KR102818201B1 - Pseudomonas aeruginosa EPR-Q6 strain with excellent oil decomposition ability and method for remediating oil-contaminated soil using the strain - Google Patents

Abstract

The present invention relates to a novel microorganism, Pseudomonas aeruginosa EPR-Q6, having excellent oil-decomposition activity, and a method for biologically restoring soil contaminated with oil by culturing the microorganism. The present invention explores the characteristics and oil-decomposition ability of the microorganism Pseudomonas aeruginosa EPR-Q6, and provides a method for biologically restoring oil-contaminated soil, which can rapidly restore oil-contaminated soil using the microorganism cultured under optimal conditions. In addition, the strain can be used to dramatically improve the oil-decomposition speed and degradation ability of land-farming and biopiles.

Description

{Pseudomonas aeruginosa EPR-Q6 strain with excellent oil decomposition ability and method for remediating oil-contaminated soil using the strain}

The present invention relates to a novel microorganism, Pseudomonas aeruginosa EPR-Q6, having excellent oil-decomposing activity, and a method for biologically restoring soil contaminated with oil by culturing the microorganism. By using the microorganism of the present invention, secondary pollutants are not emitted due to contaminated oil treatment in landfarming and biopile sites, and environmentally friendly and efficient purification is possible.

In Korea, as the use of petroleum has increased in all fields along with industrial growth due to modernization, the frequency of various types of oil spill accidents has also increased. As soil and groundwater contamination due to leakage of representative petroleum such as diesel, which is widely used in vehicles and industrial sites, has become serious, various physical, chemical, and biological methods have been applied since 2000 to restore soil contaminated with oil, focusing on military bases, gas stations, and oil storage facilities. Among these, biological methods that utilize the biodegradation mechanism of microorganisms are environmentally friendly because they are inexpensive and produce fewer secondary treatment byproducts, and are therefore widely used both domestically and internationally.

As the use of petroleum increases in all industrial fields as described above, soil contamination by petroleum is occurring on a large scale in many places due to the increase in petroleum storage facilities, transportation equipment such as automobiles, and gas stations, and petroleum leaks from old petroleum storage facilities, military facilities, and factories used in the past are seeping into the soil, causing volatile organic compounds (VOCs) emissions and groundwater contamination, and the scope of contamination is spreading widely. Crude oil, a representative fossil fuel, causes contamination by entering the soil not only through drilling, production, transportation, and storage, but also through accidental spills and old storage facilities. Various purification methods are being proposed and implemented to restore and reuse soil contaminated with petroleum. Methods for purifying soil contaminated with oil can be largely divided into physicochemical treatment technologies and biological treatment technologies. Physicochemical treatment technologies include soil vapor extraction, soil washing, solidification and stabilization methods, and biological treatment technologies include land-farming, biopiles, bioreactors, and restoration methods using plants.

In particular, among biological treatment technologies, the technology that uses the decomposition activity of microorganisms to restore soil contaminated with oil in an environmentally friendly manner is receiving attention as a major treatment method. Biological treatment technologies using microorganisms that can decompose oil components in contaminated soil are widely used because they have advantages in terms of economy and efficiency, and attempts are continuously being made to environmentally purify soil contaminated with oil by searching for new microbial strains with excellent oil decomposition capabilities. In such biological treatment technologies using microorganisms, the decomposition pathway of organic substances such as oil is carried out by oxidation or reduction reactions of enzymes existing within microorganisms (mainly bacteria), and generally, substances with small molecular weights are decomposed more easily and quickly than substances with large molecular weights. Here, organic substances, which are oil pollutants, differ in the degree and speed of decomposition by microorganisms depending on their characteristics, so biological treatment technologies using microorganisms cannot be applied to all oil pollutants, and are currently effectively applied to low-molecular-weight oil pollutants that are volatile and highly water-soluble. Diesel is the fuel with the largest consumption in Korea and accounts for the majority of soil oil pollution-causing substances. Most of its components are relatively nonvolatile organic compounds, of which paraffin-based substances account for approximately 70%. These substances are composed of components that are relatively easy to decompose by microorganisms existing in the soil. Accordingly, there is a need for the development of strains that can efficiently decompose high-concentration diesel and for the development of more effective treatment technologies for oil-contaminated soil using these strains.

