JP2710007B2 - Microbial separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for establishing a conversion process using microorganisms in a two-phase system comprising an organic solvent containing a hydrocarbon substrate which is hardly soluble or insoluble in water and an aqueous medium. Specifically, the present invention provides an optimal organic solvent for efficient conversion and a method for efficiently separating the microorganism.

[0002]

2. Description of the Related Art There are many compounds that are hardly soluble or insoluble in water, such as steroids, terpenoids and petroleum hydrocarbons, and these are also industrially useful compounds. In addition, it can be said that most organic compounds that are industrially handled are compounds that are hardly soluble or insoluble in water, regardless of gas, liquid and solid.

Conventionally, in order to convert these liquid and solid organic compounds or derivatives thereof by microorganisms, these substrates are directly supplied to an aqueous solution to be cultured or reacted. Feed directly and stir vigorously to promote dispersion. Add a surfactant to promote dispersion. Emulsify using a homogenizer and supply. And other methods have been used.

[0004] In particular, a method for converting a solid organic compound is generally referred to as a crystal fermentation method. However, the usual method of supplying a substrate such as these is only an extension of the method of supplying a water-soluble substrate in that the substrate is directly supplied in a crystalline state, and is not effectively dissolved in a medium, so that an effective supply method is used. However, it has not been possible to efficiently improve the value of solid hydrocarbons as a raw material for microbial conversion.

[0005] There have already been many conversions by a two-phase system consisting of liquid organic compound-water, which are formed by supplying a large amount of a liquid hydrocarbon as a substrate, and these are subsequently converted by culture or reaction. However, the concept of an efficient method for supplying solid hydrocarbons was far from the concept of an efficient method for supplying solid hydrocarbons in that it was changed to an aqueous system and there was no organic compound as a solvent.

Research on enzymatic reactions in organic solvents suggests rules for optimization of biocatalysts in organic solvents using, for example, 20β-hydroxysteroid dehydrogenase and cholesterol oxidase (Laane, et. al.,
Biotechnol. Bioeng. , 30.81
1987), but is biased toward a specific reaction system such as a hydrolysis reaction.

[0007] In addition to bioreactors using organic solvents, most of those that have been put into practical use so far perform only a "single reaction with a single enzyme". The reaction system was also biased as described above, and was limited to hydrolases, isomerases and lyases. On the other hand, a development example of a transfer reaction requiring another coenzyme regeneration system, particularly a multi-step oxidation reaction in a redox reaction, is based on a crystal fermentation method.

[0008] The reason is that free coenzyme which needs to be supplied from outside the system is NAD at a price per mole.
(Nicotinamide adenine dinucleotide) about $ 1,000, ATP (adenosine triphosphate) about 8
The process of supplying these, said to be $ 00, can be very costly.

Therefore, when a coenzyme regeneration system is required and a multi-step oxidation reaction system is to be developed and an organic solvent conversion process is to be considered, the biocatalyst itself obtains energy in terms of process difficulty and cost. It is most likely that a viable cell line is used.

[0010] In recent years, Inoue, A an
d Horikoshi, K .; , Nature, 33
8, 264, 1989) and the isolation of toluene-tolerant microorganisms by Nakashima (Harushi, N., et. Al.,
Biosci. Biotech. Biochem. , 5
6 (11), 1872, 1992) reported a method for efficiently separating such microorganisms. This fact suggests that microorganisms survive even in the presence of large amounts of organic solvents and that there are bacteria that can survive even in the presence of highly toxic organic solvents. It can be said that the concept of being usable in two-phase systems was spread.

With the widespread use of this concept, a large amount of an organic solvent is interposed, and there are various types of reactions that are outside the extension of the established concept of a reaction system of a water-soluble substrate in a two-phase system using an organic solvent and are not limited to a specific reaction system. Microbial conversion of the reaction system has become realistic.

[0012] For the organic solvent-resistant microorganism,
Although there is no clear definition at present, microorganisms that can survive in an organic solvent having a LogP value of 3 or less, such as toluene and benzene, seem to correspond to this.

However, the growth limit of many conventionally known microorganisms is that the LogP value is 3 or more.
If the microorganism can survive in an organic solvent having a P value of about 4,
It is considered that the microorganism may be referred to as an organic solvent-resistant microorganism.

