JPH0472515B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) This invention relates to clostridium
The present invention relates to a method for producing acetic acid from carbon dioxide and hydrogen using a novel bacterium belonging to the genus.
Acetic acid is used in fields such as food and industrial raw materials. (Prior Art) Several microorganisms are known that assimilate carbon dioxide and hydrogen and accumulate acetic acid in the growth medium.
Among them, there are four types of bacteria belonging to the genus Clostridium that have been reported so far. And our newly created Clostridium sp No. 68-2 Microtechnical Research Institute No. 7367,
Clostridium sp
No. 307 Microtechnical Research Institute No. 7487, Clostridium
Clostridium sp No.484
There are 7 types including the 3 types listed in No. 7488 and the 4 types mentioned above. (Purpose of the Invention) The present inventor focused on carbon dioxide, which is a renewable resource that exists in abundance in the natural world, and is also the final waste product of various industries, and used it to generate hydrogen, which is considered as a future energy source. They investigated a biochemical method for producing acetic acid by reacting with carbon dioxide, and arrived at this invention.Although the above-mentioned bacteria are known, it has not been possible to industrially produce acetic acid from carbon dioxide and hydrogen. There are still many issues to be solved in order to implement this method, especially since the optimal growth pH is below PH6 using carbon dioxide and hydrogen as substrates, and there have been no reports of bacteria capable of producing acetic acid from carbon dioxide and hydrogen. Not yet. Under these circumstances, the present inventors aimed to provide a new method for producing acetic acid using carbon dioxide and hydrogen as substrates. (Structure of the Invention) The present invention uses carbon dioxide and hydrogen as substrates to cultivate bacteria that belong to the genus Clostridium and have the ability to assimilate carbon dioxide and hydrogen to produce acetic acid, and collect the acetic acid produced and accumulated. This is a method for producing acetic acid characterized by: The microorganism used in the present invention belongs to the genus Clostridium and is an acetic acid-producing bacterium that assimilates carbon dioxide and hydrogen, and the microorganism described in the Examples of the present invention is the first such microorganism. The microorganism used in the example is an anaerobic bacterium that produces spores, so it is thought to belong to the genus Clostridium, but it grows in an acidic region of PH4.0 to 7.0 with carbon dioxide and hydrogen. It differs from known bacteria of the same genus in various properties, which will be described in detail later, such as the absence of flagella, and is considered to be a new bacterial species. Since an official species name has not yet been assigned, this invention uses Clostridium sp.
sp) No. 672 will be indicated as Microtechnology Research Institute No. 8049. Next, we will show the creation method and mycological properties of Clostridium sp. No. 672 (hereinafter abbreviated as this bacterium). (Creation method) This bacterium was isolated from Aso mud in Kumamoto Prefecture 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. Substitute with sterilizing gas containing carbon (33%) and culture at 30℃ for about 30 minutes.
Succession was carried out every week. After subculturing twice in a liquid medium, the roll tube method (Methods in Microbiology, Vol. 3 B, p. 117 (1969) Academic Press) to isolate a single bacterium and obtain the present bacterium. (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),
“Bergey’s Manual of Determinative Bacteriology”
of Determinative Bacteriology) 8th Edition", "Bergie's Manual of Systematic Bacteriology (Volume 1)"
(Bergey's Manual of Systematic
The method and culture medium composition described in "Bacteriology (Volumel)" (1984) and "Classification and Identification of Microorganisms" (written by Takeharu Hasegawa, Gakkai Publishing Center) were used. (Microscopic findings) 1 Cell shape and size: Single or double bacillus, width 1.5 μm, length 6.0-8.0 μm 2 Flagella: None 3 Spores: Present 4 Gram staining: Negative (medium composition) 1st Examples are shown in the table. Table 1 Composition of basic medium (in deionized water 11) 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/1 Sodium bromoethanesulfonate
1ml Yeast extract 0.2g PH5.3 Vitamin solution composition (mg/l) 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/1) 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・2H ₂ O 0.006 (Growth condition) On an agar medium with the composition shown in Table 1 plus 3% agar. Growth is as follows. Shape: Circular Perimeter: Smooth Ridge: Slightly raised Surface: Smooth Color tone: White (physiological properties) Attitude towards oxygen: Obligately anaerobic Growth range (PH) Optimum 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 utilization Add 5 ml of liquid medium containing the following carbon source (1%) to the basic medium in Table 1 to a 18 mm diameter test tube to create a sterile medium, inoculate this bacteria, and change the gas phase to nitrogen (67%). and sterilizing gas containing carbon dioxide (33%) and heated to 30°C.
The cells were cultured statically for 14 days. Growth was measured by measuring turbidity at 600 nm using a spectrometer (Spectronik 20, manufactured by Shimadzu Corporation). If the difference in turbidity at 600 nm from the control containing no carbon source is less than 0.1, it is considered "not assimilated".
0.2 to "slightly utilize" anything greater than or equal to 0.1 and less than 0.2
The above-mentioned items were defined as ``capitalizing''. Assimilated: D-glucose, D-fructose, sorbose, xylose, D-ribose,
Rhamnose, galactose, lactose, cellobiose Carbon dioxide is also assimilated when hydrogen is used instead of nitrogen in the above test. Things that cannot be assimilated: sorbose, arabinose,
Maltose, sucrose, melibiose, trehalose, raffinose, melezitose, mannitol, methanol, ethanol (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 is nitrogen (67
%) and carbon dioxide (33%), and this bacteria was inoculated and cultured stationary at 30°C. For all carbon sources, acetic acid and n are present as organic acids in the medium.
-Butyric acid was produced. Also, when peptone/yeast extract medium or peptone/yeast extract/glucose medium was used, acetic acid and n-butyric acid were produced as organic acids in the medium. (Comparison with similar native species, etc.) From the above mycological properties, No. 672 is an obligate anaerobic Gram-negative spore bacillus, and its main fermentation metabolites are acetic acid, carbon dioxide and hydrogen. This strain is characterized by the fact that the other carbon sources it assimilates are acetic acid and butyric acid. Because of this property, Bergey's Manual of Determinative Bacteriology - 8th Edition, Bergey's Manual of Deterministic Bacteriology
According to a search based on Systematic Bacteriology (Volume 1) and Aerob Laboratory Manual 4th Edition, it is thought to be a strain that belongs to Clostridium. Therefore, I identified Clostridium phalatucus (C.
fallax). Also, Virgie's Manual of Determinative Bacteriology, 8th Edition, Virgie's. The Manual of Systematic Bacteriology (Volume 1) does not list any bacterial species whose properties match those of No. 672. When we compared the properties of No. 672 and Clostridium falutux, we found that they were both obligately anaerobic Gram-negative sporobacilli.
The characteristics of both bacteria were different in the points shown in the table. The strain of the present invention is a bacterium belonging to the genus Clostridium that grows in carbon dioxide and hydrogen to produce acetic acid. Seven types of bacteria are known to grow in carbon dioxide and hydrogen, but all of these are in the near-neutral PH range. It was distinguishable from the present invention in that it grew in the acidic region and could not grow in the acidic region.

