Degradation of 2，4-Dichlorophenol Catalyzed by Ultrasound-Assisted Laccase

GUO Huiwen ()， ZHANG Shuqin ()， REN Dajun ()*， DENG Zhiqun()， HUANG Chaofan ()， KANG Chen( )

1 College of Resource and Environmental Engineering， Wuhan University of Science and Technology， Wuhan 430081， China2 Hubei Key Laboratory for Efficient Utilization and Agglomeration of Metallurgic Mineral Resources， Wuhan University of Science and Technology， Wuhan 430081， China

Abstract: Laccase possesses a good degradation effect on organic pollutants， but it is too long to achieve the desired effect. In order to improve the treatment effect of laccase on degradation of organic pollutants， 2，4-dichlorophenol (2, 4-DCP) was selected as a treatment target in the study. This study investigated a new technique for catalyzing the degradation of 2， 4-DCP， that is， ultrasound-assisted laccase catalysis. The optimal experimental parameters such as pH， ultrasonic power， duty cycle and laccase concentration were determined under optimized experimental conditions. The results showed that the optimum conditions for degradation of 2,4-DCP were that pH=5.5， the input power was 105 W， the duty cycle was 50% and the laccase concentration was 0.4 U/mL. The degradation rate of 2， 4-DCP reached 77.5% under the optimum conditions at 4 h. When in ultrasonic environment， the enzymatic activity of laccase could be stimulated and improved. Compared with conventional methods， this technique significantly promoted the degradation rate of 2, 4-DCP while reduced action time. Furthermore， no new pollutant was introduced into the degradation process. Therefore， ultrasound-assisted laccase catalysis is an environmentally friendly technique to degrade pollutants.

Key words: ultrasonic； laccase； catalysis; 2，4-dichlorophenol(2, 4-DCP)；oxidation； degradation

Introduction

Chlorinated organics are a class of persistent pollutants that are toxic and hardly biodegradable. Surface and underground water are contaminated because of its widely use and emissions of chlorinated organics in the manufacturing， cleaning， organic solvents， pesticides， chemical production and other industries[1-2]. Chlorophenols have been widely used in wood preservative， rust production， fungicides and other pesticides， causing great pressure on natural environment protection. In addition， the toxicity of chlorophenols increases with the degree of chlorination. It is imperative to remove chlorophenolic compounds (CPs) from natural environment. 2，4-dichlorophenol (2, 4-DCP)， 2，4，6-trichlorophenol and pentachlorophenol are highly toxic substances， they are listed in blacklist by the US Environmental Protection Agency (EPA) as priority control pollutants[3]. Among them， 2，4-DCP is most widely distributed in water and it can cause damage to environment and human bodies even its concentration is extremely low. The study on the method of removing 2，4-DCP has attracted researchers’ attention. The main treatment methods of chlorophenols include adsorption， coagulation， extraction， chemical oxidation， photochemical oxidation， ultrasonic chemical， hydrogenolysis technology and radiolysis technology[4].

Laccase is a class of oxidoreductase containing Cu2+， and laccase， plant ascorbate oxidase and mammalian plasma ceruloplasmin belong to the same family[5-6]. As polyphenol catalytic oxidoreductase， laccase is capable of “eco-friendly” enzymes which can reduce the oxygen molecules to H2O. In the action process， laccase extracts an electron from the molecules of the oxidized substrate and turns the oxidized substrate into an unstable free radical which can undergo further polymerization and depolymerization reactions[7].