[0014]

The idea of supplying solid hydrocarbons dissolved in an organic solvent is to supply a solid substrate continuously and in large quantities, and thereby to realize a process capable of mass production. Although the possibility can be considered, there is no case where the microorganism and the production process which can be applied to such a special system have been put to practical use, and it can be said that there is a situation where the demonstration of the possibility is just desired.

[0015] Further, in a production field from a laboratory production experiment to an industrial production experiment, foaming is always a problem in an aqueous system. This is a very big concern because it always causes a decrease in the dissolved oxygen concentration and a restriction on the amount of aerated gas and the stirring speed.

As a countermeasure, an antifoaming agent such as silicone or soybean oil is usually used, but the effect is not long-lasting, so that the process becomes unnecessarily complicated or hinders improvement in productivity. Many factors were not satisfactory.

Further, even if a method of dissolving and supplying a solid hydrocarbon in an organic solvent is adopted, it is necessary to select an industrially effective organic solvent. The required properties of organic solvents include those that do not volatilize in terms of safety and economics in operation and equipment, and those that have moderate microbial toxicity and microbial Are not assimilated.

On the other hand, when considering microorganisms to be used in such an organic solvent system, all microorganisms having resistance to the organic solvent can be used in a conventional aqueous crystal fermentation environment in the absence of an organic solvent. It is not always the case that the function of efficiently converting the specific hydrocarbons that are expressed can be efficiently expressed in the presence of a resistant organic solvent.

That is, even if a microorganism capable of converting specific hydrocarbons has the ability to withstand organic solvents, it often cannot express the conversion ability of specific hydrocarbons dissolved in organic solvents. In particular, such a tendency is remarkable in a microorganism isolated from a separation method based on a conventional crystal fermentation method.

In other words, in a conventional separation method, microorganisms having conversion ability in the presence of an organic solvent and microorganisms having no conversion ability are mixed and separated, so that a large number of organic solvents having poor resistance to organic solvents or having a low conversion ability. However, there is a problem that microorganisms having no function as described above become noise and extremely hinder the separation efficiency of microorganisms having a desired conversion ability.

In addition, there has also been a problem of how to efficiently obtain a microorganism having a conversion ability in a selected industrially effective organic solvent system.

As described above, in the two-phase system of organic solvent-water,
In order to set up a conversion reaction process using microorganisms, it is extremely important to determine which organic solvents are effective and how to efficiently separate and acquire microorganisms that can be used in this organic solvent system. It can be said that this is an important key for the development of the organic solvent-water two-phase conversion reaction process.

[0023]

That is, the present invention relates to an efficient method for separating microorganisms capable of efficiently converting a substrate contained in an organic solvent in the presence of an organic solvent, and an efficient culture method or reaction method. Once a series of processes have been established, bioreactors based on organic solvent systems containing large amounts of organic solvents can be industrially feasible even in redox reactions requiring coenzyme regeneration, which has never been seen before. It can be said that it becomes possible. Therefore, it will also be of great use to the petroleum and petrochemical industries as a new organic compound conversion process in an environmentally friendly and environmentally friendly form.

Under these circumstances, the present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the present inventors have found that an efficient, environmentally-friendly, safe and inexpensive microorganism-producing substance can be industrially effective. The present inventors have found a method for selecting an organic solvent and an efficient method for separating microorganisms which can be converted with these organic solvent systems, which enables a simple conversion process, and have accomplished the present invention.

The present invention relates to the selection of an organic solvent and the use of the hydrocarbon dissolved in the organic solvent, which enables an industrially effective conversion process for the production of substances by microorganisms in a safe, inexpensive and efficient manner. Can be grown or converted as a carbon source, an organic solvent-an efficient method for separating biphasic conversion microorganisms of water, taking as an example the microbial conversion of 2,6-dimethylnaphthalene as a solid hydrocarbon, An object of the present invention is to provide a multi-step oxidation reaction process in a two-phase system, which has not been demonstrated so far.

According to the method of the present invention, a large amount of an organic solvent can be used in the conversion of a substrate by a microorganism, so that foaming in the system can be strongly suppressed and the amount of aeration and stirring speed can be drastically increased. be able to. In particular, it can be a very effective method for a microbial reaction requiring a high dissolved oxygen concentration. In addition, since the substrate has been solid until now,
The continuous substrate supply, which was impossible, becomes possible, and the toxicity of the organic solvent greatly reduces the probability of bacterial contamination. Further, effects such as an increase in the reaction speed are expected. This method is naturally effective not only for solid hydrocarbons but also for liquid hydrocarbons.

