KR100421654B1 - Psychrophilic microorganism for the degradation of diesel and process for preparation thereof - Google Patents

Psychrophilic microorganism for the degradation of diesel and process for preparation thereof Download PDF

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KR100421654B1
KR100421654B1 KR10-2000-0066990A KR20000066990A KR100421654B1 KR 100421654 B1 KR100421654 B1 KR 100421654B1 KR 20000066990 A KR20000066990 A KR 20000066990A KR 100421654 B1 KR100421654 B1 KR 100421654B1
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oil
strain
strains
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soil
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KR20020036902A (en
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송훈
정욱진
오영숙
문혜준
주춘성
김윤관
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주식회사 에코솔루션
정욱진
오영숙
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

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Abstract

본 발명은 유류분해 저온균주 및 그 제조방법에 관한 것으로, 저온에서도 유류를 분해할 수 있는 균주를 오염된 토양으로부터 순수분리한 후, 그 중에서 성장 및 유류분해능이 우수한 유류분해 저온균주를 제공함으로써 동절기에도 오염된 토양현장에 적용할 수 있는 뛰어난 효과가 있다.The present invention relates to an oil-degrading low temperature strain and a method for manufacturing the same, wherein the strain capable of decomposing oil even at low temperature is purely separated from contaminated soil, and thereafter, the oil-degrading low temperature strain having excellent growth and oil decomposing properties is provided in the winter season. In addition, there is an excellent effect that can be applied to the contaminated soil site.

Description

유류분해 저온균주 및 그 제조방법{Psychrophilic microorganism for the degradation of diesel and process for preparation thereof}Pychrophilic microorganism for the degradation of diesel and process for preparation

본 발명은 유류분해 저온균주 및 그 제조방법에 관한 것으로, 보다 상세하게는 저온에서도 유류를 분해함으로써 동절기에도 현장에 적용할 수 있는 유류분해 저온균주 및 상기 유류분해 저온균주를 제조하는 방법에 관한 것이다.The present invention relates to an oily low temperature strain and a method for manufacturing the same, and more particularly, to a oily low temperature strain and a method for producing the oily low temperature strain which can be applied to the field during winter by decomposing oil even at low temperature. .

최근 토양의 오염이 심각한 현안으로 대두되면서 오염된 토양의 정화기술 개발에 대한 필요성이 인식되고 있으며, 또한 이에 대한 많은 연구가 수행되고 있다. 그러나, 대부분의 토양정화기술이 이차오염 문제를 유발하고 있으며, 비용이 많이 소요되는 물리화학적 처리에 편재되어 있는 실정이다.Recently, as soil pollution is a serious issue, the necessity of developing a technology for purification of contaminated soil has been recognized, and many studies have been conducted. However, most soil purification techniques cause secondary pollution problems and are ubiquitous in costly physical and chemical treatments.

반면에, 미생물을 이용한 생물학적인 토양정화기술은 환경친화적이며 효율적이어서 최근 이에 대한 연구가 활발히 진행되고 있다. 대부분의 유류 및 각종 유기오염물질이 분해되기까지 소요되는 시간과 분해량은 제한되어 있으나, 생물 특히 미생물에 의해 오염물질들이 자연계에서 완전히 분해된다는 사실을 고려한다면, 오염된 토양 또는 지하수는 다양하고 새로운 미생물학적 처리법의 개발에 의해 보다 안전하고 간단하게 그리고 경제적으로 회복될 수 있다.On the other hand, biological soil purification technology using microorganisms is environmentally friendly and efficient, and research on this has been actively conducted recently. Although the time and amount of decomposition of most oils and various organic pollutants are limited, the contaminated soil or groundwater is diverse and new considering the fact that the pollutants are completely degraded in nature by living organisms, especially microorganisms. By the development of microbiological treatments it can be recovered more safely, simply and economically.

