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Identification of optimum fatty acid extraction methods for two different microalgae Phaeodactylum tricornutum and Haematococcus pluvialis for food and biodiesel applications

Analytical and Bioanalytical Chemistry volume 409, pages 4659–4667 (2017)Cite this article

Abstract

Microalgae have the potential to synthesize and accumulate lipids which contain high value fatty acids intended for nutrition and biodiesel applications. Nevertheless, lipid extraction methods for microalgae cells are not well established and there is not a standard analytical methodology to extract fatty acids from lipid-producing microalgae. In this paper, current lipid extraction procedures employing organic solvents (chloroform/methanol, 2:1 and 1:2, v/v), sodium hypochlorite solution (NaClO), acid-catalysed hot-water extraction and the saponification process [2.5 M KOH/methanol (1:4, v/v)] have been evaluated with two species of microalgae with different types of cell walls. One is a marine diatom, Phaeodactylum tricornutum, and the other a freshwater green microalga, Haematococcus pluvialis. Lipids from all types of extracts were estimated gravimetrically and their fatty acids were quantified by a HPLC equipped with Q-TOF mass spectrometer. Results indicated significant differences both in lipids yield and fatty acids composition. The chloroform and methanol mixture was the most effective extraction solvent for the unsaturated fatty acids such as DPA (C22:05), DHA, (C22:06), EPA (C20:05) and ARA (C20:04). While acid treatments improved the saturated fatty acids (SFAs) yield, especially the short chain SFA, lauric acid (C12:0), whose amount was 64% higher in P. tricornutum and 156% higher in H. pluvialis compared to organic solvent extractions.

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References

  1. Sanghvi AM, Lo YM. Present and potential industrial applications of macro- and microalgae. Recent Pat Food Nutr Agric. 2010;2(3):187–94.

    Article  Google Scholar 

  2. Choi S-A, Jung J-Y, Kim K, Lee J-S, Kwon J-H, Kim S, et al. Acid-catalyzed hot-water extraction of docosahexaenoic acid (DHA)-rich lipids from Aurantiochytrium sp. KRS101. Bioresour Technol. 2014;161:469–72.

    CAS  Article  Google Scholar 

  3. Lei A, Chen H, Shen G, Hu Z, Chen L, Wang J. Expression of fatty acid synthesis genes and fatty acid accumulation in Haematococcus pluvialis under different stressors. Biotechnol Biofuels. 2012;5(1):18. doi:10.1186/1754-6834-5-18.

    CAS  Article  Google Scholar 

  4. Cavonius LR, Carlsson NG, Undeland I (2014) Quantification of total fatty acids in microalgae: comparison of extraction and transesterification methods. Anal Bioanal Chem 406 (28): 7313-22.

  5. Folch J, Lees M, Sloane-Stanley G. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497–509.

    CAS  Google Scholar 

  6. Adarme-Vega TC, Lim DK, Timmins M, Vernen F, Li Y, Schenk PM. Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production. Microb Cell Factories. 2012;11:96. doi:10.1186/1475-2859-11-96.

    CAS  Article  Google Scholar 

  7. Saha SK, McHugh E, Hayes J, Moane S, Walsh D, Murray P. Effect of various stress-regulatory factors on biomass and lipid production in microalga Haematococcus pluvialis. Bioresour Technol. 2013;128:118–24.

    CAS  Article  Google Scholar 

  8. Bligh E, Dyer W. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–7.

    CAS  Article  Google Scholar 

  9. Yongmanitchai W, Ward OP. Growth of and omega-3 fatty acid production by Phaeodactylum tricornutum under different culture conditions. Appl Environ Microbiol. 1991;57(2):419–25.

    CAS  Google Scholar 

  10. Cho SC, Choi WY, Oh SH, Lee CG, Seo YC, Kim JS, Song CH, Kim GV, Lee SY, Kang DH, Lee, HY (2012) Enhancement of lipid extraction from marine microalga, Scenedesmus associated with high-pressure homogenization process. J Biomed Biotechnol(6 pageshttp://dx.doi.org/10.1155/2012/359432): p. 359432.

  11. Lohman EJ, Gardner RD, Halverson L, Macur RE, Peyton BM, Gerlach R. An efficient and scalable extraction and quantification method for algal derived biofuel. J Microbiol Methods. 2013;94(3):235–44.

    CAS  Article  Google Scholar 

  12. Li Y, Ghasemi Naghdi F, Garg S, Adarme-Vega TC, Thurecht KJ, Ghafor WA, et al. A comparative study: the impact of different lipid extraction methods on current microalgal lipid research. Microb Cell Factories. 2014;13:14. doi:10.1186/1475-2859-13-14.

    Article  Google Scholar 

  13. Liu K-S. Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials. JAOCS. 1994;71(11):1179–80.

    CAS  Google Scholar 

  14. Park J-Y, Choi S-A, Jeong M-J, Nam B, Oh Y-K, Lee J-S. Changes in fatty acid composition of Chlorella vulgaris by hypochlorous acid. Bioresour Thechnol. 2014;162:379–83.

