JPS6123992B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Methylococcus alkalophilus DM-1, which is characterized by assimilating methanol and having alkaliphilic properties.

Conventionally, when microorganisms are cultivated industrially, natural products such as molasses and sulfite pulp waste liquid, and saccharides derived from natural products have generally been used as carbon sources. However, the supply of these natural products is unstable, they are not always available in large quantities, and their prices fluctuate widely and frequently, which poses problems as industrial raw materials. In addition to these natural products, we discovered microorganisms that can assimilate synthetic organic compounds such as methanol.
If this can be used, microorganisms can be cultured stably at a low cost. Furthermore, if a microorganism that grows under very specific conditions that other microorganisms cannot grow in is discovered, the problem of bacterial contamination, which is often a problem in the microbial industry, may be solved. These microbial cells can be used as a protein source, and furthermore, completely new enzymes, enzymatic reactions, physiologically active substances, and metabolites that have not been found to date will be discovered from such microbial cells. The possibilities are huge.

In order to achieve many of the objectives mentioned above at the same time, we were able to grow using methanol as the only carbon source.
Growth pH is on the alkaline side. As a result of extensive searches for very special microorganisms that were completely unknown up to now, this strain was discovered, leading to the present invention.

The present invention belongs to the genus Methylococcus, and the growth pH is
The present invention relates to Methylococcus alkalophillus DM-1 (FER Deposit No. 7018), which is characterized by having an optimum pH of 8.5 to 9.5.

The soil collection site and mycological properties of this bacterial strain are described below.

Collection site: Soil near Tokachigawa Onsen, Obihiro City, Hokkaido Mycological properties The mycological properties of this strain are shown below.

1. Microscopic morphology The cells were cultured at 30°C for 2 to 4 days in a methanol-containing alkaline liquid medium and a methanol-containing agar plate medium (PH8.5 to 9.5), respectively.

Cell shape and size Spherical 0.7-1.0μ Population Unicell or bicellular Motility None Spore shape None Gram staining Negative Extracellular adhesives None 2 Culture properties in various media Broth agar plate culture (PH7.0, 30℃, (cultured for 1 day) No growth Colony on methanol-containing agar plate medium (PH 8.5 - 9.5, 30℃, cultured for 4 - 6 days) Colony morphology and characteristics External shape Circular size 1 - 3 mm Elevation Hemispherical structure Homogeneous surface Surface Smooth, full edge color Tone Yellow-white Transparency Opaque Broth agar slant culture (PH 7.0, 30℃, 7 days culture) No growth Methanol-containing slant culture medium (PH 8.5-9.5,
30℃, 3 days culture) Meat juice liquid culture (PH7.0, 30℃, 7 days culture) No growth Peptone water (PH7.0, 30℃, 7 days culture) No growth Methanol-containing liquid culture (PH8.5-9.5 ,
30℃, 3 days culture) Grow vigorously Meat juice agar puncture culture (PH7.0, 30℃, 7 days culture) No growth Puncture culture in methanol-containing medium (PH
8.5-9.5, cultured at 30℃ for 4 days)
Juicy gelatin high-rise culture growing in small papillary shapes (PH7.0, 30℃,
7-day culture) No growth 3 Physiological properties Tests were conducted at pH 8.5 to 9.5, except for tests on the growth pH range.

Reduction of nitrate Reduce to nitrite.

MR reaction Negative VP reaction Negative Formation of indole Negative Hydrolysis of casein Negative Formation of hydrogen sulfide Negative Hydrolysis of starch Negative Use of citric acid Negative Use of nitrogen sources Use ammonium salts, nitrates, and peptone as nitrogen sources Formation of pigments None Urease positive Catalase positive Growth range a PH 7.5-11.0, preferably 8.5-9.5 b Temperature 25-37°C, preferably 30°C Oxygen requirement Aerobic Oxidase positive O-F test (Hugh Lefson) negative Sugar assimilation Production of acids and gases The following sugars are not assimilated, and acids or gases are not produced from each sugar.

Raffinose, arabinose, xyrose, glucose, mannose, fructose, maltose, sucrose, lactose, trehalose, sorbitol, mannitol, inosyte, glycerin, starch, dextrin,
Assimilation of sugars other than galactose and inulin Assimilates methanol. Methane, methylamine, dimethylamine, n-propanol, n-butanol, formaldehyde,
Does not assimilate acetaldehyde, ethyl alcohol, formic acid, isopropanol, succinic acid, oxalic acid, citric acid, glycine, β-alanine and cyclohexane.

