An Investigation on Kinetics of Photo Catalysis , Characterization , Antibacterial and Antimitotic Property of Electrochemically Synthesized ZnS and 2 ZrS ZnS Nano Photocatalysts

Zinc sulphide is one of the commercially important II-VI semiconductors having a wide band gap, rendering it a very attractive material for optical application especially in nanocrystalline form. Nanocomposites of ZnS and ZrS2/ZnS were prepared by simple electrochemical method; their photocatalytic properties had been investigated. The structure, composition and optical property of the product were characterized by X-ray diffraction (XRD), FESEM (EDAX), UV-VIS and IR techniques. The UV-VIS spectra exhibited a blue-shift with respect to that of bulk material due to quantum confinement effect. Kinetics of photocatalytic degradation of Indigo Carmine dye has been studied. The photocatalytic decolourization of the dye follows first order kinetics. The antimitotic and antibacterial activity of these nanoparticles was investigated.


Introduction
Over the past few decades, environmental problems associated with harmful organic pollutants in waste water are driving force for sustained fundamental and applied research in the area of environmental remediation [1].Semiconductorassisted photo catalysis has received considerable attention as a promising tool for implementing the purification of waste water and hydrogen energy production [2].Last two decades have witnessed a rapid advancement in various techniques for the fabrication of nanoparticles [3] [4].Among the various semiconducting materials, ZnS is a wide-band gap semiconductor of 3.8 eV having luminescent properties and photocatalytic applications.ZnS is one of the II-VI semiconducting material finding application in Cathode ray tubes, IR windows, injection lasers, ultraviolet light-emitting diodes and flat panel displays [5] [6].Doping of metal ions into ZnS can influence the performance of these photocatalysts.ZnS has been extensively studied with the aim of controlling the size, morphology and crystallinity in order to obtain desired physical properties [7] [8].Many methods have been used to synthesize ZnS nanoparticles such as sol-gel, hydrothermal, solvothermal and mechanochemical methods.ZnS obtained by this techniques has a wide band gap of 4 -4.6 eV [9].An electrochemical procedure based on the dissolution of a metallic anode in a protic solvent, has been used to obtain nanoparticles ranging from 10 to 20 nm with reduced band gap [10] [11].
Zr is a transition metal mainly used as refractory and opacifier and in small amount as an alloying agent for its strong resistance to corrosion.Zirconium containing compounds are used in many biomedical applications, including dental implants and other restorative practices, knee and hip replacements, although Zr has no known biological role [12] [13] [14].However, a few papers have been reported on photocatalytic and biological applications of Zirconium doped nanoparticles.Doping of Zr to ZnS is quite attractive, since the dissolution potential of Zr (−1.45 eV) is more negative than Zn (−0.7618 eV) and also the radius of Zr 4+ (0.86 Å) is almost similar to that of Zn 2+ (0.88 Å).Keeping in view, ZnS and 2 ZrS ZnS nanocomposites were fabricated by the novel electrochemical method and their catalytic effects on photodegradation of Indigo carmine dye and antibacterial activity using Gram negative bioluminescent Photobacterium leiognathi and antimitotic activity using Allium cepa have been reported here.

Synthesis of ZnS Nanoparticles
Zinc Sulphide nanoparticles are synthesized electrochemically using Zinc and platinum electrodes as shown in Figure 1.The electrolytic cell consisting of 0.5 M aqueous Na 2 S solution and electrodes are separated by 1 cm.The experiment was run for 3 hrs with continuous stirring using 30 mA current and a potential of 20 V.During the electrolysis Zinc electrode acts as anode starts to dissolve and gives Zinc ions which are electrochemically reacted with sulphide ions furnished by Na 2 S to give ZnS nanoparticles.The obtained nanoparticles are washed repeatedly with distilled water till complete removal of Sodium Sulphide, centrifuged and calcinated at 750˚C for 2 hrs to remove Sodium and hydroxide impurities.

