JP5860243B2 - Solid medium plate and method for screening cyanide-degrading microorganisms using the plate - Google Patents

Description

  The present invention relates to a solid medium plate and a screening method for cyanide-degrading microorganisms using the same. More specifically, a solid medium plate in which solid fine particles of an iron cyano complex are dispersed in a medium, a method for screening a cyanide-decomposing microorganism by forming a halo on the plate, and the cyanide-decomposing microorganism, and containing a cyanide compound The present invention relates to a method for purifying soil, drainage or groundwater.

  Cyanide has been designated as a second-type toxic substance in the Soil Contamination Countermeasures Law enacted in 2003, and recently, bioremediation by microorganisms has been developed as a purification technique. In order to improve the purification performance essentially by bioremediation, not only the nutrients are added to the system to activate the resident microorganisms, but also the microorganisms with high degradation performance are acquired and introduced into the system. It is effective.

  As a method for obtaining cyanide-degrading microorganisms, a culture in which soil is suspended and accumulated in a medium containing a cyanide compound such as a metal cyano complex or a nitrile compound as a nitrogen source is cultured on an agar plate containing the cyanide compound, A method for selecting high-performance microorganisms by culturing microorganisms forming a single colony in a medium containing cyanide for 10 to 40 days and then measuring the residual total cyanide concentration to evaluate cyanide resolution (Patent Document) 1: Japanese Patent Laid-Open No. 2001-245655) and colonies formed by culturing the supernatant of physiological saline in which soil is suspended on a ferrocyan agar plate, then inoculating the ferrocyan liquid medium and inoculating 30 A performance evaluation test was conducted to analyze the ferrocyan concentration by HPLC of the culture broth cultured for 2 weeks at ℃. Cleaning method of (Patent Document 2: JP 2000-270853 JP) are known.

  However, neither of Patent Documents 1 and 2 is a method that can directly determine the presence or absence or superiority or inferiority of cyanide degradation performance of a target microorganism on a plate. Screening for microorganisms having high degradation performance is performed individually for all colonies to be compared. Cyanide concentration must be measured by liquid culture, which is time consuming and labor intensive.

  As a method for detecting a microorganism having a cyanide decomposition ability other than the method of measuring the concentration by liquid culture, a method for detecting a rhodanase (thiosulfate transferase) gene involved in cyanogen purification from a nucleic acid in the microorganism (Patent Document 3) : JP 2008-283890 A). Although this method is effective for judging the suitability for purification of microorganisms and monitoring the purification activity in the purification process, it involves steps such as gene preparation and amplification, and detection of amplification products, which takes time and effort. Therefore, screening for cyanide-decomposing microorganisms cannot be performed efficiently.

JP 2001-245655 A JP 2000-270853 A JP 2008-283890 A

As described above, since all of the conventional methods for obtaining microorganisms effective for decomposing cyanide compounds require time and labor, establishment of a technique capable of efficiently obtaining cyanide-decomposing microorganisms has been desired.
One of the objects of the present invention is to provide a solid medium plate capable of efficiently obtaining cyanide-degrading microorganisms and a screening method for cyanide-degrading microorganisms using the same. Another object of the present invention is to provide a cyanide-decomposing microorganism and a method for purifying cyanide-containing soil, wastewater or groundwater using the microorganism.

  As a result of intensive research to solve the above-mentioned problems, the inventors applied and cultured microorganisms on a solid medium plate in which solid fine particles of a dark blue iron cyano complex were dispersed in a medium. The inventors have found that cyanide-decomposing microorganisms can be screened by observing the halo (clearzone) of the plate formed by decomposition of the complex, thereby completing the present invention.

