Green versus Chemical Precipitation Methods of Preparing Zinc Oxide Nanoparticles and Investigation of Antimicrobial Properties

Bekele, Bulcha; Degefa, Anatol; Tesgera, Fikadu; Jule, Leta Tesfaye; Shanmugam, R.; Priyanka Dwarampudi, L.; Nagaprasad, N.; Ramasamy, Krishnaraj

doi:https://doi.org/10.1155/2021/9210817

Journal of Nanomaterials

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Abstract Introduction Results and Discussion Conclusions Data Availability Conflicts of Interest References Copyright Related Articles

Special Issue

Green Route Synthesis of Antimicrobial Nanoparticles

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Research Article | Open Access

Volume 2021 | Article ID 9210817 | https://doi.org/10.1155/2021/9210817

Green versus Chemical Precipitation Methods of Preparing Zinc Oxide Nanoparticles and Investigation of Antimicrobial Properties

Bulcha Bekele,¹Anatol Degefa,²Fikadu Tesgera,¹Leta Tesfaye Jule,^1,3R. Shanmugam,⁴L. Priyanka Dwarampudi,⁵N. Nagaprasad,⁶and Krishnaraj Ramasamy^3,7

Academic Editor: Shanmugam Rajeshkumar

Received16 Jul 2021

Revised05 Aug 2021

Accepted03 Sept 2021

Published20 Sept 2021

Abstract

Comparison of green and chemical precipitation method syntheses of zinc oxide nanoparticles (ZnO NPs) was performed, and antimicrobial properties were investigated. Avocado, mango, and papaya fruit extracts were carried out for the green synthesising methods, while the chemical precipitation method was chosen from chemical synthesis methods. Zinc nitrate was used as a salt precursor, whereas leaf extract was served as a reducing agent for green synthesising methods. In addition, sodium hydroxide, polyvinyl alcohol, and potassium hydroxide were used as reducing agents in the case of chemical precipitation synthesis methods. ZnO NPs were characterised by characterizing techniques such as Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The antimicrobial activities of prepared nanoparticles were evaluated on Bacillus subtilis (B. subtilis), Staphylococcus aureus (S. aureus), and Salmonella typhimurium (S. typhimurium). The particle sizes of the prepared samples which were evaluated by the Scherrer equation were in the range of 11-21 nm for green synthesis, while 30-40 nm for chemical precipitation synthesis methods. Small agglomerations were observed from SEM results of prepared ZnO NPs from both methods. Prepared ZnO NPs were showed strong antimicrobial properties. From the result, the inhibition zone was in the range of 15-24 mm for the green route and 7–15 mm for chemical precipitation methods, where the standard drugs have 25 mm of the zone of inhibition. A green synthesised method of preparing ZnO NPs gives promising antimicrobial properties compared to chemical synthesis and is also eco-friendly and safe compared to the chemical synthesis.

1. Introduction

In the previous period, nanoscience and technology of nanocomposite materials are emerging in material science fields. It manipulates matter at the atomic scale that produces a nanoproduct of new novel properties [1]. Nanomaterials are manufacturing and engineering materials that are characterised at least in one dimension in nanoscale (1 nm–100 nm). It is useful to develop structures and devices of various materials [2]. Nanoparticles are the parts of nanometres widely used in the application of medicine [3], environmental protection [1, 3], sunscreen [4], and cosmetics technology. Biomaterials make challenges while researchers are studying material science fields. Researcher loses a lot of time to generate a new idea, especially on biomaterials in medicine applications on antibiotic resistance microorganism [1, 3, 4]. Physical and chemical differences of characterisation of nanocomposite materials include mechanical and biological properties [5]. A nanoparticle gave greater properties than bulk materials and had an appreciation application in human life. It has a lower surface area to volume ratio and shows essential properties like versatility, high strength, and electrical conductivity; in comparison to bulk material with the same chemical makeup, this material has superior strength and affinity [6]. New materials with innovative purposes can be created by manipulating the shape and size of atoms and molecules on the nanoscale. [7] Nanoparticles (NPs) have a wide range of applications along with memory schematics and cordless electronic logic as well as chemical sensing and electrometers as well as computer transistors. They also have antibacterial and catalytic behaviour as well as magnetic characteristics, mechanical resistance, and conductivity. Those nanoparticles can also be used in various other fields, such as tumour heating, medicine administration, filtration, nanocomposites, and diagnostic scanning, to name a few. Since the last few generations, there has been much interest in inorganic NPs with increased characteristics [8]. Through the use of NP characterisation, it is possible to determine the direction, fractal sizes, crystallinity, and a number of the interactivities.

