KR0154295B1 - Novel pseudomonas putida bu34, and treatment method for soil - Google Patents

Abstract

The present invention relates to a new strain Pseudomonas putida Bu34 (KCTC 8719P) and a method for treating contaminated soil using the same, and more particularly, chlorination by separating from soil and degrading chlorinated phenolic compounds, especially pentachlorophenol The present invention relates to Pseudomonas putida Bu34 (KCTC 8719P), which can be useful for treating soil contaminated with phenolic compounds.

Description

[Name of invention]

New strain Pseudomonas putida Bu34 (KCTC 8719P) and treatment method of contaminated soil using the same

Detailed description of the invention

The present invention relates to a new strain Pseudomonas putida Bu34 (KCTC 8719P) and a method for treating contaminated soil using the same, and more particularly, is isolated from the soil and chlorinated phenol compounds, in particular pentachlorophenol (hereinafter referred to as PCP) The present invention relates to Pseudomonas putida Bu34 (KCTC 8719P), which can be useful for the treatment of soil contaminated with chlorinated phenolic compounds.

Chlorinated phenolic compounds, such as dichlorophenol, trichlorophenol, tetrachlorophenol and pentachlorophenol, have a higher chlorination degree and have a higher effect of inhibiting growth of living organisms. In particular, the most chlorine-substituted pentachlorophenol (PCP) among chlorinated phenolic compounds has been widely used as herbicides, insecticides, fungicides, wood preservatives, etc. (Wild et al., Chemosphere, 24, 833, 1992).

PCP is acutely toxic to most fish at low concentrations of 0.6 ppm, 40 times more toxic than phenol (Liu et al., Bull. Environ. Contam. Toxicol., 27, 289, 1981), photosynthesis and water It also inhibits the growth of algae (Gotham Rhee, J. Great Lakes Res., 8, 328, 1982). Because of these side effects, the US Environmental Protection Agency selected 10 chlorinated phenolic compounds, including PCPs, as organic priority pollutants and restricted their use (Freeman, Standard handbook of hazardous waste treatment and disposal, McGraw-Hill). , NY, 1989).

However, since the PCP was developed in the 1930's, 100,000 tons were produced and used in 1985 alone, and they remain in groundwater, aquifers, soil, rivers and lakes.

If the chlorinated phenol compound such as PCP is incinerated, the possibility of the generation of secondary environmental pollutants increases. Thus, there is a need to reduce or detoxify residual amounts of biological treatments using microorganisms capable of degrading chlorinated phenolic compounds, including PCP.

Chlorinated phenolic compounds, in particular, microorganisms that degrade PCP, can be isolated from PCP-degrading microorganisms using PCP as a carbon source because the hydrochloric acid produced during the decomposition changes the blue color of the PCP-MS (mineral salts) medium (Kennes). Lemma, Biotech. Lett., 16, 759, 1994), as a method for treating soil contaminated with PCP using microorganisms, a slurry treatment method and the like have been devised (US Pat. No. 4,923,125, WO 94-22,605). .

On the other hand, U.S. Patent No. 5,364,787 has cloned the pcpA, pcpB, pcpC genes involved in PCP degradation of Flavobacterium (Flavobacterium sp. ATCC 39723) as a microorganism that degrades chlorinated phenolic compounds, especially PCP, Arthrobacter Phanerochaete, a bacterium and white rot, has been reported, but their PCP concentration is only 400 ppm (Topp Hanon, Appl. Environ. Microbiol., 56, 541, 1990; Edgehill Finn). , ibid, 45, 1122, 1983; Otto et al., Appl. Microbiol. Biotechnol., 40, 926, 1990; Lamar Dietrich, Appl. Environ.Microbiol., 56, 3093, 1990).

Therefore, in order to solve the problems of the conventional chlorinated phenolic compounds, in particular, microorganisms having PCP resolution, the present inventors have isolated microorganisms growing at 1,000 ppm or more from PCP concentrations in soil collected at timber yards, The present invention was completed by separating growing microorganisms and identifying them as Pseudomonas putida.

An object of the present invention is to provide a new strain Pseudomonas putida Bu34 (KCTC 8719P) having a high concentration of chlorinated phenolic compounds, particularly PCP, and a method for treating soil contaminated with PCP using the same. .

Hereinafter, the present invention will be described in detail.

