雨生红球藻培养和产油脂工艺的优化
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摘要
为解决生物柴油生产中油脂短缺的问题,利用实验室筛选到的高产油脂藻株——雨生红球藻SCTCC-600012,进行培养条件优化和产油脂工艺研究,分别比较不同浓度的NaNO3、K2HPO4、α-萘乙酸(NAA)、3-吲哚丁酸(3-IBA)、Mg2+、葡萄糖对雨生红球藻SCTCC-600012生长及产油的影响.结果显示,其最适生长条件分别为NaNO3 1.0 g/L,K2HPO4 1.0 g/L,NAA 7 mg/L,3-IBA 16 mg/L,Mg2+2.5 mg/L.最适产油条件分别为NaNO3 0.125 g/L,K2HPO4 0.1 g/L,NAA 7 mg/L,3-IBA 1.0 mg/L,Mg2+2.5 mg/L,葡萄糖为1.0 g/L.优化后的油脂含量为32.8%,比基础发酵培养基中的油脂含量(19.59%)提高了1倍左右.利用GC-MS对油脂进行脂肪酸成分分析,发现主要成分为软脂酸和油酸,与植物油相似,适合用于生产生物柴油.本研究通过优化培养条件和产油工艺,提高了雨生红球藻的生长速率和油脂含量,可为选择生物柴油原料提供参考.
This study aimed to solve the problem of oil shortage during the production of biodiesel, and to explore its culture technology. A microalgae strain named Haematococcus pluvialis SCTCC-600012 was screened from our laboratory due to its high oil production rate. We investigated the effects of different concentration of NaN03, K2HPO4, NAA, 3-IBA, Mg2+ and glucose on the biomass and oil content of Haematococcus pluvialis. The optimum condition for growth was NaNO3 1.0 g/L, K2HPO4 1 g/L, NAA 7 mg/L, 3-IBA 16 mg/L, Mg2+ 2.5 mg/L, and glucose 1.0 g/L, while the optimum condition for oil production was NaNO3 0.125 g/L, K2HPO4 0.1 g/L, NAA 7 mg/L, 3-IBA 1 mg/L, Mg2+ 2.5 mg/L, and glucose 1.0 g/L. The oil content after optimization was 32.8%, nearly double that in the normal fermentation medium. In addition, through the fatty acid composition analysis by GC-MS, we found that the fatty acid composition of the lipid was palmitic acid and oleic acid, similar to that of plant oil, suitable for biodiesel production. This experiment improved the growth rate and oil content of Haematococcus pluvialis. To some extent, it can ease the problem of oil shortage and provide reference for new material selection for biodiesel production.
This study aimed to solve the problem of oil shortage during the production of biodiesel, and to explore its culture technology. A microalgae strain named Haematococcus pluvialis SCTCC-600012 was screened from our laboratory due to its high oil production rate. We investigated the effects of different concentration of NaN03, K2HPO4, NAA, 3-IBA, Mg2+ and glucose on the biomass and oil content of Haematococcus pluvialis. The optimum condition for growth was NaNO3 1.0 g/L, K2HPO4 1 g/L, NAA 7 mg/L, 3-IBA 16 mg/L, Mg2+ 2.5 mg/L, and glucose 1.0 g/L, while the optimum condition for oil production was NaNO3 0.125 g/L, K2HPO4 0.1 g/L, NAA 7 mg/L, 3-IBA 1 mg/L, Mg2+ 2.5 mg/L, and glucose 1.0 g/L. The oil content after optimization was 32.8%, nearly double that in the normal fermentation medium. In addition, through the fatty acid composition analysis by GC-MS, we found that the fatty acid composition of the lipid was palmitic acid and oleic acid, similar to that of plant oil, suitable for biodiesel production. This experiment improved the growth rate and oil content of Haematococcus pluvialis. To some extent, it can ease the problem of oil shortage and provide reference for new material selection for biodiesel production.
