Green Synthesis of ZnO Nanostructures Using Pyrus pyrifolia: Antimicrobial, Photocatalytic and Dielectric Properties

Abstract

In this study, zinc oxide nanostructures (ZnO NS) were synthesized using Pyrus pyrifolia fruit extract. Biophysical characterization results confirmed that the synthesized materials are crystalline wurtzite ZnO structures. Field emission scanning electron microscopy (FESEM) revealed that the ZnO NS are cubical, and the sizes range 20–80 nm. Transmission electron microscopy (TEM) and XRD results revealed a crystal lattice spacing of 0.23 nm and (101) the crystalline plane on ZnO NS. UV-Visible spectrophotometer results showed an absorbance peak at 373 nm. The ZnO NS demonstrated significant antibacterial activity analyzed by metabolic activity analysis and disc diffusion assay against Escherichia coli and Staphylococcus aureus. FESEM analysis confirmed the bacterial membrane disruption and the release of cytoplasmic contents was studied by electron microscopy analysis. Further, ZnO NS achieved good photocatalytic activity of decolorizing 88% of methylene blue (MB) in 60 min. The dielectric constant and loss of ZnO were found to be 3.19 and 2.80 at 1 kHz, respectively. The research findings from this study could offer new insights for developing potential antibacterial and photocatalytic materials.

1. Introduction

Nanotechnology involves the application of materials in the range of 1–100 nm. The applications span from coatings [1], gas sensing devices [2], solar cells [3], batteries [4], environmental catalysts and antimicrobials [5,6]. Metal and metal oxide nanomaterials have improved the efficiency and performance of such devices. Consequently, researchers have given ZnO much attention due to its biocompatibility, low cost, high photocatalytic efficiency and antimicrobial potential. Indeed, it is recognized as a multifunctional material, as it has played a significant role in various fields such as biomedical (e.g., antimicrobials anticancer, tissue engineering) [7,8,9], cosmetics industries [10] and photocatalysts [11]. ZnO nanostructures are II–VI semiconductors with a wide bandgap energy of 3.3 eV and a high excitation binding energy of 60 meV. Thus, the materials are suitable for large electrical fields, high temperatures and high-power functionalities such as photovoltaic cells and chemical sensors. ZnO nanocrystals mostly show a wurtzite structure with lattice parameters of a = 0.325 nm and c = 0.520 nm [12]. The scarcity of clean water has been a serious issue debated globally. Water contamination is severely inflicted everywhere. This is due to the irresponsible and uncontrolled development of industries that release chemicals into streams [13]. For example, textile industries have used large amounts of dyes and water in their textile coloring process. The release of dye wastewater from this process substantially pollutes the aqueous environment, which was predicted to be 15–20% of the total industrial pollution [14]. The introduction of carcinogenic dye pollutants to the environment cause lethal side effects to human and aquatic organisms [15]. Therefore, finding ways to remove wastewater dyes before discharging them into the environment is necessary.

In recent years, ZnO has been intensively researched as a potential material to treat dye effluents. In this process, ZnO absorbs UV light with a wavelength equal to or less than 385 nm to generate radicals that, in turn, degrade the dyes. Recent studies demonstrated that the photodegradation of methylene blue (MB) was up to 98.3% [16]. Factors such as morphology [17], shape, size and concentrations of the ZnO can directly affect the efficiency of the photocatalytic activities [18]. For instance, Barnes et al. reported that lowering the concentration of ZnO from 1 to 0.1 g/L reduced the photodegradation performance from 18 to 7% [19]. Recognizing the simplicity and wide potential of ZnO as a photocatalytic agent, numerous approaches are being used to synthesize ZnO nanostructures. However, green synthesis is more advantageous than the conventional approach and hazardous chemical-free procedures [20,21] because it is eco-friendly. This technique is similar to chemical reduction, except that the extracts of natural products replace the reducing and stabilizing agent. Furthermore, studies have shown that the nature of biological elements and the concentrations of extracts could influence the size, shape, and optical properties of nanostructures [22,23,24,25].

