KR20090030897A - Liquid composition of microorganisms for bioremediation of hydrocarbon-contaminated soil, method of preparing the same, and bioremediation using the same - Google Patents

Abstract

A microorganism liquid composition for cleaning oil-contaminated soil with enhanced oil-decomposing activities is provided to improve a decomposition rate of polluted materials and reduce a necessary time for cleaning the oil-contaminated soil. A microorganism liquid composition for cleaning oil-contaminated soil with enhanced oil-decomposing activities is characterized by mixing a culture fluid of a high growth strain and a culture fluid of a slow growth strain in a volume ratio of 1 to 1 or 1 to 9. The high growth strain is Acinetobacter, Acinetobacter haemolyticus, Pseudomonas aeruginosa, Acinetobacter radioresistens, Stenotrophomonas maltophilia, or Bacillus sphaericus. The slow growth strain is Nocardia nova, Cellulomonas turbata, or Flavobacterium johnsoniae.

Description

Liquid composition of microorganisms for bioremediation of hydrocarbon-contaminated soil, method of preparing the same, and Bioremediation using the same}

The present invention relates to a microbial liquid composition for the purification of oil-contaminated soil, a preparation method thereof and a method for the purification of oil-contaminated soil using the same, and more specifically, to separate microorganisms having oil resolution separated from the oil-contaminated soil according to the growth rate. By using cultured mixed strains, oil degradation activity that can occur due to the difference in growth rate is excellent, but the degradation time of contaminants is improved and effectively decomposed without degradation of microorganisms with slow growth rate, thereby effectively purifying oil contaminated soil. It relates to a microbial liquid composition for the purification of oil-contaminated soil that can be shortened, a preparation method thereof and a method for the purification of oil-contaminated soil using the same.

Rapid industrialization faces severe environmental pollution due to increased fossil fuel consumption, soaring pollutant emissions, population concentration in large cities, and natural destruction due to unreasonable development plans. Such environmental pollution not only has a rapid and direct effect on human life through the air or water, but also is exposed slowly through soil and groundwater for a long time. In particular, soil contamination due to improper disposal of hazardous substances, damage to old facilities, and sudden environmental accidents has been neglected for a long time due to wide range of pollution and huge disposal costs.

Pollutants that cause soil pollution can be oils such as light oil, diesel oil, heavy oil, lubricating oil, and chlorophenol, polychlorinated biphenyl, polycyclic aromatic hydrocarbons, heavy metals, among which organic compounds based on hydrocarbons. Is used as a carbon source and energy source for certain microorganisms, and has biodegradation properties without any environmental pollution.

Biodegradation is a phenomenon in which certain microorganisms of natural ecosystems decompose harmful pollutants under appropriate conditions and use them as harmless or nutrient sources.By using these functions of microorganisms, biodegradation is used to purify polluted areas such as soil, groundwater, ocean and rivers. An engineering technique is called bioremediation. If biological restoration techniques are effective, the end product must be converted into a harmless substance, such as water or carbon dioxide, to harm the environment or the organism. Such biological restoration technology is economical, and it has been recently spotlighted because there is an advantage that there is no secondary pollutant by using the purification ability of nature.

Bioremediation can be broadly classified into active restoration and passive restoration, depending on whether external techniques are applied to accelerate natural degradation in contaminated areas. Active restoration technology can be classified into on-site technology, which is treated on-site as it is for convenience, and off-site technology to which technology is applied by moving pollution mediators off-site for convenience.

For example, landfarming is a technique that can be applied in situ on contaminated soil near the surface. To promote the growth of microorganisms, nutrients, including nitrogen and phosphorus, are added to control water, and lime is used to control pH. In order to smoothly supply oxygen, a method of periodically inverting is used.

In addition, micro-stimulation method (Biostimulation) that promotes the activity of these microorganisms while using microorganisms already existing in the contaminated soil is applied to the soil cultivation technology, and microbial input method of adding foreign microorganisms having excellent resolution to specific pollutants ( Bioaugmentation may be applied. Soil cultivation technology can be used as a post-treatment of soil treated by other methods and has the great advantage that the treatment cost is the lowest. However, there is a disadvantage that the processing time is long and the decomposition rate is slow.

