KR100712425B1 - High molecular weight polycyclic aromatic hydrocarbon degrading bacteria for bioremediation of polycyclic aromatic hydrocarbon contaminated environment, the method for preparation thereof and decomposing oil composition comprising the degrading bacteria - Google Patents

Abstract

The present invention relates to a microorganism which decomposes a high molecular polycyclic hydrocarbon, the microbacterium sp. (Mycobacterium sp.) Of Accession No. KCTC8975P having the characteristic of decomposing 3-ring or higher high molecular weight polycyclic aromatic hydrocarbon (PAH). C2-3 and a method for preparing the same, and the microbacterium sp. By providing an oil decomposition composition comprising C2-3 as an active ingredient, it is possible to clean the environment contaminated with oil.

Mycobacterium sp. C2-3, high molecular weight polycyclic aromatic hydrocarbons (PAH), pyrene

Description

High molecular weight polycyclic aromatic hydrocarbon degrading bacteria for bioremediation of polycyclic aromatic hydrocarbon contaminated environment, the method for preparation according and decomposing oil composition comprising the degrading bacteria}

1 is a view showing the formation of a clear zone (central black portion) by the microorganism of the present invention.

2 is a graph showing the optimum temperature at which the microorganisms of the present invention can grow.

3 is a bar graph showing the decomposition rate of pyrene at various concentrations.

4 is a bar graph showing the decomposition rate of phenanthrene at various concentrations.

5 is a graph showing the decomposition rate of phenanthrene under various inducing conditions.

6 is a graph showing the decomposition rate of Fluoranthene under various induction conditions.

7 is a graph showing the decomposition rate of pyrene under various induction conditions (●: not inducing, ○: phenanthrene induction, ▼: fluoranthene induction, ▽: pyrene induction).

8 is a graph showing the effect of humic acid on the decomposition rate of pyrene (●: control, ○: 500 ppm, ▼: 1000 ppm).

9 is a graph showing the effect of the surfactant on the decomposition of pyrene (300 ppm) (●: control, ○: Triton X-100, ▼: Tween 80,?: SDS).

10 is a graph showing the effect of showing the toxicity of Triton X-100.

FIG. 11 is a graph depicting the effect of phenanthrene as an auxiliary substrate on the degradation of pyrene (300 ppm) (30 ° C., 150 rpm, 6 days).

12 is a graph depicting the effect of phenanthrene as an auxiliary substrate on the degradation of pyrene (300 ppm) (30 ° C., 150 rpm, 6 days).

FIG. 13 is a graph showing the effect of phenanthrene as an auxiliary substrate on the decomposition of pyrene (200 ppm) (●: control, ○: 50 ppm, ▼: 100 ppm).

FIG. 14 is a graph showing the effect of fluoranthene as an auxiliary substrate on the decomposition of pyrene (200 ppm) (•: control, ○: 50 ppm, ▼: 100 ppm).

FIG. 15 shows phenane in culture mixed with Mycobacterium sp. C2-3 and Sphingomonas sp. 1-21 against mixed polyaromatic hydrocarbon (PAH). It is a graph showing the decomposition rate of tren (■: 2 days, ??: 6 days).

FIG. 16 is a pie of culture mixed with Mycobacterium sp. C2-3 and Sphingomonas sp. 1-21 on mixed polyaromatic hydrocarbon (PAH). FIG. It is a graph showing the decomposition rate of rene (■: 2 days, ??: 6 days).

The present invention relates to a polymer polycyclic hydrocarbon decomposition microorganism for bio purification of polycyclic hydrocarbon contaminated soil, and more particularly, it is possible to decompose high molecular weight polycyclic aromatic hydrocarbon (PAH) contained in soil without cofactor or auxiliary substrate. To degrade microorganisms that are present.

High molecular weight polycyclic aromatic hydrocarbons (PAH) are pollutants produced by the burning of fossil fuels or by industrial activities. Large amounts of PAH are detected in freshwater and marine sediments in industrialized regions around the world. PAH is strongly adsorbed to suspended solids and settles to the bottom of the water system and accumulates in low quality soil. High concentrations of PAH have the effect of causing or killing aquatic mutations (Varanasi and Stein, 1991). Because exposure to PAHs poses a risk to human health, many countries have made great efforts to eliminate these substances (White, 1986).

Microorganisms play a very important role in the biodegradation of chemicals in the natural environment. Biodegradation of chemicals is achieved by metabolism for each strain or by complex metabolism by microbial communities.

