KR101332347B1 - Pseudomonas sp. nr1 and oil degradation method using the same - Google Patents

Abstract

The present invention relates to a novel Pseudomonas genus NR1 strain having an oil resolution and an oil degradation method using the same. According to the present invention as described above, the Pseudomonas genus NR1 strain, which is an oil degradation strain, exhibits excellent oil resolution in diesel-contaminated soil. It is more efficient and economical than the conventional physical and chemical treatment methods, and has an effect of removing oil by an environmentally friendly method without secondary pollution.

Description

Nr1 strain of Pseudomonas genus having oil resolution and oil degradation method using the same {PSEUDOMONAS SP. NR1 AND OIL DEGRADATION METHOD USING THE SAME}

The present invention relates to a Pseudomonas sp. NR1 strain having an oil resolution and an oil degradation method using the same, and more particularly, an oil degradation strain having an excellent oil resolution by separating microorganisms from soil contaminated with oil. The present invention relates to a method of decomposing oil using the Pseudomonas genus NR1 strain and the oil degradation strain.

Soil pollution caused by oil has a lower diffusion rate than air or water pollution, while the extent of damage can be widespread, and the damage is persistent. In addition, unlike gas or liquid, solid soils are difficult to handle engineeringally (transferring into the process, chemical or biological reactions, etc.). In addition, approaches must be approached differently from air and water pollution.

There are many sources of pollution, but the main pollutants of interest include oils refined from heavy metals and crude oil, BTEX (Benzene, Toluen, Ethylbenzene, Xylene; benzene, toluene, ethylbenzene, xylene), Aliphatic and aromatic chlorine compounds derived from organic solvent use industries and products, herbicides and pesticides, and poly aromatic hydrocarbons (PAHs) from crude oil refining and paper industry.

Among these, soil pollution caused by oil is the most widespread and has been pointed out as a big problem in recent years. The conventional method for removing oil components contaminated with soil has its own problems. For example, the physical treatment of commonly used spilled oils is not expected to be fully effective, but it is expensive to control, and the use of chemical oil dispersants poses a problem of secondary pollution due to toxicity. Because.

In addition, the incineration method not only pollutes the air, but also causes other energy costs and secondary pollution, and marine dumping of oil-contaminated soils has a problem of destroying marine ecosystems by polluting the ocean. In addition, underground or ground reclamation not only causes the generation of toxic gas and contamination of groundwater, but also uses methods of cleaning by chemical synthetic emulsifiers, microbial emulsifiers, and soil and electrode using emulsifiers. These methods, too, are difficult to effectively remove the polluting oil.

Due to the rapid growth of the current industry, the use of oil is exploding, and the frequency of oil spills is increasing rapidly, and the pollution of oil storage tanks such as US military bases or military units is also serious.

On the other hand, the mass consumption of petrochemicals requires facilities such as underground storage tanks and transfer pipes, and the number of facilities such as underground storage tanks and oil pipelines in gasoline stations that store and transport oil nationwide rapidly increased. However, the leakage of petroleum and petrochemicals due to the lack or management of these facilities continues to increase, and soil pollution is intensifying.

In the case of land developed by the withdrawal of US troops and military bases, oil pollution has been in progress for several decades, and the oil pollution area and concentration are high. The problem of soil pollution due to oil leakage is serious now, but in the future, it is a major problem not only for groundwater contamination but also for land development and other uses.

Accordingly, the inventors have focused on the fact that most oils and various organic pollutants are decomposed by microorganisms, and biological oil decomposition is a safe, economical and effective purification method without secondary pollution. As a result of diligent efforts to find microorganisms having high oil resolution from the separation, the present invention confirmed that a novel strain of Pseudomonas effectively degrades oil in contaminated soil and completed the present invention.

An object of the present invention is to provide a novel microbial strain Pseudomonas genus NR1 strain having oil resolution to soil contaminated with oil.

It is also an object of the present invention to provide a method for decomposing oil from soil contaminated with oil using the new microorganism.

It is also an object of the present invention to provide a method for separating microorganisms having excellent oil resolution from soil contaminated with less oil.

The present invention provides a Pseudomonas sp . NR1 strain (Accession No. KCTC 18225P) having oil resolution.

In addition, the present invention provides a method for decomposing the oil of the oil-contaminated soil by dispensing Pseudomonas sp . NR1 strain (Accession No. KCTC 18225P) having oil-degrading ability to the oil-contaminated soil.

In the above, the oil is petroleum oil selected from the group consisting of aviation oil, gasoline, kerosene, diesel oil, heavy oil and diesel, more preferably diesel is used as the petroleum oil.

