Green synthesis and antimicrobial activity of ZnO nanostructures Punica granatum shell extract

2.1 Materials

Pomegranates (P. granatum) were purchased from a greengrocer located in Çankırı/Turkey. Deionized water (D.I. H₂O) was used in all experiments. Zinc nitrate hexahydrate [Zn(NO₃)₂·6H₂O] was bought from Sigma-Aldrich (Germany). P. granatum shells were washed thoroughly 4–5 times under tap water and with D.I. H₂O and dried. Then, 10 g of dry shells were boiled in 100 ml D.I H₂O for 60 min at 100°C. Afterwards, the aqueous extract was cooled to room temperature and filtered using filter paper (Whatman No. 1) to remove large particles. The color of the extract was light yellow. The extract was saved in a refrigerator at 4°C for subsequent experiments.

2.2 Synthesis of ZnO-NPs

The sample preparation of ZnO-NPs is shown in Figure 1. This consisted of 5 g Zn(NO₃)₂·6H₂O being mixed in 50 ml of the extract under vigorous stirring at 100°C until the solvent (H₂O) was completely removed, and then it was annealed at 400°C for 120 min. Finally, the ZnO-NPs sample was obtained in powder form. The obtained light yellow colored powder was used for further studies.

Figure 1:

Sample preparation process for zinc oxide nanoparticles (ZnO-NPs).

2.3 Characterization

The ZnO-NPs sample was characterized structurally, morphologically, electrically and optically by X-ray diffraction (XRD) (Bruker AXS D8), field emission-scanning electron microscopy (FE-SEM) (Zeiss Ultra Plus Gemini), a four probe dc system and a UV-visible spectrophotometer (Rayleigh UV-2601), respectively. In addition, the antimicrobial activity and electrical conductivity were examined.

3.1 XRD and structural study

Based on previous results, the predicted reactions for the formation of the GS ZnO-NPs using the aqueous extract of P. granatum shell are shown in Figure 2. The gallic acid, fatty acids, catechin, EGCG, quercetin, rutin and other flavonols; flavones and flavonones; and anthocyanidins molecules can interact with Zn²⁺ to nucleate the zinc/gallic acid, fatty acids, catechin, EGCG, quercetin, rutin and other flavonols; flavones and flavonones; and anthocyanidins complex molecules. After the reactions, the complex solution was heated and dried. Throughout the heating, the complex solutions transformed into GS nanostructured ZnO particles [22]. The flavones and organic acids were the main phytochemical compounds present in leaf extract responsible for the direct reduction of zinc ions to ZnO-NPs [14], [15], [16], [17], [18], [19], [20], [21], [22]. In addition, the H₂O(g) vapor and CO₂(g) released into the environment were non-toxic. For this reason, the chemical vapor deposition (in gaseous media) production method was environmentally friendly.

Figure 2:

Possible reaction for production of zinc oxide nanoparticles (ZnO-NPs) [22].

The XRD diffraction peaks of the green synthesis ZnO-NPs sample are shown in Figure 3. The XRD patterns illustrate the notable peaks of the ZnO-NPs at 31.90°, 34.57°, 36.41°, 47.70°, 56.76°, 62.97°, 66.58°, 68.07°, 69.21°, 72.75°, 76.98° and 81.51°, which correspond to the lattice patterns of (1 0 0) (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4), (2 0 2) and (1 0 4), respectively, and can be indexed to the hexagonal wurtzite structure (JCPDS Cardno.89-1397). The lattice constants of the sample were determined at room temperature experimentally from the XRD results and the lattice constant a parameter was 3.2346 Å and the c parameter was 5.2120 Å for the GS ZnO-NPs. No extra diffraction peaks were detected, and this indicated the sample was fully composed of a ZnO hexagonal structure. The narrow and strong diffraction peaks (especially (1 0 0) (0 0 2) and (1 0 1)) illustrate the crystalline quality with high intensity of peaks. The 2θ peak angles, percentage of I/I0, lattice spacing of d and hkl miller indices of the GS ZnO-NPs pattern are illustrated in Table 1. Using the XRD analysis data, the estimated size of the ZnO-NPs was obtained using Debye–Scherrer’s formula given by the following equation:

Figure 3:

X-ray diffraction (XRD) patterns of GS zinc oxide nanoparticles (ZnO-NPs) sample.

Here, D is the average crystallite size (usually taken as 0.89), λ is k=0.15406 nm for CuKα, β is the FWHM (the line width at half-maximum height) and θ is the diffraction angle. The average crystallite size of the GS ZnO-NPs pattern was found to be in the range of 60–70 nm. Nanosized value of ZnO suggests that the pomegranate (P. granatum) shells extract can be employed as the hydrolytic agent for the formation of ZnO-NPs. We can conclude that the GS ZnO-NPs were easily manufactured using the aqueous extract of pomegranate (P. granatum) shells via a green synthesis method.

3.2 FE-SEM and morphological study

The FE-SEM morphology of the GS ZnO-NPs pattern is a crucial factor for defining its structural features in technological applications. Figures 4A and B show distinct magnification of the FE-SEM micrograph of the provided GS ZnO-NPs. Figure 4A illustrates the microstructure of the aqueous shell extract of P. granatum obtained via a GS method for ZnO-NPs and the random distribution of the agglomeration of smaller NPs inside the matrix. The enlarged morphology of the GS ZnO-NPs sample established the erythrocyte-like form NPs with a particle size between 30 nm and 180 nm (as illustrated in Figure 4B), which is essentially consistent with the conclusions by Nasrollahzadeh et al. [14] and Rostami-Vartooni et al. [19]. The agglomeration of smaller NPs occurred due to the relationship of the green synthesis with the biological materials. The SEM analysis proved the existence of even bigger NPs than the XRD analysis results. The bigger, about 180 nm, GS ZnO-NPs in the sample may be caused by the agglomeration of smaller ZnO-NPs, the existence of which was proved by the XRD analysis results, which permitted even smaller GS ZnO-NPs being defined.