However, if soil contaminated with oil is purified using biological treatment technology using microorganisms, it does not cause secondary environmental pollution, but biological treatment technology has vulnerabilities such as a relatively long treatment period, low treatment efficiency, and limitations in treatment methods depending on oxygen demand, so improvements are needed. Accordingly, the inventors of the present invention have taken note of the fact that in order to increase the economic feasibility of actual remediation of oil-contaminated soil, it is necessary to shorten the treatment period, thereby considering excellent oil decomposition ability and a dramatic improvement in the oil decomposition speed and treatment process, and have endeavored to find microorganisms with excellent oil decomposition ability and oil decomposition speed from oil-contaminated soil. As a result, they confirmed that Pseudomonas aeruginosa EPR-Q6, a novel strain of the genus Pseudomonas, effectively decomposes oil in contaminated soil, and completed the present invention, which dramatically improves the oil decomposition speed and decomposition ability of land-farming and biopile using the above strain.

Patent Publication No. 2002-0060009 (July 16, 2002) Patent Publication No. 2004-0098213 (November 20, 2004) Patent Registration Publication No. 10-1558945 (October 12, 2015)

The present invention provides a Pseudomonas aeruginosa EPR-Q6 strain having excellent decomposition ability for oil contaminated in soil, a method for decomposing oil in contaminated soil using the strain, and a method for selecting the strain.

To achieve the above purpose, the present invention provides a novel Pseudomonas aeruginosa EPR-Q6 strain (accession number KCTC 19206P) having excellent oil decomposition ability.

In the present invention, it may be characterized in that the fuel is any one selected from the group consisting of kerosene, gasoline, and diesel fuel.

In addition, the present invention provides a biological oil-contaminated soil remediation method characterized by inoculating the oil-contaminated soil with Pseudomonas aeruginosa EPR-Q6 strain.

In the present invention, the oil decomposition ability can be promoted and enhanced by adding a biostimulant to the Pseudomonas aeruginosa EPR-Q6 strain.

Additionally, the biostimulant in the present invention may be humic acid or fulvic acid.

The present invention not only provides a biological remediation method for oil-contaminated soil, which explores the characteristics and oil-degrading ability of Pseudomonas aeruginosa EPR-Q6 microorganism and can rapidly remediate oil-contaminated soil using microorganisms cultured under optimal conditions, but also can dramatically improve the oil-degrading speed and degrading ability of land-farming and biopiles using the above strain.

Figures 1a and 1b show the results of a homology test performed by aligning the 16S rRNA gene base sequence of the Pseudomonas aeruginosa EPR-Q6 strain according to the present invention.

The terminology used herein is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms “form”, “have”, “include” or “have” are intended to specify the presence of a feature, number, step, operation, component, sub-part or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, sub-parts or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined in commonly used dictionaries, such as those defined in common usage, should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and shall not be interpreted in an idealized or overly formal sense unless expressly defined herein.

In one embodiment of the present invention, a novel Pseudomonas aeruginosa EPR-Q6 strain (accession number KCTC 19206P) having excellent oil decomposition ability is provided.

In another embodiment according to the present invention, the fuel may be characterized as being any one selected from the group consisting of kerosene, gasoline, and diesel fuel.

In another embodiment of the present invention, the present invention provides a method for biologically remediating oil-contaminated soil, characterized by inoculating Pseudomonas aeruginosa EPR-Q6 strain into oil-contaminated soil.

In another embodiment of the present invention, a biostimulant may be added to Pseudomonas aeruginosa EPR-Q6 strain to promote and enhance oil degradation ability.

In another embodiment of the present invention, the biostimulant may be humic acid or fulvic acid.

Hereinafter, the present invention will be described in more detail through specific examples. These examples are only for the purpose of illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples. In addition, the unit of additives not specifically described in the specification may be weight %.