The present invention also provides a method for decomposing hydrocarbons by dispensing Pseudomonas sp . NR1 strain (Accession No. KCTC 18225P) having oil resolving power to hydrocarbon contaminants.

In the above, the hydrocarbon is an aromatic hydrocarbon, wherein the aromatic hydrocarbon is selected from the group consisting of fluorene, phenanthrene and pyrene.

According to the present invention, the NR1 strain of Pseudomonas, an oil-decomposing strain, exhibits excellent oil-degrading ability in oil-contaminated soils, which is more efficient and economical than conventional physical and chemical treatment methods, and removes oil in an environmentally friendly way without secondary pollution. can do.

In addition, the strain NR1 of Pseudomonas, the oil-decomposing strain of the present invention, also shows the possibility of decomposition of aromatic hydrocarbons, and thus can be used for the purification of soil contaminated with aromatic hydrocarbons.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The present invention provides a Pseudomonas genus NR1 strain having oil resolution to soil contaminated with oil.

The NR1 strain of the genus Pseudomonas was deposited on November 25, 2011 at the Korea Collection for Type Cultures (KCTC) of the Korea Biotechnology Research Institute, an international depository institution under the Budapest Treaty on Microbial Deposits (Accession No. KCTC 18225P). , Morphology and physiological properties shown in Table 1.

The process of isolating, identifying and identifying the oil resolution of Pseudomonas genus NR1 strain, which is an oil digestion strain of the present invention, preferably comprises the steps of (1) harvesting contaminated soil from an oil-contaminated region, and (2) decomposing oil. Separating the strain, (3) selecting and identifying the selected strain, (4) measuring the oil resolution of the strain through liquid culture, and (5) measuring the oil resolution in the oil-contaminated soil. It includes, but is not limited to such. For example, some of the above steps may be omitted in some cases, and the detailed method of each step may be changed and is not limited by the present patent. The above method is described in detail.

First, in order to isolate microorganisms having high oil decomposition efficiency, soil contaminated with oil, such as soil around a gas station, is collected.

Next, the collected contaminated soil is added to the minimum nutrient medium (Minimal Salt Medium, MSM) containing the oil and cultured. At this time, preferably, the minimum nutrient medium is replaced at 48 hour intervals, cultured for 10 days, and then cultured on tryptic soy agar medium (Tryptic Soy Agar, TSA) to spread the strain present in oil-contaminated soil. Check them.

In addition, each of the strains formed on the plate is inoculated in the least nutrient medium containing the oil to select strains showing the oil resolution. In order to identify strains selected to have oil resolution, an API Test Kit can be used, but is not limited thereto. The API Test Kit method is a method of inferring and identifying unknown microorganisms by determining the metabolism of a substance by color change using a biochemical test, an assimilation test, and a fermentation test.

Then, the oil resolution of each strain is tested in the liquid culture to select the optimum strains that can be used for the purification of oil contaminated soil. At this time, the oil resolution of each strain in the minimum nutrient medium containing a certain amount of oil is measured. In addition, for a strain considered to have oil resolution in the minimum nutrient medium, its hydrocarbon resolution may be measured. For example, the resolution for poly aromatic hydrocarbons (PAH) generated in the crude oil refining and paper industry can be measured. In addition, the resolution of the hydrocarbon can be determined by adding a mixed solution of specific hydrocarbons to the culture to incubate the strain for a certain time, and then measuring the residual amount of hydrocarbon in the culture.

Finally, oil resolution through oil contaminated soils is tested by confirming that strains with oil resolution in liquid cultures containing oil can exhibit effective oil resolution even in actual oil contaminated soils. The test includes inoculating a strain suspension cultured in a minimal nutrition medium for 24 hours in a predetermined amount of sterile soil, and adding a test solution containing a minimum nutrition medium and oil thereto to check oil resolution after a certain time.

According to one embodiment of the present invention, in order to know the characteristics of the Pseudomonas genus NR1 strain of the present invention, as a result of the hydrophobicity test and emulsification test process of the cell, Pseudomonas genus NR1 strain is high in hydrophobicity, which is the diesel resolution of the strain It appears to have a quantitative correlation with (see Example 2). In contrast, the emulsification capacity of the strain is observed to be low (see Example 3).

In addition, the present invention provides a method for decomposing oil of soil contaminated with oil by dispensing the Pseudomonas genus NR1 strain into oil contaminated soil.

