JPH0746984A - Method for treating volatile organic matter - Google Patents

Abstract

PURPOSE:To conduct treatment of volatile organic matter without causing environmental pollution through such processes that a volatile organic matter-contg. gas is introduced into a reaction vessel containing gene recombinant bacteria imparted and reinforced with volatile organic matter-decomposing ability and a culture medium to decompose the organic matter followed by removing the bacteria from the gas treated and then discharging the gas out of the system. CONSTITUTION:A liquor 6 to be treated containing volatile organic matter (e.g. trichloroethylene) is introduced, via a flow channel 5a, into an evaporation column 1 and allowed to stay there; a gas is introduced, via a flow channel 7a, into the column and diffused through a gas feeder 8 into the liquor to evaporate the volatile organic matter in the liquor and make the matter transfer to the gas side. Subsequently, the gas treated containing the volatile organic matter is fed, through a flow channel 7b, an inlet filter 2 and a flow channel 7c, into a reaction vessel 3. This reaction vessel 3 contains (A) gene recombinant bacteria containing phenol hydroxylase gene derived from Pseudomonas putida KW-1-9 strain capable of decomposing volatile organic matter and (B) nutrients therefor. With this system, the volatile organic matter in the gas to be treated is decomposed, the gas after treated is sent via a flow channel 7d to an outlet filter 4 where the gas is freed from bacteria or sterilized and then discharged out of the system.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for treating volatile organic substances with genetically modified bacteria.

[0002]

2. Description of the Related Art Since halogenated hydrocarbons such as chloromethanes, chloroethylenes, and chlorobenzenes, which are pollutants of groundwater and soil, are hardly biodegradable, they are difficult to be treated by ordinary biological treatment methods such as activated sludge method. Cannot be disassembled. As a method for treating such a halogenated hydrocarbon, JP-A-64-34499 discloses a method of decomposing a halogenated hydrocarbon by using a bacterium having an aromatic decomposing ability and utilizing an aromatic decomposing route. It has been proposed that it is possible to use a genetically modified bacterium as the bacterium.

However, in this method, it is necessary to add an inducer such as phenol in order to maintain the aromatic decomposition route, and since the treatment is carried out by contacting water or soil with the fungus in the liquid phase, the added inducer is added. Need to be removed from the treated water and discharged.

On the other hand, in Japanese Patent Publication No. 2-503866, Pseudomonas mendocina KR-1 ( Pseudomo
It has been described that a toluene monooxygenase gene (toluene monooxygenase decomposes trichloroethylene) derived from Nas mendocina KR-1) is introduced into a host cell, and this transformant is used to decompose trichlorethylene.

However, even in this method, since the water or soil is treated in contact with the genetically modified bacteria in the liquid phase, the treated water is treated in order to prevent the recombinant bacteria from flowing out together with the treated water and contaminating the environment. It is necessary to sterilize or sterilize with a sterilization filter or the like, which causes a problem of high processing cost.

[0006]

The object of the present invention is to decompose volatile organic matter efficiently by treating the volatile organic matter in contact with the genetically modified bacteria in the gas phase, and
It is an object of the present invention to propose a method for treating volatile organic substances, which can reduce the carry-out amount of recombinant bacteria in the treated gas and reduce the cost for sterilizing or sterilizing.

[0007]

The present invention is the following method for treating volatile organic substances. (1) A closed reaction vessel is constructed so as to contain a genetically modified bacterium imparting or enhancing the ability to decompose volatile organic matter and its nutrient source, and introducing the gas containing the volatile organic matter into this reaction vessel. Volatile, characterized in that the volatile organic substances in the gas are decomposed by the recombinant bacterium by contact with the recombinant bacterium, and the treated gas after contact is sterilized or sterilized and discharged to the outside of the system. Method of treating organic substances. (2) A liquid or a solid containing a volatile organic substance is brought into contact with a gas to transfer the volatile organic substance to the gas side, and a gas containing the volatile organic substance thus obtained is introduced into a reaction tank for treatment. The method according to (1) above.

The volatile organic substance to be treated in the present invention may be volatile, and halogenated hydrocarbons such as chloromethanes, chloroethylenes and chlorobenzenes are suitable for treatment, but aldehydes and others It may be a volatile substance. Such a volatile organic substance is preferably a biodegradable one, but may be an easily biodegradable one.

