WO2013157556A1 - Decomposition accelerator for volatile organic halogen compound - Google Patents

Description

Decomposition accelerator for volatile organic halogen compounds

The present invention relates to a decomposition accelerator for volatile organic halogen compounds and a decomposition acceleration method, and more specifically, a decomposition accelerator capable of promoting the decomposition of volatile organic halogen compounds by microorganisms, and the decomposition acceleration using the same. Regarding the method.

Land soil and groundwater may be contaminated with various natural or artificial chemicals. Such contamination of the land becomes a serious problem not only when the land is used as farmland for food production, but also when it is intended for residence or commercial use.

However, unlike foods and drinks that are directly in the mouth and clothing, cosmetics, and accessories that come into contact with the skin, land contamination, especially soil contamination, is not paid much attention. There were also cases where it was processed.
Such purification of land pollution has recently become a major problem, and various purification treatment methods have been proposed and implemented.

This land purification method can be roughly classified into physical treatments such as decomposition of chemical substances by high temperature heating and adsorption on activated carbon, chemical treatment to detoxify chemical substances by chemical reaction, and the ability to decompose chemical substances. It can be classified into four types: a microorganism purification method using microorganisms, and a plant purification method using plants having the ability to absorb or adsorb chemical substances.

Among them, the method using microorganisms and plants is attracting attention recently because it can reduce environmental considerations and costs. The purification method using microorganisms is “bioremediation”, and the purification method using plants. Is said to be “phytoremediation”, and various research and development are being carried out, and because the volume of soil that can be treated is large, research and development of bioremediation has become active.

There are two technologies for bioremediation. One is a technique for applying to a contaminated place a microorganism that has been confirmed in advance to exhibit an effect on the degradation of the target pollutant, and is called bioaugmentation. The other is a technique that activates the action of microorganisms by providing oxygen and nutrients to the indigenous microorganisms in the contaminated area and promotes the purification action, and is called biostimulation.

There are two methods of bioremediation. One is a method of removing contaminated soil and groundwater and treating it at another location, which is called “facility-type treatment”. The other is a method of purifying soil and groundwater contaminated at the site, which is called “in-situ purification”.

Organic pollutants represented by organic chlorine compounds such as tetrachloroethylene, trichloroethylene, dichloroethylene, dioxins, polychlorinated biphenyls and the like are pollutants that have become a major problem in recent years. In particular, volatile organic halogen compounds such as tetrachloroethylene, trichlorethylene, and dichloroethylene are often concerned with the effects on the human body through breathing, so that there are many occasions where immediate action is required.

This volatile organic halogen compound easily penetrates into the soil, reaches the groundwater vein, and easily spreads contamination over a wide area. There are soil microorganisms that decompose volatile organohalogen compounds, but generally, the closer to the surface, the more organic matter and soil microorganisms exist, but the organic matter and soil microorganisms decrease from the surface to the depth. When the depth is 1 m or more, the activity of the microorganisms is reduced to 1/100 or less of the surface layer. Under these circumstances, there is a problem that land once contaminated with volatile organic halogen compounds remains contaminated over a long period of time with a relatively low concentration.

Since such remediation of land contaminated widely at a relatively low concentration is an effective means of bioremediation, various proposals have been made for the purpose of remediation of contamination by volatile organic halogen compounds.

For example, biostimulation using a composition containing an ester of polylactic acid and a polyfunctional alcohol such as glycerin, xylitol, sorbitol, or pentaerythritol (see, for example, Patent Document 1), a composition containing yeast, fatty acid, carbohydrate, etc. Biostimulation (for example, refer to Patent Document 2) using a biotin, and biostimulation (for example, refer to Patent Document 3) using a condensation reaction product of an amino acid and oxycarboxylic acid have been proposed. Non-Patent Document 1 describes a method in which various degradation promoters are brought into contact with soil and / or groundwater in which microorganisms are present.

On the other hand, anaerobic bacteria, in particular, Dehalococcides spp. Are known as microorganisms used for the purification of land contaminated with volatile organic halogen compounds. In the absence of these microorganisms, volatile organic halogen compounds are not decomposed to the final ethylene, and may be stopped by dichloroethylene, which is an intermediate substance. It is possible that nothing will happen. However, even if it is a bacterium belonging to the genus Dehalococcides, a compound that can be decomposed is usually determined depending on the type, and several types of bacteria belonging to the genus Dehalococcides are involved in the degradation from tetrachloroethylene to ethylene. It is also known that (See Non-Patent Document 2) Therefore, the degradation rate of various volatile organohalogen compounds of the genus Dehalococcides is not always fast, and the volatile organohalogen compounds are present even if the genus Dehalococcides is present. It is not necessarily involved in the degradation of.

Therefore, bioaugmentation using a mixed strain of a plurality of Dehalococcides bacteria (see, for example, Non-Patent Document 3) or a consortia whose main cells are Dehalococcides bacteria and streptococcus are used. Bioaugmentation (see, for example, Patent Document 4) has been proposed. However, the method using this mixed strain also has a problem that the purification rate becomes slow depending on the soil contamination status, pH, and organic content.

