JPH0378110B2 - - 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 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 and Clostridium strain CV-AA1 were known. Also, under high temperature conditions, there are Acetogenium kivii, Clostridium thermoautotrophicum, and Clostridium thermoaceticum. (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. 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 microorganism of the present invention belongs to the genus Clostridium,
Clostridium sp. No. 307 is a slightly curved rod that grows on carbon dioxide and hydrogen, has no methanol assimilation ability or gelatin liquefaction ability, shows a positive indole production test, and has flagella. This bacterium is considered to belong to the genus Clostridium in that it is an anaerobic bacterium that produces spores, but it grows on carbon dioxide and hydrogen, and is different from known congener bacteria in various properties that will be described in detail later. Therefore, it is considered to be a new bacterial species. Since the official species name has not yet been assigned, this invention uses Clostridium sp.
It will be displayed as No.307. Next, we will show the creation method and mycological properties of Clostridium sp. No. 307 (hereinafter abbreviated as this bacterium). (Creation method) This bacterium was isolated from the soil of Hiraito Plain in Hokkaido 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. (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 the “Anaerobe Laboratory Manual”.
Manual) 4th Edition” (The VIP Anaerobe
Laboratory Virgini a Polytechnic Institute
and State University, Blacksburg (1972)) and Bergey's Manual of Determinative Bacteriology.
Manual of Determinative Becteriology) No. 8
The method and culture medium composition described in "Classification and Identification of Microorganisms" (written by Takeharu Hasegawa, Gakkai Publishing Center) were used. (Microscopic findings) 1 Cell shape and size: Slightly curved rod, singly or in pairs. Width 0.6-0.8μm, length 2.0~
3.4 μm 2 Flagella: 3 periflagella Spores: present, terminal 4 Gram staining: negative (medium composition) Examples are shown in Table 1.

【table】

[Table] (Growth status) Growth on an agar medium containing the composition shown in Table 1 plus 3% agar is as follows. Shape: Circular Rim: Smooth Raise: Slightly raised Surface: Smooth Color Tone: White to cream color (Physiological properties) Attitude towards oxygen: Obligately anaerobic Growth range (PH) Optimal PH: 7.7 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 Production: - (Assimilation of carbon source) Add 5 ml of a liquid medium containing the following carbon source (1%) to the basic medium shown in Table 1 to a test tube with a diameter of 18 mm to create a sterile medium and inoculate this fungus. Replace the gas phase with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%) and heat at 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 600nm from the control that does not contain a carbon source is less than 0.1, it is "not assimilated," if it is 01 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: glucose, fructose, xylose, ribose, arabinose, rhamnose, maltose, melibiose, trehalose,
Cellobiose, mannitol Also, when hydrogen is used instead of nitrogen in the above test, carbon dioxide is also assimilated. Slightly assimilated: Not applicable Slightly assimilated: Sorbose, galactose,
Satucarose, lactose, raffinose, melezitose, sorbitol, methanol, starch (generation of acids from sugars, etc.) 1% of the sugars that were confirmed to be assimilated in the above test were added to the basic medium shown in Table 1, and the gas phase Nitrogen (67
%) and carbon dioxide (33%), and this bacteria was inoculated and cultured stationary at 30°C. Acetic acid was produced as an organic acid in the medium for all carbon sources. Also, when peptone/yeast extract medium or peptone/yeast extract/glucose medium was used, acetic acid was produced as an organic acid in the medium. (Comparison with similar native species, etc.) Based on the above mycological properties, No. 307 is an obligate anaerobic, slightly curved Gram-negative sporobacillus that shows a positive indole production test. This is a strain whose fermentation metabolite is acetic acid. Because of this property, Virgie's Manual of Determinative Bacteriology 8th edition and Aerob.
A search based on the 4th edition of the Laboratory Manual suggests that it is a strain belonging to Clostridium. So, when I followed the genus identification key in the 4th edition of the Aerob Laboratory Manual, I came across Clostridium sphenoides. In addition, Virgie's Manual of Determinative Bacteriology, 8th edition, does not list any bacterial species whose characteristics match those of No. 307. No.
A comparison of the properties of 307 and Clostridium sphenoides revealed that both were in agreement that they were obligate anaerobic spore-forming 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. There are four known types of bacteria belonging to the genus Clostridium that grow on carbon dioxide and hydrogen. Of these, three are Orthobacillus and the remaining one (Clostridium strain CV-
AA1) was distinguishable from the present invention in that it had methanol assimilation ability and gelatin liquefaction ability.

[Table] From 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. 307. Furthermore, this strain was designated as "FERM-P No. 7487 (FERM-P No. 7487)" by the Institute of Microbial Technology, Agency of Industrial Science and Technology.
7487). (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, sodium molybdate, boric acid, or biotin It is common practice to add vitamins such as yeast extract and yeast extract. The present invention will be explained below using specific examples. Example 1 Clostridium sp. No. 307 strain was cultured as follows. A culture solution obtained by dispensing 5 ml of the medium shown in Table 1 into a test tube, sterilizing it, and then culturing in the same medium.
100μ in an anaerobic glove box (Farma,
The cells were added to the cellulose solution (anaerobox) and sealed with a butyl rubber stopper, and the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), followed by static culture 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, 1.70 g/acetic acid was produced during 10 days of static culture. (The product was confirmed using a gas chromatograph-mass spectrometer.) Example 2 Clostridium sp. No. 307 strain was cultured with shaking using an L-shaped test tube prepared in the same manner as in Example 1. Ta. The measurement method was the same as in Example 1, and the product was analyzed. As a result, 2.50 g/acetic acid was produced in 10 days.

Claims (1)

[Claims] 1 Clostridium sp. No. 307, which belongs to the genus Clostridium, grows on carbon dioxide and hydrogen, has no ability to assimilate methanol or liquefy gelatin, shows a positive indole production test, has flagella, and has a slightly curved rod.