微藻细胞油脂含量的快速检测方法

中国生物工程杂志

2012, Vol. 32

Issue (01): 64-72

技术与方法

微藻细胞油脂含量的快速检测方法

张敬键¹, 吕雪娟², 李爱芬¹, 万凌琳¹, 吴洪³, 尹顺吉³, 张成武¹

1. 暨南大学生命科学技术学院热带亚热带水生态工程教育部工程研究中心广州 510632
2. 华南农业大学测试中心广州 510642
3. 新奥科技发展有限公司煤基低碳能源国家重点实验室廊坊 065001

Rapid Estimation of Lipids in Microalgae Cells

ZHANG Jing-jian¹, LV Xue-juan², LI Ai-fen¹, WAN Ling-lin¹, WU Hong³, YIN Shun-ji³, ZHANG Cheng-wu¹

1. Institute of Hydrobiology, Jinan University, Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering,Ministry of Education,Guangzhou 510632,China;
2. Instrumental Analysis & Research Center, South China Agricultural University, Guangzhou 510642, China;
3. State Key Laboratory of Coal-based Low Carbon Energy, Xinao Scientific & Technological Developmental Co.Ltd.,Langfang 065001,China

全文: PDF(710 KB) HTML

摘要：

以斜生栅藻(Scenedesmus obliquus JNU20)为实验材料,采用尼罗红(NR)荧光光谱法和傅里叶变换红外光谱法(FTIR)测定该藻细胞中的油脂含量。研究结果表明NR的最佳染色条件为:染色前微波处理40 s,二甲基亚砜体积分数1%, NR最终质量浓度1.5 μg/ml,染色时间5 min,染色温度40℃。比较了NR荧光光谱法、FTIR与传统重量法测定的该藻在不同时相的油脂积累情况,利用激光共聚焦显微镜观察了细胞中油体形成的动态过程。实验结果表明NR荧光光谱法、FTIR与传统重量法测定的结果显著相关(R²=0.9258, R²=0.9844),但NR荧光光谱法和FTIR更简便快速,研究结果为规模化筛选高含油量藻株及跟踪产油微藻油脂积累过程奠定了基础。

关键词： 斜生栅藻; 油脂; 尼罗红; 荧光光谱; 傅里叶变换红外光谱

Abstract:

The Nile red(NR)fluorescence and Fourier transform infrared micro-spectroscopy (FTIR)was used to determine lipid content over time in freshwater microalgae Scenedesmus obliquus JNU20. The optimum staining conditions for S. obliquus JNU20 were as follows:pretreated 40s with microwave before staining, the volume fraction of dimethylsulfoxide was 1%, the final concentration of Nile red dye was 1.5 μg/ml and incubation 5 min at 40℃. At the different growth phases, fluorescence spectrometer and FTIR spectrometer and gravimetric methods were used to determine lipid content in S. obliquus JNU20 grown in limiting concentration of nitrogen (0.5 g/L NaNO₃), the dynamic process of lipid body formation was also observed by laser scanning confocal microscopy, the results of spectrofluorimetric and FTIR spectrometric methods were very strong correlated with the results determined by gravimetric method (R²=0.925 8, R²=0.984 4), respectively. This strong correlations thus validate that the spectrofluorimetric and FTIR spectrometric methods described provide rapid, easy manipulated and reliable methods for quantification of lipids in microalgae. It was the basis of wide-scale screening of oleaginous algal strains and tracking the process of lipid accumulation in oleaginous algae.

Key words: Scenedesmus obliquus Lipid Nile red Fluorescence spectra FTIR

收稿日期: 2011-09-20 出版日期: 2012-01-25

ZTFLH:

Q949

基金资助:

国家"863"计划(2009AA06440)、国家"973"计划(2011CB2009001)、国家自然科学基金(31170337、41176105)、广东省教育部产学研结合项目 (2010A090200008)、珠海市科技重点项目 (PC20081008)资助项目

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章

引用本文:

张敬键, 吕雪娟, 李爱芬, 万凌琳, 吴洪, 尹顺吉, 张成武. 微藻细胞油脂含量的快速检测方法[J]. 中国生物工程杂志, 2012, 32(01): 64-72.

