Effect of Size and pH Variation on Antibacterial Activities of ZnS-ZnO Nanocomposite Towards Application in Water Treatment.

INTRODUCTION
Nanotechnology has the great potential application because of tunable size and it can be manipulated in desired level. The smaller in size of semiconductor has shrinking effect and shows high chemical reactivity and good physical properties. When the confined dimension is less than de Broglie wavelength, quantum confinement occurs and shows unique optical properties that have led to opened the door for various application. 
Among the various material the mix compound nanocomposite shows attractive properties in good conductivity and efficient antibacterial activities [1]. ZnO -ZnS are low toxic material and leading them to good interesting candidates for multiple applications in optoelectronic and biomedical field. The ZnS-ZnO nanocomposite shows large surface to volume ratio and exhibits efficient antimicrobial activity. ZnO and ZnS semiconductor has direct and wide range band gap with mix phase. Both the shape has different structure and different bandgap energy making them exciting properties. The ZnS-ZnO nanocomposite shows in lower wave length optical absorption and red shift in photoluminescence (PL) properties [2]. 
Bacterial existance in drinking water becomes a major issue in developing countries. To remove the microbial agent though lots of effort has done yet composite semiconductor nanoparticles is the promising area for removal of microorganism because of high chemical affinity and photocatalytic activity [3]. 
The present work is mainly optimizing the synthesis technique with various parameter like reaction time, composite ratio, pH of the sample and photon induced time etc. The optimized sample were analyzed by various instrument like XRD, UV- Visible spectrometer, HRTEM, HRSEM, AFM and PL to investigate their structural, optical and morphological properties along with activity in microbial effect in human pathogens Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae and Escherichia coli.

MATERIALS AND METHODS
Materials 
ZnO, ZnS, NaOH , ZnCl2 and Na2S were used from Merck, India. All the materials were used in high analytic grade. ZnCl2 and ZnSO4was mixed at different concentration (1, 2, 3, 4 wt%) under a high stirring rate (250 rpm) condition. The constant temperature 70oC  in 5 hours was maintained during the process of stirring. The sample under preparation was kept for 12 h for complete dissolution to get a transparent solution. To this solution, 2 wt% Na2S was added until the complete solution turns into milky color and concentrated nitric acid to change the pH of the sample and zinc sulphate and sodium sulphide were added in different molar concentration ratio. 
The samples were prepared in both powdered and thin film form and quantum dot formation is mainly responsible of Zinc ion exchanged reaction.
The growth of film is mostly considered the result of the surface aggregation of colloid particle (cluster- by -cluster growth) in the given experimental conditions. With change in pH the color changes from light milky to deep milky and optimum value for better adhesion in case of thin film form is 0.8 - 2. At the higher value of pH the size of nanoparticles increases due to the slow nucleation growth. 

Preparation of antibacterial activity 
Escherichia coli (MTCC 739), Staphylococcus aureus (MTCC-740), Klebsiella pneumonia (MTCC 432) and Pseudomonas aeruginosa (MTCC-424) were prepared for study of antibacterial efficacy in composite material. The microbial cultures were used from the “Microbial Gene Bank” (MTCC). 
The bacteria were cultured at -4°C. Antibacterial process were done at optimized smallest particle at different pH value. The nutrient agar plates were applied for 3.0 ml of inoculums and swab over the region. 6mm diameter were cut on the agar plates and the wells are placed with ZnO-ZnS nanoparticles. Standard antibiotic (Tetracyclin) was maintained for control the positive effect. 
All samples were kept overnight at 370C.The inhibition diameters were measured with sophisticated travelling microscope. 

