JPH01164498A - Removal of ethyl trimethyl ammonium compound - Google Patents

Abstract

PURPOSE:To make waste liquid contg. an ethyl trimethyl ammonium compd. harmless with high efficiency by allowing the waste liquid to contact with mycelia of a specified bacterium belonging to Mycobacterium genus. CONSTITUTION:Nutrient such as carbon source, nitrogen source, inorganic component, and other nutrient components are added to a liquid to be treated contg. ethyl trimethyl ammonium hydroxide and/or ethyl trimethyl ammonium salt. The water to be treated is made harmless by cultivating bacteria belonging to Mycobacterium genus capable of assimilating and decomposing the ethyl trimethyl ammonium compd, at 5-43 deg.C and 5-9pH aerobically using the above described treated liquid as medium. On one band, a medium contg. the mycelia of previously cultivated bacteria of this invention may be allowed to contact with the liquid to be treated, and treated liquid having a concn. of ethyl trimethyl ammonium compd. below an allowable value after the treatment is discharged.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides ethyltrimethylammonium hydroxide and/or ethyltrimethylammonium salt (
Regarding a method for removing ethyltrimethylammonium compound from a liquid containing ethyltrimethylammonium compound (hereinafter, both may be collectively referred to as ethyltrimethylammonium compound),
More specifically, the present invention relates to a method of decomposing and removing ethyltrimethylammonium compounds in a liquid containing ethyltrimethylammonium compounds using bacteria.

[Prior art and problems to be solved by the invention] In the deethylation reaction of organic synthesis reactions, trimethylamine is generally used as an ethyl group acceptor. Ethyltrimethylammonium salts such as trimethylammonium iodide and ethyltrimethylammonium bromide are converted to ethyltrimethylammonium salts, which are discharged from the reactor. However, these effluents containing ethyltrimethylammonium salt are harmful and must be treated to render them harmless before being discharged from the viewpoint of environmental hygiene.However, conventional activated sludge treatment It is extremely difficult to render it harmless.

Ethyltrimethylammonium compound is a stable and difficult to decompose substance, but if a microorganism that can efficiently assimilate or decompose this substance can be found, this microorganism can be used to treat wastewater containing ethyltrimethylammonium compound. It becomes possible to make it harmless efficiently.

[Means and effects for solving the problem] The present inventors,
As a result of a wide search in the natural world, we found a bacterium belonging to the genus Mycobacterium that can actively assimilate or strongly decompose ethyltrimethylammonium compounds and grow and multiply, and the present invention uses this bacterium. completed.

That is, in the present invention, a solution containing an ethyltrimethylammonium compound is brought into contact with bacterial cells belonging to the genus Mycobacterium that assimilate and decompose the ethyltrimethylammonium compound. This is a method for removing an ethyltrimethylammonium compound, which is characterized by decomposing and removing the compound.

The bacteria used in the present invention are not particularly limited as long as they belong to the genus Mycobacterium and have the ability to assimilate and decompose ethyltrimethylammonium compounds.

A representative example of a strain belonging to the genus Mycobacterium is Mycobacterium methanoli, which the present inventors isolated from soil.
cap-70 (Feikoken Bibori No. 9464), Tl+-
30 (Feikoken Bibori No. 9465) and the same Tl[-35
(Feikoken Bibori No. 9497) (Patent applications have been filed for these strains themselves - Japanese Patent Application No. 62-208206 and Japanese Patent Application No. 62-212874).

The mycological properties of these strains are shown below.

■, 30°C for each of the form broth liquid medium and broth agar medium.
The cells were cultured for 3 days.

■ Usually short bacilli. Width 0.5~0.8- Length 1~
3tIrn. ■ type of dividing cells are observed.

■ Motility None.

■ Presence or absence of spores Not produced.

■ Durham staining positive.

■ Anti-acidity positive.

