JPH10323543A - Method for separating product in microbial reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently separate a product from the producing system in microbial reaction without killing the microbes by bringing the producing system containing the product obtd. by the reaction of a starting material in the presence of microbes into contact with a separation membrane and separating the product from the producing system. SOLUTION: In the production of chemical industrial products or intermediates by using metabolism of microbes, and when microbes and the broth component for the microbes are present as mixed with the product and unreacted starting material in the producing system, the microbes and the broth are separated from others. Namely, such a device is used that is equipped with a treating tank 1 and a collecting tank 2 divided by a separation membrane 3. The treating tank 1 is filled with the liquid 4 to be treated, while the collecting tank 2 is filled with a collecting liquid 5. These liquids are left to stand to carry out separation of the product or the like in the liquid 4 to be treated. With time during leaving the liquids to stand, separation of the product proceeds as the product in the liquid 4 moves through the separation membrane 3 to the collecting tank 2. When the producing system is brought into contact with the separation membrane, the collecting agent is made present in the opposite side of the separation membrane 3 to the side of the producing system.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for efficiently separating a product from a production system in a microbial reaction.

[0002]

2. Description of the Related Art In recent years, production of various chemical products and intermediates utilizing metabolism of microorganisms has been attempted. In the reaction using these microorganisms, since the microorganisms and their medium components are mixed together with the products and unreacted starting materials in the production system, generally, the production system is extracted by an organic solvent or the like. The product and unreacted starting material are separated and taken out of the reaction.

However, in the extraction method, it is not easy to select and separate only a product or an unreacted starting material. Also,
There is a disadvantage that microorganisms are killed due to the toxicity of the organic solvent used for extraction. A solution to this has not yet been proposed.

[0004]

An object of the present invention is to provide a method for efficiently separating a product from a production system in a microbial reaction without killing the microorganism.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems of the prior art, and as a result, achieved the above object by bringing a production system in a microbial reaction into contact with a separation membrane. They have found that they can do this and have completed the present invention.

That is, the present invention is characterized in that a production system containing a product obtained by reacting a starting material in the presence of a microorganism is brought into contact with a separation membrane to separate the product from the production system. The present invention relates to a method for separating products in a microbial reaction.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The object of the separation method of the present invention is a production system containing a product obtained by reacting a starting material in the presence of a microorganism. The reaction using a microorganism that produces such a production system is not particularly limited, and includes various reactions. For example, a method for producing hydroxybiphenyls from biphenyl using microorganisms of the genus Cunninghamella or Absidia (RDScgwartz, A. et al.
l.Williams and DBHutchinson, Applied and Environ
mental Microbiology, 1980, pp. 702-708), a method for producing naphthalenecarboxylic acid from methylnaphthalene using microorganisms of the genus Pseudomonas, and producing indanols from indane using microorganisms of the genus Bacillus. (JP-A-8-266293), a method for producing naphthol from naphthalene using a microorganism of the genus Bacillus, a method for producing glycine from ethanolamine using a microorganism of the genus Corynebacterium, and the like. Asymmetric reduction of ethyl acetoacetate using beaker's yeast (Org. Synth. Col., Vol. 7, p. 215)
And various hydrolysis reactions such as a method of hydrolyzing cyclic acetate using a microorganism of the genus Bacillus (Journal of the Society of Synthetic Organic Chemistry, vol. 41, p. 1054, 1983). Note that various reactions using a microorganism can be applied to various compounds, and the products in the production system differ depending on the selected reaction and starting material.

[0008] The reaction using a microorganism is usually carried out in distilled water or a liquid medium, but is preferably carried out in a liquid medium from the viewpoint of the yield of the reaction. Therefore, usually, a liquid medium is included in the production system in the reaction using the microorganism. The liquid medium is usually prepared by adding nutrients necessary for culturing microorganisms to distilled water. As a nutrient source, meat extract, peptone and the like are used as a carbon source and a nitrogen source. In addition, phosphorus, magnesium, iron, manganese,
Inorganic substances such as sodium chloride and vitamins are appropriately mixed. Examples of such a medium include a liquid medium obtained by dissolving fish (bonito) meat extract, polypeptone, and sodium chloride in distilled water. The medium needs to be sterilized, and can be performed by ordinary high-pressure steam sterilization or the like.

The microorganisms cultured in the liquid medium can be used as they are, but they can also be used by immobilizing them on a support. Examples of the support material include various gelling bases including polysaccharides obtained from seaweed, such as carrageenan, alginic acid, and agar, or gelling ability of polyacrylamide, polyvinyl alcohol, polyacrylic acid (salt), and the like. And a water-absorbing polymer having: Among these support materials, it is particularly preferable to use carrageenan and alginic acid.

