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

Bioprocess and Biosystems Engineering volume 43, pages 785–796 (2020)Cite this article

Abstract

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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References

  1. Varela JC, Pereira H, Vila M, León R (2015) Production of carotenoids by microalgae: achievements and challenges. Photosynth Res 125:423–436. https://doi.org/10.1007/s11120-015-0149-2

    CAS  Article  PubMed  Google Scholar 

  2. Johnson EJ (2002) The role of carotenoids in human health. Nutr Clin Care 5:56–65. https://doi.org/10.1046/j.1523-5408.2002.00004.x

    Article  PubMed  Google Scholar 

  3. Fiedor J, Burda K (2014) Potential role of carotenoids as antioxidants in human health and disease. Nutrients 6:466–488. https://doi.org/10.3390/nu6020466

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Eggersdorfer M, Wyss A (2018) Carotenoids in human nutrition and health. Arch Biochem Biophys 652:18–26. https://doi.org/10.1016/j.abb.2018.06.001

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.3390/md9030319

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Guedes AC, Amaro HM, Malcata FX (2011) Microalgae as sources of carotenoids. Mar Drugs 9:625–644. https://doi.org/10.3390/md9040625

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Borowitzka MA (2013) High-value products from microalgae-their development and commercialisation. J Appl Phycol 25:743–756. https://doi.org/10.1007/s10811-013-9983-9

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/j.procbio.2008.01.004

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1104/pp.91.3.1040

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Mulders KJM, Lamers PP, Martens DE, Wijffels RH (2014) Phototrophic pigment production with microalgae: biological constraints and opportunities. J Phycol 50:229–242. https://doi.org/10.1111/jpy.12173

    CAS  Article  PubMed  Google Scholar 

  11. Siegel BZ, Siegel SM (1973) The chemical composition of algal cell walls. CRC Crit Rev Microbiol 3:1–26. https://doi.org/10.3109/10408417309108743

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1046/j.1529-8817.1998.340779.x

    CAS  Article  Google Scholar 

  13. Fernández-Sevilla JM, Fernández AFG, Grima ME (2010) Biotechnological production of lutein and its applications. Appl Microbiol Biotechnol 86:27–40. https://doi.org/10.1007/s00253-009-2420-y

    CAS  Article  PubMed  Google Scholar 

  14. Saini RK, Keum YS (2018) Carotenoid extraction methods: a review of recent developments. Food Chem 240:90–103. https://doi.org/10.1016/j.foodchem.2017.07.099

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1039/c3ay26295b

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1038/srep35663

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1038/s41598-018-23340-3

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1016/j.biortech.2016.10.027

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1042/bst0110591

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1080/09670262.2012.692816

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1021/jf100020s

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1021/jf2025456

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.4172/2158-7544.1000158

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/j.foodchem.2012.11.133

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1016/j.foodchem.2015.07.071

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1016/j.algal.2014.12.002

    Article  Google Scholar 

  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. https://doi.org/10.1111/j.1529-8817.2009.00660.x

    CAS  Article  PubMed  Google Scholar 

  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

    Google Scholar 

  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. https://doi.org/10.3354/meps195029

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/j.foodres.2018.11.043

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1021/ja00034a077

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/S0958-6946(02)00038-9

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/j.biortech.2011.07.046

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1016/j.biortech.2013.04.074

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1021/jf00070a006

    CAS  Article  Google Scholar 

  36. Rivera S, Canela R (2012) Influence of sample processing on the analysis of carotenoids in maize. Molecules 17:11255–11268. https://doi.org/10.3390/molecules170911255

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1016/j.marchem.2006.05.003

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1007/BF02279480

    CAS  Article  Google Scholar 

  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. https://doi.org/10.3354/meps077183

    CAS  Article  Google Scholar 

  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

    Google Scholar 

  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. https://doi.org/10.1016/j.foodchem.2014.05.107

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1021/jf00015a013

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/j.biortech.2015.10.071

    CAS  Article  PubMed  Google Scholar 

  44. Efsa ANS (2012) Scientific opinion on the re-evaluation of butylated hydroxytoluene BHT (E 321) as a food additive. EFSA J 10:2588. https://doi.org/10.2903/j.efsa.2012.2588

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1007/s10811-012-9804-6

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/j.algal.2013.01.004

    Article  Google Scholar 

  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. https://doi.org/10.1007/s10811-014-0333-3

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1002/bit.20660

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1002/bit.22725

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1186/s13068-016-0633-8

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1007/s00216-012-6687-y

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1016/S0044-8486(96)01416-0

    Article  Google Scholar 

  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. https://doi.org/10.1016/j.biortech.2017.04.008

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1038/srep41215

    Article  Google Scholar 

  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. https://doi.org/10.1002/bit.25014

    CAS  Article  PubMed  Google Scholar 

  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. https://doi.org/10.1080/02757540903193148

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1016/j.ejar.2016.10.006

    Article  Google Scholar 

  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. https://doi.org/10.1016/0305-1978(95)00075-5

    CAS  Article  Google Scholar 

  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. https://doi.org/10.1186/s12944-016-0378-1

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1016/j.jbiotec.2015.06.400

    CAS  Article  PubMed  Google Scholar 

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Funding

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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Authors and Affiliations

  1. Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal

    Lisa M. Schüler, Katkam N. Gangadhar, Paulo Duarte, Chloé Placines, João Varela & Luísa Barreira

  2. Department of Chemistry, Biochemistry, University of Huelva, Avda de las Fuerzas Armadas s/n, 21071, Huelva, Spain

    Ana María Molina-Márquez & Rosa Léon-Bañares

  3. CIMA, Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, 8005-139, Faro, Portugal

    Vânia S. Sousa

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  1. Lisa M. Schüler
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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 43, 785–796 (2020). https://doi.org/10.1007/s00449-019-02273-9

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Keywords

  • Marine microalgae
  • Glass beads
  • Lutein
  • RP-HPLC
  • Wet biomass