KR101867957B1

KR101867957B1 - Microbial consortia for weathered crude oil of C25 or higher

Info

Publication number: KR101867957B1
Application number: KR1020170122242A
Authority: KR
Inventors: 고성환; 송영호; 정홍배; 이송이; 이아람; 심두섭
Original assignee: (주)에코필
Priority date: 2017-08-09
Filing date: 2017-09-22
Publication date: 2018-06-20
Also published as: CN109385376A

Abstract

This paper presents a new microbial agent with superior ability to decompose oil, especially decomposition of weathered oil of carbon (C) 25 or more. Wherein the microorganism preparation is selected from the group consisting of KSS-1 (Deposit No. KCTC 12877BP), KSS-2 (Deposit (registered trademark)), No. KCTC 12863BP), KSS-7 (accession numbers KCTC 12864BP), KSS-8 (accession numbers KCTC 12865BP), KOL-1 (accession numbers KCTC 12874BP), KOL-4 No. KCTC 12876BP).

Description

Technical Field [0001] The present invention relates to a microbial agent for purifying a weathered oil having a carbon (C) content of 25 or more (Microbial consortia for weathered crude oil of C25 or higher)

The present invention relates to a microbial agent for oil purification, and more particularly, to a microbial agent for purifying a weathered oil of carbon 25 or more of middle-eastern oil contaminated with oil.

Petroleum hydrocarbon compounds (hereinafter referred to as oil) are major pollutants that pollute the environment. Oil flows into the soil due to production processes, transportation and storage as well as outflows due to accidents. Biodegradation methods have been attracting attention by using microbial degradation activity and restoring soil contaminated with oil with less cost and environmentally friendly. BACKGROUND ART Bioregradation methods using microorganisms such as bacteria, fungi, yeast and microalgae capable of decomposing oil components have advantages in economical efficiency and efficiency and are widely used. Recently, microorganisms having excellent ability to degrade oil components in oil contaminated areas have been separated and used. Accordingly, attempts have been made to develop a microorganism preparation for purifying soil contaminated with oil by searching for a new microorganism strain having excellent oil decomposition ability.

One of the most important factors when biologically purifying soil contaminated with oil is the activity of oil-degrading microorganisms. However, more than 2,000 compounds have been identified to date. Some representative hydrocarbon compounds should be defined before selecting the oil-borne microorganisms. After that, the oil-degrading microorganisms capable of decomposing each specific hydrocarbon compound are separated and identified and mixed.

The released oil is immediately weathered by a number of biochemical and physical reactions. Specifically, it is subjected to biological and nonbiological weathering in soil and groundwater, and the weathering process works with strain rates associated with environmental factors including temperature, moisture, organic matter and oxygen content. In addition, the particle size distribution of soil is also an important parameter to control the weathering of oil pollutants in soil. Major non-biological reactions include hydrolysis, dehydrogenation, oxidation and polymerization, and these reactions are often closely related to the microbial deformation of the soil profile. Biotic weathering of oil pollutants depends on two interdependent mechanisms: microbial uptake and metabolic degradation. These processes occur sequentially, producing alcohols, phenols, aldehydes and carboxylic acids.

Conventionally, Journal of Basic Microbiology, vol. 284 ~ 291 (2002). However, in the purification of oil pollutants using conventional microorganisms, a relatively simple structure and a low molecular weight oil were to be decomposed. Accordingly, the conventional method has not been able to purify the weathered oil which is complicated in structure and stable. This weathered oil is the physical, chemical, and biological weathering of crude oil that has flowed out due to the bombing of the Gulf War, and it accounts for most of the oil pollutants in the Middle East.

The object of the present invention is to provide a novel microorganism strain capable of decomposing to a weathered oil having a carbon (C) content of 25 or more.

A microorganism preparation for purifying a weathered oil of carbon (C) 25 or more to solve the problem of the present invention is a microorganism preparation for purifying a weathered oil of carbon (C) 25 or more, KC-12863BP), KSS-7 (Deposit No. KCTC 12864BP), KSS-8 (Deposit No. KCTC 12865BP), KOL-1 (Accession No. KCTC 12874BP), KOL-4 (Accession No. KCTC 12875BP), and KOS-1 (Accession No. KCTC 12876BP).

