148 ISSN CN /Q Chin J Biotech January 25, 2007 Vol.24 No.1 content and yield was 6.23% and mg/l during mixotrophic culture with 1

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1 生物工程学报 Chin J Biotech 2008, January 25; 24(1): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2008 Institute of Microbiology, CAS & CSM, All rights reserved. 研究简报刘晓娟, 段舜山, 李爱芬, : 研究了 3 种有机碳对三角褐指藻生长胞内物质和脂肪酸组分的影响结果表明, 三角褐指藻具有利用有机碳进行兼养生长的能力, 生长速率加快, 倍增时间缩短, 生物量显著提高, 100 mmol/l 甘油兼养的生物量最高 (713 mg/l), 是自养 (460 mg/l) 的 1.60 倍, 乙酸钠和葡萄糖兼养的生物量分别是自养的 1.28 倍和 1.21 倍兼养下蛋白质含量较自养明显下降, 碳水化合物和总脂含量高于自养, 乙酸钠和甘油兼养的总脂含量分别是自养的 1.43 倍和 1.20 倍, 葡萄糖兼养的总脂含量与自养无明显差异 3 种有机碳兼养的饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的比例增大, 多不饱和脂肪酸比例降低, EPA(eicosapentaenoic acid) 比例降低, 乙酸钠兼养的胞内 EPA 含量 (6.23%) 和产量 (36.59 mg/l) 均高于自养, 分别是自养的 1.10 倍和 1.40 倍, 甘油和葡萄糖兼养的 EPA 含量和产量均低于自养 : 有机碳, 三角褐指藻, 兼养, 生长, 总脂, 脂肪酸, EPA Effects of Organic Carbon Source on Growth, Biochemical Components and Fatty Acid Composition of Phaeodactylum tricornutum Xiaojuan Liu, Shunshan Duan, and Aifen Li Institute of Hydrobiology, Jinan University, Guangzhou , China Abstract: We examined the effects of three organic carbon sources on mixotrophic growth, biochemical components and fatty acid composition of Phaeodactylum tricornutum. Mixotrophically, P. tricornutum grew faster and had shorter doubling time. The biomass of P. tricornutum was greatly enhanced under mixotrophic condition with its highest biomass of 713 mg/l after 16 days, in medium containing 100 mmol/l glycerol. This was 1.60-fold of that obtained under autotrophic condition. The biomass during mixotrophic culture with 100 mmol/l glucose and 100 mmol/l acetate was 1.28-fold and 1.21-fold of that obtained under autotrophic condition, respectively. Compared with autotrophic condition, the content of soluble protein decreased obviously, whereas the content of soluble carbohydrate and total lipid increased. The content of lipid during mixotrophic culture with 100 mmol/l acetate and 100 mmol/l glycerol was 1.43-fold and 1.20-fold of that obtained under autotrophic condition, respectively. There was no difference between lipid content of mixotrophic growth with 100 mmol/l glucose and that of autotrophic condition. The eicosapentaenoic acid (EPA) Received: April 13, 2007; Accepted: July 9, 2007 Supported by: the National Natural Science Foundation of China (No ), the Guangdong Key Guiding Project of Science and Technology Planning (No. 2005B ) and the Zhuhai Science and Technology Planning Project Contract Research (No. PC ). Corresponding author: Shunshan Duan. Tel: ; Tssduan@jnu.edu.cn (No ), (No. 2005B )(No. PC )

2 148 ISSN CN /Q Chin J Biotech January 25, 2007 Vol.24 No.1 content and yield was 6.23% and mg/l during mixotrophic culture with 100 mmol/l acetate. These were 1.10-fold and 1.40-fold of that obtained under autotrophic condition, respectively. The EPA content and yield with glycerol and glucose were lower than that obtained under autotrophic condition. These results indicated that mixotrophic cultivation with acetate was beneficial to produce EPA. Keywords: organic carbon source, Phaeodactylum tricornutum, mixotrophic, growth, lipid, fatty acid, EPA, (polyunsaturated fatty acids, PUFAs) ( β-), (Eicosapentaenoic acid, EPA) (Docosahexaenoic acid, DHA), EPA,,, EPA EPA,, ph [1,2,3], EPA, Phaeodactylum tricornutum UTEX-64, Phaeodactylum tricornutum Böhlin [4,5],,, (Phaeodactylum tricornutum),, EPA 1 材料与方法 1.1 藻种 (Phaeodactylum tricornutum), 1.2 微藻的培养 f/2, 30, (20±1), 50 μmol m 2 s 1, 12L:12D,,, 100 mmol/l, 300 ml 500 ml, 0.15 g/l 1.3 生长的测定 20 μl (CASY-TT, Schärfe-System, ) Y( )=0.0223X( ) μ (lnn2-lnn1)/(t 2 t 1 ) (t ),, N 2 N 1 t 2 t 1, =ln2/μ,, μ 1.4 藻体的收获, g 10 min, 3, ALPHR 2-4(), 胞内物质含量的测定 [6], [7], Bligh-Dyer( mbd) [8] 1.6 脂肪酸的提取 Bigogno [9] 25 mg, 2.0 ml 2% H 2 SO 4, 80 1 h, 1.0 ml 1.0 ml,, 3500 r/min 5 min,, 100 μl, 1.7 脂肪酸的气相色谱分析 -, ThermoFinnigan TRACE GC-MS : DB-5 (30 m 0.25 mm); 260 ;, 60 2 min, 30/min 120, 1.5/min min;, 1.2 ml/min EI, 70 EV 2 结果与分析 2.1 有机碳对三角褐指藻生长的影响 3

