JPH0472510B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing isobutyric acid from carbon dioxide and hydrogen using bacteria belonging to the genus Eubacterium. Isobutyric acid is used in the field of synthetic chemistry, including as a raw material for fragrances. (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 these bacteria produce isobutyric acid from carbon dioxide and hydrogen. The only microorganism that can produce isobutyric acid from carbon dioxide and hydrogen is Clostridium sp. No. 68-2, which we previously created, No. 7367. The bacteria to which it belongs have not been reported so far. (Purpose of the Invention) The present inventor focused on carbon dioxide, which is a renewable resource that exists in abundance in the natural world, and is also the final waste product of various industries, and has focused on carbon dioxide, which is considered as a powerful energy source in the future. They investigated a method of biochemically producing carboxylic acid by reacting it with hydrogen, and arrived at this invention. There are still many issues to be solved in order to carry out the production industrially.In particular, the bacteria that has the ability to produce isobutyric acid using carbon dioxide and hydrogen as substrates is Clostridium S.B. No. 68, which we discovered earlier. -2, and no bacteria belonging to the genus Eubacterium have been reported so far. Under these circumstances, the present inventor aims to provide a new method for producing isobutyric acid using carbon dioxide and hydrogen as substrates. (Structure of the Invention) The present invention uses carbon dioxide and hydrogen as substrates to culture a bacterium belonging to the genus Eubacterium that has the ability to assimilate carbon dioxide and hydrogen to produce isobutyric acid. This is a method for producing isobutyric acid characterized by recovering isobutyric acid. The microorganism used in the present invention belongs to the genus Eubacterium and is an isobutyric acid producing bacterium that assimilates carbon dioxide and hydrogen. It's my first time. The microorganism used in the examples is an anaerobic Gram-positive non-spore bacillus that is thought to belong to the genus Eubacterium and produces isobutyric acid and acetic acid, but it grows on carbon dioxide and hydrogen,
It is different from known bacteria of the same genus in various properties, which will be described in detail later, such as the absence of flagella, 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 and Eubacterium sp. No. 8045 in the present invention. Next, we will show the creation method and mycological properties of Eubacterium S.B. No. 477 (hereinafter abbreviated as this bacterium). (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. Carbon (33
%), and cultured at 30°C, and subcultured approximately every 3 weeks. After sub-planting twice in a liquid medium, the roll tube method (Methods in Microbiology, Volume 3 B,
117 (1969) Academic Press), a single bacterium was isolated and the present bacterium was obtained. (Mycological properties) The mycological properties of the bacterial cells of the present invention are shown. For examination of this mycological property, please refer to the “Anaerobe Laboratory Manual”.
Manual) 4th Edition” (The VIP Anaerobe
Laboratory Virginia Polytechnic Institute
and State University Blacksburg (1972), Bergey's Manual of Determinative Bacteriology.
Determinative Bacteriology) 8th Edition", "Bergey's Manual of Systematic Bacteriology (Volume 1)"
(Bergey's Manual of Systematic
The method and culture medium composition described in ``Bacteriology (Volume 1) (1984)'' and ``Classification and Identification of Microorganisms'' (by Takeharu Hasegawa, Gakkai Publishing Center) were used. (Microscopic findings) 1 Cell shape and size: Single or double bacilli. Width 0.8μm, length 2.0~2.5μm. 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 deionized water 11) 0.1% resazurin 1 ml 10% NH ₄ Cl 10 ml 1MKH ₂ PO ₄ (PH 7.0) 5 ml 20% MgSO _{4.7H 2} O 0.5 ml Vitamin solution 20 ml Mineral solution 40 ml Cysteine hydrochloride (monohydrate) 0.5g Na ₂ S 0.25g NaHCO ₃ 10g 0.06% sodium bromoethanesulfonate 1ml Yeast extract 0.2g NaCl 30g Vitamin solution composition (mg/1) Biotin 2 Folic acid 2 Pyridoxine Hydrochloride 10 Thiamin 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/1) 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 AIK(SO ₄ )2・12H ₂ O 0.009 H ₃ BO ₄ 0.005 NaMoO ₄・2H ₂ O 0.006 (Growth condition) Growth on an agar medium containing the composition shown in Table 1 plus 3% agar is as follows. Shape: Circular Perimeter: Smooth Ridge: Slightly raised Surface: Smooth Color tone: White (physiological properties) Attitude towards oxygen: Obligately anaerobic Growth range (PH) Optimal PH: 7.3 Growth PH: 6.0-8.0 (Temperature) ) Optimum 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 liquid medium containing the following carbon source (1%) to the basic medium shown in Table 1 to a 18 mm diameter test tube to create a sterile medium, inoculate this bacteria, and change the gas phase to nitrogen (67%). and sterilizing gas containing carbon dioxide (33%) and heated to 30°C.
The cells were cultured statically for 14 days. Growth was measured by measuring turbidity at 600 nm using a spectrometer (Spectronic 20, manufactured by Shimadzu Corporation). If the difference in turbidity at 600 nm from the control containing no carbon source is less than 0.1, it is considered "not assimilated".
0.2 to "slightly utilize" anything greater than or equal to 0.1 and less than 0.2
The above-mentioned items were defined as ``capitalize''. What is assimilated: D-fructose, arabinose, raffinose Also, when hydrogen is used instead of nitrogen in the above test, carbon dioxide is also assimilated. Things that cannot be assimilated: D-glucose, sorbose,
Xylose, D-ribose, rhamnose, galactose, maltose, sucrose, lactose, melibiose, trehalose, cellobiose, melezitose, mannitol, methanol, ethanol (generation of acids from sugars, etc.) Add 1% of sugar, which has been confirmed to cause oxidation, and nitrogen (67
%) and 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. Also, when peptone/yeast extract medium or peptone/yeast extract/glucose medium was used, acetic acid and isobutyric acid were produced as organic acids in the medium. (Comparison with similar native species, etc.) From the above mycological properties, No. 477 is an obligate anaerobic Gram-positive non-spore bacillus, and its main fermentation metabolites are acetic acid and isobutyric acid. This is a characteristic strain. Due to this property, Virgie's Manual of
Determinative Bacteriology 8th Edition and Aerob Laboratory Manual 4th Edition
Based on the edition, it appears to be a strain belonging to Eubacterium. Therefore, Eubacterium limosal (E.limosum) was identified according to the genus identification key in the 4th edition of the Aerob Laboratory Manual.
I come across it. Also, Birdsey's Manual of Determinative Bacteriology No. 8
The edition did not mention any 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. . The strain of the present invention grows on carbon dioxide and hydrogen to produce acetic acid and isobutyric acid. A bacterium belonging to the genus Clostridium that grows on carbon dioxide and hydrogen is Clostridium S.B. No. 68, which we previously created.
-2, but this bacterium was distinguishable from the present invention in that it belonged to the genus Clostridium.

