KR20160150391A - Pseudomonas sp. sdy3 being abled to resolve oil and oil degradation method using the same - Google Patents

Abstract

The present invention relates to a novel Pseudomonas sp. SDY3 strain having oil decomposition ability. The strain is obtained by isolating, identifying, and screening a large number of strains isolated from various oil contaminated soils. The strain is obtained by selecting oil-decomposing strains showing the most rapid growth among the selected strains, shows fast decomposition rate in high concentration diesel of 2% (v/v) and has excellent decomposing abilities. In addition, the strain is stable in oil-contaminated soils, is economical, and has an effect of removing oil by an eco-friendly method without secondary contamination.

Description

A novel Pseudomonas sp. SDY3 strain having oil resolution and a method of oil degradation using the same. {PSEUDOMONAS SP. SDY3 BEING ABLE TO TO RESOLVE OIL AND OIL DEGRADATION METHOD USING THE SAME}

The present invention relates to novel Pseudomonas sp. sp . ) SDY3 strain and a method for degrading oil using the same. More particularly, the present invention relates to a strain of Pseudomonas sp. Which selects the best one by measuring the degradation rate of oil and the degradation rate of microorganisms isolated from oil- sp . ) SDY3 strain (Accession No. KCTC18365P) and a method for decomposing the oil using the oil degrading strain.

Along with rapid industrial growth, the use of oil has exploded and the frequency of oil spill accidents has also increased rapidly. Especially, in relation to oil pollution in soil, it is necessary to prevent oil leaks in past US military units or domestic military bases, leakage of petrochemicals due to inadequate management of facilities such as underground storage tanks and oil pipelines, Soil contamination is intensifying. In addition, in the case of land developed due to the withdrawal and movement of US military units and domestic military bases, oil pollution has progressed for several decades and the area and concentration of polluted areas are high.

Soil contamination by oil is characterized by a low rate of contamination diffusion compared to air or water pollution, but with varying patterns of damage and persistent appearance, accompanied by additional problems associated with groundwater contamination and land development for other uses. In addition, unlike gas or liquid, solid soil is difficult to be engineered when considering the transfer into the process and the chemical or biological reaction, and it takes a lot of time and money to completely clean the contaminated soil In terms of cleanup technology, it must be approached in a different way from air and water pollution.

The method of purification of soil polluted by oil can be roughly divided into biological treatment method, physical or chemical treatment method, and thermal treatment method. Among these methods, there are cleaning methods, cleaning methods, solvent extraction methods, solidification methods and the like. Thermal processing methods include thermal desorption, incineration, etc. The cleaning method is a method in which secondary contamination is caused by a cleaning solution such as a surfactant In addition, there is a disadvantage in that the cost of the detergent is low due to the high cleaning agent cost and the diffusion of contaminants may be caused. In addition, the cleaning method is costly for excavation and transportation of contaminated soil, And waste water / waste disposal costs. Therefore, there is a disadvantage in relatively low economic efficiency. In the solvent extraction method, there is a fear of secondary contamination by the extraction solvent. In the solidification method, it is difficult to immobilize the volatile organic substance in the solidified material There is a disadvantage that separate disposal work is necessary. In addition, the thermal desorption method requires a device for treating a gaseous secondary product and has a disadvantage in that it requires a high energy processing cost. The incineration method requires disposal of the ash material and decomposes soil microorganisms and organic substances, There are disadvantages.

The use of biological treatment methods has attracted attention due to the problems of treatment of such secondary products, the concern of secondary pollution, and the cost of purification. In addition, by supplying oxygen and nutrients to soil such as biological decomposition method and biological ventilation method, A method of promoting the biodegradation of the biosensor is used. Biodegradation is a phenomenon in which specific microorganisms of natural ecosystem decompose harmful pollutants under appropriate conditions and use them as a nutrient source. These biological treatment methods are advantageous because they are economic and have no secondary pollution source have.

