JPH10150979A - Biodegradation-activating agent for halogen-substituted hydrocarbon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject activating agent useful for purification of water or soil contaminated with halogen-substituted hydrocarbons using microorganisms by blending an alicyclic hydrocarbon and a chain hydrocarbon containing a functional group as effective ingredients. SOLUTION: This activating agent for biodegrading halogen-substituted hydrocarbons used for purifying water or soil contaminated with halogen- substituted hydrocarbons (e.g.; trichloroethylene, etc.) using microorganisms is obtained by blending an alicyclic hydrocarbon such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, etc., and/or a chain hydrocarbon containing a functional group such as an alcohol, a ketone, an organic acid, etc., (e.g. cyclohexanol, etc.) as effective ingredients. By using this activating agent, microorganisms capable of degrading halogen-substituted hydrocarbons, degradation-enzymes of the organism or manifestation of genes which code the enzymes can be activated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for treating microorganisms in water or soil contaminated with halogen-substituted hydrocarbons.

[0002]

2. Description of the Related Art In recent years, organic solvents, especially halogen-substituted hydrocarbons, have been used in large quantities as cleaning agents in advanced industries,
Currently, it is a compound that is gaining interest as a source of groundwater pollution. Among them, trichloroethylene and the like, which are suspected of carcinogenicity, are contained, and it is difficult to decompose biologically and non-biologically in soil and groundwater as contaminated places.

[0003] As a conventional treatment method, air is blown into contaminated soil to volatilize compounds, and the compound is volatilized and adsorbed and removed with activated carbon or the like.
A vacuum extraction method of vaporizing and removing by vacuum suction is known. In the case of groundwater, a method of adsorbing water pumped from a well with activated carbon or the like is well known.

On the other hand, research on a so-called biological purification method for efficiently decomposing contaminants by microorganisms and rendering them harmless has been rapidly progressing. Since this method uses a mechanism of decomposing microorganisms, it is considered that a large amount of energy is not required and the pollutants can be completely decomposed without secondary contamination as compared with the above method. Furthermore, it has the advantages of being able to purify even low-concentration contamination and being able to perform a wide-range treatment in situ, and is highly expected.

[0005] Among the halogen-substituted hydrocarbons, aliphatic halogenated hydrocarbons, particularly trichloroethylene (TCE), are important as pollutants. TCE degradation by anaerobic bacteria, microorganisms that inhabit soil or groundwater, has been reported, but the degradation products include harmful substances such as dichloroethylene and vinyl chloride. It is said that aerobic bacteria, methane assimilating bacteria and aromatic compound assimilating bacteria can decompose TCE without producing harmful by-products as described above. The mechanism of TCE degradation by these aerobic bacteria is that various oxygenases such as methane monooxygenase and toluene monooxygenase epoxidize TCE, and then the epoxide is converted to harmless substances such as inorganic chlorides and carbon dioxide via dichloroacetic acid and the like. It is thought to be done.

However, to activate these biological pathways of TCE degradation, it has been necessary to add compounds such as methane gas and aromatic compounds. As the prior art in this area, there is used a substituted benzene typified by phenol, toluene and cresol (JP-A-64-34499) in addition to methane, but it is extremely difficult to use it in practice because it is an environmental pollutant. It is. on the other hand,
In consideration of the burden on the environment, tryptophan (Japanese Patent Application Laid-Open No. 4-502277), which is a non-toxic oxygenase inducer, has been reported, but has the disadvantage that its effect is low and it is expensive to apply to the environment.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a low-toxicity and inexpensive activator capable of activating a halogen-substituted hydrocarbon capable of degrading a microorganism having the capability of degrading the same. A biodegradation method for halogen-substituted hydrocarbons utilizing

[0008]

In order to solve the above-mentioned problems, the present invention relates to a halogen containing an aliphatic or alicyclic hydrocarbon having 3 or more carbon atoms, an organic acid or an alcohol as an active ingredient. A substituted hydrocarbon biodegradation activator is provided. The present invention is also characterized by using the above-mentioned activator. A method for activating the expression of a halogen-substituted hydrocarbon-degrading microorganism, a degrading enzyme of the microorganism, or a gene encoding the enzyme is provided.

The present invention also provides a method for biodegrading halogen-substituted hydrocarbons in the environment, wherein the activator is sprayed in the environment to activate microorganisms having the ability to degrade halogen-substituted hydrocarbons that inhabit the environment. A method comprising: The present invention also provides a method for activating or dispersing the microorganism by culturing or incubating a microorganism having the ability to degrade a halogen-substituted hydrocarbon in the presence of the activator, and dispersing the microorganism in the environment, or the microorganism and the activity. And dispersing the agent into the environment.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The substances to be decomposed in the process of the present invention are halogen-substituted hydrocarbons, for example, halogen-substituted aliphatic hydrocarbons, and of particular importance in practical use is trichloroethylene (TCE). However, other halogenated hydrocarbons such as 1,1-dichloroethylene, cis-
1,2-dichloroethylene, vinyl chloride and the like can also be subject to decomposition. According to the present invention, various substances such as soil, waste, wastewater and the like containing the above contaminants are to be treated, and in the present invention, these various objects to be treated may be collectively referred to as "environment". .

