KR20100031858A - Microbial consortia for the remediation of petroleum oil - Google Patents
Microbial consortia for the remediation of petroleum oil Download PDFInfo
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Abstract
Description
본 발명은 유류 오염 정화용 복합 미생물제제에 관한 것이다.The present invention relates to a complex microbial agent for oil pollution purification.
자연 환경 내에서 미생물들은 독자적으로 역할을 수행하는 경우는 극히 드물며, 미생물, 다른 생물, 또는 주변 환경과 상호관계를 이루며, 이러한 특성은 폐수처리 및 정수시스템, 병원성 미생물의 생존과 항생제 내성, 오염 환경 복원, 금속의 부식 등 우리 주변에서 발견되는 거의 모든 미생물 현상에서 발견되고 있다. 복합 미생물제제는 미생물 한 종으로써는 완전하지 못한 특별한 기능을 서로 보완함으로써 단일 미생물로서 수행하기 어려운 역할들을 효과적으로 수행할 수 있다. 이를 위해 각각의 미생물의 기능과 서로 보완하는 기능 연구 등의 복합적 정보 분석이 필요하다. In the natural environment, microorganisms rarely play their own role, and they interact with microorganisms, other organisms, or the surrounding environment, and these characteristics are associated with wastewater treatment and water purification systems, survival of pathogenic microorganisms, antibiotic resistance, and contaminated environments. It is found in almost all microbial phenomena found around us, including restoration and corrosion of metals. Complex microbial agents can effectively play roles that are difficult to perform as a single microorganism by complementing one another with special functions that are not complete with one species of microorganism. To this end, complex information analysis, such as research on the function of each microorganism and complementary functions, is required.
현재 국내에서는 중요한 생물자원 및 세포간의 상호작용 연구모델로서 복합 미생물의 중요성은 점차로 인식되고 있으나, 본격적인 복합 미생물 제제의 확보 및 연구는 아직 수행되지 못하고 있고, 여전히 오염물질의 환경 복원 기술은 단일 물질에 대한 단일 미생물 균주를 이용한 분해대사의 연구가 주를 이루고 있으며, 이 를 이용한 분해 미생물세포의 재설계 및 바이오모니터링을 위한 프로브 개발 연구 등이 주로 수행되고 있다. At present, the importance of complex microorganisms is increasingly recognized as a research model of important biological resources and interactions between cells, but the full-fledged acquisition and research of complex microbial agents has not been carried out yet. The study of degradation metabolism using a single microbial strain is mainly focused on the redesign of the degraded microbial cells and the development of probes for biomonitoring.
M Vinas는 5개의 미생물 복합체를 이용하여 방향족 화합물을 분해하는 실험을 통하여 원유 분해에 있어 미생물 복합체의 유용성을 확인하였다[J of Industrial Microbiol. and Biotechnol.2002, 28:252-260]. M Vinas confirmed the usefulness of microbial complexes in crude oil degradation through experiments using five microbial complexes to decompose aromatic compounds [J of Industrial Microbiol. and Biotechnol. 2002, 28: 252-260.
Fatima Menezes Bento는 디젤로 오염된 토양을 미생물 복합체를 이용하여 분해실험을 실시하여 디젤로 오염된 토양을 미생물 복합체가 효과적으로 복원하는 것을 확인하였다[Braz. J. Microbiol. 2003, 34:65-68 ].Fatima Menezes Bento conducted microbial complex degradation experiments on soils contaminated with diesel and confirmed that microbial complexes effectively restored soils contaminated with diesel [Braz. J. Microbiol. 2003, 34: 65-68.
집적 배양 후 분리된 균들 중 잘 자라는 종은 16S rDNA 염기서열 분석 결과, 엔테로박터(Enterobacter)와 오크로박테리움(Ochrobacterium)이였으며, t-RFLP 분석 결과에서도 동일종들이 발견되었다. 또한, 이들이 분해 활성에도 중요한 영향을 미침을 밝혔다[Wat. Air Soil Poll. 2003, 3: 103-115].Among the isolates after the incubation culture, the well-growing species were Enterobacter and Ochrobacterium as a result of 16S rDNA sequencing, and the same species were found in the t-RFLP analysis. In addition, they were found to have a significant effect on degradation activity [Wat. Air Soil Poll. 2003, 3: 103-115.
