Comparative study of zinc oxide nanocomposites with different noble metals synthesized by biological method for photocatalytic disinfection of Escherichia coli present in hospital wastewater

The pollution of natural water by chemical and biological contaminants is being recognized as a major global concern. These pollutants are highly hazardous not only to the health of humans but also to the entire ecosystem. Moreover, there has been a growing occurrence of antibiotic-resistant bacteria in water sources (Odonkor & Addo 2018). The prevalence of multidrug-resistant Escherichia coli in water sources is reported as 49.48% among other species. In addition, E. coli is susceptible to almost all antimicrobial agents, but it has a great capacity to accumulate antimicrobial resistance genes (Abu-Sini et al. 2023). Multidrug resistance in E. coli has become a worrying issue and is increasingly observed (Poirel et al. 2018). Hospital wastewater acts as a reservoir for antibiotic resistance genes rather than other wastewater systems (Zhang et al. 2020).

Advanced oxidation processes (AOP) are one of the most effective methods for the elimination of organic compounds and bacteria from water and wastewater by oxidation (Sadeghfar et al. 2021). This is due to the fact that these processes mainly use hydroxyl radicals (^•OH), which have a high oxidation potential, for the remediation of organic contaminants and hazardous pollutants into carbon dioxide and water. Photocatalysis is a heterogeneous AOP process using ultraviolet (UV) light irradiation and a semiconductor photocatalyst to produce ^•OH radicals, which has been an eco-friendly process for the removal of organic pollutants from water (Ahmed & Haider 2018), especially emerging contaminants such as pharmaceutical and personal care products (Çifçi et al. 2016; Oluwole et al. 2020). Several metal oxide materials have been used as photocatalysts, such as titanium dioxide (TiO₂) and zinc oxide (ZnO), to accelerate the degradation and disinfection of chemical and biological compounds (Roy & Chakraborty 2021; Shintre et al. 2022). ZnO nanoparticles have shown some additional advantages, such as being relatively non-toxic, cheap, safe, biocompatible, and easily available, which has allowed them to be widely used in photocatalytic and biological applications (Moezzi et al. 2012).

Generally, when ZnO is irradiated with UV light at a wavelength less than 385 nm, electrons in the valence band (VB) jump to the conduction band (CB), subsequently producing positive holes (h⁺) and electrons (Moezzi et al. 2012). The VB holes react with the water molecules and hydroxide ions to form hydroxyl (^•OH) radicals, whereas the electron reacts with oxygen molecules and forms superoxide anion (⁠⁠) radicals. The ^•OH radical is a powerful oxidizing agent that reacts with and decomposes most organic compounds. However, ZnO has a fast recombination of photoinduced electron and hole pairs, which in turn reduces its photocatalytic efficiency (Nagaraju et al. 2017). Therefore, many studies have been focused on the hybridization of ZnO with noble metals to improve charge separation, thereby increasing the photocatalytic activity of ZnO (Bloh et al. 2014; Liu et al. 2017; Nagaraju et al. 2017; Manríquez et al. 2018; Pathak et al. 2019; Shintre et al. 2022). These active noble metals for modification of ZnO, for example, are silver (Ag), ruthenium (Ru), palladium (Pd), and gold (Au). Most reports have paid more attention to the comparison of the photocatalytic activity between pure ZnO and metal-modified ZnO (Bloh et al. 2014; Adeel et al. 2021; Shintre et al. 2022). However, it is worthwhile studying the influence of different noble metals on metal-modified ZnO. The catalytic activity of noble metal-ZnO nanostructures has been found to rely on not only the species of noble metal but also the architecture of the catalyst material (Liu et al. 2017).

Pathak et al. (2019) have reported that the morphology and optical properties of Pd-doped ZnO, Au-doped ZnO, and Ag-doped ZnO were similar, whereas Ag–ZnO provided higher antimicrobial activity and a lower minimum inhibitory concentration (MIC) than the others. This is due to the strong antimicrobial effect of Ag. On the other hand, RuO₂ has been studied as a catalyst showing strong oxidation and antimicrobial properties (Kannan & Sundrarajan 2015; Manríquez et al. 2018). However, Ag–ZnO and Ru–ZnO have not been studied comparatively yet. Typically, the synthesis of ZnO and ZnO nanocomposites utilizes chemical methods. In recent years, green synthesis has attracted significant attention as a simple, cost-effective, and eco-friendly alternative route to conventional chemical and physical methods for the preparation of nanoparticles and metal oxides. Bioactive constituents in plant extracts are utilized as natural reducing, stabilizing, and capping agents for synthesizing nanoparticles (Basnet et al. 2018).

