KR101896428B1 - Pseudomonas aeruginosa TSKW-S5 strain having oil resolution and oil-releasing ability, a method for purifying contaminated soil using the same, and a method for selecting the strain - Google Patents

Abstract

The present invention relates to a Pseudomonas aeruginosa TSKW-S5 strain having oil decomposition and oil release functions. The strain is obtained by undergoing separation, selection, and identification for numerous strains isolated from oil contaminated soil. The strain shows excellent decomposition functions for high concentration oil. In addition, the strain is capable of releasing light oil components adsorbed in pores of soil out of the pore. The strain is effective in removing oil via an environmentally friendly method which does not cause secondary contamination and is economically feasible and stable in oil contaminated soil.

Description

[0001] The present invention relates to a Pseudomonas aeruginosa TSKW-S5 strain having oil degradation ability and oil tar separation ability, a contaminated soil purification method using the same, and a method for selecting the strain, a method for purifying contaminated soil using the same, and a method for selecting the strain}

The present invention relates to a Pseudomonas aeruginosa strain TSKW-S5 having oil degradation ability and oil releasing ability, a method for purifying contaminated soil using the same, and a method for selecting the strain, and more particularly, And the best resolution of Pseudomonas aeruginosa ( Pseudomonas aeruginosa) aeruginosa TSKW-S5 strain and a method for purifying contaminated soil using the strain.

Soil pollution in environmental problems is relatively slow compared to air pollution or water pollution, but the side effects on contaminated neighboring areas are very persistent. In addition, associated with groundwater contamination can affect a wide area.

The causes of soil pollution are very diverse, but in Korea, oil pollution of gas stations, various oil storage facilities, and soil in the plant area is recognized as a serious problem. There are many types of the above-mentioned oil, but most of them can be referred to as fuel. Gasoline, diesel oil and coal oil are the main types of fuel oil. Among them, light oil is the main cause of soil pollution.

Light oil generally has a boiling point of 190 to 350 ° C, a specific gravity of 0.82 to 0.84, and a long chain hydrocarbon mixture of 15 to 30 carbon atoms. The hydrocarbons constituting the light oil are 74 to 79% (w / w) of saturated hydrocarbons, 0.6 to 1.5% of unsaturated hydrocarbons and 19 to 24% of aromatic hydrocarbons. As can be seen from this composition, light oil is less volatile than gasoline, has a high specific gravity and high viscosity, has low mobility in soil, and is low in hydrophilicity and water solubility. Therefore, removal of diesel oil from soil particles is difficult with physico-chemical treatment methods.

Bioremediation is a method of biologically treating oil contaminated soils. The technology utilizes microorganisms and can be classified into Biostimulation (supply of nutrients and electron acceptors) and Bioaugmentation (supply of microorganisms) depending on whether the microorganisms responsible for degradation of pollutants are supplied externally. Biostimulation is a process to induce decomposition of contaminants by activating indigenous microorganisms present in contaminated soil by supplying nutrients and oxygen, and the treatment time is longer than that of bioaugmentation and the cost is also higher. Bioaugmentation, on the other hand, can shorten the treatment time by injecting microorganisms with oil resolution directly into the contamination site. However, the success or failure of the treatment is determined by the contamination site adaptation mode or the oil degradation ability of the infecting microorganism.

The degradation of oil by microorganisms is caused by the oxidation or reduction of enzymes present in the cells. In general, a substance with a low molecular weight is decomposed more rapidly than a substance with a large molecular weight. However, since the degree of degradation by microorganisms varies depending on the characteristics of the oil, the biological treatment method can not be applied to all the oils. At present, it is limitedly applied to low molecular organic contaminants with high volatility and water solubility. Therefore, a microorganism having an excellent resolution with respect to the fuel oil is required.

In addition, since the diesel oil has a small mobility in the soil as described above, a microorganism having a characteristic of desorbing diesel particles adsorbed in the soil pore to the outside of the pore is needed.

Accordingly, the inventors of the present invention focused on the fact that in order to improve the purification efficiency in the purification of oil contaminated soil, the oil resolution of microorganisms and the degree of desorption of oil from soil pores should be taken into consideration, microorganisms are separated from soil contaminated with oil As a result of efforts to find a microorganism having excellent oil resolution and oil desorption, it was confirmed that the strain of Pseudomonas sp. Effectively removes the oil of contaminated soil, and the present invention has been completed.

KR 2002-0060009 A KR 2004-0098213 A

An object of the present invention is to provide a strain having excellent oil resolution.

Another object of the present invention is to provide a strain having a high degree of desorbing diesel oil adsorbed in a soil pore outside the pores.

It is another object of the present invention to provide a method for removing oil from contaminated soil using the strain.

It is another object of the present invention to provide a method for selecting microorganisms having excellent oil resolution from soil contaminated with oil.

