JPS63152980A - Immobilization of microbial cell - Google Patents

Abstract

PURPOSE:To maintain pH of a reaction system to neutral state during the reaction and keep the activity of microorganism to high level, by polymerizing a water-soluble suspension liquid containing a specific main resin, a crosslinking agent, a polymerization initiator, a pH buffering agent and microbial cells at <=40 deg.C. CONSTITUTION:A water-soluble suspension liquid containing a main resin, a crosslinking agent, a radical polymerization initiator, a polymerization accelerator, a pH buffering agent and microbial cell is prepared beforehand. The liquid is polymerized at a low temperature (<=40 deg.C) to immobilize the microbial cells. The main resin used in the above process is an unsaturated monoester selected from methoxypolyethylene glycol acrylate and methoxypolyethylene glycol methacrylate and the crosslinking agent is polyethylene glycol dimethacrylate which is an unsaturated diester. The polymerization initiator is a persulfate and the polymerization accelerator is dimethylaminopropionitrile.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Natural Application] The present invention relates to a method for producing an entrapping gel of immobilized microorganisms that is useful for improving a method for purifying wastewater by an activated sludge method.

[Conventional technology]

Conventionally, carrier binding methods, crosslinking methods, entrapment methods, etc. are known as methods for immobilizing microbial cells.

The carrier binding method includes a physical adsorption method in which microorganisms are attached to a carrier such as activated carbon, porous glass, kaolinite, or silica gel. In the field of wastewater purification, this method is called the attached microorganism method, and the bonding force between microorganisms and carriers is weak and detachment occurs easily due to factors such as biofilm formation and water flow, so it may be difficult to collect microorganisms. there were. Further, as a carrier binding method, there is a method called an ionic bonding method.

This is a method of ionically bonding bacterial cells to an ion exchange resin such as DBAB cellulose or polyaminostyrene, but depending on the type of salt in the wastewater, the bacterial cells may be ion-exchanged and eluted, so it cannot be used. Among the carrier bonding methods, the covalent bonding method exhibits the strongest bonding force. This method uses amino groups, carboxyl groups, hydroxyl groups, etc. of polysaccharide derivatives, polyacrylamide derivatives, styrene derivatives, etc. to covalently bond to bacterial cell membrane components, and is the most popular immobilization method among immobilized enzyme methods. However, although the binding force is strong, it is difficult to maintain activity under reaction conditions.

The crosslinking method is a method of crosslinking using geltaraldehyde, hexamethylene diisocyanate, etc., and is characterized by not using an insoluble carrier, but it is not used for immobilizing microbial cells.

The most popular method for immobilizing microbial cells is the blanket method. There are two comprehensive methods, and the grid type is the most adopted method. Typical examples of lattice types include polyacrylamide, polyvinyl alcohol, calcium alginate,
These include carrageenan, collagen, and agar. These gels and microbial cells are mixed, the gel is polymerized using a method such as photoradicals, and the microbial cells are included in the lattice. Another comprehensive method is the microcapsule method, in which bacteria and cells are wrapped in a porous film such as nylon, polyurea, or polystyrene. It is not suitable for covering all types of substances, and development issues remain in terms of membrane permeation control.

Among these methods, the present invention relates to improvements in comprehensive method lattice type carriers.

Microbial cells generally have poor stability against heat. Although there are some heat-resistant bacteria that can grow even at 70 to 80°C, they usually die or lose their activity when the temperature exceeds 40°C. Due to the high reaction temperature of conventional methods, a large percentage of the bacteria may be lost or killed. Next, the following conditions are required for the carrier, especially in the field of wastewater purification. Unlike immobilized enzymes, they are used outdoors for long periods of time rather than in the production process, so they are durable, have mechanical strength that can withstand the forces of air bubbles and water flow, and are used underwater to prevent bacterial cell growth. The carrier is hydrophilic, water-permeable,
It has porosity, and it is a carrier that maintains the activity of activated sludge, which is said to be useful for purifying wastewater.