Callistemo viminalis, commonly known as bottlebrush, is widely distributed across the world. It has been reported to have medical importance, such as antibacterial, antifungal, antioxidant, and other pharmaceutical and insecticidal properties (Ahmad & Athar 2017). C. viminalis (CV) extract is rich in phenolics and flavonoids (Salem et al. 2017), which are involved in the stabilization, formation, and bioreduction of metal oxides and metal nanoparticles (Basnet et al. 2018). In this research, two ZnO-based nanocomposites with different noble metals (Ag and Ru) were prepared by green synthesis. The leaf extract of CV was used as a bio-reducing agent for nanoparticle formation through a precipitation process. The characteristics, photocatalytic disinfection, and degradation efficiencies of silver–zinc oxide (Ag–ZnO) and ruthenium–zinc oxide (Ru–ZnO) nanocomposites were studied. The photocatalytic disinfection was tested against Escherichia coli present in hospital wastewater, while the photocatalytic degradation was evaluated using methylene blue (MB) dye. MB is widely used in hospital activities for various diagnostic and therapeutic purposes (Muttaqin et al. 2022); thus, it has been reported as a hospital wastewater model pollutant as well. The novelty of this work was to reveal the influence of different noble metal incorporation on the physicochemical properties and photocatalytic performance between Ag–ZnO and Ru–ZnO nanocomposites.

Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), ruthenium (III) chloride (RuCl3), and silver nitrate (AgNO₃), provided by Sigma-Aldrich (St. Louis, MO, USA), and sodium hydroxide (NaOH) pellets from Carlo Erba, France, were applied, respectively, as precursors and precipitating agents for the synthesis process. MB was purchased from Sigma Aldrich, USA. Nutrient agar (NA), Muller–Hilton agar (MHA), and peptone were purchased from Difco Laboratories, USA. All the chemicals used were of analytical grade, and all solutions were prepared in deionized (DI) water.

The fresh leaves of CV were collected from a local garden. Later, they were cleaned with DI water, air-dried for 48 h, and cut into small pieces. To produce the extract, 30 g of the small leaf pieces were added to 250 mL of DI water and heated at 60 °C for 20 min. Then, the aqueous extract was cooled down and filtered through Whatman filter paper no. 1. The obtained extract (a pale yellow) was kept in a refrigerator at 4 °C for experimentation.

The pure ZnO, Ag–ZnO, and Ru–ZnO nanocomposites were synthesized via a precipitation method using CV extract. Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), ruthenium (III) chloride (RuCl₃), and silver nitrate (AgNO₃) were used as Zn, Ru, and Ag precursors, respectively, and prepared in DI water.

To synthesize ZnO nanoparticles (NPs), a solution (100 mL) containing 70 mM zinc nitrate was mixed with 10 mL of CV leaf extract under stirring. The solution was then adjusted to pH 12 by adding 1 M NaOH dropwise. Next, the reaction solution was heated to 60 °C and kept under constant stirring for several hours until the color of the solution stopped changing further. The precipitates settled at the bottom of the beaker. After this, the system was allowed to cool down to room temperature. The supernatant was discarded, and the precipitates were collected and air-dried for 24 h. Lastly, they were thoroughly ground into a fine powder and calcined for 3 h at 300 °C.

