JP2542766B2 - Immobilization method of microorganisms and non-aqueous microbial conversion reaction method by immobilized microorganisms - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immobilizing microorganisms and a method for microbial conversion reaction of water-insoluble or sparingly soluble organic compounds using immobilized microorganisms.

[0002]

2. Description of the Related Art Conventionally, when an enzyme or microbial cell is allowed to act on a water-insoluble substrate, a surfactant is used to emulsify the substrate in the enzyme or microbial cell dispersion to prepare a reaction system. It was common to carry out a reaction with an emulsion state that is a liquid interface system. But in this case,
Due to the small difference in specific gravity between oil droplets and enzymes or cells,
It is difficult to separate the enzyme or bacterial cells after the reaction, and to avoid the toxicity of the substrate to the enzyme or microorganism, lower the input substrate mass or the substrate concentration, and as a result, it is possible to avoid the low reaction rate and low yield. could not. Further, there are many problems such as requiring a large amount of aeration and stirring force for improving the substrate dispersibility and supplying oxygen.

On the other hand, a method of adding a divalent cation (J. Gen. Microbiol., In order to avoid the toxicity which occurs when a water-insoluble or sparingly soluble organic compound harmful to the microorganism is brought into contact with the microorganism. Vol.11
9, 155 (1980)), crystal fermentation method (J. Ge.
n. Appl. Microbiol. Vol. 7,11
3 (1961)), a method of utilizing an organic solvent resistant microorganism (Nature Vol. 338, 264 (198).
9); Japanese Unexamined Patent Publication No. 63-216473) has been tried.

However, the addition of divalent cations has not been so effective, and the crystal fermentation method and the method utilizing organic solvent-resistant microorganisms have a problem that the types of applicable microorganisms or conversion reactions are limited. .

Furthermore, in order to avoid the toxicity of organic compounds to microorganisms, entrapment of microorganisms has been attempted (Biocatalysis in Organic).
Media P51, Elsevier, Amste
rdam (1986)). Regarding the immobilization of microorganisms, a method of using an immobilized enzyme or immobilized microbial cells in a non-aqueous enzyme reaction or a microbial conversion reaction has been widely performed, and various methods for immobilizing enzymes or microbial cells have been proposed. . The reaction using immobilized enzymes or immobilized microbial cells has many cost advantages such as high reaction rate and high yield, long-term continuous operation, and the ability to repeatedly use expensive enzymes. Immobilized enzymes or immobilized cells have been attracting attention as catalysts for bioreactors.

However, in the entrapping immobilization method, toxicity is often exhibited due to poor permeation and diffusion of the organic compound and its microbial conversion product in the immobilization carrier, and
In order to inhibit the growth of microorganisms, there are many examples of using immobilized enzymes in non-aqueous reactions, while on the other hand, examples of using immobilized microbial cells as immobilized non-proliferating cells. However, there is no example in which immobilized cells are grown and used.
Immobilized microbial cells can utilize reactions based on the physiology, metabolism, etc. peculiar to the cells, unlike immobilized enzymes in which enzymes extracted and purified from organisms are immobilized, but in non-growing cells Only a small portion of these cell activity is available.

Therefore, it is necessary to develop an effective culture method for proliferating immobilized microbial cells while protecting the microorganisms from the toxicity of organic compounds that are toxic to the microorganisms, and to immobilize the immobilized microorganisms in a non-aqueous reaction. It has been strongly desired in recent years to develop a non-aqueous bioreactor in which the activity peculiar to growing cells can be fully utilized in the industrial field handling water-insoluble products. Development of the non-aqueous microbial conversion reaction method used
It is strongly desired as a next-generation bioreactor.

[0008]

Means for Solving the Problems As described above, the present inventors have described a method for growing immobilized microbial cells and a microbial conversion reaction using the immobilized microbial cells, which are problems in non-aqueous reactions. As a result of extensive studies, when a hydrophilic carrier to which microbial cells have been adhered and immobilized is brought into contact with an organic solvent in the presence of an aqueous medium containing a nutrient source, microorganisms are present at the interface between the organic solvent and the immobilized carrier. The inventors have found that the bacterial cells proliferate vigorously to form a bacterial cell phase on the carrier, and that this bacterial cell phase can catalyze a microbial conversion reaction specific to the bacterial cells, and have completed the present invention.