However, biological treatment methods using biodegradation have a disadvantage of a long treatment period, so it is important to ultimately maximize biodegradation through investigation and experiments on factors that can enhance biodegradation of pollutants. In particular, in advanced countries, microbial purification technology has been put into practical use by many studies on contaminated soil, and microorganism input method in which foreign microorganisms having excellent resolution for specific pollutants are applied is also applied.

 In this study, the isolation and screening of strains having excellent oil resolving power have priority, and Korean Patent Publication No. 2002-0060009 discloses a method for treating soil contaminated with crude oil 1% (v / w) using Pseudomonas spp. CU1 for 7 days It has been reported that 57.17% of the crude oil was decomposed in the soil, and Korean Patent Publication No. 2004-0098213 describes Pseudomonas geneco-1 showing 67% diesel degradation rate in a soil having 10,000 ppm TPH. And the like.

However, in biological treatment methods, it is an important task to acquire a high oil fractionation efficiency in a shorter period of time. However, consideration of the oil fractionation speed is somewhat lacking in the selection of the strain, Is emerging.

Accordingly, the inventors of the present invention focused on the fact that, in order to shorten the treatment period in the purification of a substantially oil contaminated soil, it is necessary to take into account the two functions of the oil resolution and the oil degradation rate, and the microorganisms are separated from the oil- As a result of efforts to find a microorganism having excellent oil resolution and oil degradation rate, it has been confirmed that a novel strain of Pseudomonas sp. Efficiently decomposes the oil of the contaminated soil and completed the present invention.

An object of the present invention is to provide a novel Pseudomonas sp. sp . ) SDY3 strain.

Another object of the present invention is to provide a method for decomposing oil pollutants in soil using the strain.

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

In order to achieve the object of the present invention, there is provided a Pseudomonas sp. sp . ) SDY3 strain.

In the above description, the oil is a petroleum-based oil selected from the group consisting of jet fuel, gasoline, kerosene, case, heavy oil and diesel, and more preferably Pseudomonas sp . ) SDY3 strain.

The Pseudomonas sp. sp . ) The strain SDY3 shows the ability to decompose hydrocarbons, preferably the hydrocarbons are aromatic hydrocarbons and the aromatic hydrocarbons are selected from the group consisting of fluorene, phenanthrene and pyrene.

The present invention also relates to the use of the Pseudomonas sp. sp . ) SDY3 strain into oil-contaminated soil to provide a method for decomposing the oil of the oil-contaminated soil.

The Pseudomonas sp. sp . ) The SDY3 strain has excellent oil resolving power, and is a particularly good oil-degrading strain with high resolving power for diesel and hydrocarbons and high decomposition rate. The method of decomposing the oil-contaminated soil using the strain is more environmentally friendly than the physical, chemical and thermal treatment methods, and exhibits excellent oil degradation rate and is more efficient and economical than conventional biological treatment methods.

Another advantage of the present invention is that the strain is selected from domestic oil-contaminated soils, and has excellent self-sufficiency and does not cause confusion in the ecosystem when it is distributed in domestic soil.

Furthermore, Pseudomonas species (Pseudomonas oil decomposition strains of the invention sp . ) The SDY3 strain has an ability to decompose aromatic hydrocarbons, and can be used for purification of soil contaminated with such aromatic hydrocarbons, or for purification of contaminated soil complexed with oil such as diesel and aromatic hydrocarbons.

Pseudomonas species of this invention (Pseudomonas sp . ) The SDY3 strain is a new microbial strain deposited on March 31, 2015 in the KTC (International Collection Service for Type Cultures), an international depository organization under the Budapest Treaty on the Deposit of Microorganisms. Explain.

1. Pseudomonas sp. Pseudomonas sp . ) SDY3  Strain

Pseudomonas species of this invention (Pseudomonas sp . ) The SDY3 strain is characterized by the shape and biochemical characteristics shown in Table 1 below.