[Table] * Virgie's Manual Determination of Bacteriology (8th Edition)
Based on 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. 672. Furthermore, this strain was deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERM-P No. 8049. 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 does not matter. However, in addition to the commonly used stirring mixing tank, there are single-stage or multi-stage bubble column types,
A draft tube type fermenter 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 yeast, as necessary. Adding vitamins such as extracts is a common practice. The present invention will be explained below using specific examples. Example 1 Clostridium sp. No. 672 strain was cultured as follows. Transfer the culture medium shown in Table 1 to the test tube.
Culture solution cultured in the same medium after 5ml dispensing and sterilization
Add 100 μl in an anaerobic glove box, seal with a butyl rubber stopper, replace the gas phase with sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and incubate at 30°C.
The cells were cultured statically. Bacterial strains 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, 0.78 g/l of acetic acid was produced during 10 days of static culture. Example 2 Clostridium sp. No. 672 strain was cultured with shaking using an L-shaped test tube prepared in the same manner as in Example 1. The measurement method was the same as in Example 1, and the product was analyzed. As a result, 0.98 g/l of acetic acid was produced in 10 days.

Claims (1)

[Claims] 1 Using carbon dioxide and hydrogen as substrates, cultivate the bacterium Clostridium sp. No. 672, which belongs to the genus Clostridium and has the ability to assimilate carbon dioxide and hydrogen to produce acetic acid, and collect the produced and accumulated acidic acid. A method for producing acetic acid characterized by: