KR100873054B1 - Complex bacteria(kacc 91316p) for degradation of volatile aromatic hydrocarbons - Google Patents

Abstract

A composite strain BTEX 1, and a method for decomposing soil-polluting materials by using the composite strain BTEX 1 are provided to decompose benzene, toluene, ethylbenzene, xylene or their mixture biologically and rapidly. A composite strain BTEX 1 is the composite strain BTEX 1 (KACC 91316P) of Rhodococcus sp. and Ochrobactrum intermedium and decomposes benzene, toluene, ethylbenzene, xylene or their mixture. Benzene, toluene, ethylbenzene, xylene or their mixture is decomposed by injecting the composite strain BTEX 1 (KACC 91316P) into the medium polluted with benzene, toluene, ethylbenzene, xylene or their mixture.

Description

Complex bacteria (KACC 91316P) for degradation of volatile aromatic hydrocarbons}

The present invention relates to a composite strain BTEX 1 (KACC 91316P) that is a representative of soil pollutants BTEX (benzene, toluene, ethylbenzene, xylene) is completely biodegradable and harmless.

BTEX is an aromatic compound in which one benzene ring is free of methyl and ethyl groups or one or two are attached. BTEX is an aromatic hydrocarbon containing one benzene ring that is difficult to decompose and is relatively more water soluble than other light hydrocarbons. These pollutants are classified as carcinogens or potential carcinogens by the US Environmental Protection Agency. 80% of petroleum is made up of BTEX, and it is a representative substance of volatile organic compounds (VOCs) that occur in groundwater or soil by management alone, oil storage tanks, spills to oil pipelines, and many industries not only in vehicles but also in industry. The released BTEX pollutes the surrounding environment, and when inhaled into the human body through the respiratory system, leukemia, lymph cancer, blood cancer, lesions, and cell tumors occur.

According to the 2005 Ministry of Environment's data, 56 of the nation's soil pollution surveys exceeded the standard 56 out of 3,902 sites (1.4%) exceeded the soil pollution concern standards, and 22 of them (0.6%) met the standard. Exceeded. In factories and industrial areas, Cu, Zn, BTEX, etc. were polluted due to oil leakage from manufacturing process and oil tanks, and in the transportation related facilities, soil pollution was relatively high due to oil handling carelessness and tank aging. Appeared.

In some industrial complexes, the BTEX pollution level was 6230mg / kg, which was much higher than 80mg / kg of concern and 200mg / kg of countermeasure. Techniques for removing organic contaminants from these contaminated soils can be divided into physical, chemical and biological methods. Physical removal techniques include soil washing, thermal desorption, activated carbon fixation, solidification stabilization, soil vapor extraction, and sequestration after soil excavation. Chemical removal methods include incineration of soil to completely oxidize contaminants, soil vitrification, and solvent extraction. The biological methods include the on-site method of biodegradation after excavation and the in-situ method of decomposing and removing contaminants by activating microorganisms by injecting microorganisms or injecting air without excavation. There is this.

Among these various treatment methods, biological treatment methods are known as environmentally friendly and economical methods, and in fact, the amount of soil treatment by biological treatment methods is known to increase in the United States. Degradation of organic substances by microorganisms is carried out by oxidation or reduction reactions of enzymes present in bacteria, and in general, substances having a low molecular weight are faster than those of a large substance. Since the degree of degradation by microorganisms varies depending on the nature of the organic material, biological treatment methods may not be applicable to all organic pollutants. At present, the present invention is limitedly applied to low molecular organic pollutants having high volatile and high water solubility.

Since BTEX is relatively volatile and water-soluble and is known as a toxic substance, the development and study of strains that can efficiently decompose BTEX are in progress.

Therefore, the present invention is to solve the problems of the prior art as described above, by separating the strain having BTEX resolution by collecting soil samples contaminated with BTEX, using the isolated strain after identifying the strain It is to provide microorganisms capable of biologically degrading BTEX.