Figure 4:

Field emission-scanning electron microscopy (FE-SEM) images of zinc oxide nanoparticles (ZnO-NPs): (A) GS and (B) enlarged GS samples.

3.3 Electrical conductivity study

The temperature dependence of the electrical characteristics of the GS ZnO-NPs sample was measured by a standard four probe dc system. The conductivity of the GS ZnO-NPs sample was determined using the following equation:

where V loading is a direct measurement of the voltage drop and G is the geometric correction constant [23]. The variation in the electrical conductivity (σ) with the applied temperature in the range of 25–650°C for the GS ZnO-NPs was determined and depicted in Figure 5. As can be seen from this figure, the logarithmic values of the electrical conductivity were found to be in the range of −6.15 (7.07×10⁻⁷) with −3.48 (3.31×10⁻⁴) Ω⁻¹·cm⁻¹. It can be clearly seen that Figure 5 shows the conductivity increases with an increase in temperature. This demonstrates the semiconducting properties of the ZnO-NP material. In addition, ZnO particles are a non-stoichiometric and n-type semiconductor owing to the presence of O₂ holes and an interstitial Zn matrix [23]. The local defects in the ZnO result in the electrical conductivity and cause temperature dependence in the electrical conductivity [24]. Generally, for n-type semiconductor materials, the electrical conductivity grows exponentially with annealing temperature. This characteristic shows thermally activated properties. The full electrical conductivity is shown as in the following:

Figure 5:

Electrical conductivity graph for GS zinc oxide nanoparticles (ZnO-NPs) sample.

where σ is the electrical conductivity, σ₀ is the electrical conductivity at the applied temperature (T), Ea is the activation energy and k is the Boltzmann constant [25].

The activation energy of the GS ZnO-NPs sample is determined using the following equation:

The Arrhenius graph is the gradient of the linear part of the electrical conductivity and the –Ea/k values are equal to the logσ-10³/T. For the applied temperature values, the GS ZnO-NPs have Arrhenius-type conductivity behavior. Figure 5 illustrates that there are separate parts matching different Ea values. These values are divided into two different zones: first zone between 25°C and 300°C, which is the low temperature zone, and second zone between 300°C and 650°C, which is the high temperature zone [25]. The low temperature area (between 25°C and 300°C) has an Ea value of 0.143 and the high temperature area (between 300°C and 650°C) has an Ea value of 0.486 eV. For the low temperature area (up to 300°C), an increase in the electrical conductivity in the lower temperature area can be attributed to the increase in the charge mobility and the Ea value decreases, which is because a little thermal energy is enough for the activation of the carriers to take part in the conduction process. By contrast, for the high temperature area (up to 650°C), the Ea is elevated more than that of the low temperature region. For the high temperature, the electrical conductivity is primarily decided via the internal atom defects, and for this reason it is called internal conduction or high temperature. The high Ea values provided for this region could connect the energy needed to form defects, which is much larger than the energy required for its drift. In addition, the internal failures due to the thermal vacillations establish the electrical conductivity characteristics of the GS ZnO-NPs at increased applied temperatures [26]. Similar results have also been reported elsewhere [23], [26].

3.4 Optical study

Figure 6 shows the room temperature optical transmittance spectrum of the ZnO-NPs sample synthesized using a P. granatum shell extract. Figure 6 illustrates that between 350 nm and 1000 nm there is very high optical transparency. The optical transparency displays characteristic absorption peaks of ZnO at the 370 nm wave length, which detects the fundamental band-gap absorption of the ZnO-NPs for the electron jumping between the valence and conduction band [2p (O₂)→3d (Zn)] [27].

Figure 6:

Optical transmittance graph for GS zinc oxide nanoparticles (ZnO-NPs) sample.

Moreover, the GS ZnO-NPs pattern has an optical transparency of over 85%. This high optical transmittance spectrum displays high quality optical transparency, which is connected with a high morphological homogeneity and crystallinity of the GS ZnO-NPs [26].

The optical band gap (Eg) was estimated from the absorption spectra in Figure 6, for which the variation in the absorption coefficient with the photon energy hν can be indicated as follows:

Here, E_g is the optical energy gap between the conduction and the valence band [28]. Figure 7 displays the plots of (αE)² versus photon energy (E, eV) for the GS ZnO-NPs sample. The optical band gap (E_g) value was determined from the plot and found to be 3.4 eV, which is in agreement with those reported by KhorsandZak et al. [27].

Figure 7:

(αE)² versus photon energy (eV) for zinc oxide nanoparticles (ZnO-NPs) sample.

3.5 Antimicrobial activity

The disk agar diffusion technique was used to determine the antimicrobial activity of the samples against a Gram-positive bacterium (Bacillus thuringiensis). Disk agar cultures were established and swabbed by an aseptic L-shaped glass tube with 200 ml of 24 h grown chicken broth culture of the Gram-positive bacterial family. This work used 500 mg/well concentrations of the GS ZnO-NPs to detect the antibacterial activity. The ZnO-NPs did not show any antimicrobial effects against the Gram-positive B. thuringiensis bacterium.