2. Growth status in each of the following media (cultured at 37°C for 3 days unless otherwise specified) ■ Medium growth on broth agar plate medium.

Colony morphology and properties: External shape: circular, 2-3 mm in size.

Ridges - hemispherical, structure - homogeneous, surface - rough.

Margin: Wavy 5 colors: Yellowish white, no gloss.

Transparency - opaque, hardness - buttery.

■ Methanol-containing agar plate medium Same as in broth agar plates.

■ Broth agar slant medium Grows vigorously and uniformly along the inoculation line.

Colony morphology and properties: Raised - Medium 1 Surface - Rough 2 Near Green - Wavy.

Color: Yellow-white, non-glossy; Transparency: Opaque.

Hardness - buttery.

■ Methanol-containing agar slant medium Same as in broth agar slants.

■ Meat juice liquid medium Forms a white cream-colored bacterial ring. It also forms a film.

■ Peptone water liquid medium Same as in broth liquid medium.

■ Grows vigorously in liquid medium containing methanol. Forms a white cream-colored fungal ring. It also forms a film.

■ Meat juice gelatin puncture culture Cultured at 20°C for 4 weeks.

Grow. However, it does not liquefy gelatin.

■ Ridmus milk Cultured at 37°C for 4 weeks.

The culture medium grows alkaline (pl+ 6.8 →
pH 8.3), but not peptonization.

[Phase] 1χ Ogawa medium Grows vigorously. The colony is smooth in appearance.

([1) I (A medium (hydroxylamine hydrochloride 500μ
Cultured at 37°C for 5 days.

P-70 grows vigorously.

TH-30 and Tl-35 grow weakly.

@ PAS medium (bara aminosalidylate sodium 2
■/wdl supplemented 1χ Ogawa medium) Cultured at 37°C for 7 days.

It grows vigorously and the medium turns black.

[Phase] Picric acid medium (modified 5auton agar medium supplemented with 0.2x picric acid) Cultured at 37°C for 2 weeks.

It grows vigorously and the medium turns reddish brown.

Q PNB medium (1χ Ogawa medium supplemented with paranitrobenzoic acid 500./d) Cultured at 37°C for 7 days.

Grows vigorously.

o EB medium (5 μg/ml ethambutol added 1
χ Ogawa medium) Cultured at 37°C for 7 days.

Grows vigorously.

3. Physiological properties ■ Reduction of nitrate Reducing nitrate to nitrite.

■ MR test Negative.

■ VP test Negative.

■ Indole production Negative.

■ Production of hydrogen sulfide Positive.

■ Starch hydrolysis Negative.

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

■ Pigment generation　　　　　　　　No generation.

■ Urease positive.

[Phase] Catalase Positive.

■ Production of ammonia Produce.

@ Denitrification reaction Negative.

■ Oxidase Negative.

Q O-F test (Hyu Lifeson llugh
(by Leifson method) Negative.

■Growth range: Grows in the range of pl+5 to 9. pl+ is preferably in the range of 6 to 8.

It does not grow at temperatures of 5°C and 943°C. Temperature 10-40
゛C is preferred.

[Phase]　Attitude towards oxygen　　　Aerobic.

(Margin below) ■Sugar assimilation and acid production (a) Assimilation ability (b) Acid production + (what): It is weak, but it generates.

■ Assimilation of carbon sources other than sugars B: The culture solution turns black.

[Phase] Salt resistance Grows vigorously in a medium containing 3wtχNacl.

It does not grow vigorously in a medium containing 6 wt% NaCl.

[Phase] Vitamin requirement Absolutely does not require vitamins.

■ Photochromic test Negative.

@Dark color test Negative.

0 Tween 80 hydrolysis test P-70 negative.

T) I-30, Tl1-35 patency.

[Phase] Contains mycolic acid. Positive.

[Phase] GC (guanine + cytosine) content P-7067
, 2molχ T) I-3066, 0molχ Tl1-35 66.4molχ [phase] Main bacterial cell fatty acid composition Straight chain fatty acid C10:.

