JPH09149786A - Microorganism capable of decomposing halogenated hydrocarbon and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject new microorganism belonging to Pseudomonas pickettii, capable of decomposing halogenated hydrocarbons in high efficiency and useful e.g. for the microbial cleaning treatment of water or soil contaminated with halogenated hydrocarbons. SOLUTION: This new microorganism Pseudomonas pickettii B-1 (FERM BP-5288) is a microorganism belonging to Pseudomonas pickettii capable of decomposing halogenated hydrocarbons and useful for the efficient microbial cleaning treatment, etc., of water or soil contaminated with halogenated hydrocarbons (e.g. trichloroethylene). The microorganism was obtained by collecting activated sludge from a waste-water treating facility of Aichi Prefecture, Japan, inoculating the sludge to a medium put into a vial, adding trichloroethylene and toluene to the medium, sealing the vial, subjecting to static culture at 30 deg.C transplanting to a plate medium, culturing on the medium and repeating the culture of the developed colony.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for microbial purification treatment of water or soil contaminated with halogenated hydrocarbons.

[0002]

2. Description of the Related Art In recent years, with the increase in industrial use of organic solvents, particularly halogenated hydrocarbons, measures against soil pollution by these substances or wastewater containing these substances have been desired. There are physical treatment methods and biological treatment methods as the purification technology for contaminated soil. The physical methods include blowing air into the excavated contaminated soil to volatilize the organic halide and adsorbing it with activated carbon.The air stripping method is used to drive the pipe into the contaminated soil and reduce the pressure to produce volatile organic halogen. There is a vacuum extraction method in which the compound is vaporized and extracted from the soil.

These methods require a great deal of power for blowing air, the former requires excavation of the soil, and the latter requires a lower concentration of pollutants to lower extraction efficiency and prevent purification. There is a drawback that. Furthermore, in any of the methods, the pollutant is simply adsorbed on the activated carbon or the like, so that the pollutant must be detoxified separately. In recent years, it has been reported that biological treatment methods under development can completely decompose and detoxify pollutants by utilizing the ability of microorganisms to decompose substances, and input energy is less than physical methods. is there. Furthermore, even low-level pollution can be purified, so there are great expectations as a low-cost soil purification technology.

As a method for purifying contaminated soil, a solid-phase treatment method for promoting decomposition of pollutants by mixing microorganisms with nutrient sources such as phosphorus and nitrogen in excavated soil, water in excavated soil, A slurry treatment method that promotes the decomposition of pollutants by mixing microorganisms with nutrient sources and treating the soil in a liquid state, injecting air, nutrient sources, etc. into the soil without excavating the contaminated soil There are in-situ treatment methods that promote the decomposition of pollutants by existing microorganisms.

Among the conventional biological treatment techniques, the solid-phase treatment method and the slurry treatment method require excavation of soil and have a narrow application range, and the treatment and facility costs are relatively high. On the other hand, the in-situ processing method has a lower processing cost and equipment cost than the above method, and can perform processing over a wide range. However, the purification rate is slow because the absolute number of soil microorganisms is small. Especially in the case of persistent compounds such as halogenated hydrocarbons, it is possible that microorganisms capable of decomposing pollutants do not live in the soil, and in that case purification is impossible. In these cases, it is possible to improve the cleaning speed and to clean the soil in which microorganisms capable of degrading pollutants do not live by obtaining and inoculating the soil with microorganisms capable of degrading halogenated hydrocarbons.

[0006] As a known microorganism that decomposes trichlorethylene, which is important as a halogenated hydrocarbon pollutant, is a methane-utilizing bacterium, Methylosinus tricosporium.
ium) OB3 (Tokuhoku Hyo 4-501667, Japanese Patent Laid-Open No. H5-50167)
212371) and Methylosynaceus trichosporium (M
(Ethylosinus tricosporium)
TUKUBA (JP-A-2-92274, JP-A-3-29)
2970), a Pseudomonas genus
Putida (Pseudomonus putida) F1
(JP-A 64-34499), Pseudomonus putida BH (Fujita et al .; Chemical Engineering, 39, 6, p494-).
498, 1994), Pseudomonas putida (Pse
udomonus putida) UC-R5, UC-
P2 (JP-A-62-84780), Pseudomonus putida KWI
-9 (JP-A-6-70753),