The nutrient is a mixture of ammonium sulfate and potassium phosphate. The mixed strain has a hydrophobic property of 30 to 70% and an emulsifying property of 0.5 to 1.1. The mixed strain may have a hydrophobic property of 40 to 70% and an emulsifying property of 0.7 to 1.0. The KSS-1, the KSS-2, the KSS-7, the KSS-8, the KOL-1, the KOL-4 and the KOS-1 are repeatedly observed in terms of hydrophobicity and emulsification Class. The mixed strain can cross-resolve the oil and the second decomposition product.

According to the microorganism preparation for purifying the weathered oil of carbon (C) 25 or more according to the present invention, KSS-1 (Deposit No. KCTC 12877BP), KSS-2 (Deposit No. KCTC 12863BP), KSS-7 KCTC 12876BP), KOS-8 (Accession No. KCTC 12865BP), KOL-1 (Accession No. KCTC 12874BP), KOL-4 (Accession No. KCTC 12875BP), and KOS-1 (Accession No. KCTC 12876BP) It has excellent ability to decompose oil, and especially has ability to decompose to weathered oil.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing a classification of microorganisms according to the hydrocarbon uptake mode of the oil-degrading microorganisms according to the present invention. FIG.
FIG. 2 is a graph showing trends of changes in hydrophobicity and emulsifying ability of the oil-degrading microorganism according to the present invention over time. FIG.
FIGS. 3 to 5 are graphs showing changes in TPH, number of heterotrophic microorganisms, and number of oil-degrading microorganisms in the experimental group and the control group, respectively, according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. The embodiments described below can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. The embodiments of the present invention are provided to enable those skilled in the art to more fully understand the present invention.

In order to purify the C weathered oil over C25 according to the embodiment of the present invention, the selected microorganism preparations were KSS-1 (Deposit No. KCTC 12877BP), KSS-2 (Deposit No. KCTC 12863BP), KSS-7 (Deposit No. KCTC 12864BP) , KSS-8 (Accession No. KCTC 12865BP), KOL-1 (Accession No. KCTC 12874BP), KOL-4 (Accession No. KCTC 12875BP) and KOS-1 (Accession No. KCTC 12876BP). The seven oil-degrading microorganisms are optimized to decompose the C25-weathered oil.

The KSS-1 (Deposit No. KCTC 12877BP) is Yarrowia sp., Gram positive, aerobic, optimum growth temperature is 25-30 ° C, pH is 6 ~ 8. The KSS-2 (Deposit No. KCTC 12863BP) is Corynebacterium sp., Gram positive, aerobic, optimum growth temperature is 25-30 ° C, pH is 6 to 8. The KSS-7 (Accession No. KCTC 12864BP) is Microbacterium sp., Gram positive, aerobic, optimal growth temperature is 25-30 ° C, pH is 6 ~ 8. The KSS-8 (Deposit No. KCTC 12865BP) is Micrococcus sp., Gram positive, Aerobic, Optimum Growth Temperature is 25 ~ 30 ℃, pH is 8 ~ 9.

The above KOL-1 (Accession No. KCTC 12874BP) is Rhodococcus sp., Gram positive, aerobic, optimum growth temperature is 25 ~ 30 ℃, pH is 6 ~ 8. The KOL-4 (Accession No. KCTC 12875BP) is Bacilus sp., Gram positive, aerobic, optimal growth temperature is 25-30 ° C, pH is 6-8 to be. The KOS-1 (accession number KCTC 12876BP) is Rhodococcus sp., Gram positive, aerobic, optimum growth temperature is 25-30 ° C, pH is 6 ~ 8.

On the other hand, the process of weathering by oil can be evaluated by analyzing total petroleum hydrocarbon (TPH). In the case of crude oil, n-alkane such as pristine (2,6,10,14-tetramethylpentadecane), phytane (2,6,10,14-tetramethylhexadecane) and TRH of some isoprenoid alkanes Resolvable Hydrocarbons. However, the major part of the crude oil is not characterized by TPH analysis, which appears in the form of a hump on the chromatogram. This is called UCM (Unresolved Complex Mixture), which is judged to include branched and cyclic alkanes and polar transformation products. Thus, total petroleum hydrocarbon (TPH) is defined as the sum of Total Resolvable Hydrocarbons (TRH) and Unresolved Complex Mixture (UCM).

