JP6246072B2 - Novel microorganism and method for decomposing metal cyano complex - Google Patents

Description

  The present invention relates to a novel microorganism and a method for decomposing a metal cyano complex.

Cyanide is an inorganic substance composed of carbon and nitrogen, and is classified as a heavy metal and classified as a second-type specific hazardous substance in the Soil Contamination Countermeasures Law (hereinafter referred to as the soil-to-ground law). The cyan compound includes a group of compounds called cyanide and metal cyano complex. The cyanide is also called “free cyan” and is represented by the general formula An (CN) x, where A is hydrogen (H), sodium (Na), potassium (K), ammonium (NH ₄ ). ), Calcium (Ca), etc., which are the most toxic forms of cyanide compounds. The metal cyano complex is a compound in which a metal salt of hydrogen cyanide and a metal are combined with an excess of cyanide ion (CN ⁻ ), and is represented by the general formula An [M (CN) x]. Here, M corresponds to metals such as silver (Ag), gold (Au), cadmium (Cd), cobalt (Co), copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn). However, it is dissolved or suspended in the solution.

  The cyanide compound may be contained in industrial wastewater or the like, and is a substance having a property that should be removed by purification treatment. Factories that discharge cyanide include plating plants, beneficiation refineries, steel heat treatment plants, and coke production plants. In these factories, cyanide wastewater treatment equipment is installed in the factories because cyanide compounds are by-produced in the process of building baths such as copper, zinc, nickel, and gold, and in the product manufacturing process. According to “Survey results on the status of implementation of the 2008 Soil Contamination Countermeasures Law and Soil Contamination Surveys / Countermeasures Cases: February 2010: Ministry of the Environment” (hereinafter referred to as “Soil vs. Case Example Results”) The industry categories that exceeded the soil environmental standards by are in the order of metal products manufacturing industry, gas industry, and chemical industry. In Japan, there are many small and medium plating plants, and there are more than 2,000 companies nationwide. Most of them were built around urban areas during the post-war high economic growth period, potentially leaking from cyanide compounds during operation, and leaks from plating baths and piping due to aging of equipment, resulting in factory sites that could potentially exceed the survey results. The soil is expected to be contaminated with cyanide.

  Many studies have been conducted on the presence of cyanide compounds in solution (wastewater) in the wastewater treatment field, and it has been reported that most of the forms are the free cyanide and the metal cyano complex. . Treatment methods for wastewater containing these cyanides include alkali chlorine method, ozone oxidation method, electrolytic oxidation method, bitumen method (precisely soluble complex compound precipitation method), acid decomposition combustion method, boiled method (boiled high temperature combustion method) In addition, a wet heat decomposition method and an adsorption method are known. However, these treatment methods do not apply to highly stable metal cyano complexes, such as iron, cobalt, silver, and gold cyano complexes, or require severe equipment under severe reaction conditions, There was a problem that further processing of the product was necessary. In view of this, a technique for restoring the environment using biological functions, so-called bioremediation, has attracted attention, and a search for microorganisms having the ability to decompose cyanide compounds has been conducted.

  Currently, regarding the microbial degradation of free cyanide such as potassium cyanide and sodium cyanide, Pseudomonas putida, Pseudomonas sp. Acinetobacter sp. Fusarium sp. Klebsiella sp. There are reports that microorganisms such as these have been isolated and identified.

Regarding the metal cyano complex, Fusarium solani and Trichoderum polysporum have been reported as metal cyano complex-decomposing microorganisms that decompose nickel cyano complex ([K ₂ Ni (CN) ₄ ]). Moreover, Fusarium oxysporum, Cytalidium thermophilum, and Penicillium miczynski have been reported as degrading bacteria of potassium ferrocyanide ([K ₄ Fe (CN) ₆ ]), which is an iron cyano complex (all of which are Non-Patent Document 1). Also, the applicant of the present application has isolated and identified various metal cyano complex-decomposing microorganisms that decompose iron cyano complexes (for example, Patent Documents 1 and 2 and the like, Patent Document 3 and the like as identification examples other than the applicant of the present application). ).

