JPH0472513B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) This invention is based on Bacteroides.
The present invention relates to a method for producing acetic acid from carbon dioxide and hydrogen using bacteria 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 the Clostridium genus that we newly created.
Clostridium sp No. 68-2, Clostridium sp No. 7367, Clostridium sp No. 307, Clostridium sp No. 7487, Clostridium sp No.
There are three types: 484 Microtechnical Research Institute No. 7488, and the 4 mentioned above.
There are seven types including seeds. No bacteria in the genus Bacteroides that produce acetic acid from carbon dioxide and hydrogen have been reported so far. (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, and in particular, there are no reports of bacteria capable of producing acetic acid by growing in an acidic region such as PH4.0 to 7.0 using carbon dioxide and hydrogen as substrates. It has not been. Under these circumstances, the present inventor aims 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 culture a bacterium belonging to the genus Bacteroidetes that has the ability to assimilate carbon dioxide and hydrogen to produce acetic acid.
This is a method for producing acetic acid, characterized by recovering generated and accumulated acetic acid. The microorganism used in the present invention belongs to the genus Bacteroides 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 a Gram-negative, non-spore bacillus and is thought to belong to the genus Bacteroides in that it produces not only acetic acid but also n-butyric acid and isovaleric acid from sugars; It is considered to be a new bacterial species because it grows in an acidic region of .0 to 7.0 and has no flagella, as it differs from known bacteria of the same genus in various properties that will be described in detail later. Since the official species name has not yet been assigned, this invention uses Bacteroides sp.
sp) No. 669 and Bacteroides sp. No. 671 and Bacteroides sp. No. 8048. Next, Bacteroides sp. No. 669 and Bacteroides sp. No. 671 (hereinafter abbreviated as this bacterium)
The method of creation and mycological properties of this drug are shown. (Creation method) Both bacteria were isolated from mud at Beppu Hot Springs in Oita Prefecture by the following method. That is, the liquid medium shown in Table 1
After dispensing 5 ml 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 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 Stath University, Blacksburg (1972),
“Bergey’s Manual of Determinative Bacteriology”
of Determinative Bacteriology) 8th Edition", "Bergey'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) No. 669 No. 671 1 Cell shape Single or 2 Single or 2 And size: ~ 3 straight rods ~ 3 straight rods (μm) 1.0 × 4.5 ~ 5.0 1.1 ~ 1.4 × 6.0 ~10.0 2 Flagella: None None 3 Spores: None None 4 Gram staining: Negative Negative (medium composition) Examples are shown in Table 1. 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/sodium bromoethanesulfonate
1ml Yeast extract 0.2g PH5.3 Vitamin solution composition (mg/) 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/) 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 ₃ BO ₄ 0.005 NaMoO ₄・2H ₂ O 0.006 (Growth condition) Growth on an agar medium with the composition shown in Table 1 plus 3% agar is as follows. It is as follows. No.669 No.671 Shape: Circular Circular Periphery: Smooth Smooth Protuberance: Slightly elevated Slightly elevated Surface: Smooth Smooth Color: White White (physiological properties) No.669 No.671 Attitude toward oxygen: Unbiased Sexually anaerobic Obligately anaerobic Optimum growth PH: 6.5 Optimum PH: 5.8 Range (PH) Growth PH: 4.7-7.0 4.7-7.0 (Temperature) Optimum temperature: 30℃ Optimum temperature: 30℃ Growth temperature: 25-40℃ 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 shown 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: No.669 D-glucose, D-fructose, xylose, D-ribose, arabinose, galactose, maltose, sucrose, lactose, cellobiose No.669 D-glucose, D-fructose, xylose, rhamnose, galactose, Sucrose, lactose, trehalose, cellobiose, melezitose, mannitol Carbon dioxide is also assimilated when hydrogen is used instead of nitrogen in the above test. Things that cannot be assimilated: No.669 Sorbose, rhamnose, melibiose,
Trehalose, raffinose, melezitose, mannitol, methanol,
Ethanol No. 671 Sorbose, D-ribose, arabinose, maltose, melibiose, raffinose, methanol, ethanol (generation of acid from sugar, etc.) Sugars that were confirmed to be assimilated into the basic medium in Table 1 in the above test Add 1% of
%) and carbon dioxide (33%), and this bacteria was inoculated and cultured stationary at 30°C. Acetic acid, n-butyric acid, isovaleric acid, and succinic acid were produced as organic acids in the medium using all carbon sources. Also, when peptone/yeast extract medium or peptone/yeast extract/glucose medium was used, acetic acid, n-butyric acid, isovaleric acid, and succinic acid were produced as organic acids in the medium. (Comparison with similar native species, etc.) From the above mycological properties, No. 669 and No. 671 are obligate anaerobic Gram-negative non-spore bacilli whose main fermentation metabolites are carbon dioxide and hydrogen. acetic acid from carbon sources, n-butyric acid, isovaleric acid, and isovaleric acid from other assimilated carbon sources.
Because of this property, Virgies produces succinic acid.
A search based on the Manual of Determinative Bacteriology, 8th Edition, Virgie's Manual of Systematic Bacteriology (Volume 1), and Aerob Laboratory Manual, 4th Edition results in Bacteroides ( It is thought to be a strain belonging to Bacteroides. Therefore, according to the genus fixed key in the 4th edition of the Aerob Laboratory Manual, we identified B. ruminicola subspense ss.
ruminicola). Also, Bargie's
The Manual of Determinative Bacteriology, 8th edition, Virgie's Manual of Systematic Bacteriology (Volume 1) does not contain descriptions of bacterial species whose characteristics match No. 669 and No. 671. Nakatsuta. When we compared the properties of Bacteroides ruminicola subspecis ruminicola in No. 669 and No. 671, we found that they were both obligate anaerobic nonspore bacilli, but as shown in Table 2, the properties of both bacteria were It was different. The strain of the present invention grows on carbon dioxide and hydrogen to produce acetic acid. Bacteria that grow in carbon dioxide and hydrogen among the Bacteroidetes layer have not been reported so far.