Oil-degrading microorganisms generally degrade branched alkanes and isoprenoid compounds at a much slower rate than straight-chain alkanes. Therefore, when there are many branched alkanes and isoprenoid compounds, the UCM hump increases and the TRH peaks decrease. On the other hand, hydrocarbons constituting the weathered crude oil mainly consist of isoprenoid alkanes and UCM. Biodegradation of oil contaminated soils is strongly influenced by the structure of the hydrocarbons and the weathering time. In other words, biodegradation is closely related to the degree of weathering, and as weathering becomes more severe, biodegradation by microorganisms becomes more difficult.

The weathering process is the result of biological, chemical and physical processes that can affect the types of hydrocarbons remaining in the soil. This process improves the adsorption of hydrophobic organic pollutants (HOCs) on the soil matrix, thereby reducing the rate of biodegradation and degradation. In general, only a portion of the HOCs dissolved in the liquid can be used for biological degradation, and the adsorbed HOCs have low biological resolution. Moreover, soils contaminated with weathered oils generally consist of high molecular weight hydrocarbons (above C25) compounds that can not be degraded by indigenous microorganisms.

Representative products In the case of imported diesel, gasoline, kerosene and aviation oils, the metabolites of high molecular weight hydrocarbons (C25 and higher) compounds are almost non-existent. However, analysis of the components of the residual oil in the weathered contaminated soils reveals a considerable amount of metabolic resin and asphaltene-bound asphalts. These asphalt is a polar material generated in the weathering process of oil, and ketone series, carboxylic acid series, and alcohol series are observed in the oxidation process of oil. The polar material is similar to the metabolites produced as a result of biological degradation of the oil, and when the metabolites accumulate in the soil, it disturbs the oil resolution of the oil-degrading microorganisms.

Thus, the metabolic products must be removed in order to promote the biological degradation of the oil-borne microorganisms. That is, in order to biologically purify the weathered oil contaminated soil, various kinds of oil-degrading microorganisms are required, and it is necessary to mix the microorganisms so that they complement each other well.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing a classification of microorganisms according to a hydrocarbon uptake mode in which oil-degrading microorganisms according to an embodiment of the present invention ingest oil. FIG. Here, FIG. 1 refers to Korean Biotechnology Association, 13 (5), pp 606-614, and shows the relationship between hydrophobicity and emusifying activity. At this time, the hydrophobicity and emulsifying ability were based on the definition described in "Growth Characteristics of Selective Oil-Borne Microorganisms" of Korean Biotechnology Association, 13 (5), p607.

1, the microorganism group is divided into four classes. First, it is a class (a) having an intake mechanism by the interaction of microbial cells with hydrocarbons dissolved in a liquid. On the other hand, most hydrocarbons are not soluble in water, but naphthalene, bibenzyl, and phenanthrene molecules have a certain solubility in the liquid phase. The first class of oil-degrading microorganisms is slower in growth rate than the other classes. Second, a class (b) with an intake mechanism by direct contact of hydrocarbon droplets with microbial cells. At this time, the microorganisms directly contact the hydrocarbon droplet surface, which is much bigger than the cells, and ingest the oil. The second class of oil-degrading microorganisms coagulate and grow on each other during the period of oil distillation because the cell surface has little hydrophobicity and emulsification ability.

Third, it is a class (c) that has an intake mechanism by the interaction of microbial cells with hydrocarbons that are solubilized by biosurfactants. The oil-degrading microorganisms belonging to the third category secrete the biosurfactant to make the oil smaller than itself, and take pseudo-solubilized oil. The cell surface has low hydrophobicity and high emulsification ability. Fourth, it is a class with all three of these ingestion mechanisms (Ko, SH, and JMLebeault 1997. Effect of a mixed culture on co-oxidation during the degradation of saturated hydrocarbon mixture J. Appl. Microbiol., 86: 1008-1016, d). The hydrophobic and emulsifying ability of the oil-degrading microorganism belonging to the fourth class continuously changes with the lapse of the incubation time. That is, the characteristics of the first to third classes of the oil-degrading microorganisms are periodically repeated. FIG. 2 shows the trend of the hydrophobicity and emulsifying ability of the microorganism belonging to the fourth class over time.