The present invention relates to the new strain Pseudomonas putida Bu34 (KCTC 8719P).

In addition, the present invention includes the method used in the treatment of the new strain to the soil contaminated with chlorinated phenolic compounds.

The present invention will be described in more detail as follows.

The present invention relates to a chlorinated phenolic compound, in particular Pseudomonas putida Bu34 (KCTC 8719P) having a high concentration of PCP resolution, the isolation and identification of the new strain is as follows.

[Isolation of mycobacteria]

(a) Take 1g of each soil collected from wood yards in Busan, Yeosu, Gunsan, and Incheon, inoculate it into PCP liquid medium containing 100 ppm of PCP, and incubate for 10-20 days at 30 ℃. . Here, the PCP liquid medium is 0.65 g of K ₂ HPO ₃ , 0.12 g of KG ₂ PO ₄ , 0.1 g of MgSO ₄ , 0.5 g of NaNO ₃ , and 100 ppm of PCP (Fluka Co., Ltd.) in 1 L of distilled water. The pH is 7.3. to be.

1 ml of the shake culture solution is taken and inoculated into a PCP-MS solid medium and incubated at 30 ° C. for 3 to 20 days. When PCP is decomposed, hydrochloric acid is produced, so it is yellow in the vicinity of colony formation. Here, PCP-MS solid medium is prepared by adding 20 mg of bromothymol blue (BTB) and agar 1.5% to the PCP liquid medium.

The yellowish colonies were purely separated, inoculated in a PCP-MS (mineral salt) liquid medium, and shaken at 30 ° C. to confirm that the cells were changed to yellow. Here, the PCP-MS liquid medium is a BTB 20mg is added to the PCP liquid medium.

Through the above process, 70 kinds of microorganisms decomposing 100 ppm of PCP were isolated.

(b) The concentration of PCP was increased to 1,000 ppm, and the method of (a) was repeated to isolate 12 microorganisms. In addition, the PCP concentration was increased to 3,000 ppm to isolate a single microorganism, which was named Bu34.

Increasing the isolated Bu34 PCP concentration to 4,000 ppm did not inhibit growth.

On the other hand, as a result of testing the degradability of the microorganisms in the same manner as described above for the dichlorophenol, trichlorophenol and tetrachlorophenol compounds, it was confirmed that the degradation ability for these chlorinated phenol compounds.

[Sympathy with mycobacteria]

When the microorganism Bu34 isolated above was incubated for 24 hours at 30 ° C. in TSA (Trypticase Soy Agar; product of BBL), white colonies were formed, and the cell size was 1.5 to 2.0 × 0.5 to 1 μm.

Motility is positive, Gram staining is negative.

Identification of microorganisms was carried out at the Genetic Resources Center of the Korea Institute of Science and Technology.

The results of examining the physiological and biochemical characteristics using a biolog system (product of US MicroStation) applied to Gram-negative bacteria are as follows.

The above results were compared with Pseudomonas putida KCTC 1644 as a control, and the characteristics were well agreed except that King B agar was not grown at 4 ° C.

Meanwhile, the quinone and intracellular fatty acid composition of the new strain were compared according to the method of Oyaizu Komagata. J. Gen. Appl. Microbiol., 29, 17, 1983.

The new strain was inoculated into 300 ml of Nutrient Broth (manufactured by Diffco Co.), shaken at 30 ° C., recovered, and washed with 30 mM phosphate buffer. 20 ml of chloroform / methanol (2: 1 v / v) were added and shaken for 10 minutes. 2 After filtering with filter paper, the process of concentrating the filtrate with a vacuum concentrator is repeated three times to completely extract the isoprenoid quinone.

2 ml of nucleic acid was added to the flask by a vacuum concentrator, and then stirred. The nucleic acid layer, which is the supernatant, is transferred to another vacuum concentration flask and concentrated three times.

Concentrate, dissolve in a small amount of acetone, transfer to thin layer chromatography (TLC) consisting of silica gel (Kiesel gel 60 F254, Merck), and develop with petroleum benzene / diethyl ether (9: 1 v / v) developing solution. Let's do it.