  However, when decomposing the iron cyano complex, the above metal cyano complex decomposing microorganisms work under aerobic conditions, so the application form of the metal cyano complex decomposing microorganism is limited, and almost no oxygen supply is possible. Since it has various technical problems in actual use, such as being unable to decompose the iron cyano complex in non-soil, it has not been put into practical use. The metal cyano complex-decomposing microorganism described in Patent Document 3 has the possibility of decomposing an iron cyano complex under anaerobic conditions. However, the degrading bacterium Klebsiella pennumoniae is a pathogenic bacterium of BSL2, It is not suitable for the iron cyano complex decomposition treatment under anaerobic conditions by the augmentation method of the position treatment method, and since it still has various technical problems, it has not been put into practical use.

In view of the circumstances as described above, it is desirable that the purification of the iron cyano complex is applied to soil, particularly contaminated soil reaching the saturated layer.
As the actual condition of the treatment of contaminated soil, excavation and removal for the purpose of complete purification are often selected in consideration of the psychological factors of the landowner and the economic aspects of real estate transactions. In particular, heavy metals such as lead and cadmium cannot be further decomposed, and despite the high cost of 50,000 yen / m ³ or more due to excavation and removal, the second class specified harmful Excavation removal is adopted in 83% of the cases of excess substances (according to “Soil vs. Case Results”). The reason why the excavation and removal ratio is very high at 83% is also because the in-situ purification technology has not been established. In particular, for cyanide compounds, the range where in-situ purification technology can be applied is limited. Insolubilization requires long-term monitoring, and land development is limited during development. Chemical addition treatment (Fenton method) and ozone injection treatment are limited to some metal cyano complex compounds, such as being inapplicable to stable ferrocyan (iron-cyan complex). In the case of excavation and removal, it is necessary to detoxify the excavated soil separately, heat treatment represented by cement raw materials, insolubilization treatment that is chemical treatment, chemical addition treatment, ozonolysis treatment, classification cleaning treatment that is physical treatment There is. As described above, metal cyano complexes are not easily decomposed and heat treatment is often employed. In addition, regarding the heat treatment, acceptance standards in the case of cement raw materials are strict, and there is no choice but to entrust the treatment to a dedicated heat treatment facility. Therefore, the processing cost of the metal cyano complex is higher than that of the normal heavy metal processing cost, and application of the in-situ purification technology is required as a lower cost processing method.

  Patent Document 4 describes anaerobic iron cyano complex-decomposing microorganisms that have the ability to decompose iron cyano complexes under mildly alkaline growth conditions. These are pathogenic bacteria or opportunistic bacteria, and can be used for soil purification in open systems. There was an unsuitable problem.

The current bioremediation of cyanide-contaminated soil is carried out by an “injection method” in which nutrients, dissolved oxygen water and air are injected into the soil to promote microbial activity. However, there are conditions for the application of the “injection method” and the contaminated site
<1> It is a soil contaminated with free cyanide or copper cyano complex that is relatively easy to decompose, and
<2> Formation conditions suitable for the injection method (saturated layer with good water permeability and easy transfer of injected material)
In such a case, it remains at the empirical research stage where purification was confirmed.
Cyan compounds have a wide variety of forms, and particularly iron cyano complexes are very stable substances. It is known that most of cyanide compounds contained in cyanide-contaminated soil derived from coal gas, which is a problem in the steel industry and gas business, are iron cyano complexes. Cyan-contaminated soil at plating plants is a copper cyano complex with heavy metals used in the plating bath, but since the soil contains a large amount of iron, cyanide that permeates and diffuses into the ground can also be used as an iron cyano complex. In order to achieve environmental standards, it is necessary to decompose iron cyano complexes that are difficult to decompose.

JP 2000-270847 A JP 2003-275791 A JP 2006-281053 A JP 2001-269166 A

Knowles C.I. J. et al. et. al, Enzyme and Microbiology 22: 223-231, (1998).

  Further, in order to decompose and remove the cyanide that has permeated and diffused into the ground, the present inventors have established a novel microorganism Clostridium chromoreductance ER-CL-1 strain (deposit number NITE P-1434). Has been found to be useful.

  However, the above microorganisms have the property of anaerobically decomposing iron cyano complexes, and in order to apply the above microorganisms to actual soil, a device for maintaining the environment anaerobically is required. Therefore, in reality, the handling tends to be difficult, and there is a demand for microorganisms that have the property of decomposing iron cyano complexes and that are easy to handle.

  In view of the above circumstances, an object of the present invention is to provide a novel microorganism Brevundimonas sp. The present invention provides a method for decomposing a metal cyano complex capable of effectively decomposing and removing the metal cyano complex from 1015-6 strain (deposit number NITE P-01856) (hereinafter referred to as 1015-6 strain) and waste water, soil, etc. that may contain an iron cyano complex. There is.