Laurie et al.은 PAH로 오염된 뉴질랜드의 와이카토 지역의 토양 두 곳과 오염되지 않은 시베리아 토양, 안타틱로스 섬의 토양으로부터 nahAc, phnAc (phenanthrene dioxygenase)의 양을 정량하였다[Appl. Environ. Microbiol. 2000, 66: 1814-1817].Laurie et al. Quantified the amount of nahAc and phnAc (phenanthrene dioxygenase) from two soils in Waikato, New Zealand, PAH-contaminated, uncontaminated Siberian and Antarctic islands [Appl. Environ. Microbiol. 2000, 66: 1814-1817.
Yeates et al.은 오스트레일리아의 웨일즈의 네 지역 중 방향족 탄화수소로 오염된 지역 두 곳과 오염되지 않은 지역 두 곳의 토양 시료를 대상으로 기존의 알려진 방향족 탄화수소 분해 유전자들로부터 만들어진 프라이머를 이용하여 PCR과 하이브리다이제이션(hybridization)법으로 새로운 유전자 탐색 및 이들의 기능을 관찰하였다[Environ. Microbiol. 2000, 2: 644-653].Yeates et al. Conducted PCR and hive using primers made from known aromatic hydrocarbon degradation genes for soil samples from two aromatic hydrocarbon-contaminated and two non-contaminated soils in four of Wales, Australia. New gene search and their function were observed by the hybridization method [Environ. Microbiol. 2000, 2: 644-653.
그리고, Jeon 등은 콜타르가 오염된 지역에서 나프탈렌 분해 미생물을 방사선동위원소 기술을 이용하여 in situ로 분석하여 미생물의 계통학적 분석과 기능적인 특징을 서로 연결하고자 하였다[Jeon et al., 2003].In addition, Jeon et al. Attempted to analyze the naphthalene-degrading microorganisms in situ using radioisotope techniques in coal tar-contaminated areas and to link the phylogenetic analysis and functional characteristics of the microorganisms [Jeon et al., 2003].
이렇게 현재까지 알려진 미생물의 유류 분해 실험은 단일 미생물을 사용함으로써 분해물질의 완전 분해가 이루어지지 않으며, 환경적응이 어렵다는 문제점이 야기되었고, 이러한 문제점을 해결하기 위해 복합 미생물제제가 절실히 요구되고 있다.The oil degradation experiments of the microorganisms known to date are not completely decomposed by the use of a single microorganism, and the problem of difficult environmental adaptation has been caused, and a complex microbial agent is urgently required to solve these problems.
이에, 본 발명자들은 자연계에 존재하는 미생물들이 단일 미생물로 역할을 하기보다는 복합적으로 수행하고 있으나, 미생물에 관련된 연구들이 단일 미생물에 집중되어 왔기 때문에 유용 복합 미생물 연구에 관련된 실험이 부족한 가운데 해양 원유를 생분해하는 미생물을 확보하여 기능적 특성을 확인한 후, 상기 미생물을 혼합한 복합 미생물 제제와 단일 미생물의 원유 분해활성을 비교하여 복합 미생물제제의 현저히 월등한 유류 분해 활성을 확인함으로써 본 발명을 완성하게 되었다.Therefore, the present inventors are performing a combination of microorganisms in nature rather than acting as a single microorganism, but since biotechnology has been concentrated on a single microorganism, biodegradation of marine crude oil is lacking in experiments related to research on useful microorganisms. After securing the microorganisms to confirm the functional characteristics, the present invention was completed by confirming the significantly superior oil degradation activity of the complex microbial agent by comparing the crude oil decomposition activity of the complex microbial agent and the single microorganism mixed with the microorganism.
