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Improvement of carotenoid extraction from a recently isolated, robust microalga, Tetraselmis sp. CTP4 (chlorophyta)


In recent years, there has been increasing consumer interest in carotenoids, particularly of marine sustainable origin with applications in the food, cosmeceutical, nutritional supplement and pharmaceutical industries. For instance, microalgae belonging to the genus Tetraselmis are known for their biotechnologically relevant carotenoid profile. The recently isolated marine microalgal strain Tetraselmis sp. CTP4 is a fast-growing, robust industrial strain, which has successfully been produced in 100-m3 photobioreactors. However, there are no reports on total carotenoid contents from this strain belonging to T. striata/convolutae clade. Although there are several reports on extraction methods targeting chlorophytes, extraction depends on the strength of cell coverings, solvent polarity and the nature of the targeted carotenoids. Therefore, this article evaluates different extraction methods targeting Tetraselmis sp. CTP4, a strain known to contain a mechanically resistant theca. Here, we propose a factorial experimental design to compare extraction of total carotenoids from wet and freeze-dried microalgal biomass using four different solvents (acetone, ethanol, methanol or tetrahydrofuran) in combination with two types of mechanical cell disruption (glass beads or dispersion). The extraction efficiency of the methods was assessed by pigment contents and profiles present in the extracts. Extraction of wet biomass by means of glass bead-assisted cell disruption using tetrahydrofuran yielded the highest amounts of lutein and β-carotene (622 ± 40 and 618 ± 32 µg g−1 DW, respectively). Although acetone was slightly less efficient than tetrahydrofuran, it is preferable due to its lower costs and toxicity.

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  1. 1.

    Varela JC, Pereira H, Vila M, León R (2015) Production of carotenoids by microalgae: achievements and challenges. Photosynth Res 125:423–436.

  2. 2.

    Johnson EJ (2002) The role of carotenoids in human health. Nutr Clin Care 5:56–65.

  3. 3.

    Fiedor J, Burda K (2014) Potential role of carotenoids as antioxidants in human health and disease. Nutrients 6:466–488.

  4. 4.

    Eggersdorfer M, Wyss A (2018) Carotenoids in human nutrition and health. Arch Biochem Biophys 652:18–26.

  5. 5.

    Vílchez C, Forján E, Cuaresma M, Bédmar F, Garbayo I, Vega JM (2011) Marine carotenoids: biological functions and commercial applications. Mar Drugs 9:319–333.

  6. 6.

    Guedes AC, Amaro HM, Malcata FX (2011) Microalgae as sources of carotenoids. Mar Drugs 9:625–644.

  7. 7.

    Borowitzka MA (2013) High-value products from microalgae-their development and commercialisation. J Appl Phycol 25:743–756.

  8. 8.

    Sánchez JF, Fernández JM, Acién FG, Rueda A, Pérez-Parra J, Molina E (2008) Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis. Process Biochem 43:398–405.

  9. 9.

    Ben-Amotz A, Shaish A, Avron M (1989) Mode of action of the massively accumulated β-carotene of Dunaliella bardawil in protecting the alga against damage by excess irradiation. Plant Physiol 91:1040–1043.

  10. 10.

    Mulders KJM, Lamers PP, Martens DE, Wijffels RH (2014) Phototrophic pigment production with microalgae: biological constraints and opportunities. J Phycol 50:229–242.

  11. 11.

    Siegel BZ, Siegel SM (1973) The chemical composition of algal cell walls. CRC Crit Rev Microbiol 3:1–26.

  12. 12.

    Becker B, Melkonian M, Kamerling JP (1998) The cell wall (theca) of Tetraselmis striata (chlorophyta): Macromolecular composition and structural elements of the complex polysaccharides. J Phycol 34:779–787.

  13. 13.

    Fernández-Sevilla JM, Fernández AFG, Grima ME (2010) Biotechnological production of lutein and its applications. Appl Microbiol Biotechnol 86:27–40.

  14. 14.

    Saini RK, Keum YS (2018) Carotenoid extraction methods: a review of recent developments. Food Chem 240:90–103.

