JPH0465674B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) This invention relates to a novel bacterium belonging to the genus Bacteroides. This bacterium can be used in a method for producing acetic acid from carbon dioxide and hydrogen. (Prior art) As a microorganism that assimilates carbon dioxide and hydrogen under room temperature conditions and accumulates acetic acid in the growth medium,
Clostridium aceticum, Acetobacterium uthudei, Eubacterium limosum,
Butylobacterium methylotrophicum, Clostridium strain CV-AA1, and Clostridium sp. No. 68-2, which we created.
FERM-P No. 7367, Clostridium sp. No. 307, FERM-P No. 7487
No. (FERM-P No. 7487), Clostridium sp.
7488) and Acetobacterium sp. No. 446 FERM-P No. 7563 (FERM-P No. 7563). In addition, under high temperature conditions, there are Acetogenium kivii 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. However, no bacteria that grows in the acidic pH range of 4.0 to 7.0 and has the ability to produce acetic acid from carbon dioxide and hydrogen has yet to be reported. Based on these objectives, the present invention aims to obtain a new microorganism that assimilates carbon dioxide and hydrogen and accumulates acetic acid in a culture medium. (Structure of the Invention) The present invention uses anaerobic bacteria with a width of 1.1 to 1.4 μm and a length of 6.0 to 10.0 μm, singly or in groups of 2 to 3 Gram-negative nonspore bacilli, to convert sugars into acetic acid, n-butyric acid, isokylate, etc. Since it produces grass acid and succinic acid, it is thought to belong to the genus Bacteroidetes, but it grows on carbon dioxide and hydrogen, is Gram stain negative, and has a pH of 4.0.
It is a straight rod bacterium without flagella that grows in the range of ~7.0 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 Bacteroides sp. No. 671 in the present invention. Next, we will show the method for creating Bacteroides sp. No. 671 and its mycological properties. (Creation method) This bacterium was isolated from the mud of Beppu Hot Springs in Oita Prefecture by the following method.
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,
1173 (1969) Academic Press), a single bacterium was isolated and the present bacterium was obtained. (Mycological properties) The mycological properties of the bacterial strain of the present invention are shown below. For examination of this mycological property, please refer to "Anaerobe Laboratory Manual 4th Edition".
Manual，，The VIPAnaerobe 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 "Microbial Classification and Identification" (written by Takeharu Hasegawa, Gakkai Publishing Center) and the culture medium composition were used. (Microscopic findings) 1 Cell shape and size: Single or 2-3 rows of straight rods Width: 1.1~
1.4 μm Length 6.0-10.0 μm 2 Flagella: None 3 Spores: None 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% MgSO _{4.7H 2} O solution 0.5 ml Vitamins Solution 20ml Mineral solution 40ml Cysteine hydrochloride (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 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・2H2O 0.006 (Growth condition) Growth on an agar medium with the composition shown in Table 1 plus 3% agar is as follows. It is. Shape: Circular Perimeter: Smooth Ridge: Slightly raised Surface: Smooth Color tone: White (physiological properties) Attitude towards oxygen: Obligately anaerobic Growth range (PH) Optimal PH: 5.8 Growth PH: 4.0-7.0 (Temperature) ) Optimum temperature: 30℃ Growth temperature: 25-40℃ Indole production: - Gelatin liquefaction: - Catalase production: - Starch hydrolysis: - Aesculin hydrolysis: - Pigment production: - (Carbon source assimilation ) 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 prepare a sterile medium, inoculate this bacteria, and replace the gas phase with nitrogen. The cells were replaced with a sterilizing gas containing (67%) and carbon dioxide (33%), and cultured 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 turbidity at 600 nm differs from the control containing no 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." And so. Assimilated: D-glucose, D-fructose, xylose, rhamnose, galactose,
Syuclok, lactose, trehalose, cellobiose, melezitose, mannitol Substances that cannot be assimilated: sorbose, D-ribose, arabinose, maltose, melibiose, raffinose, methanol, ethanol (generation of acids from sugars, etc.) Add the above to the basic medium in Table 1. Adding 1% or 0.5% of carbon sources known to be assimilated in tests,
The gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and this bacterium was inoculated and cultured stationary at 30°C. Acetic acid, butyric acid, isovaleric acid, and succinic acid were produced as organic acids in the medium using all carbon sources. (Comparison with similar native species, etc.) Based on the above mycological properties, No. 671 is an obligately anaerobic Gram-negative non-spore bacillus, and its main fermentation metabolites are carbon dioxide and hydrogen, acetic acid, and other This strain is characterized by the fact that the carbon sources it assimilates are acetic acid, butyric acid, isovaleric acid, and succinic acid. Due to this property, Virgies Manual of Systematic Bacteriology (Volume 1) and Aerob Laboratory Manual Vol.
Based on the edition, it appears to be a strain belonging to Bacteroides. Therefore, according to the genus identification key in the 4th edition of the Aerob Laboratory Manual, we identified B.ruminicola subspecis ruminicola (B.ruminicola ss.ruminicola).
I come across it. In addition, Virgie's Manual of Systematic Bacteriology (Volume 1) does not list any bacterial species whose symptoms match those of No. 671. When we compared the symptoms of No. 671 and Bacteroides ruminicola subspecis ruminicola, we found that they were both obligate anaerobic nonspore bacilli, but the characteristics of both bacteria were different in the points shown in Table 2. .

[Table]

[Table] Produces grass acid and succinic acid
give birth to
Virgie's Manual of Systematic Bacterology (Volume 1) Based on the above, this strain is considered to be a new bacterial species belonging to the genus Bacteroides, and therefore it was named Bacteroides sp. No. 671. Furthermore, this strain was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERM-P No. 8048. As in the above case, it is necessary to prevent oxygen from entering, and in the laboratory, a method is used in which the incubator is left standing or shaken in an incubator sealed tightly with a rubber stopper.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 raw material gas.The type of fermenter is not particularly important. However, in addition to commonly used stirring mixing tanks, single or multi-stage bubble column and draft tube fermenters can also be used.The carbon source used for culture is usually supplied as carbon dioxide gas, but the culture medium It can also be added as dissolved carbon dioxide, carbonate, or hydrogen carbonate.As the nitrogen source, various nitrogen compounds that can be used in normal fermentation, such as ammonium salts such as ammonium chloride and nitrates such as sodium nitrate, can be used. 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 molybutate, boric acid, etc. can be added as necessary. The addition of a vitamin source such as yeast extract or yeast extract is a common practice.The present invention will be explained below with reference to specific examples.Example 1 Bacteroides sp. No. 671 strain was cultured as follows. 1. Transfer 5 ml of the culture medium shown in Table 1 to a test tube.
After dispensing and sterilizing, 100μ of culture solution was cultured in the same medium.
was added in an anaerobic glove box, and after sealing with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and cultured at 30°C. 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, 0.72 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, Bacteroides sp. No. 671 strain was cultured with shaking. The measurement method was the same as in Example 1, and the product was analyzed. As a result, 1.24 g/acetic acid was produced in 10 days.

Claims (1)

[Claims] 1 Bacteroides sp., which belongs to the genus Bacteroides, grows on carbon dioxide and hydrogen, has a growth pH range of 4.0 to 7.0, and does not assimilate ribose.
No.671