JP5352850B2 - Novel microorganisms that degrade amino-s-triazine compounds - Google Patents

Description

  The present invention relates to a novel microorganism belonging to the genus Nocardioides having the ability to degrade at least one substance selected from the group consisting of amino-s-triazines, in particular melamine, ammelin and ammelide, and a complex microbial system comprising the microorganisms Further, the present invention relates to a method for decomposing an amino-s-triazine compound using the microorganism or the complex microorganism system.

  The s-triazine compound is a general term for compounds having a symmetric triazine ring composed of 3 carbon atoms and 3 nitrogen atoms as a basic skeleton, and is manufactured and used for a wide range of applications such as resins, paints, fertilizers, herbicides and fungicides. It's being used. These substances are relatively stable in the environment and accumulate in the environment with use. In addition, some s-triazine compounds such as melamine, atrazine, and cimetrin are listed in the “Matters for Investigation for Water Environment Conservation” (June 5, 1998) Press release) ”.

  As an example of damage caused by s-triazine-based compounds accumulated in the environment, for example, in the United States, contamination of groundwater for drinking due to large-scale use of atrazine has been reported. Also, in recent years, health hazards to human livestock such as dogs and cats that ate pet food mixed with melamine die due to renal failure, and infants who drink milk mixed with melamine developed kidney stones. Many examples have been reported. In addition, it is estimated that these health hazards are caused by the combined action of melamine and its metabolite cyanuric acid. From such a point, when s-triazine compounds are added to fertilizers, feeds or foods, or remain and are discharged in large quantities in the environment such as soil or water, there is a possibility that it becomes a serious problem.

  The s-triazine compounds, particularly melamine and its hydroxyl-substituted cyanuric acid, are industrially produced in large quantities compared to other s-triazine compounds, are contained in industrial waste, Since it is hardly degradable, it can be said that it is highly likely to accumulate when released into water and soil environments.

  Once the s-triazine compound containing melamine and its hydroxyl-substituted product is released into the environment, it is not practical to recover it and decompose it by a normal chemical reaction. Therefore, means for biodegrading these compounds released into the environment with microorganisms have been tried. As such a method, Patent Document 1 discloses a method of biologically degrading melamine by contacting an aqueous solution or suspension of melamine with a microorganism or enzyme agent having melaminease activity under anaerobic conditions. ing. Patent Document 2 discloses a method of degrading ammeline or ammelide into cyanuric acid by contacting a specific strain of the genus Pseudomonas under aerobic conditions. Patent Document 3 discloses a method for biodegrading a chlorinated triazine or hydroxytriazine into a corresponding ammelide by contacting a specific strain of the genus Rhodococcus under aerobic conditions. Furthermore, Patent Documents 4 and 5 disclose methods for decomposing triazine-based pesticides under aerobic conditions. Furthermore, Patent Document 6 discloses a method for degrading melamine by a strain belonging to the genus Agrobacterium, Microbacterium, Okrobactrum, or Philobacterium.

However, the bacteria and methods described in these documents cannot hydrolyze melamine to cyanuric acid, or decompose melamine to cyanuric acid but cannot decompose cyanuric acid, and cyanuric acid accumulates, or There are problems such as concern about the remaining of the decomposition product and the residual.
JP 54-163892 A JP-A-57-86293 JP 60-114191 A JP-A-2005-27536 JP2007-6714 JP2007-282631

  Accordingly, an object of the present invention is to provide a method for assimilating and decomposing amino-s-triazine compounds, particularly melamine and hydroxyl-substituted products thereof, contained in fertilizers, feeds, industrial waste materials, industrial wastewater, environmental water, or soil, and It is to provide a novel microorganism that can be used in this degradation method.

  The present inventors have isolated bacteria of the genus Nocardioides having the ability of amino-s-triazine compounds from paddy soil and have reached the present invention. This bacterium was a novel bacterium from 16S rRNA gene sequence analysis and mycological properties. This bacterium is a single bacterium that has the ability to degrade melamine to amnellin, ammelide, and cyanuric acid.In addition, this strain is a complex microbial system co-cultured with known simazine-degrading bacteria. The present invention was completed by discovering that it has the ability to be completely degradable via cyanuric acid and that the complex microbial system can significantly improve the degradation rate of cyanuric acid.