Monounsaturated fatty acids C,,,,C,,.

10-methyl fatty acid 10-methyl CI
91゜0 Quinone type Menaquinone MK-
9 (H[phase] Contains cell wall structure meso-diaminopimelic acid.

[Phase] Separation source Soil.

``Birshi's Manual of Systematics Inc.
Bacteriology [“Bergey”’s Manual
of Systematic Bacteriology
y'' Volume 2 1 Editor Snea th,
Mair, Sharpe and Ilolt: Williams & Wilkins
(1986)), these strains are bacilli, non-motile, Durham positive, acid-fast, contain mycolic acid, and are aerobic.
It was determined that the bacteria belonged to the genus Mycobacterium.

This is further supported by various aspects such as GC content, cell fatty acid composition, quinone type, and cell wall structure.

Furthermore, these strains have been disclosed in a patent application (Japanese Patent Application No. 61-15156) based on the invention made by one of the present inventors.
It is very similar to Mycobacterium mechnorica disclosed in 5), and was identified as Mycobacterium mechnorica.

Although these strains were not known at the time of the earlier patent application, subsequent research revealed that they further utilize ethyltrimethylammonium compounds as a carbon source.

In the present invention, the experimental method for investigating mycological properties is described in "Bergey's Manual of Systematic Bacteriology".
l of Systematic Bacteriol
ogy” Volume 1 Editors: Krieg and Ilo lt: Williams &
Wilkins (Williams & Wilkins)
Co., Ltd. (1984)) J., ``Birshi's Manual of Systematic Hackeriology'' B.
ergey's Manual of Systema
tic Bact-eriology'' Volume 2'', Institute of Medical Science alumni venue ``Bacteriology Practice Summary J (1958) and Edited by Takeharu Hase [@Classification and Identification of Organisms J (1)
975).

A methanol-containing agar plate medium and a methanol-containing agar slant medium were prepared as follows.

That is, (NIL)zscL 3g, KIzPOn
],,44g Nazllll"042.1g,
MgSO4'711zOO,2g, CaC1z'2H
z030mg.

FeC, Il, Ot'XI1.030mg, MnC
1z'4Hz05mg, ZnSO4411□05m
g, CuSO4・5Hz 00.5 mg and yeast extract 0.2 g were dissolved in pure water 11, and pi+ was adjusted to 7.1. Then, 15 g/l of agar was added, and this was dissolved by heating. 8 g/l of methanol was added and then sterilized at 1 kg/c BoG for 20 minutes.

As the methanol-containing liquid medium, a medium with the same composition as the methanol-containing agar plate medium and the methanol-containing agar slant medium without adding agar was used.

In addition, the agar plate medium containing ethyltrimethylammonium salt, a slant medium containing ethyltrimethylammonium salt, and a liquid medium containing ethyltrimethylammonium salt are used to examine the assimilation ability of ethyltrimethylammonium compounds. In each of the agar-containing slant medium and methanol-containing liquid medium, 5 g/ethyltrimethylammonium chloride was added instead of methanol.
A medium supplemented with 1 was used. In addition, the aqueous solution containing ethyltrimethylammonium hydroxide is
Because it is alkaline, bacteria can efficiently assimilate ethyltrimethylammonium.

For decomposition, it is preferred to bring the pH of the aqueous solution to approximately neutral with acidic substances such as hydrochloric acid, hydroiodic acid and sulfuric acid. By adjusting the pH of this aqueous solution to neutrality, ethyltrimethylammonium hydroxide is converted into ethyltrimethylammonium salts such as ethyltrimethylammonium chloride, ethyltrimethylammonium iodide and ethyltrimethylammonium sulfate, It is presumed that the ethyltrimethylammonium hydroxide in this aqueous solution is more easily removed because it is more easily assimilated and decomposed by bacteria.

