JPH06261771A - Method for decomposing organic aminocarboxylic acid utilizing microorganism - Google Patents

Abstract

PURPOSE:To obtain a readily usable method for decomposing an organic aminocarboxylic acid with a microorganism in which the organic aminocarboxylic acid can efficiently be decomposed without exerting any damaging action on humans and the new microorganism, capable of decomposing the organic aminocarboxylic acid and extremely suitable for carrying out this method. CONSTITUTION:This method for decomposing an organic aminocarboxylic acid utilizing a microorganism is characterized by bringing a bacterium which has the ability to decompose the organic aminocarboxylic acid and belong to the genus Bacillus into contact with the organic aminocarboxylic acid, its metallic complex or a salt thereof. Furthermore, this microorganism (Bacillus editabidus) is capable of decomposing the organic aminocarboxylic acid.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for decomposing an organic aminocarboxylic acid, which is used in large amounts in various industries, by a microorganism and a novel organic aminocarboxylic acid decomposing bacterium.

[0002]

2. Description of the Related Art Decomposition of ethylenediaminetetraacetic acid, which is one of organic aminocarboxylic acids, by microorganisms has already been described in JP-A-58-43782. In this method, a bacterium belonging to the genus Pseudomonas or the genus Alcaligenes that grows in a medium containing ethylenediaminetetraacetic acid as a single carbon source and a single nitrogen source is used as the ethylenediaminetetraacetic acid-degrading bacterium. However, as is well known, the bacteria belonging to the genus Pseudomonas or the genus Alcaligenes have a narrow temperature, a proper range of salt concentration, etc., and are not necessarily suitable as pollution-controlling microorganisms that have to withstand a wide variety of environments. Further, some Pseudomonas bacteria, such as Pseudomonas aeruginosa , are harmful to the human body, and their use in pollution control poses a problem from the viewpoint of public health.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a method for degrading an organic aminocarboxylic acid by a microorganism, which has no harmful effect on humans, is easy to use, and can efficiently decompose an organic aminocarboxylic acid. The purpose is to provide a method. Another object of the present invention is to provide a novel organic aminocarboxylic acid-degrading bacterium which is extremely suitable for carrying out this method.

[0004]

DISCLOSURE OF THE INVENTION The present invention is a bacterium that is different from the bacteria belonging to the genus Pseudomonas and the genus Alcaligenes, and that the bacteria belonging to the genus Bacillus have excellent decomposition characteristics for organic aminocarboxylic acids. It was made based on. That is, the present invention is characterized in that a bacterium belonging to the genus Bacillus having the ability to decompose an organic aminocarboxylic acid is brought into contact with an organic aminocarboxylic acid, a metal complex thereof, or a salt thereof, by the microbial decomposition of the organic aminocarboxylic acid. Provide a processing method. The present invention also provides an organic aminocarboxylic acid degrading bacterium, Bacillus editorvidus ( Bacillus) .
s editabidus ). The seed of this Bacillus editor <br/> Bidas (Bacillus editabidus ) is Bacillus edita
bidus-1 (Microtechnology Research Institute, Microbiology Research Institute No. 13449: FERMP-1344
9) belongs to the species. Bacteria belonging to the genus Bacillus having the ability to decompose organic aminocarboxylic acid used in the present invention include Bacillus editabidas.
idus ), Bacillus subtilis ),
Bacillus megaterium (Bacillus megaterium), and the like, such as Bacillus sphaericus (Bacillus sphaericus). These are, for example, Bacillus edtabidus-1
(Microbiology Research Institute No. 13449), Bacillus subtilis
NRIC 0068, B. megaterium NRIC 1009, B. sphaericu
s It can be easily obtained as NRIC 1013.

