JP2000270847A - New microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a bacterium belonging to the genus Ochrobactrum capable of decomposing a ferrocyanide compound contained in a wastewater or soil efficiently since it has an iron-cyano complex-decomposing activity and useful for an environmental maintenance, 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 Ochrobactrum is Ochrobactrum anthropi having decomposing activities of a metal-cyano complex such as a nickel-cyano complex and the iron-cyano complex, and also a new strain, Ochrobactrum anthropi 14SA3-2-5BI strain (FERM P-17320) having the cyanide-decomposing activity. Further it is preferable to decompose and remove the ferrocyanide in a soil or wastewater by using Ochrobactrum anthropi 14SA3-2-5B strain.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to a novel microorganism belonging to the genus (Ochrobactrum), which is capable of decomposing a cyanide compound, in particular, an iron cyano complex under a weak alkaline growth condition.

[0002]

2. Description of the Related Art Cyanide compounds include a group of compounds called cyanide and metal cyano complexes. The cyanide is also called “free cyanide” and has a general formula A _n
Represented by (CN) _x , A includes hydrogen (H), sodium (Na), potassium (K), ammonium (NH ₄ ), calcium (Ca), etc., and is the most toxic form of cyanide It is.
The metal cyano complex is obtained by combining a metal salt of hydrogen cyanide and a metal with an excess of cyanide ion (CN ⁻ ), and is represented by a general formula _An [M (CN) _x ]. Where M is silver (A
g), gold (Au), cadmium (Cd), cobalt (Co), copper (Cu),
Metals such as iron (Fe), nickel (Ni), and zinc (Zn) are applicable, and are dissolved or suspended in a 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 wastewater containing the iron cyano complex,
Under weakly acidic conditions, the iron cyano complex is present in the form of a hardly soluble salt, so that the iron cyano complex in the wastewater is likely to precipitate in a flow path or a treatment tank during the treatment. 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 Ochrobactrum capable of decomposing a cyanide, and specifically, is a novel microorganism of the genus Ochrobactrum anthropi.
bactrum anthropi) 14SA3-2-5B1 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, the colonies formed on the ferrocyan agar plate were collected, and each colony was again inoculated on a sterilized ferrocyan agar plate and cultured at 30 ° C. Colonies formed on the medium were separated. Further, 10 ml of a sterilized ferrocyanide liquid medium was dispensed into a sterilized test tube, and one platinum loop of the above-mentioned isolated strain was inoculated and shake-cultured at 30 ° C. for 2 weeks (150 minutes).
１７０170 rpm). Then, the culture was centrifuged to remove bacterial cells, and the concentration of ferrocyanin remaining in the supernatant was determined by HPLC.
And a strain having a reduced ferrocyanin concentration in the medium after the culture was selected. As a result, a strain having ferrocyanide resolution was obtained, and this strain was named 14SA3-2-5B1 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 sterilized separately at 121 ° C. for 15 minutes, and filter sterilization were added. 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.
A solution sterilized at 15 ° C. for 15 minutes was used.

Table 2 shows the bacteriological properties of the 14SA3-2-5B1 strain.
It writes in.

[0016]

[Table 2]

The taxonomic position of the 14SA3-2-5B1 strain having the above-mentioned mycological properties is described in "Bergey'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.), "Bergey's Manual of Deterministic Bacteriology 9th Edition" (Holt.
Krieg, NR, Sneath, PHA, Staley, jT and William
s, ST: ゛ Bergery's Manual of Determinative Bacter
iology ″ Ninth Edition, (1994) Williams & Wilkin
s.) and Holmes, B., Popoff, M., Kiredjian, M. and
Kersters, K. (Int.J.Syst.bacteriol., 38, 406-416 (198
8)). As a result, the 14SA3-2-5B1
The strain is Ochrobactrum anthropi.
thropi). However, there has never been a report that a known Ochrobactrum anthropi can decompose a cyanide compound.
The 2-5B1 strain is a new strain that is distinguished from known strains. The strain determined to be Ochrobactrum anthropi 14SA3-2-5B1 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-17320.

