JPH07102355B2 - How to remove dimethylformamide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for decomposing and removing dimethylformamide in a liquid containing dimethylformamide, and more specifically, to dimethylformamide in a liquid containing dimethylformamide. It relates to a method for decomposing and removing formamide using bacteria.

[Prior Art, Problems to be Solved by the Invention] Dimethylformamide is hydrophilic, stable to heat, has a large polarity, and has a large dissolving power for many organic compounds and inorganic compounds. Since it is an excellent solvent, it is a compound whose use amount is increasing remarkably in recent years.

The main uses thereof are solvents for producing synthetic fibers and synthetic leather, solvents for acrylic synthetic resins and vinyl synthetic resins, raw materials for reagents, and solvents for paints and pigments. These effluents containing dimethylformamide after being used for these applications are harmful to fishes such as aquatic animals, microorganisms and algae. Must be released. However, since dimethylformamide is generally highly toxic to microorganisms, it cannot be detoxified by conventional activated sludge treatment.

On the other hand, as a method for decomposing and removing dimethylformamide in the effluent containing dimethylformamide, for example,
There is a method described in Japanese Patent Publication No. 54-1792. This method is a method of decomposing by using an enzyme in the cells of a microorganism belonging to the genus Micrococcus.
Insufficient for practical use.

If stable, difficult-to-decompose dimethylformamide can be found to be a microorganism that can efficiently assimilate or decompose, it is possible to efficiently detoxify the effluent containing dimethylformamide using this microorganism. Become.

[Means and actions for solving problems]

As a result of extensively searching the natural world, the present inventors have found a microorganism that can actively assimilate dimethylformamide, or can strongly decompose it, and completed the present invention using this microorganism.

That is, the present invention comprises contacting a liquid containing dimethylformamide with bacterial cells of a bacterium belonging to the genus Pseudomonas that can assimilate and decompose dimethylformamide and / or a treated product of bacterial cells of the bacterium, and A method for removing dimethylformamide, which comprises decomposing and removing dimethylformamide.

The bacterium used in the present invention belongs to the genus Pseudomonas,
There is no particular limitation as long as it is a strain having the ability to efficiently assimilate and decompose dimethylformamide.

A representative example of these strains is Pseudomonas aminovorans DM-81 (Ministry of Industrial Research, No. 9948).

This strain is a strain isolated from the soil by the present inventors.

The monetary value of this strain is shown below.

1. Morphology 1 at 30 ℃ in both broth liquid medium and broth agar medium
Cultured for a day.

 Direct bacillus Width 0.9 to 1.2 μm Flowing 1.5 to 4 μm Population, single cell or bicellular.

 There is mobility. Has polar flagella.

 Presence or absence of spores Not produced.

 Gram stain Negative.

 Anti-acid negative.

 Extracellular adhesive Not produced.

2. Growth conditions in each of the following media (cultivation at 30 ° C for 3 days unless otherwise specified) Morphology and properties of broth agar: colony-circle, size-2-3 mm, ridge-hemisphere, structure-homogeneous , Surface-smooth, edge-smooth on all edges, color-white or yellow-white, transparency-opaque, hardness-butter.

 Agar plate containing monomethylamine Same as in broth agar plate.

 Broth agar slope medium It grows vigorously evenly on the inoculation line.

Colony morphology and properties: ridge-moderate, surface-smooth, marginal-smooth, color-white or yellow-yellow, transparency-opaque, hardness-buttery.

 Agar slant medium containing monomethylamine Same as in broth agar slant medium.

 Broth liquid medium Grows throughout. There is a precipitate and no bacterial ring is formed.

 Peptone water liquid medium Grows weakly throughout the medium. There is precipitation. Does not form a bacterial ring.

 Monomethylamine-containing liquid medium Grows throughout. There is precipitation. Does not form a bacterial ring.

Meat juice agar puncture culture It grows uniformly in the form of small papillae. 2-4m in diameter on the surface of the medium
It grows in a circle in the mth position.

Agar puncture culture containing monomethylamine It grows uniformly in the form of small papillae. 2-4m in diameter on the surface of the medium
It grows in a circle in the mth position.

 High-layer culture of broth gelatin Cultured at 20 ℃ for 10 days.