Pyrene is one of 16 PAHs belonging to 129 priority pollutants defined by the US EPA (Keith and Telliard, 1979). Four aromatic rings are fused together with benzo [a] pyrene, indeno- (1,2,3-CD) -pyrene (indeno- (1,2,3-cd) Because it has a structure similar to toxic carcinogens such as) -pyrene) and 1-nitropyrene, it has been studied as a model compound in the study of PAH degradation and metabolism along with lactic acid bacteria culture (Chen, 1983). Jacob et al., 1982).

PAH is known to be removed by photolysis, chemical oxidation and vaporization, but mainly by microorganisms. Therefore, the development of techniques for cleaning contaminated areas using microorganisms has great potential for the purification of contaminated areas (Muller et al., 1991). In order to develop such biological purification technology, it is necessary to secure strains with high degradability and support the experimental results of decomposing products as well as ecological and physiological studies of degrading bacteria.

The study of PAH degradation by microorganisms has been studied steadily since the 1920s. In 1928, Gray and Thornton reported the ability to metabolize aromatic hydrocarbons such as naphthalene, phenol, and cresol. have. Most studies of several species of Pseudomonas (Pseudomonas), Monastir (Aeromonas) in the Oh, Flavobacterium (Flavobacterium), Bay jerin Kea (Beijerinkia), Alcaligenes (Alcaligenes), microcode Syracuse (Micrococcus), Vibrio (Vibrio), and M. have been concentrated in Te Leeum (Mycobacterium).

Phenanthrene is composed of three rings and is less toxic than other PAHs, but is widely used in the natural environment and is used as a representative model compound of PAH. The definition of high molecular weight polycyclic aromatic hydrocarbons (PAH) is a generic term for compounds in which the benzene ring or pentacyclic moieties are linear, angular, or collectively arranged. These PAHs have chemical properties such as low solubility, thermodynamically stable structure, and strong adsorption to particles, and have cancer-causing properties in living organisms (Varanasi and Stein, 1991). Due to its unique chemical nature, exposure to the environment makes the degree of contamination durable. PAH contamination pathways can be divided into three categories: biosynthetic, geochemical and anthrophogenic factors. Among them, the pollution level is severe and the environmental cleanup program is caused by an oil spill accidentally, deliberate dumping of creosote, coal tar and petroleum compounds, and incomplete combustion of wood or fossil fuel. And so on. PAH is known to be removed by photolysis, chemical oxidation and vaporization, but mainly by microorganisms.

Pyrene is a high-molecular-weight polycyclic hydrocarbon with four-rings. Alkaligenes D. nitrophyte after Heitakemp et al.'S first discovery in 1988 that Mycobacterium sp. ( Alcaligenes denitrificans) and Rhodococcus sp. Unlike the decomposition of low-molecular-weight polycyclic hydrocarbons, very few microorganisms are believed to be involved in pyrene decomposition, which is why they are relatively durable even in soil-containing environments. Many microorganisms have already been published to decompose low molecular weight polycyclic hydrocarbons including naphthalene and phenanthrene, and research on ecology, physiology and molecular biology has been actively conducted. However, high molecular weight polycyclic hydrocarbons, including pyrene, have few species involved in decomposition and their ecology, physiology, and molecular biology are very few.

Interestingly, mycobacteria are constantly being separated from oil contaminated soils as microorganisms capable of degrading PAHs having four or more ring structures. It is believed that the hydrophobic cell surface favors adsorption to insoluble PAH particles on water. Mycobacterium PYR-1 is known to degrade pyrene and floreanthene faster than naphthalene and phenanthrene (Hietakemp and Cerniglia. 1989), and this strain is known as benzo [A] pyrene. [a] pyrene) could not be mineralized. However, another Mycobacterium strain, RJGII-135, has been reported to degrade pyrene and benzo [A] pyrene (Grosser et al., 1991), while PYR-1 and RJGII-135 strains using 16s RNA. Phylogenetic analyzes indicate that they belong to the fast-growing group of mycobacteria (Govindaswami et al., 1995).

(1) Ecological Survival and Degradation Characteristics of PAH Degrading Bacteria

Many studies have been conducted to identify various problems that can be caused by exposure of PAH to the environment and to remove them from the environment using decomposition bacteria. Many of these studies, however, were the subject of a major study using a single PAH to test the resolution and to understand its degradation characteristics. However, it is important to note that PAH is not contaminated with a single PAH but is contaminated with a mixture of two to five or more rings. Stringfellow and Altken (1995) reported that competitive metabolism is caused by naphthalene, methylnaphthylene, and fluorene during Phenanthrene degradation in Pseudomonas. Therefore, the metabolic relationship between PAHs must be examined in the modeling and regulation of PAH contaminated areas using specific strains.