Special Table 2000-511969 JP 2005-185870 A JP 2010-104962 A JP 2011-244769 A

Industrial Research Co., Ltd. “Chemical Equipment” July 2007 issue, Hiroshi Yamazaki “Soil and Groundwater Purification Technology-VOC Decomposition and Purification Technology” Sakihara Mori et al. “Analysis of behavior of bacteria belonging to the genus Dehalococides in in situ biomediation for chloroethenes” Lecture Meeting on Groundwater / Soil Contamination and Prevention (2008) Osamu Yagi “Current Status and Future Prospects of Land Restoration Technology” “In Search of Food and Environmental Safety – Hazardous Chemical Substances in Agriculture, Forestry and Fisheries Ecosystems – Summary”, page 10 (2007)

However, the methods described in Patent Document 1 and Patent Document 3 have low activity of microorganisms in the initial administration stage, and therefore, the purification treatment rate is low particularly in contaminated soil containing a large amount of volatile organic halogen compounds, especially groundwater, There has been a problem that the time required for detoxification becomes longer. In addition, the method described in Patent Document 2 has an effect of promoting the decomposition reaction of volatile organic halogen compounds, but the activation of microorganisms is still insufficient, and there is a problem that the time required for detoxification is long. It was. On the other hand, the method using a mixed strain as disclosed in Non-Patent Document 3 and Patent Document 4 also has a problem that the purification rate may be slow depending on the soil contamination status, pH, and organic content. there were.

Accordingly, an object of the present invention is a decomposition accelerator used for purification (bioremediation) of microorganisms in a land contaminated with volatile organic halogen compounds, and in particular, by increasing the activity of microorganisms at the initial administration stage, An object of the present invention is to provide a decomposition accelerator capable of quickly detoxifying an organic halogen compound.

In addition, the purpose of the present invention is to improve the activity of microorganisms at the initial stage of administration, particularly when purifying the soil contaminated with volatile organic halogen compounds by microorganisms (bioremediation). It is an object of the present invention to provide a method for promoting the decomposition of volatile organic halogen compounds by microorganisms, which can be rendered harmless quickly.

As a result of various studies to achieve the above object, the present inventors have found that when one or more of the following (A) to (C) are used as a decomposition accelerator for volatile organic halogen compounds, The present inventors have found that the decomposition of volatile organic halogen compounds by microorganisms is effectively promoted, and have completed the present invention.

That is, the decomposition accelerator for volatile organic halogen compounds of the present invention is characterized by containing one or more of the following (A) to (C).
(A) Citrus fruit or extract obtained from the fruit (B) Citrus fruit skin or extract obtained from the fruit skin (C) Formulation containing all of the following (1) to (3) (1) Glycerin (2) Milk protein and / or yeast extract (3) Vitamin B12

In addition, when the decomposition accelerator of the present invention contains at least one of (A) and (B), the extract is preferably an extract obtained using water as a solvent.

When the decomposition accelerator of the present invention contains (C), 0.1 to 3 parts by mass of the milk protein and / or yeast extract of (2) as a solid content with respect to 1 part by mass of glycerin of (1) It is preferable to contain.
Further, when the decomposition accelerator of the present invention contains (C), it contains 0.00001 to 0.001 parts by mass of the vitamin B12 of (3) with respect to 1 part by mass of glycerin of (1). Preferably there is.

The decomposition accelerator composition of the present invention is characterized by containing a decomposition accelerator as an active ingredient.

The method for promoting the decomposition of a volatile organic halogen compound by a microorganism according to the present invention comprises contacting any one of the above decomposition accelerators or the above decomposition accelerator composition with soil and / or groundwater containing the volatile organic halogen compounds. It is characterized by making it.

In the method of the present invention, the volatile organic halogen compound is preferably an organic chlorine compound.

In the method of the present invention, the organochlorine compound is carbon tetrachloride, chloroform, dichloromethane, monochloromethane, 1,2-dichloroethane, 1,1-dichloroethylene, cis-1,2-dichloroethylene, trans-1, It is preferably at least one selected from the group consisting of 2-dichloroethylene, 1,3-dichloropropene, tetrachloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, and vinyl chloride.

In the method of the present invention, the microorganism is at least one member selected from the group consisting of Clostridium genus bacteria, Dehalobacter genus bacteria, Dehalococcoides genus bacteria, Dehalospirilum genus bacteria, Desulfobacterium genus bacteria, Desufomonas genus bacteria, and Desulfomonile genus bacteria. Preferably there is.

The decomposition accelerator of the present invention is easy to handle, and can be used for the purification (bioremediation) of microorganisms contaminated with volatile organic halogen compounds, thereby quickly detoxifying contaminated soil and groundwater. It is possible and it is inexpensive and not burdened on the environment.
In addition, the method of the present invention can promote the purification of soil and groundwater contaminated with volatile organic halogen compounds by microorganisms at low cost and without burdening the environment.

FIG. 3 is a graph showing changes in c-DCE, VC, and ethylene content in Example 1. FIG. 6 is a graph showing changes in c-DCE, VC, and ethylene content in Example 2. FIG. 6 is a graph showing changes in c-DCE, VC, and ethylene content in Comparative Example 1. 6 is a graph showing changes in c-DCE, VC, and ethylene content in Comparative Example 2. FIG. FIG. 9 is a graph showing the transition of trichlorethylene, c-DCE, VC, and ethylene content in Example 7. FIG. 6 is a graph showing the transition of trichlorethylene, c-DCE, VC, and ethylene amount in Comparative Example 3. In Example 15, it is a graph showing transition of the amount of trichlorethylene, dichloroethylene, vinyl chloride, and ethylene.

