Journal of Applied Phycology

, Volume 17, Issue 5, pp 413–422 | Cite as

Cryopreservation of the coccolithophore, Emiliania huxleyi (Haptophyta, Prymnesiophyceae)

  • Aude Houdan
  • Benoît Véron
  • Pascal Claquin
  • SéBastien Lefebvre
  • Jean-Marc Poncet


We studied the cryopreservation of the most common coccolithophore, Emiliania huxleyi which is considered as one of the main global carbon cycle participants. Both stages of this complex life cycle species were submitted to gradual addition of three distinct cryoprotectants: dimethylsulfoxide (7.5% v/v), methanol (5% v/v) and proline (0.5 M). They were then control-rate cooled (−5 °C min−1) to −50 °C before plunging into liquid nitrogen. Free radical oxygen species have been proposed to occur in cells subjected to pre-freezing manipulation or to cooling. Therefore, catalase (preventing accumulation of hydroxyl radicals) was evaluated for its ability to improve cell viability before and after freezing-thawing challenge.

With the exception of proline which induced a decrease in diploid cell proliferation, cryoprotectants had no deleterious effects. On the contrary, growth of the haploid stage was enhanced by each CPA treatment, suggesting mixotrophic growth. Cryopreservation succeeded when dimethylsulfoxide was used, and the late exponential phase was obtained as soon as the 15th post-thawing day. Cell densities were then similar to the unfrozen controls. Catalase had no beneficial effect on the ability of cells to grow, neither prior freezing nor after thawing. In comparison with former attempts to cryopreserve E. huxleyi in other culture collection centers, our protocols allowed faster recovery.

Key words

cryopreservation Emiliania huxleyi life cycle quantum efficiency of PSII viability 


