JP2580539B2 - How to remove phenols in water - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an improved method for removing phenols in water. More specifically, the present invention suppresses enzyme inactivation by oxidizing phenols in water with immobilized peroxidase obtained by adsorbing peroxidase from the aqueous solution to particulate magnetite and hydrogen peroxide. The present invention also relates to a method for removing phenols in water, in which oxidation products of phenols are adsorbed and removed by an immobilized enzyme.

[0002]

2. Description of the Related Art Various phenols are contained in wastewater from a chemical plant, a coke plant, or a heavy oil cracking treatment process. Since these phenols cause environmental pollution, strict standards are set for their content when discharging wastewater into rivers.

Conventionally, as a method for treating wastewater containing phenols, for example, an adsorption method, an extraction method, an ion exchange method, an oxidation method, a biological treatment method, and the like are known. Practically biological treatment method (activated sludge method)
Only has been done. However, even in this biological treatment method, phenols have low biodegradability and are toxic, so their biological activity is inhibited, and the treatment is not performed sufficiently. There are many. Therefore, development of a method for easily and efficiently treating phenols in wastewater at low cost is desired.

As an inexpensive and simple method for treating phenols, a method utilizing the catalytic action of an enzyme is considered, and various methods utilizing an enzyme catalyzing the oxidation reaction with hydrogen peroxide, such as peroxidase, have been considered. Have been tried. However, this method has drawbacks in that enzymes such as peroxidase bind to and inactivate oxidation products of phenols, and the oxidation products remain dissolved in water and cannot be removed.

On the other hand, a method is known in which magnetite is used as a carrier and various enzymes are immobilized on the carrier. However, this immobilization requires multiple steps involving complicated operations of first treating magnetite with 3-aminopropyltriethoxysilane, then reacting with glutaraldehyde, and then binding an enzyme thereto. Therefore, it cannot always be said that the method is industrially practiced.

[0006]

SUMMARY OF THE INVENTION The present invention overcomes the drawbacks of the conventional methods for treating phenols in water using such enzymes, suppresses enzyme deactivation, It is an object of the present invention to provide a method for immobilizing an enzyme by a simple means so that oxidation products of a kind can be easily removed from water.

[0007]

The present inventors have conducted various studies on immobilization of an enzyme. As a result, when peroxidase is used as an enzyme, the enzyme can be easily adsorbed on magnetite simply by adsorbing it from an aqueous solution thereof. When used in combination with hydrogen peroxide to oxidize phenols in water, it is less inactivated than conventional immobilized enzymes, and is superior in adsorbing oxidation products. This led to the present invention based on this finding.

[0008] That is, the present invention provides a method for removing phenols in water by oxidation treatment with peroxidase and hydrogen peroxide, wherein the peroxidase is adsorbed from an aqueous solution thereof to powdered magnetite and immobilized. And a method for removing phenols in water.

The phenols that can be removed by the method of the present invention include various hydroxyl-containing aromatic compounds.
For example, there are phenol, chlorophenol, methoxyphenol, cresol, hydroxyphenol and the like. The concentration of these phenols is 0.001.
It is preferably in the range of about 10 to 10 mM.

In the method of the present invention, peroxidase (EC 1.1.1.7) is used as the enzyme. This peroxidase is an enzyme that catalyzes a reaction generally represented by H ₂ O ₂ + AH ₂ → 2H ₂ O + A (AH ₂ : substrate), and is used for animals, plants,
It is widely distributed in the microbial kingdom, especially in horseradish. In the present invention, this peroxidase is used as a catalyst for oxidizing phenols in the presence of hydrogen peroxide. The origin of the peroxidase used in the present invention is not particularly limited, and may be any of animals, plants, and microorganisms.

In the method of the present invention, it is necessary to use the peroxidase immobilized on powdered magnetite. The particulate magnetite i.e., the average particle diameter of iron oxide black (Fe ₃ O ₄₎ is 0.1~1000μm
Is suitable.

[0012] The immobilization of peroxidase on magnetite is carried out, for example, by adding 0.001 to 10 g of peroxidase to the magnetite.
Magnetite is suspended in an aqueous solution containing a concentration of about 1 liter at a ratio of about 0.001 to 1000 g / liter, and then stirred at a temperature of usually 0 to 50 ° C.
Peroxidase is adsorbed on magnetite, then
It is advantageous to carry out the process by recovering magnetite according to a conventional method, since the operation is simple, no special chemicals need to be used, and an immobilized peroxidase with high removal efficiency can be obtained.

In the present invention, phenols in water are oxidized using the thus obtained immobilized peroxidase and hydrogen peroxide. As a method of the oxidation treatment, for example, a method in which immobilized peroxidase is dispersed in the water to be treated and hydrogen peroxide is added, or the column is filled with the immobilized peroxidase, and the water to be treated is passed along with hydrogen peroxide. A method of liquefying can be used.

