Horseradish peroxidase immobilized on aluminum-pillared interlayered clay for the catalytic oxidation of phenolic wastewater

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40 (2006) 283 – 290

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Horseradish peroxidase immobilized on aluminum-pillared interlayered clay for the catalytic oxidation of phenolic wastewater Jun Cheng, Shao Ming Yu, Peng Zuo College of Chemical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China

art i cle info

A B S T R A C T

Article history:

Horseradish peroxidase (HRP) was successfully immobilized on aluminum-pillared

Received 29 May 2005

interlayered clay (Al-PILC) to obtain enzyme–clay complex for the treatment of wastewater

Received in revised form

polluted with phenolic compounds. The immobilized HRP exerted a perfect phenol removal

27 September 2005

by precipitation or transforming to other products over a broader pH range from 4.5 to 9.3.

Accepted 2 November 2005

The addition of polyethylene glycol (PEG) could significantly enhance the phenol removal efficiency, and reduce the amount of immobilized enzyme required to achieve a high

Keywords:

removal efficiency of over 90%. When the mass ratio of PEG/phenol and the molar ratio of

Aluminum-pillared interlayered clay

hydrogen peroxide/phenol were 0.4 and 1.5, respectively, the oxidation of phenol could be

Horseradish peroxidase

completed within short retention time after the initiation of reaction in the absence of

Immobilization

buffer. HRP immobilized on Al-PILC had better storage stability compared with free

Phenolic wastewater

enzyme. However, the reusability of the immobilized enzyme was not very satisfactory. In

Polyethylene glycol

the fourth repeated test, the immobilized enzyme lost its catalytic performance. Further

Catalytic oxidation

research should focus on the improvement of reusability. & 2005 Elsevier Ltd. All rights reserved.

1.

Introduction

The phenolic compounds were present in wastewater from many industries, such as coal conversion, resins and plastics processing, petroleum refining, iron and steel, textiles, timber, soaps, detergents, dyes and other organic chemicals (Klibanov et al., 1980, 1983; Liu et al., 2002). All these phenolic streams were toxic pollutants because they were potential danger to human health (suspected carcinogens). Several available conventional methods including adsorption, chemical oxidation, solvent extraction and biodegradation had been extensively applied. These methods were practicable and effective, but they had several drawbacks such as incomplete removal, the potential formation of toxic residual products which were even more toxic than the original ones, and applicability to a limited concentration range (Aitken, 1993; Corresponding author. Tel.: +86 551 2901452; fax: +86 551 2901450.

E-mail address: [email protected] (S. Ming Yu). 0043-1354/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.11.017

Nicell et al., 1993; Liu et al., 2002). Enzymes were promising candidates for catalysis, sensor, and separation applications owing to their excellent catalyses with super chemo-, regio-, stereo-, and chiral-selectivity (Bornscheuer, 2003). Enzymatic methods had become increasingly important in the removal of phenolic contaminants since the initial work of Klibanov et al. (1980). Horseradish peroxidase (HRP) was effective for the removal of a broad spectrum of aromatic compounds such as phenols, biphenols, anilines and enzidines by catalyzing the oxidation of phenols in the presence of hydrogen peroxide (H2O2) to phenoxy radicals over relatively wide ranges of pH, temperature, contaminant concentration and salinity (Nicell et al., 1992, 1993). The free radicals combined in different ways and formed insoluble high molecular weight polymers that could be easily removed by filtration or sedimentation (Klibanov and Morris, 1981; Dura´n and Esposito, 2000). But its

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application was currently hampered by its high cost, lack of long-term stability under reaction conditions and the difficulty in recovering active enzyme for recycling. Improvement in the active life of the enzyme, and thereby a reduction in treatment cost, had been accomplished through different approaches. One of most common and promising was enzyme immobilization that exhibited the following advantages: enhanced stability, repeated or continuous use, easy separation from the reaction mixture, possible modulation of the catalytic properties, prevention of protein contamination, easier prevention of microbial contaminations (Bornscheuer, 2003). In addition, enzyme usually exhibited higher catalytic activity and improved stability against denaturation as a result of immobilization procedure (Fishman et al., 2002). Various natural or synthetic supports for enzyme immobilization had been reported in the literatures (Dumitriu et al., 2003; Le´tant et al., 2004). Pillared interlayered clays (PILCs) comprised a class of porous, large surface area, biocompatible two-dimensional materials which had attracted considerable attention owing to their potential technological applications. Several comprehensive reviews of the subject were available (Kloprogge, 1998; Ding et al., 2001). PILCs were prepared by exchanging the charge-compensating cations between the clay layers with larger inorganic hydroxyl cations, which were polymeric or oligomeric hydroxyl metal cations formed by hydrolysis of metal salts. Upon calcinations, dehydration and dehydroxylation occured from these metal hydroxyl cations and the structural OH groups, thus formed stable metal oxide pillars to keep the clay layers apart and create interlayer and interpillar spacings of molecular dimension (Hutson et al., 1999). PILCs possessed abound of terminal silanol groups and aluminum hydroxyl on the surface of PILCs, which should increase the hydrophobicity of PILCs and facilitate immobilization of enzymes via hydrogen bonding and other forces. However, studies on enzyme immobilized on PILCs were almost nonexisting in the open literature. We selected Al-PILC as the support for immobilization of HRP due to its easy of preparation. As it was known, during the enzymatic treatment, the interaction of phenoxy radicals with the active center of the enzyme and the entrapment of HRP by polymers which generated in the catalytic cycle leaded to the relatively short catalytic lifetime of the enzyme, which was attributed to the inactivation of the enzyme (Klibanov et al., 1983; Nakamoto and Machida, 1992; Baynton et al., 1994). However, several researchers had suggested that highly hydrophilic additives such as polyethylene glycol (PEG) and gelatin could be used to protect the enzyme from inactivation during phenol removal (Nakamoto and Machida, 1992; Wu et al., 1993; Cooper and Nicell, 1996; Wu et al., 1998). Moreover, PEG was a nontoxic organic compound, which was fit for human consumption and had relatively low environmental impact. It might have a greater affinity for the phenoxy radicals than HRP (Cooper and Nicell, 1996). Thus, the presence of PEG could increase the lifetime of the expensive enzyme, therefore, increase the potential economic feasibility of the enzymatic process. Nakamoto and co-workers had declared PEG with molecular weight in excess of 1000 g/mol was most effective in protecting the enzyme (1992). PEG with different average molecular

40 (2006) 283– 290

weight had been selected by different research groups. In our study, we used PEG with molecular weight of 4000 g/mol. In this study, we synthesized Al-PILC to immobilize HRP and characterized these complexes. Specifically, phenol was used as model substrate to assess the performance of immobilized enzyme and the protective effect of PEG on activity of immobilized enzyme. In addition, we quantified the appropriate amounts of immobilized HRP and PEG required to achieve a high level of phenol removal, and studied the conditions of the emzymatic reaction such as pH, the concentration of H2O2 and phenol. Storage stability and changes in the enzyme activity after repeated use were also investigated.

2.

Experimental

2.1.

Materials

HRP (E.C.1.11.1.7, A4250 units=mg) was purchased from Yuan Ju Bio-Tech Co. Ltd. (Shanghai, China). The desiccated enzyme was stored at
4 1C until used. Na-montmorillonite was obtained from Sigma Chemical Co. (St Louis, MO). All other chemicals used in the work were of analytical grade and were used without further purification, aqueous solutions were prepared with deionized water. Diluted solution of H2O2 was prepared daily.

2.2.

Methods

2.2.1.