Thus, according to the first aspect of the present invention, a microorganism is attached to a hydrophilic immobilization carrier and is substantially insoluble or sparingly soluble in water in the presence of an aqueous medium containing a nutrient source for the microorganism. A method for immobilizing a microorganism, which comprises contacting an organic solvent with the microorganism on the carrier, growing the microorganism at a contact interface between the carrier and the organic solvent, and forming an immobilized bacterial cell phase on the carrier. Will be provided.

Further, according to the second aspect of the present invention, microorganisms are adhered and immobilized on a hydrophilic immobilization carrier, and a substantially water-insoluble or sparingly soluble organic compound as a substrate is used as a nutrient source for the microorganisms. There is provided a method for converting an organic compound, which comprises contacting with an immobilized bacterial cell phase on the carrier in the presence of an aqueous medium containing.

The feature of the first aspect of the present invention is that a microorganism is attached to a hydrophilic immobilization carrier in a state in which it can grow, and an aqueous medium (hereinafter referred to as a culture solution) containing a nutrient source for the microorganism is supplied thereto. At the same time, a substantially water-insoluble or sparingly soluble organic solvent is brought into contact with the microorganisms on the carrier, and the microorganisms are grown at the interface between the carrier and the organic solvent to form an immobilized bacterial cell phase on the carrier. In point.

It goes without saying that it is preferable that the organic solvent to be brought into contact with the microorganisms on the above-mentioned carrier is one that does not substantially show toxicity to the adherent microbial cells, but according to the immobilization method of the present invention, Surprisingly, it became clear that the microbial cells actively grow at the interface between the organic solvent and the carrier even if the organic solvent contains an organic compound harmful to the microorganism, and as a result, by the method of the present invention, It has been clarified that it is possible to avoid the toxicity that occurs when a harmful substance for a microorganism comes into contact with the microorganism and protect the microorganism from the microbial inhibition of the harmful substance.

The feature of the second aspect of the present invention is that, for example, as in the method of the first aspect, microorganisms are adhered and immobilized on a hydrophilic immobilization carrier in a proliferative state, and the nutrients of the microorganisms are immobilized. An immobilized microbial phase is formed on the carrier while supplying an aqueous medium (culture solution) containing a source and the microorganism is grown there, and the immobilized microbial phase is converted into substantially water as a substrate. The point is that it is brought into contact with an insoluble or sparingly soluble organic compound or an organic solvent solution thereof to convert the organic compound of the substrate into a microorganism at the interface between the immobilized bacterial cell phase and the organic liquid phase.

If the immobilized bacterial cell phase formed by the method of the present invention is used, even if the organic compound as the substrate is toxic by the microorganism, the toxicity of the harmful substance to the microorganism is as described above. It has been found that the substrate can be contacted with the immobilized bacterial cell phase at a high concentration because it can be avoided and protected from microbial inhibition by harmful substances.

The organic solvent and the substrate or the organic solvent solution thereof brought into contact with the immobilized bacterial cell phase may be hereinafter referred to as an organic liquid phase.

The supply of the culture solution to the immobilized cells can be carried out by preliminarily including it in the carrier as long as the carrier can sufficiently contain and retain the culture solution, such as agar. And / or for example, the culture liquid may be added to the organic liquid phase, and the microorganism-immobilized carrier may be interposed at the interface between the organic liquid phase and the culture liquid phase to be formed.

The immobilization carrier usable in the present invention is not particularly limited as long as it is hydrophilic, and it is supplied to a microorganism existing at the interface with an organic solvent by impregnating or contacting it with an aqueous solution containing a nutrient source. Any material can be used as long as it can be used. Specifically, for example, natural polymers such as alginic acid, carrageenan, starch matrix, agar, and cellulose materials such as filter paper; polyvinyl alcohol, Examples include synthetic polymers such as urethane polymers, polyacrylamide, and polyacrylic acid; inorganic materials such as foam glass and silica gel.

