JPH0378099B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel bacterium belonging to the genus Acetobacterium. 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,
Known species included Clostridium aceticum, Acetobacterium uthudei, Butylobacterium methylotrophicum, Eubacterium limosum, and Clostridium strain CV-AA1. Also under high temperature conditions are Acetogenium kivyi and Clostridium thermoautotrophicum. Among these, one species belongs to the genus Acetobacterium, A. utsudei. (Object 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 these, it is important to obtain bacteria that have a high accumulation concentration and production rate of acetic acid. 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. With this intention in mind, 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 is known in the art in that it is a single or double bacillus with a width of 0.6-1 μm and a length of 2-5 μm, and a mixture of straight bacilli and slightly curved or bulge-shaped bacilli. This is a new microorganism, Acetobacterium sp. No. 446, which can be distinguished from the same species of bacteria. This microorganism is an obligate anaerobic, Gram-positive, non-spore bacillus that metabolizes and produces acetic acid when cultured under anaerobic conditions in a medium containing carbon dioxide and hydrogen, and is of the same genus as the known Acetobacterium uthudei. However, it differs in some mycological properties, especially in morphology, and is considered to be a new bacterial species. Since the official species name has not yet been assigned, it is designated as Acetobacterium sp. No. 446 in the present invention. Next, we will show the method for creating Acetobacterium sp. No. 446 and its mycological properties. (Creation method) This bacterium was isolated from field soil in Nakatane-cho, Tanegashima, by the following method. That is, 5 ml of the liquid medium shown in Table 1 was dispensed into test tubes and sterilized. Approximately 0.3 g of soil was added in an anaerobic glove box. 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 30 weeks. After subculture twice in liquid medium, roll tube method (Methods in Microbiology, Volume 3B, 117)
(1969) Academic Press), a single bacteria was isolated on an agar medium prepared by adding 3% agar to the medium shown in Table 1 to obtain this bacterium. (Mycological properties) The mycological properties of the strain of the present invention are shown. In order to investigate this mycological property, the Aerob Laboratory
Manual 4th edition (Anaerobe Laboratory
Manual，The VIPAnaerobe Laboratory
Virginia Polytechnic Institute and State
University, Blacksburg (1972)). The method and culture medium composition described in Classification and Identification of Microorganisms (written by Takeharu Hasegawa, published by Gakkai Publishing Center) were used. (Microscopic findings) 1 Cell shape and size: single or double rods, with a mixture of straight rods and slightly curved or bulge-shaped rods. Figure 4 shows an electron micrograph (4000x magnification) of this bacterium. Width 0.6-1μm,
Length 2-5 μm 2 Flagella: present, subterminal 3 Spores: absent 4 Gram staining: positive, becomes negative in late culture. Metachromatic substances are seen within the cells. (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 Edge: Smooth ridge Rise: Slightly raised Surface: Smooth Color Tone: White to cream color Does not grow on gravy agar media. (Physiological properties) Attitude towards oxygen: static anaerobic Growth range (PH) Optimum PH: 7.7 Growth PH: 5.5-8.5 (Temperature) Optimum temperature: 30℃ Growth temperature: 20-40℃ Indole production: - Liquefaction of gelatin: - Production of catalase: - Hydrolysis of starch: - Hydrolysis of esculin: - Production of pigments: - Vitamin requirement: thiamine, pantothenic acid (generation of acids from sugars, etc.) Table 1 shows the basic medium 1% or more of the following carbon sources
Add 0.5% and replace the gas phase with sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), inoculate this bacteria,
It was statically cultured at 30°C. Fructose (1%), DL
-Lactic acid (1%), methanol (0.5%), formic acid (0.5
%) and ribose (1%) as a carbon source, acetic acid was produced as an organic acid in the medium. (Assimilation of carbon sources) Add 5 ml of a liquid medium containing the following carbon sources (1% unless otherwise specified) to the basic medium shown in Table 1 to a test tube with a diameter of 18 mm to create a sterile medium. 446 was inoculated, the gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and this bacterium was inoculated and statically cultured at 30°C for 14 days. Growth was measured by measuring turbidity at 600 nm using a spectrometer (Spectronic 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 considered "not assimilated," if it is 0.1 or more and less than 0.2, it is "slightly assimilated," and if it is more than 0.2, it is "assimilated." did. Assimilated: D-fructose, sorbose,
DL-lactic acid, methanol (0.5%) Slightly assimilated: D-ribose, formic acid (0.5%) Not assimilated: arabinose, cellobiose,
Galactose, D-glucose, lacnose,
Maltose, mannose, melezitose, melibiose, raffinose, rhamnose, sucrose, trehalose, xylose, erythritol, inositol, mannitol, starch, sorbitol, ethylene glycol, glycerol, acetic acid (0.5%), propionic acid (0.5%)
%), ethanol (0.5%), propanol (0.5
%), succinic acid, fumaric acid, pyruvate, malic acid, casamino acids, glutamic acid, aspartic acid, alanine, glycine, serine, adonyl, salicin, hippuric acid, amygdalin, peptone, yeast extract. (Comparison with conventional similar species, etc.) Based on the above mycological properties, No. 446 is a strain characterized by being an obligately anaerobic Gram-positive non-spore bacillus whose main fermentation metabolite is acetic acid. be. Based on this property, a search based on Virgie's Manual of Determinative Bacteriology, 8th edition and Aerob Laboratory Manual, 4th edition suggests that it belongs to Eubacterium, but Eubacterium In the genus, there was no description of a bacterial species whose characteristics matched those of No. 446. On the other hand, Acetobacterium (type strain) has been newly discovered as an obligate anaerobic, Gram-positive, non-spore bacillus that accumulates acetic acid from carbon dioxide and hydrogen as the main fermentation product.
Acetobacterium woodii) was proposed in 1977. (International Journal of Systematic Bacteriology, Volume 27,
When we compared the properties of No. 446 and Acetobacterium utsudei, we found that they were both obligately anaerobic, Gram-positive, non-spore bacilli that accumulate acetic acid as the main fermentation product, but the points shown in Table 2 The properties of both bacteria were different.