[Table] Do
do
* Virgies Manual of Systematics Bacteriology (Volume 1)
Based on the above, both strains are considered to be new bacterial species belonging to the genus Bacteroides, so they were named Bacteroides SB No. 671. Furthermore, this strain was designated as "FERM-P No. 8046 (FERM-P No. 8046)" by the Institute of Microbial Technology, Agency of Industrial Science and Technology.
8046)” and “FERM-
PNo.. 8048". (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, carbonate, or 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 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 and Bacteroides sp. No. 671 strains were cultured as follows.
After dispensing 5 ml of the medium shown in Table 1 into test tubes and sterilizing them, 100 μl of the culture solution cultured in the same medium was added in an anaerobic glove box (Anaerobox, Pharma Co., Ltd.), and the tube was sealed with a butyl rubber stopper. The phase was replaced with a sterilizing gas containing hydrogen (67%) and carbon dioxide (33%), and the cells were incubated statically 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, after 10 days of static culture, Bacteroides
Bacteroides sp. No. 669 produced 0.95 g/acetic acid, and Bacteroides sp. No. 671 produced 0.72 g/acetic acid. Example 2 Using an L-shaped test tube, prepared in the same manner as in Example 1, Bacteroides sp. No. 669 and Bacteroides sp. No. 671 strains were cultured with shaking.
The measurement method was the same as in Example 1, and the product was analyzed. As a result, Bacteroides sp. No.
669 produced 1.30 g/acetic acid, and Bacteroides sp. No. 671 produced 1.24 g/acetic acid.

Claims (1)

[Claims] 1. Acetic acid, which is characterized by culturing bacteria belonging to the genus Bacteroides and capable of assimilating carbon dioxide and hydrogen to produce acetic acid, using carbon dioxide and hydrogen as substrates, and recovering the produced and accumulated acetic acid. manufacturing method.