The wastewater sample was gathered from the wastewater treatment plant of Songklanagarind Hospital (Songkhla, Thailand). E. coli was isolated from wastewater, according to Maal et al. (2015). One liter of wastewater was filtered through a nitrocellulose filter (pore size 0.22 μm). The filtrate was mixed into 500 mL of nutrient broth medium (HiMedia Laboratories Pvt. Ltd, Mumbai, India) and incubated for 24 h. After incubation, the nutrient broth medium containing cultured bacteria was streaked on MacConkey agar (Becton, Dickson, and Company, USA) and incubated for 24 h. The pink colonies were observed and collected from the MacConkey agar, and they were re-streaked on eosin methylene blue (EMB) agar (HiMedia Laboratories Pvt. Ltd, India). E. coli bacteria were found in the metallic green sheen colonies on EMB agar (Maal et al. 2015). A single colony of E. coli was picked, and it was subcultured in NA media (HiMedia Laboratories Pvt. Ltd). Isolated E. coli from NA plates was transferred to LB broth and incubated at 37 °C overnight for use in further experiments.

The photocatalytic disinfection of biosynthesized ZnO, Ag–ZnO, and Ru–ZnO was evaluated against E. coli present in hospital wastewater. The suspensions of pure ZnO, Ag–ZnO, and Ru–ZnO were prepared at concentrations of 1 mg/mL). For this, 10 mL of E. coli stock suspension was added to the flask containing 90 mL of a 0.9% saline solution and incubated for 2 h. Subsequently, 100 mL of bacterial solution was taken and added to 900 mL of the sterile DI water to form the stock solution (2.03 × 10⁷ CFU/mL). Next, 1 mL of pure ZnO, Ag–ZnO, and Ru–ZnO suspensions (1 mg/mL) were added to 99 mL of the E. coli solution. The final concentration of biosynthesized nanomaterials in the E. coli solution was 1 mg/100 mL. The suspension was constantly stirred and subjected to UVA illumination for 60 min at 25 °C. An aliquot (3 mL) of the suspension was taken at 15-min intervals and immediately diluted to an appropriate dilution. The diluted samples were spread on NA and incubated at 37 °C for 24 h. The number of colonies formed was counted to determine the number of viable cells. All the above experiments were conducted in triplicate. The percentage of disinfection (%) was determined by Equation (2). The disinfection kinetics was described by a pseudo-first-order reaction using Equation (3).

The agar disc diffusion assay was carried out to evaluate the antibacterial properties of the biosynthesized ZnO, Ag–ZnO, and Ru–ZnO. Initially, an isolated E. coli inoculum (2.8 × 10⁷ CFU/mL) was spread on the MHA agar plate, and then sterile discs loaded with 40 μL of pure ZnO, Ag–ZnO, and Ru–ZnO suspensions (two concentrations of 1 and 10 mg/mL) were placed on the MHA plates. The plates were then incubated at 37 °C. After 24 h, the inhibition zone formed around each disc was measured. Tests were performed in triplicate.

The microbial reduction efficiency of ZnO, Ag–ZnO, and Ru–ZnO was further determined using a standard test method (ISO 27447:2019 Test Method for Antibacterial Activity of Semiconducting Photocatalytic Materials). For this, 2 g of ZnO, Ag–ZnO, and Ru–ZnO were dispersed in 100 mL of sterile DI water using ultrasonication to give a uniform suspension. Subsequently, the dispersed suspension (1 mL) was drop-coated onto the surface of the filter paper (Whatman no. 1, 5 × 5 cm) and allowed to dry at 100 °C for 30 min. The determined concentration of ZnO, Ag–ZnO, and Ru–ZnO on the coated paper was 0.8 mg/cm². The antimicrobial activity was performed by pouring about 1 mL (4 × 10⁴ CFU/mL) on the photocatalyst-coated and noncoated papers and then covering them with a glass coverslip. Next, the samples were placed in the absence or presence of UVA irradiation for 8 h. After the desired time, the paper specimens were washed with sterile DI water and immediately diluted. The density of living cells was then also determined by counting the CFU/mL on NA.

The leaf extract of CV is reported to be rich in polyphenols and flavonoids (Salem et al. 2017). Polyphenolic compounds, mainly phenolic acids, present in the CV extract acted as both reducing and stabilizing agents to synthesize nanoparticles (Dwivedi et al. 2021). The presence of OH groups in flavonoids and phenolic compounds is responsible for the reduction of metal ions into nanoparticles, and the C = O − C, C = C, and C = O groups in leaf extract may act as stabilizers (Basnet et al. 2018).