引文
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5 李涛.营养对雨生红球藻Haematococcus pluvialis CG-11生长的影响及培养基优化研究[D].广州:暨南大学,2009[Li T.Studies on the effects of nutrition on the growth of Hematocoecus pluvialis CG-11 and its medium optimization[D].Guangzhou:Jinan University,2009]
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7 曹健,汪晨辉,曾实,赵文杰,白爱英.卷枝毛霉Mucorcircinelloides3 .2208油脂几种提取方法的比较[J].中国油脂,2004,29(4):38-40[Cao J,Wang CH,Zeng S,Zhao WJ,Bai AY.Comparison of several methods for extracting oil from Mucor circinelloides 3.2208[J].China Oils&Fats,2004,29(4):38-40]
8 Cohen Z,Norman HA.Potential use of substituted pyridazinones for selecting polyunsaturated fatty acid over-producing cell lines of algae[J].Phytochemistry,1993,32:259-264
9 孙博,侯冬岩,回瑞华.螺旋藻中脂肪酸的GC-MS分析[J].鞍山师范学院学报,2010,12(4):36-38[Sun B,Hou DY,Hui RH.Analysis on fatty acids in Spirulina by gas chromaogrphy-mass spectrometry[J].J Anshan Norm Univ(鞍山师范学院学报),2010,12(4):36-38
10 石娟,潘克厚.不同培养条件对微藻总脂含量和脂肪酸组成的影响[J].海洋水产研究,2004,25(6):79-85[Shi J,Pan KH.Effects of different culture conditions and growth phases on lipid of microalgae[J].Mar Fish Res,2004,25(6):79-85]
11 杨佳.高油脂微藻的筛选及其在再生水中的产油特性研究[D].北京:清华大学,2009[Yang J.Isolation of high-lipid microalgae and study on the properties of lipid production in reclaimed water[D].Beiing:Tsinghua University,2009]
12 Feng DN,Chen ZA,Xue S,Zhang W.Increased lipid production of the marine oleaginous microalgae Isochrysis zhangjiagensis(Chrysophyta)by nitrogen supplement[J].Bioresour Technol,2011,102:6710-6716
13 Rodolfi L,Zittelli GC,Bassi N,Padovani G,Biondi N,Bonini G,Tredici MR.Microalgae for oil:Strain selection,induction of lipid synthesis and outdoor mass cultivation in a low-cost photo-bioreactor[J].Biotechnol&Bioengin,2008,10(2):1002-1023
14 张桂艳,温小斌,梁芳,欧阳峥嵘,耿亚红,梅洪,李夜光.重要理化因子对小球藻生长和油脂产量的影响[J].生态学报,2011,31(8):2076-2085[Zhang GY,Wen XB,Liang F,Ou YZR,Geng YH,Mei H,Li YG.The effects of physical and chemical factors on the growth and lipid production of Chlorella[J].Acta Ecol Sin,2011,31(8):2076-2085]
15 Zhao YD.The role of local biosynthesis of auxin and cytokinin in plant development[J].Plant Biol,2008,11:16-22
16 侯和胜,吴超元.藻类中植物激素的研究进展[C]//中国科学院海洋研究所.海洋科学集刊:第40集.北京:科学出版社,1998.167-176[Hou HS,Wu CY.Advances in research on plant hormones in algae[C]//Institute Oceanology,Chinese Academy of Sciences.Studia Marina Sinica:40.Beijing:Science Press,1998.167-176]
17 牟晓真,范晓,韩丽君.藻类中植物生长素的研究[C]//中国科学院海洋研究所.海洋科学集刊:第42集.北京:科学出版社,2000.65-73 [Mou XZ,Fan X,Han LJ.Reviews of research on auxins algae[C]//Institute Oceanology,Chinese Academy of Sciences.Studia Marina Sinica:42.Beijing:Science Press,2000.65-73]
18 曾正兵,郭金耀,杨晓玲.NAA对盐藻生长与物质积累的影响[J].江苏农业科学,2008,1:182-184[Zeng ZB,Guo JY,Yang XL.Effect of NAA on growth and material accumulation of Dunaliella salina[J].Jiangsu Agric Sci,2008,1:182-184]
