KR20010081145A - A novel microorganism Rhodococcus pyridinovorans PDB9 degrading aromatic compounds - Google Patents

Abstract

PURPOSE: A novel microorganism, Rhodococcus pyridinovorans PDB9, decomposing aromatic compounds is provided, thereby the polluted environments can be effectively purified without causing second pollution. CONSTITUTION: The novel microorganism, Rhodococcus pyridinovorans PDB9 (KCTC 0647BP), having the base sequence of 16S rRNA represented by SEQ ID NO: 1 decomposes aromatic compounds comprising pyridine, 2-ethylpyridine, 2-methylpyridine, 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, phenol, aniline, benzene, o-chloroaniline, o-cresol, cyclohexanol, 3,4-dihydroxybenzoic acid, 2-nitrobenzoic acid, quinoline, and 4-quinoline. The aromatic compounds are decomposed by incubating Rhodococcus pyridinovorans PDB9 (KCTC 0647BP) at 30 to 37 deg. C and pH 7.5 to 8.5.

Description

A novel microorganism Rhodococcus pyridinovorans PDX9 degrading aromatic compounds}

The present invention relates to waste water, sewage, or a soil treatment agent containing decomposition method and the microorganism of the aromatic compound by using also a novel microorganism capable of decomposing aromatic compounds Lactococcus flutes Dino borane's PDB9 (Rhodococcus pyridinovorans PDB9) with them, More specifically , Rhodococcus pyridinoboranth PDB9, a new microorganism capable of degrading aromatic compounds that have a toxic effect on the human body and other living organisms, is isolated and identified, and optimization of pyrodine, which is highly toxic among aromatic compounds, is optimized. A treatment agent that can be used to purify wastewater, sewage or soil containing pyridine.

Aromatic compounds are introduced into the natural environment every year by the progress of industrialization, but most of them are recalcitrants, which do not biodegrade naturally and accumulate in the environment, polluting soil and water systems, and destroying ecosystems. Ultimately, it will have a toxic effect on humans. Such aromatic compounds may be naturally degraded by biological purification such as photooxidation and particulate absorption, but such natural purification does not result in complete mineralization of aromatic compounds. Only partial decomposition is known to occur. As an alternative to this, the activity of microorganisms having an activity of degrading aromatic compounds has been suggested. As a result, in the past decades, the degradation activity of microorganisms has been studied as the most powerful means to remove the toxicity of various aromatic compounds polluting ecosystems such as soils and water systems. It is recognized.

In particular, pyridine, which is a by-product of industrial activities, is one of the toxic aromatic compounds that threaten the ecosystem and human survival itself. Pyridine is mainly generated as a by-product from coal liquefaction plants, petrochemical plants that manufacture petroleum refining or liquefied petroleum gas, pesticide manufacturing plants, and other plants using these compounds, or by using raw materials containing these compounds. It is used as a solvent for the synthesis of other compounds in many industries and research fields, such as textile mills. Pyridine, which is discharged from wastewater during this industrial activity, pollutes surrounding rivers, surface water, soil, and air, and is used as a herbicide and an insecticide to be released into soil and the natural environment. Moreover, pyridine is well soluble in water, so it is known to diffuse well into the natural environment and to increase toxicity even with slight modifications to the ring structure (Jori et al., Ecotoxicol. Environ. Safety, 7, 251, 1983; Leenheer & Stuber, Environ. Sci. Technol., 15, 1467, 1981; Stuermer et al., Environ. Sci. Technol ., 16, 582, 1982).

Pyridine not only destroys the ecosystem by harmful effects on the natural environment, but also has a fatal effect on the human body because it is known as a mutagen and carcinogen (Sims &O'Loughlin, CRC. Crit. Rev. Environ. Control ., 19, 309, 1989). Thus, in addition to being very harmful to workers in the industry and those around them, if pyridine is present in the wastewater and sewage, it may be involved in the natural purification process or may be toxic to artificially administered microorganisms and interfere with the purification process.