In recent years， the use of ultrasonic technology to treat various refractory organic pollutants in water is more effective. Ultrasonic treatment is not only a clean， efficient pollution control and treatment technology， but also a high-level catalytic oxidation technology. During ultrasonic treatment process， there is an ultrasonic cavitation phenomenon which can create a unique environment that can cause the chemical reaction rate to surge. It has been shown that ultrasound exhibits a strong ability to degrade organic， so ultrasound has a strong removal effect on general organic pollutants which can achieve complete mineralization after sustained ultrasonic action. However， the degradation effect of some hardly degradable organic pollutants such as chlorophenol wastewater is not obvious[8-9]. Studies showed that laccase possessed a good degradation effect on chlorophenols， but they showed a long degradation period to remove chlorophenols. The present research aimed at the exploration of ultrasonic effect on the enzymatic activity of laccase and the degradability of organic pollutants. In the study， we took 2，4-DCP for example and applied ultrasonic technology to assist laccase for the degradation 2，4-DCP. In ultrasonic process， on one hand the cavitation phenomenon produce free radicals with strong oxidizing properties which have a certain effect on the degradation of 2，4-DCP， on the other hand strong shock waves and micro-jets accelerate the mass transfer between laccases and contaminants. At the same time， it was found that the activitiy of the laccase was improved during ultrasonic process. Therefore， ultrasound-assisted laccase catalysis can improve the degradation rate and degradation degree of organic pollutants.

1 Material and Methods

1.1 Main instruments and materials

UV-Vis spectrophotometer UV2550(Shimadzu, Japan), High Performance Liquid Chromatography UltiMate 3000 Series( Diana， American) and Ultrasonic cell grinder SCIENTZ-950E (Ningbo Xinzhi， China) were used in the study.

Laccase from Trametes versicolor， laccase from Agaricus bioporus， 2，2′-azino-bis-(3-ethylbenzothiazo line-6-sulfonic acid)(ABTS) and 2，4-DCP used in this study were purchased from Sigma Company. The other chemical reagents were prepared in China and analytically pure.

1.2 Determination of 2，4-DCP concentration

First， the solution containing 2，4-DCP was filtered through a 0.45 μm membrane， followed by the analysis of 2，4-DCP with High Performance Liquid Chromatography(HLPC). The measurement conditions were shown as follows. Column: thermo SCIENTIFIC C18 reverse column (4.6 mm×250 mm)； mobile phases: methanol and water (volume ratio was 75∶25)； detection wavelength: 285 nm， 280 nm and 275 nm； detection temperature: 35 ℃； flow velocity: 1.0 mL/min； loading quantities of samples: 20 μL. The peak time of 2，4-DCP was about 6.5 min.

1.3 Experimental method

About 50 mL of 10 mg/L 2，4-DCP solution was placed in a beaker and the pH of the solution was controlled by acetic acid-sodium acetate buffer solution (0.1 mmol/L). Then， add a certain concentration of laccase， followed by mixing the solution in opened ultrasound equipment for ultrasonic degradation. It should be noted that the ultrasonic probe to be at least 1 cm away from the liquid surface and at least 2-3 cm from the bottom of the beaker. The experimental temperature was about 25 ℃. During the reaction， the concentration of 2，4-DCP in reaction solution was measured every half an hour or one hour.

2 Results and Discussion

2.1 Effect of laccase species on degradation of 2，4-DCP

Fig.1 Effect of laccase species on degradation of 2， 4-DCP

In this study， laccases from Trametes versicolor and Agaricus bioporus were selected respectively. The preparation concentration of the enzyme solution with pH value of 3 was 0.8 U/mL. The initial 2，4-DCP concentration was 10 mg/L and the treatment time was set to 7 h. During the degradation process of 2，4-DCP， laccase transferred the electrons in substrate to oxygen molecules， and in oxidation process， two free radicals randomly combined to form a dimer. Then， the dimer was isomerized to produce a stable aryl chemical[10]. In Ref.[11], the study on the nature， function and catalytic mechanism of laccases showed that different laccases exhibited distinct biochemical properties， though their activity sites were basically same[11]. In addition， the activity of laccase could also be affected by copper ion chelating agents， halides， cysteines and other substances[11]. As shown in Fig.1， laccase from Trametes versicolor exhibited greater potential for the degradation of 2，4-DCP than that from Agaricus bioporus. This may be due to that 2，4-DCP belongs to halide and different types of laccase have different tolerance to 2，4-DCP. The laccase from Trametes versicolor was chose as reactant for the following experiments. In Fig. 1， the initial concentration and pH of the solution were set to 10 mg/L and 3， respectively. Add a certain amount of laccases to the identically configured 2，4-DCP solution to ensure that the concentration of the two kinds of laccases in the solution is 0.8 U/mL. The 2，4-DCP concentration was measured every one hour. Each result was the average of the experiments that were performed in quadruplicates.

