KR20040002268A - Novel cellulomonas sp. with degradable activity of pahs and clarifying method of pollutants having pahs using the same - Google Patents

Abstract

PURPOSE: Provided are novel Cellulomonas sp. with degradable activity of PAHs and a purification method of pollutants having PAHs using the same, thereby effectively purifying pollutants having PAHs. CONSTITUTION: A purification method of pollutants having PAHs is characterized by isolating and identifying Cellulomonas sp. B4(KCTC 10259BP) having degradable activity of PAHs from oil contaminated soil, conforming the activity of Cellulomonas sp. B4(KCTC 10259BP), and purifying pollutants having PAHs using the Cellulomonas sp. B4(KCTC 10259BP).

Description

Novel Cellulomonas Ｂ4 Strain with PHAs Degradation and Purification Method of PHAs Contaminant Using the Same {Novel Cellulomonas sp. with degradable activity of PAHs and clarifying method of pollutants having PAHs using the same}

The present invention relates to a novel polyaromatic hydrocarbons (PAHs) decomposition strain and a method for purifying pollutants using the same, and more particularly, from the soil contaminated by oil, Cellulomonas sp. B4 (KCTC). 10259BP) isolates and strains to purify polyaromatic hydrocarbons (PAHs) contaminants using the strain.

Polyaromatic hydrocarbons (PAHs), also known as polycyclic aromatic hydrocarbons, are potentially harmful substances that cause cancer or mutations in two or more benzene rings. In the US EPA, 16 PAHs compounds among 129 priority pollutants Is specified. PAHs are hydrocarbons composed of carbon and hydrogen that contain other elements such as nitrogen, sulfur, and oxygen, and have several benzene ring structures, which can be formed when incomplete combustion of fossil fuels or organic materials. It is also caused by. Today, due to industrial development, the use of fossil fuels increases, the generation of PAHs is increasing, and the generation of PAHs by automobiles is known to be very serious. In particular, it is reported that the concentration of PAHs in the air is higher in the urban area or the airport area where the relevant factories are located, or the vehicle area is higher than other areas. PAHs material diffused into the natural environment may exist in the gaseous state at high temperature above 150 ℃, but condensate at room temperature and adhere to small particles such as fly ash and enter the human respiratory system through various paths such as water, soil, and air, and adversely affect health. .

PAHs in nature are produced, transported, and extinguished by various causes, immobilized in soil by physical adsorption and volatilization, diffused into the atmosphere, and decomposed by chemical reactions. In particular, it is known to be eliminated by photolysis, chemical oxidation and vaporization, but mainly by microorganisms.

PAHs in soil can accumulate in the atmosphere or by biosynthesis of bacteria, algae or higher plants. These PAHs are reported to be contained in lettuce, tomatoes, cooking oil, bok, coffee, smoked meat, etc. PAHs in the water are found in the soil with rainwater, air pollutants, industrial wastewater, etc. It is known to enter the path. Contaminants introduced through these pathways can be concentrated through the food chains of plankton, small meats, and large meats, affecting the human body. PAHs are nonpolar, hydrophobic, and chemically stable environmental pollutants, whose properties are determined by the arrangement of benzene rings. The more benzene rings, the less water solubility or volatility. Therefore, in the ecosystem, they are mainly absorbed by organic matter or fine particles in the soil. The biodegradation rate of PAHs varies widely and depends not only on the structure of the PAHs compound, but also on the growth and morphology of the microorganisms present and the physicochemical parameters, and their desorption rate greatly affects the degradation rate of the microorganisms. In addition, these substances are concentrated in living organisms, including microorganisms, or completely decomposed into CO ₂ and H ₂ O by mineralization.

Recognizing the necessity of technology development for the purification of the environment contaminated by PAHs, many studies have been conducted, most of which focus on physicochemical treatment, which requires a lot of cost and equipment, and causes secondary pollution. This is not the ultimate environmental cleanup technology in this respect. However, biodegradation of PAHs by microorganisms is a technology that can maximize the activity of specific microorganisms isolated from nature and safely and economically handle contamination by PAHs.