Hereinafter, the decomposition accelerator for volatile organic halogen compounds of the present invention will be described in detail. In the present specification, the decomposition of a volatile organic halogen compound means dehalogenation of a volatile organic halogen compound.
The decomposition accelerator for volatile organic halogen compounds of the present invention is characterized by containing one or more of the following (A) to (C).
(A) Citrus fruit or extract obtained from the fruit (B) Citrus fruit skin or extract obtained from the fruit skin (C) Formulation containing all of the following (1) to (3) (1) Glycerin (2) Milk protein and / or yeast extract (3) Vitamin B12

First, the component (A) and the component (B) will be described.
The kind of the citrus fruits of the component (A) and the component (B) is not particularly limited and may be any plant as long as it belongs to the citrus family, citrus genus or genus Kumquat. Plants produced by crossing using citrus or kumquat are preferred. Specific examples of citrus fruits are, for example, Valencia orange, Navel, Blood orange, Grapefruit, Lemon, Yuzu, Lime, Wenzhou orange, Yawata, Sweet summer, Bundan, Kinkan, Tachibana, and more There are tangoles such as Taiyo, Kiyomi, Shiranui, and Tan Zero such as Seminole and Mineola. Among these, it is preferable to use at least one of Valencia orange, grapefruit, lemon, and Satsuma mandarin since it is easily available and available in large quantities at low cost.

In the above component (A) and component (B), the whole citrus fruit or a part of the fruit may be used. However, since the decomposition promoting effect is high, the skin is preferable.

The form of the component (A) and the component (B) is not particularly limited. For example, fruit, pericarp or flesh as it is, fruit, pericarp or flesh dried, fruit, pericarp or flesh Examples include those dispersed in water, fruits, fruit skins and pulps, fruit juices, and the like. Moreover, the extract extracted with water, such as warm water thru | or hot water, from the fruit, especially fruit peel, polar solvents, such as ethanol, acetone, ethyl acetate, and nonpolar solvents, such as hexane, may be sufficient.

Among them, an extract extracted from the skin with water such as warm water or hot water is preferable in that it has an immediate effect when applied to soil and a high effect can be obtained. The temperature of water during extraction is preferably 30 to 100 ° C., more preferably 60 to 95 ° C., and still more preferably 60 to 80 ° C.

The extraction / separation apparatus used for the extraction may be any apparatus that can efficiently acquire the extract constituting the decomposition accelerator for the volatile organic halogen compound of the present invention. For example, a continuous centrifugal apparatus, a membrane separation apparatus, a supercritical apparatus An extraction device etc. can be mentioned.

In addition, the citrus peel is produced as a by-product in the production of primary processed citrus fruits (juice, canned sweets, etc.), and has been mostly discarded without promising use. The use of volatile organic halogen compound decomposition accelerators as a raw material in this way not only can provide products at a lower cost than the conventionally known volatile organic halogen compound decomposition accelerators, but also effectively use resources. It is also meaningful from the aspect.

Furthermore, the extract as one form of the component (A) and the component (B) may be extracted from a residue obtained by extracting components such as pectin, aroma component, pigments, and hesperidin from fruits. Other components may be included.

Citrus fruits have been edible for a long time, and the decomposition accelerator for volatile organic halogen compounds of the present invention using this as a raw material is highly safe and relatively stable to heat. It is easy to handle.

As the above components (A) and (B), among the citrus fruits, the peel particularly exhibits high decomposition promoting effects, and the effect of the extract obtained by water extraction such as warm water and hot water is high. Therefore, although details are not necessarily clear, as one possibility, the component contained in the citrus fruit that brings about the effect of the decomposition accelerator of the present invention may be a mixture of water-soluble saccharides, salts, and organic acids. Conceivable.

Next, the component (C) will be described.
The component (C) is a blend containing all of the following (1) to (3).
(1) Glycerin (2) Milk protein and / or yeast extract (3) Vitamin B12

The glycerin used in the blend which is the component (C) is a microbial carbon source and a hydrogen source for substituting the chlorine atom of the organochlorine compound, that is, a hydrogen donor. Alternatively, it may be in the form of glyceride in which 1 to 3 fatty acids are bonded, but preferably glycerin itself is used. When using commercially available glycerin, the purity is not limited to 100% or more than 99% (for example, reagent grade), but glycerin from Japan Pharmacopoeia (purity 80-90%), purified glycerin D, food additive glycerin Further, concentrated glycerin for cosmetics (both manufactured by Lion Corporation) can be used.

In the compound which is said (C) component, milk protein and / or a yeast extract are used as a nitrogen source of microorganisms.
The milk protein may be either whey protein only, casein protein only, or a combination of casein protein and whey protein, but it is more preferable to use whey protein and casein protein in combination.
The milk protein is preferably water-soluble. When using a commercially available product, any product that contains milk protein at a high concentration and that is harmless to the human body for food and cosmetics (or that does not significantly inhibit the growth of microorganisms) may be used. For example, sodium caseinate, potassium casein, whey powder, WPC (whey protein concentrate), WPI (whey protein isolate), total milk protein (TMP), protein concentrated whey powder, whole milk powder, skim milk powder, lactose whey Non-lactose whey powder, buttermilk powder, sweetened powdered milk, prepared milk powder, milk protein concentrate (MPC) and the like. In the present invention, total milk protein (TMP) and / or nonfat dry milk is preferable, and nonfat dry milk is more preferable in terms of low lipid content and high storage stability.