(2) Effect of additives on PAH degradation

PAH has hydrophobic properties. This hydrophobicity increases with increasing molecular weight. Research into the use of biologically or chemically produced surfactants to increase the degree of use or degradation by microorganisms is ongoing. These studies suggest that surfactants promote the biodegradation of PAH in water or soil systems during microbial PAH degradation (Guerin and Jones, 1988; Aronstein, et al., 1991; Lantz et al., 1995; Thiem, 1994). . In contrast, the addition of surfactants does not affect or reduce the degree of PAH degradation by microorganisms (Aronstein, et al., 1991; Efroymson and Alexander, 1991; Laha, and Luthy, 1991, 1992; Thiem, 1994; Volkering, et al., 1995; Liu, et al., 1995). In addition, when surfactants are used, processing above the critical micelle concentration (CMC) value is toxic to soil or aquatic microorganisms (Aronstein, et al., 1991; Laha, and Luthy, 1992; Liu et al. , 1991; Edward et al. 1991). However, only concentrations above CMC are known to increase the motility or solubility of PAH (Vigon and Rubin, 1989; Liu et al., 1991; Edward et al. 1991). These findings can be explained for the following reasons.

1) elevated toxicity due to destruction of bacterial cell membranes or increased permeability (Helenius and Simons, 1975; Cserhati et al., 1991; Heipiper et al., 1994),

2) Physicochemical effects due to the interaction of bacteria with surfactants, such as preventing bacteria from adhering to hydrophobic substrates (Efroymson and Alexander, 1991; Neu, 1996),

3) toxicity due to increase of PAH concentration in water layer under the influence of surfactant,

4) reduction in the utility of surfactant-micelle-solubilized PAH resulting from slow PAH elution from micelles (Volkering, et al., 1992, 1995; Guha and Jaffe, 1996 Zhang, et al., 1997) and

5) Factors such as competition between the surfactant and PAH as substrates (Liu, et al., 1995; Thiem, 1994) may act in one or a combination, and the possibility of acting depending on the strain applied cannot be excluded.

(3) Degradation Pathway and Molecular Biology of PAH Degrading Bacteria

Many types of bacteria have been isolated that have the ability to break down PAHs as carbon and energy sources. The catabolic pathway of PAHs has been described in detail in Pseudomonas putida G7 for naphthalene, compared with partial studies for other PAHs (Yen and Serdar, 1988; Cerniglier, 1992; Pothuluri and Cerniglia, 1994). Naphthalene metabolism by P. putida G7 was determined by naphthalene deoxygenase and 1,2-dihydroxy-1,2-dihydronaphthalene in one ring. Hydrogenation is carried out to produce). This naphthalene deoxygenase is composed of one reductase ( nahAa ), one ferredoxin ( nahAb ), and two iron-sulphur proteins ( nahAc and nahAd ). It is a multimeric enzyme. After 1.2-dihydroxy-1,2-dihydronaphthalene is dehydrated to 1,2-dihydroxynaphthalene by dehydroxygenase ( nahB ), the hydroxylated ring is 1,2-di It is broken by hydroxynaphthalene deoxygenase ( nahC) ( 1,2-dihydroxynaphthalene dioxygenase ( nahC )). The salicylate and pyruvate are then produced by isomerase ( nahD ), hydratase-aldolase ( nahE ) and dehydrogenase ( nahF ). (Eaton and Champman, 1992; Eaton, 1994). The resulting salicylate is transformed into catechol, ortho and meta cleavage depending on the strain, this step is called the lower pathway (Williams and Sayers, 1994). The catabolic pathway from the naphthalene to salicylate, also called the upper pathway, is also found in other types of PAHs. In the case of P. fluorecence 5R, phenanthrene or anthracene is 1-hydroxy- An example is the conversion of 2-naphthoic acid to 2-hydroxy-3-naphthoic acid (Menn et al., 1993). . In P. putida G7, the genes encoding the lower and upper pathways are arranged in two polycistronic operons, sal and nah, located in the NAH7 plasmid. Regulation of the expression of these two operons is regulated by the products of nahR. The activity of NahR is determined by the concentration of salicylates in cells (Schnell, 1985).

(4) Application to biological purification in natural environment

There is a widespread use of the method to clean the living environment by pollutants. It is considered to be a very useful tool to remove contaminants from the environment or to increase the resolution by administering microorganisms with the resolution of the pollutant to the contaminated area (Atlas, 1991). However, reports of successful treatment of refractory materials by administration of microorganisms (Briglia et al., 1990; Brodkorb and Legge, 1992; Grosser et al., 1991; Heitakemp and Cerniglia, 1989; Middledorp et al., 1990) (Goldstein, et al., 1985; Harkness, et al., 1993; Liu, et al., 1990) is compatible.