ZnO NPs are used as supercapacitors because of their high energy density, electrochemical activity, environmental friendliness, abundant availability, and inexpensive cost. Because of its large surface area, ZnO nanoparticles (ZnO NPs) have been used to eliminate arsenic and sulphur from water. ZnO nanopowders are commonly employed in sectors such as blue laser diodes, solar cells, and conductive thin films [2, 4–8]. When compared to green synthetic methods, these methods have some disadvantages, such as being challenging to operate, costly, emitting radiation, requiring very high pressure, and being toxic [9]. The medical uses of some technologies are harmed by the adsorption of hazardous chemical species on the surface. Green synthesis has been developed to prevent chemical toxicity and an intense environment of physical and chemical processes. They are suitable for biomedical applications due to their characteristics. The shape of the NPs is influenced by a variety of factors during green synthesis, including plant extract content, pH, temperature, reaction duration, and solvent [10]. Because of the existence of useful photochemicals such as ascorbic acids, phenols, and carboxylic acids, plant biodiversity was being extensively employed in the production of green synthesis. Therefore, the aims of this study are to synthesise ZnO NPs by green and chemical precipitation methods and examine its antimicrobial properties.

2. Experimental Details

2.1. Materials and Chemical Used in Green Synthesis

Here, avocado, papaya, and mango fruits were collected from the land farm of Kellem Wollega Zone, Mugi Woreda. In addition, zinc nitrate hexahydrate Zn(NO₃)₂.6H₂O, zinc acetate dihydrate (Zn(CH₃COO)₂.2H₂O), zinc sulphate heptahydrate (ZnSO₄.7H₂O) of 99% pure, sodium hydroxide (NaOH), polyvinyl alcohol (PVA), potassium hydroxide (KOH), and ethanol were purchased from Addis Ababa Chemical Shop, Piazza. Different types of glasses of double-distilled water were used for washing.

2.2. Green Synthesis/Methods of Extractions

The green synthesis methods were developed from the procedure developed in Safavinia et al. (2021). Avocado fruit sizes were reduced using a knife by cutting and washed six times by double distilled and three times by ethanol. 16 g of avocado was mixed with 170 mL of double-distilled water and then continuously heated for the duration of 30 min on a magnetic stirrer. The aqueous solution of avocado extractions was obtained and filtered by Whatman paper and stored at normal temperature for later use. 10 g of Zn(NO₃)₂.6H₂O were measured by triple bean balance and dissolved in 30 mL of double-distilled water by using a high-speed magnetic stirrer at constant temperature for 30 min. Then, 1.75 M of avocado extract were added to Zn(NO₃)₂.6H₂O combination mixture and continuously stirred for 1 hr. The solutions were now settled for 24 hr at normal temperature. Similar procedures were repeated for papaya and mango.

2.3. Chemical Precipitation Methods of Zinc Oxide Nanoparticles

The chemical precipitation methods used in the chemical synthesis were Zn(NO₃)₂.6H₂O, Zn(CH₃COO)₂.2H₂O, and ZnSO₄.7H₂O as salt precursor, while NaOH, PVA, and KOH are reducing agents. According to the procedure developed in Bekele et al. (2021), zinc oxide nanoparticles were prepared. All zinc salts and reducing agents were measured by beam balance. 12 gm of the sodium hydroxide (NaOH) solution was blended in 70 mL of twice distilled water and agitated. Under gentle magnetic stirrer for 30 min. Again, 4 gm of Zn(NO₃)₂.6H₂O was dissolved into double-distilled water of 30 mL and stirred continuously for 20 min. Slowly drop by drop, Zn(NO₃)₂.6H₂O solution was added to NaOH solution and stirred continuously for 2 hr at 60°C. At this stage, gel-like solutions were formed and left to cure in an oven at a temperature of 160°C for 10 hr overnight. Then, the sample was taken furnace (Model: MC2-5/5/10-12, BIOBASE, China) and calcinated at 300°C for 6 hrs. Similar procedures were followed for other salt precursors, zinc acetate (Zn (CH₃COO)₂.2H₂O), and zinc sulphate hydrate (ZnSO₄.7H₂O).