[Table] From the above, No. 446 is considered to be a new bacterial species belonging to the genus Acetobacterium, and therefore it was named Acetobacterium sp. No. 446. Furthermore, this strain was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERMP No. 7017. (Culture method) The culture method is basically the same as for general microorganisms. However, 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 tightly closed with a rubber stopper.On a slightly larger scale, it is usually used. The fermentation tank 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 fermentation tank is not particularly limited, but In addition to the commonly used stirred mixing tank, single-stage or multi-stage bubble column type, and draft tube type fermenters can also be used.The carbon source used for culture is usually supplied as carbon dioxide gas, but it is not dissolved in the medium. It can also be added as carbon dioxide, carbonate, or hydrogen carbonate.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 a common practice to add vitamins such as yeast extract or the like.The present invention will be explained below using specific examples.Use Example 1 Acetobacterium sp. No. 446 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 ml dispensing and sterilization
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, and the supernatant was injected into a high-performance liquid chromatograph (Shimadzu Corporation, model LC4A) equipped with an Ionpack C811 (Showa Denko) column. 0.1% phosphoric acid aqueous solution was flowed as the mobile phase at a flow rate of 1 ml/min, and detection was performed at 210 n.
When this was carried out using the absorption of m, the chromatogram shown in FIG. 1 was obtained. The retention times of the two peaks A and B in this chromatogram were confirmed to match those of the medium and acetic acid, respectively, by comparison with standard products. The growth of bacterial cells was observed by measuring turbidity at 600 nm with a spectrometer, and the amount of gas phase components absorbed by the microorganisms was determined by inserting a syringe needle attached to one end of a U-shaped tube into a culture test tube using butyl rubber. Insert it through the stopper,
It was measured from the fluctuation of the water level inside the U-shaped pipe. When the change over time was measured using the above experimental method,
As shown in Figure 2, this strain has a 5.1
g/g of acetic acid was produced. The arrow in the figure indicates the date when the gas phase was replaced with new gas (H ₂ /CO ₂ = 2/1). Use Example 2 An L-shaped test tube was prepared in the same manner as in Use Example 1, and Acetobacterium SB No. 446 was cultured with shaking. The measurement method was the same as in Use Example 1, and the changes over time were measured. As shown in FIG. 3, this strain produced 9.2 g/acetic acid after 24 days of culture. The arrows in the figure change the gas phase to new gas (H ₂ /
The date of replacement with CO ₂ = 2/1) is shown. Usage Example 3 Acetobacterium S.B. No. 446 was statically cultured in the same manner as in Usage Example 1, acetic acid was measured, and carbon dioxide in the gas phase was collected using a Unibeads C (Gas Kuro Kogyo) column. Measured using a gas chromagraph equipped with Flow helium as the mobile phase at a flow rate of 80 ml/min, and set the column temperature to 60 °C.
Detected by TCD. The amount of bicarbonate and dissolved carbon dioxide contained in the culture medium were measured by the method described in Applied and Environmental Microbiology, Vol. 33, p. 1270 (1977). When the carbon yield was determined by the above method, the third
As shown in the table, it was 82% after 12 days of culture.
The yield was defined as 100% when one molecule of acetic acid was produced from two molecules of carbon dioxide.

【table】

【table】 [Brief explanation of drawings]

Figure 1 is a chromatogram of the culture solution obtained in Use Example 1, and Figures 2 and 3 show the daily changes in bacterial growth, acetic acid production, and gas absorption in Use Examples 1 and 2, respectively. It is a graph. FIG. 4 is an electron micrograph showing the morphology of Acetobacterium sp. No. 446.

Claims (1)

[Claims] 1 Acetobacterium sp. No. 446, a single or double bacillus with a width of 0.6-1 μm and a length of 2-5 μm, containing a mixture of straight bacilli and slightly curved or bulge-shaped bacilli.