The temperature in the oxidation treatment is preferably in the range of 10 to 50 ° C., and the pH is usually 4 to 9, preferably 4 to 9.
~ 7. Since the amount of the immobilized peroxidase depends on the concentration of the phenol, it is preferable to conduct a preliminary test in advance and determine the amount. However, the concentration of the phenol is 0.001 to 10 mM, and the concentration of the enzyme is 0.01 to 100 mg. Per liter is preferred. The amount of hydrogen peroxide used is usually 1 phenol.
It is selected in the range of 0.5 to 1.5 equivalents to the equivalents. This hydrogen peroxide may be initially added in its entirety, may be sequentially added little by little, or may be generated in the system by a chemical reaction or the like.

In the case of phenols which are hardly oxidized with hydrogen peroxide by an immobilized peroxidase catalyst, an auxiliary agent is added to promote the reaction, and the oxidation product is polymerized with the phenols. You may. At this time, phenol or the like can be used as an auxiliary agent to be added.

In the case where the immobilized peroxidase is suspended and oxidized in the water to be treated, after the oxidation, the immobilized peroxidase to which the oxidation product of phenols is adsorbed is separated and removed. The separation and removal method is not particularly limited, and may be, for example, separation by magnetic force, removal by precipitation, or removal by filtration, centrifugation, flotation, or the like.

[0017]

According to the present invention, oxidization of phenols in water with immobilized peroxidase obtained by adsorbing peroxidase from its aqueous solution to magnetite and hydrogen peroxide suppresses peroxidase inactivation. At the same time, the oxidation products of the phenols readily bind to the immobilized peroxidase and are completely removed. In addition, if the enzyme adsorption method is used, there is no need to use expensive chemicals, and the immobilization can be performed by a simple operation.
In addition, since magnetite is used as the carrier, the separation of the treatment solution after the reaction and the immobilized enzyme is extremely easy, so that the processing cost can be greatly reduced as compared with the conventional method.

The method for removing phenols in water according to the present invention comprises:
Because of these excellent characteristics, it is extremely practical and can be suitably used for, for example, water treatment, industrial water treatment, wastewater treatment, waste leachate treatment, and the like.

[0019]

Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Reference Example 1 Magnetite powder having an average particle size of about 5 μm (manufactured by Hanoi Chemical Co., Ltd.)
To 50 mg, 25 ml of an aqueous solution of peroxidase (manufactured by Wako Pure Chemical Industries, wasabi origin) was added, and the mixture was stirred at 20 ° C. for 15 hours to separate magnetite. The peroxidase concentration of the aqueous solution after separation was measured, and the adsorption equilibrium was determined. The amount of adsorption at an equilibrium concentration of 1 mg / liter was 8.5 mg.
/ G. The adsorption from tap water and aqueous sodium chloride solution (10 mM) was almost the same.

Reference Example 2 Magnetite powder having an average particle size of about 5 μm (manufactured by Hanoi Chemical Co., Ltd.)
1 g peroxidase (Wako Pure Chemical Industries, wasabi origin)
Add 3 ml of an aqueous solution having a concentration of 0.33 mg / ml, and add
, And washed with water. When the enzyme concentration in this wash water was measured and the yield of immobilization was determined,
The activity was about 99%, and the activity yield was about 100% [hereinafter referred to as immobilized enzyme (A)]. This immobilized enzyme was hardly desorbed by water, was stable at room temperature, and retained its activity at 100% after 3 weeks.

Reference Example 3 Peroxidase was immobilized by a carrier cross-linking method using glutaraldehyde, which is well known as an enzyme immobilization method.

1 g of magnetite powder having an average particle size of about 5 μm
2 ml of a 0.1% by weight aqueous solution of 3-aminopropyltriethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) was added thereto, and the mixture was reacted at 45 ° C. for 15 hours to carry an amino group, followed by washing with water. Then, 0.1 ml of a 5% by weight aqueous solution of glutaraldehyde (a 25% by weight aqueous solution, manufactured by Hanui Chemical Co., Ltd.) was added thereto, reacted for 3 hours, and washed with water. This was combined with a 0.3 mg / ml aqueous solution of peroxidase 0.3
After adding 3 ml and stirring at 20 ° C. for 15 hours, the mixture was washed with water. The enzyme concentration in the washing water was measured and the immobilization yield was determined to be about 99%, and the activity yield was about 1
00% [hereinafter referred to as immobilized enzyme (B)].

Example 1 A phenol oxidation experiment was performed using the immobilized enzyme (A) obtained in Reference Example 2, the immobilized enzyme (B) obtained in Reference Example 3, and dissolved peroxidase.