In the above description, the oil is not particularly limited, but may be petroleum oil, and may include, without limitation, aviation oil, gasoline, kerosene, diesel oil, heavy oil, diesel, and the like. According to one embodiment of the present invention, the Pseudomonas genus NR1 strain of the present invention not only shows excellent diesel resolution in an in vitro environment (see Tables 2 and 3), but also 80% within one month for actual diesel-contaminated soils. The above diesel decomposition rate is shown (see Table 4).

The present invention also provides a method for decomposing hydrocarbons by dispensing the Pseudomonas genus NR1 strain to hydrocarbon contaminants.

The hydrocarbon is not particularly limited but is an aromatic hydrocarbon, and exhibits excellent resolution of fluorene, phenanthrene or pyrene, as well as benzene, naphthalene and xylene anthracene 뽄. According to one embodiment of the invention, the Pseudomonas genus NR1 strain of the invention exhibits excellent resolution for aromatic hydrocarbon mixtures in an in vitro environment (see Table 5).

Hereinafter, the present invention will be described in more detail with reference to Examples.

This embodiment is intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1 Polluted Soil Collection and Strain Separation

After collecting oil-contaminated soil from a diesel reservoir at a US military base being demolished in Gyeonggi-do, the collected soil samples were filtered out to remove large particles and stored in a 4 ° C refrigerator. 10 g of minimal salt medium (MM) containing 0.3 g (v / v) of diesel in the soil; K ₂ HPO ₄ : 5.8 g, KH ₂ PO ₄ : 4.5 g in 1 liter distilled water , MgC1 ₂ ; 0.16, CaC1 ₂ : 0.02g, NaNo ₄ ; 0.002g, FeSO ₄ ; 0.001g, MnC1 ₂ ; including 0.001g) and concentrated culture. MSMs of the cultures were replaced at 48 hour intervals and cultures were plated on Tryptic Soy Agar (TSA) after 10 days. After incubating the medium for 48 hours at 30 ° C, 25 bacterial colonies were obtained.

Three strains showing diesel resolution were selected by inoculating MSM containing diesel, as shown in Experimental Example 1, to each strain collected from the bacterial colonies. Each of the three strains was incubated for 24 hours in TSA (Tryptic Soy Agar: tryptic soy agar medium), and then strains were identified through the reaction using the API Test method. As a result, the three strains were named Pseudomonas genus NR1 strain, Acinetobacter sp. NR2 strain and Pseudomonas genus NR3 strain, respectively. Table 1 shows the morphology and biochemical properties of the Pseudomonas genus NR1 strain.

Example 2 Cellular Hydrophobicity of Pseudomonas Genus NR1 Strains

In order to know the cell hydrophobicity of the oil degradation strain, Pseudomonas genus NR1 strain according to the present invention, 50 ml of MSM (minimum nutrient medium) containing 2% (v / v) diesel was inoculated with the strain of the present invention for 5 days. After incubation, the cells obtained by centrifugation of the culture were suspended in MSM. After the suspension and hexadecane were mixed and stirred at 10: 1, the resulting aqueous layer was left to stand and the absorbance was measured. The absorbance was expressed in% compared to the initial value. The hydrophobicity of NR1 strain of Pseudomonas was 97%, and it was determined that the cell hydrophobicity correlated with the oil resolution of the strain.

Example 3 Emulsification Capacity of Pseudomonas Genus NR1 Strain

In order to know the emulsification capacity of NR1 strain of Pseudomonas, 2 ml MSM containing 2% (v / v) diesel was inoculated with the strain of the present invention and incubated for 5 days, and the remaining solution was filtered through a filter paper and the remaining solution was phosphoric acid. It was added to Potassium Phosphate Buffer. Nucledecane was added to the solution at a ratio of 1/10, stirred and allowed to stand for 10 minutes, and then hexadecane emulsified in the aqueous layer was measured by absorbance. As a result, the emulsification capacity of Pseudomonas genus NR1 was 11%.

Example 4 Measurement of Oil Resolution Through Liquid Culture

In 20 ml MSM containing 2% (v / v) diesel, concentrations of 3% (v / v) of Pseudomonas genus NR1 strain, Acinetobacter NR2 strain and Pseudomonas genus NR3 strain were incubated for 24 hours in nutrient medium. Inoculated at 5 ° C. for 5 days at 25 ° C., pH 7, 150 rpm. In addition, in the same manner, the oil resolution when the strains were cultured in combination was also measured. The oil resolution was analyzed by gas chromatography GC-FID (HP 6890) by extracting residual diesel with 15 mL hexane. Diesel resolution of a single strain is shown in Table 2, and diesel resolution of the mixed strain is shown in Table 3 below.