The microbial strains according to the examples of the present invention were selected from the strains belonging to the fourth class (d) of the class shown in Fig. 1 with reference to the hydrophobic and emulsifying ability values shown in Table 2 below.

Specifically, the first to third classes of oil-degrading microorganisms are excellent in oil resolution at each ingestion mechanism. By the way, in order to induce higher oil resolution, mixing of the respective groups may in some cases impair oil resolution. That is, when the groups having high emulsifying ability and the group having high hydrophobicity are mixed, the inhibitory effect is caused by the difference of the intake mechanism. On the other hand, the microorganisms of the fourth class (d), which take a mixed intake mechanism, are well matched to any kind of group, thus increasing the oil resolution. In addition, they complement each other well with various kinds of indigenous microorganisms. Accordingly, the microbial formulation according to the embodiment of the present invention has the characteristics of the fourth class (d).

The embodiments of the present invention have investigated microorganisms in various regions in order to separate the weathered oil of Kuwait into microorganisms using carbon and energy sources for biological restoration of oil polluted areas. The isolates were selected by investigating the hydrolysis of hydrocarbons and Kuwait weathered oils with crude oil. In addition, a microcosm experiment was carried out to select the Kuwait weathered oil degrading strains to be added to the oil degrading microorganism mixture to measure the oil resolution. Here, the Kuwait weathered oil refers to the oil remaining after evaporating the hexane after extracting the Weathered Kuwait Crude Oil (WKCO) from the Kuwait weathered oil-contaminated soil WKCO.

In order to explain the characteristics of the microbial formulation of the present invention in detail, the following examples are presented. However, the present invention is not particularly limited to the following examples. For this purpose, a process for extracting a microorganism preparation for purifying a weathered oil of carbon (C) 25 or more and a process of analyzing characteristics of a microorganism preparation for purifying a weathered oil of carbon (C) 25 or higher will be described .

&Lt; Extraction of microorganism preparation for purifying carbonized (C) weathered oil of 25 or more >

In order to isolate microbial strains that can grow by using Kuwait weathered oil as a carbon source, samples of oil spill area, oil lake and oil sludge were collected from the area where the contamination of the soil is expected to be high. The collected samples were collected, mixed well, then refrigerated, transferred to the laboratory, and cultured. On the other hand, if only a high concentration of weathered oil from Kuwait grows to a substrate, microorganisms that can not use Kuwait weathered oil as a carbon source are gradually culled and microorganisms that can use Kuwait weathered oil as a substrate will survive.

Samples collected under these conditions were shaken at 30 ° C and 180 rpm in a mineral medium supplemented with Kuwait weathered oil, and 1 mL of the culture medium was inoculated into the new medium three times at intervals of one week. The culture was serially diluted in sterile distilled water, plated on a mineral agar medium, and cultured at 30 ° C for 5 days. The microorganism colonies formed on the medium were classified into morphological characteristics, and they were subcultured by pure separation until they were judged to be a single strain. The morphological characteristics of the grown colonies and optical microscopic observation were observed. The isolated strains were suspended in 20% glycerol solution and stored at -70 ° C.

Separated Kuwait weathered oil degrading microorganisms were inoculated into 100 ml of mineral medium supplemented with 1% of Kuwait weathered oil and cultured at 30 ° C and 180 rpm for 1 week with shaking. The resolution was determined by hexane extraction of the whole flask, And analyzed by TPH weighing method according to EPA Method 9071B. In the above method, strains of high weathering degree and resolution of Kuwait weathered oil were selected. The sample was pretreated with concentrated hydrochloric acid and then chemically dried using magnesium sulfate or sodium sulfate. Magnesium sulfate is used to dry the sludge, and sodium sulfate is used to dry the soil and sediments. The dried samples were extracted with EPA METHOD 9071B (n-hexane extract (HEM) for sludge, sediment and solid samples) with n-hexane using a Soxhlet apparatus. The dry weight of the HEM was measured. If necessary, the fraction and concentration of HEM relative to the dry weight of the soil, sediment or waste were calculated.

The colony morphology and growth characteristics of the colonies on the medium showed that 12 strains, 10 strains from oil lake and 5 strains from oil sludge were collected from soil samples collected from oil spill accident area through optical microscope observation , And a total of 27 species of candidate strains for weathering oil degradation in Kuwait were selected as shown in Table 1. All of the 27 strains had a circular form.