[Configuration 1]
In order to solve the above object, the present invention provides a novel microorganism Brevundimonas sp. Strain 1015-6 (deposit number NITE P-01856).

[Function 1]
The present inventors aerobically enriched culture at neutral pH using ferrocyan as a sole nitrogen source, glucose as a carbon source, and general soil or cyanogen-contaminated soil as a separation source. An aerobic ferrocyan decomposition consortium was obtained that decomposes chemically.
A colony obtained by developing the obtained aerobic ferrocyanate-degrading consortium on a normal nutrient agar medium is subjected to liquid culture using ferrocyan as a nitrogen source, thereby obtaining a strain that decomposes and grows ferrocyan. It was. As a result of analysis of this strain, it was revealed that the strain was a novel strain 1015-6 from the results of 16S rDNA base sequence analysis, morphology observation, and physiological and biochemical tests. This strain is at the level of BSL-1 and can be safely used in a contaminated soil purification program.

[Configuration 2]
Moreover, the characteristic structure of the decomposition | disassembly method of the metal cyano complex of this invention exists in the point which applies 1015-6 stock | strain to the soil containing a metal cyano complex in an aerobic environment, and decomposes | disassembles the said metal cyano complex.

  Since strain 1015-6 has the ability to decompose an iron cyano complex under an aerobic environment, the metal cyano complex can be decomposed by applying it to soil containing the metal cyano complex under an aerobic environment, The soil can be purified and detoxified. Here, since the 1015-6 strain is an aerobic bacterium, it is not necessary to maintain the part which becomes a supply channel of the bacterium and nutrient source or the soil itself in an anaerobic environment, and it can be applied to the soil with good handleability. it can.

[Configuration 3]
The metal cyano complex may be an iron cyano complex.

  Since strain 1015-6 can decompose an iron cyano complex, it can be expected that even a metal cyano complex stabilized as an iron cyano complex in soil can be decomposed and purified.

  Therefore, according to the present invention, even a cyan complex waste liquid containing a hardly decomposable iron cyano complex or a cyanide-contaminated soil can be suitably purified.

Graph showing accumulation culture results of metal cyano complex degrading microorganisms (CN1 + 2 + 0.1 mM ferro) novel Brevundimonas sp. Graph showing the 1015-6 strain culture results

  The inventors performed aerobic enrichment culture at neutral pH using ferrocyan as the sole nitrogen source, glucose as the carbon source, general soil or cyanogen-contaminated soil as the separation source, as follows, An aerobic ferrocyan decomposition consortium was obtained that decomposes ferrocyan aerobically. A colony obtained by developing the obtained aerobic ferrocyanate-degrading consortium on a normal nutrient agar medium is subjected to liquid culture using ferrocyanine as a nitrogen source, thereby obtaining a strain capable of decomposing and proliferating ferrocyan. It was. The following is the new Brevundimonas sp. The screening method for the 1015-6 strain will be specifically described.

<Experiment method>
Accumulation culture of metal cyano complex degrading microorganisms was performed from cyan contaminated soil by the following procedure.

(1) 1 g of each collected soil, 49 mL of basic synthetic medium, and 1 mL of 50 mM ferrocyanine aqueous solution are added to a vial, and glucose is added to 1 M, and then sealed with argon gas. Further, 20 mL of gas is extracted from the gas layer substituted with argon by a gas tight syringe and 20 mL of oxygen is added.
(3) Shake culture for 1 week at 30 ° C.
(3) 0.5 mL of the culture solution after 1 week of culture is added to 50 mL of the second synthetic medium and cultured at 30 ° C. (Another week)
(4) Further, 0.5 mL of the culture solution after culturing for one week is added to 50 mL of ferrocyan medium and cultured at room temperature.

