JPS623787A - Immobilization of microorganism - Google Patents

Abstract

PURPOSE:To suppress the lowering of the microbial activity, by forming particles of an insoluble salt of alginic acid from a liquid mixture of a microbial suspension, a polyurethane prepolymer and a water-soluble salt of alginic acid, and polymerizing the polyurethane prepolymer. CONSTITUTION:A liquid mixture containing a polyurethane prepolymer, a water- soluble salt (e.g. sodium salt) of alginic acid and microorganisms is added dropwise to an aqueous solution of a compound (e.g. calcium chloride) which forms an insoluble salt with alginic acid. Particles of insoluble salt of alginic acid including the microorganisms are produced. The polyurethane prepolymer held in the particle is polymerized to obtain the objective immobilized microorganism. The toxic effect of the chemicals such as monomer, modifying agent, plasticizer, etc., to the microorganism is suppressed and the lowering of the microbial activity is prevented.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for entrapping and immobilizing microorganisms in an active state on polyurethane.

Conventionally (2) In the case of entrapping and immobilizing microorganisms, naturally occurring substances such as carrageenan and alginic acid, and synthetic polymeric substances such as polyacrylamide and polyurethane have been proposed as carriers. When natural substances are used as carriers, since natural substances have low toxicity to microorganisms, the activity of immobilized microorganisms does not decrease much, but their physical strength is small,
There is a problem with durability. When microorganisms are immobilized inside polyacrylamide, the resulting immobilized microorganisms have high physical strength and good durability, but the acrylamide monomer, crosslinking agent, polymerization initiator, etc. used for immobilization may have a toxic effect on the microorganisms. , which tends to lead to a decrease in activity.

JP-A-52-38084 discloses that a polyurethane prepolymer having isocyanate groups at the terminals is brought into contact with an enzyme to polymerize and immobilize the enzyme on the polyurethane. It is also known that this method can be applied to microorganisms by adding a polyurethane prepolymer solution to a microorganism suspension, stirring it, pouring it into a block-shaped or film-shaped mold, and polymerizing it to immobilize the microorganisms on polyurethane. ing.

When microorganisms are immobilized inside polyurethane, the resulting immobilized microorganisms have very high physical strength and good durability, but the The polyurethane prepolymer solution has a toxic effect and is likely to lead to a decrease in activity.

Therefore, the present invention solves the problems of the prior art, and
The object of the present invention is to provide a method for producing immobilized microorganisms using a polyurethane prepolymer, which causes less reduction in microorganism activity.

. In the present invention, a mixture of a microorganism suspension, a polyurethane prepolymer, and a water-soluble salt of alginic acid is added to an aqueous solution of a compound capable of forming an insoluble salt with alginic acid, and the microorganisms are encapsulated. The above problems are solved by forming particles of an insoluble salt of alginic acid and polymerizing the polyurethane prepolymer held inside the particles.

That is, in the present invention, a mixed solution containing a polyurethane prepolymer having an isocyanate group at the terminal, a water-soluble salt of alginic acid, and a microorganism is dropped into an aqueous solution containing a compound capable of forming an insoluble salt with alginic acid, and polymerization is carried out. , is characterized by forming polyurethane in which microorganisms are entrappingly immobilized.

The polyurethane prepolymers used in the invention are prepared in a known manner by reacting diisocyanates, triisocyanates or other polyisocyanates with active hydrogen-containing compounds, in particular glycols, polyglycols, polyester polyols or polyether polyols. can do. The polyurethane prepolymer used in the present invention has an average molecular weight of 3,000 to 4,000. NG
Preferably, the O content is between 2.5 and 4%. It is preferable that the amount of polyurethane prepolymer added to the microorganism/alginic acid water-soluble salt mixture is 5 to 16%.

In the present invention, as the water-soluble salt of alginic acid, for example, a sodium salt, a potassium salt or an ammonium salt can be used.

