JPH0358782A - Obtaining of clostridium bacterium mutant, same mutant and production of butanol using thereof - Google Patents

Abstract

NEW MATERIAL:A Clostridium pasteurianum CA 101 stock (FERM P-10817) as a mutant of genus Clostridium bacterium having analog resistance to fermented intermediate of butanol and butanol producibility. USE:Used for production of butanol. PREPARATION:For instance, Clostridium pasteurianum DSM 525 stock as a mother stock of genus Clostridium bacterium is anaerobically cultured in a medium in a nitrogen atmosphere at 37 deg.C for 12hr and collected by centrifuge, then subjected to mutation treatment with adding mutagen such as N-methyl-N'- nitro-N-nitrosoguanidine, thus resultant mutant is cultured in a medium containing crotonyl alcohol to afford a mutant of genus Clostridium bacterium having colony of crotonyl alcohol-resistant stock and butanol producibility.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for obtaining a mutant strain of a Clostridium bacterium,
The present invention relates to the mutant strain and a method for producing butanol using the mutant strain. The mutant strain of the present invention is a mutant strain that has the ability to produce a significant amount of butanol, and the butanol produced by the production method of the present invention can be obtained from solvents, combinations, or raw materials. It is used as fuel in the solvent industry, organic chemical industry, etc.

[Prior art and problems to be solved by the invention] Butanol is useful as a solvent, raw material, fuel, etc., and can be produced by petrochemistry, but there are problems such as a decrease in petroleum resources. Therefore, there is a desire for an inexpensive production method using a fermentation method using 1) [ as a raw material. Conventionally, Clostridium acetobutylicum and the like have been used to produce butanol by fermentation, but attempts have been made to isolate various mutant strains in order to improve the productivity of butanol. The fermentation production method for butanol using a butanol-producing mutant strain is as follows:
JP-A-58165786 using a butanol-resistant mutant strain
The method described in Biotechnol Lett.
+ 9, 175 (1987), 7, 711 (1.
985), the method described in Appl. Environ.
Microbiol. , 53, 2854 (1987
) Toj] Catty Q-. 1.238 (1985). Same base-j
), 477 (1985) and the like are known. In addition, as a fermentation production method of butanol using Ku[':I Stridium bastrianum, JP-A-63
1.57989 Bow Bulletin and Riotechnol Lett
．． , Dan, p. 889 (1986) and the like are known. However, both force methods have the problem that the yield of butanol is insignificant.

[Means for Solving the Problems] Therefore, the present inventors conducted a search for suitable bacterial strains for the purpose of producing butanol effectively using fermentation methods, and found that cross 1. We have found that mutant strains obtained by mutating genus bacteria are suitable, and have completed the present invention.

That is, the present invention is characterized in that after mutation treatment of Cross I.Rhydium bacteria, a mutant strain of Cross I.Rhydium bacteria is generated by analog resistance of a butanol fermentation intermediate. acquisition power method of mutant strains,
Analog resistance of butanol fermentation intermediate 111,
Cross I. risou l, genus mutant strain having the ability to produce bukunol, and culturing the mutant strain to produce butanol 1.1
The present invention provides a method for producing butanol, which involves collecting the same.

The Clostridium genus bacterial mutant strain of the present invention is obtained by mutating a parent strain of Clostridium genus bacteria and separating it based on the stiffness of the butanol fermentation intermediate relative to the analogue.

Here, as Cross I and Rhydium bacteria, Cross 1
- Clostridium
pasteur. ianum) DSM 525 strain. In addition, mutation treatment of the parent strain can be carried out using known methods, such as ultraviolet irradiation, X-ray irradiation, radioactive irradiation such as gamma rays, treatment with mutagenic agents, etc. I can do it. In addition, as a mutagenic agent, ethylmethanesulfoun-1-N-methyl-N''-nitro-N-2I-rosoguanidine dimethyl sulfate, 2-aminobuline, acriflavine, acridine orange, hydrazine 4-nitroquinoline-N-oxyto , manganese chloride, etc.

Next, analogs of butanol fermentation intermediates refer to, for example, crotonyl alcohol and allyl alcohol acrylic acid, preferably crotonyl alcohol. Furthermore, the analog resistance of a butanol fermentation intermediate refers to the property that it is possible to produce butanol in a medium containing the analog without suffering growth damage.

Furthermore, six specific methods for isolating mutant strains will be explained. First, Clostridium passi Lianum DSM5
The 25 strains were cultured anaerobically in the CM3 medium shown in Table 1 at 37°C under a nitrogen atmosphere for 12 hours. After the completion of culture, centrifugation was performed to collect the bacteria, and 50mM 1.
(Tris-Malate) buffer (p.+1 5.
Wash twice at step 5. Then, N-methyl-N) N-21 Rosoguanicine 0.2 / mp
After suspending this bacterial suspension in the same buffer solution containing 11 L of
Mutation treatment is carried out by shaking at °C for 15 minutes. After the mutation process is completed, collect the bacteria and obtain 1・lisma l/−1
- Wash 3 times with buffer.

