JPS6142557B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention involves culturing a bacterium belonging to Pseudomonas fructomethanolica, which can assimilate methanol and fructose well, and slightly monomethylamine and glucose, in a medium containing methanol as the main carbon source, and extracting bacteria from the culture solution. This is a method for producing bacterial cells, characterized by separating the cells. Conventionally, molasses, sulfite pulp waste liquid, n-paraffin, etc. have been used as raw materials for producing microbial cells. However, these fermentation feedstocks have problems in terms of supply and price. Fermentation methods in which microorganisms are used to utilize hydrocarbons such as kerosene, naphtha, light oil, and n-paraffin are also being considered as a solution to food problems that are predicted to occur in the near future, but these raw materials contain carcinogens and other substances. However, there are still many practical problems, such as how to treat fermentation waste liquid. The present inventors searched for bacteria that strongly assimilate methanol as a suitable fermentation raw material because it is obtained purely in large quantities through the chemical industry and has high water solubility. They discovered a new bacterium that does not exist in Japan, and completed the present invention. A new bacterial species belonging to the genus Pseudomonas used in the present invention, Pseudomonas fructomenorica sp.
The mycological properties of fructomethanolica sp.nov.C-66) (NIFE deposit acceptance number 4019) are as follows. 1 Morphological properties Inorganic salt medium supplemented with 1% methanol* ¹ at 37℃2
Cultured for 1 day. The cells are rod-shaped, 0.3 to 0.7 x 1 to 3 microns in size, and usually move singly or in pairs, with a single flagellum. Gram staining is negative, no spores are formed, and acid-fast staining is negative. 2. Cultivation properties in various media (Culturing was carried out at 37°C for 3 days unless otherwise specified) Meat juice agar culture: No growth. Methanol-containing inorganic salt* ¹ Plate culture: Good growth. Colony: round, convex, full edge, smooth surface, glossy, white, translucent, slightly viscous. Methanol-containing inorganic salt *Slope culture: Good growth Linear growth, smooth surface, glossy, smooth edges, white (slightly pale yellow or reddish when cultured for longer than 5 days) Translucent, slightly viscous, medium There is no formation of water-soluble pigments. Methanol-containing broth agar slant culture: Good growth. The culture properties are almost the same as those of methanol-containing inorganic salt medium. Meat juice liquid culture: No growth. Methanol-containing inorganic salt *Liquid culture: Good growth. It becomes uniformly cloudy and forms a ring. (During long-term culture, this species may form a thin film depending on the strain.) Methanol-containing gelatin multilayer puncture culture: Grows on the surface. Do not liquefy the medium. Litmus milk culture: No change. (No growth) 3 Physiological properties Nitrate reduction: Reduces. (A modified method was used in which about 1% methanol was added to the conventional culture medium.) Denitrification reaction: None MR test: Negative VP test: Negative Indole production: Negative Hydrogen sulfide production: Negative Starch hydrolysis: Negative Citrate availability (Koser and
(using Christensen medium): Negative Availability of inorganic nitrogen sources: Use nitrates and ammonium salts. Formation of dye: No water-soluble dye is formed. (As mentioned above, when cultured on an inorganic salt agar medium containing methanol for a long period of time, the bacterial cells turn slightly bright yellow.) Urease: positive Oxidase: positive Catalase: positive Growth range: Grows in the pH range of 5 to 9. PH6-8 is preferred. Grows at temperatures between 10° and 42°C. 30-41°C is preferred. Attitude towards oxygen: aerobic. Hemolytic: Negative Penicillin sensitivity (2.5Iu/ml): Negative Oxytetracycline sensitivity (10γ/
ml): Negative Salt tolerance: Grows in medium containing 1.5% salt, but cannot grow in 2% or more salt. Fermentability of sugars (by Hugh & Leifson method)

[Table] The results were negative for gas generation from each sugar. Assimilation of carbon sources* ² : Grows assimilates fructose and methanol well. In addition, glucose and monomethylamine showed slight growth during long-term culture (about 2 weeks or more). The assimilation ability of formaldehyde depends on the test method and differs depending on the strain, but
One of the representative strains of the present invention, strain C-66, is slightly assimilated. Many of the carbon sources listed below were tested, but none were found to be assimilated. Sugars: L-arabinose, D-xyrose, D-
