JPS5976507A - Prevention of stagnation of foam - Google Patents

Abstract

PURPOSE:To continue reaction stably by incorporating a surfactant in immobilized microorganism complex and releasing slowly the same during reaction. CONSTITUTION:A carrier incorporated with a surfactant is beforehand prepd. and microorganisms are deposited thereon to make immobilized microorganism complex. The surfactant to be used in this case is more particularly preferably a nonionic surfactant, and the surfactant of which the HLB value indicating the ratio between a hydrophobic group and a hydrophilic group ranges 1-10 provides a satisfactory preventive effect against stagnation of foam. The ratio of the surfactant with respect to the immobilized microorganisms is preferably 0.01-10wt% in general. For example, polyoxyethylene alkyl ether or the like is used as the surfactant. On the other hand, for example, an ion exchange resin, agar-agar, etc. are used as the carrier. The surfactant is gradually released from the immobilized matter during the reaction using such immobilized microorganism complex, whereby the various troubles occuring in the stagnation of foam are eliminated, the growth of the microorganisms is improved and the growing power of metabolite is maintained for a long period of time.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for preventing air bubbles from accumulating in a reaction using an immobilized microbial complex, and more specifically, the present invention relates to a method for preventing air bubbles from accumulating in a reaction using an immobilized microbial complex. Concerning a method for preventing air bubble retention, which comprises incorporating a surfactant into the immobilized microbial complex to be used and allowing it to be released during the reaction.Microorganisms are used in fields such as the food industry and pharmaceutical industry as a means of producing various useful substances. It is used in Conventional microbial reactions have been carried out batchwise. However, the microorganisms suspended in the reaction solution are extremely small, and recovering and reusing them requires a high-performance separator.
Because it is difficult to recover without contaminating bacteria, it is usually discarded after the reaction is completed. In order to improve this method of use with low utilization efficiency, there is active development of technology that immobilizes living microorganisms on water-insoluble carriers, molds them into a size and shape that is easy to handle, and then uses them for reactions. It began to be carried out in

The immobilized microorganisms take in nutrients in the reaction solution and grow. After the reaction is completed, the microorganism can be easily recovered and used again in the reaction. It is also possible to carry out a continuous fixed bed reaction by filling a reactor with microorganisms immobilized on a carrier.

Focusing on such advantages, various σ immobilization methods have been developed.

The present inventors have also proposed a novel immobilization method that involves stably enclosing microorganisms in water-insoluble polymers such as cellulose triacetate (Japanese Patent Application Laid-open No. Shojj-/J3!).
99/).

Conventionally, reactions using immobilized microbial complexes have been carried out either by suspending them in a reactor or filling them as a fixed bed. However, when this reaction is carried out under aeration, or when a large amount of carbon dioxide gas is produced as a product of the reaction, such as in alcoholic fermentation, (b) air bubbles remain inside and around the chemotactic microbial complex. This prevents the supply of nutrients necessary for the growth of microorganisms, or accumulates waste products that are unsuitable for growth.

As a result, the ability to produce desired metabolites often decreases. For example, when the reaction is carried out under aerobic conditions, air and gas are supplied, and the supplied gas forms a layer on the surface of the immobilized microbial complex. Furthermore, when microorganisms themselves produce substances that are easily gasified, such as carbon dioxide, gas retention is observed not only on the surface of the carrier on which the microorganisms are immobilized, but also near the microorganisms. In particular, in fixed-bed packed reactions, the air bubbles that stay around the immobilized microbial complex stick together and create even larger bubbles, which may prevent the reaction liquid from flowing uniformly or, in extreme cases, cause the reaction solution to flow inside the packed bed. Problems such as hollowing out frequently occur. When such a trouble occurs, the contact efficiency between the immobilized microorganism and the reaction solution is significantly reduced, and as a result, the growth of the microorganism is inhibited and the ability to produce metabolites is reduced.

Conventionally, when reacting with microorganisms suspended in a reaction solution,
Addition of a surfactant is mainly used to prevent foaming of the reaction solution. However, in reactions using aggregates of immobilized microorganisms, simply adding a surfactant to the reaction solution can prevent foaming of the reaction solution itself; is insufficient. In particular, in a reaction in a fixed bed, bubbles remain attached to the periphery of the immobilized material and do not easily collapse. As a result of this observation and intensive research into a method to easily release the gas adhering to the surface of the carrier, it was surprisingly discovered that the gas retention around the carrier where microorganisms were immobilized disappeared, allowing a stable reaction to continue. Heading This invention has led to this invention.

