Published: Vol 8, Iss 1, Jan 5, 2018 DOI: 10.21769/BioProtoc.2676 Views: 8482
Reviewed by: Maria SinetovaRoman A. SidorovAgnieszka Zienkiewicz
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Abstract
Triacylglycerols (TAGs) are esters formed from one glycerol and three fatty acids. TAGs are induced to accumulate in algal cells under environmental stress conditions including nutrient-limitation, hyperosmosis, and low temperature, for the storage of metabolic energy and carbon, and also for the consumption of excess energy (e.g., Hirai et al., 2016; Hayashi et al., 2017). Beside their physiological significance, the commercial utilization of algal TAG has been expected for the production of biodiesel, the methyl esters of fatty acids, from the aspect of carbon-neutral conception. The amounts of TAGs can be determined through quantitative measurement of their constituent fatty acids. This protocol consists of the following three parts: the first is the extraction of total lipids from algal cells with the use of organic solvents, chloroform and methanol, according to the method of Bligh and Dyer (1959), the second is the separation of TAG from the other lipid classes by thin-layer chromatography (TLC), and the third is the production of methyl-esterified derivatives of their constitutive fatty acids and subsequent quantitation of them by capillary gas-liquid chromatography (GLC). This protocol adapted from Sato and Tsuzuki (2011) is used for TAG analysis in a green alga, Chlorella kessleri.
Keywords: Chlorella kessleriBackground
Several methods have been used for determination of the fatty acid content of TAG. Simple and convenient protocols, e.g., include conversion of TAG to glycerol on treatment with a lipase, and subsequent measurement of the glycerol content through enzymatic generation of a product that reacts with a color- or fluorescence-generating probe (McGowan et al., 1983; Mendez et al., 1986). However, this enzymatic reaction based quantitation of TAG, inevitably, gives no information about the composition of constituent fatty acids. Meanwhile, HPLC provides information on TAG molecular species through their separation based on the numbers of carbon atoms and double bonds of constituent fatty acids, and enables their respective quantitation when combined with tandem mass spectrometer like in LC-MS/MS (Mu et al., 2000; Dorschel, 2002; MacDougall et al., 2011). The LC-MS/MS instrument, however, is very expensive. In this context, TLC/GLC based protocol for the measurement of the fatty acid content of TAG is introduced here, in view of the requirement of less expensive equipment than LC-MS/MS and definite information that can be obtained on quality and quantity of the constituent fatty acids.
Materials and Reagents
Equipment
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Acknowledgments
This work was supported by Tokyo University of Pharmacy and Life Sciences. The authors have declared that no competing interests exist.
References
Article Information
Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Aoki, M. and Sato, N. (2018). Fatty Acid Content and Composition of Triacylglycerols of Chlorella kessleri. Bio-protocol 8(1): e2676. DOI: 10.21769/BioProtoc.2676.
Category
Plant Science > Plant biochemistry > Lipid
Microbiology > Microbial biochemistry > Lipid
Biochemistry > Lipid > Lipid measurement
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