ZrS ZnS Nanoparticles
The experimental set up is similar to synthesis of ZnS.Here Zinc, platinum and Zirconium electrodes were used.The rate of electrochemical reaction is not same for Zr 4+ and Zn 2+ , as the redox potential of Zr 4+

Determination of Photocatalytic Activities
The photo reactivity of nanocatalysts are influenced by variables such as the do- A calculated amount of catalyst was added to the dye solution, stirred in dark for The adsorption and photocatalytic conversion (g%) was calculated as follows [17]: ( ) 8000 vol of FAS in blank vol of FAS in dye soln normality of FAS COD .Sample volume

− =
The mineralization of dye was measured by the decrease of chemical oxygen demand (COD) of the solution.The COD was measured according to the standard dichromate titration method [18] [19].The mineralization efficiency of dye was estimated by the following expression: The decrease in COD (mg/l) and increase in % T of the dye solution with colour removal was observed as follows: 2 ZnS nanoparticles ZrS ZnS nanoparticles > .
The mechanism of photodegradation can be represented as follows [20] [21].
ts CO , H O, NH and other degradation products → Scheme 2. Mechanism of dye degradation by OH radical.

X-Ray Diffraction
The XRD pattern of synthesized ZnS and

Optical Absorption Spectra
The UV-Visible spectrum of ZnS and 2 ZrS ZnS nanoparticles (Figure 3) over the range 200 -600 nm showed that the synthesized nanoparticles are photoactive under UV light radiation.ZnS nanoparticles showed three absorption peaks where as 2 ZrS ZnS nanoparticles showed two peaks in the UV region.There is no absorption peak in the visible region.The band gaps of the samples are calculated using Tauc's plot [23] [24] and was found to be 2.7 eV for ZnS and 3.9

EDX of ZnS and
2

ZrS ZnS Nanoparticles
The EDAX analysis confirmed the presence of Zinc and Sulphur in ZnS and Zirconium, Zinc and Sulphur in 2 ZrS ZnS nanoparticles (Figure 5 and Figure 6).The surface morphology of ZnS and 2 ZrS ZnS nanoparticles was observed by FE-SEM analysis.The SEM images showed that the synthesized nanoparticles consisted of agglomerated particles (Figure 7).

Effect of Concentration of Dye
To ensure the optimum dye concentration, photodegradation is carried out with different concentration of Indigo carmine with constant weight of catalyst (Table 1 and Figure 8).As the optimum concentration of catalyst for ZnS nano particle is 0.02 g, keeping this as standard the same amount of radicals [26].

Effect of Catalyst Loading
The experiments were performed by taking different amount of catalyst varrying from 0.02 to 0.06 g in order to study the effect of catalyst loading (Table 2 and Figure 9).Photocatalytic rate initially increases with catalyst loading and then decreases at high values because of light scattering and screening effects [17] [27].The tendency toward agglomeration also increases at high solid concentration, resulting in a reduction in the surface area available for light absorption and a decrease in photocatalytic degradation rate.The number of active sites in solution will increase with catalyst loading, a point appears to be reached where light penetration is compromised because of excessive particle concentration [28].A further increase in catalyst loading beyond the optimum will result in non-uniform light intensity distribution, so that the reaction rate would indeed be lower with increased catalyst dosage.

Effect of Temperature
Temperature is one of the important factor which effects the rate of photodegradation.Increase of temperature is an indication of slighter increase in the rate of photodegradation as raise in temperature results in number of effective collisions leading to higher rate of reaction.However, the photodegradation efficiency is not much affected (Table 3 and Figure 10).

Reuse of Catalyst
The possibility of reusing the photocatalyst was tested to see the cost effectiveness of the method used.After degradation of the dye, the dye solution was kept overnight and then the supernatant liquid was decanted.The photocatalyst was thoroughly washed with double distilled water and then reused for the photodegradation by taking fresh IC dye solution.The reuse sample has shown almost same degradation efficiency compared to the fresh samples (Table 4 and    Figure 11), while an obviously decrease in photoactivity was noticed with the reuse cycles [29].This indicates the nano samples can be regenerated and reused with very low or significance change in the efficiency.An obviously decrease in rate of reaction was observed with the second use of catalyst.Reuse cycles might cause the aggregation of photocatalyst and the decrease in specific surface area and the losses of catalyst, resulting in a loss of catalytic activity [30].