[0005] Among these, organic aminocarboxylic acids degrading bacterium Bacillus Editabidasu (Bacilluseditabidus) are new species, Bacillus editabidus-1 (BikokenkinYadoriki 13
No. 449) has the following mycological properties. I. Morphological properties (1) Bacterial form: bacillus (2) Size: 0.8-0.9 μm × 2.3-2.7 μm (normal broth culture at 27 ° C for 24 hours) (3) Spores Shape: Ellipsoid (4) Location of spores: Centrality (5) Gram stain: Gram positive II. Culture properties (1) Normal agar medium: Good growth This fungus forms a round, light brown colony, and the surrounding area. Is wavy, and the degree of ridge is centrally convex.

III. Physiological properties (1) Pigment production :-( 2) Melanin production: + (3) Catalase test: + (4) Urea hydrolysis: + (5) Starch hydrolysis: + ( 6) Casein hydrolysis: + (7) Esculin hydrolysis: + (8) Chitin hydrolysis: + (9) Tributyrin hydrolysis :-( 10) Oxidase test: + (11) Paranitrophenyl phosphate Hydrolysis of: + (12) Hydrolysis of alginic acid:-(13) Production of hydrogen sulfide:-(14) Growth pH: pH6-7 (15) Growth temperature: 10.5-39.0 ° C (16) Growth Salt concentration: 1.5-6.0% (17) Oxygen requirement: Aerobic (18) Aggregation of skim milk:-(19) Peptonization of skim milk: + (20) Acid and gas formation: D-glucose , L-arabinose, D-xylose and D-mannitol were examined for acid formation. Produced a D- glucose only acid. However, no gas was generated. Based on the above mycological properties, Bergey's Manual of Systema
According to tic Bacteriology (Volume 2), this strain was
It was identified as a strain belonging to the genus us. In addition, since the hydrolysis of urea is +, it is clearly distinguished from Subtilis, firmus, and Coagulans. The positive hydrolysis of starch and the negative motility clearly distinguish it from Sphaericus. Acid production from xylose is negative and Oxid
A positive ose test clearly distinguishes it from Circulans. Also, since the size is width 0.8 to 0.9, me
Clearly distinguished from gaterium. It can also be referred to that Motility is megaterium + in most cases. Therefore,
Certified as a new strain.

A method for culturing this strain will be described below.
The composition of the medium used for culturing this strain is such that the strain to be used grows well, and a suitable carbon source, a nitrogen source or an organic nutrient inorganic salt, etc. is used to smoothly decompose organic aminocarboxylic acids such as EDTA. Become. As a carbon source, an organic aminocarboxylic acid metal complex (eg, EDTA-Fe or EDT)
A-Na, etc.) can be used. Further, as the nitrogen source or the organic nutrient source, for example, polypeptone, yeast extract,
Meat extract etc. are mentioned. It is preferable to use an organic nutrient source of about 0.1 to 1%. As the inorganic salt, various phosphates and magnesium sulfate can be used. Furthermore, a trace amount of heavy metals is used, but it is not always necessary to add it in a medium containing a natural product. The preferred medium is Fuji No.
1 medium (polypeptone 0.5%, yeast extract 0.1%, ED
TA iron ammonium salt 0.01%, agar 2.0%, phosphate buffer (pH 5.8)). For culturing, the medium is sterilized by heating or the like, and then inoculated with bacteria, and the temperature is set at 25 to 39 ° C.
It may be left standing for a day, shaken, or aerated with aeration. The pH is preferably about 6-8. The decomposition of EDTA can be confirmed by ion chromatography. That is, the remaining rate can be observed by subjecting the solution after culturing to a suitable dilution of the solution obtained by filtering the solution after filtration with a 0.45 μm Millipore filter and subjecting it to ion chromatography.