The above-mentioned volatile organic matter is treated by contacting it with a genetically modified bacterium in a state of being contained in gas, but when it is contained in liquid or solid (including slurry, gel, etc.) such as water or soil. Is a process in which a liquid or solid containing volatile organic matter is contacted with a gas to dissipate the volatile organic matter and transfer it to the gas side, and the resulting gas containing volatile organic matter is contacted with a recombinant bacterium. To do.

The genetically modified bacterium used in the treatment of the present invention is a bacterium to which the ability to decompose volatile organic substances is imparted or enhanced by genetic recombination. Such a recombinant bacterium includes a gene constitutively expressing a volatile organic compound-degrading enzyme from another bacterium, a bacterium transformed by introducing an expression promoter or the like that constantly expresses such a gene, or a repressor. Examples include bacteria with a deleted gene. Even if the host bacterium itself has the above gene in its chromosome,
By introducing a plasmid in which such a gene is combined with a gene necessary for autonomous replication and a broad host range replication region containing a replication initiation site, volatile organic compound degrading performance can be imparted or enhanced.

When treating halogenated hydrocarbons such as chloroethylenes, genetically engineered bacteria into which the toluene monooxynase gene described in the above-mentioned JP-A-2-503866 is introduced, and other halogenated hydrocarbons. The treatment can be carried out by using a genetically modified bacterium having imparted or enhanced resolution. In this case, preferred recombinant bacteria include Pseudomonas putida KWI-9 ( Pseudomon).
as putida KWI-9) strain chromosomal DNA-derived phenol hydroxylase gene, a drug resistance gene, a gene necessary for autonomous replication in Gram-negative bacteria and a broad host range replication region containing a replication initiation site, An example is a genetically modified bacterium in which a recombinant plasmid capable of replication in Gram-negative bacteria has been introduced into a host bacterium, and this bacterium can be used to efficiently decompose halogenated hydrocarbons such as trichlorethylene.

The above-mentioned phenol hydroxylase gene is contained in the DNA fragment represented by the following restriction enzyme map [1] which is isolated from the chromosomal DNA of Pseudomonas putida KWI-9 strain and the like.

[0013]

[Chemical 1] (In the map, C23O is a part of the catechol 2,3-oxygenase gene, and PH is the phenol hydroxylase gene.)

The Pseudomonas putida KWI-9 strain was transferred to the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry,
It has been deposited under the microorganism accession number of FERM P-13109, and its bacterial properties and culture method are described in Japanese Patent Application No. 4-228169.

The DNA fragment is a fragment cleaved at the restriction enzyme Stu I or Sma I site and has the following cleavage sites for the following restriction enzymes. Restriction enzyme number of cleavage sites Sac I 1 Sal I 1 Hin dIII 1 Eco RI 1 The relative cleavage sites of the above restriction enzymes are completely digested with a plurality of restriction enzymes, and the generated DNA fragment is analyzed by agarose gel electrophoresis. Can be obtained by

The above-mentioned DNA fragment is an 8.5 kb product obtained by ligating the following two DNA fragments obtained by cleaving the chromosomal DNA of Pseudomonas putida KWI-9 strain with a restriction enzyme.
Present in the DNA fragment (see FIG. 5). DNA fragments of 10kb obtained by cutting with Hin dIII (see Fig. 3): Eco of 2.3kb of DNA fragment
The catechol 2,3 oxygenase (C23O) gene is present between the RI site and the Sal I site of 4.8 kb. The transformant containing this DNA fragment was C23.
Since catechol turns yellow by O, it can be easily detected.

6.0k obtained by cutting with Eco RI
DNA fragment of b (see FIG. 4): 3.7 of this DNA fragment
It does not Hin dIII site of kb 6.0kb of Eco R
I site part, of the DNA fragment Hin of 0kb dII
It overlaps with the I site or the 2.3 kb Eco RI site part. That is, the 0 kb Eco of the DNA fragment
RI site to Hin dIII site of 3.7kb is,
Is an upstream portion of the DNA fragment of DNA fragment of
It can be separated by Southern hybridization using the overlapping portion of both as a probe.

8.5 kb DNA containing the above DNA fragment
The fragment binds the 0 kb Hin dIII site to the 4.8 kb Sal I site fragment of the above DNA fragment to the 0 kb Eco RI site to the 3.7 kb Hin dIII site fragment of the upstream DNA fragment Downstream of the DNA fragment of, or, the 2.3 kb Eco RI site or 4.8 kb of the DNA fragment of
It can be obtained by ligating Sal I site fragments.
As the DNA fragment, the 8.5 kb DNA fragment described above is St
Obtained as a fragment cleaved at the u I and Sma I sites.

