JPH0465676B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel bacterium belonging to the genus Eubacterium. This bacterium can be used in a method for producing acetic acid and isobutyric acid from carbon dioxide and hydrogen. (Prior Art) Several microorganisms are known that assimilate carbon dioxide and hydrogen and accumulate acetic acid in the growth medium.
Among them, there are four types of bacteria that belong to the genus Clostridium, but it is not known that they produce isobutyric acid from carbon dioxide and hydrogen. Clostridium sp. No. 68-2, which we previously created, is the only microorganism that can produce isobutyric acid from carbon dioxide and hydrogen, but no bacteria belonging to the genus Eubacterium have been reported to date. Bacteria that have the ability to assimilate sugars and produce isobutyric acid include Clostridium stichlandii, Clostridium propionicum, and Clostridium gonii. (Purpose of the Invention) The present inventor focused on carbon dioxide, which is a renewable resource, abundantly present in nature, and is also the final waste product of various industries, and has developed a carbon dioxide system that is considered to be a powerful energy source in the future. This invention was arrived at after studying a method for biochemically producing carboxylic acid by reacting it with hydrogen. Although the above-mentioned bacteria are known, there are still many issues to be solved in order to industrially produce carboxylic acid from carbon dioxide and hydrogen. The bacterium that has the ability to produce butyric acid is Clostridium, which we created earlier.
SP No. 68-2 (FERM No. 7367)
- P No. 7367)), and no bacteria belonging to the genus Eubacterium have been reported so far. Based on these objectives, the present invention aims to obtain a new microorganism that assimilates carbon dioxide and hydrogen and accumulates acetic acid and isobutyric acid in a culture medium. (Structure of the Invention) The present invention is directed to Eubacterium, which is an anaerobic bacterium with a width of 0.8 μm and a length of 2.0 to 2.5 μm, and is a Gram-positive non-spore bacillus, singly or in groups of 2 to 3, that produces isobutyric acid in addition to acetic acid. Although it is thought to belong to the genus, it is a straight bacillus that grows on carbon dioxide and hydrogen and has no flagella, and is different from known bacteria due to the following properties, and is considered to be a new bacterial species. Since the official species name has not yet been assigned, it is designated as Eubacterium sp. No. 477 in the present invention. Next, we will show the creation method and mycological properties of Eubacterium sp. No. 477. (Creation method) This bacterium was isolated from the seabed mud of Tagonoura, Shizuoka Prefecture, by the following method. That is, 5 ml of the liquid medium shown in Table 1 was dispensed into test tubes, sterilized, approximately 0.3 g of soil was added in an anaerobic glove box, and after sealing with a butyl rubber stopper, the gas phase was mixed with hydrogen (67%) and carbon dioxide. The cells were replaced with a sterilizing gas containing (33%) and cultured stationary at 30°C, and subcultured approximately every 3 weeks. After sub-planting twice in liquid medium, add 3% agar to the medium shown in Table 1.
The roll tube method (Methods in Microbiology, Volume 3B,
117 (1969) Academic Press), a single bacterium was isolated and the present bacterium was obtained. (Mycological properties) The mycological properties of the strain of the present invention are shown. For examination of this mycological property, please refer to "Anaerobe Laboratory Manual 4th Edition".
Manufl，，The VIP Anaerobe Laboratory
Virgini a Polytechnic Institute and State
University, Blacksburg (1972)) and Bergey's Manual of Determinative Bacteriology (8th edition).
Manual of Determi native Bacteriology)
(EIGHTH EDITION) (1974)) and Virgies Manual of Systematics.
Bacteriology (Volume 1) (Bergey's
Manual of Systematic Bacteriology
(Volume 1) (1984)) and "Classification and Identification of Microorganisms" (written by Takeharu Hasegawa, published by Gakkai Publishing Center) and the culture medium composition were used. (Microscopic findings) 1 Cell shape and size: Single or 2-3
Straight bacilli, width 0.8 μm, length 2.0-2.5 μm 2 Flagella: None 3 Spores: None 4 Gram staining: Positive (medium composition) Examples are shown in Table 1. Table 1 Composition of basic medium (in 1 part deionized water) 0.1% resuthrin solution 2 ml 10% NH ₄ Cl solution 10 ml 1MKH ₂ PO ₄ (PH 7.0) solution 5 ml 20% MgSO _{4.7H 2} O solution 0.5 ml Vitamin solution 20ml Mineral solution 40ml Cysteine hydrochloride (1H ₂ O) 0.5g Sodium sulfide 0.25g Sodium hydrogen carbonate 1g 600mg/1 Sodium bromoethanesulfonate
1ml Yeast extract 0.2g NaCl 30g Vitamin solution composition (mg/) Biotin 2 Folic acid 2 Pyridoxine hydrochloride 10 Thiamine hydrochloride 5 Riboflavin 5 Nicotinic acid 5 Ca pantothenate 5 Vitamin B12 0.01 P-aminobenzoic acid 5 Thioctic acid 1 Mineral solution composition ( g/) Nitrilotriacetic acid 0.25 MgSO ₄・7H ₂ O 0.1 MnSO ₄・4H ₂ O 0.28 NaCl 0.5 FeSO ₄・7H ₂ O 0.05 CoCl ₂・6H ₂ O 0.09 CaCl ₂・2H ₂ O 0.07 ZnSO ₄・7H ₂ O 0.09 CuSO ₄ 0.03 AlK (SO ₄ ) ₂・12H ₂ O 0.009 H ₃ BO ₄ 0.005 NaMoO4・2H ₂ 0 0.006 (Growth condition) Growth on an agar medium with the composition shown in Table 1 plus 3% agar is as follows. That's right. Shape: Circular Perimeter: Smooth Ridge: Slightly raised Surface: Smooth Color tone: White (physiological properties) Attitude towards oxygen: Knitted anaerobic Growth range (PH) Optimum PH: 7.3 Growth PH: 5.3-8.0 (Temperature) ) Optimal temperature: 30℃ Growth temperature: 25-40℃ Indole production: - Gelatin liquefaction: - Catalase production: - Starch hydrolysis: - Aesculin hydrolysis: - Pigment production: - (Carbon source utilization Add 5 ml of a liquid medium containing the following carbon source (1% unless otherwise specified) to the basic medium in Table 1 to a test tube with a diameter of 18 mm to create a sterile medium, inoculate this bacteria, and inoculate the gas phase. The cells were replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and cultured stationary at 30°C for 14 days. For growth, measure the turbidity at 600 nm using a spectrometer.
20, Shimadzu Corporation). If the turbidity at 600 nm differs from the control with no change in carbon source by less than 0.1, it is "not assimilated," if it is 0.1 or more and less than 0.2, it is "slightly assimilated," and if it is 0.2 or more, it is "assimilated." ”. Assimilated: D-fructose, arabinose, raffinose Not assimilated: D-glucose, sorbose, xylose, D-ribose, rhamnose, galactose, maltose, sucrose, lactose, melibiose, trehalose, cellobiose, melezitose, mannitol, methanol ,
Ethanol (generation of acid from sugar, etc.) Add 1% or 0.5% of the carbon source known to be assimilated in the above test to the basic medium shown in Table 1, and add nitrogen (67%) to the gas phase. The air was replaced with a sterilizing gas containing carbon dioxide (33%), and this bacteria was inoculated and cultured stationary at 30°C. Acetic acid and isobutyric acid were produced as organic acids in the medium using all carbon sources. (Comparison with native similar species, etc.) Based on the above mycological properties, No. 477 is an obligately anaerobic Gram-positive non-spore bacillus, and its main fermentation metabolites are acetic acid and isobutyric acid. This is a bacterial strain. Due to this property, Birdsey's Manual of Determinative Bacteriology No. 8
According to a search based on the 4th edition of Versey's Manual of Systematic Bacteriology (Volume 1) and the 4th edition of Aerob Laboratory Manual, it is thought that the strain belongs to Eubacteroim. Therefore, according to the genus identification key in the 4th edition of the Aerob Laboratory Manual, we will identify E. limosum (E.limosum).
I come across it. Also, Birdsey's Manual of Dataminative Bacteriology No. 8
Edition and Versey's Manual of Systematic Bacteriology (Volume 1)
There was no description of a bacterial species whose characteristics matched those of No. 477. When we compared the properties of No. 477 and Eubacterium limosum, we found that they were both obligate anaerobic Gram-positive nonspore bacilli, but the properties of both bacteria were different in the points shown in Table 2. .

