JP2007282631A - Microorganism having degrading and assimilating ability for triazine-based compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide novel microorganisms having a degrading and assimilating ability for triazine-based compounds, particularly melamine derivatives such as melamine and melamine resin, contained in liquid such as waste water. <P>SOLUTION: Disclosed are microorganisms having a degrading and assimilating ability for triazine-based compounds and belonging to genera Agrobacterium, Microbacterium, Ochrobactrum, or Phyllobacterium. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel microorganism having an ability to decompose and assimilate triazine compounds which are hardly decomposed in wastewater treatment and the like.

  Conventionally, industrial wastewater such as paint wastewater discharged from paint factories and wastewater containing a large amount of organic matter such as agricultural wastewater are subjected to physical treatment such as coagulation separation treatment and ultrafiltration, and then aerobic conditions using the activated sludge method. In general, biological treatment is performed under the condition (see, for example, Patent Document 1). However, in the normal activated sludge method, there is a limit to the ability of microorganisms to decompose organic matter, and in this method, difficult-to-decompose organic matters such as triazine compounds remain in the treated water as they are and remain in the environment as pollutants. .

  In general, triazine-based compounds are contained in industrial products such as paints, photopolymerization initiators, ultraviolet absorbers, metal treatment agents, and water reducing agents for concrete products. It is also abundant in agricultural and general household products such as glazes. However, because triazine compounds are difficult to decompose, they remain in treated water such as factory wastewater, agricultural wastewater, and household wastewater discharged from the factory, and the used agricultural chemicals and herbicides penetrate into the soil. As a result, it remains in the groundwater, and it becomes a problem that it remains in the environment or diffuses as a pollutant.

  In addition, triazine compounds are considerably stable in water, are not decomposed in an aqueous solution at pH 5-9 at 20 ° C., and are said to have endocrine disrupting effects. .

As a method for decomposing triazine compounds, particularly melamine derivatives such as melamine and melamine resins, Patent Document 2 discloses that an aqueous solution or suspension of melamine is brought into contact with a microorganism or enzyme agent having melaminease activity under anaerobic conditions. Thus, a method for biological degradation of melamine is disclosed. Patent Document 3 and Patent Document 4, a method of decomposing by contacting the triazine compound in certain strains of Pseudomonas (Pseudomonas) genus or Rhodococcus (Rhodococcus) genus under aerobic conditions is disclosed . Furthermore, Patent Document 5 discloses a method for degrading triazine-based pesticides that are frequently used all over the world, using bacteria belonging to beta-proteobacteria and / or complex microbial systems containing the same.

JP-A-6-55172 JP 54-163892 A JP-A-57-86293 JP 60-114191 A Japanese Patent Laying-Open No. 2005-27536

  An object of the present invention is to provide a novel microorganism having the ability to assimilate triazine-based compounds contained in liquids such as waste water, particularly melamine derivatives such as melamine and melamine resins.

The present inventors focused on microorganisms that can be grown using triazine compounds as a nitrogen source, that is, microorganisms that have the ability to assimilate triazine compounds, and conducted various screenings. This time, triazine compounds obtained from a paint wastewater treatment reactor From the group of bacteria that decompose melamine, the melamine-degrading microbial cells were isolated and the obtained microbial cells were identified. The genus Agrobacterium , Microbacterium , The inventors have found that microorganisms belonging to the four genera of the genus Ochrobactrum and Phyllobacterium have excellent ability to degrade melamine, and have completed the present invention.

Thus, the present invention provides a microorganism having a decomposition utilization ability of Agrobacterium (Agrobacterium) genus-mycobacterial (Microbacterium) genus Ochrobactrum (Ochrobactrum) genus or Filo Agrobacterium (Phyllobacterium) triazine compounds belonging to the genus To do.

  The microorganism of the present invention can decompose and assimilate triazine compounds contained in liquids such as factory wastewater, agricultural wastewater, and household wastewater, and is very useful from the viewpoint of environmental conservation.

  In the present specification, the “triazine-based compound” is a compound having at least one triazine ring in one molecule. For example, melamine, melamine resin, cyanuric acid, isocyanuric acid, melamine sulfate, melamine polyphosphate, ammelin, Melam, melam sulfate, benzguanamine, acetoguanamine, phthalodiguanamine, succinoguanamine, melamine cyanurate, melamine phosphate, melamine pyrophosphate, butylenediguanamine, norbornene diguanamine, triguanamine, benzoguanamine, acetoguanamine, methylenedimelamine , Ethylene dimamine, trimethylene melamine, tetramethylene dimamine, hexamethylene dimamine, 1,3-hexylene melamine, tris (β-cyanoethyl) isocyanurate, etc. Gerare, it is particularly preferred melamine and melamine resins. Here, the melamine resin includes a resin obtained by reacting melamine with an aldehyde, particularly formaldehyde and / or alcohol, and examples thereof include alkyl ethers such as methyl etherified melamine, ethyl etherified melamine, and butyl etherified melamine. Melamine or methylol melamine can be mentioned.

