Green Synthesis, Characterization, and Antibacterial Activity of CuO/ZnO Nanocomposite Using Zingiber officinale Rhizome Extract

. Te synthesis of metal oxide nanocomposite by using the green method has gotten special consideration due to a cheaper and eco-friendly approach. Decreasing antibiotic efectiveness calls for the fast advancement of other alternative antimicrobials. CuO, ZnO, and CuO/ZnO nanocomposites were successfully synthesized using Zingiber ofcinale rhizome extract as a mild, renewable, and nontoxic reducing agent and profcient stabilizer with the nonappearance of hazardous and toxic chemicals. UV-Vis spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, and X-ray difraction (XRD) were used to characterize CuO, ZnO, and CuO/ZnO nanocomposites. Te UV-Visible result showed the absorbance peak at 270nm, 355 nm, 365nm, and 370nm corresponding to the characteristic band of CuO NPs, ZnO NPs, 10% CuO/ZnO, and 20% CuO/ZnO nanocomposites, respectively. FT-IR confrmed the nature of bonds and the presence of diferent functional groups in the Zingiber ofcinale rhizome extract, CuO, ZnO, and CuO/ZnO nanocomposites. Te XRD analysis revealed that all the synthesized particles have a crystalline nature with a particle size of 4.35nm, 14.54nm, 18.41 nm, and 20.50nm of CuO NPs, ZnO NPs, 10% CuO/ZnO, and 20% CuO/ ZnO NCs, respectively. Te synthesized nanoparticles and nanocomposites showed inhibition against Gram-positive and Gram-negative bacteria up to a concentration of 12.5 mg/mL. Te highest inhibition against Staphylococcus aureus ATCC 25926 and Escherichia coli ATCC was 20 ± 0.7 mm and 16 ± 0.5mm in diameter, respectively, by 50 mg/mL of 20% CuO/ZnO NCs. In general, the biosynthesized nanoparticles and nanocomposites showed efective antibacterial activity.