As reported by far decomposition pyridine microorganism is Brevibacterium (Brevibacterium) bacteria belonging to the genus (Shukla & Kaul, Ind. J. Biochem. Biophysics., 12, 326, 1975), no carboxylic Dia (Nocardia) bacteria belonging to the genus and the like (Watson & Cain, Biochem. J. , 146, 157, 1975), Corynebacterium (Corynebacterium) bacteria belonging to the genus (Shukla & Kaul, Ind. J. Biochem. Biophysics., 11, 201, 1974), Attempts have been made to find microorganisms that degrade pyridine and aromatic compounds more efficiently.

The present inventors attempted to search for new microorganisms capable of more efficiently decomposing aromatic compounds in addition to the microorganisms thus far identified, and confirmed that the strain isolated from the wastewater around the dyeing plant has an excellent ability to decompose pyridine, one of the aromatic compounds. As a result of analyzing the bacteriological properties and the resolution of other aromatic compounds in order to identify the strain, the present invention was completed by confirming that it is a new unknown species that can efficiently decompose an aromatic compound including pyridine.

An object of the present invention is to provide an industrial process, human activity and Lactococcus flutes Dino borane's PDB9 (Rhodococcus pyridinovorans PDB9) also novel microorganism to break down the material of the aromatic compound toxic to all organisms that escapes to the environment from.

In addition, the present invention isRhodococcus pyridinoborancePDB9 (Rhodococcus pyridinovoransIt is an object of the present invention to provide a method for decomposing various aromatic compounds including pyridine using PDB9), and to provide wastewater, sewage or soil treatment containing the microorganisms.

Figure 1 shows the results of pyridine decomposition of different concentrations by Rhodococcus pyridinoboranth PDB9.

In order to achieve the above object, the present invention can decompose an aromatic compound.RhodococcusNew microorganisms in the genusRhodococcus pyridinoboranceProvide PDB9.

The present invention also uses Rhodococcus pyridinoborance PDB9, 2-ethylpyridine with 2-pyridine, 2-methylpyridine, 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, phenol, aniline, benzene Provided are methods for extensively decomposing aromatic compounds, such as o-chloroaniline, o-cresol, cyclonucleosides, 3,4-dihydroxybenzoic acid, 2-nitrobenzoic acid, quinoline, and 4-quinolinol.

The present invention also provides a wastewater, sewage or soil treatment containing the microorganisms.

Hereinafter, the present invention will be described in detail.

The bacteriological properties centering on the physiological, chemical taxonomic and molecular biological properties of Rhodococcus pyridinoboranth PDB9, a novel microorganism provided by the present invention, are as follows.

1) Morphological Physiological Characteristics

Rhodococcus pyridinoboranth PDB9 is a gram-positive aerobic bacterium that does not form spores, has no motility, and has the form of a bacterium or eggplant at the beginning of its growth, and is divided into monobacterium or cocci as growth progresses. . As the growth conditions of the strain, 30 to 37 ℃, pH 6.0 to 9.0 is preferred, particularly pH 7.5 to 8.5 is more preferred.

The enzyme activity was oxidase negative and catalase positive. Tween 80, esculin, arbutin, urea and tyrosine It can hydrolyze and does not hydrolyze starch, casein, nucleic acid (DNA), xanthine and hypoxanthine. In addition, in terms of the degradability of the carbon source, D-fructose, glycerol, D-mannitol, D-mannose, D-mannose, and D-ribose Decomposes salicycin, D-sorbitol and starch to produce acids, but L-arabinose, D-cellobiose, D- Galactose (D-galactose), D-glucose (D-glucose), inulin, lactose, maltose, D-raffinose, L-rhamnose ), Trehalose and D-xylose are not degradable and do not produce acids.

