极端嗜盐绿色杜氏藻混合培养技术的研究
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摘要
极端嗜盐绿色杜氏藻(D.viridis)是一种单细胞海洋微藻,具有较为独特的生物特性,对温度、盐度的适应能力很强,尤其是嗜盐性在生物界中是最强的。极端嗜盐绿色杜氏藻中含有多种营养成分和生物活性物质,具有很好的开发和应用前景,对其进行大规模培养意义重大。
以往,对极端嗜盐绿色杜氏藻大都采用光能自养培养的方式,培养效果不大好。为提高效率,本论文提出通过添加不同的有机盐—葡萄糖、蛋白胨、卤虫干粉及发酵液对其进行混合营养培养的方法,并对培养过程中的藻形、藻的生物量等方面进行考察与分析。
实验分两阶段进行:首先往18oBé盐度下的培养液中加入不同浓度的营养盐对绿色杜氏藻进行培养,以找出该盐度下营养盐的最适范围,再以此浓度为参考值,改变培养液的盐度对藻进行培养,从而找出有利于极端嗜盐绿色杜氏藻生长的最佳条件。
通过以上系统的研究工作,不仅验证了极端嗜盐绿色杜氏藻的混合培养的设想和可能性,更为极端嗜盐绿色杜氏藻的混合培养提供了较为系统的基础数据。混合培养能大大提高藻的生物量使藻提前进入生长指数期,缩短培养时间,增加产量,大幅降低产品成本,增强绿色杜氏藻的国际市场竞争力,从而会取得可观的经济效益和社会效益。
The extreme halotolerant green alge Dunaliella viridis (D. viridis) is a kind of unicellular ocean algae,and it has special biological characteristic and stronger adaptable ability to temperature and salinity.Especially its characteristic of tolerance to salinity is the strongest among all other biology. Because it contains manifold nutrimental components and biological active substances, it has good foreground of exploiture and appliance and the large-scale culture of D. viridis is of great importance.
In the past, extreme halotolerant green alge Dunaliella viridis (D. viridis) was only cultured by the phototrophic way ,but the result wasn't very good.In order to raise efficiency, this thesis put forward a method by which we increased different organic nutriments—glucose,peptone , ferment liquid and power of Artemia to D. viridis to carry on mixotrophic culture and during the period of culture we could investigate and analyse the form and biomass of D. viridis.
The experiment had two stages:first we put different organic nutriments to culture medium of D.viridis under 18oBésalinity to find out the most suitable concentration of nutriment,second we took former concentration as a reference , then changed salinity of culture medium to culture D.viridis and find the best condition .
The above study not only proved the assumption and possibility but also provided the basic date of mixotrophic culturing extreme halotolerant D.viridis. The mixotrophic culture of extreme halotolerant D.viridis improved the biomass of algae greatly and brought forwards the exponential period of algae,consequently it shortened the time,increased the production, decreased the manufacturing cost,strengthened our product competition in the international market and obtained considerable economic and social benefits.
以往,对极端嗜盐绿色杜氏藻大都采用光能自养培养的方式,培养效果不大好。为提高效率,本论文提出通过添加不同的有机盐—葡萄糖、蛋白胨、卤虫干粉及发酵液对其进行混合营养培养的方法,并对培养过程中的藻形、藻的生物量等方面进行考察与分析。
实验分两阶段进行:首先往18oBé盐度下的培养液中加入不同浓度的营养盐对绿色杜氏藻进行培养,以找出该盐度下营养盐的最适范围,再以此浓度为参考值,改变培养液的盐度对藻进行培养,从而找出有利于极端嗜盐绿色杜氏藻生长的最佳条件。
通过以上系统的研究工作,不仅验证了极端嗜盐绿色杜氏藻的混合培养的设想和可能性,更为极端嗜盐绿色杜氏藻的混合培养提供了较为系统的基础数据。混合培养能大大提高藻的生物量使藻提前进入生长指数期,缩短培养时间,增加产量,大幅降低产品成本,增强绿色杜氏藻的国际市场竞争力,从而会取得可观的经济效益和社会效益。
The extreme halotolerant green alge Dunaliella viridis (D. viridis) is a kind of unicellular ocean algae,and it has special biological characteristic and stronger adaptable ability to temperature and salinity.Especially its characteristic of tolerance to salinity is the strongest among all other biology. Because it contains manifold nutrimental components and biological active substances, it has good foreground of exploiture and appliance and the large-scale culture of D. viridis is of great importance.