Accordingly, the present invention includes the following [1] to [18].
[1]
A bacterium belonging to the genus Nocardioides characterized by the ability to resolve amino-s-triazine compounds.
[2]
The bacterium according to [1], wherein the amino-s-triazine compound is melamine, ammelin, or ammelide.
[3]
The bacteria of the genus Nocardioides are the following (A) or (B):
(A) DNA comprising the base sequence set forth in SEQ ID NO: 1
(B) DNA having 95% or more identity with the base sequence set forth in SEQ ID NO: 1,
The bacterium according to [1] or [2], which has a 16S rRNA gene comprising
[4]
A bacterium belonging to the genus Nocardioides is Nocardioides sp. The bacterium according to any one of [1] to [3], which is ATD6 (Accession No. FERM P-21710) or a mutant thereof.
[5]
[1] A microbial preparation for degrading an amino-s-triazine compound containing the bacterium according to any one of [4] as an active ingredient.
[6]
The microorganism preparation [7] according to [5], wherein the amino-s-triazine compound is melamine, ammelin, or ammelide.
[1] A complex microbial preparation for degrading an amino-s-triazine compound containing the bacterium according to any one of [4] and one or more coexisting bacteria as active ingredients.
[8]
As the coexisting bacteria, the following (C) or (D):
(C) DNA comprising the base sequence set forth in SEQ ID NO: 2
(D) DNA having 95% or more identity with the base sequence set forth in SEQ ID NO: 2,
The complex microbial preparation according to [7], comprising a bacterium belonging to a betaproteobacterium having a 16S rRNA gene comprising
[9]
As the coexisting bacteria, the following (C) or (D):
(C) DNA comprising the base sequence set forth in SEQ ID NO: 2
(D) DNA having 95% or more identity with the base sequence set forth in SEQ ID NO: 2,
A bacterium belonging to beta-proteobacteria having a 16S rRNA gene comprising
And the following (E) or (F):
(E) DNA comprising the base sequence set forth in SEQ ID NO: 3
(F) DNA having 95% or more identity with the base sequence set forth in SEQ ID NO: 3,
The complex microbial preparation according to [7], comprising a bacterium of the genus Bradyrizobium having a 16S rRNA gene comprising
[10]
[8] or [9], wherein the bacterium belonging to the beta proteobacterium is beta proteobacterium CDB21 (Accession No. FERM P-19395; Accession No. FERM BP-10403) or a mutant strain thereof.
[11]
The complex microorganism according to [9] or [10], wherein the bacterium of the genus Bradyrizobium is Bradyzobium japonicum CSB1 (Accession No. FERM P-19394; Accession No. FERM BP-10402) or a mutant strain thereof. Formulation.
[12]
[7] The composite microbial preparation according to any one of [7] to [11], wherein the amino-s-triazine compound is melamine, ammelin, or ammelide.
[13]
[1] A method for degrading an amino-s-triazine compound, which comprises using the bacterium described in any one of [4].
[14]
[5] A method for decomposing an amino-s-triazine compound characterized by using the microbial preparation according to any one of [6].
[15]
[7] A method for decomposing an amino-s-triazine compound, comprising using the composite microbial preparation according to any one of [12] to [12].
[16]
Use of the bacterium according to any one of [1] to [4] for degrading an amino-s-triazine compound.
[17]
Use of the microorganism preparation according to any one of [5] to [6] for decomposing an amino-s-triazine compound.
[18]
Use of the composite microorganism preparation described in any one of [7] to [12] for decomposing an amino-s-triazine compound.

  The present invention also relates to a microbial preparation and a composite microbial preparation containing the above bacteria, and further to a microbial preparation for degrading an amino-s-triazine compound and a complex microbial preparation for degrading an amino-s-triazine compound. Furthermore, the present invention relates to an amino-s-triazine-based compound decomposing agent, and further relates to a microbial catalyst for decomposing amino-s-triazine-based compounds and a composite microbial catalyst for decomposing amino-s-triazine-based compounds.

  ADVANTAGE OF THE INVENTION According to this invention, the triazine type compound contained in a fertilizer, feed, industrial waste material, factory wastewater, environmental water, and soil, especially a melamine and its hydroxyl substitution product can be decomposed | disassembled rapidly. Furthermore, by using the complex microorganism of the present invention, complete degradation can be rapidly performed under a wide range of conditions.