Characteristics Result Gram stain (-)
Cell size 0.5x1.75um
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 +

The Pseudomonas sp. sp . ) The SDY3 strain exhibits excellent oil resolution, and the oil may be a petroleum oil selected from the group consisting of jet fuel, gasoline, kerosene, light oil, heavy oil and diesel. More preferably, the strain exhibits excellent diesel resolution and fast degradation rate of diesel. Specifically, the Pseudomonas sp. sp . ) SDY3 strain was inoculated at 3% (v / v) concentration in a minimal nutrient medium (MSM) containing 2% (v / v) diesel and the diesel resolution after incubation at 25 ° C, 93%, and the oil degradation rate in the soil contaminated with 2% (w / w) diesel is 91% after 28 days, and the decomposition rate is fast and the resolution is excellent.

The Pseudomonas sp. sp . ) The SDY3 strain is characterized by high cell hydrophobicity and low emulsifying ability. The cell hydrophobicity can be confirmed by measuring the absorbance of the water layer formed in the mixed solution of the suspension suspension and hexadecane. The cell hydrophobicity of the strain enables a wide contact surface with the contaminated oil, It can be determined that they have a correlation.

In addition, the above Pseudomonas sp. sp . ) The strain SDY3 shows an ability to decompose aromatic hydrocarbons, and is preferably useful for aromatic hydrocarbons having 16 or less carbon atoms. Experimental Example 3 of the present invention shows excellent resolution of 28.8% in fluorene, but the characteristics of the present invention are not limited thereto.

2. Pseudomonas sp. Pseudomonas sp . ) SDY3  Isolation, Identification and Screening of Strain

The Pseudomonas sp. sp . The process of isolating, identifying, selecting and isolating the SDY3 strain is preferably (1) collecting the contaminated soil from the oil-contaminated area, (2) decomposing the oil using the solidification medium, (3) measurement of the oil degradation rate and degradation rate of the strain through the selection of liquid culture using minimal media, (4) the step of measuring the oil resolution and degradation rate in the diesel contaminated soil, (5) Selecting the finally selected excellent strain and further identifying whether or not it can exhibit effective oil resolution even in actual oil contaminated soil. However, the present invention is not limited thereto. For example, some of the above steps may be omitted in some cases, the detailed method of each step may vary, and the present invention is not limited to this patent. The configuration of the present invention will be described in detail as follows.

The first step involves collecting contaminated soil in the oil, such as the surrounding soil contaminated with low-oil or leached oil, to isolate microorganisms with high efficiency of oil degradation. Preferably, the soil contaminated with oil is collected from a diesel storage site of a demolished army base or a gas station around the country, and may include diesel as a main component of the pollution source.

The second step involves diluting the soil contaminated with the oil and then streaking on a minimal nutrient solidification medium containing diesel to select strain colonies with oil resolution.

The third step involves concentrating the selected strain colonies by adding them into a Minimal Salt Medium (MSM) containing diesel and hydrocarbon components. At this time, in order to select strains having excellent oil-resolving ability, strains to be screened are alternately screened by replacing the culture medium at intervals of 1 to 5 days. After 10 days of concentration, the culture broth was streaked on Tryptic Soy Agar (TSA) to identify excellent oil degradation strains present in the oil contaminated soil. The identified strains were identified as containing diesel and hydrocarbon components Inoculation is carried out on the minimal nutrient medium and the oil-degrading strains having excellent growth rate of the strain are crossed and selected. That is, the oil-degrading strain having a high decomposition rate of diesel and hydrocarbon and having a high decomposition rate is finally selected.

The fourth step involves testing the oil degradation of each strain in a liquid culture to select the best strains available for purification of oil contaminated soils. This step may be a step of measuring the diesel resolving power and growth rate of each strain in a minimal nutrient medium containing a predetermined amount of diesel and hydrocarbon, and may include a step of measuring the hydrocarbon resolution of the selected strain. The hydrocarbons may include polyaromatic hydrocarbons (PAH) generated in the crude oil refining and paper industry. The decomposition ability of hydrocarbons can be determined by adding a mixture of specific hydrocarbons to the culture solution, culturing the strain for a certain period of time, and then measuring the residual amount of hydrocarbons in the culture solution.