Pseudomonas mendocina (Pseud)
MONUS mendocina) KR1
503866, 5-502593), Pseudomonus cepaci
a) G4 (Japanese Patent Laid-Open No. 4-502277), Pseudomonus cepaci
a) KK01 (JP-A-6-296711), other Alcaligenes Utrohus
eutropus) JMP134 (AR Hark)
er Appl. Environ. MIcrobio
l. , 56, 4, 1179-1181, 1990), Alcaligenes Utrohus
S eutropus) KS01 (Japanese Patent Laid-Open No. 7-1239)
76), an ammonia-oxidizing bacterium, Nitrosomonas europaea (Nitrosomonas europae).
a) (D. Arciero et al. Bioche
m. Biophys. Res. Commun. , 15
9, 2, 640-643, 1989) and the like are known.

[0008] Many of these known microorganisms are not so high in the ability to decompose trichlorethylene, and at most decompose to 5 ppm of trichlorethylene in the culture solution as an initial concentration. In addition, since it is necessary to decompose trichlorethylene under a special environment such as soil, not only can microorganisms used for biological purification have sufficient trichloroethylene decomposition activity, but it can also effectively demonstrate its decomposition ability in soil. However, many of the known microorganisms do not have sufficient ability.

Pseudomonas cepacia (Pse
udomonus cepacia) KK01 has an initial concentration of 30 ppm of trichlorethylene of 15 ppm in the culture solution.
It has been reported that trichloroethylene with an initial concentration of 5 ppm is decomposed to about 1 ppm in a soil environment up to (50%) (JP-A-6-296711). In addition, Alcaligenes eutrohus
pus) KS01 has been reported to decompose trichloroethylene with an initial concentration of 50 ppm in the culture solution to below the detection limit, and trichlorethylene with an initial concentration of 1 ppm to below the detection limit in a soil environment (JP-A-7-123976).

These microorganisms have a higher decomposing ability than conventional microorganisms in a culture solution, and it has been confirmed that the decomposing ability in a soil environment is exerted. However, in order to exert the degrading ability, an inducer (inducing agent) is used. It is necessary to add at least one kind of aromatic compound to the soil environment. Since the aromatic compound itself is a pollutant, it may cause secondary pollution. Even if an aromatic compound is added, it is completely decomposed and removed together with trichlorethylene, or trichloroethylene is added without adding an aromatic compound. Acquiring a microorganism capable of decomposing urine is a major problem to be solved in practice.

Therefore, in order to put into practice the biological purification method of trichlorethylene, it has a high decomposition ability and is completely decomposed / removed together with trichlorethylene even if an aromatic compound is added, or the aromatic compound is removed. It is desired to obtain a microorganism that can decompose trichlorethylene in soil without adding it. Furthermore, in the environment such as contaminated soil, due to the effects of predation by protozoa and competition with other indigenous microorganisms, the density of the microorganisms that cause the scattered decomposition is remarkably suppressed, so that it is necessary to meet the required treatment capacity. Increasing the density is often extremely difficult.

[0012] In order to increase the density of the microorganisms, there may be mentioned a method of pumping air or nutrients into the soil. However, both methods require a great amount of energy,
It is difficult to increase the cell density by itself, and the processing capacity remains at a low level as a whole. In addition, a large amount of energy such as nutrient supply and aeration is required to maintain a high cell density even when processing is performed in an open system and a closed system such as in a reactor.

If the decomposing ability per unit cell amount can be increased, a sufficient decomposing ability can be obtained even if the cell density is low, so that it is not necessary to input a large amount of energy to maintain the cell density. A microorganism that decomposes trichlorethylene decomposes trichlorethylene by expressing an enzyme that can decompose the substance, but an inducer is required to express this enzyme. It has already been known that microorganisms can exert the ability to decompose trichlorethylene by adding an inducer to the microorganism during contact, but focusing on the time of contact with the inducer, the ability to decompose per unit cell mass There is no conventional example that examined the method of increasing the.

