KR20020003461A - Bacterium with higher environmental resistance level and ethylbenzene degradation activity - Google Patents
Bacterium with higher environmental resistance level and ethylbenzene degradation activity Download PDFInfo
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Abstract
Description
본 발명은 유독 물질인 에틸벤젠을 효과적으로 분해시킬 수 있는 로도코쿠스 로도크러스 (Rhodococcus rhodochrous) 균주에 관한 것이다.The present invention is a rodococcus rhodochrus (Rhodococcus rhodochrous) It is about a strain.
오염물질의 정화 방법은 물리적, 화학적, 생물학적 방법 등을 이용할 수 있다. 이 중에서 생물학적 방법은 오염물질을 분해하는 활성을 가진 미생물을 이용하는 방법으로, 물리화학적 정화 방법과는 달리 일반적으로 2차 적인 오염 물질을 발생하지 않는 장점을 가지고 있다. 그러나, 어떤 미생물이 단순히 오염물질을 분해하는 활성을 가졌다고 해서 곧바로 오염 지역에 적용할 수 있는 것은 아니다. 환경 정화용 미생물은 오염 지역의 환경에서 지속적으로 생장 가능하여야 하며 가능하면 다양한 환경 조건에서 생장 가능한 생태학적 특성을 보여주어야 한다. 바람직한 생태학적 특성 중 대표적인 것으로는 산성도(pH)에 대한 최적 생장 범위가 있다. 즉, 국내 토양 조건이 대개 약 산성임을 고려할 때, 약산성의 pH에서 생존가능한 미생물이 바람직한 환경 정화용 미생물이 될 수 있는 것이다.The pollutant purification method may use a physical, chemical, biological method and the like. Among these, biological methods use microorganisms having activity to decompose pollutants, and unlike physical and chemical purification methods, biological methods generally do not generate secondary pollutants. However, just because some microorganisms have the activity of breaking down pollutants does not mean they can be applied immediately to contaminated areas. Environmental microorganisms should be capable of sustained growth in the environment of the contaminated area and, where possible, show ecological characteristics that can be grown under various environmental conditions. Representative of the preferred ecological properties is the optimum growth range for acidity (pH). That is, considering that domestic soil conditions are usually weak acid, microorganisms viable at weakly acidic pH may be desirable microorganisms for environmental purification.
한편, 전세계적으로 석유탄화수소는 산업현장에 널리 쓰이고 있으며, 많은 양의 원유 또는 정제된 제품들이 생산, 운반, 저장, 이용 등의 과정에서 환경에 유출되고 있다. 이들 제품은 산업 발달과 더불어 꾸준히 사용량이 증가하고 있어 이로 인한 환경오염이 더욱 심화되고 있으며, 주유소, 공장 또는 각종 사업체 등에서탄화수소에 의한 환경오염 문제의 심각성이 국내외적으로 크게 대두되고 있다. 특히, 전체 기름 중 특히 가솔린에 많이 포함되어 있는 에틸벤젠(ethylbenzene)은 벤젠, 톨루엔, 자일렌 등과 함께 발암물질로 잘 알려져 있으며 비교적 높은 용해도와 휘발성으로 인해 대기와 수질에 매우 큰 오염을 일으키고 있다. 따라서 이를 분해할 수 있는 방법, 특히 미생물을 이용하여 에틸벤젠을 생분해할 수 있는 정화 방법의 개발이 시급하며, 이에 이용가능한 미생물의 분리와 동정이 절실히 필요한 실정이다.On the other hand, petroleum hydrocarbons are widely used in industrial fields all over the world, and a large amount of crude oil or refined products are leaked to the environment during production, transportation, storage and use. These products have been steadily increasing with the development of the industry, resulting in deeper environmental pollution. The seriousness of hydrocarbon pollution at gas stations, factories, and businesses is increasing at home and abroad. In particular, ethylbenzene, which is included in gasoline, in particular, is well known as a carcinogen along with benzene, toluene, and xylene. Due to its relatively high solubility and volatility, air and water are highly polluted. Therefore, it is urgent to develop a method for decomposing this, in particular, a purification method for biodegrading ethylbenzene using microorganisms, and there is an urgent need for separation and identification of available microorganisms.