2.2 Effect of laccase dosage on degradation of 2， 4-DCP

Briefly， the initial 2， 4-DCP concentration was 10 mg/L and the treatment time was set to 7 h. The concentrations of laccase added in 2，4-DCP solution were set to 0.2， 0.4 and 0.8 U/mL， respectively. As shown in Fig. 2， the degradation effect of laccase with the low concentration of 0.2 U/mL on 2，4-DCP was not ideal. However， the degradation of 2，4-DCP was improved when the concentration of laccase was 0.4 U/mL， and it was close to saturation. When the concentration of laccase was increased to 0.8 U/mL， the degradation tendency was similar to that when the concentration of laccase was 0.4 U/mL， and the degradation rate did not increase significantly. Therefore， 0.4 U/mL was the most appropriate laccase concentration for the degradation of 2，4-DCP when considering economical efficiency and treatment effect. In Fig.2， the initial concentration and pH of the solution were set to 10 mg/L and 3， respectively. Different masses of laccase were added into three identical 2，4-DCP solutions. The 2，4-DCP concentration was measured once an hour. Each result was the average of the experiments that were performed in quadruplicates.

Fig.2 Effect of laccase dosage on degradation of 2，4-DCP

2.3 Effect of pH on degradation of 2，4-DCP

Laccase is a protein， so pH of solution is an important parameter that affects the degradation of 2，4-DCP by laccase. If pH is not appropriate， the structure of laccase may be changed， thus causing laccase deactivation. Acetic acid-sodium acetate (0.1 mol/L HAc-NaAc) buffer was used to control pH(3，4，5，6) for degradation of 2，4-DCP at 25 ℃. The concentration of laccase was set to 0.4 U/mL. As shown in Fig. 3， when the concentration of laccase and the buffer pH were set to 0.4 U/mL and 5-6， respectively, the degradation of 2，4-DCP was more effective. However， when used 0.1 U/mL laccase solution for enzymatic activity， the optimum pH is between 4 and 5. Different pollutants may change the properties of solution to some extent， resulting in changes in the optimal pH of laccase. Therefore， in the present study， the selected optimum pH was 5.5. In Fig.3， the initial concentration of the solution and the concentration of laccase were set to 10 mg/L and 0.4 U/mL， respectively. The pH of the 2，4-DCP solution was adjusted with buffer， and 0.4 U/mL laccase was added into the 2，4-DCP solution. The 2，4-DCP concentration was measured every one hour. Each result was the average of the experiments that were performed in quadruplicates.

Fig.3 Effect of pH on degradation of 2，4-DCP

2.4 Effect of ultrasonic power on degradation of 2，4-DCP

Fig.4 Effect of ultrasound power on degradation of 2，4-DCP

In previous studies， it has been reported that ultrasonic power could affect the enzymatic reaction and excessive ultrasonic power could lead to denaturation of laccase[12]. Accordingly， ultrasonic power is an important factor for the degradation of 2，4-DCP catalyzed by laccase. During the experiment， the ultrasonic frequency was 20-25 Hz and the ultrasonic power ranged from 75 to 120 W (75 W， 90 W， 105 W and 120 W). With the increased of ultrasonic power from 75 W to 105 W， the degradation rate of 2，4-DCP also increased from 65.2% to 77.5% (Fig. 4). In the studies on the ultrasound-assisted laccase catalyzed degradation of ciprofloxacin hydrochloride[13]， it was found that the increase of ultrasound power could make the implosion of bubbles more active， thus causing the improvement of the degradation rate of ciprofloxacin hydrochloride. In Dey S and Meral Dükkanci’s studies[9，14]， β-carotene and Orange II were treated with ultrasound， as the ultrasonic power continued to increase， excess cavitation bubbles emerged， thus resulting in the inhibition of propagation of sound waves or the formation of some large bubbles and weak implosion. Overall， the degradation effect of 2， 4-DCP could be reduced slightly. Therefore， the maximum degradation rate could be achieved when the power was 105 W. In Fig.4， the initial concentration of the solution was set to 10 mg/L. The concentration of laccase and the pH in the solution were set to 0.4 U/mL and 3， respectively. Four equal volumes of 2，4-DCP solution were taken and the reactions were performed at different powers. In the first two hours， the 2，4-DCP concentration was measured every half an hour， and then it was measured every hour. Each result was the average of the experiments that were performed in quadruplicates.

