JPS62224290A - Method for immobilizing mold of bacterium - Google Patents

Abstract

PURPOSE:To obtain a stable immobilized mold of bacterium keeping optimum activity simply, by polymerizing aqueous suspension containing a bacterium mold in the presence of a specific resin, a crosslinking agent, a radical polymerization initiator and a pH buffer agent at low temperature. CONSTITUTION:10-30wt% polyethylene glycol monomethacrylate having an average number m of ethylene oxide addition of >=4 as an unsaturated polyester resin, 0.3-1.5wt% N,N'-methylenebisacrylamide as a crosslinking agent, >=0.1wt% K2S2O3 as a radial polymerization initiator and >=0.2wt% dimethylaminopropionitrile as a polymerization promotor are blended with a pH buffer agent and aqueous suspension containing a bacterium mold and polymerization reaction is carried out at pH6-8 at <=40 deg.C. Then, the inclusion gel is immobilized to give an immobilized bacterium mold.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing an entrapping gel of immobilized microorganisms useful for improving the purification method of tJt-water by 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 method of attaching micropores to a carrier such as activated carbon, porous glass, kaolinite, silica gel, etc. as a physical adsorption method. This method is υ1. In the field of water purification, it is called the +1 microorganism method, and it is effective in controlling microorganisms and microorganisms because the binding force between microorganisms and carriers is weak (biofilm formation, water flow, etc., and they can easily separate). Furthermore, there is a method called ionic bonding method in the carrier bonding method.This method involves ionic polymerization of bacterial cells on ion-exchanged polar fats such as DEAR cellulose and polyaminostyrene, but the PI of salts in wastewater is In this case, the bacterial cells may be ion-exchanged and eluted, so they cannot be used.
There is a combination method. Polysaccharide derivative, polyacrylamide 4
amino group, carboxyl group of styrene derivatives,
A method of covalently bonding to bacterial cell membrane components using hydroxyl groups, etc.
This is the most popular immobilized enzyme method. Although the binding force is strong, it is difficult to maintain activity under reaction conditions.

The crosslinking method is a method in which crosslinking is performed using gel taraldehyde, hexamethylene diisocyanate, etc., and is unique because it does not use an insoluble carrier, and 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 polyacrylic acid, polyvinyl alcohol, calcium alginate,
These include carrageenan, collagen, and agar. These gels and microbial cells are mixed, and the gel is polymerized using methods such as photoradicals, so that the microbial cells are included in the lattice. Another comprehensive method is the microcapsule method, in which bacterial bodies and cells are wrapped in a porous film such as nylon, polyurea, or polystyrene, or, as it is a high-grade method, activated sludge for wastewater purification is used. However, it is not suitable for comprehensively including other products, and there is still some development work to be done 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. In the conventional method, the reaction temperature is high, so in some cases, a large percentage of the bacteria contained in it may be killed. Since it is used outdoors for a long period of time, not in the production process like immobilized enzymes, it must be durable and have sufficient mechanical strength to withstand forces such as air bubbles and water flow.
The carrier, which is used in water and is a place for bacterial growth, is hydrophilic,
It has water permeability and porosity, and 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]

In the conventional method of performing a polymerization reaction by mixing hydrophilic low-molecular-weight monomers such as acrylamide and hydroxyethyl methacrylate with an aqueous suspension of microbial cells, the reaction temperature may exceed 40°C or There is a concern about biotoxicity due to residual low-molecular-weight monomers from the reaction. Furthermore, there are cases where 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 keeps the gel polymerization reaction temperature below 40°C, select a resin monomer that has no problems with biotoxicity and femininity, and maintain a neutral PH range. This was completed as a result of selection of a possible PH buffer system, selection of a crosslinking reaction system with sufficient mechanical strength, etc.

[Means for solving problems]

Useful materials for the hydrophilic gel include unsaturated polyester, unsaturated acrylic resin, unsaturated epoxy, unsaturated acrylic-urethane resin, unsaturated polyester-urethane resin, and unsaturated urethane resin. Among them, those considered suitable as resins for bacterial gel are polyethylene glycol monomethacrylate, formalin cross-linked polysaccharide,
Probably pentaesothritol, polymethacrylate, etc. Polyethylene glycol monomethacrylate (abbreviated as PgGMA) was selected as the unsaturated polyester resin from the viewpoints of gel strength, reaction temperature, and biotoxicity. As a crosslinking agent, N,N''-methylenebisacrylamide (abbreviated as BIS) was selected according to a conventional method.As a redox polymerization initiator system, H80, -iron(II), persulfate-sulfite, cumene hydroxyperoxide- Iron (■),
Is there a system such as benzoyl peroxide-dimethylaniline?
Potassium persulfate was selected as the radical polymerization initiator and dimethylaminopropionitrile (abbreviated as DMAPN) was selected as the polymerization accelerator in terms of reaction time and reaction temperature, and the optimal conditions for the microbial cell gel phase were determined. Ta.