ZnO is also gaining interest due to its electronic polarizability. This allows ZnO to be researched as a dielectric material. Dielectric materials are mainly applied in developing flexible electronic devices [26]. In addition, the dielectric properties of the developed material strongly depend on the synthesis conditions [27]. Lanje et al. reported that the ZnO obtained via the precipitation method has a dielectric constant of 14.52 at 100 kHz [28]. On the other hand, ZnO synthesized using starch as a stabilizing agent reported a dielectric constant in the range of 4–5 at the same frequency [27]. At the same time, numerous research studies have investigated the phytosynthesis of ZnO, but very few have discussed its dielectric or electrical properties. In the present work, we develop a facile method for the synthesis of ZnO nanostructures by using a fruit extract of Pyrus pyrifolia. To the best of our knowledge, this is the first study of ZnO NS synthesis using P. pyrifolia fruits. To study the interaction between ZnO nanostructures and the bacteria and the decolorization ability of MB, we studied antimicrobial assays and the photocatalytic reaction. Notably, the dielectric properties of ZnO NS have also been reported in this study.

2. Materials and Methods

2.1. Chemicals and Reagents

All chemicals used in this study were of analytical grade and used without further purification. The zinc nitrate (Zn(NO₃)₂.6H₂O), Ethidium bromide (EB) and MB were purchased from Sigma Aldrich (Burlington, MA, USA). Acridine orange (AO) was obtained from VWR AMRESCO Life science, Radnor, PA, USA. Distilled water (DW) was used in all experiments.

2.2. Preparation of Extracts

Fresh fruits of Pyrus pyrifolia were obtained from a supermarket in Kuala Lumpur, Malaysia. The fruits were washed twice with DW to remove any dust and impurities, then 100 g of the fruit was cut into small pieces and ground with 100 mL DW. The resulting saturated extract was filtered through the Buchner funnel by vacuum filtration and further filtration procedures were carried out (e.g., using gravity filtration) to ensure a clear extract was attained.

2.3. Synthesis of ZnO Nanostructures

For the synthesis of ZnO nanostructures, 0.1 M Zn(NO₃)₂.6H₂O was prepared by dissolving 7.43 g of zinc nitrate in 250 mL DW and sonicated for 30 min to achieve complete dissolution. Aqueous fruit extract in the amount of 50 mL was introduced into the above solution, magnetically stirred for 15 min and left at room temperature for 24 h. Afterwards, the reaction solution was heated at 80 °C till the volume was reduced to 3/4 of its original volume and the color changed to a deep yellow paste [29]. Next, the paste was collected in a clean crucible and calcined at 450 °C for 60 min using a small benchtop muffle furnace (Barnstead/Thermolyne furnace 1400, Thermo Scientific, Waltham, MA, USA). The calcined materials were washed with DW and ethanol to remove the impurities. Finally, the resultant materials were dried for 12 h in a vacuum oven at 70 °C to obtain the white zinc oxide powder.

2.4. Characterization of the ZnO

X-ray diffraction (XRD) analysis was performed using Ultima IV (Rigaku, Tokyo, Japan) at a scan speed of 2° min⁻¹ and a wavelength of 1.5406 Å in the 2θ range of 20–90 degrees. The crystallite size of the resultant ZnO NS was calculated using the Scherrer Equation (1).

$$D=\frac{\kappa \lambda }{\beta cos\theta }$$

(1)

where D is the average crystallite size (in Å), κ is the shape factor, λ is the X-ray wavelength of X-ray (1.5406 Å) Cu-Kα radiation, β is the full width at half maximum (FWHM) of the diffraction peak and θ is the Bragg angle [30].

The UV-Vis absorbance spectra were obtained using a UV-1700 Spectrometer (Shimadzu, Kyoto, Japan) with measurements in the wavelength range of 300–500 nm. Fourier transform infrared (FT-IR) spectroscopy analysis was carried out to detect the possible functional groups involved in the synthesis of ZnO nanostructures. A PerkinElmer, Waltham, MA, USA, Frontier FT-IR Spectrophotometer in the attenuated total reflectance (ATR) mode in the range of 4000–500 cm⁻¹ was used. Raman spectra were recorded in the backscattering geometry using a 632 nm HeNe laser with a LabRAM HR Evolution spectrometer, Kyoto, Japan. The morphological properties were characterized using FESEM (JEOL JSM 6701-F, Peabody, MA, USA) equipped with EDX analysis. EDX tests were carried out to identify the element and obtain the weight/atomic ratio of each element in the synthesized nanomaterials. Transmission electron microscopy (TEM) analysis was carried out on a (JEOL 2010, Peabody, MA, USA) instrument operated at an accelerating voltage of 200 kV. The Brunauer-Emmett-Teller (BET) nitrogen adsorption-desorption (Nova 2000E) was used to calculate the specific surface areas using desorption data. The sample was prepared using 200 mg of ZnO NS, which was subsequently degassed at 200 °C for 2 h to remove the moisture. Nitrogen gas was introduced as an adsorbent into the sample cell and the pressure changes due to the adsorption process were monitored via pressure transducers. When the saturation pressure was achieved, the sample was removed from the nitrogen atmosphere and heated to release the adsorbed nitrogen, which was then quantified.

