湛江等鞭金藻培养基和培养模式优化的研究
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摘要
湛江等鞭金藻是中国的经济藻类,含有多不饱和脂肪酸,是具有大规模生产多不饱和脂肪酸潜力的藻种。为了找到适合湛江等鞭金藻扩大培养的条件,本研究针对培养基中的氮源及其浓度、氮磷比设计一个三因素五水平的正交实验L_(25)(5~6),找到最适氮源及其浓度为尿素75 mg/L,最适氮磷比为15:1。另外设计光照强度不同的3组实验,以探讨最适光照强度,实验结果表明湛江等鞭金藻在光照强度为4 028 Lx时有较大的生长速率。通过结合半连续培养模式和分批补料培养模式的优点,自主设计半连续分批补料培养模式,结果表明此培养模式在促进湛江等鞭金藻生长过程中表现出明显的优势。光生物反应器的培养结果说明优化条件适用于湛江等鞭金藻的扩大培养,希望能对湛江等鞭金藻的工业化生产提供一个有利的理论基础。
Isochrysis galbana, an economic microalgae in China, contains polyunsaturated fatty acids. It is a kind of algae with the potential of large-scale production of polyunsaturated fatty acids. In order to find suitable conditions for the expansion of Isochrysis galbana, this study designed three-factor and five-level orthogonal tests L25(56) for the nitrogen source, its concentration and nitrogen/phosphorus ratio of the medium. The results showed that the optimum nitrogen source was urea and its concentration was 75 g/L, the optimum nitrogen/phosphorus ratio was 15: 1. In addition, three groups of experiments with different illumination intensity were designed to explore the optimum illumination intensity. The results showed that there was higher growth rate when the light intensity is 4 028 Lx. By combining the advantages of semi-continuous cultivation mode and batch feeding cultivation mode, we independently designed a semi-continuous batch feeding model. The results showed that the cultivation model showed obvious advantages in promoting the growth of Isochrysis galbana. The results of the photobioreactor showed that the optimization conditions applied to expand culture of Isochrysis galbana. And it was hoped that it would provide a favorable theoretical basis for the industrial production of Isochrysis galbana.
Isochrysis galbana, an economic microalgae in China, contains polyunsaturated fatty acids. It is a kind of algae with the potential of large-scale production of polyunsaturated fatty acids. In order to find suitable conditions for the expansion of Isochrysis galbana, this study designed three-factor and five-level orthogonal tests L25(56) for the nitrogen source, its concentration and nitrogen/phosphorus ratio of the medium. The results showed that the optimum nitrogen source was urea and its concentration was 75 g/L, the optimum nitrogen/phosphorus ratio was 15: 1. In addition, three groups of experiments with different illumination intensity were designed to explore the optimum illumination intensity. The results showed that there was higher growth rate when the light intensity is 4 028 Lx. By combining the advantages of semi-continuous cultivation mode and batch feeding cultivation mode, we independently designed a semi-continuous batch feeding model. The results showed that the cultivation model showed obvious advantages in promoting the growth of Isochrysis galbana. The results of the photobioreactor showed that the optimization conditions applied to expand culture of Isochrysis galbana. And it was hoped that it would provide a favorable theoretical basis for the industrial production of Isochrysis galbana.
