JP5930796B2 - Method for treating water containing iron cyano complex compound and method for soil purification - Google Patents

Description

  The present invention relates to a method for treating iron cyano complex compound-containing water and a method for soil purification.

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 above-mentioned cyanide resolution 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.

As for the metal cyano complex, Fusarium solani and Trichoderum polysporum have been reported as decomposing microorganisms of the nickel-cyano complex ([K ₂ Ni (CN) ₄ ]). In addition, 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 microorganisms that degrade 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 present applicant).

  However, when decomposing the iron cyano complex, since all the microorganisms work under aerobic conditions, the application form of the microorganisms is limited, and the iron cyano complex is reduced in soil with almost no oxygen supply. It has not been put into practical use because it has various technical problems in actual use, such as being unable to be decomposed. In addition, although the microorganisms described in the said patent document 3 have a possibility of decomposing | disassembling an iron cyano complex under anaerobic conditions, Klebsiella pnenumoniae of decomposing bacteria is a pathogenic bacterium of BSL2, It is not suitable for the treatment of anaerobic cyano complex compounds by the augmentation 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. The chemical addition process (Fenton method) and ozone injection process are limited to some cyanide compounds because they cannot be applied 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, cyanide is 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 treatment cost of the cyanide compound is higher than the normal heavy metal treatment cost, and application of the in-situ purification method is required as a lower cost treatment method.

  In addition, microorganisms capable of decomposing free cyanide, microorganisms producing cyanide-degrading enzymes, and gene sequences encoding cyanide-degrading enzyme proteins are known from Non-Patent Documents 2 and 3, and can be obtained from the following DNA database. .

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

Knowles C.I. J. et al. et. al, Enzyme and Microbiology 22: 223-231, (1998). Stephen Ebbs, Current Opinion in Biotechnology 2004, 15: 231-236 Robert D. Fallon, Applied and Environmental Microbiology, 1992 3163-3164

[DNA database]
Genbank (http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide)
DDBJ (http://www.ddbj.nig.ac.jp/Welcome-j.html)
EMBL (http://www.ddbj.nig.ac.jp/Welcome-j.html)

The current bioremediation of cyan contaminated soil uses an “injection method” that injects nutrients, dissolved oxygen water and air into the soil to promote microbial activity. However, there are conditions for applying the “injection method”, and the contaminated site is <1> soil contaminated with free cyanide or copper cyano complex that is relatively easy to decompose cyanide, and <2> formation conditions suitable for the injection method. In the case of (saturated layer with good water permeability and easy transfer of injection material), it remains in the empirical research stage where purification was confirmed.
Cyanide compounds have a wide variety of forms. Ferrocyan is a very stable substance. It is known that most of the cyan-contaminated soil derived from coal gas, which is a problem in the steel industry and gas business, is ferrocyan. Cyan-contaminated soil in plating plants is a copper cyano complex with heavy metals used in the plating bath, but since the soil contains a lot of iron, cyanide that has penetrated and diffused into the ground also exists as ferrocyan. In order to achieve environmental standards, it is necessary to decompose ferrocyan that is difficult to decompose.

  Accordingly, in view of the above circumstances, an object of the present invention is to provide a method for treating water containing an iron cyano complex compound that can efficiently decompose and purify a hardly decomposable cyano compound such as the iron cyano complex even under anaerobic conditions. And providing a soil purification method.

[Configuration 1]
The configuration of the present invention for achieving the above object is that glucose is added to iron cyano complex compound-containing water, and the iron cyano complex compound-containing water to which glucose is added is decomposed by an anaerobic microorganism utilizing glucose. Thus, free cyanide is generated, and the free cyanide is decomposed by a microorganism expressing free cyanide-degrading enzyme.

[Operation effect 1]
The present inventors have clarified that the microorganisms expressing free cyanide degrading enzyme capable of decomposing free cyanide described later purify the water containing iron cyano complex compound under anaerobic conditions. Furthermore, when the present inventors analyzed the decomposition process of the iron cyano complex compound of this microorganism, it has become clear that the iron cyano complex is once converted into free cyan and then decomposed by the action of cyanidase.

