KR101424855B1 - Method for selection high-lipid content microalgae using fluorescence activated cell sorter and nile red dye - Google Patents
Method for selection high-lipid content microalgae using fluorescence activated cell sorter and nile red dye Download PDFInfo
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- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000001044 red dye Substances 0.000 title claims abstract description 19
- 150000002632 lipids Chemical class 0.000 claims abstract description 30
- 238000004043 dyeing Methods 0.000 claims abstract description 8
- 230000001172 regenerating effect Effects 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000000523 sample Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims 1
- 238000002955 isolation Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 42
- 240000009108 Chlorella vulgaris Species 0.000 description 10
- 235000007089 Chlorella vulgaris Nutrition 0.000 description 10
- 238000010186 staining Methods 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000003960 organic solvent Substances 0.000 description 6
- 238000000265 homogenisation Methods 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- 239000003225 biodiesel Substances 0.000 description 3
- 210000003763 chloroplast Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
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- 244000005700 microbiome Species 0.000 description 2
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- 230000000243 photosynthetic effect Effects 0.000 description 2
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- 239000002028 Biomass Substances 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
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- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- AJDUTMFFZHIJEM-UHFFFAOYSA-N n-(9,10-dioxoanthracen-1-yl)-4-[4-[[4-[4-[(9,10-dioxoanthracen-1-yl)carbamoyl]phenyl]phenyl]diazenyl]phenyl]benzamide Chemical compound O=C1C2=CC=CC=C2C(=O)C2=C1C=CC=C2NC(=O)C(C=C1)=CC=C1C(C=C1)=CC=C1N=NC(C=C1)=CC=C1C(C=C1)=CC=C1C(=O)NC1=CC=CC2=C1C(=O)C1=CC=CC=C1C2=O AJDUTMFFZHIJEM-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- 241000894007 species Species 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000001043 yellow dye Substances 0.000 description 1
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Abstract
본 발명은 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)와 나일레드(Nile red) 염색약을 이용하여 높은 지질함량을 가지는 미세조류 균주를 신속하게 분리하는 방법 및 이를 재배양하는 방법에 관한 것으로서, 본 발명의 미세조류 균주 분리 방법에 의하면, 초음파파쇄기 (sonicator)를 이용하여 세포를 균질화 함으로써 나일레드(Nile red) 염색약의 사용 농도를 1/10 수준으로 줄이고도 균질한 염색이 가능하게 유도하였으며, 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 이용함으로써 상대적으로 높은 지질을 함유하는 미세조류 균주를 보다 빠르고 정확하게 분리할 수 있으며, 각각 하나의 세포로 분리할 수 있는바, 이로부터 세포의 재배양이 가능하다.The present invention relates to a method for rapidly isolating a microalgae strain having a high lipid content using a fluorescence activated cell sorter (FACS) and a Nile red dye, and a method for regenerating the microalgae. According to the microalgae strain isolation method of the present invention, the cells are homogenized using an ultrasonic sonicator to induce homogeneous dyeing even when the concentration of nile red dye is reduced to 1/10 level, By using a fluorescence activated cell sorter (FACS), it is possible to separate microalgae strains containing relatively high lipids more rapidly and accurately, and they can be separated into individual cells. From this, Amount is possible.
Description
본 발명은 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)와 나일레드(Nile red) 염색약을 이용하여 높은 지질함량을 가지는 미세조류 균주를 신속하게 분리하는 방법 및 이를 재배양하는 방법에 관한 것이다.The present invention relates to a method for rapidly isolating a microalgae strain having a high lipid content using a fluorescence activated cell sorter (FACS) and a Nile red dye, and a method for regenerating the microalgae.