[0014]

Therefore, the present invention can exhibit a high halogenohydrocarbon decomposition ability in water or soil, and it is not always necessary to add an aromatic compound or the like as an inducer to the soil to be treated. It is intended to provide a microorganism capable of decomposing an unnecessary microorganism or an added inducer, and a method for purifying water or soil pollution using the microorganism.

[0015]

Accordingly, the present invention is directed to Pseudomonas pickettii.
Provided are microorganisms belonging to the species and having the ability to decompose halogenated hydrocarbons. The microorganism is preferably able to assimilate aromatic compounds and is activated in the presence of aromatic compounds. As a specific example of such a strain, Pseudomonas picetti B-1 (FERM BP-52
88).

The present invention further provides a method for purifying water or soil containing halogenated hydrocarbons, which comprises adding the above microorganisms to water or soil containing halogenated hydrocarbons. In this method, it is preferable, but not essential, to add an aromatic compound assimilable to the microorganism to the water or soil to be treated together with the above microorganism. In particular, before adding to water or soil, the microorganism can be activated by pre-culturing the microorganism in the presence of an aromatic compound, and when such activated microorganism is added to water or soil. In particular, it is not necessary to add aromatic compounds to water or soil.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The microorganism of the present invention is Pseudomonus pi.
ckettii ), and any microorganisms capable of decomposing halogenated hydrocarbons may be used, and one example thereof is Pseudomonas picetti B-1 strain. The isolation method and taxonomic characteristics of this strain are specifically described in Example 1. This microorganism is P. pic
KETTI B-1 was deposited on November 9, 1995 as FERM BP-5288 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

The microorganism of the present invention is a halogenated hydrocarbon,
In particular, it has the ability to decompose at least one of chlorinated hydrocarbons such as trichloroethylene, dichloroethylene, monochloroethylene, etc., and particularly to decompose trichlorethylene. The microorganism of the present invention is also preferably capable of assimilating and decomposing aromatic compounds, and culturing the microorganism of the present invention in the presence of an aromatic compound activates the ability to decompose halogenated hydrocarbons. To be done. Aromatic compounds that can be assimilated by the microorganism of the present invention are especially aromatic hydrocarbons such as benzene, toluene, phenol and the like, especially toluene.

The microorganism of the present invention can be cultivated in the presence of a conventional carbon source and nitrogen source, if necessary, in a medium containing trace elements such as inorganic salts and vitamins.
The carbon source is preferably a carbohydrate, particularly glucose, which is preferably assimilated by the microorganism of the present invention, but is not limited to this and, for example, maltose, fructose and the like can be used. The concentration of the carbon source in the medium varies depending on the type of carbon source, but is preferably 0.1 g / L to 0.5 g / L. As the nitrogen source, an organic nitrogen source such as yeast extract,
Peptone, meat extract, etc. can be used, and as the inorganic nitrogen source, ammonium salt, nitrate, etc. can be used. The concentration of nitrogen source depends on its type,
It is preferably 0.1 g / L to 1.4 g / L.

The inorganic salts include metal ions such as potassium ion, calcium ion, magnesium ion, iron (II) ion, manganese ion, cobalt ion and nickel ion, and anions such as hydrochloric acid ion, sulfate ion and phosphate ion. The salts consisting of are preferred. The culturing is preferably carried out aerobically, and in shaking culture or large-scale culturing, hyperaeration / agitation culture is preferable. Culture temperature is 2
0 ° C to 37 ° C, especially around 30 ° C is preferable.

The present invention also relates to a method for purifying water or soil, characterized in that the above-mentioned microorganism is added to water or soil containing a halogenated hydrocarbon. In this method, the cells of the microorganism of the present invention cultured as described above may be added to the water or soil to be treated.
The cells may be added in the form of a culture solution, or may be separated from the culture solution and added as cells (cells). The amount of microbial cells (cells) added is
Yl the amount or the like of the halogenated hydrocarbon in water or soil to be treated, 10 ⁶ cells / g to 10 ⁹ cells / g, preferably 10 ⁶ cells / g to 10 ⁸ cells / g.