  Therefore, the present inventors, even if it is a hardly decomposable iron cyano compound, first, if a condition capable of decomposing to the stage of generating free cyanide is prepared, if it is a microorganism expressing free cyanide degrading enzyme, it is difficult to decompose. I thought that there is a possibility that the iron cyano compound can be purified to a harmless state, and further research was conducted.

  As a result, when glucose-assimilating anaerobic microorganisms are cultured in the iron cyano complex compound-containing water to which glucose is added, the anaerobic microorganisms decompose the iron cyano complex compound to produce free cyanide. In addition, the present inventors have found that an operation for greatly changing the liquidity such as pH of the iron cyano complex compound-containing water is not necessary.

  Conventionally, it has been known that when the pH of the iron cyano complex compound-containing water is lowered by an acid, free cyan is likely to be generated from the iron cyano complex compound. However, cyan gas is generated as free cyan is generated. In fact, there is a situation that it is difficult to adopt such an operation because it is necessary to be careful. In contrast, the production of free cyanide accompanying the cultivation of anaerobic microorganisms has the effect of lowering the pH of the iron cyano complex compound-containing water, but it is unlikely that cyanide gas will be generated, and a mild reaction will proceed. And is considered particularly preferable.

Then, when glucose is added to iron cyano complex compound-containing water and the iron cyano complex compound-containing water added with glucose is treated with a glucose-assimilating anaerobic microorganism, the anaerobic microorganism converts the iron cyano complex compound into water. Decompose to produce free cyanide.
When the resulting free cyanide is decomposed by a microorganism expressing free cyanide degrading enzyme, the free cyanide is rendered harmless and the iron cyano complex compound is decomposed and purified as a whole.

  Therefore, conventionally, when purifying soil or the like containing a hard-to-decompose iron cyano complex compound, it has been necessary to excavate the soil and separately purify it. It has become possible to purify at a non-existing in-situ position, and can be applied simply, safely and at low cost.

[Configuration 2]
Further, a genetically modified microorganism into which a gene encoding a cyanidase protein is incorporated as the free cyanide-degrading enzyme-expressing microorganism can also be used.

[Configuration 3]
Furthermore, the gene encoding the cyanidase protein is a gene encoding cyanidase derived from the Bacillus pumilus NBRC12092 strain.

[Operation effect 2] [Operation effect 3]
That is, since the free cyanide-degrading enzyme-expressing microorganism can decompose free cyanogen produced by a glucose-utilizing anaerobic microorganism, it can be used for purification of iron cyano complex compound-containing water in an anaerobic environment. Can do. Here, when using genetically modified microorganisms, by selecting and using harmless microorganisms such as E. coli as a host, it can be applied to the purification of iron cyano complex compound-containing water more safely. Become. As a gene encoding a cyanidase protein to be incorporated into such a microorganism, a gene encoding a cyanidase derived from Bacillus pumilus NBRC12092 strain is effectively used. It has become clear that As such a microorganism, for example, CDH-pET26b-Escherichia coli BL21 (DE) (accession number NITE P-1278) obtained by genetic recombination of Escherichia coli by the present inventors can be used.

[Configuration 4]
In the soil purification method of removing the iron cyano complex compound contained in the soil subject to purification by using microorganisms, the characteristic configuration of the soil purification method of the present invention,
Cyan liberation step of adding cyanine and glucose-assimilating anaerobic microorganisms to soil pore water in the soil to be purified to liberate cyanogen in the soil pore water in the soil to be purified, and liberation in the cyan liberation step The cyanide decomposing step of decomposing cyanide with a free cyanide degrading enzyme-expressing microorganism at the in situ position of the soil to be purified.

[Configuration 5]
The free cyanolytic enzyme-expressing microorganism may be a genetically modified microorganism into which a gene encoding a cyanidase protein has been incorporated.