In order to detoxify a liquid containing ethyltrimethylammonium compounds (hereinafter also referred to as the liquid to be treated), various nutritional components are added to the liquid to be treated, and this is used as a medium to grow Mycobacterium spp. There is a method of culturing bacteria (hereinafter referred to as the "bacteria") that can assimilate and decompose ethyltrimethylammonium compounds.

That is, the nutritional components added to the liquid to be treated are not particularly limited as long as they can be assimilated by the present bacteria used (including carbon sources, nitrogen sources, inorganic components, and other components).

As a carbon source, only the ethyltrimethylammonium compound in the liquid to be treated may be used, but other carbon sources that can be assimilated by the present bacteria used, such as D-glucose, D-flag-1-ose, and trehalose, may also be used. tJj class, D
Sugar alcohols such as -sorbitol, []-mannitol, inositol and glycerol, alcohols such as methanol and ethanol, and alkylamines such as monomethylamine, dimedylamine and trimethylamine, etc. can also be used in combination.

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

In addition, the ethyltrimethylammonium compound is also
Since it is also a nitrogen compound, all of these bacteria assimilate the ethyltritylammonium compound and grow sufficiently without adding any other nitrogen sources.

It can grow and decompose ethyltrimethylammonium compounds.

In addition, 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, boromine compounds, iodine compounds, and the like are used.

Furthermore, when using a bacterial strain that requires nutritional substances such as vitamins, the nutritional substances required by the strain are added.

The concentration of ethyltrimethylammonium compound in the gutter treatment solution is not particularly limited as long as it can be assimilated and decomposed by the bacteria used, but it is usually 3 wt% as ethyltrimethylammonium hydroxide. It is preferably at most 2 wt%, particularly preferably at most 2 wt%.

The culture conditions may be any conditions that allow the bacteria used to grow and multiply, but generally the temperature is, for example, 5 to 43°C.
C1 is preferably 10-40°C, and pl+ is 5-40°C.
9, preferably 6-8.

Culture is carried out aerobically under these conditions.

In addition, the dissolved oxygen concentration of the culture solution is low enough for this bacterium to grow.

There is no particular restriction on the dissolved oxygen concentration as long as it allows proliferation, but it is usually about 0.5 to 20 ppm.

In order to achieve such a dissolved oxygen concentration, it is necessary to adjust the amount of aeration gas, stir it, use oxygen gas or a mixed gas of oxygen and air as the aeration gas, and adjust the pressure inside the culture tank. Measures such as increasing the

Although the growth and proliferation of this bacterium is relatively poor and the efficiency of removing and decomposing ethyltrimethylammonium compounds is relatively reduced, this does not preclude culturing under these conditions.

Furthermore, 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 or amine is consumed for bacterial cell production during the culture period, resulting in a decrease in the pH of the culture solution. In this case, ammonia, caustic potash, caustic soda, and an alkali such as amine are added to maintain the PTL of the culture solution at a predetermined value.
Alternatively, it is preferable to add an amine.

In addition to the method of making the liquid to be treated harmless by culturing the bacteria using the liquid to be treated as a medium as described above,
Pre-cultured cells of this bacterium and processed materials of this bacterium - for example, culture solution containing cells of this bacterium, crushed cells of this bacterium, and cells of this bacterium are prepared using synthetic resin, etc. Immobilized microbial cells (hereinafter sometimes referred to as "microbial cells") can also be brought into contact with the liquid to be treated.

For example, (a) adding microbial cells to the liquid to be treated; (c) bringing the liquid to be treated into contact with activated sludge mixed with the bacterial cells, culture solution, and crushed bacterial cells; and (c) There are methods such as passing the liquid to be treated through a column filled with immobilized bacterial cells.

Note that wastewater containing ethyltrimethylammonium compounds often also contains other substances, so when treating such wastewater, use a strain of this bacterium that can decompose other substances. It is possible and preferable to use them together.