The time required for the treatment depends on the hydrogen halide decomposing ability of the microbial cells to be used, the amount of halogenated hydrocarbons in the water or soil to be treated, the amount of microbial cells to be added, etc., but it is 1 to 10 days. , For example, 2 to 7 days. According to a preferred embodiment of the method of the present invention, when a microorganism is added to water or soil to be treated, the aromatic compound which can be assimilated and decomposed by the microorganism as an inducer of a halogenated hydrocarbon decomposing activity. Preferably, benzene, toluene and phenol are added. Thus, the use of an inducer causes secondary pollution when the microorganism does not have the ability to decompose aromatic compounds or when the microorganism has a low ability to decompose aromatic compounds, but it uses microorganisms having a strong ability to decompose aromatic compounds. As long as there is no worry. The amount of the aromatic compound added as an inducer, such as toluene, is 0.04 g / kg to the amount of water or soil to be treated.
It is 0.1 g / kg.

In a further preferred embodiment, as mentioned above, when the aromatic compound is added as an inducer, another carbon source which is preferably assimilated by the microorganism used, particularly a carbohydrate, particularly a monosaccharide, such as glucose, is added. To do. This addition enhances the ability of the microorganism to decompose halogenated hydrocarbons. The added amount of this carbon source such as glucose is, for example, 0.18 g / kg, relative to the amount of water or soil to be treated.

In another preferred embodiment of the invention, the microorganism is activated by an inducer before it is added to the water or soil to be treated. The inducer in this case is the same as that described above as an inducer added to water or soil, and in a further preferred embodiment, the carbon source described above is added as a carbon source added to water or soil. To do. As a method of activating the microorganism, in the culture for the production of microbial cells to be added to water or soil, the aromatic compound as a carbon source,
Alternatively, the aromatic compound and glucose or the like may be used,
Alternatively, after the microbial cells are produced, they may be pre-cultured in a medium containing the inducer before adding them to water or soil.

When an inducer is added in the process of producing microbial cells, an aromatic compound such as toluene is added to the medium in an amount of 0.04 g / L to 0.1 g / L, preferably 0.092 g. To be included in. More preferably, in addition to the aromatic compound, a carbohydrate such as glucose is added in an amount of 0.1 g / L to 0.5 g / L, preferably 0.1.
18g is contained. The pre-culture period is 10 hours or more,
It is preferably 12 hours to 30 hours. The other components in the preculture medium are, for example, those described above as the components of the culture medium for microbial culture.

In the water or soil purification treatment method of the present invention, the addition of the inducer to the water or soil and / or the activation of the microorganism by the inducer prior to the addition of the microorganism to the water or soil is performed by the method of the present invention. Although not essential to the method, performing one or both of these steps increases the concentration of treatable halogenated hydrocarbons in the water or soil. In particular, by subjecting the microorganism to activation treatment with an inducer before adding it to water or soil, without adding the inducer to water or soil,
The advantage is that high concentrations of halogenated hydrocarbons in water or soil can be decomposed.

Halogenated hydrocarbons which can be decomposed by the process of the present invention include, among others, chlorinated hydrocarbons such as trichloroethylene, dichloroethylene, monochloroethylene and the like. The method of the present invention can be applied to the solid-phase treatment method and the slurry treatment method described above, but it is not always necessary to use these methods that require excavation of the soil, and the microorganism of the present invention can be applied to the soil. The soil or the like can be purified by simply adding or inoculating the above.

[0028]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG. Isolation and Identification of Microorganisms Microorganisms according to the present invention were isolated from activated sludge collected from wastewater treatment plants in Aichi Prefecture by the following method. 0.1 ml of the collected activated sludge was placed in a 30 ml capacity vial.
ml NMS medium was inoculated and 0.5 ppm trichlorethylene and 1 mM toluene were added. The vial was capped with a Teflon-coated butyl rubber, sealed with an aluminum cap, and then allowed to stand at 30 ° C. for a predetermined period.