In addition, low molecular weight components such as naphthalene in PAHs are known to be degraded by microorganisms, but high molecular weight components with high benzene rings such as penanthrene and pyrene are known to be decomposed by microorganisms. It does not disintegrate well and natural purification is not easy. Therefore, it is urgent to isolate microorganisms capable of degrading high molecular weight PAHs, and many strains have been found through many studies. Previous studies on the decomposition of PAHs have tested the resolution using a single PAHs, but in actual contaminated areas, there is not a single PAHs but a mixture of two to five or more benzene rings. Is contaminated Therefore, in view of this, there is a need for research on the biological restoration technology of the contaminated environment by PAHs in a mixed state.

Therefore, in this study, microorganisms that decompose PAHs, which are environmental pollutants, are isolated, the optimum conditions for degradation are examined, and fluorene, pyrene, benzo [a] pyrene, etc. are used. To determine the degradation activity by the microorganism.

Benzo [a] pyrene, also known as 3,4-benzopyrene, is a carcinogenic substance and has a boiling point of 310 to 312 ° C (10 torr) as a yellow crystal having a formula C20H12, melting point of 179.9 to 180.3 ° C.

Accordingly, an object of the present invention is to provide a novel Cellulomonas sp. B4 (KCTC 10259BP) strain having a resolution of polynuclear aromatic hydrocarbons (PAHs). Another object of the present invention is to provide a method for purifying a material contaminated by polynuclear aromatic hydrocarbons (PAHs) using a novel Cellulomonas sp. B4 (KCTC 10259BP) strain.

The object of the present invention is to isolate and identify strains having PAHs resolution from soil contaminated with oil, and to decompose PAHs homogeneous or admixture of isolated Cellulomonas sp. B4 (KCTC 10259BP) strains. After confirming the activity and degradation activity according to the concentration of PAHs, it was achieved by the purification of soil contaminated with PAHs using the novel Cellulomonas sp. B4 (KCTC 10259BP) strain of the present invention.

Figure 1 is a graph showing the PAHs single substance resolution of the cellulomonas ( Cellulomonas sp.) B4 (KCTC 10259BP) strain of the present invention.

Figure 2 is a graph showing the resolution of the PAHs complex of the cellulomonas ( Cellulomonas sp.) B4 (KCTC 10259BP) strain of the present invention.

Figure 3 is a graph showing the degradation activity according to the concentration of polycyclic aromatic hydrocarbons (PAHs) of the present invention Cellulomonas sp. B4 (KCTC 10259BP) strain.

Figure 4 is a graph showing the PAHs resolution in soil contaminated with PAHs single material (200ppm) of the Cellulomonas sp. B4 (KCTC 10259BP) strain of the present invention.

5 is a graph showing PAHs resolution in soil contaminated with PAHs complex (200ppm) of the present invention Cellulomonas sp. B4 (KCTC 10259BP) strain.

The present invention contaminates soil contaminated with oil with PAHs and concentrates culture of contaminated soil microorganisms, and then isolates and identifies a novel Cellulomonas sp. B4 (KCTC 10259BP) strain having PAHs resolution. Doing; Inoculating MSM medium containing Cellulomonas sp. B4 (KCTC 10259BP) strain with PAHs to confirm the degradation activity of PAHs single substance or mixture; cellulomonas sp. B4 of the present invention (KCTC 10259BP) inoculating the MSM medium added to the PAHs concentration strain strain to measure the degradation activity; After contaminating the soil with PAHs single or complex material, the present invention consists of inoculating and purifying Cellulomonas sp. B4 (KCTC 10259BP) strain.

Hereinafter, the present invention will be described in detail.

The soil used for the present invention was collected from Yasan, Yongin, Gyeonggi-do, and used after passing a 2 mm sieve. In order to understand the physical properties of the soil, pH, water content, moisture retention, specific gravity, particle size distribution, and organic substance content were measured. The pH and moisture content were measured according to the method suggested by the Korean Soil Pollution Process Test, and the soil particle size distribution was determined by sieving 100 g of uncontaminated dried natural soil by mesh size (0.075mm, 0.2mm, 0.425mm, 1mm). , 2mm), and separated according to the size determined based on the Unified Soil Classification System (USCS).

In this study, the pH and moisture content of soils collected for soil cleaning experiments were measured by soil pollution process test method. The pH was about 5.4, the moisture content was about 16.5%, and the water retention was 37.3%. The particle size distribution of was 51.7% of coarse in sand and 46.3% of fine, which accounted for more than 98% of the total soil. Was%.