현재의 에틸벤젠 분해 세균의 분리는 실험실 조건에서, 즉 낮은 에틸벤젠 농도 (최대 800 ppm)에서 실시되었다. 그러나 오염지의 환경과 유사한 고농도의 에틸벤젠 조건 (1000 ppm 이상)에서 분해율을 측정한 사례는 없다 (Mallakin and ward 1996 J of Industrial Microbiology 16, 309-318; Heldeman 1994 Applied and environmental microbiology 60:2697-2703; Haack 등 1994 Applied and environmental microbiology 60:2480-2493; Robert 등. 1998 Applied and environmental microbiology 64:3961-3165; Norman 등. Plant diesease 1997 81:847-850; Jaunisses 등 97 Jaunisses 등 Phytoma la defence des vesetanx 49:17-22). 따라서 실제 오염 환경과 비슷한 조건에서 분해율을 측정하기 위한 균주 선발, 동정과 가시적인 세균 활력 검정을 통해서, 새롭고 강력한 에틸벤젠 분해 세균을 동정하는 것이 보다 바람직하다.The isolation of current ethylbenzene degrading bacteria was carried out under laboratory conditions, ie at low ethylbenzene concentrations (up to 800 ppm). However, no degradation rate has been measured under conditions of high concentrations of ethylbenzene (more than 1000 ppm) similar to the environment of contaminated sites (Mallakin and ward 1996 J of Industrial Microbiology 16, 309-318; Heldeman 1994 Applied and environmental microbiology 60: 2697-2703 Haack et al. 1994 Applied and environmental microbiology 60: 2480-2493; Robert et al. 1998 Applied and environmental microbiology 64: 3961-3165; Norman et al. Plant diesease 1997 81: 847-850; Jaunisses et al. 97 Jaunisses et al. Phytoma la defence des vesetanx 49: 17-22). Therefore, it is more desirable to identify new and potent ethylbenzene-degrading bacteria through strain selection, identification and visible bacterial vitality assays to measure degradation rates in conditions similar to the actual contaminated environment.
이를 위하여 본 발명에서는 500ppm의 고농도 에틸벤젠을 효과적으로 분해할 수 있는 미생물을 선별하여 분리 및 동정한 후, 이 균주가 기존의 에틸벤젠 분해용 미생물에 비해 훨씬 높은 농도의 에틸벤젠을 분해할 수 있음을 확인하였을 뿐 아니라 약산성 조건에서도 지속적 생장이 가능함을 확인함으로써 본 발명을 완성하였다.To this end, in the present invention, after selecting, separating and identifying microorganisms capable of effectively decomposing 500 ppm of high concentration ethylbenzene, the strain can decompose much higher concentrations of ethylbenzene compared to conventional microorganisms for decomposition of ethylbenzene. In addition to confirming, the present invention was completed by confirming that it is possible to grow continuously in weakly acidic conditions.
본 발명의 목적은 에틸벤젠을 빠르게 다량 분해할 수 있으면서도 실제 환경에서 높은 생장력을 갖는 균주를 선별하는 것이다.An object of the present invention is to select strains having high growth ability in real environment while being able to rapidly decompose large amounts of ethylbenzene.
상기 목적을 달성하기 위해, 본 발명에서는 고농도의 에틸벤젠을 분해하는 활성을 가진 로도코쿠스 로도크러스 (Rhodococcus rhodochrous) 균주를 제공한다.In order to achieve the above object, the present invention provides a Rhodococcus rhodochrous strain having an activity of decomposing high concentration of ethylbenzene.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 고농도의 에틸벤젠을 분해하는 활성을 가진Rhodococcus rhodochrous균주를 제공한다.The present invention provides a Rhodococcus rhodochrous strain having an activity of decomposing high concentration of ethylbenzene.