There are no particular restrictions on the shape of these immobilization carriers, and they can be formed into any shape such as fibrous, film-like, and granular shapes, and can be in the form of cloth, non-woven fabric, paper, cardboard, etc. It may be molded into.

The organic solvent or the organic solvent for preparing the substrate solution in the organic phase which is in contact with the immobilized bacterial cell phase is preferably one which does not show substantial toxicity to the adherent microbial cells, and specifically, Normal paraffins or liquid paraffins represented by methane hydrocarbons having 6 to 20 carbon atoms such as hexane, heptane, octane, nonane, and decane;
Isoparaffins such as isooctane; Pentylbenzene, hexylbenzene, heptylbenzene, octylbenzene and other normal chain alkyl chains having 5 to 15 carbon atoms; Isoalkylbenzenes such as kyumen; Alicyclic hydrocarbons such as cyclohexane Examples thereof include ethers such as diethyl ether and the like.

Microorganisms which can be attached to the above-mentioned immobilized carrier and can grow at the interface between the carrier and the organic liquid phase include bacteria, fungi,
It may be any microorganism such as yeast or actinomycete, and may be either aerobic or anaerobic. Specifically, for example, Pseudomonas (Pse u domonas) genus Gluconobacter (Gluconobacter) genus Acetobacter (Acetobacter) genus, Alice Arthrobacter (Arithrobactor) genus Corynebacterium (Corynebactrium) genus Rhodococcus (Rhodococ c us ) Genus, Alcaligenes genus, Candida
Examples thereof include microorganisms belonging to the genus ida, the genus Hansenula, the genus Aspergillus, and the like.

In the present invention, these microorganisms can be grown at the interface between the carrier and the organic liquid phase even when the organic solvent with which they come into contact contains harmful substances for the microorganisms. For example, Pseudomonas fluorescens (Ps
eudomonas fluorescens and Pseudomonas put
In ida), a highly toxic organic compound such as benzene can grow even if it is contained at 5% with respect to the normal paraffin of the organic solvent to be contacted. As for the carbon tetrachloride and toluene chloride, to strongly toxic organic compounds in such as styrene, it is possible to grow even at high concentration of 10% to 25%. Furthermore, in the case of long-chain fatty acid esters and aliphatic hydrocarbons, which belong to the least toxic category, 1
It can be used as an organic solvent having a concentration of 00%, that is, forming an organic liquid phase by itself.

According to the method of the present invention, a substantially water-insoluble or sparingly water-soluble organic compound or a solution of the organic solvent as a substrate is used as an organic liquid phase, and the substrate is immobilized at the interface between the immobilized bacterial cell phase and the organic liquid phase. When converting an organic compound into a microorganism, the microorganism that can be attached and immobilized on the carrier is not particularly limited as long as it has the ability to convert the organic compound as a substrate into a desired product, and is aerobic or anaerobic. Any of these may be used, and any of bacteria, fungi, yeasts, actinomycetes, etc. may be used. Specifically, microorganisms belonging to the above-mentioned genus, more specifically Pseudomonas frassii (Ps.
i), Micrococcus varians (Mi.varia
ns), Pseudomonas fluorescens (Ps.f) having the ability to synthesize an ester of an organic carboxylic acid and an alcohol
luorescence), Gluconobacter oxydans oxidation bacteria for oxidizing a carbinol group to Ca Ruboki <br/> sill group (Gl.oxydans), Pseudomonas putida (Ps. p utida), to the carboxyl group of an aldehyde group performing an oxidation reaction Gluconobacter-Gurukonikasu (Gl.gluc o nicus), 2 grade oxidation reaction capable Acetobacter Asechigenasu alcohol (Ac.acetigenus), similarly, Acetobacter which may perform oxidation reaction of the secondary alcohol Ac.acetosus, Alysobacter pacens, which selectively oxidizes the 1-hydroxyl group of glycols, and Alcaligenes, which selectively oxidizes the 1-hydroxyl group of glycols.
Alfa facalis, aldehyde,
Candida utilis (Ca. utilis), which has the ability to reduce ketones to alcohols, hansenula anomala (Ha. Anomala), which has the ability to reduce aldehydes and ketones and the hydrogenation of double bonds, and hydroxylation of double bonds Aspergillus oryzae (As.oryza)
e), Arislovobacter simplex (Ar. simplex) which has the ability to cleave oxidative side chains of cholesterol
x), Rhodococcus Exports Lee (Rh.equi), Nocardia transformer Valentin cis (No.transba
lens) and the like.