The fifth step is to identify strains showing diesel resolving ability selected in the above step, and includes use of API Test Kit. The API Test Kit method is a method to determine the presence or absence of metabolism by color change using biochemical test, assimilation test, fermentation test, and to infer unknown microorganisms.

Additionally, testing may include testing whether a strain having oil resolution in a liquid culture containing diesel can exhibit effective oil resolution in actual oil contaminated soils. The test includes a step of inoculating a sterilized suspension of the strain cultivated in a minimal nutritive medium for 24 hours in a sterilized amount of soil, and adding a test solution in which a minimal nutrient medium and diesel are mixed to confirm the diesel resolution after a certain period of time.

3. Pseudomonas spp. Pseudomonas sp . ) SDY3  A method of purifying contaminated soil by dispensing the strain into the soil.

The present invention relates to the aforementioned Pseudomonas sp. sp . ) A method of decomposing the oil of soil contaminated with oil by distributing the strain SDY3 to oil contaminated soil.

In the above, the oil path is not particularly limited, but it is a petroleum-based oil, and may include aviation oil, gasoline, kerosene, case, heavy oil, diesel and the like. According to Experimental Example 2 of the present invention, Pseudomonas sp. sp . ) The SDY3 strain exhibits fast diesel resolution with fast speed when decomposing more than 90% of the diesel in 28 days for soil contaminated with actual 2% (w / w) diesel, and dispensing to diesel contaminated soil.

In addition, the oil contaminated soil may contain hydrocarbon pollutants. The hydrocarbon is not particularly limited but is an aromatic hydrocarbon and includes benzene, naphthalene, xylene anthracene, as well as fluorene, phenanthrene, or pyrene. Pseudomonas species of this invention (Pseudomonas sp ) The SDY3 strain exhibits excellent resolving power for hydrocarbon contaminants, especially for aromatic hydrocarbons having up to 16 carbon atoms. According to Experimental Example 3, the Pseudomonas sp. sp . ) Since the SDY3 strain shows a resolving power against fluorene, phenanthrene, or pyrene, in particular, fluorene exhibits an excellent resolving power of 28% or more. Therefore, in the case of not only oil contaminated soil but also contaminated soil of oil and hydrocarbon and soil contaminated with hydrocarbon, The soil can be purified by dispensing the strain.

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> Collect contaminated soil and isolate strain

After collecting oil contaminated soil from diesel storage sites in Gyeonggi Province and Korea Army bases and disposal sites around the country, sampled soil samples were collected by filtration and stored in a refrigerator at 4 ℃.

1 g of the above soil was suspended in 10 ml of Minimal Salt Medium (MSM) containing 5.8 g of K ₂ HPO ₄ , 4.5 g of KH ₂ PO _{4 in} 1 liter of distilled water, containing 0.3% (v / v) MgCl ₂ : 0.16 g, CaCl ₂ : 0.02 g, NaNO ₄ : 0.002 g, FeSO ₄ : 0.001 g, and MnCl ₂ : 0.001 g). MSM of the culture broth was replaced at intervals of 48 hours. In order to effectively select a strain having excellent oil resolving ability, diesel and hydrocarbons were added for selective culture. This procedure was repeated 5 times, and the culture broth was plated on a tryptic soy agar (TSA). The medium was incubated at 30 DEG C for 48 hours and 17 colonies were obtained.

Each of the strains collected from the above bacterial colonies was inoculated into MSM containing diesel to select four strains exhibiting the best diesel resolution. Specifically, 20 ml of MSM containing 2% (v / v) diesel was inoculated at a concentration of 3% (v / v) for 24 hours in a nutrient medium and cultured at 25 ° C, pH7, 150 rpm for 5 days. The strain is selected by measuring the resolution. The four selected strains were cultured in a Tryptic Soy Agar (TSA) for 24 hours, respectively, and strains were identified using the API Test method.

The four strains were designated Pseudomonas sp. sp . ) SDY1, Acinetobacter sp . ) SDY2, Pseudomonas sp . ) SDY3, Pseudomonas sp . ) SDY4.