2.5. Dielectric Studies

The dielectric studies of ZnO NS were carried out using a chemical impedance analyzer (Model: Hioki Im3590, Nagano, Japan). The as-synthesized ZnO nanostructure was compressed into pellets 13 mm in diameter and 0.539 mm in thickness by applying a force of 10 tons with a hydraulic press. The dielectric constant and loss were measured in the frequency range of 1 Hz–100 kHz.

2.6. Photocatalytic Decolorization of Methylene Blue

We placed 2.5 mg/L MB dye (50 mL) and 10 mg catalyst ZnO NS in a glass beaker and the suspension was magnetically stirred for 30 min in the dark to reach equilibrated adsorption between the ZnO NS and MB. Then the suspension was irradiated under UV light sources (Philips, λ = 365 nm, 6 W) with continuous stirring. Another beaker containing the same concentration of MB dye without ZnO was prepared as a control and underwent the same treatment as the one with ZnO. The samples (5 mL) were taken out at regular intervals of 0, 15, 30, 45 and 60 min, centrifuged for 5 min at 4000 rpm and the absorbance was measured using UV-Vis spectroscopy. The decolorization efficiencies of the dyes were estimated from the following equation [31]:

$$\mathrm{Decolorization}\left(\%\right)=\frac{Ao-A_t}{Ao}\times 100$$

(2)

where A_o represents the absorbance of dye before illumination and A_t denotes the absorbance of the dye after a specific irradiation time.

2.7. Antimicrobial Assay of ZnO NS

2.7.1. Disc Diffusion Assay

The antibacterial activity of the ZnO NS was studied using disc diffusion assay. Briefly, Gram-positive bacteria B. subtilis (ATCC 23857) and Gram-negative bacteria E. coli (ATCC 25922) were used to perform the antimicrobial assays. The freshly grown bacterial single colonies were spread in the Mueller-Hinton agar plate. The different concentrations (100, 200, 300 and 400 µg/mL) of ZnO NS solution (20 µL) were impregnated on the paper disc (6 mm diameter) and labelled as 1, 2, 3 and 4. The disc in the center was loaded with 20 µL DW and served as a control. The plates were then incubated for 24 h at 37 °C. The bacterial inhibition zones observed around the discs were measured and tabulated.

2.7.2. Resazurin Assay Based Minimum Inhibitory Concentration (MIC) Determination

The MICs of the ZnO NS were determined by a standard broth microdilution assay in a 96 well plate. Briefly, the concentration of overnight grown bacterial pathogens was adjusted to 1 × 10⁵ CFU/mL. The ZnO NS (500 µg/mL) were serially diluted (2-fold) in 175 µL of MHB with 10 µL of selected bacterial inoculum. The wells containing only MHB and the MHB containing bacteria were negative and positive controls, respectively. Then, the plates were incubated for 24 h at 37 °C. Next, the MIC was determined by visually observing the turbidity. The lowest concentration of ZnO NS treated bacteria wells without turbidity were considered as the MIC [32]. Meanwhile, another set of experimental wells were flooded with 10 µL of resazurin and incubated for 60 min. The sample wells turned from blue to pink, indicating bacterial viability, and the minimal dosage of ZnO NS treated wells remained blue, indicating MIC [33].

2.7.3. Analysis of Bacterial Morphological Changes

The morphological changes of bacteria caused by the ZnO NS were examined by scanning electron microscopy. Briefly, P. aeruginosa and B. subtilis were treated with ZnO NS and incubated at 37 °C for 2 h. Then the cells were washed with PBS, followed by glutaraldehyde fixation for 6 h. The samples were then washed with H₂O and dehydrated with increasing concentrations of ethanol and acetone. Lastly, the samples were processed for gold coating and viewed under a field emission scanning electron microscope (FESEM).