In order to accomplish the object of the present invention, it has been found that Pseudomonas aeruginosa ( Pseudomonas aeruginosa) aeruginosa strain TSKW-S5 (Accession No. KCTC18564P).

The oil is any one selected from the group consisting of diesel oil, gasoline, aviation oil, kerosene and vulcanized oil.

Rouge said Pseudomonas ah oil contaminated soil labor (Pseudomonas aeruginosa ) TSKW-S5 strain in the presence of a microorganism.

Collection stage of contaminated soil; Liquid phase increasing concentration screening step; A plate culture step; And confirming the efficiency of the oil degradation after culturing in a liquid medium. The Pseudomonas aeruginosa ( Pseudomonas aeruginosa) aeruginosa TSKW-S5 strains.

The strain Pseudomonas aeruginosa TSKW-S5 according to the present invention (Accession No. KCTC18564P) has excellent oil-resolving power.

The strain may desorb the light oil adsorbed in the soil pore outside the pores.

The strain may be inoculated in the oil contaminated soil to remove the oil from the oil contaminated soil.

According to the present invention, the strain can be selected from soil contaminated with oil.

The strain is selected from domestic oil-contaminated soils, and when inoculated into domestic soil, it is excellent in self-sufficiency and does not cause confusion of ecosystem.

FIGS. 1A and 1B are diagrams showing sequences of the 16S rRNA gene of the strain selected in the present invention and the known Pseudomonas aeruginosa Aeruginosa strains were sequenced and homologous to 16S rRNA.

Rouge Pseudomonas ah of the present invention, industrial (Pseudomonas aeruginosa The strain TSKW-S5 was deposited on April 20, 2017, No. 5817 in the International Depository Organization (KCTC) under the Budapest Treaty on Microbial Deposit, and its deposit number is KCTC18564P. The selection method and features will be described in detail below.

1. Pseudomonas Aarujinosa  ( Pseudomonas aeruginosa ) TSKW Characteristics of -S5 strain

Rouge Pseudomonas ah of the present invention, industrial (Pseudomonas aeruginosa TSKW-S5 strain has the 16S rRNA gene partial sequence as shown in Figs. 1A and 1B. It also has the morphology and biochemical characteristics as shown in Table 1 below.

Morphological and biochemical characteristics of marine bacterium Pseudomonas aeruginosa TSKW-S5.  Characteristics Result Gram stain (-)
Cell size 0.5 x 1.75 탆
Cell shape Coccobacilli
Oxidase -
Rhamnose -
N-acetylglucosamine -
D-ribose-
Inositol -
Sucrose -
Maltose -
Itaconate -
Suberate +
Malonate -
Acetate +
D, L-lactate +
L-alanine +
5-ketogluconate -
Glycogen -
3-hydroxy-benzoate +
L-serine-
Mannitol -
D-glucose -
Salicine -
D-melibiose -
L-fucose-
D-sorbitol-
L-arabinose -
propionate +
Caprate -
Valerate +
Citrate -
Histidine +
2-ketogluconate -
3-hydroxy-butylrate +
p-4-hydroxy-benzoate +
L-proline +

Pseudomonas aeruginosa aeruginosa) that TSKW-S5 strain exhibits excellent resolution oil, the oil may be selected from diesel, gasoline, jet fuel, kerosene, and vulcanizing the group consisting of Euro. More preferably, the strain exhibits excellent resolution against diesel. Specifically, the Pseudomonas aeruginosa aeruginosa The TSKW-S5 strain was inoculated at a concentration of 3% (w / w) in a minimal medium containing 20,000 ppm light oil and cultured at 30 ° C and 150 rpm for 96 hours. The light oil decomposition rate was 86 to 91%. In addition, the degradation rate of the strain was 92%, 91%, 90% and 87% after 4 weeks, respectively, in the soil contaminated with 5,000 ppm, 10,000 ppm, 15,000 ppm and 20,000 ppm light oil, respectively.

In addition, the Pseudomonas aeruginosa aeruginosa The TSKW-S5 strain can desorb oil particles adsorbed in the soil pore outside the soil pore. Specifically, when the culture broth of the strain was mixed with soil contaminated with light oil, the degree of desorption of light oil on the supernatant liquid was measured and the desorption efficiency was 18%.

2. Pseudomonas Aarujinosa ( Pseudomonas aeruginosa ) TSKW -S5 Strain selection and identification

The above Pseudomonas aeruginosa The process of isolating, identifying and identifying the aerobic isolate TSKW-S5 strain is preferably (1) collecting contaminated soil from oil-contaminated areas, (2) (3) a step of culturing a plate on a solidification medium to select an oil-degrading strain, (4) a step of confirming the efficiency of light oil degradation after culturing in a liquid medium containing light oil, (5) And isolating and identifying an excellent strain. However, the present invention is not limited thereto. For example, some of the above steps may be omitted in some cases, and the detailed method of each step may vary, and the present invention is not limited thereto. The configuration of the present invention will be described in detail as follows.