The recombinant plasmid has a phenol hydroxylase gene derived from Pseudomonas putida KWI-9 strain chromosomal DNA. Phenol hydroxylase formed from this gene degrades trichlorethylene, so the recombinant plasmid of the present invention is introduced into a host bacterium for transformation, and the obtained recombinant bacterium is used to halogenate carbonized trichloroethylene or the like. Can decompose hydrogen.

The above recombinant plasmid can be constructed by ligating the following DNA fragments with ligase. DNA fragment containing broad host range replication region Drug resistance gene (marker) Pseudomonas putida KWI-9 strain chromosome DN
Phenol hydroxylase gene from A

The broad host range replication region is a gene region required for the recombinant plasmid to grow independently and stably be maintained in Gram-negative bacteria.
However, it is not limited to this. RSF1010 contains the genes and origin of replication required for autonomous replication in Gram-negative bacteria,
It is concentrated in a DNA region of about 5.8 kb. This area is not only for RSF1010, but also for pKT240, pMM
It can be separated from a plasmid such as B22 using a nuclease such as a restriction enzyme.

As the drug resistance gene, chloramphenicol resistance gene, ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene and the like can be adopted, and pACYC184, pKK223-3, pTrc.
It can be isolated from plasmids such as 99A and pKT240.
Pseudomonas putida KWI-9 strain chromosomal DNA
As the derived phenol hydroxylase gene,
The DNA fragment of the present invention can be used.

The recombinant plasmid can express phenol hydroxylase via various different promoter-terminator systems. Therefore, the improvement of trichlorethylene decomposing ability can be easily performed by improving the promoter. Further, by incorporating a repressor gene such as lacIq, it becomes possible to control by an inducer such as isopropylthiogalactoside (IPTG). Therefore, addition of phenol and toluene is unnecessary. Furthermore, by deleting the repressor gene, it is not necessary to add an inducer, and it is possible to cause phenol hydroxylase to act as a constitutive enzyme. Therefore, trichlorethylene degrading ability can be maintained for a long time. The lacIq gene / promoter / operator / terminator system is pTrc99.
It can be separated from a plasmid such as A using a nucleolytic enzyme such as a restriction enzyme.

The recombinant plasmid can be introduced into the host very easily by electroporation, calcium and rubidium treatment, transfer using a helper plasmid, and the like.
Gram-negative bacteria, in which the recombinant plasmid is stably maintained, are used as the host. Pseudomonas bacteria are preferable, and Pseudomonas putida KWI-9 strain and mutants thereof are preferable.

The genetically modified bacterium transformed by introducing the recombinant plasmid in this manner can be used to decompose halogenated hydrocarbons such as trichlorethylene. Decomposition of halogenated hydrocarbons such as trichlorethylene can be carried out by a method of contacting with a gas containing halogenated hydrocarbon in a state where the recombinant bacterium is aerobically cultured in a medium containing a nutrient source, This allows complete dehalogenation.

The genetically engineered bacterium into which the recombinant plasmid has been introduced is carried out under conditions suitable for the growth of the host, and peptone, tryptone, yeast extract, etc. as carbon and nitrogen sources,
Using sodium chloride, potassium chloride, etc. as inorganic salts,
PH of medium 5 to 8.5, preferably 6 to 7, temperature 15 to
It can be cultivated aerobically at 35 ° C, preferably around 30 ° C.

By culturing in this way, the genetically modified bacterium grows. Therefore, a closed reaction vessel is constructed so that the grown recombinant bacterium and its nutritional source (including energy source) are built in and treated. To do. In this case, the above medium can be used as it is as a nutrient source. Therefore, the culture solution containing the grown bacterial cells can be directly supplied to the reaction tank, but the bacterial cells may be separated and supplied to the reaction tank.

The reaction tank may store the culture solution containing the genetically modified bacteria and the nutrient source as it is, but it is a packed bed obtained by supporting the bacterial cells on a packing material having a large porosity such as granular material or fibrous material. Can also be configured. In any case, the reaction tank should be a closed system so that the cells are not released into the atmosphere.

In the method for treating volatile organic substances, a gas containing volatile organic substances is introduced into the biological reaction tank and brought into contact with the genetically modified bacteria to decompose the volatile organic substances. At this time, the recombinant bacterium is imparted with or enhanced the ability to decompose volatile organic substances, so that the volatile organic substances are efficiently decomposed.