Table 1 shows the colonization pattern of Kuwait weathered oil degrading bacteria selected on the mineral agar medium.

number Strain name
Colony Properties Bracket
surface transparency elevation One KSS-1 Rough Opaque Convex d 2 KSS-2 Matte Opaque Convex d 3 KSS-3 Smooth Translucent Convex a 4 KSS-4 Smooth Opaque Umbonate a 5 KSS-5 Rough Opaque Umbonate c 6 KSS-6 Smooth Opaque Convex b 7 KSS-7 Smooth Opaque Convex d 8 KSS-8 Rough Opaque Umbonate d 9 KSS-9 Smooth Translucent Convex b 10 KSS-10 Smooth Opaque Convex b 11 KSS-11 Smooth Opaque Convex b 12 KSS-12 Smooth Opaque Convex b 13 KOL-1 Smooth Opaque Convex d 14 KOL-2 Smooth Transparent Pulvinate a 15 KOL-3 Smooth Opaque Convex a 16 KOL-4 Smooth Opaque Convex d 17 KOL-5 Rough Opaque Convex c 18 KOL-6 Smooth Opaque Umbonate a 19 KOL-7 Smooth Opaque Pulvinate b 20 KOL-8 Smooth Translucent Umbonate b 21 KOL-9 Smooth Opaque Convex a 22 KOL-10 Smooth Opaque Convex c 23 KOS-1 Smooth Opaque Convex d 24 KOS-2 Smooth Opaque Convex b 25 KOS-3 Smooth Opaque Convex b 26 KOS-4 Smooth Opaque Convex a 27 KOS-5 Smooth Opaque Convex a

As shown in Table 1, seven strains of the strains exhibiting a high resolution of 50% or more in combination with oil ingestion mechanism were cultured in medium supplemented with 5,000 ppm of Kuwait weathered oil, 2. These seven strains belong to the fourth class (d) in Fig. 1 with reference to the hydrophobic and emulsifying ability values as shown in Table 2 below.

Table 2 shows the decomposition (%) of the Kuwait weathered oil (5,000 ppm) compound added to the mineral medium by seven strains of weathered oil degradation in Kuwait.

number Strain name Resolution (%) Hydrophobicity (%) Emulsifying ability
(OD at 610 nm) One KSS-1 67.3 ± 3.7 47 1.0 2 KSS-2 51.8 ± 2.8 50 0.7 3 KSS-7 71.1 ± 3.3 47 1.1 4 KSS-8 64.7 ± 4.1 60 1.0 5 KOL-1 77.7 ± 3.4 42 1.0 6 KOL-4 66.7 ± 3.3 42 0.7 7 KOS-1 52.1 ± 1.9 40 0.8

Table 2 shows that KOL-1 showed the highest resolution of metabolites, 77.7 ± 3.4%, KSS-7, 71.1 ± 3.3%, and 50% -88% of all other strains . Seven strains showed 40 ~ 60% hydrophobicity and 0.7 ~ 1.0 emulsifying ability. Substantially, the strain of the fourth class (d) is 30-70% hydrophobic and 0.5-1.1 emulsifiable (OD at 610 nm).

&Lt; Characteristic Analysis of Microorganism Preparations for Purifying Carbon (C) 25 Weathered Oil >

Seven Kuwait weathering degradation strains (KSS-1, KSS-2, KSS-7, KSS-8, KOL-1, KOL-4 and KOS-1) The possibility of biodegradation was investigated by making a small experiment with a stainless steel container in the laboratory. The efficacy of the oil degradation test was divided into the natural abatability test, the indigenous microorganism activity test method and the external distillation microorganism test method. The physico-chemical and biological characteristics of the Kuwait-contaminated soils used in this study are shown in Table 3.