       < Example  2> Pseudomonas sp. Pseudomonas sp . ) SDY3  Cell hydrophobicity of strains

Oil decomposition strain according to the present invention Pseudomonas species (Pseudomonas sp . ) In order to determine the cell hydrophobicity of the strain SDY3, the strain of the present invention was inoculated into 50 ml of minimal nutrient medium (MSM) containing 2% (v / v) diesel and cultured for 5 days, The cells were suspended in minimal nutrient medium (MSM). The suspension was mixed with hexadecane at a ratio of 10: 1, stirred, and then the aqueous layer formed was taken and absorbance was measured. The absorbance was expressed in% compared with the initial value of absorbance. Pseudomonas sp . The hydrophobicity of SDY3 was 98% , and the hydrophobicity of these cells was correlated with the oil resolution of the strain.

       < Example  3> Pseudomonas sp. Pseudomonas sp . ) SDY3  Strain Emulsifying ability

Pseudomonas sp . ) To determine the emulsifying activity of the SDY3 strain, the strain of the present invention was inoculated in 2 ml of MSM containing 2% (v / v) diesel and cultured for 5 days. The culture solution was filtered with a filter paper, (Patassium Phosphate) buffer. Hexadecane was added to the solution at a ratio of 1/10, stirred, and allowed to stand for 10 minutes. The resulting aqueous layer was measured for hexadecane emulsified in the water layer by absorbance. The resulting Pseudomonas sp . ) The emulsifying ability of SDY3 strain was 13% .

       < Experimental Example  1> Measurement of oil resolution by liquid culture

20 ml of MSM containing 2% (v / v) diesel was added to the culture medium of Pseudomonas sp . ) SDY1 strain, Acinetobacter sp . ) SDY2 strain, Pseudomonas sp. sp . ) SDY3 strain, Pseudomonas sp. sp . ) SDY4 strain was inoculated at a concentration of 3% (v / v), and cultured at 25 DEG C, pH7, and 150 rpm for 5 days. Residual diesel was extracted with 15 mL of hexane and analyzed by gas chromatography GC-FID (HP6890) did. The diesel resolutions of the respective strains were as shown in Table 2 below.

Strain number Strain Diesel Resolution (%) One Pseudomonas sp. SDY1 ( Pseudomonas sp. sp SDY1) 77 2 Acinetobacter Spanish cis SDY2 (Acinetobacter sp SDY2) 66 3 Pseudomonas sp. SDY3 ( Pseudomonas sp. sp SDY3) 93 4 Pseudomonas sp. SDY4 ( Pseudomonas sp. sp SDY4) 61

In Table 2 above, Pseudomonas sp. sp . ) The highest resolution of SDY3 strain was 93%.

< Experimental Example  2> Pseudomonas sp. Pseudomonas sp . ) SDY3  Oil Resolutions in Diesel Contaminated Soils of Strain

The soil was collected from the mountains of Gunpo, Gyeonggi - do, and the characteristics of the soil were analyzed through water availability, pH and total organic matter. As a result of analysis of the collected soil, the acidity was found to be 0.58g (H ₂ O) / g (soil), 9.66% (w / v) total organic matter, and 4.28 pH. For the growth of the strain, the pH was adjusted to 7.0 by adding CaCO ₃ to the soil and then the experiment was carried out.

The soil was sieved using a 1.19 mm sieve, followed by moist heat sterilization (121 캜, 30 minutes, 3 times) and drying in a 75 캜 drier for 24 hours to prepare sterilized soil of 50 g. The soil was sprayed with 2% (w / w) of diesel and cultivated in TSB (tryptic bean medium) for 24 hours. Pseudomonas The strain SDY3 was centrifuged to obtain microbial cells, which were suspended in a minimal nutrient medium (MSM) and mixed with the soil so that the soil water holding capacity was 40%. For the growth of the strain, 0.1 g CaCO ₃ per 10 g soil was added and mixed. This experiment was carried out for 28 days, and the experimental results for diesel decomposition with the incubation time (days) are shown in Table 3 below.

