Synthesis and Enhanced Photocatalytic Activity of Zinc Oxide- Based Nanoparticles and Its Antibacterial Activity

Centre for Bioscience and Nano Science Research, 641021, Coimbatore, India Department of Civil Engineering, Kongu Engineering College, Perundurai, Erode 638060, India Department of Civil Engineering, M.Kumarasamy College of Engineering, Karur 639113, India Department of Civil Engineering, Vaagdevi College of Engineering, Warangal 506005, India Department of Civil Engineering, K.S.R. College of Engineering, Tiruchencode 637215, India Department of Hydraulic and Water Resource Engineering, Wollega University, Ethiopia Department of Construction Technology and Management, Wollega University, Ethiopia


Introduction
Nanometric zinc oxide can have many different forms. It can be found in structures that are one dimensional (1D), two dimensional (2D), and three dimensional (3D). Needles, helixes, nanorods, ribbons, belts, wires, and combs are among the most common one-dimensional structures. Nanopellets, nanosheet/nanoplate, and other twodimensional structures of zinc oxide can be found [1]. Nanotechnology is the manufacture and application of materials on the tiniest scale possible [2].
Nanoscale particles have lately emerged as possible antibacterial agents due to its higher surface area to volume ratio, which is attracting researchers, due to the development of microbial resistance to metal ions, antibiotics, and the generation of resistant strains.
The recent expansion in the field of porous and nanometric materials generated through nontraditional techniques has sparked interest in finding new uses for ZnO nanoparticles [3]. Zinc oxide is also a fascinating semiconductor material because of its use in solar cells, gas sensors, ceramics, catalysts, cosmetics, and varistors [4].
Zinc oxide nanoparticles are widely known for their antibacterial properties. Using the disc diffusion approach, Wahab et al. employed this feature to suppress Bacillus subtilis and Escherichia coli growth. The potential antibacterial activity of zinc oxide nanoparticles was then assessed [5,6].
The old known techniques such as metal organic chemical vapor deposition (MOCVD), spray pyrolysis, laser ablation, sputter deposition, ion beam-assisted deposition, template-assisted growth, and chemical vapor deposition have also been used for the zinc oxide nanostructure synthesis.
Further, literature reports [7] have demonstrated the synthesis of zinc oxide nanoparticles with sizes ranging from 50 to 200 nm using the solution-combustion method with zinc acetate dihydrate and ethylene glycol, as well as the synthesis of zinc oxide nanoparticles using the solvothermal method at 180°C for 24 hours with zinc acetate dehydrate and urea. When compared to inorganic antibacterial agents, antibacterial researches on organic materials are frequently unstable, especially at high temperatures and/or pressures [8].
It has been claimed that eco-friendly plant extracts can be used to synthesis nanoparticles. Because ZnO is nontoxic, it can be employed as a photocatalytic degradation material for contaminants in the environment [9].
Zinc oxide has additional properties that make it an efficient antibacterial agent. That Zn2+, in addition to being a nontoxic metal, is a necessary nutrient found in practically every cell, is required for a variety of physiological processes, and must be eaten in the diet to maintain good health [10]. This study focuses on the chemical synthesis of ZnO nanoparticles, as well as their characterization and antibacterial activity against the human bacterial pathogen Staphylococcus aureus [11].
Germination tests on maize and rice were used to assess the phytotoxicity of seven metal oxide NPs: titanium oxide (nTiO2), silicon di oxide (nSiO2), cerium dioxide (nCeO2), magnetite (nFe3O4), aluminium oxide (nAl2O3), zinc oxide (ZnO), and copper oxide (nCuO). As toxicity markers, root length and shoot length were chosen since they are susceptible to an unfavourable environment [12]. This study contributed to the application of metal oxide NPs in agriculture and the assessment of environmental safety. . Zinc oxide nanoparticle was prepared from 0.1 M of zinc nitrate (1.487 g weighed and 50 ml distilled water dissolved) separated into two portions each of 25 ml. Then, 0.2 M of 0.04 g weighed and 5 ml water and sodium hydroxide (NaOH) and 0.2244 g weighed and 5 ml of potassium hydroxide (KOH) were prepared.

