JPH0358714B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing ethanol. As a result of various studies on methods for producing ethanol using methanol as a carbon source, the present inventors discovered that microorganisms belonging to the genus Hyphomicrobium have the ability to convert methanol into ethanol, and arrived at the present invention. That is, the gist of the present invention is to produce ethanol, which is characterized by culturing a microorganism belonging to the genus Hyphomicrobium and having the ability to produce ethanol, using methanol as a carbon source, and obtaining ethanol from the culture. It's in the manufacturing method. The present invention will be explained in detail below. The microorganisms used in the present invention belong to the genus Hyphomicrobium and have the ability to produce ethanol, such as Hyphomicrobium barriervir 42-3-1.
(Hyphomicrobium variabile42-3-1)
(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 Cultured on agar plate medium containing 1.5 methanol at 30℃ for 5 days (colony morphology) a) External shape: circular b) Size: 1.0 to 2.0 mm c) Surface protuberances: convex lens-like h ) Surface shape: smooth E) Gloss: None Color tone: Cream to light yellow T) Transparency: Semi-transparent T) Periphery: Full edge Liquid medium containing 1.5% methanol and agar plate medium, 30°C, 2 Morphological properties for ~5 days a) Cell morphology: oval to round rods b) Cell size: 0.5 to 1.0 x 1.0 to 3.0 μm c) Pleomorphism: None d) Motility: Yes, single (1) Spore formation: 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℃;
Growth status for 5 days. 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℃,
Growth status for 5 days. It grows only along the inoculation line. Growth on the surface is vigorous. c) Liquid static culture containing 1.5% methanol, 30
°C, growth conditions for 5 days. Medium growth. It becomes cloudy. No film was formed. D) Gelatin medium containing 1.5% methanol, 30
°C, growth conditions for 5 days. Medium growth. Does not liquefy gelatin. e) Growth conditions on gravy agar slant medium at 30℃ for 5 days. Growth conditions are similar to those on methanol-containing slants. f) Growth condition in gravy layer medium, 30℃, 5 days. Same as growth on methanol-containing strata medium. g) Meat juice liquid static culture, growth condition at 30℃ for 5 days. Same as growth in methanol-containing liquid culture. h) Growth condition in meat juice/gelatin medium at 30℃ 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.
Manual of Determinative Bacteriology 8th
It was found that it belonged to the genus Hyphomicrobium, which is described in ed. (1974)). According to the 8th edition of Virgy's Manual, three genera of Budding Bacteria are described: Hyphomicrobium, Hyphomonas, and Pedomicrobium. Hyphomonas does not utilize C ₁ compounds, Pedomicrium does not utilize C ₁ compounds, and has polymorphic forms.
It has been identified from the genus Hyphomicrobium. Species-level identification The genus Hyphomicrobium includes H. neptinium, H. vulgare,
H. indicum, H. variabile (Japanese Unexamined Patent Publication No. 14589/1989) and H.
Coagulans (H.coagulans) (Takada,
1974) are known. Of these five species, the former three live in brackish water to seawater.
Known as Marine Bacteria, 50~
It is a microorganism that can grow 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. This strain, 42-3-1
A comparative study of various microbial properties of the reference strain of H. variabil (NCIB 10517) revealed that carbon source assimilation and other physiological Both strains were very similar in properties. Regarding both strains
When the G-C content of DNA was measured, H. variabil (NCIB 10517) was 60.2-60.7 mol%.
The present strain 42-3-1 showed a measured value of 60.2 to 60.5 mol%, and the two bacteria were in good agreement in this respect as well. Therefore, this bacterial strain 42-3-1 was identified as Hyphomicrobium variabil. The medium used in the present invention includes methanol as the main carbon source,
There are no particular restrictions. As a carbon source, in addition to methanol, various carbohydrates, organic acids, etc. may be further added.
As the 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, both proliferating microbial cells and dormant microbial cells can be used. When collecting and purifying ethanol 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
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) in which MnCl ₂ .4H ₂ O (0.04 mg) was dissolved and the pH was adjusted to 7.0 was dispensed into a 500 ml shoulder-mounted container and sterilized at 120° C. for 10 minutes. Hyphomicrobium barrierville 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 bacteria was inoculated into the above-mentioned Kolben, and cultured for 6 days at 30°C 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 days to obtain 1.5 mg of ethanol (at the same time, acetic acid, acetone, and isopropanol were obtained). 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), the bacterial cells were collected, and the cells were further cultured in 10 ml of the same medium A. The suspension was suspended and statically cultured at 30°C for 2 days to obtain 3 mg of ethanol (acetic acid, acetone, and isopropanol were obtained at the same time).

Claims (1)

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