That is, the present invention includes the following [1] to [5] solid medium plate, [6] cyanide-decomposing microorganism screening method, [7] to [10] cyanide-decomposing microorganism, and [11] to [11] 12] of the method for purifying cyanide-containing soil, drainage or groundwater.
[1] A solid medium plate, wherein solid fine particles of an iron cyano complex are dispersed in and / or on a solid medium.
[2] The solid medium plate according to [1], wherein a solid medium in which solid fine particles of the iron cyano complex are dispersed is laminated on a support medium not including the iron cyano complex.
[3] The iron cyano complex is Fe (III) ₄ [Fe (II) (CN) ₆ ] ₃ , Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ , and Fe (II) ₂ [ The solid medium plate according to [1] or [2] above, which is one or more selected from Fe (II) (CN) ₆ ].
[4] The solid medium plate according to any one of [1] to [3] above, wherein the concentration of the solid fine particles of the iron cyano complex is 1 to 10 g / L.
[5] The solid medium plate according to any one of [1] to [4], wherein the solid medium in which solid fine particles of the iron cyano complex are dispersed has a thickness of 0.1 to 3 mm.
[6] A step of applying a microorganism on a solid medium in which solid fine particles of the iron cyano complex of the solid medium plate according to any one of [1] to [5] are dispersed, a step of culturing the microorganism, And a screening method for cyanide-degrading microorganisms, comprising a step of selecting microorganisms forming halo.
[7] A cyanide-degrading microorganism selected by the screening method described in [6] above.
[8] The cyanide-decomposing microorganism according to [7], which belongs to the genus Rhodococcus, Bacillus, Arthrobacter, Klebsiella, or Acinetobacter.
[9] A cyanide-degrading microorganism which is Rhodococcus aetherivorans BH0213 strain (Accession number: FERM P-21791) or Rhodococcus aetherivorans BH0234 strain (FERM P-21719).
[10] Bacillus megaterium BH040101 strain (Accession number: FERM P-21790), Bacillus megaterium BH040204 strain (Accession number: FERM P-21722), or Bacillus subtilis (Bacillus subtilis) Cyanide-degrading microorganism which is BH040102 strain (Accession number: FERM P-21721).
[11] A method for purifying cyanide-containing soil, drainage or groundwater, which comprises adding at least one of the cyanide-decomposing microorganisms according to any one of [7] to [10] to the cyanide-containing soil, drainage or groundwater. .

  If the screening method using the solid medium plate of the present invention is used, cyanide-degrading microorganisms can be efficiently obtained. In addition, cyanide-containing soil, drainage water or groundwater can be purified by using the screened cyanide-decomposing microorganism.

Cyan non-degrading bacteria Bacillus subtilis SD901 strain (left) and cyan-degrading bacteria Arthrobacter sp. It is the photograph of the blue plate which culture | cultivated 5 strains (right). It is a graph which shows the relationship between the halo diameter of a halo forming microbe, and elution total cyanide decomposition density | concentration. It is a graph which shows the time-dependent change of the elution total cyan density | concentration in soil at the time of adding the cyanide decomposition microorganisms screened by Example 2 to cyanide containing soil.

  Hereinafter, the present invention will be described in detail.

[Solid medium plate]
The solid medium plate of the present invention (hereinafter sometimes referred to as “hello plate”, “blue medium”, “blue plate”) has solid fine particles of dark blue iron cyano complex in the solid medium and / or solid medium. It is characterized by being dispersed above, and mainly comprises a medium support, solid fine particles of an iron cyano complex, and a nutrient component.

  The medium support is not particularly limited as long as it has a function of solidifying a medium solution in which components are added to water without inhibiting the growth of microorganisms. For example, agar, gelatin, gellan gum, gluco Mannan or the like can be used, and preferably agar can be used at a concentration of 10 to 30 g / L, more preferably 15 to 20 g / L.

The solid fine particles of iron cyano complex used in the present invention are precipitates of iron cyano complex that can be dispersed without dissolving in the above-mentioned medium support, and are generally named bitumen, Berlin blue, turnable blue, Prussian blue. , Milly blue, Chinese blue and Paris blue, Fe (III) ₄ [Fe (II) (CN) ₆ ] ₃ , Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ , Fe (II) ₂ It is represented by a composition formula of [Fe (II) (CN) ₆ ].