  15. 15.

    Arvayo-Enríquez H, Mondaca-Fernández I, Gortárez-Moroyoqui P, López-Cervantes J, Rodríguez-Ramírez R (2013) Carotenoids extraction and quantification: a review. Anal Methods 5:2916–2924.

  16. 16.

    Pereira H, Gangadhar KN, Schulze PSC, Santos T, de Sousa CB, Schueler LM, Custódio L, Malcata FX, Gouveia L, Varela JCS, Barreira L (2016) Isolation of a euryhaline microalgal strain, Tetraselmis sp. CTP4, as a robust feedstock for biodiesel production. Sci Rep 6:35663.

  17. 17.

    Pereira H, Páramo J, Silva J, Marques A, Barros A, Maurício D, Santos T, Schulze P, Barros R, Gouveia L, Barreira L, Varela J (2018) Scale-up and large-scale production of Tetraselmis sp. CTP4 (Chlorophyta) for CO2 mitigation: from an agar plate to 100–m3 industrial photobioreactors. Sci Rep 8:5112.

  18. 18.

    Schulze PSC, Carvalho CFM, Pereira H, Gangadhar KN, Schüler LM, Santos TF, Varela JCS, Barreira L (2017) Urban wastewater treatment by Tetraselmis sp. CTP4 (Chlorophyta). Bioresour Technol 223:175–183.

  19. 19.

    Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 11:591–592.

  20. 20.

    Couso I, Vila M, Vigara J, Cordero BF, Vargas MÁ, Rodríguez H, León R (2012) Synthesis of carotenoids and regulation of the carotenoid biosynthesis pathway in response to high light stress in the unicellular microalga Chlamydomonas reinhardtii. Eur J Phycol 47:223–232.

  21. 21.

    del Cerón-García M, Campos-Pérez I, Macías-Sánchez MD, Bermejo-Román R, Fernández-Sevilla JM, Molina-Grima E (2010) Stability of carotenoids in Scenedesmus almeriensis biomass and extracts under various storage conditions. J Agric Food Chem 58:6944–6950.

  22. 22.

    Ryckebosch E, Muylaert K, Eeckhout M, Ruyssen T, Foubert I (2011) Influence of drying and storage on lipid and carotenoid stability of the microalga Phaeodactylum tricornutum. J Agric Food Chem 59:11063–11069.

  23. 23.

    Taucher J, Baer S, Schwerna P, Hofmann D, Hümmer M, Buchholz R, Becker A (2016) Cell disruption and pressurized liquid extraction of carotenoids from microalgae. Thermodyn Catal 7:1–7.

  24. 24.

    Hu CW, Te CL, Yu PC, Chen CNN (2013) Pigment production by a new thermotolerant microalga Coelastrella sp. F50. Food Chem 138:2071–2078.

  25. 25.

    Tsai H-P, Chuang L-T, Chen C-NN (2016) Production of long chain omega-3 fatty acids and carotenoids in tropical areas by a new heat-tolerant microalga Tetraselmis sp. DS3. Food Chem 192:682–690.

  26. 26.

    Goiris K, Van Colen W, Wilches I, León-Tamariz F, De Cooman L, Muylaert K (2015) Impact of nutrient stress on antioxidant production in three species of microalgae. Algal Res 7:51–57.

  27. 27.

    Garrido JL, Rodríguez F, Zapata M (2009) Occurence of loroxanthin, loroxanthin decenoate, and loroxanthin dodecenoate in Tetraselmis species (Prasinophyceae, Chlorophyta). J Phycol 45:366–374.

  28. 28.

    Wright SW, Jeffrey SW, Mantoura RFC (1997) Evaluation of methods and solvents for pigment extraction. In: Jeffrey SW, Wright SW, Mantoura RFC (eds) Phytoplankton pigments in oceanogpaphy: guidelines to modern methods. UNESCO Publishing, Paris, pp 261–282

  29. 29.

    Zapata M, Rodríguez F, Garrido JL (2000) Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a. Mar Ecol Prog Ser 195:29–45.

  30. 30.

