JPH0378105B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to Hyphomicrobium variabile 42-3-1. As a result of various studies on methods for producing ethanol using methanol as a carbon source, the present inventors discovered that the microorganism Hyphomicrobium variabil 42-3- has the ability to convert methanol into ethanol, etc.
1 and arrived at the present invention. This strain is FERMP-
7019). Hyphomicrobium barrierville according to the present invention
42-3-1 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 ridges: convex lens shape (d) Surface shape: smooth (e) Gloss: None ( f) Color tone: cream to light yellow (g) Transparency: translucent (ch) Periphery: entire rim Morphological properties of cells in liquid medium containing 1.5% methanol and agar plate medium, 30℃, 2 to 5 days (b) 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 bent 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. (3) Physiological properties

【table】

【table】 (4) Presence or absence of acid and gas generation from various sugars

【table】 (5) Assimilation of sugars

【table】 (6) Assimilation of carbon sources other than sugars

【table】

[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 Bactericlogy 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 ethanol etc., both proliferating microbial cells and dormant microbial cells can be used. When collecting and purifying ethanol etc. from the 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 g) per 1 water.
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 mg), MuCl ₂・4H ₂ O (0.04 mg) were dissolved and 50 ml of medium (A) adjusted to pH 7.0 was poured into a 500 ml container with a shoulder. Dispense into
Sterilized at 120°C for 10 minutes. 20g of agar in this medium A
Hyphomicrobium variabil 42-3-1 was added to a methanol-containing agar slant medium supplemented with 30
℃ for 4 days, a loopful of the culture was inoculated into the above-mentioned Kolben, and cultured for 6 days at 30℃ using a reciprocating shaker at a rotation speed of 112 times/min. The obtained culture solution was further statically cultured at 30° C. for 2 to 5 days to obtain 1.5 mg of ethanol, 15 mg of acetone, 4 mg of isopropanol, and 3 mg of acetic acid. 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. Suspend at 30℃ for 2-6
After static culture for 1 day, add 3 mg of ethanol and acetone.
15 mg, isopropanol 4 mg, and acetic acid 3 mg were obtained.

Claims (1)

[Claims] 1 Has methanol assimilation ability, and has the ability to assimilate methanol.
Hyphomicrobium variabile42-3-1, deposited as No.
-3-1).