ZHANG Jing-jian, LV Xue-juan, LI Ai-fen, WAN Ling-lin, WU Hong, YIN Shun-ji, ZHANG Cheng-wu. Rapid Estimation of Lipids in Microalgae Cells. China Biotechnology, 2012, 32(01): 64-72.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I01/64

[1] Bligh E G, Dyer W J. A rapid method for total lipid extraction and purication. Can J Biochem Physiol,1959,37(8):911-917.

[2] Akoto L, Pel R, Irth H,et al. Automated GC-MS analysis of raw biological samples. Application to fatty acid profiling of aquatic micro-organisms. J Anal Appl Pyrol,2005,73(1):69-75.

[3] Greenspan P, Fowler S D. Spectrouorometric studies of the lipid probe Nile red. J Lipid Res,1985,26(7):781-789.

[4] Greenspan P, Mayer E P, Fowler S D. Nile red:a selective fluorescent stain for lipid droplets. J Cell Biol,1985, 100(3):965-973.

[5] Cooksey K E, Guckert J B, Williams S A, et al. Fluorometric determination of the neutral lipid content of microalgal cell using Nile Red. J Microbiol Meth, 1987, 6(6):333-345.

[6] Kimura K, Yamaoka M, Kamisaka Y. Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence. J Microbiol Meth, 2004, 56(3):331-338.

[7] Alonzo F, Mayzaud P. Spectrofluorometric quantification of neutral and polar lipids in zooplankton using Nile red. Mar Chem,1999, 67(3):289-301.

[8] Lee S J, Yoon B D, Oh H M. Rapid method for the determination of lipid from the green alga Botryococcus braunii. Biotechnol Tech, 1998, 12(7):553-556.

[9] Elsey D, Jameson D, Raleigh B, et al. Fluorescent measurement of microalgal neutral lipids. J Microbiol Meth,2007, 68(3):639-642.

[10] Chen W, Zhang C W, Song L, et al.A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J Microbiol Meth, 2009, 77(1):41-47.

[11] Chen W,Sommerfeld M, Hu Q.Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae. J Microbio Meth,2011,102(1):135-141.

[12] 杨文潮,邬蓓蕾,王豪,等. 近红外光谱法测定ABS树脂中氮含量.理化检验-化学分册,2011,47(3):337-338. Yang W C,Wu B L,Wang H,et al.Determination of content of nitrogen in ABS resin by NIRS.PTCA(Part B:Chem Anal), 2011,47(3):337-338.

[13] Dean A P,Sigee D C,Estrada B,et al.Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae. Bioresour Technol,2010,101(12):4499-4507.

[14] Stehfest K,Toepel J,Wilhelm C.The application of micro-FTIR spectroscopy to analyze nutrient stress-related changes in biomass composition of phytoplankton algae.Plant Physiol Biochem,2005,43(7):717-726.

[15] Soler C, Claquin P, Goutx M,et al.Impact of nutrient starvation on the biochemical composition of the marine diatom Thalassiosira weissflogii:from the whole cell to the frustule fraction. Biogeosci Discuss,2010,7(4):5953-5995.

[16] Giordano M,Kansiz M, Heraud P, et al. Fourier transform infrared spectroscopy as a novel tool to investigate changes in intracellular macromolecular pools in the marine microalga Chaetoceros muellerii (Bacillariophyceae). J Phycol. 2001,37(2):271-279.

[17] 许瑾,张成武,李爱芬,等.华南地区淡水产油微藻藻株的分离与筛选.可再生能源,2011,1(29):66-72. Xu J,Zhang C W,Li A F,et al.Isolation and selection of freshwater microalgae for oil Production in south china.Renewable Energy,2011,1(29):66-72.

[18] Khozin-Goldberg I,Shrestha P,Cohen Z.Mobilization of arachidonyl moieties from triacylglycerols into chloroplastic lipids following recovery from nitrogen starvation of the microalga Parietochloris incisa.J Biochem Biophy Acta,2005,1738(1):63-71.

[19] Moellering E R, Benning C.RNA interference silencing of a major lipid droplet protein affects lipid droplet size in chlamydomonas reinhardtii. Eukaryot Cell,2010,9(1):97-106.

[20] Coates J. Interpretation of infrared spectra, a practical approach.In:Meyers R A. Encyclopedia of Analytical Chemistry. Chichester:John Wiley and Sons Ltd, 2000.1-23.

[21] Murdock, J N, Wetzel D L.FT-IR microspectroscopy enhances biological and ecological analysis of algae. Appl Spectrosc Rev,2009, 44(4):335-361.