The first step involves collecting contaminated soil around the oil spill, such as around the gas station or around the oil reservoir, in order to isolate microorganisms with high efficiency of oil fractionation. Preferably, it is taken from soil around a closed petrol storage tank exposed to oil pollution for a long time and around an old oil reservoir of a military unit, and may include diesel as a main component of the pollution source.

The second step includes the step of screening for an oil-degrading strain by liquid-phase-increasing concentration screening. The oil contaminated soil is cultivated in a minimal nutrient broth containing light oil for a certain period of time, and then a portion of the culture is inoculated into another minimal nutrient broth containing a higher concentration of light oil and cultured. Repeat this process by increasing the concentration of light oil.

The third step includes a step of plating a part of the culture medium, which is finally cultured in the second step, on a solidification medium, culturing a plate, and selecting an oil-degrading strain.

The fourth step includes the step of confirming the light oil decomposition efficiency after inoculation of the selected strains in the third step into a liquid medium containing light oil.

The fifth step includes a step of sorting 16S rRNA by selecting strains identified as having excellent degradation efficiency in the fourth step.

3. Pseudomonas Aarujinosa ( Pseudomonas aeruginosa ) TSKW -S5 strain by inoculating the oil contaminated soil.

The present invention relates to the aforementioned Pseudomonas aeruginosa aeruginosa TSKW-S5 strain is inoculated on soil contaminated with oil to provide a method for removing oil from contaminated soil.

The oil is not particularly limited, but may include light oil, gasoline, aviation oil, kerosene, and vulcanized oil. According to Experimental Example 3 of the present invention, the oil degradation rate of the Pseudomonas aeruginosa strain TSKW-S5 in the soil contaminated with light oil of 5,000 ppm, 10,000 ppm, 15,000 ppm and 20,000 ppm, respectively, 92%, 91%, 90% and 87%, respectively.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

< Example  1> Collection of contaminated soil and selection of strain

Soil samples were collected around the storage tanks of the closed gas station in Anyang City, Gunpo City, and Uiwang City in Gyeonggi Province, and around the old oil reservoirs of military units, and then sieved and stored in a refrigerator at 4 ℃.

After that, a liquid phase increasing concentration screening method was developed and carried out in order to quickly select excellent oil degrading bacteria. First, 10 g of the oil-contaminated soil sample stored in the refrigerator was placed in a 500 mL flask, and the minimum medium (Yeast extract 0.1 g / L, (NH ₄ ) ₂ SO ₄ 1 g / L, NaH ₂ PO ₄ 0.2 g / _{4 · 7H 2 O 0.25g / L} , MnSO 4 · H 2 O 0.02g / L, CaCl 2 0.05g / L) into the via to 5,000ppm in 200mL value was 1 days 24 hours of incubation with 30 ℃, 180rpm. Secondly, the primary culture solution was mixed by shaking, 20 mL was taken, inoculated in 200 mL of the minimum medium containing 10,000 ppm of light oil, cultured at 30 DEG C and 180 rpm for 24 hours, and allowed to stand for 1 day. Third, these repetitive processes were sequentially performed at a concentration of 15,000 ppm and 20,000 ppm, and cultured at 30 ° C. and 180 rpm for 24 hours.

0.3 mL of the above-mentioned final culture was taken, and a 1.5% agar minimum medium (agar 15 g / L, Yeast extract 0.1 g / L, (NH ₄ ) ₂ SO ₄ 1 g / L NaH ₂ PO ₄ 0.2 g / L, MgSO ₄ · were plated in _{7H 2 O 0.25g / L, MnSO} 4 · H 2 O 0.02g / L, CaCl 2 0.05g / L). Three strains (S2, S5, and H11) were selected by observing the strains growing while cultivating the agar-agar-agar petri dishes in a 30 ° C incubator while forming colonies over time.

Strain 48hr 72hr 96hr 120hr 144hr 168hr S2 ++ +++ +++ S3 + + ++ ++ + + S5 ++ +++ ++++ S8 + + ++ ++ + + H4 - + ++ + + + H6 - + ++ + + + H9 - + ++ + + + H11 ++ +++ +++ H13 - + ++ + + +

In the above table, + means growth, and +++ was selected.

< Experimental Example  1 > of the strain through liquid culture Oil resolution  Measure

To a 500 mL flask was added a minimal medium (yeast extract 0.1 g / L, (NH ₄ ) ₂ SO ₄ 1 g / L, NaH ₂ PO ₄ 0.2 g / L, MgSO ₄ .7H ₂ O 0.25 g / L, MnSO ₄ .H ₂ O _{0.02g / L, CaCl 2 0.05g /} L) was inoculated to the cultured species strain after filling into 200mL be a light oil 20,000ppm ro 3% (w / w), remaining after the 96 hour incubation with 30 ℃, 150rpm The diesel oil degradation rate of each strain is shown in Table 3 below.

