JPH0870855A - Method for decomposing organic chlorine compound by microorganism - Google Patents

Abstract

PURPOSE: To provide a method for decomposing an organic chlorine compound with a microorganism without causing the production of a halo acid. CONSTITUTION: This method for decomposing an organic chlorine compound is to bring the organic chlorine compound into contact with a microorganism having the ability to decompose the organic chlorine compound under alkaline conditions. Either of trichloroethylene or dichloroethylene is used as the organic chlorine compound and the alkaline conditions are within the range of pH 8-11. A bacterium belonging to the genus Pseudomonas, Acinetobacter, Xanthobacter, Corynebacterium, etc., is cited as the microorganism.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for biodegrading / detoxifying an organochlorine compound, which comprises decomposing and purifying an organochlorine compound released into the environment by a microorganism degrading the organochlorine compound.

[0002]

2. Description of the Related Art In recent years, environmental pollution caused by organic chlorine compounds, which are harmful to living organisms and hardly decomposed, has become a serious problem. In particular, it is said that contamination by organochlorine compounds such as tetrachloroethylene (PCE), trichlorethylene (TCE) and dichloroethylene (DCE) has spread to a considerable extent in the soil of paper and pulp industry and precision machinery related industrial areas in Japan and overseas. This is considered, and many cases have been reported that were actually detected in environmental surveys. It is said that those organic chlorine compounds that remain in the soil dissolve in groundwater due to rainwater and spread to the entire surrounding area. Since such compounds are suspected to be carcinogenic and are extremely stable in the environment, pollution of groundwater used as a water source for drinking water is a major social problem.

From the above, purification of groundwater and the like by removing and decomposing organic chlorine compounds is an important issue from the viewpoint of environmental protection, and techniques necessary for purification have been developed.

For example, adsorption treatment with activated carbon and decomposition treatment with light and heat have been studied, but it is not always practical in terms of cost and operability.

On the other hand, a microbial organic chlorine compound such as TCE, which is stable in the environment, has recently been reported to be decomposed by microorganisms, and studies for its practical use have begun.
That is, in the biodegradation treatment using microorganisms, it is possible to decompose organic chlorine compounds into harmless substances by selecting the microorganisms to be used, basically no special chemicals are required, and maintenance labor and cost can be reduced. And so on.

For example, more than ten species of TCE-degrading bacteria have been discovered and isolated so far. Among them, the representative ones can be roughly divided into two types, methane-utilizing bacteria and aromatic-utilizing bacteria, depending on the type of substrate. A typical example of the former methanotrophs is Methylocystis sp., Which has methane monooxygenase . strai
n M (Agric. Biol. Chem., 53, 2)
903 (1989), Biosci. Biotech.
Biochem. , 56, 486 (1992), 5
6,736 (1992)), Methylosinus
trichosporium OB3b (Am. Ch
em. Soc. Natl. Meet. Dev. Envi
ron. Microbiol. , 29, 365 (198
9), Appl. Environ. Microbio
l. 55, 3155 (1989), Appl. Bio
chem. Biotechnol. , 28,877 (1
991)) and so on. Further, as the latter aromatic compound-assimilating bacterium, Ps having phenol hydroxylase
eudomonas Putida BH (Sewer Association, 24, 27 (1987)), Acinetobacter sp. having toluene monooxygenase . s
trainG4 (Appl. Environ. Micr
obiol. , 52, 383 (1986), ibid. 53, 9
49 (1987), ibid 54, 951 (1989), ibid 5
6,279 (1990), 57,193 (199)
1)), Pseudomonas mendocina
KR-1 (Bio / Technol., 7, 282)
(1989)), P having toluene dioxygenase
seudomonas putida F1 (App
l. Environ. MIcrobiol. , 54, 1
703 (1988), ibid. 54,2587 (1988)).
Can be mentioned. In addition, Nitrosom
Ammonia-utilizing bacteria such as onas europaea have also been reported to degrade TCE by ammonia monooxygenase (Appl. Environ.
Microbiol. , 56, 1169 (199
0)).

[0007]

As described above, the TCE
Various strains have been isolated and identified as degrading bacteria, but all of these strains are isolated by T
It is said that CE is epoxidized and then biodegraded or biodegraded. At this stage, T
Protonation of the CE epoxide results in dichloroacetic acid alongside glyoxylic acid.

[0008] Dichloroacetic acid is one of the substances that have been problematic as a chlorine disinfection by-product of drinking water and is designated as an environmental monitoring substance as a substance that has neurotoxicity and is suspected of causing eye damage. (Guideline value 0.04 mg /
l). In addition, these are the 23rd Japan Society for Water Environment Seminar / Analytical method with revision of water quality standards (Japan Society for Water Environment) Proceedings p55-p64 (1993)
Month).