Hereinafter, the present invention will be described in detail by illustrating embodiments of the present invention.
The present invention relates to an amino-s-triazine compound, more preferably a bacterium of the genus Nocardioides having a resolution of melamine, ammelin or ammelide, and a microorganism for degrading an amino-s-triazine compound containing the bacterium as an active ingredient Also related to formulation.

  In a preferred embodiment, the bacterium of the genus Nocardioides according to the present invention is such that the 16S rRNA gene does not impair the base sequence described in SEQ ID NO: 1 in the sequence listing, or the ability to degrade melamine, ammelin, and ammelide. The base sequence described in SEQ ID NO: 1 in the sequence listing is 95% or more, preferably 96% or more, more preferably 96.5% by deleting, substituting or adding one or more bases as a limit. Or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.5% or more, more preferably 99.8% or more, more preferably 99.9% or more. A base sequence having homology, and an amino-s-triazine compound, more preferably melamine, ammeline, or amme It can be Nocardioides bacteria of the genus with de resolution.

  In a preferred embodiment, specific examples of the amino-s-triazine degrading bacteria belonging to the genus Nocardioides include Nocardioides sp. ATD6 (Accession No. FERM P-21710) or a mutant thereof can be mentioned, and preferably Nocardioides sp. ATD6 (Accession number FERM P-21710).

  The present invention also relates to a complex microorganism for degrading an amino-s-triazine compound comprising the amino-s-triazine-degrading bacterium belonging to the genus Nocardioides and one or more coexisting bacteria. In addition, the present invention also relates to a complex microorganism preparation for decomposing amino-s-triazine compounds. In addition to the amino-s-triazine compound, the hydroxyl-substituted compound can be decomposed by using such a complex microbial system or complex microbial preparation. Therefore, the amino-s-triazine compound can be completely decomposed. Can be promoted.

  Examples of the coexisting bacteria include strains having a cyanuric acid-degrading ability and strains that help the growth thereof. In one embodiment of a preferred embodiment, for example, a simazine-degrading bacterium belonging to beta-proteobacteria can be contained as a coexisting bacterium. In another preferred embodiment, the coexisting bacteria include, for example, in addition to simazine-degrading bacteria belonging to beta-proteobacteria, Bradyrizobium bacteria, Rhodococcus rhodochrous, Nocardioides jenseni, Nocardioides fulbus, Nocardio One or more bacteria selected from the group consisting of Ides Simplex, Pseudomonas aeruginosa, and Sphingomonas sp. Can be contained, preferably, in addition to the simazine-degrading bacteria belonging to the beta proteobacteria, Bacteria can be included.

  In a preferred embodiment, the simazine-degrading bacterium belonging to the beta-proteobacteria according to the present invention is a degradation of the base sequence described in SEQ ID NO: 2 in the sequence listing of the 16S rRNA gene, or the complex microbial melamine, ammelin, and ammelide. 95% or more, preferably 96% or more of the base sequence described in SEQ ID NO: 2 in the sequence listing by deleting, substituting or adding one or more bases as long as the ability is not impaired. Preferably it is 96.5% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.5% or more, more preferably 99.8% or more, more preferably Simazine content belonging to beta proteobacteria having a base sequence having a homology of 99.9% or more It can be a bacterium. In addition to the bacterium belonging to the genus Nocardioides, the complex microbial system and the complex microbial preparation containing the simazine-degrading bacterium belonging to the beta-proteobacterium include the hydroxyl-substituted compound in addition to the amino-s-triazine compound. Can also be decomposed, so that complete decomposition of the amino-s-triazine compound can be promoted.

  In a preferred embodiment, examples of the simazine-degrading bacteria belonging to the beta proteobacteria include rhodocycleraceae bacteria. Specific examples include the known beta proteobacteria CDB21 (Independent Administrative Institution Deposited on June 13th, 2003 at the Biological Depositary Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture) and transferred to International Deposit on 22nd August 2005 Domestic deposit accession number FERM P-19395; international deposit accession number FERM BP-10403) or a mutant thereof, preferably beta proteobacterium CDB21 (FERM P-19395; FERM BP-10403).

