In situ facile green synthesis of Ag–ZnO nanocomposites using Tetradenia riperia leaf extract and its antimicrobial efficacy on water disinfection

In this work, Ag–ZnO nanocomposites were prepared by a green synthesis route using aqueous leaf extract of Tetradenia riperia and investigated for antibacterial activity against Escherichia coli and Staphylococcus aureus. To optimize the synthesis of the Ag–ZnO, the effects of precursor concentrations, pH, and temperatures were studied. The Ag–ZnO nanocomposites were characterized by XRD, ATR-FTIR, FESEM, and TEM. Results show that the concentration of 8% Ag, the temperature of 80 °C, and a pH of 7–8 were optimal for the synthesis of Ag–ZnO nanocomposites. The XRD analysis showed the decrease in particle size of Ag–ZnO from 23.6 to 14.8 nm with an increase in Ag concentrations, which was further supported by FESEM analysis. TEM image of 8% Ag provides more information on the coexistence of Ag on ZnO where an average particle size of 14.8 nm was determined. The ATR-FTIR analysis confirmed the presence of phenolic compounds, which work as reducing and stabilizing agents. The antimicrobial activity results show that Ag–ZnO nanocomposite demonstrated a higher antimicrobial potency on E. coli than on S. aureus. Therefore, Tetradenia riperia leaf extract is a viable route for the synthesis of Ag–ZnO nanocomposites to be used for various applications, including water disinfection.

Preparation of Tetradenia riperia (TR) leaves extract. The collected leaves were washed several times with tap water followed by double distilled water (DD) to remove any dust particles from the surface. Then fresh leaves were chopped into small pieces and ground by using an electric mortar and blender into fine particles. About 20 g of fresh, ground Tetradenia riperia leaves were mixed in 100 ml of double-distilled water followed by shaking on a mechanical shaker for 24 h to allow effective extraction of phytocompounds from TR leaves. The obtained aqueous extract was centrifuged at 4000 rpm for 15 min, then the supernatant (brown color) was filtered using Whatman No.1 filter paper and stored at 4 °C ready for further use as a reducing and stabilizing agent during the green synthesis of Ag-ZnO nanocomposites (Fig. 1). The pH of Tetradenia riperia aqueous leaves extract was 5.10.

Synthesis of Ag-ZnO nanocomposites and ZnO nanoparticles. Biosynthesis of ZnO nanoparti-
cles. The synthesis of ZnO nanoparticles was carried out based on the literature 27 with modification. For the synthesis of ZnO NPs, 2.9748 g of ZnO (NO 3 ) 2 ·6H 2 O was dissolved in 100 ml of Tetradenia riperia leaf extract in a 250 ml Erlenmeyer flask, then stirred for 30 min. This lowered the pH of the media to 4.9. Then 0.1 M NaOH was added dropwise while stirring to adjust the pH of the media to 7-8. The batch was set under continuous stirring on magnetic stirring plate at room temperature (30 °C) for 3 h. The resultant mixture was incubated to age for 24 h, followed by centrifugation at 4000 rpm for 15 min. The supernatant was discarded, and the precipitate was redispersed in an ethanol-water mixture at a ratio of 1:1 (v/v), and then recentrifuged. The centrifugation and redispersion processes were repeated three times. Similar procedures were adopted for the batches prepared at 60 and 80 °C. The purified precipitates were dried at 80 °C in a hot air oven for 4 h, then calcined at 450 °C for 2 h at a heating rate of 5 °C/min. The white-coloured residues formed were ground into powder by using a Hargett mortar and pestle, then stored in an airtight container ready for further analysis and use.