引文
Chang Y.Q.,Song J.,Wang G.D.,Ding J.,Yan S.G.,and Wang X.F.,2002,Seeds breeding of hard clam Mercenaria mercenaria,Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),9(1):43-47(常亚青,宋坚,王国栋,丁君,阎世国,王喜福,2002,硬壳蛤的人工育苗技术,中国水产科学,9(1):43-47)
Chen Y.M.,Shi Y.,Wang M.Z.,and Chen B.L.,2015,The proceeding on exploiting and utilization of feed microalgae in mariculture,Shengwu Jishu Tongbao(Biotechnology Bulletin),31(9):60-65(陈艳梅,石阳,王明兹,陈必链,2015,海产养殖饵料微藻开发利用进展,生物技术通报,31(9):60-65)
Chisti Y.,2007,Biodiesel from microalgae,Biotechnol.Adv.,25(3):294-306
Das U.N.,2002,Estrogen,statins,and polyunsaturated fatty acids:similarities in their actions and benefits-is there a common link?Nutrition,18(2):178-188
Duplus E.,and Forest C.,2002,Is there a single mechanism for fatty acid regulation of gene transcription?Biochemical Pharmacology,64(5-6):893-901
Ferreira M.,Seixas P.,Coutinho P.,Fábregas J.,and Otero A.,2011,Effect of the nutritional status of semi-continuous microalgal cultures on the productivity and biochemical composition of Brachionus plicatilis,Marine Biotechnology,13(6):1074-1085
Gim G.H.,Ryu J.,Kim M.J.,Kim P.II,and Kim S.W.,2016,Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions,Journal of Industrial Microbiology&Biotechnology,43(5):605-616
Huang Y.M.,Yang X.H.,Lan L.B.,and Zhan J.T.,Luo H.,Jiang L.M.,2017,Studies on the diversity of marine microalgae and the strains with high polysaccharides,lipids and proteins along Zhanjiang coastal areas,Shuisheng Shengwu Xuebao(Acta.Hydrobiologica Sinica.),41(5):1080-1090(黄永梅,杨晓红,兰柳波,詹静婷,罗辉,江黎明,2017,湛江沿海海洋微藻及其多糖、脂类和蛋白藻株多样性研究,水生生物学报,41(5):1080-1090)
Jiang H.M.,Zhang F.Z.,Gu H.Y.,Zhai J.,and Gao K.S.,2004,Effects of nitrogen sources on the growth and fatty acid composition of Isochrysis galbana(Prymnesiophyceae),Shipin Yu Fajiao Gongye(Food and Fermentation Industries),30(10):5-10(蒋汉明,张凤珍,顾洪雁,翟静,高坤山,2004,氮源对等鞭金藻生长和脂肪酸组成的影响,食品与发酵工业,30(10):5-10)
Jiang L.L.,Zhang Y.J.,Li X.P.,and Guo B.R.,2010,Research developments in cultivation modes for microalgae,Kezaisheng Nengyuan(Renewable Energy Resources),28(1):56-62(蒋礼玲,张亚杰,李潇萍,郭荣波,2010,微藻培养模式研究进展,可再生能源,28(1):56-62)
Liang Y.,Mai K.S.,Sun S.C.,Pan J.H.,and Zhou H.C.,1999,Effects of different nutrient concentrations on the growth of Phaeodactylum tricornutum,Haiyang Huzhao Tongbao(Transactions of Oceanology and Limnology),4:43-47(梁英,麦康森,孙世春,潘敬洪,周华财,1999,不同的营养盐浓度对三角褐指藻生长的影响,海洋湖沼通报,4:43-47)
Liu D.Y.,Sun J.,Gong J.,and Qian S.B.,2002,The effects of different N/P ratios on the growth of Isochrysis galbana,Haiyang Shuichan Yanjiu(Marine Fisheries Research),23(1):29-32(刘东艳,孙军,巩晶,钱树本,2002,不同氮、磷比例对球等鞭金藻生长的影响,海洋水产研究,23(1):29-32)
Liu J.,Sommerfeld M.,and Hu Q.,2013,Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production,Applied Microbiology and Biotechnology,97(11):4785-4798
Marta C.,Picardoa M.C.,de Medeirosa J.L.,de Araújoa O.D.Q.F.,and Chaloub R.M.,2013,Effects of CO2 enrichment and nutrients supply intermittency on batch cultures of Isochrysis galbana,Bioresource Technology,143:242-250