The XRD analysis of ZnO, Ag–ZnO, and Ru–ZnO nanocomposites prepared using CV extract (Figure 3) reveals ZnO in a hexagonal wurtzite structure. The diffraction pattern clearly shows peaks corresponding to ZnO located at 31.65°, 34.32°, 36.00°, 47.49°, 56.46°, 62.83°, 66.32°, 67.83°, 69.00°, 72.5°, and 77°, which corresponded to crystal faces of (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), and (202) planes. The sharp and strong peaks of the XRD patterns showed that the prepared ZnO, Ag–ZnO, and Ru–ZnO particles had high crystallinity and were in the nanoscale range. The XRD pattern of Ag–ZnO also reveals the additional peaks of Ag–ZnO at 38° (111), 44.6° (200), and 64.1° (220), which correspond to cubic metallic Ag. The result indicated that Ag⁺ could get precipitated as an Ag metallic phase, confirming the successful formation of an Ag–ZnO nanocomposite. On the other hand, two extra peaks positioned at 28° (110) and 54.2° (211), which corresponded to tetragonal RuO₂ nanoparticles, could be observed for Ru–ZnO. No metallic Ru peak (Ru°) was detected by XRD analysis, which agrees well with the previous report (Bloh et al. 2014). The result confirmed the formation of ZnO with a small quantity of crystal RuO₂. The diameter of the ZnO crystallite was calculated using the Debye–Scherrer formula. The ZnO crystallite sizes were in order of ZnO (25.4 nm) < Ag–ZnO (29.8 nm) < Ru–ZnO (34.4 nm). Similar results were found in previous works showing that the crystallite sizes of Ru–ZnO and Ag–ZnO were larger than those of pure ZnO (Aranganayagam et al. 2013; Nagaraju et al. 2017). An increase in crystallite size with Ag and Ru incorporation indicated ZnO lattice expansion.

An earlier report (Michael et al. 2014) suggested that noble metal can be incorporated into the ZnO crystal either as a substituent or as an interstitial atom, thereby introducing defects in the ZnO lattice. If Zn²⁺ is substituted by metal ions, a corresponding peak would be expected. However, no shift in the ZnO peaks was observed in the present study, which agrees with the previous work of Peng et al. (2019). Ag atoms can easily diffuse through the ZnO crystal structure and lead to implantation as Ag interstitial atoms (Masoumi et al. 2017). X-ray photoelectron spectroscopy (XPS) study previously confirmed the binding energies of metallic Ag in Ag–ZnO (Peng et al. 2019). On the other hand, the chemical oxidation states of Ru in the Ru–ZnO samples were investigated using the XPS technique (Manríquez et al. 2018). They found that two oxidation states exist on the surface of the Ru–ZnO solids: major Ru⁴⁺ (RuO₂) and minor Ru⁶⁺ (RuO₃). Several reports revealed that the Ru ions are incorporated into the ZnO lattice as either Ru⁶⁺ ions (Bloh et al. 2014), Ru⁴⁺ ions (Kumar et al. 2014), or Ru³⁺ ions (Aranganayagam et al. 2013). In the present work, the major oxidation state of the Ru ions in the Ru–ZnO nanocomposites is probably Ru⁴⁺ (RuO₂). RuO₂ was observed in XRD patterns, but other Ru species were not detected.

Figure S1 presents FESEM images of the pure ZnO and Ag/ZnO nanocomposites. The spherical or block-like morphology, having approximately less than 100 nm in size with a smooth surface, was shown. Contrary to this, Ru–ZnO nanocomposites showed an irregular shape and tended to agglomerate in plagues. The particle size of Ru–ZnO was greater than that of pure ZnO and Ag–ZnO. This is probably due to the aggregation of RuO₂ on the surface of ZnO nanoparticles. The EDS spectra indicated that ZnO is the host material for Ag–ZnO and Ru–ZnO nanocomposites. The weight percentages of Ag and Ru elements achieved from the EDS studies were 17.07% Ru and 4.30% Ag (Figure S1).

