KR101114426B1 - Novel strain Chlamydomonas pitschmannii YSL03 - Google Patents
Novel strain Chlamydomonas pitschmannii YSL03 Download PDFInfo
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- KR101114426B1 KR101114426B1 KR20100064306A KR20100064306A KR101114426B1 KR 101114426 B1 KR101114426 B1 KR 101114426B1 KR 20100064306 A KR20100064306 A KR 20100064306A KR 20100064306 A KR20100064306 A KR 20100064306A KR 101114426 B1 KR101114426 B1 KR 101114426B1
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- 241000195608 Chlamydomonas pitschmannii Species 0.000 title claims description 23
- 241000195585 Chlamydomonas Species 0.000 claims abstract description 36
- 150000002632 lipids Chemical class 0.000 claims abstract description 32
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- 241000195493 Cryptophyta Species 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 17
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- C12N1/125—Unicellular algae isolates
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
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- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
본 발명은 신균주 클라미도모나스 피트쉬만니 YSL03[KCTC 11715BP] 더욱 상세하게는 신균주 클라미도모나스 피트쉬만니 YSL03[KCTC 11715BP]는 높은 지질 함량을 가지고 있어 바이오디젤 생산에 매우 효과적이고, 또한 폐수 내성이 강하여 폐수 내 질소와 인을 제거할 수 있으므로 폐수 처리와 동시에 바이오 에너지 생산이 가능하다.The present invention is Mycobacterium Klimidomonas Fitschmanny YSL03 [KCTC 11715BP] More specifically, the new strain Chlamydomonas pitschmanny YSL03 [KCTC 11715BP] has a high lipid content, which is very effective for biodiesel production and strong wastewater resistance, which can remove nitrogen and phosphorus in the wastewater.
Description
본 발명은 폐수 내성 및 지질 함량이 높은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주에 관한 것이다.
The present invention, Chlamydomonas pitschmanny high in wastewater resistance and lipid content YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.
지구상의 에너지 사용 증가는 지속적으로 증가하여 많은 환경오염을 초래하며, 결과적으로 지구온난화의 문제를 가져왔다[Chisti, Y. 2007. Biodiesel from microalgae. Biotechnology Advances, 25: 294-306.]. 바이오매스는 온실 효과에 작용을 하지 않는 신재생에너지원으로[Widjaja, A., Chao-Chang Chien, Yi-Hsu Ju. 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J. Taiwan Inst. Chem. Eng.40, 13-20.] 대량 생산 시 에너지로 활용이 되어 지속적으로 개발되고 있다. 바이오디젤은 트리글리세리드 오일(triglyceride oil)과 모노하이드릭 알콜(monohydric alcohols)에서 얻어지며, 바이오매스에 속하는 에너지원으로 주로 콩, 사탕수수, 동물지방 등에서 생산되어왔다[Lang, X., Dalai, A.K., Bakhshi, N.N., Reaney, M.J., Hertz, P.B., 2002. Preparation and characterization of biodiesels from various Bio-Oils. Biores. Technol. 80, 53-62.]. 그러나, 인류식량난 이론에 부딪혀 개발에 어려움이 있다. Increasing use of energy on the planet continues to increase, resulting in many environmental pollutions, resulting in global warming [Chisti, Y. 2007. Biodiesel from microalgae. Biotechnology Advances, 25: 294-306.]. Biomass is a renewable energy source that does not affect the greenhouse effect [Widjaja, A., Chao-Chang Chien, Yi-Hsu Ju. 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J. Taiwan Inst. Chem. Eng.40, 13-20.] It is continuously developed as it is used as energy in mass production. Biodiesel is obtained from triglyceride oils and monohydric alcohols. It is an energy source belonging to biomass and has been mainly produced from soybeans, sugar cane and animal fats [Lang, X., Dalai, AK. , Bakhshi, NN, Reaney, MJ, Hertz, PB, 2002. Preparation and characterization of biodiesels from various Bio-Oils. Biores. Technol. 80, 53-62.]. However, it is difficult to develop due to the food shortage theory.
