KR100817256B1 - Microorganisms for degrading crude oil components and their metabolites, and use and separation thereof - Google Patents

Abstract

A novel strain degrading oil ingredients and a metabolite thereof and a microbial agent comprising the same are provided to restore oil contaminated soil more rapidly, stably and environment-friendly when used with a conventional microbial agent. A Micrococcus sp. SBS-8 isolated from oil contaminated soil and degrading oil ingredients and resin, a metabolite thereof, is deposited as a deposition no. KCTC 11071BP. A microbial agent for degrading the oil ingredients and resin comprises at least one strain selected from the Micrococcus sp. SBS-8(deposition no. KCTC 11071BP) as an effective ingredient. A method for purifying the oil contaminated soil comprises a step of applying the microbial agent to the oil contaminated soil. A method for selecting the strain comprises the steps of: (a) shake-culturing the oil contaminated soil in a resin-added mineral culture medium at a temperature of 25-35 deg.C; (b) smearing a mineral agar culture medium with the cultured bacteria to isolate resin degrading strains; and (c) selecting the Micrococcus sp. SBS-8 strain based on the emulsification degree and degrading rate regarding the resin.

Description

Microorganisms for Degrading Oil Components and Their Metabolites, and Uses and Separation Methods thereof {Microorganisms for degrading crude oil components and their metabolites, and use and separation}

1 shows a TLC / FID analysis chromatogram of oil components.

2 shows the metabolic pathways of aliphatic hydrocarbons by microorganisms.

3 shows the major pathways of microbial metabolism of polycyclic aromatic hydrocarbons.

4 shows the gas oil component analysis by TLC / FID method.

5 shows the analysis of residual oil components in diesel contaminated soil.

6 shows the carbon flux due to the biodegradation of oil.

Figure 7 shows the classification according to the hydrocarbon intake mechanism of the oil decomposition microorganisms.

FIG. 8 illustrates the process of ingestion of the oily microorganisms in which the oily microorganisms secrete biosurfactants to make the oil smaller than itself and consume the solubilized oil.

FIG. 9 illustrates the growth process of lyolytic microorganisms that attach directly to an oil surface to ingest oil.

10 shows residual oil analysis by TLC / FID.

FIG. 11 shows the percent degradation of metabolites (resin 200 ppm) added to the mineral medium by selected metabolite compound degrading bacteria.

FIG. 12 shows the degradation rate of aliphatic mixed compounds (SHM, 2,000 ppm) added to the mineral medium by selected metabolite compound degrading bacteria.

FIG. 13 shows the degradation rate of the polycyclic aromatic hydrocarbon mixed compound (AHM, 2,000 ppm) added to the mineral medium by selected metabolite compound degrading bacteria.

FIG. 14 shows the degradation rate of metabolite (resin 200 ppm) added to mineral medium by selected metabolite compound degradation bacteria.

15 shows the degradation rate of dibenzothiophene (DBT, 200 ppm) added to the mineral medium by selected metabolite compound degraded bacteria.

FIG. 16 shows the degradation rate of Arabian light crude oil (2,000 ppm) added to the mineral medium by selected metabolite compound degrading bacteria.

Figure 17 shows the contaminated soil morphology collected for Example 2 of the present invention.

18 shows contaminant component analysis of collected samples by TLC / FID method.

19 shows a biodegradation feasibility study experimental model (Microcosm Test).

20 shows residual oil analysis by PetroFLAG.

FIG. 21 shows the total bacterial count (dilution smear) and oil degradation bacteria count (optimal spread method) tests present in soil samples.

Figure 22 shows the amount of TPH change by bioremediation.

Figure 23 shows the change in total bacterial counts by biopurification.

24 shows the change in the number of lyolytic bacteria by biopurification.

FIG. 25 shows oil component changes (GC / FID) during the biodegradation period of contaminated samples.

One of the most important factors when biologically purifying oil contaminated soil is the activity of the oil-degrading microorganisms. However, there are more than 2,000 kinds of oil compounds known to date, but only a part of them. Therefore, a method commonly used when selecting oil-degrading microorganisms has been to take a method of selecting a representative material from the oil components and separating and mixing microorganisms that can decompose it.

The classification used to classify the representative components of oil can be classified into four types: aliphatic hydrocarbon compound (Aliphatic Fraction Compound), aromatic hydrocarbon compound (Aromatic Fraction Compound), resin (Resin) and asphaltene (Asphaltene). ). Representative components of these groups are taken to select degraded microorganisms. This is because the microbial paths of each group are different and one microorganism does not have a system capable of simultaneously decomposing these groups.

For example, there was the case of aliphatic hydrocarbons comprises 30 ~ 50% of oil components, mainly normal alkane (n -alkane), isoalkanes (branched-chain alkane), alkene (alkene), cyclo alkanes (cycloalkane) such as Can be classified as (Table 1). These aliphatic hydrocarbons are decomposed in nature by various microorganisms, and Table 2 shows microorganisms that can use aliphatic hydrocarbons. These microorganisms are somewhat different in the dominant community distribution according to the contaminated soil or the type of hydrocarbon, and are known to occupy about 20% of the total microbial community. In addition, the more soil contaminated with oil compounds are known to be high that the distribution ratio (Britton, LN 1984. Microbial degradation of aliphatic hydrocarbons. P 89-129. In DT Gibson (ed.). Microbial degradation of organic compounds . Marcel Dekker Inc. New York, NY).