Analysis item Analysis Moisture content 2.5% to 3.4% pH 6.45 ~ 7.04 Water Capability (WHC) 28.6%
Soil type

sand 85.9% mass 6.7% clay 3.4% Soil type Good quality Sato TPH (gravimetric method, EPA METHOD 9071B) 21,899 mg / kg Total heterotrophic microorganisms (x10 ⁶ CFU / g-soil) 0.39 Number of oilseed microorganisms (x10 ⁶ MPN / g-soil) 0.41

According to Table 3, the optimal pH for microbial growth and activity was about 7, and the acceptable range for biological pH was 6 to 8 for soil pH. Soil pH ranged from 6.45 to 7.04 and the water holding capacity was 28.6%. Soils were classified into sand, silt, and clay depending on their size. The contents of sand, silt and clay were 85.9%, 6.7% and 7.4%, respectively. According to TPH analysis (EPA METHOD 9071B), the TPH concentration of the soil used in the examples of the present invention was 21,899 mg / kg. The total number of heterotrophic microorganisms was 3.88 × 10 ⁶ (CFU / g, soil dry weight), and the number of oilseed microorganisms was 4.1 × 10 ⁵ (MPN / g, soil dry weight).

The first step in performing a characterization experiment is to establish laboratory scale control and experimental groups to determine the potential for biodegradation of contaminated soil. The conditions for the experiment are as follows. A stainless steel container (7.87 inches x 7.87 inches x 7.87 inches) was used as the experimental sphere. 6 kg of Kuwait contaminated soils were divided into three experimental groups. The control conditions of the three experimental groups were control (no control), experimental group Ⅰ (Microcosm I, biostimulation), nutrient and WKCO microorganism (Microcosm II, bioaugmentation) in which KSS-1, KSS-2, KSS-7, KSS-8, KOL-1, KOL-4 and KOS-1 were injected. The ratio of C: N: P for microbial metabolism was set at 100: 10: 1, and ammonium sulfate and potassium phosphate were used for nutrient input. WKCO degrading microorganism mixture was mixed with 7 strains and cultured up to 1.0 × 10 ⁶ cells / g soil level.

At this time, Biostimulation is a biological stimulation method, and Bioaugmentation is a biological stimulation method. The biological stimulation method (experimental group I) is a purification method that stimulates the activity of the naturally occurring native oil-degrading microorganisms in the soil by regulating nutrient supply, moisture, temperature, and oxygen conditions in oil contaminated soil. Therefore, biostimulation may not be effective in the absence of an effective indigenous oil-degrading microorganism in the soil. Biological Promotion Method (Experiment Group Ⅱ) is a purification method that supplies both nutrients and oil-degrading microorganisms to oil-contaminated soil and promotes (maximizes) the effect of microorganisms on the oil by controlling moisture, temperature and oxygen conditions.

Experimental group II was the only experimental group to which the microorganism was injected in the whole experimental group, and the microorganism used in this experiment was a mixture of the above seven strains. Since the control group is intended to compare the experimental results and the degradation effects by the actual microorganisms, only moisture, temperature and oxygen conditions are the same, and no nutrients and oil-degrading microorganisms are supplied at all. In addition, experimental group I was supplied only with nutrients to determine the degradation effect of native oil - degrading microorganisms, that is, the possibility of bio - stimulation.

On the other hand, the lower the microbial diversity of the mixed microorganism preparation, the less the effect of oil degradation can be. This is because, in the process of biodegradation of the oil, a secondary degradation product may be generated, and the secondary degradation product may inhibit the decomposition activity of the whole oil-degrading microorganism. At this time, the higher the diversity of the microorganisms belonging to the fourth class (d), the more different microbial strains have different decomposition cycles, and the oil pollutants and the second degradation products are cross-decomposed. The mixed strain of seven strains according to the embodiment of the present invention considers the decomposition of the secondary degradation product.

The experiment was carried out in an incubator at 30 ° C. I mixed well once a day (tilling) and adjusted the moisture content once every three days. In addition, TPH analysis by EPA METHOD 9071B was carried out once a week by collecting the soil of each experiment. The analysis of total petroleum hydrocarbons (TPH) is the same as EPA METHOD 9071B. For microbial activity analysis, the total number of heterotrophic microorganisms is analyzed by the CFU method, and the number of microorganisms for oil degradation is analyzed by the MPN method.

Put 1 g of soil sample in a test tube with 9 mL of sterile physiological saline. After mixing evenly, 0.1 mL of the previous step dilution is added to 0.9 mL of sterile physiological saline and the solution is sequentially diluted to 10 ^-9 . The total number of heterotrophic microorganisms is prepared by preparing three nutrients agar plates for each dilution. Then, 0.1 mL of the diluted solution is taken on a nutrients agar plate and stained. And cultured at 30 ° C for 3 days. Three days after the culture, the microorganism colonies are identified and the number of colonies of each agar plate is counted to calculate the average number of microorganisms.