RESULTS AND DISCUSSION

UV-Vis Spectra analysis
The ZnO-ZnS nanocomposite prepared by chemical process in optimum conditions showed sharp absorbance beyond its band edge wave length 350 nm. Absorption spectra also show that the optimum pH value for larger blue shift and correspondingly for smaller dot. 
It is observed that absorptions were shifted towards the shorter wavelength (or higher energy) region with decrease in pH. Moreover, the larger blue shift corresponding to smaller particle size indicated that strong confinement of nanocomposite. The photon induced time was also recorded and found that exposing time in sun light shows small size and effective microbial effect. 
The energy band gap was calculated from Fig. 2. using a graph graph (αhʋ)2 versus hʋ .From the graph band gap were calculated extrapolating the function straight-line to to (αhʋ )2 = 0. From the shifting of band gap, radius of the particles  is calculated using mass approximation model(EMA) [2]. 

where E(R)=Enp- Eg, Eng is the bandgap of the composite ZnO-ZnS nanoparticle, Eg the average band gap value of bulk ZnS and ZnO , me* and mh* is the effective mass of electron and hole and r the radius of the particle. Using the above equation, particle size was estimated at different pH in optimum condition. The calculated particle size shows exitonic effect because it was less than de Broglie wave [4-5]. 
Table 1 Blue shift energy and particle size from optical model of ZnO-ZnS at different pH. Optical absorption and band gap estimation suggest that the composite material consist of single-phase cubic structure. 
The shift of the absorption edge, which provides band gap energy, of the semiconductor to higher energy provides experimental evidence for such quantum confinement.

Structural Analysis by XRD
Fig. 3. shows four different spectra of ZnO-ZnS nanocomposite thin film form prepared at different pH of the solution ranging from 0.7 to 2.0. The XRD spectrum of A contain broad peaks at 2θ =29.370, 47.324 indicating growth of nanostructure at pH =0.7. B also shows the peaks at 2θ =29.131, 47.164,C at 2θ =28.942,46.763 and D at 2 θ =28.752,46.457 for pH value 1.2, 1.6 and 2.0 respectively. The spectrum shows diffraction peaks corresponding to (111), (220) planes of Zinc blende(sphalerite) phase (JCPDS card, No. 5-0566) and (100),(002),(102),(110) for composite ZnO-ZnS. The XRD spectrum of all the spectra exhibit weak and broad peaks, suggesting small crystallite size. Again, it was observed that pH decreases more broadening take place with decreasing in crystallite size [4-6]. 
The increasing value of diffraction angle(2θ) suggest lattice contraction. The lattice contraction created decreasing crystallite size and increase in strain. The ratio of ZnO-ZnS maintained at 5:3 with expose in sun light. The effect of pH can be explained that in lower pH OH- act as a catalyst for growing in composite nanoparticle. OH- may increase the ionization strength of Zn2+ for faster reaction with S2- and O2- to accelerate the formation of nuclei. As formation of nuclei increases smaller particle coalescence and produce bigger particle [4]. 
Since we used a simple chemical process for composite thin film so possibility oftwins formation can be neglected. Therefore, XRD peak broadening was considered only for   size. The lattice parameter ‘a’ was determined with corresponding planes and systematic errors in 2 θ were eliminated by Nelson and Riley plot in from three peaks. The corrected value of lattice constant “a” is calculated by F(θ) to zero. The lattice constant was explained in Fig. 4. 
The average size of nanocrystal was determined using Scherer formula [2].
             

Where, qB is the Bragg angle in radian and K=0.9 for spherical shape. The average crystalline was calculated 27 nm which is quite bigger than the size obtained in TEM and optical model. 

FTIR Analysis
The interaction between ZnS and ZnO was analyzed by FTIR spectra. In Fig. 5. two characteristic bands at 3352 and 1092­cm-1 were observed. The band at 3352 cm-1 in all spectra was due to O-H vibration band and another band at 1092 cm-1 was due to hydroxy C-O stretching band. The OH- group arises due to ZnO and ZnS nanoparticles. The band at 1700 cm-1 probably appears due to water absorption while handling the sample in air [7]. 
The absorption peak at 2074 cm?1 is the functional and can be assigned to -CH stretching mode  of -CH3 and -CH2. The band 1638 cm-1 agrees with  C=O group. Weak band at 1018 cm-1 can be form due to C-N vibrations of aliphatic amines. The main bands absorbed at 3443.95 cm-1, 1638.18 cm-1 and 669.97 cm-1indicates the existence of alcohols and phenols (O-H).