Biosynthesis of Ag-ZnO nanocomposite. The Ag-ZnO nanocomposites were synthesized using a green synthesis method, adopted from the literature 41   Antibacterial assay. The antibacterial activity of the synthesized ZnO nanoparticles and Ag-ZnO nanocomposites was assessed against gram-positive (Staphylococcus aureus-ATCC 6538P) and gram-negative (Escherichia coli-ATCC 9677) strains using the disc diffusion method 47 . The prepared and sterilized nutrient agar media (15-20 ml) was poured into the sterilized petri dishes and allowed to solidify. After solidification of the nutrient agar medium, each bacterial strain was inoculated onto individual agar dishes and spread uniformly by using a sterilized swab. The sterilized absorbent discs of about 6 mm were soaked in a colloidal solution of ZnO and Ag-ZnO (1, 3, 5, and 8% Ag) with different concentrations (minimum inhibitory concentration-MIC) (0.5, 1.0, and 1.5 mg/ml). The soaked discs were placed on the inoculated Petri dishes along with Ciprofloxacin discs as standard and Tetradenia riperia extract discs as control. Each plate is comprised of three test discs, one standard disc, and one control disc. Thereafter, all dishes were incubated at 37 °C for 24 h. The antimicrobial activity of ZnO nanoparticles and Ag-ZnO nanocomposites was determined by measuring the zone of inhibition (ZOI), which appeared as clear areas around the discs.

Results and discussion
X-ray diffraction.  www.nature.com/scientificreports/ concentration increases, the peak intensity of ZnO decreases, implying a decrease in the crystallinity and particle size in Ag-ZnO nanocomposites 65 44 . However, a subsequent decrease in the average crystal size of Ag-ZnO with an increase in Ag content might be ascribed to the dispersion of Ag nanoparticles in or near the boundary of ZnO lattice, which limits the alliance and diffusion of ZnO, thus hinders the growth of the nanocomposite. A similar scenario was reported elsewhere 65,66 . No further peaks were identified, suggesting no linked work function between Ag and ZnO and thus the synthesized Ag-ZnO nanocomposite is pure. This provides insights that doping Ag on the surface of ZnO-NPs was more successful than into the ZnO lattice, attributed to the larger ionic radius for Ag + (1.26 Å) compared to Zn 2+ (0.74 Å) 44 . Furthermore, an increase in the synthetic temperature from 30 to 80 °C exhibited additional peaks in the Ag-ZnO nanocomposite (Fig. 2b). Apart from, hexagonal wurtzite structure peaks of ZnO nanoparticle, new five peaks at 38.36°, 44.20°, 64.55°, 77.74°, and 81.74° indexed to the crystal planes of (111), (200), (220), (311) and (222) respectively were observed. These peaks correspond to the face center cubic (fcc) structure of Ag nanoparticles according to JCPDS, card No. 04-0783 63 , confirming the existence of metallic Ag in Ag-ZnO nanocomposites. The intensity and sharpness of Ag peaks gradually increase with the increase in the synthesis temperature, providing insight that more metallic Ag forms in the matrix at higher temperatures. This might be ascribed to the high nucleation rate leading to the formation of smaller crystalline nuclei 67 ) and distinct nanoparticles.
where D is the average crystallite size in nm, K is the Scherrer constant; equal to 0.9, λ is the specific wavelength of X-ray used (0.154 nm), θ is the diffraction Bragg angle and β 2θ is the angular width in radians at an intensity equal to full width and half maximum.
Optical properties. ATR-FTIR study. Figure 3a shows the FTIR spectrum of the active biomolecules from Tetradenia riperia leaves that work as reducing and capping agent during the biosynthesis of nanomaterials. The absorption peaks at 3292, 2931, and 1709 cm −1 correspond to the O-H stretching, C-H sp 3 stretching and C=O stretching mode of the carbonyl group respectively, suggesting the presence of alcohol, polyphenols, amides, esters and acids that signifies the availability flavonoids, saponins, tannins, alkaloids, and reducing sugars [34][35][36]68 .