The Ochrobactrum anthropi
The 14SA3-2-5B1 strain was prepared using the following SCDA medium (pH 7.3 ± 0.
2, 25 ° C., manufactured by DIFCO) and YG medium (pH 7.2 ± 0.
Using 2), the cells can be cultured 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%

The Ochrobactrum anthropi 14SA3-2-5B1 strain according to the present invention is a new microorganism classified as a bacterium. Generally, bacteria are neutral to slightly alkaline p
Growing in the H region, the inventors have determined by the growth test described below that Ochrobactrum anthropi 14S
The strain A3-2-5B1 has also been shown to be able to grow by decomposing the iron cyano complex under neutral to slightly alkaline growth conditions. As described above, the Ochrobactrum anthropi 14SA3-2-5B1 strain can be used for wastewater treatment by bioremediation because it can degrade cyanogen compounds, particularly iron cyano complexes, in the neutral to weakly alkaline pH range. It can be said that it is a microorganism suitable for. 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 cyano form in the soil is ferrocyan which is a complex of iron and cyanide. Therefore, the Ochrobactrum anthropi strain 14SA3-2-5B1 according to the present invention is used. By decomposing and removing ferrocyanin in the soil using, it is considered that soil contaminated with cyanide can be purified.

Embodiments of the present invention will be described below.

Example 1 Measurement of Ferrocian Degradability of Ochrobactrum Anthropi 14SA3-2-5B1 Strain The above-mentioned sterilized ferrocyanide liquid medium was placed in a sterilized test tube.
Dispense 10 ml and add Ochrobactrum anthropi
One platinum loop was inoculated with 14SA3-2-5B1 strain. Then, the test tube inoculated with the strain is shake-cultured at 30 ° C. (150 to 1).
70 rpm), and ferrocyanide in the medium at regular intervals.
(Total cyan) content was measured. Here, the ferrocyanide (total cyanide) content in the culture solution can be determined by referring to the `` Cyan pre-distillation operation method (in soil Method of heating and distilling all the cyanide) ''
In accordance with the above, the distillate obtained by distilling the whole amount of the culture (culture solution, bacterial cells) was subjected to a fully automatic cyan measurement device (T-CN501 manufactured by Anatech Yanaco) and measured. The result is shown in FIG.
Shown in

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

As shown in FIG. 1, the Ochrobactrum anthropi 14SA3-2-5B1 strain degraded 3.0% of ferrocyanin in the liquid medium two weeks after the start of culture.
It can be seen that 30.1% was decomposed after a week.

[Example 2] Dependence of ferrocyanide resolution on culture medium pH The Ochrobactrum anthropi 1 was prepared using the ferrocyanide liquid medium prepared at various pH values and sterilized.
We investigated the pH dependence of ferrocyanide resolution of 4SA3-2-5B1 strain. Specifically, pH 7.0, 8.0, 9.0,
After adjusting to 10.0, 11.0 or 12.0, 10 ml of sterilized ferrocyanide liquid medium was dispensed into each sterilized test tube, and Ochrobactrum anthropi 14SA3-2-5B1 was added.
The inoculated strains were inoculated with one platinum loop and designated as experimental plots AF. Shaking culture (150 to 170 rpm) was performed at 30 ° C. for each of these six experimental plots, and the ferrocyanide (total cyan) content in the medium and the pH of the medium were measured at regular intervals. The ferrocyanide (total cyan) content in the culture solution was measured using the same method as in [Example 1]. Less than,
FIG. 2 shows the time-dependent change in the ferrocyanin content in the medium, and FIG. 3 shows the time-dependent change in the medium pH.

As is clear from FIG. 2, the degradation of ferrocyanide by the Ochrobactrum anthropi strain 14SA3-2-5B1 was good in the experimental plots A to D, that is, in the weak alkaline pH condition. It is remarkable in ward A. As is clear from FIG. 3, the Ochrobactrum anthropi 14SA3-2-5B1 strain decomposes 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 Ochrobactrum anthropi 14SA3-2-5B1 strain is considered to be suitable as a bacterial strain used for purification treatment of industrial wastewater or the like because it can decompose solubilized ferrocyanin. .

[Brief description of the drawings]

FIG. 1 is a graph showing the time course of ferrocyanide degradation by Ochrobactrum anthropi 14SA3-2-5B1 strain.

[Figure 2] Ochrobactrum anthropi 14SA3-2-5B1
Graph showing pH dependence of ferrocyanide degradation by strains

[Fig. 3] Ochrobactrum anthropi 14SA3-2-5B1
Graph showing medium pH during strain culture

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 1/20 C12R 1:01) (C12N 1/20 C12R 1:01) (C12N 1/20 C12R 1:01) F term (reference) 4B065 AA01X AC20 BA23 BB12 BC02 BC18 CA56 4D004 AA41 AB05 CA19 CC07 4D040 DD03 DD14 DD20

Claims (4)

[Claims] 1. A bacterium belonging to the genus Ochrobactrum having iron cyano complex decomposability. 2. Ochrobactrum anthropi having the ability to decompose a metal cyano complex. 3. Ochrobactrum anthropi having iron cyano complex decomposability. 4. A new strain Ochrobactrum anthropi 14S capable of decomposing cyanide
A3-2-5B1 strain.