The microorganisms can be adhered and immobilized on the carrier by, for example, coating or spraying the microbial cell dispersion on a carrier previously containing an aqueous medium containing a nutrient source, or immersing the carrier in the microbial cell culture solution. Or, after attaching the microbial cells to the carrier by a method such as attaching the microbial cells to the carrier by an appropriate means and then supplying an aqueous medium containing the nutrient source to the carrier, the carrier is previously fed with the nutrient source. It is also possible to culture in an aqueous medium containing, but usually, in the state of contacting with an organic solvent with or without an organic compound as a substrate, the adhered microbial cells of the carrier and the organic solvent It can be carried out by growing at the interface and forming an immobilized bacterial cell phase on the carrier. By this culturing, the microorganisms adhere strongly to the surface of the carrier, and the immobilized bacterial cell phase hardly peels off from the surface of the carrier.

As the nutrient source of the microorganisms that can be used in the above culture, an optimum one can be selected depending on the type of bacterial cells used, and for example, a carbon source such as glucose or nitrogen such as urea. The source can be a general one consisting of a trace metal salt such as magnesium sulfate and a trace nutrient such as yeast extract.

Culturing can generally be carried out in a culturing apparatus such as a thermostat or an incubator, or the carrier is immersed in an organic solvent containing or not containing a substrate, and optionally an aqueous medium containing a nutrient source is added. It may be carried out while controlling the temperature in the reaction vessel. The culture conditions such as the culture temperature and the culture time can be selected according to the type of the microorganism used and suitable for each microorganism.

The organic compound as a reaction substrate may be added from the initial stage of the above-mentioned culture, or may be added after the microorganisms have sufficiently grown to form an immobilized bacterial cell phase, or fixed from the early stage of the culture. It may be added at any time during the formation of the modified bacterial cell phase. When the organic compound as a substrate does not show substantial toxicity to the cells, the organic compound can be used as an organic liquid phase instead of the organic solvent.

In this way, the immobilized microbial cell phase can be formed on the carrier by continuously culturing the microbial cells attached on the carrier in contact with the organic solvent.

Further, the microorganism conversion reaction of the organic compound is carried out by continuing the culture of the immobilized bacterial cell phase on the carrier in contact with the organic compound as the substrate or the organic liquid phase consisting of the organic solvent solution. Can be made.

As the substantially water-insoluble or insoluble organic compound that can be used as a substrate for this microbial conversion reaction, various compounds can be used depending on the conversion ability of the immobilized microorganisms, and there is no particular limitation. Without, for example, benzene, toluene, xylene, ethylbenzene, styrene,
Aromatic hydrocarbons such as naphthalene and phenanthrene;
Aliphatic hydrocarbons such as tridecane and tetradecane; alicyclic compounds such as cyclohexanol and cyclohexanone; heterocyclic compounds such as methylimidazole, collidine and picoline; lauric acid, palmitic acid, stearic acid, oleic acid, linole Higher fatty acids such as acids; higher alcohols such as octyl alcohol, decyl alcohol, lauryl alcohol, cetyl alcohol, stearyl alcohol; fatty acid esters such as ethyl caprylate and ethyl caproate.

On the other hand, the organic solvent for dissolving these organic compounds is preferably one which is not substantially toxic to adherent cells, and specifically, the above-mentioned normal paraffins, alkylbenzenes and alicyclic rings are used. Formula hydrocarbon ethers etc. are mentioned.