Synthesis and characterizations of Al-PILC

The starting montmorillonite was suspended in water in a 1.0 wt% concentration for 24 h. A 0.2 M AlCl3 solution treated with a 0.2 M NaOH solution as hydrolyzing agent in an OH/Al molar ratio of 2.4 at 353 K was vigorously stirred to form the Keggin ion of [Al13O4(OH)24(H2O)12]7+, and then aged for 24 h at 303 K. The solution was used as Al-pillaring agent and was added drop-wise to the clay suspension with agitation for 8 h. The mixture was allowed to age for 24 h at 303 K. The slurry was repeatedly filtrated and suspended using fresh deionized water untill chloride free tested by 0.1 M AgNO3 solution. The solid was air dried at 333 K for 6 h. The resultant material was called Al-crosslinked clay (Al-CLC). The sample was ground, sieved (200 meshes) and calcined at 773 K for 2 h before being used as the support for immobilizing enzyme. And the final product was identified as Al-PILC. Al-PILC provided useful properties such as porosity, large specific surface area and weak acidity for embedding the enzymes. To investigate the microstructures of the matrix, X-ray diffraction was experimented using a Rigaku D/max-rB powder diffractometer with CuKa radiation at a speed of 41/ min and a step size of 0.021. The specific surface area of the matrix, which was measured applying the BET method by N2 adsorption at 77 K in a Beckman coulter SA 3100 instrument, was 183.39 m2/g, while that of the starting clay was 37.30 m2/ g. FT-IR spectrum were generally obtained at ambient temperature using a Bruker Vector 22 spectrometer. The samples were prepared using the standard KBr disk method.

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2.2.2.

Enzyme activity measurements

HRP enzyme activity was tested according to the method of Worthington (Decker, 1977). This assay used phenol, 4aminoantipine (4-AAP) and H2O2 as substrates. The assay solution contained 1.4 mL mixture of phenol and 4-AAP of which concentrations were 16.2 and 0.5 mg/mL respectively, 1.5 mL 1.96 mM H2O2 prepared with 100 mM sodium phosphate buffer pH 7.0, and 0.1 mL 0.05–0.25 U/mL enzyme solution. The rate of reaction was proportional to the concentration of active enzyme, which could be measured by monitoring the rate of formation of the colored nonprecipitating products that absorbed light at wavelength of 510 nm. Color development and absorbance were tested using a Shimadzu UV-2550 UV-visible spectrophotometer (wavelength range 200–900 nm with a 1 nm resolution) and distilled water was used as reference. The optical pathlength of quartz cuvettes were 1 cm. One unit of activity (U) was defined as the amount of enzyme required hydrolyzing 1 mmol H2O2 converted per minute at 25 1C and pH 7.0.

2.2.3.

Immobilization of HRP on Al-PILC

The immobilization of enzyme on Al-PILC was carried out as follows, about 0.8 g Al-PILC was added to 100 mL 2.5 U/mL HRP solution in a vessel in the absence of buffer, open to the atmosphere. The mixture was agitated using magnetic stirrers and Teflon-coated stir bars at room temperature for 120 min. After that, suspension was filtered and washed with deionized water for five times. The enzyme activity of the supernatant and washing solution was measured using the method of Worthington as described above. The amount of immobilized HRP was calculated by the difference of the concentration of the enzyme before and after adsorption, from which it was estimated that the immobilization yield of the enzyme was more than 99%.

2.2.4.

Catalytic reaction experiment

Batch reactor consisted of a vial containing 50 mL of a mixture of aromatic substrate, additive (PEG), and immobilized enzyme, and been covered to prevent evaporation. H2O2 used as oxidant was added to initiate the reaction. Generally, the reaction was stirred vigorously using magnetic stirrer and Teflon-coated stir bar and allowed to proceed in the absence of buffer for 4 h at room temperature. At the end of the reaction, no leaching of HRP was found by means of immediately measuring the enzyme activity of the supernatant. Then, the pH value of the solution was adjusted to less than 2 by addition of 1:1 H2SO4 solution to stop reaction.

2.2.5.

Analysis of the concentration of phenol

The concentration of phenol was determined according to the assay reported by Wu et al. (1998). The assay contained 2.0 mL of sample or sample diluted with distilled water and 0.25 mL of two reagents: potassium ferricyanide reagent (83.4 mM K3Fe(CN)6 in 0.25 M NaHCO3) and 4-AAP reagent (20.8 mM 4AAP in 0.25 M NaHCO3). The extent of color generation at 510 nm after 10 min versus reagent blank was proportional to the total concentration of phenol. Total phenol concentration was determined from a calibration line based on pure phenol.