In the present invention, Fe (III) ₄ [Fe (II) (CN) ₆ ] ₃ , Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ , and Fe (II) ₂ [Fe (II) ( One or more selected from CN) ₆ ] can be used at a concentration of 1 to 10 g / L in the solid medium. When the concentration of the solid fine particles of the iron cyano complex is less than 1 g / L, the blue color of the dispersed fine particles becomes light. When the concentration of the solid fine particles exceeds 10 g / L, the blue color of the dispersed fine particles becomes dark. It becomes difficult to discriminate.

As the solid fine particles of the iron cyano complex, a commercially available product can be obtained. For example, 3K ₄ [Fe (II) (CN) ₆ ] + 2Fe ₂ (SO ₄ ) ₃ → Fe (III) ₄ [Fe (II) ( CN) ₆ ] ₃ ↓ + 6K ₂ SO ₄ , 2K ₃ [Fe (III) (CN) ₆ ] + 3FeSO ₄ .7H ₂ O → Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ ↓ + 3K ₂ SO ₄ + 21H ₂ O, K ₄ [Fe (II) (CN) ₆ ] + 2FeSO ₄ .7H ₂ O → Fe (II) ₂ [Fe (II) (CN) ₆ ] ↓ + 2K ₂ SO ₄ + 14H ₂ O It is preferable to use an iron cyano complex added with excess iron ions and precipitated at a concentration of 1 to 10 g / L, preferably 3 to 7 g / L, because it can be sterilized in a solution state before the precipitation reaction. .

Nutrient components include, for example, nutrient broth, peptone, yeast extract, meat extract, yeast extract, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, urea, glucose, sucrose, fructose, phosphoric acid 0.1-50 g of phosphates such as potassium monohydrogen phosphate and potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, calcium acetate, manganese chloride, biotin, thiamine, etc. / L, preferably at a concentration of 5 to 15 g / L.
A liquid in which each of the above components is added and dispersed is prepared as a solid medium liquid.

  Examples of the sterilization of the solid medium include, but are not limited to, methods such as autoclave sterilization and sterilization filter filtration. Each medium component can be sterilized by all components, all components divided, or partially divided. Examples of solid medium liquid sterilization and halo plate production methods are shown below.

  The shape of the halo plate in which the solid fine particles of the iron cyano complex are dispersed is not particularly limited. For example, the sterilized solid medium solution aseptically as described above in a sterile petri dish is, for example, 0.1 to 3 mm in thickness, preferably 1 It can be added to ˜2 mm and allowed to harden at room temperature. If the thickness is less than 0.1 mm, the plate may be easily damaged when a liquid containing microorganisms is applied to the plate. If the thickness is more than 3 mm, it is dispersed in a medium separated from the surface layer on which the microorganisms are growing. The blue color of the fine iron cyano complex particles makes it difficult to confirm halo formation (color loss) on the surface.

  When the thickness of the plate is small, the strength of the halo plate is reinforced as a two-layer structure in which the halo plate is laminated on a support medium obtained by solidifying a sterilized solution by adding a medium support not containing solid particles of iron cyano complex to water. can do. In addition, since the solid medium in which the solid fine particles of the iron cyano complex are dispersed has a deep blue color, a colorless or white medium support in which the solid fine particles of the iron cyano complex are not dispersed is laminated on the lower layer. Compared to the configuration, it is easier to check for halos.

  As another method for preparing a hello plate, a sterilized iron cyano solution is prepared on a surface obtained by aseptically adding a medium support and nutrient components to water and adding it to a sterile petri dish and allowing it to cool and solidify. The suspension of solid fine particles of the complex can be suspended aseptically and spread evenly with a sterilized rod or the like to form a precipitate layer.