Materials and Methods
First, a portion of 25 ml of zinc nitrate and 5 ml of prepared sodium hydroxide (NaOH) (NaOH was added to enhance the formation of the alkoxide phase during the zinc oxide nanoparticle synthesis) are added drop by drop and stirred constantly with magnetic stirrer (100 rpm) for 2-3 hrs and incubated at room (37°C) temperature without light contact for overnight. After the incubation period, zinc oxide nanoparticles are formed [13,14].
A second portion of 25 ml of zinc nitrate and 5 ml of prepared potassium hydroxide (KOH) are added drop by drop and constant stirred with magnetic stirrer for 2-3 hrs and incubated at room temperature without light contact for 24 hours. After the incubation, white color zinc oxide nanoparticles are formed.    Journal of Nanomaterials sodium hydroxide (NaOH) and 0.2244 g weighed and 5 ml of potassium hydroxide (KOH) were prepared [15].
To compare the best catalytic agent, first, a portion of 25 ml of zinc sulphate and 5 ml of sodium hydroxide (NaOH) are added drop by drop and constant stirred with magnetic stirrer for 2-3 hrs and incubated at room temperature without light contact for overnight. After the incubation, the white color formed the presence of zinc oxide nanoparticle.
A second portion of 25 ml of zinc sulphate and 5 ml of potassium hydroxide (KOH) are added drop by drop and constant stirred with magnetic stirrer for 2-3 hrs and incubated at room temperature without light contact for over-night. After the incubation, the white color formed the presence of zinc oxide nanoparticle [16].
2.3. Characterization of Nanoparticle. UV-visible spectroscopy, FTIR, and SEM were used to analyse the produced ZnO nanoparticles (among the two). To acquire the structural figure of the created nanoparticles and to determine their size, the liquid solution is utilized for UV-visible spectroscopy, and the powder form of zinc oxide nanoparticles was used for FTIR, SEM, and EDX images.     Journal of Nanomaterials using Labotronic LT 291 UV-VIS and microprocessor spectrophotometer with 5 nm interval.

FTIR (Fourier Transform Infrared Spectroscopy).
Pellets of samples were made into 1 mm pellets using a hydraulic press and scanned more than a wave number range of 4000 cm −1 to 400 cm −1 using Shimadzu FTIR spectrometer.

SEM (Scanning Electron Microscopy)
. Characteristic Xrays are produced when one electron is removed from the sample's inner shell, prompting a higher-energy electron to occupy the shell and release energy in the form of X-rays. These distinctive X-rays are then utilized to determine the material's composition as well as to determine the presence of components and contaminants in the sample. Magnification in a scanning electron microscopy technique can be adjusted across 6 orders of magnitude range, from around 10 to 500,000 times.

EDX (Energy-Dispersive X-Ray).
Energy-dispersive Xray spectroscopy (EDS or EDX) also known as energy dispersive X-ray analysis is a chemical characterization and elemental analysis technique.
2.3.5. Antimicrobial Activity. The antimicrobial activity of produced zinc oxide nanoparticles was tested using the Agar well diffusion assay method in Mueller Hinton agar, which was produced by diluting 28 gm in 1000 ml distilled water and sterilized under autoclave. After sterilization, media was poured to Petri dish and allowed for solidification. Pure culture of Staphylococcus aureus and Escherichia coli (Sneha Clinical Laboratory, Coimbatore) bacteria was swabbed in two different Petri dishes. Make four wells using a well maker. Then, the wells are filled with ZnO NP (1), ZnO NP (2), ZnO NP (3), and ZnO NP (4), and antibiotic disc was used as a positive control, and the Mueller Hinton agar plates are kept for incubation at 37°C for 24 h [17].
2.3.6. Photocatalytic Study. Photocatalysis is well-established for the efficient and long-term elimination of organic and inorganic contaminants from water, and this technology is especially valuable for industrial water treatment. The percentage of dye decolorization was calculated by the following formula [18]: where C is the control and T is the absorbance value.