최근 바이오디젤을 안정적이며 경제적으로 생산할 수 있는 에너지원인 조류를 개발하고 있으며, 이는 조류의 다음과 같은 특징 때문이다. 1) 조류는 독립, 종속 영양 광합성균으로 태양에너지와 물을 비롯 이산화탄소가 있으면 어느 곳(바다, 호수, 강 등)에서 성장되는 특성을 지니고 있다. 2) 다른 물질(예: 콩, 옥수수, 사탕수수 등) 보다 생산성이 좋고[Becker, 1994. Measurement of algal growth. In: Microalgae biotechnology and microbiology. Cambridge University Press, P 56-62.], 3) 바이오디젤의 생산물질인 지질 함량을 높게 갖고 있다고 보고된 바 있다[Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998. A look back at the U.S. Department of Energy’s Aquatic Species Program: Biodiesel from Algae. Close-Out report. National Renewable Energy Lab, Department of Energy, Golden, Colorado, U.S.A. Report number NREL/TP-580-24190.]. 그러나, 최근까지 연구된 대부분 조류는 해양에 서식하는 해양 조류이며, 재배 시 질소-인 등의 원소를 인위적으로 공급해야 하는 단점을 지닌다. 하천, 호소수에 서식하는 조류 경우 폐수에 함유된 미량의 성분 또는 하천과 강에 포함된 질소ㆍ인을 이용하여 성장한다. 따라서, 폐수 내성을 갖는 미세조류 균주종을 분리한다면 미세조류 배양기에 공급되는 폐수에서 영양분을 섭취하고 있으므로 질소-인의 인위적 주입을 고려하지 않아 공정의 경제성을 향상시킬 수 있을 것이다. 그러나, 현재 균주 은행에 기탁된 대부분의 일반 미세조류 균주종은 이러한 폐수에서 생육이 보고가 미미하며 지질 함량 및 생장 속도에 대한 자료가 부족하므로 새로운 미세조류의 분리가 절실히 필요하다.Recently, algae, which is an energy source capable of producing biodiesel in a stable and economical manner, are being developed due to the following characteristics of algae. 1) Algae are independent, heterotrophic photosynthetic bacteria that grow in the presence of solar energy, water, and carbon dioxide (sea, lake, river, etc.). 2) Better productivity than other materials (eg soybeans, corn, sugar cane, etc.) [Becker, 1994. Measurement of algal growth. In: Microalgae biotechnology and microbiology. Cambridge University Press, P 56-62.], 3) has been reported to have a high lipid content, which is a biodiesel product [Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998.A look back at the US Department of Energy ’s Aquatic Species Program: Biodiesel from Algae. Close-Out report. National Renewable Energy Lab, Department of Energy, Golden, Colorado, U.S.A. Report number NREL / TP-580-24190.]. However, most of the algae studied until recently are marine algae inhabiting the ocean, and have the disadvantage of artificially supplying elements such as nitrogen and phosphorus during cultivation. Algae that live in rivers and lakes grow by using trace elements in wastewater or by nitrogen and phosphorus in rivers and rivers. Therefore, if the microalgae strains having wastewater resistance are separated, the nutrient is ingested from the wastewater supplied to the microalgae incubator, and thus the economic efficiency of the process may be improved without considering the artificial injection of nitrogen-phosphorus. However, most of the common microalgal strains currently deposited in the strain banks are very poorly reported in these wastewaters and lack of data on lipid content and growth rate, so the isolation of new microalgae is urgently needed.
이에, 본 발명자들은 폐수에 대해 내성을 갖는 미세조류 균주를 확보하기 위해 연구를 진행하였다. 그 결과, 폐수에 내성을 가지며, 폐수에서 성장이 빠르고, 바이오디젤 생산 물질인 지질 함량 50% 이상을 가지는 미세조류 종을 자연 환경으로부터 분리하여 바이오디젤 생산에 유용함을 알게됨으로써 본 발명을 완성하게 되었다. Thus, the present inventors conducted a study to secure microalgae strains resistant to wastewater. As a result, microalgae species having resistance to wastewater, growing rapidly in wastewater, and having a lipid content of 50% or more of biodiesel production material are separated from the natural environment, and thus, are useful for biodiesel production. .
따라서, 본 발명은 신규한 미세조류를 폐수 처리에 사용하여 환경오염 문제를 해결하고, 그 과정에서 생성된 미세조류를 바이오디젤 생산용 원료로 활용하여 대체에너지를 해결하는 방법을 제공하고자 한다.Accordingly, the present invention is to solve the environmental pollution problem by using a novel microalgae in wastewater treatment, and to provide a method for solving the alternative energy by using the microalgae generated in the process as a raw material for biodiesel production.
상기 목적을 달성하기 위하여, 본 발명은 In order to achieve the above object, the present invention
본 발명은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주 및 이의 배양액을 그 특징으로 한다.The present invention Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strains and cultures thereof are characterized.
또한, 본 발명은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주 또는 이의 배양액으로부터 지질을 분리하고, 또한 상기 분리된 지질을 이용하여 바이오디젤을 생산하는 방법을 다른 특징으로 한다.In addition, the present invention Chlamydomonas Fitschmann Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strains or cultures thereof, and the method for producing biodiesel using the separated lipids are another feature.
또한, 본 발명은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주 또는 이의 배양액을 포함하는 폐수 처리용 미생물제제를 또 다른 특징으로 한다.In addition, the present invention Chlamydomonas Fitschmann Another feature is a microbial agent for wastewater treatment comprising a strain YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] or a culture thereof.
또한, 본 발명은 폐수에서 상기 신균주를 배양하는 방법을 포함한다.
The present invention also includes a method for culturing the new strain in wastewater.