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3.

Results and discussions

3.1.

Characterization of support

XRD patterns of the five clay samples were illustrated in Fig. 1, exhibiting a first-order peak in the region of 2yo101 and revealing the main structural differences between the untreated, crosslinked and pillared clay. The parent montmorillonite showed a relatively intense XRD peak at 2y ¼ 7:201 ˚ after desiccation at 333 K for 6 h with basal spacing of 12.267 A ˚ after calcinaand at 2y ¼ 9:141 with basal spacing of 9.668 A tion at 773 K for 2 h. This was due to the loss of interlayer water. After pillaring, a shoulder reflection appeared around ˚ due to the successful 2y ¼ 4:501 with basal spacing of 19.621 A 7+ intercalation of Al7+ 13 (abbreviation of [Al13O4(OH)24(H2O)12] ). A small shift of peak to 2y ¼ 4:661 with basal spacing of ˚ after calcination at 773 K for 2 h resulted from 18.947 A conversion of Al7+ 13 to meatl oxide pillars. The intensity of the d001 peak increased, which showed the montmorillonite could obtain ordered and oriented silicate layer structure after thermal treatment. Mokaya and Jones reported the calcination temperature range between 400 and 500 1C was crucial. In this temperature range, dehydroxylation occurs from the pillars and from the structural OH groups (Mokaya and Jones, 1995). The remaining aluminum atoms on the pillars existed in a variety of coordination states. Most of these sites possessed imcompletely coordinated aluminum. On the other hand, Al7+ 13 species decomposed to yield protons. The produced protons migrating into the clay octahedral layer increased the hydrophobicity of the PILC. After immobilization of HRP on Al-PILC, no change appeared in the XRD pattern. The average molecular diameter of HRP was about ˚ . The rough dimensions of HRP were far larger than the 48 A basal spacing of the support, which made HRP diffuse into the Al-PILC galleries impossible. Abundant silanol groups and weakly acidic hydroxyl groups existed on the surface and interlayer pillars after pillaring and calcinations (Mokaya and Jones, 1995). Mostly HRP inhabited on these sites on the external surfaces and at the edges of the interlayer sheets

Al-PILC after immobilization of HRP Al-PILC Al-CLC Original clay (773K) Original clay (333K)

Relatively intensity (a.u.)

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2.5

10

20 2θ degree

30

40

Fig. 1 – XRD patterns of original montmorillonite (333 K and 773), Al-CLC, Al-PILCs before and after immobilization of HRP.

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through hydrogen bonding, Van Der Waals and electrostatic force interactions with hydrophilic residues of the enzyme without reduction of activity of enzyme. Entrapment of the enzymes through silanation prevented enzyme loss into solution. The interaction between Al-PILC and HRP was examined by Fourier transform infrared as shown in Fig. 2. The absorption band at 3631 cm
1 was correlated with stretching vibration of free hydroxyl groups, which indicated that there were an abundance of free hydroxyl groups on the surface of Al-PILC. After immobilization of HRP, the band nearly disappeared, and the intensity of adsorption bands associated with frame asymmetric and symmetric flexible vibrations (799 and 1054 cm
1) decreased, suggesting that there might be intermolecular interaction between enzyme and some specific sites of matrix, and apparently the Al-PILC was a good immobilization matrix for enzyme loading. HRP had specific adsorption bands at 1541 and 1450 cm
1 corresponding to –NH2 and
CH2 deformation vibrations, but these specific adsorption bands did not exist in the IR spectra of Al-PILCHRP obviously, which resulted from the low content of HRP in the complex (mass ratio of HRP/PILC less than 1%).

3.2.

