JPH0378991B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application The present invention relates to a method for producing microbial cells, and more specifically, the present invention relates to a method for producing microbial cells, and more specifically, the present invention relates to a method for producing microbial cells. This invention relates to a method for producing microbial cells by culturing and collecting them.

Conventionally, when trying to collect microbial cells by culturing microorganisms that can use carbon dioxide gas as a carbon source,
Hydrogen bacteria are said to be advantageous.

The reason is that the raw materials are clean with no impurities, so when using microbial cells as feed,
Among the autotrophic bacteria, the growth rate of hydrogen bacteria is particularly fast, and there are few toxicity problems.

B. Means for solving the problem Therefore, the present inventors previously provided a method for creating hydrogen bacteria that belong to the genus Xanthobacter and can grow under conditions of high concentration of oxygen (Japanese Patent Publication No. 51995/1983). Furthermore, as a result of repeated research on how to effectively utilize the created microorganisms, we focused on growing them using nitrogen gas as the only nitrogen source, and completed the present invention.

That is, a microorganism characterized in that hydrogen bacteria belonging to the genus Xanthobacter are cultured in the presence of a mixed gas consisting of oxygen gas, hydrogen gas, and carbon dioxide gas, using nitrogen gas as the sole nitrogen source. It concerns the method of manufacturing the body.

Conventionally, there have been no reports of cell production of hydrogen bacteria belonging to the genus Xanthobacter using nitrogen gas as the sole nitrogen source, and the present invention is the first.

As a result, the present invention not only makes it possible to effectively utilize nitrogen gas in the air as the only nitrogen source in place of conventional inorganic nitrogen sources, but also uses oxygen gas and carbon dioxide gas in the air as an energy source and carbon dioxide source. This makes it possible to significantly reduce the cost of producing microbial cells.

To specifically cultivate this bacterium, microorganisms are inoculated into a sealable container after sterilizing an aqueous solution containing inorganic compounds such as phosphates, magnesium salts, iron salts, etc., which are commonly used for culturing microorganisms. Next, a mixed gas consisting of hydrogen gas, oxygen gas, carbon dioxide gas, and nitrogen gas is aerated and stirred. In addition, the composition ratio of these gases is usually 10-63% hydrogen bacteria and 1-63% oxygen gas.
23%, carbon dioxide gas 3-10%, and nitrogen gas 11-83%. Culture temperature is usually 25-37℃, PH
is cultured in the range of 6.0 to 9.5.

In the present invention, a representative microorganism suitably used is Xanthobacter strain Y-38 (FERM P-6274), which was previously created by the present inventors.

The mycological properties of this strain are as shown below.

a Morphology (1) Cell shape and size, broth agar medium 30℃, 2
Cultured for 7 days: Bacillus of 0.6-0.8 x 1.5-3.0μ, straight or curved (2) Cells polymorphic, becomes short bacillus after 7 days of culture.

(3) No motility (4) No sporulation (5) Gram-negative (6) Non-acid-fast (7) Branched cells are seen when cultured in succinic acid-supplemented medium.

(8) Accumulates poly-β-hydroxybutyric acid (PHB) as a storage substance.

b Growth status (1) Broth agar plate culture: yellow, smooth, shiny;
Forms circular colonies. No diffusion of pigment (2) Meat juice agar slant culture: Same as meat juice agar plate culture (3) Meat liquid culture: No surface growth, viscosity of culture medium increases slightly due to mucilage production.

(4) Meat juice gelatin puncture culture: No growth, no liquefaction (5) Lithomal milk: No coagulation, no liquefaction, becomes alkaline when cultured for a long time.

c Physiological properties (1) Nitrate reduction + (2) Denitrification reaction − (3) MR test − (4) VP test − (5) Indole formation − (6) Hydrogen sulfide formation − (7) Starch hydration Decomposition − (8) Utilization of citric acid (Koser medium, Christensen medium) + (9) Utilization of inorganic nitrogen source
(Nitrate, ammonium salt) + (10) Pigment production Water-insoluble yellow pigment production (11) Urease + (12) Oxidase + (13) Catalase + (14) Growth range PH6.0-9.5 Temperature 25-37℃ (15) Attitude towards oxygen Aerobic (16) O-F test
(by Hugh Leifson method) − (17) Generation of acids and gases from sugars − (18) Growth in solid or liquid medium containing only inorganic compounds in the coexistence of hydrogen gas, oxygen gas, and carbon dioxide gas (19) Methanol , ethanol, n-propanol, n-butanol, formic acid, acetic acid, propionic acid, succinic acid, gluconic acid, and other alcohols and organic acids as the sole carbon source (20) Capable of fixing nitrogen gas (21) Water Produces the insoluble carotenoid pigment zeaxanthin deirhamnoside.