In the reaction, when the starting material is hardly soluble in water, it may be added after dissolving the starting material in a small amount of an organic solvent. As the organic solvent, those having high hydrophilicity such as methanol and acetonitrile, and those having low hydrophilicity such as acetone and dimethyl sulfoxide can be appropriately selected and used.

In the present invention, the production system in the reaction using the microorganism is brought into contact with a separation membrane to separate a product from the production system. In the case where the production system contains a starting material remaining unreacted in addition to the product, such a starting material is also separated from the production system together with the product.

Various commercially available separation membranes can be used. For example, polyvinyl alcohol film, Teflon film, polyolefin film, polysulfone film, regenerated cellulose film, polypropylene film, nitrocellulose film,
Cellulose acetate membrane, polyethylene membrane, acetylcellulose membrane, polytetrafluoroethylene membrane, ceramic membrane,
Examples thereof include a cellulose mixed ester film, a polyvinyl chloride film, a polyvinylidene fluoride film, a polyamide film, a nylon 6 film, a nitrocellulose film, a polyacrylonitrile film, a polyimide film, and a polyethersulfone film. Among them, the separation membrane is preferably a cellulose membrane. Note that a suitable separation membrane differs depending on the type of starting material, product, microorganism, and the like. Therefore, the separation membrane is appropriately selected and used according to the type of the microbial reaction. Further, as the separation membrane, one having a MWCO (Molecular Weight Cut Off: molecular weight separation) function is preferable.

When the production system in the microbial reaction is brought into contact with the separation membrane, a product or the like separated from the production system is collected on the opposite side of the production system with the separation membrane interposed therebetween.
Usually, a collector is present. As the collecting agent, water, a buffer, an organic solvent or the like or a mixture thereof can be used.
The product and the like separated by the separation membrane and contained in the collecting agent are isolated by an appropriate known means.

As a method for bringing the production system into contact with the separation membrane, for example, a stationary method and a flow method can be adopted.

In the stationary method, as shown in FIG. 1, an apparatus in which a processing tank 1 and a collecting tank 2 are separated by a separation membrane 3 is used. At the same time, the collection tank 2 is filled with the collection liquid 5 and allowed to stand to separate products and the like in the processing liquid 4. In other words, the products and the like in the treatment liquid 4 move to the collection tank 2 through the separation membrane 3 with the elapse of the standing time, and are separated. The standing time varies depending on the type of the separation membrane and the type of the product to be separated. As the standing time, a time at which the separation of the product or the like occurs at 80% or more is preferable.

Further, in the flow method, as shown in FIG. 2, an apparatus having a collection tank 2 filled with a collection liquid 5 on the outer surface of a separation membrane tube 3 in which the separation membrane 3 is formed into a tube is used as a treatment liquid. The product 4 and the remaining starting material in the processing liquid 4 are separated by passing the solution 4 through the separation membrane tube 3. The inner diameter and length of the separation membrane tube can be appropriately selected depending on the scale. The circulation of the treatment liquid 4 is desirably in a range where a value (liquid hourly space velocity) obtained by dividing the volume when the treatment liquid 4 is caused to flow for 1 hour by the volume of the separation membrane tube is about 0.01 to 100 hours -1. . In view of the economics that the processing time is long, the lower limit of the liquid hourly space velocity is desirably 1 hour-1. In order to efficiently perform the treatment by the separation membrane, the upper limit is 30 hours-1. It is desirable.

In the stationary method, the processing solution 4 is supplied to the processing tank 1, while in the flow method, the separation membrane tube 3 is supplied.
As shown in FIG. 1 or FIG. 2, the supply of the processing liquid 4 from the reactor 6 (which may be taken out from the reactor once) to the supply line 7 by operating a pump or the like, as shown in FIG. Through. Also,
The treatment liquid 4 that has been treated by the separation membrane 3 (separation membrane tube 3) and from which products and the like have been taken out is collected by a collection unit 9 by a return line 8. When a product or the like is contained in the collected processing liquid 4, the processing liquid 4 can be brought into contact with the separation membrane 3 again to perform a separation process. Further, the processing liquid 4 from which the products and the like have been taken out can be returned to the reactor 6 and used again for the microbial reaction. By such a circulation step, the reaction can be repeatedly performed with a certain amount of the microorganism and the medium composition, and the reaction yield is increased. Note that the supply line 7 and the return line 8 are preferably made of a fluororesin with little adhesion of microorganisms, culture media, and the like.

The separation treatment is usually carried out at a temperature not lower than the temperature at which the processing liquid 4 and / or the collecting liquid 5 does not freeze and at a temperature lower than the temperature at which the processing liquid 4 and / or the collecting liquid 5 does not boil or the microorganisms do not die. It is desirable to carry out at room temperature or normal temperature from an economic viewpoint. In addition, the processing solution 4 used for the separation process
Is not limited to the one after the completion of the microbial reaction, and may be the one in which the microbial reaction is in progress.