19 韩春梅,刘建国,张勇.不同激素配伍对雨生红球藻(Haematococcus pluvialis)细胞生长和虾青素累积的调节作用、藻株差异及应用[J].海洋与湖沼,2009,40(4):430-436[Han CM,Liu JG,Zhang Y.Cell growth and astaxanthin accumulation of four strains of Heematococcus pluvialis exposed to different plant regulators[J].Oceanol Limnol Sin(海洋与湖沼),2009,40(4):430-436]
20 崔宝霞,钟方旭.植物生长调节剂对雨生红球藻细胞增殖及虾青素积累的影响[J].水产科学,2008,27(9):478-482[Cui BX,Zhong FX.Effects of plant growth regulators on growth and astsxanthin content in green alga Haemataccus pluvialis[J].Aquat Sci,2008,27(9):478-482]
21 刘世名,梁世中.植物激素在小球藻异养培养中的作用[J].植物学通报,1999,16(6):696-700[Liu SM,Liang SZ.Roles of phytohormones in the growth of Chlorella vulgaris in heterotrophic culture[J].Chin Bull Bot,1999,16(6):696-700]
22 李方芳.微藻固定CO2生产生物柴油的研究[D].武汉:武汉科技大学,2012[Li FF.Production of biodiesel coupling with co2 fi xation by microalgae[D].Wuhan:Wuhan University of Science and Technology,2012]
23 荆家海.植物生理学[M].西安:陕西科技出社,1994[Jing JH.Plant physiology[M].Xi’an:Shaanxi Science and Technology Press,1994]
24 王镜岩,朱圣庚,徐长法.生物化学[M].3版.北京:高等教育出版社,2002[Wang JY,Zhu SG,Xu CF.Biochemistry[M].3rd ed.Beijing:Higher Education Press,2002]
25 刘建康.高级水生生物学[M].北京:科学技术出版社,1999[Liu JK.Advanced aquatic biology[M].Beijing:Science and Technology Press,1999]
26 孙茜.有机碳源和植物生长调节剂对铜绿微囊藻生长及产毒的影响[D].武汉:华中师范大学,2006[Sun Q.The effect of organic carbon source and plant growth regulator on growth and microcystin production of Microcysis aeruginosa[D].Wuhan:Central China Normal University,2006]
27 贺国强,邓志平,陶丽,陈三凤.高油脂产率微藻的筛选及发酵条件的优化[J].农业生物技术学报,2010,18(6):1046-1053[He GQ,Deng ZP,Tao L,Chen SF.Screen and fermentation optimization of microalgaes with high lipid productivity[J].J Agric Biotechnol,2010,18(6):1046-1053]
28 卢丽娜,孙利芹,田焕玲,任蒙,王长海.32株海洋微藻总脂含量及其脂肪酸组成的研究[J].中国油脂,2009,34(11):68-73[Lu LN,Sun LQ,Tian HL,Ren M,Wang CH.Total lipid content and fatty acid composition of 32 strains of marine microalgae[J].China Oils Fat,2009,34(11):68-73]
29 李涛,李爱芬,桑敏,吴洪.富油能源微藻的筛选及产油性能评价[J].中国生物工程杂志,2011,31(4):98-105[Li T,Li AF,Sang M,Wu H.Screening oleaginous microalgae and evaluation of the oil-producing characteristic[J].China Biotechnol,2011,31(4):98-105]
30 Liliana R,Graziella CZ,Niccolo B,Giulia P,Natascia B,Gimena B,Mario RT.Microalgae for oil:strain selection,Induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor[J].Biotechnology,2009,102(1):100-112
31 杜晓凤.微绿球藻藻油提取工艺及富油培养条件的优化[D].烟台:鲁东大学,2012[Du XF.Optimization of the lipid extraction method and oil-rich culture conditions of Nannochloropsis sp.[D].Yantai:Ludong University,2012]
32 冀颐之,刘杨平,黄迎春.梅尼小环藻产油培养工艺的优化[J].安徽农业科学,2012,40(11):6360-6362[Ji YZ,Liu YP,Huang YC.The optimization of culture conditions on lipid produced by Cyclotella meneghiniana[J].J Anhui Agric Sci,2012,40(11):6360-6362]
