JP2000270849A - New microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a bacterium belonging to the genus Pseudomonas capable of easily and efficiently decomposing a cyanide compound contained in a waste water or soil under a weak alkaline condition, since it has a metal-cyano complex-decomposing activity, and useful for an environmental purification, etc. SOLUTION: This new microorganism has a decomposing activity of an iron-cyano complex such as potassium ferrocyanide. Further, the bacterium belonging to the genus Pseudomonas is Pseudomonas sp 14SA3-2-5B2 (FERM P-17321) having decomposing activity of the cyanide compound. Further it is preferable to perform a purification treatment of an industrial wastewater containing the cyanide compound by using the above strain. The new microorganism is obtained by suspending 1 g soil collected from various places in a sterilized 0.9% saline, inoculating its supernatant on a ferrocyanide agar plate medium, collecting the formed colonies, again inoculating them on the sterilized ferrocyanide agar plate medium, incubating at 30 deg.C and then separating the formed colonies.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a Pseudomonas (P)
The present invention relates to a bacterium belonging to the genus seudomonas, which relates to a novel microorganism having a metal cyano complex, particularly an iron cyano complex decomposability under a weak alkaline growth condition.

[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 oxys is a decomposing bacterium of potassium ferrocyanide ([K ₄ Fe (CN) ₆ ]) which is an iron cyano complex.
porum, Scytalidium thermophilum, Penicillium miczy
nski have been reported (both Knowles CJ et. al,
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 metal cyano complex, particularly an iron cyano complex, in view of the above-mentioned disadvantages.

[0008]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors conducted screening on soil and succeeded in isolating a novel microorganism having a metal cyano complex decomposability, and completed the present invention. I let it.

[0009] That is, the present invention is a novel microorganism of the genus Pseudomonas capable of decomposing a metal cyano complex, and specifically, a microorganism of the genus Pseudomonas sp.
SA3-2-5B2. Further, the metal cyano complex is 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 cells were 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. Further, 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 (1).
50-170 rpm). Then, the culture was centrifuged to remove the cells, and the concentration of ferrocyanin remaining in the supernatant was measured.
Analysis using HPLC was performed to select strains in which the ferrocyanin concentration in the medium was reduced after culturing. As a result, a strain having ferrocyanide resolution was obtained, and this strain was named 14SA3-2-5B2 strain. 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 14SA3-2-5B2 strain.
It writes in.

[0016]

[Table 2]

The taxonomic position of the 14SA3-2-5B2 strain having the above-mentioned mycological properties is described in "Barjay's Manual of Systematic Bacteriology Volume 1" (K
rieg, NR and Holt, JG: ゛ Bergery's Manual of Sys
tematic Bacteriology ″ Vol.1, (1984) Williams & Wilk
ins.) and "Bergey's Manual of Deterministic Bacteriology 9th Edition" (Holt.
JG, Krieg, NR, Sneath, PHA, Staley, jT and W
illiams, ST: ゛ Bergery's Manual of Determinative
Bacteriology ″ Ninth Edition, (1994) Williams & Wil
kins.). As a result, the 14SA3-2-
The 5B2 strain was determined to be a bacterium belonging to the genus Pseudomonas. However, as a result of examining the bacteriological properties of the 14SA3-2-5B2 strain in light of the above literature, there was no known species of the genus Pseudomonas having these properties. Further, there is no report that a known Pseudomonas bacterium has iron cyano complex decomposability. Therefore, the Pseudomonas sp. 14SA3-2-5
B2 is considered to be a new strain that is distinguished from known strains.

In this manner, Pseudomonas sp.
The strain determined to be A3-2-5B2 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-17321.

The above Pseudomonas sp. 14SA3-2-5B
2 is the following SCDA medium (pH 7.3 ± 0.2, 25 ° C.,
It can be cultured using a YG medium (manufactured by DIFCO) and YG medium (pH 7.2 ± 0.2) at a temperature of 30 ° C.

