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Bio-production of eicosapentaenoic acid from the diatom Nanofrustulum shiloi via two-step high performance countercurrent chromatography

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Abstract

Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid of nutritional and physiological relevance for humans. It is commercially produced mainly from fish oil, but new sustainable sources are required. In this study, pure EPA ethyl ester was produced from Nanofrustulum shiloi biomass by integrating two separation steps via high performance countercurrent chromatography (HPCCC). The HPCCC first step consisted of a 10 separation cycles (1200 mg sample per cycle) process, each cycle consisting of three stages comprising EPA fraction separation in reversed phase elution mode (lower polar phase used as mobile phase); with stationary phase extrusion (by switching the pumping of the mobile phase to the stationary phase, while maintaining the column rotation); and hydrodynamic equilibrium achievement inside the column. The process led to 982.1 mg of a fraction rich in EPA with a purity of 71%. To improve the purity of the EPA fraction, an HPCCC second step was performed leading to 698.97 mg of EPA ethyl ester with a purity of 99%, and a recovery of 98%. To avoid unnecessary waste of solvents, liquid phases were formulated based on a priori NMR measurement. The HPCCC process throughput was 0.936 g h–1, while the efficiency per gram of EPA was 0.054 g h–1. Environmental risk and process evaluation factors were used for the evaluation of the separation process. Overall, this isolation approach may represent a useful model for the efficient production of pure EPA from microalgae.

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Acknowledgements

Daniela Bárcenas-Pérez gratefully acknowledges the research supervision of José Cheel (Centre Algatech–Czech Academy of Sciences) during the doctoral study as well as the University of South Bohemia for a doctoral scholarship. We thank Brenda Priscila Portuguez Solano for microalgae cultivation (Proyecto Fiscal del CIBNOR—PAC01). The authors thank Leslie Brown for critical review and English proofreading of the article.

Funding

This work has received funding from the Bio Based Industries Joint Undertaking (JU) under grant agreement No 887227. The JU receives support from the European Union's Horizon 2020 research and innovation programme and the Bio Based Industries Consortium (J.C., D.B-P., and J.K.). The work was also supported by the Technology Agency of the Czech Republic (NCK grant TN010000048/03, J.C., D.B-P., M.L., and P.H.) and the National Programme of Sustainability I of the Ministry of Education, Youth and Sports of the Czech Republic (ID: LO1416, J.C, P.H, J.K).

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Daniela Bárcenas-Pérez: conceptualization, methodology, investigation, formal analysis, writing—review & editing. José Cheel: conceptualization, methodology, writing—original draft, supervision. Martin Lukeš: methodology, formal analysis. Pavel Hrouzek: methodology, formal analysis. Jakub Zápal: methodology, formal analysis. Marek Kuzma: methodology, formal analysis. Jiří Kopecký: formal analysis. David Kubáč: formal analysis. Bertha O. Arredondo Vega: methodology, formal analysis. All authors reviewed the manuscript.

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Correspondence to José Cheel.

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Bárcenas-Pérez, D., Lukeš, M., Hrouzek, P. et al. Bio-production of eicosapentaenoic acid from the diatom Nanofrustulum shiloi via two-step high performance countercurrent chromatography. J Appl Phycol 34, 2995–3010 (2022). https://doi.org/10.1007/s10811-022-02816-w

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