Solvents for dissolving the solid hydrocarbon substrate include, for example, aliphatic hydrocarbons such as n-pentane and n-pentane.
-Hexane, n-octane, n-decane, 2-pentene, 2-hexene, 1-octene and the like, cycloaliphatic hydrocarbons, cyclopentane, cyclohexane, methylcyclohexane, cyclooctane, tetralin, decalin, etc. Among aromatic hydrocarbons, benzene, toluene, p-
Xylene, ethylbenzene, chlorobenzene, bromobenzene, styrene, etc., alcohols, ethanol, butanol, heptanol, octanol, etc.,
Examples of ethers include diethyl ether, n-hexyl ether, diphenyl ether, benzyl ether and the like.

On the other hand, as the properties of the organic solvent required for culturing or reacting the microorganisms, those which do not volatilize are safe in equipment and operation and are economical. In addition, if the microbial toxicity is too strong, it may affect the health of the operator and decrease the efficiency of separation of useful bacteria.If the microbial toxicity is too weak, there is a risk of contamination of various bacteria during culture or reaction. Therefore, the advantage as an organic solvent-based bioreactor is reduced by half.

Then, as a result of repeating various investigations and microbial tests on these matters, ethers were not so preferable, and aliphatic hydrocarbons and alicyclic hydrocarbons were good. In view of the above reasons in selecting a solvent, those selected as preferred organic solvents by the present inventors include alicyclic hydrocarbons, more preferably decalin and tetralin, and even more preferably decalin.

Decalin (decahydronaphthalene) has a boiling point of 197.54 ° C. (cis) and 187.2 ° C. (tra).
ns), having a flash point of 57.8 ° C (cis, closed), and tetralin (tetrahydronaphthalene) having a boiling point of 207.6 ° C and a flash point of 71.1 ° C.

From these properties, it can be said that these two types of organic solvents are suitable for long-term culture such as separation culture and production culture of microorganisms among nonpolar solvents. Regarding the effect on productivity, the productivity was favorable among the solvents studied and examined. Polarity values were used as indicators of the toxicity of organic solvents. This polarity value is represented by a common logarithmic value (Log P value) of the partition coefficient value in the aqueous phase and the octanol phase in a two-phase system of an organic solvent, water and octanol. The smaller the value of the LogP value, the stronger the polarity.
That is, the toxicity increases. This polarity value is also used as an indicator of microbial toxicity.

The microbial toxicity (polarity value) was found to be higher for tetralin than for decalin in tests with microorganisms.
It was also found that it was not as toxic as toluene and benzene.

In order to confirm the growth limit of microorganisms by various organic solvents, the degree of resistance to organic solvents was evaluated. This is a medium in which the microorganism grows well, each organic solvent having a known polarity value is overlaid, the growth limit when cultured is expressed by the polarity value of the organic solvent, and used to determine the presence or absence of organic solvent resistance. .

That is, the present invention provides, when separating microorganisms, a two-phase solution comprising an aqueous medium containing no carbon source and an alicyclic hydrocarbon such as decalin or tetralin containing a hydrocarbon substrate. The basic technical concept of the present invention is a method for separating microorganisms, which comprises culturing, applying this culture solution to a plate medium, and further culturing by supplying a substrate.

Therefore, according to the separation method of the present invention,
The target microorganism can be isolated by dissolving only the substrate as a carbon source in decalin or tetralin or the like, and screening the microorganisms grown by the two-phase culture to confirm the production ability of the produced substance. In a two-phase system using an organic solvent such as tetralin and / or the like, when transforming a substrate by culturing or reacting with a microorganism, microorganisms having a conversion ability can be separated in these systems. Furthermore, by arbitrarily selecting a hydrocarbon as a substrate which is a carbon source, it becomes possible to efficiently separate a microorganism having a conversion ability for a specific hydrocarbon.

The details of the separation method of the present invention are as follows.
-An example of a microorganism having a conversion ability using dimethylnaphthalene and decalin as an effective solvent is described below. Moreover, the microorganisms are not limited to the exemplified microorganisms as long as they are separated by the separation method of the present invention.