최근 국제유가의 지속적인 상승으로 인해 신재생 에너지 개발에 대한 관심이 높아지고 있으며, 이에 따라 미세조류를 이용한 바이오에너지 개발에 대한 연구가 국내외 적으로 활발히 진행되고 있다. 특히, 미세조류는 대표적인 광합성 생물로서 높은 광합성 효율을 가지며, 일반 육상 식물에 비해 단위면적당 바이오매스 및 바이오디젤 생산이 1헥타르 당 130여 배에 이른다 (Chisti, Y. 2007. Biodiesel from microalgae. Biotechnol. Adv. 25: 294-306). 현재까지 전 세계적으로 알려져 있는 미세조류 종(species)은 약 40,000종으로 추정되고 있으나, 이들 중 4%에 해당되는 1,600여 종만이 각국의 컬쳐콜렉션 (Culture Collection)에 의해 유지, 보존되는 것으로 알려지고 있다. 따라서 아직까지 알려지지 않은 더 많은 종류의 미세조류를 확보하고 이 중 높은 지질 함량을 지니고 바이오디젤 생산에 적합한 우량 미세조류 주(strain)의 분리 및 동정 작업이 필요하다 (오희목. 2010. 녹색기술동향보고서 2010 제 4호). Recently, interest in the development of renewable energy has been increasing due to the continuous rise of international oil prices, and research on the development of bio-energy using microalgae has been actively carried out both at home and abroad. In particular, microalgae have high photosynthetic efficiency as typical photosynthetic organisms, and produce biomass and biodiesel per unit area of 130 times per hectare (Chisti, Y. 2007. Biodiesel from microalgae. Biotechnol. Adv. 25: 294-306). It is known that about 40,000 species of microalgae are known worldwide, but only about 1,600 of them (4%) are known to be maintained and preserved by the Culture Collection of each country have. Therefore, it is necessary to acquire more kinds of microalgae that are not yet known, and to isolate and identify high-quality microalgae strains suitable for biodiesel production with high lipid content (Oh, Hee Mok, 2010. Green Technology Trend Report 2010 fourth).
미세조류로부터 지질함량을 측정하는 방법으로는 일반적으로 클로로포름과 메탄올을 이용한 용매추출법 (Bligh E.G. and W.J. Dyer.1959. A rapid method of total lipid extraction and purification.Can. J. Biochem . Physiol 37(8):911-917)이 많이 사용되고 있으나, 이는 다양한 현장으로부터 채취된 미세조류 주(strain)의 지질함량을 신속하게 측정하기에는 다소 어려움이 따르며, 신속하게 지질함량을 측정할 수 있는 방법이 필요하다. 현재 개발되어 있는 신속한 미세조류의 지질 정량법은 Nile red 염색약으로 염색한 후 형광을 측정하여 지질을 정량함으로써 단시간 내에 간편하고 신속하게 지질 함량을 측정하는 방법이 있다 (한국특허출원 제10-1998-0020708호).A method for measuring the lipid content from microalgae is generally a solvent extraction method using chloroform and methanol (Bligh EG and WJ Dyer.1959. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol 37 (8) : 911-917) is widely used, but it is difficult to quickly measure the lipid content of microalgae strains collected from various sites, and a method for measuring lipid content rapidly is needed. The lipid determination method of the rapid microalgae that is currently being developed is a method of measuring the lipid content easily and quickly in a short time by measuring the fluorescence after dyeing with Nile red dye and measuring the lipid (Korean Patent Application No. 10-1998-0020708 number).
상기 미세조류 지질 정량법은 신속한 지질함량의 측정은 가능하나, 실시간으로 높은 지질함량을 지니는 각각의 균주 및 세포만을 따로 분리해 내기에는 어려움이 있고, Nile red 염색약을 녹여 사용하는 유기용매인 아세톤의 독성으로 인해 염색 후 세포의 생존여부가 불투명하며, Nile red 염색약이 250 ppm의 높은 농도로 첨가된다는 약점이 있다. The microalgae lipid determination method can measure the lipid content rapidly. However, it is difficult to separate only the strains and cells having high lipid content in real time separately. Toxicity of acetone, an organic solvent used for dissolving nile red dye, , It is unclear whether the cells survive after dyeing, and Nile red dye is added at a high concentration of 250 ppm.
본 발명은 상기와 같은 문제점을 해결하기 위한 것으로서, 본 발명의 목적은 기존 방법의 Nile red 염색약 사용 농도를 줄이고, Nile red 염색약을 녹이기 위한 유기용매의 독성을 낮추어 균주 및 세포의 생존률을 높이며, Nile red 염색 후 지질염색 정도에 따른 선별적인 균주 분리와 해당 균주의 재배양 방법 등을 제공하고자 하는 것이다.The object of the present invention is to reduce the concentration of Nile red dye used in conventional methods and to lower the toxicity of organic solvents for dissolving Nile red dye to increase the survival rate of strains and cells, red staining, and the method of culturing the strain according to the degree of lipid staining.