In general, the biodegradation of aliphatic hydrocarbons is initiated by the oxidation of the terminal methyl group to an alcohol group in the presence of an enzyme called molecular oxygen monomonogenase (monooxygenase). The oxidized alcohol group is converted back to a carboxyl group via an aldehyde group to form a fatty acid. The fatty acids thus formed are metabolized by beta oxidation as a common energy source in the metabolism of organisms. The resulting compound of C ₂ units (acetyl-CoA) is finally broken down into carbon dioxide and water via the TCA circuit (FIG. 2). Biodegradation of aliphatic hydrocarbons includes alpha oxidation (α-oxidation) and omega oxidation (ω-oxidation) in addition to beta oxidation. Biodegradation of unsaturated aliphatic hydrocarbons is accomplished by dihydroxylation of unsaturated double bonds, epoxidation of unsaturated bonds, oxidation of saturated methyl groups at the end, and the like.

Oxidation systems of aliphatic hydrocarbons exhibit various substrate specificities depending on the species of the bacteria. For example, Pseudomonas putida ) PbG6 (Oct) is known to have high growth rate in hydrocarbons having 6 to 10 carbon atoms and Acinetobacter sp. HO1-N in longer chain hydrocarbons (Nieder, M. and Shapiro J.). 1975. Physiological function of Pseudomonas putida PpG6 ( Pseudomonas oleovarans) alkane hydroxylase:... monoterminal oxidation of alkanes and fatty acids J. Bacteriol 122, 93-98; Singer, ME and Finnerty WR 1984. Genetics of hydrocarbon-utilizing microorganisms In Petroleum Microbiology , ed.RM Atlas, pp. 299-354.New York: Macmillan).

Table 1. Microbial Degradation Paths and Typical Microbial Examples According to Oil Components

division Structure example Representative resolution path Core enzymes Degrading microorganisms Aliphatic Components (Alkanes) n-

β-oxidation Monooxygenase Pseudomonas (Pseudomonas) no carboxylic Dia (Nocardia) Acinetobacter (Acinetobacter) Corynebacterium Rhodococcus Candida (Candida), Yarrow subtotal (Yarrowia) Iso

α, ω-oxidation Cyclo-

Hydroxylation Aromatic components Mono

Hydroxylation Deoxygenase Pseudomonas (Pseudomonas) no carboxylic Dia (Nocardia) Pseudomonas aero (Aeromonas) Alcaligenes Micrococcus Mycobacterium Sphingomonas D-

Hydroxylation PAH

Hydroxylation

Table 2. Microorganisms Metabolizing Aliphatic Hydrocarbons

Germ leaven Achromobacter (Achromobacter) Acinetobacter (Acinetobacter) evil Martino My process (Actinomyces) Aero Pseudomonas (Aeromonas) alkali Regensburg (Alcaligens) Bacillus (Bacillus) benek Kea (Beneckea) Brevibacterium (Brevibacterium) Corynebacterium ( Corynebacterium) Flavobacterium (Flavobacterium) methyl bakteo (Methylobacter) methyl tumefaciens (Methylobacterium) methyl Rhodococcus (Methylococcus) as a city seutiseu (Methylocystis) methyl Pseudomonas (Methylomonas) methyl Sinners (Methylosinus) micro mono Spokane La (Micromonospora) Mycobacterium (Mycobacterium) no carboxylic Dia (Nocardia) Pseudomonas (Pseudomonas) Vibrio (Vibrio) Candida (Candida) Cryptococcus Rhodococcus (Cryptococcus) debari Oh, my process (Debaryomyces) endo My process (Endomyces) Hanse Cronulla (Hansenula) My coat Lula (Mycotorula) blood teeth (Pichia) also torulra (Rhodotorula) saccharide in my process (Saccaromyces) Selenium Nautili Sporidiobolus Sporobolomyces Torulopsis Trichosporon

Aromatic hydrocarbons enter the natural environment through various natural or artificial pathways, and are gradually extinguished in nature by various methods. In the first place, a small amount of decomposition is caused by photooxidation or autooxidation. Get up. However, most of them are degraded by various microorganisms that live in polluted environments. Generally, small molecular weight materials such as naphthalene, anthracene, and phenanthrene are reported to be able to decompose many kinds of microorganisms, but benzene rings are linked to four or more. microorganism indicating the resolution in the high-molecular substance is not known (Mueller, JG, CE Cerniglia, and PH Prichard 1996. Bioremediation of environments contaminated by polycyclic aromatic hydrocarbons in Crawford, RL and DL Crawford (ed) Bioremediation....: Principles and applications.Cambridge Univ.Press.p 125-194). Table 3 shows the different types of microorganisms that degrade aromatic hydrocarbons (Cerniglia, CE 1992. Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation . 3, 351-368).

