KR100312007B1 - New species Gordonia nitida LE31 which can degrade 3-ethylpyridine and 3-methylpyridine - Google Patents
New species Gordonia nitida LE31 which can degrade 3-ethylpyridine and 3-methylpyridine Download PDFInfo
- Publication number
- KR100312007B1 KR100312007B1 KR1019990028370A KR19990028370A KR100312007B1 KR 100312007 B1 KR100312007 B1 KR 100312007B1 KR 1019990028370 A KR1019990028370 A KR 1019990028370A KR 19990028370 A KR19990028370 A KR 19990028370A KR 100312007 B1 KR100312007 B1 KR 100312007B1
- Authority
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- South Korea
- Prior art keywords
- ethylpyridine
- methylpyridine
- gordonia
- nitida
- present
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
Abstract
본 발명은 3-에틸피리딘과 3-메틸피리딘을 분해할 수 있는 새로운 종인 고르도니아 니티다 (Gordonia nitida)에 관한 것으로, 구체적으로 본 발명의 새로운 세균 종인 고르도니아 니티다는 인체 및 다른 생물체에 유독한 성질을 나타내는 3-에틸피리딘과 3-메틸피리딘을 분해할 수 있는 유용한 특징을 가지고 있으므로, 3-에틸피리딘 또는 3-메틸피리딘이 부산물로 발생하는 화학공장 등의 주변 폐수, 하수 또는 토양에 가하면 미생물을 이용한 생분해 과정을 통하여 환경의 2차 오염 문제를 일으키지 않고 3-에틸피리딘과 3-메틸피리딘을 효율적으로 분해할 수 있다.The present invention relates to Gordonia nitida , a new species capable of decomposing 3-ethylpyridine and 3-methylpyridine. Specifically, the new bacterial species of the present invention, Gordonia nitida , are toxic to humans and other organisms. It has a useful feature to decompose 3-ethylpyridine and 3-methylpyridine, which show their properties. Therefore, when 3-ethylpyridine or 3-methylpyridine is added to surrounding wastewater, sewage or soil, such as a chemical plant where by-products occur Through biodegradation process, 3-ethylpyridine and 3-methylpyridine can be efficiently decomposed without causing secondary pollution problem.
Description
본 발명은 3-에틸피리딘과 3-메틸피리딘을 분해할 수 있는 고르도니아(Gordonia) 속 미생물과 이를 이용하여 3-에틸피리딘 또는 3-메틸피리딘을 분해하는 방법에 관한 것으로, 보다 상세하게는 폐수로부터 분리된 신규 미생물로서 3-에틸피리딘과 3-메틸피리딘을 분해하는 고르도니아 니티다 (Gordonia nitida), 상기 미생물을 이용하여 3-에틸피리딘 또는 3-메틸피리딘을 분해하는 방법 및 상기 미생물을 함유하는 폐수 또는 하수 처리제에 관한 것이다.The present invention relates to a microorganism of genus Gordonia capable of decomposing 3-ethylpyridine and 3-methylpyridine and a method of decomposing 3-ethylpyridine or 3-methylpyridine using the same, and more specifically, wastewater. Gordonia nitida , which decomposes 3-ethylpyridine and 3-methylpyridine as a new microorganism isolated from the method, a method of decomposing 3-ethylpyridine or 3-methylpyridine using the microorganism, and containing the microorganism. It relates to wastewater or sewage treatment agent.
3-에틸피리딘 (3-ethylpyridine)과 3-메틸피리딘 (3-methylpyridine)은 알킬피리딘 종류의 화합물로서 다른 종류의 피리딘과 함께 산업활동에 이용되거나 그 부산물로서 발생한다. 특히 알킬피리딘은 석유정제, 액화석유가스 제조 등의 석유화학공장, 농약제조공장 및 이들 화합물을 이용하는 공장 또는 이들 화합물이 들어 있는 원료를 이용하는 공장에서 부산물로 발생한다. 따라서 알킬피리딘은 상기 산업활동 과정에서 폐수를 통해 유출될 수 있으며 주변의 강, 지표수, 토양, 대기 등을 오염시킬 뿐 아니라, 물에 잘 녹는 특성이 있으므로 자연환경으로의 확산이 잘 이루어 진다.3-ethylpyridine and 3-methylpyridine are alkylpyridine-type compounds that, together with other pyridine species, are used in industrial activities or occur as by-products. In particular, alkylpyridine is generated as a by-product from petrochemical plants such as petroleum refining and liquefied petroleum gas production, pesticide manufacturing plants and factories using these compounds, or factories using raw materials containing these compounds. Therefore, the alkylpyridine can be discharged through the wastewater during the industrial activities, and not only pollute the surrounding river, surface water, soil, air, etc., but also has good water-soluble characteristics, so that it can be easily diffused into the natural environment.