Examples of compounds that form insoluble salts with alginic acid include water-soluble calcium salts, barium salts, or aluminum salts, methylene blue, or ferric salts.

When carrying out the method of the invention, since the polyurethane prepolymer solution has a high viscosity, it is preferable to add, for example, methyl ethyl ketone, dioctyl phthalate, dibutyl phthalate, etc. as a plasticizer. Also, various modifiers can be added, for example toluene diisocyanate to increase strength.

Okina U1 The decrease in microbial activity that occurs when microorganisms are immobilized using polyurethane prepolymer is due to the toxic effects of chemicals such as low-molecular-weight polyurethane prepolymer, residual monomers, modifiers, and plasticizers in the polyurethane prepolymer solution. it is conceivable that.

According to the method of the present invention, a polyurethane prepolymer and ′,
A mixed solution containing a water-soluble salt of alginic acid (explained below using sodium salt as an example) and microorganisms is dropped into an aqueous solution of a compound that forms an insoluble salt with alginic acid (explained using calcium chloride as an example). At the same time, the polyurethane prepolymer held within the particles is polymerized.

In this method, when the liquid mixture is dropped into an aqueous calcium chloride solution, low-molecular substances leak from the pores of calcium alginate and are dispersed in the aqueous calcium chloride solution. In particular, low-molecular residual monomers, modifiers, plasticizers, etc. are instantly dispersed, and low-molecular polyurethane prepolymers are also easily dispersed in the calcium chloride aqueous solution. The high molecular weight polyurethane prepolymer polymerizes inside the calcium alginate particles without leakage. Therefore, the time that microorganisms come into contact with chemicals such as 7-mer 1 modifiers and plasticizers is shortened.
, it is less susceptible to toxic effects, so microbial activity can be maintained at a high level. If a modifier such as toluene diisocyanate is mixed into the liquid mixture immediately before dropping, the contact time between the microorganism and the chemical will be further shortened, and the activity will be increased.

Nitrifying bacteria 4 was immobilized by the method of the present invention while changing the concentration of sodium alginate, and the activity amount was measured from the amount of oxygen absorbed by the nitrifying bacteria. The results are shown in FIG.

As is clear from Figure 1, sodium alginate was
．． It is preferable to add 3 to 1.2%, preferably 0.3 to 0.6%. This is thought to be because when the concentration of sodium alginate is high, it is difficult for the above-mentioned toxic substances to leak into the calcium chloride solution.

Using methyl ethyl ketone as a plasticizer and varying the methyl ethyl ketone concentration in the polyurethane prepolymer solution, nitrifying bacteria were immobilized by the method of the present invention, and the residual activity rate was measured from the amount of oxygen absorbed by the nitrifying bacteria. The results are shown in Figure 2. show. In this case, 12% polyurethane prepolymer solution and 0.5% sodium alginate were used. As is clear from FIG. 2, the amount of methyl ethyl ketone added is preferably 30% or less. Since the polyurethane prepolymer solution has a high viscosity, it is better to add methyl ethyl ketone, preferably in an amount of 5 to 30%.

Furthermore, in the method of the present invention, toluene diisocyanate is used as a modifier, the concentration of toluene diisocyanate in the polyurethane prepolymer solution is varied, the nitrifying bacteria are kept constant, and the residual activity rate is measured from the amount of oxygen absorbed by the nitrifying bacteria. , the results are shown in Figure 3. In this case as well, the polyurethane prepolymer solution was 12% and the sodium alginate was 0%.
．． 5% was used. As is clear from Figure 3, toluene diisocyanate is 1.5% or less, preferably 0.8%.
The following is good.

Note that if the polyurethane prepolymer solution does not contain a small amount of toluene diisocyanate, the solution itself will react with moisture in the air and polymerize, so it is preferable to contain 0.2 to 0.8% toluene diisocyanate. In addition, methyl ethyl ketone is used for cleaning piping and reaction tanks in the polyurethane prepolymer solution production line, and is essential for industrial production, and is contained in small amounts in polyurethane prepolymer solutions. .