Introduction
Nanoscience and technology play a crucial part in numerous scientifc felds with their particular features. Nanotechnology helps in reducing the particle size of materials as an efcient and reliable tool for enhancing their biocompatibility and can universally alter the view concerning science [1]. Nanoparticles are small particles made through nanotechnology. Tey have sizes from 1 to 100 nm [2]. Te study of nanomaterials has greatly increased. Tey have unique qualities like being very hard, conducting electricity, staying chemically stable, having the ability to speed up chemical reactions, and fghting against microbial and antioxidants. Nanomaterials with the ability to kill microbial have been widely used in medicine [3].
Te fast growth of industry and population greatly impacts the environment and raises many concerns and challenges about creating a sustainable and healthy ecosystem. Nanoparticles are tiny particles that are used to make the environment clean and safe. Tey help improve technology in industries [4]. Diseases that can be easily spread are a big danger to people everywhere. In the past few years, scientists have been using nanobiotechnology to create new drugs and fnd ways to control diseases [5]. Metal oxide nanoparticles are very important in nanotechnology because they are used a lot in diferent industries and pharmaceuticals. Tey are used as disinfectants, catalysts, fllers, and drugs that fght against bacteria [6]. Moreover, metal oxide nanomaterials have diferent reactions against microbes depending on their size [7].
Inorganic metal oxide nanoparticles are interesting because they are chemically stable, safe, and efective at killing bacteria [8]. Metal oxide nanoparticles have been used in many areas such as sensors, photocatalysts, protecting against UV rays, carrying drugs, making cosmetics, flling materials in medicine, and killing bacteria agents [9]. One of these metal oxides is zinc oxide nanoparticle (ZnO) as an ntype semiconductor. It is a special type of material that conducts electricity in a particular way. It has a lot of good qualities, like being environmentally friendly, cheap, very stable, and easy to prepare [10]. On the other hand, copper oxide nanoparticles (CuO) are considered another type of ptype semiconductor that is commonly used. Tey have low band gap energy, are chemically stable, friendly for the environment, and have properties that can reduce infammation and antibacterial activities [11]. ZnO and CuO NPs are considered the most common nanoparticles because they have great chemical, physical, and mechanical properties. Some of these properties include a low melting temperature, a bigger surface area, structural stability, high difusion, and high surface energy [12].
Tere are many expensive ways to synthesize CuO/ZnO nanocomposites using chemical and physical methods. Tese ways also involve using toxic organic solvents and hazardous reagents, high pressure, and risks to the environment and living things. Because of this, that restricts their use in medical applications [13]. Tere are several papers on the green synthesis of CuO, ZnO, and CuO/ZnO nanocomposite for use in biological applications utilizing plant extracts. Biological approaches have a number of advantages over chemical and physical ones for synthesizing metal oxide nanocomposite. Based on the previous literature reports, ZnO NPs, CuO NPs, and CuO/ZnO NCs have been synthesized from various plant extracts. Te green synthesis of CuO NPs using the Syzygium guineense (SyG) leaf extract on bacteria with the evaluation of electrochemical properties has been reported [14]. Te extract from the medicinal plant, Syzygium guineense leaf, was used to synthesize copper and its oxide (SyG-CuO) nanostructures, and the process was successful. Te green copper oxide NPs showed great potential for antimicrobial and electrochemical applications.
ZnO nanoparticles were synthesized using a natural and environmentally friendly method using an extract from Zingiber ofcinale rhizome. Te synthesized ZnO nanoparticles were incorporated into a glucose biosensor. Te prepared biosensor exhibited good electrocatalytic ability for the determination of glucose [15]. In addition, ZnO nanoparticles were synthesized using the extract of Ranunculus multifdus plant. Terefore, the antibacterial activity of synthesized ZnO NPs was tested against Gramnegative and Gram-positive bacterial strains such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. Te fnding showed that the synthesized ZnO NPs had a strong ability against bacterial strains, especially Gram-positive pathogenic bacterial strains [16]. However, the ZnO material has a large energy band gap as reported previously [17] which can decrease the antibacterial properties. Te addition of CuO to ZnO could form the CuO/ZnO nanocomposite, which increases particle size and decreases the band gap energy [18]. CuO/ZnO nanocomposite was biosynthesized by mixing copper and zinc precursors with a type of fungus, Penicillium corylophilum strain, for photocatalytic activity. Te synthesized CuO/ZnO nanocomposite has higher stability than either of the ZnO and CuO nanoparticles [19].
Te ZnO and Cu-doped ZnO nanocomposites (NCs) were synthesized by the solution combustion synthesis (SCS) method, which is fast and saves energy, to synthesize the materials. Te doping and heterojunction method made the properties of NCs better than just using the individual nanoparticles, and this increased the capabilities of the materials to transfer charge and absorb light better [20]. When two metal oxides are combined, they have some important characteristics, they are very stable when exposed to heat, and they have a large surface area. Tis helps them react better because they have more active sites on their surface and they work more efectively [21].
Many researchers have worked on green synthesis, characterization, and photocatalytic activities of nanocomposite. However, up to the knowledge of the authors, there is a gap in the synthesis of CuO/ZnO binary nanocomposites using Zingiber ofcinale rhizome extract. Zingiber ofcinale (ginger) is a medicinal plant; it belongs to the Zingiberaceae family. Ginger is a common spice that is used in many diferent countries, particularly in Asia and Africa [22]. Because Zingiber ofcinale rhizome contains a lot of phytochemicals, it is selected for the synthesis of CuO/ZnO NCs. Terefore, in this study, the CuO/ZnO nanocomposite is selected as the target material to be synthesized through a plant-mediated routine and to further investigate their antibacterial activity. CuO/ZnO nanocomposite exhibited enhanced antibacterial activity compared with singlecomponent CuO and ZnO NPs. Tis study aims to address a lack of the synthesis of CuO/ZnO binary nanocomposites using the extracts from Zingiber ofcinale rhizome for enhanced antibacterial properties than single CuO and ZnO nanoparticles.