  In a preferred embodiment, the bacterium belonging to the genus Bradyrizobium according to the present invention is capable of decomposing the 16S rRNA gene in the base sequence described in SEQ ID NO: 3 in the sequence listing, or in the complex microbial melamine, ammelin, and ammelide. The base sequence described in SEQ ID NO: 3 in the sequence listing is 95% or more, preferably 96% or more, more preferably, by deleting, substituting or adding one or more bases as long as they do not impair Is 96.5% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.5% or more, still more preferably 99.8% or more, more preferably 99 It can be a bacterium of the genus Bradyrizobium having a base sequence having a homology of 9% or more. By adding the bacteria of the genus Bradyrizobium, it helps the growth of simazine-degrading bacteria belonging to beta-proteobacteria, and complete degradation of amino-s-triazine compounds by the complex microbial system and the complex microbial preparation according to the present invention. Can be promoted.

  In a preferred embodiment, a specific example of a bacterium belonging to the genus Bradyrizobium is Bradyzobium japonicum CSB1 (Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, 1-chome Higashi 1-chome Tsukuba, Ibaraki No. 1 Central 6) Deposited on June 13, 2003 and transferred to International Deposit on August 22, 2005; Domestic Deposit Accession Number FERM P-19394; International Deposit Accession No. FERM BP-10402) or a mutant thereof, preferably Bradyrizobium japonicum CSB1 (FERM P-19394; FERM BP-10402). Bradyrizobium japonicum CSB1 (FERM P-19394; FERM BP-10402) helps the growth of the betaproteobacterium CDB21 (FERM P-19395; FERM BP-10403), known as simazine-degrading bacteria, to the present invention. The complete degradation of the amino-s-triazine compound by the complex microbial system and the complex microbial preparation can be promoted. The use of beta-proteobacterium CDB21 and Bradyrizobium japonicum CSB1 is described in Japanese Patent Application Laid-Open No. 2005-27536, and this description is incorporated as a part of this specification.

で表される化合物である。
式Ｉにおいて、Ｘ１は、アミノ基、アルキルアミノ基、ジアルキルアミノ基、ヒドロキシアルキルアミノ基、ビス（ヒドロキシアルキル）アミノ基からなる群から選択される基であり、特に好ましくはアミノ基であり、
Ｘ２、Ｘ３は、それぞれ独立に、アミノ基、水酸基、ハロゲン原子、水素原子、アルキル基、アルキルチオ基、アルキルアミノ基、ジアルキルアミノ基、アミノアルキル基、ヒドロキシアルキルアミノ基、ビス（ヒドロキシアルキル）アミノ基からなる群から選択される基である。 In the present invention, the amino-s-triazine compound means the following formula (I):

It is a compound represented by these.
In Formula I, X1 is a group selected from the group consisting of an amino group, an alkylamino group, a dialkylamino group, a hydroxyalkylamino group, and a bis (hydroxyalkyl) amino group, and particularly preferably an amino group,
X2 and X3 are each independently an amino group, hydroxyl group, halogen atom, hydrogen atom, alkyl group, alkylthio group, alkylamino group, dialkylamino group, aminoalkyl group, hydroxyalkylamino group, bis (hydroxyalkyl) amino group A group selected from the group consisting of

Examples of the alkyl group include a methyl group, an ethyl group, a propan-1-yl group, a propan-2-yl group (isopropyl group), an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. Preferably, a methyl group, an ethyl group, and particularly preferably a methyl group can be mentioned.
As an alkylthio group, a methylthio group and an ethylthio group can be illustrated, for example, Preferably a methylthio group can be mentioned.
Examples of the alkylamino group include a methylamino group, an ethylamino group, a propan-1-ylamino group, a propan-2-ylamino group (isopropylamino group), an isobutylamino group, a sec-butylamino group, and a tert-butylamino group. And preferably an ethylamino group.
Examples of the dialkylamino group include a dimethylamino group and a diethylamino group, preferably a dimethylamino group.
Examples of the aminoalkyl group include aminomethyl group, aminoethyl group, aminopropan-1-yl group, aminopropan-2-yl (aminoisopropyl group), aminoisobutyl group, amino sec-butyl group, and amino tert-butyl. Examples of the group include aminomethyl groups.
As a hydroxyalkylamino group, a hydroxymethylamino group and a hydroxyethylamino group can be illustrated, for example, Preferably a hydroxymethylamino group can be mentioned.
Examples of the bis (hydroxyalkyl) amino group include a bis (hydroxymethyl) amino group and a bis (hydroxyethyl) amino group, preferably a bis (hydroxymethyl) amino group.
Examples of the halogen atom include a fluorine atom or a chlorine atom, preferably a chlorine atom.

