JPH10324861A - Biodegradation activator for halogenated hydrocarbon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel low-toxicity biodegradation activator for chlorinated hydrocarbons. SOLUTION: This activator contains, as the active ingredient, a compd. comprising an arom. ring substd. by a carboxyl or alcoholic hydroxyl group or by a substituent having a carboxyl or alcoholic hydroxyl group. Microorganisms capable of decomposing halogenated hydrocarbons, their decomposition enzymes, and a method for activating the expression of genes coding the enzymes are provided, all being based on the activator. Halogenated hydrocarbons can be decomposed in the presence of this low-toxicity activator.

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. These include trichlorethylene, which is suspected of ring toxicity, and are difficult to degrade biologically and non-biologically in contaminated soil and groundwater.

[0003] As a conventional treatment method, air is blown into contaminated soil, a compound is exerted, and a soil aeration method in which activated carbon or the like is adsorbed and removed, a pipe is driven into contaminated soil,
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, in order to activate these biological pathways of TCE degradation, there is the use of, for example, methane gas or substituted benzene represented by phenol, toluene and cresol (Japanese Patent Application Laid-Open No. 64-34499). Since it is an environmental pollutant, it is extremely difficult to use it. On the other hand, in consideration of the burden on the environment, tryptophan, which is a non-toxic oxygenase-inducing substance (Japanese Unexamined Patent Publication No.
277) was reported, but had the drawback of being ineffective and expensive to apply to the environment.

When the substituted benzene of the prior art as described above is used as an activator, as shown in FIG. 4 and FIG. There is a problem that the decomposition of the halogenated hydrocarbon is not started, so that the time for decomposing the halogenated hydrocarbon is short, and thus the decomposition of the halogenated hydrocarbon is not efficient. Furthermore, known substituted benzene activators have high toxicity.

[0008]

Accordingly, the present invention is an activator for the biodegradation of halogenated hydrocarbons,
An object of the present invention is to provide an activator having low toxicity and capable of biodegrading a halogenated hydrocarbon even in the presence of the activator. The present invention also provides a method for biodegrading a halogen-substituted hydrocarbon using such an activator.

[0009]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have searched various compounds and found that they have been substituted with a carboxyl group or an alcoholic hydroxyl group, or have been substituted with a carboxyl group or an alcoholic hydroxyl group. A compound consisting of an aromatic ring substituted by a substituent containing a group and having the common characteristic of low toxicity activates the biodegradation of a halogen-substituted hydrocarbon, and is substituted with a halogen even in the presence of the compound. The present inventors have obtained a new finding that hydrocarbons are decomposed, and have completed the present invention.

Accordingly, the present invention provides a halogenated hydrocarbon containing as an active ingredient a compound comprising an aromatic ring substituted by a carboxyl or alcoholic hydroxyl group or by a substituent containing a carboxyl or alcoholic hydroxyl group. A biodegradation activator is provided. The present invention also provides 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, characterized by using the above-mentioned activator.

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 the 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.

[0012]

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 target of activation is a microorganism having the ability to degrade a halogen-substituted hydrocarbon, an enzyme involved in the decomposition of the halogen-substituted hydrocarbon of 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.

The enzymes include various oxygenases such as methane monooxygenase, phenol hydroxylase, toluene monooxygenase, and toluene dioxygenase. In the present invention, the aromatic ring constituting the compound which is the active ingredient of the activator is a preferred benzene ring.

The compound comprising an aromatic ring substituted by a carboxyl group or by a carboxyl group-containing substituent includes, for example, benzoic acid, aminobenzoic acid, for example, 3
-Aminobenzoic acid, aminosalicylic acid such as p-aminosalicylic acid, homogentisic acid, anthranilic acid, hydroxybenzoic acid such as o-, p- or m-hydroxybenzoic acid, hydroxyanthranilic acid such as 5
-Hydroxyanthranilic acid, aminohydroxybenzoic acid, anisic acid, cinnamic acid, gallic acid, phthalic acid, phenylmalonic acid, syringic acid, shikimic acid, phenylacetic acid, toluylacetic acid, such as o-, m- or p-toluylacetic acid, Methylphenylacetic acid such as o-, m- or p
-Methylphenylacetic acid, benzoic acid, such as p-aminobenzoic acid. Reference to an acid in the present invention also includes a salt of the acid, for example, a metal salt, for example, an alkali metal salt, for example, a salt such as sodium or potassium, or an ammonium salt.