Decomposition of PAHs contaminants with the novel Cellulomonas sp. B4 (KCTC 10259BP) strain of the present invention and extraction of PAHs from the liquid phase to determine their degradation activity was carried out in 50 mL of dichloromethane in the reaction flask. After the addition, the mixture was mixed well using a separatory funnel, and left to stand for 15 minutes. The solvent layer was collected, separated into an extraction flask, and repeated twice to recover the remaining PAHs. The recovered solvent is volatilized using a rotary evaporator (EYELA, Japan), and then concentrated to 1 mL of dichloromethane and analyzed using HPLC (Waters, USA). In addition, the extraction of PAHs from soil uses EPA 3550M. Anhydrous sodium sulfate (Na ₂ SO ₄ ) equal to 10 g of contaminated soil is mixed to remove moisture from the soil, and 20 mL of extraction solvent n-hexane (n-hexane) is added, followed by ultrasonic desorption for 30 ° C. for 30 minutes. This is repeated twice. The extracted solvent was analyzed by GC / FID (HP 6890, USA) by volatilizing the solvent using a rotary evaporator and then concentrating the residual oil with 1 mL of n-hexane.

In order to isolate and identify microorganisms with high PAHs decomposition efficiency, the soil contaminated with oil-contaminated soil with PAHs, followed by enrichment of the contaminated soil microorganisms for 1 month, and 0.1g of contaminated soils enriched for 1 month, 50mL minimum nutrient medium (MSM : Minimal Salt Medium) and incubated at 25 ℃ for 7 days at 150rpm. After diluting 1 mL of the solution incubated for 7 days in MSM medium, spread the plate on nutrient agar medium, incubate at 25 ° C. for 3 days to separate pure colonies, and observe the size, shape, color, and the like. The PAHs resolution of the isolated degradation strain was confirmed by the color change caused by the reduction reaction of 2,6-DCPIP (Dichlorophenol-Indophenol). The isolated strain is incubated in TSBA (Trypticase soy broth agar) for one day, and then extracted and identified by fatty acid by MIDI (Microbial Identification System, Microbial ID Inc., Newark, Del.) Method. As a result, the isolated strain was identified as Cellulomonas sp. And it was Gram-positive bacteria, and Cellulomonas sp. Named B4 (KCTC 10259BP). The novel strain of the present invention was deposited with the accession number KCTC 10259BP dated May 23, 2002 to the Gene Center in the Biotechnology Research Institute, an international depository.

In the present invention a novel cellulose Pseudomonas (Cellulomonas sp.) B4 by (KCTC 10259BP) strain cellulose to investigate the decomposing activity of PAHs Pseudomonas (Cellulomonas sp.) B4 of single PAHs and mixed PAHs by (KCTC 10259BP) strains Degradation activity is measured. Degradation activity of single PAHs was determined by dissolving each PAHs in ether, placing them in a flask, evaporating solvents, and using them as energy and carbon source for inoculating microorganisms, and adding MSM medium, followed by Cellulomonas sp. B4 (KCTC 10259BP). ) Degradation activity is measured by inoculating strains. As a result, the degradation activity of a single PAHs by the novel Cellulomonas sp. B4 (KCTC 10259BP) strain of the present invention was 20 than that of the control without the addition of Cellulomonas sp. B4 (KCTC 10259BP) strain. It has a high decomposition rate of more than%. In fact, since PAHs exist in the form of complex mixtures, the degradation activity of mixed PAHs is also measured. Various PAHs dissolved in ether are mixed and added to MSM medium, and the degradation activity is measured by inoculating Cellulomonas sp. B4 (KCTC 10259BP) strain. In general, composite PAHs tend to have lower degradation rates than single PAHs, but degradation of some PAHs in composite PAHs tends to be somewhat higher.