In the past, extreme halotolerant green alge Dunaliella viridis (D. viridis) was only cultured by the phototrophic way ,but the result wasn't very good.In order to raise efficiency, this thesis put forward a method by which we increased different organic nutriments—glucose,peptone , ferment liquid and power of Artemia to D. viridis to carry on mixotrophic culture and during the period of culture we could investigate and analyse the form and biomass of D. viridis.
The experiment had two stages:first we put different organic nutriments to culture medium of D.viridis under 18oBésalinity to find out the most suitable concentration of nutriment,second we took former concentration as a reference , then changed salinity of culture medium to culture D.viridis and find the best condition .
The above study not only proved the assumption and possibility but also provided the basic date of mixotrophic culturing extreme halotolerant D.viridis. The mixotrophic culture of extreme halotolerant D.viridis improved the biomass of algae greatly and brought forwards the exponential period of algae,consequently it shortened the time,increased the production, decreased the manufacturing cost,strengthened our product competition in the international market and obtained considerable economic and social benefits.
引文
[1] T. P. Moulton,M. A. Burford,The mass culture of Dunaliella viridis (Volvocales, Chlorophyta) for oxygenated carotenoids : laboratory and pilot plant studies,Hydrobiology,1990,204-205(1):401-408
[2] 张福,马若欣,石良,彭小雪,极端嗜盐绿色杜氏藻培养技术的研究[J],海湖盐与化工,2004,33(2):13-16
[3] МАСЮК Н П. Морфология, систематика, экология географическое распространение Рода Dunaliella Teod[M]. Издательство Киев: Науково думка, 1973
[4] Mordhay Avron & Ami Ben –Amotz . Dunaliella:Physiology,Biochemistry,and biotechnology[M].United Statesof America:CRC Press,inc,1992.2-10
[5] LEWIN,Ralph A,盐田生物技术国际研讨会论文集[C],天津:轻工业部制盐工业科学研究所出版,1991
[6] Jiménez C,Berl T,Rivard CJ,Edelstein CL,Phosphorylation of MAP kinase-like proteins mediate the response of the halotolerant alga Dunaliella viridis to hypertonic shock, Biochimica et biophysica acta,2004,1644(1):61-90
[7] Borowitzka L J . The microflora , adaptations to life in extremely saline lakes[J ]. Hydrobiologia , 1981 , 81(1)
[8] 游兰英,刘广发,林建明,杜氏藻同步化生长及核裂分析[J],厦门大学学报(自然科学版),1997,36(2):276-281
[9] 吕爱玲,张福,马若欣,姜润林,中国渤海湾滩涂极端嗜盐绿色杜氏藻生物学特性研究(二)[J], 海湖盐与化工,2005,34(3):19-23
[10] Brown A.D.,Microbial water stress,Bacteriological Reviews,1976,40:803-846
[11] Pollard A. and Wyn Jones R.G.,Enzyme activities in concentrated solutions of glycine, betaine and other solutes,Planta ,1979,144:291-298
[12] Carlos Jiménez , F. Xavier Niell , Growth ofDunaliella viridis Teodoresco: effect of salinity, temperature and nitrogen concentration,Journal of Applied Phycology,1991,3(4):319-327
[13] Francisco J. L. Gordillo, Carlos Jiménez, Félix L. Figueroa, F. Xavier Niell,Influence of elevated CO2 and nitrogen supply on the carbon assimilation performance and cell composition of the unicellular alga Dunaliella viridis,Physiologia Plantarum,2003,119(4):513-518
[14] Gordillo F.J.L., Jiménez C., Chavarría J., Xavier Niell F,Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis,Photosynthesis Research,2001,68(3):225-235
[15] Kosmakova V.E,Prozumenshchikova L.T.,Growth and biochemical composition of the algae Dunaliella saline and Platymonas viridis fed on organic and inorganic forms of nitrogen,Biologiya Morya,1983,1:42-46