That is, the present invention is a method for preventing bubble retention, which is characterized by slow release of a surfactant contained in an immobilized microbial complex in a reaction using an immobilized microbial complex.

In the present invention, the immobilized microorganism complex does not only refer to immobilized microorganisms, but also refers to microorganisms and a carrier used for immobilization, or when a suspension of microorganisms in water etc. is enclosed in a carrier. It also includes everything contained in the carrier.

As the surfactant used in the present invention, cationic or anionic surfactants can be used, but nonionic surfactants are particularly preferably used because they have a low growth inhibiting effect on microorganisms. Examples of the nonionic surfactants include polyoxyethylene alkyl ethers, keroxyethylene alkyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, sorbitan fatty acid esters, Examples include glycerin fatty acid esters, sugar fatty acid esters, and block polymers such as polyethylene oxide and polypropylene oxide. H indicating the ratio of
It is particularly preferable to use a material having an LB value in the range of 7 to IO. These materials may be used in combination.

These surfactants are contained in one immobilized microbial complex. This microorganism is kept alive on a carrier,
There are no particular limitations, as long as it can be used in a living state or while being propagated.

To support microorganisms on a carrier, for example, they may be supported on the surface of an ion exchange resin carrier by ionic bonding, or agar, carrageenan, collagen, polyacrylamide, various photocurable polymers, cellulose triacetate, polyacrylonitrile, It may also be incorporated into various water-insoluble polymers such as polymethacrylic acid. For example, when microorganisms are to be immobilized on the surface of a carrier, the microorganisms may be supported after preparing a carrier containing a surfactant in advance. Furthermore, when microorganisms are encased and immobilized using a water-insoluble polymer, a surfactant can be added during the immobilization process. That is, when enclosing it in agar, carrageenan, etc., it can be achieved by dissolving it in warm water, then mixing microorganisms and a surfactant, and then cooling this mixture to gel it.

When using collagen, this can be achieved in the same manner by adding a surfactant to a solubilized collagen solution, air-drying this, and then crosslinking with glutaraldehyde or the like. In addition, when using photocurable +f IJ polymers such as polymerizable monomers such as polyacrylamide and urethanized prepolypolymers (average molecular weight approximately s, ooo), surfactant may be added to the mixture of these compounds and microorganisms. After adding the agent, the polymerization and crosslinking reactions may be carried out.

Furthermore, when it is to be incorporated into a water-insoluble sieve molecule such as cellulose triacetate, it can be achieved by the same immobilization method as described in, for example, JP-A-5S-I3SSQI. That is, a surfactant is dispersed in an organic solvent in which cellulose triacetate, etc. is dissolved, and then mixed with ice grains containing microorganisms. An immobilized product containing microorganisms and microorganisms is obtained.

During the reaction, the surfactant is gradually released from the immobilized product. While the surfactant is being released in a sustained manner, the effect of preventing bubble retention continues. It is preferable to contain this surfactant in as large a quantity as possible without interfering with the growth of microorganisms or the production of desired metabolites, in order to prevent the retention of air bubbles for a long period of time. For θQ
/-10% by weight is preferred. In the present invention, a surfactant may also be added to the reaction solution in order to further enhance the effect of preventing bubble retention. The additive company may add enough solder to reach a concentration that substantially enhances the effect, but
Generally, 0.007 Nl 2 O weight percent is a preferred range. Adding a surfactant to the reaction solution can also be expected to have a defoaming effect on the reaction solution itself, and in this case as well, the HLB value is 7.
-IO is preferably used.

In the reaction, the immobilized microorganisms may be suspended in the reactor or may be packed as a fixed bed. In either case, the various troubles caused by the accumulation of bubbles, which have been problems in the past, are solved, the microorganisms grow well, and the ability to produce metabolites can be maintained for a long period of time. Especially in fixed-bed packed continuous reactions,
Almost no air bubbles are observed around the immobilized microbial complex, and the generated air bubbles sequentially move upward within the packed bed. As a result, the efficiency of contact between the immobilized microorganisms and the reaction solution is improved, allowing the microorganisms to fully demonstrate their capabilities, allowing stable reactions to continue for a long period of time, and most of the conventional problems can be solved.

This will be explained in detail below using an actual example.