[Operation effect 4] [Operation effect 5]
When glucose is added to iron cyano complex compound-containing water, and the iron cyano complex compound-containing water added with glucose is treated with a glucose-assimilating anaerobic microorganism, the anaerobic microorganism decomposes the iron cyano complex compound. To produce free cyanide.
That is, by adding glucose and glucose-assimilating anaerobic microorganisms to soil pore water in the soil to be purified, the iron cyano complex compound-containing water is decomposed to perform a cyan liberation step that generates free cyanide. it can.
When the resulting free cyanide is decomposed by a microorganism expressing free cyanide degrading enzyme, the free cyanide is rendered harmless and the iron cyano complex compound is decomposed and purified as a whole.
Therefore, when a cyanogen-degrading enzyme-expressing microorganism is allowed to act on cyan liberated in the cyan liberation step, a cyan degradation step for degrading the free cyan can be performed, so that it can be performed in situ on the soil to be purified as a whole. become.

  In addition, glucose-assimilating anaerobic microorganisms can use conventional ones that exist in the soil, and may be added separately if the work of the conventional ones is considered insufficient. it can. When added separately, it may be any microorganism of BSL1, and various known microorganisms such as soil-derived microorganism consortia, lactic acid bacteria, actinomycetes, and photosynthetic bacteria can be used.

  The amount of glucose added is not particularly limited, but is preferably 0.1% or more for the purpose of maintaining the activity of the anaerobic microorganism and generating a sufficient amount of acid from the assimilated glucose. From the viewpoint that the effect is saturated even if it is added too much, it is preferable to be 10% or less.

  As the free cyanide-degrading enzyme-expressing microorganism, a genetically modified microorganism into which a gene encoding a cyanidase protein is incorporated is used from the following embodiments, and can be used safely and easily. It is also possible to obtain a free cyanide-degrading enzyme-expressing microorganism by screening from the soil.

  Therefore, the contaminated soil, which has been difficult to clean up in the past, can be cleaned in-situ safely and at low cost.

Flow diagram of the method for treating water containing iron cyano complex compound of the present invention Graph showing the effect of glucose addition in the cyanide release process Graph showing the effect of lactose and sucrose addition in the cyan release process SDSPAGE photograph showing expression of free cyanide degrading enzyme by microorganisms expressing free cyanide degrading enzyme Graph showing the effect of cyanide degradation on free cyanide by E. coli expressing cyanide Graph showing total cyan reduction effect by soil remediation verification test Graph showing ferrocyan (iron cyano complex compound) reduction effect by soil purification verification test Graph showing the effect of free cyanide generation by soil purification verification test

  Below, the processing method of the iron cyano complex compound containing water of this invention is demonstrated. Preferred examples are described below, but these examples are described in order to more specifically illustrate the present invention, and various modifications can be made without departing from the spirit of the present invention. The present invention is not limited to the following description.

[Method of treating water containing iron cyano complex compound]
As shown in FIG. 1, the method for treating iron cyano complex compound-containing water of the present invention comprises adding glucose to iron cyano complex compound-containing water, and using the iron cyano complex compound-containing water to which glucose has been added, A cyan liberation step is performed in which a free anaerobic microorganism decomposes to produce free cyanide, and the free cyanide is decomposed by a free cyanolytic enzyme-expressing microorganism.

  Here, as iron cyano complex compound-containing water, iron cyano complex contained in soil is promising, and particularly in the saturated layer, iron cyano complex compound-containing water that exists in an oxygen-free state together with groundwater Become.

[Cyan liberation process]
When glucose was added to the ferrocyanine-containing culture solution and the microorganism was anaerobically cultured, it was examined that cyan was liberated from ferrocyan.

  Those inoculated and not inoculated with E. coli in 20 mL of glucose-added LB medium shown in Table 1 were anaerobically cultured (Ar-100%, 30 ° C.). Thereafter, the free cyanide concentration was measured by liquid chromatography.

