微藻這類細小的生物通常生活在淡水和海洋生態系統，生長形式包括單獨生長、鏈狀或是團狀生長(Thurman HV, Burton EA. Introductory oceanography: Prentice Hall New Jersey; 1997)。微藻生物多樣性複雜，估計微藻大約有20-80 萬種，其中已發現並記錄的僅有5 萬種(Borowitzka MA. Commercial production of microalgae: ponds, tanks, tubes and fermenters. Journal of biotechnology 1999;70:313)，微藻幾乎是為一個幾乎未積極開發的資源。 脂質為微藻之次級代謝產物，具保持細胞膜通透性及因應環境變化作為細胞信號傳導途徑之功能。微藻生產之油脂量與組成會隨周遭環境產生變化(Borowitzka MA. (1999) 及Thompson PA, Harrison PJ, Whyte JN. Influence of irradiance on the fatty acid composition of phytoplanktonl. Journal of Phycology 1990;26:278。因此隨著培養環境的條件包括光強度、生長階段、光週期、溫度、鹽度、CO
2濃度、氮和磷濃度等，油脂之含量、組成和各種脂肪酸比例亦會隨之改變(Dunstan G, Volkman J, Barrett S, Garland C. Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture. Journal of Applied Phycology 1993;5:71 及Wu H, Volponi JV, Oliver AE, Parikh AN, Simmons BA, Singh S. In vivo lipidomics using single-cell Raman spectroscopy. Proceedings of the National Academy of Sciences 2011;108:3809)。微藻富含三酸甘油脂、雙酸甘油脂、磷脂和醣脂、碳氫化合物和其他脂類，含油總量可佔乾重的1至90％，取決於藻種和培養條件(Spolaore P, Joannis-Cassan C, Duran E, Isambert A. Commercial applications of microalgae. Journal of Bioscience and Bioengineering 2006;101:87 及Chisti Y. Biodiesel from microalgae. Biotechnology Advances 2007;25:294 )。 微藻作為一個可持續發展的綠色能源之一，相較於其他油脂植物像是棕梠、油菜、大豆及甘蔗作為生物燃料生產者時，其操作性更高，在短時間內生產生物柴油、生物乙醇、生物氫及生物質的產量更大。又微藻可使用非耕地、苦鹹水及民生廢水等進行生產，減少使用農耕土地與淡水資源，進而減少與糧食和經濟作物的競爭。因此各國已投入微藻及其衍生物包括生物燃料、化學品及高價商品的商業化。 微藻可用來生成一系列的可再生燃料，包括生質柴油(Tran D-T, Chen C-L, Chang J-S. Effect of solvents and oil content on direct transesterification of wet oil-bearing microalgal biomass of
Chlorella vulgarisESP-31 for biodiesel synthesis using immobilized lipase as the biocatalyst. Bioresource Technology 2013;135:213；Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M,
et al. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. The Plant Journal 2008;54:621 及Cheng H-H, Whang L-M, Chan K-C, Chung M-C, Wu S-H, Liu C-P,
et al. Biological butanol production from microalgae-based biodiesel residues by
Clostridium acetobutylicum. Bioresource Technology 2015;184:379 )、生物乙醇(Ho S-H, Li P-J, Liu C-C, Chang J-S. Bioprocess development on microalgae-based CO
2fixation and bioethanol production using
Scenedesmus obliquusCNW-N. Bioresource Technology 2013;145:142；Ho S-H, Huang S-W, Chen C-Y, Hasunuma T, Kondo A, Chang J-S. Bioethanol production using carbohydrate-rich microalgae biomass as feedstock. Bioresource Technology 2013;135:191 及Harun R, Danquah MK. Influence of acid pre-treatment on microalgal biomass for bioethanol production. Process Biochemistry 2011;46:304)、生物氫(Sambusiti C, Bellucci M, Zabaniotou A, Beneduce L, Monlau F. Algae as promising feedstocks for fermentative biohydrogen production according to a biorefinery approach: A comprehensive review. Renewable and Sustainable Energy Reviews 2015;44:20；Oncel S, Kose A, Faraloni C, Imamoglu E, Elibol M, Torzillo G,
et al. Biohydrogen production from model microalgae
Chlamydomonas reinhardtii: A simulation of environmental conditions for outdoor experiments. International Journal of Hydrogen Energy 2015;40:7502 及Batista AP, Ambrosano L, Graça S, Sousa C, Marques PA, Ribeiro B,
et al. Combining urban wastewater treatment with biohydrogen production–An integrated microalgae-based approach. Bioresource Technology 2015;184:230)、甲烷(Caporgno M, Taleb A, Olkiewicz M, Font J, Pruvost J, Legrand J,
et al. Microalgae cultivation in urban wastewater: Nutrient removal and biomass production for biodiesel and methane. Algal Research 2015;10:232；Ajeej A, Thanikal JV, Narayanan C, Kumar RS. An overview of bio augmentation of methane by anaerobic co-digestion of municipal sludge along with microalgae and waste paper. Renewable and Sustainable Energy Reviews 2015;50:270 及Kim J, Kang C-M. Increased anaerobic production of methane by co-digestion of sludge with microalgal biomass and food waste leachate. Bioresource Technology 2015;189:409)及合成氣(Raheem A, WAKG WA, Yap YT, Danquah MK, Harun R. Optimization of the microalgae Chlorella vulgaris for syngas production using central composite design. RSC Advances 2015;5:71805；Raheem A, Sivasangar S, Azlina WW, Yap YT, Danquah MK, Harun R. Thermogravimetric study of
Chlorella vulgarisfor syngas production. Algal Research 2015;12:52 及Hu Z, Ma X, Li L. The synergistic effect of co-pyrolysis of oil shale and microalgae to produce syngas. Journal of the Energy Institute 2015 )。然而，在微藻生物燃料的商業化之技術和經濟量產較為困難，需突破之處包括能耐受室外培養、光合效率更好及生長更快之藻種，以及在大規模生產時採收、萃取等油品製造之成本。 預計到2050 年時，世界人口將達到90 億，這將導致全球糧食之供應面臨挑戰(Foley JA, Ramankutty N, Brauman KA, Cassidy ES, Gerber JS, Johnston M,
et al. Solutions for a cultivated planet. Nature 2011;478:337 及Tilman D, Balzer C, Hill J, Befort BL. Global food demand and the sustainable intensification of agriculture. Proceedings of the National Academy of Sciences 2011;108:20260)[23, 24]。近年來微藻已成為可持續生產糧食及飼料、燃料和化學品的明星選項。相較於傳統糧食作物，微藻產量是高等植物的6-7倍，是可靠的蛋白質、碳水化合物和脂質來源(Becker E. Micro-algae as a source of protein. Biotechnology advances 2007;25:207)。許多藻種生產脂質是以蓄積三酸甘油酯(triacylglycerol，TAG)形式為主，具有類似於植物油之脂肪酸組成(Draaisma RB, Wijffels RH, Slegers PE, Brentner LB, Roy A, Barbosa MJ. Food commodities from microalgae. Current Opinion in Biotechnology 2013;24:169 及Gunstone F. Vegetable oils in food technology: composition, properties and uses: John Wiley & Sons; 2011)，此外還能生產一些高價脂肪酸例如二十碳五烯酸(Eicosapentaenoic Acid，EPA)和二十二碳六烯酸(Docosahexaenoic acid，DHA)( Guschina IA, Harwood JL. Algal lipids and effect of the environment on their biochemistry. Lipids in aquatic ecosystems: Springer; 2009, p.1 及Mühlroth A, Li K, Røkke G, Winge P, Olsen Y, Hohmann-Marriott MF,
et al. Pathways of lipid metabolism in marine algae, co-expression network, bottlenecks and candidate genes for enhanced production of EPA and DHA in species of
Chromista. Marine drugs 2013;11:4662 )。又人體必需脂肪酸包括n6系列之亞麻油酸(C18:2)與n3系列之次亞麻油酸(C18:3)，通常藉由飲食攝取，部分微藻能生產這些碳鏈油脂(Lang I, Hodac L, Friedl T, Feussner I. Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC plant biology 2011;11:124)。美國Solazyme 於2013 年通過美國FDA 對於其藻油產品GRAS 之認證(FDA U. RE: High Lipid
Chlorella protothecoidesS106 Flour GRAS 2013)，說明藻油能夠運用在日常烹調與烘焙。 人類文明發展活動例如燃燒化石燃料、毀林和能源生產，進而造成了強烈的溫室氣體排放。二氧化碳其在大氣中的濃度，這是自工業革命以前到2013年，已從280升至390 ppm (Rahaman MSA, Cheng L-H, Xu X-H, Zhang L, Chen H-L. A review of carbon dioxide capture and utilization by membrane integrated microalgal cultivation processes. Renewable and Sustainable Energy Reviews 2011;15:4002及Singh UB, Ahluwalia A. Microalgae: a promising tool for carbon sequestration. Mitigation and Adaptation Strategies for Global Change 2013;18:73)。二氧化碳能在大氣中存在50-200 年，造成全球變暖有52％是歸咎於二氧化碳(Wilbanks TJ, Fernandez S, Backus G, Garcia P, Jonietz KK. Climate Change and Infrastructure, Urban Systems, and Vulnerabilities: Technical Report for the US Department of Energy in Support of the National Climate Assessment: Island Press; 2014)。微藻固定二氧化碳的效率是陸生植物的10-50倍(Cheng J, Huang Y, Feng J, Sun J, Zhou J, Cen K. Improving CO2 fixation efficiency by optimizing
ChlorellaPY-ZU1 culture conditions in sequential bioreactors. Bioresource technology 2013;144:321及Lam MK, Lee KT, Mohamed AR. Current status and challenges on microalgae-based carbon capture. International Journal of Greenhouse Gas Control 2012;10:456)。陸生植物預計只能削減約3-6％全球二氧化碳排放量(Ho S-H, Chen C-Y, Lee D-J, Chang J-S. Perspectives on microalgal CO2-eission mitigation systems—a review. Biotechnology advances 2011;29:189及Kao C-Y, Chen T-Y, Chang Y-B, Chiu T-W, Lin H-Y, Chen C-D,
et al. Utilization of carbon dioxide in industrial flue gases for the cultivation of microalga
Chlorellasp. Bioresource Technology 2014;166:485)。微藻每生產1 公斤微藻生物質約可固定1.83 公斤的二氧化碳(Jiang Y, Zhang W, Wang J, Chen Y, Shen S, Liu T. Utilization of simulated flue gas for cultivation of
Scenedesmus dimorphus. Bioresource Technology 2013;128:359)。因此微藻已被公認為生產生物燃料最有前途的替代方案。由於微藻光合效率在生物轉化二氧化碳時表現出高生產效率、高量的脂質蓄積，又能降低大氣二氧化碳濃度，可生產有價值的原料，亦可生產可再生能源及有價值的非燃料副產品(Xie Y-P, Ho S-H, Chen C-Y, Chen C-NN, Liu C-C, Ng I-S,
et al. Simultaneous enhancement of CO
2fixation and lutein production with thermo-tolerant
Desmodesmussp. F51 using a repeated fed-batch cultivation strategy. Biochemical Engineering Journal 2014;86:33)。微藻光合過程中除了可使用大氣中的二氧化碳，對於電廠煙道氣中之二氧化碳亦可進行捕捉利用，從而減少排碳量(Doucha J, Straka F, Lívanský K. Utilization of flue gas for cultivation of microalgae
Chlorellasp.) in an outdoor open thin-layer photobioreactor. Journal of Applied Phycology 2005;17:403及Maeda K, Owada M, Kimura N, Omata K, Karube I. CO
2fixation from the flue gas on coal-fired thermal power plant by microalgae. Energy Conversion and Management 1995;36:717)。 以富含胺基酸、有機酸的食品工業廢水而言，結合微藻養殖的廢水處理系統似乎是一個不錯的選擇(Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: a review. Renewable and sustainable energy reviews 2010;14:217)。利用藻類廢水處理具有很多優點，包括廢水中豐富的營養物質，例如氮和磷等，以及適量的重金屬離子，提供藻類生長所需，達到去除化學、有機污染物及部分重金屬的效益。另外可回收藻體之生物質作為肥料，可以抵消部分運轉費用(Munoz R, Guieysse B. Algal–bacterial processes for the treatment of hazardous contaminants: a review. Water research 2006;40:2799)。可應用在廢水養殖之微藻例如
Chlorococcumsp. RAP13以異營的方式處理乳製品廠污水，其所生成之油脂含量可達到42% (Ummalyma SB, Sukumaran RK. Cultivation of microalgae in dairy effluent for oil production and removal of organic pollution load. Bioresource technology 2014;165:295)。
Scenedesmussp.處理富含果糖、葡萄糖和乙酸之廢水，其所生成之油脂含量更可達到52.6% (Ummalyma SB, Sukumaran RK. Cultivation of microalgae in dairy effluent for oil production and removal of organic pollution load. Bioresource technology 2014;165:295)。
Chlorella pyrenoidosaFACHB-9處理大豆加工廢水，其所生成之油脂含量達到37% (Hongyang S, Yalei Z, Chunmin Z, Xuefei Z, Jinpeng L. Cultivation of
Chlorella pyrenoidosain soybean processing wastewater. Bioresource Technology 2011;102:9884)。 根據AlgaeBase資料庫(http://www.algaebase.org/)記載，
