JP2570313B2 - New microorganism - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to novel microorganisms, and more particularly, to novel bacteria that assimilate dialkylformamide.

[Prior art and problems to be solved by the invention]

Dimethylformamide is a solvent that is hydrophilic, stable to heat, highly polar, and highly soluble in many organic and inorganic compounds. An organic compound.

The main uses are, for example, solvents for the production of synthetic fibers and synthetic leather, solvents for acrylic synthetic resins and vinyl synthetic resins, pharmaceutical raw materials, and solvents for paints and pigments. Since the effluent containing dimethylformamide after being used for these applications is harmful to aquatic animals such as fish, microorganisms, algae, etc., it cannot be discharged as it is and must be detoxified. . However, due to its toxicity to microorganisms, conventional activated sludge treatment could not be performed.

As a method for decomposing and removing dimethylformamide in the effluent containing dimethylformamide, for example,
Japanese Patent Publication No. 54-1792 describes a method for decomposing dimethylformamide using an enzyme in a microbial cell belonging to the genus Micrococcus, but the processing capacity is still insufficient for practical use. is there.

If microorganisms that can efficiently assimilate or decompose this hardly decomposable dimethylformamide can be found, it is possible to treat the effluent containing dimethylformamide using the microorganism and efficiently detoxify it. .

[Means and actions for solving the problem]

The present inventors have extensively searched for microorganisms capable of vigorously assimilating dialkylformamide or capable of strongly decomposing, and as a result, have found novel microorganisms which assimilate dialkylformamide vigorously and grow and proliferate.

That is, the present invention is a novel microorganism belonging to Mycobacterium methanolica and characterized by utilizing at least dimethylformamide as dialkylformamide.

The bacteriological properties of Mycobacterium metanolica TH-35 (No. 9497, manufactured by B.I.) are shown below.

That is, 1. Form Each of the broth liquid medium and the broth agar medium was cultured at 37 ° C. for 3 days.

Cell shape and size Usually bacilli. 0.5-0.8 μm in width and 1-3 μm in length. V-type dividing cells are observed.

 Mobility None.

 Presence or absence of spores Not produced.

 Gram staining Gram positive.

 Acid-fast positive.

2. Growth condition in each of the following media (cultured at 37 ° C for 3 days unless otherwise noted) Broth agar plate medium Shows moderate growth.

Colony morphology and properties: external shape-round, size-2 to 3 mm, raised-hemispherical, structure-homogeneous, surface-rough, margin-wavy, color-yellowish white, no gloss, transparency-opacity, hardness- Butter quality.

 Methanol-containing agar plate Same as in broth agar plate.

 Gravy agar slant culture medium Shows moderately uniform growth on the inoculation line.

Raised-moderate, surface-rough, margin-wavy, color-yellowish white and dull, transparency-opaque, hardness-buttery.

 Agar slant containing methanol Same as in gravy agar slant.

Liquid broth forms white cream colored bacterial rings. In addition, a film is formed.

 Peptone water broth Same as in broth broth.

Liquid medium containing methanol Grows vigorously. Forms a white cream colored bacterial ring. In addition, a film is formed.

 Broth gelatin puncture culture Cultured at 20 ° C for 4 weeks.

 Grow. However, there is no gelatin liquefaction.

 Litmus milk culture Cultured at 37 ° C for 4 weeks.

It grows and the culture solution changes to alkaline (pH 6.8 → pH 8.3), but does not peptone.

 1% Ogawa medium grows vigorously. The settlement is smooth.

HA medium (hydroxylamine hydrochloride 500μg / ml added 1
(Ogawa medium) Cultured at 37 ° C for 5 days.

 Grows weakly.

PAS medium (sodium paraaminosalicylate 2mg / ml
Cultured at 37 ° C for 7 days.

 It grows vigorously and the medium turns black.

Picric acid medium (modified Sauton with 0.2% picric acid)
(Agar medium) Cultured at 37 ° C for 2 weeks.