2.5 Effect of duty cycle on degradation of 2，4-DCP

Duty cycle is also one of the important factors in the analysis of enzymatic reactions. Avhadetal.[15]studied the impact of ultrasonic power， duty cycle and time on enzyme and it showed that laccase could degenerate due to the continuous exposure to sound waves. Therefore， in the control reaction process， there should be suitable duty cycle， which can be controlled by adjusting the opening and closing time of the ultrasonic wave. In addition， considering unnecessary electrical energy consumption as well as circumvent denaturation of laccase at higher duty cycle due to excessive heating， the opening and closing time of ultrasonic wave should be also appropriate[16]. In this experiment， the proportion of duty cycle was set to 50.0% and 66.7%， respectively. As shown in Fig. 5， when the duty cycle was 50.0%， the degradation rate of 2，4-DCP was higher. The concentration of 2，4-DCP solution was 10 mg/L， the concentration of laccase in the solution was 0.4 U/mL and pH=5.5. The control power was set to 105 W. The 2， 4-DCP concentration was measured every hour at different duty cycles. Each result was the average of the experiments that were performed in quadruplicates.

2.6 Effects of laccase， ultrasound and ultrasound-assisted laccase on degradation of 2，4-DCP

In this experiment， the concentration of laccase， pH， the ultrasonic power and the duty cycle were set to the optimal value， respectively， and the initial 2，4-DCP concentration was 10 mg/L. Then， 2，4-DCP solutions were treated with laccase， ultrasound， and ultrasound-assisted laccase， respectively. When 2，4-DCP was treated with ultrasound only， the treatment effect of laccase was better during 0 to 1.5 h， then， the effect tended to be stable and the degradation rate of 2，4-DCP was only 45.3% after 4h. However， in the treatment with ultrasound-assisted laccase， the concentration of 2，4-DCP kept gradual decreasing and the degradation rate of 2，4-DCP could reach 77.5% at 4 h. In Fig. 6， under the conditions of the laccase concentration 0.4 U/mL， pH=5.5， the ultrasonic power is 105 W， and the duty ratio is 50%. The solution of 2, 4-DCP was treated in different ways. Each result was the average of the experiments that were performed in quadruplicates.

Fig.6 Effect of laccase， ultrasound and ultrasound-assisted laccase on degradation of 2，4-DCP

2.7 Effects of ultrasound on enzymatic activity

Fig.7 Effect of ultrasound on enzymatic activity

In the research on degradation of pollutants catalyzed by ultrasound-assisted laccase， it was necessary to explore the effect mechanism of ultrasound on laccase. In this experiment， the concentration of laccase， the ultrasound power and the duty cycle were set to the optimal value， respectively. The concentration of laccase was 0.4 U/mL and the pH of laccase solution was adjusted to 3， 4， 5 and 6， respectively. The enzymatic activity was measured by ultraviolet spectrophotometry. As shown in Fig. 7， when pH was adjusted to 3， the relative enzymatic activity of laccase was only 31% after 4 h， and it was still not ideal when pH=4. However， when pH is 5 and 6， the relative enzymatic activity of laccase has achieved more than 120%. Therefore， when pH=5.5， the relative enzymatic activity of laccase could maintain higher levels and stability by ultrasonic assistance. In Fig.7， the laccase solution was sonicated at a power of 105 W and a duty cycle of 50%. The relative enzyme activity was measured every hour at different pH. Each result was the average of the experiments that were performed in quadruplicates.