Effect of PEG concentration

PEG could improve the efficiency of phenol removal by the meaning of formation protection layer in the vicinity of the active centers of HRP to restrict the attack of free phenoxy radicals formed in the catalytic cycle. On the other hand, PEG had a greater affinity with the polymer products than the enzyme, most of the polymers was preferentially coupled with PEG so that the enzyme was prevent from being adhered and entrapped by precipitation products (Nakamoto and Machida, 1992; Wu et al., 1998). The apparent inactivation of enzyme was alleviated and the useful life of enzyme could be extended. The mechanism had been illustrated in detail in literatures (Nakamoto and Machida, 1992; Wu et al., 1993, 1998). To access the effectiveness of PEG as an additive to enhance immobilized enzyme performance during the treat-

Transmittance [%]

a b

c

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100 Efficiency of phenol removal (%)

286

500

0

Wavenumber cm-1 Fig. 2 – FT-IR spectrum of (a) Al-PILC, (b) free HRP and (c) immobilized HRP on Al-PILC.

80 70 60 50 40 30 20 0.0

0.3 0.6 0.9 1.2 Mass ratio of PEG/phenol

1.5

Fig. 3 – Effect of PEG on the efficiency of phenol removal as a function of initial phenol concentrations. Symbols: (’) initial phenol concentrations of 105.5 mg/L; (K) initial phenol concentrations of 253.5 mg/L. The reactions were allowed to proceed for 4 h with [H2O2]/[Phenol] molar ratio of 1.0 and immobilized HRP dose of 0.4 U/mL.

ment of phenolic solution and to quantify the optimum amount of PEG to achieve specific levels of treatment, effect of the amount of PEG on the phenol removal efficiency as a function of phenol concentrations was studied as shown in Fig. 3. As for both phenol concentrations, the phenol removal efficiencies were only around 20% in the absence of PEG. However, they were significantly enhanced with the increase of the amount of PEG added before the mass ratio of PEG/ phenol up to 0.4, then gradually reach the maximum values as the mass ratio of PEG to phenol near to 1.0. The further addition of PEG would result in slightly reduction of the phenol removal efficiency, which was in correspondence with the result reported by Wu et al. (1997). The relationships indicated the PEG requirements were directly linked to the total amount of phenol in the solution. The concentration of phenol being high, the amount of phenoxy radicals and polymers increased, the addition of PEG should be enlarged enough to disperse them, as confirmed by Nakamoto and Machida (Nakamoto and Machida, 1992). From the results expressed above, the improvement of the phenol removal efficiency was negligent when the mass ratio exceeded 0.4. Moreover, the phenol removal efficiencies were over 92% at this time. In account of the concentration of organic compound in the drainage and the treatment cost, the PEG requirement in the further experiments depended on the mass ratio of PEG/phenol being about 0.4.

3.3.

4000 3500 3000 2500 2000 1500 1000

90

Effect of immobilized HRP dose

Figure 4 illustrated the dependence of phenol removal efficiency over a range of immobilized HRP dose in terms of two concentrations of phenol. It was showed clearly that immobilized HRP could catalyze oxidation of phenol effectively. The mechanism of oxidation was as the same as that of free enzyme as documented in many reports (Dura´n and Esposito, 2000; Klibanov and Morris, 1981). In order to

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100 Efficiency of phenol removal (%)

Efficiency of phenol removal (%)

100 80 60 40 20 0 0.0

95

90

85

80 0.2

0.4

0.6

0.8

1.0

4

Immobilized HRP dose (U/mL) Fig. 4 – Effect of immobilized HRP dose on the efficiency of phenol removal as a function of initial phenol concentrations. Symbols: (’) initial phenol concentrations of 105.5 mg/L; (K) initial phenol concentrations of 253.5 mg/L. The reactions were allowed to proceed for 4 h with [H2O2]/[Phenol] molar ratio of 1.0 and PEG/phenol mass ratio of 0.4.