[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%

Pseudomonas sp. 14SA of the present invention
3-2-5B2 is a novel microorganism classified as a bacterium. In general, bacteria grow in a neutral to weakly alkaline pH range.The inventors have found that Pseudomonas sp.14SA3-2-5B2 also has a neutral to weakly alkaline pH by a growth test described below. It has been revealed that the iron cyano complex can be decomposed and grown under growth conditions. As described above, Pseudomonas sp.14SA3-2-5B2 is suitable for use in wastewater treatment by bioremediation because it can decompose metal cyano complexes, particularly iron cyano complexes, in a neutral to weakly alkaline pH range. Microorganisms. When this strain is used for treating wastewater containing a metal cyano complex, 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 form of cyanide in the soil is ferrocyan which is a complex of iron and cyanide. Therefore, Pseudomonas sp.
It is considered that soil contaminated with cyanide can be purified by decomposing and removing ferrocyanin in soil using 4SA3-2-5B2.

Embodiments of the present invention will be described below.

Example 1 Pseudomonas sp. 14SA
3-2-5 Measurement of ferrocyanide resolution of B2 In a sterilized test tube, the sterilized ferrocyanide liquid medium
Dispense 10 ml and add Pseudomonas sp.14SA3-2-
One loopful of 5B2 was inoculated. Then, the test tube inoculated with the strain was shake-cultured (150 to 170 rpm) at 30 ° C., and the ferrocyan (total cyan) content in the medium was measured at regular intervals. Here, the ferrocyanide (total cyan) content in the culture solution is described in `` Method for measuring CN content of low-quality soil (Rinus water pipe 127 No. 14.2) '' `` Cyan distillation pre-operation method (in the soil) According to the method for heating and distillation of all cyan)), a distillate obtained by distilling the entire culture (culture solution, bacterial cells),
Fully-automatic cyan measurement device (T-CN501 manufactured by Anatech Yanaco)
And measured. 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 Pseudomonas s
p.14SA3-2-5B2 degrades ferrocyanin in the liquid medium by 12.2% two weeks after the start of the culture, and 3% after four weeks.
It turns out that it decomposed by 5.2%.

Example 2 Dependence of Ferrocian Degradability on Medium pH The above Pseudomonas sp. 14SA3-2-5B2 was prepared using the ferrocyanide liquid medium prepared at various pH values and sterilized.
The pH dependence of ferrocyanide resolution was investigated.
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 inoculated loop of Pseudomonas sp. 14SA3-2-5B2 was inoculated. Experimental groups 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 apparent from FIG. 2, the decomposition of ferrocyanide by the Pseudomonas sp. 14SA3-2-5B2 was good in the experimental plots AD, that is, in the weak alkaline pH condition. Significant in A. FIG.
As is clear from the above, the Pseudomonas sp.
-5B2 decomposes ferrocyanin in the 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.

Thus, the Pseudomonas sp.
Since SA3-2-5B2 can decompose solubilized ferrocyanin, it is considered to be suitable as a microorganism used for purification treatment of industrial wastewater and the like.

[Brief description of the drawings]

FIG. 1 is a graph showing the change over time of ferrocyanide degradation by Pseudomonas sp. 14SA3-2-5B2.

FIG. 2 is a graph showing the pH dependence of ferrocyanide degradation by Pseudomonas sp. 14SA3-2-5B2.

FIG. 3 is a graph showing the pH of the medium during the culture of Pseudomonas sp. 14SA3-2-5B2.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B065 AA41X AC12 AC20 BA23 BB01 BC01 BC02 BC26 BC31 CA56 4D040 DD03 DD14

Claims (3)

[Claims] 1. A bacterium belonging to the genus Pseudomonas having the ability to decompose a metal cyano complex. 2. A bacterium belonging to the genus Pseudomonas having iron cyano complex decomposability. 3. Pseudomonas sp. 14SA3-2-5B2 having cyanide decomposability.