Plant Material Collection and Identifcation.
Te rhizome of Zingiber ofcinale was purchased from Nekemte, Oromia region, East Wollega Zone, in March 2022. Ten, it was identifed and authenticated at the National Herbarium of Ethiopia, which is part of Addis Ababa University.

Preparation of Zingiber ofcinale Rhizome Extract.
Te Zingiber ofcinale rhizome was cleaned with tap water and then with distilled water to get rid of any dirt. It was dried and turned into a fne powder. Lastly, it was kept at room temperature for later use. 10 g of Zingiber ofcinale dried powder was mixed with 100 mL of double distilled water in a 250 mL Erlenmeyer fask, and a magnetic heater stirrer was used for stirring at 70°C for 30 min to get the extract. Te obtained extract was then centrifuged at 6000 revolutions per minute and passed through a flter ( Figure 1). Te fltered extract was stored in a refrigerator at 4°C for future experimental use [23].

Phytochemical
Screening of the Zingiber ofcinale Rhizome Extract. Te qualitative phytochemical test was done using standard and previously reported steps to check various phytochemicals in the extract [24]. Phytochemical Screening of Zingiber ofcinale rhizome extract was done at Chemistry Laboratory, Wallaga University. Terefore, the presence of phytochemicals such as alkaloids, phenols, saponins, favonoids, and tannins in the extract was checked (Table 1).

Optimization Methods for Green Synthesis of CuO, ZnO
NPs, and CuO/ZnO NCs. Optimization is very important to obtain nanoparticles and nanocomposites with high quality, stability, and desired size. Based on previous reports, different parameters were adjusted as being optimum conditions for nanoparticle synthesis by changing one parameter and keeping the other constant at a time to get the optimum results. Terefore, the efects of metal salt concentration, the amount of Zingiber ofcinale extract, pH, and temperature were optimized [27].

Synthesis of the CuO NPs, ZnO NPs, and CuO/ZnO NCs
Using the Aqueous Extract of Zingiber ofcinale Rhizome. CuO NPs, ZnO NPs, and CuO/ZnO NCs were synthesized using a method that had been described before with some improvement.

Synthesis of CuO Nanoparticles.
Copper oxide nanoparticles were synthesized by using 0.1 M Cu (NO 3 ) 2 .3H 2 O ( Figure S1). In brief, 100 mL of the copper nitrate trihydrate was taken in a 250 mL Erlenmeyer fask and 25 mL of the Zingiber ofcinale rhizome extract was added slowly to reduce copper ions to its copper oxide nanoparticles. Ten, 10 mL of 2 M NaOH solution was added to adjust pH to 11 while stirring it constantly. Te solution was stirred continuously at 80°C for 2 h. Te blue-colored solution turned green immediately and after about 2 h, a dark brown precipitate was formed. Te dark brown precipitate formed indicated that all the copper ions have been reduced and CuO NPs have been formed [28]. Te obtained precipitate was centrifuged at 10000 rpm for 10 min and washed several times using distilled water and ethanol for removal of impurities, and the sample was dried at 80°C for 2 h.

Synthesis of ZnO Nanoparticles.
Zinc oxide nanoparticles were successfully synthesized using Zingiber ofcinale rhizome extracts ( Figure S1). 0.1 M zinc (II) acetate dihydrate (Zn (CH 3 COO) 2 .2H 2 O) was prepared by adding 2.195 g of zinc (II) acetate dihydrate in 100 mL deionized water in a 250 mL beaker. 25 mL of Zingiber ofcinale rhizome extract was added and stirred for a few minutes. Te pH of the solution was adjusted to 11 by using 2 M NaOH while stirring. Ten, the solution was stirred continuously at 90°C for 2 h, and the white-colored precipitate was observed in the solution. Te precipitate was collected and centrifuged at 10000 rpm for 10 min. Finally, the obtained precipitate was cleaned with deionized water and absolute ethanol several times, the precipitate was collected, dried in an oven at 90°C for 2 h, and ground to a powder by using mortar and pestle, and it was kept for further physical characterizations and biological applications [29].