[16] BOROWITZKA L J . The microflora , adaptations to life in extremely saline lakes[J ]. Hydrobiologia , 1981 , 81 :33 - 46
[17] Aharon Oren,A hundred years of Dunaliella research: 1905–2005,Saline Systems,2005,1:2
[18] 方希修,胡新岗,王冬梅,黄银去。海藻饲料的应用研究。中国饲料行业信息网
[19] Thomas W.H.,Seivert D.I.R.,Alder M.,Neori A.and Eldridge P.,Yields,photosy- snthetic efficiencies and proximate composition of dense marine microalgal cultures,Ⅱ.Dunaliella primolecta and Tetraselmis suecica experiments,Biomass ,1984,5:211-225
[20] Ben-Amotz A.,Avron M.,Glycerol,β-carotene and dry algal meal production by commercial cultivation of Dunaliella,North Holland Biomedical Press,1980
[21] 张建民,刘新宁,可利用微藻的种类及其应用前景[J],资源开发与市场,2005,21(1):65-66
[22] 江孟鳞,张淑美,李弘调,膳食鱼油对大白鼠的血浆脂蛋白及血糖浓度之影响[J],中华营养学杂志,1995,20:201-214
[23] 汪本凡,杜氏盐藻纯化与生物学特性研究,安徽农业大学硕士生论文,20040501
[24] Neilson,A.H.,Lewin,R.A., The uptake and utilication of organic carbon by algae:an essay in compararive biochemixtry,phycologia, 1974,13:227-264
[25] Sherwood, Keith ,Craig Idso, Is the Growth of Phytoplankton Limited by the Current Low Concentration of Atmospheric CO2,CO2 Science,2004,7(21):26
[26] Gladue,R., Heterotrophic microalgae production potential for application to aquaclture feeds,In:Fulks,W.,Main,K.L.(Eds),Rotifer and Microalgae culture sy- stems.Poceedings of a U.S.-Asia workshop.Honolulu,Hawaii January, 1991,28(31):275-286
[27] Droop,M.R., W.D.P.stewart , Algal physiology and Biochemistry,Botanical Monographs,1974,10:530-559
[28] Tan,C.K.,Johns,M.R., Fatty acid Production by heterotrophic Chlorella saccharophila.Hydrobiologia, 1991,215:13-19
[29] 张海滨,孙世春,麦康森等,微藻异养培养技术的研究进展[J],海洋湖沼通报,2000,3:51-59
[30] Carlos Jiménez, F. X. Niell,Influence of high salinity and nitrogen limitation on package effect and C/N ratio in Dunaliella viridis,Hydrobiology,2003,492(1-3):201-206
[31] Francisco J. L. Gordillo,Madeleine Goutx, Felix L. Figueroa, F. Xavier Niell,Journal of Applied Phycology,1998,10(2):135-144
[32] Chen F, Johns M R. A strategy for high cell density culture of hetertrophic microalgae with inhibitory substrates[J ].J Applphycol,1996,7:43-46
[33] 王永红,李元广,施定基等,集胞藻 6803 的混合培养—光照强度和葡萄糖的影响[J ],生物工程学报,2000,16(2):193-197
[34] 张永起,开发卤虫资源创沿海特色产业,河北渔业,1999,4:38-39
[35] 何为,1999~2000 年度我国沿海卤虫产业调查与分析[J ],上海水产大学学报,2000,9(3):264-267
[36] M M Shirdhankar, P C Thomas,S K Barve,Phenotypic estimates and heritability values of Artemia franciscana,Aquaculture Research,2004,35(1):35
[37] 吕志华,于广利,王远红,赵侠等,卤虫卵壳营养成分分析[J ],水产科学 2004(23),1:42-44
[2] 张福,马若欣,石良,彭小雪,极端嗜盐绿色杜氏藻培养技术的研究[J],海湖盐与化工,2004,33(2):13-16
[3] МАСЮК Н П. Морфология, систематика, экология географическое распространение Рода Dunaliella Teod[M]. Издательство Киев: Науково думка, 1973
[4] Mordhay Avron & Ami Ben –Amotz . Dunaliella:Physiology,Biochemistry,and biotechnology[M].United Statesof America:CRC Press,inc,1992.2-10
[5] LEWIN,Ralph A,盐田生物技术国际研讨会论文集[C],天津:轻工业部制盐工业科学研究所出版,1991
[6] Jiménez C,Berl T,Rivard CJ,Edelstein CL,Phosphorylation of MAP kinase-like proteins mediate the response of the halotolerant alga Dunaliella viridis to hypertonic shock, Biochimica et biophysica acta,2004,1644(1):61-90
[7] Borowitzka L J . The microflora , adaptations to life in extremely saline lakes[J ]. Hydrobiologia , 1981 , 81(1)
[8] 游兰英,刘广发,林建明,杜氏藻同步化生长及核裂分析[J],厦门大学学报(自然科学版),1997,36(2):276-281
[9] 吕爱玲,张福,马若欣,姜润林,中国渤海湾滩涂极端嗜盐绿色杜氏藻生物学特性研究(二)[J], 海湖盐与化工,2005,34(3):19-23
[10] Brown A.D.,Microbial water stress,Bacteriological Reviews,1976,40:803-846