Growth of the fungus is observed, but gelatin is not liquefied.

 Cultured litmus milk at 30 ℃ for 4 weeks.

Bacteria grow, but alkali is not produced. It grows vigorously.

3. Physiological properties Reduction of nitrates Nitrate is reduced to nitrite.

 MR test negative.

 VP test negative.

 Formation of doll Negative.

 Generation of hydrogen sulfide Negative.

 Hydrolysis of starch Negative.

Do not use citric acid (Kosa Koser medium and Christensen medium are used together).

Use of nitrogen source Ammonium salt, nitrate and peptone are used as nitrogen sources, respectively.

 Dye formation No formation.

 Urease positive.

 Catalase positive.

 Ammonia generation Generates.

Growth range It grows in the range of pH 6-8. A pH range of 6.5-7.5 is preferred.

It grows at a temperature of 5 to 35 ° C. 10 to 32 ° C is preferable.

 Attitude toward oxygen Aerobic.

 Oxidase negative.

OF test (Hugh Leifson
The method decomposes sugars oxidatively but not fermentatively.

Assimilation of sugars and formation of acid and gas Assimilation of carbon sources other than sugars Salt tolerance Does not grow in medium containing 3 wt% NaCl.

 Vitamin requirement Never ask for vitamins.

 Denitrification reaction is negative.

GC (guanine + cytosine) content 63.2mol% Main cell fatty acid composition Monounsaturated fatty acid C _{18: 1} Main hydroxy acid 3-hydroxy acid C _{12: 0} Quinone type Ubiquinone Q ₁₀ Separation source Soil.

Among the mycological properties, this strain has polar flagella, forms white or yellowish white colonies, is a gram-negative monomethylamine-assimilating bacterium, and has a GC content of 63.2 mol%,
Monounsaturated fatty acid C _{18: 1} is the major bacterial fatty acid composition,
Since 3-hydroxy acid C _{12: 0} is the main hydroxy acid and the quinone type is ubiquinone Q ₁₀ ,
Urakami and classification by the Komagata [ "J.Gen.Microbiol." 32, 31
7-341 (1986)], a Pseudomonas aminoborans Pseudo belonging to the group 1 monomethylamine-assimilating bacterium.
It was identified as a strain belonging to monas aminovorans.

In the present invention, the experimental method for investigating the mycological properties is described in "Bergey's Manual of Systema".
tic Bacteriology, Volume 1, Editors Krieg and Holt: Williams & Wilkins, Inc. (1984)], Institute of Medical Science, "Annual Review of Bacteriology" (1958) And Takeshi Hasegawa, “Classification and Identification of Microorganisms” (1975).

Agar plates containing monomesylamine and agar slants containing monomethylamine were prepared as follows. That is, (NH ₄ ) ₂ SO ₄ 3g, KH ₂ PO ₄ 1.4g, Na ₂ HPO ₄ 2.1g, Mg
_{SO 4 · 7H 2 O 0.2g,} CaCl 2 · 2H 2 O 30mg, FeC 6 H 5 O 7 · XH 2 O 30m
g, MnCl ₂・ 4H ₂ O 5mg, ZnSO ₄・ 7H ₂ O 5mg, CuSO ₄・ 5H ₂ O 0.5m
g, yeast extract 0.2 g and monomethylamine hydrochloride 5 g were dissolved in pure water 1 and the pH was adjusted to 7.1, then agar 15 g / was further added and dissolved by heating to 1 kg / cm ² G At 20
Sterilized for a minute.

As the liquid medium containing monomethylamine, a medium containing no agar in the composition of the agar plate medium containing monomethylamine and the slant medium containing monomethylamine was used.

Further, the dimethylformamide-containing agar plate medium and the dimethylformamide-containing agar slant medium were prepared as follows. That is, (NH ₄ ) ₂ SO ₄ 3g, KH ₂ PO ₄ 1.4g, Na ₂ HP
_{O 4 2.1g, MgSO 4 · 7H} 2 O 0.2g, CaCl 2 · 2H 2 O 30mg, FeC 6 H 5 O 7
・ XH ₂ O 30mg, MnCl ₂・ 4H ₂ O 5mg, ZnSO ₄・ 7H ₂ O 5mg, CuSO ₄
· 5H ₂ O 0.5 mg, and the yeast extract 0.2g was dissolved in pure water 1 was adjusted to pH 7.1. To this, 15 g / of agar was added and dissolved by heating, and then dimethylformamide 5 g /
Was added and sterilized at 1 kg / cm ² G for 20 minutes.