The treated gas after contact is sterilized by a sterilization filter to remove the genetically modified bacteria, or sterilized by a sterilizer and discharged to the outside of the system. Examples of the sterilization filter include a membrane filter such as a millipore filter and a fiber filter, and examples of the sterilization device include a heating or ultraviolet irradiation sterilization device.

When the genetically engineered bacterium is contained in a closed reaction tank and brought into contact with gas, the bacterial cells are confined in the reaction tank. The amount of bacterial cells taken out is small, the sterilization or sterilization operation is simpler than the case of contacting with liquid or solid, and the processing cost is also low.

When the treatment is carried out in a closed system, the activity of the genetically modified bacterium is reduced, but it is possible to continue the treatment by supplementing the nutrient source (particularly the energy source) to maintain the activity. At this time, the metabolites and the proliferated bacterial cells are taken out by gradually withdrawing the liquid in the tank, sterilized and the like and discharged to the outside of the system.

[0033]

Embodiments of the present invention will be described below with reference to the drawings. 1 and 2 are flow sheets showing a processing method according to different embodiments. In the figure, 1 is a diffusion tower,
2 is an inlet filter, 3 is a reaction tank, and 4 is an outlet filter.

In FIG. 1, the liquid 6 to be treated is introduced into the stripping tower 1 from the flow path 5a and retained therein, and gas is introduced from the flow path 7a to diffuse gas into the liquid from the gas supply device 8 to volatilize the liquid. Disperse the organic organic matter and transfer it to the gas side. The processing liquid is discharged from the system through the flow path 5b. The gas to be treated containing volatile organic substances passes through the flow path 7b, the inlet filter 2, and the flow path 7c to the reaction tank 3.
Supply to. The inlet filter 2 is provided so as to remove bacteria when the liquid from the reaction tank 3 flows backward.

The reaction tank 3 is a closed system, and the culture solution containing the genetically modified bacteria and the nutrient source is introduced and retained through the channel 9a, and then the nutrient source is continuously supplied from the channel 9a. A part of the internal solution 11 is drawn out from the flow path 9b, and the gas to be treated is diffused from the gas supplier 12 and brought into contact with the bacterial cells in the culture solution. At this time, the bacterial cells ingest the nutrient source in the culture solution and proliferate, but at the same time, decompose the volatile organic substances that migrate from the gas to be treated to the liquid phase. The treated gas after the contact is sterilized from the channel 7d through the outlet filter 4 and discharged from the channel 7e to the outside of the system. The liquid in the tank drawn out from the flow path 9b is sterilized by an autoclave or the like and discharged to the outside of the system.

In FIG. 2, a packing material layer 15 is provided inside the stripping tower 1, a liquid to be treated is sprinkled from a sprinkler 16 and is brought into contact with a gas supplied from a gas supply device 8 to generate a volatile organic substance. Disperse. In addition, a packing material layer 17 is provided in multiple stages in the reaction tank 3, the packing material is made to carry the genetically modified bacteria, and a part of the tank liquid 11 containing the nutrient source is replenished from the channel 9a. Along with the nutrient source, it is circulated by the pump 19 from the circulation path 18 to be sprinkled from the sprinkler 20 to raise the gas to be treated supplied from the flow path 7c, and the genetically modified bacteria carried on the packing material. Contact it for processing. Other operations are the same as in the case of FIG.

By the above operation, the volatile components in the liquid to be treated are diffused into the gas in the stripping tower 1, and the gas containing the volatile components is decomposed into the genetically modified bacteria in the reaction tank 3. Further, since the processing gas can be easily separated into gas and liquid or gas and solid, the number of bacterial cells mixed in the processing gas is smaller than that in the case of processing in a liquid or solid state. Further, since the operating pressure of the sterilization filter is lower in the case of gas than in the case of liquid or solid, sterilization can be easily performed, and the treatment cost is lower than that in the case of treatment in the liquid or solid state.

Although the above description has described the case where a volatile substance is contained in the liquid or solid, when the volatile organic substance is discharged from the pollution source in the state of being contained in the gas, the genetically modified bacterium is directly used. Can be contacted with and treated.