3. Results and Discussion

3.1. ZnO NS Synthesis and UV-Vis Spectrum

The phytochemical constituents in the P. pyrifolia fruit extract chelate with the metallic zinc ions (Zn²⁺) and form yellow-colored zinc coordinated complex sediment in the reaction medium [34]. Then, the obtained Zn complex was decomposed by calcining it at 450 °C for 60 min to get the nanostructured zinc oxide materials. The schematic diagram of P. pyrifolia mediated ZnO NS synthesis is represented in (Figure 1a).

Generally, UV-Vis spectral analysis is used to confirm the formation of metal and metal oxide nanoparticles. The synthesized aqueous ZnO NS solution showed that the synthesized nanoparticles exhibit an excitation wavelength of 373 nm (Figure 1b), which is almost similar to the range of results in the literature [11,22]. The band gap energy (E) of the synthesized ZnO nanostructure was estimated by applying the peak at 373 nm, using Equation (2).

$$E=\frac{hc}{{\lambda }_{max}}$$

(3)

where h is the Planck’s constant and c is the speed of light in vacuum. E of the resultant ZnO NS was calculated to be 3.32 eV. This result is in accordance with the previous report on the synthesis of ZnO nanoparticles [35].

3.2. X-ray Diffraction Analysis of ZnO NS

The X-Ray diffraction peaks of the synthesized ZnO NS are shown in Figure 2. The X-ray diffraction patterns of the resultant ZnO NS showed different diffraction peaks at the 2θ values of 31.87°, 34.49°, 36.36°, 47.49°, 56.75°, 62.94°, 66.29°, 68.06°, 69.29°, 75.58° and 76.85°, which can be indexed to the (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) and (2 0 2) planes, respectively. These observed diffraction peaks are highly matched with the hexagonal phase of the wurtzite ZnO structure (JCPDS card number: 36–1451). The sharp diffraction peaks demonstrate the high crystalline nature of the formation of ZnO particles. No obvious presence of unknown peaks implies a high purity of the synthesized ZnO NS. Our results are in accordance with an earlier report [36]. The crystallite size of the resultant ZnO NS was calculated using the diffraction peak of 36.36° and found to be 17 nm.

3.3. FESEM and EDX Pattern of ZnO NS

The FESEM images showed that the diameters of the samples of ZnO nanostructures are in the range of 20–80 nm (Figure 3a,b). The ZnO is cubical, and the shape is almost uniform throughout the sample. The formation of clusters by the nanoparticles is mostly likely attributed to agglomeration, which is common in nano-sized materials. Furthermore, the elemental analysis spectrum results revealed the presence of zinc and oxygen in the densely populated nanoparticles region (Figure 3c). The elemental weight percentage is shown in the inset of (Figure 3c).

3.4. TEM and BET Measurement of Resultant Nanomaterials

To study the structure and morphology of the synthesized ZnO NS, the TEM analysis was employed, as shown in (Figure 4a), and thus the mean size of the particles can be analyzed. It is apparent from the TEM image that the shape of the ZnO particles is modified from a spindle structure to a nearly spherical form due to the involvement of the extract in the crystallization process. This further asserts the findings of Bayrami et al. [37]. The results showed the lattice fringes spacing was measured to be 0.23 nm, which can be attributed to the space between two planes (101) of the wurtzite ZnO NS [38]. Moreover, this result corroborates the crystal lattice spacing determined from the above XRD peak 2θ value of 36.36° with a corresponding lattice plane of (101).