Synthesis of CuO/ZnO
Nanocomposites. CuO/ZnO nanocomposite has been successfully synthesized in an ecofriendly manner using Zingiber ofcinale rhizome extract. Te same procedure was used for CuO and ZnO nanoparticles ( Figure S1). For the synthesis of 20% CuO/ZnO nanocomposite, 1.76 g zinc (II) acetate dihydrate and 0.4832 g copper nitrate trihydrate were added to 100 mL deionized water and 25 mL of the Zingiber ofcinale rhizome extract. An aqueous solution of NaOH (2 M) was added drop-wise to adjust the pH of the solution to 11 and the formed precipitate was centrifuged at 10000 rpm for 10 min, and the precipitate was cleaned and dried in the same way as ZnO NPs [23]. In the synthesis of the 10% CuO/ZnO nanocomposite, 1.98 g of zinc acetate and 0.2416 g of copper nitrate were used ( Figure 2).

Characterization of Green Synthesized CuO NPs, ZnO
NPs, and CuO/ZnO NCs. Te visual property of CuO NPs, ZnO NPs, and CuO/ZnO NCs was studied, by using UV-Vis absorption spectra from 200 to 800 nm range of wavelength. FT-IR was used to identify which functional groups are Journal of Chemistry involved in reducing the metal ions to nanoparticles and nanocomposites. Te size, crystallinity, and purity of the nanoparticles and nanocomposites were characterized using XRD [30].

Antibacterial Activity Test. Te test was done using
Muller-Hinton agar media by the disc difusion method. 1 mL of actively growing bacterial inoculums (from the logarithmic growth phase) that have approximately 10 7 CFU·mL −1 (colony forming units) (0.5 McFarland Standard) was uniformly spread using a swab on the agar media. Te inoculated plates were put at room temperature for 6 min to allow for any surface moisture to be absorbed before applying the samples. Simultaneously, 50 mg·mL −1 of the green synthesized NPs and NCs was prepared in a 10% DMSO, and the autoclaved flter paper discs (diameter: 6 mm) were loaded with synthesized NPs and NCs. When the flter paper absorbed the green synthesized NPs and NCs, these were put on the Mueller-Hinton agar plates that had been inoculated. Erythromycin (15 μg) was used as the positive control, and a flter paper disc soaked with 10% DMSO was used as the negative control. Te zones of inhibition were measured in mm after incubation of plates for 24 h at 37°C [31].

Minimum Inhibitory Concentration (MIC).
Te green synthesized nanoparticles and nanocomposites that showed a positive efect in a bacteria test for the disc difusion were used to determine the minimum inhibitory concentration (MIC) by using the dilution method with little modifcation. Serial dilutions of the green synthesized nanoparticles and nanocomposites were prepared in the 10% DMSO with concentrations of 6.25, 12.50, 15, 25, and 50 mg/mL. Ten, flter paper discs containing the samples at the desired concentration were placed on top of the agar surface.
Generally, antimicrobial agents were difused into the agar, and zones of inhibition were measured [32].

Data Analysis.
Te data related to antibacterial activities were done by using one-way ANOVA using statistical analysis software (SAS 14.1) at α � 0.05.

Phytochemical Screening of Zingiber ofcinale (ZO)
Rhizome Extract. In this study, Zingiber ofcinale (ZO) has been extracted by using deionized water and then the presence of its phytochemicals such as tannins, phenols, favonoids, alkaloids, and saponins was tested. During synthesis, the phytochemicals found in the extract are used as the reducing agent to change metal ions to metal oxide nanoparticles and nanocomposites and simultaneously used as capping agents to protect the NP agglomeration. Te results of the qualitative phytochemical analysis of the Zingiber ofcinale (ZO) extract are shown in Table 2. Te result represented in Table 2 indicates the presence of favonoids, phenols, alkaloids, and saponins confrming the availability of polyols which serve as the capping agent and reducing agent.

