Green synthesis of ZnO nanoparticles by pineapple peel extract from various alkali sources

ABSTRACT Zinc oxide nanoparticles (ZnO NPs) are concerned as potential materials due to their wide-ranging applications. The green synthesis of ZnO NPs using of plant extract as capping agent has been attracted much of interest of reserachers. Pineapple peel wastes are aboundance in Thailand and its extract contained high levels of phytochemical compounds (flavonoids and their derivatives). In this study, pineapple extract was used as a capping agent in ZnO NP synthesis, and KOH and a lye solution were used as reducing agents for comparison. The XRD patterns exhibit pure-phase ZnO with high crystallinity. The averages of the most petite crystalline sizes obtained from the Scherrer equation calculation of the prepared ZnO powder are 64.61 and 65.41 nm for KOH solution and lye use, respectively. Fourier transform infrared (FTIR) spectroscopy confirmed the presence of ZnO particles and pineapple extract residue in the as-received powder. Scanning electron microscope (SEM) images and transmission electron microscope (TEM) images showed the nano-size of the synthesized particles. The flower-like ZnO-NPs from a 0.06 M zinc precursor and KOH solution exhibited fascinating optical properties. Finally, all the results showed that lye from wood ash could be applied in ZnO nanoparticle synthesis using pineapple peel extract.

Physical and chemical processes dominate the synthesis of ZnO nanoparticles. The physical processes concerned require high vacuum and energy consumption, while the chemical processes are environmentally unfriendly. Therefore, the green synthesis approach is gaining increasing attention today as an environmentfriendly, cost-effective, and safe alternative. The nanoparticle green synthesis method is an approach to synthesizing nanoparticles using extractions from plants, bacteria, fungi, algae, etc. These plant extracts make excellent phytochemical sources due to the presence of different classes of phytochemicals such as polyphenols, terpenoids, flavonoid alkaloids, and sugar that act as both reducing agents and capping or stabilization agents. Numerous parts of the plants, including the roots, leaves, stems, seeds, fruits, and peels, have also been applied to ZnO nanoparticle synthesis [7][8][9][10][11][12]. The current Sustainable Development Goals (SDGs) are concerned with economic growth and environmental balance; making the use of fruit peel biowaste is an attractive target. There are a few reports on the use of fruit peel biowaste as a phytochemical source examples include the use of dragon fruit (Hylocereus polyrhizus) peel [13], Garcinia mangostana pericarp [14], Citrus sinensis (orange), Citrus paradisi (grapefruit) and Citrus aurantifolia (lemon) [15].
Pineapple (Ananas cosmosus) is the popular fruit that is most widely planted in Thailand. Countless tons of pineapple waste are generated each year, creating disposal problems due to the fruits wide range of applications in the food industry, such as in beverages, jams, purees, and pastes. The present of phytochemicals such as total phenolic and alkaloid content in dried pineapple peel [16], wet pineapple peel [17], and pineapple slice [18] extracts were reported. Subsequently, the pineapple peel extract was used and studied with respect to ZnO NPs synthesis [19,20]. Nevertheless, the resulting green synthesis of ZnO nanoparticles was not exactly a green method, because sodium hydroxide (NaOH) or potassium hydroxide (KOH) pellets or solution were still employed as the alkali source and reducing agent in the reaction. Traditionally, lye is an alkaline liquid obtained by leaching wood ash with water. Lye most commonly refers to metal hydroxides such as sodium hydroxide or potassium hydroxide, which are highly soluble in water to produce basic caustic solutions. The major elements of wood ash are calcium (7-33%), potassium (3-4%), magnesium (1-2%), manganese (0.3-1.3%), phosphorus (0.3-1.4%), and sodium (0.2-0.5%) [21]. Moreover, the synthesis of ZnO nanoparticles from pineapple peel waste using lye from wood ash has not been reported previously.
Therefore, in this work, the lye from wood ash and KOH solution was used as a reducing agent in the green synthesis of ZnO nanoparticles with pineapple peel extract acting as the capping agent. The ZnO NPs prepared from the two alkaline sources were investigated by X-ray diffraction analysis, scanning electron microscopy, and transmission electron microscopy for physical properties. Fourier transform infrared spectroscopy was employed to confirm the presence of pineapple peel extract and the existence of ZnO in the synthesized NPs.