Chlorella lewinii之模式生物(type species)為CCAP 221/90，對此藻種目前尚未有太多研究，僅於分類學研究(Bock C, Krienitz L, Proeschold T. Taxonomic reassessment of the genus
Chlorella(Trebouxiophyceae) using molecular signatures (barcodes), including description of seven new species. Fottea 2011;11:293.)及產氫研究(Pongpadung P, Liu J, Yokthongwattana K, Techapinyawat S, Juntawong N. Screening for hydrogen-producing strains of green microalgae in phosphorus or sulphur deprived medium under nitrogen limitation. ScienceAsia 2015;41:97)中，作為比對之用。關於油脂生產、固碳效率及異營生長特性等，目前尚未有學術文獻或專利發表。 無論是經濟發展或民生所需，對於乾淨的空氣與水源的需求日益重要。為減緩溫室效益與對環境的破壞，對於CO
2及工業所產生的廢水，兩者的排放標準越來越嚴格，儼然成為各國政府與企業巨大的挑戰。因此，新的概念或工程系統日趨重要，例如混合廢水與廢棄的微藻處理系統可以是一個有效的污染物去除的方法，廢水中含有可生物降解的有機物，在處理廢水的同時又可生產能源，此混合系統之發展已成為一種趨勢。期待未來能夠發展出適合的技術來因應環保與能源問題。
Small organisms such as microalgae usually live in freshwater and marine ecosystems, and growth forms include individual growth, chain or cluster growth (Thurman HV, Burton EA. Introductory oceanography: Prentice Hall New Jersey; 1997). The microalgae is complex in biodiversity, with an estimated 200-800,000 species of microalgae, of which only 50,000 have been discovered and recorded (Borowitzka MA. Commercial production of microalgae: ponds, tanks, tubes and fermenters. Journal of biotechnology 1999) ; 70:313), microalgae is almost a resource that is hardly developed. Lipids are secondary metabolites of microalgae, which have the function of maintaining cell membrane permeability and responding to environmental changes as a cell signaling pathway. The amount and composition of oil produced by microalgae varies with the surrounding environment (Borowitzka MA. (1999) and Thompson PA, Harrison PJ, Whyte JN. Influence of irradiance on the fatty acid composition of phytoplanktonl. Journal of Phycology 1990;26:278 Therefore, as the conditions of the culture environment include light intensity, growth stage, photoperiod, temperature, salinity, CO 2 concentration, nitrogen and phosphorus concentration, the content, composition and various fatty acid ratios of the oil will also change (Dunstan G , Volkman J, Barrett S, Garland C. Changes in the lipid composition and maximisation of the polyunsaturated fatty acid content of three microalgae grown in mass culture. Journal of Applied Phycology 1993; 5:71 and Wu H, Volponi JV, Oliver AE, Parikh AN, Simmons BA, Singh S. In vivo lipidomics using single-cell Raman spectroscopy. Proceedings of the National Academy of Sciences 2011;108:3809). Microalgae rich in triglycerides, diglycerides, phospholipids and sugars Fats, hydrocarbons and other lipids, the total oil content can account for 1 to 90% of the dry weight, depending on the algae species and culture strips (Spolaore P, Joannis-Cassan C, Duran E, Isambert A. Commercial applications of microalgae. Journal of Bioscience and Bioengineering 2006; 101:87 and Chisti Y. Biodiesel from microalgae. Biotechnology Advances 2007; 25:294). One of the sustainable green energy sources is more operative than other oily plants like palm stalks, canola, soybeans and sugar cane as biofuel producers, producing biodiesel, bioethanol, and biomass in a short period of time. The production of hydrogen and biomass is greater. Microalgae can be produced using non-cultivated land, brackish water and people's livelihood wastewater, reducing the use of agricultural land and fresh water resources, thereby reducing competition with food and cash crops. Therefore, countries have invested in the commercialization of microalgae and its derivatives including biofuels, chemicals and high-priced commodities. Microalgae can be used to generate a range of renewable fuels, including Tan DT, Chen CL, Chang JS. Effect of solvents and oil content on direct transesterification of wet oil-bearing microalgal biomass of Chlorella vulgaris ESP-31 for biodiesel Bioresource Technology 2013;135:213;Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, et al . Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. The synthesis uses lipase as the biocatalyst. Plant Journal 2008;54:621 and Cheng HH, Whang LM, Chan KC, Chung MC, Wu SH, Liu CP, et al . Biological butanol production from microalgae-based biodiesel residues by Clostridium acetobutylicum . Bioresource Technology 2015;184:379 ) Bio-ethanol (Ho SH, Li PJ, Liu CC, Chang JS. Bioprocess development on microalgae-based CO 2 fixation and bioethanol production using Scenedesmus obliquus CNW-N. Bioresource Technology 2013; 145:142; Ho SH, Huang SW, Chen CY, Hasunuma T, Kondo A, Chang JS. Bi Oethanol production using carbohydrate-rich microalgae biomass as feedstock. Bioresource Technology 2013;135:191 and Harun R, Danquah MK. Influence of acid pre-treatment on microalgal biomass for bioethanol production. Process Biochemistry 2011;46:304), biohydrogen ( Sambusiti C, Bellucci M, Zabaniotou A, Beneduce L, Monlau F. Algae as promising feedstocks for fermentative biohydrogen production according to a biorefinery approach: A comprehensive review. Renewable and Sustainable Energy Reviews 2015;44:20; Oncel S, Kose A, Faraloni C, Imamoglu E, Elibol M, Torzillo G, et al . Biohydrogen production from model microalgae Chlamydomonas reinhardtii : A simulation of environmental conditions for outdoor experiments. International Journal of Hydrogen Energy 2015;40:7502 and Batista AP, Ambrosano L, Graça . S, Sousa C, Marques PA , Ribeiro B, et al Combining urban wastewater treatment with biohydrogen production-An integrated microalgae-based approach Bioresource Technology 2015; 184:. 230), Alkyl (Caporgno M, Taleb A, Olkiewicz M, Font J, Pruvost J, Legrand J, et al Microalgae cultivation in urban wastewater: Nutrient removal and biomass production for biodiesel and methane Algal Research 2015; 10:.. 232; Ajeej A, Thanikal JV, Narayanan C, Kumar RS. An overview of bio augmentation of methane by anaerobic co-digestion of municipal sludge along with microalgae and waste paper. Renewable and Sustainable Energy Reviews 2015;50:270 and Kim J, Kang CM. Increased anaerobic Production of methane by co-digestion of sludge with microalgal biomass and food waste leachate. Bioresource Technology 2015;189:409) and syngas (Raheem A, WAKG WA, Yap YT, Danquah MK, Harun R. Optimization of the microalgae Chlorella vulgaris For syngas production using central composite design. RSC Advances 2015;5:71805;Raheem A, Sivasangar S, Azlina WW, Yap YT, Danquah MK, Harun R. Thermogravimetric study of Chlorella vulgaris for syngas production. Algal Research 2015;12:52 And Hu Z, Ma X, Li L. The synergi Stic effect of co-pyrolysis of oil shale and microalgae to produce syngas. Journal of the Energy Institute 2015). However, the technical and economic mass production of microalgae biofuels is difficult, and the breakthroughs include algae that can withstand outdoor culture, better photosynthetic efficiency and faster growth, and harvest in large-scale production. The cost of oil production such as extraction. It is estimated that by 2050, the world population will reach 9 billion, which will lead to global food supply challenges (Foley JA, Ramankutty N, Brauman KA, Cassidy ES, Gerber JS, Johnston M, et al . Solutions for a cultivated planet. Nature 2011;478:337 and Tilman D, Balzer C, Hill J, Befort BL. Global food requirements and the sustainable sustainable intensification of agriculture. Proceedings of the National Academy of Sciences 2011; 108:20260) [23, 24]. In recent years, microalgae has become a star option for the sustainable production of food and feed, fuels and chemicals. Compared to traditional food crops, microalgae production is 6-7 times higher than that of higher plants, and is a reliable source of protein, carbohydrate and lipid (Becker E. Micro-algae as a source of protein. Biotechnology advances 2007; 25:207) . Many algae produce lipids based on the accumulation of triacylglycerol (TAG), which has a fatty acid composition similar to vegetable oils (Draaisma RB, Wijffels RH, Slegers PE, Brentner LB, Roy A, Barbosa MJ. Food commodities from Microalgae. Current Opinion in Biotechnology 2013; 24:169 and Gunstone F. Vegetable oils in food technology: composition, properties and uses: John Wiley &Sons; 2011), in addition to producing some high-priced fatty acids such as eicosapentaenoic acid ( Eicosapentaenoic Acid, EPA) and Docosahexaenoic acid (DHA) (Guschina IA, Harwood JL. Algal lipids and effect of the environment on their biochemistry. Lipids in aquatic ecosystems: Springer; 2009, p.1 and Mühlroth A, Li K, Røkke G, Winge P, Olsen Y, Hohmann-Marriott MF, et al . Pathways of lipid metabolism in marine algae, co-expression network, bottlenecks and candidate genes for enhanced production of EPA and DHA in species of Chromista . Marine drugs 2013;11:4662 ). The essential fatty acids include the n6 series of linoleic acid (C18:2) and the n3 series of linoleic acid (C18:3), which are usually produced by dietary intake, and some microalgae can produce these carbon chain fats (Lang I, Hodac). L, Friedl T, Feussner I. Fatty acid profiles and their distribution patterns in microalgae: a comprehensive analysis of more than 2000 strains from the SAG culture collection. BMC plant biology 2011; 11: 124). In 2013, Solazyme of the United States passed the FDA certification for its algae product GRAS (FDA U. RE: High Lipid Chlorella protothecoides S106 Flour GRAS 2013), indicating that algae oil can be used in daily cooking and baking. Human civilization development activities such as burning fossil fuels, deforestation