【table】

[Table] Based on the above, this strain is considered to be a new bacterial species belonging to the genus Eubacterium, and therefore it was named Eubacterium sp. No. 477. Furthermore, this strain was designated as "FERM-P No. 8045" by the Institute of Microbial Technology, Agency of Industrial Science and Technology.
8045)". (Cultivation method) The cultivation method is basically the same as that for general microorganisms, but it is necessary to prevent oxygen from entering, and in the laboratory, culture is carried out in an incubator tightly closed with a rubber stopper. , leaving it still or shaking it. 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 the device with an inert gas such as nitrogen or a raw material gas. The type of fermentation tank is not particularly limited, but in addition to commonly used stirring and mixing tanks, general or multistage 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. Adding vitamins such as extracts is a 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 dispensing 1ml and sterilizing
Add 100 μl in an anaerobic glove box, seal with a butyl rubber stopper, replace the gas phase with sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and incubate at 30°C.
The cells were cultured statically. 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/l of acetic acid and 0.32 g/l of 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 the same as in Example 1, and the product was analyzed. As a result, 1.25 g/l of acetic acid and 0.49 g/l of isobutyric acid were produced in 10 days.

Claims (1)

[Claims] 1 Using carbon dioxide and hydrogen as substrates, cultivate a bacterium belonging to the genus Eubacterium that has the ability to assimilate carbon dioxide and hydrogen to produce isobutyric acid,
A method for producing isobutyric acid, which comprises recovering produced and accumulated isobutyric acid.