In the present invention, the bacterium having the ability to decompose the organic aminocarboxylic acid of the genus Bacillus is brought into contact with the organic aminocarboxylic acid, its metal complex or its salt to decompose them. Therefore, the method of the present invention can be effectively used as a method for treating wastewater containing an organic aminocarboxylic acid such as EDTA. Organic aminocarboxylic acids such as EDTA are mainly used for paper (bleaching), fibers (dyeing), detergents, plating, foods, photographs and processing liquids thereof, cosmetics, pharmaceuticals, agricultural chemicals,
It is used in fields such as synthetic rubber (polymerization agent) and vinyl chloride resin (heat stabilizer), and there are severe restrictions on the wastewater and wastewater of these plants. Therefore, the present invention will be described below by taking as an example a method for treating EDTA-containing wastewater.
The EDTA-containing wastewater is preferably subjected to treatment with EDTA-decomposing bacteria after first decomposing components that can be decomposed by ordinary biological treatment. If no components other than EDTA that are decomposed by biological treatment are included, biological treatment is not necessary. Here, the type of biological treatment is not particularly limited. Examples of the biological treatment method include microbial suspension method such as activated sludge method, biological filtration method, immersion filter method, fluidized bed method, rotary disk method, sprinkling filter method and other biofilm methods, and self-granulation method. Etc. can be used. It may be either aerobic or anaerobic or a combination thereof. For more specific methods of these biological treatments, see “Biological water treatment technology and equipment” edited by the Society of Chemical Engineering (Baifukan), “Microbiology for environmental purification” edited by Ryuichi Sudo (Kodansha Scientific). Have been described.
The wastewater thus pretreated is brought into contact with the organic aminocarboxylic acid-decomposing bacteria. The contact time and temperature may be arbitrary, but it is preferable that the contact is carried out for a time at which a desired decomposition rate is obtained at a suitable temperature of the organic aminocarboxylic acid-decomposing bacterium. Usually, the organic aminocarboxylic acid is 0.01 to 1
% Aqueous solution of 25 to 39 ° C. is preferably contacted with the organic aminocarboxylic acid-decomposing bacterium for about 12 to 240 hours.

[0009] At this time, it is preferable to bring the organic aminocarboxylic acid-decomposing bacterium into contact with the immobilized one. The immobilization method may be of any type as long as it is an immobilization method that prevents the organic aminocarboxylic acid-decomposing bacteria from flowing out of the treatment tank.
As a specific immobilization method, for example, a biofilm method using a contact material with which a microbial film is formed and attached, an entrapping immobilization method in which cells are confined in a gel, and the like can be used.
As the contact material in the biofilm, for example, porous ceramics,
Activated carbon, sponge, string carrier, plastic, honeycomb carrier, corrugated carrier, anthracite, gravel, sand, etc. 1
One kind or two or more kinds can be used. As the immobilization method, acrylamide method, agar-acrylamide method,
PVA-boric acid method, PVA-freezing method, photocurable resin method,
Acrylic synthetic polymer resin method, sodium polyacrylate method, sodium alginate method, K-carrageenan method, etc.
Any type of cell may be used as long as the cell can be confined, the cell activity is maintained in the treatment tank, and the cell has a large physical strength and can be used for a long time. More specific methods of these immobilization methods are described in “Wastewater Treatment by Microbial Immobilization Method” edited by Ryuichi Sudo (Industrial Water Research Committee). For the treatment using the organic aminocarboxylic acid decomposing bacterium, the contact material as described above, the immobilized gel or the like may be floated and fluidized in the treatment tank, or the biological filtration method, the immersion filter bed method, the fluidized bed method, It may be used as a carrier for the rotating disc method, the sprinkling filter method, or the like.