알킬피리딘은 자연 환경에 유해한 영향을 미쳐서 생태계를 파괴하며, 돌연변이 유발체 및 발암물질로 알려져 있기 때문에 인체에도 치명적인 영향을 미친다. 이와 같이 알킬피리딘은 관련 산업의 종사자 및 주변 사람들의 건강에 매우 유해한 영향을 미칠 뿐 아니라, 알킬피리딘이 폐수 및 하수에 존재하고 있을 경우 자연 정화 과정에 관여하거나 인공적으로 투여된 미생물에도 독성을 나타내기 때문에 정수 과정을 방해할 수도 있다.Alkylpyridine has a detrimental effect on the natural environment and destroys the ecosystem, and because it is known as a mutagen and a carcinogen, it has a fatal effect on the human body. As such, alkylpyridine not only has a very detrimental effect on the health of workers in the industry and those around it, but also when it is present in wastewater and sewage, it is toxic to microorganisms involved in natural purification processes or artificially administered. This may interfere with the water purification process.
따라서, 알킬피리딘을 분해 또는 제거하기 위한 방법이 다양하게 연구되어 왔으며, 특히 다른 난분해성 물질의 경우에서처럼 2차적인 오염 물질을 발생하지 않는 미생물을 이용하는 생분해 방법이 연구되어 왔다. 하지만 알킬피리딘 화합물 중에서 3-에틸피리딘과 3-메틸피리딘을 분해하는 것으로 보고된 미생물에 관한 보고는 거의 없다. 3-메틸피리딘을 분해하는 것으로 보고된 미생물로는 슈도모나스속 세균 (Hesset al.,Apple. Environ. Microbiol.,56, 1551, 1990) 등이 보고되고 있으나, 3-에틸피리딘을 분해하는 미생물에 대해서는 알려진 바가 없다. 따라서 상기 물질을 생분해하기 위해서는, 3-에틸피리딘과 3-메틸피리딘을 분해하는 미생물을 탐색하고 그 분해 조건을 최적화하는 연구가 시급히 요구되어 왔다.Therefore, various methods for decomposing or removing alkylpyridine have been studied, and in particular, biodegradation methods using microorganisms which do not generate secondary pollutants as in the case of other hardly degradable substances have been studied. However, there are few reports of microorganisms reported to decompose 3-ethylpyridine and 3-methylpyridine among alkylpyridine compounds. As microorganisms reported to degrade 3-methylpyridine, Pseudomonas bacteria (Hess et al ., Apple. Environ. Microbiol. , 56 , 1551, 1990) have been reported, but microorganisms that degrade 3-ethylpyridine are reported. It is not known. Therefore, in order to biodegrade the material, research has been urgently required to search for microorganisms that decompose 3-ethylpyridine and 3-methylpyridine and to optimize their degradation conditions.
이에 본 발명자들은 3-에틸피리딘과 3-메틸피리딘을 분해할 수 있는 새로운 미생물의 탐색을 시도한 결과, 대구의 폐수 처리장에서 채취한 폐수로부터 분리한 한 균주가 3-에틸피리딘과 3-메틸피리딘을 함께 분해하는 성질을 가지고 있다는 것을 확인하였으며, 상기 균주로 3-에틸피리딘과 3-메틸피리딘에 대한 분해성 실험과 미생물 동정을 한 결과, 이 균주가 3-에틸피리딘과 3-메틸피리딘을 효율적으로 분해할 수 있는 기존에 알려지지 않은 새로운 종이라는 것을 확인함으로써 본 발명을 완성하였다.Therefore, the present inventors attempted to search for new microorganisms capable of decomposing 3-ethylpyridine and 3-methylpyridine, and as a result, one strain isolated from wastewater collected from a wastewater treatment plant of Daegu was used to determine 3-ethylpyridine and 3-methylpyridine. As a result of degrading experiments and microorganism identification of 3-ethylpyridine and 3-methylpyridine, the strain efficiently decomposed 3-ethylpyridine and 3-methylpyridine. The present invention has been completed by confirming that it is a new, unknown paper.
본 발명의 목적은 산업활동 과정에서 자연환경에 유출되는 인체 및 모든 생물체에 유독한 물질인 3-에틸피리딘과 3-메틸피리딘을 분해하는 미생물을 제공하는 것이다.An object of the present invention is to provide a microorganism that decomposes 3-ethylpyridine and 3-methylpyridine, which are toxic to the human body and all living organisms, which are released into the natural environment during industrial activities.
도 1a는 고르도니아 니티다 LE31에 의한 3-에틸피리딘의 농도별 분해도를 나타낸 그래프이고,Figure 1a is a graph showing the degree of decomposition of 3-ethylpyridine by Gordonia nitida LE31,
도 1b는 고르도니아 니티다 LE31에 의한 3-메틸피리딘의 농도별 분해도를 나타낸 그래프이다.Figure 1b is a graph showing the degree of decomposition of 3-methylpyridine by Gordonia nitida LE31.