Decomposition mechanism of the aromatic hydrocarbon shows a difference depending on the type of microorganism as shown in FIG. In the case of bacteria and algae, cis -dihydrodiol is produced from an aromatic ring using two oxygen atoms by deoxygenase, and then catechol is generated by dehydrogenase. To form. Since phenanthrene, anthracene, pyrene, and benzozopyrene (banzo [a] pyrene) are composed of multiple benzene rings, bacteria are metabolized at various positions to produce cis-dihydrodiol. Will be The catechol produced through this process forms a metabolite, an aliphatic hydrocarbon that is finally available to different microorganisms through ring-cleavage. On the other hand, the benzene ring is ortho decomposition - and the number of the division (meta -cleavage) two, ortho-cleavage (ortho -cleavage) and metadata via the multiple paths by the division finally acetaldehyde and pyruvic acid is formed , meta- by division by being finally succinate and acetyl -CoA is to be formed becomes metabolism is achieved (Mueller, JG, CE Cerniglia, Prichard and PH 1996. Bioremediation of environments contaminated by polycyclic aromatic hydrocarbons in Crawford, RL.. and DL Crawford. (ed.) Bioremediation: Principles and applications.Cambridge Univ.Press.p 125-194).

Fungi and cyanobacteria use mechanisms different from those of bacteria. In most molds, oxidation is initiated using cytochrome P-450 monooxygenase, that is, only one oxygen atom, and the reaction mainly forms arene oxide. In addition, most arene oxides are unstable and thus form trans -dihydrodiol or non-enzymatic rearrangements by enzymatic hydration or epoxide hydrolase. The enzymatic rearrangement produces phenols that can bind to glucose, sulfates, xylose, and glucuronic acid. However, few fungi can decompose aromatic hydrocarbons into carbon dioxide, most of which are bioconverted or cometabolized with weakly toxic products (Mueller, JG, CE Cerniglia, and PH Prichard. 1996. Bioremediation . of environments contaminated by polycyclic aromatic hydrocarbons In Crawford, RL and DL Crawford (ed.) Bioremediation:.... Principles and applications Cambridge Univ Press p 125-194).

Table 3. Microorganisms Metabolizing Aromatic Hydrocarbons

organism organism Bacterial Achrobacter Acinetobacter Aero Monastir (Aeromonas) Alcaligenes (Alcaligenes) are Tropez bakteo (Arthrobacter) Bacillus (Bacillus) The Bay rinkeu Kea (Beijerinckia) Corynebacterium (Corynebacterium) cyanobacteria (Cyanobacteia) Flavobacterium (Flavobacterium) morak Cellar (Moraxella) mycobacteria (Mycobacteria) no carboxylic Dia (Nocardia) also Pseudomonas (Pseudomonas) Rhodococcus (Rhodococcus) Streptomyces (Streptomyces) Fungal ascorbic Mai Kota (Ascomycota) kiteuri also my three tests (Chytridomycetes) Bar Sidi Oh, my Kota (Basidiomycota) Dew Tero Mai Kota (Deuteromycota) Siu Mai-year-old Tess (Oomycetes) Here is my Cotta (Zygomycota) microalgae Cloud Mai also Thira ( Chlamydomonas) chlorella (chlorella) dia Toms (Diatoms) two analytic Ella (Dunaliella) Petal Catalonia (Petalonia) formate flutes Stadium (Porphyridium) Ulva (Ulva)

Next, metabolite compound (resin), which is a hardly decomposable compound, is a polar compound containing a small amount of nitrogen, sulfur, or oxygen in crude oil, and the decomposition rate is very slow and attached to rocks after oil spill accident. Remain black, stimulating visual contamination (Rotani, JF, F. Bosser-Joulak, E. Rambeloarisoa, JC Bertrand, G. Giusti, and R. Faure. 1985. Analytical study of ASTHART crude oil: asphaltenes biodegradation Chemosphere. 14: 1413-1422). The component analysis of metabolite compounds in crude oil is composed of a wide variety of substances like crude oil, and the analysis of detailed components by polymerized polymer compounds is very rare, but the nitrogen in the metabolite component by Athabasca Bitumen Carbazole-containing, thiophene-containing sulfur, furan-containing and fluorenol, fluorenol, fluorenone, carboxylic acid, Sulfoxide has been reported (Mackay. D. 1987. Formation and stability of water-in-oil emulsions. DIWO-Report No. 1). Gawel, I. 1987. Structural investigation of asphalts produced from paraffinic-base crude oil by different methods. Fuel 66: 618-621 discloses that the average molecular formula of the metabolite component in USSR paraffinic crude oil is C _78.9 H _105.11 N _0.74 S _1.25 O _0.99 , see Ali, MF, A. Bukhari, and M. Hassan. 1989. Structural characterization of Arabian heavy crude oil residue. Fuel Sci. Technol. Int . 7: 1179-1208, it is reported that the metabolite components of Arabian Light crude oil are mostly aromatic compounds with alkyl groups or polar groups (Venkateswaran. K, Hoaki. T, Kato. M, and Maruyama. T. 1995. Microbial degradation of resins fractionated from Arabian light crude oil.Can.J. Microbiol. 41: 418-424). Among these, the typical thiophene-based dibenzothiophene (DBT) is a model compound of polycyclic aromatic sulfur heterocycle, and has been studied for biodegradation of this substance for more than 20 years. Condensed thiophene is more difficult to decompose than polycyclic aromatic hydrocarbons (PAH) and is concentrated in organisms for a long time after a crude oil spill, which is known to be more toxic than polycyclic aromatic hydrocarbons (PAH) (Kropp, KG, and PM Fedorak. 1998. A review of the occurrence, toxicity, and biodegradation of condensed thiophenes found in petroleum. Can.J. Microbiol. , 44: 605-622). Nevertheless, the biological reports on these substances are extremely small, and all of them are studied by Korea Maritime Research Institute. The reason for this is that these substances are difficult to decompose biologically and are not classified as environmentally regulated substances.

