JP2000270848A - New microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a bacterium belonging to the genus Rhodococcus capable of easily decomposing an iron-cyano complex contained in a wastewater or soil efficiently under a weak alkaline condition, since it has the iron-cyano complex-decomposing activity and useful for an environmental purification, etc. SOLUTION: This new microorganism has a decomposing activity of an iron-cyanate complex such as potassium ferrocyanide. Further, the bacterium belonging to the genus Rhodococcus is Rhodococcucus rhodochrous having decomposing activities of a metal-cyano complex such as a nickel-cyano complex and the iron-cyano complex, and also a new strain, Rhodococcus rhodochrous KR9-4 strain (FERM P-17322) having the cyanide compound-decomposing activity. Further it is preferable to perform a purification treatment of a soil or wastewater containing the cyanide compound by using the Rhodococcus rhodochrous KR9-4 strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to Rhodococcus (Rhococcus).
A bacterium belonging to the genus dococcus, which relates to a novel microorganism capable of decomposing a cyanide compound, in particular, an iron cyano complex under weak alkaline growth conditions.

[0002]

2. Description of the Related Art Cyanides include cyanide and metal silicon.
A group of compounds called ano complexes are included. The cyanation
The product is also called "free cyan" and has the general formula
A_n(CN) _xA represents hydrogen (H), sodium (Na),
Lium (K), ammonium (NH_Four), Calcium (Ca), etc.
And the most toxic form of cyanide.
Further, the metal cyano complex may be a metal salt of hydrogen cyanide and gold.
Genus and excess cyanide ion (CN^-)
And the general formula A_n [M (CN)_x]. Where M is silver (A
g), gold (Au), cadmium (Cd), cobalt (Co), copper (C
u), iron (Fe), nickel (Ni), zinc (Zn), etc.
And dissolved or suspended in the solution.

[0003] The above-mentioned cyanide compound may be contained in industrial wastewater or the like, and is a substance having a property to be removed by a purification treatment. Regarding the form of cyanide present in the solution (drainage), much research has been conducted in the field of wastewater treatment, and it is reported that the form is mostly the free cyanide and the metal cyano complex. . As a method for treating wastewater containing these cyanide compounds, an alkali chlorine method, an ozone oxidation method, an electrolytic oxidation method, a navy blue method (a sparingly soluble complex compound precipitation method), an acid decomposition combustion method, a boiling method (a boiling high temperature combustion method) ,
A wet thermal decomposition method and an adsorption method are known. However, in these treatment methods, highly stable metal cyano complexes, such as iron, cobalt, silver, and gold cyano complexes are not applied, or the reaction conditions are severe, and large-scale equipment is required, There has been a problem that further processing of the product is required. Therefore, a technique for restoring the environment using a biological function, so-called bioremediation, has attracted attention, and a search for microorganisms having the cyanide decomposability has been conducted.

At present, regarding the biodegradation of free cyanide such as potassium cyanide and sodium cyanide, Pseudomonas putida, Pseudomo
nassp., Acinetobacter sp., Fusarium sp., Klebsiell
There are reports that microorganisms such as a sp. have been isolated and identified.

With respect to the metal cyano complex,
Fusarium solani and Trichoderum polysporum have been reported as microorganisms that decompose nickel-cyano complex ([K ₂ Ni (CN) ₄ ]). Fusarium ox is a decomposing bacterium of potassium ferrocyanide ([K ₄ Fe (CN) ₆ ]) which is an iron cyano complex.
ysporum, Scytalidium thermophilum, Penicillium mic
zynski has been reported (Knowles CJ et.
l, Enzyme and MicrobialTechnology 22: 223-231, (199
8)). Incidentally, reports on the decomposition of the metal cyano complex,
All are caused by fungi, and no cases of decomposition by bacteria including actinomycetes have been reported.

[0006]

However, since the iron cyano complex-decomposing bacteria are all classified as fungi, their growth pH range is limited to a weakly acidic range. Here, considering the treatment of the waste water containing the iron cyano complex, under weakly acidic conditions, the iron cyano complex is present as a hardly soluble salt, and therefore, the iron cyano complex in the waste water is In the course of the above-mentioned processing, it is easy to settle in a channel or a processing tank.
Therefore, even if the wastewater treatment is performed using the iron cyano complex-decomposing bacteria, it is considered that the decomposition efficiency is low or an operation such as vigorous stirring is required to increase the efficiency. Therefore, it was substantially difficult to apply the iron cyano complex-decomposing bacteria to the treatment of wastewater containing the iron cyano complex.

Accordingly, an object of the present invention is to provide a novel microorganism capable of decomposing a cyanide compound, particularly an iron cyano complex, in view of the above-mentioned drawbacks.

[0008]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have conducted screening on soil and succeeded in isolating a novel microorganism having the ability to decompose cyanide, and have completed the present invention. Was.