상기 목적을 달성하기 위하여 본 발명에서는 3-에틸피리딘과 3-메틸피리딘을분해할 수 있는 고르도니아 속의 새로운 미생물 고르도니아 니티다를 제공한다.In order to achieve the above object, the present invention provides a new microorganism Gordonia nitida of the genus Gordonia which can decompose 3-ethylpyridine and 3-methylpyridine.
또한 본 발명에서는 상기 미생물을 이용하여 3-에틸피리딘 또는 3-메틸피리딘을 분해하는 방법을 제공한다.The present invention also provides a method for decomposing 3-ethylpyridine or 3-methylpyridine using the microorganism.
또한 본 발명에서는 상기 미생물을 함유하는 폐수, 하수 또는 토양 처리제를 제공한다.The present invention also provides a wastewater, sewage or soil treatment containing the microorganisms.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에서는 3-에틸피리딘과 3-메틸피리딘을 분해할 수 있는 고르도니아 속의 새로운 미생물 고르도니아 니티다를 제공한다. 상기 고르도니아 니티다는 3-에틸피리딘 및 3-메틸피리딘을 분해하는 미생물을 탐색한 결과 새로운 종으로 분리, 동정된 세균이다.The present invention provides a new microorganism Gordonia nitida of the genus Gordonia which can decompose 3-ethylpyridine and 3-methylpyridine. The Gordonia nitida is a bacterium that has been isolated and identified as a new species by searching for microorganisms that degrade 3-ethylpyridine and 3-methylpyridine.
구체적으로, 3-에틸피리딘과 3-메틸피리딘을 분해하는 미생물을 탐색하는 과정은 하기와 같이 수행할 수 있다. 즉, 폐수 시료를 채취하여, 분리용 배지 (0.1% K2HPO4, 0.05% NaH2PO4·2H2O, 0.025% KCl, 0.025% MgSO4·7H2O, 1 ml 미량원소용액)에 희석하고 그 중 일부를 3-에틸피리딘과 3-메틸피리딘이 포함된 플라스크에 옮긴 후, 약 30℃에서 진탕배양하였다. 일정시간이 지난 후 3-에틸피리딘과 3-메틸피리딘이 완전히 분해된 것을 확인하고 배양액 일정량을 다시 신선한 배지에 옮겨 배양하였다. 상기 과정을 반복 시행한 후 마지막 배양액의 일정량을 3-에틸피리딘과 3-메틸피리딘이 들어있는 고체 배지에 도말·배양하여, 나타나는 균주 중에서 3-에틸피리딘과 3-메틸피리딘을 분해하는 활성이 우수한 균주를 선별하여 #LE31로 명명하였다.Specifically, the process of searching for microorganisms to decompose 3-ethylpyridine and 3-methylpyridine can be performed as follows. That is, wastewater samples are taken and separated into a medium for separation (0.1% K 2 HPO 4 , 0.05% NaH 2 PO 4 2H 2 O, 0.025% KCl, 0.025% MgSO 4 · 7H 2 O, 1 ml trace element solution). Diluted and some of them were transferred to a flask containing 3-ethylpyridine and 3-methylpyridine, and then shaken at about 30 ° C. After a certain time, it was confirmed that 3-ethylpyridine and 3-methylpyridine were completely decomposed, and the culture was transferred to a fresh medium and cultured. After repeating the above process, a predetermined amount of the last culture was smeared and cultured in a solid medium containing 3-ethylpyridine and 3-methylpyridine, and thus the activity of degrading 3-ethylpyridine and 3-methylpyridine was excellent among the strains. Strains were selected and named # LE31.
# LE31 균주를 동정하기 위하여, 상기에서 선별된 균주를 배양한 후, 그 형태; 탄소 및 에너지 원으로서 이용할 수 있는 기질의 활용 특성 등을 포함하는 생리학적 특성; 세포벽을 이루는 디아미노피멜산 (Diaminopimelic acid; 이하 'DAP'라 약칭함)의 종류, 이소프레노이드 퀴논 (isoprenoid quinone)의 종류, 미콜산 (mycolic acid)의 탄소수 분석, 지방산의 종류 및 구아닌·시토신 조성 등의 화학분류학적 특성; 16S rRNA 유전자의 염기 서열 결정 및 분석; 및 DNA-DNA 상동성 실험 등을 수행하였다.# In order to identify LE31 strain, after culturing the strain selected above, the form; Physiological properties, including utilization characteristics of substrates that can be used as carbon and energy sources; Type of diaminopimelic acid (hereinafter abbreviated as 'DAP') that forms the cell wall, type of isoprenoid quinone, carbon number analysis of mycolic acid, type of fatty acid and guanine and cytosine Chemical classification properties such as composition; Base sequencing and analysis of 16S rRNA genes; And DNA-DNA homology experiments.