Methyl Blue.
Methyl blue was prepared by adding 40 ml methyl blue and 20 ml of distilled water that are mixed well. Separate each 30 ml dyes in two beakers. First beaker is taken as a control, and in the second beaker, zinc oxide nanoparticles were added and then place the beakers in the sunlight (or) UV light. Color change is notified in every 1 hour, and OD value is notified.

Methyl Red.
Methyl red was prepared on 40 ml methyl red and 20 ml of distilled water that are mixed well. Then, separate each 30 ml dyes in two beakers. The first beaker was control, and the second beaker was zinc oxide nanoparticles and then placed it under sunlight (or) UV light. After one hour, color changes were observed, and OD values were observed.   Journal of Nanomaterials 2.6. Orange G. 0.025 mg of Orange G and 120 ml distilled water were taken and mixed well, and it was divided into three portions. First portion as 60 ml of Orange G was taken as control. Second portion as 20 ml of Orange G was taken and added with 5 mg of zinc oxide nanoparticles. Third portion as 40 ml of Orange G was taken and added with 5 mg of zinc oxide nanoparticles and then put under sunlight (or) UV light. After one hour, color changes were observed, and OD values were taken.

Seed Germination.
For germination, the green gram seeds were rinsed with sterile water several times for surface sterilization. Four cups were taken the seed germination, and plant growth was processed in the respective cups. In all the four cups, soil was added in half of the size which was essential for seed germination. The soil was added with tap water, control Orange G, treated Orange G, and the nanoparticle, respectively, in each cup. Then, the 10 seeds were soaked in all the cups. Incubate the cups under the sunlight for germination. The seed germination and the plant growth were observed within 2-3 days of incubation. After that, the plant grown in each cup was picked up from the soil, and then, the leaf counting and the length of the stem and root were measured [19].

Result and Discussion
Zinc nanoparticle was prepared from 0.1 M of 1.487 g zinc nitrate and dissolved in 50 ml distilled water. Then, 0.2 M of 0.04 g sodium hydroxide (NaOH) was prepared. Figures 1 and 2, zinc nitrate and 5 ml of sodium hydroxide (NaOH) are added drop by drop and stirred constantly with magnetic stirrer for 2-3 hrs and incubated at room temperature without light contact for overnight. After the incubation, the presence of zinc oxide nanoparticles is observed by the formation of white color.

FTIR (Fourier Transform Infrared Spectroscopy).
To determine the quality and composition of the metal nanoparticles, infrared examinations were conducted. Interatomic vibrations cause absorption bands in metal oxides in the fingerprint area, which is below 1000 cm -1 . Peaks at 3286.70 cm -1 and 1041.56 cm -1 represent O-H stretching and deformation due to water adsorption on the metal surface ( Figure 4). The peaks at 1634.00 cm -1 correspond to stretching and deformation vibrations in ZnO, respectively [22,23].

SEM (Scanning Electron Microscopy).
A scanning electron microscope (SEM) is a microscopy technique that creates a high beam of electrons to view a material. Zinc oxide nanoparticle was synthesized from the chemical reduction method on powdered sample placed under the sunlight and dried, and the morphology was observed under the scanning electron microscopy [24]. SEM (ZEISS) shows that lateral dimension of particles (including the reducing agent, which may be agglomerated      Journal of Nanomaterials of the nanoparticles) is of the order of 310 nm synthesized zinc oxide nanoparticles that are spherical, crystal, and cuboids in nature; external morphology of synthesized nanoparticles in the range of 3 m can be seen ( Figure 5).

EDX (Energy-Dispersive X-Ray Analysis).
The EDX profile shows the chemical analysis of synthesized ZnO nanopattern. EDX pattern shows major emission energy at 1 kg which is the binding energy for zinc with the weight of 51.43% and the atomic range of 20.72%, whereas the 0.5 Kiev which is the binding energy for oxygen with the weight of 47.74% and atomic range of 78.58% which confirms that ZnO has been correctly identified ( Figure 6).
3.6.2. Escherichia coli. Antibacterial activity is defined as anything that kills bacteria or inhibits their growth or reproduction. The four compounds produced from Mueller Hinton agar (38.0 g in 1000 ml distilled water, HiMedia) were utilized as the medium in antibacterial investigations of ZnO using the well diffusion method. The organism used is Escherichia coli bacteria (Figure 8), and the antibiotic is   (Table 2) in the Petri plates and measured [25].