The present inventors dispensed an aqueous medium (hereinafter, referred to as a medium) containing no carbon source from among the components necessary for the growth of microorganisms in soil collected from all over the country into test tubes and the like.
Further, after adding an organic solvent in which 2,6-dimethylnaphthalene is dissolved, for example, decalin to which 2,6-dimethylnaphthalene is added and sterilizing, soil is added, and culture is performed using a test tube shaker or the like. This culture solution is previously used for the same
-After passing the organic solvent in which dimethylnaphthalene is dissolved and the medium to another test tube or the like into which mixed and dispensed, the cells are further cultured by a test tube shaker or the like. This culture solution or a culture solution diluted with sterile water or the like is applied to an agar medium or the like, and 2,6-dimethylnaphthalene dissolved in an organic solvent is supplied to further culture. After the culture, the formed colony may be isolated.

Further, for example, when isolating a microorganism that produces 2,6-naphthalenedicarboxylic acid, the ability to produce 2,6-naphthalenedicarboxylic acid for each strain is confirmed. Producing microorganisms can be selected.

As a medium for culturing these microorganisms, general medium components can be used. That is, as the nitrogen source, for example, ammonium chloride, ammonium phosphate,
Ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, sodium nitrate, urea and the like, as inorganic salts, for example, potassium, sodium, iron,
Salts such as magnesium, manganese, copper, and calcium can be used. In addition, the culture conditions may be any culture conditions in which microorganisms generally do not die, for example, a pH of about 5 to 9,
It is performed aerobically at a temperature of about 20-40 ° C.

The culturing or reaction conditions may be such that the separated microorganisms can be grown or reacted without killing them. For example, the culturing temperature is about 15 to 37 ° C., and more preferably about 25 to 35.
C., the pH of the medium is about 4.2 to 8.9, more preferably about 6.0 to 8.0, and the culture or reaction may be performed aerobically for about 1 to 30 days.

The step of producing a culture product from 2,6-dimethylnaphthalene using the microorganism may be a batch type or a continuous type using a bioreactor or the like.

Further, the microbial cells can be washed with a solution such as a phosphate buffer and suspended in the solution before use.

Next, an example of the 2,6-dimethylnaphthalene assimilating microorganism obtained by the separation method of the present invention will be described.

As a result of conducting various microbiological tests and a test using API20NE (BioMérieux) for identification of non-fermentative bacteria other than intestinal bacteria, they had the mycological properties shown in Table 1.

[0045]

[Table 1]

The mycological properties shown in Table 1 were
Y'S MANUAL OF SYSTEMATIC
BACTERIOLOGY, vol. 1, P198 (1
984), INTERNATIONAL JOURNA
L OF SYSTEMATIC BACTERIOL
OGY, vol. 40, no. 3, P320-321
(1990), classified by API20NE (BioMérieux) profile index, it was recognized as belonging to Sphingomonas paucimovilis.

Based on the above bacteriological properties, the present inventors determined that the bacterium was a new strain belonging to the genus Sphingomonas, and determined that the bacterium was Sphingomonas paucimobilis AK2M16 (Sph
ingomonas paucimobilis AK
2M16) strain and deposited (FERM P-13996) with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

The microorganism obtained by the above separation method is
Although it has the ability to oxidize the methyl group at the 2,6-position, the separation method of the present invention is not limited to microorganisms belonging to the species Sphingomonas sp.

[0049]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.

[0050]

Example 1 Using 2,6-dimethylnaphthalene as the sole carbon source, it was dissolved in decalin, and screening was performed to separate microorganisms that grow in a heterogeneous system (two-phase system) using this and an aqueous medium. The medium used had the components shown in Table 2. When these were dissolved in deionized water, the pH became 7.

[0051]

[Table 2]

(Separation of 2,6-dimethylnaphthalene-assimilating microorganisms) For soil collected from all over the country, among the components necessary for the growth of microorganisms, a medium containing the components shown in Table 2 and containing no carbon source was used. Place 8 ml into a test tube with an inner diameter of 21 mm, add 2 ml of decalin containing 1 wt% of 2,6-dimethylnaphthalene, sterilize at 121 ° C. for 20 minutes, cool at room temperature, and add 1-2 tablespoons of soil to test. Shaking culture was performed at 30 ° C. and 270 rpm using a tube shaker.

The culture solution was added to another test tube in which a similar medium containing 1 wt% of 2,6-dimethylnaphthalene (1 wt%) and a medium were previously mixed and dispensed with a pipette.
and subcultured again, followed by further culturing by a test tube shaker.