[Problem that the invention seeks to solve]

Conventional methods of mixing acrylamide, hydroxyethyl methacrylate, etc. with a water-soluble suspension of microbial cells to perform a polymerization reaction as hydrophilic low-molecular weight polymers require a reaction temperature of 40°C or higher. There are concerns about biotoxicity due to residual unreacted low molecular weight substances. moreover,
In some cases, the reaction conditions tend to be alkaline or acidic and deviate from the optimum pH range. In order to improve these problems, the present invention aims to select a reaction system that allows the gel polymerization reaction temperature to be 40°C or less, select a resin monomer that does not have any biotoxicity or safety problems, and maintain a neutral pH range. This was completed as a result of selection of a PH buffer system, selection of a crosslinking reaction system with sufficient mechanical strength, etc.

[Means for solving problems]

Unsaturated polyester is used as the material for the hydrophilic gel, and the main resin is methoxypolyethylene glycol acrylate, which is a monofunctional or monoester, or methoxypolyethylene glycol methacrylate, which is a derivative thereof, and the crosslinking agent is a difunctional, or diester. Polyethylene glycol diacrylate or its g^ derivative, polyethylene glycol dimethacrylate, was selected. These monoesters and diesters are derivatives of ethylene glycol, have high reactivity, and easily undergo crosslinking reactions. As a polymerization catalyst, when immobilizing microorganisms, its temperature and pH are mild (
For example, pH 5 to 8, temperature 40°C or less), and a redox catalyst is used, in which potassium persulfate (K, S, O) is used as a radical polymerization initiator.

dimethylaminopropionitrile (abbreviated as DMAPN) was selected as the polymerization accelerator.

Using the above compositions of monoester, diester, and catalyst, the optimal conditions for use as a carrier for microbial cells were determined.

In Figures 1 and 2, the amount of resin is constant, and the polymerization accelerator D
The gel initiation time and peak reaction temperature are shown when the amounts of *S t O* added to MAPN and the polymerization initiator are changed. In the figure, the solid line plots the reaction characteristics of methoxypolyethylene glycol methacrylate and polyethylene glycol dimethacrylate;
The reaction characteristics of methoxypolyethylene glycol acrylate and polyethylene glycol diacrylate, and of methoxypolyethylene glycol acrylate and polyethylene glycol dimethacrylate are within the ranges shown within the chain lines. Is the temperature important for microorganisms? Initial temperature 2
At 5°C, the peak reaction temperature is 27°C and the temperature rise ΔT is 2 degrees. In either case, gels are formed at temperatures below 30°C, which is convenient for sanitary products. In gelation of acrylamide, when the initial temperature is 25°C, the peak reaction temperature is 40°C or higher, which causes a significant decrease in activity. The gel initiation time varies depending on the amount added, but when considering actual microorganism immobilization operations, gelation occurs within 10 minutes, which is practical if it is too long.

From these, DMAPNao, Ks8wO, which can generate sufficient radicals to initiate radical polymerization in a short time,
The amount of s needs to be 0.2% or more, o, and 1% or more in terms of total type i ratio.

Next, the monomers serving as the resin base and the crosslinking agent need to be soluble in water, and the polymer is also required to be hydrophilic. As a result of investigating the water solubility of Nanatsuma, it was found that methoxypolyethylene glycol acrylate and methoxypolyethylene glycol methacrylate have ethylene oxide (abbreviated as EO) in the molecule, and the average number of moles of EO added is 9 or more for the former and 23 or more for the latter, making them completely water-soluble. Met. In addition, for polyethylene glycol diacrylate and polyethylene glycol dimethacrylate, the average MO
When the number of moles added was 14 or more for the former and 23 or more for the latter, they were completely water-soluble. Figure 1, l? It was found that the gel as a polymer obtained in +2 did not exclude water by gelation, but contained all the water within the gel, and was an extremely highly hydrophilic gel.

