Photocatalytic dye degradation and antimicrobial activities of Pure and Ag-doped ZnO using Cannabis sativa leaf extract.

A facile green route has been employed for the synthesis of ZnO and Ag-doped ZnO using Cannabis sativa as a reducing and stabilizing agent. The as-synthesized nanoparticles were characterized and tested for photocatalytic dye degradation and antimicrobial activity. The results suggested that nanoparticles have shown antimicrobial activity against different human pathogenic bacteria (Escherichia coli, Klebsiella pneumonia, MRSA, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus) and fungal strains (Fusarium spp. and Rosellinia necatrix). Ag-doped nanoparticles comparatively have shown better removal Congo red and methyl orange under visible light. Therefore, green synthesized nanoparticles could have beneficial applications in environmental science and biological field.

Antibacterial Assay. Antimicrobial activity was done with the help of agar well diffusion method 46 . 100 µl of the bacterial inoculums was spread over plates containing nutrient agar and then 6 mm wells were created with the help of puncture on the plates and check the antimicrobial activity by taking 100 µl of nanoparticle extracts against all pathogenic bacteria. Two controls were included in the test i.e., the antibiotic ampicillin, which was considered as positive control and 10% DMSO which was taken as a negative control. The plates were kept in the incubator for 18-24 hrs at 37 °C. All the tests were performed in triplicate. The inhibition zones obtained around the wells were measured. The inhibition zones were measured by taking the amount of 100 µl of nanoparticle extract in a different well. The antibiotic ampicillin (+ve control) showed the inhibition zone by taking the amount of 10 µl at the concentration of 100 mg/ml. 10% DMSO (−ve control) showed no zone against all bacteria.
Antifungal Assay. Nanoparticles extract were prepared at concentrations 1%, were added in 25 ml of sterilized potato dextrose agar in petri plates. A 6 mm diameter of the actively growing mycelium disc of the pathogen of 6-7 day old culture was placed in the center of the petri dish. Plates without extract served as the negative control. Plates were incubated at 25 °C. Radial growth of mycelium was measured after seven days of incubation. The growth results were compared with the negative control. The experiment was repeated three times, and the mean of the readings was taken for further calculations. The percent inhibition of the fungus in the experiment was calculated using the following formula; Where L is the percent inhibition; C is the colony radius in the control plate, and T is the radial growth of the pathogen in the presence of nanoparticles extracts 47 . photodegradation analysis. The photocatalytic activity of ZnO and Ag doped ZnO was evaluated for the photodecomposition of congo red and methyl orange listed in Table 1 under solar light irradiation. To confirm degradation process, small volume of aliquot was taken and centrifuged for 5 min to completely remove nanoparticles. The concentration of azo dyes was detected using UV-Vis spectrophotometer. The self-degradation of congo red and methyl orange under solar irradiation is not significant.

Results and Discussion
XRD Analysis. The Phase identification and purity of the samples were confirmed by X-ray diffraction measure-   48 . The particle size listed in Table 2 was calculated using the Scherrer formula 49 , Where D is the average crystallite size, λ is the X-ray wavelength, θ is the Bragg angle, and β is the FWHM of the experimental data. Dislocation density S was calculated using crystallite size (D) which represents the amount and defects present in the crystal system, given by the formula 50 and listed in Table 1:- Where β is full width at half maximum (FWHM), D is the crystallite size, and ε is the strain Plotting βcosθ against 4εsinθ from the linear fit of the data, we determined the lattice strain from the slope and crystallite size using the intercept as listed in Table 2.
The crystallite sizes obtained from Sherrer method are smaller in comparison to William-hall method because Sherrer method measures the cohesion length of the X-rays any defects and vacancies will cause the measured size to be lower than the actual size whereas William-hall method takes micro-strain into consideration.