상기 균주가 가진 에틸벤젠의 분해 활성은, 기질에 500 ppm의 농도로 존재하는 에틸벤젠을 48시간 이내에 100% 분해할 수 있는 활성으로 정의된다. 그러나 상기 균주는 800 ppm 내지 1500 ppm의 고농도 에틸벤젠이 포함되어 있는 경우에도 분해 활성을 보유하는 특징을 보여 준다.The decomposition activity of ethylbenzene possessed by the strain is defined as the activity capable of 100% degradation of ethylbenzene present in the substrate at a concentration of 500 ppm within 48 hours. However, the strain shows a characteristic that retains its decomposition activity even when containing 800 ppm to 1500 ppm high concentration of ethylbenzene.
상기 균주는 약산성 조건에서 생장 가능한 것을 또다른 특징으로 한다. 구체적으로, 상기 균주는 pH 5 내지 pH 7에서도 생장이 우수하며 가장 바람직하기로는 pH 7에서 생장 가능하다. 또한, 상기 균주는 혐기 조건 및 호기 조건에서 생장 가능하다.The strain is another feature that can be grown in weakly acidic conditions. Specifically, the strain is excellent in growth even at pH 5 to pH 7 and most preferably can be grown at pH 7. In addition, the strain can be grown under anaerobic and aerobic conditions.
상기 균주는 자연적으로는 공업폐기물처리장 주변 토양에 서식하고 있다. 본 발명의 일 실시 예에서는 대한민국 경기 남부지역, 수원 동부, 수원 지역의 공업폐기물처리장 주변 토양을 채취한 후 이로부터 에틸벤젠 분해 활성이 우수한 균주를 선발, 분리하였다.The strain naturally resides in soil around an industrial waste treatment plant. In one embodiment of the present invention, after extracting the soil around the industrial waste treatment plant in southern Gyeonggi, Suwon East, Suwon region of South Korea, the strain was selected and separated excellent ethylbenzene decomposition activity.
상기 균주의 기타 생리학적 특성, 형태학적 특성은 일반적인Rhodococcus rhodochrous의 특성과 일치한다. 본 발명의 일 실시예에서는 상기 분리된 균주를 미생물 생리/생태조사인 MIDI (Mallakin and ward 1996 J of Industrial Microbiology 16, 309-318)로 동정한 결과, 상기 균주가Rhodococcus rhodochrous임을 확인하고 이 균주를Rhodococcus rhodochrousHPLEB-1 로 명명한 후, 2000년 5월 18일에 한국종균협회에 기탁하였다 (수탁번호 KFCC-11170).Other physiological and morphological properties of the strain are consistent with those of the general Rhodococcus rhodochrous . In one embodiment of the present invention as a result of identifying the isolated strain by the microbial physiology / ecology MIDI (Mallakin and ward 1996 J of Industrial Microbiology 16, 309-318), confirming that the strain is Rhodococcus rhodochrous After being named Rhodococcus rhodochrous HPLEB-1, it was deposited with the Korean spawn association on May 18, 2000 (Accession No. KFCC-11170).
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
<실시예 1> 에틸벤젠 분해 균주의 분리Example 1 Isolation of Ethylbenzene Degrading Strains
경기도 용인 지역에 오랫동안 다양한 기름으로 오염된 토양을 접종 시료로 사용하였다. 기름오염지 토양을 지하 0~50cm 부위에서 선발한 후 즉시 비닐봉투에 투입하고 4℃에서 냉장 보관하였다. 구체적인 토양 채취 사항은표 1에 기재한 바와 같다.Soils contaminated with various oils were used as inoculation samples in Yongin, Gyeonggi-do. Oil stained soil was selected from 0 ~ 50cm underground and immediately put into plastic bag and refrigerated at 4 ℃. Specific soil collection details are shown in Table 1 .