2.4. Characterisation Techniques

In order to report the presence of functional groups attached to a surface of the synthesised nanoparticles, the Perkin Elmer FT-IR spectrum was used in the scanning region of 4000-400 cm^-1 and a resolution of 4 cm^-1 for the analysis of the connected functional groups to the exterior of the synthesised ZnO NPs. The X-ray diffraction (XRD) distribution of ZnO nanoparticles was acquired utilising an XPERT-PROX-ray diffractometer that generated Cu-K radiation (with an angular resolution of 1.5418 angstroms). It is being employed to evaluate the crystalline particle size that has been manufactured. For the purposes of characterisation, a tiny quantity of powder samples was used. At room temperature, when the X-ray generators were working at 40 kV and transmitting a charge of 30 mA to the target, it was considered a successful hit. Properties were measured at room temperature in steps of 0.02, over an ambient temperature of 100 to 800 degrees Celsius, with the diffractometer connected to a computer for the collection of data and presentation of characterisation results. It was necessary to authenticate the structure of the crystal by comparing its peak positions with those of the existing standard data. For each sample, about 0.4 g of produced ZnO NPs were grinded to tiny powders and quantified using a beam balance before being transferred to a metal plate. Once the nanoparticles had been formed, it was discovered that the nanoparticle morphology might be investigated using a scanning electron microscope (Hitachi, H-7600), which functions under extreme vacuum as well as have magnifications varying between 20x to around 30,000x, with resolutions varying between 50 and 100 nm. Increased magnification of the ZnO NPs was achieved by reducing the raster width of the specimen, and vice versa, for the ZnO NPs with fixed sizes. A quartz cuvette with a diameter of 1 cm has been used to examine the absorbance spectra of produced ZnO NPs utilising UV-vis spectroscopy (Perkin Elmer Lambda 950), which was run over a wavelength range of 200-500 nm. The ZnO NPs were combined using double-distilled water until being placed in a quartz cuvette to produce their solutions, which were then analysed. After that, the cuvette was placed in an ultraviolet-visible spectrophotometer, where the absorption spectra of ZnO NPs were determined.

2.5. Antimicrobial Activity

The green and hydrothermal methods of synthesised zinc oxide nanoparticles were evaluated for antibacterial activities through disc-diffusion methods as developed in literature [11–14]. A growing media was created, autoclaved, and then moved to a putrefaction Petri plate in order to execute the technique. It was necessary to prepare the growing media, autoclave it, and transfer it aseptically to sterilised Petri plates. Slants of test tube media were transferred to Petri plate media, and sterile and dried paper discs (6 mm) were inoculated with 10 L of freshly prepared ZnO NPs, either through a 0.45-millipore filter or through a 0.45-millipore filter with freshly prepared ZnO NPs for both the synthetic and the synthesis methods. The discs that had been impregnated were cured in a laminar flow cabinet. Controls and discs were planted on newly seeded microbial lawns in conjunction with the experimental discs. Negative controls included sterile water (10 L per disc) and streptomycin (1 per cent per disc), while positive controls included 1 per cent streptomycin (10 L per disc). Several investigations were carried out in triplicate to ensure accuracy. With a zone reader, the regions of inhibition or antimicrobial activities were counted in millimetres (mm) on the Petri plates after they had been incubated at their respective temperatures.

3. Results and Discussion

3.1. FT-IR Spectral Analysis of ZnO NPs

ZnO NPs were prepared by green and chemical precipitation synthesised techniques. Green synthesising methods of ZnO NPs from avocado, mango, and papaya extracts and chemical synthesis by hydrothermal methods were depicted in Figures 1(a) and 1(b), respectively. ZnO NPs were prepared by hydrothermal methods from zinc nitrate hexahydrate (Zn (NO₃)₂.6H₂O) and sodium hydroxide (NaOH), zinc sulphate heptahydrate (ZnSO₄.7H₂O) and polyvinyl alcohol (PVA), and zinc acetate dihydrate (Zn(CH₃COO)₂.2H₂O) and potassium hydroxide (KOH) as S1, S2, and S3, respectively. The FT-IR spectrum shows the transmission band around 487, 808, 1425, 1594, 2341, 2919, and 3435 cm^-1 in the region of 400–4000 cm^-1. The transmission band observed at 487 and 808 cm^-1 shows the presence of ZnO NPs for all extracts [13]. The peak observed at 1425, 1594, and 2341 cm^-1 shows the presence of carbonyl (-C=O) groups are stretching vibration, CN stretching, and COO- antis metric stretching, respectively [14]. The peak observed around 2919 and 3435 cm^-1 depicts the presence of C-H stretching vibrations to CH₂ and CH₃ [15]. Bioactive compounds were also found in the region between 808 and 1425 cm^-1. Similarly, broad transmission peaks were depicted from chemical synthesis by the hydrothermal method. FT-IR spectrum shows transmission peaks at 498, 895, 1892, 2279, 2689, 3321, 3689, and 3896 cm^-1 for all chemical synthesis of S1, S2, and S3. ZnO NPs were observed at and around the 498 and 895 cm^-1, where the textile bond and oxygen vacancies were observed [15]. The peaks observed at 1892 and 2279 cm^-1 represents symmetrical stretching of zinc carboxylate for all samples. The peaks observed at 2689 and 3321 cm^-1 represents the O-H hydroxyl group’s fluctuation [16]. In addition, the transmission band observed at 3689 and 3896 cm^-1 depicts the valence vibrations of water molecules. The broad peaks of ZnO NPs were observed in chemical synthesis methods than in the green synthesis methods. As the dimension of zinc oxide nanoparticles increases, the content of the impurities attached to the samples were decreased for both synthesising methods.