  The steps (1) to (4) were performed, and the change in ferrocyan concentration in the step (4) was measured. Moreover, the process of (1)-(4) is performed also about the thing which did not add glucose to the nutrient source in the process of (1), and the thing which did not add soil, and the ferrocyan concentration in the process of (4) The change of was measured. The result is as shown in FIG. (Indicated with glucose, without glucose, and control, respectively)

Basic synthetic (syn) medium Na ₂ HPO ₄ 0.1%
KH ₂ PO ₄ ... 0.05%
NaCl ··· 0.05%
MgSO ₄ ... 1 mM
CaCl ₂ ... 0.1 mM
Glucose ... 10 mM
Trace element solution ... 1/1000 amount pH 7.0

Second synthetic medium Basic synthetic medium Growth factor solution ... 1/1000 volume

Ferrocyanic medium Second synthetic medium Potassium ferrocyanide 0.5 mM

  As a result, it was found that in the presence of glucose, a consortium with a high ferrocyan degradation ability was formed. After culturing this consortium, the bacterial flora was analyzed, and as a result, many Nocardioides and Brevundimonas were observed. Therefore, it was decided to isolate bacteria having the ability to decompose iron cyano complexes using these as targets.

  The consortium was cultured in an agar medium at 30 ° C. for 3 days, colony PCR was performed using primers for 16S rRNA gene, base sequence analysis was performed, and a strain was estimated. Further, each strain was cultured in the second synthetic medium for 14 days at 30 ° C., and the cyanolytic ability of each strain was determined from the residual cyan concentration in the second synthetic medium after 14 days of cultivation (FIG. 2). Four strains with high resolution were extracted. For these, 16S rDNA nucleotide sequence analysis, morphology observation, and physiological / biochemical tests were performed.

  As a result, among each strain, 1015-6 strain is not a known genus Brevundimonas. Brevundimonas sp. Closely related to B. aurantica (Brevundimonas sp.).

<Homology with Brevundimonas sp.>
As a result of the BLAST homology search for Apollon DB, BA9.0, the 16S rDNA base sequence of strain 1015-6 showed high homology to the 16S rDNA base sequence of Brevandimonas sp. . There is a possibility that it belongs to Aurantia. However, the difference of 6 bases was confirmed between the 16S rDNA base sequences excluding the primer portion, and all were clearly different, and it was found that the possibility that they were different as species could not be denied.
Therefore, as a result of 16S rDNA analysis, the isolated microorganism was identified as Brevundimonas sp. It was confirmed that this was a new strain belonging to The new microorganism is obtained from the National Institute for Product Evaluation Technology Patent Microorganisms Depositary (NPMD), and the new microorganism Brevundimonas sp. It has been deposited as strain 1015-6 (deposit number NITE P-01856).

The mycological properties of this new microorganism are as follows.
(In the following description, + indicates positive,-indicates negative, and + w indicates weak positive)

Cell morphology: curved rods,
Gram staining:-,
Presence or absence of spores:-,
Mobility: +

Colony morphology
Color: Light yellow,
Shape: round,
Raised state: lenticular,
Perimeter: all edges,
Surface shape: smooth,
Transparency: opaque,
Viscosity: Butter-like growth temperature test 37 ° C:-
45 ° C:-

Catalase reaction: +,
Oxidase reaction:-+
Acid / gas production from glucose: + w / −,
O / F test: +/-
Nitrate reduction:-,
Indole production:-,
Glucose acidification:-,
Arginine dihydrolase:-,
Urease:-,
Esculin hydrolysis: +,
Gelatin hydrolysis:-,
β-galactosidase: +,
Glucose utilization: +,
L-arabinose utilization:-,
D-mannose utilization:-,
D-mannitol assimilation:-,
N-acetyl-D-glucosamine utilization:-,
Maltose utilization: +,
Potassium gluconate utilization:-,
n-Capric acid assimilation:-,
Adipic acid utilization:-,
d1-Assimilation of malic acid: +,
Sodium citrate assimilation:-,
Assimilation of phenyl acetate:-,
Cytochrome oxidase: +
Growth under anaerobic conditions:
Starch hydrolysis:-,
Ethanol utilization:-

  The novel microorganism and the method for decomposing a metal cyano complex according to the present invention can be applied to industrial wastewater and cyanogen-contaminated soil in a factory site where cyanide exists as a chemically stable and hardly decomposable iron cyano complex such as Prussian blue or ferrocyan. It can be used for purification.

Claims (3)

  A novel microorganism Brevundimonas sp. 1015-6 strain (deposit number NITE P-01856).   A method for decomposing a metal cyano complex, wherein the novel microorganism according to claim 1 is applied to soil containing a metal cyano complex in an aerobic environment to decompose the metal cyano complex.   The method for decomposing a metal cyano complex according to claim 2, wherein the metal cyano complex is an iron cyano complex.

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