Photocatalytic Treatment of Microcystin-LR-Containing Wastewater Using Pt/WO 3 Nanoparticles under Simulated Solar Light

This study investigates the photocatalytic degradation of microcystin-LR (MC-LR) under simulated solar light using Pt modified nano-sized tungsten trioxides (Pt/WO3). Photocatalytic activity was higher during the degradation of MC-LR with Pt/WO3 than with pure WO3 or TiO2. The catalyst loading greatly affect the degradation performance. The rate of degradation is influenced by the initial pH of the reaction solution. This study also investigates the photocatalytic inactivation of cyanobacteria. The results show that the algal growth was successfully controlled by the Pt/WO3. This study suggests Pt/WO3 photocatalytic oxidation with solar light is a promising treatment for water containing MC-LR.


Introduction
An intensification of agricultural and industrial activities resulting from an increase in population has led to eutrophication in superficial freshwater bodies and has therefore induced more frequent cyanobacteria blooms worldwide.The toxins released into freshwater by cyanobacteria are well-documented [1].The most commonly occurring toxins released by cyanobacteria are called Microcystins.
Microcystins are a family of cyclic heptapeptides heaptotoxins containing the unique C 20 amino acid, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-d ienoic acid, which is abbreviated Adda.The most abundant and frequently detected microcystin is microcystin-LR (MC-LR), which has leucine (L) and arginine (R) in the variable positions.Microcystins are strongly heaptotoxic because they disrupt protein phosphatases 1 and 2A [2], which may promote primary liver cancer and cause the death of animals and humans.The World Health Organization (WHO) has determined a provisional guideline value of 1.0 μg/L for MC-LR in drinking water.
Various water treatment processes have been evaluated to determine their efficacy in degrading these toxins because microcystins are considered a threat to human health.However, MCs are chemically stable across a range of pH values and temperatures, due to a cyclic structure; consequently, traditional water treatment processes are unsuccessful in removing MCs [3][4][5].
Photocatalytic oxidation is an advanced oxidation technology that has been deemed an environmentally friendly water treatment option in recent years [6][7][8][9][10].When a photocatalyst is illuminated with a light of an appropriate wavelength, pairs of electrons (e -) and electron holes (h + ) are generated on the surface of the catalyst by photons.These pairs react with oxygen and water molecules or hydroxyl groups adsorbed on the surface of the catalyst and form highly reactive oxygen species, such as hydroxyl radicals (•OH), superoxide ions ( 2 ) or hydroperoxyl radicals (•OOH) [11].Reactive oxygen species can nonselectively oxidize a large number of organic wastes, including dyes, pesticides, bacteria and herbicides [6][7][8]12].Previous research proved that photocatalytic oxidation with TiO 2 photocatalyst could effectively destroy MCs, even at extremely high toxin concentrations [13,14].However, TiO 2 has a large absorption band gap (E g ) of 3.2 eV that restricts its universal use because it can only absorb UV light [15].Conversely, with an E g between 2.4 eV and 2.8 eV, tungsten oxide (WO 3 ) is a photocatalyst that absorbs visible light irradiation up to 480 nm [11].Compared with mixed metal oxides and doped oxides, WO 3 is inexpensive to prepare and stable in acidic and oxidative conditions, which makes it a promising material for photocatalytic applications.Previous research showed that WO 3 degradation of organic species under visible light intensified in the presence of suitable co-catalysts, such as Pt, Pd and CuO [16][17][18].However, there is no literature on the photocatalytic degradation of MCs under visible light with WO 3 -based catalysts.
The antimicrobial activity of photocatalyic reaction was first demonstrated by Matsunaga and coworkers [19], since then, photocatalysis has been shown to be capable of killing a wide range of organisms including Gramnegative and Gram-positive bacteria, including endospores, fungi, algae, protozoa and viruses, and has also been shown to be capable of inactivating prions [20][21][22].Moreover recently works also reported that TiO 2 -photocatalysis has the ability to inhibit the growth of the filamentous algae, Oedogonium and Cladophora [23,24].
In our study, the photocatalytic degradation of MC-LR, a model toxin, was investigated using Pt/WO 3 under simulated solar irradiation.Variations in sample parameters, such as catalyst loading and initial pH, were present in this study and are discussed later.To investigate the photocatalytic inactivation of cyanobacteria, Microcystis aeruginosa (M.aeruginosa) was selected as test species, for it is the most common blue-green algae and easily causes eutrophication.
Pt-loaded WO 3 sample (Pt/WO 3 ) (0.5% w/w) was prepared by a photo deposition method [18].An aqueous suspension containing the particulate WO 3 and H 2 PtCl 6 •6H 2 O was exposed to visible light (λ > 400 nm) provided by a 300 W Xe lamp (LX-300F, Cermax, CA) fitted with a cutoff filter (L-42, HOYA, Japan).After 2 h of irradiation, methanol (10 vol%) was added and the suspension exposed to further irradiation for 2 h.The as-prepared sample was collected by centrifugation and washed twice with Milli-Q water and finally dried at 105˚C for 2 h.The amount of deposited Pt was determined by analyzing the concentration of unused chloroplatinic acid remaining in the centrifuged solution after photo-deposition.The chloroplatinic acid concentration was analyzed by a inductively coupled plasma mass spectrometry (ICPS-8100, Shimadzu Co. Ltd., Japan).The prepared sample was characterized.UV-visible spectrum of the sample was recorded on a spectrophotometer (UV-2550, Shimadzu Co. Ltd., Japan).X-ray powder diffraction (XRD) measurement was carried out by using a X-ray diffractometer (Rigaku Smartlab).