유류로 오염된 토양을 생물학적으로 처리하기 위해서는 먼저, 유류분해능을 보유한 미생물을 토양으로부터 탐색하고 그 중에서 우수균주를 선별해야 한다. 유류분해능이 뛰어난 우수균주를 확보하는 것만으로도 이를 유류로 오염된 토양 또는 지하수의 정화에 이용할 수 있다.In order to biologically treat soil contaminated with oil, first of all, microorganisms having oil resolution should be searched from the soil, and excellent strains are selected from them. Just by obtaining excellent strains with excellent oil resolution, they can be used for the purification of soil or groundwater contaminated with oil.

그러나, 상기 선별된 유류분해 균주를 오염된 토양으로 적용함에 있어서, 토양특성, 오염원, 접종시기 등에 따라 그 영향이 매우 크므로 상기 인자들을 고려해야 한다. 특히 미생물은 적용하는 시기의 온도에 따라 미생물의 활성 등에 큰 차이가 있으므로 현장적용시기의 온도가 생물학적 토양정화기술에 가장 중요한 요소라고 할 수 있다. 중온에서 유류를 분해하는 미생물에 대한 연구가 있었으나, 이를 이용한 오염토양의 정화는 하절기 등에 편중되어 왔으며, 동절기에도 적용할 수 있는 저온균주에 관한 연구는 극히 미미한 실정이다.However, in applying the selected oil degradation strains to contaminated soil, the above factors should be taken into consideration because the effects are very large depending on soil characteristics, pollutants, inoculation time, and the like. In particular, since microorganisms have a big difference in the activity of microorganisms depending on the temperature at the time of application, the temperature at the time of application is the most important factor in the technology of biological soil purification. There have been studies on microorganisms that degrade oil at mid temperature, but the purification of contaminated soil using this has been concentrated in summer, and there are very few studies on low temperature strains that can be applied in winter.

따라서, 본 발명의 목적은 동절기에도 적용할 수 있도록 저온에서도 유류를분해할 수 있는 균주를 제공하는 것이다.Accordingly, it is an object of the present invention to provide a strain capable of decomposing oil even at low temperatures so that it can be applied in winter.

본 발명의 다른 목적은 상기 유류분해 저온균주를 제조하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing the oil-degrading low temperature strain.

상기 목적을 달성하기 위하여, 본 발명에서는 유류분해 저온균주를 오염된 토양으로부터 순수분리하고, 유류분해능이 우수한 균주를 선별하여 유류분해능 및 특성을 조사하였다. 본 발명에 따라 토양에서 분리한 유류분해균주 슈도모나스 CX3A(Pseudomonas sp.CX3A)는 5℃에서도 50% 이상의 유류분해능을 나타내어 저온에서도 오염된 토양현장에 적용할 수 있는 우수한 균주임을 확인하였다.In order to achieve the above object, in the present invention, the oil-degrading low temperature strain was purified purely from the contaminated soil, and the oil-degrading strains were selected to investigate the oil-degrading ability and characteristics. Pseudomonas sp. CX3A ( Pseudomonas sp. CX3A), isolated from the soil according to the present invention, exhibited an oil resolution of 50% or more even at 5 ° C., which was confirmed to be an excellent strain applicable to contaminated soil sites even at low temperatures.

이하, 본 발명의 구성 및 작용을 설명한다.Hereinafter, the configuration and operation of the present invention.

도 1은 본 발명에 따라 토양에서 분리한 슈도모나스 CX3A의 성장곡선을 나타낸 그래프이며, 상기 균주는 5℃ 및 4% (v/v) 디젤을 함유하는 최소영양배지에서 성장하였다.Figure 1 is a graph showing the growth curve of Pseudomonas CX3A isolated from the soil according to the present invention, the strain was grown in a minimal nutrient medium containing 5 ℃ and 4% (v / v) diesel.