The Pseudomonas aeruginosa EPR-Q6 strain of the present invention was deposited with the Korean Collection for Type Cultures (KCTC), an international depository institution under the Budapest Treaty on the Deposit of Microorganisms, on June 21, 2024, and its deposit number is KCTC 19206P. The selection method and characteristics thereof are described in detail below.

Example 1. Isolation of strains with excellent oil decomposition ability

The inventors collected soil samples from various oil-contaminated sites to isolate microorganisms with excellent oil-decomposing ability. After collecting soil samples from contaminated sites around gas station storage tanks and military base oil storage facilities that had been exposed to oil contamination for a long period of time, contaminants were removed using a 2 mm diameter sieve, and the samples were stored in a refrigerator at 4℃ and used as samples. The minimum medium shown in Table 1 below was used.

[Table 1]

The strain selection method is as follows. First, 5,000 ppm of kerosene was added to 100 ml of the prepared minimal medium after sterilization, and 10 g of the sample stored in a refrigerator at 4°C was added and shaken at 150 rpm at 25°C for 72 hours, and then stationary culture was performed at 30°C for 48 hours. 5 ml of the cultured sample was added to 100 ml of the minimal medium containing 10,000 ppm of kerosene, and shaken at 150 rpm at 25°C for 72 hours, and cultured using the same method while increasing the kerosene concentration to 15,000 ppm and 20,000 ppm. 0.1 mL of the final culture solution was smeared on a solid medium, and 0.2 ml of kerosene was applied. The colonies formed during culture in an incubator at 30°C were selected. The selected strains were confirmed to have the ability to decompose and grow in high-concentration diesel fuel, and these were repeatedly separated by the streak method on TSA medium until pure strains were isolated. The final 6 strains were selected through colony shape and microscopic examination, which are shown in Table 2 below. In Table 2, 0 indicates growth ability, and strains with a score of 000 or higher were selected.

[Table 2]

Example 2. Measurement of the oil-degrading ability of the strain

The decomposition rate of the six selected strains was measured while culturing them in a minimal medium containing 20,000 ppm of kerosene for 20 hours. Specifically, the six finally selected strains were each cultured in TSB (Tryptic Soy Broth) for 24 hours, prepared as spawn, inoculated 1 ml into 100 mL of the minimal medium containing 20,000 ppm of kerosene, and cultured at 25°C and 150 rpm for 120 hours. The decomposition ability of each strain was measured by analyzing the remaining kerosene, as shown in Table 3 below. Three flasks were used for each strain, and each average was calculated to finally select the Pseudomonas aeruginosa strain with the best decomposition ability, and the strain was finally named Pseudomonas aeruginosa EPR-Q6.

[Table 3]

At this time, the concentration of the remaining fuel was confirmed by measuring the concentration of TPH, and the TPH concentration was measured by a gas chromatograph (HP-5890 Plus II, Hewlett Packard) equipped with a FID detector. A DB-5 GC column (30 m × 0.53 mm, 1.5 μm) was used as the column, and the temperature of the inlet was 250 °C, the temperature of the detector was 280 °C, and the oven temperature was maintained at 40 °C for 1 minute and then increased at 15 °C/min and maintained at 280 °C for 5 minutes.

Example 3. Identification of strains with excellent oil decomposition ability

The minimal medium was prepared by dispensing 100 ml each into 300 ml flasks and sterilizing in an autoclave at 121℃ for 15 minutes, and the kerosene was added at each concentration before the experiment after 0.2 um syringe filtering. In order to rapidly and efficiently select oil-decomposing bacteria with excellent oil-decomposing ability, 6 strains that formed morphological superiority were selected while increasing the kerosene concentration in the minimal medium according to the concentration, and the 16s rRNA base sequence of the Q6 strain with the best decomposition ability was analyzed and identified, which is shown in Figures 1a and 1b.