[Screening method]
Screening performed on a halo plate can be carried out by directly applying a water suspension of a soil containing microorganisms to the halo plate and cultivating the culture solution once cultured in a plate medium and / or a liquid medium as appropriate. It can also be diluted with a solution and applied to a halo plate for culturing.

The medium components of the plate medium and / or liquid medium can be selected from cyan compounds and nutrient sources as appropriate, but preferably potassium ferricyanide (K ₃ [Fe (III) (CN) ₆ ]) as the only nitrogen source. And a cyanide compound such as potassium ferrocyanide (K ₄ [Fe (II) (CN) ₆ ]) is added in an amount of 0.1 to 10 g / L, in addition to carbon sources such as glucose, sucrose, fructose, and KH ₂ PO _4. , NaH ₂ PO ₄ , Na ₂ HPO ₄ , K ₂ HPO ₄ and other phosphates, and a minimal medium supplemented with minerals such as MgSO ₄ , FeSO ₄ and MnSO ₄ are used.

  From the viewpoint of sufficiently growing the microorganism, for example, the temperature of the halo plate can be 30 to 40 ° C., and the time can be 1 to 7 days, but is not limited thereto. Selection of colonies grown on a halo plate can be performed in the order of formation of halos, starting from the largest halo.

[Evaluation of cyanide resolution of selected strains]
Evaluation of the cyanide-decomposing ability of the selected strain can be carried out by confirming the decrease amount of the cyanide after inoculating the culture solution of the strain on soil or liquid medium containing the cyanide and cultivating it. Although the culture solution of the inoculated strain can be grown on an appropriate nutrient medium, from the viewpoint of comparing the superiority and inferiority of multiple strains, the number of bacteria in the culture solution is measured with an appropriate nutrient medium plate, and the final concentration after addition is determined. Can be aligned. The final concentration can be set to an arbitrary numerical value of 10 ⁸ to 10 ¹⁰ pieces / g, for example.

  As the cyanide compound used for the evaluation culture, for example, a metal cyanide complex such as an iron cyano complex, a copper cyano complex or a nickel cyano complex, an inorganic cyanide such as potassium cyanide or sodium cyanide, an organic cyanide compound containing a nitrile group, or the like Two or more kinds can be used in combination at a concentration of 0.1 to 50 g / L. Nutritional components include, for example, nutrient broth, peptone, yeast extract, meat extract, ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, urea, glucose, sucrose, fructose, potassium monohydrogen phosphate , Phosphates such as potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, calcium acetate, manganese chloride, biotin, thiamine, etc., in combination of one or more, 0.1 to 50 g / L, Preferably, it can be used at a concentration of 5 to 15 g / L. Evaluation culture conditions can be performed, for example, at a temperature of 30 to 40 ° C. and a time of 1 to 60 days, but are not limited thereto.

  Cyanide concentration is the soil elution total cyanide concentration (elution total cyanide) measurement method used in the soil contamination countermeasure method in the case of soil evaluation culture samples. It can be analyzed by the “measurement method”. The unit of elution total cyanide is mg / L, and the soil contamination countermeasure law designation standard is <0.1. In the case of a culture sample in a liquid medium, the total cyan concentration can be measured by a pyridine-pyrazolone spectrophotometric method or a pyridinecarboxylic acid-pyrazolone spectrophotometric method.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention does not receive any limitation by these description.

Example 1 Halo Formation Experiment of Solid Medium Plate Both components were added and dispersed in water so that the nutrient broth (NB) was 8.9 g / L and the agar was 22 g / L. A solution sterilized at 20 ° C. for 20 minutes (solution A), a 11.1 g / L aqueous solution of K ₃ [Fe (III) (CN) ₆ ] sterilized with a 0.45 μm sterilization filter, and a concentration of 14.1 g / A solution (Liquid B) containing 10.0 g / L of Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ precipitate was prepared by mixing equal amounts of L FeSO ₄ .7H ₂ O aqueous solution. Thereafter, the B liquid and water are aseptically added to the A liquid before cooling and solidifying at the ratios shown in Table 1, and mixed to obtain Fe (II) ₃ [Fe (III) (CN) ₆ ] _2. Medium solutions (= blue medium) containing precipitates at concentrations of 1 g / L, 5 g / L, and 10 g / L were prepared.