  In one preferred embodiment, X1 can be an amino group, and X2 and X3 are each independently an amino group, a hydroxyl group, a halogen atom, an aminoalkyl group, a hydroxyalkylamino group, or a bis (hydroxyalkyl) amino group. Group, or a hydrogen atom, preferably an amino group, a hydroxyl group, a halogen atom, or a hydrogen atom, more preferably an amino group, a hydroxyl group, or a hydrogen atom, more preferably an amino group or a hydroxyl group. it can.

  In one preferred embodiment, the amino-s-triazine compound is a compound (melamine) in which X1, X2 and X3 are amino groups in the above formula I, X1 and X2 are amino groups and X3 is a hydroxyl group. A certain compound (ammelin) and a compound (ammelide) in which X1 is an amino group and X2 and X3 are hydroxyl groups can be obtained.

  The present invention also relates to a method for degrading an amino-s-triazine compound by using a bacterium belonging to the genus Nocardioides having amino-s-triazine-degrading ability and a microbial preparation containing the bacterium as an active ingredient. Also related.

  Further, the present invention uses a complex microbial preparation having an amino-s-triazine-degrading bacterium belonging to the genus Nocardioides and at least one coexisting bacterium, and thereby decomposing an amino-s-triazine-based compound. Regarding the method. Preferred examples of the amino-s-triazine compound include melamine, ammelin, and ammelide. Such a complex microbial preparation can decompose the hydroxyl-substituted compound in addition to the amino-s-triazine compound, and thus can promote complete decomposition of the amino-s-triazine compound. . Examples of the hydrogen-substituted compound of the amino-s-triazine compound include cyanuric acid.

  In addition, the composite microbial preparation of the present invention helps bacteria that belong to the genus Nocardioides having amino-s-triazine-degrading ability, and degrading bacteria of hydroxyl-substituted amino-s-triazine compounds and the growth of the degrading bacteria. It may be a preparation containing bacteria, and preferably contains a carrier that allows growth of the complex microorganism system. Examples of the carrier include soil, activated sludge, culture medium, charcoal, bamboo charcoal and the like. By adding, mixing or adhering the bacteria according to the present invention to such a carrier, and adding or spraying it to feed, fertilizer, soil or water system, the decomposition method according to the present invention can be suitably carried out. it can.

  Bacteria belonging to the genus Nocardioides having amino-s-triazine, preferably melamine, ammelin, or ammelide resolution of the present invention include those having the following mycological characteristics.

A. Morphological properties (Medium used: R2A agar (Daigo, Japan), culture temperature 30 ° C., culture time 48 hours)
(1) Cell morphology: Polymorphic koji mold (2) Size: 0.8-1.0 × 2.0-4.0 μm
(3) Spore:-
(4) Motility:-

B. Colony morphology (use medium: R2A agar, culture temperature 30 ° C., culture time 48 hours)
(1) Colony diameter: 1.0 mm or less (2) Color: Cream color (3) Shape: Circular (4) Raised state: Convex lens shape (5) Periphery: Full edge (6) Surface shape: Smooth (7) Transparency : Opaque (8) Viscosity: Butter

C. Physiological properties (1) Gram staining: + (undefined)
(2) Growth at 37 ° C: +
(3) Growth at 45 ° C .: −
(4) Catalase: +
(5) Oxidase: −
(6) Acid / gas production from glucose:-/-
(7) O / F test:-/-
(8) Growth under anaerobic conditions: −
(9) Formation of aerial hyphae: −
(10) Antiacid staining: −

D. Biochemical property test (by API Coryne manufactured by BioMérieux)
(1) Nitrate reduction:-
(2) Pyrazine amidase: −
(3) Pyrrolidonyl allylamidase: −
(4) Alkaline phosphatase: −
(5) β-glucuronidase: −
(6) β-galactosidase: −
(7) α-Glucosidase: +
(8) N-acetyl-β-glucosaminidase: +
(9) Esculin:-
(10) Urease:-
(11) Gelatin hydrolysis: −
(12) Glucose assimilation: −
(13) Ribose utilization: −
(14) Xylose utilization: −
(15) Utilization of mannitol: −
(16) Maltose utilization: −
(17) Lactose utilization: −
(18) Sucrose utilization:-
(19) Glycogen utilization: −

  As a specific example of the subject amino-s-triazine-degrading bacterium, Nocardioides sp. There is ATD6. Nocardioides sp. ATD6 has been deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary, under the name “Nocardioides sp. ATD6” and the deposit number “FERM P-21710”. Have

  The present inventors have found a novel amino-s-triazine degrading strain ATD6 by the method described in Example 1 described later. When this microorganism was experimentally searched for a medium suitable for degrading each amino-s-triazine, it was found to be the medium shown in Table 1 below.