In the phylogenetic relationship, Rhodococcus pyridinoborance PDB9 differs in physiological characteristics from standard strains of Rhodococcus as well as its ability to degrade aromatic compounds, especially 2-ethylpyridine, including pyridine, 2-methylpyridine, 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, phenol, aniline, benzene, o-chloroaniline, o-cresol, cyclonucleosanol, 3,4-dihydroxybenzoic acid, 2 Decomposes aromatic compounds such as nitrobenzoic acid, quinoline and 4-quinolinol. Rhodococcus genus Among Rhodococcus ohpae carcass (Rhodococcus opacus) and Rhodococcus buffer coke tooth (Rhodococcus percolatus) only indicate the ability to decompose the aromatic compound, Rhodococcus flutes Dino borane's PDB9 is Rhodococcus ohpae carcass (Rhodococcus opacus) thereof and also it shows the excellent aromatics resolution than Lactococcus buffer coke tooth (Rhodococcus percolatus).

2) Chemical classification

Rhodococcus pyridinoboranth PDB9 has meso-type diaminopimelic acid (DAP) on the cell wall, and mainly isaquinone quinone (menaquinone (MK) -8 (H2)). In addition, the carbon number of mycolic acid in Rhodococcus pyridinoboranth PDB9 is between 36 and 46 carbon atoms, and the main fatty acids are C16: 0, C18: 1 cis9 and 10-methyl-C18: 0 (TBSA). ), And the composition of guanine and cytosine in DNA is 66%.

3) Molecular Biology

The base sequence of the 16S rRNA gene of Rhodococcus pyridinoborance PDB9 is set forth in SEQ ID NO: 1 .

In addition, the flexible closely with Rhodococcus flutes Dino borane scan results of testing the DNA-DNA homology PDB9, Rhodococcus flutes Dino borane's PDB9 the 16S rRNA base sequence by the analysis also to the grid taxonomic Lactococcus flutes Dino borane's PDB9 Showing DNA-DNA homology of 8.3% to 43.3% with standard strains of the genus Rhodococcus . Rhodococcus pyries based on international standards (Wayne et al., Int. J. Syst. Bacteriol. 37, 463, 1987) that classify DNA-DNA homology with less than 70% taxonomically. Dino's PDB9 borane was identified as a new species that do not exist.

The present inventorsRhodococcus pyridinoborancePDB9 (Rhodococcus pyridinovoransPDB9) and was deposited with the Biotechnology Research Institute Gene Bank on July 21, 1999 (Accession Number: KCTC 0647BP).

The present invention also provides a method for decomposing an aromatic compound including pyridine using Rhodococcus pyridinoborance PDB9. In particular, the present invention provides the optimum conditions that can effectively decompose the toxic pyridine in the aromatic compound using Rhodococcus pyridinoborance PDB9. In the above decomposition method, the optimum conditions for Rhodococcus pyridinoborance PDB9 to decompose pyridine are preferably 30 to 37 ° C., pH 6.0 to 9.0, and particularly preferably pH 7.5 to 8.5. Rhodococcus pyridinoboranth PDB9 of the present invention is a pyridine as well as 2-ethylpyridine, 2-methylpyridine, 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, phenol, aniline, It decomposes various aromatic compounds such as benzene, o-chloroaniline, o-cresol, cyclonucleosides, 3,4-dihydroxybenzoic acid, 2-nitrobenzoic acid, quinoline, 4-quinolinol.

The present invention also provides a wastewater, sewage or soil treatment containing Rhodococcus pyridinoboranth PDB9.

The treating agent of the present invention can be used to purify waste water, sewage or soil contaminated with aromatic compounds, and includes the microorganism of the present invention and a pharmaceutically acceptable additive. The treatment agent including the microorganism and the additive of the present invention may be directly smeared or sprayed with the culture medium of the microorganism, or the method of spraying the culture medium of the microorganism by adsorbing diatomaceous earth, perlite, or the like may be used. In addition, a known wastewater purification method using microorganisms may also be used. The treatment agent includes Rhodococcus pyridinoboranth PDB9, a novel strain of the present invention, as an active ingredient, but may be supplied as an additive, but may be stored separately for long-term storage and mixed before use. To this end, the microorganisms to be used in the treatment agent are stored at -80 ° C, including glycerol components, or suspended in sterile 10% skim milk to be freeze-dried for long-term stable preservation. Other additives included in the treatment agent may include excipients, stabilizers, and additives generally known in the biological treatment agent manufacturing field using microorganisms, or may use an adsorbent such as diatomaceous earth, perlite, loess, and the like.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited to the examples.