To examine the surface properties of resultant ZnO NS, the Brunauer-Emmett-Teller (BET) nitrogen adsorption-desorption isotherms were measured, as shown in (Figure 4b). In both cases, it is evident that the volume of the sample increases monotonously with increasing relative pressure. Particularly, at low pressure (between 0 and 0.1 P/P₀), the adsorption can be largely attributed to its microporous filling. A nearly similar trend is noticed for desorption. However, with the enhancement in pressure, a gentle hysteresis loop ensues, which is a habitual hallmark of mesoporous materials [39]. The BET measurements estimate the surface area of ZnO to be 21.4 m²/g. Furthermore, relating the ZnO NS surface area with their photocatalytic performances, two trends of results have been reported, as the photocatalytic activity of ZnO nanoparticles increased with both an increase in specific surface area [40] and a decrease in specific surface area [41]. Moreover, it has been reported that, although the ZnO NS have a larger surface area and it has no significant effect on the pore diameter, it may result in a larger active site [42]. The obtained data curve denotes typical type II isotherms, which relate to the nonporous characteristic of solids [37].

3.5. FTIR and Raman Measurement of ZnO NS

FTIR spectra of the P. pyrifolia fruit extract and synthesized ZnO nanostructures before and after calcination were showed in (Figure 5a, A,B,C). A vibration band of fruit extract showed peaks at 3382, 1638, 1488, 1381 and 845 cm⁻¹. The peak at 3382 cm⁻¹ corresponds to the O-H stretching functional group [43]. The peaks at 1638 and 1488 cm⁻¹ can be attributed to the carbonyl (C=O) functional group and bending vibration of the sp² C=C aromatic ring [44], respectively. The obtained peaks at 1381 and 845 cm⁻¹ are probably due to the alkene group of C-H stretching and C-N amine [45], respectively. Phenolic contents in the extract of pear may also be involved in the formation of ZnO NS. Momeni et al. suggested that the peaks that range from 3500 to 3100, 1720, 1605, 1395 and 1100 cm⁻¹ can be linked to the free OH in the extract, thus forming hydrogen bonds, a carbonyl group (C=O), a stretching C=C aromatic ring and C-OH and C-H stretching vibrations. Correspondingly, these indicate the presence of phenolic structures in the plant extract [46]. The FTIR spectrum of synthesized zinc complex before calcination showed peaks at 3412, 1635 and 1385 cm⁻¹. A comparison of FTIR spectra revealed a slight shift in the peaks of the extract and the zinc complex. This is anticipated due to the adsorption of the plant extract (C-H stretching vibration, O-H and carbonyl groups) onto the zinc surface, which may be involved in the synthesis of the nanoparticles. Further, the FTIR spectrum of the ZnO NS revealed no significant peaks due to the decomposition of bioactive functional groups during calcination [5].

The Raman spectrum of the ZnO nanostructures from 290 cm⁻¹ to 990 cm⁻¹ is shown in Figure 5b. The obtained spectrum has a Raman peak comparable to ZnO nanocrystals presented in an earlier report [47] with a slight shift (in the range of 1–2 cm⁻¹) caused by different crystal sizes [48]. Theoretically, the wurtzite crystal structure of ZnO belongs to the C6v4, possessing 2 formula units in each primitive cell with all the atoms lodging the C3V sites [49]. The major, sharp peak labelled as E2 at 439 cm⁻¹ is recognized as Raman active optical phonon mode, which is the characteristic of the wurtzite hexagonal phase ZnO. Raman modes at 333.6 and 439 cm⁻¹ are denoted as 2E2 and E2 modes, respectively [47,48].

3.6. ZnO NS Photocatalytic Decolorization of Methylene Blue

The photocatalytic decolorization efficiency of ZnO NS on MB was observed using different amounts of ZnO catalyst (1 mg, 5 mg, 10 mg and 20 mg) in 50 mL of 2.5 mg/L MB solution under UV light for 60 min. The decolorization was analyzed by measuring the absorbance peak of the MB. As shown in Figure 6a, a lower absorbance peak indicates that more MB is decolorized and vice-versa. These results showed that by using 10 mg of ZnO, the absorbance peak was the lowest compared to the other tested ZnO concentrations, inferring the highest amount of MB decolorized at this particular concentration. This may be attributed to the increase of the catalyst concentration, which subsequently increases the production of ROS and accelerates the number of active sites on the ZnO NS for the reaction.