Materials
Analytical grade chemicals were used in the green synthesis of ZnO nanoparticles. Zinc nitrate hexahydrate (Zn(NO 3 ) 2 · 6H 2 O, 98% purity) was purchased from Millipore Sigma (USA) and used as a precursor to synthesize ZnO nanoparticles. The "Phulae" pineapple cultivars (Ananas comosus L. Merr) were planted, harvested, and sold by the Agri Coffee Pavilion shop, The School of Agriculture and Natural Resources, at the University of Phayao. The pineapple peels were collected from October to November 2020. Potassium hydroxide (KOH) pellets were purchased from Millipore Sigma (USA) and a reducing agent for comparison. Wood ash was collected from a dried longan factory in Lamphun province, Thailand. De-ionized (DI) water and 95% ethanol were used in the synthesis or solution preparation.

Methods
In this study, the procedures were divided into three parts, as follows:

Preparation of pineapple extract solution
The fresh pineapple peels were washed with water three times to remove dirt, then cut into pieces with a knife and crushed and mixed with a blender. The resulting 600 g crushed pineapple peel was soaked and boiled in 3 L of the DI water for 60 minutes, and the DI water was then drained three times. The received crushed pineapple peel was soaked in 95% ethanol in a 1:3 ratio for 24 hours at ambient temperature. The extracts were filtered using Whatman No. 1 filter paper and the filtrates were then concentrated by evaporation using a rotary evaporator. The filtered solution was collected and stored in a clean, dry beaker at 4 •C for further use.

Preparation of the ZnO NPs
The ZnO nanoparticles were synthesized using 2 different alkali sources, 5 M KOH solution and lye. For KOH use, the zinc precursor was prepared by combining 0.02, 0.04, 0.06, 0.08, and 0.1 M in 10 ml. of the prepared-pineapple peel extract to achieve optimized conditions. The 0.06 M zinc nitrate solution was prepared in 10 ml. of the prepared-pineapple peel extract for lye employment. The pH of the solution was maintained at around pH 12. The solution was refluxed at boiling temperature continuously for 1 h until a white precipitate was observed. The precipitate was centrifuged at 15,000 rpm for 2 minutes and washed several times with distilled water and then with 95% ethanol. The precipitate was further dried in an oven at 80 •C overnight.

Characterization 2.2.3.1. Total phenolic content analysis. The total
phenolic content of the crude pineapple peel extract (PPE) was determined using the method described by Singleton [22] with some modifications. Gallic acid was used as a standard calibration curve. Briefly, into 0.20 mL of extract (1 mg/mL) was added 1.00 mL Folin-Ciocalteu reagent (10-fold dilution in deionized water), mixed thoroughly, and kept in the dark for 5 min. Then, 0.40 mL of 7.5% (w/v) sodium carbonate solution was added, mixed well, and incubated in the dark for 60 min. The absorbance was measured at 765 nm (Thermo Fisher Scientific UV-Vis Spectrophotometer) using deionized water as a blank. The above procedure was conducted in triplicate. TPC was calculated from the linear equation of a gallic acid standard curve and expressed as milligrams of gallic acid equivalent (GAE) per 1 g of dry weight (mg GAE/g).

Total carbohydrate content. A total sugar
assay was performed according to the method of Nielsen [23] with minor modifications. Glucose was used to produce a calibration curve. A series of test tubes containing 0, 10, 20, 40, 60, 80 µg/mL of standard solution in a total volume of 2 mL were filled with glucose stock solution (100 mg/L). To each test tube, 0.05 mL of 80%w/v phenol solution was added and shaken well. A direct stream of 5 mL concentrated sulfuric acid was then added and shaken well. The tubes were set aside for 10 min and kept in a water bath at 25°C for 20 min. Absorbance was read at 490 nm using DI as a blank. The same procedure was repeated with PPE before and after ZnO NP synthesis at a concentration of 5 mg/mL each time.