(a)

(b)

3.2. XRD Spectral Analysis

ZnO NPS were synthesised through green and chemical precipitation methods (as shown in Figures 2(a) and 2(b)). The XRD pattern of ZnO NPs was generated using avocado, mango, and papaya extracts in green synthesis methods and S1, S2, and S3 samples of zinc nitrate, zinc acetate, and zinc sulphate salts with their corresponding reducing agents of sodium hydroxide, polyvinyl alcohol, and potassium hydroxide, respectively. In accordance with JCPDS card no. 36-1451, many of the XRD diffraction patterns of ZnO NPs are in perfect agreement with the hexagonal wurtzite structure (hexagonal phase, crystal structure P63mc) with lattice parameters of and as reported in the recovered products [17]. Nine well-defined spectral peaks were observed at 31°, 34°, 36°, 47°, 56°, 62°, 66°, 68°, and 69° with their corresponding reflection (100), (002), (101), (102), (110), (103), (200), (112), and (201) crystal planes, respectively, for green synthesis methods for mango and papaya extracts. There is a shift towards a smaller angle in the case of the avocado extract by ten degrees when compared to the other extracts. From the graph, the narrow and sharp peaks of diffractions were observed that the prepared nanoparticles are well crystallised. The diffraction peaks of chemical precipitation synthesising methods for samples S1, S2, and S3 of zinc salt precursors correspond to Bragg reflections with two theta values of 17.29°, 35.08°, 38.39°, 40.12°, 47.95°, 53.93°, 62.95°, 70.77°, and 76.10° to their corresponding Miller Bravais indices (100), (002), (101),(102), (110), (103), (200), (112), and (201) planes of hexagonal structure phases of ZnO, respectively. These XRD patterns of the samples are in good agreement with the standard data of the hexagonal ZnO wurtzite structure of standard JCPDS card 36-1451. Sharp and intense peaks of chemically prepared nanoparticles were indicated that ZnO NPs with highly crystalline were successfully prepared. The average particle sizes of green and chemically prepared ZnO NPs were calculated by the Scherrer equation. whereby is the mean dimension of nanoparticles, is the frequency of radiation, is the whole width half peak in radians, and is the degree diffraction [15–18]. Avocado, mango, and papaya extracts of green methods have been used to synthesis nanoparticles with mean sizes of 11 nm, 16 nm, and 21 nm, respectively. The average sizes of the nanoparticles produced with avocado, mango, and papaya extracts are tabulated in Table 1.

(a)

(b)

The average sizes of the zinc oxide nanoparticles produced by chemical precipitation methods are seen in Table 2. Based on the foregoing findings, it can be inferred that throughout the instance, green synthesising methods gave smaller nanoparticles which is more applicable for biomedical application [18, 19].

3.3. SEM Analysis of ZnO NPs

Figure 3(a) shows the scanning electron microscope (SEM) morphology of green synthesised ZnO NPs from avocado, mango, and papaya extracts. An avocado extract of SEM image depicts that the prepared nanoparticle is rod-like images, while flower images were observed in the case of mango extract with small agglomerations. It is noticed that small agglomeration of green synthesis methods were probability obtained from the escape of volatile substances or gases during the calcination process. Nanotube-like images without any agglomeration were also seen from SEM results of papaya extracts, and the same results were reported in the literature of [20].

Figure 4 depicts SEM morphology of ZnO NPs by chemical precipitation method from zinc nitrate hexahydrate, zinc acetate dehydrate, and zinc sulphate heptahydrate from sodium hydroxide, polyvinyl alcohol, and potassium hydroxide as reducing agents, respectively. Highly magnified semispherical shape of prepared nanostructured was seen in case of S1 and uniform distributed spherical shape in S2 prepared sample. In addition, an almost nearly spherical shape was observed in the case of sample S3. In both green and chemical synthesising methods, SEM results confirm the crystalline nature of the ZnO NPs [21].