The yeast extract is an extract extracted by subjecting a yeast culture to treatment such as autolysis, enzyme, hot water, physical disruption, acid decomposition, alkali decomposition, and freeze-thaw method. The kind of yeast used for the production of the yeast extract is not particularly limited, and baker's yeast, brewer's yeast, wine yeast, torula yeast and the like can be used without particular limitation. Among them, yeast belonging to the genus Saccharomyces is preferably used. The yeast extract may be pasty, powdery or granular.

In the present invention, among the milk protein and yeast extract, only the milk protein may be used, or only the yeast extract may be used, but preferably only the milk protein is used, or more preferably the milk protein. And yeast extract.
Here, the mixing ratio when used in combination is preferably 0.1 to 2 parts by mass, more preferably 0.3 to 1 part by mass, based on 1 part by mass of milk protein, as yeast solid content.

In the blend which is the component (C), the content ratio of the glycerin (1), the milk protein (2) and / or the yeast extract of (2) is as described in (2) above with respect to 1 part by mass of the glycerin (1). The milk protein and / or yeast extract is preferably 0.1 to 3 parts by mass, more preferably 0.1 to 1 part by mass as a solid content.

Vitamin B12 is used in the blend which is the component (C).
Vitamin B12 is a generic term for vitamins containing cobalt, and there are hydroxocobalamin, adenosylcobalamin, methylcobalamin, cyanocobalamin, sulfitocobalamin, and the like, which are water-soluble vitamins, and any of them can be used in the present invention. it can.
In the present invention, the purified product may be used, or a food containing a large amount of vitamin B12 may be used. For example, vitamin B12 is abundant in the liver of seaweed, shellfish and animal foods.

In the composition which is the component (C), the content ratio of the glycerin (1) and the vitamin B12 (3) is preferably the vitamin B12 (3) with respect to 1 part by mass of the glycerin (1). Is 0.00001 to 0.001 parts by mass, more preferably 0.00002 to 0.0001 parts by mass.

The decomposition accelerator for volatile organic halogen compounds of the present invention can contain other components other than the above-mentioned component (A), component (B), and component (C).
Examples of the other components include glucose, fructose, ammonium sulfate, urea, ammonium salts, sulfur compounds, phosphorus compounds, potassium compounds such as potassium chloride, magnesium compounds such as magnesium chloride and magnesium sulfate, yeast You may use with extract or peptone. Moreover, it is good also as a decomposition accelerator composition which added the appropriate amount of the said additive with respect to the decomposition accelerator of this invention. When the decomposition accelerator composition is used, the amount of each additive is not particularly limited. For example, when using powdered yeast extract or fructose, the solid content of the fruit extract is 100 parts by mass. The solid content is preferably 1 to 200 parts by mass, more preferably 10 to 100 parts by mass.
However, when the component (C) is used as the present invention, as the above-mentioned other components, components that can be nutrients of microorganisms, such as glucose, fructose, ammonium sulfate, urea, ammonium salts, sulfur compounds, phosphorus compounds, potassium chloride, etc. It is preferable that no potassium compound, magnesium compound such as magnesium chloride or magnesium sulfate, or peptone is contained. In particular, when sulfate-reducing bacteria are present in soil or groundwater, they compete with the sulfate-reducing bacteria in the presence of sulfate ions, so volatile organic halogen compounds will not be decomposed, so sulfates such as ammonium sulfate and magnesium sulfate Is preferably not included.

The form of the decomposition accelerator for the volatile organic halogen compound of the present invention is not particularly limited, and various forms such as solid (including powder and granules) and liquid (including paste) can be adopted. Moreover, it can also be used in the state diluted with solvents, such as water.

The decomposition accelerator of the present invention promotes the decomposition of volatile organic halogen compounds by microorganisms by contacting with soil, groundwater, and other samples contaminated with volatile organic halogen compounds. The volatile organic halogen compound that is the subject of the present invention is preferably an organic chlorine compound such as carbon tetrachloride, chloroform, dichloromethane, monochloromethane, 1,2-dichloroethane, 1,1-dichloroethylene, cis- Examples include 1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,3-dichloropropene, tetrachloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, vinyl chloride, and the like.

Especially, the decomposition accelerator of this invention can accelerate | stimulate suitably decomposition | disassembly of chloroethenes, such as tetrachloroethylene, trichloroethylene, dichloroethylenes, vinyl chloride.
For example, tetrachloroethylene is sequentially decomposed by microorganisms into trichlorethylene, dichloroethylene, monochloroethylene (vinyl chloride), and ethylene.

The decomposition accelerator of the present invention accelerates the decomposition of volatile organic halogen compounds by microorganisms, and may utilize microorganisms originally present in soil or groundwater to be purified, and decomposes volatile organic halogen compounds. It may be used together with useful microorganisms. Moreover, you may use with the composition containing such microorganisms. That is, when the soil or groundwater to be purified contains sufficient microorganisms that decompose volatile organic halogen compounds, the decomposition accelerator or decomposition accelerator composition of the present invention may be applied to the target soil as it is. On the other hand, when the amount of microorganisms in the soil is small or when it is desired to accelerate decomposition, the decomposition accelerator or decomposition accelerator composition of the present invention may be applied together with a microorganism or a composition containing microorganisms prepared in advance. .