Total flavonoid content.
Total flavonoid content (TFC) was determined by the aluminum chloride colorimetric method as reported by Woisky and Salatino [24] with minor modifications. Quercetin was used to produce a standard calibration curve. A series of 0, 20, 40, 60, 80 and 100 mg/L samples of quercetin standard solution were prepared from 100 mg/L quercetin stock solution. Into 0.5 mL of each standard solution was entered 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. After incubation at room temperature for 30 min, the absorbance was read at 415 nm using ethanol as a blank. The actions were conducted in triplicate. The above procedure was repeated with PPE before and after production of the ZnO NP synthesis solutions (5 mg/mL). TFC was calculated from the linear equation of the quercetin standard curve and expressed as milligrams of quercetin equivalents (QUE) per 1 g of dry-weight crude extract (mg QUE/g).

Material characterization. The functional
groups of the synthesized-ZnO particles and the residue of the extract were characterized by Fourier transform infrared (FTIR) spectroscopy in the frequency range from 400 to 4000 cm-1 using a Thermo Nicolet Nexus FTIR spectrophotometer (iD5-ATR, USA). X-ray diffraction (XRD) patterns were collected by Rigaku X-ray diffractometer (MiniFlex500, Japan). Data were recorded in the 2θ range from 20• to 80• (diffraction angle 2θ) using Cu-Kα radiation (λ = 1.5418 Å) generated at 30 kV and 30 mA. The as-received ZnO particles were investigated by scanning electron microscope (SEM) to obtain morphological information. The entire sample was coated with gold before SEM analysis.
A TEM micrograph was obtained using a JEM 2010 (JEOL) by dropping a droplet of sonicated dispersion of the sample directly onto a carbon-coated copper grid that was subsequently dried. Finally, the optical properties of the as-received ZnO NPs were examined by diffused reflectance spectroscopy (DRS).