The substrate concentration in the organic solvent is not particularly limited and can be determined depending on the toxicity to the bacterial cells. For example, when the substrate is the above-mentioned aromatic hydrocarbon having strong toxicity, it can be added at a concentration of about 5% to 30% with respect to normal paraffin which is a methane hydrocarbon having 10 to 15 carbon atoms. Further, when the substrate is a fatty acid ester having relatively weak toxicity, it can be added in an amount of 50% or more with respect to normal paraffin.

Among the substrates, the higher fatty acid esters, which are particularly less toxic, do not mix with normal paraffin and
It can be used as a conversion substrate as well as an organic solvent which is brought into contact with the bacterial cell phase on the immobilized carrier at a concentration of 00%, that is, as a conversion substrate.

Similar to higher fatty acid esters, aliphatic hydrocarbons are also particularly less toxic when used as a substrate, and are themselves used as an organic solvent for contacting the bacterial phase on the immobilization carrier and as a conversion substrate. To be

According to the above-mentioned method of the present invention, esterification, hydrolysis, ester conversion, redox, amination and desorption of organic compounds such as aliphatic, aromatic, alicyclic and heterocyclic compounds. The amination reaction and the like can be carried out extremely efficiently using immobilized microorganisms. In that case, if a side reaction may occur, it should be blocked by the addition of an appropriate metabolic or conversion inhibitor, or a microorganism improved to prevent such side reaction should be used. it can.

According to the method of the present invention, microbial cells are vigorously proliferated at the contact interface between the hydrophilic carrier and the organic liquid phase to obtain an immobilized proliferating microbial cell phase, and microbial conversion specific to the microbial cells is obtained. Although the detailed reason why the reaction can be performed is unknown, it is considered as follows . That is, it is known that not only microorganisms but also those that adhere to the support grow vigorously as compared with the free ones in the culture and growth of cells. Also solid
Microorganisms attached to the body surface activate oxidation reaction and DNA synthesis
It is also known to turn into. Therefore, in the present invention, it is considered that the microorganism grows stably by attaching it to the hydrophilic carrier. It is possible that the interfacial energy generated at the interface between the carrier and the organic liquid phase contributes to the stabilization of the microorganism.

Further, as compared with the case of the microbial conversion reaction at the liquid / liquid interface such as an emulsion system, the microbial conversion at the immobilized bacterial cell phase formed at the interface between the carrier and the organic liquid phase in the present invention. In the reaction, a very high concentration of the conversion substrate or product can be present in the organic liquid layer, and the toxicity of the conversion substrate or product, which is harmful to the cells, to the cells due to the presence of the conversion substrate or product in the organic liquid layer. It may be reduced. Furthermore, since the enzyme solubility of the organic liquid phase is several to ten times greater than that of water, it is considered that a growth promoting effect or a reaction promoting effect may occur.

When the substrate and the product of the microbial conversion reaction are hydrophobic, the reaction product is accumulated at a high concentration in the organic liquid layer. Thus, according to the method of the present invention, the frequency of contact between the immobilized bacterial cell phase and the substrate is dramatically increased by collecting the reaction product accumulated in the organic liquid layer and supplementing the substrate. It is possible to increase the reaction rate, yield, and yield substantially the same, and continuous operation becomes possible.

On the other hand, when the substrate for the microbial conversion reaction is hydrophobic and the reaction product is hydrophilic, the reaction product is accumulated inside the hydrophilic carrier on the side of the aqueous layer. In this case, if the reaction product is toxic to the growing cells, in order to prevent product inhibition,
A device to collect accumulated products on the water layer side, for example, by growing microbial cells on only one side of the membranous carrier, contacting the growth surface with the organic liquid layer side and the opposite surface with the water layer side, It is desirable to use a method such as recovery from the water layer side. However, even if the product is not toxic, the microbial conversion reaction may be carried out by this method, whereby the product can be recovered from the aqueous layer side.

The microbial conversion reaction in which an organic compound as a substrate is brought into contact with the immobilized vegetative bacterial cell phase according to the present invention is compared with a conventional microbial conversion reaction in which an emulsion system is used as a substrate,
It is possible to set the substrate concentration 10 times or more, and in some cases 100 times or more, without the need for vigorous aeration and agitation for substrate dispersion and oxygen supply, and the reaction product with high reaction rate and high yield. Can be obtained, and various industrially advantageous advantages can be obtained.