상기의 목적을 달성하기 위하여, 본 발명은 a) 초음파 파쇄기를 이용하여 미세조류 세포를 균질화 하는 단계; b) 상기 균질화 된 미세조류 세포를 나일레드(Nile red)로 염색하는 단계; 및 c) 상기 염색된 미세조류 세포로부터 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 이용하여 지질함량이 높은 세포를 분리하는 단계를 포함하는 미세조류 균주의 분리 방법을 제공한다.In order to accomplish the above object, the present invention provides a method for producing microbial cells, comprising the steps of: a) homogenizing microalgae cells using an ultrasonic crusher; b) staining the homogenized microalgae cells with Nile red; And c) separating the high-lipid-rich cells from the stained microalgae cells using a fluorescence activated cell sorter (FACS).
또한, 본 발명은 상기 c) 단계에서 분리된 지질함량이 높은 세포를 재배양하는 단계를 더 포함할 수도 있다.In addition, the present invention may further include a step of re-culturing the cells having a high lipid content separated in step c).
본 발명에 있어서, 상기 미세조류 세포를 균질화 하는 단계는 미세조류를 2000 ~ 4000 rpm에서 1 ~ 5분 간 원심분리한 후 초음파 파쇄기를 이용하여 미세조류 세포를 균질화 할 수 있다.In the present invention, the microalgae are homogenized by centrifuging the microalgae at 2000 to 4000 rpm for 1 to 5 minutes, and then homogenizing the microalgae cells using an ultrasonic crusher.
또한 본 발명에서, 상기 초음파 파쇄 조건은 duty cycle 50 ~ 70%, Micro-tip Limit 3 ~ 5, 프로브(probe) 직경 2 ~ 4 mm에서 8 ~ 12초 간 균질화 하는 것을 특징으로 한다.In the present invention, the ultrasonic disintegration conditions are characterized by homogenization at a duty cycle of 50 to 70%, a Micro-tip Limit of 3 to 5, and a probe diameter of 2 to 4 mm for 8 to 12 seconds.
또한 본 발명에 있어서, 상기 나일레드(Nile red) 염색약은 무독성 유기용매에 녹여 사용하는 것을 특징으로 하며, 상기 무독성 유기용매는 알코올인 것을 특징으로 하며, 바람직하게는 메탄올 또는 에탄올을 포함할 수 있으나, 이에 한정되지 않는다.In the present invention, the Nile red coloring agent is dissolved in a non-toxic organic solvent, and the non-toxic organic solvent is an alcohol, preferably methanol or ethanol, , But is not limited thereto.
본 발명에서 상기 나일레드(Nile red) 염색약은 미세조류 샘플 1 ㎖ 당 0.05 ~ 0.15 ppm 농도로 첨가하는 것이 바람직하다.In the present invention, the Nile red dye is preferably added at a concentration of 0.05 to 0.15 ppm per 1 ml of the microalgae sample.
또한 본 발명에 있어서, 상기 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 이용하여 지질함량이 높은 세포를 분리하는 단계에서 형광활성 세포분류기의 FITC-a 필터의 exite 파장 영역이 400 ~ 500 nm, emite 파장 영역이 600 ~ 700 nm인 형광파장신호를 검출하는 것을 특징으로 한다.In the present invention, in the step of separating cells having high lipid content using the fluorescence activated cell sorter (FACS), the exited wavelength region of the FITC-a filter of the fluorescence activated cell sorter is 400 to 500 nm , and a fluorescent wavelength signal having an emite wavelength range of 600 to 700 nm is detected.