In order to achieve the above object, the present invention provides a composite strain BTEX 1 (KACC 91316P) that effectively decomposes BTEX.

In another aspect, the present invention provides a method for decomposing BTEX, wherein the complex strain BTEX 1 (KACC91316P) is used in a method for decomposing biologically polycyclic aromatic hydrocarbons.

The present invention is to isolate the complex strain BTEX 1 from oil contaminated soil, biologically easy to decompose volatile aromatic hydrocarbons benzene, toluene, ethylbenzene, xylene (BTEX), and BTEX is mixed and contaminated It can be usefully used for biological purification of media (such as soil or water).

The complex strain was isolated from soil contaminated with BTEX. The collected soil was put in 200 ml of C-medium, and bacterial growth was confirmed through turbidity. Strains that were confirmed to grow were again intended to isolate the desired strain through subculture. After repeating the passage at least 10 times, the desired complex strain was obtained by separating, streaking, or spreading the complex strain decomposing BTEX. The isolated complex strain consisted of two strains, and inoculated the conjugate strain into the C-medium medium that can use BTEX as a carbon source, and isolate BTEX 1 with resolution by changing turbidity, color, and BTEX concentration. Could. BTEX 1 was deposited with KACC 91316P, a Korean National Institute of Agricultural and Biotechnology, KACC.

In addition, the present invention is a microorganism KACC 91316P (complex strain BTEX 1) can be decomposed if the presence of benzene, toluene, ethylbenzene, xylene, respectively in the aqueous solution, and the strain having the property of simultaneously decomposing when BTEX is present in the mixed substrate It provides the decomposition characteristics of. In addition, the strain KACC 91316P is characterized in that the decomposition rate of ethylbenzene, toluene, benzene is much faster than the decomposition rate of xylene and decomposes in the order of ethylbenzene, toluene, benzene.

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

Example 1 Isolation of Complex Strain BTEX 1

Take soil samples and place 1.0 g of soil in a liquid medium (200 ml) containing 200 ul of carbon-minimal medium, carbon-minimal medium (see Table 1), which is a carbon source-deficient medium, and incubate for several weeks in a shake-dry incubator. The culture is inoculated in fresh fresh medium to carry out subculture. Next microbial growth is confirmed by turbidity. When the growth of bacteria is confirmed through turbidity, the culture medium is inoculated again at 1% in the same conditions. In the same way, at least 10 passages are carried out in a C-medium digestion test, and then plated on LB solid medium (see Table 2). Carried out. Figure 1 is an optical micrograph of gram staining magnified 1000 times of the complex strain BTEX 1 (KACC 91316P) of the present invention.

Table 1

(NH ₄ ) ₂ SO ₄ 5.0 g Trace element solution KH ₂ PO ₄ 1.0 g MoO ₃ 1.0 mg K ₂ HPO ₄ 2.0 g ZnSO ₄ 7H ₂ O 7.0 mg MgSO ₄ 7H ₂ O 0.2 g CuSO ₄ 5H ₂ O 0.5 mg NaCl 2.0 g H ₃ BO ₃ 1.0 mg CaCl ₂ 10 mg MnSO ₄ 5H ₂ O 1.0 mg FeSO 4 _· 7H ₂ O 10 mg CoCl _{2 ·} 6H ₂ O 1.0 mg Yeast extract 0.2 g NiSO ₄ 7H ₂ O 1.0 mg Trace elements solution 2.0 mL Deionized water 1.0 L Deionized water 1.0 L pH 7.0

TABLE 2

 Tryptone 10 g  Yeast extract 5 g  NaCl 10 g  Agar 15 g

Example 2 Identification of Isolated Strains

Strains isolated by the above method were first subjected to morphological observation under gram staining and 16S rRNA sequencing and then to sequencing. Eggplant microorganisms were identified. Morphology of the isolates was observed using an optical microscope (Olympus x40) (see Figure 1). After 16S rRNA sequencing, it was confirmed that Rhodococcus sp. And Ochrobactrum intermedium sp.