본 발명은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주를 이용하여 폐수 내성과 바이오디젤 생산원인 지질 함유를 검토하여 바이오 디젤 생산용 신균주로서의 가능성을 확보하였다. The present invention Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain was used to examine wastewater resistance and lipid content as a biodiesel production source, thus securing the potential as a new strain for biodiesel production.
본 발명의 신균주는 폐수 내성이 강하며, 폐수에 함유된 N, P의 직접 적용이 가능하여 배양의 경제성을 크게 향상시키는 효과를 가진다.The new strain of the present invention has a strong wastewater resistance, and can directly apply N and P contained in the wastewater, thereby greatly improving the economics of the culture.
또한, 성장된 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP]는 높은 함량(50% 이상) 지질을 함유하고 있어 바이오디젤 생산에 기여토록 하는 효과를 가진다.
Also, grown Chlamydomonas Fitschmann. YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] contains high content (more than 50%) lipids, which contributes to biodiesel production.
도 1은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03) [KCTC 11715BP]의 사진이다.
도 2는 클라미도모나스 피트쉬만니 YSL03[KCTC 11715BP]의 계통수를 나타낸 것이다.
도 3은 폐수와 BBM에서 클라미도모나스 피트쉬만니 YSL03[KCTC 11715BP]의 질소 및 인 제거 효과를 나타낸 것이다.
도 4는 클라미도모나스 피트쉬만니 YSL03[KCTC 11715BP]의 지방산 분포를 나타낸 것이다 [리놀렌산 (C18:3n6), 리놀레닉산(C18:3n 3), 올레인산(C18:1n9c), 스테아린산(C18:0), 헵타데칸산(C17:0), 팔미토레익산(C16:1), 팔미틴산(C16:0), 미리스틱산(C14:0), 라우릴산(C12:0)].
1 is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP].
Fig. 2 Chlamydomonas Fitschmann The phylogenetic tree of YSL03 [KCTC 11715BP] is shown.
Figure 3 Chlamydomonas Fitschmann in wastewater and BBM YSL03 [KCTC 11715BP] shows the effect of nitrogen and phosphorus removal.
Figure 4 Chlamydomonas Fitschmann Fatty acid distribution of YSL03 [KCTC 11715BP] [linolenic acid (C18: 3n6), linolenic acid (C18: 3n 3), oleic acid (C18: 1n9c), stearic acid (C18: 0), heptadecanoic acid (C17: 0 ), Palmitoleic acid (C16: 1), palmitic acid (C16: 0), mystic acid (C14: 0), lauryl acid (C12: 0)].
이와 같은 본 발명을 더욱 상세히 설명하면 다음과 같다. Hereinafter, the present invention will be described in detail.
본 발명의 신균주인 클라미도모나스 피트쉬만니 YSL03 은 본 발명자들에 의해 최초로 분리 동정된 것으로서, 하ㆍ폐수 처리장에서 BBM 배지를 이용하여 균주를 분리하였으며, 분리한 균주를 28s rDNA 시퀀싱 서열 검색에 의해 분자 유전학적인 방볍으로 분석한 결과, 표준 균주인 클라미도모나스 피트쉬만니 AF183462과 99% 유사한 신균주임을 확인하였다.Chlamydomonas pitschmanny, a new strain of the present invention YSL03 was first identified and identified by the present inventors, and strains were separated using BBM medium in sewage and wastewater treatment plants. As a result of analyzing the isolated strains in a molecular genetic manner by 28s rDNA sequencing sequence search, a standard strain was identified. Inn Klamidomonas Fitschmann It was confirmed that the strain was 99% similar to AF183462.
본 발명자들은 상기 분리 동정된 신균주를 클라미도모나스 피트쉬만니 YSL03라 명명하였으며, 2010년 6월 18일자로 한국생명공학연구원 생물자원센터에 기탁하여 수탁번호 KCTC 11715BP를 부여받았다.The present inventors named the isolated and identified mycobacterium Klamidomonas Fitschmanny YSL03, and deposited on June 18, 2010 to the Korea Institute of Bioscience and Biotechnology Biological Resource Center was given accession number KCTC 11715BP.