The success of bioremediation using microorganisms can depend on a variety of factors. The factors are the type of inoculum, the characteristics of the soil, and the amount and amount of substrate present in the soil.

At present, domestic laws and regulations on polluted environment are not systematic, and there are few kinds of compounds that can be regulated. For this reason, it is not possible to determine the extent of contamination by PAH in soils and water systems, especially domestic soils. Currently, the United States and many other countries have developed and commercialized PAH contamination treatment programs using microorganisms. However, at present, there is a lack of research on a method for treating the microorganisms made with domestic microorganisms.

Therefore, the present invention has been made to solve the above problems, by separating the bacteria decomposing high molecular weight PAH from the domestic soil and grasping the ecological and physiological characteristics of the domestic soil used in the biopurification of the soil and its efficiency To provide a degrading microorganism that can increase the.

The present invention to achieve the object as described above,

Provided is Mycobacterium sp. C2-3 having the resolution of a polycyclic aromatic hydrocarbon having at least 3-ring of Accession No. KCTC8975P.

The 3-ring or more polycyclic aromatic hydrocarbon is a polycyclic aromatic hydrocarbon selected from the group consisting of pyrene, phenanthrene, and fluoranthene.

In addition, the present invention Mycobacterium sp. (Mycobacterium sp.) Mycobacterium sp. C2-3 has the resolution of the normal-alkane (n-alkane). Provide C2-3.

In another aspect, the present invention provides an oil degradation composition comprising a microbacterium sp. C2-3, an accession number KCTC8975P as an active ingredient.

The oil degradation composition may further comprise a surfactant and / or humic acid.

In addition, the present invention

a) take samples from soil contaminated with oil,

b) incubate at least 1 week at room temperature by adding the collected samples to a minimal medium containing pyrene as the only carbon source,

c) concentration of the culture solution inoculated with fresh medium under the same conditions as the culture conditions of b), followed by isolation of C2-3 strains,

d) incubating the C2-3 strain isolated in c) for at least one week on a minimal solid medium coated with pyrene,

e) a method for producing Mycobacterium sp. C2-3, characterized in that it is identified as Mycobacterium sp.

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

In order to isolate pyrene-decomposing strains, chronic oil-contaminated soil around the oil storage facility is added to a minimal medium containing pyrene as the only carbon source and incubated for 1 week at room temperature. The concentrated cultures inoculated with fresh medium are separated by Kiyohara's method. The isolated C2-3 strains can observe a pronounced degradation ring after one week of incubation on minimal solid medium to which pyrene has been applied.

C2-3 strains are Gram positive and have been identified as Microbacterium sp. In addition, as compared to other microbacterium strains using the maximum possible phylogenetic tree using PHYLIP based on the base sequence of C2-3, it can be seen that it belongs to the fast-growing Mycobacterium group.

Microbacterium sp. The optimal temperature for culture of C2-3 is 20-30 ° C. in Tryptic Soy Broth (TSB), at which temperature it can produce visually observable colonies on solid medium for several days and the shape of colonies is medium in size. It is soft in shape and has a radiant character. The color is yellow.

Microbacterium sp. Hydrocarbons that C2-3 can decompose are shown in Table 2. M. sp. Of the present invention can decompose high molecular weight PAH such as pyrene and fluoranthene, but poorly decompose low molecular weight PAH such as naphthalene. On the other hand, normal-alkanes such as hexadecane and heptadecane can be degraded.

M. sp. In order to investigate the effect of humic acid on pyrolysis of C2-3, pyrene when treated with humic acid as shown in FIG. 8 showing the change of decomposition degree when 500 ppm and 1000 ppm humic acid were treated. It can be seen that the decomposition degree of is high.

One method of increasing the degree of decomposition of hydrocarbons is the addition of surfactants. Therefore, the addition of the surfactant is M. sp. Triton X-100 (manufactured by Junsei Chemical, Japan), Tween 80 (manufactured by Sigma, USA), and SDS (manufactured by sigma, USA) to determine the effect of C2-3 on pyrene decomposition . In the experimental group treated with Tween 80, 50% resolution was observed in 2 days of culture, and the pyrene added after 3 days decomposed more than 95%. In the case of SDS, the degree of degradation is slightly lower than that of Tween 80, which is about 30%, and more than 95% of pyrene is degraded after 4 days of culture. However, it can be seen that Triton X-100 shows no resolution.