After the cultivation, 15 g of agar was added to the medium containing the components shown in Table 2 and the culture solution or the culture solution diluted with sterile water.
/ L, and then spread on a plate medium added to
-An ether in which dimethylnaphthalene was dissolved was sprayed, precipitated and further cultured. After culturing for 5 to 7 days, a colony formed on the plate medium and growing by forming a zona pellucida indicating that 2,6-dimethylnaphthalene was assimilated was isolated. Among them, B-2 strain, E-1 strain, G-1 strain, G
2-1 strain, AK2M16 strain, and more than a dozen strains were isolated.

(Test for Resistance to Organic Solvents) Next, five arbitrarily selected strains among the confirmed 2,6-naphthalenedicarboxylic acid-producing bacteria were tested for the resistance to various organic solvents.

The culture for the test for the degree of resistance to organic solvents is carried out as follows.
L medium showing good growth (1% bactotryptone, 0.
5% yeast extract, 1% sodium chloride, 0.2% glucose, 10 mM magnesium sulfate, pH 7.0) and an equal amount of each organic solvent, and after sterilizing and cooling, inoculate one platinum loop of each strain, 30 ° C, The cells were cultured with reciprocal shaking at 300 rpm for 2 days, and the presence or absence of growth in each organic solvent was evaluated.
Table 3 shows the results.

[0057]

[Table 3]

From the results of the organic solvent resistance test, when viewed in terms of the polarity value (Log P) of each organic solvent, the microbial toxicity of decalin (polar value Log P about 4.5) or the microbial toxicity of tetralin (polar value Log P about 4. 0) It can be seen that only microorganisms having the above-mentioned organic solvent resistance were obtained.

(Evaluation of Conversion Ability of 2,6-Dimethylnaphthalene in Two-Phase Culture) Further, even if it has sufficient organic solvent resistance, it has the ability to convert a substrate dissolved in an organic solvent in two-phase culture. Therefore, the conversion ability of 2,6-dimethylnaphthalene in the following two-phase system culture was evaluated.

5 ml of a medium containing the components shown in Table 2 and 2,6-
5 ml of decalin containing 1 wt% of dimethylnaphthalene
Was dispensed into a test tube having an inner diameter of 21 mm.
After sterilization for 0 minutes and cooling at room temperature, the separated B-2 and E-
One platinum loop was inoculated for each of the microorganisms 1, 1, G-1, G2-1, and AK2M16 strain, and cultured with shaking at 30 ° C and 270 rpm for 7 days. Table 4 shows the results.

[0061]

[Table 4]

(Measurement of Amount of Residual 2,6-Dimethylnaphthalene by Concentrated Cell Reaction) Since microorganisms other than AK2M16 strain showed good growth, the ability to convert 2,6-dimethylnaphthalene, which is the only carbon source, was confirmed. Was confirmed,
For the AK2M16 strain in which no growth was observed, the amount of 2,6-dimethylnaphthalene remaining in the organic solvent phase after the concentrated cell reaction was measured.

In a 500 ml baffled flask, 100 ml of the medium shown in Table 2 and 100 mg of 2,6-dimethylnaphthalene were placed, sterilized at 121 ° C. for 20 minutes, cooled at room temperature, and one loop of AK2M16 strain was planted. ,
Shaking culture was performed at 30 ° C. and 160 rpm for 5 days. After the culture, the proliferating cells were collected and separated by a centrifugal separator, and the collected
The suspension was suspended in a two-phase system consisting of 4 ml of the same medium in a 1 mm test tube and 4 ml of decalin containing 1% by weight of 2,6-dimethylnaphthalene, followed by shaking culture at 30 ° C. and 270 rpm for 2 days. After the cultivation, the decalin phase was separated and analyzed by gas chromatography to determine the amount of the remaining 2,6-dimethylnaphthalene. Table 5 shows the results.

[0064]

[Table 5]

From these results, it was found that although the AK2M16 strain did not grow in the biphasic culture, it had the ability to convert 2,6-dimethylnaphthalene in the biphasic culture. Therefore, it can be understood that all of the microorganisms can express the function of assimilating 2,6-dimethylnaphthalene in the two-phase culture.

Hereinafter, comparative examples will be described in order to demonstrate the remarkable effects of the present invention.

