JP3513715B2 - Microorganisms and water or soil purification methods - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microorganism that decomposes trichlorethylene efficiently and a method for purifying water or soil using the microorganism.

[0002]

As is well known, it has been pointed out that groundwater and soil are contaminated with trichlorethylene used as a cleaning agent and the like in advanced industries, and a treatment method for trichlorethylene suspected of being carcinogenic is urgently required. .

As a method of treating groundwater contaminated with trichlorethylene, there has been proposed a method of separating trichlorethylene by a physical treatment such as a vacuum suction method or an air stripping method.

[0004]

However, these methods require a secondary treatment such as activated carbon treatment from the viewpoint of preventing secondary pollution such as prevention of air pollution. However, even if such treatment is performed, At present, it cannot be completely removed.

On the other hand, in recent years, studies have been conducted on a so-called biological purification method in which trichlorethylene is decomposed by microorganisms extremely efficiently to render it harmless. Since this biological purification method uses the function of degrading microorganisms, it does not require much energy compared to the above physical treatment methods, does not cause secondary pollution, and spreads over a wide range in situ. It is an excellent method with the advantages of biotechnology such as being processable.

The present invention has been made from the above viewpoint, and an object thereof is to provide a microorganism capable of decomposing trichlorethylene efficiently. Another object of the present invention is to provide a method for purifying water or soil that can efficiently purify water or soil contaminated with trichlorethylene without causing secondary pollution.

[0007] Microorganisms of the present invention, in a medium group containing toluene 1-10 ppm, with the ability to resolve 50ppm about trichlorethylene, alcaligenes Eurotorofusu (Alcaigenes eurotrophus) KS01
(FERM P-13761) strain .

Further, the method for purifying water or soil according to the present invention comprises inoculating and mixing the microorganisms according to claim 1 into water or soil contaminated with trichlorethylene to remove trichlorethylene contained in the water or soil. A method of purifying water or soil to be decomposed, which comprises adding and mixing toluene when inoculating and mixing the microorganisms into the water or soil.

[0009]

The microorganism according to the present invention decomposes trichlorethylene in water or soil in the presence of an aromatic compound. Therefore, when inoculating / mixing this microorganism into water or soil contaminated with trichlorethylene, by adding and mixing at least one kind of aromatic compound, trichlorethylene can be safely and reliably decomposed and removed.

[0010]

Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Microorganisms according to the present invention, artificially adjusted contaminated water containing trichlorethylene exposed Isehara Kuroboku soil for a long time, put a portion of the soil in a vial, the following composition (per 1 liter of medium) ) Inorganic medium, a predetermined amount of trichlorethylene and toluene were added, and after stoppering with Teflon-coated butyl rubber, sealing with an aluminum cap, shaking culture at 30 ° C, and after a certain period of time, the gas phase in the doubled bottle was changed. Gas chromatograph analysis was performed, repeating accumulation while diluting the soil amount for the test system in which attenuation of trichlorethylene was observed, and LB (Luria-
Bertani) broth was applied on a plate medium containing agar, and colonies that appeared were repeatedly cultured and isolated. In addition, for culturing the microorganism, it is possible to arbitrarily select optimal conditions such as LB broth and trypticase soy broth.

K ₂ HPO ₄ 1.0 g, KH ₂ PO ₄ 1.0 g NH ₄ NO ₃ 1.0 g, MgSO ₄ .7H ₂ O 0.2 g Fe ₂ (SO ₄ ) ₃ 5 mg, Na ₂ MoO ₄ .H ₂ O 5 mg MnSO ₄ · H ₂ O 5 mg, yeast extract 5 mg

Then, a strain having a particularly strong trichlorethylene decomposing ability was isolated from the results of analysis by gas chromatography, and its morphological and physiological properties were examined. The following results were obtained.