#LE31의 형태 및 생리학적 특성을 조사한 결과, #LE31은 그람양성의 호기성 세균으로 포자를 형성하지 않고 운동성이 없으며, 세포의 형태는 성장초기에는 간균이며 성장이 진행됨에 따라 구균으로 변함을 알 수 있었다. 상기 균주의 성장 조건으로는 30∼37℃, pH 6.0-11.0 이 바람직하며, 특히 pH 8.0-9.0 이 바람직하다. 생리학적 특징으로는 옥시다제 (oxidase) 음성, 카탈라제 (catalase) 양성이며, 트윈 80 (tween 80), 에스쿨린 (esculin), 핵산 (DNA), 요소 (urea)를 분해하며, 녹말 (starch), 카제인 (casein), 알부틴 (arbutin), 엘라스틴 (elastin)은 분해하지 못한다. D-글루코오스 (D-glucose), D-리보오스 (D-ribose), 수크로오스 (sucrose), 투라노즈 (turanose), D-트레할로스 (D-trehalose), 시트르산 삼나트륨 (trisodium citrate), 글루콘산 나트륨 (sodium gluconate), L-알라닌 (L-alanine), L-프롤린 (L-proline)을 탄소 및 에너지원으로 이용할 수 있으나, D-갈락토오스 (D-galactose), 미오-이노시톨 (myo-inositol), L-람노오스 (L-rhamnose), D-아라비톨 (D-arabitol), L-아스파르트산 (L-aspartate), 벤조산 나트륨 (sodium benzoate), 숙신산 이나트륨 (disodium succinate), L-류신 (L-leucine), L-발린 (L-valine), 아세트아미드 (acetamide)는 탄소 및 에너지원으로 이용하지 못한다. 또한, D-글루코오스 (D-glucose), 수크로오스 (sucrose), D-트레할로스 (D-trehalose)로부터 산을 생성하지만, D-갈락토오스 (D-galactose), 미오-이노시톨 (myo-inositol), D-솔비톨 (D-sorbitol), L-람노오스 (L-rhamnose)로부터는 산을 생성하지 않는다. 상기 #LE31의 생리학적 특징은 고르도니아속의 다른 종과 비교해 볼 때 상이하였으며, 특히 3-에틸피리딘과 3-메틸피리딘를 분해하는 특징은 고르도니아속의 다른 종들에서는 발견되지 않았다.As a result of investigating the morphology and physiological characteristics of # LE31, # LE31 is a gram-positive aerobic bacterium that does not form spores and has no motility, and the cell shape is bacilli at the early stage of growth and changes to cocci as it progresses. there was. As growth conditions of the said strain, 30-37 degreeC and pH 6.0-11.0 are preferable, pH 8.0-9.0 is especially preferable. Physiological features are oxidase negative, catalase positive, break down tween 80, esculin, nucleic acid (DNA), urea, starch, Casein, arbutin, elastin do not break down. D-glucose, D-ribose, sucrose, turanose, D-trehalose, trisodium citrate, sodium gluconate ( Sodium gluconate, L-alanine and L-proline can be used as carbon and energy sources, but D-galactose, myo-inositol, L L-rhamnose, D-arabitol, L-aspartate, sodium benzoate, disodium succinate, L-leucine leucine, L-valine and acetamide are not available as carbon and energy sources. It also produces acids from D-glucose, sucrose, D-trehalose, but D-galactose, myo-inositol, D- It does not produce acid from sorbitol, L-rhamnose. The physiological characteristics of # LE31 were different compared to other species of the genus Gordonia, and in particular, the characteristic of degrading 3-ethylpyridine and 3-methylpyridine was not found in other species of the genus Gordonia.
#LE31의 화학분류학적 특성으로는, 세포벽에 meso형의 DAP를 가지고 있으며, 이소프레노이드 퀴논은 주로 메나퀴논 (menaquinone) (MK)-9(H2)를 가지고 있다. #LE31에 존재하는 미콜산 (mycolic acid)의 탄소수는 47개에서 55개 사이이다. 또한, #LE31에 존재하는 주요 지방산은 C16:0, C18:1 w9c, 10-methyl-C18:0 (TBSA)형태이며, DNA의 구아닌·시토신의 조성은 67%이다.The chemical classification of # LE31 has meso-type DAP on the cell wall, and isoprenoid quinone mainly contains menaquinone (MK) -9 (H 2 ). Mycolic acid in # LE31 has between 47 and 55 carbon atoms. The major fatty acids present in # LE31 are C16: 0, C18: 1 w9c and 10-methyl-C18: 0 (TBSA) forms, and the composition of guanine and cytosine in DNA is 67%.
