JPH10234358A - Culture of bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable fermentation in the presence of an organic solvent in high concentration in a high state of bacterium activity, namely in a large number of viable cells, to prevent contamination with various germs and to improve fermentation productivity. SOLUTION: A bacterium is cultured under restriction of oxygen in a two phase-based culture solution comprising an aqueous solution (water phase) containing an electron acceptor compound (preferably 10-100mM addition concentration) such as a sulfate or a carbonate in anaerobic respiration and a component such as glucose required for proliferation of bacterium and a slightly water-soluble organic solvent (organic solvent phase). Preferably the culture solution contains >=2mM of sodium nitrate and >=0.2(W/V)% of glucose.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for culturing microorganisms.

[0002]

2. Description of the Related Art When a compound that is hardly soluble in water is metabolized by a microorganism or a metabolite produced by the microorganism is poorly soluble in water, the microorganism may be cultured in a culture solution containing an organic solvent. In such a case, since many organic solvents are generally toxic to microorganisms, it is necessary to supply the organic solvent by vapor or to culture the microorganisms while maintaining the concentration at such a low level that it is not toxic. . Therefore, the concentration of the compound to be metabolized by the microorganism or the metabolite of the microorganism in the culture solution is kept low, and the productivity in the metabolic reaction is not always satisfactory.

[0003]

SUMMARY OF THE INVENTION If cultivation in the presence of a high concentration of an organic solvent in a state where the activity of microorganisms is high, that is, in a state where the number of viable bacteria is large, it becomes possible to use a compound which is hardly soluble in water. In addition to being effectively used for fermentation production related to metabolism, it can also be used as a method for preventing contamination by various germs that occur during culture of microorganisms. For this reason, there has been a demand for the development of a microorganism culturing method capable of maintaining a high viable count of microorganisms in the presence of a high-concentration organic solvent.

[0004]

Under such circumstances, the present inventors have conducted intensive studies and as a result, have found that an aqueous solution (water) containing an electron acceptor compound and components necessary for the growth of microorganisms in anaerobic respiration. Phase) and a two-phase culture medium comprising an organic solvent (organic solvent phase) that is poorly soluble in water, wherein the microorganism is cultured under oxygen limitation (hereinafter referred to as the culture of the present invention). The law).

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
Microorganisms to which the culture method of the present invention is applied include, for example, nitrate-respiring bacteria. Specifically, for example, genus Aquaspirillum (Aquaspirillum), Agrobacterium (Agrobact
erium), Acinetobacter, Alcaligenes, Escherichia
a) genus, Campylobacter genus, Xanthomonas genus, Kingella genus, Gluconobacter genus, Chromobacterium
(Chromobacterium), Corynebact
erium), thermothrix (Thermothrix), Pseudomonas, Cytophaga, Spyrilum, Serratia, Thiobacillus, and Nitrosomonas
nas), Neisseria, Hyphomicrobium, Bacillus, Paracoccus, Halobacte
rium), Flavobacterium, Branhamella, Propionibacterium
Examples include microorganisms belonging to the genus (Propionibacterium), the genus Rhizobium, the genus Rhodopseudomonas, and the like, but the culture method of the present invention is not limited to these microorganisms.

The term "electron acceptor compound in anaerobic respiration" used in the culture method of the present invention generally means a compound which is an electron acceptor except for molecular oxygen, for example, sulfur oxides such as nitrate, Sulfates, carbonates, fumaric acid, iron salts and the like. The addition concentration of the electron acceptor compound is, for example, about 2 mM to about 200 mM, preferably about 10 mM to about 10 mM.
0 mM.

[0007] The term "components necessary for the growth of microorganisms" used in the culture method of the present invention means the components of a medium suitable for culturing the microorganisms used. For example, glucose, sucrose, Xylose, starch, sugars such as starch hydrolysates, alcohols such as methanol and ethanol, hydrocarbons such as methane and ethylene, succinic acid,
Organic acids such as salicylic acid and acetic acid can be mentioned, and nitrogen sources include meat extract, peptone, yeast extract, dried yeast, corn steep liquor, casamino acid, urea, sodium nitrate and the like. In addition, inorganic salts such as phosphates, magnesium salts, calcium salts, iron salts, potassium salts, copper salts, and manganese salts can be added as appropriate. The concentration of glucose to be added is, for example, about 0.2 (W / V)% to about
10 (W / V)%, preferably about 0.3 (W / V)% to about 2
(W / V)%. When the above-mentioned component acts as the above-mentioned electron acceptor compound, both the above-mentioned component and the above-mentioned electron acceptor compound may be included alone without distinction.