[0067]

Comparative Example 1 (Separation of 2,6-dimethylnaphthalene assimilating microorganism)
In order to isolate bacteria that grow using 2,6-dimethylnaphthalene as the sole carbon source, screening was performed by the following method.

In the same manner as in Example 1, 10 ml of a medium containing the components shown in Table 2 containing no carbon source among the components necessary for the growth of microorganisms was put into a test tube having an inner diameter of 21 mm for soil collected from all over Japan. Further, 2 mg of 2,6-dimethylnaphthalene was added, sterilized at 121 ° C. for 20 minutes, cooled at room temperature, added with 1 to 2 spoonfuls of soil, and cultured with shaking at 270 rpm at 30 ° C. using a test tube shaker. Was performed.

0.1 ml of this culture was subcultured into 10 ml of a medium having the components shown in Table 2 and 10 mg of 2,6-dimethylnaphthalene added thereto, and then shake-cultured again under the same conditions. did. One week later, a part of the culture solution was diluted with sterilized physiological saline, and applied to a plate medium obtained by adding agar to the medium having the components shown in Table 2 at a concentration of 15 g / l. A diethyl ether solution in which dimethylnaphthalene was dissolved was sprayed onto the plate medium to precipitate out, and the cells were cultured at 30 ° C for 5 to 7 days.

After the cultivation, the 2,6
-A colony indicating utilization of dimethylnaphthalene was isolated. Among these, K113-2 strain, K118-
2 strains, T374-29 strain, T44-1 strain, T272-4
8, T272-81, T272-31, A-7
And more than 200 other strains.

(Test for Resistance to Organic Solvents) Next, the resistance of various organic solvents to seven strains selected arbitrarily from among the confirmed 2,6-dimethylnaphthalene assimilating bacteria was determined in Examples. Tested in the manner described in 1. Table 6 shows the results.

[0072]

[Table 6]

As is evident from the above results, the bacterial strain obtained by the isolation method of this comparative example has a function of assimilating 2,6-dimethylnaphthalene, but a large number of strains having weak organic solvent resistance are isolated. You can see that.

(Evaluation of Conversion Ability of 2,6-Dimethylnaphthalene in Heterogeneous Culture) Further, even if it has sufficient organic solvent resistance, it has a conversion ability of a substrate dissolved in an organic solvent in heterogeneous culture. Therefore, the conversion ability of 2,6-dimethylnaphthalene was evaluated in the same manner as described in Example 1 for the strains showing resistance to decalin. Table 4 shows the results.

(Measurement of Amount of Residual 2,6-Dimethylnaphthalene by Concentrated Cell Reaction) As in Example 1, no growth was observed in strains T374-29, T44-1, and T272.
-48 strain, T272-81 strain, and T272-31 strain, remaining 2,6 in the organic solvent phase by the concentrated cell reaction.
-An attempt was made to measure the amount of dimethylnaphthalene. Table 5 shows the results.

From the above results, it is clear that according to the separation method of the comparative example, even if the organic solvent resistance is strong, there are a lot of substances which cannot express conversion ability in the two-phase culture.

[0077]

According to the present invention, hydrocarbons which are hardly soluble or insoluble in water, such as 2,6-dimethylnaphthalene, are dissolved in decalin or tetralin as a sole carbon source, and this is dissolved in a medium or medium. Microorganisms that can be cultured or reacted in a two-phase system composed of a reaction aqueous solution can be efficiently separated.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Naohisa Uemura 1-113-32 Namiki, Kawaguchi-shi, Saitama Prefecture Everlean Kawaguchi 609 (72) Inventor Yutaka Sakai 2-36 Fujigaoka, Midori-ku, Yokohama-shi, Kanagawa Prefecture 66 Green Hill Fujigaoka CII-114 Examiner Kazuo Hirata

Claims (4)

(57) [Claims] When a microorganism is separated, the microorganism is cultured in a two-phase system solution comprising an aqueous medium not containing a carbon source and an alicyclic hydrocarbon containing a substrate of hydrocarbons. A method for separating microorganisms, which comprises applying a substrate, and further culturing by supplying a substrate. 2. The method according to claim 1, wherein the alicyclic hydrocarbon is decalin and / or tetralin. 3. The method according to claim 1, wherein the microorganism is a microorganism belonging to the genus Sphingomonas. 4. The method according to claim 1, wherein the substrate is 2,6-dimethylnaphthalene.