    Di Lena G, Casini I, Lucarini M, Lombardi-Boccia G (2019) Carotenoid profiling of five microalgae species from large-scale production. Food Res Int 120:810–818.

  31. 31.

    Petrier C, Jeunet A, Luche JL, Reverdy G (1992) Unexpected frequency effects on the rate of oxidative processes induced by ultrasound. J Am Chem Soc 114:3148–3150.

  32. 32.

    Geciova J, Bury D, Jelen P (2002) Methods for disruption of microbial cells for potential use in the dairy industry: a review. Int Dairy J 12:541–553.

  33. 33.

    Schwenzfeier A, Wierenga PA, Gruppen H (2011) Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp. Bioresour Technol 102:9121–9127.

  34. 34.

    Spiden EM, Yap BHJ, Hill DRA, Kentish SE, Scales PJ, Martin GJO (2013) Bioresource technology quantitative evaluation of the ease of rupture of industrially promising microalgae by high pressure homogenization. Bioresour Technol 140:165–171.

  35. 35.

    Khachik F, Beecher GR, Whittaker NF (1986) Separation, identification, and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. J Agric Food Chem 34:603–616.

  36. 36.

    Rivera S, Canela R (2012) Influence of sample processing on the analysis of carotenoids in maize. Molecules 17:11255–11268.

  37. 37.

    van Leeuwe MA, Villerius LA, Roggeveld J, Visser RJW, Stefels J (2006) An optimized method for automated analysis of algal pigments by HPLC. Mar Chem 102:267–275.

  38. 38.

    Zapata M, Garrido JL (1991) Influence of injection conditions in reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids. Chromatographia 31:589–594.

  39. 39.

    Wright S, Jeffrey S, Mantoura R, Llewellyn C, Bjornland T, Repeta D, Welschmeyer N (1991) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser 77:183–196.

  40. 40.

    Porra RJ, Pfündel EE, Engel N (1997) Metabolism and function of photosynthetic pigments. In: Jeffrey SW, Wright SW, Mantoura RFC (eds) Phytoplankton pigments in oceanogpaphy: guidelines to modern methods. UNESCO Publishing, Paris, pp 85–126

  41. 41.

    Ahmed F, Fanning K, Netzel M, Turner W, Li Y, Schenk PM (2014) Profiling of carotenoids and antioxidant capacity of microalgae from subtropical coastal and brackish waters. Food Chem 165:300–306.

  42. 42.

    Craft NE, Soares JH (1992) Relative solubility, stability, and absorptivity of lutein and β-carotene in organic solvents. J Agric Food Chem 40:431–434.

  43. 43.

    Chen CY, Jesisca HC, Lee DJ, Chang CH, Chang JS (2016) Production, extraction and stabilization of lutein from microalga Chlorella sorokiniana MB-1. Bioresour Technol 200:500–505.

  44. 44.

    Efsa ANS (2012) Scientific opinion on the re-evaluation of butylated hydroxytoluene BHT (E 321) as a food additive. EFSA J 10:2588.

  45. 45.

    Goiris K, Muylaert K, Fraeye I, Foubert I, De Brabanter J, De Cooman L (2012) Antioxidant potential of microalgae in relation to their phenolic and carotenoid content. J Appl Phycol 24:1477–1486.

  46. 46.

    Batista AP, Gouveia L, Bandarra NM, Franco JM, Raymundo A (2013) Comparison of microalgal biomass profiles as novel functional ingredient for food products. Algal Res 2:164–173.

  47. 47.

    Mulders KJM, Weesepoel Y, Bodenes P, Lamers PP, Vincken J, Martens DE, Gruppen H, Wijffels RH (2015) Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study. J Appl Phycol 27:125–140.

  48. 48.

    León R, Vila M, Hernánz D, Vílchez C (2005) Production of phytoene by herbicide-treated microalgae Dunaliella bardawil in two-phase systems. Biotechnol Bioeng 92:695–701.

  49. 49.

    Lamers PP, van de Laak CCW, Kaasenbrood PS, Lorier J, Janssen M, De Vos RCH, Bino RJ, Wijffels RH (2010) Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnol Bioeng 106:638–648.