The surfactant is M. sp. To investigate the effect of C2-3 on cell growth, Tween 80 and SDS promoted cell growth, but the surfactants were added to the minimal medium supplemented with glucose as a carbon source. (Triton) X-100 was completely suppressed. This is thought to act as a cofactor, rather than being used as a carbon source for Tween X-100 or SDS. The reason for the increased decomposition rate during pyrene decomposition is that these surfactants act as cofactors and the solubility of pyrene. It is thought that the resolution was increased by increasing. However, for Triton X-100, M. sp. The growth of C2-3 itself is believed to be toxic.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided to better understand the present invention, and the present invention is not limited to the following examples.

Example

[Isolation of PAH Degrading Strains]

Isolation of the strain was selected where the oil is contaminated around the soil around the oil tank. The collected soil samples were inoculated with 0.5 g each of 20 ml vials containing 5 ml of medium and 500 ppm of pyrene, and enriched for 1 week at 28 ° C and 130 rpm. The concentrated samples were inoculated with 100 μl of fresh medium under the same conditions, transferred, and cultured for 1 week. This transfer was repeated three times. The components of the minimal medium used are as follows. 30 mg of (NH ₄ ) ₂ SO ₄ , NaCl 2 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ -7H ₂ O 50 mg, CaCl ₂ -H ₂ O 20 mg, KNO ₃ 10 mg, trace metals per liter of distilled water 1 ml of trace metal solution.

After three consecutive concentrations, inoculate 100 μl by diluting stepwise in a minimum medium solidified by adding 1.5% agar, and then spraying by dissolving pyrene in acetone to 2% by the method of Kiyohara (1982). It was. The culture was incubated at 28 ° C. and observed from time to time to isolate colonies in which a clear zone was formed around the colonies. About 150 colonies were obtained and streaked in Tryptic soy agar (Difco), which was twice dilute for pure culture, to obtain pure single colonies.

[Checking Pyrene Degradation by Concentration of Isolated Strain]

Pyrene was dissolved in acetone and added to 50, 100, 250, 500, and 1000 ppm in 5 ml of the minimum liquid medium in order to find out the concentration of the isolated strain to degrade pyrene. The inoculation of the bacteria was incubated in TSB 24 hours before centrifugation (8000 rpm, 4 ℃, 10 minutes) to harvest the cells and washed three times using a minimal liquid medium. The harvested cell solution was inoculated to OD ₄₄₀ 0.1 in the medium to which pyrene was added. Incubation was carried out at 28 ℃, 130 rpm, residual pyrene analysis was extracted with ethyl acetate corresponding to three times the amount of the medium, the ethyl acetate was evaporated in a distillation and dissolved in 2 ml of acetonitrile LC-PAH (Supelco) column ( HPLC (Shimazu) equipped with colume) was used, and the analysis conditions were 100 ml of acetone, 0.5 ml per minute, 20 µl of the sample was injected, and the detector was measured at 254 nm using a UV detector.

[Identification of Bacteria with Resolution]

Degradable strains are identified by physiological biochemistry experiments, analysis of the composition of FAME and by examining the sequence of 16S rRNA. Genomic DNA of the cells was extracted by modifying the method of Giovannoni et al. (1990). The primer used for amplification of the 16S rRNA gene is 27F ( E. coli numbering 8 to 27: 5'-AGAGTTTGATCMTGGCTCAG-3 'which specifically attaches and amplifies the 16S rDNA domain of eubacteria. ) And 1492R ( E. coli numbering 1492-1510: 5'-GGTTACCTTGTTACGACTT-3 ') were used (Lane, 1991). The composition of the PCR reaction was 10-fold reaction solution (100 mM Tris.HCl, 400 mM KCl, 500 μg / ml BSA, pH 8.3), 1.5 mM MgCl ₂ , 250 M dNTPs, 100 pmol primer, and purified DNA. (50 ng / μl) and 3U Taq polymerase were added to make a mixture of 50 μl in total, and 30 μl of mineral oil was layered to proceed with the reaction.

PCR reaction conditions were performed after the initial heat treatment at 94 ℃ for 2 minutes, 2 minutes at 94 ℃, 1 minute at 50 ℃, 2 times at 72 ℃ 30 times and finally the reaction was stopped after treatment for 20 minutes at 72 ℃ . The amplified PCR reactions were separated by electrophoresis on an agarose gel. Direct sequencing was performed using a DNA sequencing system (ABI377). The determined nucleotide sequences were compared with databases such as GenBank and EMBL (European molecular biology laboratory) to confirm the taxonomic location.

[Environmental Factors on Decomposition]

Effect of temperature and pH

In order to determine the optimal temperature in the decomposition of pyrene isolated isolates were confirmed the resolution at 4 ℃, 15 ℃, 25 ℃, 30 ℃, 37 ℃, 45 ℃.