[Table] Furthermore, this strain has been designated by the Institute of Microbiology, Agency of Industrial Science and Technology as "Feikoken Bacteria Collection No. 8045 (FERM-P No. 8045)".
8045). (Cultivation method) The cultivation method is basically the same as for general microorganisms, but it is necessary to prevent oxygen contamination. In this case, the method of leaving it still or shaking it is used. On a slightly larger scale, a commonly used fermenter can be used as is, and an anaerobic atmosphere can be created by replacing the oxygen in equipment farming with an inert gas such as nitrogen or raw material gas. The type of fermentation tank is not particularly limited, but in addition to commonly used stirring and mixing tanks, single-stage or multi-stage bubble column type, and draft tube type fermentation tanks can also be used. The carbon source used for culture is usually supplied as carbon dioxide gas, but it can also be added to the medium as dissolved carbon dioxide, carbonate, or bicarbonate. As the nitrogen source, various nitrogen compounds that can be used in normal fermentation can be used, such as ammonium salts such as ammonium chloride and nitrates such as sodium nitrate. Other inorganic compounds such as potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, iron sulfate, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, alum, sodium molybdate, boric acid, or yeast, as necessary. Addition of vitamin sources such as extracts is common practice. The present invention will be explained below using specific examples. Example 1 Eubacterium sp. No. 477 strain was cultured as follows. Transfer the culture medium shown in Table 1 to the test tube 5
Culture solution cultured in the same medium after ml dispensing and sterilization
After adding 100μ in an anaerobic glove box and sealing it with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%).
The cells were incubated statically at ℃. Bacterial cells were separated from a portion of the culture solution using a centrifuge, the supernatant was made acidic with phosphoric acid, and the product was quantified by gas chromatography. As a result, 0.95 g/acetic acid and 0.32 g/isobutyric acid were produced during 10 days of static culture. Example 2 Using an L-shaped test tube, prepared in the same manner as in Example 1, Eubacterium sp. No. 477 strain was cultured with shaking. The measurement method was carried out in the same manner as in Example 1, and the product was analyzed. As a result, 1.25 g/acetic acid and 0.49 g/isobutyric acid were produced in 10 days.

Claims (1)

[Claims] 1. Eubacterium sp. No. 477, which belongs to the genus Eubacterium, grows in carbon dioxide and hydrogen, grows in 3% NaCl, assimilates arabinose, and produces isobutyric acid.