  Hereinafter, the isolation culture and identification of microorganisms having the ability to assimilate the triazine compound of the present invention will be described in more detail.

Isolation and identification of microorganisms capable of assimilating triazine compounds The microorganisms having the ability to assimilate triazine compounds of the present invention are obtained by screening as follows.

  Prepare a basic medium (composition: sodium chloride 5 g, dipotassium hydrogen phosphate 1 g, potassium dihydrogen phosphate 1.18 g, magnesium sulfate heptahydrate 200 mg, potassium chloride 2.23 g, melamine 100 mg, distilled water 1 L), Also, succinic acid 590 mg / L (medium 1-1), or succinic acid 590 mg / L and n-butanol 555 mg / L (medium 1-2), or succinic acid 590 mg / L and ethylene glycol monobutyl ether 590 mg / L (medium 1-3) or four types of media supplemented with 590 mg / L of succinic acid and 900 mg / L of glucose (medium 1-4) as a carbon source were prepared and sterilized by filtration using a filter having a pore size of 0.20 μm.

On the other hand, sludge collected from a wastewater treatment facility at a paint manufacturing plant was inoculated into 100 ml of each of the above four types of medium, and then cultured with shaking at 25 ° C. and 150 rpm. The melamine concentration in each medium after culturing for 7 days was as shown in FIG. In these cultures, a decrease in melamine was confirmed, and at the same time an increase in ammonia nitrogen concentration was confirmed. By this cultivation, four types of fungal groups that decompose melamine were obtained. Each fungal group is referred to as MD-1, MD-2, MD-3, MD-4.

  MD-1, MD-2, MD-3, and MD-4 melamine-degrading bacterial groups obtained by the above method were cultured by the following method, and melamine-degrading bacterial strains were isolated from the degrading bacterial group.

Medium 2 (composition: meat extract 5 g, peptone 10 g, sodium chloride 5 g, agar 15 g, distilled water 1 L) was prepared, and the culture solution of each degrading bacterium group was applied to medium 2 and allowed to stand at 30 ° C. for 2 days. did. Culture medium 3 (composition: sodium chloride 5 g, dipotassium hydrogen phosphate 1 g, potassium dihydrogen phosphate 1 for catching the colonies grown in this culture and confirming the colonies of bacteria considered to have melamine degradability 18 g, magnesium sulfate heptahydrate 200 mg, potassium chloride 2.23 g, melamine 100 mg, agar 15 g, distilled water 1 L), and static culture was performed at 30 ° C. for 7 days. As a result of the culture, the presence of strains capable of growing on both media was confirmed from each group of degrading bacteria, and these strains were isolated by a conventional method.

  The melamine-degrading strain thus isolated was identified by the following method.

The morphological properties, growth aspects and physiological properties of the melamine-degrading strains isolated as described above were evaluated by conventional methods. Culture was performed at 30 ° C. unless otherwise specified. The existence of four strains was confirmed from the morphological properties of the grown strains, the mode of growth in the medium and the physiological properties. The mycological properties of these four strains are shown in Tables 1 to 4 below. Moreover, the analysis of the 16S-rRNA gene of these four strains was requested to NC Japan, and the base sequence of the 16S-rRNA gene was determined. The base sequences of about 1500 bases excluding the PCR primer sequences at both ends of the base sequences of the 16S-rRNA genes of these four strains are shown in SEQ ID NOs: 1 to 4 in the sequence listing. Furthermore, when a phylogenetic tree was prepared by the neighborhood joining method (NJ method) and compared, it was estimated that the four strains were bacteria classified into the genus Agrobacterium , Microbacterium , Ochrobacterium, and Phyllobacterium .