In order for these bioremediation techniques to succeed, that is, for microorganisms to effectively decompose contaminants in the soil, the presence of nutrients necessary for the growth of microorganisms, the presence of electron acceptors such as oxygen, the delivery of pollutants as carbon and energy sources, Several conditions must be met, such as the activation of enzymes that can degrade contaminants. In order to solve this problem, fertilizers containing nitrogen and phosphorus which may be insufficient in the soil may be added, and physical oxygen supply such as soil inversion or oxygen-inducing substances such as hydrogen peroxide may be added, and surfactants may be used to promote the delivery of pollutants. It may also be added. Biochemical degradation of contaminants takes several steps, eventually transforming them into carbon dioxide, water, and microbial organisms. Each step requires the proper activation of enzymes. In particular, when contaminated with a high concentration of oil, the microbial living environment is very poor, so it can be said that it is essential to inject microorganisms from the outside and make an appropriate living environment.

Meanwhile, Patent Documents 1, 2, 3 and 4 describe microbial strains having oil resolution, and Patent Documents 5, 6 and 7 describe microbial preparations in which strains having oil resolution are mixed.

However, in the case of introducing the decomposing microorganism in the existing biological restoration technology, the oil degradation strain is usually added after simple cultivation, and there is no consideration of how to prepare the inoculation strain by appropriately culturing the mixed strains. Particularly, when a mixed strain is used, some strains that grow slowly during mass culturing are rarely present in the final inoculation strain due to nutrient competition. In the case of heavy oil, the microorganisms have very low availability, and thus, microorganisms with slow growth rate are often more actively decomposed by direct contact.

Therefore, the existing culture method is very inefficient when there is a slow growth rate of the mixed strain, but a high degradation efficiency strain. As a result, the introduced foreign microorganisms often disappear due to inadequate adaptation to the site soil environment, and even if the microorganism grows smoothly, the decomposition of the contaminated material is often very slow, so that soil pollution cannot be solved quickly. There was a problem.

Patent Document 1: Korean Registered Patent No. 421654

Patent Document 2: Korean Patent No. 421655

Patent Document 3: Korean Registered Patent No. 469480

Patent Document 4: Korean Registered Patent No.

Patent Document 5: Korean Patent No. 464107

Patent Document 6: Korean Patent No. 535936

Patent Document 7: Korea Patent Publication No. 2005-43506

Therefore, the present invention has been conducted to solve the above-mentioned problems, as a result of the separation of microorganisms having oil resolution from the oil-contaminated soil, these are separated and incubated in accordance with the growth rate, respectively, mixed to the oil-contaminated soil Treatment has been found that the rate of decomposition of pollutants can be accelerated and various pollutants can be effectively decomposed, thereby reducing the purification time of contaminated soil, and the present invention has been completed based on this.

Accordingly, it is an object of the present invention to provide a microbial liquid composition for oil-contaminated soil purification with enhanced decomposition activity of oil.

Another object of the present invention to provide a method for preparing a liquid composition for microbial soil contaminated soil.

It is another object of the present invention to provide a method for biological purification of oil contaminated soil which can quickly clean up oil contaminated soil.

The microbial liquid composition for oil-contaminated soil purification for achieving the object of the present invention comprises a culture medium of high growth strain reaching a stationary phase within 10 hours having oil resolution separated from the oil-contaminated soil; And a culture medium of a low growth strain having a slower growth rate than the high growth strain.

According to another aspect of the present invention, there is provided a method of preparing a microbial liquid phase composition for cleaning oil-contaminated soil, comprising: selecting and separating strains having oil-degrading ability from oil-contaminated soil; Separating the strain into a high growth strain, the strain reaching the stop phase within 10 hours according to the growth rate, and separating the strain reaching the stop phase after 10 hours into a low growth strain; Culturing the high growth strain and the low growth strain in a culture medium, respectively; And mixing the culture medium of the high growth strain and the low growth strain culture solution.

The biological purification method of the oil-contaminated soil to achieve another object of the present invention is the microbial liquid composition for oil-contaminated soil purification in the range of 1 to 200g / ㎏ relative to the oil-contaminated soil, the final microbial population is 10 ⁶ per 1g soil It consists of processing to be more than an object.

According to the present invention, if strains having oil resolution are separated and cultured according to the growth rate and then mixed and used, they can increase the decomposition rate of pollutants and effectively decompose various pollutants, thereby shortening the purification time of contaminated soil. Since it can be, there is an effect that can reduce the purification cost.

Hereinafter, the present invention will be described in more detail.