An anaerobic microorganism is preferable as a microorganism useful for decomposing volatile organic halogen compounds, and examples include microorganisms such as Clostridium genus, Dehalobacter genus, Dehalococcoides genus, Dehalospiririum genus, Desulfobacterium genus, Desulfomonas genus, and Desulmonas genus.

When using the decomposition accelerator of the present invention, it is preferable to measure in advance the abundance of anaerobic microorganisms, for example, Dehalococides bacteria in a sample containing a volatile organic halogen compound. A known method such as a real-time PCR method can be used for quantifying Dehalococcides genus bacteria (see, for example, Non-Patent Document 1).

The method of bringing the decomposition accelerator of the present invention into contact with soil and / or groundwater is not particularly limited, and may be “facility-type treatment” in which contaminated soil or groundwater is removed and treated at another place, In-situ purification may be used to purify contaminated soil and groundwater at the site, but microorganisms that decompose volatile organic halogen compounds exert their effects under anaerobic conditions to the fullest extent. It is preferable that it is position purification.

In the case of institutional purification, there is no particular limitation on the method of contacting the soil and / or groundwater in which the microorganism of the present invention is present, for example, a method in which excavated contaminated soil is piled up and directly injected into this, Examples thereof include a method of mixing and stirring with soil, and a method of adding water to contaminated soil as a fluid or liquid form.

In the case of in-situ purification, there is no particular limitation on the method of contacting the soil and / or groundwater in which the microorganism of the present invention is present, for example, a method of burying directly in soil, an injection well in groundwater or soil. It may be a direct injection method in which injection is performed or a method using a permeable reaction purification wall utilizing the flow of groundwater, but the direct injection method is preferred.
It should be noted that the supply amount of the decomposition accelerator of the present invention is not limited as long as a sufficient purification effect can be obtained, and should be determined in advance by confirming the range of the contaminated area, the degree of contamination, the type of contaminants, etc. It ’s fine.

<Decomposition experiment 1 of chloroethenes>
[Production of decomposition accelerator 1]
<Manufacture of citrus extract>
[Production Example 1]
The Satsuma mandarin orange was washed well with water, peeled off, and the peel (dry weight 100 g) was pulverized with a disc mill, followed by stirring and extraction with 2000 ml of 60 ° C. warm water for 1 hour. This was filtered, and the filtrate was concentrated with a rotary evaporator and then dried with a vacuum drier to obtain a hot water extract (about 10 g) of Unshu mandarin orange peel. The obtained pericarp warm water extract was directly used as the decomposition accelerator A of the present invention.

[Production Example 2]
50 parts by mass of powdered yeast extract and 50 parts by mass of fructose were added to 100 parts by mass of the pericarp warm water extract obtained in Production Example 1 and mixed homogeneously, and this was used as the decomposition accelerator B of the present invention.

[Production Example 3]
The decomposition accelerator C of the present invention was obtained in the same manner as in Production Example 1 except that hot water of 98 ° C. was used instead of hot water of 60 ° C. used for extraction.

[Production Example 4]
A decomposition accelerator D of the present invention was obtained in the same manner as in Production Example 1 except that Valencia orange was used instead of Wenzhou orange.

[Production Example 5]
A decomposition accelerator E of the present invention was obtained in the same manner as in Production Example 1, except that grapefruit was used instead of Wenzhou mandarin.

[Production Example 6]
A decomposition accelerator F of the present invention was obtained in the same manner as in Production Example 1 except that lemon was used instead of Wenzhou mandarin.

[Evaluation Method 1 for Decomposition Accelerator]
<Creation of bacterial fluid>
(About decomposition accelerators A to F)
50 mL of a medium obtained by adding yeast extract to 0.1 g / L to the mineral base medium shown below was taken in a 100 mL glass vial, purged with nitrogen, sterilized with an autoclave, and then contaminated with chloroethenes. After adding 25 mL of groundwater collected from the soil and substituting with nitrogen, 2.5 mL of hydrogen and 0.58 μL of cis-1,2-dichloroethylene (corresponding to 10 mg / L) were enclosed, and the mixture was statically cultured at 20 ° C. in the dark. Chloroethenes in the headspace were measured periodically, and 1 mL was collected when chloroethenes were no longer detected, and was transferred to 75 mL of an autoclaved mineral basal medium supplemented with 0.1 g / L of yeast extract. This subculture was performed 6 times as a “bacterial fluid” and used for the following chloroethene degradation experiment.

<Manufacture of mineral basal medium (pH 7.0-7.5)>
10 ml of the following salt stock solution, 1 ml of the following trace element solution A, 1 ml of the following trace element solution B, 50 μl of resazurin sodium solution (0.5% w / v), 0.1 g of sodium acetate, L-cysteine 0.3 ml of hydrochloride monohydrate, 2.52 g of sodium hydrogen carbonate and 0.048 g of sodium sulfide nonahydrate were filled up to 1000 ml, and this was used as a mineral basal medium.

