JPH0465673B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel bacterium belonging to the genus Clostridium. This bacterium can be used in a method for producing acetic acid from carbon dioxide and hydrogen. (Prior art) As a microorganism that converts carbon dioxide and hydrogen under room temperature conditions and accumulates acetic acid in the growth medium,
Clostridium aceticum, Acetobacterium uthudei, Eubacterium limosum,
Butybacterium methylotrophicum, Clostridium strain CV-AA1, Clostridium sp. No. 68-2 that we created, FERM-P No. 7367, Clostridium sp. No. 307 FERM-P No. 7487 (FERM-P No. 7487), Clostridium sp. No. 484 FERM-P No. 7488 (FERM-P No.
7488) and Acetobacterium sp. No. 446 FERM-P No. 7563 (FERM-P No. 7563). Also under high temperature conditions are Acetogenium kivyi and Clostridium thermoautotrophicum. (Objective of the invention) Although the above-mentioned bacteria are known, there are still many problems to be solved in order to industrially produce acetic acid from carbon dioxide and hydrogen, among them the accumulation concentration of acetic acid and It is important to obtain bacteria with a high production rate. To this end, in addition to breeding and improvement based on known bacterial species, the creation of new bacterial species that can produce acetic acid from carbon dioxide and hydrogen is an extremely important means. Furthermore, no bacterium has been reported that has an acidic pH range of 4 to 7 and has the ability to produce acetic acid from carbon dioxide and hydrogen. The present invention aims to obtain a new microorganism that accumulates acetic acid in a culture medium by converting carbon dioxide and hydrogen. (Structure of the Invention) The present invention is an anaerobic bacterium with a width of 0.6 to 1.0 μm and a length of 5.0 to 9.0 μm, and is a straight rod bacterium, either singly or in groups of 2 to 3, and is a bacterium belonging to the genus Clostridium in that it produces spores. However, it is a straight bacillus without flagella that grows on carbon dioxide and hydrogen, is negative for Gram staining, and grows in the pH range of 4.0 to 7.0, and is different from known bacteria due to the following properties. It is thought to be a bacterial species. Since the official species name has not yet been assigned, this invention uses Clostridium sp. No.
Display as 670. Next, we will show the creation method and mycological properties of Clostridium sp. No. 670. (Creation method) This bacterium was isolated from mud at Beppu Hot Springs in Oita Prefecture by the following method. That is, the liquid medium 5 shown in Table 1
After dispensing ml into test tubes and sterilizing them, 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 (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 strain of the present invention are shown. For examination of this mycological property, please refer to "Anaerobe Laboratory Manual 4th Edition".
Manual,,TheV.IPAnaerobe Laboratory
Virginia Polytechnic Institute and State
University, Blacksburg (1972)) and Bergey's Manual of Determinative Bacteriology (8th edition).
Manual of Determinative 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
Direct rods in series, width 0.6-1.0 μm, length 5.0-9.0 μm 2 Flagella: None 3 Spores: Present, terminal 4 Gram staining: Negative (medium composition) Examples are shown in Table 1. Table 1 Composition of basic medium (in 1 part deionized water) 0.1% indigo carmine solution 2 ml 10% NH ₄ Cl solution 10 ml 1MKH ₂ PO ₄ (PH 7.0) solution 5 ml 20% MzgSO _{4.7H 2} O solution 0.5 ml Vitamins Solution 20ml Mineral solution 40ml Cysteine hydrochloric acid (1H ₂ O) 0.5g Sodium sulfide 0.25g Sodium hydrogen carbonate 1g 600mg/sodium bromoethanesulfonate 1ml Yeast extract 0.2g PH5.3 Vitamin solution composition (mg/) Biotin 2 Folic acid 2 Pyridoxine hydrochloride 10 Thiamine hydrochloric acid 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 -2H2O 0.006 (Growth condition) Growth on an agar medium containing the composition shown in Table 1 with 3% agar added is as follows. Shape: Circular Perimeter: Smooth Ridge: Slightly raised Surface: Smooth Color tone: White (physiological properties) Attitude towards oxygen: Knitted anaerobic Growth range (PH) Optimum PH: 6.5 Growth PH: 4.0~
7.0 (Temperature) Optimal temperature: 30°C Growth temperature: 25°C to 40°C Indole productivity: - Gelatin liquefaction: - Catalase productivity: - Starch hydrolysis: - Aesculin hydrolysis: + Pigment formation: - (Characterization of carbon source) 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 and incubate this bacterium. After inoculation, the gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and the cells were incubated statically at 30°C for 14 days. For growth, measure the turbidity at 600 nm using a spectrometer.
20, manufactured by Shimadzu Corporation). If the difference in turbidity at 600 nm from the control that does not contain a carbon source is less than 0.1, it is "not refined", if it is 0.1 or more and less than 0.2, it is "slightly refined", if it is 0.2 or more, it is "qualified". And so. What to qualityify: D-glucose, D-fructose, xylose, D-lipose, arabinose, galactose, maltose, lactose,
Cellobiose, melezitose, mannitol Substances that do not convert into substances: sorbose, rhamnose, sucrose, nuribiose, raffinose, methanol, ethanol (generation of acids from sugars, etc.) Known to be assimilated into the basic medium shown in Table 1 in the above test Add 1% or 0.5% of carbon source,
The gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and this bacterium was inoculated and cultured statically at 30°C. Acetic acid and butyric acid were produced as organic acids in the medium using all carbon sources. (Comparison with similar native species, etc.) From the above mycological properties, No. 670 is an obligately anaerobic Gram-negative spore bacillus, and its main fermentation products are acetic acid, carbon dioxide and hydrogen. This strain is characterized by the fact that the other carbon sources it assimilates are acetic acid and butyric acid. Based on this property, a search was made based on Versey's Manual of Determinative Bacteriology 8th Edition, Versey's Manual of Systematic Bacteriology (Volume 1), and Aerob Laboratory Manual 4th Edition. It is thought to be a strain belonging to Clostridium. Therefore, I identified Clostridium innoquium (C.
innocuum). Also, Virgie's Manual of Determinative Bacteriology 8th Edition and Virgie's Manual of Determinative Bacteriology, 8th Edition.
The Observatory of Systematic Bacteriology (Volume 1) does not list any bacterial species whose characteristics match those of No. 670. No.670 and Clostridium
A comparison of the properties of the two bacteria revealed that they were both obligate anaerobic spore-forming bacilli, but the properties of the two bacteria were different in the points shown in Table 2.

[Table] produces acetic acid and butyric acid
do.
* Virgie's Manual of Determinative Bacteriology (8th edition) Based on the above, this strain is considered to be a new bacterial species belonging to the genus Clostridium, and therefore it was named Clostridium sp. No. 670. Furthermore, this strain was given to the Institute of Microbial Technology, Agency of Industrial Science and Technology as "FERM Stock Submission No. 8047 (FERM-P No.
8047). (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, 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 as dissolved carbon dioxide, carbon salt, or bicarbonate during culture. 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 Clostridium sp. No. 670 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 sterilization in ml aliquots
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, 0.7 g/acetic acid was produced during 10 days of static culture. Example 2 Using an L-shaped test tube, prepared in the same manner as in Example 1, a strain of Clostridium sp. No. 670 was cultured with shaking. The measurement method was the same as in Example 1, and the product was analyzed. As a result, 1.35 g/acetic acid was produced in 10 days.

Claims (1)

[Claims] 1 Clostridium sp. No. 670, which belongs to the genus Clostridium, grows on carbon dioxide and hydrogen, has a growth pH range of 4.0 to 7.0, and assimilates maltose.