Antibacterial activity
The antibacterial activity was tested in four bacterial pathogenic bacteria E. coli, B. subtilis, K. pneumoniae and S. aureus. Fig. 6 represents the zone of bacterial inhibition (diameter) 18mm, 16mm, 12mm and 14mm in E. coli, B. subtilis, K. pneumoniae and S. aureus bacterial suspension respectively. When the positive control, amoxicillin (1mg/ml) was tested for microbial activity against S. aureus, B. subtilis, K. pneumoniae and E. coli, the diameter of zone was 33mm, 35mm, 33mm and 38mm respectively [8-10].
Tetracyclin of 1mg/ml, concentration was perform control antibacterial agent. The results in Table-2 depict that Fe doped ZnO-ZnS nanoparticles are efficiently giving zone of inhibition. Table 2. Antibacterial efficacy of ZnO-ZnS nanopareticles with standard antibiotic in bacterial strains

PL Studies
For PL analysis the wavelengths were applied in between 250 nm to 400 nm. Fig. 7. Shows result of luminescence of ZnO-ZnS composite at 5:3 ratio and at different pH A-0.7,B-1.2 and C-1.6. All luminescence spectra clearly show that prepared samples have emission peak with high intensity at longer wave length.  Another peak of smaller intensity is also observed at higher wave length
The peaks centered at 425 to 530 nm arises due to zinc and Sulphur vacancies in the lattice [11-15]. It is observed from the Fig.7 that peak position changes with the pH, which indicate the dependence on the particle size. The sample prepared at pH 0.7 showed greater emission intensity (at 425nm) then the sample prepared at other pH value. Thus, the pH takes an optimization factor for enhancement in PL. It is found that under the acidic condition, the Zn2+ and S2- resides in a neighborhood of high OH- ion  and  OH- ions may take part in the lattice formation of ZnO-ZnS nanocomposite. The study reveals that pH takes an important parameter for improvement of emission intensity. Broad emission bands was due to the wide distribution of trap distance [16-20]. The stock shift energy was calculated from absorption and transmittance peak. 
Table 2. represents stokes energy of a representative samples at different conditions. The large Stokes shift energy represents the strong coupling to the lattice phonons. The strength of electron-phonon coupling with large particle size. 

TEM , SEM and AFM studies
Fig.8. AFM direct image of ZnO-ZnS at 5:3 ratio and pH-0.7 in with sun light our optimum antimicrobial effect. The TEM image shows that the size of the particle was spherical in shape and found in the range 5-8nm which was a good similarity as size obtained in optical model. Fig. 9 represent the SEM photograph of ZnO:ZnS nanocomposite at optimum size and antimicrobial effect. It was found that some grains are overlap and showed different shape. The SEM photo images were not very sharp because of lower operating voltage (15KV). The images were observed at lower operating voltage  to avoid sample damage. Higher operating voltage could have improved the image sharpness and resolution [20-25]. Fig.10 showed the AFM (contact mode) images of the ZnO-ZnS nanocomposite at different pH ranging. 
Images showed that with decreasing pH the sharp smoothness takes place i.e roughness decreases. The Fig represent the ZnO:ZnS-5:3 at pH 2.0,1.2 and 0.7 from left to right and observed that at lower pH pyramid shape becomes clear and it is evidence of Zinc blende (Sphalerite) structure. 

CONCLUSION
ZnO-ZnS nanocomposite in optimum condition has been establish for better efficient in antimicrobial activity. Again, small and spherical dot exbibit better antimicrobial effect. The size was confirmed by XRD, TEM, optical model and SEM. Antimicrobial effect was calculated with the important pathogens Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae and Escherichia coli. UV Visible and PL properties shows the pH takes a major role for formation of small particle and desired band gap. PL analysis showed the emission were red shifted and optical study showed that absorption was blue shifted. The lattice parameter was confirmed by Nelson Relay plot with XRD.AFM image represents the surface topography with change of pH. 

ACKNOWLEDGMENTS
I would like to acknowledge Department of Biotechnology (DBT), Ministry of Science & Technology, Government of India for financial assistance through star college and Biotech HUB scheme. We would also like to acknowledge ASTEC for financial support. We would like to acknowledge Department of Molecular Biology and Biotechnology, Tezpur University for providing FTIR facilities.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.