Test Example Next, a test example of the present invention will be described. The plasmids and microorganisms used in the test examples are as follows. 1) An expression vector used for expressing the pKK223-3 protein. It has a strong tac promoter that can be regulated by the lac repressor and induces transcription when an inducer such as IPTG inactivates the lac repressor. Multiple cloning site (MCS) immediately downstream of tac promoter
And a strong rrn ribosomal terminator is present. This plasmid is commercially available from Pharmacia and has the product code number 27-4935-01.

2) A derivative of pTrc99A pKK233-2, which has a long multi-cloning site and a strong terminator (rrB) immediately downstream of the strong trc promoter.
Also no lacIq (powerful ones of the lac repressor) in pKK233-2 for containing the gene, E the lac repressor is lost. E. coli can be used as a host. The trc promoter is driven by the addition of IPTG. N of the multi-cloning site
The coI restriction enzyme cleavage site has a translation initiation codon ATG, and it can be expressed by inserting a gene lacking this codon. This plasmid is commercially available from Pharmacia and has a product code number of 27-5007-.
01.

3) pACYC184 E. coli cloning vector having a size of 4244 bp. The first guanine (G) on the Eco RI site
Is the 1st in the nucleotide sequence, it has a tetracycline resistance gene at 1580 to 2770th position and a chloramphenicol resistance gene (Cm) at 3804th to 219th position.

4) It has a broad host range replication region derived from pKT240 plasmid RSF1010 and can be stably maintained in many Gram-negative bacteria.
It is a 2.9 kb plasmid. It has an ampicillin resistance gene and a kanamycin resistance gene. ATCC
Is commercially available from. ATCC No. 37258 (A
TCC Catalog of Recombin
ant DNA Materials 2nd edi
Tion, 1991).

5) E. coli DH5α Easily transformed by a large plasmid. Commercially available from Bethesda Research, Inc. (BRL). Product number is 8
262 SA. 6) Pseudomonas putida KWI-9 strain A trichlorethylene-degrading strain that has a phenol-utilizing ability and not a toluene-utilizing ability, and has been deposited under the above-mentioned microorganism deposit number. Degradability of trichlorethylene is significantly inhibited by the coexistence of phenol.

Test Example 1 It is considered that the phenol hydroxylase produced by Pseudomonas putida KWI-9 strain is degrading trichlorethylene, and the C23O gene coexists with the phenol hydroxylase gene in one operon and the C23O gene is present. It was considered that the phenol hydroxylase gene was present in the upper stream of the enzyme, and an attempt was made to separate the phenol hydroxylase gene using C23O as a marker. In addition, C23O is catechol as 2-hydroxymuconic semialdehyde (2-Hydroxymuc).
It oxidizes to onic semidehyde) and turns yellow.

LB medium was used for liquid culture of Escherichia coli DH5α, and the culture temperature was set at 37 ° C. Antibiotics are ampicillin and chloramphenicol 100 μg /
ml, used at a concentration of 50 μg / ml. E. coli DH5
The method of Hanahan was used for the introduction of the plasmid into α. The Southern hybridization method, the ligation method, and other basic methods related to molecular biology are
Second of all "Molecular cloning"
According to the edition.

[0046] was cleaved with Hin dIII, 5 'to the ends were dephosphorylated processing pKK223-3, Pseudomonas putida KWI-9 limits the chromosome of strain enzyme Hin dIII
The DNA fragment cleaved with was incorporated. This plasmid was introduced into Escherichia coli DH5α and transformed. As a result, about 1
2000 colonies were obtained, of which 9 colonies turned yellow by spraying with catechol. All of these transformants incorporated the same 10 kb DNA fragment. A restriction enzyme map of the plasmid containing this DNA fragment is shown in FIG. This DNA fragment contains 2.3 kb of Ec
o The C23O gene was present between the RI site and the Sal I site.

[0047] The pKK223-3 incorporating the Hin dIII DNA fragments of the above 10kb was introduced into E. coli DH5α, were subjected to the decomposition test of phenol by this E. coli, phenol was not degraded at all. Therefore, a further upstream DNA fragment was obtained by the following procedure.