  In the present embodiment, the measurement of free cyanide concentration by liquid column chromatography was performed under the following conditions.

Column: Shodex IEC DEAE-825
(8.0mmID * 75mm)
Elution solvent: (10 mM sodium carbonate + 1 mM
Ethylenediamine) aq. / CH ₃ OH = 90/10
Elution rate: 1.0 mL / min
Detector: EC (Electrode; Ag 0 mV SCE)
Column temperature: 40 ° C

  As a result, as shown in FIG. 2, the amount of free cyanide dissociation from ferrocyan was significantly increased as compared with the case of culturing in the LB medium without addition. At this time, when cultured in an LB medium containing glucose, the pH at the end of the culture was greatly lowered. Thus, it was found that the dissociation of free cyanide from ferrocyan could be affected by a decrease in the pH of the medium. (In the present specification and drawings, LB is an LB medium composed of three basic components in the medium shown in Table 1, Ecoli is E. coli, Glc is glucose, Lac is lactose, and Suc is sucrose.)

  In addition, the effect of using lactose or sucrose instead of glucose was also investigated. As a result, as shown in FIG. 3, it was found that even when lactose was used, the generation of free cyanide was observed, and as shown in Table 2, the pH of the medium was lowered. In particular, it can be seen that the amount of free cyanide produced based on glucose and lactose is large, and in the case of glucose, no decrease in the generation rate of free cyanide is observed particularly over a long period of time.

  Next, it was confirmed that the generation of free cyan was influenced by a decrease in the pH of the medium.

  Each of those inoculated with E. coli with 20 mL of pH-adjusted LB medium shown in Table 3 (control) was anaerobically cultured (Ar-100%, 30 ° C.). Thereafter, the free cyanide concentration was measured by liquid chromatography.

As a result, free cyanide dissociation from ferrocyan was observed in the LB medium having a low pH without inoculation with E. coli. Therefore, it can be concluded that the free cyanide dissociation from ferrocyan has no direct relationship with the activity of microorganisms and occurs when the pH decreases.
However, in the actual treatment to be purified, there is a problem that making the pH acidic with a chemical or the like has a risk of rapid cyan gas generation and costs. In order to avoid this, it can be said that the method of adding glucose to the culture solution and gradually releasing cyanide by the action of microorganisms as described above is effective when considering the safety of work and adverse effects on the environment.

[Preparation of cyanidase-expressing E. coli]
Cyanidase, Rhodanesee, Glutathione S-transferase (GST) A gene encoding a protein was incorporated into an expression vector (pET26b) and expressed in Escherichia coli BL21 (DE).

  First, a microorganism having the above enzyme is searched from a DNA sequence database, Pseudomonas stutzeri ATCC17588 as a microorganism having a gene encoding rhodanese, Pseudomonas putida KT2440, a gene encoding cyanidase as a microorganism having a gene encoding GST As a microorganism having, Bacillus pumilus NBRC 12092 was obtained, and the target gene was amplified from the genome by PCR to obtain a cyanolytic gene. Each gene was incorporated into the expression vector pET26b and transformed into E. coli BL21 (DE3) to prepare each microorganism expressing free cyanide degrading enzyme.

  When three types of microorganisms expressing free cyanolytic enzymes were cultured and subjected to SDSPAGE under the conditions of 12% polyacrylamide gel, 150 V, and 1 hr, it was confirmed that cyanogen degrading enzyme proteins were produced (FIG. 4). reference). In SDSPAGE, cyanidase (Cyanidase) is detected at a position corresponding to 37.3 kDa, rhodanese (Rhodanese) at 29.8 kDa, and GST is detected at a position corresponding to 24.4 kDa.