Milledge J.J.,2011,Commercial application of microalgae other than as biofuels:a brief review,Reviews in Environmental Science and Bio/Technology,10(1):31-41
Palakurthi S.S.,Fluckiger R.,Aktas H.,Changolkar A.K.,Shahsafaei A.,Harneit S.,Kilic E.,and Halperin J.A.,2000,Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid,Cancer Res.,60(11):2919-2925
Richmond A.,ed.,Huang H.,Gao Z.,and Song P.,trans.,2014,Handbook of microalgal culture biotechnology and applied phycology,Science Press,Beijing,China,pp.30-123(里士曼A.,主编,黄和,高振,宋萍,主译,2014,微藻培养指南:生物技术与应用藻类学,科学出版社,中国,北京,pp.30-123)
Sun Y.Y.,2007,Effect of environmental factors on growth of Isochrysis galbana,Dessertation for Ph.D.,Dalian University of Technology,Supervior:Wang C.H,pp.15-35(孙颖颖,2007,环境因子对球等鞭金藻生长的影响,博士学位论文,大连理工大学,导师:王长海,pp.15-35)
Wang H.T.,Liu Y.N.,Cao X.P.,Xue S.,and Wang W.L.,2014,Nile red fluorescence method for rapid measurement of neutral lipids during the cultivation of the marine microalgae Isochrysis zhangjiangensis,Shengwu Jiagong Guocheng(Chinese Journal of Bioprocess Engineering),12(6):78-83(王海涛,刘亚男,曹旭鹏,薛松,王伟良,2014,尼罗红荧光法快速检测培养过程中湛江等鞭金藻的中性脂,生物加工过程,12(6):78-83)
Wang J.,Cui L.J.,Lan L.B.,and Jiang L.M.,2015,Diversity of culturable extracellular proteases producing marine fungi isolated from the intertidal zone of Naozhou island in south China sea,Weishengwuxue Tongbao(Microbiology China),42(2):238-253(王洁,崔丽娇,兰柳波,江黎明,2015,中国南海硇洲岛潮间带产胞外蛋白酶的可培养海洋真菌多样性(英文),微生物学通报,42(2):238-253)
Wang M.Q.,Chi X.Y.,Qin S.,Yang G.P.,Jiang P.,and Pang S.J.,2008,Total lipids and fatty acid composition of marine microalgae,Zhongguo Youzhi(China Oils and Fats),(11):67-70(王明清,迟晓元,秦松,杨官品,姜鹏,逄少军,2008,海洋微藻总脂含量和脂肪酸组成的测定,中国油脂,(11):67-70)
Wang X.Y.,and Huang X.X.,2016,Effect of different carbon sources on growth and biochemical composition of Isochrysis galbana,Shandong Nongye Daxue Xuebao(Ziran Kexue ban)(Journal of Shandong Agricultural University(Natural Science Edition)),47(4):506-513(王星宇,黄旭雄,2016,不同碳源对球等鞭金藻生长和细胞组成的影响,山东农业大学学报(自然科学版),47(4):506-513)
Wu D.Y.,Zou N.,Chang L.,and Cong S.,2010,The culture research progress and application prospects of Isochrysis galbana parke,Keji Xinxi(Science and Technology Information),(33):28-29(吴电云,邹宁,常林,丛山,2010,球等鞭金藻(Isochrysis galbana parke)的培养研究进展及应用前景,科技信息,(33):28-29)
Yang X.H.,Huang Y.M.,Lan L.B.,Zhan J.T.,Jiang L.M.,and Luo H.,2016,The biodiversity of culturable lipid-rich microalgae from Xuwen coral reef nature reserve,Shengtai Kexue(Ecological Science),35(4):52-61(杨晓红,黄永梅,兰柳波,詹静婷,江黎明,罗辉,2016,徐闻珊瑚礁自然保护区可培养富脂海洋微藻的生物多样性,生态科学,35(4):52-61)
Yehuda S.,Rabinovitz S.,Carasso R.L.,and Mostofsky D.I.,2002,The role of polyunsaturated fatty acids in restoring the aging neuronal membrane,Neurobiology of Aging,23(5):843
Zhang C.W.,and Richmond A.,2003,Sustainable,high-yielding outdoor mass cultures of Chaetoceros muelleri var.subsalsum and Isochrysis galbana in vertical plate reactors,Marine Biotechnology,5(3):302-310
Zhu Y.F.,and Guo X.Q.,2000,Effects of different nitrogen,phosphorus and silicon concentrations and inoculation density on the growth of Phaeodactylum tricornutum,Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),7(4):47-51(朱艺峰,郭小强,2000,不同氮磷硅含量和接种密度对三角褐指藻生长的影响,中国水产科学,7(4):47-51)