In addition, when looking at the pH that affects microorganisms, the optimum pH range for activated sludge, which generally has a mixed microbial group, is 6 to 6.
It is in 8. The monoester of horseradish resin is weakly alkaline and will not exceed the optimum pH range even when added to an aqueous suspension of microorganisms, so there is no need to worry about it, but polymerization accelerator D is strongly alkaline.
The final pH of a mixed suspension aqueous solution in which S, O, and S are added to MAPN, a weakly acidic polymerization initiator, is on the alkaline side, and depending on the amount added, may exceed the optimum pH range and reach about 8 to 9. Therefore, when mixing each gel with molar phosphate buffer instead of physiological saline, the pH before and during the reaction is
The pH did not change significantly and was around 7.3. P in neutral range
Addition of a PH buffer is necessary to maintain H6-8.

Next, Table 1 shows the gelation status of the suspension aqueous solution of each combination of monoester and diester.

00 molar ratio indicates the diester/monoester ratio.

In either combination, a gel containing water is easily formed. When applying the gel formed here to a bioreactor, the shape of the microorganism immobilization carrier may be pellet-like, sheet-like, cylindrical, etc., depending on the bioreactor format. In particular, in the form of pellets, there is collision between pellets and the strength is low. It becomes necessary. Sheet-like and cylindrical shapes are considered to have resistance to water flow, and need to have high elasticity for wear resistance. Therefore, it is desirable that the microbial immobilization carrier has high compressive strength and high elasticity.

As can be seen from Table 1, the conditions for satisfying these characteristics include a resin amount of 15% or more and a molar ratio of diester to monoester of 0.02 or more.

If the concentration is lower than this, gelation will occur, but the gel will be significantly deformed and it will be difficult to maintain the shape, and it will be easily avoided.

When the amount of resin is 309b or more and the molar ratio of diester to monoester is 0.2 or more, the compressive strength increases but the elasticity is lost, resulting in a hard and brittle gel. Because of this, the total amount of resin is f! 15-30% in quantity ratio
．． The molar ratio of diester to monoester is 0.02.
It is necessary to immobilize microorganisms in the range of ~0.2. When I tried to compare the compressive strength with other gels, I found that the acrylamide gel was 1kg/cfl! , calcium alginate gel, and carrageenan gel are said to have a strength of about 0.8 kg/cd, and the strength is equivalent to or higher than that of these gels.

Figure 3 shows the effects of monoester monomers on microbial respiration activity, that is, biotoxicity. The microorganism used was activated sludge from a sewage treatment facility, which was washed with water, monomer was added to it, and air was blown into it for aeration. It is shown as a ratio.At the same time, the results for acrylamide monomer are shown.Although microbial inhibition is observed, the effect is less than that of acrylamide monomer, and therefore it can be said that the decrease in activity after immobilization is small.

〔Example〕

Example 1. In a glass test tube 1, add 8 m+ of phosphate buffer, 20% resin amount of methoxypolyethylene glycol methacrylate, monomer composition such that the molar ratio of polyethylene glycol dimethacrylate to methoxypolyethylene glycol methacrylate is 0.05, and polymerization accelerator DMAPN ( 5% solution) 1 ml, polymerization initiator,
Add 1 ml of 8.0° (2.5% liquid) one by one while stirring well.

A thermocouple is inserted into the center of the test tube to track the reaction temperature, and the induction period from the initial stage to the start of temperature rise is called the gel start time.The test tube is immersed in a temperature-controlled water bath and the temperature of the water bath is Let it be the initial reaction temperature. After performing the above operations, the combination of methoxypolyethylene glycol acrylate and polyethylene glycol dimethacrylate was placed in test tube 2, the combination of methoxypolyethylene glycol methacrylate and polyethylene glyfur diacrylate was placed in test tube 3, and the combination of methoxypolyethylene glycol acrylate and polyethylene was placed in test tube 3. A combination of glycol diacrylate was used in test tube 4, and conventional acrylamide was used as a comparison material: 8 mK of phosphate buffer, 1.5 g of acrylamide, and 0.12 g of N-N' methylene bisacrylamide as a crosslinking agent.