Rietveld refinement of both the XRD patterns was done using Full prof program with space group P63mc. The background was fitted with linear interpolation, and peak patterns were described by pseudo-Voigt profiles. First, global parameters such as background, instrumental and scale factors were refined and further cell parameters, FWHM parameters, shape parameters, preferred orientation and atomic positions were refined in sequence. There is a slight decrease in the lattice parameters of Ag-doped ZnO but lack of peak shift in the experimental data suggests that there may be the segregation of AgO and Ag species on the grain boundaries of ZnO crystal or an inadequate amount of silver atoms would have incorporated in ZnO crystal 52 . Figure 2(a,b) shows the Rietveld analysis of ZnO and Ag-ZnO nanoparticles, respectively.
It can be seen in Fig. 2 that the observed and calculated profiles are in good resemblance to each other and all calculated parameters along with reliability factors R factors (R p , R wp , R exp , R b ) as listed in Table 2.
XpS Analysis. The determination of elements with chemical bonding states of ZnO and Ag doped ZnO was employed by analyzing XPS spectra shown in Fig. 3.
The full scan spectra shown in Fig. 3(a) identifies the presence of Zn, O, C and Ag in both the samples. The presence of carbon peak C(1 s) at 283.8 eV is presumably due to organic contaminants adsorption on sample surface or acetate vestige 53,54 .
The core-shell level XPS spectra of O (1 s), Zn (2p) and Ag (3d) are shown in Fig. 3(b-d). In Fig. 3(b) asymmetric O(1 s) curves were fitted with two symmetrical Gaussian curves (i and ii) for both nanoparticles. Peak (i) with lower binding energy in comparison to peak (ii) is attributed to O 2− ions of ZnO www.nature.com/scientificreports www.nature.com/scientificreports/ bonding and adsorption of hydroxyl group which plays a major role in increasing photocatalytic activity by preventing recombination 53 . Silver doping shifted O(1 s) spectrum to lower binding energy which is due to reduced oxygen vacancy 53 .
In Fig. 3(c) two symmetrical peaks at 1022.4 eV,1045.4 eV for pure ZnO and 1022.05 eV, 1045.14 eV for Ag doped ZnO are ascribed to Zn (2p 1/2 ) and Zn (2p 3/2 ). These peaks are in good agreement with binding energy of stoichiometric ZnO, i.e., 1045.1 eV for Zn (2p 1/2 ) and 1022.1 eV for Zn (2p 3/2 ) which is attributed to vacancy driven Zn 2+ to O 2− charge transfer In Fig. 3(d) the Ag (3d) levels are shown for examining the chemical state of Ag element. The Ag(3d 3/2 ) peak at 374.08 eV and Ag (3d 5/2 ) peak at 368.08 which can be attributed to metallic silver AgO and Ag-Zn-O ternary www.nature.com/scientificreports www.nature.com/scientificreports/ compound which is consistent with the XRD. Comparatively there is significant shift towards lower binding energy upon doping with binding energy of bulk silver i.e. 368.3 eV for Ag(3d 3/2 ) and 374.3 eV for Ag(3d 5/2 ) which is because binding energy of unit valency silver (AgO) is much lower than zero valency silver and the formation of oxide layer on surface of silver 53 . feSeM. Surface morphology of the samples was investigated through FESEM and are shown in Fig. 4(a,b).
Pure ZnO nanoparticles are relatively homogeneous, which can be attributed to the uniform distribution of Zn cations in a three-dimensional structure. The agglomeration is due to densification caused by the narrow space between the particles. For Ag-doped ZnO, the particles are much more agglomerated with the formation of small particles on the larger clusters which may be due to the creation of AgO nanoparticles 55 . teM Analysis. Figure 5(a,b) presents the TEM micrographs of with inset showing particle size distribution as well as the SAED pattern for pure ZnO and Ag doped ZnO nanoparticles, respectively. It is seen that the nanoparticles are highly agglomerated with almost spherical shape. The ImageJ software was used to analyze the micrographs to calculate the particle size as well as to study the shape of the pure and doped ZnO nanoparticles. Particles of 34 nm and 38 nm as maximum has been observed in the TEM of micrographs of pure and Ag doped ZnO. In an SAED, we witness circular ring patterns, which is a signature to confirm the high crystalline order of the samples. In our studied samples, both the samples have shown bright circular patterns, therefore confirming presence of high crystalline order. Figure 6(a,b) presents the elemental mapping of ZnO and Ag doped ZnO nanoparticles using EDS technique. The EDS analysis confirms the presence of all the elements without any additional impurity phase.