After the treatment is completed, the treated liquid in which the concentration of ethyltrimedelammonium compound in the treated liquid has become below the permissible concentration,
It is discharged as it is, or after removing the bacterial cells and/or the treated bacterial cells as required.

[Example] The present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to the examples.

Example 1 Per pure water tp, (N114)2S043g, Kll
□PO41.4g.

NaztlPOn 2.1 g, MgSO4°7
Hz00.2g, CaCIz°2H2H2O30
, FeCbllsOt'X1lzO30mg, Mn
CIz°4H205mg, ZnSO4711z05
mg, C1ISO4'511ZO0.5mg, yeast extract 0.2g and ethyltrimethylammonium iodide at concentrations of 1tχ, 2tχ, 4-tχ,
6-tχ, 8wtχ and 10wt% were added, and the pH of each medium was adjusted to 7.0.

Mycobacterium methanolica P-70, Mycobacterium TH-30, and Mycobacterium TH-35 were inoculated into each of these media, and cultured at 30°C for 7 days.

The results are shown in Table 1.

Table 1 Example 2 Per 11 pure water, (NHa)zsO43g, KIIz
POn 1.4g.

NazllPO42,1g, MgSO4'711zO
0.2g, CaC1z'21(z.

30mg, FeC6H50t・XH2O30mg,
MnCIz゛4H, 05mg, ZnSO4711z0
Add 5mg, Cu5On・58z00.5mg, yeast extract 0.2g and ethyltrimethylammonium hydroxide 10g and pl+7 using hydrochloric acid.
．． 200+nN of medium adjusted to 1! The mixture was placed in an Erlenmeyer flask and sterilized at 120°C for 20 minutes.

This medium was inoculated with a pre-culture of Mycobacterium methanolica Tl1-30 (bacteria cell concentration 0D6107-1-0) obtained by pre-culture in the same medium as above at a concentration of 1 vol. After culturing at °C for 65 hours, the bacterial cell concentration (OD, expressed as □) and pl+ of the culture solution were 1.8 and 5.1, respectively.
Met. Furthermore, no ethyltrimethylammonium compound was detected in the culture supernatant.

Note that the generation time was approximately 8 hours.

Example 3 1w of ethyltrimethylammonium hydroxide
Nutrient components were added to the factory wastewater containing tχ and pi (12,0) so that the medium composition would be the same as in Example 2 except (NI+4)2SO4, and further pu7
．． 1 g of Mycobacterium methanolica TH-3 () isolated from a preculture solution obtained by culturing in advance in the same medium as in Example 2 was suspended in a solution If adjusted to 0. The pH and temperature of the culture solution were maintained at 7.0 and 30°C, respectively, with aeration and stirring.

It took about 60 hours before the ethyltrimethylammonium compound in the factory effluent was no longer detected.

In each of the above Examples, the ethyltrimethylammonium compound in the liquid was analyzed using Toso ion chromatography.

Pump: Toso CCPD Detector: Toso CM-8000 Column: TSKgel IC-Cation (To
so) [Effect of the invention] From the present invention, it becomes possible to efficiently decompose and remove ethyltrimethylammonium compound, which is a stable, difficult to decompose, and harmful substance, and thus it becomes possible to efficiently decompose and remove ethyltrimethylammonium compound containing ethyltrimethylammonium compound. Harmful waste liquid can be effectively rendered harmless, and the present invention has extremely high value in terms of environmental health preservation.

Patent applicant: Mitsubishi Gas Chemical Co., Ltd. Representative Nagano Japanese book

Claims (1)

[Claims] A liquid containing an ethyltrimethylammonium compound,
An ethyltrimethylammonium compound characterized in that the ethyltrimethylammonium compound in the liquid is decomposed and removed by contacting with bacterial cells belonging to the genus Mycobacterium that assimilate and decompose the ethyltrimethylammonium compound. Removal method.