The culture solution in which a slight turbidity was observed was subcultured to the same medium and left standing. This transfer was repeated 3 times in total. After completion of the third stationary culture, the culture solution is appropriately diluted to N
The YG medium was spread on a plate medium containing 1.5% agar, and the colonies that appeared were repeatedly cultured to isolate the microorganism. In addition to NYG medium described above, microbial cultures include Nutrient Medium (Nutrient Med).
This can be done by arbitrarily selecting the optimum conditions such as ium).

[0030]

[Table 1]

[0031]

[Table 2]

[0032]

[Table 3]

The isolated microorganism was added to 6 ml of NYG medium in a 30 ml volume vial, and 1.5% to NYG medium.
Inoculate with a platinum loop to inoculate a colony of this strain from a plate medium containing the above agar, or inoculate 0.06 ml of a preculture liquid obtained by shaking culture of this strain overnight in NYG medium at 30 ° C. did. Simultaneously with the inoculation, 10ppm trichlorethylene and 1
mM toluene was added. Use a Teflon-coated butyl rubber stopper for the vial and seal it with an aluminum cap at 30 ° C.
After shaking culture for 3 days at room temperature, the gas phase in the vial was
It was analyzed by gas chromatography equipped with a CD detector.

Based on these results, a strain having a particularly strong trichlorethylene decomposing ability was selected, and the morphological and physiological properties of the strain were examined. The results shown below were obtained.

[0035]

[Table 4]

[0036]

[Table 5]

From the above results, the literature (NR Krieg and J.
G. Holt, “Bergey's Manual of Systermatic Bacteri
ology "Vol.1 (1984) Williams ans Wilkins and J.
G. Holt, NR Krieg, PHA Senath, JT Stale
y and ST Williams, "Bergey's Manual of Detamina
tive Bacteriology "Ninth edition (1994) Williamsan
s Wilkins), the strains with high trichlorethylene degrading ability were identified as Pseudomonas.
Piquette (Pseudomonus pickett)
i) and identified as B-1 strain.

Since the known Pseudomonas piketti has not been reported to have the activity of degrading halogenated hydrocarbons, the strain B-1 was identified as a novel microorganism. In addition, Pseudomonas picketti (Pseudomonus pi)
ckettti) was Burkholderi in 1992.
a has been transferred to a new genus (E. Yabuuch, Y. Kosak
a, H. Oyaizu, I. Yano, H. Hotta, Y. Hashimoto, T.
Ezaki and M. Arakawa (1992) Microbiol. Immunol., 3
6, 1251).

Example 2 Trichlorethylene in medium
Decomposition of 6 ml of NYG medium in a 30 ml volume vial,
Inoculate the colony of this strain with a platinum loop from a plate medium prepared by adding 1.5% agar to NYG medium, or inoculate
This strain was shake-cultured overnight in a YG medium at 30 ° C. and inoculated with 0.06 ml of a preculture solution (about 10 ⁶ cells / ml in terms of the number of cells). Simultaneously with the inoculation, 10 or 30 ppm trichlorethylene and 1 mM toluene were added. The vial was covered with a Teflon-coated butyl rubber stopper, sealed with an aluminum cap, and cultured by shaking at 30 ° C. The gas phase in the vial was periodically analyzed by gas chromatography with an ECD detector.

The results are shown in FIG. As shown in the figure, trichlorethylene in the culture solution was
It was confirmed that 0% was decomposed and that this microorganism had a high trichlorethylene decomposing ability. Compared with the known Pseudomonas, Pseudomonas Putida F1 and Pseudomonas Sepasia G4 (both JP-A-64-344)
99), Pseudomonas mendocina KR1 (JP-A-2-503866), Pseudomonas putida KWI.
-9 (JP-A-6-70753) and Pseudomonas
Sepacia KK01 (JP-A-6-296711) is known as Pseudomonas that decomposes trichlorethylene, but Pseudomonas cepacia KK01 is 30 pp.
About 15ppm of m trichloroethylene (50
%, But only the decomposition of trichlorethylene at a low concentration of 10 ppm or less is reported.