 It grows vigorously and the medium becomes reddish brown.

PNB medium (1% Ogawa medium supplemented with 500 µg / ml of paranitrobenzoic acid) Cultured at 37 ° C for 7 days.

 It grows vigorously.

EB medium (1% Ogawa medium supplemented with 5 μg / ml of etamptorol) Cultured at 37 ° C. for 7 days.

 It grows vigorously.

3. Physiological properties Nitrate reduction Nitrate is reduced to nitrite.

 MR test negative.

 VP test negative.

 Indole formation Negative.

 Generation of hydrogen sulfide positive.

 Starch hydrolysis Negative.

Use of nitrogen sources Ammonium salts, nitrates, urea and peptone are used as nitrogen sources, respectively.

 Dye formation Not generated.

 Urease positive.

 Catalase positive.

 Ammonia production Produces.

 Denitrification reaction Negative.

 Oxidase reaction negative.

OF Test (Hugh Leifson
Negative).

 Growing range Growing in the pH range of 5-9. pH 6-8 is preferred.

It does not grow at temperatures of 5 ° C and 43 ° C. Temperatures of 25-40 ° C are preferred.

 Attitude to oxygen Aerobic.

 Sugar assimilation and acid production.

Assimilation of carbon sources other than sugars Salt tolerance It grows vigorously in a medium containing 3% by weight of NaCl.

 It does not grow on a medium containing 6% by weight of NaCl.

 Vitamin requirement None.

 Photochromic test negative.

 Dark color test Negative.

 Tween 80 water dissolution test weakly positive.

 Mycolic acid content positive.

GC (guanine + cytosine) content 66.4 mol% Major fatty acid composition of bacterial cells Linear fatty acids C _{16: 0} Monounsaturated fatty acids C _{16: 1} , C _{18: 1} 10 methyl fatty acids 10-methy1 C _{19: 0} Quinone type Menaquinone MK-9 (H ₂₎ containing the structural meso- diaminopimelic acid of the cell wall.

 Source Soil.

The microorganism of the present invention (hereinafter referred to as “the bacterium”) is referred to as “Bergey's Manual of Systematic Bacterology [(Bergey's Menual of Systematic Bacter
iology, Volume 2, editors Sneath, Mair, Sharpe, and Holt:
Williams and Wilkins (Williams &
According to Wilkins), 1986)], these strains are rod-shaped, non-motile, gram-positive, acid-fast, contain mycolic acid, and are aerobic. Was determined to be a bacterium belonging to the genus Mycobacterium. This is further supported by various points such as GC content, bacterial cell fatty acid composition, quinone type and cell wall structure.

Furthermore, the bacterium is very similar to Mycobacterium methanolica disclosed in a patent application (Japanese Patent Application Laid-Open No. 61-151565) based on an invention made by one of the present inventors. Identified. However, Mycobacterium methanolica BT-84 (Japanese Patent No. 8823) disclosed in this patent application and the like,
-143 (Pierce No.8824), P-23 (Pierce No.8825), P-26 (Pierce No.8826), P-85 (Pierce No.88) Bacteria No. 8827), P-70 (Microtechnical Lab. No. 9464) and TH-30 (Microtechnological Lab. No. 9465)
No. 3) did not have the assimilation property of dialkylformamide, while the bacterium had the assimilation property of dialkylformamide. Therefore, the bacterium was determined to be a novel microorganism belonging to Mycobacterium methanolica.

In the present invention, an experimental method for examining mycological properties is described in “Bergey's Manual of Systematic Bacteriology”.
c Bacteriology) Volume 2 Editor Sneath,
Mair, Sharpe, and Holt: Williams and Wilkins, (1984), “Study of Bacteriology,” edited by the Institute of Medical Science Alumni Association (1958) and Hasegawa
Based on "Classification and Fixation of Microorganisms", edited by Takeji (1975).