상기 클라미도모나스 피트쉬만니 YSL03은 폐수에서 배양이 가능하며, 특히, 25 내지 27 ℃, pH 7.2 내지 7.6에서 배양하는 것이 바람직하다. 또한, BBM 배지(KH2PO4, CaCl2ㆍ2H2O, MgSO4ㆍ7H2O, NaNO3, K2HPO4, NaCl, H3BO3 및 미량원소를 함유하는 배지)에서 배양하는 것이 보다 바람직하다. 상기 미량원소는 ZnSO4ㆍ7H2O, MnCl2ㆍ4H2O, MoO3, CuSO4ㆍ5H2O 및 Co(NO3)2ㆍ6H2O로 이루어진 군에서 선택된 하나 이상일 수 있다.The Chlamydomonas pitschmanny YSL03 can be cultured in the waste water, in particular, it is preferable to incubate at 25 to 27 ℃, pH 7.2 to 7.6. In addition, culturing in BBM medium (KH 2 PO 4 , CaCl 2 · 2H 2 O, MgSO 4 · 7H 2 O, NaNO 3 , K 2 HPO 4 , NaCl, H 3 BO 3 and a trace element containing trace elements) More preferred. The trace element may be at least one selected from the group consisting of ZnSO 4 .7H 2 O, MnCl 2 .4H 2 O, MoO 3 , CuSO 4 .5H 2 O, and Co (NO 3 ) 2 .6H 2 O.
본 발명에 따른 클라미도모나스 피트쉬만니 YSL03은 고농도의 질소, 인(800 mg/L, 10 mg/L) 조건 하에서도 생육이 가능하므로 폐수의 희석 배율을 크게 낮추지 않고도 배양이 가능하다.Chlamydomonas pitschmanny YSL03 according to the present invention can be grown under high concentrations of nitrogen and phosphorus (800 mg / L, 10 mg / L) conditions can be cultured without significantly reducing the dilution ratio of the waste water.
또한, 폐수 내 오염원인 질소, 인 제거 효과를 나타내므로 폐수 처리에 활용 가능하다.In addition, since it shows the effect of removing nitrogen, phosphorus, which is a pollution source in wastewater, it can be used for wastewater treatment.
국내의 축산 농가는 11만 가구 이상으로 많은 양의 질소, 인의 성분이 폐수처리장으로 유입된다. 기존 질소, 인 제거는 물리, 화학, 생물학적 방법이 전부 이용되며 경제적인 부분에 많은 단점이 있으나, 조류 재배 시 광합성에 필요한 빛은 자연 상태인 태양으로부터 섭취하며 부가적 성장 요소인 질소, 인을 폐수에서 자연스럽게 섭취하므로 처리 공정이 단순하다. 따라서 공정 시설에 필요한 투자비와 운전비용이 대단히 낮아 상용화 가능성은 매우 높다.Domestic livestock farms have more than 110,000 households, and large amounts of nitrogen and phosphorus flow into the wastewater treatment plant. Existing nitrogen and phosphorus removal are all physical, chemical, and biological methods, and there are many disadvantages in the economic part. However, the light needed for photosynthesis during algae cultivation is taken from the natural sun and wastewater is added to the additional growth factors nitrogen and phosphorus. Ingestion naturally, so the process is simple. Therefore, the investment cost and operation cost required for the process facility is very low, so the possibility of commercialization is very high.
상기와 같이, 본 발명은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주 또는 이의 배양액을 포함하는 폐수 처리용 미생물제제를 제공할 수 있다.As described above, the present invention is Chlamydomonas Fitschmann Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strain or culture medium thereof may be provided.
한편, 폐수를 활용하여 배양시킨 본 발명에 따른 클라미도모나스 피트쉬만니 YSL03은 빠른 성장 속도와 동시에 지질이 풍부한 종(균체 건조 중량의 지질 함유량 50% 이상)으로서, 지질 추출로 인한 바이오 디젤을 생산할 수 있는 성분 제공 요소로 사용 가능하다.On the other hand, Chlamydomonas pitschmanny according to the invention cultured using wastewater YSL03 provides fast growth rates and lipid-rich species ( Lipid content of 50% or more), which can be used as a component providing element capable of producing biodiesel from lipid extraction.
따라서, 본 발명은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주 또는 이의 배양액으로부터 지질을 분리하는 방법과 이로부터 분리된 지질을 이용하여 바이오디젤을 생산하는 방법도 포함한다.Therefore, the present invention, Chlamydomonas Fitzmann Chlamydomonas pitschmannii YSL03 (KCTC 11715BP) strain or a culture medium thereof, and a method for producing biodiesel using the lipids separated therefrom are also included.
조류의 지질을 분석하는 방법으로는 Sulfophosphovanillin 법, Kunkel 법, Brugdon 법 등을 실시하여 추출된 지질을 분석해 낼 수 있다.The lipid analysis of algae can be carried out using the Sulfophosphovanillin method, Kunkel method, and Brugdon method.
본 발명의 신균주 역시 상기와 같은 통상의 지질 분석법으로 지질을 분석하였다.The new strain of the present invention was also analyzed for lipids by the conventional lipid analysis method as described above.
또한, 이렇게 분리한 지질 내 지방산을 분석한 결과, 팔미틴산(palmitic acid)와 올레인산(oleic acid)가 총 지방산 함량에 대하여 30 중량% 이상 포함되어 있음을 확인하였고, 분리된 지질(지방산)을 사용하여 바이오디젤을 생산할 수 있는 바, 본 발명은 상기 지질을 이용한 바이오디젤 생산방법도 포함할 수 있다.