Organic influence

Heitkamp and Cerniglia (1988) report that PAH degradation bacteria increase the resolution of PAH when small amounts of organic matter such as peptone, yeast extract, or starch are added. In this experiment, we determined whether the isolated strain can increase the degree of degradation by grasping the effect of organic matter on pyrene decomposition.

Effect of Humic Acids

Humic acid is common in soil and groundwater, which acts as a surfactant (Chiou et al., 1987). Therefore, the isolated strain was identified the effect of humic acid on the decomposition of pyrene.

Degradation of induced and non-induced cells

Enzymes involved in PAH degradation are inducible catabolic enzymes encoded in plasmids (Williams, 1981). Therefore, the purpose of this study was to investigate the effects of enzymatic induction and protein synthesis on the degradation of PAH. Induced starter culture was harvested cells by centrifugation (4 ℃, 4,000 rpm) after pre-culture (5 days, 30 ℃, 130 rpm) in TSB and washed three times with minimal medium (minimal medium). After washing, the cells were transferred to 50 ml of minimal medium containing 50 ppm of PAH, and then used after incubation for one week under the same conditions as the previous culture. For cells not induced, cells were harvested and washed in the same manner after incubation in TSB.

[Identify the decomposition route]

Tracking metabolites of pyrene may be the basis of physiological or enzymatic studies in degradation studies and may suggest ways to increase pyrene degradation by tracking the steps up to pyrolysis and CO ₂ . have. In addition, PAH degradation must be identified, not only because of its own toxicity, but also because its metabolites can react with DNA and become strong mutagens or carcinogens (AAFP, 1993). Pyrene decomposition pathways known to date are mycobacterium sp.ga pyrene cis-4,5-dihydrodiol-cis-4,5-dihydrodiol (pyrene cis-4,5-dihydrodiol), Degrading into 4-hydroxyperinaphthenone, 4-phenanthroic acid, phthalic acid, cinnamic acid (Heitakemp et al., 1988) Rhodococcus sp. Is different from Mycobacterium sp. 1,2 and 4,5-dihydroxypyrene, cis-2-hydroxy-3- (ferry). Naphthenone-9-yl) propenic acid (cis-2-hydroxy-3- (perinaphthenone-9-yl) propenic acid) and 2-hydroxy-2 (phenanthrene-5-one-4-enyl) acetic acid ( Decomposes to 2-hydroxy-2 (phenanthrene-5-one-4-enyl) acetic acid (Walter et al., 1991). Analysis of metabolites during pyrene degradation by isolated bacteria was performed by Heitakamp et al. (1988).

[Competitive Decomposition of Pyrene with Other PAHs]

In general, contamination of PAH is not contamination of a single substance, but contamination of a mixture of PAHs. Therefore, low molecular PAH (3-ring or less) such as phenanthrene was added to the co-substrate to determine the kinetics of pyrene decomposition. The removal efficiency of high pyrene was examined.

[Decomposition in the Experimental Laboratory]

The uncontaminated soil was made by using a polyethylene box having a width × length × height of 30 × 30 × 15 cm. The amount of pyrene, a substrate, was added at a rate of 300 mg per kg of soil, and the isolated strain was added at 10 ⁶ / g, and 1 g of three soils were collected at 3 days intervals, and the remaining pyrene was mixed. Analyzed. Soil pH, particle size analysis, soil moisture content, ammonia salt nitrogen, nitrite nitrogen, and nitrate nitrogen were performed according to the Methods of soil analysis.

The following results were obtained for the above experiment.

1. Isolation of Strains

The isolated C2-3 strain was able to observe a distinct degradation ring after one week of culture on the minimal solid medium to which pyrene was applied (FIG. 1). Strain C2-3 is Gram-positive and 16S rRNA sequencing analysis revealed a total of 1,474 bp. Comparing the determined 1,474 bp sequence with GenBank's database, C2-3 was identified as Mycobacterium sp. Mycobacterium gilvum was the strain having the most identical nucleotide sequence to C2-3, and 1,421 bp of the 1,428 bp sequence was identical.

The results of comparative analysis of 16S rRNA sequences with Mycobacterium sp. 1,421 bp of overlapping regions of BB1 and 1,428 bp overlapped, indicating a similarity of 0.9950. M. sp. PAH135 is consistent with 1,423 bp of 1,451 bp, showing a similarity of 0.9807, and known to degrade 3-ring anthracene M. Anthracene was matched with 1,404 bp of 1,448 bp, showing the lowest similarity, 0.9696 (Table 1).