examine the influence of the support on phenol removal, the experiments with the same conditions expect using Al-PILC (about 10 g/L) instead of immobilized enzyme as catalyst were done. The results revealed that phenol removed by Al-PILC via adsorption and catalysis was negligent. Therefore, the phenol removals were attributed to immobilized HRP. When the immobilized enzyme dose was 0.1 U/mL for phenolic solution of 105.5 mg/L, 0.2 U/mL for that of 253.5 mg/L, the phenol removal efficiencies were around 80%. That was to say, one unit of immobilized HRP could remove one milligram of phenol. The result was comparable with the conclusion drawn by Klibanov et al. that one molecule of peroxidase (MW ¼ 44 KDa for HRP) could remove approximately 103 molecules of phenol (Klibanov et al., 1983; Nicell and Wright, 1997). The residual phenol could be eliminated completely by increasing the dose of the immobilized enzyme. However, the quantity of phenol removed per unit amount of immobilized HRP diminished. The price of commercially available HPR was very high. The cost of enzyme had always been the bottleneck of application of enzymatic process on the treatment of wastewater. Therefore, in practice, the reasonable dose of immobilized enzyme should be applied to obtain higher catalytic efficiency as soon as possible.

3.4.

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Effect of pH

Nicell et al. reported the optimum working pH value of free HRP for phenolic compounds was from 6 to 9 (Nicell et al., 1992). In order to determine that of immobilized HRP, a series of experiments were performed in the presence of buffer of different pH value as shown in Fig. 5. The appropriate operating pH shifted to the range between 4.5 and 9.3 as a consequence of immobilization. The microenvironment effect could explain the phenomenon. The presence of protonated, un-reacted Si–OH on the surface of Al-PILC might repel protons from the region in the vicinity of the surface and

5

6

7 pH

8

9

10

Fig. 5 – Effect of pH value on the efficiency of phenol removal. The reactions were allowed to proceed for 4 h in the presence of buffer with immobilized HRP dose of 0.4 U/mL, [H2O2]/[Phenol] molar ratio of 1.5 and PEG/phenol mass ratio of 0.4.

create a higher pH at the boundary layer between the support and the bulk solution and thus lead to the lowering of apparent optimal pH. The microenvironment of the enzyme attached to the surface of the support had been found to be quite different from that of the bulk solution. The real wastewaters had pHs in the range of slightly acidic to slightly basic. The optimum pH range of immobilized enzyme was benefit to apply it in the real industrial wastewater. This might decrease costs associated with the initial pH adjustment of wastewaters, corrosion of hardware during treatment and pH neutralization of wastes prior to their release. Since the pH value of the reaction solution without addition of buffer was around 7 and in the middle of the optimum working range of immobilized HRP. Based on these results, all experiments could be performed in the absence of buffer.

3.5.

Effect of H2O2 concentration

The removal efficiencies of phenol at different level of experiments involving a range of H2O2 to phenol molar ratio as a function of initial phenol concentration were investigated as shown in Fig. 6. In these experiments, the dose of immobilized HRP was fixed at 0.4 U/mL phenolic wastewater. The amount could ensure that the conversion of phenols was mainly limited by the availability of the H2O2. The gradual addition of the H2O2 could lead to the lower possibility of enzyme inactivation (Wagner and Nicell, 2002), therefore, the experiments were conducted by drop-wise addition of at most 2.0 mL 0.30% H2O2 at a time every period of 30 min until to the different designed amount. From the initiation of the first addition of H2O2, the reactions were allowed to proceed for 4 h. All samples were separated after termination of these reactions, and these clarified solutions were allowed to stay overnight with addition of a small quantity of MnO2 to remove the excessive H2O2. A conclusion could be made according to the experimental results. When the molar ratio of H2O2 to phenol was less than 1.0, as for the two phenolic solutions of different concentration, the quantity of H2O2

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100 Efficiency of phenol removal (%)

Efficiency of phenol removal (%)

100 80 60 40 20 0 0

1

2

3

4

5

6

7

90 80 70 60 50 40

8

0

Molar ratio of H2O2 /phenol Fig. 6 – Effect of H2O2 concentration on the efficiency of phenol removal as a function of initial phenol concentrations. Symbols: (’) initial phenol concentrations of 105.5 mg/L; (K) initial phenol concentrations of 253.5 mg/L. The reactions were allowed to proceed for 4 h with immobilized HRP dose of 0.4 U/mL and PEG/phenol mass ratio of 0.4.