Results and discussion
Bioorganic molecules in the crude pineapple peel extract (PPE) before and after ZnO NPs synthesis were assessed. Total phenolic content (TPC) was determined based on the method described by Singleton [22]. The gallic acid standard curve is shown in Figure 1. The TPC valueds for the PPE before and after ZnO NP synthesis were determined to be 3.71 and 0.3 mgGAE/g of the crude extract. Total carbohydrate content (TCC) was determined based on a phenol-sulfuric colorimetric assay [23] using glucose as a standard and the calibration curve is shown in Figure 2. The TCC values for the PPE before and after ZnO NPs synthesis were 1.12% and 0.33% glucose equivalent. Total flavonoid content (TFC) was determined by aluminum trichloride colorimetric assay using quercetin as standard [24] and the calibration curve is shown in Figure 3. The TFC values for PPE before and after ZnO NPs synthesis were 0.09 and 0.01 mgQUE/g of the crude extract.
The IR spectrum of PPE ( Figure 4) showed a broad peak from at 3369 cm −1 corresponding to the hydroxyl group (OH). The peaks at 2927, 2853, and 1462 cm −1 correspond to aliphatic CH (CH 2 ; CH 3 ), the peaks at 1604 cm −1 and 1514 cm −1 belong to the aromatic ring (C = C) and the peaks at 1030 cm −1 belong to the C-O group of alcohol, ether, or ester [25]. The  prepared ZnO powders were examined for residue of the pineapple extract by Fourier-transform infrared (FTIR) spectroscopy. The FTIR spectra of the synthesized ZnO nanoparticles with different zinc precursor concentrations and alkaline sources are displayed in Figure 5. The prominent broadband corresponding to stretching vibrations of the hydrogen bond (O-H) of phenol appears in the range of 3600-3400 cm -1 in all infrared spectras. The small peaks at 2977.50 cm −1 correspond to alkane C-H stretch vibrations. The bands at 1645.68 cm −1 were attributed to the C = C stretching vibration of the carbon bond in the aromatic ring. The strong band located at about 897.70 cm -1 is a signal of C-H bending in the aromatic ring [26]. The peaks below 400-600 cm −1 are in the region known as the characteristic region of the intrinsic adsorption bands of metal oxides [27]. The strong bands located at about 559.26 cm -1 and 420.41 cm −1 are signals of the intrinsic adsorption bands of metal oxides corresponding to the Zn-O bond of the zinc oxide phase [28,29]. The FTIR results show the phytochemical residues from pineapple peel extract and the formation of ZnO nanoparticles. The mechanism of ZnO-NP formation from aqueous zinc nitrate has been shown to occur via layered zinc salts through zinc nanoparticles [30], while biogenic synthesis of ZnO-NPs is not well elucidated. Two plausible mechanisms of ZnO-NP formation using zinc ion precursors via biogenic synthesis have been proposed. In both mechanisms, bioorganic molecules such as carbohydrates, phenolic compounds, and flavonoids play a crucial role in ZnO NP formation [31]. In the first mechanism, bioorganic molecules form complexes with Zn 2+ ions, which are then under high pH conditions; these complexes then collapse to generate zinc hydrate. This pathway requires a further calcination process to obtain ZnO NPs. Another mechanism occurs via bioreduction of Zn 2+ ions to Zn metal by bioorganic substances, followed by oxidation of metallic zinc, probably with dissolved oxygen molecules in the media to form ZnO nuclei. Bioorganic molecules act as stabilizers to prevent the ZnO NPs from agglomerating [31,32]. The PPE comprised diverse chemical entities, mainly carbohydrates and phenolic compounds with trace amounts of flavonoids. These bioorganic substances contain hydroxyl groups (OH), carbonyl (C = O), aromatic (C = C), and aliphatic (C-H) (Figure 6), which are required to facilitate the biogenic synthesis of ZnO-NPs [31][32][33]. These molecules donate electrons from hydroxyl groups to the Zn 2+ ions to form metallic zinc. The metallic zinc reacts with an oxidizing substance, presumably oxygen, in the solution to form ZnO NPs, and bioorganic molecules then act as a capping agent to stabilize the resulting ZnO-NPs. The investigation of PPE before and after ZnO NP synthesis showed that the carbohydrate and phenolic contents were reduced by 70% and 91%, respectively. This could imply the involvement of these bioorganic molecules in the biogenic synthesis of the ZnO NPs by PPE.
X-ray diffraction spectroscopy was employed for phase characterization of the as-synthesized ZnO nanoparticles using pineapple peel extract as a capping agent with a reducing agent of KOH solution or lye. All the results indicate that pure ZnO nanoparticles were successfully synthesized. The peak patterns of the as-prepared ZnO nanoparticles are shown in well-known Scherrer equation: D = 0.9λ/(βcosθ), where D represents crystallite size (Å), λ is the wavelength of CuKα radiation (Å), and β is the corrected full width at half maximum (FWHM) of the diffraction peak. Basically, the defect and residual strain in crystal are directly related to the crystalline size because they obstruct crystal growth. The reduction in crystalline size reflects the higher degree of internal defect and strain. The strain (ε) and dislocation density (δ) can be further studied by applying XRD data using the following equations ε = (βcosθ)/4 and δ = 1/(L) 2 . As shown in Table 1  This finding confirms the assumption that residual stress causes low crystal growth. Similarity, lye-prepared ZnO exhibits a crystallite size of 65.41 nm with values of 0.00053 and 0.00023 line/ m 2 for strain and dislocation density, respectively. This result clearly shows that lye is a good reducing