채취한 토양 시료를 각각 1g 씩을 250㎖ 크기의 삼각플라스크에 담긴 기본 영양 배지 (MSM, Minimum Salt Medium; Na2HPO4ㆍ12H20 9g/ℓ, KH2PO41.5g/ℓ, MgSO4ㆍ7H20 0.2g/ℓ, NH4Cl 1.0g/ℓ, FeSO4ㆍ7H20 0.1g/ℓ, CaCl2ㆍ2H20 0.1g/ℓ, trace elemental solution (ZnSO4ㆍ7H20 10mg/ℓ, MnClㆍ4H20 3mg/ℓ, H3BO330mg/ℓ, CoCl2ㆍ6H20 20mg/ℓ, CuClㆍ6H20 0.56mg/ℓ, NiClㆍ6H20 2mg/ℓ, NaMoO4ㆍ2H20 10mg/ℓ) 3㎖/ℓ) 50㎖에 첨가하였다. 탄소원 및 에너지원으로 에틸벤젠 1000 ppm을 첨가한 후 테프론으로 처리한 고무마개로 막아 30℃, 170rpm에서 2~3일간 농후 배양하였다. 균주가 자라 배지가 탁해지면 계대배양을 하였다. 2, 3회 계대배양 후 1.5% 한천을 첨가한 LA (Luria's Agar media) 플레이트에 평판도말하였다. 이틀 후 균체가 많이 형성된 균주를 LA 플레이트에 선발하여 500, 1000, 1500 ppm의 에틸벤젠이 포함된 MSM에서 배양시킨 후 에틸벤젠의 분해율을 측정하였다.Basic nutrient medium (MSM, Minimum Salt Medium; Na 2 HPO 4 12H 2 0 9g / ℓ, KH 2 PO 4 1.5g / ℓ, MgSO 4 ㆍ) containing 1 g each of the collected soil samples in a 250 ml Erlenmeyer flask. 7H 2 0 0.2g / ℓ, NH 4 Cl 1.0g / ℓ, FeSO 4 ㆍ 7H 2 0 0.1g / ℓ, CaCl 2 ㆍ 2H 2 0 0.1g / ℓ, trace elemental solution (ZnSO 4 7H 2 0 10mg / l, MnCl 4 H 2 0 3 mg / l, H 3 BO 3 30 mg / l, CoCl 2 6H 2 0 20 mg / l, CuCl 6H 2 0 0.56 mg / l, NiCl 6H 2 0 2 mg / l, NaMoO 4 2H 2 0 10 mg / L) 3 mL / L) 50 mL. 1000 ppm of ethylbenzene was added as a carbon source and an energy source, and the mixture was sealed with a rubber stopper treated with Teflon and concentrated incubated at 30 ° C. and 170 rpm for 2 to 3 days. As the strain grew and the medium became turbid, subculture was performed. Plates were plated on LA (Luria's Agar media) plates to which 1.5% agar was added after two or three passages. Two days later, a large number of cells were formed on the LA plate, incubated in MSM containing 500, 1000, and 1500 ppm of ethylbenzene, and the degradation rate of ethylbenzene was measured.
<실시예 2><Example 2> Rhodococcus rhodochrousRhodococcus rhodochrous HPLEB-1 균주의 분리 및 동정Isolation and Identification of HPLEB-1 Strains
상기 실시예에서 분리된 균주들을 에틸벤젠 포함 배지에서 배양한 후 106~107/㎖로 많이 생성된 콜로니를 선택하여 각각 순수분리하였다. 각각의 균주는 접종 후 24시간과 48시간 째에 에틸벤젠 분해도를 측정하여 분해능이 우수한 균주를 최종 선발하고 동정에 들어갔다. 균주 동정은 생리/생태조사로서 MIDI로 동정하였으며,Rhodococcus rhodochrousHPLEB-1 균주로 명명한 후, 2000년 5월 18일 한국미생물보존센터에 기탁하였다 (수탁번호 KFCC-11168).The strains isolated in the above examples were cultured in an ethylbenzene-containing medium, and 10 6 ~ 10 7 / ml of the colonies were produced by selecting a lot of pure separation. Each strain was determined 24 hours and 48 hours after the inoculation to determine the ethylbenzene decomposition degree was finally selected and identified strains with excellent resolution. Strain identification was identified by MIDI as a physiological / ecological survey, named Rhodococcus rhodochrous HPLEB-1 strain, and deposited on May 18, 2000 at the Korea Microbiological Conservation Center (Accession No. KFCC-11168).