A reaction solution (pH 7.0) containing 1 mM of phenol, a predetermined concentration of each peroxidase, 1.2 mM of hydrogen peroxide and 10 mM of a buffer solution was stirred at room temperature.
The phenol concentration after one minute was measured and the removal rate was determined. The reaction-terminated liquid was centrifuged at 3000 rpm for 10 minutes, and the absorbance of the separated liquid was measured at 400 nm.

As a result, the phenol removal rate at an enzyme amount of 4 mg / liter was almost 100% for the immobilized enzyme (A),
It was 95% for the immobilized enzyme (B) and 60% for the lytic enzyme. In the case of the immobilized enzyme, the separated solution after the reaction was colorless and transparent.

On the other hand, in the lytic enzyme, phenol
The amount of the enzyme required to be removed was 20 mg / liter, and the reaction-terminated liquid had an absorbance of 400 nm of 0.1, was brown, and had no precipitate.

Example 2 Using the immobilized enzyme (A) obtained in Reference Example 2, the immobilized enzyme (B) obtained in Reference Example 3, and dissolved peroxidase, an oxidation experiment of o-chlorophenol was performed. Was.

1 mM o-chlorophenol, a predetermined concentration of each peroxidase, 1 mM hydrogen peroxide and 10 mM buffer
The reaction solution (pH 7.0) containing mM was stirred at room temperature, and the o-chlorophenol concentration after 30 minutes was measured to determine the removal rate. In addition, the reaction completed solution was rotated at 3000 rpm.
m, and centrifuged at 10 m for 10 minutes, and the absorbance of the separated solution at 400 nm was measured.

As a result, when the enzyme amount was 2 mg / liter, o-
The chlorophenol removal rate was almost 1 for the immobilized enzyme (A).
00%, 90% for immobilized enzyme (B), 48% for lytic enzyme
Met. In the case of the immobilized enzyme, the separated solution after the reaction was colorless and transparent.

On the other hand, in the case of the lytic enzyme, an enzyme amount of 4 mg / liter is required to remove 100% of o-chlorophenol, and the reaction-terminated liquid has an absorbance of 400 nm of 1.
No. 2 and no precipitate.

Example 3 Using the immobilized enzyme (A) obtained in Reference Example 2, the immobilized enzyme (B) obtained in Reference Example 3, and dissolved peroxidase, an oxidation experiment of m-chlorophenol was performed. Was.

1 mM of m-chlorophenol, each peroxidase at a predetermined concentration, 1 mM of hydrogen peroxide and 10 mM of buffer
The reaction solution (pH 7.0) containing mM was stirred at room temperature, and the concentration of m-chlorophenol after 30 minutes was measured to determine the removal rate. In addition, the reaction completed solution was rotated at 3000 rpm.
m, and centrifuged at 10 m for 10 minutes, and the absorbance of the separated solution at 400 nm was measured.

As a result, when the amount of enzyme was 4 mg / liter, m
-The chlorophenol removal rate was 94% for the immobilized enzyme (A).
%, 85% for the immobilized enzyme (B) and 38% for the lytic enzyme. In the case of the immobilized enzyme, the separated solution after the reaction was colorless and transparent.

On the other hand, in the case of the lytic enzyme, an amount of 20 mg / liter of enzyme was required to remove 100% of m-chlorophenol.

Example 4 Using the immobilized enzyme (A) obtained in Reference Example 2, the immobilized enzyme (B) obtained in Reference Example 3, and dissolved peroxidase, an experiment for oxidizing p-chlorophenol was performed. Was.

1 mM p-chlorophenol, each peroxidase at a predetermined concentration, 1 mM hydrogen peroxide, and 10 mM buffer
The reaction solution (pH 7.0) containing mM was stirred at room temperature, and the p-chlorophenol concentration after 30 minutes was measured to determine the removal rate. In addition, the reaction completed solution was rotated at 3000 rpm.
m, and centrifuged at 10 m for 10 minutes, and the absorbance of the separated solution at 400 nm was measured.

As a result, p at an enzyme amount of 1 mg / liter
-The chlorophenol removal rate was almost 100% for the immobilized enzyme (A) and the immobilized enzyme (B), and 35% for the lytic enzyme. In the case of the immobilized enzyme, the separated solution after the reaction was colorless and transparent.

On the other hand, in the case of the lytic enzyme, an amount of 4 mg / liter of enzyme is required to remove 100% of p-chlorophenol, and the reaction-terminated solution has an absorbance of 400 nm at 0.0 nm.
It was colored at 42.

Example 5 Using the immobilized enzyme (A) obtained in Reference Example 2, the immobilized enzyme (B) obtained in Reference Example 3, and dissolved peroxidase, a phenol oxidation experiment in a short time was carried out. went.