and energy production have resulted in strong greenhouse gas emissions. The concentration of carbon dioxide in the atmosphere, which has risen from 280 to 390 ppm since the Industrial Revolution to 2013 (Rahaman MSA, Cheng LH, Xu XH, Zhang L, Chen HL. A review of carbon dioxide capture and utilization by Renewable and Sustainable Energy Reviews 2011; 15:4002 and Singh UB, Ahluwalia A. Microalgae: a promising tool for carbon sequestration. Mitigation and Adaptation Strategies for Global Change 2013;18:73). Carbon dioxide can exist in the atmosphere for 50-200 years, and 52% of global warming is attributed to carbon dioxide (Wilbanks TJ, Fernandez S, Backus G, Garcia P, Jonietz KK. Climate Change and Infrastructure, Urban Systems, and Vulnerabilities: Technical Report for the US Department of Energy in Support of the National Climate Assessment: Island Press; 2014). The efficiency of fixing carbon dioxide by microalgae is 10-50 times that of terrestrial plants (Cheng J, Huang Y, Feng J, Sun J, Zhou J, Cen K. Improving CO2 fixation efficiency by optimizing Chlorella PY-ZU1 culture conditions in sequential bioreactors. Bioresource Technology 2013; 144:321 and Lam MK, Lee KT, Mohamed AR. Current status and challenges on microalgae-based carbon capture. International Journal of Greenhouse Gas Control 2012; 10:456). Terrestrial plants are expected to cut only about 3-6% of global carbon dioxide emissions (Ho SH, Chen CY, Lee DJ, Chang JS. Perspectives on microalgal CO2-eission mitigation systems—a review. Biotechnology advances 2011; 29:189 and Kao CY , Chen TY, Chang YB, Chiu TW, Lin HY, Chen CD, et al . Utilization of carbon dioxide in industrial flue gases for the cultivation of microalga Chlorella sp. Bioresource Technology 2014; 166: 485). Microalgae can fix 1.83 kg of carbon dioxide per 1 kg of microalgae biomass (Jiang Y, Zhang W, Wang J, Chen Y, Shen S, Liu T. Utilization of simulated flue gas for cultivation of Scenedesmus dimorphus . Bioresource Technology 2013 ;128:359). Microalgae has therefore been recognized as the most promising alternative to producing biofuels. Because the photosynthetic efficiency of microalgae shows high production efficiency, high lipid accumulation in bioconversion of carbon dioxide, and lower atmospheric carbon dioxide concentration, it can produce valuable raw materials, and can also produce renewable energy and valuable non-fuel by-products ( Xie YP, Ho SH, Chen CY, Chen C-NN, Liu CC, Ng IS, et al . Simultaneous enhancement of CO 2 fixation and lutein production with thermo-tolerant Desmodesmus sp. F51 using a repeated fed-batch cultivation strategy. Engineering Journal 2014; 86:33). In addition to the use of atmospheric carbon dioxide in the photosynthetic process of microalgae, carbon dioxide in power plant flue gas can also be captured and utilized to reduce carbon emissions (Doucha J, Straka F, Lívanský K. Utilization of flue gas for cultivation of microalgae Chlorella sp.) in an outdoor open thin-layer photobioreactor. Journal of Applied Phycology 2005;17:403 and Maeda K, Owada M, Kimura N, Omata K, Karube I. CO 2 fixation from the flue gas on coal-fired thermal Power plant by microalgae. Energy Conversion and Management 1995;36:717). In the case of food industry wastewater rich in amino acids and organic acids, it seems to be a good choice for wastewater treatment systems that combine microalgae farming (Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: a review Renewable and sustainable energy reviews 2010;14:217). The use of algae wastewater treatment has many advantages, including rich nutrients in wastewater, such as nitrogen and phosphorus, and moderate amounts of heavy metal ions, which provide the necessary growth of algae to remove the benefits of chemical, organic pollutants and some heavy metals. In addition, the biomass of the algae can be recovered as a fertilizer, which can offset part of the operating cost (Munoz R, Guieysse B. Algal-bacterial processes for the treatment of hazardous contaminants: a review. Water research 2006; 40: 2799). Microalgae that can be used in wastewater culture, such as Chlorococcum sp. RAP13, to treat dairy wastewater in a heterogeneous manner, which produces a fat content of up to 42% (Ummalyma SB, Sukumaran RK. Cultivation of microalgae in dairy effluent for oil production And removal of organic pollution load. Bioresource technology 2014;165:295). Scenedesmus sp. treats wastewater rich in fructose, glucose and acetic acid, which produces a lipid content of up to 52.6% (Ummalyma SB, Sukumaran RK. Cultivation of microalgae in dairy effluent for oil production and removal of organic pollution load. Bioresource technology 2014;165:295). Chlorella pyrenoidosa FACHB-9 treats soybean processing wastewater with a 37% oil content (Hongyang S, Yalei Z, Chunmin Z, Xuefei Z, Jinpeng L. Cultivation of Chlorella pyrenoidosa in soybean processing wastewater. Bioresource Technology 2011; 102: 9884). According to the AlgaeBase database (http://www.algaebase.org/), the type species of Chlorella lewinii is CCAP 221/90, which has not been studied much so far, only in taxonomic studies ( Bock C, Krienitz L, Proeschold T. Taxonomic reassessment of the genus Chlorella (Trebouxiophyceae) using molecular signatures (barcodes), including description of seven new species. Fottea 2011; 11:293.) and hydrogen production studies (Pongpadung P, Liu J , Yokthongwattana K, Techapinyawat S, Juntawong N. Screening for hydrogen-producing strains of green microalgae in phosphorus or sulphur deprived medium under nitrogen limitation. ScienceAsia 2015; 41:97), for comparison. There are no academic literature or patent publications on oil production, carbon sequestration efficiency and heterogeneous growth characteristics. Whether it is for economic development or for the needs of the people, the need for clean air and water is increasingly important. In order to alleviate greenhouse efficiency and damage to the environment, the emission standards for both CO 2 and industrial wastewater are becoming more and more stringent, which has become a huge challenge for governments and enterprises. Therefore, new concepts or engineering systems are becoming more and more important. For example, mixed wastewater and waste microalgae treatment system can be an effective method for removing pollutants. The wastewater contains biodegradable organic matter, which can also produce energy while treating wastewater. The development of this hybrid system has become a trend. I look forward to developing suitable technologies to meet environmental and energy issues in the future.
本發明可藉由下述實施方式中所揭示之各種發明態樣、實施例及表列之相關敘述所瞭解。除非在本文中另作定義，否則與本發明關聯使用之術語(包含技術及科學術語)應具有本發明所屬技術領域中具有通常知識者所瞭解之含義。且當可瞭解，除非本文中提供之定義另作說明，在任何潛在歧義之情況，術語之定義應與該等普遍使用之術語(如詞典中所定義)一致。可進一步瞭解者，本案所使用的術語僅係用作描述特定實施態樣之目的，而非用於限定。 必須注意的是，除非有清楚的相反指示，於說明書或申請專利範圍使用之單數格式「一」、「一種」及「該」亦包含複數表示。因此，除非上下文另有需要，單數術語應包含複數，而複數術語亦包含單數。 本發明的範圍以「自一『約』特定數值及/或至另一『約』特定數值」表示。當範圍藉上述方式表示時，其包含自一特定數值及/或至另一特定數值之範圍。同樣地，當數值可藉由術語「約」以表示近似值，將可了解其為一特定值的另一個態樣。可進一步了解，當提及有關其它端點及其他端點本身而言，每一範圍的兩端點皆為有意義的。根據本發明，「約」可表示±20%，較佳為±10%，更佳為±5%。 於本發明中，術語「經分離」或「分離」意謂使物質自其原始環境(若天然存在則為天然環境)中移出。術語"經分離"或"分離"並不一定指物質係經純化者。 本發明之一目的係提供一種綠球藻 (
Chlorella lewinii)分離藻株，其中該綠球藻分離藻株包含SEQ ID NO: 1所示核苷酸序列的18S rDNA序列及SEQ ID NO: 2所示核苷酸序列的ITS區域序列。於本發明較佳實施態樣中，該綠球藻分離藻株為寄存於財團法人食品工業發展研究所且寄存編號為BCRC 980043之藻株，或為與寄存於財團法人食品工業發展研究所且寄存編號為BCRC 980043之藻株具有實質上完全相同識別特徵之變異株。 上述術語「變異株」意謂涵蓋全體細胞遺傳組成已藉由如化學突變誘發、自發突變、遺傳工程、轉化或轉染而改變，以致影響其物理或生物化學特性之任何綠球藻株。然而，該變異株應具有寄存於財團法人食品工業發展研究所且寄存編號為BCRC 980043之綠球藻分離藻株的所有分類學識別特徵。 本發明之一目的係在於提供一種製備綠球藻培養產物之方法。於本發明之實施態樣中，該方法包含將本發明之綠球藻分離藻株接種於培養基中，及進行培養以獲得該培養產物。 於本發明中，術語「培養產物」意謂將微藻置於培養基中培養後，所獲得富含該微藻細胞的產物。於本發明中，該培養產物中之微藻細胞可不必與培養基分離，且該培養產物可呈液態、固態或黏稠狀。 於本發明中，用於培養綠球藻分離藻株之「培養基」可為任何容許綠球藻生長、繁殖並製造三酸甘油酯及/或脂肪酸之培養基，例如C培養基[每100 mL中包含15 mg Ca(NO
3)
2•4H
2O、10 mg KNO
3、5 mg β-甘油磷酸二鈉•5H
2O、4 mg MgSO
4•7H
2O、0.01 μg 維生素B12、0.01 μg 生物素 (Biotin)、1 μg 噻胺(Thiamine) HCl、0.3 mL PIV微量元素溶液(每100 mL中包含100 mg Na
2EDTA•2H
2O、19.6 mg FeCl
3•6H
2O、3.6 mg MnCl
2•4H
2O、1.04 mg ZnCl
2、0.4 μg CoCl
2•6H
2O、0.25 μg Na
2MoO
4•2H
2O及水)、50 mg Tris (hydroxymethyl) aminomethane及水]、胰蛋白酶大豆培養液(Tryptic Soy Broth；TSA)、馬鈴薯葡萄糖培養液(Potato Dextrose Broth；PDA)及營養培養液(Nutrient Broth；NA)。若要製備洋菜固體培養基，可於液態培養基中加入1.5% (w/v) 的洋菜膠，經滅菌冷卻後即可獲得洋菜固體培養基。本技術領域之人士可根據既有知識針對培養基的成分作調整。於本發明之一較佳實施態樣中，該培養基為液態培養基。 本發明中用於培養綠球藻分離藻株之條件意指如培養基之pH值、鹽度、培養溫度、照光、通氣條件及培養時間等條件，其可容許該綠球藻分離藻株生長、繁殖並製造三酸甘油酯及/或脂肪酸。本技術領域之人士可根據既有知識針對培養條件作調整。 於本發明之實施態樣中，綠球藻分離藻株可在不照光下(即24小時黑暗)下進行異營性生長、12小時光照12小時黑暗之光週期下進行混營性生長或持續光照下進行自營性生長。照光量可為約100 lux至約4,000 lux，較佳為約2,000 lux之亮度 於本發明之實施態樣中，綠球藻分離藻株之培養溫度可為約10℃至約60℃ (例如約10℃、約15℃、約20℃、約25℃、約30℃、約35℃、約40℃、約45℃、約50℃、約55℃或約60℃)，較佳為約20℃至約40℃，更佳為約30℃。 於本發明之實施態樣中，用於培養綠球藻分離藻株之培養基的pH值可為約pH1至約pH14 (例如約pH1、約pH1.5、約pH2、約pH2.5、約pH3、約pH3.5、約pH4、約pH4.5、約pH5、約pH5.5、約pH6、約pH6.5、約pH7、約pH7.5、約pH8、約pH8.5、約pH9、約pH9.5、約pH10、約pH10.5、約pH11、約pH11.5、約pH12、約pH12.5、約pH13、約pH13.5或約pH14)，較佳為約pH4至約pH10，更佳為約pH5至約pH7。 於本發明之實施態樣中，可視需要調整培養綠球藻分離藻株之培養基的鹽度。本文中所謂「鹽度」意旨溶解於培養基中之鹽類含量。本發明中培養基之鹽度可為0% (w/w)至約6% (w/w) (例如0% (w/w)、約0.5% (w/w)、約1% (w/w)、約1.5% (w/w)、約2% (w/w)、約2.5% (w/w)、約3% (w/w)、約3.5% (w/w)、約4% (w/w)、約4.5% (w/w)、約5% (w/w)、約5.5% (w/w)或約6.0% (w/w))，較佳為0% (w/w)至約3% (w/w)，更佳為1.5% (w/w)。 本發明製備綠球藻分離藻株培養產物之方法中，可視需要包含分離該培養產物的步驟，而該分離步驟可為如離心及/或過濾等習知的方法步驟。 本發明亦提供由上述方法所獲得之培養產物。本發明之培養產物中富含三酸甘油酯及/或脂肪酸，故可用作獲得三酸甘油酯及/或脂肪酸的原料，進而分別用於製作健康油脂及/或生質燃料。 本文中之「三酸甘油酯」意旨具有1個甘油分子及3個脂肪酸分子之酯類化合物，其中該3個脂肪酸分子可具有完全相同、部份相同或完全相異之碳數及不飽和鍵。 本文中之「脂肪酸」意旨具有8至30個碳原子及0至6個不飽和鍵的羧酸化合物，其較佳為具有12至20個碳原子及0至5個不飽和鍵的羧酸化合物，更佳為具有16至18個碳原子及0至3個不飽和鍵的羧酸化合物。 三酸甘油酯及脂肪酸之獲得可使用本技術領域所熟知的任何萃取及分離方法，例如Folch等人(Folch, J.