Comprehensive analysis of the morphological properties, growth behavior in the medium, physiological properties and results of 16S-rRNA gene base sequence of the above four strains, the microorganisms of the genus Agrobacterium are Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), Microbacterium sp. ( Microbacterium sp.) Strain, Ochrobacterium sp. Strain, Ochrobactrum anthropi strain, and Phyllobacterium sp. Has been identified as a strain of Phyllobacterium myrsinacearum . These strains are respectively Agrobacterium tumefaciens MD5-YMTK, Microbacterium sp. ( Microbacterium sp.) MD7-MNTY, Ochrobactrum anthropi MD1-MTJY and Phylobacteria As U.M.-Millsilarum ( Phyllobacterium myrsinacearum ) MD6-JKRH, International Patent Organism Depository, National Institute of
Deposited to Advanced Industrial Science and Technology as deposit numbers FERM P-20719, FERM P-20721, FERM P-20718 and FERM P-20720 on November 24, 2005 (original deposit), and in 2006 On October 23rd, based on BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE, accession numbers FERM BP-10709, FERM BP-10711, FERM, respectively
BP-10708 and FERM BP-10710 were transferred to the International Deposit.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited only to a following example.

In addition, the composition of the culture medium 3 used in the examples is 5 g of sodium chloride, 1 g of dipotassium hydrogen phosphate, 1.18 g of potassium dihydrogen phosphate, 200 mg of magnesium sulfate heptahydrate, 2.23 g of potassium chloride, 100 mg of melamine, Agar 15g, distilled water 1L, basic medium composition: sodium chloride 5g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1.18g, magnesium sulfate heptahydrate 200mg, potassium chloride 2.23g, melamine 100 mg, 1 L of distilled water.

Example 1: Degradability of cyanuric acid A medium 3 (hereinafter referred to as medium 3-1) containing 100 mg of cyanuric acid was prepared, and Agrobacterium tumefaciens MD5-YMTK
FERM BP-10709, Microbacterium sp. MD7-MNTY FERM BP-10711, Ochrobactrum anthropi MD1-MTJY FERM BP-10708 and Phyllobacterium myrsin 6 -JKRH FERM
Four strains of BP-10710 were inoculated and subjected to static culture at 30 ° C. for 7 days. As a result of culturing each strain, colonies were confirmed in the medium 3-1.

  As a result, it was confirmed that all four strains had triazine ring decomposition activity.

Example 2: Degradability of melamine resin Medium 3 (hereinafter referred to as medium 3-2) containing 100 mg of melamine resin (Cymel 325, manufactured by Cytec Industry Co., Ltd., methyl etherified melamine resin, solid content 80%) was prepared. Agrobacterium tumefaciens MD5-YMTK FERM BP-10709, Microbacterium sp. ( Microbacterium sp.) MD7-MNTY FERM BP-10711, Ochrobactrum anthropi MD1-MTJY Four strains of FERM BP-10708 and Phyllobacterium myrsinacearum MD6-JKRH FERM BP-10710 were inoculated and cultured at 30 ° C. for 7 days. As a result of culturing each strain, colonies were confirmed in the medium 3-2.

  As a result, it was confirmed that all four strains had triazine ring decomposition activity.

Example 3
The aforementioned degrading bacteria group MD-1, MD-2, MD-3 and MD-4 were each centrifuged (10000 rpm for 10 minutes at 4 ° C). The fungus paste thus obtained was washed with a basic medium and centrifuged again (at 4 ° C. for 10 minutes, 10,000 rpm). The supernatant liquid after centrifugation was taken out and filtered through a filter having a pore size of 0.20 μm to obtain a filtrate from which cells were completely removed from the supernatant liquid. The bacterial paste and filtrate thus obtained were suspended in a basic medium and cultured with shaking at 25 ° C. and 150 rpm, and the daily change in melamine concentration was followed. The results are shown in Table 5 below.

  As a result, it was confirmed that the melamine-degrading activity of the bacteria was not expressed by extracellular enzymes but expressed by intracellular enzymes.

FIG. 1 is a graph showing melamine degradability in MD-1, MD-2, MD-3, and MD-4 culture systems.

Claims (6)

Agrobacterium (Agrobacterium) genus-mycobacterial (Microbacterium) genus Ochrobactrum (Ochrobactrum) genus or Filo Agrobacterium (Phyllobacterium) microorganism having a decomposition utilization ability of triazine compounds belonging to the genus. The microorganism according to claim 1, which is Agrobacterium tumefaciens MD5-YMTK FERM BP-10709. The microorganism according to claim 1, which is Microbacterium sp. ( Microbacterium sp.) MD7-MNTY FERM BP-10711. The microorganism according to claim 1, which is Ochrobactrum anthropi MD1-MTJY FERM BP-10708. The microorganism according to claim 1, which is Phyllobacterium myrsinacearum MD6-JKRH FERM BP-10710.   The microorganism according to any one of claims 1 to 5, wherein the triazine compound is melamine or a melamine resin.