한편, #LE31의 16S rRNA 유전자의 염기 서열은서열번호 1로 기재된다 [미국 진뱅크 (GenBank)에 수탁 번호 (accession number) AF148947 로서 등록됨].On the other hand, the nucleotide sequence of the 16S rRNA gene of # LE31 is described as SEQ ID NO: 1 (registered as Accession number AF148947 in GenBank, USA).
또한, #LE31의 DNA-DNA 상동성을 조사한 결과, #LE31은 고르도니아속의 기존 종들의 표준 균주들과 비교해 볼 때, 3.7%-44.5%의 DNA-DNA 상동성을 보여, 국제적으로 70% 이하의 DNA-DNA 상동성을 보이는 경우 서로 다른 종으로 판단하는 기준에 근거했을 때 기존에 존재하지 않는 새로운 종으로서 동정되었다. 상기 신규 종을 고르도니아 니티다 (Gordonia nitida)로 명명하고, 한국과학기술연구원 부설 생명공학연구소 유전자은행에 1999년 5월 3일자로 기탁하였다 (수탁번호: KCTC 0605BP).In addition, DNA-DNA homology of # LE31 showed that # LE31 showed 3.7% -44.5% DNA-DNA homology compared to the standard strains of existing species of the genus Gordonia, which is less than 70% internationally. DNA-DNA homology was identified as a new species that did not exist on the basis of different criteria. The new species was named Gordonia nitida and was deposited on May 3, 1999 to the Genetic Bank of Korea Research Institute of Science and Technology (accession number: KCTC 0605BP).
또한, 본 발명은 상기 신규의 미생물 종을 이용하여 3-에틸피리딘 또는 3-메틸피리딘을 분해하는 방법을 제공한다. 상기 분해 방법에서, 고르도니아 니티다가 3-에틸피리딘과 3-메틸피리딘을 분해하기 위한 조건으로는 30-40℃, pH 6.0-9.0 이 바람직하고, 특히 33-37℃, pH 7.0-8.0 이 바람직하다.The present invention also provides a method for digesting 3-ethylpyridine or 3-methylpyridine using the novel microbial species. In the above decomposition method, 30-40 ° C., pH 6.0-9.0 is preferable, and especially 33-37 ° C., pH 7.0-8.0 is preferable for gordonia nitida to decompose 3-ethylpyridine and 3-methylpyridine. Do.
구체적으로, 본 발명의 균주 고르도니아 니티다 #LE31은 접종을 위한 세포 농도를 최소로 하였을 경우 3-에틸피리딘은 300 ppm (mg/l)을 3-메틸피리딘은 200 ppm (mg/l)을 완전히 분해할 수 있었다. 3-에틸피리딘과 3-메틸피리딘의 분해를 위한 최적 배양온도는 35℃이었으며 최적 pH는 7.5이었다.Specifically, the strain Gordonia nitida # LE31 of the present invention exhibited 300 ppm (mg / l) of 3-ethylpyridine and 200 ppm (mg / l) of 3-methylpyridine when the cell concentration for inoculation was minimized. Could be disassembled completely. The optimum incubation temperature for the decomposition of 3-ethylpyridine and 3-methylpyridine was 35 ° C and the optimum pH was 7.5.
또한, 본 발명은 상기 신규의 미생물 종을 함유하는 폐수, 하수 또는 토양 처리제를 제공한다.The present invention also provides wastewater, sewage or soil treatments containing the novel microbial species.
상기 처리제는 3-에틸피리딘 또는 3-메틸피리딘으로 오염된 폐수, 하수 또는 토양을 정화하기 위해 사용할 수 있다.The treatment may be used to purify wastewater, sewage or soil contaminated with 3-ethylpyridine or 3-methylpyridine.
상기 처리제는 본 발명의 미생물을 장기간 안정적으로 보존하기 위해 글리세롤 성분을 포함하여 -80℃에 보관하거나 멸균된 10% 탈지유에 현탁하여 동결건조하는 것이 바람직하다.In order to preserve the microorganism of the present invention for a long time stable, the treating agent is preferably stored at −80 ° C. including a glycerol component or suspended in sterile 10% skim milk to be lyophilized.
이하 실시예에 의하여 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples.
단, 하기 실시예들은 본 발명을 예시하는 것으로, 본 발명의 내용이 실시예에 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the content of the present invention is not limited to the examples.