2 Gouveia L,Oliveira AC.Microalgae as a raw material for biofuels production[J].J Ind Microbiol Biotechnol,2009,36:269-274
3 庄惠如,陈必链,王明兹,卢海声,施巧琴.雨生红球藻混合营养与异养培养研究[J].微生物学通报,2000,27(3):198-201[Zhuang HR,Chen BL,Wang MZ,Lu HS,Shi QQ.Mixotrophic and heterotrophic growth of Heematococcus pluvialis[J].Acta Microbiol Sin,2000,27(3):198-201]
4 刘建国,殷明众,张京浦,孟昭才,Bourne WF.雨生红球藻的细胞周期初探[J].海洋与湖沼,2000,31(2):145-150[Liu JG,Yin MZ,Zhang JP,Meng ZC,W F Bourne.Studies of cell cycle in Hematocoecus pluvialis[J].Oceanol Limnol Sin,2000,31(2):145-150]
5 李涛.营养对雨生红球藻Haematococcus pluvialis CG-11生长的影响及培养基优化研究[D].广州:暨南大学,2009[Li T.Studies on the effects of nutrition on the growth of Hematocoecus pluvialis CG-11 and its medium optimization[D].Guangzhou:Jinan University,2009]
6 Hu Q,Sommerfeld M,Jarvis E,Ghirardi M,Posewitz M,Seibert M,Darzins A.Microalgal triacylglycerols as feedstocks for biofuel production Perspectives and advances[J].Plant J,2008,54:621-639
7 曹健,汪晨辉,曾实,赵文杰,白爱英.卷枝毛霉Mucorcircinelloides3 .2208油脂几种提取方法的比较[J].中国油脂,2004,29(4):38-40[Cao J,Wang CH,Zeng S,Zhao WJ,Bai AY.Comparison of several methods for extracting oil from Mucor circinelloides 3.2208[J].China Oils&Fats,2004,29(4):38-40]
8 Cohen Z,Norman HA.Potential use of substituted pyridazinones for selecting polyunsaturated fatty acid over-producing cell lines of algae[J].Phytochemistry,1993,32:259-264
9 孙博,侯冬岩,回瑞华.螺旋藻中脂肪酸的GC-MS分析[J].鞍山师范学院学报,2010,12(4):36-38[Sun B,Hou DY,Hui RH.Analysis on fatty acids in Spirulina by gas chromaogrphy-mass spectrometry[J].J Anshan Norm Univ(鞍山师范学院学报),2010,12(4):36-38
10 石娟,潘克厚.不同培养条件对微藻总脂含量和脂肪酸组成的影响[J].海洋水产研究,2004,25(6):79-85[Shi J,Pan KH.Effects of different culture conditions and growth phases on lipid of microalgae[J].Mar Fish Res,2004,25(6):79-85]
11 杨佳.高油脂微藻的筛选及其在再生水中的产油特性研究[D].北京:清华大学,2009[Yang J.Isolation of high-lipid microalgae and study on the properties of lipid production in reclaimed water[D].Beiing:Tsinghua University,2009]
12 Feng DN,Chen ZA,Xue S,Zhang W.Increased lipid production of the marine oleaginous microalgae Isochrysis zhangjiagensis(Chrysophyta)by nitrogen supplement[J].Bioresour Technol,2011,102:6710-6716
13 Rodolfi L,Zittelli GC,Bassi N,Padovani G,Biondi N,Bonini G,Tredici MR.Microalgae for oil:Strain selection,induction of lipid synthesis and outdoor mass cultivation in a low-cost photo-bioreactor[J].Biotechnol&Bioengin,2008,10(2):1002-1023
14 张桂艳,温小斌,梁芳,欧阳峥嵘,耿亚红,梅洪,李夜光.重要理化因子对小球藻生长和油脂产量的影响[J].生态学报,2011,31(8):2076-2085[Zhang GY,Wen XB,Liang F,Ou YZR,Geng YH,Mei H,Li YG.The effects of physical and chemical factors on the growth and lipid production of Chlorella[J].Acta Ecol Sin,2011,31(8):2076-2085]
15 Zhao YD.The role of local biosynthesis of auxin and cytokinin in plant development[J].Plant Biol,2008,11:16-22