  The trace element solution in Table 1 has the composition shown in Table 2 below.

[Example]
Hereinafter, the present invention will be described with reference to the following examples of the isolation and classification of the melamine-degrading bacteria of the present invention, the classification and identification, and the experimental examples of amino-s-triazine compounds such as melamine using the present-degrading bacteria and the present complex microorganism system. The present invention will be described in more detail and specifically, but the present invention is not limited to the following examples.

Example 1 Isolation and Identification of Melamine Degrading Bacteria The inventors conducted the following novel melamine degrading bacterium Nocardioides sp. ATD6 strain was isolated. FIG. 1 is a schematic view of a reflux apparatus used for accumulation of bacteria. 3 g of wood carbonized material and 1 g of paddy soil in Japan are put in the soil layer tank 1, and betaproteobacterium CDB21, bradyrizobium japonicum CSB1, nocardioides sp. MTD22 (FERM P-20989; FERM BP-10849), Rhodococcus SP. FJ1117YT (FERM P-20535) and the like were inoculated. Using the pump 3 from the storage tank 2 to the soil layer tank 1 using 200 ml of the sterilized medium shown in Table 1 added with 5 mg / L, 20 mg / L or 40 mg / L melamine as a carbon source and a nitrogen source as a reflux liquid And refluxed.

  The melamine concentration in the reflux liquid after the start of reflux was measured by HPLC. The results are shown in FIG. FIG. 2 is a diagram showing the transition of the melamine concentration during the accumulation of bacteria. The vertical axis in FIG. 2 indicates the melamine concentration (mg / L) in the reflux liquid, and the horizontal axis indicates the reflux time (days). The downward arrows in FIG. 2 indicate the time when the total amount of the reflux liquid was replaced with fresh one (reflux liquid replacement timing). As can be seen in FIG. 2, a decrease in the melamine concentration in the reflux liquid was observed from the 21st day after the start of reflux. This rapid decrease in melamine concentration was observed after replacement of the reflux.

Therefore, 1 ml of the reflux solution taken out from the reflux apparatus was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of the medium shown in Table 1 (melamine concentration 20 mg / L), and cultured with shaking at 30 ° C. and 210 rpm. Decreased and increased ammelin concentration. Therefore, the culture solution was subcultured by limiting dilution for the purpose of increasing the composition ratio of bacteria having melamine-degrading ability in the culture solution. That is, one part of the culture broth was diluted with 9 times the amount of medium, and further one part of the diluted culture broth was diluted with 9 times the amount of medium. 0.9 ml from each dilution series was inoculated into 20 ml of the new medium shown in Table 1 (melamine concentration 10 mg / L, 39 mg / L, or 50 mg / L), and cultured with shaking at 210 rpm at 30 ° C. If necessary, measure the melamine concentration in the culture solution using HPLC, collect a portion of the culture solution from the culture solution inoculated with the highest dilution series in which melamine degradation has been observed, and carry out transplantation after dilution by the same procedure. It was. The above operation was repeated 3 times approximately every 10 days.
By repeating the subculturing operation by limiting dilution method, the melamine concentration decreased rapidly even in the subculture medium of the higher dilution, and the composition ratio of melamine degrading bacteria present in the culture medium was increased. Since it was thought, it shifted to the isolation work using an agar medium.