<실시예 1> 폐수 시료로부터 3-에틸피리딘과 3-메틸피리딘을 분해하는 미생물의 분리Example 1 Isolation of Microorganisms Degrading 3-ethylpyridine and 3-methylpyridine from Wastewater Samples
대구의 염색공장에서 폐수 시료를 채취하여, 분리용 배지 (0.1% K2HPO4, 0.05% NaH2PO4·2H2O, 0.025% KCl, 0.025% MgSO4·7H2O, 1ml 미량원소용액)에 10분의 1로 희석하여 접종하였다. 희석된 용액 50 ml을 0.5 mM 3-에틸피리딘 및 0.5 mM 3-메틸피리딘이 들어있는 250 ml 플라스크에 옮긴 후 30℃에서 진탕배양하였다. 일정시간이 지난 후 분광광도계 (spectrophotometer)를 사용하여 200 nm 내지 400 nm 에서 배양액의 흡광도를 측정하여 3-에틸피리딘과 3-메틸피리딘이 완전히 분해되었는지 확인하고, 배양액 25 ml을 다시 신선한 배지에 옮겨 배양하였다. 상기 과정을 3회 연속적으로 시행한 후 마지막 배양액을 모두 0.5 mM 3-에틸피리딘 및 3-메틸피리딘이 들어있는 고체 배지에 도말·배양하여, 나타나는 균주중에서 3-에틸피리딘과 3-메틸피리딘을 분해하는 활성이 우수한 균주를 선별하였다. 분리된균주의 동정 전까지 상기 균주는 잠정적으로 #LE31로 명명하였다.A wastewater sample was collected from a dyeing plant in Daegu, and the medium was separated (0.1% K 2 HPO 4 , 0.05% NaH 2 PO 4 2H 2 O, 0.025% KCl, 0.025% MgSO 4 7H 2 O, 1ml trace element solution). ) Was inoculated with a tenth dilution. 50 ml of the diluted solution was transferred to a 250 ml flask containing 0.5 mM 3-ethylpyridine and 0.5 mM 3-methylpyridine and shaken at 30 ° C. After a certain period of time, the absorbance of the broth was measured at 200 nm to 400 nm using a spectrophotometer to confirm that 3-ethylpyridine and 3-methylpyridine were completely decomposed, and 25 ml of the broth were transferred to fresh medium. Incubated. After the procedure was performed three times in succession, the final culture was plated and cultured in a solid medium containing 0.5 mM 3-ethylpyridine and 3-methylpyridine to decompose 3-ethylpyridine and 3-methylpyridine among the strains shown. Strains with good activity were selected. Prior to identification of the isolated strain, the strain was tentatively named # LE31.
<실시예 2> 분리한 #LE31의 동정Example 2 Identification of Isolated # LE31
실시예 1에서 분리된 #LE31 균주를 영양 배지 (tripticase soy 배지, BBL사 제품)에서 30℃ 조건으로 배양하였다. #LE31의 형태 및 생리학적 특성은 윤 등 (Yoonet al., Int. J. Syst. Bacteriol.,47, 904, 1997)의 방법에 기준하여 결정하였다. 단, 탄소 및 에너지 원으로서 이용할 수 있는 기질의 활용 특성 및 당으로부터의 산의 생성은 다케우치와 하타노의 방법 (Takeuchi & Hatano,Int. J. Syst. Bacteriol.,48, 907, 1998)을 이용하였다. 한편, 세포벽의 펩티도글리칸 (peptidoglycan)을 이루는 DAP의 종류, 이소프레노이드 퀴논의 종류, 미콜산 (mycolic acid)의 탄소수 분석, 지방산의 종류 및 구아닌·시토신의 조성 등의 균주의 화학분류학적 특성과 16S rRNA 유전자의 염기 서열 결정 및 분석은 윤 등 (Yoonet al.,Int. J. Syst. Bacteriol.,47, 933, 1997)의 방법을 사용하였다. 또한, DNA-DNA 상동성 실험을 위해서 #LE31과 고르도니아속의 종들의 DNA를 윤 등 (Yoonet al.,Int. J. Syst. Bacteriol., 46, 502, 1996)의 방법을 이용하여 추출하였고, 이들 DNA들은 에자키 등 (Ezakiet al.,Int. J. Syst. Bacteriol., 39,224-229)의 방법을 이용하여 서로의 결합 정도를 측정하였다.# LE31 strain isolated in Example 1 was incubated in nutrient medium (tripticase soy medium, BBL company) at 30 ℃ condition. Morphological and physiological properties of # LE31 were determined based on the method of Yoon et al., Int. J. Syst. Bacteriol. , 47 , 904, 1997. However, the utilization characteristics of the substrate which can be used as a carbon and energy source and the generation of acid from sugar were used by Takeuchi & Hatano, Int. J. Syst. Bacteriol. , 48 , 907, 1998. . On the other hand, chemical taxonomics of strains such as DAP, peptidoglycan of cell wall, isoprenoid quinone, carbon number analysis of mycolic acid, type of fatty acid and composition of guanine and cytosine Characterization and sequencing and analysis of the 16S rRNA gene were performed by Yoon et al. , Int. J. Syst. Bacteriol. , 47 , 933, 1997. For DNA-DNA homology experiments, DNAs of # LE31 and Gordonia were extracted using Yoon et al. , Int. J. Syst. Bacteriol., 46 , 502, 1996. These DNAs were measured using the method of Ezaki et al ., Int. J. Syst. Bacteriol., 39, 224-229.