3 : 149 1, 16, 100 mmol/l (713 mg/l), (460 mg/l) 1.6, (587 mg/l)(555 mg/l) , ( 1), 39.24% 17.72% 15.19%,, 2.2 有机碳对三角褐指藻胞内组分的影响 ( 2), 54.57% 19.69% 59.83%, , 图 1 三角褐指藻在自养和兼养下的生长曲线 Fig. 1 Growth curves of autotrophic and mixotrophic cultivation of Phaeodactylum tricornutum The concentration of glycerol, acetate and glucose of mixotrophic growth is 100 mmol/l 表 1 三角褐指藻在自养和兼养下的生长参数 Table 1 Growth parameters of Phaeodactylum tricornutum under autotrophic and mixotrophic cultivation Autotrophic Glycerol Acetate Glucose Specific growth rate/d ± ± ± ±0.002 Doubling time/d 8.75± ± ± ±0.21 Maximum biomass/mg/l 460±3 713±11 587±8 555±10 图 2 三角褐指藻在自养和兼养下的胞内组分变化 Fig. 2 Biochemical components of autotrophic and mixotrophic cultivation of Phaeodactylum tricornutum The concentration of glycerol, acetate and glucose of mixotrophic growth is 100 mmol/l 2.3 有机碳对三角褐指藻脂肪酸组分的影响,,, C16:0, C16:1 C20:5, C16:0 C16:1 表 2 三角褐指藻在自养和兼养下的脂肪酸含量 Table 2 Fatty acids composition of autotrophic and mixotrophic cultivation of Phaeodactylum tricornutum Fatty acids of total fatty acids Autotrophic Glycerol Acetate Glucose C14: C16: C16: C16: C16: C18: C18: C18: C18: C18: C20: C20: Others TSFA TMUFA TPUFA EPA content (%dry weight) EPA yield/mg/l TFA, total fatty acids; TSFA: total saturated fatty acids; TMUFA: total mono-saturated fatty acids; TPUFA: total polyunsaturated fatty acids; : not detected.

4 150 ISSN CN /Q Chin J Biotech January 25, 2008 Vol.24 No.1 3, EPA 3,, C18 (C18:0, C18:1, C18:2, C18:3 C18:4) 3,, EPA EPA (6.23%) (36.59 mg/l), , EPA 3 讨论 3,,,, [10], µ [11,12],, 3 (Platymonas subcordiformis) (Nannochloropsis sp.) 6 1.4, (Chlamydomonas humicola) 9.2, (Phaeodactylum tricornutum) 1.60, (Chlorella pyrenoidosa) [18] (Chlorella Vulgaris) [19] (Phaeodactylum tricornutum),, (Chlamydomonas humicola) [17] Ogbonna [20],,,,, (Chlorella pyrenoidosa) 1.47, 16% [18] (Nannochloropsis sp.) 1.21 [21],,, EPA EPA, EPA, 18:2ω6 Δ6 Δ15(ω3), 18:3ω6 18:3ω3, 20:4ω6 20:5ω3 (Rhodomonas salina), (Nitzschia sp.)(navicula saprophila) EPA [12], (Navicula saprophila) Δ15(ω3) EPA, 11.2% 18.6% [22] 表 3 不同微藻在自养和兼养下的生物量 Table 3 Biomass of different microalgae under autotrophic and mixotrophic cultivation Biomass/(g/L) Microalgae References Autotrophic Mixotrophic Spirulina platensis (Glucose) Marquez et al, Chlorella sp. VJ (Glucose) Lalucat et al, Nannochloropsis sp (Glucose) Xu et al, (Ethanol) Platymonas subcordiformis (Glucose) Xie et al, Chlamydomonas humicola (Acetate) Lalibert et al, Phaeodactylum tricornutum (Glycerol) This paper (Acetate) (Glucose)