FIG. 1 shows the initial temperature, gel initiation time, and peak reaction temperature.

For polyacrylamide gels, if the reaction is started at 35°C, the peak reaction temperature at the center of the gel is 50°C.
Since the concentration increases above this level, there is a concern that the activity of microbial cells will be significantly degraded. On the other hand, in the case of PBGM human gel, even if the reaction is started at 35°C, the peak reaction temperature at the center of the gel increases by only +2°C, and the gelation reaction can be carried out at 40°C or lower. The gel initiation time is also within 1 minute, which is practical.

Figures 2 and 3 show the effects of DMAPN, a polymerization accelerator,
It shows an effect of *8*Os on potassium persulfate, which is a nail metal initiator. The reaction conditions etc. are as shown in the figure. The amount of D M APN, x * s * o * fil, which can generate sufficient radicals to initiate radical polymerization in a short time, is preferably 0.2% or more and 0.1% or more, respectively, in terms of total weight ratio. is necessary.

FIG. 4 shows the I'EGMA elasticity and gel compressive strength.

A sample piece molded into a cylindrical shape of 13φ×15L was compressed with Tensilon, and the load at which the gel was destroyed was determined. The properties of PBGMA gel are highly elastic. First of all, as shown in the molecular formula of polyethylene glycol monomethacrylate (% formula %), polyethylene glycol monomethacrylates with different average numbers of added moles of ethylene oxide (GO) are on sale. m=2, m=
4-5, m=7-9, or a representative one thereof. As shown in Figure 4, the number of moles of EO added is 11 = 2 P B
GM gel does have high compressive strength, but its hydration properties deteriorate, so it cannot be used as the gel of the present invention. ) As the number of moles added to EO increases, the crosslinked structure becomes coarser and the hydration property improves, and sufficient gel compressive strength can be obtained.
is preferably 4 or more. Regarding the amount of PFiGMA resin, the higher the resin amount ratio, the better the compressive strength, but on the other hand, when the resin amount ratio is 25
%, there is a risk that the gel will collapse during the drying-hydration process.

Therefore, there is naturally a practical range for the PFGMA resin ratio, and it should be used in a range of 10 to 30%. FIG. 5 shows the crosslinking agent ffi (BIS) ratio and gel compressive strength. Practically speaking, the appropriate BIB ratio is 0.3% to 1.5% in terms of total weight ratio. Since the gel compressive strength of carrageenan and calcium alginate is about lKy/cJ,
PgGMA shows higher strength.

Figure 6 shows the amount of PgGM resin and gel swelling properties. When a gel is soaked in water from a dry state, it absorbs water and swells. At this time, the larger the amount of water absorbed, the better the gel's hydration, retention, and water permeability, and can provide a favorable environment for the survival of microorganisms. It has been shown that when the amount of resin is high, the water absorption rate tends to decrease, and the gel material can contain more than four times its own weight of water. The gel swelling properties of the PEGM gel were good and comparable to those of the acrylic acid gel.

Fig. 7 and Fig. 8 show the influence of the seven-layer polymer and the polymer (gel) on microbial respiration activity, that is, the biotoxicity. The microorganisms used were activated sludge from a sewage treatment facility, washed with water, mixed with monomer and polymer at the mixing ratio shown in the figure, and aerated by blowing air. It is expressed as a ratio to the oxygen utilization rate of activated sludge when no sludge is added. Acrylamide monomer clearly shows biotoxicity, and PEGMA has no biotoxicity at all in both the monomer and polymer (gel).

Furthermore, reaction reagents P BGMA, BIS, DMAP
When NSK*5tOs is mixed with physiological saline, the pH is 9.
-10, which is not a favorable environment for the survival of microbial cells.