Meanwhile, studies on biodegradation of metabolite compounds in crude oil are described in Venkateswaran. K, Hoaki. T, Kato. M, and Maruyama. T. 1995. Microbial degradation of resins fractionated from Arabian light crude oil. Can. J. Microbiol. 41: 418-424, but has generally been known to be difficult to biodegrade, and reports of these degraded microorganisms are insignificant and the pathway of degradation is unclear. Known microbial degradation pathways have been reported to either be degraded by co-metabolism or by microorganisms with a wide range of aromatic compounds degradation pathways (Venkateswaran. K, Hoaki. T, Kato. M, and Maruyama. T). . 1995. Microbial degradation of resins fractionated from Arabian light crude oil Can J. Microbiol 41:... 418-424).

SUMMARY OF THE INVENTION An object of the present invention is to provide a strain having excellent resolution of oil components and metabolites thereof, in particular metabolites, and a method for biologically purifying oil contaminated soil using such strains.

In the case of diesel, gasoline, kerosene, and aviation, which are typical product oils, these metabolite components are hardly present (FIG. 4). However, when analyzing the components of residual oils in chronic contaminated soils, a large part of the metabolite compound parts (asphalt) Asphalts) = Resin + Asphaltene) can be seen (FIG. 5).

These materials are polar substances derived from the weathering of oil, and detailed structural analysis is not carried out. However, these substances are usually obtained from oxidation of oil, and may be assumed to be ketone series, carboxylic acid series, or alcohol series. This is similar to metabolites generated as a result of biodegradation of oil, and when these metabolites accumulate in the soil, it becomes a factor that inhibits the oil-degrading ability of the oil-degrading microorganism (FIG. 6).

Therefore, these metabolites must be removed from the soil in order to enhance the biological oil degradation of the oil-degrading microorganisms.

Therefore, various kinds of oil-degrading microorganisms are required for the biological purification of oil-contaminated soils, and a technique for mixing them to achieve a complementary relationship between these microorganisms is required.

On the other hand, according to the mechanism of ingesting oil by the oil-degrading microorganisms (Hydrocarbon Uptake Mode), the microbial group can be classified into four types (Go Sung-hwan, Hong-geum Lee, and Sang-jin Kim. 1998. Effect, Korean Journal of Biotechnology and Bioengineering, 13 (5), 606-614; The first is a class of lyolytic microorganisms that secrete biological surfactants to make oil smaller than themselves and consume the solubilized oils. These microorganisms are low in hydrophobicity and high in oilability. 8). The second is the direct contact with the oil surface to ingest the oil (Direct contact), these microorganisms are hydrophobic cell surface and little emulsification capacity, so they coagulate and grow with each other during the oil decomposition period (Fig. 9). The third is to consume only oil dissolved in water, and the growth rate is slower than other classes. Finally, these three intake mechanism can be divided into classes taken by all (Ko, SH, and JM Lebeault . 1997. Effect of a mixed culture on co-oxidation during the degradation of saturated hydrocarbon mixture. J. Appl. Microbiol., 86: 1008-1016).

It is interesting to note that the oil-degrading microorganisms of each of these classes are very good in oil-resolving ability, but in some cases, when the mixing of the classes is carried out in order to induce higher oil-degrading, a phenomenon in which the oil-degrading ability is rather inhibited is observed. have. In other words, when the microbial group of the high emulsifying group and the high hydrophobic group is mixed, the inhibitory effect is caused by the difference in intake mechanism. On the other hand, microbial groups that take a mixed intake mechanism have been well matched with any kind of group, resulting in increased resolution, and are known to be well matched with various indigenous microbial groups.

The inventors have isolated microorganisms in various regions to isolate microorganisms using metabolites as carbon and energy sources for final biological recovery of oil contaminated regions. Strains were selected by investigating the degradability of hydrocarbons and metabolites with crude oil, and microscale experiments were performed to measure the degradation rate of oil by adding the selected metabolite degradation strains to the oil degradation microbe mixture. It was.

Example
In this embodiment, the metabolite refers to the "resin" compound and was tested with "dibenzothiophene (DBT)" as a representative material of the "resin" compound.

Example 1 Metabolite Degradation Microbial Isolation and Selection of Excellent Metabolite Degradation Strains

1.1 Metabolite Degradation Microbial Isolation

1.1.1 Materials and methods

1.1.1.1 Research Area and Sampling

In order to isolate the bacteria that can grow by using metabolites as the sole carbon source, soil samples were collected from oil spill areas, gas stations, and refineries. The collected samples were collected, mixed well, refrigerated, transferred to a laboratory, and cultured.

1.1.1.2 Isolate Metabolite Degrading Strains

end. Rich culture using metabolite as carbon source

When only high concentrations of metabolites are grown as substrates, microorganisms that cannot use metabolites as carbon sources are gradually eliminated, and only microorganisms that can use metabolites as substrates survive. Samples collected under this premise were shaken at 30 ° C. and 120 rpm in mineral medium to which metabolites were added, and 1 ml of the culture solution was inoculated in fresh medium three times a week. The culture solution was serially diluted in sterile distilled water and plated on mineral agar medium and incubated at 30 ° C. for 5 days.