에 기탁하였다 (수탁번호 KFCC-11170).Was deposited (Accession No. KFCC-11170).
<시험예 1> 에틸벤젠 분해율 측정<Test Example 1> Determination of ethylbenzene decomposition rate
멸균된 7㎖ MSB (Na2HPO4 12H2O, 9g; KH2PO4, 1.5g; MgSo4 7H2O, 0.2g; NH4Cl, 1g; FeSO4 7H2O, 0.1g; CaCl2 2H2O, 0.1g; Trace element, 소량)에 단일 탄소원으로 에틸벤젠을 500 ppm, 1000 ppm 또는 1500 ppm 씩 첨가한 배지에 A660에서 0.12로 조절한 3㎖의Rhodococcus rhodochrousHPLEB-1 균주를 혈청병에 2반복으로 접종하였다. 그 후 24~48시간동안 30℃에서 170rpm으로 진탕배양하였다. 그 후 혈청병의 상부 공기층에 존재하는 에틸벤젠을 500㎕ gas tight syringe로 100㎕ 채취하여 가스크로마토그래피로 잔류 에틸벤젠량을 분석하고, 미생물을 접종하지 않은 대조구와 비교하였다. 그 결과는표 2에 나타내었다.Ethylbenzene as a single carbon source in sterile 7 ml MSB (Na2HPO4 12H2O, 9g; KH2PO4, 1.5g; MgSo4 7H2O, 0.2g; NH4Cl, 1g; FeSO4 7H2O, 0.1g; CaCl2 2H2O, 0.1g; Trace element, small amount) In a medium added 500 ppm, 1000 ppm or 1500 ppm, 3 ml of Rhodococcus rhodochrous HPLEB-1 strain adjusted from A 660 to 0.12 was inoculated twice in serum. Then, shaking culture was performed at 30 ° C. at 170 rpm for 24 to 48 hours. Thereafter, 100 μl of ethylbenzene in the upper air layer of the serum bottle was collected with a 500 μl gas tight syringe, and the residual ethylbenzene amount was analyzed by gas chromatography, and compared with a control without inoculation of microorganisms. The results are shown in Table 2 .
<시험예 2> 배양액 pH에 따른 생장 저해도<Test Example 2> Growth inhibition according to the culture medium pH
Rhodococcus rhodochrousHPLEB-1 균주를 하루밤동안 배양한 다음, 1.0M HCl 및 NaOH를 이용하여 pH를 4, 5, 6, 7로 조절한 LB 배양액에 이를 접종한 후 48시간 동안 배양하였다. 균주의 생장 정도는 UV 분광광도계(휴렛패커드 8453)를 이용하여 550nm에서 분석하였다. 그 결과는표 3에 나타내었다. Rhodococcus rhodochrous HPLEB-1 strains were incubated overnight, and then inoculated into LB culture medium adjusted to pH 4, 5, 6, 7 using 1.0M HCl and NaOH, and then cultured for 48 hours. The growth of the strain was analyzed at 550 nm using a UV spectrophotometer (Hewlett Packard 8453). The results are shown in Table 3 .