et al., A simple method for the isolation and purification of total lipids from animal tissue. The Journal of Biological Chemistry 1957; 23:497-509)、Balasubramanian等人(Balasubramanian S.
et al., Oil extraction from
Scenedesmus obliquususing a continuous microwave system – design, optimization, and quality characterization., Bioresource Technology, 2011; 102:3396-3403)及Sajilata等人(Sajilata M. G.
et al., Supercritical CO
2extraction of γ-linolenic acid (GLA) from
Spirulina platensisARM 740 using response surface methodology. Journal of Food Engineering 2008; 84: 321–326)所述的方法。簡而言之，該方法可包含將綠球藻細胞以如研磨法或超音波法等方式擊碎，藉由適當的溶劑萃取綠球藻細胞中之三酸甘油酯及/或脂肪酸，再藉由如HPLC及/或離子交換樹脂的技術獲得三酸甘油酯及/或脂肪酸。 本發明之綠球藻培養產物可經化學轉化(如氫化、轉酯化、水熱碳化(hydrothermal carbonisation)、發酵或裂解等)或萃取而成固態、液態或氣態之生質燃料，其包括(但不限於)生質柴油、生物甲醇、生物乙醇、生物丁醇、生物甲烷、生物氫或生質煤炭。 上述生質燃料之獲得可使用本技術領域所熟知的任何方法，諸如Tran D. T.等人(Tran D. T.
et al., Effect of solvents and oil content on direct transesterification of wet oil-bearing microalgal biomass of
Chlorella vulgarisESP-31 for biodiesel synthesis using immobilized lipase as the biocatalyst. Bioresource Technology 2013; 135:213-221)、Cheng H. H.等人(Cheng H. H.,
et al., Biological butanol production from microalgae-based biodiesel residues by
Clostridium acetobutylicum. Bioresource Technology 2015; 184:379-385)、Sambusiti C.等人(Sambusiti C.,
et al., Algae as promising feedstocks for fermentative biohydrogen production according to a biorefinery approach: A comprehensive review. Renewable & Sustainable Energy Reviews2015; 44:20-36)及Heilmann S. M.等人(Heilmann S. M.
et al., Hydrothermal carbonization of microalgae. Biomass & Bioenergy 2010; 34(6):875–882及Heilmann S.M.
et al., Hydrothermal carbonization of microalgae II. Fatty acid, char, and algal nutrient products. Applied Energy 2011; 88(10):3286–3290)所述之方法。 本文所述之所有公開案、專利及專利文獻均以全文引用的方式併入本文中。 提供以下實例以輔助熟習此項技術者實施本發明。即使如此，不應將該等實例視為本發明之限制，因為本發明所屬技術領域中具有通常知識者在不背離本發明之精神或範疇的情況下對本文所討論之實施例進行的修改及變化，而仍屬於本發明之範圍。
實施例 材料與方法1. 培養基配方 1.1 C培養基 將Ca(NO
3)
2•4H
2O 15 mg、KNO
310 mg、β-甘油磷酸二鈉•5H
2O 5 mg、MgSO
4•7H
2O 4 mg、維生素B12 0.01 μg、生物素 (Biotin) 0.01 μg、噻胺(Thiamine) HCl 1 μg、PIV微量金屬溶液0.3 mL與Tris 50 mg混合後將其體積補水至100 mL，調整pH至7.5後進行高壓滅菌。若為1.5% (w/v)洋菜固體培養基則需加入15 g的洋菜膠一同滅菌。 PIV微量金屬溶液的配製為依序加入Na
2EDTA•2H
2O 100 mg、FeCl
3•6H
2O 19.6 mg、MnCl
2•4H
2O 3.6 mg、ZnCl
21.04 mg、CoCl
2• 6H
2O 0.4 μg與Na
2MoO
4•2H
2O 0.25 μg，隨後將其體積補水至100 mL後進行高壓滅菌。 測試不同鹽度生長特性時，使用C培養基組成，另外加入NaCl調整鹽度，並使用鹽度計測量鹽度使達到所需的鹽度。測試不同pH 生長特性時，使用C 培養基組成，另外加入HCl 或NaOH 調整pH 值，並使用酸鹼量測計測量使達到所需的pH值。 1.2 TSB 培養基(胰蛋白酶大豆培養液(Tryptic Soy Broth)) 依序加入15.0 g的胰水解蛋白(Tryptone)、5.0 g的大豆水解蛋白(Soytone)及5.0 g的NaCl水中，隨後將其體積補水至1 L，調pH至7.3後進行高壓滅菌。 1.3 PDB培養基(馬鈴薯葡萄糖培養液；Potato Dextrose Broth) 依序加入200.0 g的切丁馬鈴薯及20.0 g的葡萄糖，隨後將其體積補水至1 L，調pH至7.3後進行高壓滅菌。 1.4 NB 培養基(營養培養液；Nutrient Broth) 依序加入3.0 g的牛肉萃取物及5.0 g的蛋白腖(Peptone)，隨後將其體積補水至1 L，調pH至7.0後進行高壓滅菌。 2. 藻樣採集、分離及培養 取台灣桃園的有機稻田之水樣與土樣均勻混合後取出約10 mL置於50 mL的離心管中，加入約30 mL的C培養基，於25℃照光培養。培養期間以顯微鏡觀察是否有藻體生長，之後取出適量含藻體的培養液，將其轉至平板培養基，於25℃照光培養。待藻體生長後取單一藻種將其於平板培養基中塗開，以上步驟需重覆至篩選獲得單一藻體為止。平板培養則取單一藻落塗至C平板培養基，於25℃照光培養。 3. 油脂染色分析 將培養好的藻體取20 μL與1 μL Nile Red (於二甲基亞碸中0.1 mg/mL)混合以進行油滴染色，染色後於室溫靜置5分鐘，再利用螢光顯微鏡進行觀察(Chen, W.
et al., A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. Journal of Microbiological Methods 2009; 77:41-47及Huang, G. H.,
et al., Rapid screening method for lipid production in alga based on Nile red fluorescence. Biomass & Bioenergy 2009; 33:1386-1392)。 4. 藻種之分子鑑定 4.1 藻體基因體(genomic) DNA的抽取 自平板培養基上刮取適量之新鮮培養的藻體，將其收集在2 mL微量離心管中，依照ZYMO RESEARCH的ZR Fungal/Bacterial DNA MiniPrep™ kit說明書操作取得基因體DNA，並以NanoDrop (ND-1000分光光度計)檢測DNA濃度。 4.2 PCR增幅、定序及親源分析 將藻體基因體DNA作為PCR模板，以18S rRNA與ITS區域(包含18S核糖體RNA的後端、內轉錄間隔區(internal transcribed spacer) 1、5.8S 核糖體RNA、內轉錄間隔區2與28S核糖體RNA的前端等序列)的相關引子組(http://biology.duke.edu/fungi/mycolab/primers.htm)來擴增其基因片段。PCR反應溶液如下：適量的基因體DNA溶液作為PCR模板，於含10 mM dNTP 8 µL、10X PCR緩衝液10 µL、10 pmole的5¢端引子與3¢端引子與
TaqDNA 聚合酶5U。PCR反應條件為96℃/30秒、50℃/30 秒、72℃/150 秒；補齊增殖段共1 循環72℃/10 分鐘；最後保持在4℃。取5 μL 產物進行電泳跑膠分析。 將PCR產物純化後以適當引子(http://biology.duke.edu/fungi/mycolab/primers.htm)進行定序，將定序結果以Vector NTI Suite 10軟體(VNTI)與NCBI/Blastn (http://www.ncbi.nlm.nih.gov/BLAST/)進行序列重組與序列相似性比對分析。另，分別將定序所得的C40序列經NCBI/Blastn後所得相近的藻株與數個藻種中心較接近的藻株及藻屬列為比較範圍，進行演化樹分析，以MEGA 6.0軟體做比對，接著利用最大概似法(Maximum Likelihood)以GTR+G+I的方式繪製演化樹，Bootstrap則為100次，分析之結果可作為分類地位上之鑑別參考。 5. 藻體分析 5.1 藻體含油量分析 C40以800 mL C培養基，培養於1L血清瓶中，並通入無菌空氣，於30℃照光培養一個月。收集藻體後將其冷凍乾燥成藻粉，秤取定量之藻粉，萃取其油脂。油脂萃取方法參考Folch等人的方法(Folch, J.