The adsorption peaks at 1589, and 1375 cm −1 are due to the C=C stretching of alkene present in the aromatic ring structure and C-C stretching from the flavonoids respectively 34,69 . The adsorption peaks at around 1247 and 1028 cm −1 are assigned to C-N stretching groups of amines 35,49,70 , whereas the 859 cm −1 peak is attributed to N-H bending vibration of amine 35 . Moreover, the absorption bands at 817 and 772 cm −1 are due to C=C bending for vinylidene and trisubstituted alkene, whereas, band at 536 cm −1 represents the aromatic C-H out of plane bending in polyphenols 68 . The amine C-N stretching and N-H bending from the TR extract confirm the presence of alkaloids 36,39 . Alkaloid compounds work as weak base due to presence of nitrogen atoms in cyclic rings which provide electrons pairs to react with water molecules to produce − OH ions which hydrolyses or reduces the metal ions 34,36,38,39 . On the other hand, the − OH bending and − OH stretching depict the presence of flavonoids, tannins, and saponins, in TR extract which act as capping agents to prevent agglomeration and thus control the particle size 34,36,39,71 . During nanoparticles formation the − OH hydrophilic head of phytocompounds interact with metal ions whereas hydrophobic part provides a steric hindrance that prevent the agglomeration of nanoparticles 36,72 . Apart from the OH groups, studies have shown that the C=C and C=O groups from the phytocompounds can work as a capping agents 48 . Furthermore, it has been observed that some TR extract peaks (3292, 2931, 1709, 1589, 1247,859, 817, and 771 cm −1 ) disappeared after the formation of ZnO nanoparticles UV-Vis spectrum. Figure 3b shows the UV-Vis absorption spectra of pure ZnO nanoparticles and 1% Ag, 3% Ag, 5% Ag, and 8% doped ZnO nanocomposites. Results show that pure ZnO nanoparticles showed an absorption peak at 395 nm, due to the excitonic absorption, while the Ag-doped ZnO nanocomposites with 1%, 3%, 5%, and 8% Ag content exhibited absorption peaks attributed to surface plasmon resonance at 397, 380, 377 and 368 nm, respectively. The increase in peak from 395 to 397 nm (redshift) can be ascribed to the increase in particle size attributed to Ag doping on the ZnO matrix 73 . This increases the distance between the valence bands, which lowers the frequency of electromagnetic emission. Furthermore, the intensity of the absorption of doped ZnO decreases from 397 to 368 nm with an increase in Ag content (blue shift). This can be explicated by a reduction in particle size of the Ag-ZnO nanocomposites attributed to anchored Ag nanoparticles and the formation of smaller nuclei on the ZnO surface 43,74 , which obstructs the movement and diffusion of ZnO as evinced by the XRD study. Similar results were also reported elsewhere 43 .
Morphological study of nanomaterials.  www.nature.com/scientificreports/ a decrease in particle size with Ag loading. On the other hand, to confirm the coexistence of Ag on ZnO nanoparticles, TEM analysis was performed on the nanocomposite with 8% Ag. The TEM image in Fig. 4f shows a dispersion of the spherical Ag nanoparticles anchored on the surface of ZnO nanoparticles, as was reported elsewhere 75 . This might be attributed to the large ionic size of Ag, which hindered the incorporation of doped Ag into the crystal lattice of ZnO 76 . The average particle size of the Ag-ZnO nanocomposite depicted by TEM analysis with 8% Ag was 12.3 nm, which is in good agreement with XRD results of the same composition as was reported elsewhere 77 . Figure 5 shows the EDX spectra for the Ag-ZnO nanocomposite in which Ag, O, and Zn signals were observed; this confirms the anchoring of Ag on ZnO on the surface. Furthermore, to ascertain the amount of Ag doped on ZnO at various concentrations, EDX analysis of Ag-ZnO with different Ag content was performed. The results of Fig. 5 show that the amount of doped Ag (1, 3, 5, and 8%) corresponds with the elemental analysis shown in the table (inset), indicating that Ag was successfully doped on ZnO. The presence of elemental sodium in the EDX spectra is ascribed to the NaOH used for pH adjustment during the synthesis of the nanomaterials.