[0008] The "poorly water-soluble organic solvent" used in the culture method of the present invention is, for example, a compound having a solubility in water of about 0.2.
(W / V)% or less means an organic solvent such as an aliphatic hydrocarbon, an alicyclic hydrocarbon, an aromatic hydrocarbon, an alcohol, an ether, and a ketone, which alone may be 2 or more. It may be a mixture of more than one species. Specifically, pentane, hexane, heptane, octane, isooctane,
Nonane, decane, dodecane, tetradecane, hexadecane, 1-octene, 1-dodecene, 1,5-hexadiene, 1,7-
Aliphatic hydrocarbons such as octadiene, cyclohexane,
Alicyclic hydrocarbons such as methylcyclopentane and methylcyclohexane, aromatic hydrocarbons such as benzene, toluene, xylene, styrene, chlorobenzene, ethylbenzene and propylbenzene, 1-octanol, 1-nonanol, 1-decanol, 1 -Alcohols such as undecanol and 1-dodecanol; ethers such as dibutyl ether, dipentyl ether and diphenyl ether; and ketones such as 2-nonanone, 2-decanone and benzophenone.

A two-phase cultivation comprising an aqueous solution (aqueous phase) containing an electron acceptor compound and components necessary for the growth of microorganisms in anaerobic respiration, and an organic solvent (organic solvent phase) that is poorly soluble in water. In the liquid, the mixing ratio varies depending on the type of microorganisms, the type of organic solvent which is hardly soluble in water, and the like, and for example, from about 95: about 5 (volume ratio) to about 5: 5.
0: about 50 (volume ratio).

The term "under oxygen limitation" in the culture method of the present invention means, for example, that the oxygen concentration of the gas phase in the culture tank is about 10 (V / V)% or less, preferably about 5 (V / V)% or less, more preferably. Means a state of about 2 (V / V)% or less. For example, a method of replacing the gas phase in the culture tank with an inert gas such as nitrogen gas, carbon dioxide, or argon can be used.

The culturing conditions, such as culturing temperature and pH, used in the culturing method of the present invention may be those which are in accordance with ordinary culturing conditions for microorganisms. Further, depending on the type of metabolite produced by the microorganism, the above various culture conditions may be adjusted so that the metabolic reaction is promoted.

[0012]

EXAMPLES Next, examples of the present invention will be described, but the present invention is not limited to these examples.

Example 1 (culturing example) LBGMg medium (1% Bacto-trypton) was placed in a 500 mL Erlenmeyer flask.
e, 0.5% yeast extract, 1.0% NaCl, 0.1% glucose, 10
mM MgSO ₄ .7H ₂ O) 100 mL was dispensed and sterilized at 121 ° C. for 5 minutes. To this medium was added 50 mM sodium nitrate or 0.5% (W / V) glucose, and the oxygen concentration in the gas phase was 2%.
The degassing of the culture tank and the replacement with nitrogen were repeated three times so as to be (V / V)% or less. E. coli W
After inoculating the pre-culture solution of 3110, n-hexane 20m
L was overlaid. After sealing the Erlenmeyer flask with a butyl rubber stopper, main culture was performed at 37 ° C. while shaking at 160 rpm. During the main culture, the culture solution was sampled over time, and the diluted solution was spread on an LBGMg plate medium, and the number of viable bacteria in the culture solution was examined by the number of colonies that appeared. For comparison, a system (comparative section) in which neither sodium nitrate nor glucose was added to the culture solution and culture was performed under oxygen limitation was used. The results are shown in Figure 1. In the case of the group to which sodium nitrate was added under oxygen limitation (invention section) and the section to which glucose was added under oxygen limitation (invention section), the viable cell count was 6-7 × 10 ⁸ / m 2.
L, increased to 1-2 × 10 ⁸ / mL. On the other hand, in the comparative plot, the viable cell count decreased to 1 × 10 ⁵ / mL after 24 hours of culture. Therefore, the viable cell count after 24 hours of culture in the group of the present invention was increased to about 1000 times in the group with added glucose under oxygen limitation and about 10,000 times in the group with added sodium nitrate under oxygen limitation as compared with the comparative group.

[0014]

Industrial Applicability According to the present invention, it is possible to culture in a state in which the activity of microorganisms is high in the presence of a high concentration of an organic solvent, that is, in a state where the number of viable bacteria is large, and the metabolism of a compound which is hardly soluble in water is possible. Can be effectively used for the fermentation production according to the present invention, and also can be used as a method for preventing various bacterial contaminations that occur during the culture of microorganisms.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a time-dependent change in the number of viable cells when E. coli W3110 is cultured in a medium containing n-hexane. Black circles indicate the sodium nitrate-added section under oxygen limitation (invention section), black triangles indicate the glucose-added section under oxygen limitation (invention section), open circles indicate that neither sodium nitrate nor glucose was added and The system (comparative section) for culturing under oxygen restriction is shown.

Claims (3)

[Claims] 1. A two-phase system comprising an aqueous solution (aqueous phase) containing components necessary for the growth of an electron acceptor compound and microorganisms in anaerobic respiration, and an organic solvent (organic solvent phase) hardly soluble in water. A method for culturing a microorganism, comprising culturing the microorganism in a culture solution under oxygen limitation. 2. The method according to claim 1, wherein the culture solution contains sodium nitrate at a concentration of 2 mM or more. 3. A culture solution containing 0.2 (W / V)% glucose.
The method for culturing a microorganism according to claim 1, wherein the microorganism is contained at the above concentration.