[11] Pollard A. and Wyn Jones R.G.,Enzyme activities in concentrated solutions of glycine, betaine and other solutes,Planta ,1979,144:291-298
[12] Carlos Jiménez , F. Xavier Niell , Growth ofDunaliella viridis Teodoresco: effect of salinity, temperature and nitrogen concentration,Journal of Applied Phycology,1991,3(4):319-327
[13] Francisco J. L. Gordillo, Carlos Jiménez, Félix L. Figueroa, F. Xavier Niell,Influence of elevated CO2 and nitrogen supply on the carbon assimilation performance and cell composition of the unicellular alga Dunaliella viridis,Physiologia Plantarum,2003,119(4):513-518
[14] Gordillo F.J.L., Jiménez C., Chavarría J., Xavier Niell F,Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis,Photosynthesis Research,2001,68(3):225-235
[15] Kosmakova V.E,Prozumenshchikova L.T.,Growth and biochemical composition of the algae Dunaliella saline and Platymonas viridis fed on organic and inorganic forms of nitrogen,Biologiya Morya,1983,1:42-46
[16] BOROWITZKA L J . The microflora , adaptations to life in extremely saline lakes[J ]. Hydrobiologia , 1981 , 81 :33 - 46
[17] Aharon Oren,A hundred years of Dunaliella research: 1905–2005,Saline Systems,2005,1:2
[18] 方希修,胡新岗,王冬梅,黄银去。海藻饲料的应用研究。中国饲料行业信息网
[19] Thomas W.H.,Seivert D.I.R.,Alder M.,Neori A.and Eldridge P.,Yields,photosy- snthetic efficiencies and proximate composition of dense marine microalgal cultures,Ⅱ.Dunaliella primolecta and Tetraselmis suecica experiments,Biomass ,1984,5:211-225
[20] Ben-Amotz A.,Avron M.,Glycerol,β-carotene and dry algal meal production by commercial cultivation of Dunaliella,North Holland Biomedical Press,1980
[21] 张建民,刘新宁,可利用微藻的种类及其应用前景[J],资源开发与市场,2005,21(1):65-66
[22] 江孟鳞,张淑美,李弘调,膳食鱼油对大白鼠的血浆脂蛋白及血糖浓度之影响[J],中华营养学杂志,1995,20:201-214
[23] 汪本凡,杜氏盐藻纯化与生物学特性研究,安徽农业大学硕士生论文,20040501
[24] Neilson,A.H.,Lewin,R.A., The uptake and utilication of organic carbon by algae:an essay in compararive biochemixtry,phycologia, 1974,13:227-264
[25] Sherwood, Keith ,Craig Idso, Is the Growth of Phytoplankton Limited by the Current Low Concentration of Atmospheric CO2,CO2 Science,2004,7(21):26
[26] Gladue,R., Heterotrophic microalgae production potential for application to aquaclture feeds,In:Fulks,W.,Main,K.L.(Eds),Rotifer and Microalgae culture sy- stems.Poceedings of a U.S.-Asia workshop.Honolulu,Hawaii January, 1991,28(31):275-286
[27] Droop,M.R., W.D.P.stewart , Algal physiology and Biochemistry,Botanical Monographs,1974,10:530-559
[28] Tan,C.K.,Johns,M.R., Fatty acid Production by heterotrophic Chlorella saccharophila.Hydrobiologia, 1991,215:13-19
[29] 张海滨,孙世春,麦康森等,微藻异养培养技术的研究进展[J],海洋湖沼通报,2000,3:51-59
[30] Carlos Jiménez, F. X. Niell,Influence of high salinity and nitrogen limitation on package effect and C/N ratio in Dunaliella viridis,Hydrobiology,2003,492(1-3):201-206
[31] Francisco J. L. Gordillo,Madeleine Goutx, Felix L. Figueroa, F. Xavier Niell,Journal of Applied Phycology,1998,10(2):135-144
[32] Chen F, Johns M R. A strategy for high cell density culture of hetertrophic microalgae with inhibitory substrates[J ].J Applphycol,1996,7:43-46
[33] 王永红,李元广,施定基等,集胞藻 6803 的混合培养—光照强度和葡萄糖的影响[J ],生物工程学报,2000,16(2):193-197
[34] 张永起,开发卤虫资源创沿海特色产业,河北渔业,1999,4:38-39
[35] 何为,1999~2000 年度我国沿海卤虫产业调查与分析[J ],上海水产大学学报,2000,9(3):264-267
[36] M M Shirdhankar, P C Thomas,S K Barve,Phenotypic estimates and heritability values of Artemia franciscana,Aquaculture Research,2004,35(1):35
[37] 吕志华,于广利,王远红,赵侠等,卤虫卵壳营养成分分析[J ],水产科学 2004(23),1:42-44