Photocatalytic Study
3.7.1. Methyl Blue. In this study, chemically synthesized zinc oxide nanoparticle was added to the methyl blue dye, and the control was maintained. The control and the nanoparticle added dye were placed under the sunlight for the activity of zinc oxide nanoparticle against the dyes and maintained as such for 48 hrs. After 24 hrs, the absorbance was measured under the UV-Vis spectroscopy at a time interval of 1 hour. After 48 hrs, the absorbance value was taken at an interval of about 2 hrs for the discoloration of dye by the zinc oxide nanoparticles synthesized, and the result was observed by the taken absorbance value (Table 3).

Methyl Red.
In this study, chemically synthesized zinc oxide nanoparticle was added to the methyl red dye, and the control was maintained. The control and the nanoparticle added dyes were placed under the sunlight for the activity of zinc oxide nanoparticle against the dye and maintained as such for 48 hrs. After 24 hrs, the absorbance was measured under the UV-Vis spectroscopy at a time interval of 1 hour. After 48 hrs, the absorbance value was taken at an interval of about 2 hrs for the discoloration of dye by the zinc oxide nanoparticle, and the result was observed by the taken absorbance value (Table 4).
3.8. Orange G. In this study, chemically synthesized zinc oxide nanoparticle was added to the Orange G dye, and the control was maintained. The control and the nanoparticle added dye were placed under the sunlight for the activity of zinc oxide nanoparticle against the dye and maintained as such for 48 hrs. After 24 hrs, the absorbance was measured under the UV-Vis spectroscopy at a time interval of 1 hour. After 48 hrs, the absorbance value was taken at an interval of about 2 hrs for the discoloration of dye by the zinc oxide nanoparticle, and the result was observed by the taken absorbance value (Table 5).
3.9. Seed Germination 3.9.1. Before Seeding. The toxicity study was conducted using seed germination assay and found that the treated water seeds grown well on par with the control seed and corelated that the treated water does not contain any toxic elements. In these cups, 10 seeds are soaked for seed germination process with respective samples and allowed for growth under sunlight of about 2 weeks (Figures 9 and 10).
3.9.2. After 1 Week. After 1 week of incubation, seed germination was observed in all the cups. In each cup, the number of plant seed germination occurred was observed as follows: The control nanoparticle contained plant has a good seed germination and good root, shoot, and growth compared to the treated Orange G and the control Orange G. It has high chlorophyll content compared with control tap water plant. It showed that the nanoparticle can be easily eliminated soil toxic compounds and helped for plant seed germination and growth development process [26]. Recently, [27] studied the photocatalytic studies of methylene blue using zinc oxide nanoparticles and found the degradation of 85%.

Conclusion
In the present study, zinc oxide nanoparticle was synthesized using chemical reduction method. The nanoparticle was characterized using UV-visible spectroscopy, and the plasma peak was found to be 355.2 nm, confirmed the zinc oxide nanoparticle synthesis. The FTIR was also carried out to characterize the zinc oxide nanoparticle. The morphology of the nanoparticle was studied using SEM. The round spherical with 80 to 110 nm size of the nanoparticle was obtained. Further, the EDX was carried out, and the zinc 51.43% confirmed zinc oxide nanoparticles.
The antibacterial study was carried out against E.coli and Staphylococcus aureus, and the zone of inhibition was measured. The zone of inhibition was maximum in E. coli. The photocatalytic study was carried out against methyl blue, methyl red, and Orange G under sunlight. The maximum percentage of dye decolorization after two days in methyl blue is 94%, in methyl red is 66%, and in Orange G is 90%.
The seed germination assay was also studied using zinc oxide nanoparticles of treated water, tap water, control dye, and treated dye. Among the control dye, the treated one showed the maximum chlorophyll content.

Data Availability
The data used to support the findings of this study are included within the article.