The object of the present invention is to produce an organochlorine compound under such a condition that dichloroacetic acid, which is a harmful substance that is universally produced in the decomposition of such an organochlorine compound by an oxygenase system, is not produced or is significantly lower than the standard value. An object of the present invention is to provide a method for decomposing / detoxifying an organic chlorine compound, which carries out biological decomposition and purification of

[0010]

As the method of the present invention,
Media conditions for microbial degradation of organochlorine compounds by the oxygenase system lead to the production of dichloroacetic acid,
The purpose is to carry out microbial decomposition and purification of organic chlorine compounds by setting so that TCE epoxide is not protonated. For that purpose, microbial decomposition is performed in an alkaline, specifically, specific pH range, and the pH is 8
The range of from 11 to 11 is desirable, but more preferably from 8.5 to 9.
The microbial decomposition and purification of the organic chlorine compound is limited to the range of 5. Under such conditions, protonation of the TCE epoxide does not occur, water (hydroxyl ion) is added to form carbon monoxide and formic acid, and finally converted into harmless carbon dioxide.

The microorganism used in the method of the present invention may be a microorganism which decomposes an organic chlorine compound through an epoxide of the organic chlorine compound and does not decompose a halo acid such as dichloroacetic acid which is a by-product. The TCE-degrading bacteria listed in the above-mentioned conventional techniques can be used without limitation. Therefore, it can be expected that even such unidentified microorganisms can be used as long as they have the above-mentioned properties. Therefore, non-isolated microorganisms, symbiotic microbial groups, and isolated / identified microorganisms can also be used. As the microorganisms that have been identified, Pseudomonas spp, Acinetobacter spp, Xanthobacter spp, Corynebacterium spp., And the like having the above-mentioned properties can also be used, and in particular Pseudomonas cepacia, Pseudomonas putida, Pseudomonas fluorescens. , Pseudomonas arginosa and the like are effective, and for example, Pseudomonas cepacia strain KK01 (FERMBP-
4235; hereinafter referred to as KK01 strain), or Corynebacterium sp. J1 strain (FERMBP-14332
No .; hereinafter referred to as J1 strain) and the like.

Organochlorine compounds to be decomposed include
Examples thereof include chlorinated ethylene such as TCE and DCE.

The method of the present invention is applied to wastewater treatment, soil treatment, etc.
It can be applied to both closed and open systems. Further, the microorganisms may be immobilized on a carrier or the like, or various methods for promoting growth may be used in combination.

[0014]

The present invention will be described in more detail with reference to the following examples. The M9 medium used in each example has the following composition.

M9 medium composition (in 1 liter); Na ₂ HPO ₄ 6.2 g KH ₂ PO ₄ 3.0 g NaCl 0.5 g NH ₄ Cl 1.0 g Water balance (pH 7.0) Measurement of all TCE concentrations Was carried out by the headspace-gas chromatography method, that is, 15 ml of M9 medium prepared to a predetermined TCE concentration by the method described later was added to a 50 ml vial bottle, 100 μl of the bacterial solution was added, and a rubber stopper was added. After sealing with an aluminum cap and shaking culture at 30 ° C. for a certain period of time, 0.1 ml of the gas phase was sampled and analyzed by gas chromatography. In addition, for the measurement of dichloroacetic acid, the TCE-decomposed culture solution was adjusted to pH 2 with diluted sulfuric acid,
The centrifuged supernatant was extracted with diethyl ether and ethyl acetate, dried on a rotary evaporator and then redissolved in deionized water to obtain 95% 0.01N sulfuric acid aqueous solution / acetonitrile = 95/5 (v / v). Was used as a solvent to perform high performance liquid chromatography. It was confirmed by the GC / MS method that the peak eluting at the same retention time as that of the dichloroacetic acid sample was indeed dichloroacetic acid.

Example 1 Degradation of TCE in strains KK01 and J1 at various pHs pH was adjusted to 4, 5, 6 (adjusted with diluted sulfuric acid), 7 (not adjusted), 8, 9, 10 (diluted sodium hydroxide). TCE was added to M9 medium adjusted to 10 ppm so as to be 10 ppm, and phenol 100 ppm and yeast extract 0.
KK with enhanced TCE in degradation activity in M9 medium containing 05%
The 01 strain and the J1 strain were collected and the bacterial concentration was 6 to ⁸ × 10 ^8.
cells / ml at 30 ° C, 120
The cells were cultivated with shaking at rpm, and after 24 hours, the TCE concentration was measured by the method as shown in the preceding stage of the example.

As a result, the pH of both KK01 strain and J1 strain was
From 4 to 9, 10 ppm of TCE was decomposed to below the detection limit, while at pH 10, neither strain of TCE was decomposed at all. Based on the above results, it was decided to carry out the dichloroacetic acid production amount control experiment in the range of pH 4 to 9.

Example 2 Control of pH of Dichloroacetic Acid Production in TCE Degradation by KK01 and J1 Strains In the same manner as in Example 1, 1 liter of M9 medium adjusted to pH 4, 5, 6, 7, 8, 9 was used. TCE was added to 10 ppm, and the KK01 strain and J1 strain whose decomposition activity was enhanced by TCE were collected in advance in M9 medium containing 100 ppm of phenol and 0.05% of yeast extract, and the bacterial concentration was 6-8.
x10 ⁸ cells / ml, add 30
The cells were cultivated with shaking at 120 ° C. at 24 ° C., and after 24 hours, the dichloroacetic acid concentration was measured by the method as described in the previous section of the examples.