As described above, the present invention selects microbial strains having oil resolution from oil-contaminated soil, separates them according to growth rate, incubates them, and then mixes and applies them to oil-contaminated soil, thereby simplifying the microorganisms having conventional oil resolution. It is possible to purify the oil contaminated soil efficiently and quickly by preparing a microbial liquid composition without oil degradation due to the disappearance of the high oil degradation efficiency but slow growth rate that can occur when mixed and cultured.

According to the present invention, first, strains having oil resolution are selected and separated from oil-contaminated soil, and the strains are separated into high growth strains and low growth strains according to the growth rate. Thereafter, the high growth strain and the low growth strain are each cultured in a culture medium containing an enzyme expression promoter, so that the culture medium and the low growth strain culture solution of the high growth strain are preferably in a volume ratio of 1: 1 to 1: 9, more preferably. Mixing at a volume ratio of 1: 1 to provide a microbial liquid composition. Deviation from the mixing ratio tends to lower the decomposition efficiency.

According to the present invention, the criteria for judging high growth strains and low growth strains may be determined as high growth strains when entering a stationary phase before and about 10 hours after culture, and low growth strains thereafter. Both high growth strains and low growth strains can secure a population of 10 ^{9-10 10} / ml after completion of culture, and can be maintained at a similar level even after mixing them.

Strains with oil resolution can be selected, for example, from strains that grow on minimally nutrient media containing oil by collecting oil-contaminated soil, and through 16S rDNA sequencing or fatty acid analysis. I can sympathize.

According to one embodiment of the present invention, the soil contaminated with oil in the industrial area to select a population of about 10 kinds of microorganisms grown on a minimal culture medium containing heavy oil, these were 16S rDNA sequencing or fatty acids (fatty) acid) method. Consequently, they each Acinetobacter (Acinetobacter) sp., Acinetobacter Mori's Curse Tee (Acinetobacter haemolyticus ), Pseudomonas aeruginosa , Acinetobacter radioresistens ), Stenotrophomonas maltophilia ), and Bacillus spalicus sphaericus ), Nocardia nova ), Cellulomonas turbata ), and Flavobacterium johnsoniae .

Here, Acinetobacter (Acinetobacter) sp., Acinetobacter by Mori T carcass (Acinetobacter haemolyticus ), Pseudomonas aeruginosa , Acinetobacter radioresistens ), Stenotrophomonas maltophilia , and Bacillus sphaericus are high growth strains, Nocardia nova ), Cellulomonas turbata ), and Flavobacterium johnsoniae are low growth strains.

Thus, strains having oil resolution separated from oil-contaminated soil are separated into high growth strains and low growth strains according to the growth rate. In the case of low growth strains with relatively slow growth rates, when there are strains with higher oil degradation activity than the high growth strains, they may be rarely present in the final culture when they are mixed with the high growth strains. Because of its low availability, microorganisms with slow growth rate can be actively degraded by direct contact. Therefore, in the present invention, microorganisms having oil resolution are separated into high growth strains and low growth strains, and these are cultured separately, and then mixed and used.

In the present invention, a culture medium including an enzyme expression promoter is used to promote the expression of an enzyme having oil degradation activity in the culture of the high growth strain and the low growth strain. The enzyme expression promoter is, for example, benzoic acid and / or n-octane and the like. The amount of these used is 0.001 to 1 g / l based on the medium, preferably about 0.2 g / l. If the amount of these used exceeds 1g / l microbial growth can be inhibited, if less than 0.001g / l has little addition effect.

Meanwhile, the high growth strain solution and the low growth strain solution, which are separated and cultured, respectively, may be mixed in a volume ratio of 1: 1 to 1: 9 and applied to oil soils.

When the microbial agent of the present invention is treated to the soil, it is preferable from the viewpoint of treatment efficiency and economical efficiency to treat the final microbial population to be 10 ⁶ or more per gram of soil within the range of 1 to 200 g / kg relative to the treated soil. The use volume of the microbial strain solution should be prepared and added to the volume of water within the range where the soil moisture content is not saturated after the population condition is adjusted.

As such, when the microorganisms are separated and cultured separately according to the growth rate, the microorganisms are separated and mixed with each other according to the growth rate. Since the substance can be effectively decomposed, the purification time of the contaminated soil can be shortened, thereby reducing the purification cost.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Experimental Example 1

Isolation and Identification of Lipolytic Microorganisms

The soil sample used in the present invention was used to collect soil contaminated with oil in the industrial area. Soil samples were taken from the top 15 cm of the soil, filtered through a 2 mm diameter sieve, and stored at 4 ° C. until use. 10 g of soil samples were placed in 100 ml minimal nutrient medium containing 1 ml of Bunker C oil and incubated at 25 ° C. for 150 weeks at 150 rpm. 1 ml of the upper layer of the cultured sample was taken and inoculated into 10 ml of the minimal nutrient medium containing 0.1 ml of Bunker C and incubated for 1 week. The culture samples were diluted and plate cultured in nutrient agar medium (Nutrient Broth Agar, Difco), colonies showing different forms were formed.