According to the present invention, various organic aminocarboxylic acids can be decomposed. Examples of the organic aminocarboxylic acid serving as a decomposition control include the following. B-1 Ethylenediaminetetraacetic acid (EDTA) B-2 Ethylenediaminetetraacetic acid disodium salt B-3 Ethylenediaminetetraacetic acid diammonium salt B-4 Ethylenediaminetetraacetic acid tetra (trimethylammonium) salt B-5 Ethylenediaminetetraacetic acid tetrapotassium salt B- 6 Ethylenediaminetetraacetic acid tetrasodium salt B-7 Ethylenediaminetetraacetic acid trisodium salt B-8 Diethylenetriaminepentaacetic acid B-9 Diethylenetriaminepentaacetic acid pentasodium salt B-10 Ethylenediamine-N- (β-oxyethyl)
-N, N ', N'-triacetic acid B-11 ethylenediamine-N- (β-oxyethyl)
-N, N ', N'-triacetic acid trisodium salt B-12 ethylenediamine-N- (β-oxyethyl)
-N, N ', N'-triacetic acid triammonium salt B-13 1,2-diaminopropanetetraacetic acid B-14 1,2-diaminopropanetetraacetic acid disodium salt B-15 nitrilotriacetic acid

B-16 Nitrilotriacetic acid trisodium salt B-17 Nitrilodiacetic acid monopropionic acid B-18 Nitrilomonoacetic acid dihydroxypropionic acid B-19 Nitrilomonoacetic acid dipropionic acid B-20 Nitrilodiacetic acid monohydroxypropionic acid B-21 1,2 -Diaminocyclohexanetetraacetic acid B-22 1,2-Diaminocyclohexanetetraacetic acid disodium salt B-23 Iminodiacetic acid B-24 Dihydroxyethylglycine B-25 Ethyletherdiaminetetraacetic acid B-26 Glycoletherdiaminetetraacetic acid B-27 Ethylenediamine Tetrapropionic acid B-28 1,3 Diaminopropane tetraacetic acid B-29 Ethylenediaminediacetic acid Dipropionic acid B-30 Ethylenediaminediacetic acid

B-31 Ethylenediaminedioltohydroxyphenylacetic acid B-32 Ethylenediaminedipropionic acid B-33 Hydroxyethylethylenediaminetriacetic acid B-34 Nitrilotripropionic acid B-35 Butylenediaminetetraacetic acid B-36 Metaphenylenediaminetetraacetic acid B-36 -37 Triethylenetetramine hexaacetic acid B-38 Metaxylylenediamine tetraacetic acid B-39 Anisidine blue B-40 Chromazurol S B-41 Fluoxin B-42 Methylthymol blue B-43 Methylxylenol blue B-44 Sircosin cresol Red B-45 Stilbene Fluoro Blue S B-46 N, N-Bis (2-hydroxyethyl) glycine

[0013]

[Chemical 1]

[0014]

[Chemical 2]

[0015]

[Chemical 3]

[0016]

[Chemical 4]

[0017]

[Chemical 5]

In the present invention, various metal complexes of the above-mentioned organic aminocarboxylic acids, for example, metal complexes with iron, calcium, magnesium, cobalt, manganese, gold and the like, salts with alkali metals, ammonium and alkanolamines are also decomposed. Can be listed as a target. The method for decomposing an organic aminocarboxylic acid of the present invention by a microorganism can be carried out by the above-mentioned method, but it is preferably carried out in the presence of soluble iron, and particularly preferably in the presence of 30 to 3000 ppm of soluble iron. Good. Examples of the soluble iron include ferrous sulfate, ferric chloride, ferric nitrate and the like. Further, it is preferable that the liquid to be treated is decomposed while being in constant contact with the organic aminocarboxylic acid-decomposing bacteria while stirring. Next, the present invention will be described with reference to examples.

[0019]

【Example】

Example 1 100 ml of the following culture solution containing EDTA-salts was sterilized in an autoclave at 120 ° for 20 minutes, and the following strains were inoculated into this medium, followed by static culture at 37 ° C. for 5 days. I didn't block light. Types of strains The following four types were used. Bacillus editabidus-1
The Bacillus subtilis NRIC 0068 B. megaterium NRIC 1009 B. sphaericus NRIC 1013 fungus was isolated from the mixed soil around the fields and river mouths in the Seisho area, Kanagawa prefecture. Isolation of this strain from the source soil
A medium containing EDTA was dispensed into a test tube, sterilized, soil was added, and the mixture was shake-cultured at 27 ° C. After that, monospores were separated using an agar medium to obtain the bacterium. In addition, culture of Bacillus bacteria other than this bacterium can be performed in the same manner as above. All of the above-mentioned bacteria are preserved strains of Tokyo University of Agriculture. After stationary culture, the residual degree and the decomposition rate of EDTA-Fe were determined by the ion chromatography method. The results are shown in Table 1.
() Shows the decomposition rate.

