JPH035799B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) This invention relates to a method for producing formic acid from carbon dioxide and hydrogen using microorganisms. Formic acid is a useful material such as a dyeing aid, and is industrially produced from caustic soda and carbon monoxide. (Prior art) Regarding microorganisms that produce formic acid from carbon dioxide and hydrogen, it was known that spore-producing anaerobic bacteria (possibly belonging to the genus Clostridium) accumulate formic acid as a byproduct of acetic acid. Applied and Environmental
Microbiology Vol. 33, p. 1270 (1977)). On the other hand, Acetobacterium utsudei produces acetic acid by culturing it under anaerobic conditions in a medium containing carbon dioxide and hydrogen (International
Journal of Systematic Bacteriology Volume 27 355
(1977)), it was completely unknown that formic acid could be obtained. (Purpose of the Invention) The present inventor focused on carbon dioxide, which is a renewable resource, exists in abundance in nature, and is the final waste product of various industries, and aims to use it as a powerful energy source in the future. This invention was achieved by studying a biochemical method for producing formic acid by reacting it with hydrogen. (Structure of the Invention) In researching a method for producing carboxylic acid from carbon dioxide using microorganisms belonging to the genus Acetobacterium, the present inventor discovered that when 1-iodopropane is present in the culture medium, normal production The present invention was completed by discovering that not only the production of acetic acid, which is a chemical, is suppressed, but also formic acid is produced as a new product and accumulated in the culture medium. That is, the present invention provides a method for producing carboxylic acid by culturing microorganisms belonging to the genus Acetobacterium and assimilating carbon dioxide and hydrogen. This is a characteristic method for producing formic acid. (Microorganism) The microorganism used in the present invention is a microorganism belonging to the genus Acetobacterium that can assimilate carbon dioxide and hydrogen. sp. No. 446, FERM-P No. 7017). The former is a known bacterium from the above-mentioned literature. On the other hand, the latter is a microorganism discovered by the present inventors, and its mycological properties are as described in the specification of Japanese Patent Application No. 58-53644, but this will be described in a separate section. (Additional culture of 1-iodopropane) Formic acid is produced by adding 1-iodopropane to a medium containing carbon dioxide and hydrogen and culturing the above-mentioned microorganisms belonging to the genus Acetobacterium under anaerobic conditions. Can be done. If the amount of 1-iodopropane present in the medium is too small, acetic acid will be produced together with formic acid, and if it is too large, the amount of formic acid accumulated will decrease. The appropriate amount of 1-iodopropane to be added can be determined experimentally depending on the culture conditions, but the concentration in the culture solution should be between 0.1 and 1-iodopropane.
5mM, especially about 0.5 to 2mM is preferable. Regarding the method for culturing the above-mentioned microorganisms, the points other than the addition of 1-iodopropane are the same as those for culturing to obtain acetic acid from carbon dioxide and hydrogen, and the outline will be described in a separate section. Next, Acetobacterium sp. No. 446 (hereinafter referred to as this bacterium) used in the examples of the present invention
The method of creation and mycological properties of (Creation method of No. 446) This bacterium was isolated from field soil in Nakatane-cho, Tanegashima, by the following method. That is, 5 ml of liquid medium shown in Table 1.
After dispensing 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 converted into a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%). Replace the plate and culture it statically at 30℃ for about 3
Transplanting was carried out every week. . After subculture twice in liquid medium, roll tube method (Methods in Microbiology, Vol. 3 B, p. 117 (1969))
The present bacterium was isolated using an agar medium prepared by adding 3% agar to the medium shown in Table 1 (Academic Press). (Mycological properties of No. 446) For examining the mycological properties of this bacterium, please refer to "Aerob Laboratory Manual 4th Edition".
(Anaerobe Laboratory Manual The VIP
Anaerobe Laboratory Virginia Polytechnic
Institute and State University, Blacksburg
(1972)) and "Classification and Identification of Microorganisms" (Takeharu Hasegawa, Gakkai Publishing Center, 1975) and the method and medium composition described. Microscopic findings (1) Cell shape and size: Single or double bacilli, with a mixture of straight bacilli and slightly curved or bulge-shaped bacilli Width 0.6-1 μm, length 2-5 μm (2 ) Flagella: present, subterminal (3) Spores: absent (4) Gram staining: positive, becomes negative in the late stage of culture. Metachromatic substances are seen within the cells. Medium composition is illustrated in Table 1.