Figure 5 displays the FTIR spectra of CV-mediated ZnO, Ag–ZnO, and Ru–ZnO samples. This was attributed to the interaction between functional groups in CV extract and prepared metal oxides. Noticeable vibration bands of biosynthesized ZnO were observed at 835, 840, and 1,355 cm⁻¹, whereas Ag–ZnO and Ru–ZnO nanocomposites had slightly different peaks at 696, 840, 1,153, 1,409, and 1,427 cm⁻¹. The bands at 1,355, 1,409, and 1,427 cm⁻¹ were assigned to the hydroxyl group (O–H bonds), which indicates the presence of O − H bending vibration in phenolic compounds absorbed onto the metal oxide surface. Additionally, the vibration peaks at 1,153 cm⁻¹ (C–O bonds), 835 and 840 cm⁻¹ (C = C bonds), and 696 cm⁻¹ (C–H bonds) were presented. The interaction between biological molecules and a metal oxide surface can create a layer that prevents agglomeration and increases the stability of nanoparticles (Faisal et al. 2021).

The incorporation of Ag during the ZnO synthesis process caused an increase in the absorbance value of Ag–ZnO in comparison to pure ZnO (Figure 6). A similar result was reported in a previous study (Kadam et al. 2018). The maximum adsorption value depends on the surface plasmon resonance (SPR) (Zare et al. 2019). Furthermore, a broad peak of Ag–ZnO was reported due to a plasmon band of Ag atoms, or a cluster of Ag atoms located on the surface of ZnO nanocrystals (Michael et al. 2014). Conversely, the addition of Ru caused a negative effect on the UV absorption of Ru–ZnO. This showed that the noble metals have a substantial effect on the optical properties of ZnO. The UV absorption capacity was found to be in the order Ag–ZnO > ZnO > Ru–ZnO. UV absorption is a first step in the semiconductor photocatalysis process. If Ru–ZnO exhibited poor UV absorption, the activation of the Ru–ZnO photocatalyst would be less.

ZnO can absorb UV light with a wavelength equal to or less than 385 nm to produce ^•OH radicals, which attack the pollutants adsorbed on the surface of ZnO (Moezzi et al. 2012). Çifçi et al. (2016) reported that UVB irradiation (315 nm) provided a faster photocatalytic reaction rate than that under UVA irradiation. However, in the present work, the biosynthesized ZnO, Ag–ZnO, and Ru–ZnO samples exhibited good absorbance for UVA, but poor absorbance for UVB wavelengths (Figure 6). The photodegradation of MB dye was done under UV-A irradiation because of the strong absorbance of ZnO, Ag–ZnO, and Ru–ZnO at the UVA wavelength (Figure 6).

Modification of ZnO with noble metals effectively inhibits electron–hole recombination and thereby prolongs the lifetime of the electron–hole pairs and increases the amount of and ^•OH radicals (Fang et al. 2020). The present study first revealed the excellent photocatalytic performance of Ag–ZnO over Ru–ZnO. This might be attributed to the higher crystallinity, greater UV light adsorption capacity, and smaller particle sizes of biosynthesized Ag–ZnO, as shown in Figures 4, 6, and S1. High crystallinity of ZnO resulted in more generation of e–h pairs because of more adsorption of photons, and smaller particle sizes could generate more active sites for dye absorption and degradation (Supin et al. 2023).

Peng et al. (2019) investigated the role of active species (^•OH and ⁠) in the photocatalytic degradation of phenol over ZnO and Ag/ZnO using electron paramagnetic resonance (EPR) and quenching tests. They found that the intensity of ^•OH and signals in energy dispersive spectroscopy (EDS) spectra was significantly increased in Ag–ZnO in comparison to ZnO, which was consistent with the photocatalytic activity. The quenching test using the scavengers for ^•OH and revealed both ^•OH and contributions to the decomposition of phenol (Peng et al. 2019). On the other hand, Ding et al. (2020) reported that the EPR spectra of Ag/ZnO showed the existence and abundance of •OH radical peaks, whereas the radical peak was not clear and insufficient, and the results all corresponded to quenching experiments. They suggested that the ^•OH radical might play a more critical role than in the photocatalytic degradation of metronidazole as an antimicrobial drug for Ag/ZnO.