Some studies have shown that ultrasound was helpful for the improvement of enzymatic activity. In the course of ultrasonic radiation， the local liquid produced the cavitation phenomenon which could generate an environment of extreme temperature and pressure， thus stimulating the enzymatic activity of laccase[17]. At the same time， the transmission of enzyme active site can be greatly improved in ultrasonic environment， thus promoting the catalytic efficiency of laccase.

2.8 Effect and products of 2，4-DCP degradation catalyzed by ultrasound-assisted laccase

In general， liquids generate cavitation bubbles under ultrasonic radiation. Ultrasonic cavitation means that the tiny nucleus in the liquids is excited by the action of ultrasonic waves and the dynamic process of the appearance， growth， contraction and rupture of the foam core oscillation. Foam in the instant outbreak of space is very small， which can result in high temperature and high pressure， strong shock wave and other extreme conditions， thus promoting organic matter “water phase combustion reaction”[11]. During the process of cavitation bubble burst， due to the dissociation of the vapor confined in the cavitation bubbles， reactive free radicals with strong oxidizing property are produced to degrade 2，4-DCP to a certain extent.

In Ref.[18]， it was shown that chlorophenols could be degraded via oxidation by laccase. During the process of degradation， laccase acted as a single electron oxidoreductase to deoxidize oxygen into water without producing hydrogen peroxide or other secondary products. Furthermore， the oxidation of chlorophenols was achieved by electron transfer， that is， the Cu2 +in the active center of laccase was first reduced， and then， electrons were transferred from substrate to oxygen to degrade chlorophenols.

To combined ultrasound-laccase assisted degradation of chlorophenols， on one hand， ultrasound itself can act as strong oxidative free radicals， on the other hand， it can produce strong shock waves and micro-jet to strengthen the vibration and stirring of solution， thus promoting the contact of laccase and 2，4-DCP.

The peak time and the reductive dechlorination route of the degradation products of 2，4-DCP were shown in Figs. 8-9， respectively.

As presented in Fig.8， only a small amount of 2-dichlorophenol (2-CP)， 4-dichlorophenol (4-CP) and phenol in the 2，4-DCP solution could be observed before the reaction (0 min). The peak corresponding to 2，4-DCP continued to decrease at the reaction time ranging from 60 min to 180 min. At the reaction time of 60 min， the peak corresponding to 2-CP， phenol and 4-CP increased， and at 180 min， the first two still kept increasing. However， the peak intensity of phenol did not increase significantly and even declined in later period. The reason for the reduction of phenol may be due to that phenol continued to be catalytically oxidized by laccase to form free radicals or reactive quinines which can be polymerized with each other and play a role in reducing their toxicity.

The degradation speed of o-substituted chlorophenol catalyzed by laccase was faster than that of para-substituted chlorophenol. In addition， the ortho and para chlorine atoms of 2，4-DCP could be replaced and the ortho-dechlorination was first catalyzed by laccase. The reduction and dechlorination pathway of 2，4-DCP was shown in Fig.9.

Fig.8 Reductive dechlorination products of 2，4-DCP

Fig.9 Reductive dechlorination of 2，4-DCP

3 Conclusions

The pH of the solution， ultrasonic power and the type of laccase have a great influence on the degradation of 2，4-DCP. The degradation of 2，4-DCP was the most effective when pH=5.5， the input power was 105 W， the duty cycle was 50% and the laccase concentration was 0.4 U/mL. Compared to the degradation of 2，4-DCP catalyzed by single ultrasound or single laccase， it could be greatly promoted by ultrasound-assisted laccase in catalytic effect and processing time. Moreover， other pollutants were not produced during the degradation process and the toxicity of intermediates and final products of 2，4-DCP degradation were lower than that of 2，4-DCP.