JPH02242665A - Method for culturing microorganism - Google Patents

Abstract

PURPOSE:To enable culture of a microorganism belonging to the genus Achromobacter, etc., in the presence of an organic solvent in a high concentration by containing the organic solvent having a specific value of logarithmic partition coefficient (logP) in an amount of >=0.3% in a culture medium. CONSTITUTION:In a culture medium containing a carbon source, such as glucose, and a nitrogen source, such as peptone, is contained >=0.3% organic solvent selected from aliphatic hydrocarbons, such as hexane, alicyclic hydrocarbons, such as cyclopentane, aromatic hydrocarbons, such as benzene, alcohols, such as 1-heptanol, and ethers, such as n-hexyl ether, having a greater logarithmic value (limit logP value) than that of the minimum partition coefficient capable of growing a microorganism. The resultant culture medium is then used to culture a microorganism, belonging to the genus Achromobacter, Agrobacterium, Alcaligenes, etc., and having tolerance to the organic solvent at 20-40 deg.C. Thereby, the culture can be carried out in the presence of the organic solvent in a high concentration.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for culturing microorganisms that are resistant to organic solvents, and more specifically, a method for culturing the microorganisms in a medium containing an organic solvent that satisfies specific conditions. Regarding.

[Prior Art and Problems to be Solved by the Invention] Several methods of culturing microorganisms in media containing organic solvents have been reported, but generally, due to the toxicity of organic solvents, low concentrations or It was intended to be cultured by contacting it in the form of steam.

In recent years, organic solvent-resistant bacteria have been discovered by Bengami, and these microorganisms have been cultured in the presence of various organic solvents (aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, alcohols, ethers, etc.). A method was proposed. According to this method, cultivation can be carried out in the presence of highly concentrated organic solvents, which has advantages such as prevention of bacterial contamination and the ability to use substrates that are poorly soluble in water. It was limited.

[Means for Solving the Problems] Therefore, the present inventors have conducted repeated studies to develop a method for culturing known species of general microorganisms in the presence of a silently concentrated organic solvent. They discovered that the objective could be achieved by selecting a specific organic solvent, and completed the present invention.

That is, the present invention relates to Achromobacter (Achromobacter).
bacter) genus, Agrobacterium (＾groba)
cterium), Alcaligenes (AIcalig)
enes) H, Acromonas (＾chromonas
) genus, Acinetobacter
) genus, Urwinia genus, Enterobacter genus, Brevibacterium genus, Citrobacter genus, Flavobacterium genus, Paracoccus genus, Proteus
eus) genus, Klebsiella
Genus, Chromobacterium
ium), Rhodococcus
), Microbacterium
Genus Ler i um, Nocardia
a) A microorganism that belongs to the genus Bacillus, Corynebacterium, or Saccharomyces and is resistant to organic solvents is determined by the partition coefficient of the organic medium tolerated by the microorganism. The present invention relates to a method for culturing microorganisms, characterized in that the microorganisms are cultured in a medium containing 0.3% or more of an organic solvent having a log P value of "-" or more than the logarithm (P) of (P).

Examples of microorganisms that can be used in the present invention include Achromobacter pulicarasu and delica Lul.
us) IAM 1433 Agrobacterium tumefaciens (A, tumefaciens NI 0)
305)3. Alcaliges faecalis Ca, fasalis-) JCM 1474. Acromonas hyophila (A, -j1 row-tomochil↓bow) JC Sen1021.
−? Citroctor calcoaceticus (A, cal
coaceticus) IFO12552, Bacillus
cereus (B, cereus) IFO3131 Bacillus suffutilis (dust, sut and one basket jyu river) l (
U 1219. Brevibacterium paraminoriticum (B.

dnoli ticum) 8 TCC21195, Fiberbi Hacteri 1 Sim-) Revolume (Jiao, helvo
lum) 8TCC21342, Flavobacterium lactofermentum (B, Iactofermentum)
um) ATCC 21420, Brevibacterium ammoniagenes (Dan, u dish 1 Bvj 5) 8 TCC 137
46, Flavobacterium ammoniagenes IFO1
2072, Brevibacterium roserum (B, ro
seum) ATCC 13825, Flavum (B, flavum) ATCC 1382
6. Chromobacterium cocolatum (C, choco
latum) TFO3758, Corynebacterium glutamicum (Iodan amlc, tomo) JC gate 1318. Corynebacterium flavesiens (C, flaves)
cens) TAM 1642+ Corynebacterium hydrocarboxidans (C.

h drocarboox dans) ^TCC217
67, Corynebacterium herculis (C, her
culis) ATCC13868 + Erwinia herbicola, blood 0 differences 1" Yu^TCC21434 Citrobacter freundii ((,, freundii) A
TCC6750, Microbacterium ammoniagenesum (k.

L sister shouting stud, qe) IFO3318, Rhodococcus erythropolis (Ill, rainbow L1 ru exuberance) IFO1232
0, Rhodococcus suicidal (R, Plate ox) IFO3730,
Nocardia 1 Suhy (N, H) 8TCC2+145
．． Paracoccus denitrificans (P, denit)
rificans) IFO13301, Proteus mirabilis (P, m1rabilis) II70384
9. Satucharomyces uharum (Σ, ■arum)
There are ATCC26G02, etc.