Phenol 1 mM, peroxidase 2 m
g / liter, 1.2 mM hydrogen peroxide and 10 m buffer
Stir the reaction solution (pH 7.0) containing M at room temperature,
After 0.5, 1, 2, 5, 15, and 30 minutes, 0.5
The reaction was stopped by immediately adding 0.5 ml of a 0.8 mg / ml aqueous solution of catalase, and the phenol concentration was measured.

As a result, in the case of the lytic enzyme, the enzyme was inactivated after 2 minutes and the reaction was stopped (removal rate: 26%), whereas in the case of the immobilized enzyme (A), the removal rate was 84.5%, 30% after 15 minutes. After 5 minutes, it was 95.2%, and for the immobilized enzyme (B), it was 73.6% after 15 minutes, and 84.6% after 30 minutes. Also,
No reaction occurred when the immobilized enzyme (A) was boiled for 30 minutes. This indicates that the reaction was an enzyme-catalyzed reaction, and that there was no adsorption of phenol to the immobilized enzyme.

Example 6 Using the immobilized enzyme (A) and dissolved peroxidase obtained in Reference Example 2, an experiment for oxidizing 2,6-dichlorophenol was performed.

0.2 mM 2,6-dichlorophenol,
Peroxidase 2 mg / liter, hydrogen peroxide 0.2
A reaction solution (pH 5.
5) was stirred at room temperature, and the concentration of 2,6-dichlorophenol after 30 minutes was measured to determine the removal rate of 2,6-dichlorophenol.

As a result, the removal rate after 30 minutes was 85% for the immobilized enzyme (A), whereas it was 55% for the lysed enzyme.
%Met. The removal rates after 3 hours were 10
0% and 70%.

Example 7 Using the immobilized enzyme (A) and the dissolved peroxidase obtained in Reference Example 2, an experiment of oxidizing a mixed aqueous solution of chlorophenol was performed.

P-chlorophenol 0.2 mM, 2,4
-Dichlorophenol 0.2 mM, 2,4,5-trichlorophenol 0.1 mM, 2,4,6-trichlorophenol 0.1 mM, 2,3,4,6-tetrachlorophenol 0.02 mM and pentachlorophenol 0 .
Reaction solution containing 01 mM, 2 mg / liter peroxidase, 1 mM hydrogen peroxide and 10 mM buffer (pH
5.5) was stirred at room temperature, and the chlorophenol concentration after 30 minutes was measured to determine the removal rate.

As a result, in the immobilized enzyme (A), the chlorophenol removal rates were 100, 100 and 9 respectively.
For the lytic enzyme, 56.9, 81.3, 41.3, and 9.1, 100, 100, and 100%, respectively.
9.6, 97.6, 92.7 and 92.2%.
The removal rates of total organic carbon (TOC) and activated carbon-adsorbing organic chlorine (AOX) were 85% and 89% for the immobilized enzyme (A), while 54% and 68% for the lytic enzyme.
% Stayed.

Comparative Example A phenol oxidation experiment was performed using magnetite without immobilized peroxidase and dissolved peroxidase. Magnetite 10 mg, phenol 1 mM,
Peroxidase 2 mg / liter, hydrogen peroxide 1.2
Reaction solution containing 10 mM mM and 10 mM buffer (pH 7.
0) was stirred at room temperature, and the phenol concentration of the reaction solution after 30 minutes was measured. The result was 0.74 mM, and the removal rate was only 26%.

From the above results, the immobilized peroxidase (A) obtained by adsorbing peroxidase from its aqueous solution to magnetite shows a far superior effect in each case than the case of dissolved peroxidase. Conventionally, it was found that an effect superior to that of immobilized peroxidase (B) immobilized by a carrier cross-linking method using glutaraldehyde, which is often used as an enzyme immobilization method, was obtained.

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Claims (3)

(57) [Claims] 1. A method for removing phenols in water by oxidation treatment with peroxidase and hydrogen peroxide, wherein the peroxidase is adsorbed from an aqueous solution of the aqueous solution to particulate magnetite and immobilized. 2. An amount of 0.001 to 10 g of peroxidase.
/ Magnetite is suspended in an aqueous solution containing at a concentration of 0.001 to 1000 g / liter in an aqueous solution containing the peroxidase at a temperature of 0 to 50 ° C. until the peroxidase is sufficiently adsorbed on the magnetite. The method according to claim 1, wherein the conversion is performed. 3. An amount of 0.5 to 1.5 equivalents of hydrogen peroxide based on immobilized peroxidase and phenols at an enzyme concentration of 0.01 to 100 mg / liter with respect to water containing phenols at a concentration of 0.001 to 10 mM. The method according to claim 1, wherein the method is performed using: