JPH0421476B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing acetone. The present inventors have conducted various studies on methods for producing acetone using methanol as a carbon source, and as a result, have discovered that microorganisms belonging to the genus Hyphomicrobium have the ability to convert methanol into acetone, and have arrived at the present invention. That is, the gist of the present invention is to obtain acetone from the culture by culturing a microorganism belonging to the genus Hyphomicrobium and having the ability to produce acetone using methanol as a carbon source. It's in the manufacturing method. The present invention will be explained in detail below. The microorganism used in the present invention belongs to the genus Hyphomicrobium and has the ability to produce acetone. For example, Hyphomicrobium variabil 42-3-1
(Hyphomicrobium variabile 42-3-1)
(FERM P-7019). Hyphomicrobium Barrierville 42-3-
No. 1 was isolated from soil collected in Australia in December 1981, and its bacteriological properties are as follows. (1) Microscopic characteristics Agar plate medium containing 1.5% methanol, 30℃5
Culture properties during the day (colony morphology) (a) External shape: circular (b) Size: 1.0 to 2.0 mm (c) Surface protuberances: convex lens shape (d) Surface shape: smooth (e) Gloss: no ( f) Color tone: cream to pale yellow (g) Transparency: translucent (ch) Periphery: entire edge Morphological properties of 1.5% methanol-containing liquid medium and agar plate medium at 30°C for 2 to 5 days (b) Cell Morphology: Oval to circular rods (B) Cell size: 0.5 to 1.0 x 1.0 to 3.0 μm (C) Pleomorphism: None (D) Motility: Yes, single polar Ben hair (E) Sporulation: None (F) Gram staining: Negative (G) Acid-fastness: Negative (H) Division mode: Proliferates by budding. Its germination mode is reminiscent of the germination of mold spores. The tip of the germ tube-like hyphae swells and daughter cells are formed. (2) Growth status in each medium (a) Slant medium containing 1.5% methanol, 30℃, 5
Daily growth status. Vigorous growth, growing uniformly along the inoculation line. Colony color is cream to pale yellow. The surface is smooth. The surrounding area is completely smooth. Opacity. (b) High-rise culture medium containing 1.5% methanol, 30℃, 5
Daily growth status. It grows only along the inoculation line. Growth on the surface is vigorous. (c) Static culture in liquid containing 1.5% methanol, 30℃,
Growth status for 5 days. Medium growth. It becomes cloudy. No film was formed. (d) Gelatin medium containing 1.5% methanol, 30℃,
Growth status for 5 days. Medium growth. Does not liquefy gelatin. (e) Growth condition on broth agar slant medium at 30℃ for 5 days. Growth conditions are similar to those on methanol-containing slants. (f) Growth condition in gravy multilayer medium at 30°C for 5 days.
Same as growth on methanol-containing strata medium. (g) Growth status of meat juice liquid static culture at 30°C for 5 days. Same as growth in methanol-containing liquid culture. (H) Growth condition in meat juice/gelatin medium at 30°C for 5 days. Same as growth in gelatin medium containing methanol. Does not liquefy gelatin.

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[Table] (7) Chemical composition analysis of bacterial cells The content of guanine + cytosine in DNA (as determined by melting temperature measurement) was 60.2 to 60.5 mol%. Identification at the genus level This strain, 42-3-1, exhibits a budding-type division mode and has motile polar bent hairs.
It was found that it belonged to the genus Hyphomicrobium, which is described in Determinative Bacteriology 8th ed. (1974)). According to the 8th edition of Virgy's Manual, three genera of Budding Bacteria are described: Hyphomicrobium, Hyphomonas, and Pedomicrobium.
Hyphomonas do not utilize _C1 compounds;
Pedomicrobium does not utilize _C1 compounds and has a polymorphic form.
It has been identified from the genus Hyphomicrobium. Species-level identification The genus Hyphomicrobium includes H. neptinium, H. vulgare, H.
H. indicum, H. variabil (H.
variabile) (JP 47-14589) and H. coagulans (Takada, 1974).
species are known. Of these five species, the former three are known as Marine Bacteria, which live in brackish water to seawater, and are microorganisms that can grow 50 to 100% in seawater. On the other hand, H. variabile and H. coagulans are known as soil bacteria. A comparison of this strain, 42-3-1, with the original description of H. coagulans revealed that it grows weakly in peptone water, does not form a thin film (pellicle), and is distinguished from H. coagulans by its carbon source assimilation pattern. It was done. When we compared various microbial properties of this strain, 42-3-1, and the reference strain of H. variabil (NCIB 10517), we found that they are carbon sources, as shown in test items (3) to (8). The two strains were very similar in their assimilation ability and other physiological properties. Furthermore, when we measured the G-C content of the DNA of both strains, we found that H. variabil (NCIB)
10517) is 60.2-60.7 mol%, this strain 42-3-1
showed a measured value of 60.2 to 60.5 mol%, and both bacteria were in good agreement in this respect as well. Therefore, this strain 42
-3-1 was identified as Hyphomicrobium variabil. The medium used in the present invention is not particularly limited as long as it contains methanol as a main carbon source. As a carbon source, in addition to methanol, various carbohydrates, organic acids, etc. may be further added, and as a nitrogen source, organic ammonium salts, inorganic ammonium salts, urea, etc. can be used. Furthermore, various phosphates, sulfates, etc. can be used as inorganic substances, and various organic nutrients can also be added as necessary. Cultivation is usually carried out under aerobic conditions for about 12 hours to 10 days, but part of the culturing (the latter stage) can also be carried out under anaerobic conditions. The pH of the medium is selected from 4-10, and the temperature is selected from about 20-40°C. In the production of acetone, both proliferating bacterial cells and dormant bacterial cells can be used. When collecting and purifying acetone from culture,
Methods generally used for collecting and purifying organic compounds can be employed. The present invention will be further explained below with reference to Examples. The substances in the Examples were identified by comparing them with standard samples using gas chromatography/mass spectrometry and the like. Example 1 Methanol (15 g), NH ₄ Cl (4
g), K ₂ HPO ₄ (1 g), Na ₂ HPO ₄ (1 g),
MgSO ₄ 7H ₂ O (0.5 g), pyridoxine hydrochloride (1
mg), biotin (0.01 mg), thiamine hydrochloride (1 mg),
Riboflavin (1 mg), sodium D-pantothenate (1 mg), folic acid (0.01 mg), FeSO ₄ 7H ₂ O (1
mg), ZnSO ₄・7H ₂ O (1 mg), CuSO ₄・5H ₂ O (0.1
50 ml of medium (A) prepared by dissolving MnCl ₂ .4H ₂ O (0.04 mg) and adjusting the pH to 7.0 was dispensed into a 500 ml container with a shoulder strap and sterilized at 120° C. for 10 minutes. Hyphomicrobium variabil 42-3-1 was added to a methanol-containing agar slant medium prepared by adding 20 g of agar to this medium A.
The bacteria were cultured at 30°C for 4 days, a loopful of the culture was inoculated into the above-mentioned Kolben, and cultured for 4 days at 30°C using a reciprocating shaker at a rotation speed of 112 times/min. The obtained culture solution was further left to stand at 30° C. for 2 days to obtain 15 mg of acetone (ethanol, acetic acid, and isopropanol were obtained at the same time). Example 2 Hyphomicrobium barriervir 42-3-1 was cultured in the same manner as in Example 1. The culture solution was centrifuged (12,000 rpm for 15 minutes) to collect the bacteria, and further cultured in 10 ml of the same medium A. The suspension was suspended and statically cultured at 30°C for 3 days to obtain 15 mg of acetone (ethanol, acetic acid and isopropanol were obtained at the same time).

Claims (1)

[Claims] 1 Hyphomicrobium
1. A method for producing acetone, which comprises culturing a microorganism belonging to the genus Acetone and having the ability to produce acetone using methanol as a carbon source, and obtaining acetone from the culture.