Galactose, maltose, sucrose, lactose, trehalose, and starch. Alcohols: ethanol, n-propanol,
n-butanol, inositol, D-sorbitol, D-mannitol. Organic acids: formic acid, acetic acid, pyruvic acid, succinic acid,
Citric acid, lactic acid, malonic acid, propionic acid, gluconic acid, fumaric acid, malic acid, benzoic acid,
2,5-dihydroxybenzoic acid, p-hydroxybenzoic acid, m-hydroxybenzoic acid, p-aminobenzoic acid, anisic acid, anthranilic acid, oxalic acid. Amino acids: DL-glycine, L-alanine, L-
Leucine, DL-isoleucine, L-arginine, L-lysine, DL-methionine, DL-aspartic acid, L-glutamic acid, 1-asparagine, glutamine, tryptophan. Amines: dimethylamine, trimethylamine,
Monoethylamine. Amides: formamide, acetamide, hydrocarbons: methane, ethane, propane, butane. Separation source: Land transfer and sewage *1 Composition of inorganic salt medium with 1% methanol addition: 2 g of sulfuric acid, 1.5 g of potassium phosphoric acid, 2 potassium phosphoric acid 2
g, magnesium sulfate 0.3 g, small amount of inorganic salt solution (magnesium sulfate 6 g, calcium chloride 0.015
g, zinc sulfate 0.14g, ferrous sulfate 0.028g, copper sulfate 0.025g, sodium molybdate 0.024g, cobalt chloride 0.024g, manganese sulfate 0.084g dissolved in 100ml of distilled water), 1ml to 490ml of tap water Dissolve in water to obtain the first liquid. Separately, 500 g of agar 15g to 20g
Dissolve in ml of tap water to make the second solution, and separately add 1Kg/
After sterilizing for 10 minutes under pressure of cm ² and cooling to 40-50 °C,
Combine both liquids aseptically and add methanol10 inorganically.
Add ml. *2 The carbon source assimilation test was carried out using the composition of the above-mentioned 1% methanol-added inorganic salt medium, excluding methanol, using distilled water instead of tap water, and containing agar as the basic medium. Among the above carbon sources, sugars and amino acids account for 0.5%
(W/V) amount, 0.1% (W/V) amount of organic acids, 0.5-0.1% amount of methylamines, and 0.1% amount of amides in a small amount of distilled water. Separately, it was added aseptically to a basal medium that had been sterilized and cooled to 40 to 50°C to obtain the prescribed amount, which was then used as an agar plate medium. Furthermore, tests were also conducted on the addition of 0.01% of acetaldehyde, acetone, benzene, phenol, and organic acids such as formic acid, oxalic acid, and propionic acid. The organic acids were neutralized with sodium hydroxide and treated aseptically before use. Gaseous hydrocarbons were cultured and tested in a gas mixed with the same amount of air. The inoculum was inoculated by suspending bacterial cells cultured one day earlier in a methanol-added inorganic salt medium in sterile water. Culture was carried out at 30°C for 10 days or more, and the presence or absence of growth was judged. Furthermore, for those that were difficult to judge, they were cultured in a liquid medium with the same composition to confirm. For some tests of physical properties such as nitrate reducing ability and hydrolysis of starch and gelatin, when using a medium that cannot grow as it is, add about 1% methanol to grow the bacteria. did. Many methanol-assimilating bacteria have been isolated and reported so far. According to Virgie's Manual of Determinative Bacteriology, 8th edition, 1974, there is no suitable genus name to which bacteria with these mycological properties should belong. However, in addition to using _C1 compounds such as methanol and methylamine, it can be grown using fructose among sugars and only a small amount of glucose. Considering the various properties, it is considered most appropriate to assign it to the genus Pseudomonas. Among methanol-assimilating bacteria
The inventors have already reported that it would be more appropriate to classify bacteria that only assimilate _C1 compounds into the genus Methylomonas [Journal of General Aplytic Microbiology, Vol. 19, p. 11] In 1973, the International Conference on Microbiology Utilizing C1 Compounds (Tokyo) and Microbial Growth on Sea _One Compounds (Microbial Growth on Sea One Compounds) were also held.
Growth on C ₁ - Compounds, p. 11, 1975], the bacterium of the present invention uses not only C ₁ compounds but also fructose, a C ₆ compound, and exhibits the same growth rate, making it distinct from Methylomonas bacteria. It should be. Many academic reports and patent applications have been filed to date regarding methanol-assimilating bacteria, and many new bacterial species have been reported in related academic journals, patent publications, and patent publications. I can't find anything that has the same properties as . Therefore, this bacterium is considered to be a new species of the genus Pseudomonas, and is classified as Pseudomonas fructomethanolyca sp.