Therefore, it was found that the microorganism of the present invention has a higher trichlorethylene decomposing ability than known Pseudomonas. It should be noted that, when compared to known microorganisms other than Pseudomonas, Alkagenes Eutrohus KS01 (JP-A-7-123976) is reported to completely decompose 50 and 25 ppm of trichlorethylene within 4 days, but it was inoculated into the culture solution at the time of decomposition of trichlorethylene. The amount of the microorganisms to be used is 10 ⁸ / ml, which is about 100 times the amount of the microorganisms of the present invention. Therefore, the microorganism of the present invention can significantly reduce the amount of inoculated cells required for decomposing a predetermined amount of trichlorethylene, as compared with Alkagenes eutrofus KS01, and thus has the advantage of reducing the cell culture cost.

Example 3 In an aqueous solution in the presence of toluene
Decomposition of trichlorethylene To 4 ml of NYG medium placed in a 20 ml volume vial,
Inoculate the colony of this strain with a platinum loop from a plate medium prepared by adding 1.5% agar to NYG medium, or inoculate
The preculture liquid obtained by culturing this strain overnight in YG medium at 30 ° C. was 1/100 the volume of the NYG medium (about 1
( ⁶ cells / ml). Simultaneously with the inoculation, 10 ppm trichlorethylene and 1 mM toluene were added. The vial was covered with a Teflon-coated butyl rubber stopper, sealed with an aluminum cap, and cultured by shaking at 30 ° C. The gas phase in the vial was periodically analyzed by gas chromatography with an EID detector.

The results are shown in FIG. As shown in the figure, trichloroethylene and toluene in the culture solution were 12
It is decomposed to less than the detection limit of the gas chromatography by culturing for a long time, the trichlorethylene decomposition rate of the microorganism of the present invention is extremely fast, and the mixed and added toluene is also completely decomposed. It was also confirmed that there was no concern about contamination. Although not shown in the figure, trichloroethylene is certainly decomposed even with 1 mM benzene or 500 ppm phenol instead of toluene, and benzene is decomposed at the same time as the detection limit of the gas chromatography at the same time as trichlorethylene in the culture for 5 days. It was confirmed that

Example 4 Microbiota activated by toluene
Decomposition of Trichlorethylene in Soil by Material Next, a method for purifying soil contaminated with trichlorethylene using the microorganism of the present invention will be described. Volume 30
Into a ml vial, 10 g of artificially contaminated Kuroboku soil (obtained only from an upland field in Aichi prefecture and subjected to air-drying treatment) of 0.7 mg / kg was placed.

[0045] This strain was shake-cultured in NYG medium supplemented with 1 mM toluene for 2 days at 30 ° C, and the culture solution was collected by centrifugation and then resuspended in NYG medium to inoculate a solution having a adjusted bacterial cell concentration. The amount of cells was 10 ⁸ to 10 ⁹ cells / g soil, water was added so that the water content after addition of medium was 40%, a screw-type cap with Teflon-coated packing was sandwiched, and the mixture was stirred at 30 ° C. It was left still for 7 days. After static culture for 30 days at 30 ℃, weigh 10g of soil into an Erlenmeyer flask with a stopper and aerated with activated carbon to pass 90ml.
Of ion-exchanged water, 5 ml of phosphoric acid and 10 ml of n-hexane were added, and the vessel was sealed. It was sonicated for 20 minutes in an ultrasonic cleaner, then shaken for 5 minutes on a shaker, and then the aqueous phase and n-hexane phase were transferred to a colorimetric tube with a stopper. The colorimetric tube was sealed and sonicated, and the separated n-hexane was washed with E.
It was analyzed by gas chromatography equipped with a CD detector.

The results are shown on the left side of FIG. Initial density 0.7
It was confirmed that mg / kg of trichlorethylene was decomposed to 0.3 mg / kg in 7 days, and that the present microorganism exerts trichlorethylene decomposing ability even in a natural environment and in a state where an aromatic compound is not added at the same time. Therefore, although it is effective in a sterilized culture solution, it is effective against existing microorganisms that are often unable to exert their decomposing ability by being exterminated by indigenous microorganisms in the natural world. In contrast to existing microorganisms that cannot often be exerted, the present microorganisms exhibit their ability to decompose even in the natural environment.Therefore, by contacting the contaminated water with the microorganisms, or contaminated soil with water containing the microorganisms. It is possible to purify them by putting them in suspension.