Chen Y.M.,Shi Y.,Wang M.Z.,and Chen B.L.,2015,The proceeding on exploiting and utilization of feed microalgae in mariculture,Shengwu Jishu Tongbao(Biotechnology Bulletin),31(9):60-65(陈艳梅,石阳,王明兹,陈必链,2015,海产养殖饵料微藻开发利用进展,生物技术通报,31(9):60-65)
Chisti Y.,2007,Biodiesel from microalgae,Biotechnol.Adv.,25(3):294-306
Das U.N.,2002,Estrogen,statins,and polyunsaturated fatty acids:similarities in their actions and benefits-is there a common link?Nutrition,18(2):178-188
Duplus E.,and Forest C.,2002,Is there a single mechanism for fatty acid regulation of gene transcription?Biochemical Pharmacology,64(5-6):893-901
Ferreira M.,Seixas P.,Coutinho P.,Fábregas J.,and Otero A.,2011,Effect of the nutritional status of semi-continuous microalgal cultures on the productivity and biochemical composition of Brachionus plicatilis,Marine Biotechnology,13(6):1074-1085
Gim G.H.,Ryu J.,Kim M.J.,Kim P.II,and Kim S.W.,2016,Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions,Journal of Industrial Microbiology&Biotechnology,43(5):605-616
Huang Y.M.,Yang X.H.,Lan L.B.,and Zhan J.T.,Luo H.,Jiang L.M.,2017,Studies on the diversity of marine microalgae and the strains with high polysaccharides,lipids and proteins along Zhanjiang coastal areas,Shuisheng Shengwu Xuebao(Acta.Hydrobiologica Sinica.),41(5):1080-1090(黄永梅,杨晓红,兰柳波,詹静婷,罗辉,江黎明,2017,湛江沿海海洋微藻及其多糖、脂类和蛋白藻株多样性研究,水生生物学报,41(5):1080-1090)
Jiang H.M.,Zhang F.Z.,Gu H.Y.,Zhai J.,and Gao K.S.,2004,Effects of nitrogen sources on the growth and fatty acid composition of Isochrysis galbana(Prymnesiophyceae),Shipin Yu Fajiao Gongye(Food and Fermentation Industries),30(10):5-10(蒋汉明,张凤珍,顾洪雁,翟静,高坤山,2004,氮源对等鞭金藻生长和脂肪酸组成的影响,食品与发酵工业,30(10):5-10)
Jiang L.L.,Zhang Y.J.,Li X.P.,and Guo B.R.,2010,Research developments in cultivation modes for microalgae,Kezaisheng Nengyuan(Renewable Energy Resources),28(1):56-62(蒋礼玲,张亚杰,李潇萍,郭荣波,2010,微藻培养模式研究进展,可再生能源,28(1):56-62)
Liang Y.,Mai K.S.,Sun S.C.,Pan J.H.,and Zhou H.C.,1999,Effects of different nutrient concentrations on the growth of Phaeodactylum tricornutum,Haiyang Huzhao Tongbao(Transactions of Oceanology and Limnology),4:43-47(梁英,麦康森,孙世春,潘敬洪,周华财,1999,不同的营养盐浓度对三角褐指藻生长的影响,海洋湖沼通报,4:43-47)
Liu D.Y.,Sun J.,Gong J.,and Qian S.B.,2002,The effects of different N/P ratios on the growth of Isochrysis galbana,Haiyang Shuichan Yanjiu(Marine Fisheries Research),23(1):29-32(刘东艳,孙军,巩晶,钱树本,2002,不同氮、磷比例对球等鞭金藻生长的影响,海洋水产研究,23(1):29-32)
Liu J.,Sommerfeld M.,and Hu Q.,2013,Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production,Applied Microbiology and Biotechnology,97(11):4785-4798
Marta C.,Picardoa M.C.,de Medeirosa J.L.,de Araújoa O.D.Q.F.,and Chaloub R.M.,2013,Effects of CO2 enrichment and nutrients supply intermittency on batch cultures of Isochrysis galbana,Bioresource Technology,143:242-250
Milledge J.J.,2011,Commercial application of microalgae other than as biofuels:a brief review,Reviews in Environmental Science and Bio/Technology,10(1):31-41