The minimum nutritional medium composition is shown in Table 1 below.

ingredient content NH ₄ Cl 5.35g NaH ₂ PO ₄ 0.6g Na ₂ HPO ₄ 1.6 g EDTA 0.38 g KCl 0.75 g Na ₂ SO ₄ 0.28 g MgSO _{4 ·} 7H ₂ O 0.31 g Mineral solution 5 ml Secondary distilled water 1ℓ

As a result of the experiment, 10 heavy oil digested strains were isolated, and these were named SK49, SK60, O4, D4, D7, D9, C1, C2, C4, and C6, respectively. Each strain was analyzed by 16S rDNA sequencing or fatty acid analysis. As a result, SK49 and SK60 belong to Acinetobacter , and O4 to Acinetobacter. haemolyticus ), D4 is Pseudomonas aeruginosa , D7 is Nocardia nova nova ), D9 is Cellulomonas turbata ), C1 is Acinetobacter radioresistens ), C2 is Stenotrophomonas maltophilia ), C4 was identified as Bacillus sphaericus , and C6 was identified as Flavobacterium johnsoniae .

Experimental Example 2

Isolation by Growth Rate of Oil-Degrading Microorganisms

Ten kinds of oil-degrading microorganisms according to Experimental Example 1 were examined for growth rate in Nutrient Broth. 8 ml of sterile nutrient medium was added to a 15 ml test tube, and each strain was inoculated, and then the optical density (OD) was measured at 600 nm over time while incubating in a reciprocal shaker 150 times per minute at 25 ° C. Table 2 shows OD values after 9 hours and after 24 hours.

As a result, SK49, SK60, O4, D4, C1, C2 and 4C grew rapidly and reached stationary phase within 9 hours.

C4 had a low OD at maximum growth, but grew rapidly, reaching a quiescence in the early 9 hours. D7, D9 and C6 grew slowly and continued to grow from 9 hours to 24 hours. Based on the results of this experiment, SK49, SK60, O4, D4, C1, C2, and C4 were classified as high growth strain groups and D7, D9 and C6 were low growth strain groups.

division Strain name OD (9h) OD (24h) Classification One Acinetobacter sp. SK49 0.69 0.68 CF 2 Acinetobacter sp. SK60 0.54 0.58 CF 3 Acinetobacter haemolyticus O4 0.72 0.74 CF 4 Pseudomonas aeruginosa D4 0.76 0.80 CF 5 Nocardia nova D7 0.09 0.18 CS 6 Cellulomonas turbata D9 0.11 0.17 CS 7 Acinetobacter radioresistens C1 0.40 0.39 CF 8 Stenotrophomonas maltophilia C2 0.59 0.58 CF 9 Bacillus sphaericus C4 0.25 0.28 CF 10 Flavobacterium johnsoniae C6 0.27 0.43 CS

Experimental Example 3

Preparation of long-term storage strain

Strains purely separated in Experimental Example 1 were inoculated into 50 ml centrifuge tubes each containing 25 ml of nutrient medium and incubated at 25 ° C. and 150 rpm for 24 hours. 10 ml of sterile 50% glycerol was added to each tube, mixed, and then dispensed into 1 ml of microtubes and stored in a 70 ^° C. freezer.

Experimental Example 4

Extraction and analysis of bunker C oil

Add appropriate nucleic acid for extraction of bunker C oil from the medium, shake, mix, and centrifuge for 10 minutes in a sonicator, and repeat the procedure of extracting the nucleic acid layer twice. Put a small amount of Na ₂ SO ₄ in the collected nucleic acid layer. Shake well, mix, filter with 0.45㎛ filter, and use as sample solution. Analysis of bunker C oil was performed using a capillary column (HP-5, 30 m in total length, 0.25 mm in diameter; Hewlette Packard Co., USA) and gas chromatography (Agilent technologies 6890G, Agilent Co. , USA). The operating temperatures of the detector and the injector were 320 ° C and 300 ° C, respectively. The column (oven) temperature was fixed at 50 ° C. for the first two minutes and then increased to 320 ° C. at a rate of 8 ° C./min. The carrier gas was helium, and the gas flow rate was 40 ml / min.