et al., A simple method for the isolation and purification of total lipids from animal tissue. The Journal of Biological Chemistry 1957; 23:497-509)並經修飾來進行，其過程為將30 mg冷凍乾燥的藻粉(A值)置入2 mL微量離心管，加入約2.0 mL氯仿/甲醇(v:v=2:1)與適量大顆玻璃珠，以撞擊式細胞破碎儀(Retsch® MM400)振盪約5分鐘，重複兩次。以10,000 rpm離心5分鐘後，取出上清液並將其加入拋棄式15 mL離心管中，隨即於2 mL微量離心管內加入約2.0 mL氯仿/甲醇(v:v=2:1)，再以超音波振盪與離心處理，取出上清液並將其加入另一拋棄式15 mL離心管中，重複上述萃取離心步驟直到萃取液無色為止。於裝有萃取液的15 mL離心管中加入等體積的145 mM NaCl溶液後，以試管旋轉混合器混和均勻後，經離心管在4,500 rpm下離心10分鐘。以玻璃吸管取下層液體到已秤重的玻璃瓶(B值)中。將此玻璃瓶內液體隔夜風乾再秤重(C值)，計算藻乾含油量的百分比(D值)。藻乾含油量計算公式:
5.2 脂肪酸圖譜分析 將抽取的藻油樣品以HPLC分析其油脂組成，HPLC分析條件：分離管柱為德國Merck公司製造之Silica gel (4.6 mm id × 250 mm, 5 μm particle size)；沖提溶劑A：hexane；沖提溶劑B：hexane/ethyl acetate/iso-propanol = 80：10：10 (v/v)，在0分鐘溶劑A/B =98：2 (v/v)，在8分鐘線性增加至溶劑A/B =50：50 (v/v)，在8.5分鐘線性增加至溶劑A/B =2：98 (v/v)，15分鐘維持相同梯度，20分鐘線性減少至溶劑A/B =98：2 (v/v)；流速：1.2 mL/min；蒸發光散射檢測器(ELSD；Evaporative Light Scattering Detector)條件；氣體流量2.6 L/min; 蒸發器溫度：40℃ (詹國靖, 黃啟紋, 范少怡, 朱燕華. 以甘油與植物油利用脂解酶之轉酯化反應生產 1, 3-雙醯甘油. 臺灣農業化學與食品科學 2010;48:19)。 5.3 脂肪酸圖譜分析方法 刮取適量乾燥藻體置於玻璃試管中，加入1 mL solution 1 (NaOH 45g, methanol 150 mL, ddH2O 150 mL)，震散藻體。於100℃加熱5分鐘，再將所有藻體震散，續加熱25分鐘。加入2 mL solution 2 (6N HCl 325 mL, methanol 200 mL)，於80℃加熱10 分鐘，完成後迅速冷卻。加入1.25 mL solution 3 (hexane 200 mL, tert-butyl methyl ether 200 mL)，緩慢混合10分鐘，以玻璃吸管尖吸取下層液體並丟棄。將上層液體加入3 mL solution 4 (NaOH 10.8 g, ddH
2O 900 mL)，混合5分鐘後，吸取上層液體以GC/MS (HP 5973 GC/MS System)分析其脂肪酸含量。GC/MS分析方法參考2007年Valencia, I.等人的方法(Valencia I, Ansorena D, Astiasarán I. Development of dry fermented sausages rich in docosahexaenoic acid with oil from the microalgae
Schizochytriumsp.: Influence on nutritional properties, sensorial quality and oxidation stability. Food Chemistry 2007;104:1087)，GC/Mass分析條件為：毛細管管柱：SP-2560, 75 m x 0.18 mm I.D., 0.14 μm。注入口温度：Inj, 250℃。離子源温度：FID, 250℃。管柱烘箱溫度：起始溫度140°C，保持5分鐘後以4℃/min 之昇温速率昇温至240℃，保持2分鐘。Carrier gas：He。Column流量：40 cm/sec@175℃。Injection: 1 μL。split ratio：1/100。脂肪酸標準品：37-Component FAME Mix (Cat. 18919-1AMP, Sigma-Aldrich)。設定好條件後，先分析標準品確認圖譜正確後再進行樣品分析。樣品層析數據利用質譜數據與標準品位置進行比對，以確認脂肪酸組成與比例。 6. 藻種培養特性分析 6.1 不同培養溫度測試 放入含20%二氧化碳之密封袋中，以不同溫度20℃、30℃與40℃進行照光培養，光照週期為12小時照光：12小時黑暗(12L:12D)。於培養第0小時、24小時及96小時觀察生長情形。 6.2 同鹽度培養基測試 以C培養基作為基礎，以此製作0％、1.5％及3％鹽度之培養基平板。藻種均勻塗佈於培養基平板上，放入含20%二氧化碳之密封袋中，以30℃進行照光培養，光照週期12L:12D。於培養第0小時、24小時及96小時觀察生長情形。 6.3 不同pH值培養基測試 以C培養液作為基礎，以此製作pH 3-10之培養基。因酸性固體培養基製作較困難，洋菜不易凝固，故使用液體方式進行培養。作法為添加酸液或鹼液並充分溶解後，以pH測定儀測量pH值，培養液以高溫高壓進行滅菌。將藻種均勻接種於各pH值之培養液中，做三重複。以30℃進行照光培養，於培養第0天、第4天及第19天觀察生長情形。 6.4 固碳效率測定 C40藻株於二氧化碳固碳篩選平台，以體積為1公升之C培養基(含氮量為原培養基之2倍：添加20 mg KNO
3)，在30℃全日照光條件下進行監測。固碳篩選平台以通氣量為0.1 vvm持續通入5%二氧化碳至潛力藻株藻液，再利用二氧化碳自動監測系統持續監測進流及出流之二氧化碳濃度（濃度單位為%，1% = 10,000 ppm），經由氣體濃度轉換公式之計算將二氧化碳濃度單位轉換為毫克/立方米，計算每日固定二氧化碳毫克數(Ya-jun H. Brief Discussion on conversion coeficient between the concentration units ppm and mg/m3 of nitrogen oxides (NO
X). Sichuan Environment 2010;1:006)： 二氧化碳氣體濃度轉換之計算公式：
分子量(二氧化碳)：44.01 t：測定溫度(30℃) 固碳效率計算公式：
R：每日固定毫克數(mg) V：微藻培養體積(m
3) Q：氣體通氣量(vvm，volume per volume per minute) T：每日通氣時間(min) C
in：二氧化碳進流濃度(mg/m
3) C
out：二氧化碳出流濃度(mg/m
3) 6.5 有機培養基培養測試 測試之有機培養基包括TSA、PDA及NA共三種。C40藻體均勻塗佈於有機培養基平板上，各有機培養基塗佈兩片，一片置於30°C光照培養箱，光照條件12L：12D。另一片置於30°C黑暗培養箱，於培養第0小時、第24小時及第96小時觀察生長情形。
實例一、藻株鑑定於台灣桃園有機稻田土壤與水樣樣品，分離純化得到藻株C40。以1,000X 顯微鏡觀察，此藻以單一藻體存在，细胞為圓球狀，直徑約為5~10 μm，經Nile Red染色後，以螢光顯微鏡觀察到藻體內部有大量明顯且呈現黃色之油滴分佈，顯示其藻體內可以蓄積油滴(圖1A及B)。 18S序列分析：分析C40藻株之18S rDNA序列(SEQ ID NO: 1)和NCBI的nr 資料庫比對後，發現與小球藻 (
Chlorella sorokiniana) UTEX 2714 (LK021940.1)及綠球藻(
Chlorella lewinii) CCAP 211/90 (FM205861.1)，覆蓋率為100％，相似度99％。以親源分析結果得知，C40 與
Chlorella lewiniiCCAP 211/90關係最近，結果如圖2A所示，但由於18S序列鑑別度不高，故再進行ITS序列比對。 ITS 序列分析：將C40 的ITS序列(SEQ ID NO: 2)和NCBI 的nr 資料庫比對後，發現與藻株
Chlorella lewiniiKU220 (KM061464.1)、
Chlorella lewiniiKU201 (KM061450.1)及
Chlorella lewiniiKU213 (KM061460.1)之ITS序列相近，故以KU220、KU201、KU213及上述CCAP 211/90之ITS序列與C40之ITS序列進行比對。以親源分析結果各藻種ITS序列得知，C40與
Chlorella lewiniiCCAP 211/90關係最近，結果如圖2B所示。利用VNTI AlignX軟體比對，結果發現比對覆蓋率100％時，相似度為分別為99.3%、99.1%、98.8%及98.8%，比對結果如圖3所示，分析發現C40 之ITS 序列與上述各藻株之序列至少有5個核酸序列位置不同。 綜合上述序列分析結果得知C40 應為
Chlorella lewinii，且與目前被發表之最相近藻種仍有差異。
Chlorella lewiniiC40已於2016年12月2日寄存於財團法人食品工業發展研究所，寄存編號為BCRC 980043。
實例二、 C40 之組成分析油脂分析 C40以800 mL C培養基，培養於1L血清瓶中，並通入無菌空氣，於30℃照光培養一個月。收集藻體後將其冷凍乾燥成藻粉，秤取定量之藻粉萃取其油脂，發現其含油量為40.2%的藻體乾重。分析其油脂成分，三酸甘油酯(TAG)含量為99.3% (表1)。脂肪酸組成則為C16:0佔22.3％、C18:1佔42.5％、C18:2佔20.3％、C18:3佔14.9％。飽和脂肪酸比例為22.3％，單元不飽和脂肪酸比例為42.5％，多元不飽和脂肪酸比例為35.2％。歐盟生質柴油標準之DU值經計算為112.9，優於標準值(表2)。又其C18:1為ω-9形式、C18:2為ω-6形式，C18:3為ω-3形式，皆是食用油脂中對人體較好的油脂，在此藻體中含量豐富，因此本藻種亦可作為食用油。根據上述結果顯示，C40之油脂可作為食用油及生質柴油。 表1
<TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td><b>油脂類別</b></td><td><b>油脂組成百分比</b><b>(%) (w/w)</b></td></tr><tr><td><b>TAG</b></td><td> 99.3 </td></tr><tr><td><b>FA</b></td><td> - </td></tr><tr><td><b>1,3-DAG</b></td><td> 0.4 </td></tr><tr><td><b>1,2-DAG</b></td><td> 0.4 </td></tr><tr><td><b>MAG</b></td><td> - </td></tr></TBODY></TABLE>註:
TAG: 三酸甘油酯(triacylglycerol)
FA: 脂肪酸(fatty acid)
1,3-DAG: 1,3-雙醯基甘油酯(1,3-diacylglycerol)
1,2-DAG: 1,2-雙醯基甘油酯(1,2-diacylglycerol)
MAG: 單醯基甘油酯 (monoacylglycerol) - : 低於檢測極限 表2
<TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td><b>脂肪酸類別</b></td><td><b>組合物百分比</b><b>(%) (w/w)</b></td></tr><tr><td><b>C16:0</b></td><td> 22.3 </td></tr><tr><td><b>C18:1</b></td><td> 42.5 </td></tr><tr><td><b>C18:2</b></td><td> 20.3 </td></tr><tr><td><b>C18:3</b></td><td> 14.9 </td></tr><tr><td><b>飽和</b></td><td> 22.3 </td></tr><tr><td><b>單元不飽和</b></td><td> 42.5 </td></tr><tr><td><b>多元不飽和</b></td><td> 35.2 </td></tr><tr><td><b>不</b><b>飽和度</b><b>(DU)</b></td><td> 112.9 </td></tr></TBODY></TABLE>- DU：不飽和度 (Degree of Unsaturation) = (單不飽和, w% + 2 (多不飽和, w%) (Ramos, M. J.,
et al., Influence of fatty acid composition of raw materials on biodiesel properties. Bioresource Technology2009; 100:261-268)
實例三、 C40 的培養特性分析1. 不同溫度培養測試 將藻體均勻接種於C 培養基中，各平板放入含20%二氧化碳之密封袋中，以不同溫度20℃、30℃與40℃進行照光培養。結果顯示C40 可生長在20-40℃，以30℃培養生長最好，20℃及40℃亦生長良好(圖4)。 2. 不同鹽度培養基測試 比較C40於不同鹽度培養基之生長狀況，結果顯示在0-3％鹽度之環境皆可生長(圖5)，以1.5％鹽度生長最好，故此藻種能夠以淡水與海水進行培養。 3. 不同pH值培養基測試 比較C40 於不同pH 值培養液中之生長狀況，結果顯示C40 在pH4-10之C培養液中均可生長(圖6)，以pH5-7之間生長最好。故此藻種可生長於pH4-10之範圍。 4. 有機培養基培養測試結果 C40以TSA、PDA及NA培養之結果分述如下： TSA 培養：混營條件下，於30°C 且給予12 小時光照12小時黑暗之光週期條件下，觀察C40呈現快速的生長，且約在24 小時後即可觀察到明顯的藻體生長狀態。異營條件下，於30°C 之黑暗條件下，於24小時後也可觀察到明顯的藻體生長狀態(圖7)。故C40以TSA培養時，可行混營與異營生長。 以PDA 培養：混營條件下，於30°C 且給予12 小時光照12 小時黑暗之光週期條件下，於24 小時及96小時觀察C40有生長。異營條件下，於30°C之黑暗條件下，於24小時及96小時觀察C40有生長(圖7)。故C40以PDA培養時，也可以混營與異營方式生長。 NA培養：混營條件下，於30°C且給予12小時光照12 小時黑暗之光週期條件下，於24 小時及96 小時觀察C40有生長。異營條件下，於30°C之黑暗條件下，於24小時及96小時觀察C40也有生長(圖7)。故C40以NA培養時，可以混營與異營方式生長。 由上述結果得知C40 在混營與異營條件下，分別以TSA、PDA 及NA 培養，皆可快速生長，其中以TSA 混營條件下生長最快。TSA 培養基中含有胰水解蛋白(Tryptone)及大豆水解蛋白(Soytone)兩種成分，C40可以利用此兩種氮源生長，故C40可能具有處理乳清或是大豆廢水，降低廢水中含氮量之潛力。 5. 藻株固碳效率測試 C40藻株以1公升之體積利用5%之二氧化碳氣體，以0.1vvm之通氣量培養10天後，共固定6.3克之二氧化碳生成微藻之藻體。利用第6~10天固碳穩定期進行評估，C40藻株之固碳效率為595.78 ± 37.79 mg/L/day (圖8)。 根據以上測試結果發現，C40 可生長在20-40℃，以30℃培養生長最好，20℃及40℃生長良好。又其可生長在0-3％鹽度之環境，以1.5％鹽度生長最好。又其可生長在pH4-10 之環境，以pH5-7 之間生長最好。C40 亦可行自營、混營與異營性生長。以C培養基通入空氣培養一個月後其油量佔微藻體乾重的40.2％，其脂肪酸組成適合作為生質柴油與食用油。又其固碳效率為595.78 ± 37.79 mg/L/d，可作為固碳之用。
結論由台灣分離一株產油微藻C40，經由鑑定為
Chlorella lewinii。本藻種可生長於溫度20-40°C、鹽度0-3％及pH4-10。又本藻株可行混營及異營培養。C40 藻株的培養條件測試結果得知，本藻株在高低溫、淡水海水、各種pH (pH4~10)及自營、異營與混營皆可生長，是一個在任何環境下皆可生長的優良藻株。此藻株的乾燥藻體之含油量在40.2%。油脂的脂肪酸組成以C16~C18的脂肪酸為主，佔總組成100％，其中C18:1 佔42.5％，可作為食用油脂。其固碳效率為595.78 ± 37.79 mg/L/d。由以上的結果顯示
Chlorella lewiniiC40藻株可以做為生產食用油脂及生質柴油的原料，也可做為二氧化碳及廢水減廢之用。
The present invention can be understood from the various aspects of the invention, the embodiments and the description of the embodiments disclosed herein. Unless otherwise defined herein, terms (including technical and scientific terms) used in connection with the present invention shall have the meaning as understood by those of ordinary skill in the art. And, as will be appreciated, unless the definitions provided herein are otherwise specified, in the case of any potential ambiguity, the definition of terms should be consistent with such commonly used terms (as defined in the dictionary). It is to be understood that the terminology used herein is for the purpose of describing the particular embodiments It must be noted that the singular forms "a", "an" and "the" are used in the <RTIgt; Therefore, unless the context requires otherwise, the singular terms shall include the plural, and the plural terms also include the singular. The scope of the present invention is expressed by "a particular value" and / or another "about" specific value. When the range is expressed in the above manner, it includes a range from a particular value and/or to another particular value. Similarly, when a value can be approximated by the term "about", it will be understood that it is another aspect of a particular value. It can be further appreciated that when referring to other endpoints and other endpoints themselves, the endpoints of each range are meaningful. According to the present invention, "about" can mean ± 20%, preferably ± 10%, more preferably ± 5%. In the present invention, the term "separated" or "isolated" means that the substance is removed from its original environment (or natural environment if it is naturally present). The term "isolated" or "isolated" does not necessarily mean that the material is purified. One object of the present invention is to provide a Chlorella lewinii isolated strain, wherein the Chlorella isolated strain comprises the 18S rDNA sequence of the nucleotide sequence shown in SEQ ID NO: 1 and the SEQ ID NO: 2 The sequence of the ITS region showing the nucleotide sequence. In a preferred embodiment of the present invention, the green algae isolated algae strain is an algae plant deposited in the Food Industry Development Research Institute of the Corporation and registered as BCRC 980043, or is deposited with the Food Industry Development Research Institute. The strain with the accession number BCRC 980043 has a variant with substantially identical identification characteristics. The term "variant strain" is intended to encompass any Chlorella strain in which all cytogenetic compositions have been altered by chemical mutation induction, spontaneous mutation, genetic engineering, transformation or transfection, such that they affect their physical or biochemical properties. However, the variant strain should have all the taxonomic identification features of the isolated strain of Chlorella isolated deposited in the Food