따라서, 본 발명은 유류 오염 갯벌로부터 확보된 원유 분해능을 가지고 있는 단일 미생물 및 이들을 혼합하여 복합 미생물제제를 제공하는데 그 목적이 있다.Accordingly, an object of the present invention is to provide a single microorganism having a crude oil resolution obtained from oil contaminated tidal flat, and a combination of these microorganisms.
본 발명은 유류 분해 활성을 갖는 마이크로박테리움 옥시단스(Microbacterium oxydans) M101[KCTC 11366BP], 로도박테라세애 박테리움(Rhodobacteraceae bacterium) M116[KCTC 11367BP], 슈도모나스 마린콜라(Pseudomonas marincola) M157[KCTC 11368BP] 각각을 그 특징으로 한다.The present invention is a microbacterium oxydans M101 [KCTC 11366BP] having oil degradation activity, Rhodobacteraceae bacterium M116 [KCTC 11367BP], Pseudomonas marincola ( Pseudomonas marincola ) Each is characterized by M157 [KCTC 11368BP].
또한, 본 발명은 상기 미생물 2종 이상 혼합된 복합 미생물제제를 또 다른 특징으로 한다. In addition, the present invention is another feature of the complex microbial agent mixed with two or more microorganisms.
이와 같은 본 발명을 더욱 상세하게 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명은 유류 오염 갯벌에서 원유 분해 활성을 갖는 미생물을 분리하였고, 분리한 미생물의 유류 분해 활성이 우수함을 확인하였고, 또한 상기 미생물들을 혼합한 복합 미생물제제이 단일 미생물에 비해 월등한 유류 분해 활성을 가짐을 확인함으로써 유류 오염 정화에 유용한 기능성 미생물제제에 관한 것이다.The present invention isolated microorganisms having crude oil decomposition activity in oil-contaminated tidal flats, it was confirmed that the oil-degrading activity of the separated microorganisms is excellent, and the complex microbial agent mixed with the microorganisms has superior oil degradation activity compared to a single microorganism The present invention relates to a functional microbial agent useful for the purification of oil pollution.
본 발명에서 고농도 유류로 오염된 갯벌로부터 유류 분해 미생물을 확보하기위해 사용된 배지는 브쉐넬-하스(Bushnell-Haas; BH)를 인공해수에 녹인 액체 배지를 사용하였고, l%(w/v)의 멸균된 원유를 첨가한 배지를 사용하였다. 준비된 배지에 집적배양을 통해 기능성 미생물 커뮤니티를 확보하고, 그 커뮤니티 안에서 미생물을 순수 분리하였다. 순수 분리한 미생물들을 원유 분해능 실험을 통해 우수한 유류 분해 미생물을 선별하였고, 선별한 미생물에서 추출한 DNA를 PCR 기법을 통해 유전자 확인 실험과 순수 분리 미생물을 동정하였다. 순수 분리한 유류 분해 미생물은 마이크로박테리움 옥시단스(Microbacterium oxydans) M101[이하, M101로 명명], 로도박테라세애 박테리움(Rhodobacteraceae bacterium) M116[이하, M116으로 명명]및 슈도모나스 마린콜라(Pseudomonas marincola) M157[이하, M157로 명명]로 동정되었고, 한국생명공학연구원 유전자은행에 2008년 7월 23일자로 기탁하였으며, 기탁번호는 각각 KCTC 11366BP, KCTC 11367BP, KCTC 11368BP로 부여받았다. In the present invention, a medium used to secure oil-degrading microorganisms from tidal flats contaminated with high concentration of oil was used as a liquid medium obtained by dissolving Bushnell-Haas (BH) in artificial seawater and l% (w / v). A medium to which sterilized crude oil was added was used. A functional microbial community was secured through integrated culture in the prepared medium, and microorganisms were purely separated therein. Pure oil-separated microorganisms were screened for crude oil-degrading microorganisms, and DNA extracted from the selected microorganisms was identified for genetic identification and pure-separated microorganisms. Purely isolated oil-degrading microorganisms include Microbacterium oxydans M101 (hereinafter referred to as M101), Rhodobacteraceae bacterium M116 (hereinafter referred to as M116) and Pseudomonas marincola ) It was identified as M157 [hereinafter referred to as M157], and was deposited with the Korea Biotechnology Research Institute Gene Bank on July 23, 2008. The accession numbers were assigned to KCTC 11366BP, KCTC 11367BP and KCTC 11368BP, respectively.