In addition, as a result of analyzing the fatty acids in the lipids thus separated, it was confirmed that palmitic acid and oleic acid contained more than 30% by weight relative to the total fatty acid content, using the separated lipid (fatty acid) The biodiesel can be produced, and the present invention may also include a biodiesel production method using the lipid.
이하, 본 발명을 실시예에 의해 더욱 상세히 설명한다. 단, 하기의 실시예는 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 제한되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by way of examples. It should be noted, however, that the following examples are illustrative of the invention and are not intended to limit the scope of the invention.
실시예Example 1: One: 신균주의Neomycology 분리 및 배양 Isolation and Cultivation
강원도 원주 지역의 하ㆍ폐수처리장 주변에서 시료를 채취하였다. 채취 시료는 200 mL 시험관에 다음 표 1에 나타낸 바와 같은 BBM 액체 배지를 넣고 약 2주간 정치 배양하였다. 폐수의 농도는 질소-인이 각각 800 mg/L, 10 mg/L로 공급하며 형광등을 광원으로 하여 배양 시험관 표면에서 조도를 약 24 watt로 조절한 후 직접 배양하였다. 예비 배양한 시료 1 mL를 10 mL의 멸균 증류수가 포함된 30 mL시험관에 넣고 잘 혼합하여 미세조류 세포를 분산시켰다. 이로부터 마이크로 피펫을 사용하여 0.1 mL의 시료를 취한 후 멸균 증류수가 포함된 시험관에 넣고 혼합하였다. 이러한 조작을 5회 반복한 후 각각의 시험관에서 약 0.1 mL의 시료를 취하고, 이를 다음 표 2에 나타낸 바와 같은 조성의 BBM 배지가 놓인 페트리디쉬(Petri dish)에 접종한 다음, 온도 25 ~ 27 ℃ 및 조도 50 μmol/㎡-sec의 배양기에서 2~3주간 정치 배양하였다. 미세조류의 군락(Colony)이 나타나면 현미경으로 확인하면서 백금이를 사용하여 각각의 군락을 웰 세포 배양 플레이트(well cell culture plate)에 옮겨 분리를 시작하였다. 각각의 홀에는 조류군락과 BBM배지를 1:1로 하여 투여 후 10 내지 14일간 배양하였다. 일부 미생물의 성장을 억제하게 위해 본 연구에서는 항생제 스트렙토마이신을 사용하여 조류만을 성장하도록 하였다. 이때 사용된 항생제의 양은 배지 1L 당 0.15 μm/ mL를 사용하였다. 일정 기간 후 각각의 홀에 배양된 군락을 현미경 관찰한다. 이때 대부분의 홀에서는 특성 미세조류만 성장하며, 같은 모양을 지닌 종을 BBM 배지로 준비된 페트리디쉬에서 배양을 하였다. 일정 기간 후 배양된 조류 군락은 한 종류의 군락으로 우세하게 배양되며, 이 군락을 채취하여 100 m의 BBM 배지가 들어있는 250 mL 삼각 플라스크에 이식하여 본 배양을 시작하였다. Samples were collected from the sewage and wastewater treatment plant in Wonju, Gangwon-do. The collected sample was placed in a BBM liquid medium as shown in Table 1 in a 200 mL test tube and left to incubate for about 2 weeks. Nitrogen-phosphorus was fed at 800 mg / L and 10 mg / L, respectively, and the concentration of wastewater was adjusted to about 24 watts on the surface of the culture tube using a fluorescent light source, followed by direct incubation. 1 mL of the pre-cultured sample was placed in a 30 mL test tube containing 10 mL of sterile distilled water and mixed well to disperse the microalgal cells. From this, a 0.1 mL sample was taken using a micro pipette, and then mixed into a test tube containing sterile distilled water. After this procedure was repeated five times, about 0.1 mL of the sample was taken from each test tube, which was inoculated in a Petri dish containing BBM medium having a composition as shown in Table 2 below, followed by a temperature of 25 to 27 ° C. And stationary culture for 2-3 weeks in an incubator of
실시예Example 2: 2: 신균주New strain 배양 culture
상기 표 1의 BBM 배지를 이용하여 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03) 균주를 25 내지 27 ℃, pH 7.2 내지 7.6 하에서 250 mL 삼각플라스크에 10 mL의 상기 균주를 넣고 형광 교반 배양기에서 약 14일간 배양을 하였다. 교반은 150 rpm으로 유지하였으며, 조도 50 μmol/㎡-sec를 유지하였다.