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  1. Andersen RA (1996) Algae. In Hunter-Cevra JC, Belt A (eds), Maintaining Cultures for Biotechnology and Industry. Academic Press, San Diego, pp. 29–64.Google Scholar
  2. Baumber J, Ball BA, Linfor JJ, Meyers SA (2003) Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. J. Androl. 24: 621–628.Google Scholar
  3. Brand JJ, Diller KR (2004) Application and theory of algal cryopreservation. Nova Hedwigia 79: 175–189.Google Scholar
  4. Cañavate JP, Lubian LM (1994) Tolerance of six marine microalgae to the cryoprotectants dimethyl sulfoxide and methanol. J. Phycol. 30: 559–565.Google Scholar
  5. Cañavate JP, Lubian LM (1995) Some aspects on the cryopreservation of microalgae used as food for marine species. Aquaculture 136: 277–290.Google Scholar
  6. Chen THH, Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr. Op. Plant Biol. 5: 250–257.Google Scholar
  7. CODENET (2004) Part 2 : Physiology and bloom studies; In Thierstein HR, Young JR (eds), Coccolithophores – From molecular processes to global impact. SpringerVerlag, Germany, pp. 75–249.Google Scholar
  8. Cros L, Kleijne A, Zeltner A, Billard C, Young JR (2000) New examples of holococcolith-heterococcolith combination coccospheres and their implications for coccolithophorid biology. Mar. Micropalaent. 39: 1–34.Google Scholar
  9. Crutchfield ALM, Diller KR, Brand JJ (1999) Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta). Eur. J. Phycol. 34: 43–52.Google Scholar
  10. Day JG, Fleck RA, Benson EE (2000) Cryopreservation-recalcitrance in microalgae: Novel approaches to identify and avoid cryo-injury. J. Appl. Phycol. 12: 369–377.Google Scholar
  11. Edvardsen B, Eikrem W, Green JC, Andersen RA, Moon-van der Staay SY, Medlin LK (2000) Phylogenetic reconstructions of the Haptophyta inferred from 18S ribosomal DNA sequences and available morphological data. Phycologia 39: 19–35.Google Scholar
  12. Fleck RA, Benson EE, Bremmer DH, Day JG (2000) Studies of free radical-mediated cryoinjury in the unicellular green alga Euglena gracilis using a non-destructive hydroxyl radical assay: A novel approach for developing protistan cryopreservation strategies. Free Rad. Res. 32: 157–170.Google Scholar
  13. Fleck RA, Benson EE, Bremmer DH, Day JG (2003) A comparative study of antioxidant protection in cryopreserved unicellular algae. Cryo-Lett. 24: 213–228.Google Scholar
  14. Fleck RA, Day JC, Clarke KJ, Benson EE (1999) Elucidation of the metabolic and structural basis for the cryopreservation recalcitrance of Vaucheria sessilis. Cryo-Lett. 20: 271–282.Google Scholar
  15. Fujiwara S, Tsuzuki M, Kawachi M, Minaka N, Inouye I (2001) Molecular phylogeny of the Haptophyta based on the rbcl gene and sequence variation in the spacer region of the rubisco operon. J. Phycol. 37: 121–129.Google Scholar
  16. Geisen M, Billard C, Broerse ATC, Cros L, Probert I, Young J (2002) Life cycle associations involving pairs of holococcolithophorid species: Intraspecific variation or cryptic speciation? Eur. J. Phycol. 37: 531–550.Google Scholar
  17. Green JC, Course PA, Tarran GA (1996) The life cycle of Emiliania huxleyi: A brief review and a study of relative ploidy levels analysed by flow cytometry. J. Marine Syst. 9: 33–44.Google Scholar
  18. Helliot B, Mortain-Bertrand A (1999) Accumulation of proline in Dunaliella salina (Chlorophyceae) in response to light transition and cold adaptation. Effect on cryopreservation. Cryo-Lett. 20: 287–296.Google Scholar
  19. Houdan (2003) Les cycles de vie et les stratégies de développement chez les coccolithophores (Haptophycée). Implications écologiques. Thesis, Université de Caen Basse-Normandie, p. 246.Google Scholar
  20. Houdan A, Billard C, Marie D, Not F, Sáez AG, Young JR, Probert I (2004a) Holococcolithophore-heterococcolithophore (Haptophyta) life cycles: Flow cytometric analysis of relative ploidy levels. Systemat. Biodiv. 1: 453–465.Google Scholar
  21. Houdan A, Bonnard A, Fresnel J, Fouchard S, Billard C, Probert I (2004b) Toxicity of coastal coccolithophores (Prymnesiophyceae Haptophyta). J. Plankton Res. 26: 875–883.Google Scholar
  22. Hubalek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiol. 46: 205–229.Google Scholar
  23. Joseph I, Panigrahi A, Chandra PK (2000) Tolerance of three marine microalgae to cryoprotectants dimethylsulfoxide, methanol and glycerol. Indian J. Mar. Sci. 29: 243–256.Google Scholar
  24. Keller MD, Selvin RC, Claus W, Guillard RRL (1987) Media for the culture of oceanic ultraphytoplankton. J. Phycol. 23: 633–638.Google Scholar
  25. Klaveness D (1972) Coccolithus huxleyi (Lohmann) Kamptner. I. Morphological investigations on the vegetative cell and the process of coccolith formation. Protistologica 8: 335–346.Google Scholar
  26. Klaveness D (1973) The microanatomy of Calyptrosphaera sphaeroidea, with some supplementary observations on the motile stage of Coccolithus pelagicus. Norw. J. Bot. 20: 151–162.Google Scholar