Therefore, when each gel was mixed with 1/15 molar phosphate buffer instead of physiological saline, the pH before and during the reaction remained unchanged at pH 6.8.

Addition of a pH buffer is essential to maintain a neutral pH range of 5 to 8.

〔Example〕

Example 1 Physiological saline/8 m4 PEG in a 16φ glass test tube 1
MA@2g%BISO” 12g, KxS
sOs (2.5% solution) ・Q, 8mt, DMA P
Add 1 mt of N (5% solution) and mix. test tube 2
3 mt of physiological saline, 1.5 g of acrylamide, B
IsO, 12g, Kt8*Os (2.5% solution)Q
, 8 mt, and 1 mt of DMAPN (5% solution) are added and mixed.

A thermocouple is inserted into the center of the test tube to track the reaction temperature, and the period from mixing 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 determined as the initial reaction temperature. shall be. The results are shown in Table 1.

Table 1 Initial temperature, gel start time, peak reaction temperature Example 2゜P EGMA: NOF Bremmer PR-9
Q, PH-350 was used. According to the catalog, the average EO
The number of moles added is lfi! 2, m=7-9.

Put 2 g and 4 g of PEGMAPE-90 and PG-350 in test tubes 1 to 4, respectively, and add 8 mt of physiological saline.
BIS O, 12g each, 0.24g each, Kx
SsOs (2.5% solution)1. QmtDMAPN (
59b solution) Add 1 and Qmt and mix to gel. 1
Table 2 shows the results of molding into a 3φ×15L shape and measuring the compressive strength using Tensilon.

Table 2 Amount of PH0M resin and gel compressive strength [Effects of the invention] The present invention achieves the following effects with the above configuration.

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

2) The reaction can be carried out at a temperature of 4Q'C or less, causing little damage to the microbial cells and requiring no cooling operation during the reaction.

3) Under standard gelation conditions, gel compressive strength is 1h/
c1 or higher is possible, which is equivalent to or higher than other gel materials.

4) The pH during the reaction can be kept neutral by the action of the PH buffer, and the activity of the microorganism can be optimally maintained.

5) Since neither the monomer nor the polymer of PBGM A exhibits biotoxicity, it inhibits the activity of microorganisms to a lesser extent than polyacrylamide gel.

6) Since the water absorption amount of the gel is 4 g/g gel or more, it is highly water absorbent and is comparable to that of polyacrylamide gel, so it can provide a better microbial environment.

[Brief explanation of drawings]

Figure 1 shows the initial temperature, gel initiation time, and peak reaction temperature. Figure 2 shows the effect of the polymerization accelerator. Figure 3 shows the effect of the polymerization initiator. Figure 4. is pgoN+
A [j Diagram showing the amount of fat and gel pressure cord 1 strength, Figure 5 is BI
Figure 6 shows the amount of S-crosslinking agent and gel compressive strength, Figure 6 shows the amount of resin and gel swelling properties, Figure 7 shows the relationship between the seven months and respiration activity, and Figure 8 shows the relationship between polymer and respiration. It is a figure showing activity. 1/TXIO (1/Ju1 anti/L-QJ'l) I/
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Claims (1)

[Claims] 1. Polyethylene glycol monomethacrylate of unsaturated polyester resin as the main resin material and N as a crosslinking agent.
, N'-methylenebisacrylamide, persulfate as a radical polymerization initiator, dimethylaminopropionitrile as a polymerization accelerator, a PH buffer, and an aqueous suspension containing microorganism cells at 40°C. A microbial cell immobilization method characterized by polymerizing at a low temperature below to immobilize microbial cells. 2. The method for immobilizing microorganisms according to claim 1, characterized in that the average number m of added moles of ethylene oxide in polyethylene glycol monomethacrylate is 4 or more, and the total weight ratio is 10% to 30%. . 3. The method for immobilizing microorganisms according to claim 1, which contains 0.3% to 1.5% of the total weight of 3, N,N'-methylenebisacrylamide. 4. The method for immobilizing microorganisms according to claim 1, which contains K_2S_2O_■ as a persulfate in a total weight ratio of 0.1% or more. 5. The method for immobilizing microorganisms according to claim 1, which contains dimethylaminopropionitrile in a total weight ratio of 0.2% or more. 6. Immobilization of microorganisms according to claim 1, characterized in that the pH of the aqueous suspension is maintained in the neutral range of 6 to 8 before and during polymerization using a pH buffer. Law.