유류 분해에 효과적이라고 알려져 있는 미생물들과 선별된 각 미생물, 복합 미생물의 유류 분해능을 분석한 결과, 복합 미생물제제가 92%로 가장 분해능이 우수했으며, 선별된 유류 분해 미생물들도 58 ~ 72%의 분해능이 있는 것을 확인하였다. 2종을 혼합한 미생물제제의 경우도 단일 미생물보다 다소 우수한 80% 정도의 분해능을 보여주었다. 그러나, 다른 대부분의 미생물들은 해양 환경과 낮은 온도에서 생장조차 하지 않았다. As a result of analyzing oil resolution of microorganisms known to be effective for oil decomposition, selected microorganisms and complex microorganisms, the compound microorganisms had the highest resolution at 92%, and the selected oil-degrading microorganisms also had resolutions of 58 to 72%. It was confirmed that there is. In the case of the mixed microbial agent, the resolution of 80% was slightly better than that of a single microorganism. However, most other microorganisms did not even grow in marine environments and at low temperatures.
선별된 미생물의 배양을 통한 유류 분해능 유무와 유류 분해능 유지를 위한 최적 배양조건을 탐색한 결과, 마린 아가(Marine agar), R2A 아가에서 모두 생장이 가능했으나, 2회 계대 배양한 후에는 유류 분해능이 현저히 낮아지는 것을 확인하였다. 선별된 3개의 미생물들은 원유 이외의 다른 유기 탄소원을 이용하며 생장할 수 있는 미생물이었고, 탄소원으로 원유가 없는 경우 원유 분해능을 금방 상실하였다. 따라서, 유류 분해능을 유지하기 위해 원유를 탄소원으로 포함하는 배지에서 배양하였으며, 원유 분해능이 양호하게 유지되는 것을 확인하였다.After cultivating the selected microorganisms, it was possible to grow oil in marine agar and R2A agar. It was confirmed that it is significantly lowered. The three selected microorganisms were microorganisms that could grow by using organic carbon sources other than crude oil, and crude oil resolution was quickly lost in the absence of crude oil as a carbon source. Therefore, in order to maintain oil resolution was cultured in a medium containing crude oil as a carbon source, it was confirmed that the crude oil resolution is maintained well.
복합 미생물 제제 제작에는 선별된 유류 분해 미생물 3종을 각각 배양한 후, 같은 균체수의 양으로 혼합하고, 해양 환경의 적응을 위해 인공해수를 이용하여 혼합 현탁하였다. 혼합시 각각의 미생물은 전체 균주량에 대하여 24 ~ 40%가 바 람직하다.In the preparation of the complex microbial preparation, each of three selected oil-degrading microorganisms was cultured, mixed in the same amount of the cells, and mixed and suspended using artificial seawater for adaptation to the marine environment. When mixed, each microorganism is preferably 24 to 40% of the total strain amount.
따라서, 본 발명에 따른 복합 미생물제제는 해양 환경에서 적응과 원유 분해능이 우수한 것으로 확인되었으며, 그 유용성도 검증된 바, 해양유류 유출 사고에 매우 유용하게 사용되리라 기대된다.Therefore, the composite microbial agent according to the present invention was found to have excellent adaptation and crude oil resolving power in the marine environment, and its usefulness was also verified, and thus, it is expected to be very useful for marine oil spill accidents.