Industry Development Institute of the consortium and registered under the number BCRC 980043. It is an object of the present invention to provide a method of preparing a culture product of Chlorella. In an embodiment of the present invention, the method comprises inoculating the chlorella isolated strain of the present invention into a culture medium, and culturing to obtain the culture product. In the present invention, the term "culture product" means that after the microalgae are cultured in a medium, a product rich in the microalgae cells is obtained. In the present invention, the microalgae cells in the culture product may not necessarily be separated from the culture medium, and the culture product may be in a liquid state, a solid state or a viscous state. In the present invention, the "medium" for culturing the chlorella isolated strain may be any medium that allows chlorella to grow, multiply and produce triglyceride and/or fatty acid, such as C medium [included per 100 mL) 15 mg Ca(NO 3 ) 2 •4H 2 O, 10 mg KNO 3 , 5 mg β-glycerophosphate disodium • 5H 2 O, 4 mg MgSO 4 •7H 2 O, 0.01 μg vitamin B12, 0.01 μg biotin ( Biotin), 1 μg Thiamine HCl, 0.3 mL PIV trace element solution (100 mg Na 2 EDTA•2H 2 O, 19.6 mg FeCl 3 •6H 2 O, 3.6 mg MnCl 2 •4H 2 per 100 mL) O, 1.04 mg ZnCl 2 , 0.4 μg CoCl 2 •6H 2 O, 0.25 μg Na 2 MoO 4 •2H 2 O and water), 50 mg Tris (hydroxymethyl) aminomethane and water], Tryptic Soy Broth ; TSA), Potato Dextrose Broth (PDA) and Nutrient Broth (NA). To prepare a solid medium for acacia, 1.5% (w/v) of acacia gum can be added to the liquid medium, and the solid medium of the agar can be obtained after sterilization and cooling. One skilled in the art can adapt the composition of the culture medium based on prior knowledge. In a preferred embodiment of the invention, the medium is a liquid medium. The condition for cultivating Chlorella sp. isolates in the present invention means conditions such as pH value, salinity, culture temperature, illumination, aeration conditions, and culture time of the medium, which allow the growth of the algae strain by the Chlorella Reproduction and manufacture of triglycerides and/or fatty acids. Those skilled in the art can adapt to the culture conditions based on prior knowledge. In the embodiment of the present invention, the chlorella isolated algae strain can carry out mixed growth or continuous irradiation under the light cycle of 12 hours of light and 12 hours of dark light under no light (ie, 24 hours darkness). Self-supporting growth under light. The amount of illumination may range from about 100 lux to about 4,000 lux, preferably about 2,000 lux. In embodiments of the invention, the culture temperature of the chlorella isolated strain may range from about 10 ° C to about 60 ° C (eg, about 10 ° C, about 15 ° C, about 20 ° C, about 25 ° C, about 30 ° C, about 35 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C or about 60 ° C), preferably about 20 ° C It is about 40 ° C, more preferably about 30 ° C. In an embodiment of the present invention, the pH of the medium for cultivating the chlorella isolated strain may be from about pH 1 to about pH 14 (eg, about pH 1, about pH 1.5, about pH 2, about pH 2.5, about pH 3). , about pH 3.5, about pH 4, about pH 4.5, about pH 5, about pH 5.5, about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8, about pH 8.5, about pH 9, about pH 9.5, about pH 10, about pH 10.5, about pH 11, about pH 11.5, about pH 12, about pH 12.5, about pH 13, about pH 13.5 or about pH 14), preferably about pH 4 to about pH 10, more Preferably, it is from about pH 5 to about pH 7. In the embodiment of the present invention, the salinity of the medium in which the chlorella isolated strain is cultured can be adjusted as needed. As used herein, "salinity" means the salt content dissolved in the medium. The culture medium may have a salinity of from 0% (w/w) to about 6% (w/w) (e.g., 0% (w/w), about 0.5% (w/w), about 1% (w/). w), about 1.5% (w/w), about 2% (w/w), about 2.5% (w/w), about 3% (w/w), about 3.5% (w/w), about 4 % (w/w), about 4.5% (w/w), about 5% (w/w), about 5.5% (w/w) or about 6.0% (w/w), preferably 0% ( w/w) to about 3% (w/w), more preferably 1.5% (w/w). In the method for preparing the culture product of the isolated strain of the genus Chlorella by the present invention, the step of isolating the culture product may be included as needed, and the separation step may be a conventional method step such as centrifugation and/or filtration. The present invention also provides a culture product obtained by the above method. The culture product of the present invention is rich in triglyceride and/or fatty acid, and thus can be used as a raw material for obtaining triglyceride and/or fatty acid, and is used for producing healthy fats and/or biofuels, respectively. As used herein, "triglyceride" means an ester compound having one glycerol molecule and three fatty acid molecules, wherein the three fatty acid molecules may have identical, partially identical or completely different carbon numbers and unsaturated bonds. . The "fatty acid" herein means a carboxylic acid compound having 8 to 30 carbon atoms and 0 to 6 unsaturated bonds, preferably a carboxylic acid compound having 12 to 20 carbon atoms and 0 to 5 unsaturated bonds. More preferably, it is a carboxylic acid compound having 16 to 18 carbon atoms and 0 to 3 unsaturated bonds. The triglyceride and the fatty acid can be obtained by any extraction and separation methods well known in the art, such as Folch, J. et al ., A simple method for the isolation and purification of total lipids from animal tissue. The Journal of Biological Chemistry 1957; 23: 497-509), Balasubramanian S. et al. , Oil extraction from Scenedesmus obliquus using a continuous microwave system – design, optimization, and quality characterization., Bioresource Technology, 2011; 102:3396-3403) and Sajilata et al. (Sajilata MG et al. , Supercritical CO 2 extraction of γ-linolenic acid (GLA) from Spirulina platensis ARM 740 using response surface methodology. Journal of Food Engineering 2008; 84: 321–326 The method described. Briefly, the method may comprise pulverizing the Chlorella cells in a manner such as grinding or ultrasonication, extracting the triglyceride and/or fatty acid in the Chlorella cells by a suitable solvent, and then borrowing Triglycerides and/or fatty acids are obtained by techniques such as HPLC and/or ion exchange resins. The chlorella culture product of the present invention can be chemically converted (such as hydrogenation, transesterification, hydrothermal carbonisation, fermentation or cleavage, etc.) or extracted into a solid, liquid or gaseous biomass fuel, including But not limited to) biodiesel, biomethanol, bioethanol, biobutanol, biomethane, biohydrogen or raw coal. The above biomass fuel can be obtained by any method known in the art, such as Tran DT et al. , Effect of solvents and oil content on direct transesterification of wet oil-bearing microalgal biomass of Chlorella vulgaris ESP- 31 for biodiesel synthesis using immobilized lipase as the biocatalyst. Bioresource Technology 2013; 135:213-221), Cheng HH, et al ., Biological butanol production from microalgae-based biodiesel residues by Clostridium acetobutylicum . Bioresource Technology 2015 184:379-385), Sambusiti C., et al. , Algae as promising feedstocks for fermentative biohydrogen production according to a biorefinery approach: A comprehensive review. Renewable & Sustainable Energy Reviews 2015; 44:20- 36) and Heilmann SM et al. (Heilmann SM et al. , Hydrothermal carbonization of microalgae. Biomass & Bioenergy 2010; 34(6): 875–882 and Heilmann SM et al. , Hydrothermal carbonization of microalgae II. Fatty acid, char, And algal nutrient pr Oducts. Applied Energy 2011; 88(10): 3286–3290). All publications, patents and patent documents mentioned herein are hereby incorporated by reference in their entirety. The following examples are provided to assist those skilled in the art in practicing the invention. </ RTI></RTI></RTI></RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt;</RTI><RTIgt; Changes are still within the scope of the invention. EXAMPLES Materials and Methods 1. Medium Formulation 1.1 C medium will be Ca(NO 3 ) 2 • 4H 2 O 15 mg, KNO 3 10 mg, β-glycerophosphate disodium • 5H 2 O 5 mg, MgSO 4 • 7H 2 O 4 mg, vitamin B12 0.01 μg, biotin (Biotin) 0.01 μg, thiamine (HCl) HCl 1 μg, PIV trace metal solution 0.3 mL and Tris 50 mg were mixed, and the volume was hydrated to 100 mL, and the pH was adjusted to 7.5. Autoclave. If it is 1.5% (w/v) acacia solid medium, it should be sterilized by adding 15 g of vegetable gum. The PIV trace metal solution was prepared by sequentially adding Na 2 EDTA•2H 2 O 100 mg, FeCl 3 •6H 2 O 19.6 mg, MnCl 2 •4H 2 O 3.6 mg, ZnCl 2 1.04 mg, CoCl 2 • 6H 2 O 0.4 Gg and Na 2 MoO 4 •2H 2 O 0.25 μg, and then conditioned to a volume of 100 mL and then autoclaved. When testing different salinity growth characteristics, the composition of the C medium was used, NaCl was additionally added to adjust the salinity, and the salinity was measured using a salinity meter to achieve the desired salinity. When testing different pH growth characteristics, use C medium composition, add HCl or NaOH to adjust the pH value, and use the acid-base meter to measure to achieve the desired pH. 1.2 TSB medium (Tryptic Soy Broth) Add 15.0 g of Tryptone, 5.0 g of Soytone and 5.0 g of NaCl water, then replenish the volume to 1 L, autoclaved after adjusting the pH to 7.3. 1.3 PDB medium (potato glucose broth; Potato Dextrose Broth) 200.0 g of diced potato and 20.0 g of glucose were sequentially added, and then the volume was hydrated to 1 L, and the pH was adjusted to 7.3, followed by autoclaving. 1.4 NB medium (nutrient medium; Nutrient Broth) 3.0 g of beef extract and 5.0 g of peptone were added in sequence, then the volume was hydrated to 1 L, adjusted to pH 7.0 and autoclaved. 