Synthesis of CuO, ZnO Nanoparticles, and CuO/ZnO
Nanocomposites. Te green method was applied to synthesize CuO, ZnO NPs, and CuO/ZnO NCs using Zingiber ofcinale rhizome extract. Te phytochemicals found in the extract acted as reducing agents to reduce the metal ions to their corresponding nanoparticles. When the Zingiber ofcinale extract was added to blue copper nitrate, greenish color was formed; after 2 h of heating, dark brown solid (precipitate) formed indicating the formation of CuO nanoparticles. When the Zingiber ofcinale extract was added to colorless zinc acetate, a white precipitate was formed indicating the formation of ZnO nanoparticles. When the Zingiber ofcinale extract was added to the mixed solution of blue copper nitrate and zinc acetate, a light greenish blue precipitate was formed indicating the formation of CuO/ZnO NCs (Figure 3).   Journal of Chemistry Accordingly, 100 mL of metal precursor with diferent concentrations was used. So, 0.01, 0.03, 0.05, 0.1, and 0.2 M concentrations were used. As the concentration of the solution metal precursor was increased from 0.05 M to 0.1 M, the sharpness and intensity of the absorption peak were increased. However, when the metal precursor concentration was further increased to 0.2 M, the intensity of the absorption peak decreased. As a result, it was determined that further increasing the concentration of metal precursor more than the optimized value made a decrease in the yield in the synthesis of nanoparticles. Tis is likely due to the fact that the higher concentrations of Cu (NO 3 ) 2 and Zn (CH 3 COO) 2 act in favor of agglomeration of the CuO and ZnO particles rather than in the formation of capped nanoparticles and nanocomposites in a colloidal solution [34]. Ten, 0.1 M was taken as the optimum concentration for the synthesis of CuO, ZnO, and CuO/ZnO NCs.

Efect of Volume of Zingiber ofcinale Rhizome
Extracts. Te process of synthesis of NPs and NCs using plant extracts relies on the specifc phytochemicals found in plant extracts and the amount used [35]. Te amount of plant extracts used in the preparation of nanoparticles infuences how long it takes to synthesize them. Previous report has found that using more extracts can speed up the synthesis process of nanoparticles. Tis is because there are more chemical ingredients available in the solution which binds with the precursor to synthesize nanoparticle form rapidly and stabilize them [36]. In this study, 10 mL, 15 mL, 20 mL, 25 mL, and 30 mL aqueous solutions of Zingiber ofcinale rhizome extract were used with 100 mL of 0.1 M precursor salt solutions. It was noticed that the absorption and peak prominence got better when the amount of extract increased from 10 mL to 25 mL. Ten 25 mL was optimized because a sharp peak with high intensity was obtained.  When the volume was higher or smaller than this amount, the absorption peak was decreased. Tis shows that when the amount of extracts is higher, the biomolecules act as reducing agents and cap the nanoparticle surfaces preventing them from aggregation [37].

Efect of pH. Te pH value measures how acidic or basic a solution is. Te varying pH values of the solution infuenced the synthesis of CuO and ZnO nanoparticles and
CuO/ZnO nanocomposites. As previously reported, the pH level can greatly impact the size and texture of certain nanoparticles synthesized using plant extracts [33]. In addition, changes in pH levels have been used to control the shape and size of the synthesized nanoparticles [38]. Te pH of the solution varied from 5 to 12. When the pH level of the solution was raised, the absorption peak of nanoparticles and nanocomposites increased. Te characteristic absorption peak was obtained at pH 10 and 11, so pH 11 was optimized because a better peak and maximum absorption peak intensity were found at pH 11 with 25 mL of plant extracts. At pH 5-7, no absorption peaks were seen, indicating that the acidic pH is not good for efcient synthesis due to the slow reaction rate. At pH 12, the absorption peak was reduced due to NP aggregation, indicating that the basic pH of 11 is best for producing CuO/ZnO NCs and CuO and ZnO NPs [39].

