JPH0465675B2 - - 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, Clostridium strain CV-AA1, and Clostridium sp. No. 68-2, which we created.
FERM-P No. 7367, Clostridium sp. No. 307, FERM-P No. 7487
No. (FERM-P No. 7487), Clostridium sp.
7488) and Acetobacterium sp. No. 446 FERM-P No. 7563 (FERM-P No. 7563). Also under high temperature conditions are Acetogenium kivyi and Clostridium thermoautotrophicum. (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. Furthermore, no bacteria has been reported that has an optimal growth range below pH 6 and is capable of producing acetic acid from carbon dioxide and hydrogen. 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 present invention is an anaerobic bacterium with a width of 1.5 μm and a length of 6.0 to 8.0 μm, and is considered to be a bacterium belonging to the genus Clostridium in that it is a single or two or three straight rod bacterium and produces spores. However, it grows on carbon dioxide and hydrogen, is negative for Gram staining, grows in the pH range of 4.0 to 7.0, and is a straight bacillus without flagella.It is different from known bacteria due to the following properties, and is a new bacterial species. It is thought that.
Since the official species name has not yet been assigned, it is designated as Clostridium sp. No. 672 in the present invention. Next, we will show the method for creating Clostridium sp. No. 672 and its mycological properties. (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 globe 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.
Transplanting was carried out every week. After subculture 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 "Anaerobe Laboratory Manual 4th Edition".
Manual,,The VIPAnaerobe Laboratory
Virginia Polytechnic Institute and State
University, Blackburg (1972)) and Burgess Manual of Determinative
Bacteriology (8th edition) (Bergy's Manual
of Determinftive Bacteriology (EIGHTH
EDITION (1974)) and Bergey's Manual of Systematic Bacteriology (Volume 1).
Systematic Bacteriology (Volume 1) (1984))
and “Classification and Identification of Microorganisms” (written by Takeharu Hasegawa).
The method and culture medium composition described in the Japan Society Publishing Center) were used. (Microscopic findings) 1 Cell shape and size: Single or 2-3
Direct rods in series, width 1.5 μm, length 6.0-8.0 μm 2 Flagella: None 3 Spores: Present, terminal 4 Gram staining: Negative (medium composition) Examples are shown in Table 1. Table 1 Composition of basic medium (in 1 part deionized water) 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 PH5.4 Vitamin solution composition (mg/) Bithion 2 Folic acid 2 Pyridoxine hydrochloride 10 Thiamin hydrochloride 5 Riboflavin 5 Nicotinic acid 5 Ca pantothenate 5 Vitamin B12 0.01 p-Aminobenzoic acid 5 Thioctic acid 1 Mineral 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・1H ₂ O 0.009 H ₃ BO ₄ 0.005 NaMoO4・2H2O 0.006 (Growth condition) Growth on an agar medium with the composition shown in Table 1 plus 3% agar is as follows. . Shape: Circular Periphery: Smooth Ridge: Slightly raised Surface: Smooth Color tone: White (Physiological properties) Attitude towards oxygen: Obligate anaerobic Growth range (PH) Optimum PH: 5.8 Growth PH: 4.0-7.0 (Temperature) ) Optimum temperature: 30℃ Growth temperature: 20-40℃ Indole production: - Gelatin liquefaction: - Catalase production: - Starch hydrolysis: - Aesculin hydrolysis: + Pigment production: - (Carbon source utilization Add 5 ml of a liquid medium containing the following carbon source (1% unless otherwise specified) to the basic medium in Table 1 to a test tube with a diameter of 18 mm to create a sterile medium, inoculate this bacteria, and inoculate the gas phase. The cells were replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and cultured stationary at 30°C for 14 days. Growth was measured using a spectrometer (Spetronik 20,
(manufactured by Shimadzu Corporation). If the turbidity at 600 nm differs from the control containing no carbon source by less than 0.1, it is "not assimilated," if it is 0.1 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: D-glucose, D-fructose, xylose, D-ribose, rhamnose,
Galactose, lactose, and cellobiose that cannot be assimilated: sorbose, arabinose, maltose, sucrose, melibiose, trehalose, raffinose, melezitose, mannitol, methanol, ethanol (generation of acids from sugars, etc.) The above test is applied to the basic medium in Table 1. Adding 1% or 0.5% of carbon sources known to be assimilated by
The gas phase was replaced with a sterilizing gas containing nitrogen (67%) and carbon dioxide (33%), and this bacterium was inoculated and cultured statically at 30°C. Acetic acid and butyric acid as organic acids were produced in the medium with all carbon sources. (Comparison with similar native species, etc.) From the above mycological properties, No. 672 is an obligately anaerobic Gram-negative spore bacillus, and its main fermentation acid metabolites are acetic acid, This strain is characterized by producing butyric acid from other assimilated carbon sources. Due to this property, Virgies Manual
Of Determinative Bacteriology 8th Edition, Versey's Manual of Systematic Bacteriology (Volume 1) and Aerob Laboratory Manual Volume 4
Based on the edition, it appears to be a strain belonging to Clostridium. So, when I tried to identify it according to the 4th edition of the Aerob Laboratory Manual, I came across Clostridium fallax (C. fallax).
In addition, Virsey's Manual of Determinative Bacteriology, 8th edition, and Virsey's Manual of Systematic Bacteriology (Volume 1) have No. 1 properties.
There were no descriptions of bacterial species that matched 672. When we compared the properties of No. 672 and Clostridium falutux, it was found that they were both obligate anaerobic spore-bearing bacilli, but the properties of both bacteria were different in the points shown in Table 2.

[Table] produces acetic acid and butyric acid
do.
*Bergie's Manual of Determinant Bacteriology (8th edition) Based on the above, this strain is considered to be a new bacterial species belonging to the genus Clostridium, so 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. (Culture method) In principle, the cultivation method is 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 The fermentation tank used 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. However, in addition to commonly used stirring mixing tanks, single 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 It can also be added as dissolved carbon dioxide or as a carbonate or hydrogen carbonate.The nitrogen source can be an ammonium salt such as ammonium chloride or a nitrate such as sodium nitrate.
Various nitrogen compounds that can be used in normal fermentation can be used. 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 Clostridium sp. No. 672 strain 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 sterilization in ml aliquots
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, 0.82 g/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, 1.15 g/acetic acid was produced in 10 days.

Claims (1)

[Claims] 1. Chromatridium sp. No. 672, which belongs to the genus Clostridium, grows in carbon dioxide and hydrogen, has a growth pH range of 4.0 to 7.0, and does not assimilate maltose.