Algal Culture
M. aeruginosa was obtained from National Institute for Environment Studies (NIES) (Ibaraki, Japan), The composition of the MA medium used in algal growth tests was listed in Table 1. 1 L of MA medium was added to a 3 L conical flask and was autoclaved at 121˚C for 20 min.The cultivation was carried out in the cultivating box with illumination for 10 days.The continuous light was provided by a fluorescence lamp with an automated 12 h/12 h light/dark cycle.The light intensity during the light phase was 1500 lx.The temperature was controlled at 25˚C ± 1˚C.

Photocatalytic Tests
The reactor was a 6-mL vessel equipped with a magnetic stirrer.A solar lamp (XC-100B, SERIC Ltd., Japan) was used as the irradiation source, and the light intensity was measured with a photometer (LI-250A, LI-COR Inc., USA).The photoemission spectrum was measured with an optical fiber spectrometer (USB4000, Ocean Optics Inc., USA).
An aliquot of stock MC-LR solution (50 mg/L) was added to the test solution to achieve an initial concentration of 1 mg/L.A suspension with catalyst particles was transferred to the reactor containing MC-LR to obtain a final volume of 5 ml.The catalyst loading was varied depending on the experiment condition.The initial pH was adjusted with H 2 SO 4 or NaOH.Before irradiation, the suspension was stirred for 60 min in the dark to equilibrate the solution.During irradiation, samples were taken and centrifuged every 30 minutes for analysis.Copyright © 2012 SciRes.

OJAppS
Photocatalytic inhibition of M. aeruginosa was carried out in a 200-mL beaker equipped with a magnetic stirrer.The irradiation source was the same solar lamp with an automated 12 h/12 h light/dark cycle.All the experiment equipments were placed in a clean bench to prevent the interference of dust and microorganism brought by air.150 ml of algal solution and a suspension with catalyst particles were added to the beaker for irradiation.Samples were taken every 2 days for analysis.counts.Then the samples were dripped to a hemocytometer and covered with a clear cover for counting.The enumeration was achieved by a microscope.

Characterization of Phoctocatalyst Samples
Figure 1 shows the absorption (100-reflectance) spectra for WO 3 and Pt/WO 3 powders.The absorption of WO 3 increased at approximately 460 nm, which is consistent with previously reported value [16].For Pt/WO 3 sample, with the contribution of Pt doping the spectrum shows stronger broad absorption in the visible light region.The XRD patterns of WO 3 and Pt/WO 3 samples are illustrated in Figure 2. Compared to tungsten oxide JCPD files (No. 43-1035) and those reported by others [23,24], the diffraction patterns of the samples assigned those of WO 3 monoclinic structure.As shown, no sign of crystallite Pt is detected in the patterns with Pt/WO 3 .It can be related to the fact that lower Pt concentrations lie below XRD instrumental detection limit and indicated that the Pt doping did not influence the crystal structures of WO 3 .