Large Plate Next, the present invention will be described in detail based on Examples, but the present invention is not limited thereto.

Example 1 Activated sludge from a sewage treatment plant in Matsudo City was used as seed bacteria, and the activated sludge was incubated with nitrifying bacteria in ammonia-containing synthetic wastewater, methane bacteria anaerobically acclimatized with glucose-containing wastewater,
Pseudomonas isolated from activated sludge
onas sp, ) and Flavobacterium (Fla
vobacterium sp.) was used. These bacteria were each immobilized with the composition shown in the table below.

The activated sludge was concentrated to 40,000μ/1, and this concentrated liquid 10
Add 5 ml of 2% sodium alginate to m1 and 0.24 ml of methyl ethyl ketone (hereinafter abbreviated as MEK).
and toluene diisocyanate (hereinafter abbreviated as TDI) 15μ! 2.4g of polyurethane prepolymer containing
was added, stirred thoroughly, and dropped into a 2.5% calcium chloride solution using a syringe to obtain spherical immobilized microorganisms with a diameter of 2 to 3.

Add 5 ml of water to 10 ml of the above concentrate, MEKo, 2
2.4 g of polyurethane prepolymer containing 4 ml and 115 μl of TD was added, stirred well, poured onto a polyethylene film, fixed in the form of a film with a thickness of 2
It was molded into a ml square.

Similarly, nitrifying bacteria, methane bacteria, pseudomonas bacteria, and Flavobacterium bacteria were immobilized using the method of the present invention and the conventional method.

The activity of each immobilized microorganism was measured. For activated sludge and nitrifying bacteria, activity is determined by the respiration rate (unit: ■
02/h), and the number of viable bacteria for methane bacteria, Pseudomonas bacteria, and Flavobacterium bacteria. The results are shown in the table below.

The respiration rate was measured from the decrease in dissolved oxygen by placing the entire amount of the immobilized microorganisms prepared in a 110 ml sample.

As is clear from the table, the method of the present invention is superior to the conventional method in that both the respiration rate and the number of viable bacteria are higher.

Nitrifying bacteria were immobilized in a film at the same bacterial cell concentration using a conventional method, and the effects of MEK and TDI were also measured, as shown in FIGS. 2 and 3. This result also shows that the method of the present invention is superior to the conventional method.

(Hereinafter, blank space) Hikari 1 Kyo B whole fruit According to the present invention, microorganisms can be comprehensively immobilized in polyurethane in a highly active state.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between the amount of sodium alginate and the respiration rate of immobilized nitrifying bacteria, and Figure 2 is a graph showing the relationship between the amount of methyl ethyl ketone in the polyurethane prepolymer and the respiration rate of immobilized nitrifying bacteria. The graph, FIG. 3, is a graph showing the relationship between the amount of toluene diisocyanate in a polyurethane prepolymer and the respiration rate of immobilized nitrifying bacteria.

Claims (4)

[Claims] (1) A mixed solution containing a polyurethane prepolymer having an isocyanate group at the end, a water-soluble salt of alginic acid, and microorganisms is dropped into an aqueous solution containing a compound that can form an insoluble salt with alginic acid, polymerization is performed, and microorganisms are removed. A method for immobilizing microorganisms, characterized by forming polyurethane that is comprehensively immobilized. (2) The method for immobilizing microorganisms according to claim 1, wherein a modifier, such as toluene diisocyanate, is mixed into the mixed liquid immediately before dropping. (3) The method for immobilizing microorganisms according to claim 1, which uses, for example, methyl ethyl ketone, dioctyl phthalate, dibutyl phthalate, etc. as the plasticizer. (4) Immobilization of microorganisms according to claim 1, in which a polyurethane prepolymer containing 5 to 30% of methyl ethyl ketone and 0.2 to 0.8% of toluene diisocyanate is used to form a polyurethane in which microorganisms are comprehensively immobilized. method.