Chlamydomonas pitschmanny using the BBM medium of Table 1 above YSL03 ( Chlamydomonas pitschmannii YSL03) strain was placed in a 250 mL Erlenmeyer flask at 25 to 27 ° C., pH 7.2 to 7.6, and 10 mL of the strain was incubated in a fluorescent stirred incubator for about 14 days. Stirring was maintained at 150 rpm and
실시예Example 3: 3: 신균주의Neomycology 동정 Sympathy
클라미도모나스 피트쉬만니 YSL03균주의 염기서열 분석은 28s rDNA 염기서열 시퀀스분석법으로 분석하였다. 샘플의 상태는 클라미도모나스 피트쉬만니 YSL03 집락이 있는 평판배지 상태로 의뢰하였으며, 의뢰 내역으로는 Extraction-gDNA, PCR Amplication에 의한 PCR 생성물을 시퀀싱(sequencing) 반응을 통해 분석하였다. 분석에 사용된 프라이머는 다음과 같다.Chlamydomonas Fitzmanny The sequencing of the strain YSL03 was analyzed by 28s rDNA sequencing. The state of the sample is Chlamydomonas Fitschmann. YSL03 colonies were plated, and as a request, PCR products by Extraction-gDNA and PCR Amplication were analyzed by sequencing reaction. Primers used for analysis are as follows.
정방향 프라이머: 5'-AGCGGAGGAAAAGAAACTA-'3 (서열번호 2)Forward primer: 5'-AGCGGAGGAAAAGAAACTA-'3 (SEQ ID NO: 2)
역방향 프라이머: 5'-TACTAGA-AGGTTCGATTAGTC-'3 (서열번호 3)Reverse primer: 5'-TACTAGA-AGGTTCGATTAGTC-'3 (SEQ ID NO: 3)
28s rDNA 염기서열 분석 결과를 첨부된 서열번호에 기재하였으며, 계통학적 결과는 쥬크-캔터(Jukes-Cantor) 모델을 적용한 네이버-조이닝(Neighbour-joining) 방법에 따라 계통수(phylogenetic tree) 결과를 도 2에 나타내었다.The 28s rDNA sequencing results are described in the attached SEQ ID NO., And the phylogenetic results are shown in the phylogenetic tree results according to the neighbor-joining method using the Jukes-Cantor model. 2 is shown.
클라미도모나스 피트쉬만니 YSL03균주의 염기서열[서열번호 1]은 NCBI에서 상동성을 조사한 결과, 다음 표 3에 나타낸 바와 같이 클라미도모나스 피트쉬만니로 분류되었다.Chlamydomonas Fitzmanny The base sequence [SEQ ID NO: 1] of the strain YSL03 was classified as Chlamydomonas pitschmanny as shown in Table 3 below when the homology was examined in NCBI.
이상에서와 살펴본 바와 같이, 본 발명에서 분리한 미세조류균은 폐수의 내성과 높은 함량의 지질을 함유하는 점을 제외하고는 형태 및 최적 온도, pH 등의 특성을 종합 고려할 때 클라미도모나스 피트쉬만니 에 속하는 것으로 밝혀졌다.As described above, the microalgal bacteria isolated from the present invention, when considering the characteristics of morphology, optimal temperature, pH, etc., except that it contains wastewater resistance and high content of lipids, Chlamydomonas Fitsch Manni Turned out to belong.
이에, 본 발명에 따른 신균주를 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)으로 명명하였고, 2010년 6월 18일자로 한국생명공학연구원 생물자원센터에 기탁하였고, 수탁번호 KCTC 11715BP를 부여받았다.
Thus, the new strain according to the present invention Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03), and it was deposited on June 18, 2010 at the Korea Institute of Bioscience and Biotechnology, and was assigned accession number KCTC 11715BP.
실시예Example 4: 폐수 내성 및 질소, 인 제거 확인 4: Check wastewater resistance and nitrogen and phosphorus removal
MF(Micro Filtration)을 거친 가축폐수에 상기 실시예 2에 의해 확보된 조류 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)에서 질소와 인 제거를 검토하기 위하여 각각 다른 유입원에 따라 조류를 씨딩(seeding)하였다. 각각의 반응조는 20 내지 22 ℃의 실내 온도를 유지하였으며, pH는 7.7 내지 7.9로 자연 상태로 유지하였고 회분 실험 형태로 진행하였다. 처음 식종 후 조류가 적응하는 데까지 걸리는 시간은 10일 내외로 그 이후 폐수와 BBM의 초기농도에 관계없이 질소와 인의 제거가 발생되었다. Alga Chlamydomonas pitschmanny secured by Example 2 in livestock wastewater after MF (Micro Filtration) YSL03 ( Chlamydomonas pitschmannii In YSL03, algae were seeded according to different sources for consideration of nitrogen and phosphorus removal. Each reactor maintained a room temperature of 20-22 ° C., pH was maintained at 7.7-7.9 in nature and proceeded in the form of batch experiments. After the first planting, algae took about 10 days to acclimate, after which the removal of nitrogen and phosphorus occurred regardless of the initial concentrations of wastewater and BBM.