Efect of Temperature.
Temperature is an important factor that afects the synthesis of metal oxide nanoparticles. Te temperature suggested for the biosynthesis of metal oxide nanoparticles is between 25°C and 100°C [40]. To identify the infuence of temperature on the synthesis of CuO/ZnO NCs, ZnO, and CuO NPs, optimization was done at six diferent temperatures (room temperature, 50°C, 70°C, 80°C, 90°C, and 100°C) to obtain optimum temperature synthesis and keep the amount of the precursor constant (25 mL). 80°C for CuO NPs and 90°C were optimized for ZnO NPs and CuO/ZnO NCs. According to the results, the intensity of NP and NC absorbance peak increased with temperature. Tis observation may be due to the fact that at higher temperatures, the reduction of metal ions to its nanoparticles is rapid [39]. However, when further increased in temperature beyond the optimized value, the absorption peak became less intense. Tis observation may also be due to the agglomeration of the NPs and NCs, possibly because the heat destroyed the reducing agents and capping agents found in the plant extracts [39]. respectively. 10% and 20% were optimized because the absorption peak with high intensity and the largest wavelength was obtained at these ratios ( Figure S2).  Journal of Chemistry change, the reduction of metal ions to nanoparticles and nanocomposites was confrmed by measuring the UV-Visible spectrum, for the detection of surface plasmon resonance (SPR) by taking a small amount of the synthesized sample and diluting it in deionized water. UV-Vis spectrum was measured in the wavelength range of 200-800 nm. An aqueous extract of the Zingiber ofcinale rhizome has been used to synthesize NPs and NCs. Te green synthesized CuO, ZnO, 10% CuO/ZnO, and 20% CuO/ZnO nanocomposites exhibited maximum absorption bands at 270 nm, 355 nm, 365 nm, and 370 nm, respectively (Figure 4). Te result matches with values that were reported previously [41][42][43]. Te addition of CuO NPs improved the energy band gap compared to ZnO NPs without the addition of any material. Te value of the energy band gap was calculated using the Tauc equation [44]. Te adsorption edges of the ZnO NPs were blue-shifted when compared to the wavelength of bulk ZnO which was seen at 385 nm [45]. Te absorption peak shift towards blue was because of a decrease in the particle sizes for the ZnO NPs synthesized by using Zingiber ofcinale rhizome extract, and this change is due to the quantum confnement efect [46]. Te energy band gaps were 1.65 eV, 2.90 eV, 2.76 eV, and 2.58 eV for CuO NPs, ZnO NPs, 10% CuO/ZnO, and 20% CuO/ZnO NCs, respectively ( Figure 5). Te band gap for ZnO-NPs is higher compared to CuO/ZnO NCs, which indicates that an increase in CuO amount in the nanocomposites results in a decrease in the energy band gap. On the other hand, Zingiber ofcinale rhizome extract shows a UV-Visible absorption peak of 275 nm in the ultraviolet region ( Figure 4). Tis is in line with the previously reported value [23].