  50. 50.

    Huang JJ, Bunjamin G, Teo ES, Ng DB, Lee YK (2016) An enclosed rotating floating photobioreactor (RFP) powered by flowing water for mass cultivation of photosynthetic microalgae. Biotechnol Biofuels 9:218.

  51. 51.

    Castro-Puyana M, Herrero M, Urreta I, Mendiola JA, Cifuentes A, Ibáñez E, Suárez-Alvarez S (2013) Optimization of clean extraction methods to isolate carotenoids from the microalga Neochloris oleoabundans and subsequent chemical characterization using liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 405:4607–4616.

  52. 52.

    Lourenço SO, Marquez UML, Mancini-Filho J, Barbarino E, Aidar E (1997) Changes in biochemical profile of Tetraselmis gracilis I. Comparison of two culture media. Aquaculture 148:153–168.

  53. 53.

    Dahmen-Ben Moussa I, Chtourou H, Karray F, Sayadi S, Dhouib A (2017) Nitrogen or phosphorus repletion strategies for enhancing lipid or carotenoid production from Tetraselmis marina. Bioresour Technol 238:325–332.

  54. 54.

    Sansone C, Galasso C, Orefice I, Nuzzo G, Luongo E, Cutignano A, Romano G, Brunet C, Fontana A, Esposito F, Ianora A (2017) The green microalga Tetraselmis suecica reduces oxidative stress and induces repairing mechanisms in human cells. Natl Publ.

  55. 55.

    Abiusi F, Sampietro G, Marturano G, Biondi N, Rodolfi L, D’Ottavio M, Tredici MR (2014) Growth, photosynthetic efficiency, and biochemical composition of Tetraselmis suecica F&M-M33 grown with LEDs of different colors. Biotechnol Bioeng 111:956–964.

  56. 56.

    Borghini F, Colacevich A, Bergamino N, Micarelli P, Dattilo AM, Focardi S, Focardi S, Loiselle SA (2009) The microalgae Tetraselmis suecica in mesocosms under different light regimes. Chem Ecol 25:345–357.

  57. 57.

    El-Kassas HY, El-Sheekh MM (2016) Induction of the synthesis of bioactive compounds of the marine alga Tetraselmis tetrathele (West) Butcher grown under salinity stress. Egypt J Aquat Res 42:385–391.

  58. 58.

    Egeland ES, Eikrem W, Throndsen J, Wilhelm C, Zapata M, Liaaen-Jensen S (1995) Carotenoids from further prasinophytes. Biochem Syst Ecol 23:747–755.

  59. 59.

    Dammak M, Hadrich B, Miladi R, Barkallah M, Hentati F, Hachicha R, Laroche C, Michaud P, Fendri I, Abdelkafi S (2017) Effects of nutritional conditions on growth and biochemical composition of Tetraselmis sp. Lipids Health Dis 16:41.

  60. 60.

    Maadane A, Merghoub N, Ainane T, El Arroussi H, Benhima R, Amzazi S, Bakri Y, Wahby I (2015) Antioxidant activity of some Moroccan marine microalgae: Pufa profiles, carotenoids and phenolic content. J Biotechnol 215:13–19.

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The authors are indebted to the Foundation for Science and Technology (FCT), Portugal, for funding through UID/Multi/04326/2019 research program and a doctoral research grant (SFRH/BD/115325/2016) awarded to LS. Further funding was provided by the 0055 ALGARED + 05 INTERREG V-A–España Portugal project. KNG also is thankful to the FCT and University of Algarve for financial support under the transitional rule of Decree-Law no. 57/2016 as amended by Law No 57/2017.

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Correspondence to Luísa Barreira.

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Schüler, L.M., Gangadhar, K.N., Duarte, P. et al. Improvement of carotenoid extraction from a recently isolated, robust microalga, Tetraselmis sp. CTP4 (chlorophyta). Bioprocess Biosyst Eng (2020).

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  • Marine microalgae
  • Glass beads
  • Lutein
  • Wet biomass