【table】

[Table] Growth conditions Growth on an agar medium containing the composition shown in Table 1 with 3% agar added is as follows. Shape: Circular Edge: Smooth Ridge: Slightly raised Surface: Smooth Color tone: White or cream Physiological properties that do not allow it to grow on meat juice agar Attitude towards oxygen: Obligate anaerobic Growth range (PH) Optimum PH: 7.7 Growth PH :5.5~8.5 (Temperature) Optimal temperature: 30℃ Growth temperature: 20~40℃ Indole production: - Gelatin liquefaction: - Catalase production: - Starch hydrolysis: - Aesculin hydrolysis: - Pigment production: - Vitamin requirement: Production of acid from thiamine, pantothenic acid sugar, etc. Add 1% or more of the following carbon sources to the basic medium shown in Table 1.
0.5 was added, the gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and this bacteria was inoculated.
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 source Prepare 5 ml of a sterile liquid medium containing the following carbon source (1% unless otherwise specified) and the basic medium composition in Table 1 in a test tube with a diameter of 18 mm, and replace the gas phase with nitrogen (67 %)
and replaced with sterilizing gas containing carbon dioxide (3%),
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. The difference in turbidity at 600nm from the control without carbon source is 0.1
Anything less than 0.2 was considered ``not to be utilized,'' 0.1 or more but less than 0.2 was ``slightly utilized,'' and 0.2 or more was ``available.'' 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, lactose, 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, pyruvic acid, malic acid, casamino acid, glutamic acid, aspartic acid, alanine,
Glycine, serine, adonitol, salicin, hippuric acid, amygdalin, peptone, yeast extract (cultivation method) The cultivation method is basically the same as for general microorganisms, but it is necessary to prevent oxygen contamination. In the laboratory, a method is used in which the culture vessel is left standing or shaken in an incubator sealed with a rubber stopper or the like. 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 equipment with an inert gas such as nitrogen or raw material gas (carbon dioxide and hydrogen). It is possible. The type of fermentation tank is not particularly limited, but in addition to commonly used stirring and mixing tanks, single- or multi-stage bubble column or draft tube 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, carbonate, 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, as necessary. It is common practice to add vitamins such as yeast extract and yeast extract. Formic acid accumulated in the culture solution can be recovered using known techniques. The present invention will be explained below using specific examples. Example 7 ml of the culture medium shown in Table 1 was dispensed into L-shaped test tubes, and after sterilization, 1-iodopropane was added in the amount shown in Table 2. This was inoculated with 140 μl of a culture solution of Acetobacterium sp. No. 446 cultured in the medium shown in Table 1. After sealing, the gas phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and shaking culture was performed at 30°C for 9 days. The supernatant of the culture solution was analyzed by high performance liquid chromatography using absorption at 210 nm. The retention times of the two peaks in the obtained chromatogram were confirmed to be those of the medium and formic acid, respectively, by comparing them with standard products. Further, the analytical values of product concentration are shown in Table 2.

[Table] Comparative Example Culture was carried out in exactly the same manner as in Example except that the amount of 1-iodopropane added was 0. When the culture solution was analyzed by high-performance liquid chromatography, the accumulated concentration of acetic acid was 1.7 g/min, and no formic acid was produced.

Claims (1)

[Claims] 1. A method for producing carboxylic acid by culturing microorganisms belonging to the genus Acetobacterium and assimilating carbon dioxide and hydrogen, characterized by producing and accumulating formic acid by allowing 1-iodopropane to exist in the medium. Method for producing formic acid.