도 3에서는 조류 적응이 진행된 후로 10 ~ 12일 내로 질소와 인의 저감이 빠르게 진행되었고, 반응기에 정상 상태(steady-state)의 조류를 적용시 빠른 인, 질소의 제거가 발생될 것으로 판단된다.
In FIG. 3, the reduction of nitrogen and phosphorus proceeded rapidly within 10 to 12 days after the algae adaptation proceeded, and it is determined that rapid removal of phosphorus and nitrogen occurs when the steady-state algae is applied to the reactor.
실시예Example 6: 지질 생산 및 지방산 추출 6: lipid production and fatty acid extraction
조류는 탄수화물, 단백질, 지질 등으로 구성되어 있는 종속/독립영양으로 광합성 작용을 통해 성장한다. Algae are heterotrophic / independent nutrients composed of carbohydrates, proteins, and lipids that grow through photosynthesis.
지방산 함량 및 조성은 Lepage와 Roy[Lepage, G., C.C. Roy (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification, Journal of Lipid Research, 25, 1391-1396]의 방법을 변형하여 분석하였다. Fatty acid content and composition were determined by Lepage and Roy [Lepage, G., C.C. Roy (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification, Journal of Lipid Research, 25, 1391-1396.
표준물질로 지방산 메틸 에스테르 혼합물인 Mix RM3, Mix RM5, GLC50, GLC70[Supelco, USA]와 헵타데칸산(Heptadecanoic acid), 감마-리놀렌산(gamma-Linolenic acid)[Supelco, USA]를 사용하였다. 테프론 마개를 가진 유리 튜브[11 mL, DH.GL28020, Daihan Scientific, Korea]에 질량을 측정한 미세조류 지질 시료 넣고 클로로포름-메탄올(2:1, vol/vol) 2 mL을 주입한 후 상온에서 10분간 볼텍스 믹서(vortex mixer)[Vorex Genius 3. Ika, Italy]로 섞었다. A mixture of fatty acid methyl esters Mix RM3, Mix RM5, GLC50, GLC70 [Supelco, USA], heptadecanoic acid and gamma-linolenic acid [Supelco, USA] was used as a standard. Place a mass of microalgal lipid sample in a glass tube [11 mL, DH.GL28020, Daihan Scientific, Korea] with a Teflon stopper, and inject 2 mL of chloroform-methanol (2: 1, vol / vol) at room temperature. It was mixed for a minute with a vortex mixer (Vorex Genius 3. Ika, Italy).
내부표준물질인 노나데칸산(nonadecanoic acid)[Sigma Co., USA]를 함유한 클로로포름 1 mL (500 ㎍/L), 메탄올 1 mL, 황산 300 ㎕를 순차적으로 유리튜브에 첨가한 후 5분간 믹서로 섞었다. 튜브를 항온 수조에 넣고 100 ℃에서 10분간 반응시켰다. 튜브를 상온까지 냉각시킨 후 증류수 1 mL을 주입하고, 믹서로 5분 정도 격렬히 섞은 후 4,000 rpm에서 10분간 원심분리하여 층 분리를 시켰다. 아래층 (유기상)을 1회용 PP 재질 주사기(Norm-ject, Germany)로 뽑아 1회용 0.22 ㎛ PVDF 실린지필터[(Millex-Gv, Millipore, USA)로 여과 후 자동 주입기를 가진 가스크로마토그래피[Model 7890, Agilent, USA]로 분석하였다. 1 mL (500 μg / L) of chloroform, 1 mL of methanol, and 300 μl of sulfuric acid containing nonadecanoic acid (Sigma Co., USA), an internal standard, are sequentially added to the glass tube, followed by a mixer for 5 minutes. Mixed into. The tube was placed in a constant temperature water bath and reacted at 100 ° C for 10 minutes. After the tube was cooled to room temperature, 1 mL of distilled water was injected, mixed vigorously with a mixer for 5 minutes, and centrifuged at 4,000 rpm for 10 minutes to separate layers. The lower layer (organic phase) was extracted with a disposable PP syringe (Norm-ject, Germany) and filtered with a disposable 0.22 μm PVDF syringe filter (Millex-Gv, Millipore, USA), followed by gas chromatography with an automatic injector [Model 7890 , Agilent, USA].
본 발명에 따른 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03) 균주는 균체 내에 건조 중량 약 50 중량%의 지질이 함유되어 있는 것으로 확인되었다[표 4].Chlamydomonas pitschmanny according to the present invention The strain YSL03 ( Chlamydomonas pitschmannii YSL03) was found to contain about 50% by weight of dry weight lipids in the cells [Table 4].
또한, 구성하고 있는 지방산 중 팔미틴산(16:0), 스테아린산(C18:0), 올레인산(C 18:1n9c), 및 리놀레닉산(C18:3n3)은 바이오디젤 생산화를 위한 지방산으로 알려져 있다. Among the fatty acids, palmitic acid (16: 0), stearic acid (C18: 0), oleic acid (C 18: 1n9c), and linolenic acid (C18: 3n3) are known as fatty acids for biodiesel production.