Example 1 Isolation of Microorganisms Degrading Aromatic Compounds from Wastewater Samples

In order to isolate microorganisms capable of degrading aromatic compounds, wastewater samples around the dyeing plant were taken and 100 ml of separation medium containing 0.1% of pyridine, which is highly toxic of aromatic compounds, was used (0.1% K2HPO4, 0.05% NaH2PO4, 2H2O). , 0.025% KCl, 0.025% MgSO4.7H2O, 1 ml of trace element solution (Lee et al., Appl. Microbiol. Biotechnol ., 35, 824, 1991), and then inoculated with a 10/10 shaking culture at 30 ℃. . After a certain time, the absorbance of the culture solution was measured using a spectrophotometer at 200 nm to 400 nm to confirm that pyridine was completely decomposed, and 10 ml of the culture solution was transferred to fresh medium and cultured. After the procedure was performed three times in succession, strains having superior activity for degrading pyridine were selected from the strains grown by smearing and incubating the final culture in a solid medium containing 0.1% pyridine.

Example 2 Screening Rhodococcus pyridinoborance Identification of PDB9

(2-1) Morphological and Physiological Characterization of Rhodococcus Pyridinoboranth PDB9

In order to identify the Rhodococcus pyridinoboranth PDB9 strain isolated in Example 1, it was incubated at a temperature of 30 ℃ using BBL nutrient medium (tripticase soy media). Morphological and physiological properties of Rhodococcus pyridinoborance PDB9 were determined based on the method of Yoon et al. (Yoon. Et al., Int. J. Syst. Bacteriol. , 47, 904, 1997). However, acid production from carbohydrates was used by Hugh & Leifson, Int. J. Syst. Bacteriol. , 48, 907, 1998.

As a result of examining the morphology and growth conditions of Rhodococcus pyridinoborance PDB9 , Rhodococcus pyridinoborance PDB9 was a gram positive aerobic bacterium which did not form spores and did not have motility. In addition, the cell morphology was characterized as the bacillus or eggplant in the early stage of growth, but fractionated into monobacterium or cocci as the growth progressed, and grew best at the growth conditions of 30 to 37 ℃, pH 7.5 to 8.5.

Also in a select strain Lactococcus flutes Dino borane's general physiological characteristics and carbon source utilization characteristics existing Rhodococcus genus Rhodococcus nose profile Russ (Rhodococcus coprophilus) strains known in the PDB9, Rhodococcus Rodney (Rhodococcus rhodnii), Rhodococcus also Rhodococcus rhodochrous, Rhodococcus ruber and Rhodococcus zopfii are shown in Tables 1 and 2 below.

< Table 1> Comparison of physiological characteristics of the standard strains of the genus Rhodococcus and selected strains ( Rhodococcus pyridinoborance PDB9)

As shown in Table 1 , Rhodococcus pyridinoboranth PDB9 is oxidase negative and catalase positive in enzymatic activity, tween 80, esculin, arbutin ), Urea and tyrosine can be hydrolyzed, while starch, casein, nucleic acid (DNA), xanthine and hypoxanthine cannot.

<Table 2> also comparison of the acid generated from the carbon atom of the genus Rhodococcus strains of the strain (Rhodococcus flutes Dino borane's PDB9) screening and

Also in the analysis results of the carbon source, as shown in Table 2 above, D-fructose. Glycerol, D-mannitol, D-mannose, D-ribose, D-ribose, salicin, D-sorbitol and starch Decomposes to produce acid, but L-arabinose, D-cellobiose, D-galactose, D-glucose, and inulin ), Lactose, maltose, D-raffinose, L-rhamnose, trehalose and D-xylose ) Do not decompose and do not produce acid. The physiological characteristics of Rhodococcus pyridinoborance PDB9 were different from the physiological characteristics of standard strains of Rhodococcus as shown by 16S rRNA sequencing.