Bergey's Manual of Determinative Bacteriology
Manual of Determinative Bacteriology) No. 8
No bacterial species corresponding to this strain is found in the edition. In other words, since this strain is a coccus that does not assimilate anything other than methanol, it is considered to belong to the genus Methylococcus; however, when comparing this strain with Methylococcus capsulatus, the only bacterial species that belongs to the genus Methylococcus, we find that A decisive difference can be seen in the growth pH. In other words, Methylococcus capsulatus grows vigorously near neutrality, but this strain does not. For example, Methylococcus capsulatus Texas strain, Methylococcus capsulatus NCIB 11132 strain, etc. grow vigorously in a methane- or methanol-containing medium adjusted to pH 7.0.
The pH is too low for this strain to grow. This strain also differs from Methylococcus capsulata in that it does not produce extracellular adhesives. Based on these facts, this strain was determined to be a new strain and named Methylococcus alkalophilus DM-1.

Experimental methods are described in the above-mentioned Virgies Manual and in the ``Bacteriology Practice Summary'' edited by the Institute of Medical Science Alumni Association.
(1958). Furthermore, the compositions shown in Table 1 were used as a methanol-containing agar medium, a methanol-containing slant medium, and a methanol-containing puncture medium. Furthermore, as the methanol-containing liquid medium, the present medium not containing agar was used.

For example, the method for isolating this bacterial strain from soil is as follows. That is, the soil for isolation was thoroughly suspended in sterilized water, and a small amount of the resulting suspended supernatant was smeared onto the methanol-containing alkaline agar plate shown in Table 1, and the mixture was incubated for about 1 week for 30 minutes. Incubate at ℃. The colonies thus obtained are isolated, spread again onto the same plate medium, and cultured at 30°C for about one week. The obtained colony was re-isolated and grown on a slant medium with the same composition to obtain an isolated strain.

Table 1 Na ₂ HPO ₄ 2.1g KH ₂ PO ₄ 1.4g (NH ₄ ) ₂ SO ₄ 3.0g MgSO ₄・7H ₂ O 0.2g Anhydrous soda 4.0g *1 Mineral mixture 1.0ml *2 Vitamin mixture 1.0ml Methanol 12.5ml Distilled water 1 PH 9.5 *1 Mineral mixture CuSO ₄・5H ₂ O 0.15g MuCl ₂・4H ₂ O 1.5g ZnSO ₄・7H ₂ O 1.5g Ferric citrate・XH ₂ O 9.0g CaCl ₂・2H ₂ O 9.0g Distilled water 1 *2 Vitamin mixture biotin 20mg Calcium pantothenate 4g Folic acid 20mg Inositol 20mg Nicotinic acid 4g Pyridoxine hydrochloride 4g Thiamine hydrochloride 4g Riboflavin 2g Para-aminobenzoic acid 2g Distilled water 1 The medium used for culturing this bacteria is Any medium may be used as long as it contains methanol as a carbon source and appropriate amounts of a nitrogen source, inorganic substances, etc. The methanol concentration is preferably 3% by weight or less for good bacterial growth and proliferation. Inorganic nitrogen sources such as ammonium salts, nitrates, etc. and/or organic nitrogen-containing substances such as peptone, yeast extract, casein, etc. are used as nitrogen sources. Examples of inorganic components include calcium salts, magnesium salts,
Potassium salts, sodium salts, phosphates, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, boron compounds, iodine compounds, and the like are used. Culture conditions are temperature 25-37℃, preferably
30℃, and PH7.5~11.0, preferably PH8.5~9.5
It is. Culture is carried out aerobically under these conditions. When cultured outside of these conditions, the growth of this bacterium becomes poor. The oxygen concentration in the culture solution is not particularly limited, but is preferably 0.2 to 2.0 ppm. The culture method may be either batch culture or continuous culture. After culturing the bacteria in this manner, the bacterial bodies are separated from the culture solution. For separation, ordinary solid-liquid separation means are used.

When cultivating the novel microorganism of the present invention in large quantities, there is no need to stop operations due to contamination with bacteria, the raw materials are stably supplied, the culture can be stably carried out, and the resulting microorganisms are not contaminated with other microorganisms. There isn't.