After the development, 245 ml of ultraviolet light is checked to check the band, the band is scraped off, washed with acetone, and the supernatant is taken out using an Eppendorf centrifuge. Concentrate with nitrogen gas and perform HPLC (Hitachi, Model L-5000). At this time, the column (YMC-Pack ODS-AM, manufactured by YMC Co., Ltd.) was 4.6 x 250 mm, and 1 mL of methanol / isopropyl ether (3: 1 v / v) was flown per minute. When detected with absorbance at 275 mm, the quinone of the new strain is ubiquinone-9.

Q-6 (Saccharomyces cerevisiae IAM 4206 was extracted after incubation at 25 ° C. in YM broth) as standard, Q-7 (Candida agrestis KCTC 7186 was obtained by YM Incubated at 25 ° C. in broth], Q-8 [Burkholderia cepacia KCTC 2504 is extracted by adding 0.5% sodium chloride to nutritious broth and incubated at 30 ° C.], Q-9 [Pseudomonas aeruginosa (Pseudomonas aeruginosa) KCTC 8750 extracted by incubation at 30 ℃ in a nutri broth], Q-10 [Sphingomonas paucimobilis KCTC 2346 incubated at 30 ℃ in a nutrient broth Extracted]. At this time, the ubiquinone of the strain was detected at the same time as the Q-9 standard.

And the composition of the intracellular fatty acid was examined to confirm the new strain.

The procedure for methylating fatty acids according to Miller's method (Miler, J. Clin Microbiol., 18, 861, 1982) is as follows.

The new strain was incubated in a TSA solid medium, and 50 mg of cells were added with 1 ml of 50% methanol added with 15% caustic soda, heated at 100 ° C. for 30 minutes, and cooled at room temperature. 2 ml of a mixture of 325 ml of 6.0 N hydrochloric acid and 275 ml of methanol were added, and the mixture was heated at 80 ° C. for 10 minutes and quenched. 1.25 ml of nucleic acid / t-butyl and methyl ether (1: 1 v / v) were added and stirred for 10 minutes. The mixture was left to stand, the supernatant was collected, 3 ml of caustic soda was added, and a few drops of saturated salt solution were added. Afterwards, about 2/3 of the supernatant was taken and transferred to a special vial (manufactured by Altec Corporation).

Fatty acid esters were then analyzed using Hewlett-Packard's gas chromatograph (model 6890A).

Analytical conditions were that the carrier gas was hydrogen, the column head pressure was 10 psi, the split ratio was 100: 1, the split vent was 50 ml / min, the septum sebum (septum purge) 5 ml / 1 min, flame ionization detector (FID) hydrogen 30 ml / min, FID nitrogen 30 ml / min, FID air 400 ml / min, initial temperature 170 ° C, program rate 5 ℃ / min, final temperature is 270 ℃, FID temperature is 300 ℃, injection port is 250 ℃, injection volume is 2μl, and column is 0.22mm × 25m, methylphenylsilicon fused silica open column (HP 19091B-102).

The standard was used as a standard mixture (manufactured by Microbial ID Inc.), and the data of fatty acid methyl esters (FAMEs) was used as Microbial Identification System Software (manufactured by Microbial ID Inc.).

As a result, the intracellular fatty acid composition was C16: 0, C16: 1, C18: 1, C10: 0 3OH, C12: 0 3OH. Accordingly, the test strain contains ubiquinone-9, and no growth is observed at 41 ° C, no gelatin resolution, and the use of phenylacetate as a carbon source, such as Pseudomonas putida. ) It is defined as a microorganism and identified as a new strain that has not been isolated from King B agar and the difference in its non-growth at 4 ° C. The genetic resource in the Biotechnology Research Institute of Korea Institute of Science and Technology dated December 19, 1995 It was deposited in the center and was given accession number KCTC 8719P.

Hereinafter, the present invention will be described in detail with reference to Examples. The present invention is not limited by the pentachlorophenol compound and Examples.

[Example; Isolation of mycobacteria

1 g of soil collected from a timber yard in Busan, Yeosu, Gunsan, and Incheon was inoculated into a PCP liquid medium containing 100 ppm of PCP, and shaken for 20 days at 30 ° C. 1 ml of shake culture was inoculated into PCP-MS solid medium and incubated at 30 ° C for 12 days. The yellowish colonies were purely separated, inoculated in a PCP-MS liquid medium, and shaken at 30 ° C. to confirm that the cells were changed to yellow.