<Manufacture of salt stock solution>
The following components were dissolved in water and filled up to 1000 ml to obtain a salt stock solution.
100 g NaCl, 50 g MgCl ₂・ 6H ₂ O, 20 g KH ₂ PO ₄ , 30 g NH ₄ Cl, 30 g KCl, 1.5 g CaCl ₂・ 2H ₂ O

<Manufacture of trace element solution A>
The following components were dissolved in water and filled up to 1000 ml to obtain Trace element solution A.
10 mL HCl (25% solution, w / w), 1.5 g Fe Cl ₂・ 4H ₂ O, 0.19 g CoCl ₂・ 6H ₂ O, 0.1 g MnCl ₂・ 4H ₂ O, 70 mg Zn Cl ₂ , 6 mg H ₃ BO ₃ , 36 mg Na ₂ MoO ₄・ 2H ₂ O, 24 mg NiCl ₂・ 6H ₂ O, 2 mg CuCl ₂・ 2H ₂ O

<Manufacture of trace element solution B>
The following components were dissolved in water and filled up to 1000 ml to obtain Trace element solution B.
6mgNa ₂ SeO ₃・ 5H ₂ O, 8 mg Na ₂ WO ₄・ 2H ₂ O, 0.5 g NaOH

<Decomposition experiment 1 of chloroethenes>
[Example 1]
Assuming groundwater contaminated with chloroethenes, a decomposition test was conducted by the following method.
75 ml of the mineral basal medium was put in a glass 100 ml vial, the decomposition accelerator A was added to a concentration of 0.1 g / L, sterilized by autoclaving after nitrogen substitution. After cooling, 1.5 ml of the above bacterial solution was added, and after substituting with nitrogen, cis-1,2-dichloroethylene (c-DCE) was sealed at 10 μg / ml.
This vial was subjected to stationary culture at 20 ° C. 0, 3, 10, 18, 24, 36, 45, 49, 59, 66, 75, 84, 87 days later cis-1,2-dichloroethylene (c-DCE) content in the headspace of the vial, vinyl chloride The (VC) content and ethylene content were measured by gas chromatography.

Regarding the experimental results, first, the fluctuation of cis-1,2-dichloroethylene (c-DCE) content, vinyl chloride (VC) content, and ethylene content from day 0 to 87 is shown in FIG.
In addition, since the activation of microorganisms is considered to occur in the early stage, the amount of decrease in the c-DCE content per day until the 18th day is shown in Table 1 as the initial degradation rate.

[Example 2]
A decomposition experiment of chloroethenes was conducted in the same manner as in Example 1 except that the decomposition accelerator B was added to 0.2 g / L instead of the decomposition accelerator A 0.1 g / L, and the results are shown in FIG. The results are shown in Table 1.

[Comparative Example 1]
A decomposition experiment of chloroethenes was conducted in the same manner as in Example 1 except that 0.1 g / L of the decomposition accelerator A was not added, and the results are shown in FIG.

[Comparative Example 2]
A decomposition experiment of chloroethenes was conducted in the same manner as in Example 2 except that 0.2 g / L of the decomposition accelerator B was not added, and 0.05 g / L of yeast extract and 0.05 g / L of fructose were added. 4 and Table 1.

Example 3
The decomposition experiment of chloroethenes was conducted in the same manner as in Example 1 except that the decomposition accelerator C was added to 0.1 g / L instead of the decomposition accelerator A 0.1 g / L. However, sampling is performed until the 0th, 3rd, 10th, and 18th days, and the amount of decrease in the c-DCE content per day up to the 18th day is calculated in the same manner as in Example 1, and the initial degradation rate is shown in Table 1. It was shown to.

Example 4
A decomposition experiment of chloroethenes was conducted in the same manner as in Example 1 except that the decomposition accelerator D was added to 0.1 g / L instead of the decomposition accelerator A 0.1 g / L. The sampling was performed in the same manner as in Example 3, and only the initial decomposition rate was calculated. The results are shown in Table 1.

Example 5
The decomposition experiment of chloroethenes was conducted in the same manner as in Example 1 except that the decomposition accelerator E was added so as to be 0.1 g / L instead of the decomposition accelerator A 0.1 g / L. The sampling was performed in the same manner as in Example 3, and only the initial decomposition rate was calculated. The results are shown in Table 1.

Example 6
A decomposition experiment of chloroethenes was conducted in the same manner as in Example 1 except that the decomposition accelerator F was added to 0.1 g / L instead of the decomposition accelerator A 0.1 g / L. The sampling was performed in the same manner as in Example 3, and only the initial decomposition rate was calculated. The results are shown in Table 1.

※単位＝（μｇ／ｌ／ｄａｙ）

* Unit = (μg / l / day)

As is clear from the results of Comparative Example 1 (FIG. 3), in the case of only the basic medium, c-DCE remains even on the 87th day, VC also tends to increase, and generation of ethylene is not observed. In particular, the decomposition of chloroethenes hardly proceeded.

On the other hand, as is clear from the results of Example 1 (FIG. 1) and Example 2 (FIG. 2), in the sample to which the decomposition accelerator of the present invention was added, c-DCE was almost decomposed on the 50th day, On day 87, both c-DCE and VC were almost completely decomposed.

As is clear from the comparison between Example 2 (FIG. 2) and Comparative Example 2 (FIG. 4), the nutritional component of the present invention was compared with the sample using yeast extract and fructose, which are conventional nutrients. By adding an additional decomposition accelerator, c-DCE was decomposed more rapidly, and the decomposition rate increased particularly on the 18th to 50th days. Similarly, since the day when VC shows the maximum concentration is from the 59th day (Comparative Example 2, FIG. 4) to the 24th day (Example 2, FIG. 2), the degradation promoter of the present invention is used as a conventional nutrient. It was confirmed that the initial decomposition rate can be greatly increased by additionally adding.