본 발명에 따른 높은 지질함량을 가지는 미세조류 균주의 분리 방법은 초음파파쇄기 (sonicator)를 이용하여 세포를 균질화 함으로써 나일레드(Nile red) 염색약의 사용 농도를 1/10 수준으로 줄일 수 있으며, 이에 염색약에 의한 세포의 손상을 최소화할 수 있다. 또한, 본 발명의 균주 분리 방법은 나일레드(Nile red) 염색약을 녹이는 유기용매로 아세톤 대신 메탄올을 사용함으로써 세포에 미치는 독성을 급격히 낮추어 미세조류 세포의 생존률을 향상시킬 수 있다. 또한, 본 발명은 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 이용함으로써 상대적으로 높은 지질을 함유하는 미세조류 균주를 보다 빠르고 정확하게 분리할 수 있으며, 각각 하나의 세포로 분리할 수 있는바, 이로부터 세포의 재배양이 가능하다.According to the method of the present invention for isolating microalgae strains having a high lipid content, the concentration of Nile red dye can be reduced to 1/10 level by homogenizing cells using an ultrasonic sonicator, Can be minimized. In addition, the method of isolating a strain of the present invention can improve the survival rate of microalgae cells by rapidly reducing toxicity to cells by using methanol instead of acetone as an organic solvent for dissolving Nile red dye. In addition, the present invention can more rapidly and accurately separate microalgae strains containing relatively high lipids by using a fluorescence activated cell sorter (FACS), and can separate them into individual cells, From this, it is possible to regenerate the cells.
도 1은 본 발명의 지질함량이 높은 미세조류 균주 분리 방법을 도식화한 순서도이다.
도 2는 본 발명의 일실시예에 따라 Chlorella vulgaris 균주 샘플을 원심분리한 결과이다.
도 3은 본 발명의 일실시예에 따라 Chlorella vulgaris 균주 샘플을 원심분리한 초음파 파쇄기로 균질화한 결과이다.
도 4는 본 발명의 일실시예에 따라 나일레드(Nile red) 염색약으로 염색한 Chlorella vulgaris 균주 샘플을 고속 라이브 공초점 현미경 (Ultrafast Live Scanning Microscope System, Carl Zeiss Microimaging GmbH, Germany)을 이용하여 촬영한 결과이다.
도 5는 본 발명의 일실시예에 따라 나일레드(Nile red) 염색약으로 염색한 Chlorella vulgaris 균주 샘플을 형광활성 세포분류기 (fluorescence activated cell sorter, FACS, Becton Dickinson and Company, USA)를 이용하여 분리한 결과이다.Brief Description of the Drawings Fig. 1 is a flowchart illustrating a method of isolating a microalgae strain having a high lipid content according to the present invention.
FIG. 2 is a result of centrifuging a sample of Chlorella vulgaris strain according to an embodiment of the present invention.
FIG. 3 is a result of homogenization of a sample of Chlorella vulgaris strain by an ultrasonic crusher centrifuged according to an embodiment of the present invention.
FIG. 4 is a photograph of a sample of Chlorella vulgaris strain stained with a Nile red dye according to an embodiment of the present invention using a high speed live scanning microscope (Carl Zeiss Microimaging GmbH, Germany) Results.
FIG. 5 is a graph showing the results of a method of isolating a sample of Chlorella vulgaris strain stained with a Nile red dye according to an embodiment of the present invention using a fluorescence activated cell sorter (FACS, Becton Dickinson and Company, USA) Results.
본 발명은 미세조류 세포에 포함된 지질을 균질하게 염색하기 위한 세포 균질화 과정과, 균질화된 세포의 Nile red 염색 과정, 그리고 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 이용하여 염색된 지질의 형광파장신호 (fluorescence emission signal)에 따라 높은 지질함량을 가지는 균주를 신속하게 분리하는 방법을 제공하며, 분리된 균주를 재배양하는 방법을 제공한다.The present invention relates to a method for preparing a microorganism which comprises the steps of homogenizing cells for homogeneous dyeing of lipid contained in microalgae cells, nile red staining of homogenized cells, and staining of the dyed lipids using a fluorescence activated cell sorter (FACS) A method for rapidly isolating a strain having a high lipid content according to a fluorescence emission signal is provided and a method for regenerating a separated strain is provided.