Example yeast Saccharomyces formocensis (3acch
aromyoes formoseneis) to YM
Medium (glucose IO book ratio, peptone θS nail amount%, yeast extract 03% by weight, malt extract 03% by weight, PHA
) and cultured at 17C for 2 hours.The grown yeast was centrifuged to collect the bacteria, and 2g of it was inoculated with SO Kyomai%.
Suspend the potatoes in 2 oocc of YM medium containing glycerin.

Yu! The surfactant shown in Table 1 was added to 12,900 g of methi dichloride in which fXXM% recellulose triacetate was dissolved.
After being forgiven, it was cooled to -5OC. Next, in this methylene dichloride, the above yeast suspension /! R09 was sprayed into minute water droplets and quickly frozen. During this time, the methylene dichloride was maintained at -SOC 6 This methylene dichloride solution was then cooled to -SOC n-
It was dropped into hexane 131 and coagulated into particles. By removing the organic solvent contained in this coagulated product under reduced pressure, a granular immobilized product in a frozen state was obtained. The particle size of this immobilized product was Col 3 Dragon.

After melting this immobilized material at room temperature,
A glass reactor with a length of 3 gcm was filled.

The packed bed was 100 m1.

From the bottom of this reactor, molasses medium (sugar concentration, 20wt/y)
%) was continuously fed at a reaction temperature of about 30C.

Continuous alcoholic fermentation was carried out for about 7 hours in contact with Pi-11A3 to Fu01. The accumulation of air bubbles in the immobilized yeast packed bed was observed every day after the start of liquid passage. Alcohol fermentation began to take place actively from about the third day, but the generated carbon dioxide gas gradually moved above the packed bed without forming a layer around the immobilized material or staying within the packed bed. During the test period of approximately 3 months, no troubles due to air bubble retention occurred.

comparison C1,) in immobilized microorganisms and in precision
A case in which no killing agent was included was also studied. The preparation conditions for the immobilized product and the fermentation conditions are the same. In this case, carbon dioxide bubbles form a layer around the immobilized material, and these bubbles stick together, eventually forming a fairly large empty AI number within the packed bed, or sometimes 1? There was a phenomenon in which the Y-distilled gas rose all at once in the packed bed, or (as the gas moved, the viscosity channeled and moved upward.

The results of the fermentation test were as shown in Table 0.Also, the time-course changes in alcoholic fermentation for Example 1 and Comparative Example were as shown in Figure 7. The use of such immobilized substances is effective in eliminating troubles in operating the reactor and, as a result, producing fermentation products stably over a long period of time.

Table l/) Polyoxyethylene (POE) 9 mol, polyoxy-brohy.

polyoxybutylene (POD)/?
3) Visual judgment (fermentation days 308) Example A Continuous alcohol production was performed using the immobilized yeast prepared in the same manner as in Example. 002 wt in molasses medium
% OJ Dishome BC-1/Y (NOF, Ni,
The fermentation conditions were the same as in Example 1, except that the fermentation conditions were the same as in Example 1. During the test period of approximately 6 months, there were no troubles due to bubble retention, and ethanol with a concentration of approximately 1%/Ovol/vol could be stably produced.

[Brief explanation of the drawing]

The figure shows the relationship between the number of fermentation days and the alcohol concentration produced.

Claims (1)

[Claims] / A method for preventing bubble retention, which comprises slow release of a surfactant contained in an immobilized microbial complex in a reaction using the immobilized microbial complex. U. The method for preventing bubble retention according to item 1 of the scope of Patent No. 1i74, characterized in that the surfactant has an HLB value of 7 to lθ and is nonionic. 3. Special feature #'l: BI, characterized in that the immobilized microorganism complex is a water-insoluble polymer containing microorganisms.
The method for preventing air bubble retention according to item 1 or item 1 of the scope of V requirements. The method for preventing air bubble retention according to claim 1, 2, or 3, characterized in that the immobilized microorganism complex contains 0.0/-10% by weight of a surfactant. Method. A method for preventing bubble retention according to claim 1 or 2 or 3 or 3, characterized in that a surfactant of θ00/NlO heavy storm% is added to the reaction solution. . A. The bubble retention according to claim 1, 2, 3, q, or s, characterized in that the immobilized microbial complex is packed in a reactor as a fixed bed. Prevention method: Claims 7 or 2 or 3 or 9 or 5, characterized in that the microorganism used for the immobilized microorganism complex is a microorganism capable of producing ethanol. The method for preventing σ bubbles ff1k retention described in item 6 or item 6.