본 발명에 따른 복합 미생물 제제는 단일 미생물보다 탁월한 유류 분해능을 가지고 있고, 복합 미생물 제제는 단일 미생물보다 여러 조건이 영향을 미치는 오염 환경에서 더욱 그 유용성이 높을 것이라 생각된다. 복합 미생물 제제를 이용한 해양 유류 오염 환경의 생물학적 환경정화(Bioremediation) 공정 개발을 가속화 할 것이며, 이를 통해 오염 환경의 정화라는 기술적인 성과뿐만 아니라 경제적으로도 큰 부가가치를 창출할 수 있을 것으로 기대된다.The composite microbial preparation according to the present invention has better oil resolution than a single microorganism, and the composite microbial preparation is considered to be more useful in a contaminated environment where several conditions are affected than a single microorganism. The development of bioremediation processes for marine oil pollution using complex microbial products will be accelerated, and this is expected to create economically significant added value as well as technical achievements in the purification of polluted environment.
이하, 실시예를 들어 본 발명을 상세히 기술할 것이나 본 발명의 범위를 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.
실시예Example 1: 갯벌로부터 유류 분해 기능성 미생물 커뮤니티 확보 1: Securing Oil-Degrading Functional Microbial Communities from Tidal Flats
유류로 오염된 곰소항 갯벌에서 BH 배지(표 1)를 인공해수(표 2)를 이용하여 만들고 10,000 ppm의 원유를 첨가하여 24 ℃, 120 rpm에서 10일간 배양하여 4차례 의 집적 배양을 통하여 유류 분해능이 가장 높은 기능성 미생물 커뮤니티 FMC-CR8을 확보하였다.BH medium (Table 1) was made using artificial seawater (Table 2) from Gomso Port tidal flats contaminated with oil, and 10,000 ppm of crude oil was added and cultured for 10 days at 24 ℃ and 120 rpm. This highest functional microbial community FMC-CR8 was secured.
[표 1] TABLE 1
BH 배지의 성분Ingredients of BH Medium
[표 2]TABLE 2
인공해수의 조성Composition of Artificial Seawater
실시예 2: 원유 분해 미생물의 분리 및 동정Example 2: Isolation and Identification of Crude Microorganisms
확보된 기능성 미생물 커뮤니티 FMC-CR8로부터 다음 표 3의 배지들을 이용하여 24 ℃, 48시간 배양하여 배양 가능한 미생물을 분리하였다. 육안으로 관찰하여 서로 다른 콜로니 12 종류를 순수 분리하였다. The cultured microorganisms were isolated from the obtained functional microbial community FMC-CR8 by culturing at 24 ° C. for 48 hours using the media of Table 3 below. Visually observed, 12 different colonies were purely separated.
[표 3] [Table 3]
순수 분리를 위한 배지 조성Medium composition for pure separation
분리된 미생물의 원유 분해활성을 비교 분석하였다. 각각의 시료는 107 cfu/ml 되도록 식염수에 희석하여 접종하였다. BH 배지를 인공해수(표 2)에 녹여 배지를 만들고 10,000 ppm의 원유를 첨가하여 20 ℃, 120 rpm에서 10일간으로 10일간 진탕 배양하였다. 그 결과, 3개의 분리 미생물에서 높은 원유 분해능을 확인하였다. M101은 72%, M116은 58%, M1577은 67%의 분해능을 보였다[도 1]. 3개 분리 미생물은 Genomic DNA extraction kit(intron)을 이용하여 DNA를 추출한 후, PCR을 수행한 후 솔젠트에 의뢰해서 동정하였다. 동정 결과는 다음 표 4와 같다.The crude oil degradation activity of the isolated microorganisms was analyzed. Each sample was inoculated by diluting with saline to 10 7 cfu / ml. BH medium was dissolved in artificial seawater (Table 2) to make a medium, and 10,000 ppm of crude oil was added, followed by shaking culture for 10 days at 20 ° C. and 120 rpm for 10 days. As a result, high crude oil resolution was confirmed in the three isolated microorganisms. M101 had a resolution of 72%, M116 58%, and M1577 67% [Fig. 1]. The three isolated microorganisms were identified by genomic DNA extraction kit (intron), DNA extraction, PCR, and then solgent. Identification results are shown in Table 4 below.