The compound of the present invention comprising an aromatic ring substituted by an alcoholic hydroxyl group or by a substituent containing an alcoholic hydroxyl group includes, for example,
Benzyl alcohol, dimethoxybenzyl alcohol,
For example 3,4-dimethoxybenzyl alcohol, saligenin such as o-, m- or p-saligenin, salicin, benzenedimethanol, hydroxybenzyl alcohol such as o-, m- or p-hydroxybenzyl alcohol, methoxybenzyl alcohol, for example o-, m- or p-methoxybenzyl alcohol,
Examples include aminobenzyl alcohol, for example, o-, m- or p-aminobenzyl alcohol, methylbenzyl alcohol, for example, o-, m- or p-methylbenzyl alcohol, cinnamon alcohol, phenoxyethanol and the like.

The activators of the present invention have low toxicity, for example, the LD _{50 in} mice is 0.53 g / kg for phenol, whereas it is 3.1 g / kg for benzyl alcohol. 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. Or,
The activator and the microorganism may be added to the environment to be treated, for example, by spraying. The concentration of the activator added to the culture or incubation medium for the culture or incubation, depending on the type of activator, etc.,
For example, it is in the range of 100 to 500 ppm.

When the activator is added directly into 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, it is possible to use a conventional medium for culturing bacteria, for example, an NMS medium (Table 1) to which a nitrogen source such as yeast extract or a carbon source such as glucose is added. .

[0019]

[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.

[0021]

Next, the present invention will be described more specifically. Embodiment 1 FIG. Time course of TCE degradation in liquid culture The N16-1 strain was cultured for 1 day in a liquid medium containing 0.02% yeast extract and 5 mM glucose in NMS medium. NMS medium and 0.02% in a 30 ml vial
4 ml of a solution containing yeast extract and 500 ppm of benzyl alcohol (activator) was added thereto, and 400 μl of the above culture solution was added thereto, 100 ppm of TCE was added, and the mixture was quickly sealed with a Teflon-coated silicon septum and an aluminum cap. A large number of these vials were prepared and opened over time, and the TCE concentration in the gas phase (initial value: 30 ppm), the benzyl alcohol concentration in the medium (initial value: 500 ppm), and the viable cell count (initial value: 3 × 10 ⁷ / ml) ) Was measured.

The benzyl alcohol concentration was measured by high performance liquid chromatography, the TCE in the gas phase was measured by gas chromatography, and the viable cell count was 500 pp.
m Counted by the dilution plate method using NMS agar medium containing phenol. The results are shown in FIG. Benzyl alcohol and TCE were decomposed in parallel, and TCE was completely decomposed in 3 days.

Embodiment 2 FIG . Degradation of TCE in soil over time
Put soil were autoclaved in vials of 30 ml (sandy soil) 10 g (wet weight), to which the cells 5 × 10 ⁶ CFU of N16-1 strain was cultured in the same manner as in Example 1
/ G soil, yeast extract 200 mg / kg soil and glucose 180 mg / kg soil and benzyl alcohol 100 mg / kg
1.4 ml of bacterial cell suspension prepared to be soil and TC
0.4 ml of an E aqueous solution (660 ppm) was added (the initial concentration of TCE was 30 ppm), and the mixture was quickly sealed with a Teflon-coated silicon septum and an aluminum cap.

Many vials were prepared. These vials were incubated at 30 ° C., and the TCE concentration in the gas phase, the benzyl alcohol concentration in the soil, and the viable cell count were measured in the same manner as described in Example 1. The results are shown in FIG. Even in soil, benzyl alcohol (activator) and TCE were decomposed in parallel, and TCE was completely decomposed and disappeared in two days.

Embodiment 3 FIG . Active p-hydroxybenzoic acid
TCE degradation in a liquid medium in the case of using as a activating agent A time-varying vial prepared by the same method as in Example 1 using p-hydroxybenzoic acid as an activator, TCE concentration in medium (initial value 30 ppm), p in medium
-Hydroxybenzoic acid concentration (initial value 500 ppm) and viable cell count (initial value 5 × 10 ⁷ / ml) were measured. The p-hydroxybenzoic acid concentration was measured by high performance liquid chromatography, and the number of viable bacteria was counted by a dilution plate method using an NMS agar medium containing 500 ppm phenol. The results are shown in FIG.