Example 4. Measurement of strain oil decomposition ability in oil-contaminated soil

Pseudomonas aeruginosa EPR-Q6, which was finally selected and isolated to examine the applicability in the field of land farming, was used to measure oil decomposition ability in actual soil. After collecting soil from a nearby mountain, 10 kg of the sieved soil was placed in a container (75 cm x 50 cm x 40 cm). After preparing the sample soil, 20,000 ppm of diesel was added and mixed evenly to artificially contaminate it. After inoculating the contaminated soil with the spawn cultured Pseudomonas aeruginosa EPR-Q6 to a concentration of 1.0× ¹⁰⁶ cfu/g, the moisture content was maintained at 15-20%. The soil was turned over once a day to allow for air to enter, and samples were collected at five-day intervals for 40 days to measure the concentration of residual diesel and the number of viable cells.

[Table 4]

According to Table 4 above, the oil degradation rate of Pseudomonas aeruginosa EPR-Q6 in actual soil was 95% within 40 days, confirming excellent degradation ability. The total bacterial count was initially 3.6× ¹⁰⁵ cfu/g, but after inoculation with 1.0× ¹⁰⁶ cfu/g of Pseudomonas aeruginosa EPR-Q6, it grew to 2.1× ¹⁰⁷ cfu/g on day 30 of culture, and after 40 days of culture, it grew to a final 9.2× ¹⁰⁶ cfu/g.

Example 5. Pseudomonas aeruginosa in oil-contaminated soil Pseudomonas aeruginosa ) Effect of promoting oil decomposition and enhancing microorganisms by adding biostimulator after EPR-Q6 treatment

In the method of removing oil by applying it to land farming and biopiles, it was confirmed that biostimulants have the effect of promoting and enhancing oil decomposition ability together with the growth of oil-decomposing microorganisms. Humic acid or fulvic acid can be used as a biostimulant.

Specifically, the collected soil was screened and 10 kg each was placed in two containers (75 cm x 50 cm x 40 cm), and after preparing the sample soil, 20,000 ppm of diesel was added and mixed evenly to artificially contaminate it, and Pseudomonas aeruginosa EPR-Q6 with spawn culture was inoculated into the contaminated soil to 1.0× ¹⁰⁶ cfu/g, and the moisture content was maintained at 15-20%. The soil was turned over once a day to allow air to enter, and samples were collected at five-day intervals for 40 days to measure the concentration of residual diesel and the number of viable cells. At this time, only microorganisms were added to the existing treatment group as before, and the control group was treated by spraying humic acid dissolved in water to 10 ppm relative to the soil. The results are shown in Table 5.

[Table 5]

According to Table 5 above, when humic acid, a biostimulant, was treated simultaneously with Pseudomonas aeruginosa EPR-Q6, the inoculum cultured according to the present invention, to oil-contaminated soil, the oil decomposition rate was shown to be shortened by 5 to 6 days compared to the existing method, and the growth of oil-decomposing bacteria was also confirmed to be 16 to 19% superior. This shows that when humic acid is treated simultaneously with Pseudomonas aeruginosa EPR-Q6, the oil decomposition rate and treatment period are shortened.

As described above, the best embodiment has been disclosed in the drawings and the specification. Although specific terms have been used herein, they have been used only for the purpose of describing the present invention and have not been used to limit the meaning or the scope of the present invention described in the claims. Therefore, those skilled in the art will understand that various modifications and equivalent other embodiments are possible from this. Therefore, the true technical protection scope of the present invention should be determined by the technical idea of the appended claims.

Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC) KCTC19206P 20240621

Claims (5)

A novel strain of Pseudomonas aeruginosa EPR-Q6 with excellent oil decomposition ability (Accession No. KCTC 19206P) delete A biological oil-contaminated soil remediation method characterized by inoculating Pseudomonas aeruginosa EPR-Q6 strain into oil-contaminated soil In the third paragraph, a biological oil-contaminated soil purification method characterized by adding a biostimulant to promote and enhance oil decomposition ability. In the fourth paragraph, a biological oil-contaminated soil purification method characterized in that the biostimulant is humic acid.