In particular, Rhodococcus pyridinoboranth PDB9 , unlike most Rhodococcus strains, is characterized by degrading aromatic compounds, including pyridine, and Rhodococcus opacus , which is known to have an ability to decompose aromatic compounds among Rhodococcus strains. ) and also had an excellent aromatics resolution bihaeseodo in Lactococcus buffer coke tooth (Rhodococcus percolatus).

(2-2) Chemical Taxonomic Characterization of Rhodococcus Pyridinoboranth PDB9

Peptidoglycan of the cell wall is also the type of DAP form a glycan, isoprenoid quinone discussed type, also including the composition of the kinds and guanine-cytosine in the fatty acid research Lactococcus flutes Dino borane's Chemical taxonomic properties of PDB9 result, also in the present invention Lactococcus Pyridinoborane PDB9 had meso-type DAP on the cell wall, and mainly isnaquinone ((MK) -8 (H2)) as isoprenoid quinone. Also US cholic acid having a carbon number of (mycolic acid) was between 46-36, the major fatty acids are C16 present in Lactococcus flutes Dino borane's PDB9: a 0 (TBSA): 0, C18 : 1 cis9, 10-methyl-C18 Form, and the composition of guanine and cytosine in DNA was 66%.

(2-3) Molecular Biological Properties of Rhodococcus Pyridinoboranth PDB9

Rhodococcus rhodochrous, Rhodococcus rhodochrous, Rhodococcus louver (Rhodococcus ruber) for the DNA-DNA homology experiments ; Rhodococcus pydinonorth PDB9, Rhodococcus coprophilus, Rhodococcus rhodnii, Rhodococcus rhodochrous, ) and Rhodococcus Geoff blood (Rhodococcus zopfii), such as Rhodococcus, such as the DNA of the type strain in Yoon (Yoon et al., Int. J. Syst. Bacteriol., 46, 502, were extracted using the method of 1996), Nucleotide sequencing and analysis of the 16S rRNA gene was performed using the method of Yoon et al., Int. J. Syst. Bacteriol. , 47, 933, 1997. DNA homology between the strains was analyzed using the method of Ezaki et al., Int. J. Syst. Bacteriol ., 39, 224-229.

The 16S rRNA gene of Rhodococcus pyridinoboranth PDB9 analyzed by the above method was composed of the nucleotide sequence shown in SEQ ID NO: 1 , and the genus of Rhodococcus showing a systematic taxonomy by 16S rRNA sequencing. DNA-DNA homology with standard strains showed DNA-DNA homology of 8.3% to 43.3%. Therefore, according to the international standard (Wayne et al., Int. J. Syst. Bacteriol. 37, 463, 1987) that the DNA-DNA homology value between strains in the current bacterial taxonomy is 70% or less . Rhodococcus pyridinoboranth PDB9 has been identified as a new species that does not exist previously.

Based on the above morphological physiological properties, chemical taxonomic properties and molecular biological properties, the strains selected by the present inventors were identified as new species rather than existing species belonging to the genus Rhodococcus , so the selected strains were identified as Rhodococcus pyridinoboranth PDB9. ( Rhodococcus pyridinovorans PDB9) and was deposited on July 21, 1999 with the Institute of Biotechnology Gene Bank (Accession Number: KCTC 0647BP).