[0019]

According to the method of the present invention, products and the like can be efficiently separated and extracted from a production system in a microbial reaction containing a large amount of a mixture of microorganisms and a medium in addition to the products and the like. Further, since the method of the present invention does not kill microorganisms during the separation operation, those obtained by separating products and the like from the production system are a certain amount of microorganisms, and the composition of the medium is not disrupted. Exists and can be used repeatedly for microbial reactions. Furthermore, since the method of the present invention can perform a separation treatment together with a microbial reaction, the reaction product produced by the method itself becomes poisonous and the yield of the reaction product does not increase. The problem of bacteriostasis of microorganisms due to an increase in the number of substances can be solved. As a result, a product can be obtained in a high yield.

[0020]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

(Microbial Reaction: Microbial Oxidation of Naphthalene)
Dissolve 10 g of fish meat (bonito) extract, 10 g of polypeptone and 2 g of sodium chloride in 1000 ml of distilled water,
The pH was adjusted to 7.0, and a liquid medium sterilized by high pressure at 120 ° C. for 20 minutes was prepared. In a Sakaguchi flask, 150 ml of the liquid medium and Bacillus. Cereus (Ba
cillus cereus) and shaking culture was performed at 30 ° C. for 24 hours. Then, 108 μmol of naphthalene was added to obtain 2
The reaction was carried out by shaking at 5 ° C for 24 hours to obtain naphthol. The yield of naphthol was 8% (8.64 μmol). This was used as the processing solution

(Analysis of Product) To determine the amounts of naphthalene and naphthol in the treatment solution, 10 ml of the treatment solution was extracted three times with 2 ml of ethyl acetate, and high performance liquid chromatography (column: ODS-A, 150 × 4.6 mm ID) was used. YMC C
o. , Gradient system, methanol: water = 3: 7
-9: 1, 1 ml / min).

(Separation of Product and Residual Starting Product from Production System by Separation Membrane) Separation treatment was carried out by an apparatus (flow method) shown in FIG. Biotec as separation membrane tube 3
h Dialysis Membrance (length 40cm x diameter 3.8m)
m, thickness 4mm (when dry) Spectra / Por, MWCO; 8000, Spec
torum Medical Industries Inc.), and using the separation membrane tube 3 as a glass tube (50
cm × 20 mm), and both ends of the glass tube were sealed and placed horizontally. After the collection tank 2 is filled with water (150 ml), 20 ml of the treatment liquid 4 is supplied by the operation of the liquid sending pump.
Under the conditions of room temperature and about 36 minutes (liquid hourly space velocity = 10 hours-1), the solution was allowed to flow through the separation membrane tube 3 through the supply line 7. The treatment liquid 4 treated in the separation membrane tube 3 by the return line 8 was collected in the collection device 9. Water 15 in collection tank 2
0 ml was taken out and quantitative analysis of naphthalene and naphthol was performed in the same manner as described above. Table 1 shows the calculated values of the residual rates of naphthalene and naphthol (after / before treatment) in the treatment liquid 4 after performing the membrane separation treatment from the results of the quantitative analysis. Separately, treatment liquid 4 (20 m
The same operation was performed for 1), and this operation was performed four times in total.

[0024]

[Table 1]

The microorganisms in the recovered treatment liquid 4 were not killed, and could be used again for microbial reactions.

[Brief description of the drawings]

FIG. 1 is a schematic view of a membrane separation apparatus by a stationary method.

FIG. 2 is a schematic diagram of a membrane separation device by a flow method.

[Explanation of symbols]

 1: Treatment tank 2: Collection tank 3: Separation membrane 4: Treatment liquid 5: Collection liquid 6: Reactor 7: Supply line 8: Return line 9: Recovery unit

Claims (5)

[Claims] Claims: 1. Production in a microbial reaction characterized by contacting a production system containing a product obtained by reacting a starting material in the presence of a microorganism with a separation membrane, and separating the product from the production system. How to separate things. 2. The method according to claim 1, wherein the separation membrane is a cellulose membrane.
Separation method as described. 3. The method according to claim 1, wherein the separation membrane is a MWCO (Molecular Weight).
3. A cut off (molecular weight separation) function.
Separation method as described. 4. The separation membrane is a separation membrane tube,
Claim 1, wherein the production system is circulated in the separation membrane tube.
4. The separation method according to 2 or 3. 5. A reaction using a microorganism, wherein Bacillus (Baci
The separation method according to claim 1, 2, 3, or 4, which is an oxidation reaction using the genus llus).