  Further, when each cyanide-degrading enzyme-expressing microorganism was measured for cyanide-degrading activity, as shown in FIG. 5, E. coli CDH-pET26b-Escherichia coli BL21 (DE) (accession number NITE P-1278) expressing cyanidase was used. ) (Hereinafter, cyanidase-expressing E. coli) has a free cyanolytic activity. In FIG. 5, those with cyanidase 0.1 mL and 1 mL mean those tested using culture solutions 0.1 mL and 1 mL of cyanidase-expressing E. coli cultured in Table 4.

[Soil purification method]
When the iron cyano complex compound contained in the soil to be purified is removed using the cyanidase-expressing E. coli, a glucose-containing medium and cyanidase-expressing E. coli are added to the soil interstitial water in the soil to be purified, Cyan liberation process is performed in which cyanogen is released to soil interstitial water in the soil to be purified by existing anaerobic microorganisms. Cyanide liberated in the cyan liberation process is degraded by cyanidase-expressing Escherichia coli by microorganisms expressing free cyanide degrading enzyme. By performing the cyan decomposition process, the purification target soil can be purified in situ. A glucose-assimilating anaerobic microorganism can also be added together with the glucose-containing medium. Further, cyanidase-expressing Escherichia coli can maintain free cyanide-degrading activity by periodically supplementary supplementation during the period during which the soil to be purified is being purified.

[Verification experiment]
A verification experiment was conducted to determine whether ferrocyanin could be completely decomposed by a combination of glucose-containing medium + cyanidase-expressing E. coli.

  Cyanidase-expressing Escherichia coli is added (CDH) to ferrocyanine-containing glucose nutrient medium (1% glucose nutrient medium, 0.5 mM potassium ferrocyanide) with time (twice a week, indicated by arrows in the figure). As a negative control, an experiment was also performed in parallel with the addition of cyanidase non-expressing E. coli. Measure all cyan once a week.

  As a result, it became like FIG.

From FIG. 6, it can be seen that the total cyan content tends to decrease as the addition of cyanidase-expressing Escherichia coli continues, but the control hardly changes. However, from FIG. 7 and FIG. 8, since ferrocyanine tends to decrease and free cyanide also increases in the case of control, decomposition proceeds to free cyanide even in the case of a hardly decomposable iron cyano complex compound. It has become clear.
In the case of addition of Cyanidase-expressing Escherichia coli, about 90% reduction of ferrocyanine and about 50% reduction of total cyanide were observed in one month. On the other hand, almost no accumulation of free cyanide was observed. Thus, about half of the free cyanide produced by ferrocyan degradation is degraded by cyanidase-expressing E. coli.

  According to the present invention, it can be used to purify contaminated soil, which has conventionally been difficult to purify, in situ.

Claims (5)

Add glucose to water containing iron cyano complex compound,
The iron-cyano complex compound-containing water to which glucose is added is decomposed by an anaerobic microorganism utilizing glucose, thereby generating free cyanide,
The free cyanide is decomposed by a free cyanide-degrading enzyme-expressing microorganism;
A method for treating water containing an iron cyano complex compound.   The method for treating water containing an iron cyano complex compound according to claim 1, wherein the microorganism expressing free cyanide degrading enzyme is a genetically modified microorganism into which a gene encoding a cyanidase protein is incorporated.   The method for treating water containing an iron cyano complex compound according to claim 2, wherein the gene encoding the cyanidase protein is a gene encoding cyanidase derived from Bacillus pumilus NBRC12092. In the soil purification method of removing the iron cyano complex compound contained in the soil to be purified using microorganisms,
Adding cyanide and glucose-assimilating anaerobic microorganisms to the soil pore water in the soil to be purified, and releasing cyan in the soil pore water in the soil to be purified; and
A cyanide decomposing step of decomposing cyan liberated in the cyan liberating step with a free cyanolytic enzyme-expressing microorganism,
A soil purification method performed at an original position of the soil to be purified.   The soil purification method according to claim 4, wherein the microorganism expressing free cyanide degrading enzyme is a genetically modified microorganism into which a gene encoding cyanidase protein is incorporated.