JPH0465672B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present 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 woodi, 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 No. 484 Microtechnical Research Institute 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.

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 having a width of 1.0 μm and a length of 4.5 to 5.0 μm, singly or in groups of 2 to 3 Gram-negative nonspore bacilli, to extract sugars from acetic acid, n-butyric acid, isovaleric acid, etc. Since it produces succinic acid, it is thought to belong to the genus Bacteroidetes, but it grows on carbon dioxide and hydrogen, is negative for Gram staining, grows in the pH range of 4.0 to 7.0, and is a straight rod without flagella. It 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, this invention uses Bacteroides sp. No. 669.
is displayed.

Next, we will show the method for creating Bacteroides sp. No. 669 and its mycological properties.

(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, 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 stationary at 30°C6, 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，，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 Bergey's Manual of Systematic Bacteriology (Volume 1)
Manual of Systematic Bacteriology (Volume
1) (1984)) and "Classification and Identification of Microorganisms" (written by Takeharu Hasegawa, published by Gakkai Publishing Center), the method and culture medium composition were used.

(Microscopic findings) 1 Cell shape and size: Single or 2-3
Direct rods in series 1.0 x 4.5-5.0 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% 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/l Sodium bromoethanesulfonate 1
ml 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 NaMoO ₄・2H2O 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: Obligate anaerobic Growth range (PH) Optimum PH: 6.5 Growth PH: 4.0~
7.0 (Temperature) Optimal temperature: 30℃ Growth temperature: 25-40℃ Indole production: - Gelatin liquefaction: - Catalase production: - Starch hydrolysis: - Aesculin hydrolysis: + Pigment production: - (Carbon source Assimilation ability) 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 and inoculate it with this fungus. The gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and the cells were statically cultured 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 without 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, D-ribose, arabinose, galactose, maltose, sucrose, lactose, cellobiose Not assimilated: sorbose, rhamnose, melibiose, trehalose, raffinose, 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,
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, butyric acid, isovaleric acid, and succinic acid were produced as organic acids in the medium using all carbon sources.

(Comparison with native and similar species, etc.) From the above mycological properties, No. 669 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 Volume 4
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. Also, Birdsey's Manual of Determinative Bacteriology No. 8
The edition did not mention any bacterial species whose characteristics matched those of No. 669. No.669 and Bacteroides ruminicola
When comparing the properties with Subspecis ruminicola, it was found that both were obligate anaerobic non-spore bacilli, but the properties of the two bacteria were different in the points shown in Table 2.

Table 2 B Ruminicola SS. Ruminicola No. 669 (Literature value ^* ) (Actual measurement value) Morphology: Straight bacillus Straight bacillus alone or 2 to 3 rows Single or 2 to 3 rows No flagella No flagella Size: 0.8-1.0× 0.8−8.0 1.0×4.5~5.0 (μm) Gram staining: Negative Negative pigment production: None None Carbon source assimilation Melibiose assimilation: Well assimilated None Roughinose assimilation: Well assimilated None Ribose assimilation : None Products that are commonly assimilated: In addition to acetic acid, it produces formic acid and succinic acid.
Acetic acid is produced from carbon dioxide and hydrogen, and acetic acid, butyric acid, isovaleric acid, and succinic acid are produced from other assimilated carbon sources.

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. 669. Furthermore, this strain was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERM-P No. 8046.

(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 to the medium as dissolved carbon dioxide, carbon salt, 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, molybdate leader, 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 Bacteroides sp. No. 669 strain was cultured as follows. Transfer 5 ml of the culture medium shown in Table 1 to a test tube.
After dispensing and sterilizing, 100 μl of the culture solution was cultured in the same medium.
was added in an anaerobic glove box (Anaerobox, Pharma Inc.), sealed tightly with a butyl rubber stopper, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and the mixture was left to stand still at 30°C. It was cultured in situ.

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 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 SB No. 669 strain was cultured with shaking. The measurement method was the same as in Example 1, and the product was analyzed. As a result, 1.30 g/acetic acid was produced in 10 days.

Claims (1)

[Claims] 1 Bacteroides sp. No. belongs to the genus Bacteroides, grows in carbon dioxide and hydrogen, has a growth pH range of 4.0 to 7.0, and assimilates ribose.
669.