Further, as is apparent from Table 1, it can be seen that the initial decomposition rate is greatly increased by adding the decomposition accelerator of the present invention.
A sample using yeast extract and fructose, which are conventional nutrients, has a certain degradation rate improving effect, but as is clear from comparison between Examples 1, 3 to 6 and Comparative Example 2, The decomposition acceleration effect was higher when the decomposition accelerator was used. Furthermore, it can be seen from the results of Example 2 that the initial decomposition rate is greatly increased by additionally adding the decomposition accelerator of the present invention.

[Example 7 and Comparative Example 3]
Assuming land contaminated with chloroethenes, a decomposition test was conducted by the following method.
Put 700g of soil collected from land contaminated with trichlorethylene and 300g of groundwater collected from the same place into a 1L screw mouth bottle, add 5mL of the above bacterial solution, and then add 0.2g of degradation accelerator A and yeast extract 0 .2 g was dissolved in 50 mL of distilled water, added, and purged with nitrogen, followed by stationary culture at room temperature in the dark. Periodically, the concentration of chloroethenes (trichlorethylene, c-DCE, VC) and the concentration of ethylene in the head space were measured by gas chromatography. All chloroethenes, including trichlorethylene, contained in the contaminated soil and groundwater, are environmental standards (trichlorethylene = 0.03 mg / l, dichloroethylene = 0.04 mg / l, VC = 0.002 mg / l) after 120 days. ) It became the following. The results are shown in FIG. Here, when 0.2 g of fructose was added in place of the decomposition accelerator A (Comparative Example 3), VC still remained above the groundwater environmental standard value even after 150 days had passed, and land purification was impossible. there were. The results are shown in FIG.

<Decomposition experiment 2 of chloroethenes>
[Production of decomposition accelerator 2]
[Production Examples 7 to 16]
(1) Glycerin as component, (2) skim milk powder and / or yeast extract powder as component, and vitamin B12 preparation as component (3) were mixed as described in Table 2 to obtain degradation accelerators G to P. Of the obtained decomposition accelerators, G to N and P were pasty, and O was powdery.
The content ratio of (1) :( 2) and the content ratio of (1) :( 3) are also shown in Table 2.
The obtained degradation accelerators G to P were evaluated according to the following degradation accelerator evaluation method, and the results are shown in Table 2.

[Evaluation method 2 of degradation accelerator]
<Creation of bacterial fluid>
50 mL of a medium obtained by adding yeast extract to the above mineral basal medium to a concentration of 0.1 g / L is taken in a 100 mL glass vial, sterilized by autoclaving after nitrogen substitution, and then from soil contaminated with chloroethenes. 25 mL of the collected ground water was added, and after substituting with nitrogen, 2.5 mL of hydrogen and 0.58 μL of cis-1,2-dichloroethylene (corresponding to 10 mg / L) were enclosed, and the mixture was statically cultured at 20 ° C. in the dark. Chloroethenes in the headspace were measured periodically, and 1 mL was collected when chloroethenes were no longer detected, and was transferred to 75 mL of an autoclaved mineral basal medium supplemented with 0.1 g / L of yeast extract. This subculture was performed 6 times (however, the yeast extract was not added only for the 6th time) as “bacterial fluid” and used for the following chloroethene degradation experiment.

<Decomposition experiment 2 of chloroethenes>
[Examples 8 to 14, Comparative Examples 4 to 6]
Assuming groundwater contaminated with chloroethenes, a decomposition test was conducted by the following method.
75 ml of the mineral basal medium was placed in a 100 ml glass vial, the above-mentioned decomposition accelerators G to P were added so as to be 0.3 g / L each, and after sterilization with an autoclave after substitution with nitrogen. After cooling, 1.5 ml of the above bacterial solution was added, and after substituting with nitrogen, cis-1,2-dichloroethylene (c-DCE) was sealed at 10 μg / ml.
This vial was subjected to stationary culture at 20 ° C. Periodically, c-DCE content, VC content, and ethylene content in the vial headspace were measured by gas chromatography. Regarding the experimental results, the number of days until the c-DCE content and the VC content are below the groundwater environmental standard value, that is, the c-DCE is 0.04 mg / L or less and the VC is 0.002 mg / L or less. Is listed in Table 2 as “days required for decomposition”.
The same experiment was conducted as Comparative Example 7 for the case where no decomposition accelerator was added, and the results are shown in Table 2.

As can be seen from the results in Table 2, when the decomposition accelerators of Examples 8 to 14 containing the components (1) to (3) were used, the number of days until the chloroethenes were completely decomposed was 31 days or less. Whereas (1) Comparative Example 4 containing no component is 48 days, (2) Comparative Example 5 containing no component is 45 days, and (3) Comparative Example 6 containing no component is 40 days. It can be seen that the decomposition rate is extremely low.

In the case where no decomposition accelerator was used (Comparative Example 7), the decomposition of chloroethenes was not completed even on the 100th day.

Moreover, the Example which uses skim milk powder rather than Example 11 which uses only a yeast extract so that Example 9, 11, and 12 which uses skim milk powder and / or a yeast extract as a component (2) may be understood. It can be seen that No. 9 has a higher degradation rate, and Example 12 in which skim milk powder and yeast extract are used in combination has the highest degradation rate.

As can be seen by comparing Examples 9, 13, and 14 in which the content of vitamin B12 was varied, the blending amount of vitamin B12 with respect to 1 part by mass of glycerin was within the range of 0.00001 to 0.001 parts by mass. It can be seen that there is no difference in the decomposition rate.