That is, the present invention is a novel microorganism of the genus Rhodococcus capable of decomposing a cyanide,
Specifically, Rhodococcus Rhodocros
hodochrous) KR9-4 strain. Further, the cyan compound is specifically a metal cyano complex, and more specifically, an iron cyano complex. The inventors have isolated a novel microorganism according to the present invention as follows.

1 g of soil collected from various places was sterilized.
The suspension was suspended in 9% physiological saline, and the supernatant was inoculated on a sterilized ferrocyan agar plate medium and kept at 30 ° C.
Then, colonies formed on the ferrocyan agar plate medium were collected, and each colony was again inoculated on a sterilized ferrocyan agar plate medium and cultured at 30 ° C. Colonies formed on the medium were separated. Furthermore, 10 ml of the sterilized ferrocyanide liquid medium was dispensed into a sterilized test tube, and one loopful of the above isolated strain was inoculated and shake-cultured at 30 ° C. for 2 weeks.
(150-170 rpm). Then, the culture was centrifuged to remove the cells, and the supernatant was analyzed for the concentration of ferrocyanin remaining in the supernatant using HPLC, and a strain having a reduced ferrocyanin concentration in the medium after the culture was selected. As a result, a strain having the ability to degrade ferrocyan was obtained, and was designated as strain KR9-4. The composition and preparation method of the ferrocyan agar medium and the ferrocyan liquid medium are as described below.

[0011]

[Table 1]

Table 1 shows the composition of the ferrocyan agar medium. After adjusting the pH value to 7.2 ± 0.2 by mixing the medium composition and, the medium composition sterilized at 121 ° C. for 15 minutes, the medium composition separately sterilized at 121 ° C. for 15 minutes, and filter sterilization were used. The ferrocyan agar medium was prepared by adding the medium composition.

The composition of the ferrocyan liquid medium is as follows. [Composition of ferrocyanide liquid medium] K ₄ [Fe (CN) ₆ ] · 3H ₂ O 1 mM glucose 0.25% (W / V) Na ₂ HPO ₄ 0.35% (W / V) KH ₂ PO ₄ 0.15% (W / V) _{V) MgSO 4 · 7H 2 O} 0.001% (W / V) FeCl 3 · 6H 2 O 0.001% (W / V) MEM vitamin mix (Gibco) 0.1% (W / V)

The pH of the ferrocyanide liquid medium was adjusted to 7.2 ± 0.2 unless otherwise specified.
What was sterilized at 1 ° C. for 15 minutes was used.

Table 2 shows the bacteriological properties of the KR9-4 strain.

[0016]

[Table 2]

The taxonomic position of the KR9-4 strain having the above-mentioned mycological properties is described in the "Barjay's Manual of
Systematic Bacteriology Volume 2 "(Sneat
h, PHA, Mair, NS, Sharpe, ME and Holt, JG:
゛ Bergery's Manual of Systematic Bacteriology ″ Vo
l.2, (1986) Williams & Wilkins.)
Manual of Deterministic Bacteriology 9th Edition "(Holt. JG, Krieg, NR, Sneath, PH
A., Staley, jT and Williams, ST: ゛ Bergery'sManu
al of Determinative Bacteriology ″ Ninth Edition,
(1994) Williams & Wilkins.), `` Experimental Method for Chemical Classification of Microorganisms, First Edition '' (Kazuo Komagata (1982) Society Press) (1987) Nane Publishing). As a result, the KR9-4 strain was Rhodococcus rhodochros (Rhod
ococcus Rhodochrous). However, there is no report that known Rhodococcus rhodochros can decompose a cyanide compound.
The KR9-4 strain is a new strain that is distinguished from known strains. The strain determined to be Rhodococcus rhodochros KR9-4 strain has been deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology under the accession number FERM P-17322.

The Rhodococcus rhodochros KR9-
Four strains were prepared using the following SCDA medium (pH 7.3 ± 0.2, 25
C., DIFCO) and YG medium (pH 7.2 ± 0.2) at a temperature of 30.degree.

[Composition of SCDA medium] Bacto Tryptone 15g (Pancreatic Digest of Casein) Bacto Soytone 5g (Papaic Digest of Soybean Meal) NaCl 5g Bacto Agar 15g

[Composition of YG medium] Yeast Extract 0.1% Glucose 0.1% K ₂ HPO ₄ 0.03% KH ₂ PO ₄ 0.02% MgSO ₄・ 7H ₂ O 0.02% Agar 1.5%

Rhodococcus rhodochros according to the present invention
KR9-4 strain is a novel microorganism classified as a bacterium. In general, bacteria grow in a neutral to weakly alkaline pH range, and the inventors have found that the Rhodococcus rhodochros KR9-4 strain also has a neutral to weakly alkaline growth by a growth test described below. Under these conditions, it is clear that the iron cyano complex can be decomposed and grown. As described above, the Rhodococcus rhodochros KR9-4 strain is capable of decomposing a cyanide compound, particularly an iron cyano complex in a neutral to weakly alkaline pH range. It can be said that. When this strain is used for treating wastewater containing a cyanide compound, the hardly soluble iron cyano complex can be decomposed in a state of being dissolved in the wastewater, so that the wastewater can be purified efficiently.