[표 4] [Table 4]
분리 미생물의 동정Identification of isolated microorganisms
실시예 3: 선별 유류 분해 미생물의 특성Example 3: Characteristics of Selected Oil-Degrading Microorganisms
선별된 유류 분해 미생물 특성을 검토하기 위해 유류 분해 관련 유전자를 분석하였으며, 이를 위해 각 미생물을 Genomic DNA extraction kit(intron)을 이용하여 DNA를 추출하였고, 다음 표 5의 프라이머를 이용하여 PCR을 수행하였다. M101은 alkB2와 alkB4를 M116은 alkB1, alkB2, alkB4, Rnar, bphA, nahAc,xylE를 M157은 alkB1, bphA, narB, nahAc를 각각 보유하고 있는 것으로 확인되었다[도 2]. M101은 주로 지방족 탄화수소 분해 관련 유전자를 포함하고 있으며, M116은 지방족과 방향족 탄화수소 분해 유전자를 다양하게 포함하고 있고, M157은 주로 방향족 탄화수소 분해 유전자를 포함하고 있는 것으로 확인되었다. 이 결과로부터 선별된 3개의 미생물은 복합 오염물질인 유류를 다양한 대사경로를 통해 분해할 수 있는 특성을 가지고 있음을 확인하였다.In order to examine the characteristics of selected oil degradation microorganisms, genes related to oil degradation were analyzed. For this, each microorganism was extracted with a genomic DNA extraction kit (intron), and PCR was performed using the primers of Table 5 below. . M101 has alkB2 and alkB4, M116 has alkB1, alkB2, alkB4, Rnar, bphA, nahAc, xylE, and M157 has alkB1, bphA, narB, nahAc, respectively [Fig. 2]. M101 mainly contains aliphatic hydrocarbon degradation genes, M116 contains various aliphatic and aromatic hydrocarbon degradation genes, and M157 mainly contains aromatic hydrocarbon degradation genes. From these results, it was confirmed that the three selected microorganisms are capable of decomposing complex pollutant oil through various metabolic pathways.
[표 6] TABLE 6
사용된 프라이머Primer used
DGGE를 위한 PCR 조건PCR conditions for DGGE
반응 혼합물의 구성:Composition of the reaction mixture:
DNA 1 ㎕1 μl DNA
12.5 pmol primer F 2.5 ㎕12.5 pmol primer F 2.5 μl
12.5 pmol primer R 2.5 ㎕12.5 pmol primer R 2.5 μl
10 mM dNTP 혼합물 1 ㎕1
10ㅧ PCR 완충액 5 ㎕5 μl of 10 ㅧ PCR buffer
보빈 시럼 알부민(BSA) 0.5 ㎕0.5 μl bobbin serum albumin (BSA)
Taq DNA 중합효소(TaKaRa, Japan) 0.5 ㎕0.5 μl of Taq DNA polymerase (TaKaRa, Japan)
dH2O 36 ㎕36 μl dH 2 O
반응조건(초기 변성 및 최종 연장과정을 제외하고 30회 반복):Reaction conditions (30 repetitions except for initial denaturation and final extension):
초기 변성 과정 - 95 ℃, 3분Initial denaturation process-95 ℃, 3 minutes
변성 과정 - 94 ℃, 1분Denaturation process-94 ℃, 1 minute
어닐링 과정 - 60 ℃, 1분Annealing process-60 ℃, 1 minute
중합 과정 - 72 ℃, 1분Polymerization process-72 ℃, 1 minute
최종 연장 과정 - 72 ℃, 10분Final extension process-72 ° C, 10 minutes
PCR 반응물은 아가로스 겔에서 확인하였다.PCR reactions were identified on agarose gels.