[0020]

[Table 1] Table-1 EDTA residual rate and decomposition rate No. Static culture 37 ° C 5 days ──────────────────────────── ────── B. editabidus-1 57.3ppm (42.1%) Colored in brown B. subtilis NRIC 0068 98.9 B. megaterium NRIC 1009 98.5 B. sphaericus NRIC 1013 81.4 (17. 8%) Colored brown Control (without inoculation) 99.0 ──────────────────────────────────

Example 2 The same procedure as in Example 1 was carried out except that shaking culture was carried out. Table showing the residual rate and decomposition rate of EDTA-Fe
2 shows.

[Table 2] Table-2 EDTA residual rate and decomposition rate Bacterial No. Shaking culture 37 ° C 7 days ───────────────────────────── ────── B. editabidus-1 9.8ppm (90%) B. subtilis NRIC 0068 70.6 (28%) B. megaterium NRIC 1009 52.9 (46%) B. sphaericus NRIC 1013 35.3 (64%) B. Natto 82.3 (16%) Control (without inoculation) 98.0 ──────────────────────────── ───────

Example 3 100 ml of the following culture solution containing organic aminocarboxylic acid was added to 12
After sterilizing in an autoclave for 20 minutes at 0 ° C, add Ba to this medium.
cillus editabidus-1 (Microbiology Research Institute No. 13449)
Was inoculated, and static culture (sometimes shaking) was performed at 37 ° C. for 7 days. No light was shielded. Culture liquid composition Polypeptone 0.5% Yeast extract 0.1% PDTA / Fe 0.01% Distilled water 100 ml Phosphate buffer (KH ₂ PO ₄ (1/30 mol%) 8 ml, Na ₂ HPO
_The pH was adjusted to 5.8 with ₄ (1/30 mol%) 1 ml). In addition, PDT
A.Fe was added in the form of ferric ammonium salt of 1,3-diaminopropanetetraacetic acid to the above concentration. Further, the same culture was performed after the culture in which PDTA.Fe was changed to BDTA.Fe. In addition, BDTA / Fe is
It was added in the form of butylenediaminetetraacetic acid, ferric chloride and ammonia (each equimolar amount) to the above concentration. The decomposition rates of PDTA and BDTA were evaluated in the same manner as in Example 1 (ion chromatography method).
The results are shown below. PDTA decomposition rate 52% BDTA decomposition rate 36%

[0023]

INDUSTRIAL APPLICABILITY According to the present invention, by using a bacterium belonging to the genus Bacillus having the ability to decompose organic aminocarboxylic acid, organic aminocarboxylic acid, its metal complex or its salt can be easily decomposed. . And, especially when the organic aminocarboxylic acid degrading bacterium Bacillus editorvidus is used, the organic aminocarboxylic acid can be decomposed extremely efficiently.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location (C12N 1/20 C12R 1:07) (72) Inventor Seiji Suzuki 210 Nakanuma, Minamiashigara City, Kanagawa Fuji Photo inside film company

Claims (2)

[Claims] 1. An organic aminocarboxylic acid using a microorganism, which comprises contacting a bacterium belonging to the genus Bacillus having an ability to decompose an organic aminocarboxylic acid with an organic aminocarboxylic acid, a metal complex thereof or a salt thereof. Disassembly method. 2. An organic aminocarboxylic acid-degrading bacterium Bacillus
The editor Vidas (Bacillus editabidus ).