Effect of temperature on the green synthesis of nanomaterials. In wet chemistry synthesis and engineering of nanomaterials, temperature has a significant influence on the synthesis of nanoparticles through nucleation and growth of nanoparticles. The formation of nuclei (nucleation) that yields smaller particles is favored at a higher temperature, while growth is favored at a lower temperature 67,78 . In this study, the effect of temperature on the size of the synthesized ZnO and Ag-ZnO nanoparticles was investigated at room temperature (30 °C), 60 and 80 °C. Results shown in Fig. 6 show that the size of particles becomes smaller with an increase in reaction temperature. This might be attributed to the high nucleation of metal ions, because an increase in temperature impacts more nucleation than the growth of nuclei 79,80 . Normally, at high temperature, the kinetic energy of the molecules increases, and precursors are consumed more quickly to form nuclei, thus suppressing particle growth 78 . On the other hand, at lower temperatures, the kinetic energy of nanoparticles decreases. This results in crystal growth attributed to Oswald ripening 81 . As a result, at higher temperatures, smaller particles with a more uniform size distribution are formed 78 . However, when the temperature is too high, the surface activity of the nuclei is increased, which fosters the collision and agglomeration of nuclei 79 . Therefore, temperatures above 80 °C might lead to larger particle sizes 67,79 . Similar findings have been reported elsewhere 67,78 . Effect of concentrations of the precursor salts. The effect of the concentration of precursors on the size of Ag-ZnO nanocomposite and the efficacy of the synthesis method was studied at different concentrations of silver ions (1, 3, 5, 8 mM) doped on Ag-ZnO nanocomposites. The formation of Ag-ZnO occurs in two stages: the first stage is the generation of nuclei, followed by the growth of nuclei. Results from Fig. 6 show the decrease in size of Ag-ZnO nanocomposites with an increase in concentrations of silver salts from 1 to 8 mM. This might be ascribed to the higher nucleation of ions at higher concentrations of precursor salts, which yields smaller sized nuclei 79 . However, at higher concentrations of precursor salts, an excess number of nuclei forms, which results in agglomeration of nuclei and growth of particles 82 . A similar phenomenon was also reported in the literature 79 , where an increase in the size of green synthesized copper nanoparticles was observed when the www.nature.com/scientificreports/ concentration of the precursor salts increased from 7.5 to 10 mM. In this study, a maximum concentration of 8 mM for Ag salt resulted in the smallest nanocomposite. This is close to the optimal value of 7.5 mM for copper salt reported in the literature 79 .

Effect of pH.
In the green synthesis of nanomaterials, the reducing and capping of nanoparticles depend on the charge of phytocompounds, which is affected by a change in pH 79 . pH variation affects the formation and morphology of nanoparticles. Herein, the formation of ZnO nanoparticles and Ag-ZnO nanocomposites was evaluated at acidic and slight basic conditions of the Tetradenia riperia leaves extract. It was observed that the pH of Tetradenia riperia leaves extract was 5.10, but was lowered to 4.46 after the addition of metal precursors, which might be ascribed to the release of H + ions by some Tetradenia riperia phytocompounds when they are oxidized in the presence of metal precursor ions 83 . This can be evinced from the chemical reaction of phenols with metal ions, which results in oxidized phenol, reduced metallic elements and hydrogen ions that account for the low pH of the medium. Due to the low pH (acidity) of the TR extract, the formation of nanoparticles in the medium was suppressed, which might be attributed to the inactivation of the phytocompounds 79,82 responsible for reducing and capping of metal precursors. However, when the pH was higher (7)(8), the formation of nanoparticles was observed. A similar scenario was also reported in the literature 79 where the repression in nanoparticles formation was observed at pH of 4.7, but at pH 6.6, copper nanoparticles were observed to form from Azadirachta indica leaves extract. Interestingly, even a high pH was found to be effective in the formation of nanoparticles. However, agglomeration results in large-sized nanoparticles 78 . Therefore, the optimal pH for the bio-route formation of small-sized nanoparticles might be in the neutral to slightly alkaline range.