200

400

600

800

1000

1200

Phenol concentration (mg /L) Fig. 7 – Effect of phenol concentration on the efficiency of phenol removal. The reactions were allowed to proceed for 4 h with immobilized HRP dose of 0.4 U/ mL, [H2O2]/[Phenol] molar ratio of 1.5 and PEG/ phenol mass ratio of 0.4.

exerted direct control on the extent of phenol transformation proportionally. Subsequently, the removal efficiencies of phenol increase slightly with increase of peroxide and reached the maximal value with almost 1.5 equivalent of H2O2 to phenol. Whereas W. Hewson and co-workers reported that the theoretical stoichiometry of H2O2/phenol ratio was 0.5 for peroxidase (Hewson and Dunford, 1976), Nicell reported the optimum molar ratio of H2O2 to phenol was 1.0 (Nicell et al., 1992), and that of 2.0 given in another previous study (Klibanov et al., 1983; Liu et al., 2002). The deviation might result from polymers produced in the catalytic process larger than dimmers (Klibanov et al., 1983; Nicell et al., 1992). The optimum range of H2O2 to phenol molar ratio could defined from 1.0 to 1.5 with minor discrepancy of phenol removal efficiency, which was benefical to accommodate the fluctuating phenol concentrations in treatment of actual wastewaters. The addition of the H2O2 must be limited as the inhibition of peroxidase activity by excess quantities of peroxide (Nicell and Wright, 1997), which was in agreement with our study.

3.6.

Efficiency of phenol removal (%)

100 90 80 70 60 50 40 0

50

100

150

200

250

300

Reaction time (min) Fig. 8 – The time dependence of dephenolization as a function of the initial phenol concentrations. Symbols: (’) initial phenol concentrations of 105.5 mg/L; (K) initial phenol concentrations of 253.5 mg/L. The reactions were allowed to proceed with [H2O2]/[Phenol] molar ratio of 1.5, immobilized HRP of 0.4 U/mL and PEG/phenol mass ratio of 0.4.

Effect of phenol concentration

Phenolic pollutant concentration had been reported in the range of trace quantities to hundreds of milligrams per liter (Wu et al., 1993). Different initial concentrations of phenol were examined to the catalytic performance of immobilized HRP from low to high pollution concentration. The amounts of immobilized enzyme and PEG in all experiments were fixed at 0.4 U/mL and 150 mg/L, respectively, and the total volume of reaction solution was 50 mL. Reactions were allowed to go to completion by providing a reaction period in excess of 4 h. The results were shown in Fig. 7. The relative lower phenol removal efficiencies in the case of phenol concentrations less than 300 mg/L might be as a consequence of restraint from excessive PEG. The phenol removal efficiency significantly

reduced with the further increase of phenol concentration because of the disappearance of HRP activity. The immobilization of HRP on Al-PILC could decrease the chances of collision between enzyme’s active sites and phenoxy radicals and possibility of entrapment by polymers formed during the catalytic cycle. As a result, the immobilized HRP could keep active in the high concentration of phenol. When the initial concentration of phenol increased from 50 to 1200 mg/L, the amount of phenol transformation catalyzed by 20 U immobilized HRP was 26 mg at most. This enzymatic treatment process could be found its application to remove the bulk of the phenolic compounds in the industrial wastewater as a pre-treatment process, for example, a biological treatment system.

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100 Efficiency of phenol removal (%)

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80 60 40 20

90 80 70 60 50 40 30

0 1

2

3

0

4

10

20

Cycles Fig. 9 – Performance of the immobilized HRP in repeatedbatch treatment of phenol removal. The reactions were allowed to proceed with [H2O2]/[Phenol] molar ratio of 1.5 and PEG/phenol mass ratio of 0.4. In the first batch, the dose of immobilized enzyme was 0.4 U/mL of 253.5 mg/L phenolic wastewater.