I. Isolation of Metabolite Degrading Strains

Microbial colonies formed on the medium were divided into morphological features and subcultured by pure separation until determined as a single strain, and separated through growth of colony morphology and optical microscopy. The isolated strain was suspended in 20% glycerol solution and stored at -70 ° C.

1.1.1.3 Selection of Metabolic Excellent Degrading Strains

end. Degradation Investigation for Selection of Metabolic Excellent Strains

Isolated metabolite degradation microorganisms were inoculated in 20 ml of mineral medium containing 200 ppm of metabolite and shaken for 1 week at 30 ° C. and 120 rpm. The degradation rate was analyzed by TLC / FID after extraction of the culture medium with chloroform. . In addition, after diluting the bacteria by making a solid medium added only the N / P source, the metabolite was dissolved in chloroform to spray up to 1,000 ppm and sprayed, and then cultured in a 30 ℃ incubator to observe whether colonies were formed. In the above method, strains having high degree of emulsification and good degradation rate were selected first.

I. Investigation of hydrocarbon decomposition range of selected strains

In order to select a strain that can use metabolites as a primary when several hydrocarbons are present, the range of hydrocarbon degradation of the primary starting strain was examined. Dibenzothiophene (DBT) was selected as a standard for metabolites, aliphatic mixed compounds (SHM), polycyclic aromatic hydrocarbon mixed compounds (AHM), arabian light crude oil and metabolites.

Aliphatic mixed compound (SHM), polycyclic aromatic hydrocarbon mixed compound (AHM) and Arabian Light crude oil were added to 2,000 ppm in a flask to which 20 ml of mineral medium was added, and each strain was inoculated at 30 ° C. and 120 rpm for one week. Shake culture was performed. In order to investigate the resolution of metabolites and DBT, these substances were dissolved in chloroform and added to the flask so that their final concentration was 200 ppm, and then left in a hood for one day to remove 20 ml of mineral medium when chloroform was completely removed. Each strain was inoculated after the addition and shaken at 30 ° C. and 120 rpm.

(1) TLC / FID Analysis

Analysis was done using TLC / FID to identify residual metabolites (FIG. 10).

In order to confirm the total amount of residue, 30 ml of chloroform was added to 10 g of the culture sample, and extracted for 10 minutes in a 60 ° C. ultrasonic bath. The amount of oil obtained by repeating this process five times was measured by weight, and again, stearyl alcohol, a standard substance, was added to the weight of oil to 1% (w / w) for TLC / FID analysis, and then chloroform was added. Appropriate amount was added to the TLC / FID rod for a predetermined amount, and then developed by using a solvent, and then scanned by a flame ionization detector (spot) by assaying the separated spot (spot) was possible qualitative and quantitative analysis.

The extracted oil was dissolved in chloroform to a concentration of several tens mg / ml, and then 1 μl was added to a developing glass rod (Chromarod-SIII, coated with silica, Japan) for TLC / FID. Dropped glass rods were developed about 10 cm using 100% normal hexane (n-hexane) as the primary developing solvent (takes about 30 minutes). As a secondary developing solvent, normal hexane: toluene (1: 4, v / v) was used and developed about 5 cm (about 10 minutes). After two rounds of development, the aliphatic component developed about 10 cm to form a spot, and the aromatic hydrocarbon component developed about 5 cm, and the metabolite component remained at the origin. Scanning using Iatroscan MK6 (Iatroscan Lab., Japan) to calculate the relative area of each component, it is possible to determine the components in the oil to be measured.

Analysis conditions were as follows: integrator, HP 3396 II (Hewlett Packard), attenuation = 2 ⁵ , chart speed = 10 cm / min; Detector, Flame Ionization Detector (FID), scanning speed = 30 sec / rod; Gas flow, 160 ml / min.

In this experiment, aliphatic mixed compounds (SHM), polycyclic aromatic hydrocarbon mixed compounds (AHM), and stearyl alcohol were used as the standard for Arabian light crude oil analysis, which overlapped with the metabolite portion after the third development. As a result, the oil component develops in the order of aliphatic compound, aromatic compound, stearyl alcohol, and the metabolite stays at the origin. Meanwhile, normal development was performed using normal hexadecane as a standard for metabolite and dibenzothiophene (DBT) analysis. Metabolite analysis was performed using TLC / FID, and resolution of dibenzothiophene (DBT) was analyzed using GC / FID.

(2) GC / FID Analysis

① Sample pretreatment

The samples were extracted by ultrasonic extraction according to the soil pollution process test method. Add 10 g of soil sample to the beaker, add an appropriate amount of anhydrous sodium sulfate to keep the sample in powder form, shake well, add 100 ml of dichloromethane, and extract by ultrasonic for 10 minutes using an ultrasonic extractor. The extraction solution obtained by repeating this extraction procedure two or more times was vacuum filtered with a Buchner funnel covered with a filter paper (5B), and then washed with a small amount of dichloromethane. This effluent was concentrated to 2 ml with a rotary evaporator and purified by silica gel.