Antibacterial activity of ZnO and Ag-ZnO nanocomposites. The antimicrobial activity of the biosynthesized ZnO nanoparticles and Ag-ZnO nanocomposites against E. coli (gram-negative) and S. aureus (gram-positive) bacteria strains were evaluated by determining the zone of inhibition (ZOI) and minimum inhibition concentration (MIC). Results from Table 1 and Fig. 9 show that the ZOI values of the Ag-ZnO nanocomposites are undeniably higher than for pure ZnO for both strains. This demonstrates the higher antibacterial activity of Ag-ZnO nanocomposites over ZnO nanoparticles, which is attributed to their synergic effect. Furthermore, the antibacterial activity of Ag-ZnO nanocomposites was found to increase with the increase in Ag concentrations (Fig. 7a,b. This demonstrates that doping of Ag in ZnO improves the antibacterial activity of ZnO nanoparticles. This phenomenon can be ascribed to the stronger antimicrobial effect of Ag 84,85 as well as the smaller size of the formed nanocomposite as demonstrated by XRD and FESEM results. Furthermore, Fig. 7a,b shows that the effectiveness of nanocomposites was influenced by the temperature, in which ZnO nanoparticles and Ag-ZnO nanocomposites synthesized at RT had a moderate effect (low ZOI) compared to those synthesized at 80 °C. This might be attributed to the high nucleation rate and the formation of smaller   6,43 . These results demonstrate that the incorporation of Ag in ZnO nanoparticles improves the antibacterial activity of the Ag-ZnO nanocomposite due to a synergetic effect. Therefore, this provides insight that E. coli is more susceptible to Ag-ZnO nanocomposites (Fig. 8). This might be attributed to differences in the cell wall composition of the two bacteria 43,48,86 , in which the cell wall of S. aureus bacteria is covered by a thick and rigid peptidoglycan layer crosslinked by peptide chains, which limits the penetration of   85 . On the other hand, the cell wall of E.coli contains a thin peptidoglycan layer, facilitating easy penetration of Ag-ZnO nanocomposites 86 . Similar findings have been reported elsewhere 43,86 . Various approaches have been adopted for the synthesis of ZnO NPs and Ag-ZnO nanocomposites using green methods. It is shown in Table 2 that nanomaterials from this study show better performance when compared to concentrations used in other studies.
The mechanisms of antibacterial activities of Ag-ZnO. The Ag-ZnO nanocomposites attack and cause bacterial cell lysis by several mechanisms. Firstly, the interaction of smaller-sized nanoparticles with the bacterial cell leads to membrane penetration due to disruption of the cell membrane caused by changes in membrane protein or enzyme activity 87 . This allows the entry of Ag and ZnO nanoparticles into the bacteria cell and results in the defacement of the lipid bilayer and membrane protein. This in turn causes an imbalance within the cell, which leads to cell death. Secondly, surface oxidation of the Ag-ZnO nanocomposite results in the release of silver (Ag + ), which influences the electrostatic interactions between the ions formed and the negatively charged bacterial cell wall. This results in the high antibacterial activity of the Ag-ZnO nanocomposites reported in this paper on E. coli, as was reported elsewhere 43 . Thirdly, due to the penetration of the nanoparticles inside the bacteria cell, trickling of cytoplasm occurs; this results in shrinkage of the cell membrane and death of bacteria 6 . Fourthly, Ag-ZnO nanocomposites hinder the replication of DNA by releasing Ag + , which interact with sugar-phosphate groups, thus mutating the gene and affecting the cellular functioning of bacteria 6,88 . On the other hand, illuminated ZnO nanoparticles produce reactive oxygen species (ROS) that lead to oxidative stress in the cell 89 . This affects mitochondrial activities, weakening the metabolic activities, which ultimately lead to cell death. When ZnO nanoparticles or its generated ROS obstruct the signal transduction pathway, vital cell functions such as DNA replication, transcription, and translation are halted, resulting in cell death. 18,27,89 . Therefore, the aforementioned mechanisms show that nanocomposites offer multitarget mechanisms for denaturing the bacteria strains. This suggests that Ag-ZnO nanocomposites may have superior antibacterial activities when compared to conventional antibiotics or disinfectants (Fig. 9).

Conclusion
The Ag-ZnO nanocomposites were successfully synthesized through an environmentally benign approach by using an aqueous leaf extract of Tetradenia riperia plant and were evaluated for their antimicrobial activity against E. coli and S. aureus bacteria strains. The SEM and XRD analysis revealed that biosynthesized Ag-ZnO nanocomposites were spherical and crystalline in nature and were observed to decrease in average particle size from 23.6 to 14.8 nm as a result of an increase in Ag concentrations. Different antimicrobial activities of Ag-ZnO nanocomposites were investigated and found to have higher antimicrobial activity against E. coli than S. aureus bacteria. The Ag-ZnO nanocomposites presented higher antimicrobial properties compared to ZnO nanoparticles. This provides an insight that the addition of silver (Ag) nanoparticles improves the antimicrobial activity of the Ag-ZnO nanocomposites, especially for E. coli (gram-negative bacteria). Furthermore, the antimicrobial activities of Ag-ZnO were found to increase with increasing Ag dopant concentrations and synthetic