As seen in Figure 6b, the decolorization exhibits a linear behavior indicating that degradation is directly proportional to the amount of ZnO. However, it was also observed that when the catalyst loading was further increased to 20 mg, the absorbance peak of the MB was the highest, corresponding to the lowest decolorization of MB. The decrease in the photocatalytic decolorization efficiency is probably due to the agglomeration of the catalyst particles. As a result, the specific surface area decreased and subsequently decreased the number of active sites [50]. Moreover, a high quantity of ZnO NS would lower the opacity, turbidity of the suspension and light scattering of the catalyst particles. The more significant amount of nanoparticle suspension may have increased UV shading to hinder photocatalytic activity [19,51]. This would decrease the path of irradiation through the sample [52]. Therefore, in our case, the most effective decolorization for MB was recorded with 10 mg of ZnO NS catalyst.

Figure 6c represents the photocatalytic mechanism for MB in the presence of ZnO. Adsorption and adhesion of the MB dye molecules on the surface of ZnO result in the degradation of the MB. It has been extensively discussed that the photocatalytic decolorization of MB by semiconductors, such as ZnO, can occur due to hydroxyl radicals (^●OH) [53,54]. The ^●OH can be formed either from (i) the highly hydroxylated ZnO surface or (ii) by direct oxidation of dye pollutants under UV irradiation. Moreover, there is also a possibility that the ^●OH co-occurs by both methods. The photo decolorization process starts when ZnO absorbs UV light of energy equal to or higher than its bandgap (3.37 eV). This promotes the formation of free electrons (e⁻) and holes (h⁺) in the conduction and valence bands, respectively. These electrons can either recombine with the holes (and scatter the captivated energy as heat), or the electron-hole pairs can contribute to redox reactions. In the case of participating redox reactions, the electron-hole pairs can generate ^●OH either from the reaction of h⁺ with water or with OH⁻ anions [50]. On the other hand, the response of dissolved O₂ and e⁻ will produce superoxide (•${\mathrm{O}}_2^{-}$) and may also proceed to make $\mathrm{O}\mathrm{O}\stackrel{·}{\mathrm{H}}$. All of these active oxygen species (•${\mathrm{O}}_2^{-}$) and free hydroxyl radicals (^●OH, ^●$\mathrm{OOH}$) could also be involved in the photodegradation of the MB [16].

3.7. Dielectric Studies

The dielectric constant is a measure of the capability of a material to stock electrical energy in an electric field. It is a ratio of the material permittivity to the free space permittivity. Permittivity (ε) is a measure of the ability of a material to be polarized by an electric field. An efficient dielectric material supports polarization with minimal dissipation or loss of energy. The dissipation of energy in the form of heat, as the movement of charges in an alternating electromagnetic field occurs, as polarization switches direction. This is known as the dielectric loss (D) or tan δ (loss tangent). It is proportional to the amount of energy stored and dissipated due to the presence of an applied electric field. The dielectric constant and loss were assessed in the frequency range from 1 Hz to 100 kHz in the present study. The dielectric constant, also known as relative permittivity, is determined using the equation:

$${\epsilon }_r=\frac{C x d}{{\epsilon }_{o}A}$$

where C is the capacitance of the sample, d and A are the thickness and the area of the sample pellet, respectively, and ε_o is the dielectric permittivity of vacuum (8.854 × 10 F/m). The ε_r and tan δ for the synthesized ZnO were found to be 3.19 and 2.80 at 1 kHz, respectively. A recent report of the synthesized ZnO NPs by the co-precipitation method demonstrated the value of the dielectric constant and loss to be approximately 12 and 0.01, respectively [55], while ZnO NPs synthesized using sol-gel observed the dielectric constant and loss to be 40 and 50, respectively [56]. Apart from the preparation conditions that can influence the value of the dielectric properties [27], it is noteworthy to mention that the compression force used in preparing the pellet must also be considered because different compression forces will result in different void spaces between the particles [57], and void space affects electrical measurements. Therefore, a direct comparison of the values is difficult, as many aspects and factors come into play. Figure 7a,b represents the deviation of the dielectric constant (ε) and dielectric loss (tan δ) with respect to frequency at room temperature (300 K). The values of ε_r and D were found to decline with increasing frequency. The decrement rate was observed to be quicker at a lower frequency and slower at a higher frequency. The decrease of the dielectric constant at high frequencies is typical because any species contributing to polarization will have their space charges reduced under the applied field at higher frequencies [58]. Polarization could arise from electronic dislodgment, ionic displacement, dipole orientation and space charge displacement [56].