#LE31의 형태 및 성장 조건을 조사한 결과, #LE31은 그람양성의 호기성 세균으로 포자를 형성하지 않으며 운동성은 없었다. 또한, 세포의 형태는 성장초기에는 간균이며 성장이 진행됨에 따라 구균으로 변하였으며, 30∼37℃와 pH 8.0∼9.0 에서 가장 잘 생장하였다.As a result of examining the morphology and growth conditions of # LE31, # LE31 is a gram-positive aerobic bacterium that does not form spores and has no motility. In addition, the cell morphology was bacilli at the early stage of growth and changed to cocci as the growth progressed, and grew best at 30-37 ° C. and pH 8.0-9.0.
#LE31의 생리학적 특성은 다음과 같다. #LE31은 옥시다제 음성, 카탈라제 양성이었다. 트윈 80, 에스쿨린, 핵산, 요소를 분해하며, 녹말, 카제인, 알부틴, 엘라스틴은 분해하지 못하였다. D-글루코오스, D-리보오스, 수크로오스, 투라노즈, D-트레할로스, 시트르산 삼나트륨, 글루콘산 나트륨, L-알라닌, L-프롤린을 탄소 및 에너지원으로 이용할 수 있으나, D-갈락토오스, 미오-이노시톨, L-람노오스, D-아라비톨, L-아스파르트산, 벤조산 나트륨, 숙신산 이나트륨, L-류신, L-발린, 아세트아미드는 탄소 및 에너지원으로 이용하지 못하였다. D-글루코오스, 수크로오스, D-트레할로스로부터 산을 생성하지만, D-갈락토오스, 미오-이노시톨, D-솔비톨, L-람노오스로부터는 산을 생성하지 않았다. 이러한 #LE31의 생리학적 특징은 고르도니아속의 다른 종과 비교해 볼 때 상이하였다.Physiological characteristics of # LE31 are as follows. # LE31 was oxidase negative, catalase positive. It breaks down Tween 80, esculin, nucleic acids, urea, but not starch, casein, arbutin and elastin. D-glucose, D-ribose, sucrose, turanose, D-trehalose, trisodium citrate, sodium gluconate, L-alanine, L-proline can be used as carbon and energy sources, but D-galactose, myo-inositol, L-rhamnose, D-arabitol, L-aspartic acid, sodium benzoate, disodium succinate, L-leucine, L-valine, acetamide were not available as carbon and energy sources. Acid was produced from D-glucose, sucrose, D-trehalose, but no acid was produced from D-galactose, myo-inositol, D-sorbitol, L-rhamnose. The physiological characteristics of # LE31 were different compared to other species of the genus Gordonia.
특히, #LE31은 3-에틸피리딘과 3-메틸피리딘를 분해하는 특징을 가지고 있었지만, 고르도니아속의 다른 종들은 두 화합물을 분해할 수 없었다.In particular, # LE31 was characterized by degrading 3-ethylpyridine and 3-methylpyridine, but other species of the genus Gordonia could not degrade both compounds.
#LE31의 화학분류학적 특성을 조사한 결과, 세포벽에 meso형의 DAP를 가지고 있으며, 이소프레노이드 퀴논은 주로 메나퀴논 (MK)-9(H2)를 가지고 있었다. #LE31에 존재하는 미콜산의 탄소수는 47개에서 55개 사이였다. #LE31에 존재하는 주요 지방산은 C16:0, C18:1 w9c, 10-methyl-C18:0 (TBSA)형태이며, DNA의 구아닌·시토신의 조성은 67%이었다.The chemical taxonomic characteristics of # LE31 showed meso-type DAP on the cell wall, and the isoprenoid quinone mainly contained menaquinone (MK) -9 (H 2 ). Mycolic acid in # LE31 was between 47 and 55 carbon atoms. The major fatty acids present in # LE31 were C16: 0, C18: 1 w9c and 10-methyl-C18: 0 (TBSA), and the composition of guanine and cytosine in the DNA was 67%.
한편, #LE31의 16S rRNA 유전자의 염기 서열을 분석한 결과, 상기 염기 서열은서열번호 1에 의해서 기재됨을 확인하였으며, 미국 진뱅크 (GenBank)에 수탁 번호 AF148947 로서 등록되었다.On the other hand, as a result of analyzing the nucleotide sequence of the 16S rRNA gene of # LE31, it was confirmed that the nucleotide sequence was described by SEQ ID NO: 1 , it was registered as Accession No. AF148947 with GenBank of the United States.