3.7.

Effect of reaction time

The time course of phenol removal was investigated as a function of the assay phenol concentration as shown in Fig. 8. In the case of different concentration of phenol, phenol rapidly decomposed in the initial step, around 50% of phenol disappeared in first 20 min when most of enzyme was still active. After a period of 90 min, the removal reaction was followed by very slow removal process. This slowdown could be attributed to the simultaneous decrease in the concentration of all the reacting species (phenol, H2O2) and activity of immobilized enzyme. The enzymatic reaction could be greatly accelerated by increase in HRP concentration and introducion of sonication reported by Entezari and Pe´trier (2004). Since the total treatment cost depended on the cost of the enzyme, the compromise between reaction time and immobilized enzyme should be optimized for the treatment.

3.8.

Reusability

Unlike free enzyme, immobilized enzyme could be easily separated from reaction solution and reused. To demonstrate the reusability of HRP immobilized on Al-PILC, the immobilized enzyme was recovered by filtration after each batch and rinsed with deioned water for the subsequently batch. For each batch, the reaction was allowed to proceed for 3 h. The efficiency of phenol removal reduced with the cycle of the immobilized enzyme as shown in Fig. 9. After four times of repeated tests, the immobilized HRP deteriorated and the efficiency of phenol removal decreased to 4.94%. It resulted from the fact that the activity of the immobilized enzyme fell off rapidly when reused. The polymers produced during the enzymatic reaction covered on the immobilized enzyme and entrapped the active sites of enzyme. If the molecular weight of the polymers could be reduced so that no sedimentation appeared or the polymers covered on enzyme–clay complex could be dissolved in organic solvent after reactions, the

30

40

50

60

70

Storage time (day) Fig. 10 – Efficiency of phenol removal in terms of storage time of immobilized enzyme. The reactions were conducted in 50 mL solutions of 253.5 mg/L initial phenol with [H2O2]/[Phenol] molar ratio of 1.5 and PEG/phenol mass ratio of 0.4.

reusability would be improved. The study on the subject was in progress and would be reported in another paper.

3.9.

Storage stability

Al-PILC immobilized HRP separated from suspension was immediately sealed into a glass vessel without dessication and stored in refrigeratory of 4 1C. To examine the storage stability of immobilized HRP, 20.0 U immobilized enzyme of original enzyme activity was withdrew every a period of certain time to carry out the catalytic oxidation of phenol under the optimum reaction conditions. The results were presented in Fig. 10. During 2 weeks of storage time, the efficiency of phenol removal that represented the enzyme activity remained almost constant. After 5 weeks of storage, the efficiency of phenol removal retained 81.00%, and reduced gradually to 35.94% as the storage time extended to 8 weeks. Putter and Becker reported that HRP lost about 10% of its enzymatic activity daily at room temperature or at 4 1C (Pu¨eeter and Becker, 1982). This process appeared to be decelerated drastically when the enzyme was bound on Al-PILC.

4.

Conclusions

HRP had been successfully immobilized on the Al-PILC via interaction between functional groups of support and the enzyme. Compared with free enzyme, the immobilized enzyme could be applied over a broader range of pH from 4.5 to 9.3 and had a better storage stability. In the presence of H2O2, the immobilize HRP exhibited good phenol removal over a wide range of phenol concentration between tens and hundreds of milligrams per liter, which could be expected in industry. PEG could improve the enzymatic treatment efficiency of synthetic wastewater significantly. When the mass ratio of PEG/phenol and the molar ratio of H2O2/phenol in batch reaction system were 0.4 and 1.5, respectively, 20.0 U

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immobilized HRP could rapidly decrease the phenol concentration within short retention time. Further investigations on improvement of reusability and application of the immobilized enzyme in a continuous-flow device would be in process.

Acknowledgement This study is in part supported by a Grant from the project of the youth science and technology innovation groups of HFUT. R E F E R E N C E S

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