The results are shown in Table 1. The value of the dichloroacetic acid concentration is shown as the concentration converted to the original culture solution. in this way,
Dichloroacetic acid was detected in both the KK01 strain and the J1 strain at pH 9 below the environmental guideline value of drinking water of 0.04 mg / l, and the effect of suppressing the production of dichloroacetic acid by controlling the culture medium to the alkaline side was shown. . The measurement of dichloroacetic acid produced at pH 9 was carried out by the analysis method of haloacetic acid (tert-butyl methyl ether extraction → methylation with diazomethane → ECD-G, which was determined by the Ministry of Health and Welfare.
C analysis) also showed that the value was less than the environmental guideline value. (Unit: mg / l) Example 3 Degradation of TCE at each pH of the indigenous phenol-utilizing TCE-degrading bacteria group Usually, in general soil, there are some microorganisms potentially capable of decomposing aromatic compounds such as phenol. Kind or exist
Furthermore, some of them are said to have the ability to decompose chlorinated ethylene such as TCE by cooxidation.

Brown forest soil 1 collected in Atsugi City, Kanagawa Prefecture
Into 50 ml of M9 medium (containing 100 ppm of phenol), the culture was shaken at 30 ° C. and 120 rpm for 1 week, and 1% of the medium was subcultured in the same medium and further cultured for 1 week. This operation was repeated 3 times, and yeast extract (0.
(5%) and phenol (100 ppm) in M9 agar medium, at least 4 kinds of colonies were confirmed. Therefore, these bacteria were cultivated in a mixed system, and TCE decomposition experiments were conducted in M9 medium whose pH was adjusted to 4, 5, 6, 7, 8, and 9 as in Example 1. In addition, T
The CE concentration was 2 ppm.

As a result, 10 ppm at pH 5 to 9
Of TCE was decomposed to below the detection limit, while pH4
No more than 70% was decomposed, and at pH 10, TCE was not decomposed at all. Based on the above results, it was decided that the experiment for controlling the amount of dichloroacetic acid produced by the group of indigenous phenol-assimilating TCE-degrading bacteria would be carried out within the pH range of 5 to 9.

Example 4 Control of pH of production amount of dichloroacetic acid in TCE decomposition by indigenous phenol-utilizing TCE-decomposing bacteria group by pH M9 medium 1 adjusted to pH 5, 6, 7, 8, 9 as in Example 1 TCE was added to 2 liters so as to be 2 ppm, and 100 ppm of phenol and yeast extract were added in advance.
The indigenous phenol-utilizing TCE-degrading bacteria group, whose degradation activity was enhanced by TCE, was collected in M9 medium containing 05% to collect OD =
In addition to 1.2, 30 ℃, 120ppm
The cells were cultivated with shaking at 24 hours, and after 24 hours, the concentration of dichloroacetic acid was measured by the method described in the previous section of the example.

The results are shown in Table 2. The value of the dichloroacetic acid concentration is shown as the concentration converted to the original culture solution. in this way,
PH 9 even in the indigenous phenol-utilizing TCE-degrading bacteria group
In the experiment, dichloroacetic acid was detected in an amount less than 0.04 mg / l which is the environmental guideline value of drinking water, and the effect of suppressing the production of dichloroacetic acid by controlling the culture solution to the alkaline side was shown. In addition, the measurement of dichloroacetic acid produced at pH 9 was carried out by the analysis method of haloacetic acid (t
ert-butyl methyl ether extraction → methylation with diazomethane → ECD-GC analysis) also showed that it was below the environmental guideline value. (Unit is mg / l)

[0024]

INDUSTRIAL APPLICABILITY By the method of the present invention, when groundwater or soil polluted by volatile organic chlorine compounds such as TCE is decomposed and purified by microorganisms, halo acids such as dichloroacetic acid produced as a by-product are removed. It has become possible to control the amount and perform biological purification treatment that does not cause secondary pollution.

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Claims (9)

[Claims] 1. A method for degrading an organic chlorine compound by a microorganism, which comprises contacting the organic chlorine compound with a microorganism capable of degrading the organic chlorine compound under alkaline conditions. 2. The method according to claim 1, wherein the organochlorine compound is chlorinated ethylene. 3. The method according to claim 2, wherein the chlorinated ethylene is either trichloroethylene or dichloroethylene. 4. The alkaline condition is pH 8 to pH 11.
The method according to any one of claims 1 to 3, which is in the range of. 5. The alkaline condition has a pH of 8.5 to 9.5.
The method according to claim 4, wherein 6. The method according to claim 1, wherein the microorganism belongs to the genus Pseudomonas. 7. The microorganism is Pseudomonas cepacia KK0.
The method according to claim 6, which is one strain. 8. The method according to claim 1, wherein the microorganism is a microorganism belonging to the genus Corynebacterium. 9. The microorganism is Corynebacterium sp. J1
The method according to claim 8, which is a strain.