As the dimethylformamide-containing liquid medium, a medium to which agar was not added was used in the composition of the dimethylformamide-containing agar plate medium and the dimethylformamide-containing agar slant medium.

Liquid containing dimethylformamide (hereinafter referred to as treated liquid
In some cases, various nutrients are added to the liquid to be treated, and this is used as a culture medium that belongs to the genus Pseudomonas and can assimilate and decompose dimethylformamide (hereinafter Bacteria).

That is, the nutrient component added to the liquid to be treated may be any substance that can be assimilated by the bacterium used, and is not particularly limited, and includes a carbon source, a nitrogen source, an inorganic component and other components.

As the carbon source, only dimethylformamide in the liquid to be treated may be used, but other carbon sources that can be assimilated by the bacterium used-for example, D-xylose, D-glucose, D-fructose, D-galactose, maltose. And sugars such as sucrose, sugar alcohols such as D-sorbitol and D-mannitol, organic acids such as acetic acid, alcohols such as ethanol, and alkylamines such as monomethylamine, dimethylamine and trimethylamine, and the like can be used in combination. .

As the nitrogen source, inorganic nitrogen compounds such as ammonium salts and nitrates and / or organic nitrogen-containing substances such as alkylamines, corn steep liquor, casein, peptone and meat extract are used.

Since dimethylformamide is a nitrogen compound, all of the present bacteria can sufficiently grow and proliferate and assimilate and decompose dimethylformamide without adding any other nitrogen source.

As the inorganic component, for example, calcium salt, magnesium salt, potassium salt, sodium salt, phosphate,
Manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, cobalt salt, boron compound, iodine compound and the like are used.

Further, when using a strain that requires a nutritional substance such as vitamins, the nutritional substance required by the strain is added.

The concentration of dimethylformamide in the liquid to be treated may be such that the bacterium used can assimilate and decompose,
There is no particular limitation, but usually 3 wt% or less is preferable, and 2 wt%
% Or less is particularly preferable.

The culture conditions may be such that the bacteria used can grow and proliferate, but generally, for example, the temperature is 5 to 35 ° C,
The temperature is preferably 10 to 32 ° C., and the pH is 6 to 8, preferably 6.5 to 7.5.

The culture is performed aerobically under such conditions.

The dissolved oxygen concentration of the culture medium may be any dissolved oxygen concentration that allows the bacterium to grow and proliferate, and is not particularly limited, but usually about 0.5 to 20 ppm is preferable. In order to obtain such a dissolved oxygen concentration, the amount of aeration gas is adjusted,
Means such as stirring, using oxygen gas or a mixed gas of oxygen and air as an aeration gas, and increasing the pressure in the culture tank are adopted.

Although the growth and proliferation of the bacterium is relatively poor and the efficiency of removing and decomposing dimethylformamides is relatively low, it does not prevent culturing under these conditions.

The culture method may be batch culture, continuous culture or semi-continuous culture.

When an ammonium salt or an alkylamine salt is used as a nitrogen source, ammonia and amine are consumed for the production of bacterial cells during the culture period, and the pH of the culture solution decreases. In this case, in order to keep the pH of the culture solution at a predetermined value, ammonia, amines, alkali such as caustic potash and caustic soda are added, but it is most preferable to add ammonia.

By culturing the bacterium using the liquid to be treated as a medium as described above, other than the method of detoxifying the liquid to be treated,
Pre-cultured microbial cells of the present bacterium and treated products of the bacterium of the present bacterium-for example, a culture solution containing the microbial cells of the present bacterium, a crushed product of the bacterium of the present bacterium, and a microbial cell of the present bacterium with a synthetic resin or the like. Immobilized immobilized cells (these may be collectively referred to as cells below) and the like may be brought into contact with the liquid to be treated.

For example, (a) adding the cells to the liquid to be treated, (b) contacting the liquid to be treated with the activated sludge mixed with the cells, the culture solution and the crushed product of the cells, and (c) fixing. There is a method in which the liquid to be treated is passed through a column filled with modified cells.