Experimental Example 5

Degradation of Bunker C Oil from Pure Strains

Each bunker C oil degradation test was performed on 10 degradation strains isolated from Experimental Example 1.

In a 12 ml culture tube, a minimum nutritional medium of 5 ml and a Bunker C oil concentration of 5,000 ppm were inoculated at 4% (v / v) for each strain and incubated at 25 ° C. and 150 rpm for 2 weeks. Each strain was incubated in nutrient medium for 24 hours, centrifuged at 15000 rpm for 5 minutes, and then the supernatant was removed and a minimum nutrient medium 1ml was prepared. As shown in FIG. 2, the decomposition of bunker C was confirmed for all 10 species, in particular, O4, D4, and D7 showed a decomposition rate of more than 65%, and D7 showed the highest decomposition rate with 70% decomposition rate. The low growth strain, D7, 70%, D9 48%, O6 22% degradation was significantly higher degradation rate.

Example 1

Isolation of Mixed Strains

1. Pure culture

Ten kinds of frozen strains for long-term storage prepared in Experimental Example 3 were taken out and inoculated into 15 ml tubes containing 5 ml nutrient medium for each strain and incubated at 25 ° C. 150 rpm for 24 hours.

2. Cultivation of high and low growth strains

Among the 10 pure cultured strains, the high growth strains SK49, SK60, O4, D4, C1, C2, C4 were inoculated and mixed inoculated in a centrifuge tube containing 20 ml of the active culture solution so that 5% of the total culture was added. Incubation was carried out at 25 ° C. and 150 rpm for 12 hours. In addition, low-growth strains D7, D9, and C6 were inoculated in a centrifuge tube containing 20 ml of the active culture solution in an equal amount of 5% of the total culture solution and incubated at 25 ° C. and 150 rpm for 24 hours. On the other hand, the active culture medium composition of the high growth and low growth strain is shown in Table 3 below.

Activation medium composition ingredient content Minimum nutritional medium 1ℓ Nutrient broth 8.0g Glucose 10 g Benzoic acid 0.2 g n-octane 0.2 g

Example 2

Decomposition of Bunker C Oil of Mixed Strains

The high growth strain group (7 species, hereinafter, referred to as CFG) and the low growth strain group (three, hereinafter, referred to as CSG) cultured according to Example 1, and each of them were cultured in an active culture medium mixed with bunker C oil Decomposition was investigated in a liquid medium containing.

In a 12 ml culture tube, a minimum nutrient medium of 5 ml and a Bunker C oil concentration of 5,000 ppm were added, and the CFG and CSG were inoculated in the same manner so that each pure microbial strain was 2% (v / v) of the total culture. Incubated for 1 week at 25 ℃, 150rpm.

At this time, the culture medium of CFG and CSG was inoculated into the active culture solution to be 5% (v / v) of the total culture solution and incubated at 25 ℃ for 24 hours. The cultures were each centrifuged at 15000 rpm for 5 minutes and the supernatant was removed. Then, 1 ml of minimal nutrient medium was added and suspended. The CSG and CFG cultures were mixed 1: 1 and inoculated in a liquid medium containing bunker C oil. As a result, as shown in FIG. 2, the degree of decomposition was 81.3%, and it can be seen that the degree of decomposition was higher than that of the pure strain. On the other hand, Figure 3 is not administered with the microbial composition according to the present invention (Fig. 3a) and when treated with the highest degree of degradation D7 strain among the degradation strain (Fig. 3b), the microorganisms are mixed after separation culture according to the growth rate The graph which shows the gas chromatography of bunker C oil in the case of processing (FIG. 3C) is shown. As a result, it can be seen that the decomposition degree of the bunker C oil is high when the microorganisms are mixed after separation and culture according to the growth rate.

Example 3

CFG (O4, D4, SK49, SK60, C1, C2, C4) and CSG (C6, D7, D9) cultured according to Example 1 were examined for their ability to degrade soils.