#LE31의 DNA-DNA 상동성을 조사한 결과, #LE31은 고르도니아속의 기존 종들의 표준 균주들과 비교해 볼 때, 3.7%-44.5%의 DNA-DNA 상동성을 보였다. 따라서 현재의 세균 분류학 분야에서 균주간의 DNA-DNA 상동성 값이 70% 이하일 경우 서로 다른 종으로 분류된다는 기준에 의해서, #LE31은 새로운 종으로서 동정되었다.As a result of examining DNA-DNA homology of # LE31, # LE31 showed DNA-DNA homology of 3.7% -44.5% compared with standard strains of existing species of Gordonia. Therefore, in the current bacterial taxonomy, # LE31 was identified as a new species based on the criteria that when the DNA-DNA homology value between strains is 70% or less, they are classified as different species.
이상의 형태생리학적 특성, 화학분류학적 특성 및 분자생물학적 특성에 근거하여 선별된 균주는 기존의 고르도니아속에 존재하는 종이 아닌 새로운 종으로서 동정되었으므로, 상기 선별된 균주를 고르도니아 니티다 LE31 (Gordonia nitidaLE31)로 명명하였으며, 한국과학기술연구원 부설 생명공학연구소 유전자은행에 1999년 5월 3일자로 기탁하였다 (기탁번호: KCTC 0605BP).Based on the above morphophysiological, chemical classification, and molecular biological characteristics, the selected strains were identified as new species rather than existing species in the genus Gordonia . Therefore, the selected strains were identified as Gordonia nitida LE31. It was deposited on May 3, 1999 to Gene Bank of Korea Institute of Science and Technology, Korea Institute of Science and Technology (Accession No .: KCTC 0605BP).
<실시예 3> 고르도니아 니티다 LE31의 3-에틸피리딘과 3-메틸피리딘의 분해성 조사Example 3 Investigation of Degradability of 3-ethylpyridine and 3-methylpyridine of Gordonia Nitida LE31
실시예 1 및 실시예 2에서 분리·동정한 고르도니아 니티다 LE31이 3-에틸피리딘과 3-메틸피리딘을 분해하는 활성을 가지는지 조사하기 위해서, 실시예 1의 분리용 배지에 3-에틸피리딘과 3-메틸피리딘을 최고 농도 500 ppm 까지 다양한 농도로 첨가하여 그 분해 정도를 측정하였다. 3-에틸피리딘과 3-메틸피리딘의 분해력을 결정하기 위해, 배양액을 원심분리하여 상등액을 채취한 후분광광도계(spectrophotometer)를 사용하여 200 nm 내지 400 nm 에서 상등액의 흡광도를 측정하였다. 또한, 고르도니아 니티다 LE31의 생장 정도는 분광광도계 (spectrophotometer)를 사용하여 600 nm 에서 흡광도를 측정하여 판단하였다.In order to examine whether Gordonia nitida LE31 isolated and identified in Examples 1 and 2 has an activity of degrading 3-ethylpyridine and 3-methylpyridine, 3-ethylpyridine was separated into the medium for separating in Example 1 And 3-methylpyridine were added at various concentrations up to 500 ppm to determine the degree of degradation. To determine the degradability of 3-ethylpyridine and 3-methylpyridine, the supernatant was collected by centrifugation of the culture, and then the absorbance of the supernatant was measured at 200 nm to 400 nm using a spectrophotometer. In addition, the growth degree of Gordonia nitida LE31 was determined by measuring the absorbance at 600 nm using a spectrophotometer.
그 결과, 고르도니아 니티다 LE31의 접종 농도를 2%로 하였을 경우,도 1a과도 1b에서 보는 바와 같이 3-에틸피리딘은 약 60 시간 후 약 300 ppm (mg/l)이, 3-메틸피리딘은 약 60 시간 후 200 ppm(mg/l)이 완전히 분해됨을 확인하였다.As a result, when the inoculation concentration of Gordonia nitida LE31 was 2%, as shown in FIGS . 1A and 1B , 3-ethylpyridine was about 300 ppm (mg / l) after about 60 hours, and 3-methylpyridine After about 60 hours, it was confirmed that 200 ppm (mg / l) was completely decomposed.
상기에서 살펴본 바와 같이, 본 발명의 신규 종인 고르도니아 니티다는 인체 및 생물체에 유해한 물질인 3-에틸피리딘과 3-메틸피리딘을 분해할 수 있는 유용한 특징을 가지고 있으므로, 3-에틸피리딘과 3-메틸피리딘에 오염된 폐수, 하수 및 토양에 투여하면 미생물을 이용한 생분해 과정을 통하여 환경의 2차 오염 문제를 일으키지 않고 3-에틸피리딘과 3-메틸피리딘을 효율적으로 제거할 수 있다.As described above, the new species of the present invention, Gordonia nitida, has a useful feature to decompose 3-ethylpyridine and 3-methylpyridine, which are harmful to humans and organisms, and thus, 3-ethylpyridine and 3-methyl When administered to pyridine-contaminated wastewater, sewage and soil, biodegradation using microorganisms can effectively remove 3-ethylpyridine and 3-methylpyridine without causing secondary pollution problems in the environment.
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