3.8. Antibacterial Activity of ZnO NS

3.8.1. MIC Determination and Metabolic Activity

Although industrial effluents pollute the environment, the emergence of pathogenic bacterial drug resistance epitomizes the high risk to public health. Nanomaterials are considered alternate antimicrobials due to their unique physiochemical properties. To determine the MIC level of ZnO NS against P. aeruginosa and B. subtilis, a broth microdilution assay was performed. The turbidity observation results showed that the ZnO NS significantly inhibited the growth of P. aeruginosa and B. subtilis with MIC values of 125 and 250 µg/mL (Figure 8, Row B and D), respectively. A similar pattern of greater and lesser antibacterial activity was observed against P. aeruginosa and B. subtilis, which may be due to the differential cell wall structure of Gram-positive and Gram-negative bacteria [59]. ZnO NPs demonstrated a prominent antibacterial effect; their combinations are used in food additives due to their non-toxic nature to humans within FDA approved concentrations [60]. The turbid white color appearance of increasing ZnO NS concentrations slightly interfered with the determination of MIC and whether bacterial growth caused the turbidity. Furthermore, the samples that were incubated with resazurin after 60 min showed that the metabolically active bacterial cells appeared to change colors, from blue (resazurin) to pink (resorufin), as shown in Figure 8 (Row A and C). The lowest dosage of ZnO NS (125 and 250 µg/mL) exposed P. aeruginosa and B. subtilis, remaining unchanged in its blue color, were determined as MIC. Moreover, these results are in line with the MIC of turbidity observation analysis.

3.8.2. Disc Diffusion Assay

The antibacterial activity of ZnO NS was studied against B. subtilis and P. aeruginosa by disc diffusion assay. The assay results showed clear growth inhibition on the plates against both the tested bacteria (Figure 9a,d). From the results, the DW loaded control disc in the center, and for lesser concentrations of ZnO NS impregnated discs (1. 100 μg/mL and 2. 200 μg/mL), did not show any inhibition against both the tested bacteria. In contrast, a clear zone of inhibition was observed at increasing concentrations of ZnO NS discs (3. 300 μg/mL and 4. 400 μg/mL). A higher inhibitory zone was observed at 8 mm for both bacteria at a 400 μg/mL dosage of ZnO NS. These results showed an increased bacterial inhibitory effect as ZnONS dosage was increased, which was correlated with an earlier report [61].

To study the ZnO NS effect on bacterial cells, the ZnO NS treated cells were imaged and compared with the control bacteria. Figure 9b,e shows the bacteria without any treatment, which demonstrated a rod-like shape with a smooth cell membrane surface. After 2 h ZnO NS treatment, our observations show that both bacteria underwent structural changes, including membrane damage, pits and holes on the cell membranes (Figure 9c,f). Furthermore, the leakage of cytoplasmic content was observed, which led to bacterial death. The bactericidal effect of ZnO is ascribed to multiple reasons, such as cell wall and cell membrane damage and the release of zinc ions and their ability to produce ROS, which causes oxidative stress to the bacteria [62]. It was reported that the release of Zn²⁺ ions accelerates the ROS generation in the bacterial surface and may involve oxidizing glutathione and induce lipid peroxidation, which subsequently causes bacterial lysis [63].

4. Conclusions

ZnO nanostructures were successfully synthesized using Pyrus pyrifolia fruit extract as a reducing agent by the green synthesis route. The structural, morphological and optical properties of the ZnO nanostructures were analyzed by FESEM, UV-Vis, FTIR and Raman. The XRD pattern result confirmed the wurtzite structure of ZnO nanostructures. FESEM analysis revealed the average size of ZnO NS in the range of 20–80 nm. Flavonoids/limonoids/carotenoids, proteins and other functional groups in the fruit extract are likely responsible for forming ZnO nanostructures. Further, the ZnO NS demonstrated significant antibacterial activity against B. subtilis, and P. aeruginosa, which was confirmed by metabolic assay and morphological analysis. MB dye was effectively decolorized under UV light by controlling the concentration and catalyst loading of the MB. The synthesized ZnO NS exhibits a typical pattern of dielectric constant and loss of ZnO with respect to the frequency. The results of this study may provide new insights into the utilization of green-synthesized ZnO NS for developing novel antimicrobial combinations to treat bacterial infections, and for environmental photocatalysts to remove pollutant antibiotics.