실시예 4: 유류 분해 미생물의 배양Example 4 Cultivation of Oil-Degrading Microorganisms
미생물 커뮤니티로부터 유류 분해 미생물을 분리하기 위해 사용된 마린 아가와 R2A 아가에 선별 유류 분해 미생물을 접종하여 24 ℃ 배양기에서 48시간 동안 배양하였다. 1차 계대 배양한 것과 2차 계대 배양한 것의 유류 분해능을 분석한 결과, 원유가 포함되지 않은 배지를 이용하여 배양이 진행될수록 유류 분해능이 급격히 감소하는 것을 확인하였다. 반면에, 배지에 원유를 0.5 ~ 1% 포함하여 배양하면 유류 분해능이 보존되는 것으로 확인하였다[도 3].Selected oil degradation microorganisms were inoculated on marine agar and R2A agar used to separate the oil degradation microorganisms from the microbial community and incubated for 48 hours in a 24 ° C. incubator. As a result of analyzing the oil resolution of the primary passage and the secondary passage culture, it was confirmed that the oil resolution rapidly decreases as the culture proceeds using a medium containing no crude oil. On the other hand, the culture of crude oil containing 0.5 ~ 1% in the medium was confirmed that the oil resolution is preserved [Fig. 3].
실시예Example 5: 복합 미생물 제제 제작 5: Complex microbial preparation
기능성 미생물 M101, M116, M157은 해양 저온 환경에서 생장하면서 원유를 분해하는 능력을 가지고 있다. 각 각의 미생물의 생장률은 유사하고 유류분해 관련 유전자도 보유하고 있으나, 각각의 다른 분해 대사 경로를 가지고 있는 것으로 확인되었다. 복합 미생물 제제는 액체 배양한 3개의 미생물 배양액을 원심분리를 통하여 미생물만 모으고 각각 균체수 2 × 107 ~ 5 × 107 cfu/ml이 되도록 한다. 인공해수로 상기 3개의 미생물 각각을 동일한 균체수의 양으로 혼합 현탁하여 액상 미생물 제제를 제작하였다. 이렇게 제작된 3종 복합 미생물 제제와 2종 복합 미생물 제제, 단일 유류분해 미생물의 원유 분해능을 분석하였고, 3종 복합 미생물제제가 2종 복합 미생물 제제와 단일 유류 분해 미생물 보다 높은 92%의 유류 분해능을 보여주었다[도 4]. Functional microorganisms M101, M116 and M157 have the ability to degrade crude oil while growing in marine low temperature environment. The growth rate of each microorganism is similar, and it contains genes related to oil degradation, but has been found to have different degradation metabolic pathways. The complex microbial preparation is centrifuged to collect three microbial cultures in liquid culture, so that the number of cells is 2 × 10 7 ~ 5 × 10 7 cfu / ml each. In artificial seawater, each of the three microorganisms was mixed and suspended in the same amount of bacterial cells to prepare a liquid microbial preparation. The crude oil resolution of the three complex microbial preparations, the two complex microbial preparations, and the single oil-degrading microorganisms were analyzed. [Fig. 4].
도 1은 분리한 미생물의 유류 분해능 확인한 것이다[1: M101, 2: M102, 3: M103, 4:M114, 5: M115, 6: M116, 7:M157, 8: M158, 9; M159, 10: M1510, 11: M1511, 12: M1512].1 shows the oil resolution of the separated microorganisms [1: M101, 2: M102, 3: M103, 4: M114, 5: M115, 6: M116, 7: M157, 8: M158, 9; M159, 10: M1510, 11: M1511, 12: M1512].
도 2는 분리된 미생물의 유류 분해 관련 대사 유전자를 분석한 결과이다.Figure 2 shows the results of analyzing the metabolic genes related to oil degradation of the isolated microorganisms.
도 3은 분리한 유류 분해 미생물의 배양방법에 따른 유류 분해능 변화를 나타낸 것이다.Figure 3 shows the change in oil resolution according to the culture method of the separated oil-degrading microorganisms.
도 4는 복합 미생물 제제와 단일 미생물과의 유류 분해능 비교 분석한 그래프이다[FMC167: 3종 복합 미생물 제제; MC16: M101+M116 2종 복합 미생물 제제, MC17: M101+M157 2종 복합 미생물 제제, MC67: M116+M157 2종 복합 미생물 제제].Figure 4 is a graph comparing the oil resolution of the complex microbial agent and a single microorganism [FMC167: three complex microbial agent; MC16: M101 + M116 two-combined microbial formulation, MC17: M101 + M157 two-combined microbial formulation, MC67: M116 + M157 two-combined microbial formulation].