Fourier Transform Infrared (FT-IR) Spectroscopy
Analysis. Phytochemicals that are responsible for capping, reduction, and stabilizing are identifed by using FT-IR. FT-IR characterization was conducted at Addis Ababa University. Te FT-IR peaks are assigned to the diferent functional groups of molecules found in the Zingiber ofcinale rhizome extract, nanoparticles, and nanocomposites [47]. FT-IR analysis was done to identify functional groups of biomolecules involved in the green synthesis of CuO, ZnO NPs, and CuO/ZnO NCs.
In this study, the identifcation of the functional groups in the Zingiber ofcinale rhizome extract and green synthesized nanocomposites was done using FT-IR spectroscopy ( Figure 6), which confrmed that the Zingiber ofcinale rhizome extracts contain compounds bearing the -OH, C-H (due to aldehyde), C�O, -C-C, and -C-H (methyl) functional group because of the appearance of peaks at around 3233 (broad), 2930, 1622, 1391, 1107 (C-O), and 1047 cm −1 (C-OH). Te broad -OH peak shows the presence of phenolic compounds, which is possibly responsible for the stabilization process of the nanoparticles. Te carbonyl groups appeared to confrm that compounds such as ketones, esters, and aldehydes are present [48].
Te FT-IR spectrum showed peaks at 3570, 3264, 1620, 1381, 1076, 937, and 693 cm −1 for CuO NPs, 3347, 1653, 1560 (Figure 7). Te peak at 3570 corresponds to the N-H stretching of amines, showing the involvement of amines in the stabilization of NPs and NCs [49]. Te broad peaks at 3356, 3347, 3345, and 3264 cm −1 can be attributed to O-H stretching vibration. Te presence of the -OH functional group suggests the presence of absorbed water on the surface of the synthesized nanoparticles and nanocomposites [50].
Te characteristic peaks at 1620, 1645, 1647, and 1653 cm −1 can be assigned to C�C (carbonyl group)    stretching. Te absorption band at 1559, 1560, and 1580 cm −1 could be ascribed to amine-N-H stretching aromatic compounds corresponding to the biomolecules from ZO extract during the synthesis of samples. Te broad absorption band at 1381, 1395, and 1397 cm −1 was attributed to the O-C-O stretching of esters or may be due to the C-H stretching vibration of the alkene group. Te bands between 1042 and 1076 cm −1 are assigned to the stretching of C-O of phenols [51], whereas 937, 860, and 841 cm −1 may be assigned to C-H and C�C of the alkene [52].
Te FT-IR spectra for the CuO, ZnO NPs, and CuO/ZnO NCs showed slight changes in some related peaks. Te successful synthesis of Cu-O and Zn-O in all the samples was confrmed by the appearance of peaks at low wavelengths from 420 to 691 cm −1 [53]. Te mode of vibration of Cu-O and Zn-O is in the range of 700-400 cm −1 [19]. FT-IR spectra of the green synthesized NPs and NCs showed slight changes in some related peaks and their intensities, indicating that the major biomolecules from the Zingiber ofcinale rhizome extract were capped or bound to the surface of CuO, ZnO, and CuO/ZnO NCs (Figure 7). Te absence of impurities is also refected in the XRD pattern. Te average crystalline size of CuO NPs, ZnO NPs, 10% CuO/ZnO, and 20% CuO/ZnO NCs obtained from full width at half maximum of difraction is 4. 35, 14.54, 18.41, and 20.50 nm, respectively. Te intensities of peaks for ZnO NPs are higher compared to those for CuO NPs (Figure 8). Tis confrmed that ZnO NPs have a higher percentage in the CuO/ZnO NCs and are highly crystalline. Te reason for the low peak intensities of CuO in CuO/ZnO is because of the coating role of ZnO NPs on CuO NPs [56].

X-Ray
Te average crystallite size of each sample was calculated by using the Debye-Scherrer formula.
Te average crystalline sizes of the CuO NPs obtained from the XRD data were between 1.05 nm and 9.29 nm, whereas for ZnO NPs, they were between 9.22 nm and 21.82 nm, for 10% CuO/ZnO, they were between 11.83 nm to 28.17 nm, and for 20% CuO/ZnO, they were between 16.16 nm to 31.82 nm (Table 3). Based on the XRD analysis, the average crystalline size of the CuO/ZnO increased as the ratio of CuO increased. Tis might be because the sizes of copper ions and zinc ions are diferent.