  27. Kono S, Kuwano K, Ninomiya M, Onishi J, Saga N (1997) Cryopreservation of Enteromorpha intestinalis (Ulvales, Chlorophyta) in liquid nitrogen. Phycologia 36: 76–78.Google Scholar
  28. Kono S, Kuwano K, Saga N (1998) Cryopreservation of Eisenia bicyclis (Laminariales, Phaeophyta) in liquid nitrogen. J. Mar. Biotechnol. 6: 220–223.Google Scholar
  29. Kromkamp JC, Forster RM (2003) The use of variable fluorescence measurements in aquatic ecosystems: Differences between multiple and single turnover measuring protocols and suggested terminology. Eur. J. Phycol. 38: 103–112.Google Scholar
  30. Lee PA, de Mora SJ (1999) Intracellular dimethylsulfoxide (DMSO) in unicellular marine algae: Speculations on its origin and possible biological role. J. Phycol. 35: 8–18.Google Scholar
  31. Lee PA, de Mora SJ, Gosselin M, Levasseur M, Bouillon RC, Nozais C, Michel C (2001) Particulate dimethylsufoxide in arctic sea ice algal communities: The cryoprotectant hypothesis revisited. J. Phycol. 37: 488–499.Google Scholar
  32. Malin G, Steinke M (2004) Dimethyl sulphide production: What is the contribution of the coccolithophores? In Thierstein HR, Young JR (eds), Coccolithophores – From molecular processes to global impact. SpringerVerlag, Germany, pp. 127–164.Google Scholar
  33. Mallick N, Mohn FM (2000) Reactive oxygen species: Response of algal cells. J. Plant. Physiol. 157: 183–193.Google Scholar
  34. Mazur P, Leibo S, Chu EHY (1972) A two-factor hypothesis of freezing injury. Exp. Cell Res. 71: 345–355.Google Scholar
  35. OECD (2001) Biological Resource Centers: Underpinning the future of life-sciences and biotechnology. Paris, France: Organisation for Economic Co-operation and Development. p. 68.Google Scholar
  36. Paasche E (2001) A review of the coccolithophorid Emiliania huxleyi (Prymnesiophyceae), with particular reference to growth, coccolith formation, and calcification-photosynthesis interactions. Phycologia 40: 503–529.Google Scholar
  37. Palenik B, Henson SE (1997) The use of amides and other organics nitrogen sources by the phytoplankton Emilinia huxleyi. Limnol. Oceanog. 42: 1544–1551.Google Scholar
  38. Parke M, Adams I (1960) The motile (Crystallolithus hyalinus Gaarder & Markali) and the non-motile phases in the life history of Coccolithus pelagicus (Wallich) Schiller. J. Mar. Biol. Ass. U.K. 39: 363–274.Google Scholar
  39. Parkhill JP, Maillet G, Cullen JJ (2001) Fluorescence-based maximum quantum yield for PSII as a diagnostic of nutrient stress. J. Phycol. 37: 517–529.Google Scholar
  40. Poncet J-M, Véron B (2003) Cryopreservation of the unicellular alga, Nannochloropsis oculata. Biotech. Lett. 25: 2017– 2022.Google Scholar
  41. Probert I, Houdan A (2004) The Laboratory Culture of Coccolithophores. In Thierstein HR, Young JR (eds), Coccolithophores – From molecular processes to global impact SpringerVerlag, Germany, pp. 217–249.Google Scholar
  42. Robert R, Trintignac P (1997) Microalgues et nutrition larvaire en écloserie de mollusques. Haliotis 26: 1–13.Google Scholar
  43. Rudolph AS, Crowe JH (1985) Membrane stabilization during freezing: The role of two natural cryoprotectants, trehalose and proline. Cryobiol. 22: 367–377.Google Scholar
  44. Schreiber U, Schliwa U, Bilger W (1986) Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer. Photosynt. Res. 10: 51–62.Google Scholar
  45. Takagi H, Sakai K, Morida K, Nakamori S (2000) Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 184: 103–108.Google Scholar
  46. Taylor R, Fletcher R (1999) Cryopreservation of eukaryotic algae: A review of methodologies. J. Appl. Phycol. 10: 481–501.Google Scholar
  47. Tzovenis I, Triantaphyllidis G, Naihong X, Chatzinikolaou E, Papadopoulou K, Xouri G, Tafas T (2004) Cryopreservation of marine microalgae and potential toxicity of cryoprotectants to the primary steps of the aquacultural food chain. Aquaculture 230: 457–473.Google Scholar
  48. Watanabe MM, Sawaguchi T (1995) Cryopreservation of a water-bloom forming cyanobacterium, Microcystis aeruginosa f. aeruginosa. Phycol. Res. 43: 111–116.Google Scholar
  49. Whiteley GSW, Fuller BJ, Hobbs KEF (1992) Detrioration of cold stored tissue specimens due to lipid peroxidation: Modulation by antioxidants at high subzero temperatures. Cryobiol. 29: 668–873.Google Scholar
  50. Young JR (1994) Functions of coccoliths. In Winter A, Siesser WG (eds), Coccolithophores. Cambridge University Press, Cambridge, UK. pp. 63–82.Google Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Aude Houdan
    • 1
  • Benoît Véron
    • 1
    • 3
  • Pascal Claquin
    • 1
  • SéBastien Lefebvre
    • 1
  • Jean-Marc Poncet
    • 2
  1. 1.Laboratoire de Biologie et Biotechnologies MarinesUniversité de Caen Basse-NormandieCAEN CedexFrance
  2. 2.Plateau de Cryobiologie, ISBIOUniversité de Caen Basse-NormandieCAEN CedexFrance
  3. 3.Algobank, ISBIOUniversité de Caen Basse-NormandieCAEN CedexFrance

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