Antibacterial activity. The present study revealed that ZnO and Ag-doped ZnO nanoparticles prepared using aqueous leaf extract of Cannabis sativa showed potent antibacterial activity against different bacterial strains (Gram-positive and Gram-negative) as shown in Fig. 7(a,b). The inhibition zones (in mm) of varying sizes www.nature.com/scientificreports www.nature.com/scientificreports/ were obtained as mentioned in Table 3(a,b). The inhibition zones were measured by taking the amount of 100 µl of nanoparticle extract, DMSO and ampicillin in different wells.
From the above table, it is clearly shown that Ag-doped ZnO nanoparticles indicated the maximum inhibition zone against all pathogenic bacteria as compared to ZnO nanoparticles. Ag-doped ZnO nanoparticles showed maximum zone inhibition against Salmonella typhi and minimum zone inhibition against P. aeruginosa whereas, ZnO nanoparticles showed maximum zone inhibition against Klebsiella and no zone of inhibition against P. aeruginosa as shown in Figs. 7(a,b) and 12, 13. DMSO (Negative control) showed no zone inhibition against all bacteria. The ethanol and petroleum extracts of Cannabis leaves showed the inhibitory effects on both Gram-positive and Gram-negative bacteria 55 . The effect of Cannabis sativa L. seed oil as well as petroleum ether and methanol extracts of the whole plant on two Gram (+) organisms (Bacillus subtilis, Staphylococcus aureus), and two Gram (−) organisms (Escherichia coli, Pseudomonas aeruginosa) have been reported previously 56 . In 2008, Borchardt et al. found that the stems and leaves extract was only active against Staphylococcus aureus 57-59 . 100 µl nanoparticles were used at a final concentration of 100 mg/ml (prepared in 10% DMSO), 10 µl Positive control for bacteria (Ampicillin) was used at a concentration of 100 mg/ml, Negative control (10% DMSO), produced no zone of inhibition. www.nature.com/scientificreports www.nature.com/scientificreports/ Antibacterial mechanism. Antimicrobial activity against different bacterial strains is represented by ZnO and Ag doped ZnO nanoparticles. Both Gram-positive and Gram-negative bacteria have a negatively charged cell wall, a characteristic that is hypothesized to influence the interactions between the cell walls of the bacteria and NPs or ions released from them. In this study, Zinc oxide releases Zn 2+ ions whereas, silver release (Ag + ) ions in aqueous solution contributing to the antimicrobial effectiveness. The released Zn 2+ and Ag + ions significantly contributed to the overall antibacterial effect of nanoparticles. Nanoparticles have high potential as antibacterial agents attributable to its ability to produce reactive oxygen species (ROS). The generation of reactive oxygen species inhibits the antioxidant defense system, inhibit ATP production and causes mechanical damage to the cell membrane. Recent studies have shown that this ROS generation is profoundly affected by the modification www.nature.com/scientificreports www.nature.com/scientificreports/ of band structure by introduction of different dopant materials into them. Positively charged NPs, were able to alter the function of the electron transport chain in bacteria. It has been previously proposed that the generation of hydrogen peroxide from the surface of zinc oxide as an effective mean for the inhabitation of the bacterial growth 47,48 . Silver also display antibacterial activity, it can inhibit enzymatic system of the respiratory chain, thus by modifying the DNA synthesis 60 . Different researchers additionally reported that silver nanoparticles enter through the bacterial cell membrane and prompt neutralization of the surface electric charge of the bacterial www.nature.com/scientificreports www.nature.com/scientificreports/ membrane and change its penetrability, ultimately causing massive cell damage 61,62 . This damage included nuclear fragmentation of the Ag-NPs to the DNA, probably because of the high affinity of Ag + to phosphates highly abundant in the DNA molecule (Fig. 8).    www.nature.com/scientificreports www.nature.com/scientificreports/ Anti-fungal activity of nanoparticles. The ZnO and Ag-doped ZnO nanoparticles were also found to be effective against different plant pathogenic fungi. It is essential to mention that the extract of nanoparticles was able to inhibit the growth of fungus (Fusarium spp. and Rosellinia necatrix) in the present study as well. The inhibition (in mm) of varying sizes were obtained as mentioned in Table 4   It is clearly shown that silver doped ZnO nanoparticles indicated the maximum inhibition against Fusarium species and Rosellinia necatrix as compare to ZnO nanoparticles as shown in Figs. 9(a,b) and 14. The ethanol   photodegradation analysis. Figure 10(a,b) depicts the percentage removal of azo dyes as a function of irradiation time. Nearly, 96% and 38% of Congo red and 94% and 35% of methyl orange were removed using Ag-ZnO and bare ZnO under solar light in 80 min at pH 8. Thus, the removal efficiency of photocatalyst follows the order Ag-ZnO > ZnO. The photocatalytic efficiencies of Ag doped ZnO was remarkably superior as compared to ZnO alone for the removal of azo dye. This was due to considerable adsorption of negatively charged azo dyes at lower pH onto the surface of positively charged Ag doped ZnO.