도 4는 클라미도모나스 피트쉬만니 YSL03 균주의 지방산 분포로 팔미틴산(16:0)이 가장 많이 분포되어 있다. 특히, 현재까지 보고된 모든 미세조류의 바이오디젤 생산에 가장 안정적인 지방산은 올레인산(C 18:1n9c)과 팔미틴산(16:0)으로 알려져 있다.
Figure 4 Chlamydomonas Fitschmann Palmitic acid (16: 0) is the most widely distributed fatty acid distribution of YSL03 strain. In particular, the most stable fatty acids for biodiesel production of all the microalgae reported to date are known as oleic acid (C 18: 1n9c) and palmitic acid (16: 0).
본 발명에 따른 신균주인 클라미도모나스 피트쉬만니 YSL03은 전체 지방산 중 40 중량%가 올레인산과 팔미틴산으로 구성되어 바이오디젤 생산이 가능하다.
Klimidomonas pitschmanny, a new strain according to the present invention YSL03 is made up of 40% by weight of all fatty acids composed of oleic acid and palmitic acid for biodiesel production.
서열목록 전자파일 첨부Attach an electronic file to a sequence list
Claims (12)
Chlamydomonas pitschmanny with high wastewater resistance and lipid content YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.
According to claim 1, Chlamydomonas Fitschmanny having a nucleotide sequence of SEQ ID NO: YSL03 ( Chlamydomonas pitschmannii YSL03 [KCTC 11715BP] strain.
The method of claim 1, wherein the lipid content is more than 50% by weight of the dry cell weight Chlamydomonas Fitschmanny YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] Strain
The method of claim 1, wherein Chlamydomonas Fitschmanny containing at least 30% by weight of oleic acid and palmitic acid in the total fatty acids YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain.
Chlamydomonas Fitzmanny YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain culture.
상기 조류는 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주인 것을 특징으로 하는 지질 분리방법.
In the method for separating lipids from algae or its culture,
The bird is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain, characterized in that the lipid separation method.
상기 지질은 클라미도모나스 피트쉬만니 YSL03(Chlamydomonas pitschmannii YSL03)[KCTC 11715BP] 균주로부터 분리된 지질인 것을 특징으로 하는 바이오디젤 생산방법.
In the method of producing biodiesel using lipids,
The lipid is Chlamydomonas Fitschmann YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] Biodiesel production method characterized in that the lipid isolated from the strain.
Chlamydomonas Fitschmanny of claim 1 YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain or microbial agent for wastewater treatment comprising the culture solution of claim 4.
Chlamydomonas Fitschmanny of claim 1 YSL03 ( Chlamydomonas pitschmannii YSL03) [KCTC 11715BP] strain is cultured in wastewater.
The method of claim 9, wherein the strain is incubated at 25 to 27 ℃, pH 7.2 to 7.6.
The method of claim 9, wherein the strain is added to a medium containing KH 2 PO 4 , CaCl 2 .2H 2 O, MgSO 4 .7H 2 O, NaNO 3 , K 2 HPO 4 , NaCl, H 3 BO 3, and a trace element. Culturing.
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| KR20100064306A KR101114426B1 (en) | 2010-07-05 | 2010-07-05 | Novel strain Chlamydomonas pitschmannii YSL03 |
| PCT/KR2010/006561 WO2012005410A1 (en) | 2010-07-05 | 2010-09-27 | New strain chlamydomonas pitschmannii ysl03 |
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| CN114606131B (en) * | 2022-03-30 | 2023-07-25 | 福州文泽生物科技有限公司 | Chlorella strain and application thereof in treatment of rare earth ammonia nitrogen wastewater |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR900004635A (en) * | 1988-09-20 | 1990-04-12 | 후미오 오오누끼 | Purification of Wastewater Using Chlamydomonas Single Cell Green Algae |
| KR910006476A (en) * | 1989-09-20 | 1991-04-29 | 후미오 오오누끼 | Cultivation method and culture apparatus of strain 95 for single cell green alga |
| WO2009094440A1 (en) | 2008-01-25 | 2009-07-30 | Aquatic Energy Llc | Algal culture production, harvesting, and processing |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR900004635A (en) * | 1988-09-20 | 1990-04-12 | 후미오 오오누끼 | Purification of Wastewater Using Chlamydomonas Single Cell Green Algae |
| KR910006476A (en) * | 1989-09-20 | 1991-04-29 | 후미오 오오누끼 | Cultivation method and culture apparatus of strain 95 for single cell green alga |
| WO2009094440A1 (en) | 2008-01-25 | 2009-07-30 | Aquatic Energy Llc | Algal culture production, harvesting, and processing |
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| KR20120003610A (en) | 2012-01-11 |
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