Strain No. Strain name Decomposition rate (%) S2 TSKW-S2 69 ~ 75 S5 Pseudomonas aeruginosa TSKW-S5 86 ~ 91 H11 TSKW-H11 70 ~ 75

As can be seen from the above table, the strain S5 has the highest decomposition rate up to 91%. Accordingly, naming the strain labeled as TSKW-S5 and it is identified as Pseudomonas aeruginosa by the 16S rRNA Identification Pseudomonas aeruginosa TSKW-S5.

< Experimental Example  2> The presence of oil in the soil pore Tally  Efficacy evaluation

Light oil has low volatility, high specific gravity and viscosity, low mobility in the soil, low hydrophilicity and low solubility in water. The purpose of this study was to select microorganisms that produce components that desorb the diesel oil adsorbed in the soil pore.

Masato soil was collected from the hill of Uiwang-si, Gyeonggi-do, and then sterilized (121 ℃, 30 minutes) in Cheorgo-Grotto Autoclave and dried at 80 ℃ in a hot-air drier and divided into 100g × 4 pieces and placed in an Erlenmeyer flask. 10,000 ppm of diesel was uniformly polluted to the sample soil, and the soil was sealed for 10 days so that diesel was sufficiently adsorbed in the soil, and shaken 2 to 3 times a day.

20 mL of each strain culture and 80 mL of water were added to an Erlenmeyer flask containing 100 g of diesel contaminated soil, and the mixture was shaken for 30 minutes. Then, 50 mL of the leachate was placed in a precipitation tube and the amount of light oil released from the supernatant was measured. .

Strain name Diesel Particle Amount (mL) Removal efficiency (%) Water (control) One 2 TSKW-S2 6 12 Pseudomonas aeruginosa TSKW-S5 9 18 TSKW-H11 5 10

As can be seen from the above table, the metabolites produced during the cultivation of the oil-degrading microorganisms have the property of eliminating the diesel component adsorbed in the soil pores outside the pores, and among them, Pseudomonas aeruginosa TSKW-S5 strains have higher desorption efficiency than TSKW-S2 and TSKW-H11 strains.

< Experimental Example  3> Oil contaminated soil Pseudomonas aeruginosa TSKW -S5 strain Oil resolution

The soil was sampled at the hill of Uiwang-si, Gyeonggi-do, and the soil was sterilized (121 ℃ for 30 min) in Cheorogorocho Autoclave. After drying at 80 ℃ in a hot air drier, it was divided into four 500g × 4 pieces 5 cm) to prepare sample soil. The samples were densely contaminated with 5,000, 10,000, 15,000 and 20,000 ppm of diesel oil and sealed for 10 days so that the diesel was sufficiently adsorbed in the soil. The samples were shaken 2 to 3 times a day.

The cultured Pseudomonas aeruginosa TSKW-S5 strain was inoculated at 3% (w / w), the minimum medium was sprayed to the soil in an appropriate amount, and the soil was kept at 35-40% by mixing. A total of 10 g samples were collected at intervals of one week to measure the residual diesel oil concentration and are shown in Table 5 below.

Initial concentration 1 week 2 weeks 3 weeks 4 weeks 5,000 2500 (50.0%)  800 (84.0%)  450 (91.0%)  400 (92.0%) 10,000 4800 (52.0%) 3100 (69.0%) 1800 (82.0%)  900 (91.0%) 15,000 8500 (43.4%) 5500 (63.3%) 2500 (83.3%) 1500 (90.0%) 20,000 9500 (52.5%) 5950 (70.2%) 3500 (82.5%) 2600 (87.0%)

                                                (Unit: ppm,% decomposition)

As can be seen from the table, the light oil decomposition rate of Pseudomonas aeruginosa TSKW-S5 strain in actual soil shows excellent resolution of 87% or more within 4 weeks at various initial concentrations.

Korea Biotechnology Research Institute KCTC18564P 20170420

Claims (4)

Pseudomonas aeruginosa TSKW-S5 strain (Accession No. KCTC18564P), which has the ability to degrease oil and adsorb the diesel oil adsorbed on soil pores. The Pseudomonas aeruginosa TSKW-S5 strain according to claim 1, wherein the oil is any one selected from the group consisting of gas oil, gasoline, aviation oil, kerosene and vulcanization flow. How to claim Rouge to claim 1. Pseudomonas Ah industrial oil pollution in soil (Pseudomonas aeruginosa) was inoculated TSKW-S5 strain removing oil of oil contaminated soil. delete

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