2. Algae collection, separation and culture Take the water sample of the organic rice field in Taoyuan, Taiwan and mix it with the soil sample. Take about 10 mL and put it into a 50 mL centrifuge tube. Add about 30 mL of C medium and illuminate at 25 °C. . During the culture, the growth of the algae was observed by a microscope, and then an appropriate amount of the culture medium containing the algae was taken out, transferred to a plate medium, and cultured at 25 ° C. After the algae grows, a single algae species is taken and spread in the plate medium, and the above steps need to be repeated until a single algae is obtained by screening. For plate culture, a single algal bloom was applied to the C plate medium, and cultured at 25 ° C. 3. Oil staining analysis: 20 μL of the cultured algae was mixed with 1 μL of Nile Red (0.1 mg/mL in dimethyl sulfoxide) for oil droplet staining, and then allowed to stand at room temperature for 5 minutes after dyeing. Observation by fluorescence microscopy (Chen, W. et al. , A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. Journal of Microbiological Methods 2009; 77:41-47 and Huang, GH, et al ., Rapid screening method for lipid production in alga based on Nile red fluorescence. Biomass & Bioenergy 2009; 33:1386-1392). 4. Molecular identification of algae species 4.1 Genomic DNA extraction A suitable amount of freshly cultured algae is scraped from the plate medium and collected in a 2 mL microcentrifuge tube according to ZR Fungal/ of ZYMO RESEARCH. The Bacterial DNA MiniPrepTM kit instructions were used to obtain genomic DNA and the DNA concentration was measured using a NanoDrop (ND-1000 spectrophotometer). 4.2 PCR amplification, sequencing and pro-source analysis The algal DNA was used as a PCR template, with 18S rRNA and ITS region (including the back end of 18S ribosomal RNA, internal transcribed spacer 1, 5.8S ribose) A related primer set (http://biology.duke.edu/fungi/mycolab/primers.htm) of the bulk RNA, the internal transcribed spacer 2 and the front end of the 28S ribosomal RNA is used to amplify the gene fragment. The PCR reaction solution was as follows: an appropriate amount of the gene DNA solution was used as a PCR template in a 5 mM dNTP containing 10 mM dNTP, 10 μL of 10X PCR buffer, 10 pmole, and a 3 ¢ end primer and Taq DNA polymerase 5 U. The PCR reaction conditions were 96 ° C / 30 sec, 50 ° C / 30 sec, 72 ° C / 150 sec; the proliferative section was supplemented with 1 cycle of 72 ° C / 10 min; and finally maintained at 4 ° C. Take 5 μL of the product for electrophoresis. The PCR product was purified and sequenced with appropriate primers (http://biology.duke.edu/fungi/mycolab/primers.htm), and the sequencing results were made with Vector NTI Suite 10 software (VNTI) and NCBI/Blastn (http Sequence recombination and sequence similarity alignment analysis were performed at ://www.ncbi.nlm.nih.gov/BLAST/). In addition, the similarly obtained algae strains obtained by NCBI/Blastn and the algae and algae which are close to several algae species centers are listed as comparison ranges, and evolution tree analysis is carried out, respectively, and MEGA 6.0 software is used as a comparison. Then, using the Maximum Likelihood to map the evolution tree in GTR+G+I, Bootstrap is 100 times, and the analysis results can be used as a reference for classification status. 5. Analysis of algal body 5.1 Analysis of oil content of algae C40 was cultured in a 1 L serum bottle in 800 mL of C medium, and sterilized in a sterile air, and incubated at 30 ° C for one month. After collecting the algae, it is freeze-dried into algal flour, and the quantitative algae powder is weighed to extract the oil. The method of extracting fats and oils is described in the method of Folch et al . (Folch, J. et al ., A simple method for the isolation and purification of total lipids from animal tissue. The Journal of Biological Chemistry 1957; 23: 497-509) and modified. In the process, 30 mg of freeze-dried algal flour (A value) was placed in a 2 mL microcentrifuge tube, and about 2.0 mL of chloroform/methanol (v:v=2:1) was added to the appropriate amount of large glass beads to impact The cell disrupter (Retsch® MM400) was shaken for about 5 minutes and repeated twice. After centrifugation at 10,000 rpm for 5 minutes, the supernatant was removed and added to a disposable 15 mL centrifuge tube, and then about 2.0 mL of chloroform/methanol (v:v=2:1) was added to a 2 mL microcentrifuge tube. After ultrasonic vibration and centrifugation, the supernatant was taken out and added to another disposable 15 mL centrifuge tube, and the above extraction and centrifugation steps were repeated until the extract was colorless. An equal volume of 145 mM NaCl solution was added to a 15 mL centrifuge tube containing the extract, mixed well in a test tube rotary mixer, and centrifuged at 4,500 rpm for 10 minutes through a centrifuge tube. The lower layer of liquid was taken with a glass pipette into a weighed glass bottle (B value). The liquid in the glass bottle was air-dried overnight and weighed (C value), and the percentage (D value) of the oil content of the dried algae was calculated. Algae dry oil content calculation formula: 5.2 Analysis of Fatty Acids The extracted algal oil samples were analyzed for their oil composition by HPLC. HPLC analysis conditions: Separation column was Silica gel (4.6 mm id × 250 mm, 5 μm particle size) manufactured by Merck, Germany; solvent A was extracted. :hexane; solvent B: hexane/ethyl acetate/iso-propanol = 80:10:10 (v/v), solvent A/B = 98:2 (v/v) at 0 minutes, linear increase in 8 minutes To solvent A/B = 50:50 (v/v), linearly increase to solvent A/B = 2:98 (v/v) at 8.5 minutes, maintain the same gradient for 15 minutes, linearly decrease to solvent A/B for 20 minutes =98:2 (v/v); flow rate: 1.2 mL/min; evaporative light scattering detector (ELSD; Evaporative Light Scattering Detector); gas flow rate 2.6 L/min; evaporator temperature: 40 ° C (Zhan Guojing, Huang Qi Wen, Fan Shaoyi, Zhu Yanhua. Production of 1,3-biguanide glycerol by transesterification of glycerol with vegetable oil using lipolytic enzyme. Taiwan Agricultural Chemistry and Food Science 2010; 48:19). 5.3 Fatty acid analysis method Scrape an appropriate amount of dry algae in a glass test tube, and add 1 mL of solution 1 (NaOH 45g, methanol 150 mL, ddH2O 150 mL) to shake the algae. Heat at 100 ° C for 5 minutes, then shake all the algae and continue heating for 25 minutes. 2 mL of solution 2 (6N HCl 325 mL, methanol 200 mL) was added and heated at 80 ° C for 10 minutes, and then rapidly cooled. Add 1.25 mL of solution 3 (hexane 200 mL, tert-butyl methyl ether 200 mL), mix slowly for 10 minutes, pipette the lower layer with a glass pipette tip and discard. The upper liquid was added to 3 mL of solution 4 (NaOH 10.8 g, ddH 2 O 900 mL), and after mixing for 5 minutes, the supernatant liquid was aspirated and analyzed for its fatty acid content by GC/MS (HP 5973 GC/MS System). GC/MS analysis method refers to the method of Valencia, I. et al., 2007 (Valencia I, Ansorena D, Astiasarán I. Development of dry fermented sausages rich in docosahexaenoic acid with oil from the microalgae Schizochytrium sp.: Influence on nutritional properties, sensorial Quality and oxidation stability. Food Chemistry 2007; 104:1087), GC/Mass analysis conditions: capillary column: SP-2560, 75 mx 0.18 mm ID, 0.14 μm. Injection inlet temperature: Inj, 250 °C. Ion source temperature: FID, 250 °C. Column oven temperature: starting temperature 140 ° C, after 5 minutes, the temperature was raised to 240 ° C at a heating rate of 4 ° C / min for 2 minutes. Carrier gas: He. Column flow: 40 cm / sec @ 175 ° C. Injection: 1 μL. Split ratio: 1/100. Fatty acid standard: 37-Component FAME Mix (Cat. 18919-1AMP, Sigma-Aldrich). After setting the conditions, analyze the standard and confirm the map correctly before performing sample analysis. Sample chromatographic data were compared to the standard position using mass spectrometry data to confirm fatty acid composition and ratio. 6. Analysis of culture characteristics of algae 6.1 Different culture temperature tests were placed in a sealed bag containing 20% carbon dioxide, and cultured at different temperatures of 20 ° C, 30 ° C and 40 ° C. The photoperiod was 12 hours of illumination: 12 hours of darkness (12L) :12D). Growth conditions were observed at 0, 24, and 96 hours of culture. 6.2 Same Saline Medium Test The medium plate was prepared on the basis of C medium to prepare 0%, 1.5% and 3% salinity. The algae were evenly spread on the medium plate, placed in a sealed bag containing 20% carbon dioxide, and incubated at 30 ° C for 12 n: 12 D. Growth conditions were observed at 0, 24, and 96 hours of culture. 6.3 Different pH media test The medium of pH 3-10 was prepared by using C medium as a basis. Because of the difficulty in making acidic solid medium, the amaranth is not easy to coagulate, so it is cultured by liquid method. After adding an acid solution or an alkali solution and sufficiently dissolving, the pH value is measured by a pH meter, and the culture solution is sterilized at a high temperature and a high pressure. The algae species were uniformly inoculated into the culture solution of each pH value, and three repetitions were made. Illumination was carried out at 30 ° C, and growth was observed on days 0, 4 and 19 of the culture. 