In the present invention, the microorganism to be activated is a microorganism having the ability to degrade a halogen-substituted hydrocarbon, an enzyme involved in the decomposition of the halogen-substituted hydrocarbon in the microorganism, and the expression of a gene encoding the enzyme (ie, (Transformant by gene). One example of a suitable microorganism is Burkholderia strain N16-1 (FERM BP-
5504), but is not limited thereto. In the present invention, not only such isolated microorganisms, but also halogen-substituted hydrocarbon-degrading microorganisms naturally existing (inhabiting) in the environment to be treated are to be activated. In addition, various enzymes such as methane monooxygenase, phenol hydroxylase, toluene monooxygenase, and toluene dioxygenase can be used as enzymes.

The halogen-substituted hydrocarbon-decomposing microorganism activator of the present invention includes alicyclic hydrocarbons and / or chain hydrocarbons having a functional group. The alicyclic hydrocarbon can be used without any limitation. For example, cycloalkanes such as cyclopropane, cyclobutane,
Cyclopentane, cyclohexane, cycloheptane, etc.
Alternatively, an alicyclic acid may be mentioned, and more preferred are various cycloalcohols.

Examples of the chain hydrocarbon containing a functional group include chain alcohols, ketones, and organic acids. The organic acid is preferably a carboxylic acid, for example, a saturated or unsaturated monocarboxylic acid, for example, propionic acid, butyric acid heptanoic acid, hexanoic acid, trans-3-hexenoic acid, heptanoic acid, octanoic acid, 2-octenoic acid, capric acid acid,
Undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid,
Stearic acid, nonadecanoic acid, eicosanoic acid, tetraconic acid, etc .; saturated or unsaturated dicarboxylic acids such as malonic acid, succinic acid, fumaric acid, maleic acid, glutaric acid, adipic acid, suberic acid, etc .; hydroxycarboxylic acids such as lactic acid , Malic acid, citric acid, 2-hydroxyincaproic acid, trans-β-hydroxymuconic acid, etc .; saturated or unsaturated keto acids, such as pyruvic acid, oxaloacetic acid, glutaraldehyde, 2-ketoglutaric acid, etc .; Group carboxylic acids such as 1,4-dihydroxy-2-methylbenzene acid, and the like. Organic acids other than carboxylic acids can also be used.

The alcohol can be used without any limitation. In addition, a chain hydrocarbon having a carbonyl group or another functional group can be used. Among chain hydrocarbons containing a functional group, more preferred are organic acids, especially carboxylic acids. To treat a contaminated environment according to the present invention, the halogen-substituted hydrocarbon-degrading microorganism is cultured in the presence of the activator of the present invention or the halogen-substituted hydrocarbon-degrading microorganism is cultured. After incubating the cells in the presence of the activator of the present invention to activate the microorganism, the microorganism is added to the environment to be treated, for example, sprayed. Alternatively, the activator and the microorganism may be added to the environment to be treated, for example, by spraying. The concentration is, for example, in the range of 100 to 500 ppm, depending on the concentration of the activator added to the culture medium or the incubation medium for the culture or the incubation, the kind of the activator, and the like.

When the activator is added directly to the environment to be treated, the amount added is about 10 to 1000 ppm. According to another embodiment for treating a contaminated environment with an activator according to the invention, the activator according to the invention is added to or sprayed on the environment to be treated, for example soil. In this case, the decomposition of the halogen-substituted hydrocarbon can be promoted by activating the halogen-substituted hydrocarbon-degrading bacteria living in the environment. The amount of the activator to be added depends on the type of the activator, but is about 100 to 1000 ppm.

For culturing the halogen-substituted hydrocarbon-decomposing bacteria, a conventional medium for culturing bacteria, for example, NMS medium (Table 1) to which a nitrogen source such as yeast extract or a carbon source such as glucose is added is used. be able to.

[0017]

[Table 1]

The cultivation is preferably carried out under aerobic conditions by means such as aeration and stirring. Culture temperature is 15 ° C to 37 ° C
° C, preferably 25 ° C to 35 ° C, and the culture time is 6 to
30 hours is preferred. In order to incubate the cultured cells with the activator, for example, the activator is added to a culture solution containing the cultured cells and maintained at 25 ° C. to 35 ° C., preferably under the aerobic conditions as described above. Good.