Pseudomonas sp . ) Oil Resolutions in Diesel Contaminated Soils of SDY3 Strain Processing date 3 days 7 days 14 days 21st 28th Diesel Decomposition (%) 11 34 52 71 91 Residual diesel (ppm) 17,800 13,200 9,600 5,800 1,800

In Table 3, Pseudomonas sp. sp . ) The SDY3 strain has an oil degradation rate of 90% or more within 28 days in the actual soil, showing a rapid degradation rate and excellent resolution.

< Experimental Example  3> Pseudomonas sp. Pseudomonas sp . ) SDY3  Determination of Aromatic Hydrocarbon Resolution Ability of Strain

To the cap tube was added 2 ml of the minimal nutrient medium and a mixture of polyaromatic hydrocarbon (PAH) in acetonitrile as the carbon source to a final concentration of 10 ppm. The polyaromatic hydrocarbons (PAH) consisted of a 1: 1: 1 (v / v) mixture of Fluorene, Phenanthrene and Pyrene. At 28 ℃ TSB (Tryptic soy medium) in the culture medium for 12 hours, the cultured Pseudomonas species (Pseudomonas sp . ) SDY3 strain was inoculated with 2% (v / v) and cultured for 10 days. The culture temperature was 28 ° C and the stirring speed was 150 rpm.

The remaining PAH is analyzed by extracting the residual PAH by adding 8 ml of ethanol to 2 ml of the culture solution, and centrifuging it to 10,000 g to analyze the supernatant. Quantitative analysis was performed using an HPLC system (Thermal Separation Products, USA). The column used was a C18 Vydac column, the analysis conditions were mobile phase: 75% acetonitrile, flow rate: 1 ml / min, detector: UV / VIS detector at 254 nm; Respectively.

The results of the above analysis are shown in Table 4 below.

Pseudomonas sp . ) Aromatic hydrolysis of SDY3 strain Aromatic hydrocarbons Number of benzene rings Carbon number Hydrocarbon Decomposition (%) Fluorine 2 13 28.8 Phenanthroline 3 14 19.6 Pyrene 4 16 9.1

As shown in Table 4, the novel Pseudomonas sp. sp . ) Hydrolysis ability of SDY3 strain was highest at 28.8% of fluorene decomposition ability, and resolution was decreased as the carbon number of aromatic hydrocarbon was larger overall.

As it described above, according to the present invention Pseudomonas species (Pseudomonas sp . ) The SDY3 strain shows excellent oil resolution in oil contaminated soils, especially diesel contaminated soils.

Pseudomonas species of this invention (Pseudomonas sp . ) The SDY3 strain exhibits excellent diesel resolution even at high concentration of 2% (v / v) diesel and can be used to purify the soil economically and efficiently because of its excellent decomposition rate. It also shows the decomposition ability of aromatic hydrocarbons such as fluorene, and is likely to be widely used for the purification of polluted soils including oil contaminated soil and a complex contaminated soil with oil and organic compounds.

Korea Biotechnology Research Institute KCTC18365P 20150331

Claims (7)

Pseudomonas species (Pseudomonas resolution with oil sp . ) Strain SDY3 (Accession No. KCTC18365P). The method according to claim 1, wherein the oil is a petroleum-based oil selected from the group consisting of jet fuel, gasoline, kerosene, light oil, heavy oil, and diesel. Pseudomonas sp . ) SDY3 strain. The method according to claim 2, wherein the petroleum-based oil is diesel ( Pseudomonas sp. sp . ) SDY3 strain. The method of claim 1, wherein the strain is selected from the group consisting of Pseudomonas sp. sp . ) SDY3 strain. 5. The method of claim 4 wherein the hydrocarbon is selected from the group consisting of aromatic hydrocarbons Pseudomonas sp . ) SDY3 strain. The process according to claim 5, wherein the aromatic hydrocarbon is selected from the group consisting of fluorene, phenanthrene and pyrene. Pseudomonas sp . ) SDY3 strain. The method of claim 1, wherein the Pseudomonas sp . A method of decomposing the oil of polluted soil by distributing SDY3 strain to oil contaminated soil.