② Analysis of the sample

Extracted and purified samples were analyzed by injecting 2 µl each into a gas chromatograph using a micro syringe. TPH analysis includes GC / FID (Varian 3900 GC) with a fused silica capillary column (30 m × 0.25 mm id, Chrompack CP-Sil 8CB-MS) and the Galaxy workstation program program). High purity helium was used as the transport gas, and the temperature conditions of the oven during analysis were as follows. Initial oven conditions were maintained at 50 ° C. for 2 minutes and the temperature was increased by 8 ° C. per minute from 50 ° C. to 320 ° C. and maintained at 320 ° C. for 10 minutes.

1.1.2 Results and Discussion

1.1.2.1 Separation and Degradation of Metabolite Degradation Microorganisms

27 strains were isolated from 12 strains from soil samples collected from oil spill area, 10 strains from gaseous contaminated soil, and 5 strains from soil from oil refinery. Candidate metabolite degradation strains were selected (Table 5).

Among the 27 strains, 10 strains showing high degradation rate (10% or more) were selected as a primary result after culturing for 2 weeks or more in a medium to which 1,000 ppm of metabolite was added (Table 6, FIG. 11). The highest resolution of the metabolite of strain SBS-8 was 39.4 ± 5.9%, and SBS-10 and OB-8 showed 32 ± 3.6% and 30 ± 5.2% resolution, respectively. The decomposition rate was about 10.8 ～ 15.9%.

1.1.2.2 Investigation of hydrocarbon decomposition range of selected strains

In addition to metabolites, aliphatic hydrocarbon compounds (SHM, saturated hydrocarbon mixtures), aromatic hydrocarbon compounds (AHM, aromatic hydrocarbon mixtures), Arabian light crude oil and dibenzothiophene (DBT), etc. Was used. Aliphatic hydrocarbons (SHM), aromatic hydrocarbons (AHM) and Arabian light crude oil are added to the mineral medium at concentrations of 2,000 ppm, metabolites and dibenzothiophene (DBT) at concentrations of 200 ppm. Incubated for 10 days.

As a result of the experiment, as shown in FIGS. 14 to 17, all of these strains were found to be relatively degradable for aliphatic hydrocarbon compound (SHM), aromatic hydrocarbon compound (AHM), metabolite and dibenzothiophene (DBT). It was observed to have a decomposition range. In particular, the aliphatic hydrocarbon compound (SHM) showed a high decomposition rate of 70% strain SBS-8, 66% SBS-10, 50% SBS-12 (Fig. 12), in the case of aromatic hydrocarbon compound (AHM) SBS-2 showed 50% degradation and SBS-3 showed 49% degradation (FIG. 13). The metabolite showed 49% degradation of SBS-8 and 36% of OB-8 (Fig. 14). The highest degradation rate of SBS-10 was 53% for dibenzothiophene (DBT). -8 showed 34% degradation and OB-8 showed 36% degradation (FIG. 15).

However, in the case of Arabian Light crude oil, most strains showed degradability for aliphatic and aromatic compounds. However, the SBS-8 strain degraded about 30% of the metabolite portion, about 24% for SBS-10, and about 10% for SBS-12 and was not increased by other strains, but rather increased (FIG. 16). In this way, each strain has a wide range of hydrocarbon decomposition, but it is considered that the aliphatic and aromatic compounds which are easy to use in the components of crude oil are first decomposed and then metabolites are decomposed. Therefore, long time is required for complete decomposition of crude oil. In addition, the increase of metabolite component in the result is considered to be a result of the accumulation of metabolites that can be generated by decomposition of aliphatic and aromatic compounds in crude oil. Therefore, it is concluded that the biological treatment will be faster and more stable if the microbial agent, which is a good mixture of aliphatic and aromatic compounds with good degradation ability and selected metabolite degradation strains, is applied to oil pollution area.

TABLE 5 Colony Forms of Selected Metabolite Degrading Bacteria on Mineral Agar Media.

No. Strain name Identification properties color surface shape Opacity Elevation One SBS-1 Cloudy yellow lubricity circle opacity Convex shape 2 SBS-2 White lubricity circle opacity Protruding shape (umbonate) 3 SBS-3 White lubricity circle Translucent Convex 4 SBS-4 orange color lubricity circle opacity Centering shape 5 SBS-5 orange color coarseness circle opacity Centering shape 6 SBS-6 cream color lubricity circle opacity Convex shape 7 SBS-7 orange color lubricity circle opacity Convex shape 8 SBS-8 yellow lubricity circle Transparency Convex shape 9 SBS-9 White lubricity circle Translucent Convex shape 10 SBS-10 cream color lubricity circle opacity Convex shape 11 SBS-11 White lubricity circle opacity Convex shape 12 SBS-12 Light yellow lubricity circle opacity Convex shape 13 OB-1 White lubricity circle Transparency Convex shape 14 OB-2 yellow lubricity circle Transparency Cushion Shape (Pulvinate) 15 OB-3 cream color lubricity circle opacity Convex 16 OB-4 Bright orange lubricity circle opacity Centering shape 17 OB-5 green coarseness circle opacity Convex 18 OB-6 White lubricity circle opacity Centering shape 19 OB-7 yellow lubricity circle opacity Cushion shape 20 OB-8 yellow lubricity circle Translucent Centering shape 21 OB-9 cream color lubricity circle opacity Convex 22 OB-10 cream color lubricity circle opacity Convex 23 RS-1 White lubricity circle Translucent Convex 24 RS-2 yellow lubricity circle opacity Convex 25 RS-3 cream color lubricity circle opacity Convex 26 RS-4 White lubricity circle opacity Convex 27 RS-5 cream color lubricity circle opacity Convex