On the other hand, as shown on the right side of FIG.
No significant remediation was found in contaminated soil at concentrations as high as mg / kg, so a method to solve this problem was investigated. That is, the present inventors enhance the degrading activity of microorganisms capable of degrading trichlorethylene in soil and prepare a strain having a degrading ability capable of coping with highly contaminated soil. As a result of paying attention to the culture conditions of No. 1, it was found that there is an optimum value for the culturing time of microorganisms before contact with soil, which enhances the decomposition activity after spraying. That is, the present invention was found by optimizing the culturing time of microorganisms before inoculation into soil and thereby increasing the decomposition activity after inoculation. Hereinafter, the present invention will be described in detail with reference to examples.

Example 5 Examination of activation time before application of microorganisms
In 4ml NYG medium in a 20ml vial,
Inoculate the colony of this strain with a platinum loop from a plate medium prepared by adding 1.5% agar to NYG medium, or inoculate
The preculture liquid obtained by culturing this strain overnight in YG medium at 30 ° C. was 1/100 the volume of the NYG medium (about 1
( ⁶ cells / ml). Simultaneously with the inoculation, 10 ppm trichlorethylene and 1 mM toluene were added. The vial was sealed with a Teflon-coated butyl rubber stopper and sealed with an aluminum cap, and then cultured by shaking at 30 ° C. The gas phase in the vial was periodically analyzed by gas chromatography with an EID detector.

After collecting 4 ml of the culture solution of the present microorganism from another vial which had been inoculated at the same time as the analyzed vial and had been culturing for the same time, centrifuging and collecting the cells, 4 ml of NMS was added to the cells.
The cells were resuspended in a medium and placed in a vial having a volume of 20 ml, and 10 ppm of trichlorethylene was added at the same time. The vial was capped with a Teflon-coated butyl rubber, sealed with an aluminum cap, and then cultured by shaking at 30 ° C. for 24 hours, and the gas phase in the vial was analyzed by gas chromatography with an EID detector.

The results are shown in FIG. The line graph in the figure shows the residual rate of toluene and trichlorethylene at the culture stage before inoculation, and the bar graph shows the decomposition rate of trichlorethylene of the microorganism collected. It was found that, when the toluene was completely decomposed, the microorganism-collected microorganisms exhibited the maximum decomposition activity. Therefore, before inoculating the soil, culturing is performed while monitoring the decomposition amount of toluene and trichlorethylene, and when the toluene is completely decomposed, the culture is terminated, and when inoculated into the soil, high decomposition ability is exhibited. I understood.

Example 6 Pre-spray activated microorganisms
Decomposition of high-concentration trichlorethylene in soil Soil containing 100 ml of artificially contaminated Kuroboku soil with 100 mg / kg trichlorethylene (collected from upland fields in Aichi prefecture and subjected to air-drying treatment only) 10
I put g.

This strain was shake-cultured at 30 ° C. in NYG medium supplemented with 1 mM toluene and 10 ppm trichlorethylene while monitoring the decomposition amounts of toluene and trichlorethylene. Toluene was completely decomposed (culture 2
Cultivation is completed around 6 hours), and after centrifugation and collection, NM
A solution in which the cell concentration was resuspended in S medium to adjust the cell concentration was added to the air-dried soil so that the inoculated cell amount was 10 ⁸ to 10 ⁹ cells / g soil and the water content after medium addition was 25%. .

The vial was capped with a Teflon-coated butyl rubber, sealed with an aluminum cap, stirred, and then allowed to stand at 30 ° C. for 7 days. After static culture for 7 days at 30 ° C, 50 ml of air aerated with activated charcoal passed through a vial.
10 ml of n-hexane was added to the above ion-exchanged water, and the vessel was sealed. It was sonicated in an ultrasonic cleaner for 10 minutes and then shaken on a shaker for 10 minutes, and then separated n-hexane was analyzed by gas chromatography with an ECD detector. As a control, the same experiment as above was performed, but an experiment in which the culture time before soil addition was 48 hours (2 days) was also carried out.