From the above mycological properties, Bergey's Manual of Systematic Bacteriology Vol. 1, (1984)
Systematic Bacteriology Vol. 1 (1984)), the strain was identified as Xantobacter autotrophicus.

C. Examples Example 1 Xanthobacter Y-38 strain (FERM P-
6724) was cultured in a flask.

Medium composition: 1 part distilled water, 300 parts monopotassium phosphate
mg, dipotassium phosphate 400mg, magnesium sulfate
200mg, containing ferrous sulfate 50mg, zinc sulfate 0.05mg, sodium molybutate 0.1mg, nickel sulfate 0.2mg, pH 7.0, 10ml medium in one flask.
xantobacter Y-38 strain as dry weight
Inoculate the equivalent of 0.3 mg, and fill the gas phase in the flask with ₂₀ % H2.
The mixture was replaced with a mixed gas of +O ₂ 4% + CO ₂ 10% + N ₂ 66%, and cultured with shaking at 35°C.

The gas phase in the flask is mixed with the same composition percentage every day.
Replaced with gas. After culturing for 89 hours, the bacterial cell concentration was 4.5 g/dry weight. This bacterial cell production is caused by Alcaligenes autonitrificans AJ3942.
This is about 3 times higher than the Jia culture example (Japanese Patent Publication No. 58-9672).

Example 2 Xanthobacter Y-38 strain (FERM P-
6724) was cultured in a small jar through which a mixture of air, hydrogen gas, and carbon dioxide gas was aerated.

900 ml of a medium having the same composition as in Example 1 was placed in a small glass jar (No. 2), sterilized, and then inoculated with 100 ml of a flask preculture of the Y-38 strain. Temperature 35℃, PH
7.0, an aeration rate of 1/min, and a stirring speed of 400 rpm.

Venting gas composition is H ₂ 10% + 0 ₂ 1% + at the start of culture.
CO ₂ 10% + H ₂ 79%, and as the bacteria grow, H ₂ ,
Gradually increase _O2 concentration until finally air 75%
+H ₂ 20% + CO ₂ 5%.

The bacterial cell concentration after 105 hours of culture was 7.5 g/dry weight.
It became. This bacterial cell production is about four times higher than the jar culture example of Alcaligenes autonitrificans AJ3942 (Japanese Patent Publication No. 58-9672).

Example 3 Xanthobacter Y-38 strain (FERM P-
6724) was cultured in a jar with the dissolved oxygen concentration maintained at 0.35 ppm. Same conditions as Example 2 (but stirring speed
Jar culture was carried out at 600 rpm).

Dissolved oxygen concentration is measured using a dissolved oxygen concentration controller.
Maintained at 0.35ppm. The aeration gas composition was 13% H ₂ + 1% O ₂ + 3% CO ₂ + 83% N ₂ at the start of culture, and the H ₂ concentration was gradually increased as the bacteria grew.
It was 63% H ₂ + 23% O _{2 +} 3% CO ₂ + 11% N ₂ . The final bacterial cell concentration was 13.5 as dry weight after 72 hours of incubation.
It became g/. This bacterial cell production is about 6 times higher than the jar culture example of Alcaligenes autonitrificans AJ3942 (Japanese Patent Publication No. 58-9762).

Claims (1)

[Claims] 1. When culturing hydrogen bacteria belonging to Xanthobacter in the presence of a mixed gas consisting of oxygen gas, hydrogen gas, and carbon dioxide gas, the method is characterized by culturing using nitrogen gas as the only nitrogen source and collecting it. A method for producing microbial cells.