DMAPN (5% liquid) 1m+, K15set (2,
Use 1 ml of 5% solution as test tube 5. The results are shown in Table 2.

Example 2. Methoxypolyethylene glycol methacrylate (number of moles of EO added: 23) and methoxypolyethylene glycol acrylate (number of moles of BO added: 9) were manufactured by Nippon Jushi Co., Ltd.

In addition, polyethylene glycol dimethacrylate (number of moles added with GO: 23) and polyethylene glycol diacrylate (number of moles added with EO: 14) were products manufactured by Shin-Nakamura Chemical Industry Co., Ltd. The same composition was prepared in a test tube (made of Teflon, inner diameter 15φ) in the same manner as described in Example 1. However, an activated sludge concentrate that had been acclimatized with synthetic sewage was suspended in the phosphate buffer at a fixed concentration within the gel of 10 g/l. After gelation and ripening, extrude the gel from the test tube and
Table 3 shows the results of preparing a gel in the shape of 15 L and measuring the compressive strength using Tensilon.

Table 3 [Effects of the Invention] The present invention achieves the following effects by using the above composition.

1) Since this gelation reaction practically ends within 30 minutes, the reaction can be carried out easily.

2) The reaction can be carried out at a temperature of 40° C. or lower, causing little damage to the microbial cells, and no cooling operation is required during the reaction.

3) P) (There is no change in the gelation reaction even if a buffer is used,
The pH during the reaction can be kept neutral and the activity of microorganisms can be maintained well.

4) As a synthetic polymer carrier, it is less toxic than polyacrylamide, and the activity of microorganisms is less likely to decrease due to immobilization.

5) Moisture in the reaction solution is included by gelation, making the carrier highly hydrophilic.

6) Under standard gelling conditions, it is elastic and has a compressive strength of about 1 kg/c-, which is equal to or higher than other gel materials.

[Brief explanation of the drawing]

Figure 1 shows the effect of a polymerization accelerator on the gel reaction, Figure 2 shows the effect of a polymerization initiator on the gel reaction, and Figure 3 shows the effect of gelling agent monomers on the respiratory activity of microorganisms. FIG. -＼, Hirose Chapter 1 - Niji Figure 1

Claims (1)

[Scope of Claims] 1. Unsaturated monoester methoxypolyethylene glycol acrylate or methoxypolyethylene glycol methacrylate as a resin base, unsaturated diester polyethylene glycol diacrylate or polyethylene glycol dimethacrylate as a crosslinking agent, and radiol polymerization initiation. An aqueous suspension containing persulfate as an agent, dimethylaminopropionitrile as a polymerization accelerator, a PH buffer, and microbial cells is polymerized at a low temperature of 40°C or less to immobilize the microbial cells. A method for immobilizing microorganisms characterized by the following. 2. Methoxypolyethylene glycol acrylate or methoxypolyethylene glycol methacrylate is a monoester in which the former has an average number of added moles of ethylene oxide of 9 or more and the latter 23 or more, and the average number of added moles of ethylene oxide of polyethylene glycol diacrylate or polyethylene glycol dimethacrylate is 23 or more. The method for immobilizing microorganisms according to claim 1, characterized in that the former is a diester of 14 or more and the latter is a diester of 23 or more. 3. The amount of resin in the gel synthesized from monoester and dister is 15 to 30% in total weight ratio, and the molar ratio of diester to monoester is 0.02 to 0.2.
The method for immobilizing microorganisms according to claim 1, which is within the range of . 4. Claims characterized by comprising a catalyst containing K_3S_2O_6 as a persulfate in a total weight ratio of 0.1% or more and dimethylaminopropionitrile as a total weight ratio of 0.2% or more The method for immobilizing microorganisms according to item 1.