Strain Same sex GenBank Access Number M. sp. RJGII135 M. Chlorophenolicus (PCP-1) M. sp. BB1 M. paraportuitum M. sp. PAH135 M. sp. RF002 M. anthracenicum M. sp. WF2 M. sp. GP1 M. sp. CH1 1452/1478 (0.9824) 1452/1478 (0.9824) 1421/1428 (0.9950) 1435/1456 (0.9855) 1423/1451 (0.9807) 1405/1432 (0.9811) 1404/1448 (0.9696) 1261/1276 (0.9882) 1273 / 1298 (0.9807) 1294/1331 (0.9722) U30661 X79094 X81996 X93183 X84978 U90876 Y15709 U90877 AJ012626 AF054278

Table 1 compares the known aromatic degradation strain Microbacterium sp. And 16S rRNA sequences.

The optimum temperature for Mycobacterium sp. C2-3 was 27 ° C. in TSB, showing no significant growth at 37 ° C. and 44 ° C. (FIG. 2). Also, on the solid medium, colonies were produced that can be observed with the naked eye within 7 days at 27 ℃, and the shape of the colonies showed the characteristics of medium size, smooth and shiny, the color was yellow, and the incubation time The longer it turned into darker yellow.

Hydrocarbons that Mycobacterium sp. C2-3 can decompose are shown in Table 2. Interestingly, they could not break down low molecular weight PAHs such as naphthalene, although they could break down high molecular weight PAHs such as pyrene and fluoranthene. In contrast, n-alkane, such as hexadecane and heptadecane, was found to degrade relatively well (Table 2).

Compound Dissolution rate Naphthalene fluorene phenanthrene fluoranthene pyrene n-alkane -+ (No coloring) + + + (yellow) + + (red) + + + (pale yellow) + +

Table 2 is a table showing the degree of decomposition of aromatic compounds by the isolated strain.

(+: Weak, ++: excellent, +++: very excellent, analyzed by spraying on the minimum medium method).

In the phenanthrene, fluoranthene and pyrene-coated media, yellow, red, and hazy yellow, respectively, presumably decomposed products, were observed to diffuse around the colonies.

The reason why the pollutant should be removed from the natural environment is often due to its own toxicity. Therefore, even microorganisms that degrade PAH can be inhibited by the concentration of PAH. M. sp. C2-3 degraded most of the pyrene given during 14 days of incubation up to 250 ppm. However, in case of 500 ppm, about 45% was decomposed and about 80% of pyrene was decomposed at 28 days. In addition, the addition of 1000 ppm of the substrate did not show a significant level of resolution (FIG. 3).

In the case of phenanthrene, it is known to be relatively less toxic than pyrene. M. sp. C2-3 decomposed almost all phenanthrene in 7 days up to 250 ppm. However, in the case of 500 ppm, about 50% was decomposed at 7 days and 70% at 14 days. Compared with pyrene 14 days culture, pyrene decomposed about 45% while phenanthrene decomposed about 70%, even at 1000 ppm decomposed about 23% (Fig. 4). This is the M. sp. C2-3 shows that pyrene has much lower decomposition rate at the same time and at the same concentration than phenanthrene.

After incubation in TSB, it is inoculated again with a minimum liquid medium containing 50 ppm of phenanthrene, fluoranthene and pyrene, followed by incubation for 1 week to induce an enzyme that degrades each PAH, followed by a minimum of 300 ppm of each PAH. As a result of re-inoculation into the liquid medium and measuring the degree of degradation, in the case of phenanthrene (FIG. 5), the experimental group derived from fluoranthene and fluorine showed a sharp increase in degradation from 24 hours. However, in the experimental group induced in the lactic acid bacteria culture medium, the degree of degradation increased after 48 hours. Controls incubated in TSB increased degradation after 88 hours. It was confirmed that the induction of enzymes involved in lactic acid bacteria culture medium is induced by lactic acid bacteria culture medium, but also induced by high molecular weight PAH such as pyrene and fluoranthene.

In the case of pyrene, the experimental group induced by pyrene shows a sharp increase in degradation after 24 hours of induction (lag time) (Fig. 7). However, the experimental group induced in the lactic acid bacteria culture shows the same pattern as the control induced in TSB. This is seen to be enzymes that break down pyrene means that has poor oil at all by the lactic acid bacteria culture fluid and the case was derived from pyrene-induced decomposition of the lactic acid bacteria culture fluid Mycobacterium sp. The degradation pathway of C2-3 is thought to include the degradation of lactic acid bacteria culture medium during pyrene decomposition.