[22] Dreissig I, Machill S, Salzer R, et al. Quantification of brain lipids by FTIR spectroscopy and partial least squares regression. Spectrochim Acta A,2009,71(5):2069-2075.

[23] 王金娜, 严小军, 周成旭, 等. 产油微藻的筛选及中性脂动态积累过程的检测. 生物物理学报,2010,26(6):472-480. Wang J N, Yan X J,Zhou C X,et al. Screening of Oil-Producing Microalgae and Detecting Dynamics of Neutral Lipid Accumulation.Acta Biophysica Sinica, 2010,26(6):472-480.

[1]	卫治金,李晓,王皓楠,尹永浩,郗丽君,葛保胜. 小球藻与固氮菌Mesorhizobium sp.共培养对小球藻生长和油脂积累的促进效果 ^*[J]. 中国生物工程杂志, 2019, 39(7): 56-64.
[2]	左正三,孙小曼,任路静,黄和. 微藻生产油脂培养新技术 ^*[J]. 中国生物工程杂志, 2018, 38(7): 102-109.
[3]	夏乾竣, 王飞, 李迅. 解脂耶罗维亚酵母产油脂的研究进展[J]. 中国生物工程杂志, 2017, 37(3): 99-105.
[4]	韦璇, 郝雅荞, Susanna Leong Su Jan, 吴言, 柳叶飞, 赵洪新. Saccharomyces cerevisiae和Yarrowia lipolytica对自由饱和脂肪酸的选择性吸收及胞内积累特性研究[J]. 中国生物工程杂志, 2017, 37(2): 63-73.
[5]	王雅南, 沈宏伟, 杨晓兵, 赵宗保. 不同营养元素限制对圆红冬胞酵母油脂生产的影响[J]. 中国生物工程杂志, 2016, 36(11): 16-22.
[6]	车绕琼, 黄力, 王琳, 赵鹏, 李涛, 余旭亚. 葡萄糖对单针藻异养、兼养生长及油脂合成的影响[J]. 中国生物工程杂志, 2015, 35(11): 46-51.
[7]	杨凯, 战景明, 高芬芳, 武宝利, 苏丽霞, 周文明, 薛向明, 郝杰, 赵阳. 小球藻用于生物柴油生产的研究进展[J]. 中国生物工程杂志, 2015, 35(11): 99-104.
[8]	雷学青, 卢哲, 高保燕, 张文源, 李爱芬, 张成武. 利用平板反应器大量培养高产油绿藻——尖状栅藻的生长和油脂积累规律[J]. 中国生物工程杂志, 2014, 34(11): 91-99.
[9]	许继飞, 张艳芬, 赵桂琦, 赵吉. 产油酵母利用不同基质累积油脂的研究进展[J]. 中国生物工程杂志, 2013, 33(9): 111-118.
[10]	王美玲, 薛超友, 赵方龙, 卢文玉. 混合油脂补料发酵提高多杀菌素的产量[J]. 中国生物工程杂志, 2013, 33(8): 56-60.
[11]	刘会影, 薛冬桦, 潘安龙, 徐洪章, 叶小金, 孙国英. 微生物油脂酯化工艺优化[J]. 中国生物工程杂志, 2013, 33(3): 92-98.
[12]	汪桂林, 桂小华, 邓伟, 赵志良, 姚杰, 闫云君. “异养-胁迫”分段培养对原始小球藻生物量和油脂含量影响研究[J]. 中国生物工程杂志, 2013, 33(3): 99-104.
[13]	黄力, 贺赐安, 赵鹏, 余旭亚. *碳源、氮源对异养单针藻Monoraphidium* sp. FXY-10油脂积累和脂肪酸组成的影响**[J]. 中国生物工程杂志, 2013, 33(2): 59-64.
[14]	胡文军, 罗玮, 李汉广, 顾秋亚, 余晓斌. 产油微藻筛选和鉴定及其产油性能的研究[J]. 中国生物工程杂志, 2012, 32(12): 66-72.
[15]	艾佐佐, 颜日明, 袁锦云, 张志斌, 朱笃. 响应面法优化木薯淀粉发酵生产单细胞油脂工艺[J]. 中国生物工程杂志, 2012, 32(07): 66-72.

Viewed

Full text

Abstract

Cited

Shared

Discussed