Table 6. Degradation Rate of Metabolites (Resin 200 ppm) Compounds Added to Mineral Media by Selected Metabolite Degrading Bacteria

No. Strain number Degradation rate (%) One SBS-1 10.4 ± 2.5 2 SBS-2 15.9 ± 2.1 3 SBS-3 10.8 ± 0.8 4 SBS -8 39.4 ± 5.9 5 SBS -10 32.1 ± 3.6 6 SBS-12 13.2 ± 2.4 7 OB-4 15.2 ± 1.1 8 OB -8 30.1 ± 5.2 9 RS-1 12.4 ± 2.9 10 RS-4 15.9 ± 4.8

1.1.2.3 Deposit of Strains

Of the strains selected, two strains showing particularly good resolution for metabolites were named Bacillus sp. SBS-10 (KCTC 11070BP) and Micrococcus sp. SBS-8 (KCTC 11071BP). These strains were deposited on February 15, 2007, at the Genetic Center Genetic Bank of Korea Research Institute of Bioscience and Biotechnology, and were given accession numbers KCTC 11070BP and KCTC 11071BP, respectively.

Example 2 Microcosm Experiments

Typically, the bioconverted material of the compound in the process of oil decomposition is difficult to be degraded by the microorganism that produced it, thereby inhibiting the degradation activity (feedback inhibition). Secondary degradation is required by other microorganisms (heterotrophic bacteria) to increase degradation activity. In conclusion, the conclusion is that such soils must be administered externally with other lysogenic and metabolite microorganisms. Therefore, it was necessary to observe the effect of metabolite decomposing microorganisms upon biodegradation feasibility study.

Therefore, this experiment was conducted to investigate the degradability of oil contaminated soil using selected metabolite degrading microorganisms. In a laboratory, small scale microcosm was made in a stainless steel container to examine biodegradability in a short time. The oil degradation rate is a qualitative analysis concept rather than a quantitative analysis. It is a natural reduction applicability test (control), indigenous microbial activation (biostimulation) test, and external oil-degrading microorganisms (including metabolite-degrading microorganisms). Bioaugmentation) The purpose of this study was to determine the efficiency.

2.1 Materials and Methods

2.1.1 Contaminated Soil

The characteristics of contaminated soil samples used in microcosm experiments are summarized in Table 4. Soil pH was 6.45, which was relatively suitable for microbial activity. It was 29% of water holding capacity, but water content of 7.9% ~ 9.1% was relatively poor for microbial activity. The state of the soil was in a state where masato and clay were mixed about 8: 2 (Fig. 17). Total petroleum hydrocarbons (TPH) in soil samples were 17,030 ppm as measured by PetroFLAG (USEPA Official Method, Field Measurement Kit), and similar results were obtained by TLC / FID and GC / FID. It was. Samples contaminated with oil were detected at an average of 14,000 ppm (Table 7).

Analysis of TLC / FID results showed that the metabolite component ratio of polar substances, which is a phenomenon that occurs when weathering occurs in oil, was found to be about 12.5%. In the case of pure diesel, it was observed that the weathering of the pollutant occurred considerably compared to that in which little metabolite was detected (FIG. 18).

Table 7. Characteristics of Contaminated Soil Samples

Analysis item Contaminated soil sample Water content (%) 7.9 ～ 9.1 Water retention (%) 28.6 pHw1: 1 6.45 ～ 7.04 TPH (ppm): Petroflag 17,030 TPH (ppm): GC / FID 13,218  TPH (ppm): TLC / FID Total (ppm) (excluding asphalt) 14,790 Aliphatic Compounds (%) 65.0 Aromatic compounds (%) 22.4 Asphalt (resin + asphaltenes) (%) 12.6 Total microbial count (X10 ⁶ CFU / g) 5.89 ± 0.94 Hydrolysis Microbial Count (X10 ⁶ MPN / g) 0.97

2.1.2 Experiment site installation

Contaminated soil samples prepared for this experiment were placed in a 3L stainless steel square container at 0.5 kg each, and only the moisture was adjusted to the control. Nutrients to be added to indigenous microorganisms were Ecoguard MS I produced by Ecofil Co., Ltd. and added at a rate of 350 ppm (C / N / P = 100/10/1) per 1,000 ppm of total petroleum hydrocarbon (TPH). ) Administration. And by administering the microorganism to Legitime obtain the microbial agent is mixed Co. echo filter to produce a second metabolite degrading microorganisms (SBS SBS-8 and-10) of species selected on CDM EcoGuard to 3kg / ㎥- Land ratio (10 ⁶ MPN / g-to-soil ratio) was added, and as a nutrient, EcoGuard MS I was added under the same conditions as the indigenous microorganism activator (FIG. 19).

Each experiment was placed in a 25 ℃ thermostat, mixed well twice a day, controlled once every three days, and samples were taken from each experiment at 1, 2, 5, 9, 12 and 16 days. TLC / FID and GC / FID were used to analyze the changes of oil components and residual flow.