16 侯和胜,吴超元.藻类中植物激素的研究进展[C]//中国科学院海洋研究所.海洋科学集刊:第40集.北京:科学出版社,1998.167-176[Hou HS,Wu CY.Advances in research on plant hormones in algae[C]//Institute Oceanology,Chinese Academy of Sciences.Studia Marina Sinica:40.Beijing:Science Press,1998.167-176]
17 牟晓真,范晓,韩丽君.藻类中植物生长素的研究[C]//中国科学院海洋研究所.海洋科学集刊:第42集.北京:科学出版社,2000.65-73 [Mou XZ,Fan X,Han LJ.Reviews of research on auxins algae[C]//Institute Oceanology,Chinese Academy of Sciences.Studia Marina Sinica:42.Beijing:Science Press,2000.65-73]
18 曾正兵,郭金耀,杨晓玲.NAA对盐藻生长与物质积累的影响[J].江苏农业科学,2008,1:182-184[Zeng ZB,Guo JY,Yang XL.Effect of NAA on growth and material accumulation of Dunaliella salina[J].Jiangsu Agric Sci,2008,1:182-184]
19 韩春梅,刘建国,张勇.不同激素配伍对雨生红球藻(Haematococcus pluvialis)细胞生长和虾青素累积的调节作用、藻株差异及应用[J].海洋与湖沼,2009,40(4):430-436[Han CM,Liu JG,Zhang Y.Cell growth and astaxanthin accumulation of four strains of Heematococcus pluvialis exposed to different plant regulators[J].Oceanol Limnol Sin(海洋与湖沼),2009,40(4):430-436]
20 崔宝霞,钟方旭.植物生长调节剂对雨生红球藻细胞增殖及虾青素积累的影响[J].水产科学,2008,27(9):478-482[Cui BX,Zhong FX.Effects of plant growth regulators on growth and astsxanthin content in green alga Haemataccus pluvialis[J].Aquat Sci,2008,27(9):478-482]
21 刘世名,梁世中.植物激素在小球藻异养培养中的作用[J].植物学通报,1999,16(6):696-700[Liu SM,Liang SZ.Roles of phytohormones in the growth of Chlorella vulgaris in heterotrophic culture[J].Chin Bull Bot,1999,16(6):696-700]
22 李方芳.微藻固定CO2生产生物柴油的研究[D].武汉:武汉科技大学,2012[Li FF.Production of biodiesel coupling with co2 fi xation by microalgae[D].Wuhan:Wuhan University of Science and Technology,2012]
23 荆家海.植物生理学[M].西安:陕西科技出社,1994[Jing JH.Plant physiology[M].Xi’an:Shaanxi Science and Technology Press,1994]
24 王镜岩,朱圣庚,徐长法.生物化学[M].3版.北京:高等教育出版社,2002[Wang JY,Zhu SG,Xu CF.Biochemistry[M].3rd ed.Beijing:Higher Education Press,2002]
25 刘建康.高级水生生物学[M].北京:科学技术出版社,1999[Liu JK.Advanced aquatic biology[M].Beijing:Science and Technology Press,1999]
26 孙茜.有机碳源和植物生长调节剂对铜绿微囊藻生长及产毒的影响[D].武汉:华中师范大学,2006[Sun Q.The effect of organic carbon source and plant growth regulator on growth and microcystin production of Microcysis aeruginosa[D].Wuhan:Central China Normal University,2006]
27 贺国强,邓志平,陶丽,陈三凤.高油脂产率微藻的筛选及发酵条件的优化[J].农业生物技术学报,2010,18(6):1046-1053[He GQ,Deng ZP,Tao L,Chen SF.Screen and fermentation optimization of microalgaes with high lipid productivity[J].J Agric Biotechnol,2010,18(6):1046-1053]
28 卢丽娜,孙利芹,田焕玲,任蒙,王长海.32株海洋微藻总脂含量及其脂肪酸组成的研究[J].中国油脂,2009,34(11):68-73[Lu LN,Sun LQ,Tian HL,Ren M,Wang CH.Total lipid content and fatty acid composition of 32 strains of marine microalgae[J].China Oils Fat,2009,34(11):68-73]
29 李涛,李爱芬,桑敏,吴洪.富油能源微藻的筛选及产油性能评价[J].中国生物工程杂志,2011,31(4):98-105[Li T,Li AF,Sang M,Wu H.Screening oleaginous microalgae and evaluation of the oil-producing characteristic[J].China Biotechnol,2011,31(4):98-105]
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