[0040]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. Parts and% are based on weight.

Example 1 A glass bottle (20 ml capacity) which can be tightly closed was placed in polyppetone 1
%, Yeast extract 0.2%, magnesium sulfate 0.1%, and agar 1.5%, agar medium (pH 7.0 ) of 8 m
Each aliquot was dispensed, sterilized by high pressure steam and cooled to prepare an agar plate. Various microorganisms as shown in the following Table 1 were inoculated to this using a toothpick, and 5, 10, 15, 20, 25, 30%
The carbon tetrachloride / normal paraffin solution of each concentration was overlaid to a depth of 2 to 3 mm, and static culture was performed at 30 ° C. for 3 days. After culturing, those without overlay were used as a comparison, and the presence or absence of the formation of the immobilized cell layer due to the growth of the microorganism at each carbon tetrachloride concentration was confirmed by the method of determining the colony formation. The results are shown in Table 1 below.

As can be seen in Table 1, 10-25% in the genus Pseudomonas and 10-20% in the genus Arythrobacter.
%, 20% in the genus Bacillus, 5-1 in other bacterial groups
The formation of an immobilized bacterial cell phase was confirmed under a high carbon tetrachloride concentration of 5%.

[0043]

[Table 1]

Example 2 In the same manner as in Example 1, various microorganisms shown in Table 2 below were examined for their protective effects against the toxicity of toluene at the solid / liquid interface. As a result, 1 in Pseudomonas
0-25%, Arislovobacter genus 10-20%, Bacillus genus 10-15%, other microorganisms 5-25%
It was confirmed that toluene can be added at a very high concentration.

[0045]

[Table 2]

Example 3 A calcium alginate plate coagulated with an aqueous solution of calcium chloride was immersed in a liquid medium containing 1% of calcium chloride in the same medium composition as used in Example 1 to replace the inside of the gel with the medium. The microorganisms shown in Table 3 were inoculated with a toothpick. A styrene / normal paraffin solution having each concentration of 5, 10, 15, 20, 25, and 30% was added to this to a depth of 2-3.
The cells were layered so that the thickness became mm, and static culture was performed at 30 ° C. for 5 days. After culturing, formation of an immobilized bacterial cell phase due to growth of microorganisms was confirmed by judging colony formation. The results are shown in Table 3 below. 10 to 25% of Pseudomonas, 15 to 25% of Alislovobacter, and 15 to 3 of Bacillus.
The resistance to styrene was confirmed to be 0%, and 5 to 25% for other microorganisms.

[0047]

[Table 3]

Example 4 A section of a silica gel plate for thin layer chromatography was impregnated with a liquid medium having the same composition as used in Example 1, and then Rhodococcus equi (Rhodococcus) was used.
equi) JCM 6817, Bacillus subtilis
Sub-Spithesis Nieger (Bacillus sub
tilis subsp. niger) IFO3108
And Hansenula Sir Chu Le eggplant (Hansenul
a saturnus) IFO 0809 culture medium was smeared, and these were inoculated with ethylbenzene / normal paraffin solutions having respective concentrations of 5, 10, 15, 20, 25, and 30%. After static culturing at 30 ° C. for 7 days, the formation of the immobilized bacterial cell phase was confirmed by the presence or absence of colony formation. As a result, J
CM6817 20%, IF03108 30% IFO
With 0809, colony formation was confirmed at an ethylbenzene concentration of 10%.

Comparative Example 1 Of the microorganisms tested in Example 1, Pseudomonas spp. (IFO 13696), Arislovobacter spp. (J
CM 1363), (IFO 12959) and Bacillus (IFO 3108), (IFO 14357), a total of 5 strains, were subjected to a toxicity test of carbon tetrachloride in an emulsion system. A liquid culture medium having the same composition as that used in Example 1 was inoculated with a 1-day culture solution of the microorganism at a 1% level, and carbon tetrachloride was added thereto at 0.5, 1.0, 1.5 and 2.0%. The resulting mixture was added as described above and cultured at 30 ° C. for 2 days with shaking. Wavelength 6 after culture
The presence or absence of proliferation was confirmed by measuring the absorbance at 60 nm.
As a result, in all the strains, carbon tetrachloride concentration,
It was confirmed that it could not grow at 1.0% or more.