본 발명은 오염된 토양에서 시료를 채취하는 단계; 토양시료로부터 저온에서 유류를 분해하는 균주를 분리하는 단계; 분리된 저온균주 중에서 성장 및 유류분해율이 우수한 균주를 선별하는 단계; 및 우수 유류분해 저온균주의 생물계면활성제 생산여부를 확인하는 단계로 구성되어 있다.The present invention comprises the steps of taking a sample from the contaminated soil; Separating the strain which degrades the oil at low temperature from the soil sample; Selecting strains having excellent growth and oil degradation rates among the separated low temperature strains; And confirming the production of biosurfactant of the excellent oil degradation low temperature strain.

이하, 본 발명의 구체적인 구성을 실시예를 들어 상세하게 설명하고자 하지만, 본 발명의 권리범위는 하기 실시예에만 한정되는 것은 아니다.Hereinafter, the specific configuration of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

실시예 1: 오염된 토양의 채취 및 유류분해 저온균주의 분리Example 1 Extraction of Contaminated Soil and Separation of Oil-Degraded Low Temperature

저온에서도 유류분해능을 보유한 균주를 선별하기 위하여 여러가지 종류의유류(원유, 휘발유, 등유, 디젤, 벤젠, 크실렌 등)로 오염된 여천 저유지 토양에서 채취하였다.To screen strains with oil resolution at low temperatures, they were collected from Yeocheon reservoirs contaminated with various types of oils (crude oil, gasoline, kerosene, diesel, benzene, xylene, etc.).

채취된 토양의 일정량을 멸균된 PBS(Phosphate buffer solution)에 넣고 균질화한 후, 4℃에서 1분씩 15분간 초음파처리하여 토양세균을 탈착한 다음 상기 세균용액을 농화배양시의 접종원으로 사용하였다. 토양내의 종속영양 세균수는 영양한천배지에 도말하여 배양한 후 측정하였다. 준비된 접종원을 경유 및 원유가 유일탄소원 및 에너지원으로 각각 포함된 최소영양배지에서 5℃ 및 12℃로 유지하며 4회 농화배양한 후, 영양한천배지에 도말하여 유류분해세균을 분리하였다. 분리된 유류분해균주의 유류분해능은 2,6-DCPIP(Dichlorophenol-Indophenol)의 환원반응에 의해 나타나는 색의 변화로 확인하였다.A predetermined amount of the collected soil was put into sterile PBS (Phosphate buffer solution) and homogenized. After sonication at 1 ° C. for 15 minutes at 4 ° C., the soil bacteria were desorbed and the bacterial solution was used as an inoculum during enrichment culture. The number of heterotrophic bacteria in soil was measured after incubation in nutrient agar medium. The prepared inoculum was maintained at 5 ° C. and 12 ° C. in a nutrient medium containing diesel and crude oil as the only carbon source and energy source, respectively, and concentrated for 4 times, and then plated on nutrient agar medium to separate the oil-degrading bacteria. The oil resolution of the isolated oil-degrading strains was confirmed by the color change caused by the reduction reaction of 2,6-DCPIP (Dichlorophenol-Indophenol).

유류분해 저온균주를 분리하기 위하여 유류에 의해 오염된 8개의 저유지에서 토양을 채취하여 저온에서 농화배양한 결과, 5℃에서 디젤을 분해하는 균주 36종과 원유를 분해하는 균주 25종, 12℃에서 디젤을 분해하는 균주 34종과 원유을 분해하는 균주 23종을 분리하였다.In order to isolate the oil-degrading low-temperature strains, soil was collected from eight reservoirs contaminated by oil, and concentrated at low temperature. As a result, 36 strains to decompose diesel at 5 ℃ and 25 strains to decompose crude oil and 12 ℃ 34 strains to decompose diesel and 23 strains to decompose crude oil were isolated.