The results are shown in FIG. As a result of optimizing the culture conditions before inoculation, it was confirmed that a high concentration of 17 mg / kg of trichlorethylene was decomposed to about 11 ppm, and that this microorganism exhibited a high concentration of trichlorethylene degrading ability even in a natural environment. Therefore, by optimizing the culture conditions before inoculation, the microorganism can exert higher resolution even in the natural environment.Therefore, by contacting the microorganism with water contaminated at a higher concentration or at a higher concentration. It is possible to purify the soil contaminated with by suspending it in water containing the microorganism.

In the prior art, the case of microbial decomposition of trichlorethylene in soil was 5 ppm (mg / kg) in the presence of an inducer even if the target concentration was high (JP-A-6-296711, JP-A-7-95884, etc.). ) It is below, and the possibility of microbial purification has not been examined in the higher concentration range.

[0056]

EFFECTS OF THE INVENTION According to the microorganism of the present invention, preferably, in the presence of at least one kind of aromatic compound and a carbon source such as sugar, halogenation such as high concentration of trichlorethylene contained in water or soil. Hydrocarbons can be decomposed within a few days.

The method for purifying water or soil according to the present invention does not require a large amount of energy, and the advantages of biotechnology such as the suppression of the occurrence of secondary infection can be obtained. Water or soil contaminated with trichlorethylene can be efficiently purified on an industrial scale. According to the method for purifying water or soil according to the present invention, the water or soil contaminated with trichlorethylene can be industrially discharged into the natural environment without releasing pollutants such as aromatic compounds or combustible gases such as methane. It can be purified more efficiently on a scale.

[Brief description of the drawings]

1] FIG. 1 is a graph showing the change with time of the decomposition rate of trichlorethylene in a culture solution in Example 1.

FIG. 2 is a graph showing the change over time in the decomposition rate of trichlorethylene and toluene in the culture broth in Example 2.

FIG. 3 is a graph showing the decomposition rate of trichlorethylene in soil in Example 3. The error bar in the figure shows the standard deviation of the results of three measurements.

FIG. 4 is a residual ratio of trichlorethylene and toluene at the culture stage before addition in the culture solution in Example 4, and the bacterial cells cultured for the same time as the bacterial cells used for the measurement, and the suspension and resuspension. It is a graph which shows the decomposition rate of trichlorethylene when it becomes cloudy. The error bar in the figure shows the standard deviation of the results of three measurements.

FIG. 5 is a graph showing the decomposition rate of trichlorethylene in soil in Example 5. The error bar in the figure shows the standard deviation of the results of three measurements.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C02F 3/34 ZAB B09B 3/00 ZABE // (C12N 1/20 C12R 1:38) (72) Inventor Masami Yamashita 1188 Tomimura, Tomimura, Tomada-gun, Okayama (72) Inventor Masatoshi Uchida 38-1, Nishimarunouchi, Tsu City, Mie Prefecture

Claims (9)

[Claims] 1. Pseudomonas picketti
microorganism belonging to the species Domonus pickettii ) and having the ability to decompose halogenated hydrocarbons. 2. The microorganism according to claim 1, wherein the halogenated hydrocarbon is trichloroethylene. 3. The microorganism according to claim 1, which is capable of decomposing aromatic hydrocarbons. 4. The aromatic hydrocarbon is toluene.
The microorganism according to claim 3. 5. A Pseudomonas picketti B-1 (F
The microorganism according to any one of claims 1 to 4, which is ERM BP-5288). 6. Water or soil containing a halogenated hydrocarbon, characterized in that the microorganism according to any one of claims 1 to 5 is added to water or soil containing a halogenated hydrocarbon. Purification treatment method. 7. The method according to claim 6, wherein an aromatic compound is added to water or soil together with the addition of the microorganism. 8. The method according to claim 6, wherein the microorganism is pre-cultured in the presence of an aromatic compound prior to the addition of the microorganism. 9. The method according to claim 8, wherein the preculture is carried out in the presence of an assimilable carbohydrate together with an aromatic compound.