Example 3 Growth Characteristics of Complex Strain BTEX 1

Growth characteristics were observed using a culture medium in which 0.1% (v / v) of ethylbenzene was injected into the carbon source in order to measure the activity range according to the temperature of the isolated complex strain BTEX 1 and pH. The growth temperature was adjusted to 25 ℃, 30 ℃ and 35 ℃ to proceed with the culture and the pH was adjusted to pH3, pH5, pH6, pH7 and pH8 experiment. In general, the soil is reported to have a pH range of 5-9 depending on the characteristics. Therefore, the complex strain BTEX 1 was applied to the medium prepared according to the above method to analyze the degree of proliferation. As a result of the analysis, BTEX 1 was able to grow at temperatures ranging from 25 ° C to 35 ° C and showed the best growth rate compared to growth at other temperature conditions at 30 ° C (see Figure 2). It is expected that the strain will be available.

In addition, the growth characteristics according to the pH, the growth was possible between pH5 to pH8 and the growth of the strains in the neutral pH7 was the best (see Figure 3).

Example 4 Determination of Resolution of a Single Substrate of the Complex Strain BTEX 1

BTEX 1 is a composite strain that uses benzene, toluene, ethylbenzene, and xylene as a single substrate, respectively, and observes the resolution of four substances. 5 ml of medium is added to a 20 ml vial, and the initial concentration of each substrate is After adjusting the concentration to 20ppm was observed the degradation rate of each substrate of the complex strain BTEX 1 over time. Material analysis was performed using gas chromatography (GC) analysis equipment. Experimental method was carried out as follows. First, put the cultured BTEX 1 culture medium in a centrifuge and centrifuge at 7000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and each BTEX, which was used as a substrate, was completely removed by sterile phosphate buffer solution (PBS). The washed strain was adjusted to have an absorbance (600 nm) value of 1.0 and inoculated using a sterilized syringe by 50ul in a 20ml vial injected with BTEX. BTEX is a volatile aromatic hydrocarbon that is very volatile. Vial therefore used a Mininert valve sealing system to prevent substrate loss, which favored the assay. Then, the experiment was carried out by directly injecting gas into the GC using a gas tight syringe in the upper air of the vial incubating for several hours depending on the decomposition characteristics of the substrate at 30 ° C. and 150 rpm. Separation was performed using HP-5, a non-polar column, and analysis conditions were analyzed for 5 minutes in an isothermal mode at an injection temperature of 150 ° C, an oven temperature of 60 ° C, and a detection temperature of 200 ° C.

When the benzene was used as a single substrate, the resolution of the complex strain BTEX 1 could be observed to decompose all the benzene within 50 hours after inoculation of the strain and maintain the initial concentration of benzene in the vial without microorganisms as a control. (See Fig. 4). Since benzene has no methyl group or ethyl group and has a hexagonal stable structure, it is more difficult to decompose microorganisms than general hydrocarbons. Therefore, it was considered that longer time was required for complete decomposition than other substrates. Next, when toluene was used as a carbon source, the decomposition state was observed. Unlike benzene, degradation occurred within a short time after inoculation and completely degraded within 10 hours (see FIG. 5). After that, the resolution of ethylbenzene showed the highest degradation effect among the four substrates, indicating that almost 100% degradation occurred within 5 hours (see FIG. 6). Finally, a decomposition experiment was conducted to see the decomposition effect on xylene, which shows a decomposition characteristic almost similar to that of toluene (see FIG. 7).

As a result of the decomposition of the phase, the BTEX 1 complex strain was able to decompose completely in a short time when benzene, toluene, ethylbenzene and xylene were used as single carbon sources.