Palakurthi S.S.,Fluckiger R.,Aktas H.,Changolkar A.K.,Shahsafaei A.,Harneit S.,Kilic E.,and Halperin J.A.,2000,Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid,Cancer Res.,60(11):2919-2925
Richmond A.,ed.,Huang H.,Gao Z.,and Song P.,trans.,2014,Handbook of microalgal culture biotechnology and applied phycology,Science Press,Beijing,China,pp.30-123(里士曼A.,主编,黄和,高振,宋萍,主译,2014,微藻培养指南:生物技术与应用藻类学,科学出版社,中国,北京,pp.30-123)
Sun Y.Y.,2007,Effect of environmental factors on growth of Isochrysis galbana,Dessertation for Ph.D.,Dalian University of Technology,Supervior:Wang C.H,pp.15-35(孙颖颖,2007,环境因子对球等鞭金藻生长的影响,博士学位论文,大连理工大学,导师:王长海,pp.15-35)
Wang H.T.,Liu Y.N.,Cao X.P.,Xue S.,and Wang W.L.,2014,Nile red fluorescence method for rapid measurement of neutral lipids during the cultivation of the marine microalgae Isochrysis zhangjiangensis,Shengwu Jiagong Guocheng(Chinese Journal of Bioprocess Engineering),12(6):78-83(王海涛,刘亚男,曹旭鹏,薛松,王伟良,2014,尼罗红荧光法快速检测培养过程中湛江等鞭金藻的中性脂,生物加工过程,12(6):78-83)
Wang J.,Cui L.J.,Lan L.B.,and Jiang L.M.,2015,Diversity of culturable extracellular proteases producing marine fungi isolated from the intertidal zone of Naozhou island in south China sea,Weishengwuxue Tongbao(Microbiology China),42(2):238-253(王洁,崔丽娇,兰柳波,江黎明,2015,中国南海硇洲岛潮间带产胞外蛋白酶的可培养海洋真菌多样性(英文),微生物学通报,42(2):238-253)
Wang M.Q.,Chi X.Y.,Qin S.,Yang G.P.,Jiang P.,and Pang S.J.,2008,Total lipids and fatty acid composition of marine microalgae,Zhongguo Youzhi(China Oils and Fats),(11):67-70(王明清,迟晓元,秦松,杨官品,姜鹏,逄少军,2008,海洋微藻总脂含量和脂肪酸组成的测定,中国油脂,(11):67-70)
Wang X.Y.,and Huang X.X.,2016,Effect of different carbon sources on growth and biochemical composition of Isochrysis galbana,Shandong Nongye Daxue Xuebao(Ziran Kexue ban)(Journal of Shandong Agricultural University(Natural Science Edition)),47(4):506-513(王星宇,黄旭雄,2016,不同碳源对球等鞭金藻生长和细胞组成的影响,山东农业大学学报(自然科学版),47(4):506-513)
Wu D.Y.,Zou N.,Chang L.,and Cong S.,2010,The culture research progress and application prospects of Isochrysis galbana parke,Keji Xinxi(Science and Technology Information),(33):28-29(吴电云,邹宁,常林,丛山,2010,球等鞭金藻(Isochrysis galbana parke)的培养研究进展及应用前景,科技信息,(33):28-29)
Yang X.H.,Huang Y.M.,Lan L.B.,Zhan J.T.,Jiang L.M.,and Luo H.,2016,The biodiversity of culturable lipid-rich microalgae from Xuwen coral reef nature reserve,Shengtai Kexue(Ecological Science),35(4):52-61(杨晓红,黄永梅,兰柳波,詹静婷,江黎明,罗辉,2016,徐闻珊瑚礁自然保护区可培养富脂海洋微藻的生物多样性,生态科学,35(4):52-61)
Yehuda S.,Rabinovitz S.,Carasso R.L.,and Mostofsky D.I.,2002,The role of polyunsaturated fatty acids in restoring the aging neuronal membrane,Neurobiology of Aging,23(5):843
Zhang C.W.,and Richmond A.,2003,Sustainable,high-yielding outdoor mass cultures of Chaetoceros muelleri var.subsalsum and Isochrysis galbana in vertical plate reactors,Marine Biotechnology,5(3):302-310
Zhu Y.F.,and Guo X.Q.,2000,Effects of different nitrogen,phosphorus and silicon concentrations and inoculation density on the growth of Phaeodactylum tricornutum,Zhongguo Shuichan Kexue(Journal of Fishery Sciences of China),7(4):47-51(朱艺峰,郭小强,2000,不同氮磷硅含量和接种密度对三角褐指藻生长的影响,中国水产科学,7(4):47-51)