When the microorganisms isolated above were inoculated in PCP-MS medium added with 1,000 ppm of PCP, the above-described method was repeated, and 12 microorganisms were isolated.

Thereafter, one remaining microorganism was isolated until the PCP concentration was increased to 3,000 ppm and named Bu34. The microorganism did not inhibit its growth even when the PCP concentration was increased to 4,000 ppm.

Experimental Example 1

PCP was added to the minimum medium at 200, 500, 1,000, 2,000, 3,000 ppm and inoculated with Pseudomonas putida Bu34 (KCTC 8719P), and the PCP was quantified according to absorbance over time. Here, the minimum medium is 0.065 g of K ₂ HPO ₄ , 0.017 g of KH ₂ PO ₄ , 0.1 g of MgSO ₄ · 7H ₂ O, and 29 mg of BTB in 1 L of distilled water, and the pH is 7.3.

The rate of PCP degradation per hour was 0.83 (mg / l / hr) at 200 ppm, 3 (mg / l / hr) at 500 ppm, 2 (mg / l / hr) at 1,000 ppm and 1.2 (mg / l / hr) at 2,000 ppm. ), 0.95 (mg / L / hour) at 3,000 ppm.

Experimental Example 2

Sterilized slurry, Pseudomonas putida Bu34 (KCTC 8719P) and group inoculated with 11 kinds of microorganisms that degrade PCP (Ⅰ), group utilizing microorganisms native to contaminated soil (II), contaminated soil Slurry was made by dispersing the native microorganism Pseudomonas putida Bu34 (KCTC 8719P) and the group inoculated with 11 kinds of microorganisms decomposing PCP (III). It was inoculated in a li processing apparatus. Samples were taken from the slurry treatment device over time, and PCP was extracted from the slurry, and the results are shown in the following table.

Here, the method of extracting PCP in the slurry is as follows (Mueller et. Al., Environ. Sci. Technol., 25, 1055, 1991).

25 ml was collected from the slurry treatment apparatus, the pH was adjusted to 12 with 10N caustic soda solution, and 10 ml of dichloromethane was added thereto, followed by vigorous shaking for 1 minute. Centrifuged at 3,500 rpm for 10 minutes, the supernatant was taken and transferred to a separatory funnel and the methane dichloride layer was removed. The pH of the remaining liquid was adjusted to 12 rolls, the process of extracting with methane dichloride was repeated twice, and the water layer was taken.

The pH was adjusted to 7.0 by adding 8.5% phosphoric acid solution, shaking with addition of 10 ml of methylene chloride, and centrifuged to take a water layer. This procedure was repeated twice.

PH was adjusted to 2 by adding 8.5% phosphoric acid solution, 10 ml of methylene chloride was added and shaken, centrifuged and the organic layer was taken. This procedure was repeated twice.

10 ml of distilled water was added, the organic layer was washed, dehydrated by adding 25 g of anhydrous sodium sulfate, and the PCP in the remaining organic layer was measured by gas chromatography (Varian), Model 3300.

Gas chromatography analysis conditions are as follows.

A column having an internal diameter of 0.32 mm and a length of 32 m (Scapelco, Capillary DB-1) was maintained at 80 ° C. for 3 minutes, increased by 8 ° C. per minute, and maintained at 200 ° C. for 20 minutes. Injection temperature was 200 degreeC and detection temperature was 250 degreeC, and it detected by FID. The injection gas was flowed at a pressure of 70 psi.

From the above results, it can be seen that Pseudomonas putida Bu34 (KCTC 8719P) of the present invention can remove PCP 96% after 14 days.

As described above, the present invention was identified by separating Pseudomonas putida Bu34 (KCTC 8719P) having excellent PCP resolution, and separated Pseudomonas putida Bu34 (KCTC 8719P) into soil contaminated with PCP. When administered, it can be seen that the ability to decompose the remaining PCP well.

Therefore, Pseudomonas putida Bu34 (KCTC 8719P) of the present invention can be usefully used to effectively remove chlorinated phenol compound contamination including PCP.

Claims (3)

Pseudomonas putida Bu34 (KCTC 8719P) Pseudomonas putida Bu34 (KCTC 8719P) is used to treat soil contaminated with chlorinated phenolic compounds. The method of claim 2 wherein the chlorinated phenolic compound is pentachlorophenol.