Example 15
Assuming land contaminated with chloroethenes, a decomposition test was conducted by the following method.
Put 700g of soil collected from land contaminated with trichlorethylene and 300g of groundwater collected from the same place into a 1L screw mouth bottle, add 5mL of the above bacterial solution, and then distill 0.5mL of degradation accelerator E by 50mL. After dissolving in water and adding nitrogen, the culture was allowed to stand at room temperature in the dark. Periodically, the concentration of chloroethenes (trichlorethylene, c-DCE, VC) and the concentration of ethylene in the head space were measured by gas chromatography. All the chloroethenes including trichlorethylene contained in the contaminated soil and groundwater are environmental standard values (trichlorethylene = 0.03 mg / l, dichloroethylene = 0.04 mg / l, VC = 0.002 mg / l after 150 days). ) It became the following. The results are shown in FIG.

<Decomposition experiment 3 of chloroethenes>
[Evaluation Method 3 for Decomposition Accelerator]
<Creation of bacterial fluid>
50 mL of a medium obtained by adding yeast extract to the above mineral basal medium to a concentration of 0.1 g / L was taken in a 100 mL glass vial, purged with nitrogen, sterilized by an autoclave, and then contaminated with tetrachloroethylene (PCE). After adding 25 mL of groundwater collected from the soil and substituting with nitrogen, 2.5 mL of hydrogen and 0.46 μL of PCE (corresponding to 10 mg / L) were encapsulated and statically cultured at 20 ° C. in the dark. Chloroethenes in the headspace were measured periodically, and 1 mL was collected when chloroethenes were no longer detected, and was transferred to 75 mL of an autoclaved mineral basal medium supplemented with 0.1 g / L of yeast extract. This subculture was performed three times as a “bacterial fluid” and used for the following chloroethene degradation experiment.

[Examples 16 to 18]
Assuming groundwater contaminated with chloroethenes, a decomposition test was conducted by the following method.
Take 75 ml of the above-mentioned mineral basal medium in a glass 100 ml vial, the decomposition accelerator A (Example 16), the decomposition accelerator G (Example 17), the decomposition accelerator A, the decomposition accelerator G, etc. A quantity mixture (Example 18) was added so as to be 0.2 g / L, and after sterilizing with an autoclave after nitrogen substitution. After cooling, 1.5 ml of the above bacterial solution was added, and after nitrogen substitution, tetrachloroethylene (PCE) was sealed so as to be 10 μg / ml.
This vial was subjected to static culture at 20 ° C. Various chloroethenes in the vial headspace after 0, 3, 10, 18, 24, 36, 45, 49, 59, 66, 75, 84, 87 days, ie PCE, TCE, c-DCE, t-DCE 1,1-DCE, VC content and ethylene content were measured by gas chromatography.

Regarding the experimental results, the content of chloroethenes is below the groundwater environmental standard value, that is, PCE is 0.01 mg / L or less, TCE is 0.03 mg / L or less, cDCE and t-DCE are 0.04 mg / L or less, and 1,1-DCE is The number of days until 0.02 mg / L or less and VC became 0.002 mg / L or less is shown in Table 3 as “number of days required for decomposition”.
In addition, since the activation of microorganisms is considered to occur at an early stage, the amount of decrease in PCE content per day up to the 18th day is shown in Table 3 as the initial degradation rate.

Claims (9)

A decomposition accelerator for volatile organic halogen compounds, comprising one or more of the following (A) to (C):
(A) Citrus fruit or extract obtained from the fruit (B) Citrus fruit skin or extract obtained from the fruit skin (C) Formulation containing all of the following (1) to (3) (1) Glycerin (2) Milk protein and / or yeast extract (3) Vitamin B12 The decomposition promotion of a volatile organic halogen compound according to claim 1, wherein when the decomposition accelerator contains at least one of (A) and (B), the extract is an extract obtained using water as a solvent. Agent. When the decomposition accelerator contains (C), the milk protein and / or yeast extract of (2) is contained in an amount of 0.1 to 3 parts by mass as a solid content with respect to 1 part by mass of the glycerin (1). Item 1. The decomposition accelerator according to Item 1. 3. The degradation accelerator according to claim 1 or 2, which contains 0.00001 to 0.001 part by mass of vitamin B12 of (3) with respect to 1 part by mass of glycerin of (1) when (C) is contained. Decomposition accelerator. A decomposition accelerator composition for volatile organic halogen compounds, comprising the decomposition accelerator according to claim 1 as an active ingredient. A volatile organic halogen compound produced by a microorganism, wherein the decomposition accelerator according to claim 1 or the decomposition accelerator composition according to claim 5 is brought into contact with soil and / or groundwater containing the volatile organic halogen compound. How to promote the decomposition of the. The method according to claim 6, wherein the volatile organic halogen compound is an organic chlorine compound. Organochlorine compounds are carbon tetrachloride, chloroform, dichloromethane, monochloromethane, 1,2-dichloroethane, 1,1-dichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,3-dichloropropene The method according to claim 7, which is at least one selected from the group consisting of tetrachloroethylene, 1,1,1-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, and vinyl chloride. The method according to claim 6, wherein the microorganism is at least one selected from the group consisting of Clostridium bacteria, Dehalobacter bacteria, Dehalococcoides bacteria, Dehalospirilum bacteria, Desulfobacterium bacteria, Desulfomonas bacteria, and Desulfomonile bacteria.

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