<Example 3> Rhodococcus pyridinoborance Investigation of Pyridine Resolution of PDB9

In order to investigate the activity of Rhodococcus pyridinoboranth PDB9 which was isolated and identified in Examples 1 and 2, pyridine was decomposed, pyridine was added to the separation medium of Example 1 at various concentrations up to 4000 ppm. The degree of decomposition was measured. The resolution of pyridine was determined by measuring the absorbance at 254 nm using a spectrophotometer. In addition, the growth degree of Rhodococcus pyridinoborance PDB9 in a medium containing pyridine was determined by measuring the absorbance at 600 nm using a spectrophotometer.

As a result, as shown in Fig. 1 , even when the inoculation concentration of Rhodococcus pyridinoboranth PDB9 was set to the minimum (OD value of 0.02 when measured at 600 nm with a spectrophotometer), the resolution was shown, and pyridine 2000 ppm (mg / 1) after about 7 to 8 days, 3500 ppm (mg / l) was confirmed that completely degraded after about 16 days.

<Example 4> Rhodococcus pyridinoborance Investigation of the resolution of aromatic compounds other than pyridine of PDB9

In order to investigate whether Rhodococcus pyridinoboranth PDB9 has activity to decompose aromatic compounds other than pyridine, various kinds of aromatic compounds were added to the separation medium of Example 1 at a concentration of 0.1% to investigate their degradation activity. . Degradation activity for each compound was determined by centrifugation of the culture solution and the amount of aromatic compounds remaining in the supernatant was analyzed by HPLC (high performance liquid chromatography, Waters) using a Novapak C18 column.

TABLE 3 Degradation of Aromatic Compounds of Rhodococcus Pyridinoboranth PDB9

As shown in Table 3 above, Rhodococcus pyridinoboranth PDB9 is 2-ethylpyridine, 2-methylpyridine, 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, phenol, aniline, benzene, o- Excellent resolution was shown for aromatic compounds such as chloroaniline, o-cresol, cyclonucleoside, 3,4-dihydroxybenzoic acid, 2-nitrobenzoic acid, quinoline and 4-quinolinol.

As described above, Rhodococcus genus novel microorganism of Rhodococcus flutes Dino borane's PDB9 of the present invention may be leaked to the environment from the industrial activities process humans and invading harmful effect on the organism and further substances of the aromatic compounds causing environmental pollution It can be used to effectively clean the environment without causing secondary environmental pollution problem through the biodegradation process using the microorganism when applied to waste water, sewage or soil contaminated with aromatic compounds, In particular pyridine, including 2-ethylpyridine, 2-methylpyridine, 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, phenol, aniline, benzene, o-chloroaniline, o-cresol, cyclonucleosides, It can be usefully used to decompose aromatic compounds such as 3,4-dihydroxybenzoic acid, 2-nitrobenzoic acid, quinoline and 4-quinolinol. Uh, because of its wide range of applications, it can be effectively used as wastewater, sewage, or soil treatment.

Claims (8)

Rhodococcus pyridinoborance PDB9, which decomposes an aromatic compound. The Rhodococcus pyridinoborance PDB9 according to claim 1 , which has a nucleotide sequence of 16S rRNA as set forth in SEQ ID NO: 1 . The Rhodococcus pyridinorenate PDB9 according to claim 1, wherein the accession number is KCTC 0647BP . A pyridine, 2-ethylpyridine, 2-methylpyridine, 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, phenol, aniline, benzene, o-chloroaniline, o-cresol, cyclo Rhodococcus pyridinoborane PDB9 characterized by decomposing aromatic compounds such as nucleic acidol, 3,4-dihydroxybenzoic acid, 2-nitrobenzoic acid, quinoline and 4-quinolinol. Aromatic compound decomposition method using Rhodococcus pyridinoborance PDB9 of Claim 1. The method for decomposing aromatic compounds according to claim 5, wherein Rhodococcus pyridinoborance PDB9 is incubated at 30 to 37 ° C. The method of claim 5, wherein the Rhodococcus pyridinoborance PDB9 is incubated at pH 7.5 to 8.5. A wastewater, sewage or soil treatment agent comprising the Rhodococcus pyridinoboranth PDB9 of claim 1 as an active ingredient.