The phenomena of photodegradation of dyes depends upon the zero point charge of ZnO. The zero point charge (pH zpc ) of ZnO is 9.0 63 . If the pH solution is higher than pH zpc , the surface of ZnO is become negatively charged and at lower pH solution the surface becomes positively charged 64 . Mechanism. ZnO act as photosensitizer and can generate electron-hole pair in the presence of solar radiation. The inefficient harvesting of solar light due to large band gap of ZnO of 3.37 eV and high charge carrier recombination limits practical application of ZnO to be utilized as efficient photocatalyst 65 . Thus, surface modification with a noble metal Ag is done to enhance the photo-efficiency of bare ZnO nanoparticles. As the solar light falls on the surface of Ag-ZnO nanoparticles generation of electron-hole pair take place. These generated charge carriers have very short life span and thus rapidly undergo recombination process which decrease the efficiency of the photo-catalyst. The role of Ag nanoparticle is actually to traps the photo-generated electron by acting as an electron sink and thus averting the charge carrier recombination as shown in Fig. 11 66 . The electrons in conduction band reduces adsorbed oxygen molecule into superoxide radical (˙O 2¯) and holes oxidizes hydroxide ion into hydroxyl radical (˙OH) in the valence band, respectively 67 . The generated reactive oxidation species, oxidizes both congo red and methyl orange azo dye into innocuous molecule.
The best photocatalytic activity of Ag doped ZnO were found as compared to un-doped ZnO. The deposited metal ions consume the electron and reduces the rate of recombination; thus, majority of electron were utilized to produce superoxide radical in the conduction band rather than recombining with holes.
conclusion Pure ZnO and Ag-doped ZnO were successfully synthesized by an eco-friendly method using Cannabis sativa leaves. Cannabis sativa used as a reducing and capping agent. The XRD results confirm that silver doping has not altered the structural properties of pure ZnO as have pure hexagonal wurtize structure. The Presence of AgO on the surface of Ag doped ZnO has been confirmed by TEM and XPS. The synthesized Ag-ZnO and ZnO nanoparticles have shown antimicrobial activity against different human pathogenic bacteria (both gram-positive and gram-negative) and different fungal strains. Silver doped ZnO nanoparticles showed the maximum inhibition zone against all bacteria (Escherichia coli, Klebsiella pneumonia, MRSA, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus) whereas ZnO nanoparticles showed minimum inhibition zone. The ZnO and Ag-doped ZnO nanoparticles were also found effective against two different plant pathogenic fungi (Fusarium spp. and Rosellinia necatrix) and was able to inhibit the growth of fungi. Ag-ZnO and ZnO nanoparticles removed 96% and 38% of Congo red and94% and 35% of methyl orange under solar light in 80 min. Comparatively, green synthesized Ag-ZnO nanoparticles has shown better results for antimicrobial activity and dye degradation than pure ZnO. Hence green synthesized nanoparticles could have important applications environmental science and biological fields.