Comparative Example 2 Among the microorganisms used in Example 2, Pseudomonas sp.
FO 3238), (IFO 13696), genus Arislovobacter (JCM 1363), (IFO 1295)
9), Bacillus (IFO 3108), (IFO 14
357) Corynebacterium (IFO 1268
4), (IFO 14136) and Saccharomyces (IFO 1346)), a total of 9 strains were subjected to a toxicity test of toluene in an emulsion system. The method was according to Comparative Example 1. As a result, no growth was confirmed in any of the microorganisms at a toluene concentration of 1.0% or more.

Example 5 Pseudomonas fradi IFO 34, a lipase-producing bacterium
58 was smeared on a nutrient agar plate having a surface area of 38 cm ² using a conradi stick, and 5 ml of a 40% solution of ethyl caprylate in normal paraffin (a methane hydrocarbon solvent having 10 to 15 carbon atoms) was overlaid thereon. After stationary culture at room temperature for 5 days, an immobilized bacterial cell phase was formed on an agar carrier, and ethyl caprylate was hydrolyzed. After culturing, the normal paraffin layer was analyzed by gas chromatography and found to be 4.9 g / l.
The production of caprylic acid was confirmed.

Example 6 Folded folding filter paper having a surface area of 1600 cm ² (20 × 40 cm) was placed vertically in a reaction tank (reactor), and a lipase-producing bacterium Pseudomonas fradi IFO 12049 was used.
200 ml of the culture solution for 1 day and night was poured, and the mixture was left for 5 minutes to attach the bacterial cells to the filter paper. Then, the culture solution was removed, 20 ml of a culture medium containing no bacterial cells was placed in the bottom of the reaction tank, 150 ml of ethyl caprylate was injected into the upper layer, and the mixture was cultured at 30 ° C. for 3 days while introducing air into the reaction tank, and then filtered. An immobilized bacterial cell phase was formed on and hydrolyzed with ethyl caprylate.
After completion of the culture, the ethyl caprylate layer was collected, and caprylic acid was quantified by gas chromatography. The results are shown in Table 4 below.

Comparative Example 3 200 ml of a one-day culture solution of the lipase-producing bacterium Pseudomonas fradi IFO 12049 was placed in the reaction tank (excluding filter paper) used in Example 6 so that ethyl caprylate had a substrate concentration of 5%. In addition, the reaction mixture was suspended and dispersed while introducing air into the reaction tank, and cultured for 3 days. After culturing, the ethyl caprylate layer was separated, and caprylic acid was quantified by gas chromatography. The results are shown in Table 4 below.

[0054]

[Table 4]

Example 7 The oxidation reaction of a terminal carbinol group by Gluconobacter oxidans IFO3189, which is an oxidizing bacterium of medium chain fatty alcohol, was examined. 10 g of calcium alginate beads was added to 200 ml of a culture solution of Gluconobacter oxydans IFO 3189 for one day, and left standing for 5 minutes to attach microbial cells to the surface of the beads. After that, the beads were taken out of the culture solution to remove excess water, and then the beads were put into the reaction tank of Example 2 (excluding filter paper) containing 150 ml of normal paraffin, and cultured under aeration for one day. An immobilized bacterial cell phase was formed on the surface of the beads.
Then add normal octanol to 5%,
The cells were cultured at 30 ° C. for 5 days while continuing aeration to carry out an octanol oxidation reaction. After culturing, the concentration of octylic acid in normal paraffin was analyzed by gas chromatography. The results are shown in Table 5 below. Comparative Example 4 200 ml of a one-day culture solution of Gluconobacter oxidans IFO 3189 was placed in the reaction tank (excluding filter paper) used in Example 7, and 10% normal octanol was added so that the substrate concentration became 0.5%. Normal paraffin containing is added and suspended and dispersed while introducing air into the reaction tank,
It was cultured at 30 ° C for 5 days. After culturing, the normal paraffin layer was separated and analyzed for octylic acid by gas chromatography. The results are shown in Table 5 below.