It is known that most of the cyanide existing in soil is ferrocyan which is a complex of iron and cyanide. Therefore, the Rhodococcus rhodochros KR9-4 strain of the present invention is used. By decomposing and removing ferrocyanin in the soil, the soil contaminated with cyanide can be purified.

[0023]

Embodiments of the present invention will be described below.

[Example 1] Rhodococcus rhodochros
Measurement of ferrocyanide resolution of KR9-4 strain In a sterilized test tube, the sterilized ferrocyanide liquid medium
Dispense 10 ml and add Rhodococcus rhodochros KR
One loopful of 9-4 strains was inoculated. Then, the test tube inoculated with the strain is shake-cultured (150 to 170 rpm) at 30 ° C., and ferrocyanide (all cyan) in the medium is removed at regular intervals.
The content was measured. Here, ferrocyan in the culture solution
(Total cyanide) content is described in `` Method for measuring CN content in low-quality soil.
(Circumferential water pipe No. 127, paragraph 14.2) '' described in `` Cyan Distillation Pre-operation Method (Heat Distillation Method for All Cyan in Soil) '', obtained by distilling the entire culture (culture solution, cells). Distillate was collected by a fully automatic cyanometer (T-analyte Yanaco T-analyzer).
CN501). The result is shown in FIG.

The decomposition rate of ferrocyanide was determined by the following equation. Ferrocyan (total cyanide) decomposition rate = Ferrocyan after culture
(Total cyan) content / ferrocyan (total cyan) content before culture x 100 (%)

As shown in FIG. 1, the Rhodococcus rhodochros KR9-4 strain reduced ferrocyanin in a liquid medium by 12.2% and 27.27% after 2, 4 and 7 weeks from the start of culture.
It turns out that it decomposes by 2% and 30.8%.

[Example 2] Dependence of ferrocyanase decomposability on culture medium pH The Rhodococcus rhodochros KR9-4 was prepared using the ferrocyanide liquid medium prepared at various pH values and sterilized.
The pH dependence of ferrocyanide resolution of the strain was examined. Specifically, pH 7.0, 8.0, 9.0, 10.
After adjusting to 0, 11.0 or 12.0, 10 ml of sterilized ferrocyanide liquid medium was dispensed into each sterilized test tube, and one loopful of Rhodococcus rhodochros KR9-4 strain was inoculated into each test tube. Sections A to F were used. For each of these six experimental plots, shake culture (150-1
70 rpm) and ferrocyanin in the medium at regular intervals.
The (total cyan) content and the pH of the medium were measured. The ferrocyanide (total cyan) content in the culture solution was determined according to [Example 1].
The measurement was performed using the same method as described above. Hereinafter, the time-dependent change in the ferrocyanin content in the medium is shown in FIG. 2, and the time-dependent change in the medium pH is shown in FIG.

As is clear from FIG. 2, the decomposition of ferrocyanide by the Rhodococcus rhodochros KR9-4 strain is favorable in the experimental plots A to D, that is, in the weak alkaline pH condition. Notable in.
As is clear from FIG. 3, the Rhodococcus rhodochros KR9-4 strain degrades ferrocyanin in a neutral to weakly alkaline region. At this time, it is considered that ferrocyan in the medium exists in a state of being dissolved in the medium.

As described above, the Rhodococcus rhodochros KR9-4 strain can decompose solubilized ferrocyanin, and thus is considered to be suitable as a bacterial strain used for purification treatment of industrial wastewater and the like.

[Brief description of the drawings]

FIG. 1 is a graph showing the time course of ferrocyanide degradation by Rhodococcus rhodochros KR9-4 strain.

FIG. 2 is a graph showing the pH dependence of ferrocyanide degradation by Rhodococcus rhodochros KR9-4 strain.

FIG. 3 is a graph showing the medium pH during the culturing of Rhodococcus rhodochros KR9-4 strain

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B065 AA45X AC12 AC20 BA23 BB01 BC01 BC02 BC03 BC26 BC31 CA56 4D040 DD03 DD14

Claims (4)

[Claims] 1. A bacterium belonging to the genus Rhodococcus which has iron cyano complex decomposability. 2. Rhodococcus rhodochrous having a metal cyano complex decomposability. 3. Rhodococcus rhodochrous having iron cyano complex decomposability. 4. A new strain Rhodococcus Rhodochrous KR9- capable of decomposing cyanide.
4 shares.