The organic medium used when culturing these microorganisms is determined by the logarithm (log P) (it
! Determine for each microorganism by considering In other words, the lowest logP value (limit logP value) at which microorganisms can grow
The above organic solvents are selected and used.

The partition coefficient (P) is expressed as the ratio of the activity of a substance in an equilibrium state in a system of two immiscible solvents, and when the concentration of the substance is small, the concentration can be regarded as the activity. When determining solvents that can be used in biological reactions, the polarity of the solvent or the presence of TFO3084, Enterobacter
・Cloacae (E, cloacae) Aqueousity is used as an index, but if log P 'pi is used as an index of the polarity of the medium, a good correlation with biological reactions can be obtained, and in a polar medium with log P < 2, the reactivity is It is known that the reactivity is low, medium reactivity in solvents with loB P-2 to 4, and high reactivity in nonpolar solvents with logP>4 (C, Laan
e, S. Boeren, and K. Vos;
TrendsBiot<! chno1. 3 2511
985 and C, Laane 51iocren,
K, Vos, and C, νeeger; Biote
chnoloByand Qioenzineerin
g+ 30+ 81.1987).

The present inventors have elucidated a method for identifying an organic medium that can be used as an indicator for the growth of microorganisms in the presence of a stepped container, as described above (loBP value).

The IogP value can be determined by the following method.

(1) Calculation method The log P value is expressed as the sum of the hydrophobicities of each component constituting the solute molecule.
The hydrophobic substituent constant π8 in the compound introduced into I) is represented by the following formula.

πX =log PM-1og P, IIogP is expressed as the sum of πX of the substituent constituting the substance and the logarithm (10gP11) of the distribution coefficient of the group t% substance.

Here, although it is desirable to obtain the log P or π value experimentally, the /!
i aqueous fragment constant (hydropt+obicf
ragmantal consLanL:f),
Log P can be calculated from log P = Σ[(Nys, G, G,, l1ekkerR, Fl,
Chim, Thcmp, 5.521+ 1973
; R,F, Rekker.

1'he 1lydropobic Fragmen
tal Con5tanL, 1EIsevierNe
iv York, 1977), and Table 1 lists the log P values calculated by this calculation method. However, p
- For xylene, I was determined by the method (2) below.
ogP (Direct.

(2) Experimental method (Flask shake)
ng method) Is it possible to place ζ between an organic solvent (e.g. n-octatool) and water? It is determined by finding the concentration ratio of 8￥r. Take fixed volumes of each of an organic solvent and water that have been saturated with the other phase, and place them into a Mayer. Usually, the drug is dissolved in one of the phases in advance, and the concentration of both phases after the conversion is determined by an appropriate method (usually a colorimetric method) to determine the partition coefficient P.

When measuring, the drug concentration is varied and measured, and the value in the region where the value of I) does not change depending on the concentration is adopted (Imo 1)-1
Hiroshi, Chemistry Area, Masu Isu No. 122, ■] 731979)
.

The log P value of various organic solvents is determined by the above method, and a growth test is performed for each microorganism in an organic solvent with a predetermined log P (lI'!).
Determine the P value (limit IogP value) and calculate this limit log P
Cultivate the microorganism using an organic solvent that exceeds the specified value.

11 [1. The cultivation of microorganisms is carried out using a medium that contains nutritional substances necessary for the growth of microorganisms and is compatible with the purpose of bacterial cell production, 2. Saccharides such as hydrolysates, hydrocarbons such as Hensen, toluene, p-xylene, ethylaldi-1-
carbon source selected from alcohols such as alcohol; nitrogen source selected from peptone, meat extract, yeast extract, cornstarch liquor, ammonium casamino acid sulfate, sodium nitrate, urea, etc.; contains inorganic salts and trace components Medium LB medium, peptone-yeast extract-glucose medium, etc. are used.

In addition, the n-containing solvents having a log P value or higher include aliphatic hydrocarbons such as hequinane, heptane, octane, isooctanenonane, decane, and dodecane, alicyclic hydrocarbons such as cyclopenkune, cyclohexane, and cyclooctane, Hensen, Toluene, P-xylene, styrene, ethibenzeneprobylbenzene, chlorobenzene,
It is selected from aromatic hydrocarbons such as 0-dichlorohenzene, alcohols such as 1-heptatool, ■-octatool, and 1-decanol, and ethers such as n-hexyl ether, diphenyl ether, and methoxytoluene. The concentration of the organic solvent in the medium is 0.3% or more,
It is usually used in a range of 0.5 to 50%, preferably 10 to 50%.

Cultivation is carried out at a temperature of 20 to 40°C, preferably 30 to 37°C, for 1 to 4 days, preferably 1 to 4 days, considering the properties of the microorganism.
It will be held for ~2 days.

〔Example〕

Next, the present invention will be explained by examples.