3.4. Antimicrobial Investigations of ZnO NPs

The antibacterial investigation of green and chemical precipitation methods of prepared ZnO NPs was estimated on three bacteria such as B. substilis (Bacillus subtilis), S. aureus (Staphylococcus aureus), and S. Typhimurium (Salmonella Typhimurium) as shown in Tables 3. It is known that some bacteria consist of the cell membrane, cytoplasm, and cell wall. Especially, peptidoglycan membrane cell is found in Gram-positive bacteria and has hard cell wall 20-80 nm [22]. In addition, Gram-negative bacteria have a double cell membrane, i.e., plasma and outer cell, which have 7-8 thickness [19, 21]. But ZnO NPs able to penetrate easily through this hard, strong cell membrane. ZnO NPs exert antimicrobial action and destroy bacterial growth [23].

In this study, green synthesis methods of ZnO NPs from avocado, mango, and papaya extracts were subjected to antibacterial activity to investigate antimicrobial activities which are shown in Figure 5. From all extracted samples, avocado extract has more inhibition potential on all bacterial strains. ZnO NPs applied on Bacillus subtilis show high inhibition potential than others. In other words, because the particle size of these strains has been in the nanoscale, which can easily impede the development of these strains, the inhibition of such strains did not take particle size into consideration [24]. Figure 6 illustrates the antimicrobial activities of ZnO NPs prepared by chemical precipitation methods of S1, S2, and S3 samples of zinc salt precursors. These results have good agreements with standard references of antimicrobial activities (streptomycin).

As shown in Figure 6, The sample S1 is the most effective of the inhibitors when compared to the other salt precursors. Antimicrobial activities of sample S3 were low compared to S1 and S2. Chemical precipitation methods of ZnO NPs have more inhibition potential on the bacterial strains of S. aureus. ZnO NPs prepared by both chemical and green synthesis have been found to be key interest in antimicrobial activities [25–28]. ZnO NPs prepared by green synthesis methods were found to be more active against this selected B. subtilis, S. aureus, and S. Typhimurium compared to chemical precipitation methods and have highly antibacterial agents. The results of this finding have a good agreement with previous studies [27]. Furthermore, ZnO NPs prepared from avocado, mango, and papaya extract were highly active antimicrobial properties with slight differences. Hence, fabrication of ZnO NPs from avocado, mango, and papaya extracts are highly recommended rather than chemical synthesis for microbial agents. In the green route, bioactive compounds were present as stabilising, reducing, and capping agents [28–30]. Furthermore, green extracts produce several biomolecules, including carbohydrates and proteins; accompanying prime metabolites and secondary metabolites that have been linked to the elimination of metal ions and stability has been linked to nucleic acids [31–40]. Not only antimicrobial agents but also green synthesis is also needed for environmental pollution protections by developing and adopting benign eco methods [41–50].

4. Conclusions

Comparison of green and chemical precipitation method synthesis of zinc oxide nanoparticles (ZnO NPs) was performed, and antimicrobial properties were calculated. Avocado, mango, and papaya fruit extracts were carried out for the green synthesising methods, while the chemical precipitation method was chosen from chemical synthesis methods. Zinc nitrate was used as a salt precursor, whereas fruit extracts were served as reducing agents for green synthesising methods. In addition, sodium hydroxide, polyvinyl alcohol, and potassium hydroxide were used as reducing agents in the case of chemical precipitation synthesis methods. ZnO NPs were characterised by characterizing techniques such as Fourier transform infrared (FT-IR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The antimicrobial activities of prepared nanoparticles were evaluated on E. coli and S. aureus. The particle sizes of the prepared samples which were evaluated by the Scherrer equation were in the range 11-21 nm for green synthesis, while 30-40 nm for chemical precipitation synthesis methods. Small agglomerations were observed from SEM results of prepared ZnO NPs from both methods. Prepared ZnO NPs showed strong antimicrobial properties. From the result, the inhibition zones were in the range of 15-24 mm for the green route and 7-15 mm for chemical precipitation methods, where the standard drugs have 25 mm of the zone of inhibition. A green synthesised method of preparing ZnO NPs gives promising antimicrobial properties compared to chemical synthesis and is also eco-friendly and safe compared to the chemical synthesis. The results of this finding have good agreement with previous studies. Furthermore, ZnO NPs prepared from avocado, mango, and papaya extract were highly active antimicrobial properties with slight differences. Hence, fabrication of ZnO NPs from avocado, mango, and papaya extracts is highly recommended rather than chemical synthesis for microbial agents.

Data Availability

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

Conflicts of Interest

The authors declare that there are no conflicts of interest.

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Copyright © 2021 Bulcha Bekele et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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