본 발명자들은 기존의 나일레드(Nile red) 염색 방법에서 vortex로 세포 균질화를 유도하였던 것을 초음파 파쇄기 (sonicator)를 이용하여 미세조류 세포 간 공간분포를 균질하게 만들어 줌으로써, 종래 지질 염색에 이용되던 나일레드(Nile red) 염색시약의 처리 농도를 낮추고도 균질한 염색이 가능하게 유도하고, 나일레드(Nile red) 염색약을 녹이는 유기용매로써 독성이 높은 아세톤 대신에 메탄올을 사용함으로써 독성을 낮추어 세포 생존률을 높이며, 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 이용함으로써, 상대적으로 높은 지질량을 함유하는 미세조류 균주를 선별적으로 빠르게 분리할 수 있음을 확인하였으며, 이와 같은 방법으로 분리된 균주를 재배양 할 수 있음을 확인함으로써 본 발명을 완성하였다.The inventors of the present invention induced cell homogenization using a vortex in a conventional Nile red staining method by using an ultrasonic sonicator to homogeneously distribute the intercellular micro-alveolar cell spatial distribution. As a result, Nile red (Nile red) It induces homogeneous dyeing even when the concentration of the dyeing reagent is lowered. It is an organic solvent that dissolves nile red dye, and by using methanol instead of high toxic acetone, the toxicity is lowered to increase cell viability , And fluorescence activated cell sorter (FACS), it was confirmed that microalgae strains containing a relatively high amount of microorganism can be selectively and rapidly separated, and the strains isolated by this method can be cultivated The present invention has been completed.
본 발명의 미세조류 세포 분리 방법은 도 1에 간략히 나타내었다.
The microalgae cell separation method of the present invention is schematically shown in Fig.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
실시예 1. Example 1.
Chlorella vulgarisChlorella vulgaris
의 신속한 지질염색 및 관찰Rapid lipid staining and observation
실험에 사용된 균주는 Chlorella vulgaris이다. 배양된 균주 1.5 ㎖을 3000 rpm에서 1분간 샘플의 원심분리를 진행하였으며, 원심분리된 pellet의 상태를 도 2에 나타내었다. The strain used for the experiment is Chlorella vulgaris . 1.5 ml of the cultured strain was centrifuged at 3000 rpm for 1 minute, and the state of the centrifuged pellet was shown in Fig.
상기 원심분리된 샘플은 상등액을 제거하였으며, 남아 있는 pellet에는 1/2로 희석한 BG11 배지 1.5 ㎖를 첨가하였다. 최종적으로 세포와 배지는 2 : 1 부피비가 되도록 하였으며, 그 중 300 ㎕의 샘플을 취하여 e-tube로 옮긴 후 초음파파쇄기 (sonicator, Vibra cell, Sonics & materials INC, USA)를 이용하여 세포 균질화 (sonication)를 실시하였으며, 세포 균질화 결과를 도 3에 나타내었다. The supernatant was removed from the centrifuged samples, and 1.5 ml of BG11 medium diluted in 1/2 was added to the remaining pellet. Finally, 300 μl of the sample was transferred to an e-tube and sonicated using a sonicator (Vibra cell, Sonics & materials INC, USA) ), And cell homogenization results are shown in Fig.
상기 세포 균질화 과정에서 초음파파쇄기 (sonicator)의 세부조건은 Duty cycle은 60%, Micro-Tip Limit은 4로 설정하였고, probe는 직경 3 mm (S&M 630-0418)를 사용하였으며 세포 균질화 (sonication) 시간은 10초로 유지하였다. In the cell homogenization process, the detailed conditions of the sonicator were set to 60% for the duty cycle, 4 for the micro-tip limit, 3 mm in diameter (S & M 630-0418) Was maintained at 10 seconds.
Nile red 염색약은 10 ppm 농도를 사용하였으며, 최종적으로 샘플 1 ㎖ 당 0.1 ppm의 농도가 되도록 처리하였으며, 염색반응 시간은 10분으로 진행하였다. 반응이 완료된 샘플은 더 이상의 반응을 막고자 1/2로 희석된 BG11 배지로 2회 수세하였다. 본 조건에 의해 지질염색이 잘 이루어지는 확인하기 위하여 고속 라이브 공초점 현미경 (Ultrafast Live Scanning Microscope System, Carl Zeiss Microimaging GmbH, Germany)을 이용하여 세포를 촬영하였으며, 그 결과는 도 4에 나타내었다.Nile red dye was used at a concentration of 10 ppm and finally treated to a concentration of 0.1 ppm per 1 ml of the sample and the dyeing reaction time was 10 minutes. The reaction-completed sample was washed twice with BG11 medium diluted with 1/2 to prevent further reaction. Cells were photographed using a high speed Live Scanning Microscope System (Carl Zeiss Microimaging GmbH, Germany) to confirm that lipid staining was performed well under these conditions. The results are shown in FIG.