[0013]   (A) Form   Cell shape Short bacillus   Size 1.1 × 1.6 μm   Colony characteristics Milky white, vigorous growth (on LB agar medium)   (B) Cultural properties   Growth on MacConkey agar +   (C) Physiological properties   Gram stainability +   Nitrate reduction capacity +   Generation of indole −   Oxidase test +   Urease −   Use of citric acid ＋   Carbon source utilization     Dextrose (aerobic condition)-, Dextrose (anaerobic condition)-     Maltose-, Sucrose-     Starch-, D-xylose-   (D) Other     Deamination reaction of phenylalanine −     Arginine degradation −     Decarboxylation of lysine-     Decarboxylation of ornithine −     ONPG-     Fatty acid composition 14: 0 2.49%                     14: 0 2OH 3.85%                     16: 1 35.70%                     16: 0 20.72%                     18: 1 27.11%

From the above results, it was found that the isolated strain with high degradability of trichlorethylene was Alcaligenes eurtrophus KS01.

The microorganism has a deposit number of FERM.
Deposited at Institute for Microbial Technology, Institute of Industrial Technology, P-13761.

This microorganism completely assimilates a relatively high concentration of trichloroethylene of about 50 ppm mixed in the medium,
Disassemble. When degrading trichlorethylene with this microorganism, it is necessary to add at least one aromatic compound such as toluene to the medium substrate (medium, soil, water, etc.) containing trichlorethylene. As the aromatic compound used here, toluene, phenol, o-cresol, m-cresol, or the like is used.

The microorganism according to the present invention will be described below in more detail with reference to the following experimental examples. Experimental Example 1 15 ml of the above inorganic medium was placed in a vial having a volume of 68 ml,
The concentration in the culture solution is adjusted to 100 ppm of toluene and 0.1 to 50 ppm of trichloroethylene. Then, after inoculating 10 ⁸ cells / ml (medium) of this microorganism, which had been cultivated in LB broth and washed by centrifugation, a Teflon-coated butyl rubber stopper, which was sealed with an aluminum cap, was incubated at 30 ° C. The gas phase was analyzed by a gas chromatograph equipped with an ECD detector.

The results are shown in FIG. As shown in the figure, trichloroethylene at a concentration of 0.1 to 50 ppm in the culture solution was below the detection limit of the gas chromatograph within 5 days, and it was confirmed that the present microorganism has extremely high trichlorethylene decomposing ability.

Experimental Example 2 15 ml of the above inorganic medium was placed in a vial having a volume of 68 ml,
Trichlorethylene was added so that the concentration in the culture solution would be 1 ppm. To this, toluene 1-100pp
m, Phenol 1ppm or Phenol 10ppm was added dropwise, and after inoculating 10 ⁸ cells / ml (medium) of this microorganism that had been cultured in LB broth and spun down and washed, a Teflon-coated butyl rubber stopper was placed and sealed with an aluminum cap. 30
After culturing at 0 ° C., as in Experimental Example 1, the gas phase was ECD periodically.
It was analyzed by a detector gas chromatograph.

FIG. 2 shows the results. As shown in FIG. 2, trichlorethylene was decomposed below the detection limit of the gas chromatograph within about 3 days after inoculation of the microorganism under all conditions. It was confirmed that In addition,
Although not shown in the figure, it was confirmed that trichloroethylene was surely decomposed by the present microorganism even when 1 or 10 ppm of metacresol or orthocresol was mixed in place of toluene.

Experimental Example 3 15 ml of the above-mentioned inorganic medium was placed in a vial having a volume of 68 ml, trichlorethylene was added so that the concentration in the culture solution would be 1 ppm, and 10 ppm of toluene was added dropwise to this, which was then cultured in LB broth. After inoculating the microorganism washed and spun down to 10 ⁹ cells / ml (medium), a Teflon-coated butyl rubber stopper and an aluminum cap-sealed one were cultivated at 30 ° C., and after 1 day, the gas phase was trichlorethylene. In the case of, with a gas chromatograph with an ECD detector,
In the case of toluene, it was analyzed by a gas chromatograph with an FID detector.