Initial contamination levels also play a very important role in the selection of applicable strains. Novel Cellulomonas of the Invention for Economic and Efficient Biological Treatment of PAHs ContaminantsCellulomonassp.) Degradable concentrations of B4 (KCTC 10259BP) strains should be investigated, and in the case of PAHs contaminated areas above the degradable concentrations, consideration should be given to the application of other treatment techniques. The present invention cellulomonas (Cellulomonassp.) In order to determine the minimum and maximum limit contamination levels decomposed by the B4 (KCTC 10259BP) strain, various concentrations of PAHs were contaminated in MSM medium and then cellulomonas (Cellulomonassp.) Inoculate B4 (KCTC 10259BP) to determine the degradation activity of PAHs. As a result, the present invention novel cellulose monas (Cellulomonassp.) B4 (KCTC 10259BP) strain It can be seen that it shows the highest decomposition rate at 20 ppm concentration of PAHs and can decompose at high concentration of 500 ppm.

PAHs are actually exposed to the environment due to spills caused by bottom erosion of older oil storage tanks, or accidents caused by sea and land transport. Efficient treatment of PAHs in contaminated soils has great significance in terms of economic utilization due to site utilization. In the present invention, PAHs contamination of the novel Cellulomonas sp. B4 (KCTC 10259BP) strain Soil degradation activity is measured. Soil samples inoculated with cellulomonas sp. B4 (KCTC 10259BP) were inoculated into soil samples containing several types of single PAHs in uncontaminated soil, and the degradation activity was measured in cows after controlling the water content in the soil. In addition, inoculated with cellulomonas ( Cellulomonas sp.) B4 (KCTC 10259BP) strain in soil samples containing mixed PAHs mixed with various PAHs in uncontaminated soil, and then degraded in cows after controlling the water content in the soil. As a result, the soil to which the novel Cellulomonas sp. B4 (KCTC 10259BP) strain of the present invention is added exhibits a 10-20% higher degradation rate than that of the control without the microorganisms. In addition, when the PAHs are mixed, the degradation rate is lowered, which is different from the substances that are easily degraded due to the decomposition characteristics of the isolated strains, and the novel cellulomonas ( Cellulomonas sp.) Of the present invention is contaminated with soil properties . It is presumed that the contact opportunities of B4 (KCTC 10259BP) strains and the degree of extraction from soils vary greatly according to experiments.

Example 1 Isolation and Identification of PAHs Degrading Strains

In order to separate microorganisms with high PAHs decomposition efficiency, the soil polluted with 500 ppm of fluorene, pyrene, and benzo [a] pyrene, and the microorganisms were enriched for 1 month, and the soil was cultured for 1 month. 0.1 g was added to 50 mL of minimal nutrient medium (MSM: Minimal Salt Medium) and incubated at 150 ° C. at 25 ° C. for 7 days. The composition of MSM medium used was NaNO ₃ 4 g / L, KH ₂ PO ₄ 0.15 g / L, Na ₂ HPO ₄ 0.5 g / L, MgSO ₄ 7H ₂ O 0.2 g / L, FeCl ₃ · 6H ₂ O 0.005 g / L, CaCl ₂ H ₂ O 0.01 g / L. After diluting 1 mL of the solution incubated for 7 days in MSM medium, the plate was plated on nutrient agar medium, and cultured at 25 ° C. for 3 days to separate pure colonies, and observed the size, shape, color, and the like. The PAHs resolution of the isolated strain was confirmed by the color change caused by the reduction reaction of 2,6-DCPIP (Dichlorophenol-Indophenol). DCPIP experiments were performed as follows. 10% (v / v) was inoculated with 10% (v / v) isolates in a minimum medium containing crude oil and DCPIP (3 g / L) 2.5% was added as a reaction reagent and incubated at 25 ° C. Four cultures of PAHs degrading strains were isolated from the colorless to blue sample after incubation.

After culturing the isolated strain in TSBA (Trypticase soy broth agar) for one day, the present inventors identified and extracted fatty acids by MIDI (Microbial Identification System, Microbial ID Inc., Newark, Del.) Method, Cellulomonas sp. And Gram-positive bacteria (Table 1). Cellulomonas sp. Named B4 (KCTC 10259BP). The novel strain of the present invention was deposited with the accession number KCTC 10259BP dated May 23, 2002 to the Gene Center in the Biotechnology Research Institute, an international depository.