  That is, among the culture solutions in which melamine degradation was observed, the culture solution inoculated with the highest dilution series was serially diluted to the seventh power of 10 by the above-described method, and each dilution solution was diluted with R2A agar medium (manufactured by BD-Difco). ) 100 μL each was applied on the top using a large rod and statically cultured at 30 ° C. for 2 days. A single colony formed on an agar medium is fished with a needle, inoculated into an L-shaped tube containing 10 ml of the medium shown in Table 1 (melamine concentration 40 mg / L), cultured with shaking at room temperature, and a new R2A agar medium And stir-cultured at 30 ° C. When the melamine concentration in the culture solution of the L-shaped tube was measured by HPLC, an increase in ammelin, ammelide and cyanuric acid concentrations was observed with a decrease in melamine concentration after 7 days. However, since it was thought that isolation of degrading bacteria was still insufficient, a colony having a melamine-decomposability generated on the R2A agar medium was applied to a new R2A agar medium, and a single colony was isolated after culturing. This single colony fishing operation was repeated three more times to sufficiently purify the colonies. The purified single colony was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of the medium shown in Table 1 (melamine concentration 20 mg / L) and then swirl culture at 30 ° C. The isolated strain was named ATD6 strain.

Bacteriological examination of ATD6 strain To perform mycological positioning of ATD6 strain isolated in Example 1, morphological properties of ATD6 strain, growth aspect in culture medium, physiological properties, biochemical properties Analysis was carried out. As a result, the following mycological properties were observed.

  As a result of identification according to Bergey's Manual of Systematic Bacteriology Vol. 2 (1984) based on the results, it was estimated that the ATD6 strain of the present invention is a kind of coryneform bacteria. Coryneform bacteria are a diverse group of bacteria, and it was difficult to classify and identify this strain only from the test results of the above mycological properties. Therefore, gene taxonomic examination based on the base sequence of 16S rRNA gene of ATD6 strain was performed according to the following procedure.

(1) Genomic DNA was prepared from cells of ATD6 strain using DNeasy Tissue Kit (Qiagen).
(2) Amplifying a 16S rRNA gene fragment of ATD6 strain by PCR using the above-mentioned DNA as a template DNA and a known Eubacterium 16S rRNA gene amplification primer (Pest Manag. Sci. 63: 261-268, 2007), DNA was purified using QIAquick PCR Purification Kit (Qiagen).
(3) A cycle sequence reaction is performed using a primer for analyzing the base sequence of a known Eubacterium 16S rRNA gene (Pest Manag. Sci. 63: 261-268, 2007) fluorescently labeled using the purified DNA as a template DNA, and DNA The base sequence was determined using a sequencer SQ5500E (manufactured by Hitachi).
SEQ ID NO: 1 shows the partial base sequence and 1474 bases of the 16S rRNA gene of ATD6 strain determined by the above operation. A 16S rRNA gene partial base sequence of ATD6 strain shown in SEQ ID NO: 1 using Genetics PDB (manufactured by Genetics) and the 16S rRNA gene sequences of other microorganisms registered in the DNA base sequence database (GenBank) are FASTA The homology was compared by the method. As a result, the ATD6 strain showed 96.4% homology with Nocardioides nitrofenolicus NSP41 strain and 96.1% with Nocardioides simplex ATCC15799 strain. However, since the ATD6 strain does not form aerial hyphae, it differs from the characteristics of known Nocardioides bacteria. From the above results, the inventors have identified this strain as a novel Nocardioides sp. Identified as ATD6 and registered with the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st, 1st East, 1st Street, Tsukuba, Ibaraki Prefecture) on October 29, 2008 (FERM P-21710) As deposited.

Nocardioides sp. ATD6 or Nocardioides sp. Degradation of melamine by a complex microbial system containing ATD6 Nocardioides sp. ATD6, betaproteobacterium CDB21, and Bradyrizobium japonicam CSB1 were individually plated at 30 ° C. using R2A agar medium, and contained 100 ml containing 20 ml of liquid medium (melamine concentration 5 mg / L) shown in Table 1. The Erlenmeyer flask was inoculated, swirled in the dark at 30 ° C., 210 rpm, and the concentrations of melamine, ammelin, ammelide, and cyanuric acid were measured over time using HPLC. The results are shown in FIG. FIG. 3 is a graph showing changes over time of melamine and its metabolites during the melamine-degrading bacterium and complex microbial degradation of melamine in Example 3. In FIG. 3, the concentration of melamine and metabolites shows the average value of 3 samples, and the blank shows the result of a flask not inoculated with microorganisms. Nocardioides sp. In the medium inoculated with ATD6 alone, an increase and accumulation of ammelin, ammelide and cyanuric acid concentrations were observed with a decrease in melamine concentration. On the other hand, Nocardioides sp. In the medium inoculated with ATD6 and betaproteobacterium CDB21, it was observed that the concentration of ammelin, ammelide, and cyanuric acid once increased with a decrease in melamine concentration, decreased with the passage of culture time, and further disappeared. Moreover, the period until each substance disappears was shortened by coexisting Bradyrizobium japonicum CSB1. From these facts, Nocardioides sp. A complex microbial system consisting of ATD6, betaproteobacterium CDB21, and Bradyrizobium japonicum CSB1 was found to have the ability to fully degrade melamine, ammelin, ammelide, and cyanuric acid. Beta proteobacteria CDB21 alone or a complex microbial system consisting only of betaproteobacteria CDB21 and Bradyrizobium japonicum CSB1 did not degrade melamine.