2.1.3 Residual Oil Analysis

In order to identify the residual oil in the soil, TLC / FID (Thin Layer Chromatography / Flame Ionization Detector), GC / FID and PetroFLAG were analyzed.

end. TLC / FID Analysis

Clause 1.1.1.3 of Example 1 above. Same as the assay described in the section "Investigation of Hydrocarbon Degradation Range of Selected Strains".

I. GC / FID Analysis

Clause 1.1.1.3 of Example 1 above. Same as the assay described in the section "Investigation of Hydrocarbon Degradation Range of Selected Strains".

All. PetroFLAG Analysis

In order to quickly find out what is happening at the site, the residual flow rate was analyzed using PetroFLAG equipment that can be easily analyzed at the site or indoors authorized by the US Environmental Protection Agency (USEPA) (FIG. 20).

2.1.4 Microbial Analysis

1 g of soil was added to 10 ml of sterile saline solution (0.9% NaCl solution), homogenized at 220 rpm for 3 hours, and then diluted appropriately to use as a sample for bacterial analysis. In order to detect heterotrophic bacteria, each sample was uniformly diluted in Petri Film (USA) manufactured by 3M Inc., inoculated with 1 ml of diluting solution, and then cultured at 30 ° C. for 5 days to count only colonies colored in red.

Lipolytic microorganisms were determined using the Most probable number (MPN) coefficient method (Wreen and Venosa, 1996). Bushel-hass (Difco) medium was used as the MPN medium, and normal hexadecane (n-hexadecan, 5%, w / v) was used as the only carbon source. The inoculated medium was incubated at 30 ° C. for 10 days, and 50 µl of 0.3% Iodonitrotetrazolium chloride (INT, Sigma, USA) was added and incubated for 24 hours to form a red precipitate. The number of lyolytic bacteria was counted using Thomas' simple formula (FIG. 21).

2.2 Results and Discussion

In this experiment, the control that controlled only temperature and humidity was able to reduce oil pollution by about 5%. However, this is a result obtained by artificially adjusting the environmental factors such as humidity and temperature through feasibility experiments and natural oils in the state that the pollution is very high at the initial pollution concentration of 14,000 ppm and the environmental conditions (TN, TP) in the soil are poor. It is difficult to expect a pollution reduction effect.

In the experimental group (indigenous microorganism active group) administered only nutrients, the oil degradation rate was about 33%, which was relatively higher than that of the control group (about 5%), but the microorganisms (EcoGuard CDM & metabolite decomposition microorganisms) and nutrients It was found to have lower oil degradation activity than the experimental group (59%) with the mixed agent.

In the experimental group containing the metabolite-degrading microorganism and the oil-degrading microorganism, the degradation rate was about 59% based on the initial contaminant concentration for 16 days. This resulted in very high reduction effect on oil pollution due to metabolite degradation microorganism addition, and the oil degradation rate was also shown to show fast and stable pollution reduction compared with other experimental groups for the same time (Table 8, Table 9, 22 and 25). In addition, GC / FID results showed similar trends with TLC / FID results and disproved TLC / FID results.

Hydrolyzed microorganisms and metabolite-degrading microorganisms showed higher bacterial counts than other experiments and increased up to 10 ⁸ to 10 ⁹ CFU or MPN / g-soil during the same period. It was investigated to show (FIG. 23 and FIG. 24). Lowering the initial average TPH of 14,790 ppm to 5,737 ppm only 16 days after microbial administration, on average, decreased by about 566 ppm per day, indicating a very high removal rate.

Based on the results of this experiment, the average TPH of the sample collected was about 14,000 ppm, and the indigenous microbial activity method, which adds and processes only nutrients, is twice as long as the additional method of adding oil- and microorganism-degrading microorganisms. And cost is required. Therefore, in order to perform economic biological purification, it is effective to select a method in which a microorganism with a microorganism degraded with oil is mixed.

Table 8. Final Oil Degradation Rate (%) for each experiment.

Sample No. Control Indigenous Microorganisms External microbial inlet Contaminated soil sample 4.5% 32.8% 59.2%

Table 9. Changes in oil concentrations, total bacterial counts and number of lysogenic bacteria during the experimental period.

As described above, the present invention provides a new strain capable of decomposing oil components and metabolites thereof, a separation method thereof, and a microbial agent containing the same, and in particular, such microbial agents may be used in combination with existing microbial agents to contaminate oil. The effect is to restore the soil faster, more reliably and more environmentally friendly.

Claims (7)

delete Resin-degrading strain Micrococcus sp. SBS-8 (KCTC 11071BP), an oil component and its metabolite, isolated from oil-contaminated soil. Micrococcus sp. SBS-8 (KCTC 11071BP) An oil component comprising at least one strain selected from the group consisting of an active ingredient and a microbial agent for resin degradation which is a metabolite thereof. A method for biologically purifying oil contaminated soil, comprising applying the microbial agent according to claim 3 to oil contaminated soil. A method for biologically purifying oil contaminated soil, including simultaneously applying the microbial agent according to claim 3 and Ecoguard CDM (manufactured by Ecofil Co., Ltd.), which is a microbial agent for decomposition of oil components. delete After oil-contaminated soil is concentrated in a mineral medium containing resin, which is an oil metabolite of oil, at 25 ° C. to 35 ° C., the resin degradation strains are isolated, including smearing on mineral agar medium, and the degree of emulsification of the resin. And selecting the strain according to claim 2 based on the degradation rate.

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