The chromosome of Pseudomonas putida KWI-9 strain was digested with Eco RI and electrophoresed on an agarose gel to excise an agarose gel portion containing a 6.0 kb DNA fragment. The DNA fragment contained in this was taken out by a DNA cell (DNA CELL, trademark) manufactured by Daiichi Pure Chemicals. This 6.0 kb Eco RI D
The NA fragment was incorporated into the Eco RI site of pTrc99A to obtain about 14,000 transformants. 300 of them
10 kb of the Hin dIII DNA
It does not Hin dIII site of 0kb of fragments of 2.3kb
Southern hybridization was performed using up to the Eco RI site as a probe. As a result, 6.0 kb
A transformant having a plasmid incorporating the Eco RI DNA fragment was obtained. The restriction enzyme map of this plasmid is shown in FIG. This plasmid contains 10 kb of H
The 0 kb Hin dIII site of the in dIII DNA fragment was present at the 3.7 kb position.

The above-mentioned two types of DNA fragments (10 kb of Hi
n dIII DNA fragment, Eco RI DN of 6.0kb
A fragment) is ligated as shown in FIG. 5, and the downstream site is C2.
8.5 kb D up to Sal I site containing 3O gene
The NA fragment was incorporated into pTrc99A. The restriction enzyme site in FIG. 5 was determined using a restriction enzyme that does not cleave pTrc99A.

The restriction enzyme site at the upstream site of this plasmid was deleted stepwise to prepare plasmids of various lengths, which were introduced into Escherichia coli DH5α, and colony-derived phenol hydroxylase and C2 were introduced.
An expression test of 3O was performed. C23O was 0.1 M catechol, and in the case of phenol hydroxylase, 0.1 M phenol was used. These were dropped onto the colonies, and the color change of these colonies was observed. When the phenol hydroxylase gene is correctly integrated, the C23O gene is present in the downstream, so that after phenol is oxidized to catechol by phenol hydroxylase, a yellow substance is continuously produced by C23O. Yellowing. Therefore, the detection is easy.

The expression of the gene from the Stu I site was carried out as follows. Expression of the gene from the Stu I site caused growth failure in E. coli and did not form colonies. This was due to the Nc of pTrc99A.
Since it was found that the protein synthesized from ATG at the o I site was the cause of the inhibition, a new plasmid pTrc100 obtained by removing the ATG portion from pTrc99A was used instead of pTrc99A. This plasmid was cut pTrc99A at Nco I, creating the exposed CATG Remove with Mung bean Nuclease.

The expression results are shown in FIG. From FIG. 6, Stu I
It can be seen that the phenol hydroxylase gene exists between the site and the second Sma I site, and the C23O gene exists between the second Eco RI site and the second Sal I site. It should be noted that those showing phenol hydroxylase activity also showed trichloroethylene degrading activity, and it was revealed that phenol hydroxylase degrades trichlorethylene. Locations of the phenol hydroxylase gene and the C23O gene are shown in FIG.

Next, Stu I containing the phenol hydroxylase gene and the C23O gene or the last Sal I
A recombinant plasmid incorporating the DNA fragment up to the site (see FIG. 7) was introduced into Pseudomonas putida KWI-9 strain to prepare a recombinant strain.

The following SOB medium was used for liquid culture of Pseudomonas putida KWI-9 strain. As the agar medium, SOB agar medium was used. Antibiotics were ampicillin and chloramphenicol at 100 μg / ml and 50, respectively.
Used at a concentration of μg / ml. The culture temperature was set to 30 ° C. The introduction of the plasmid into Pseudomonas putida KWI-9 strain was carried out by the electroporation method using Gene Pulser manufactured by Bio-Rad (BIO-RAD).

SOB medium: Bacto tryptone 20 g Bacto yeast extract 5 g NaCl 0.5 g 250 mM KCl 10 ml Distilled water to a total volume of 990 ml pH 7.0 Sterilized by autoclave and cooled to room temperature , 2M Mg ²⁺ solution sterilized by autoclave in another bottle (1M MgSO ₄ · 7H ₂ O + 1M M
10 ml of gCl ₂ .6H ₂ O) is added.

First, the RSF1010 replicon widely used in Pseudomonas, the chloramphenicol resistance gene of pACYC184, and the t of pTrc100.
A plasmid pRCT200 for Pseudomonas deficient in lacIq was prepared using the part from the rc promoter to the terminator.

[0057] In other words, Pvu II and P from pKT240
about 5.8kb replicon site to be cut out by st I (this replicon of RSF1010 Pvu II (bp194
8) -corresponds to Pst I (bp 7768))
And Bst BI (bp3716) of pACYC184 ~
The BsaAI (bp310) portion was treated with T ₄ DNA polymerase and then ligated with T ₄ DNA ligase to prepare a plasmid. Next, the Xmn I of RSF1010
This plasmid was cleaved using the (bp2030) site, and p was treated with T ₄ DNA polymerase at this site.
Trc100 of Pvu II (bp4096) ~ Bsp HI
(Bp793) (lacIq does not exist in this part)
Was constructed to create a new plasmid pRCT200.