Various strains were inoculated into Odor Example YuLB medium (10 g of Bacto tryptone, 5 g of Bacto yeast extract, if distilled water) and cultured overnight. This was inoculated into a solid LB medium prepared in another glass petri dish using a replica or a platinum loop for each strain, and plates corresponding to the number of organic solvents to be examined were prepared.

10 ml of the specified organic solvent shown in Table 1 was added to each plate, the plate was sealed with tape and cultured at 30"C for two nights, and the growth of the bacterial strain was examined.

As a result, each strain has a log
It was confirmed that the phyllodes can grow in organic solvents with a P value or higher.

*Strain number l Citrohakuku fureranbui ^TCC67502
Achromobacter pricallus 1^M 14333
To Erwinia herhycola TCC214344 Acinetobacter calcoaceti #0125525 Enterobacter cloaceae IFO33206 Proteus
mirabilis IF038497 Flavobacterium ritesiens IF030848 Alcaligenes faecalis JCM 14749 Achromonas hydrophyso JCM 102710 Agrobacterium tumefaciens 1Ii0305811 Nocardia sp. ATCC2114512 Bacillus subtilis A
11ll 121913 Bacillus cereus IFO
313114 Klebsiella pneumonia i03318
15 Brevibacterium balanoiharaicum AT
CC2119516 Brevibacterium ammoniagenes ^1' CC1374617 Flavobacterium roserum ATCC 1382518 Corynebacterium frangheciens IAM 164219 : I Linehacterium human IIItI Runexidasis ATCC21
76720 Corynebacterium herculis ^TCC
1386821 Flavobacterium suaveolens I
F0315222 Microbacterium ammoniagenesm ATCC1535423 Rhodococcus erythropolis IFO1232024 Corynebacterium
Glutamicum JCM 131825 Brevibacterium herborum^TCC2134226 Brevibacterium lactofermentum ATCC2142027
Brevibacterium flavum ATCC1382628
Brevibacterium ammoniagenes IPO12Q7
229 Chromobacterium cocolatum IF037
5830 Baracoccus denitrificans IP0
1330131 Rhodococcus equi IFO373
032 Satucharomyces Uharum ^TCC26602
Example 2 1.0 g of glucose, 2.5 g of yeast, 5.0 g of peptone, and 1.0 g of distilled water. Add ON and prepare the medium to pl+7.2, dispense 100 m2 into a 500 ml Erlenmeyer flask, and add Citrobacter flerambui ATCC6.
After inoculating 750 strains and adding 30 m2 of propylbenzene, they were cultured with shaking at 30°C for 48 hours.

As a result, 80 n+g of dried bacterial cells of 50 strains of Citrobacter frerumbui ATCC6 were obtained.

Example 3 The same procedure as in Example 2 was carried out except that Enterobaratarcroacet strain IP03320 was used as the bacterial strain and n-hexane was used as the organic 6 medium, to obtain 90 mg of dried bacterial cells.

Example 4 The same procedure as in Example 2 was repeated except that Acinetobacter calcoaceticus strain IF012552 was used as the bacterial strain and n-hexane was used as the organic 6 medium, and 90 microorganisms of dried bacterial cells were obtained. Ta.

Example 5 Proteus mirabilis I as the bacterial strain in Example 2
The same procedure as in Example 2 was carried out except that F03849 strain was used and n-hexane was used as the organic solvent, and 110 mg of dried bacterial cells was obtained.

Example 6 Bacillus subtilis AH was used as the bacterial strain in Example 2.
The procedure was carried out in the same manner as in Example 2, except that the U 1219 strain was used and n-hexyl ether was used as the organic solvent.
120+ng of dried bacterial cells were obtained.

Example 7 Nocardia sp. A was used as the strain in Example 2.
The procedure was carried out in the same manner as in Example 2 except that TCC21145 strain was used and isooctane was used as the organic solvent, and 80 mg of dried bacterial cells were obtained.

[Effects of the Invention] According to the present invention, microorganisms can be cultured in the presence of a highly concentrated organic solvent by selecting an organic solvent that satisfies specific requirements. Therefore,
In addition to being able to culture microorganisms without worrying about contamination (by contaminants) and using substrates that are sparingly soluble in water, it is also possible to efficiently produce products that are sparingly soluble in water.

Claims (2)

[Claims] (1) Achromobacter, Agrobacterium, Alcaligenes, Achromonas, Acinetobacter, Erwinia, Enterobacter, Brevibacterium, Citrobacter, Flavobacterium,
Microorganisms that belong to the genus Paracoccus, Proteus, Bacillus, Klebsiella, Chromobacterium, Rhodococcus, Microbacterium, Nocardia, Corynebacterium, and Saccharomyces and are resistant to organic solvents. A method for culturing a microorganism, which comprises culturing the microorganism in a medium containing 0.3% or more of an organic solvent having a logP value greater than or equal to the logarithm (logP) value of the partition coefficient (P) of an organic solvent that the microorganism can tolerate. (2) The method for culturing microorganisms according to claim 1, wherein the organic solvent is at least one of aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, alcohols, and ethers.