Detection of Hydroxyl Radicals (•OH)
Photoluminescence (PL) with terephthalic acid as a probe molecule was used to detect •OH in the photocatalytic reaction system.Terephthalic acid reacts with •OH to produce highly fluorescent 2-hydroxyterephthalic acid [25].
In a beaker, a photocatalyst powder was dispersed in 20 mL of 5 × 10 -4 M terephthalic acid aqueous solution and 2 × 10 -3 M NaOH.The solar lamp was used as a light source.Samples were centrifuged every 20 min for analysis.

Analysis
The degradation of microcystin-LR was monitored by High-performance liquid chromatography (HPLC) (Jasco-1500, Jasco, Inc., Japan) with a high-resolution diode array detector (Jasco UV-1570) set at wavelength of 238 nm.Samples were separated on a C18 column (5 m, 250 mm, 4.6 mm id) using a mobile phase of acetonitrile and Milli-Q water containing 0.01 mol/L ammonium acetate (pH 6.8; 32:68 v/v) and a flow rate of 1 mL/min.
PL spectra generated by the 2-hydroxyterephthalic acid were measured on a Hitachi F-4500 fluorescence spectrophotometer set at a wavelength of 315 nm.The pH values of the solutions were measured with a pH meter (TES-1380, TES Co., Taiwan).
The growth of M. aeruginosa was evaluated by cells enumeration.The samples were diluted with Mill-Q water to obtain an appropriate cell density for microscopic

Photocatalytic Degradation of MC-LR with Pt/WO 3
As shown in Figure 3, the concentration of MC-LR was virtually unchanged after 3 h of irradiation when there was no photocatalyst in the solution, thereby indicating MC-LR was stable under solar irradiation.After 3 h of irradiation, 19% of MC-LR was removed from a solution with only TiO 2 added, and 24% of MC-LR was removed from a solution in which only WO 3 was added.Compare to pure TiO 2 and WO 3 , the performance of modified WO 3 was much better, with 3 h of irradiation.Over 81% of MR-LR was degraded by the Pt/WO 3 composite within 90 min.Furthermore, the removal efficacy was 100% when the contact time was lengthened to 180 min.
Poor MC-LR removal efficiency by a solution containing only TiO 2 was attributable to the light source.The simulated solar lamp used in this experiment emitted light mainly with wavelengths greater than 400 nm, and the TiO 2 excitation range is less than 390 nm [15].The low photocatalytic activity of pure WO 3 is because the conduction band level of WO 3 (+0.5Vvs. NHE) is more positive than the potential for the single-electron reduction of oxygen (O 2 / 2 = −0.56V vs. NHE; O 2 /HO 2 = −0.13V vs. NHE) [17].Without co-catalysts, the high conduction band of WO 3 restricts the compound's activity with an organic compound [10].

Photocatalytic Degradation Mechanism
Pt is a co-catalysts for WO 3 -induced photocatalytic reactions that can promote O 2 reduction in a multi-electron process [17,26].In a photocatalyic reaction, the following chain reactions have been postulated [11]: Several highly reactive oxygen species, such as •OH, HOO• and 2 , are generated through the reduction of O 2 to oxidized organic compounds.Accordingly, organic compounds could be effectively degraded by WO 3 in the presence of a co-catalyst.

O  
Photocatalytic degradation of MC-LR was initiated by the attack of hydroxyl radical on the conjugated diene structure of Adda [27], thereby indicating the primary reactive species in MC-LR degradation was the hydroxyl radical.Kim et al. proved that the deposition of Pt on WO 3 facilitates the generation of OH radicals under visible light [28], and our experiments confirmed this phenomenon using photoluminescence (PL).Figure 4 shows the spectra observed during irradiation of the Pt/WO 3 sample.At approximately 425 nm, PL intensity gradually increased with an increase in irradiation time, thereby suggesting that OH radicals are formed on the photocatalyst-water interface via photocatalytic reactions [25,29].Because the photocatalytic degradation of MC-LR was initiated by an OH radical [27], Pt/WO 3 is particularly effective in the photocatalytic degradation of MC-LR.