Antibacterial Activity of Green Synthesized CuO NPs, ZnO
NPs, and CuO/ZnO NCs. Antibacterial activities of CuO NPs, ZnO NPs, 10% CuO/ZnO, and 20% CuO/ZnO NCs were tested for antibacterial activities starting from 50 mg/ mL up to 6.25 mg/mL. As shown in Table 4, as the concentration of the NPs and NCs decreases, bacterial growth inhibition decreases for all samples and there was no inhibition at 6.25 mg/mL. Te green synthesized nanoparticles and nanocomposites were active against both Gram-positive and Gram-negative bacteria using the disc difusion method. Te maximum growth inhibition was recorded by the 20%    (Table 4). At all concentration levels for all the samples, the antimicrobial potential was strong on the Gram-positive strain (S. aureus) as compared to the Gram-negative bacteria (E. coli). But at 15 mg/mL, CuO NPs and ZnO NPs showed better efectiveness on E. coli than S. aureus (Table 4). At all concentrations, CuO NPs and ZnO NPs showed lower inhibition as compared to CuO/ZnO nanocomposite for both bacterial strains. Tis is because the combination of CuO and ZnO in the nanocomposite has a more free surface that can produce a higher amount of ROS compared to CuO and ZnO alone; adding CuO on ZnO produces more surface defect on CuO/ ZnO. When the surface has more defects, it also has more ROS; this causes more bacteria to be prevented from growing [57].
From Table 4, it was concluded that the nanocomposites were found to be more efective at inhibiting the growth of Gram-positive bacteria compared to Gram-negative bacteria. Tis is because Gram-negative bacteria (Escherichia coli) typically contain thin cell walls and an outer membrane, which protects the inside of bacteria. Te protective layer on the outside of the bacterial cell stops some drugs and antibiotics from getting inside. Gram-positive bacteria have a thick layer around their cells and a layer of the cytoplasmic membrane. Tis makes them easier to be killed by antibiotics compared to Gram-negative bacteria [58]. Gram-positive bacteria have thick cell walls that can absorb nanocomposites more than Gram-negative bacteria [59]. Te most distinctive feature of Gram-positive bacteria is the thickness of its cell wall because a peptidoglycan layer is present [60]. When the energy band gap is smaller, the antibacterial activity increases because the electrons can move more easily from the valence band to the conduction band. Te antibacterial activity of all samples further improved with increased concentration (Figure 9).

Possible Antibacterial Mechanism of Synthesized CuO,
ZnO, and CuO/ZnO NCs. Te NPs and NCs damage and kill bacteria and other microorganisms by producing ROS, breaking down the outer layers of their cells, and interfering with their proteins and DNA [61]. ROS are compounds that contain oxygen, and they are made up of highly unstable oxygen radicals such as superoxide (O 2 •), hydroxyl (OH•), hydrogen peroxide (H 2 O 2 ), and singlet oxygen (O 2 ) [62]. In this process, NPs can damage diferent microbial cell components by diferent mechanisms. CuO, ZnO, and CuO/ ZnO NCs can harm diferent cell functions and harm cells and exert cytotoxicity, which makes them useful in stopping the growth of microbes ( Figure S3).
Generally, CuO, ZnO NPs, and CuO/ZnO NCs destroy the bacterial cells by producing reactive oxygen species (ROS) and by changing or attaching to the natural components in metalloproteins [63].

Conclusions
Te green and eco-friendly method was used to synthesize CuO/ZnO nanocomposites using Zingiber ofcinale rhizome extract as a reducing and stabilizing agent. A combination of two semiconductors could make the hybrid composites work better than just single material on its own. In this study, 10% CuO/ZnO and 20% CuO/ZnO nanocomposites, which have enhanced antibacterial activity than either of the CuO and ZnO nanoparticles, were green synthesized by using copper and zinc precursors with Zingiber ofcinale rhizome extract. UV-Vis, FT-IR, and XRD were used to investigate surface plasmon resonance (SPR), functional groups, and structure of samples. Te UV-Vis absorption peaks indicate the formation of CuO, ZnO NPs, and CuO/ZnO NCs. FT-IR showed the presence of main functional groups in the synthesized samples. X-ray difraction indicates the formation of the crystalline monoclinic structure of CuO NPs and the hexagonal structure of ZnO NPs and CuO/ZnO NCs. All synthesized NPs and NCs have shown good antibacterial activity on S. aureus. However, the 10% CuO/ZnO and 20% CuO/ZnO NCs revealed enhanced antibacterial activity than CuO NPs and ZnO NPs against both S. aureus and E. coli. Tis is due to the synergistic efect between the metal oxide nanoparticles in the nanocomposites. Generally, the result of this study indicates that synthesized NPs and NCs are more efcient against S. aureus compared to E. coli.

Data Availability
Te data used in this study are available from the corresponding author upon request.