Bacterial Growth Condition
The Gram negative bioluminescent Photobacterium leiognathi (accession number: KM434234), isolated from coastal area of Goa, were used in this study.Bacteria were grown in nutrient broth (NB) containing 3% sodium chloride with aeration at 25˚C for 24 h. 10 μl of the overnight culture was inoculated into 100 ml of nutrient broth and incubated under same condition until the OD 600 reaches 0.5 (approximately 12 hours) as at this OD bacteria were emitting the maximum amount of light.

Reagents
1 mg/ml stock solution nanoparticle (NPs) was prepared in sterile distilled water.
To disperse the NPs, the suspension was sonicated before use.Later NPs dilutions were prepared in sterile broth.As these solutions are later inoculated with an equal amount of bacteria in broth, the dilutions are prepared at concentration twice the desired final concentration.

Minimum Inhibitory Concentration (MIC) Assay
Broth microdilution technique has been used to determine the MIC of nanoparticle.For this purpose, two fold serial dilutions of the compound ranging from 500 to 0.4 µg/ml were performed in 96-well white microtiter plate.Initially 100 μl of bacterial inoculums was placed into the wells of the plate and later each well seeded with 100 μl of nanoparticle dilutions.Bacterial luminescent intensity was measured using a luminometer after 6 h incubation at 25˚C with 150 rpm shaking.In addition to the NP-treated well, each plate had untreated bacterial culture as control.Sterile broth containing NP and sterile broth also served as blank for the test and control respectively.NP efficacy was calculated from the blanks, control and treated luminescence values on a plate.
Percentage of inhibition = ( ) ( ) ( ) where 1 B denotes the average luminescence for sterile broth, 2 B denotes the average luminescence for sterile broth containing NP, C denotes the average luminescence for control and T denotes the average luminescence for treated wells.

Result and Discussion
The MIC is defined as the lowest concentration of NPs that inhibits the growth of a microorganism.The percentage of inhibition for ZnS and    From the above observations, the partial-c-mitosis, full-c-mitosis with partial functional spindles and comparatively normal mitotic cells phases were noticed in various cells of the same root tip between 12 -24 hrs time duration.Therefore, the antimitotic ability of ZnS and 2 ZrS ZnS was remarkable in controlling the cell division and acts as potent antimitotic agents.

Conclusion
In this study, we have reported the synthesis of ZnS and 2 ZrS ZnS nanoparticles by novel electrochemical method which is simple, cost effective and ecofriendly method.The photo degradation by these semiconductors offers a green technology for the removal of hazardous compounds present in waste water and industrial effluents.The kinetics of degradation of Indigo carmine has been studied.The complete degradation reaction was confirmed by conducting COD experiment.The synthesized nanoparticles are capable of entering into the Allium cepa cell and bacterial cell, therefore, inhibit the cell growth and hence con- firm the biological activity as a potent antimitotic and antimicrobial agents.Moreover,

1
min to establish adsorption/ desorption equilibrium between dye and nanoparticles and then illuminated under 8 W UV source to induce a photoche-mical reaction.Aliquots were taken at an interval of 2 min (in case of ZnS) and 10/15 min (in case of 2 ZrS ZnS ) and percent transmittance was determined.