agent. Green synthesis with lye solution has potential for ZnO nanoparticle production. Interestingly, lye-prepared nanostructured metal oxide production extends our understanding of the synthesizing process. SEM images (Figure 8) show aggregated nanoparticles with different morphologies of ZnO obtained under various desired conditions. These nano-sizes correspond with the above Debye-Scherrer calculation results. Especially notable are the flower-like clusters of prepared ZnO nanoparticles acquired from 0.06 M zinc precursor using KOH solution and lye. For further confirmation, the EDS spectras of the elemental analysis of ZnO NPs are shown in the inserted image. Only zinc  and oxygen signals are detected in the spectrum, confirming that the synthesized nanoparticles are pure ZnO. For transmission electron microscope analysis, the ZnO nanoparticles from 0.06 M zinc precursor were selected. The TEM image in Figure 9(a) shows clusters of irregular and hexagonal ZnO-NPs shapes whose diameter was under 100 nm. The selected area electron diffraction (SAED) pattern in Figure 9(b) exhibits the combined spotty ring pattern of the (100), (101), (102), and (103) planes of the hexagonal ZnO structure (P63m), which is consistent with JCPDS card No. 36-1451, and the above XRD results. The HRTEM image (Figure 9(c)) of single nanoparticles reveals a reticular distance of 0.24 nm. This value is consistent with the (101) crystal space distance of the ZnO hexagonal wurtzite structure (JCPDS No. . For further confirmation of the ZnO-NP purity, thermogravimetric analysis (TGA) was employed on the ZnO NPs green-synthesized via 0.06 M precursors of KOH and lye solution. For KOH use, Figure 10a show a slight 0.6% weight loss initiated at 114°C, followed by a 1% weight loss at 200°C owing to water evaporation. The slight weight loss occurred between 350°C and 600°C, which was about 3.8% of the actual weight due to the  elimination and breakdown of organic groups resent in the specimen throughout the green-synthesis process. Similarly, for lye use, water evaporation caused a 1.1% weight loss at 200°C. The disappearance of organic substances contributed to the 3.7% weight-loss at 350-600°C. Additionally, DSC results from Barzinjy's work [34] were reported to confirm that organic matter decomposes at temperatures above 365°C. These results are consistent with Barzinjy's research, but the ZnO-NPs obtained from this experiment contain fewer organic impurities since they show lower weight loss than hat obtained in Barzinjy's research.
The plots of optical absorbance (A) and reflectance (R) responding to the photon wavelength are shown in Figure 11. The absorbance of all ZnO nanostructures increases abruptly at about 350 nm as the excitation wavelength shows. At the proper energy excitation, a multiplied free electron concentration was observed. The reflectance spectra are contrary to the absorbance spectra showing a decrease of the reflectance intensity at 350 nm. The absorbance intensity of the 0.06 M KOH concentration is the highest. This finding can be attributed to the appearance of higher levels of free carriers or defects.
Diffused reflectance spectroscopy (DRS) is a nondestructive analytical technique that can be used to approximate the bandgaps of solid  nanostructured semiconductors. The obtained diffuse reflectance spectra are analyzed according to the Kubelka-Munk theory, which describes the relationship of the diffuse reflectance (R) to the absorption coefficient (α), as shown by [35]: F(R) = k/s = (1-R) 2 /2 R = α, where F(R) is the Kubelka-Munk function, k is the absorption coefficient, and s is the scattering coefficient The F(R) value is considered to replace the α amount for evaluating the optical bandgap [36]. Therefore, the tauc equation can be rewritten as follows: F(R)hν = A(hν-E g ) n , where hϑ is photonic energy, E g is the bandgap of materials, and n is an integer that indicates the transition process: n = 1/2 for direct allowed, n = 2 for indirect allowed, n = 3/2 for direct forbidden, and n = 3 for indirect forbidden transitions.
The plot of (F(R)hν) 2 as a function of hϑ is shown in Figure 12. The energy bandgap (E g ) is estimated from the slope and the linear plot to zero F(R) value. The E g values of the samples were found to be 2.81, 3.03, 3.33, 2.85, and 2.86 eV for KOH concentrations of 0.02, 0.04, 0.06, 0.08 0.10 M, respectively. The obtained value of 3.33 eV is based on the smallest particle size synthesizing condition (63.46 nm). This result is close to that obtained in the report of Ajala [37] which presented an E g of 3.20 eV for ZnO with a 52 nm particle size. An increase in E g as a decreasing particle size can be described as due to the quantum size effect. The distance of the coulomb interaction between electron and hole plays an important role because the charge carrier leads to the valence and conduction bands of semiconductor modification [38].

Conclusions
The ZnO NPs under the six desired conditions show clusters of ZnO nanoparticles. The XRD and the SAED patterns of all the synthesized ZnO NPs show singlephase patterns consistent with JCPDS card No. 36-1451. FTIR and TGA confirm the purity of all the asreceived ZnO NPs. For pH control by lye, the obtained NPs are on a nano-scale in the same phase as the KOH solution. Impressively, the ZnO NPs from the real green synthesis (using lye) exhibit a flower-like shape similar to that obtained using KOH in 0.06 M of sodium nitrate precursor. Moreover, the ZnO NPs obtained using 0.06 M zinc precursor in KOH solution show highly interesting results for physical and optical properties results.