[0019]

Next, the present invention will be described more specifically. Example 1 Each activity against N16-1 strain in liquid medium
Effect of Agent The N16-1 strain was cultured for 1 day in a liquid medium containing 0.02% yeast extract and 5 mmol glucose in NMS medium. NMS medium and 0.02% in vial
4 ml of solution containing yeast extract and 100 ppm of each activator
, And the above culture solution was inoculated in an amount of 400 μl each, 100 ppm of TCE was added, and the mixture was quickly sealed with a Teflon-coated silicon septum and an aluminum cap. The vial was left at 15 ° C. for 1 week, and the gas phase was analyzed by gas chromatography.

The results are shown in Table 2. Numerical values represent the TCE concentration when each activator is added in%, with the residual TCE concentration when no activator is added as 100%.

[0021]

[Table 2]

As described above, the activator of the present invention comprises N16
-1 showed TCE degradation activating action. When decomposing harmful substances in the environment using microorganisms, when the activator of the microorganism is administered simultaneously or separately, it is desirable that the toxicity of the activator itself is as low as possible, but these compounds are toluene, It is much less toxic than aromatic compounds such as phenol and is considered to be advantageous in environmental purification. Example 2 Each activation against soil microorganisms in liquid medium
Location A, the effect Aichi agent soil collected in B, C and D, the 0.1 g, was inoculated into NMS medium 5ml were placed in screw-top test tube 25ml volume. 500 ppm of phenol and a vitamin cocktail were added, and the mixture was cultured at 30 ° C. for 3 days. Table 3 shows the composition of the vitamin cocktail.

[0023]

[Table 3]

The microorganism obtained as described above is
Instead of one strain, the cells were cultured in the same manner as described in Example 1, and the effect of various activators on promoting TCE degradation was tested.
The effects are shown in Table 4 below.

[0025]

[Table 4]

The soil microorganism used here is a natural flora that originally inhabits the collected soil. N16-1
Compounds that were effective on the TCE-degrading activity of the strain also showed a weaker effect on the natural flora than the N16-1 strain. This is presumably because the number of microorganisms having TCE-degrading activity present in each soil was smaller than that of the N16-1 strain alone. Example 3 Decomposition Test of TCE in Soil 10 g of each of the four types of soil used in Example 2 was placed in a vial having a capacity of 30 ml, TCE was added to a concentration of 30 ppm, sealed, and allowed to stand for 2 days. The soil was made. Each activator was adjusted with NMS medium to a final concentration of 500 ppm and a water content of 25%, added to the soil, sealed with a Teflon-coated silicon septum and an aluminum cap,
It was left at 15 ° C. for 3 weeks. After the test, the headspace of the vial was sampled with a gas syringe, and the remaining TCE concentration was measured by gas chromatography analysis. Table 5 shows the results. The numerical values are expressed as% TCE concentration when each activator is added, with the remaining TCE concentration when no activator is added as 100%.

[0027]

[Table 5]

In Example 2, the liquid culture was shown to have the effect of activating TCE degradation on natural flora, but here the compounds listed here decompose TCE without adding foreign microorganisms even in soil experiments. It became clear that it promoted.

[0029]

Industrial Applicability The activator of the present invention is more practical than conventional activators such as methane and phenol, has low toxicity, is inexpensive, and is safer in microorganisms for purifying halogen-substituted aliphatic hydrocarbons in the environment. Becomes possible. In addition, the biodegradation method using the activator according to the present invention makes it possible to efficiently purify halogen-substituted aliphatic hydrocarbons in the environment on an industrial scale without using foreign microorganisms.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Arimitsu Usuki 41-cho, Yokomichi, Nagakute-cho, Aichi-gun, Aichi Prefecture Inside Toyota Central Research Laboratory Co., Ltd. (72) Inventor Osamu Asami, Nagakute-cho, Aichi-gun, Aichi No. 41, Chochu-Yokomichi, Toyota Central Research Laboratory Co., Ltd. (72) Inventor Hisato Takeuchi 41, Nagakute-cho, Aichi-gun, Aichi Prefecture, Oji-cho, Toyota Chuo Research Institute, Inc. (72) Inventor Yukio Yamada 41, Chuo-ku, Yokomichi, Nagakute-cho, Aichi-gun, Aichi Prefecture Inside Toyota Central R & D Laboratories Co., Ltd. (72) Inventor Koichi Numata Oriental Building 202, 1-98, Horakai, Midori-ku, Nagoya City, Aichi Prefecture

Claims (2)

[Claims] 1. A halogen-substituted hydrocarbon biodegradation activator comprising an alicyclic hydrocarbon and / or a chain hydrocarbon containing a functional group as an active ingredient. 2. A method for activating the expression of a halogen-substituted hydrocarbon-degrading microorganism, a degrading enzyme of the microorganism, or a gene encoding the enzyme, comprising using the activator according to claim 1.