Furthermore, after adding and dispersing in water so that the agar is 20 g / L, a liquid (support medium) sterilized by autoclaving at 121 ° C. for 20 minutes is prepared, and the support medium before solidification is sterilized with a diameter of 90 mm. Each volume listed in Table 2 was aseptically dispensed into a petri dish and allowed to stand at room temperature to solidify, then each volume of blue medium listed in Table 2 was dispensed to each concentration of blue medium before solidification. Left to solidify, and Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ concentration is 1 g / L, 5 g / L, 10 g / L, blue medium with a thickness of 1 mm, 2 mm, 3 mm A total of nine types of solid medium plates (blue plates) having layers were prepared.

  Next, cyanogen-degrading bacteria Arthrobacter sp. 5 shares (Accession No .: FERM BP-11019, on October 18, 2007, deposited domestically as Accession No. FERM P-21400 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center. On April 16, it was deposited internationally at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-11019.) And the cyan non-degrading bacteria Bacillus subtilis SD901 strain (accession number: FERM BP-7666) , Deposited on August 7, 2000 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as Life Research Deposit No. FERM P-17989, and on July 16, 2001, the National Institute of Advanced Industrial Science and Technology patent biological deposit Internationally deposited at the center under the deposit number FERM BP-7666.) Suspension suspended in 10 mL of sterilized water was cultured on NB agar medium (composition: NB 8 g / L, agar 20 g / L) for 24 hours at 35 ° C. And then incubated at 35 ° C. for 5 days.

In the blue non-degrading bacterium Bacillus subtilis SD901, formation of halo was not confirmed in any blue plate, but the cyan-degrading bacterium Arthrobacter sp. In 5 strains, the thickness was 1 mm and the Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ concentration was 1 g / L, 5 g / L, 10 g / L, and the thickness was 2 mm and Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ Formation of halo was confirmed on five types of blue plates with concentrations of 1 g / L and 5 g / L. Table 3 shows the results of evaluating the formation state of the halo according to the following criteria.
A: A halo is formed and the discrimination is good.
○: What forms a halo,
X: A halo is not formed.
In particular, the blue plate with a thickness of 1 mm and Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ concentration of 5 g / L has the clear contrast between the color tone of the plate and the color loss of the halo, and the best discrimination (See FIG. 1).

In the plate in which halo formation could not be confirmed in this experiment, the growth of the fungus was confirmed, but since the thickness and the concentration of Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ are large, It is considered that Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ exhibiting a deep blue color could not be decolored to a level where halo could be confirmed.

Example 2: Screening and cyanide compounds resolution evaluation soil samples 1g strains by solid media _{plates, KH 2 PO 4 0.7g / L} , Na 2 HPO 4 1.1g / L, MgSO 4 · 7H 2 O 0.5g / L, FeSO ₄ · 7H ₂ O 0.01 g / L, Glucose 3 g / L, K ₃ [Fe (III) (CN) ₆ ] 1 g / L medium solution (cyan complex-containing minimum medium) 100 mL Was added to a 500 mL flask and cultured at 35 ° C. for 3 days. 1 mL of this culture broth was collected in a cyanogen complex-containing minimal medium and cultured for 3 days at 35 ° C. and appropriately diluted with sterile water, and 0.1 mL of Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ was applied to a blue plate having a concentration of 5 g / L and cultured at 35 ° C. for 5 days.