    CAS  Article  Google Scholar 

  15. Park JY, Oh YK, Lee JS, Lee K, Jeong MJ, Choi SA. Acid-catalyzed hot-water extraction of lipids from Chlorella vulgaris. Bioresour Technol. 2014;153:408–12.

    CAS  Article  Google Scholar 

  16. Norris PC, Dennis EA. Omega-3 fatty acids cause dramatic changes in TLR4 and purinergic eicosanoid signaling. Proc Natl Acad Sci U S A. 2012;109(22):8517–22.

    CAS  Article  Google Scholar 

  17. Zhang R-Z, Li L, Liu S-T, Chem R-M, Rao P-F. An improved method of cholesterol determination in egg yolk by HPLC. J Food Biochem. 1999;23:351–61.

    CAS  Article  Google Scholar 

  18. Ma YH, Wang X, Niu YF, Yang ZK, Zhang MH, Wang ZM, et al. Antisense knockdown of pyruvate dehydrogenase kinase promotes the neutral lipid accumulation in the diatom Phaeodactylum tricornutum. Microb Cell Factories. 2014;13(1):100. doi:10.1186/s12934-014-0100-9.

    Google Scholar 

  19. Saha SK, Hayes J, Moane S, Murray P. Tagging of biomolecules with deuterated water (D2O) in commercially important microalgae. Biotechnol Lett. 2013;35(7):1067–72.

    CAS  Article  Google Scholar 

  20. He L, Han X, Yu Z. A rare Phaeodactylum tricornutum cruciform morphotype: culture conditions, transformation and unique fatty acid characteristics. PLoS One. 2014;9(4):e93922. doi:10.1371/journal.pone.0093922.

    Article  Google Scholar 

  21. Hagen C, Siegmund S, Braune W. Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation. Eur J Phycol. 2002;37(2):217–26.

    Article  Google Scholar 

  22. Rippka R, Deruelles J, Waterbury J, Herdman M, Stanie RY. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol. 1979;111:1–61.

    Google Scholar 

  23. Dillon JT, Aponte JC, Tarozo R, Huang Y. Purification of omega-3 polyunsaturated fatty acids from fish oil using silver-thiolate chromatographic material and high performance liquid chromatography. J Chromatogr A. 2013;1312:18–25.

    CAS  Article  Google Scholar 

  24. Woo SG, Yoo K, Lee J, Bang S, Lee M, On K, et al. Comparison of fatty acid analysis methods for assessing biorefinery applicability of wastewater cultivated microalgae. Talanta. 2012;97:103–10.

    CAS  Article  Google Scholar 

  25. Wen ZY, Chen F. Heterotrophic production of eicosapentaenoic acid by microalgae. Biotechnol Adv. 2003;21(4):273–5.

    CAS  Article  Google Scholar 

  26. Pribyl P, Cepak V, Zachleder V. Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris. Appl Microbiol Biotechnol. 2012;94(2):549–61.

    CAS  Article  Google Scholar 

  27. Ryckebosch E, Muylaert K, Foubert I. Optimization of an analytical procedure for extraction of lipids from microalgae. J Am Oil Chem Soc. 2011;89:189–98.

    Article  Google Scholar 

  28. Cuellar-Bermudez SP, Aguilar-Hernandez I, Cardenas-Chavez DL, Ornelas-Soto N, Romero-Ogawa MA, Parra-Saldivar R. Extraction and purification of high-value metabolites from microalgae: essential lipids, astaxanthin and phycobiliproteins. Microb Biotechnol. 2015;8(2):190–209.

    CAS  Article  Google Scholar 

  29. Choi SA, Jung JY, Kim K, Kwon JH, Lee JS, Kim SW, et al. Effects of molten-salt/ionic-liquid mixture on extraction of docosahexaenoic acid (DHA)-rich lipids from Aurantiochytrium sp. KRS101. Bioprocess Biosyst Eng. 2014;37(11):2199–204.

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by the Irish Research Council (Grant No. GOIPD/2015/681) and an Enterprise Ireland Innovation partnership grant (Grant No. IP/2013/0439). Paz Otero is recipient of a Government of Ireland Postdoctoral Award 2015 (GOIPD/2015/681).

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  1. Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick, Ireland

    Paz Otero, Sushanta Kumar Saha, Joanne Mc Gushin, Siobhan Moane & Patrick Murray

  2. Reagecon Diagnostics Limited Shannon Free Zone, Shannon, Co. Clare, Ireland

    John Barron

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  1. Paz Otero
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  2. Sushanta Kumar Saha
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Correspondence to Paz Otero.

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Otero, P., Saha, S.K., Gushin, J.M. et al. Identification of optimum fatty acid extraction methods for two different microalgae Phaeodactylum tricornutum and Haematococcus pluvialis for food and biodiesel applications. Anal Bioanal Chem 409, 4659–4667 (2017). https://doi.org/10.1007/s00216-017-0412-9

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  • DOI: https://doi.org/10.1007/s00216-017-0412-9

Keywords

  • Phaeodactylum tricornutum
  • Haematococcus pluvialis
  • Fatty acids
  • Microalgae extraction methods
  • Saponification
  • Liquid chromatography-mass spectrometry