Example 1 Approximately 0.1 g of soil collected near Tokachigawa Onsen, Obihiro City, Hokkaido was placed in a test tube containing 10 ml of sterile water and thoroughly suspended in a mixer. After allowing the test tube to stand for 10 minutes, add 0.1 ml of the supernatant liquid to 1 part of distilled water, 12.5 ml of methanol, 2.1 g of Na ₂ HPO ₄ , 1.4 g of KH ₂ PO ₄
g, ( _NH4 ) _2SO43.0g , _MgSO4・_{7H2O0.2g ,}
Agar plate medium adjusted to pH 9.5 by adding 4.0 g of anhydrous soda carbonate, 1.0 ml of mineral mixture, 1.0 ml of vitamin mixture, and 20 g of agar (however, the mineral mixture is 1 part distilled water, 0.15 g of CuSO ₄ 5H ₂ O, _MuCl2・
A solution containing 1.5 g of 4H ₂ O, 1.5 g of ZnSO ₄ 7H ₂ O _, 9.0 g of ferric citrate XH ₂ O, and 9.0 g of CaCl ₂
A sprayer was used to smear a solution containing 4 g of Ca pantothenate, 20 mg of folic acid, 20 mg of inositol, 4 g of pyridoxine hydrochloride, 4 g of thiamine hydrochloride, 2 g of riboflavin, 2 g of para-aminobenzoic acid, and 4 g of nicotinic acid. As a result of culturing for 6 days in a constant temperature bath at 30℃, a diameter of 3.
Four colonies of about mm size that appeared to be the same bacteria appeared. One of these colonies was isolated using a platinum loop and directly spread on three similar plates.
After culturing at 30°C for 6 days, one of the resulting colonies was re-isolated and cultured on a slant medium with the same composition at 30°C for 6 days, and the resulting culture was used as an isolated strain.

The obtained isolated bacterial strain was cultured vertically on several similar slanted media, and from one of these, 20 ml of liquid medium PH9.5 having the same composition as the above medium (excluding agar) was added using a platinum loop. The bacteria were inoculated into a 100 ml Erlenmeyer flask and cultured with shaking at 30°C for 3 days, and as a result, the bacteria showed vigorous growth. The obtained culture solution was transformed into a liquid medium 5 having the same composition as above.
The entire amount was inoculated into a 10-year fermenter containing The pH decreased as the bacteria grew, but the pH was adjusted to 8.5 by adding ammonia water.
Batch culture was carried out at 30°C while maintaining the dissolved oxygen concentration at 1.0 to 2.0 ppm by adjusting the aeration amount and stirring speed. When the residual methanol in the culture solution reached about 0.1%, a continuous culture medium having the following composition was added [distilled water 1] with the following components. KH ₂ PO ₄ 3.0g, MgSO ₄・7H ₂ O 1.0g,
(NH ₄ ) ₂ SO ₄ 1.0g, ferric citrate/XH ₂ O60
mg, _MnCl2・_4H2O10mg , _ZnSO4・_7H2O20mg ,
_CaCl2・_2H2O40mg , _CuSO4・_5H2O1mg ,
_CoCl2・_6H2O1mg , KI1mg, _{H3BO32mg ,}
(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O 1 mg, NaCl 50 mg, methanol 30 g, and vitamin mixture 2 ml (composition is the same as that used above)] were slowly started to be fed into the jar fermenter. Over a period of 24 hours, the medium feeding rate was gradually increased while ensuring that the residual methanol in the culture solution was always kept below 10 ppm, and the residence time was finally brought to 10 hours. The culture temperature during continuous culture was 30°C, the pH was adjusted to 8.5 by adding aqueous ammonia, and the dissolved oxygen concentration was maintained at 1.0 to 2.0 ppm by adjusting the aeration amount and stirring speed. Calculate the residual methanol concentration of the culture solution during continuous culture.
It was set to less than ppm. Continuous culture was performed for 14 days with a residence time of 10 hours. At this time, the methanol bacterial cell yield was measured and was 30.3%. In addition,
No other microorganisms were found to be present in this continuous culture solution.

Claims (1)

[Claims] 1 Belongs to the Methylococcus genus and has a growth pH of 7.5~
11.0, and the optimum pH is 8.5 to 9.5.
1.