  As a result of blue plate culture, as shown in Table 4, 10 strains of colonies that formed halos having a diameter of 2 to 6 mm were obtained. The obtained 10 strains of cyanide degradation were evaluated by adding each strain to the cyanide-containing soil.

First, each strain was cultured on an NB agar medium (composition: NB 8 g / L, agar 20 g / L) in an 18 mm diameter test tube at 35 ° C. for 24 hours, and 1 mL of a suspension suspended in 10 mL of sterilized water was added to peptone. 10 g / L, yeast extract 5 g / L, K ₂ HPO ₄ 5 g / L, MgSO ₄ .7H ₂ O 0.5 g / L, glucose 20 g / L, added to a 500 mL flask containing 35 mL of medium solution, and 35 ° C. Then, the culture broth was cultured for 1 day, and the appropriately diluted culture broth was cultured on an NB agar plate at 35 ° C. for 24 hours, and the number of bacteria in the culture broth was measured.

Next, 100 g of soil with an elution total cyanide (CN) of 0.9 mg / L (specific gravity: 1.7 kg / L, water content: 25% by mass) is put into a 100 mL plastic container with a lid, and the number of bacteria is 1 × 10 as the final concentration. Each component was added and mixed so that ⁸ pieces / g, yeast extract was 0.1% by mass and moisture was 30% by mass, and the total CN eluted from each soil after standing at 35 ° C. for 14 days was measured. The results are shown in Table 4 and FIG.

  In all of the obtained bacteria, the elution total cyanide decreased and the resolution was confirmed. Furthermore, the size of the formed halo and the total elution of cyanide have a positive correlation, and it was confirmed that halo formation on the blue plate is effective for screening for obtaining cyanide-degrading bacteria.

  The identification results of 5 types of cyanide-degrading bacteria (BH0213 strain, BH0234 strain, BH040101 strain, BH040102 strain, BH040204 strain) from which good results were obtained by the above screening method are as follows.

[Cyanide-degrading microorganism 1 / BH0213 strain]
Morphological observation and physiological properties of the cyanide-degrading microorganism BH0213 strain isolated according to the present invention are as follows.
Form: Polymorphic Neisseria gonorrhoeae
Gram staining: positive,
Spore: Not formed,
Mobility: None,
Attitude toward oxygen: aerobic,
Oxidase: negative,
Catalase: positive,
OF (Oxidation Fermentation) test: Does not grow on test medium,
Settlement color: Orange.
In addition, this strain was identified by analyzing the base sequence of 16S rRNA region DNA amplified by PCR using ABI PRISM 310 Genetic Analyzer [Applied Biosystems], and converting the obtained sequence into the international base sequence database (DDBJ / EMBL). / GenBank) and the homologous search with the database of MicroSeq Analysis Softwar [Applied Biosystems] and the phylogenetic tree with related species was created by the proximity binding method (NJ method) using MicroSeq Analysis Softwar. I went.
As a result, this strain was confirmed to be related to Rhodococcus aetherivorans with a base sequence mismatch rate of 0.09%.
This strain was named Rhodococcus aetherivorans BH0213 and was deposited on March 19, 2009 at the National Institute of Advanced Industrial Science and Technology as a deposit number FERM P-21791. .

[Cyanide-decomposing microorganism 2 / BH0234 strain]
Morphological observation and physiological properties of the cyanide-degrading microorganism BH0234 strain isolated according to the present invention are as follows.
Form: Polymorphic Neisseria gonorrhoeae
Gram staining: positive,
Spore: Not formed,
Mobility: None,
Attitude toward oxygen: aerobic,
Oxidase: negative,
Catalase: positive,
OF (Oxidation Fermentation) test: Does not grow on test medium,
Settlement color: Orange.
In addition, this strain was identified by analyzing the base sequence of 16S rRNA region DNA amplified by PCR using ABI PRISM 310 Genetic Analyzer [Applied Biosystems], and converting the obtained sequence into the international base sequence database (DDBJ / EMBL). / GenBank) and the homologous search with the database of MicroSeq Analysis Softwar [Applied Biosystems] and the phylogenetic tree with related species was created by the proximity binding method (NJ method) using MicroSeq Analysis Softwar. I went.
As a result, this strain was confirmed to be closely related to Rhodococcus aetherivorans with a base sequence mismatch rate of 0.07%.
This strain is named Rhodococcus aetherivorans BH0234 and deposited on November 7, 2008 at the National Institute of Advanced Industrial Science and Technology Patent Biology Depositary No. FERM P-21719 .