실시예 2: 우수 유류분해 저온균주의 선별과 동정 및 유류분해력 측정Example 2 Screening, Identification, and Oil Degradability of Good Oil-Degrading Low Temperature Strains

디젤을 유일한 탄소원으로 하는 배양조건에서 빠르게 성장하는 균주를 선별하기 위하여 분리된 균주의 성장곡선을 측정하였다. 각 균주를 4% 디젤이 포함된 최소영양배지에서 배양하면서 660 nm에서의 광학밀도(OD)를 측정하여 각 균주의 성장속도를 비교하였다. 분리된 균주들을 TSBA(Trypticase soy broth agar)에서 1일간 배양한 후 MIDI(Microbial Identification System, Microbial ID Inc., Newark, Del.)법에 따라 지방산을 추출하여 분리균주를 동정하였다.The growth curves of the isolated strains were measured to select strains that grow rapidly in culture conditions using diesel as the only carbon source. While culturing each strain in a minimal nutrient medium containing 4% diesel, optical density (OD) at 660 nm was measured to compare the growth rates of each strain. The isolated strains were cultured in TSBA (Trypticase soy broth agar) for 1 day, and the isolated strains were identified by extracting fatty acids according to the method of Microbial Identification System, Microbial ID Inc., Newark, Del.

오염된 토양내의 유류분석은 미국의 EPA method 8015B에 따라 TPH(Total Petroleum Hydrocarbon) 함량을 측정하여 정량분석하였다. 4% (v/v) 디젤이 포함된 최소영양배지(Minimal Salt Media; pH 7.0, NaNO34 g/L, KH2PO40.15 g/L, Na2HPO40.5 g/L, MgSO4·7H2O 0.2 g/L, FeCl3·6H2O 0.0005 g/L, CaCl2·2H2O 0.01 g/L in distilled water)에 분리균주를 접종하여 5℃ 및 12℃에서 120 rpm으로 배양한 후, 배지 및 동량의 헥산으로 잔류디젤을 추출한 다음 GC-FID로 유류함량을 분석하였다. GC분석에 사용된 컬럼은 HP5(Hewlett Packard, U.S.A) 컬럼이었으며, 캐리어 가스(carrier gas)로 헬륨을 사용하였다. 분석전에 시료에 황산나트륨을 첨가하여 수분을 제거하였으며, GC 분석시의 표준시료(internal standard)로 1% (v/v) 스콸란(squalane)을 사용하였다.Oil analysis in contaminated soil was quantitatively determined by measuring total petroleum hydrocarbon (TPH) content according to the US EPA method 8015B. Minimal medium containing 4% (v / v) diesel (Minimal Salt Media; pH 7.0, 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 0.0005 g / L, CaCl 2 · 2H 2 O 0.01 g / L in distilled water) were inoculated with the inoculum and incubated at 120 rpm at 5 ℃ and 12 ℃ Then, the residual diesel was extracted with the medium and the same amount of hexane, and the oil content was analyzed by GC-FID. The column used for GC analysis was an HP5 (Hewlett Packard, USA) column, and helium was used as a carrier gas. Water was removed by adding sodium sulfate to the sample before analysis, and 1% (v / v) squalane was used as an internal standard for GC analysis.

5℃ 및 12℃에서 각각 분리된 61개 및 57개의 균주를 대상으로 DCPIP 시험을 통하여 5℃에서 32개의 균주, 12℃에서 31개의 균주를 선별하였다. 선별된 분리균주를 5℃ 및 12℃에서 배양하여 상기 균주들의 성장특성과 유류분해율을 측정한 결과, 상기 선별된 분리균주 중에서 성장 및 유류분해율이 우수한 균주 슈도모나스 CX3A를 선별하였다. 상기 슈도모나스 CX3A의 성장특성 및 유류분해율은 하기 표 1과 같으며, 성장곡선은 도 1과 같다.61 strains and 57 strains isolated at 5 ° C and 12 ° C, respectively, were screened for 32 strains at 5 ° C and 31 strains at 12 ° C through the DCPIP test. As a result of culturing the isolated strains at 5 ° C. and 12 ° C., the growth characteristics and oil degradation rate of the strains were measured. As a result, strains Pseudomonas CX3A having excellent growth and oil degradation rates were selected from the selected isolates. The growth characteristics and oil decomposition rate of Pseudomonas CX3A are shown in Table 1 below, and the growth curve is shown in FIG. 1.