구체적으로, 도 4에 의하면, 죽은 세포의 경우 엽록체만 남아있어 Nile red 염색약에 의해 염색되지 않아 적색만이 관찰이 되었으며, 살아있는 세포의 경우 엽록체 (적색)와 Nile red 염색약에 의해 노란색으로 염색된 세포의 지질부위(lipid body)가 관찰되었음을 알 수 있다.
Specifically, in FIG. 4, only dead chloroplasts remain in the chloroplast, and only the red color is observed because they are not stained with Nile red dye. In living cells, chloroplasts (red) and yellow dye (Lipid body) was observed.
실시예 2. Example 2.
Chlorella vulgarisChlorella vulgaris
의 높은 지질을 가지는 균주의 신속한 분리Rapid separation of strains with high lipid content
Chlorella vulgaris C는 돌연변이 유발 화학물질 처리를 하지 않은 균주이고, 8 내지 18번의 균주는 각각 돌연변이 유발 화학물질 처리한 균주이며, Chlorella vulgaris K는 Chlorella vulgaris의 배양에 사용되는 형광등이 아닌 자외선 (UV)를 광원으로 이용하여 배양한 균주이다 (하기 표 1 참조). Chlorella vulgaris C is a strain that has not been treated with a mutagenic chemical, and strains 8 to 18 are mutagenic chemicals, and Chlorella vulgaris K is a non-fluorescent ultraviolet (UV) light used for culturing Chlorella vulgaris As a light source (see Table 1 below).
상기 실시예 1에서 Nile red 염색약에 의해 염색된 각 균주들의 세포들은 형광활성 세포분류기 (fluorescence activated cell sorter, FACS, Becton Dickinson and Company, USA)를 이용하여 FITC-a 필터를 통해 400 ~ 500 nm 사이 영역의 exite와 600 ~ 700nm 사이 영역의 emite의 형광파장신호 (fluorescence emission signal)를 검출하였으며, 그 결과를 하기 표 1 및 도 5에 나타내었다.The cells of each strain stained with Nile red dye in Example 1 were immobilized on a FITC-a filter using a fluorescence activated cell sorter (FACS, Becton Dickinson and Company, USA) The fluorescence emission signal of the emite in the region between 600 and 700 nm was detected. The results are shown in Table 1 and FIG. 5 below.
[표 1] [Table 1]
구체적으로, 상기 표 1 및 도 5에서 알 수 있는바와 같이 각각의 균주에 따라 정규분포를 이루는 피크(peak)에 차이가 나타났다. FITC-a 필터를 통해 나온 형광파장신호 중 지정된 상위 약 90% 이상의 피크 구간 (도 5의 P1 구간)에 포함되어 높은 신호의 세기 (signal intensity)를 나타내는 균주의 세포들은 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 통해 미세조류 배지가 첨가된 96-웰 플레이트(96 well plate)에 하나의 세포씩 분리할 수 있었으며, 각 웰(well)에 받아진 세포들은 재배양이 가능하였다. Specifically, as can be seen from Table 1 and FIG. 5, peaks having a normal distribution according to each strain appeared. The cells of the strain showing a high signal intensity contained in a peak region (P1 region in FIG. 5) of about 90% or more of the designated fluorescence wavelength signal emitted from the FITC-a filter are fluorescence activated Cell sorter (FACS) was used to separate each cell into 96-well plates containing microalgae culture medium, and the cells received in each well were regrown.