The Ag–ZnO exhibited the highest photocatalytic disinfection among those prepared samples. The improved photocatalytic disinfection of Ag–ZnO can be attributed to multiple synergistic effects of greater UV absorption capacity, better charge carrier separation, and additional antimicrobial Ag ability that effectively kills the bacterial population near it (Matai et al. 2014). A kinetic reaction was studied using the pseudo-first equation model. Experimental values were fitted and shown in Figure 8(b). The linear relationship indicates that the photocatalytic disinfection of E. coli follows a pseudo-first order. The k values of ZnO, Ru–ZnO, and Ag–ZnO were found to be 2.0 × 10⁻³, 6.8 × 10⁻³, and 19.3 × 10⁻³ min⁻¹, respectively (Table 1). The highest photocatalytic performance was observed for Ag–ZnO, with a rate constant nine times higher than that of ZnO and two times higher than that of Ru–ZnO. The photocatalytic disinfection efficiencies for ZnO, Ru–ZnO, and Ag–ZnO during 60 min of illumination with UVA light were 9.85, 34.98, and 75.37%, respectively.

Table 2 shows the inhibition zones of ZnO, Ru–ZnO, and Ag–ZnO against E. coli. Results show that ZnO and Ru–ZnO could not develop zones of inhibition at both concentrations (1 and 10 mg/mL). The significant antimicrobial activity of Ag–ZnO was comparable. Ag–ZnO exhibited strong activity against E. coli when increasing its concentration from 1 to 10 mg/mL. This suggests that Ag–ZnO can be applied without UVA irradiation for the inactivation of E. coli present in hospital wastewater, but it requires a high dosage (10 mg/mL). Therefore, Ag NPs could be responsible for the antibacterial action of Ag–ZnO because no inhibition of E. coli was found for ZnO NPs (Table 2). The development of an inhibition zone for high-dose Ag–ZnO is probably attributed to the increased antimicrobial effect of Ag NPs on the surface of ZnO. This result also suggested the resistance of E. coli in hospital wastewater to ZnO. On the other hand, the effect of Ag NPs against multidrug-resistant E. coli was also reported (Selem et al. 2022).

The antimicrobial performance of Ag–ZnO with and without UVA irradiation was observed based on the ISO 27447 standard test method. Table 3 shows that the percentage reduction of hospital wastewater E. coli after exposure to Ag–ZnO immobilized on filter paper for 8 h. The E. coli reduction efficiency was two times lower in the dark than that in the presence of UVA light. Under UVA irradiation, the reactive species play a dominant role in the photocatalytic disinfection process (Saravanan et al. 2021). The synergistic effect of reactive species (.OH) and Ag NPs on the surface of Ag–ZnO might considerably cause cell membrane damage, thereby automatically enhancing the inactivation of E. coli.

The present study aimed to investigate the effect of different noble metals on the characteristics and photocatalytic disinfection of biosynthesized metal-ZnO nanocomposites. The binary ZnO-based nanocomposites with silver and ruthenium metals were synthesized using CV aqueous extract. Characterizations were accomplished using XRD, FTIR, UV-Vis, FESEM, EDS, TEM, and SAED techniques, confirming the formation of Ag–ZnO and Ru–ZnO nanocomposites. Ag–ZnO and Ru–ZnO had different nanostructures, crystallinities, UV adsorption capacities, and photocatalytic efficiency. Ag–ZnO and Ru–ZnO exhibited remarkably higher photocatalytic efficiency and reaction rate than those of ZnO. Furthermore, the photocatalytic activity was improved dramatically by metallic Ag rather than Ru/RuO₂. Results also indicated that Ag–ZnO could inactivate E. coli present in hospital wastewater via photocatalytic disinfection under UVA irradiation and Ag antimicrobial itself in the dark. This work indicates the advantage of the binary ZnO nanocomposite together with metallic Ag over Ru/RuO₂.

This study was supported financially by the National Nanotechnology Center, National Science and Technology Development, Thailand (Grant No. P1751698). We extremely thank the Thailand Graduate Institute of Science and Technology, National Science and Technology Development, Thailand, for supporting graduate student scholarships.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.