[0056]

[Table 5]

Example 8 Pseudomonas fradi IFO which is a lipase-producing bacterium
12049, Pseudomonas fluorescens IFO
3903 and Bacillus subtilis subsposis niger IFO 3108 each prepared in a glass bottle capable of sealing, 10 g of polypeptone, yeast extract 2
g, magnesium sulfate 1 g, agar 15 g and distilled water 1 l
Agar plate consisting of 150 ml (pH 7.0, surface area 44
cm ² ) was inoculated with a conradi rod, 10 ml of 2-ethylhexyl acetate was layered thereon and allowed to grow by static culture for 1 day to form an immobilized bacterial cell phase on the agar carrier. Then, 2-ethylhexyl acetate was hydrolyzed by shaking culture at 30 ° C. and 70 rpm for 5 days. After culturing, the 2-ethylhexyl acetate layer was analyzed by gas chromatography, and the results shown in Table 6 below were obtained.

Comparative Example 5 10 day-and-night culture solution of the lipase-producing bacterium used in Example 8 was used.
% Composition similar to that of Example 8 (except agar)
Acetic acid 2 to avoid substrate inhibition.
-Ethylhexyl was added at the 10% level. This 3
After culturing with shaking at 0 ° C. and 120 rpm for 5 days, the organic solvent layer was analyzed by gas chromatography. The results are shown in Table 6 below.

[0059]

[Table 6]

Example 9 Pseudomonas putida IFO 13696 and Rhodococcus rodney JC capable of oxidizing various alcohols
M 3203 was inoculated on a filter plate (manufactured by Advantech, thickness 3 mm, 44 cm ² ) impregnated with a liquid medium having the same composition as in Example 8 (except for agar), and 10% 2-octanol was added thereto. 10 ml of a normal paraffin solution containing the above was layered and statically cultured at 30 ° C. for 5 days to form an immobilized bacterial cell phase on the filter plate. After culturing, the normal paraffin layer was analyzed by gas chromatography,
Accumulation of 4.4 g / l of 2-octanone with IFO 13696 and 0.6 g / l of JMC 3203 was confirmed.

[0061] Example 10 carbonyl groups each s peptone 5g Saccharomyces Yu Ba <br/> potassium JFO0565 and Roderomaisesu-Erongisuporasu IFO 1676 having reducing ability of yeast extract 3g, malt extract 3g, from glucose 10g and distilled water 11 On a silica gel plate impregnated with a liquid medium (pH 6.0), and 10 ml of an isooctane solution containing 10% 2-octanone was layered thereon,
After static culture for 5 days at 30 ° C., a solid bacterial cell phase was formed on a silica gel plate. After incubation, the isooctane layer was analyzed by gas chromatography, IFO 056
5 g at 1.6 g / 1, 1FO 1676 at 1.5 g / 1
Accumulation of 2-octanol was confirmed.

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Claims (3)

(57) [Claims] 1. A microorganism is attached to a hydrophilic immobilization carrier,
In the presence of an aqueous medium containing a nutrient source for the microorganism, a substantially water-insoluble or sparingly soluble organic solvent is contacted with the microorganism on the carrier, and the microorganism is grown at the contact interface between the carrier and the organic solvent. And a method for immobilizing a microorganism, which comprises forming an immobilized bacterial cell phase on the carrier. 2. A hydrophilic immobilization carrier on which a microorganism is adhered and immobilized, and a substantially water-insoluble or sparingly soluble organic compound as a substrate is added to the carrier in the presence of an aqueous medium containing a nutrient source for the microorganism. A method for converting an organic compound, which comprises contacting with the above immobilized cell phase. 3. The method according to claim 2, wherein the microorganisms are adhered and immobilized on the hydrophilic immobilization carrier by the method according to claim 1.