Number L Table Glucose Polypebutone Yeast Extract (Ni+-)2SO4 K 2 I+ 11 0. MgcNz. 6H20 Ops” CaCε2 FeSO, Relisulinalium (redox pigment) t.-cy
s++・Ic°C (reducing agent) 10g 5g 2g 1.3 g 5g 1g 5g 150mg 1.3mg 2mg 0.5g Total 1. ．． Op. (pl+ 7.0) * Morpholinopropanesulfonic acid Next, the bacterial cells obtained by mutation treatment are CM shown in Table 1.
4g/j of crotonyl alcohol in 3 medium! The cells were inoculated into a medium supplemented with the following, and enrichment culture was carried out at 37°C for 3 days. After culturing, the bacteria were collected and spread on an agar plate of CM3 medium supplemented with 4 g/Q of crotonyl alcohol, and cultured at 37°C for 3 days. Separate. In this way, Clostridium bacteria that are resistant to the analog of the butanol fermentation intermediate of the present invention and capable of producing butanol can be obtained. The present inventors identified this bacterium as Clostridium 7CA101.
It was named a stock. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-10817.

The mycological properties of this bacterium are shown below.

(1) Bacillus of 0.8 x 3-10 μm (2) Motile with periflagellate (3) Endospores present (4) Positive Durham staining (5) Growth temperature 30-4 (6) Sugar utilization amictalline - maltose Arabinose mannitol
− Mannose fructose +
Melecitose Charactose - Raffinose Inulin - Rhamnose Lactose
- Clostridium bastrianum (; A 101 strain (FERM
The mycological properties of Clostridium P-1.0817) are similar to the parent strain, Clostridium P-1.0817, except that it is resistant to allyl alcohol and acrylic acid in addition to crotonyl alcohol, and has the ability to ferment and produce a significant amount of butanol. It shows completely the same mycological properties as DSM 525 strain.

In the present invention, mutant bacterial strains of the genus Clostridium that are resistant to analogues of butanol fermentation intermediates and have the ability to produce butanol, such as clostrisalin sorchitol startin uclostreha υ-nuquinulose O'C Dium bastrianum CA 101 shares (FERM P
-10817) to produce butanol, which is collected to produce butanol.

The culture medium used includes a carbon source, nitrogen source, and inorganic ions. A conventional medium containing organic nutrients may be used, and carbon sources include glucose, sucrose, fructose, and maltose. Mannose, melibiose, sorbitol, mannitol, melezitose, raffinose trehalose,
II of starch etc. class is used.

The culture is carried out under anaerobic conditions at a temperature of 20 to 40°C, a pH of 5 to 8, preferably 6 to 7, and left standing or with gentle stirring. Moreover, the amount of inoculation of this bacterium is 0.1 to 10%, preferably 1 to 5%. The cultivation period may be continued until a sufficient amount of butanol is produced, usually for 1 to 10 days.
Preferably it is 2 to 5 days. Alcohols such as butanol and organic acids that have survived in the culture solution are confirmed and quantified by gas chromatography after making the culture solution acidic with phosphoric acid.

〔Example〕

Next, the present invention will be explained by examples.

Example Clostridium bastrianum strain CA 101 (FERM P-1081) was added to the CM3 medium shown in Table 1 above.
7) and cultured anaerobically at 37°C for 18 hours. This culture solution was inoculated into CM3 medium at a ratio of 2% and statically cultured anaerobically for 2 days at 37°C in a 10 mR screw test tube.

After removing the bacterial cells from the obtained culture solution by centrifugation, the yield of butanol was measured by gas chromatography and found to be 2.1 g/f (yield based on sugar: 21%).
Other raw materials were obtained in the amounts shown in Table 2.

Comparative Example In the example, Clostridium bastrianum C
The same operation as in Example was performed except that Clostridium bastrianum DSM 525 strain was used instead of A 101 strain (FERM P-10817). The raw amount of phthanol was 0.6 g/EC (sugar yield: 6%), and other raw materials were obtained in the amounts shown in Table 2.

mirror! L surface yield (g/L) Ethanol Fukuno}1 example
0.1 0.5 2.1. ．． 0.2 0
．． 10.2 1.2 0.6
0.7 0.2 [Effects of the Invention] The bacterial mutant strain of the genus Cuicos 1-Rhijiu 1 of the present invention has rigidity in analogs of butanol fermentation intermediates, and has the ability to produce butanol at low cost and in significant quantities. have.

The butanol obtained by the present invention is useful in fields such as organic chemical industry and solvent industry.

Claims (4)

[Claims] (1) A method for obtaining a Clostridium bacterium mutant, which is characterized by obtaining a Clostridium bacterium mutant strain by mutagenicity of Clostridium bacterium and analog resistance of a butanol fermentation intermediate. (2) A Clostridium bacterial mutant strain that has analog resistance to butanol fermentation intermediates and has the ability to produce butanol. (3) The mutant strain of Clostridium bacteria according to claim 2, wherein the analog of the butanol fermentation intermediate is crotonyl alcohol, allyl alcohol, or acrylic acid. (4) resistant to analogs of butanol fermentation intermediates;
1. A method for producing butanol, which comprises culturing a mutant strain of a Clostridium bacterium capable of producing butanol to produce butanol, and collecting the same.