5 : 151 (Nannochloropsis sp.) EPA, 21.9% 18.0% [23] (Phaeodactylum tricornum UTEX-640) EPA, EPA, EPA, EPA [4], EPA, EPA, EPA,,, EPA (Nannochloropsis sp.) EPA (23.4 mg/l) (21.9 mg/l) [15], EPA 56 mg/l [24] (Navicula saprophila) EPA 34.6 mg/g, 3 [22] (Nitzschia laevis) EPA 1112 mg/l, EPA [25] EPA REFERENCES [1] Nuutila AM, Aura AM, Kiesvaara M, et al. The effect of salinity, nitrate concentration, ph and temperature on eicosapentaenoic acid (EPA) production by the red unicellular alga Porphyridium purpureum. Journal of Biotechnology, 1997, 55: [2] Xu NJ, Zhang XC, Fan X, et al. Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta). Journal of Applied Phycology, 2001, 13: [3] Hoshida H, Ohira T, Minematsu A, et al. Accumulation of eicosapentaenoic acid in Nannochloropsis sp. in response to elevated CO 2 concentrations. Journal of Applied Phycology, 2005, 17: [4] Cerón García MC, Sánchez Mirón A, Fernández Sevilla JM, et al. Mixotrophic growth of the microalga Phaeodactylum tricornutum Influence of different nitrogen and organic carbon sources on productivity and biomass composition. Process Biochemistry, 2005, 40: [5] Ukeles R, Rose WE. Observations on organic carbon utilisation by photosynthetic marine microalgae. Marine Biology, 1976, 37: [6] Zhang ZL. Guidance of Phytophysiology Experiment. Beijing: Higher Education Press, 1990, :, 1990, [7] Michel D, Gilles KA, Hamilton JK, et al. Colorimeteric method for determination of sugas and related substances. Analytical Chemistry, 1956, 28(3): [8] Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 1959, 37: [9] Bigogno C, Khozin-Goldberg I, Cohena Z. Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae, Chlorophyta). Phytochemistry, 2002, 60: [10] Facundo JM, Sasaki K, Kakizono T, et al. Growth characteristics of Spirulina platensis in mixotrophic and heterotrophic conditions. Journal of Fermentation and Bioengineering, 1993, 76: [11] Kobayashi M, Kakizono T, Yamaguchi K, et al. Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions. Journal of Fermentation and Bioengineering, 1992, 74: [12] Kitano M, Matsukawa R, Karube I. Changes in eicosapentaenoic acid content of Navicula saprophila, Rhodomonas salina and Nitzschia sp. under mixotrophic conditions. Journal of Applied Phycology, 1997, 9: [13] Marquez FJ, Saski K, Kakizono T, et al. Growth characteristics of Spirulina platensis in mixotrophic and heterotrophic conditions. Journal of Fermentation and Bioengineering, 1993, 76: [14] Lalucat J, Imperial J, Pares R. Utilization of light for the assimilation of organic matter in Chlorella sp. VJ79. Biotechnology and Bioengineering, 1984, 26: [15] Xu F, Cong W, Cai ZL, et al. Effects of organic carbon sources on cell growth and eicosapentaenoic acid content of Nannochloropsis sp. Journal of Applied Phycology, 2004, 16: [16] Xie JL, Zhang YX, Li YG, et al. Mixotrophic cultivation of Platymonas subcordiformis. Journal of Applied Phycology, 2001, 13: [17] Laliberté G, de la Noüe J. Auto-, hetero-, and Mixotrophic growth of Chlamydomonas humicola (Chlorophyceae) on acetate. Journal of Phycology, 1993, 29: [18] Gui L, Shi XM, Li L, et al. Comparison of the different cultivation systems for Chlorella pyrenoidosa. Journal of Henan University of Technology (Natural Science Edition), 2005, 26(5): ,,.. : 2005, 26(5): [19] Wang HY, Guo SY, Zheng BS, et al. Growth and biochemical components of Chlorella Vulgaris under autotrophic, heterotrophic and mixotrophic cultivations. Journal of South China University of Technology (Natural Science Edition), 2004, 32(5): [20] Ogbonna JC, Masui H, Tanaka H. Sequential heterotrophic/autotrophic cultivation An efficient method of producing Chlorella biomass for health food and animal feed. Journal of Applied Phycology, 1997, 9: [21] Xu F, Hu HH, Cong W, et al. Effect of Organic Carbon Sources on Growth and Photosynthesis of Nannochloropsis sp.. The Chinese Journal of Process Engineering, 2003, 3(6):

6 152 ISSN CN /Q Chin J Biotech January 25, 2008 Vol.24 No.1,,,. EPA (Nannochloropsis sp.)., 2003, 3(6): [22] Kitano M, Matsukawa R, Karube I. Enhanced eicosapentaenoic acid production by Navicula saprophila. Journal of Applied Phycology, 1998, 10: [23] Hu HH, Gao K. Optimization of growth and fatty acid composition of a unicellular marine picoplankton, Nannochloropsis sp., with enriched carbon sources. Biotechnology Letters, 2003, 25: [24] Xu F, Hu HH, Cong W, et al. Growth characteristics and eicosapentaenoic acid production by Nannochloropsis sp. in mixotrophic conditions. Biotechnology Letters, 2004, 26: [25] Wen ZY, Chen F. Perfusion culture of the diatom Nitzschia laevis for ultra-high yield of eicosapentaenoic acid. Process Biochemistry, 2002, 38: ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ 2008 年中国微生物学会及各专业委员会学术活动计划表 lichang78@163.com

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Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