본 발명에 따라 오염된 토양에서 순수분리한 상기 슈도모나스 CX3A를 생명공학연구소 소재 유전자은행에 수탁번호 KCTC 18054P로 2000년 11월 9일에 기탁하였다.The Pseudomonas CX3A, isolated purely from contaminated soil according to the present invention, was deposited on November 9, 2000 with accession number KCTC 18054P to the Gene Bank, Biotechnology Research Institute.

유류분해 저온균주 슈도모나스 CX3A의 성장특성 및 유류분해율.Growth Characteristics and Oil Degradation Rate of Pseudomonas CX3A from Low Temperature Hydrolysis. 균주명Strain name 성장특성Growth characteristics 유류분해율(%)Oil decomposition rate (%) 성장율(1/h)Growth rate (1 / h) 최대성장Growth OD (600 nm)OD (600 nm) 시간(일)Hours 슈도모나스 CX3APseudomonas CX3A 0.01140.0114 0.4930.493 88 58.058.0

실시예 3: 생물계면활성제의 생산 유무 확인Example 3: Confirmation of Production of Biosurfactant

분리균주 슈도모나스 CX3A의 생물계면활성제 생산여부를 확인하기 위하여 상기 분리균주의 표면장력을 측정하였다. 균주는 최소영양배지에서 농도를 OD 0.2로 맞추어 사용하였다. 글루코오스, 헥사데칸, 대두유(soybean oil)가 1% 비율로 혼합된 최소영양배지에 균주를 접종한 후 21일간 배양하였고 원심분리 후 상등액을 희석하여 표면장력을 측정하였다.The surface tension of the isolated strain Pseudomonas CX3A was measured to confirm the production of biosurfactant. The strain was used by adjusting the concentration to 0.2 OD in the minimal nutrient medium. Glucose, hexadecane, and soybean oil were inoculated with the strain in a minimal nutrient medium containing 1% of the strain, and cultured for 21 days. After centrifugation, the supernatant was diluted to measure the surface tension.

슈도모나스 CX3A의 표면장력Surface tension of Pseudomonas CX3A 균주Strain 표면장력Surface tension 최종 pHFinal pH 슈도모나스 CX3APseudomonas CX3A 36.9 ±0.536.9 ± 0.5 4.404.40

이상, 본 발명의 구체적인 구성을 실시예를 통하여 상세하게 설명한 바와 같이, 본 발명은 저온에서도 유류를 분해할 수 있는 저온균주를 제공함으로써 동절기에도 유류로 오염된 토양현장에 적용할 수 있는 뛰어난 효과가 있으므로 환경산업상 매우 유용한 발명인 것이다.As described above in detail with reference to the specific configuration of the present invention, the present invention provides a low-temperature strain that can decompose oil even at low temperatures, it is excellent effect that can be applied to soil contaminated with oil even in winter Therefore, it is a very useful invention for environmental industry.

Claims (3)

5℃ 온도에서 디젤을 포함하는 유류를 분해하는 토양으로부터 순수분리한 유류분해 저온균주 슈도모나스 CX3A(수탁번호: KCTC 18054P).Pyrolytic low temperature strain Pseudomonas CX3A (Accession No .: KCTC 18054P), purely separated from soil decomposing diesel-containing oil at a temperature of 5 ° C. 삭제delete 제 1항에 있어서, 상기 유류분해 저온균주는 생물계면활성제를 생산함을 특징으로 하는 슈도모나스 CX3A(수탁번호: KCTC 18054P).The Pseudomonas CX3A (Accession No .: KCTC 18054P) according to claim 1, wherein the low-temperature hydrolysed strain produces a biosurfactant.
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