2 ZrS
ZnS nanoparticles are shown in Figure 2. The XRD for ZnS shows three main diffraction peaks at 2θ values 31.61,34.28 and 36.08.The obtained peak positions correspond to Zinc blended type patterns and the XRD pattern is well matched with standard cubic ZnS [6].The XRD of ZrS 2 /ZnS nanoparticles are compared with ZnS nanoparticles and are as follows.From the XRD data it is evident that no much change in the position of diffraction peaks were observed compared to ZnS nanoparticles.A possible reason could be that Zr 4+ entered into the crystal lattice of ZnS and suppress the growth of ZnS crystals, because radius of Zr 4+ (0.86 Å) smaller than that of Zn 2+ (0.88 Å) and Zr substitutes Zn in the lattice.The slight change of lattice parameters of 2 ZrS ZnS also proved that the Zr ions were incorporated into the ZnS lattice.From the XRD data the cell parameters are calculated for ZnS nanoparticles and it is found to be a = b ≠ c (a = 8.483 Å, b = 8.483 Å and c = 9.957 Å) and

eV for 2 ZrS
ZnS nanoparticles as shown in Figure 4.The obtained band gap value of ZnS nanoparticle is lower than that of the bulk value of ZnS (3.68 eV).This blue shift of the band gap of ZnS nanoparticle occurs due to quantum confinement effect [25].The band gap of 2 ZrS ZnS is very much higher than that of ZnS.This supports lower photocatalytic efficiency of 2 ZrS ZnS nano particles compared to ZnS nanoparticles.

Figure 8 .
Figure 8. Plot of log % T vs. time with respect to different initial concentration of dye and COD values in case of (a) ZnS and (b) ZrS2/ZnS nanoparticles.

Figure 9 .
Figure 9. Plot of log % T vs. time with respect to catalyst loading and COD values in case of (a) ZnS and (b) ZrS2/ZnS nanoparticles.

Figure 10 .
Figure 10.Plot of log % T vs. time with respect to temperature in case of (a) ZnS and (b) ZrS2/ZnS nanoparticles.

Figure 11 .
Figure 11.Plot of log % T vs. time with respect to reuse of catalyst and COD values in case of ZnS and ZrS2/ZnS nanoparticles.

Figure 13 .
Figure 13.(a) Various stages of cell divisions seen under 40× microscopic field with normal cell division stages in control (SDW) treated groups.(b) Cells seen after 12 hours of treatment and observed under A: 40× with less stages of cell divisions, B: seen under oil immersion, there is no any further stages of cell divisions.(c) Cells seen after 18 hours of treatment and observed under A: 40× with very few stages (prophase, late anaphase and early anaphase) of cell divisions, B: seen under 40× microscopic fields with only metaphase.(d) Cells seen after 24 hours of treatment observed under 40 × microscopic views with very less cell divisions showing chromosomal aberrations, nuclear disintegration and initiation of cellular autolysis with formation of ghost cells.

2 ZrS
at different time duration(12,18 and 24 hrs) exhibited changes in chromosomes and shape of the cells with elongated appearance.The change in chromosomes and cellular morphology were achieved in increasing time and concentration.ZnS and ZnS nanoparticles showed good inhibitory effect by inhibiting the cell growth.

2 ZrS 2 ZrS 2 ZrS
ZnS nanoparticles have less photo catalytic activity compared with ZnS nanoparticles towards photodegradation.The presence of Zr suppresses the rate of photo-catalytic activity.ZnS nanoparticles are very good photocatalyst compared with ZnS nanocomposite.Hence, 2 ZrS ZnS nanocomposite can be used to block UV radiation and can be used in the preparation of cosmetics.The degradation efficiency is ~95% in case of ZnS where as 83% in ZnS nanocomposites.
and Zn 2+ are different.Since the dissolution potential of Zr (−1.45 eV) is more negative than Zn (−0.7618 eV), formation of ZrS 2 takes place in competition with ZnS.The mechanism of elec-

Table 1 .
Effect of concentration of dye on the rate of degradation.

Table 2 .
Effect of catalyst loading on the rate of photo-degradation.

Table 3 .
Effect of temperature on degradation of dye.

Table 4 .
Efficiency of catalyst in second use.
ZrS ZnS nanoparticles.The results of antimitotic activity are given in Table6and the percentage inhibition of cell division by ZnS and 2 ZrS ZnS nanoparticles comparative to control is given in Figures 13(a)-(d).

Table 5 .
Inhibition of bacterial growth at different concentrations of nanoparticles (µg/ ml).