Example 5 Determination of the Resolution of the Composite Substrate of the Complex Strain BTEX 1

When experiments were conducted using a composite substrate in which benzene, toluene, ethylbenzene, and xylene were present at the same time, an experiment was carried out to determine the BTEX degradation characteristics of the conjugate strain BTEX 1 and to evaluate the possibility of simultaneous decomposition. The decomposition experiment was carried out under the same conditions as in (Example 4), and the analysis was performed under the same conditions using GC. The analysis was carried out after inoculation with the concentration of BTEX medium and the concentration of the substrate on the upper gas was equilibrated, and then inoculated with the conjugate strain BTEX 1.

As a result, BTEX composite substrate is almost 100% decomposed after 80 hours, benzene, toluene and ethylbenzene are almost completely decomposed in about 20 hours, and only xylene is decomposed relatively late. This was different from the decomposition test using only benzene and xylene as a single substrate. It can be observed that when four substances are present at the same time, the resolution of benzene is increased and the resolution of xylene is relatively decreased. In addition, in the decomposition property with time, it was shown that the first use of ethylbenzene, and then consume the substrate in the order of toluene, benzene, xylene (see Fig. 8).

Example 6 Resolution of Complex Strain BTEX 1 in Contaminated Black Paper and Gwangju Soil

In order to determine the resolution of the complex strain BTEX 1 according to soil, the resolution was measured using the composite substrate BTEX. Experimental method was carried out as follows. Two soils with different physicochemical properties, such as organic matter content, were placed in 20ml vial, 1g each, and autoclave for 20 minutes. Homogenized these soil samples with 5ml of sterilized C-medium solution. Then, each was contaminated with BTEX. Thereafter, shaking culture for 30 days at 30 ℃, 120rpm, and the contaminants reached the adsorption equilibrium, and then the bacterial strain BTEX 1 was injected to perform biological decomposition of the contaminants. After injecting the conjugate strain BTEX 1, the concentration of contaminants in the vial was measured to observe the change in concentration of BTEX by the conjugate strain BTEX 1. Contaminants were analyzed by GC analysis under the same conditions as in Example 4.

Figure 9 shows the results of decomposition after the soil was added to the microorganism-injected control group was very little loss of BTEX, but in the experiment injected with the complex strain BTEX 1 ethylbenzene was higher than 97% in two days in each soil In the case of benzene and toluene, the decomposition rate was different according to soil characteristics, and xylene showed similar decomposition rate in each soil after 7 days. Adsorption of organic contaminants on soil varies greatly depending on the physicochemical properties of the soil and organic contaminants, and microorganisms are known to be relatively difficult to decompose strongly adsorbed contaminants. The experimental results showed that BTEX absorbed a lot of black soils and slowly degraded by microorganisms.

The biological strain of BTEX 1, which is used in this experiment, can be used to effectively remove soils contaminated with benzene, toluene, ethylbenzene and xylene simultaneously. .

Figure 1 is a microscopic observation showing the strain KACC 91316P (complex strain BTEX 1) according to the present invention (ㅧ 1000)

2 is a growth characteristic according to the temperature of the complex strain BTEX 1

Figure 3 growth characteristics according to the pH of the complex strain BTEX 1

4 is a decomposition characteristic of benzene of the complex strain BTEX 1

5 is a decomposition characteristic of toluene of the complex strain BTEX 1

6 is a decomposition characteristic of ethylbenzene of the complex strain BTEX 1

7 is a decomposition characteristic of xylene of the complex strain BTEX 1

8 is a simultaneous decomposition of BTEX mixed substrate of complex strain BTEX 1

Figure 9 Measurement of the resolution of the complex strain BTEX 1 in the black soil and Gwangju soil contaminated with BTEX

Claims (2)

Rhodococcus sp., Which degrades benzene, toluene, ethylbenzene, xylene, or mixtures thereof. And complex strain BTEX 1 (KACC 91316P) of Ochrobactrum intermedium . Rhodococcus sp. On medium contaminated with benzene, toluene, ethylbenzene, xylene, or mixtures thereof. And injecting the complex strain BTEX 1 (KACC 91316P) of Ochrobactrum intermedium , benzene, toluene, ethylbenzene, xylene, or a mixture thereof.

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