[Cyanide-decomposing microorganism 3 / BH040101 strain]
Morphological observation and physiological properties of the cyanide-degrading microorganism BH040101 strain isolated according to the present invention are as follows.
Form: Neisseria gonorrhoeae
Gram staining: positive,
Spores: Form (shape: oval, position: sub-vertical, spore sac: non-bulged),
Mobility: Yes,
Attitude toward oxygen: aerobic,
Catalase: positive,
Settlement color: Yellowish.
In addition, this strain was identified by analyzing the base sequence of 16S rRNA region DNA amplified by PCR using ABI PRISM 310 Genetic Analyzer [Applied Biosystems], and converting the obtained sequence into the international base sequence database (DDBJ / EMBL). / GenBank) and the homologous search with the database of MicroSeq Analysis Softwar [Applied Biosystems] and the phylogenetic tree with related species was created by the proximity binding method (NJ method) using MicroSeq Analysis Softwar. I went.
As a result, it was confirmed that this strain was closely related to Bacillus megaterium with a base sequence mismatch rate of 0.16%.
This strain was named Bacillus megaterium BH040101 strain and was deposited with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology on March 19, 2009 as deposit number: FERM P-21790. Yes.

[Cyanide-degrading microorganism 4 / BH040204 strain]
Morphological observation and physiological properties of the cyanide-degrading microorganism BH040204 strain isolated according to the present invention are as follows.
Form: Neisseria gonorrhoeae
Gram staining: positive,
Spores: forming (shape: oval, position: neutral to sub-vertical, spore sac: non-bulging),
Mobility: Yes,
Attitude toward oxygen: aerobic,
Catalase: positive,
Settlement color: Yellowish.
In addition, this strain was identified by analyzing the base sequence of 16S rRNA region DNA amplified by PCR using ABI PRISM 310 Genetic Analyzer [Applied Biosystems], and converting the obtained sequence into the international base sequence database (DDBJ / EMBL). / GenBank) and the homologous search with the database of MicroSeq Analysis Softwar [Applied Biosystems] and the phylogenetic tree with related species was created by the proximity binding method (NJ method) using MicroSeq Analysis Softwar. I went.
As a result, this strain was confirmed to have a close relationship to Bacillus megaterium with a base sequence mismatch rate of 0.13%.
This strain is named Bacillus megaterium BH040204 strain, and deposited on November 7, 2008 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-21722. .

[Cyanide-decomposing microorganism 5 / BH040102 strain]
Morphological observation and physiological properties of the cyanide-degrading microorganism BH040102 strain isolated according to the present invention are as follows.
Form: Neisseria gonorrhoeae
Gram staining: positive,
Spores: Form (shape: oval, position: sub-vertical, spore sac: non-bulged),
Mobility: Yes,
Attitude toward oxygen: aerobic,
Catalase: positive,
Settlement color: Does not produce characteristic settlement pigments.
In addition, this strain was identified by analyzing the base sequence of 16S rRNA region DNA amplified by PCR using ABI PRISM 310 Genetic Analyzer [Applied Biosystems], and converting the obtained sequence into the international base sequence database (DDBJ / EMBL). / GenBank) and the homologous search with the database of MicroSeq Analysis Softwar [Applied Biosystems] and the phylogenetic tree with related species was created by the proximity binding method (NJ method) using MicroSeq Analysis Softwar. I went.
As a result, this strain was confirmed to be closely related to Bacillus subtilis with a base sequence mismatch rate of 0.02%.
This strain was named Bacillus subtilis BH040102 strain and deposited on November 7, 2008 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary under the accession number FERM P-21721. Yes.