Mycological characteristics of the cellulomonas sp. B4 (KCTC 10259BP) strain of the present invention shape size Color Morphology Gram Catalase Round Radius 1mm yellow Cocci + +

Comparison of PAHs Resolution of Cellulomonas sp. B4 (KCTC 10259BP) Strains and Other Microorganisms of the Invention microbe Fluorene Pyrene Benzo [a] pyrene Concentration (ppm) % Decomposition Concentration (ppm) % Decomposition Concentration (ppm) % Decomposition Cellulomonas sp. B4 (KCTC 10259BP) strain of the present invention 200 33.7 200 27.7 200 21.5 Mixed culture of several microorganisms in contaminated areas with PAHs ¹ 5 25 - - - - Bjerkandera adusta ² 5 44 - - - - Pleurotus ostreatus ³ 5 31 - - - - Penicilium simplicissimum ⁴ - - 100 18 - - Mycobacterium PYR-1 ⁵ - - 100 26 - - Rhodococcus UM1 (Rhodococcus sp. UW1) ⁶ - - - - 250 11 Incubate for 105 days Burkholderia cepacia ⁷ - - - - 50 20-30 Incubate for 63 days References ¹ SY Yuan, SH wei and BV Chang, Biodegradation of polycyclic aromatic hydrocarbons by a mixed culture. Chemosphere , 41 (9), pp. 1463-1468 (2000) ² A. Schutzendubel, A. Majcherczyk, C. Johannes and A. Huttermann, Degradation of fluorene, anthracene, phenanthrene, fluoranthene, and pyrene lacks connection to the production of extracellular enzymes by Pleurotus ostreatus and Bjerkandera adusta. International Biodeterioration & Biodegredation , 43 (3), pp. 93-100 (1999) ³ Saraswathy, Ambujom; Hallberg, Rolf, Degradation of pyrene by indigenous fungi from a former gasworks site. FEMS Microbiology Letters , 210 (2) ,, pp. 227-232 (2002) ⁴ N. Ramirez, T. Cutright and LK Ju, Pyrene biodegradatin in aqueous solutions and soil slurries by Mycobacterium PYR-1 and enriched consortium. Chemosphere , 44 (5), pp. 1079-1086 (2001) ⁵ C. Ravelet, C. Grosset, B. Montuelle, JL Benoit-Guyod and J. Alary, Liquid chromatography study of pyrene degradation by two micromycetes in a freshwater sediment. Chemosphere , 44 (7), pp. 1541-1546 (2001) ⁶ M. Wolter, F. Zadrazil, R. Martens, M. Bahadir, Degradation of eight highly condensed polycyclic aromatic hydrocarbons by Pleurotus sp. florida in solid wheat straw substrate. Applied Microbiology and Biotechnology , 48, pp. 398-404 (1997) ⁷ AL Juhasz, ML Britz, GA Stanley, Degradtion of fluoreanthene, pyrene, benz [ a ] anthracene and dibenz [ a, h ] anthracene by Burkholderia cepacia, Journal of Applied Microbiology , 83, pp. 189-198 (1997)

Example 2 Inventive Novel Cellulomonas Cellulomonas sp.) Degradation Activity of PAHs by B4 (KCTC 10259BP) Strain

Experimental Example 1: the present invention cellulose monas ( Cellulomonas sp.) Degradation of single PAHs by B4 (KCTC 10259BP) strain

To measure the PAHs degradation activity of the isolated B4 (KCTC 10259BP) strain, fluorenes, pyrenes, and benzo [a] pyrenes with different numbers of benzene rings were dissolved in ether, and the solvents were volatilized at a concentration of 200 ppm. It was used as the energy and carbon source of the inoculated microorganism, and after adding 50 mL of MSM medium, 0.5 mL of the decomposed microorganism was inoculated, and the experiment was carried out at 150 rpm for 25 days at 25 ° C.

The results are shown in FIG. 1, 34% fluorene, 28% pyrene, 21% benzo [a] pyrene. This was shown to have a degradation rate of more than 20% higher than the control group without the degradation strain.

Experimental Example 2 Degradation of Mixed PAHs by the Novel Cellulomonas sp. B4 (KCTC 10259BP) Strain

Since the actual PAHs exist in the form of a complex mixture, decomposition experiments were performed by mixing three materials at high concentrations of 200 ppm each. To determine the resolution of mixed PAHs of isolated B4 (KCTC 10259BP) strains, fluorene, pyrene, and benzo [a] pyrene were dissolved in ether, 200ppm of each PAHs were added to MSM medium, and B4 (KCTC 10259BP) After inoculating the strain, degradation experiments were performed at 150 ° C for 25 days at 25 ° C. The results are shown in Figure 2, fluorene was 23%, pyrene 21%, benzo [a] pyrene was found to decompose 24%. The composite PAHs tended to have lower degradation rates of fluorene and pyrene than single PAHs, but the decomposition rates of benzo [a] pyrene tended to be somewhat higher.