Effect of Catalyst Loading
Experiments were carried out by varying the catalyst concentration from 50 to 250 mg/L.The results are shown in Figure 5.When the catalyst loading was 50 mg/L, the degradation efficiency was low, only 58% of MC-LR was removed after 3 h irradiation.The increase in the catalyst loading from 50 to 150 mg/L sharply increased the degradation efficiency.However, when the catalyst loading further increased to 250 mg/L, the degradation efficiency decreased.The results indicate that the inactivation efficiency is not proportional to the catalyst loading.The increasing catalyst loading induces an increase in the availability of active sites on the catalyst surface and produces a proportional increase in the number of active radicals by absorbing photons.However, an increase in the catalyst concentration results in increasing the light source extinction coefficient, and subsequently reducing the reaction efficiency [21,30].Based on this result, 150 mg/L was chosen as the optimum catalyst loading.

The Effect of Initial pH on the Photocatalytic Degradation of MC-LR
The pH affects the surface condition of catalysts and MC-LR and the generation of hydroxyl radical in hydroxylation reactions.After 180 min of irradiation, the removal of MC-LR was 89%, 100% and 77% with pH values of 3, 6 and 10, respectively (Figure 6).
Although the degradation of MC-LR was initiated by the attack of hydroxyl radical [27], the number of •OH ions should be lower at an acidic pH because hydronium ions favor the presence of an electron hole (Equation ( 8)) [31].At low pH, MC-LR degradation would be adversely affected due to the lack of OH -ions.The initial pH was Experimental conditions: MC-LR concentration of 1 mg/L, simulated solar light intensity of 0.4 mW/cm 2 .

Figure 5. Efficiency of photocatalytic degradation of MC-LR as a function of catalystloading.
Experimental conditions: MC-LR concentration of 1 mg/L, Pt/WO 3 concentration of 100 mg/L and simulated solar light intensity of 0.4 mW/cm 2 .SO  ions have an adverse effect on the photocatalytic degradation rate [32].Given the evidence, we can explain the lower efficiency observed in our experiment at acid pH.
The point of zero zeta-potential (PZZP) for WO 3 occurs at approximately pH 2, and WO 3 particles are negatively charged when the pH of a solution is greater than 2 [28].At pH values between 3 and 12, the carboxylic groups of MC-LR are ionized, and the molecule is negatively charged [33].In basic conditions, negatively charged WO 3 molecules repel MC-LR and inhibit interactions between the toxins and the catalysts.Fewer interactions result in lower photocatalytic activity and a lower degradation rate.Our results are consistent with a study by Lawton et al. in which the reaction rate of photocatalytic degradation of MC-LR by TiO 2 was lowest at pH 10 [34].

Photocatalytic Inhibition of Algal Growth under Solar Light
Figure 7 shows the results of photocatalytic inhibition of algal growth under solar light.After 6 days of irradiation, M. aeruginosa in the control samples kept growing and the number of algae cells increased from 1.3 × 10 6 to 2.2 × 10 6 .However, the growth of M. aeruginosa in the samples treated by photocatalyst was inhibited and the algae cells decreased from 1.3 × 10 6 to 0.1 × 10 6 .These results show that the photocatalytic treatment had the function of inactivation of microorganisms.
It is generally believed that the inactivation of microorganisms by photocatalylic treatment is mainly due to oxidative radicals (mainly •OH) produced by photocatalyst irradiation [35].

Conclusions
Under simulated solar irradiation, WO 3 degrades MC-LR more effectively in the presence of co-catalyst compared to solutions with pure TiO 2 or pure WO 3 .The highest rate of MC-LR removal occurred in solutions containing Pt/WO 3 .Specifically, 1 mg/L of MC-LR was removed after 3 h of irradiation by 100 mg/L of Pt/WO 3 .The optimum catalyst loading was 150 mg/L.A neutral pH, such as a pH of 6, improved the efficacy of toxin removal.The experiment results show that the algal growth was successfully controlled by the Pt/WO 3 .

Figure 3 .
Figure 3.The effect of catalysts on the efficiency of photocatalytic degradation of MC-LR.Experimental conditions: MC-LR concentration of 1 mg/L, catalyst concentration of 100 mg/L and simulated solar light intensity of 0.4 mW/cm 2 .

Figure 4 .
Figure 4. PL spectral changes observed during irradiation of the Pt/WO 3 sample.

Figure 6 .
Figure 6.Efficiency of photocatalytic degradation of MC-LR as a function of initial pH.
Experimental conditions: Catalyst concentration of 150 mg/L and simulated solar light intensity of 0.8 mW/cm 2 .