Next, since there is no Bam HI site in the DNA fragment from the Stu I to Sal I sites containing the phenol hydroxylase gene and the C23O gene (see FIG. 7), Bam HI linkers are added to both ends of this DNA fragment. It was ligated and modified so that all the DNA fragments could be excised with a single enzyme. Then, replace the pRCT200 with Bam H.
It was cleaved with I, and a DNA fragment containing the phenol hydroxylase gene and the C23O gene was inserted into that portion to prepare a new plasmid pNEM201 shown in FIG.
This plasmid is lacIq deficient,
When introduced into Pseudomonas putida KWI-9 strain, it was stably maintained.

The recombinant bacterium thus obtained decomposes halogenated hydrocarbons such as trichlorethylene without adding an inducer (IPTG), and its ability is maintained for a long time. That is, when lacIq is present, an inducer is necessary. However, when this lacIq is deleted, a repressor is not produced, so that trichlorethylene and the like can be decomposed without adding an inducer.

50 μg of the recombinant bacterium thus obtained
The bacterial solution cultivated in the SOB medium containing 1 / ml of chloramphenicol was placed in a cylindrical reaction tank having a diameter of 6 cm and a height of 35 cm, and a gas containing 50 ppm trichlorethylene was blown into the air at 10 liter / hr. The cells were brought into contact with each other, and the treated gas was sterilized with a sterilization filter (Millex FG50, trademark) manufactured by Millipore. As a result, trichlorethylene as the processing gas became 4 to 5 ppm, and this processing was stably performed for 50 hours. Again 1
The treated gas was passed through sterilized water every 0 hours, and this was spread on an SOB agar medium to confirm the presence or absence of efflux bacteria, but no efflux bacteria were detected.

[0061]

INDUSTRIAL APPLICABILITY According to the present invention, by contacting a volatile organic substance with a genetically modified bacterium in a state of being contained in a gas, the volatile organic substance is efficiently decomposed and the bacterial cells are taken out into the treated gas. It can be sterilized or sterilized with a small amount and easier and cheaper than the case of liquid or solid, and it is possible to prevent environmental pollution and perform stable treatment.

[Brief description of drawings]

FIG. 1 is a flow sheet showing a processing method of an example.

FIG. 2 is a flow sheet showing a processing method of another embodiment.

FIG. 3 is a restriction enzyme map of the plasmid prepared in Test Example 1.

FIG. 4 is a restriction enzyme map of the plasmid prepared in Test Example 1.

FIG. 5 is a restriction enzyme map of a plasmid containing the DNA fragment of Test Example 1.

FIG. 6 is a diagram showing expression results of Test Example 1.

FIG. 7 is a restriction enzyme map of a DNA fragment containing the DNA fragment of Test Example 1.

FIG. 8 is a restriction enzyme map of the plasmid prepared in Test Example 1.

[Explanation of symbols]

 1 Dispersion tower 2 Inlet filter 3 Reaction tank 4 Outlet filter 5a, 5b, 7a, 7b ..., 9a, 9b Flow path 6 Liquid to be treated 8, 12 Gas feeder 15, 17 Filler layer 16, 20 Disperser 18 Circulation Road 19 pump

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] September 8, 1993

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

[Figure 1]

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 2

[Correction method] Change

[Correction content]

[Fig. 2]

Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 1/00 Q 7236-4B 1/21 7236-4B 9/02 9359-4B // (C12N 1/21 C12R (1:40) (C12N 9/02 C12R 1:40)

Claims (2)

[Claims] 1. A closed reaction vessel is constructed so as to contain a genetically modified bacterium imparting or enhancing the ability to decompose volatile organic substances and its nutrient source, and introducing the gas containing the volatile organic substances into the reaction vessel. Then, the volatile organic matter in the gas is decomposed by the recombinant bacterium, and the treated gas after contact is sterilized or sterilized and discharged to the outside of the system. Treatment method of volatile organic substances. 2. A liquid or a solid containing a volatile organic substance is brought into contact with a gas to transfer the volatile organic substance to the gas side, and the gas containing the volatile organic substance thus obtained is introduced into a reaction tank for treatment. The method of claim 1, wherein the method is performed.