has known to show promising killing effect against planktonic cultures and has proved to effectively inhibit biofilm formation by gram-positive or gram-negative microbes. The teichoic acid filaments present in the peptidoglycan layer of gram-positive bacteria are negatively charged. Also, the cell wall of gram-positive bacteria has abundant pores. Positively charged ZnONPs and their nanosize make them ideal candidates to penetrate easily, produce cellular lesions and induce cell death in gram-positive bacteria [45]. The cell wall composition of gram-negative is complicated, wherein the peptidoglycan is held together with lipopolysaccharide, lipoproteins and phospholipids which effectively form a barrier that restricts penetration of nutrients and removal of catabolism products at the level of porins [45]. We have observed the microbial static effect of ZnONPs on a broad range of microbes. The study was done in triplicates; it was seen that ZnONPs showed broad-spectrum inhibition of microbial cultures. For the analysis of antimicrobial action of the ZnONPs, a gram-positive bacteria S. aureus ATCC 6538, a gram-negative bacteria E. coli ATCC 8739 and a fungal strain C. albicans were selected as model organisms. It was seen that with the varying concentration of ZnONPs the percentage inhibition of microbes also varied. The more the concentration of ZnONPs, the more the percentage inhibition of microbes; thus, concentration of ZnONPs was directly proportional to the percentage inhibition of microbes. A graph of concentration of ZnONPs versus percentage inhibition of microbes (S. aureus, E. coli, and C. albicans) was plotted (Fig. 10). Percentage inhibition for each was calculated by the ratio of absorbance of test solution to the absorbance of control solution multiplied by 100. In the graph, it is seen that ZnONPs show a 100% inhibitory concentration (IC 100) of 1.0 mg/mL and 0.5 mg/mL for E. coli and C. albicans, respectively. However, for S. aureus ZnO NPs show 70% inhibitory concentration (IC 70) of 0.5 mg/mL. After the above concentrations of ZnONPs, the percentage inhibition of microbes decreases with the decreasing concentration of ZnONPs for all the three model microorganisms (Fig. 10).
ZnONPs were found to be very effective against gram-positive and gram-negative bacteria as well as Scheme 2 Mechanism of degradation of RB2 dye by photocatalytic oxidative degradation against fungal strain; this indicates that it can act as the broad-spectrum microstatic drug.
E. coli ATCC 8739 was treated with ZnONPs; it showed growth inhibition and leads to death in the E. coli cells at very low concentration. The MIC value of ZnONPs against E. coli was found (1.0 mg/mL IC100) (Fig. 11a) which is very low than the formerly reported values for bare ZnO [46][47][48], doped ZnO [49] and ZnO materials in combination with antibiotics. In the case of E. coli ATCC8739, ZnONPs have higher efficiency in opposition to the strain in planktonic growth. Active pathogenic growth of S. aureus ATCC 6538 was treated by ZnONPs, and it was found that cell growth was inhibited in the presence of very low concentrations. The growth inhibition and death in microbes observed in the ZnONPstreated wells and the MIC (0.5 mg/mL IC 70) (Fig. 11b) obtained against S. aureus ATCC 6538 are very low than the previously reported values [50,51]. The ZnONPs were tested for antifungal activity towards one reference pathogenic fungal strain, C. albicans. ZnONPs show the most promising antifungal activity, being active against the fungal strain in planktonic growth state (0.5 mg mL-1 IC 100) (Fig. 11c). ZnONPs obstruct membrane integrity by producing reactive oxygen species that destroy fungi [52][53][54][55][56]. Furthermore, the production of hydrogen peroxide and Zn 2? has played a key role in the antifungal activity of nanoparticles. The obtained MICs are lower than previously reported several zinc oxide-based materials ZnO [46,48], Pd-doped ZnO and ZnO-chitosan composites [49], polycarbonatebased cationic polymer and different antibiotics [48,57]. The antimicrobial activities of ZnONPs were compared with the previous studies reported by the researchers [58][59][60][61], and it was found that the ZnONPs synthesized by a green approach using Chrysopogonzizanioides grass extract showed excellent antimicrobial activities.

Conclusion
This study signifies a simple green synthetic approach towards the synthesis of ZnONPs using aqueous Chrysopogonzizanioides grass extract. The photocatalytic degradation by ZnONPs was checked on RB2 dye under solar irradiation. The catalyst is very stable and showed excellent catalytic efficiency up to five cycles. Further, the catalyst showed very good antimicrobial activities. Thus, a very stable and very effective ZnONPs catalyst was synthesized as per our hypothesis using a green approach. A plausible mechanism of synthesis of ZnONPs is proposed. Further fragmentation of RB2 dye is explained in detail using LCMS. Since the catalyst is very effective in the degradation of highly stable RB2 (an anionic) dye, it can be applied for industrial effluents also. Our future work will be doping ZnONPs in the reduced graphene oxide (rGO) to increase the efficiency of the catalyst as well as to make it economically viable, which will enable the industries to use it in effluent treatment plant (ETP). Further doping ZnONPs in the reduced graphene oxide (rGO) may enhance their antimicrobial properties also.