Example 3 Soil Purification Evaluation of Cyanide-Decomposing Microorganisms Cyanide-degrading microorganisms BH0213, BH0234, BH040101, BH040204, and BH040102 screened according to Example 2 were prepared from NB agar medium (composition: 18 mm in diameter). NB 8 g / L, agar 20 g / L), cultured at 35 ° C. for 24 hours, suspended in 10 mL of sterilized water, 1 mL of suspension was prepared by adding 10 g / L of peptone, 5 g / L of yeast extract, and 5 g / L of K ₂ HPO _4. , A culture solution that was added to a 500 mL flask containing 100 mL of a medium solution of MgSO ₄ .7H ₂ O 0.5 g / L and glucose 20 g / L and cultured at 35 ° C. for 1 day, and an appropriately diluted culture solution was prepared. The cells were cultured on an NB agar plate at 35 ° C. for 24 hours, and the number of bacteria in the culture was measured.

Next, 100 g of soil with a total elution CN of 0.9 mg / L (specific gravity: 1.7 kg / L, water content: 25% by mass) is placed in a 100 mL plastic container with a lid, and the final concentration is 1 × 10 ⁸ cells / g. In addition, each component was added and mixed so that the yeast extract was 0.1% by mass and the water content was 30% by mass, and the mixture was allowed to stand at 35 ° C., and each was kept on the 14th, 21st, 28th, 35th, and 42nd. The soil was sampled and the total CN eluted was measured. The result is shown in FIG.

  On the 14th day for the BH040102 strain, on the 21st day for the BH0213 strain, on the 28th day for the BH040204 strain, on the 42nd day for the BH0234 strain and the BH040101 strain, Reduced to below the detection limit (<0.1 mg / L), confirming that the soil was purified.

  From the above results, it was suggested that the cyanide-decomposing microorganism-containing soil, drainage, or groundwater can be purified by adding the screened cyanide-decomposing microorganism to the cyanide-containing soil, drainage, or groundwater.

Claims (7)

A solid medium plate comprising a solid medium having a thickness of 1 to 2 mm in which solid fine particles comprising an iron cyano complex are dispersed at a concentration of 3 to 7 g / L. The solid medium plate according to claim 1 , comprising a support medium that does not contain an iron cyano complex, and the solid medium stacked thereon . The iron cyano complex contains Fe (III) ₄ [Fe (II) (CN) ₆ ] ₃ , Fe (II) ₃ [Fe (III) (CN) ₆ ] ₂ , and Fe (II) ₂ [Fe (II 3) The solid medium plate according to claim 1 or 2, which is one or more selected from (CN) ₆ ]. And characterized by comprising the step of subjecting the coated microbial the solid body culture locations solid medium plate according to any one of claims 1 to 3 steps of culturing the microorganism, and a step of selecting a microorganism to form a halo For screening cyanide-degrading microorganisms.   A cyanide-degrading microorganism which is Rhodococcus aetherivorans BH0213 strain (Accession number: FERM P-21179) or Rhodococcus aetherivorans BH0234 strain (FERM P-21719).   Bacillus megaterium BH040101 strain (Accession number: FERM P-21790), Bacillus megaterium BH040204 strain (Accession number: FERM P-21722), or Bacillus subtilis BH040102 strain ( Cyanide-decomposing microorganism having the accession number: FERM P-21721). A method for purifying cyanide-containing soil, drainage or groundwater, wherein at least one of the cyanide-decomposing microorganisms according to claim 5 or 6 is added to cyanide-containing soil, drainage or groundwater.