As shown in Table 2, the Pseudomonas genus NR1 strain showed the highest diesel resolution of 89% among a single strain.

As shown in Table 3, the combination of strains showed the highest diesel resolution of 69% when the Pseudomonas genus NR1 strain (1) and the Pseudomonas genus NR3 strain (3) were combined.

Example 5 Oil Resolution of Pseudomonas Strain NR1 in Diesel-Contaminated Soils

Soil was collected from Yasan, Uiwang-si, Gyeonggi-do, and the soil characteristics were analyzed by water retention capacity, pH, and total organic matter.

As a result of analyzing the soil, the water retention was 0.58g (H ₂ O) / g (soil), total organic matter was 9.66% (W / V) and pH was 4.28. Experiments were performed by calibrating the pH to 7.0 by adding CaCO ₃ for growth of the strain.

After the soil was filtered through a 1.19 mm sieve, wet heat sterilization (121 ° C., 30 minutes, 3 times) and dried for 24 hours in a 75 ° C. drier to prepare 50 g of sterile soil. After spraying 2% (w / w) diesel into the soil and centrifuging Pseudomonas genus NR1 strain incubated for 24 hours in TSB (Triptic soybean medium), the cells were obtained, followed by MSM (minimal nutrient medium). Suspension was added to the soil so that 40% of the soil moisture retention capacity and evenly mixed. In addition, 0.1 g of CaCO ₃ per 10 g of soil was added and mixed for growth of the strain. The experiment was carried out for 28 days, and the experimental results for the diesel decomposition according to the incubation time (days) are shown in Table 4 below.

Example 6 Determination of Aromatic Hydrocarbon Degradation of Pseudomonas Species NR1

In a 2 ml minimal nutrient medium in a Cap Tube, a mixture of Poly Aromatic Hydrocarbon (PAH) in acetonitrile as a carbon source was added to a final 10 ppm. The polyaromatic hydrocarbons (PAH) consisted of a 1: 1: 1 (v / v) mixture of fluorine, phenanthrene and pyrene. The culture solution was inoculated with 2% (v / v) of Pseudomonas genus NR1 strain incubated for 12 hours at 28 ° C. in TSB (tryptic soybean medium) and cultured for 10 days. The incubation temperature was 28 ° C. and the stirring speed was 150 rpm.

Analysis of the remaining PAH was carried out by adding 8 ml of ethanol to 2 ml of the culture and extracting the remaining PAH, centrifuging it at 10,000 g to analyze the supernatant. Quantitative analysis was performed using an HPLC system (Thermal Separation Products, U.S.A.).

The column used was a C18 Vydac column, and the analysis conditions were as follows, and the analysis results are shown in Table 4 below (mobile phase: 75% acetonitrile, flow rate: 1 ml / min, sensor: UV at 254 nm). / VIS sensor).

As shown in Table 5, the hydrocarbon degradation of the novel microorganism Pseudomonas genus NR1 strain according to the present invention had the highest fluorine degradability of 22.9%, and the higher the carbon number of the aromatic hydrocarbon, the lower the resolution.

As described above, the present invention is an oil degradation strain Pseudomonas genus NR1 strain exhibits excellent oil resolution in oil-contaminated soil, more efficient and economical than conventional physical and chemical treatment methods, environmental friendly without secondary pollution The oil can be removed in such a way.

In addition, the strain NR1 of Pseudomonas, the oil-decomposing strain of the present invention, also shows the possibility of decomposition of aromatic hydrocarbons, and thus can be used for the purification of soil contaminated with aromatic hydrocarbons.

While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

Korea Biotechnology Research Institute KCTC18225 20111125

Claims (7)

Pseudomonas sp . NR1 strain with accessibility to oil (Accession No. KCTC 18225P). A method for decomposing oil in an oil-contaminated soil by dispensing the strain of Pseudomonas genus NR1 in the oil-contaminated soil. 3. The method of claim 2, wherein the oil is petroleum oil selected from the group consisting of aviation oil, gasoline, kerosene, diesel, heavy oil and diesel. 4. The method of claim 3 wherein the petroleum oil is diesel. A method of decomposing a hydrocarbon by dispensing the Pseudomonas genus NR1 strain according to claim 1 in a hydrocarbon contaminant. The method of claim 5, wherein the hydrocarbon is an aromatic hydrocarbon. 7. The method of claim 6, wherein the aromatic hydrocarbon is selected from the group consisting of fluorene, phenanthrene and pyrene.