The methanol-containing agar plate medium and the methanol-containing agar slant medium are prepared as follows. That is,
(NH ₄ ) ₂ SO ₄ 3 g, KH ₂ PO ₄ 1.4 g, Na ₂ HPO ₄ 2.1 g, MgSO ₄・ 7H ₂ O0.
_{2g, CaCl 2 · 2H 2 O30mg} , FeC 6 H 5 O 7 · XH 2 O30mg, MnCl 2 · 4H 2 O5
_{mg, ZnSO 4 · 7H 2 O5mg} , CuSO 4 · 5H 2 O0.5mg and yeast extract
0.2 g was added to pure water 1, the pH was adjusted to 7.1, 15 g / agar was further added, and the mixture was heated and dissolved, 8 ml / methanol was added thereto, and then 1 kg / cm ² G For 20 minutes.

As the methanol-containing liquid medium, a medium without the addition of agar in the above composition was used.

A dimethylformamide-containing agar plate medium and a dimethylformamide-containing agar slant medium are prepared as follows. That is, (NH ₄ ) ₂ SO ₄ 3 g, KH ₂ PO ₄ 1.4 g, N
_{a 2 HPO 4 2.1g, MgSO 4} · 7H 2 O0.2g, CaCl 2 · 2H 2 O30mg, FeC 6 H 5 O
_{7 · XH 2 O30mg, MnCl 2} · 4H 2 O5mg, ZnSO 4 · 7H 2 O5mg, CaSO 4 · 5
Dissolve 0.5 mg of H ₂ O and 0.2 g of yeast extract in pure water 1 and adjust pH
After adjusting to 7.1, further add agar 15g /
After heating and dissolving this, 5 g of dimethylformamide
/ And then sterilized at 1 kg / cm ² G for 20 minutes.

As the dimethylformamide-containing liquid medium, a medium having the above composition and no agar was used.

Isolation of the present bacterium from soil was carried out by a conventional method using the above-mentioned dimethylformamide-containing medium.

The medium used for cultivation of the bacterium needs to contain a carbon source that can be assimilated by the bacterium, and furthermore, may be any medium containing an appropriate amount of a nitrogen source and an inorganic substance.
Either a synthetic medium or a natural medium may be used.

The carbon source is not particularly limited as long as it can be assimilated by the bacterium.In addition to dimethylformamide, synthetic carbon sources such as methanol, ethanol, and methylamines can be suitably used. Carbon sources-natural products such as molasses and meat extracts, sugars such as D-glucose and D-fructose, sugar alcohols such as D-sorbitol and inositol, and
Organic acids such as succinic acid and benzoic acid can also be used. Of these, methanol is most preferred as an industrial raw material.

Further, as the dialkylformamide, there is a compound represented by the following general formula. That is, HCONR ¹ R ² Representative examples of dialkylformamide include dimethylformamide, diethylformamide, dipropylformamide and dibutylformamide, with dimethylformamide being preferred.

The concentration of these carbon sources in the medium differs depending on the type of the carbon source, and is appropriately selected so as to grow and proliferate the microorganism. For example, when the carbon source is methanol, the methanol concentration of the medium or culture solution is 6% by weight.
The following is preferable, and 2% by weight or less is preferable from the viewpoint of good growth and growth of bacteria.

As the nitrogen source, use is made of, for example, inorganic nitrogen compounds such as ammonium salts and nitrates and / or organic nitrogen-containing substances such as urea, corn stave liquor, casein, peptone and meat extract.

As the inorganic component, for example, calcium salt,
Magnesium, potassium, sodium, phosphate, manganese, zinc, iron, copper, molybdenum,
Cobalt salts, boron compounds and iodine compounds are used.

Further, when a strain that requires a nutrient such as a vitamin is used, the nutrient required by the strain is added.

Culture conditions, the temperature is 20 ~ 42 ℃, preferably 25 ~ 40 ℃
And the pH is 5-9, preferably 6-8. Culture is performed aerobically under such conditions.

Although the growth and growth of the bacterium become relatively poor, it does not prevent culturing under these conditions.