In addition, the drainage containing dimethylformamide,
Since other substances are also often contained, a strain capable of decomposing other substances can be used together with the present bacterium in the treatment of such drainage, and it is preferable.

After the treatment, the treated liquid in which the concentration of dimethylformamide in the liquid to be treated is below the permissible concentration is discharged as it is, or after removing the bacterial cells and / or the treated material of the bacterial cells as necessary. It

〔Example〕

The present invention will be described more specifically by way of examples. The present invention is not limited to the embodiments.

Example 1 (NH ₄ ) ₂ SO ₄ 3 g, KH ₂ PO ₄ 1.4 g, Na ₂ HPO ₄ per 1 pure water
2.1g, MgSO ₄・ 7H ₂ O 0.2g, CaCl ₂・ 2H ₂ O 30mg, FeC ₆ H ₅ O ₇・
XH ₂ O 30mg, MnCl ₂ 4H ₂ O 5mg, ZnSO ₄ 7H ₂ O 5mg, CuSO ₄ 5
A basal medium was prepared by adding 0.5 mg of H ₂ O and 0.2 g of yeast extract to pH 7.1, and the basal medium had dimethylformamide concentrations of 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 3 wt%. And dimethylformamide were added so that the concentration became 5 wt% and a medium was prepared. Each of these media was inoculated with Pseudomonas aminobalance DM-81 and incubated at 30 ° C for 7 days.
After culturing for one day, the assimilability of dimethylformamides was examined.

The results are shown in Table 1.

Example 2 (NH ₄ ) ₂ SO ₄ 3 g, KH ₂ PO ₄ 1.4 g, Na ₂ HPO ₄ per 1 pure water
2.1g, MgSO ₄・ 7H ₂ O 0.2g, CaCl ₂・ 2H ₂ O 30mg, FeC ₆ H ₅ O ₇・
XH ₂ O 30mg, MnCl ₂ 4H ₂ O 5mg, ZnSO ₄ 7H ₂ O 5mg, CuSO ₄ 5
H ₂ O 0.5 mg, yeast extract 0.2 g and dimethylformamide 5 g
200 ml of a medium adjusted to pH 7.0 was added to a 1-volume Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes.

A preculture solution of Pseudomonas aminoborans DM-81 (cell concentration OD _610nm 1.5) obtained by pre-culturing in a medium similar to this was ingested to this medium at a concentration of 1 vol%, and at 50C at 30 ° C. Incubated for hours.

Absorbance (OD _610nm ) of the obtained culture solution and p of the culture solution
H was 2.3 and 7.5, respectively, and dimethylformamide was not detected in the culture supernatant. In addition,
Generation time was about 6.5 hours.

Example 3 From the composition of the basal medium in Example 1, (NH ₄ ) ₂ SO ₄ was added to a factory effluent containing dimethylformamide at 1 wt% and having a pH of 6.8.
Pseudomonas aminoborans DM separated from the preculture liquid obtained by preliminarily culturing the liquid in the same medium as in Example 2 by adding nutrients to a medium composition excluding 1 g of −81 cells was suspended, and the pH and solution temperature of the culture solution were maintained at 7.0 and 30 ° C., respectively, while aerating and stirring.

It took about 30 hours before dimethylformamide could not be detected in this factory effluent.

In each of the above Examples, the analysis of dimethylformamide in the liquid was carried out by gas chromatography.

Model: Shimadzu 7A GC (FID) Column: Unisole 30T 5% Uniport HP 80/100 Glass Colimn (3φ x 2m) Column temperature: 70 ° C Flow velocity: (standard state) 30 ml / min. [Effect of the invention] From the present invention, Stable and hard to decompose, it is possible to efficiently decompose and remove dimethylformamide, which is a harmful substance, and thus harmful effluent containing dimethylformamide can be efficiently detoxified, and environmental hygiene preservation The above value is extremely high.

Claims (1)

[Claims] 1. A liquid containing dimethylformamide is brought into contact with a bacterial cell of a bacterium belonging to the genus Pseudomonas and capable of assimilating and decomposing dimethylformamide and / or a treated product of the bacterial cell of the bacterium, and A method for removing dimethylformamide, which comprises decomposing and removing dimethylformamide.