A 120 ml serum bottle was inoculated with 100 g of soil contaminated with heavy oil, tween 80 (2 g / l), 10 ml of minimal nutrient medium, 100 µl of CFG and 100 µl of CSG. At this time, the number of microorganisms administered was inoculated by adjusting the amount to be 4 × 10 ⁶ level per 1g of soil at the level of 2 × 10 ⁹ / mL. The initial number of microorganisms in contaminated soil was 3.2 × 10 ⁵ / g, which was maintained at this level in the control group, but it increased about 10 times to 2.4 × 10 ⁷ / g after 7 days when the microorganisms were administered. The oil concentration of the initial contaminated soil was 8,050ppm and the treatment efficiency was 44% after 31 days after the administration of microbial agent. Figure 4 shows the gas chromatography of the bunker C oil when the initial sample of the soil contaminated with heavy oil (Fig. 4a) and the microbial agent was treated for 31 days (Fig. 4b), in the case of Fig. 4b, Figure 4a It can be seen that the bunker C is decomposed.

As described above, according to the present invention, if the strains having oil resolution are separated according to the growth rate and then cultured and mixed, the strains may be used to enhance the decomposition rate of pollutants and effectively decompose various pollutants.

1 is a graph showing the growth rate of the high growth strain group and low growth strain group in the active culture medium according to an embodiment of the present invention.

Figure 2 is a graph showing the decomposition degree of the bunker C decomposition and mixed strain after two weeks of each microbial strain for heavy oil decomposition according to an embodiment of the present invention.

Figure 3a, 3b and 3c is a sample (b), the microbial agent treated with a liquid sample (a) without the microbial agent administered in accordance with an embodiment of the present invention and D7 strain of the highest decomposition of the decomposition strain, the microbial agent according to the growth rate It is a graph which shows the gas chromatography of bunker C oil in the sample (c) mixed and processed after separation culture.

Figure 4a and b is a graph showing the gas chromatography of the bunker C oil of the sample (b) treated with the initial sample (a) and the microbial agent of the actual soil contaminated with heavy oil according to an embodiment of the present invention.

Claims (9)

Culture of high growth strain reaching a stationary phase within 10 hours with oil resolution isolated from oil contaminated soil; And a microbial liquid composition for purifying oil-contaminated soil, characterized in that a culture solution of a low growth strain having a slower growth rate than the high growth strain is mixed. According to claim 1, wherein the high growth strain culture medium and the low growth strain culture solution is a microbial liquid composition for oil-contaminated soil purification, characterized in that mixed in a volume ratio of 1: 1 to 1: 9. The method of claim 1, wherein the high growth strain Acinetobacter (Acinetobacter) sp., Acinetobacter by Mori T carcass (Acinetobacter haemolyticus ), Pseudomonas aeruginosa , Acinetobacter radioresistens ), Stenotrophomonas maltophilia ) and Bacillus sphaericus , and the low growth strain is Nocardia nova ), Cellulomonas turbata ) and Flavobacterium johnsoniae ( Flavobacterium johnsoniae) , characterized in that the microbial liquid composition for oil pollution soil purification. Selecting and separating strains having oil resolution from oil contaminated soil; Separating the strain into a high growth strain, the strain reaching the stop phase within 10 hours according to the growth rate, and separating the strain reaching the stop phase after 10 hours into a low growth strain; Culturing the high growth strain and the low growth strain in culture medium, respectively; And Method for producing a microbial liquid composition for oil-contaminated soil purification comprising the step of mixing the culture medium and the low growth strain culture medium of the high growth strain. The method of claim 4, wherein the culture medium further comprises 0.001 to 1 g of an enzyme expression promoter for 1 L of the medium. The method of claim 5, wherein the enzyme expression promoter is benzoic acid, benzoic acid, n-octane, or a mixture thereof. The method of claim 4, wherein the high growth strain Acinetobacter (Acinetobacter) sp., Acinetobacter by Mori T carcass (Acinetobacter haemolyticus ), Pseudomonas aeruginosa , Acinetobacter radioresistens ), Stenotrophomonas maltophilia ) and Bacillus sphaericus , and the low growth strain is Nocardia nova ), Cellulomonas turbata ) and Flavobacterium johnsoniae ( Flavobacterium johnsoniae) , characterized in that the method for producing a microbial liquid composition for oil-contaminated soil purification. The method of claim 4, wherein the high growth strain culture solution and the low growth strain culture solution are mixed in a volume ratio of 1: 1 to 1: 9. The microbial liquid composition for oil contaminated soil purification according to any one of claims 1 to 3 is treated so that the final microbial population is 10 ⁶ or more per 1 g of soil within the range of 1 to 200 g / kg with respect to the oil contaminated soil. A method of purifying oil contaminated soil, characterized in that.

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