Embodiment 4 FIG . N16-1 strain in liquid medium
The effect N16-1 strain of various activators for, and cultured for one day in a liquid medium supplemented with 0.02% yeast extract and 5 mM 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. Table 2 shows the results. The numbers are
With the residual TCE concentration at the time of no activator addition as 100%,
The TCE concentration at the time of addition of each activator is indicated by%.

[0027]

[Table 2]

Embodiment 5 FIG . Against N16-1 strain in soil
To put 10g soil in a vial effect volume 30ml various activator, it was added to a the TCE to 30 ppm, sealed and allowed to stand for 2 days,
TCE contaminated soil was prepared. Final concentration of each activator is 500
ppm, adjusted with NMS medium to 25% water content,
It was added to soil, sealed with a Teflon-coated silicon septum and an aluminum cap, and 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 3 shows the results. The numerical value indicates the concentration of the remaining TCE when no activator was added to 10
The TCE concentration at the time of adding each activator was expressed in%.

[0029]

[Table 3]

Reference Example 1 Phenol activator
TCE degradation in the liquid medium in the case of aging With the use of phenol as the activator, a large number of vials were prepared in the same manner as in Example 1, and T
CE concentration (initial value 30 ppm), phenol concentration in the medium (initial value 500 ppm) and viable cell count (initial value 5 × 10 ⁶ /
ml). The phenol concentration was measured by potassium ferricyan / 4-aminoantipyrine spectrophotometry,
The number of viable cells was counted by a dilution plate method using an NMS agar medium containing 500 ppm phenol. The results are shown in FIG. Decomposition of TCE starts after phenol is decomposed, and
TCE was completely degraded in 5 days.

Reference Example 2 Phenol activator
Degradation of TCE in soil in case of time change Soil autoclave sterilized in a vial (sandy soil)
10 g (wet weight) was added, and N was cultured as in Example 1.
1.4-1 ml of a cell suspension prepared by preparing the 16-1 strain to 5 × 10 ⁶ CFU / g-soil, yeast extract 200 mg / kg-soil, glucose 180 mg / kg-soil and phenol 100 mg / kg-soil. And 0.4 ml of an aqueous TCE solution (660 ppm), and quickly sealed with a Teflon-coated silicon septum and an aluminum cap. The vial was cultured at 30 ° C., and the TCE concentration in the gas phase, the phenol concentration in the soil, and the number of viable bacteria were measured with time in the same manner as in Example 1. FIG. 4 shows the results. Reference Example 1 in soil
The same decomposition pattern was shown.

[Brief description of the drawings]

FIG. 1 is a graph showing the progress of degradation of TCE in a culture solution when benzyl alcohol is used as an activator.

FIG. 2 is a graph showing the course of degradation of TCE in soil when benzyl alcohol is used as an activator.

FIG. 3 is a graph showing the results of degradation of TCE in a culture solution when p-hydroxybenzoic acid was used as an activator.

FIG. 4 is a graph showing the progress of degradation of TCE in a culture solution when phenol is used as an activator.

FIG. 5 is a graph showing the course of decomposition of TCE in soil when phenol is used as an activator.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Osamu Asami 41-Cho, Yokomichi, Nagakute-cho, Aichi-gun, Aichi Prefecture Inside of Toyota Central Research Laboratory, Inc. (72) Inventor Yukio Yamada Nagakute-cho, Nagakute-cho, Aichi-gun, Aichi 41, Yokomichi, Toyota Central Research Laboratories Co., Ltd. (72) Inventor Arimitsu Usuki 41, Chuo-Cho, Yoji, Nagakute-cho, Aichi-gun, Aichi Prefecture 1, Toyota Motor Central Research Laboratories Co., Ltd. (72) Inventor Hisato Takeuchi No. 41 Toyota Chuo Research Institute Co., Ltd.

Claims (1)

[Claims] 1. A halogen-substituted hydrocarbon biodegradation activity comprising, as an active ingredient, a compound comprising an aromatic ring substituted by a carboxyl group or an alcoholic hydroxyl group or by a substituent containing a carboxyl group or an alcoholic hydroxyl group. Agent.