6.4 Determination of carbon sequestration efficiency C40 algae strain in a carbon dioxide carbon sequestration screening platform, with a volume of 1 liter of C medium (nitrogen content is twice the original medium: adding 20 mg KNO 3 ), under 30 ° C full day light conditions monitor. The carbon sequestration platform continuously feeds 5% carbon dioxide to the potential algae algae solution with a ventilation of 0.1 vvm, and continuously monitors the influent and outflow carbon dioxide concentration using the carbon dioxide automatic monitoring system (concentration unit is %, 1% = 10,000 ppm) ), convert the carbon dioxide concentration unit to milligrams per cubic meter by calculation of the gas concentration conversion formula, and calculate the daily fixed carbon dioxide milligrams (Ya-jun H. Brief Discussion on conversion coeficient between the concentration units ppm and mg/m3 of nitrogen oxides (NO X ). Sichuan Environment 2010;1:006): Calculation formula for carbon dioxide gas concentration conversion: Molecular weight (carbon dioxide): 44.01 t: measured temperature (30 ° C) Formula for calculating carbon sequestration efficiency: R: mg fixed daily (mg) V: microalgae culture volume (m 3 ) Q: gas per volume per minute (vvm, volume per volume per minute) T: daily ventilation time (min) C in : carbon dioxide influent concentration (mg/m 3 ) C out : carbon dioxide outflow concentration (mg/m 3 ) 6.5 Organic medium culture test The test organic medium includes three kinds of TSA, PDA and NA. The C40 algal body was uniformly coated on an organic medium plate, and two pieces were coated on each organic medium, and one piece was placed in a 30 ° C light incubator, and the light condition was 12 L: 12D. The other piece was placed in a dark incubator at 30 ° C, and growth was observed at 0 hours, 24 hours, and 96 hours of the culture. Example 1. The strain of algae was identified in the soil and water samples of organic rice fields in Taoyuan, Taiwan, and the algae strain C40 was isolated and purified. Observed by a 1,000X microscope, the algae existed as a single algae, and the cells were spherical, with a diameter of about 5-10 μm. After staining with Nile Red, a large number of obvious and yellow colored inside the algae were observed by a fluorescence microscope. Oil droplet distribution shows that oil droplets can accumulate in the algae (Figures 1A and B). 18S sequence analysis: analysis of the 18S rDNA sequence of the C40 strain (SEQ ID NO: 1) and the NCBI nr database were compared with Chlorella sorokiniana UTEX 2714 (LK021940.1) and Chlorella ( Chlorella lewinii ) CCAP 211/90 (FM205861.1), coverage is 100%, similarity is 99%. According to the results of the pro-source analysis, the relationship between C40 and Chlorella lewinii CCAP 211/90 was the closest, and the results are shown in Fig. 2A. However, since the discrimination of the 18S sequence is not high, the ITS sequence alignment is performed. ITS sequence analysis: The alignment of C40's ITS sequence (SEQ ID NO: 2) with NCBI's nr database revealed that it was associated with the strains Chlorella lewinii KU220 (KM061464.1), Chlorella lewinii KU201 (KM061450.1) and Chlorella lewinii. The ITS sequence of KU213 (KM061460.1) is similar, so the ITS sequences of KU220, KU201, KU213 and CCAP 211/90 described above are compared with the ITS sequence of C40. The relationship between C40 and Chlorella lewinii CCAP 211/90 was obtained by the ITS sequence of each algae. The results are shown in Fig. 2B. Using the VNTI AlignX software alignment, the similarity was found to be 99.3%, 99.1%, 98.8%, and 98.8%, respectively. The results of the comparison are shown in Figure 3. The analysis found that the ITS sequence of C40 was The sequence of each of the above algal strains differs in at least five nucleic acid sequence positions. Based on the above sequence analysis results, it was found that C40 should be Chlorella lewinii , and it is still different from the most similar algae species currently published. Chlorella lewinii C40 was deposited with the Food Industry Development Institute on December 2, 2016 under the registration number BCRC 980043. Example 2 : Analysis of the composition of C40 Oil and fat analysis C40 was cultured in a 1 L serum bottle with 800 mL of C medium, and passed through sterile air, and cultured at 30 ° C for one month. After collecting the algae, it was freeze-dried into algal flour, and the amount of algae powder was extracted to extract the oil, and the dry weight of the algae was found to be 40.2%. The oil component was analyzed and the triglyceride (TAG) content was 99.3% (Table 1). The fatty acid composition was 22.3% for C16:0, 42.5% for C18:1, 20.3% for C18:2, and 14.9% for C18:3. The proportion of saturated fatty acids was 22.3%, the proportion of monounsaturated fatty acids was 42.5%, and the proportion of polyunsaturated fatty acids was 35.2%. The DU value of the EU biodiesel standard is calculated to be 112.9, which is better than the standard value (Table 2). Further, C18:1 is in the form of ω-9, C18:2 is in the form of ω-6, and C18:3 is in the form of ω-3, which is a good oil for human body in edible oils and fats, and is rich in the algae. This algae species can also be used as an edible oil. According to the above results, the oil of C40 can be used as edible oil and biodiesel. Table 1 <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td><b>Fatcategory</b></td><td><b> Percentage of oil composition</b><b>(%) (w/w)</b></td></tr><tr><td><b>TAG</b></td><td > 99.3 </td></tr><tr><td><b>FA</b></td><td> - </td></tr><tr><td><b>1 ,3-DAG</b></td><td> 0.4 </td></tr><tr><td><b>1,2-DAG</b></td><td> 0.4 </td></tr><tr><td><b>MAG</b></td><td> - </td></tr></TBODY></TABLE> Note: TAG : Triacylglycerol FA : fatty acid 1,3-DAG : 1,3-diacylglycerol 1,2-DAG : 1,2-biguanylglycerol Ester (1,2-diacylglycerol) MAG : monoacylglycerol - : Below detection limit Table 2 <TABLE border="1"borderColor="#000000"width="85%"><TBODY><Tr><td><b>Fatty Acid Category</b></td><td><b>Percentage of Composition</b><b>(%) (w/w)</b></td></tr><tr><td><b>C16:0</b></td><td> 22.3 </td></tr><tr><td><b>C18:1</ b></td><td> 42.5 </td></tr><tr><td><b>C18:2</b></td><td> 20.3 </td></tr><tr><td><b>C18:3</b></td><td> 14.9 </td></tr><tr><td><b>Saturation</b></td><td> 22.3 </td ></tr><tr><td><b>unsaturated</b></td><td> 42.5 </td></tr><tr><td><b>polyunsaturated</b></td><td> 35.2 </td></tr><tr><td><b>No</b><b>Saturation</b><b>(DU)</ b></td><td> 112.9 </td></tr></TBODY></TABLE>- DU: Degree of Unsaturation = (unsaturated, w% + 2 (more) Saturated, w%) (Ramos, MJ, et al ., Influence of fatty acid composition of raw materials on biodiesel properties. Bioresource Technology 2009; 100:261-268) Example 3. Analysis of C40 culture characteristics 1. Different temperature culture tests will The algae were uniformly inoculated into C medium, and each plate was placed in a sealed bag containing 20% carbon dioxide, and illuminated at different temperatures of 20 ° C, 30 ° C and 40 ° C. The results showed that C40 can grow at 20-40 ° C, grow best at 30 ° C, and grow well at 20 ° C and 40 ° C (Figure 4). 2. Different salinity media tests compare the growth of C40 in different salinity media, and the results show that it can grow in the environment of 0-3% salinity (Figure 5), and grows best with 1.5% salinity, so the algae can Cultured in fresh water and sea water. 3. Different pH media test to compare the growth of C40 in different pH cultures, the results show that C40 can grow in C 4-10 C culture solution (Figure 6), the best growth between pH5-7. Therefore, the algae species can grow in the range of pH 4-10. 4. Organic medium culture test results The results of C40 culture with TSA, PDA and NA are as follows: TSA culture: under mixed conditions, at 40 ° C and given 12 hours light for 12 hours under dark light cycle conditions, observe C40 presentation Rapid growth, and obvious algal growth status can be observed after about 24 hours. Under different conditions, significant algal growth was observed after 24 hours in the dark at 30 °C (Fig. 7). Therefore, when C40 is cultured in TSA, it is feasible to mix and grow. Cultured with PDA: C40 growth was observed at 24 hours and 96 hours under mixed conditions at 30 ° C and 12 hours light for 12 hours. Under different conditions, C40 was observed to grow at 24 hours and 96 hours in the dark at 30 °C (Fig. 7). Therefore, when C40 is cultured with PDA, it can also be mixed and grown in a different way. NA culture: Under mixed conditions, C40 growth was observed at 24 hours and 96 hours under the conditions of a light cycle of 12 hours of light and 12 hours of light. Under different conditions, C40 was also observed to grow at 24 hours and 96 hours in the dark at 30 °C (Fig. 7). Therefore, when C40 is cultured in NA, it can be mixed and grown in a different way. It can be seen from the above results that C40 can grow rapidly under the conditions of mixed and heterogeneous conditions, TSA, PDA and NA, respectively, and the growth is fastest under TSA mixed conditions. TSA medium contains Tryptone and Soytone, and C40 can be grown by using these two nitrogen sources. Therefore, C40 may have whey or soybean wastewater to reduce the nitrogen content in wastewater. potential. 5. Algae plant carbon sequestration efficiency test C40 algae strain was incubated with 5% carbon dioxide gas in a volume of 1 liter, and after culturing for 10 days with aeration of 0.1 vvm, a total of 6.3 g of carbon dioxide was immobilized to form algae of microalgae. The carbon sequestration efficiency of the C40 strain was 595.78 ± 37.79 mg/L/day using the solid carbon stabilization period from day 6 to day 10 (Fig. 8). According to the above test results, C40 can grow at 20-40 ° C, grow best at 30 ° C, and grow well at 20 ° C and 40 ° C. It can grow in an environment of 0-3% salinity and grows best at 1.5% salinity. It can also grow in the environment of pH 4-10, and grows best between pH 5-7. C40 can also be self-operated, mixed camp and multi-growth. After C medium was introduced into the air for one month, the oil content accounted for 40.2% of the dry weight of the microalgae. The fatty acid composition was suitable as biodiesel and edible oil. It also has a carbon fixation efficiency of 595.78 ± 37.79 mg / L / d, which can be used for carbon fixation. Conclusion An oil-producing microalgae C40 was isolated from Taiwan and identified as Chlorella lewinii . The algae species can be grown at a temperature of 20-40 ° C, a salinity of 0-3%, and a pH of 4-10. The algae strain can be mixed and cultivated in different camps. The culture condition test results of the C40 algae strain showed that the algae strain can grow in high and low temperature, fresh water seawater, various pH (pH 4~10) and self-operated, different camps and mixed camps, and can grow in any environment. Excellent algae strain. The dry algae of this algae strain had an oil content of 40.2%. The fatty acid composition of fats and oils is mainly C16~C18 fatty acids, accounting for 100% of the total composition, of which C18:1 accounts for 42.5%, which can be used as edible fats and oils. Its carbon sequestration efficiency is 595.78 ± 37.79 mg / L / d. The above results show that the Chlorella lewinii C40 strain can be used as a raw material for the production of edible oils and biodiesel, and can also be used for carbon dioxide and wastewater waste reduction.