Nobu C-66 (Pseudomonas fructmethanolica
sp.nov.C−66). The medium used for culturing this bacterium must contain methanol as the main carbon source, and if the medium also contains appropriate amounts of nitrogen sources, inorganic substances, vitamins, and other growth-promoting substances, synthetic medium or Any natural medium may be used. Methanol, the main raw material, is so-called cytotoxic, and it is necessary to maintain the concentration of the medium at 6% or less. It is preferable that the ratio is 2% or less for good bacterial growth and proliferation. Since this bacterium can assimilate and grow fructose well, it is also possible to use a mixture of methanol and fructose. As nitrogen sources, for example, inorganic nitrogen compounds such as nitrates and ammonium salts, and organic nitrogen-containing substances such as urea, corn, stave liquor, casein hydrolyzate, peptone, and amino acid solutions are used. Furthermore, it is also possible to feed ammonia in small amounts according to the growth of the bacteria to the extent that it does not inhibit the growth of the bacteria. Among the inorganic salts, the most important medium components are phosphates, magnesium salts, potassium salts, iron salts, etc., and small amounts of manganese salts, zinc salts, calcium salts, copper salts, etc. are added as necessary. In addition, vitamins and amino acids may be added as essential or growth-promoting substances depending on the strain. The culture temperature is normally 20° to 40°C, but the optimum temperature for growth is 30 to 40°C, and the culture is carried out aerobically at a pH of 6 to 8. The culture method may be either batch culture or continuous culture. Without giving methanol at high concentration from the beginning,
From the viewpoint of its toxicity, it is a preferable method to add a small amount at first and then replenish as it is consumed. In addition, ammonia is consumed as a nitrogen source, and the pH of the culture solution decreases. In this case, in order to maintain the pH of the culture solution under favorable growth conditions for the bacterial strain, it is neutralized with an alkali such as caustic potash or caustic soda, or ammonia. After culturing the bacteria in this manner, the bacterial bodies are separated from the culture solution. A centrifugation method or a filtration method is used for the separation method. In carrying out the above separation method,
Isolation is easy if pretreatment is performed such as adding various flocculants to the culture solution to aggregate the bacteria, or lowering the pH of the culture solution and further heating to cause the bacteria to aggregate. This is efficient, and these methods can also be applied. The isolated microbial cells are washed and dried, and used as they are, mixed with appropriate substances, or subjected to various treatments as raw materials for feeds, foods, medicines, and these industrial applications. According to the present invention, methanol, which is easily available through industrial production, can be used as a raw material. It is an industrially advantageous method, as it does not contain any carcinogenic substances, and the risk of transport and explosion is extremely low compared to gaseous hydrocarbons. The present invention will be explained in more detail below using examples. Example 1 As fermentation medium (NH ₄ ) ₂ SO ₄ 3 g, KH ₂ PO ₄ 2
g, _K2HPO45g , _MgSO4・_7H2O0.5g , _Fe〓2 ⁺⁺
mg, M〓2 ⁺⁺ mg, 0.1 g of yeast extract was dissolved in 1000 ml of tap water, the pH was adjusted to 6.8, the solution was dispensed into a 500 ml Sakaguchi flask in a volume of 50 ml, and the mixture was sterilized at 120°C for 10 minutes and cooled. After that, 1 g of methanol was added to each flask in an aseptic manner, and Pseudomonas fructomenolica sp. knob C-66 was precultured for 24 hours at 37°C using the same medium as above. 2 ml of the preculture solution containing the bacterial cells was inoculated and cultured under shaking at 37°C for 48 hours. After culturing, the culture solution was centrifuged to collect bacteria and dried on silica gel in a vacuum desiccator at 60°C to obtain 4.8 g of dried bacterial cells per 1000 ml of culture solution. Example 2 A culture medium with the same composition as in Example 1 before addition of methanol was placed in a 10- or 20-volume diaphragm meter, and 1
Kg/cm ² Sterilize for 10 minutes and add 1 part methanol (V/V) to this.
% was added to prepare a culture solution. 3% by volume of Pseudomonas fructomenolica sp. knob C-66, which had been previously cultured at 37°C for 24 hours in a medium with the same composition, was added and cultured with aeration at 39°C. The pH was automatically maintained at 6.8 by adding aqueous ammonia, and methanol was added continuously according to its consumption and to maintain the methanol concentration in the culture solution at 0.05-0.5 (V/V)%. added to. After 40 hours of culture, the amount of methanol added is 80ml per culture solution.
reached. Stop the culture, collect the bacterial cells by centrifugation, dry them at 80℃ for 24 hours, and add 1 per culture solution.
28.5 g of dried bacterial cells were obtained.

Claims (1)

[Claims] 1. A method for producing microbial cells, which comprises culturing bacteria belonging to Pseudomonas fructomethanolica in a medium containing methanol as a main carbon source, and separating the bacterial cells from the culture solution.