<110> Korea Research Institute of Bioscience and Biotechnology <120> Microbial consortia for the remediation of petroleum oil contaminated ocean environment <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> alkB1 forward primer <400> 1 atctgggcgc gttgggattt gagcg 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> alkB1 reverse primer <400> 2 cgcatggtga tcgctgtgcc gctgc 25 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> alkB2 forward primer <400> 3 cctgctcccg atcctcga 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> alkb2 reverse primer <400> 4 tcgtaccgcc cgctgtccac 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> alkB3 forward primer <400> 5 ggtgtcgacg ctcctgcatg gc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> alkb3 reverse primer <400> 6 cgccttggtg tgaatgagct cg 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> alkB4 forward primer <400> 7 tacggtcact tctacatcga g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> alkB4 reverse primer] <400> 8 attcgcgtgg tggtcggagt 20 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Rnar forward primer <400> 9 tggacctaca gcaacacggg aag 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Rnar reverse primer <400> 10 tgccagaggc gcagcgtcag gaa 23 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> bphA forward primer <400> 11 ttcctvaacc agtgccgsca cc 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> bphA reverse primer <400> 12 ctggatytcs acccagttct c 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> narB forward primer <400> 13 acgtgcaaga aggcgcgaaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> narB reverse primer <400> 14 acgctcccgc gaggcgagaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nahAc forward primer <400> 15 agytaycacg gctggggctt 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> nahAc reverse primer <400> 16 ctgttgttcg ggaaaacsgt gc 22 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> xylE forward primer <400> 17 tgcagctgcg tgtactggac a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> xylE reverse primer <400> 18 gggtcgaaga agtagatggt c 21 <110> Korea Research Institute of Bioscience and Biotechnology <120> Microbial consortia for the remediation of petroleum oil contaminated ocean environment <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> alkB1 forward primer <400> 1 atctgggcgc gttgggattt gagcg 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> alkB1 reverse primer <400> 2 cgcatggtga tcgctgtgcc gctgc 25 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> alkB2 forward primer <400> 3 cctgctcccg atcctcga 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> alkb2 reverse primer <400> 4 tcgtaccgcc cgctgtccac 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> alkB3 forward primer <400> 5 ggtgtcgacg ctcctgcatg gc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> alkb3 reverse primer <400> 6 cgccttggtg tgaatgagct cg 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> alkB4 forward primer <400> 7 tacggtcact tctacatcga g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> alkB4 reverse primer] <400> 8 attcgcgtgg tggtcggagt 20 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Rnar forward primer <400> 9 tggacctaca gcaacacggg aag 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Rnar reverse primer <400> 10 tgccagaggc gcagcgtcag gaa 23 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> bphA forward primer <400> 11 ttcctvaacc agtgccgsca cc 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> bphA reverse primer <400> 12 ctggatytcs acccagttct c 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> narB forward primer <400> 13 acgtgcaaga aggcgcgaaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> narB reverse primer <400> 14 acgctcccgc gaggcgagaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nahAc forward primer <400> 15 agytaycacg gctggggctt 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> nahAc reverse primer <400> 16 ctgttgttcg ggaaaacsgt gc 22 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> xylE forward primer <400> 17 tgcagctgcg tgtactggac a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> xylE reverse primer <400> 18 gggtcgaaga agtagatggt c 21
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101509326B1 (en) * | 2013-08-22 | 2015-04-08 | 순천대학교 산학협력단 | Microbial agent having degradation of organic compounds and endosulfan and method for preparation thereof |
| CN113046263A (en) * | 2021-03-16 | 2021-06-29 | 青岛农业大学 | Salt-tolerant potassium-decomposing growth-promoting microbacterium oxydans 41-C8, microbial inoculum and application thereof |
| CN113046263B (en) * | 2021-03-16 | 2022-03-25 | 青岛农业大学 | Salt-tolerant potassium-decomposing growth-promoting microbacterium oxydans 41C8, microbial inoculum and application thereof |
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