FIG. 3 shows the results. As shown in FIG. 3, it was confirmed that trichloroethylene was decomposed by this microorganism and the added and mixed toluene was also disappeared. It was also confirmed that there is no concern about environmental pollution due to the substance toluene.

Next, an experimental example relating to a method for purifying soil contaminated with trichlorethylene using the microorganism of the present invention will be described. Experimental Example 4 In a vial with a volume of 124 ml, Isehara Kuroboku soil (raw soil)
10 g (dry soil) and 2 min of the above inorganic medium containing 0.1 ppm or 1 ppm of trichlorethylene.
After adding 0 ml, 10 ppm of toluene was further added dropwise to this, and the microorganism was cultured in LB broth and spun down for 10 ⁷
After inoculating cells / ml (medium + raw soil), put a Teflon-coated butyl rubber stopper into a suspended state, and further cultivate the one sealed with an aluminum cap at 30 ° C and periodically subject the gas phase to EC.
It was analyzed by a gas chromatograph with a D detector.

FIG. 4 shows the result, which is 3 when the concentration of trichlorethylene ethylene is 0.1 ppm.
Even in the case of 1 ppm per day, it was confirmed to be decomposed within the detection limit of the gas chromatograph within 10 days, and it was confirmed that the present microorganism exhibits the trichlorethylene decomposition function even in the natural environment. By this, although it shows an effect in a sterilized culture solution, in the existing microorganisms that are often unable to exert their decomposition ability by being exterminated by indigenous microorganisms,
Since this microorganism exerts its decomposition function sufficiently even in the natural environment, by contacting it with contaminated water,
Further, it is possible to purify the contaminated soil by suspending it in water containing the microorganism.

[0025]

According to the microorganism of the present invention, trichlorethylene contained in water or soil can be decomposed within a few days in the presence of at least one aromatic compound.

The method for purifying water or soil according to the present invention does not require a large amount of energy, and has the advantages of biotechnology such as the ability to suppress the occurrence of secondary pollution, as well as the natural environment. Water or soil contaminated with trichlorethylene below can be efficiently purified on an industrial scale.

[Brief description of drawings]

FIG. 1 is an experimental result showing a decomposition effect of a microorganism according to the present invention at various concentrations of trichlorethylene.

FIG. 2 is an experimental result of investigating the effect of degrading a microorganism according to the present invention when the type and concentration of an aromatic compound are changed.

FIG. 3 is an experimental result showing the ability of the microorganism of the present invention to decompose trichlorethylene and the residual amount of toluene,
(A) is the initial peak of trichlorethylene, (b) is the peak of trichlorethylene after one day, (c) is the initial peak of toluene, and (d) is the peak of toluene after one day.

FIG. 4 is an experimental result showing a decomposition effect when the microorganism according to the present invention is applied to contaminated soil.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI // (C12N 1/20 C12N 1/20 C12R 1:05) C12R 1:05 (56) References Appl Environ Micro object. (1990), Vol. 56, No. 4, p. 1179-1181 Arch Microbiol. (1990), Vol. 154, No. 4, p. 410-413 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 1/00 A62D 3/00 C02F 3/34 CA (STN) MEDLINE (STN) REGISTRY (STN) WPIDS (STN) ) JSTPlus (STN)

Claims (2)

(57) [Claims] 1. Alkali Genes Eurotrofus (Al) having an ability to decompose about 50 ppm of trichlorethylene in a medium containing 1 to 10 ppm of toluene.
caigenes eurotrophus) KS01 (FERM P-13)
761) strains . 2. A method for purifying water or soil in which the microorganism according to claim 1 is inoculated and mixed with water or soil contaminated with trichlorethylene to decompose trichlorethylene contained in the water or soil. A method for purifying water or soil, which comprises adding and mixing toluene when inoculating and mixing the water or soil with the microorganisms.