The concentration of dissolved oxygen in the culture solution is not particularly limited as long as the concentration of dissolved oxygen is such that the present bacteria can grow and proliferate, and is usually preferably about 0.5 to 20 ppm. In order to achieve such a dissolved oxygen concentration, the amount of aerated gas is adjusted, agitated, oxygen gas or a mixed gas of oxygen and air is used as the aerated gas, or the pressure in the culture tank is reduced. Means such as enhancement are employed.

The culture system may be any of batch culture, continuous culture, and semi-continuous culture.

When an ammonium salt is used as a nitrogen source,
During the culturing period, ammonia is consumed for the production of bacterial cells, and the pH of the culture solution decreases. In this case, in order to maintain the pH of the culture solution at a predetermined value, an alkali such as ammonia, potassium hydroxide, and sodium hydroxide is added, and it is preferable to add ammonia.

After culturing the bacteria in this way, the cells are separated from the culture solution. When the target substance is a substance such as an enzyme, a coenzyme, or an amino acid that is excreted from the cells, these substances are extracted from the culture solution, separated, and further purified. On the other hand, when the target substance is an intracellular substance such as coenzyme, nucleic acid, vitamins, proteins and lipids, the cells are treated by various methods, and the target substance is extracted,
Separated and further purified.

These obtained substances are effectively used as foods, pharmaceuticals, feeds, feed additives, industrial raw materials and the like.

In addition, the bacterium has the ability to strongly assimilate and degrade dialkylformamide, tetraalkylammonium salts, methylamines such as monomethylamine, dimethylamine and trimethylamine, and methanol, etc. Processing by contacting with the contained effluent, reducing the concentration of these substances,
Alternatively, these substances can be removed.

In this drainage treatment, the concentration of these substances contained in the drainage may be any concentration at which the present bacteria can assimilate or decompose.
It is preferable that dimethylformamide is 3% by weight or less, tetramethylammonium salt is 3% by weight or less as tetramethylammonium hydroxide, methanol is 6% by weight or less, and methylamines is 1% by weight or less.

The cells to be brought into contact with the drainage may be the cells themselves, but may be a culture solution or a crushed cell, or cells immobilized with a synthetic resin or the like may be used.

In addition, since the effluent containing these substances often contains other substances, the treatment of such effluents requires a strain capable of decomposing the bacterium and other substances. Can be used in combination, and is preferred.

〔Example〕

The present invention will be described more specifically with reference to examples.
Note that the present invention is not limited to the embodiments.

Example About 1 g of a soil sample collected from various places was aseptically added to 10 ml of sterilized water and suspended. 1 ml of this suspension was placed in a dimethylformamide-containing liquid medium, and culturing was carried out at 28 ° C. for 7 days. This culture solution was cultured on an agar plate medium containing dimethylformamide, and a single colony generated was inoculated on an agar slant medium containing dimethylformamide and cultured. Thus, Mycobacterium methanolica TH
-35 pure strains were obtained.

200 ml of a dimethylformamide-containing liquid medium was placed in a 1-volume Erlenmeyer flask, inoculated with the Mycobacterium methanolica TH-35, and cultured at 30 ° C. for 3 days. The absorbance (610 nm) of the culture solution obtained was Was 4.5 for each.

Note that the culture solution at this time did not substantially contain dimethylformamide.

〔The invention's effect〕

The microorganism of the present invention can also use dialkylformamide and other substances that can be obtained stably and inexpensively by industrial production as medium components, and efficiently and stably produce various fermentation products. Can be manufactured.

Furthermore, since the microorganism of the present invention is difficult to decompose, such as dialkylformamide, and can assimilate or decompose harmful substances, it is possible to detoxify harmful effluents containing these harmful substances by this microorganism. And the value in environmental hygiene is extremely high.

Claims (1)

(57) [Claims] 1. A novel microorganism belonging to Mycobacterium methanolica, wherein at least dimethylformamide is assimilated as dialkylformamide.