M. sp. The effect of humic acid on pyrene decomposition of C2-3 is shown in FIG. 8. When the 500 ppm and 1000 ppm humic acid was treated compared to the control, the degree of decomposition increased. In the experimental group to which humic acid was added, about 68% of the decomposition was already performed on the second day of culture, and almost all pyrene was removed from the medium after three days.

One method of increasing the degree of decomposition of hydrocarbons is the addition of surfactants. Therefore, the addition of surfactant is M. sp. In order to investigate the effect of C2-3 on pyrene decomposition, it was treated with Triton X-100 (manufactured by Junsei Chemical Co., Japan), Tween 80 (manufactured by Sigma, USA), and SDS (manufactured by Sigma, USA). (FIG. 9). In the experimental group treated with Tween 80, 50% resolution was observed in the 2-day culture, and more than 95% of pyrene was added after 3 days. In the case of SDS, the degree of degradation was slightly lower than that of Tween 80, about 30%, and more than 95% of pyrene was decomposed after 4 days of incubation. Triton X-100, however, showed no resolution.

These surfactants are M. sp. To investigate the effect of C2-3 on cell growth, Tween 80 and SDS promoted cell growth after adding surfactant to each medium with glucose as a carbon source. 10) In the case of Triton (X-100) completely inhibited. This is because Tween 80 or SDS acts as a cofactor rather than as a carbon source. The reason why these surfactants increased the degree of decomposition during pyrene decomposition is that these surfactants act as cofactors and pyrenes. It is thought that the solubility was increased by increasing the solubility. However, for Triton X-100, M. sp. The growth of C2-3 itself is believed to be toxic.

Pyrene digestion bacteria isolated in the present invention was identified as Mycobacterium sp. Mycobacterium sp. Is a microorganism widely present in the natural environment (Barksdale and Kim, 1977). It is normal-butane (n-butane) and 2-butanone (2-butanone) (Phillipa and Perry, 1974) and gaseous unsaturated hydrocarbons ( Debont, et al. 1980), cycloparaffinic hydrocarbons (Beam and Perry, 1974), n-alkyl-substituted cycloparaffins (Beam and Perry, 1974), methyl ketone ketone) (Beam and Perry, 1974) and aliphatic hydrocarbons (Hou, 1982).

Bacteria degrading more than 3-ring PAH have been isolated for the past 20 years, but few of them are able to decompose 4-ring PAH under conditions without surfactants or cofactors. Mycobacterium sp. PYR-1 and Mycobacterium sp. RJGII-135 strains all require cofactors in pyrene digestion. Recently, Mycobacterium sp. It has been reported that CH1 degrades pyrene in the absence of cofactors (Churchill et al., 1999). This strain of CH1 is very sensitive to the fact that the properties of C2-3 and PAH degradation in this experiment do not degrade naphthalene, and that it degrades normal-alkanes, and does not require cofactors or surfactants for 4-ring degradation. similar. However, as shown in Table 2, the sequence of the 16S rRNA is 1,2722 bp of 1,331 bp, which is very low as 0.9722. In addition, the CH1 strain did not respond to nah irradiation as a result of hybridization using the nah gene probe, which has been studied the most in PAH degradation. Implying that other systems will exist. This may indirectly explain that C2-3 does not decompose naphthalene while decomposing PAH over 3-ring.

Claims (7)

Mycobacterium of Accession No. KCTC 8975P having an optimum incubation temperature of 20 ° C. to 30 ° C., having a rapidly degrading degree after 24 hours of lag time and having a resolution of 3-ring or more polycyclic aromatic hydrocarbons. Leeum Esp. C2-3 ( Mycobacterium sp. C2-3, KCTC 8975P). The method of claim 1, The polycyclic aromatic hydrocarbon having three or more rings is Mycobacterium sp. Which is any one selected from the group consisting of pyrene, phenanthrene, and fluoranthene. C2-3. The method of claim 1, Mycobacterium sp. C2-3 is Mycobacterium sp. Which has the resolution of n-alkane. C2-3. Mycobacterium of Accession No. KCTC 8975P having an optimum incubation temperature of 20 ° C. to 30 ° C., having a rapidly degrading degree after 24 hours of lag time and having a resolution of 3-ring or more polycyclic aromatic hydrocarbons. Leeum Esp. Oil decomposition composition comprising C2-3 ( Mycobacterium sp. C2-3, KCTC 8975P) as an active ingredient. The method of claim 4, wherein The oil degradation composition in which the composition further comprises a surfactant. The method of claim 4, wherein Oil decomposition composition further comprises a humic acid (humic acid) composition. A method for purifying contaminated soil comprising decomposing a polycyclic aromatic hydrocarbon using the oil decomposition composition having a polycyclic aromatic hydrocarbon (PAH) decomposition activity according to claim 4.

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