따라서 상기 실시예 1 및 2를 통해 Nile red 염색과 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 활용하여 높은 지질함량을 가지는 균주를 신속하게 분리해 낼 수 있었으며, 분리된 각각의 세포는 재배양이 가능한 것을 확인하였다.Therefore, through the Nile red staining and the fluorescence activated cell sorter (FACS), it was possible to quickly isolate strains having high lipid content through Examples 1 and 2, and each separated cell was cultivated It was confirmed that the amount was possible.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
Claims (9)
b) 메탄올 용매에 녹인 나일레드(Nile red) 염색약을 상기 균질화 된 미세조류 세포 샘플 1 ㎖ 당 0.05 내지 0.15 ppm 농도로 처리하여 염색하는 단계; 및
c) 상기 염색된 미세조류 세포로부터 형광활성 세포분류기 (fluorescence activated cell sorter, FACS)를 이용하여 지질함량이 높은 세포를 분리하는 단계를 포함하는 미세조류 균주의 분리 방법으로,
상기 a) 단계의 초음파 파쇄 조건은 듀티싸이클(duty cycle) 50 내지 70%, 마이크로-팁 리미트(Micro-tip Limit) 3 내지 5, 프로브(probe) 직경 2 내지 4 mm에서 8 내지 12초 간 균질화하는 것을 특징으로 하고,
상기 c) 단계는 형광활성 세포분류기의 FITC-a 필터를 통해 여기(exite) 파장 영역이 400 내지 500 nm, 발광(emite) 파장 영역이 600 내지 700 nm인 형광파장신호를 검출하는 것을 특징으로 하는, 미세조류 균주의 분리 방법.a) homogenizing microalgae cells using an ultrasonic disrupter;
b) dyeing the Nile red dye dissolved in a methanol solvent at a concentration of 0.05 to 0.15 ppm per 1 ml of the homogenized microalgae cell sample; And
c) separating the microalgae from the dyed microalgae cells by using a fluorescence activated cell sorter (FACS) to separate cells having a high lipid content,
The ultrasonic disruption condition in the step a) is performed by homogenizing for 8 to 12 seconds at a duty cycle of 50 to 70%, a micro-tip limit of 3 to 5, a probe diameter of 2 to 4 mm, .
Wherein the step c) comprises detecting a fluorescence wavelength signal having an excite wavelength range of 400 to 500 nm and an emitter wavelength range of 600 to 700 nm through the FITC-a filter of the fluorescence activated cell sorter , A method for separating microalgae strains.
상기 c) 단계에서 분리된 지질함량이 높은 세포를 재배양하는 단계를 더 포함하는 것을 특징으로 하는, 미세조류 균주의 분리 방법.The method according to claim 1,
And further comprising the step of regenerating the cells having a high lipid content separated in the step c).
상기 a) 단계는 미세조류를 2000 내지 4000 rpm에서 1 내지 5분 간 원심분리한 후 초음파 파쇄기를 이용하여 미세조류 세포를 균질화 하는 것을 특징으로 하는, 미세조류 균주의 분리 방법.The method according to claim 1,
Wherein the microalgae are centrifuged at 2000 to 4000 rpm for 1 to 5 minutes and the microalgae cells are homogenized using an ultrasonic crusher.
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| WO2015152620A1 (en) * | 2014-04-01 | 2015-10-08 | 인하대학교 산학협력단 | High-performance empirical analysis of metabolic pathways in microalgae using cell-free system |
| WO2017034351A1 (en) * | 2015-08-25 | 2017-03-02 | 주식회사 엘지화학 | Negative electrode for secondary battery, and secondary battery comprising same |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009117743A2 (en) * | 2008-03-21 | 2009-09-24 | University Of Washington | Novel chrysochromulina species, methods and media therefor, and products derived therefrom |
-
2011
- 2011-06-24 KR KR1020110061691A patent/KR101424855B1/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009117743A2 (en) * | 2008-03-21 | 2009-09-24 | University Of Washington | Novel chrysochromulina species, methods and media therefor, and products derived therefrom |
| WO2009117743A3 (en) * | 2008-03-21 | 2009-12-23 | University Of Washington | Novel chrysochromulina species, methods and media therefor, and products derived therefrom |
Non-Patent Citations (2)
| Title |
|---|
| Journal of Lipid Research, 26권, 781-789면(1985.) * |
| Journal of Lipid Research, 26권, 781-789면(1985.)* |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20160056833A (en) | 2014-11-12 | 2016-05-20 | 고려대학교 산학협력단 | Micro device for selecting microalgae strains outstanding phototaxis or chemotaxis and selection method of microalgae strains using the same |
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