Example 3 Inventive Novel Cellulomonas Cellulomonas sp.) Degradation activity according to PAHs concentration of B4 (KCTC 10259BP) strain

In order to determine the minimum and maximum limit contamination concentrations that can be degraded by the novel Cellulomonas sp. B4 (KCTC 10259BP) strain, 50 mL of MSM medium was used in 20 ppm, 50 ppm, 100 ppm, 200 ppm, and 500 ppm fluorene concentrations. Cellulomonas sp. B4 (KCTC 10259BP) was inoculated with a sample of MSM medium contaminated with single PAHs of pyrene, benzo [a] pyrene, respectively. All samples were run for one week at 25 ° C. in triplicate.

The results are shown in Figure 3, the mixed PAHs showed the highest decomposition rate at 20ppm concentration and was confirmed that can be decomposed at a high concentration of 500ppm. In case of fluorene, the highest decomposition rate was 42% at 20 ppm, and the decomposition rate was sharply reduced to 25% at 50 ppm, but the decomposition rate was maintained at 19% up to 500 ppm. It was possible to find out. Pyrene and benzo [a] pyrene also showed high decomposition rates of 31% and 22% at low concentrations of 20 ppm, but the higher the concentration, the lower the decomposition rate (Fig. 3). Therefore, it was confirmed that the cellulomonas ( Cellulomonas sp.) B4 (KCTC 10259BP) strain can degrade the mixture of PAHs having three or more benzene rings.

Example 4 Inventive Novel Cellulomonas Cellulomonas sp.) PAHs Soil Degradation Activity of B4 (KCTC 10259BP) Strains

Three soil samples containing 200 ppm of fluorene, pyrene, and benzo (a) pyrene in 10 g of uncontaminated soil were inoculated with Cellulomonas sp. B4 (KCTC 10259BP) strain. In order to control the water content to 50%, 1.5 mL of distilled water was added, followed by decomposition experiments at 25 ° C. in the dark for 7 days. In addition, a strain of Cellulomonas sp. B4 (KCTC 10259BP) was added to one soil sample containing mixed PAHs containing 10 ppm of fluorene, pyrene, and benzo [a] pyrene in 10 g of uncontaminated soil. After inoculation, the experiments were performed in the same manner as the soil test in a single component.

The results are shown in FIGS. 3 and 4. When contaminated with a single PAHs 52% fluorene, 40% pyrene, 36% benzo [a] pyrene was decomposed (Fig. 3), 29% fluorene when contaminated with mixed PAHs, pi 49% lene and 15% benzo [a] pyrene were decomposed (FIG. 4). It was found to have a 10-20% higher degradation rate than the control without the degradation microorganisms. The results of the experiments show that the degradation rate decreases when PAHs are mixed, which is different from easily decomposable substances due to the decomposition characteristics of the isolated strains, and contaminants and the novel cellulomonas of the present invention ( Cellulomonas) according to the soil experimental characteristics. sp.) It is presumed that the contact opportunities of B4 (KCTC 10259BP) strains and the degree of extraction from soils vary greatly from experiment to experiment.

As described above, the present invention Cellulomonas sp. B4 (KCTC 10259BP) strain is capable of degrading not only a single substance of polynuclear aromatic hydrocarbons (PAHs) but also mixed substances and contaminated by polynuclear aromatic hydrocarbons (PAHs). It is a very useful invention for the environmental industry because it has an excellent effect of purifying the material.

Claims (3)

Cellulomonas sp. B4 (KCTC 10259BP) strain characterized by the resolution of polynuclear aromatic hydrocarbons (PAHs). A method for purifying substances contaminated by polynuclear aromatic hydrocarbons (PAHs) using the Cellulomonas sp. B4 (KCTC 10259BP) strain of claim 1. 3. The purification method according to claim 2, wherein the polynuclear aromatic hydrocarbons (PAHs) are solely or mixed with fluorene, pyrene, and benzo [a] pyrene.