To determine the number of oil-degrading microorganisms, first prepare a sterile Bushnell Hass (BH) broth to fill the wells required for the assay. Then, on the top of a 24-well disposable cell culture plate, the number for identification of the sample is indicated. Add 1.75 mL of BH broth to each well in a sterile environment. Add 0.1 mL in each well, starting with the most diluted sample in each well. Samples are added to all wells and 20 uL of filter sterilized Hexadecane is added to each well. The culture plate is incubated at 30 DEG C for 14 days. After 14 days of cultivation, 100 μL of p-iodotetrazolium purple dye (deionized water 50 mg / 10 mL) is added to each well to determine microbial growth. Positivity is purple or pink when it stays for 45 minutes. Observe the culture plate on a white background. Record the number of positive wells and the dilution factor. Enter data using the MPN Calculator software program by Thomas's formula.

According to the examples of the present invention, TPH degradation rates of 4.1%, 22.3% and 77.3% after 125 days in the control, experimental group I and experimental group II, respectively. Based on these results, it was confirmed that TPH degradation rate was enhanced by addition of nutrient and WKCO degrading microorganism mixture as shown in Table 4.

division Control group Experiment group I
(biostimulation) Experimental group II
(bioaugmentation) Oil degradation rate 4.1% 22.3% 77.3%

Table 4 shows that the average initial TPH of 21,899 ppm was lowered to 4,976 ppm within 125 days after the administration of the microorganism, which was about 135 ppm per day, which is a very high removal rate.

FIGS. 3 to 5 are graphs showing changes in TPH, number of heterotrophic microorganisms (HB) and number of oil-degrading microorganisms (PDB) in experimental group and control group, respectively, according to an embodiment of the present invention.

3 to 5, the total number of heterotrophic microorganisms was much higher in the experimental group I than in the control group. This shows that it is very effective to form conditions to improve microbial growth in oil polluted environments. These results were confirmed in Experiment Group II with addition of WKCO degrading microorganism. The total number of heterotrophic microbes and the number of microbial microbes were higher in experimental group Ⅱ, in which nutrient and WKCO degrading microbial mixture were added during the whole experiment period, than experimental group Ⅰ.

In the examples of the present invention, the average TPH of the collected samples was about 21,899 ppm. After 125 days, the TPH degradation rate was 4.1%, 22.3%, and 77.3% in the control group, the test group I and the test group II, respectively. As a result, the bio-stimulation method in which only the nutrient of Experiment Group I is injected requires more than twice the period and cost as compared with the bio-stimulation method in which the nutrient of the experimental group II and the oil-degrading microorganism are injected together. Therefore, in order to carry out an economic biological purification, it is effective to add nutrients and WKCO microorganisms in a mixed state.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but many variations and modifications may be made without departing from the spirit and scope of the invention. It is possible.

Claims

A microorganism preparation for purifying a weathered oil having a carbon (C) content of 25 or more, which is put into contaminated soil together with nutrients,
KC-12863BP), KSS-7 (accession number KCTC 12864BP), KSS-8 (accession number KCTC 12865BP), KOL-1 (accession number: KCTC 12874BP), KOL-4 (Accession No. KCTC 12875BP), and KOS-1 (Accession No. KCTC 12876BP).

The microbial formulation according to claim 1, wherein the nutrient is a mixture of ammonium sulfate and potassium phosphate.

The microorganism preparation according to claim 1, wherein the mixed microorganism has a hydrophobic property of 30 to 70% and an emulsifying property of 0.5 to 1.1.

2. The microorganism preparation according to claim 1, wherein the mixed strain has a hydrophobic property of 40 to 70% and an emulsifying property of 0.7 to 1.0.

4. The method according to claim 3, wherein the KSS-1, the KSS-2, the KSS-7, the KSS-8, the KOL-1, the KOL-4 and the KOS- Wherein the emulsifying agent is a group that cyclically repeats the emulsifying activity. The microbial agent for purifying a weathered oil of carbon (C) 25 or more.

The microorganism preparation according to claim 1, wherein the mixed strain cross-resolves the oil and the second decomposition product.