  The present invention relates to a novel bacterium that decomposes aminotriazine-based compounds mainly composed of melamine, which is manufactured and used for a wide range of applications such as resins, paints, fertilizers, herbicides and fungicides, and a complex microbial system containing the bacteria, A method for decomposing aminotriazine-based compounds mainly composed of melamine using the bacterium or the complex microbial system, and a melamine contained in fertilizer, feed, industrial waste, factory wastewater, environmental water, and soil Decomposing and detoxifying aminotriazine-based compounds, mainly contributing to the purification of materials and the environment, and enabling the provision of clean water and soil by removing contaminants and residues And has industrial applicability.

FIG. 1 is a schematic view of an apparatus used for accumulation of novel melamine degrading bacteria of Example 1. FIG. 2 is a graph showing fluctuations in melamine concentration during the accumulation process of novel melamine-degrading bacteria in Example 1. FIG. 3 is a graph showing the change over time of melamine and its metabolites during the melamine-degrading bacterium in Example 3 and the degradation process of melamine by the complex microorganism system.

Explanation of symbols

1 soil layer tank 2 reflux liquid 3 pump

[Explanation of Sequence Listing]
Sequence number 1 of a sequence table is Nocardioides sp. It is 1474 bases of the 16S rRNA gene of ATD6 (Nocardioides sp. ATD6).
Sequence number 2 of a sequence table is 1488 bases of 16S rRNA gene of beta proteobacterium CDB21 (beta proteobacterium CDB21) based on this invention.
Sequence number 3 of a sequence table is 1462 bases of 16S rRNA gene of Bradyrhizobium japonicum CSB1 (Bradyrhizobium japonicum CSB1) based on this invention.

Claims (11)

Nocardioides sp. A bacterium which is ATD6 (Accession No. FERM P-21710). A microbial preparation for degrading an amino-s-triazine compound containing the bacterium according to claim 1 as an active ingredient. The microbial preparation according to claim 2 , wherein the amino-s-triazine compound is melamine, ammelin, or ammelide. A complex microbial preparation for degrading an amino-s-triazine compound, comprising the bacterium according to claim 1 and one or more coexisting bacteria as active ingredients. As coexisting bacteria;
Baie over data-proteobacteria CDB21 (accession number FERM P-19395; accession number FERM BP-10403) bacteria belonging to the beta-proteobacteria is;
The composite microbial formulation of Claim 4 containing this . As coexisting bacteria;
Bed Radi lyso-bi um japonicum CSB1 (Accession No. FERM P-19394; accession number FERM BP-10402) and is Bradyrhizobium bacteria of the genus;
The composite microbial formulation of Claim 4 or 5 containing this . As coexisting bacteria;
A bacterium belonging to the betaproteobacterium which is betaproteobacterium CDB21 (accession number FERM P-19395; accession number FERM BP-10403); and
A bacterium of the genus Bradyrizobium which is Bradyrizobium japonicum CSB1 (Accession No. FERM P-19394; Accession No. FERM BP-10402);
The composite microorganism preparation according to any one of claims 4 to 6, comprising The complex microorganism preparation according to any one of claims 4 to 7 , wherein the amino-s-triazine compound is melamine, ammelin, or ammelide. A method for decomposing an amino-s-triazine compound, comprising using the bacterium according to claim 1 . A method for decomposing an amino-s-triazine compound, comprising using the microbial preparation according to any one of claims 2 and 3 . A method for decomposing an amino-s-triazine compound, comprising using the composite microorganism preparation according to any one of claims 4 to 8 .

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