표 3에 따르면, 본 발명의 균주는 pH 5~7 범위의 약산성 조건에서 생장함을 알 수 있었다. 따라서, 자연에서 일어날 수 있는 조건에서 생장이 우수할 것으로 판단된다.According to Table 3 , the strain of the present invention was found to grow in weakly acidic conditions of the pH range 5-7. Therefore, growth is expected to be excellent under conditions that can occur in nature.
<시험예 3> 호기/혐기 상태에서의 생장 저해도<Test Example 3> Growth inhibition in aerobic / anaerobic
브로모칠몰 블루(bromothylmol blue)를 포함시킨 루리아스 5㎖ 배양액에Rhodococcus rhodochrousHPLEB-1 균주를 생육시킨 후 하룻밤 배양한 세균 50㎕을 접종시키고 그 후 살균된 2㎖의 액체파라핀을 투여한 후 30℃에서 4일간 정치배양시켰다. 그 후 배지의 색깔 변화와 가시적 균체생장으로 호기혐기의 조건을 평가하였다. 그 결과Rhodococcus rhodochrousHPLEB-1 균주는 혐기 조건과 호기 조건에서 연청색으로 변화하며 균체가 접종지역에서 가시적으로 생장함이 파악되었다. Rhodococcus rhodochrous HPLEB-1 strains were grown in 5 ml broth containing bromothylmol blue, inoculated with 50 µl of cultured overnight, and then sterile 2 ml of liquid paraffin was injected. Incubated at 4 ° C. for 4 days. Afterwards, the aerobic anaerobic conditions were evaluated by color change and viable cell growth. As a result, the strain of Rhodococcus rhodochrous HPLEB-1 was changed to light blue color under anaerobic and aerobic conditions, and the cells grew visibly in the inoculation area.
본 발명이 제공하는Rhodococcus rhodochrous균주을 이용하면, 800 ppm 이상의 고농도 에틸벤젠 조건에서도 효과적으로 에틸벤젠을 분해할 수 있을 뿐 아니라 약산성 조건, 호기 및 혐기 조건에서도 생장 가능하므로 에틸벤젠 오염지역의 환경 정화에 크게 이바지할 것으로 평가된다.By using the Rhodococcus rhodochrous strain provided by the present invention, it can effectively decompose ethylbenzene even under high concentration of ethylbenzene at 800 ppm or more, and can grow under weakly acidic, aerobic and anaerobic conditions, thus greatly contributing to the purification of the environment of the ethylbenzene contaminated area. Is evaluated.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100469087B1 (en) * | 2002-02-18 | 2005-02-02 | 김응빈 | Rhodococcus sp. strain DK17 with metabolic versatility to degrade a wide variety of substituted benzenes and terpenes |
| KR100481404B1 (en) * | 2002-04-11 | 2005-04-08 | 윤인길 | Novel microbe, rhodococcus pyridinovorans pyj-1 (kfcc-11302) enabling degradation of aromatic compounds and methods for disposal of waste water, sewage and air using the same |
| KR20230078089A (en) | 2021-11-26 | 2023-06-02 | 대한민국(농촌진흥청장) | Method of degrading an organochlorine insecticide DDT using Paralcaligenes sp. KSB-10 |
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2000
- 2000-07-04 KR KR1020000037984A patent/KR20020003461A/en not_active Application Discontinuation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100469087B1 (en) * | 2002-02-18 | 2005-02-02 | 김응빈 | Rhodococcus sp. strain DK17 with metabolic versatility to degrade a wide variety of substituted benzenes and terpenes |
| KR100481404B1 (en) * | 2002-04-11 | 2005-04-08 | 윤인길 | Novel microbe, rhodococcus pyridinovorans pyj-1 (kfcc-11302) enabling degradation of aromatic compounds and methods for disposal of waste water, sewage and air using the same |
| KR20230078089A (en) | 2021-11-26 | 2023-06-02 | 대한민국(농촌진흥청장) | Method of degrading an organochlorine insecticide DDT using Paralcaligenes sp. KSB-10 |
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