Abstract
Long-term phenotypic and genotypic stability is a fundamental prerequisite for the successful biotechnological exploitation of any micro-organism, as without stable starter cultures productivity can not be guaranteed. In this study two biotechnological algal production strains; Porphyridium cruentum A-408, which produces zeaxanthin, and Planktothrix A-404, which produces a potent cytotoxin, were successfully cryopreserved using a two step protocol (cooling to −40 ∘C prior to plunging into liquid nitrogen). Post-thaw viability levels of 114 ± 27% were obtained for P. cruentum A-408 and 67 ± 18% for Planktothrix A-404. Unchanged productivity levels of Zeaxanthin and beta-carotene (77% and 12% of total carotenoids respectively) were obtained in batches of P. cruentum A-408 produced from standard (serial transfer) and utilizing post-thaw (ex-cryopreserved) inocula. In addition, cytotoxin production by Planktothrix A-404 was not influenced by the origin of the inoculum, with standard (serial transfer) and post-thaw (ex-cryopreserved) inocula giving high levels of activity.
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Andersen RA (2002) The Provasoli-Guillard National Center for Culture of Marine Phytoplankton: past, present and future. Abstracts of Algae 2002 satellite symposium “Culture Collection and Environmental Researches” National Institute for Environmental Studies (NIES), Tsukuba, Japan 23 July 2002.
Baudimant G, Maurice M, Landrein A, Durand G, Durand P (1996) Purification of phosphatidylcholine with a high content of DHA from squid by counter-current chromatography. J. Liq. Chrom. & Rel. Technol. 19: 1793–1804.
Bhosale P (2003) Environmental and cultural stimulants in the production of carotenoids from microorganisms. Appl. Microbiol. Biotechnol. 63: 351–361.
Bodas K, Brennig C, Diller KR, Brand JJ (1995) Cryopreservation of blue-green and eukaryotic algae in the culture collection at the University of Texas at Austin. CryoLetters 16: 267–74.
Borowitzka MA (1988) Vitamins and fine chemicals from micro-algae. In: Borowitzka MA, Borowitzka LJ (eds.), Microalgal Biotechnology. Cambridge Univ. Press Cambridge, UK, pp. 153–196.
Borowitzka MA (1995) Microalgae as sources of pharmaceuticals and other biologically active compounds. J. appl. Phycol. 7: 3–15.
Brand JJ, Diller KR (2004) Principles and applications of algal cryopreservation. Nova Hedwigia 79: 175–190.
Cannell RJP (1993) Algae as a source of biologically active products. Pesticide Sci. 39: 147–153.
Cohen Z (ed.)(1999a) Chemicals from Microalgae. Taylor & Francis Ltd, London, p. 419.
Cohen Z (1999b) Porphyridium cruentum. In: Cohen Z (ed.), Chemicals from Microalgae. Taylor & Francis Ltd, London, pp. 1–24.
Day JG (1999) Conservation strategies for algae. In: Benson EE (ed.), Plant conservation biotechnology. Taylor & Francis Ltd., London, pp 111–124.
Day JG, Brand JJ (2005) Cryopreservation Methods for Maintaining Cultures. In: Andersen RA (ed.), Algal Culturing Techniques. Academic Press, New York, pp. 165–187.
Day JG, DeVille MM (1995) Cryopreservation of algae. Methods in Molecular Biology 38: 81–89.
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.
Day JG, Watanabe MM, Morris GJ, Fleck RA, McLellan MR (1997) Long-term viability of preserved eukaryotic algae. J. Appl. Phycol. 9: 121–127.
Downing TG, Sember CS, Gehringer MM, Leukes W (2005) Medium N: P ratios and specific growth rate comodulate microcystin and protein content in Microcystis aeruginosa PCC7806 and M-aeruginosa UV027. Microbial Ecology 49: 468–473.
Fleck RA (1998) The Assessment of Cell Damage and Recovery in Cryopreserved Freshwater Protists. PhD Thesis. Univ. Abertay Dundee, p. 393.
Fleck RA, Benson EE, Bremner 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 new approach for developing protistan cryopreservation strategies. Free Radical Res. 32: 157–170.
Fleck RA, Benson EE, Bremner DH, Day JG (2003) Studies of antioxidant protection in freeze-tolerant and freeze-sensitive microalgae: Applications in cryopreservation protocol development. CryoLetters 24: 213–228.
Fleck RA, Day JG, Clarke KJ, Benson EE (1999) Elucidation of the metabolic and structural basis for the cryopreservation recalcitrance of Vaucheria sessilis. CryoLetters 20: 271–282.
Friedl T, Lorenz M (2002) The SAG culture collection: Microalgal biodiversity and phylogeny research. In: Abstracts of Culture Collections of Algae: increasing accessibility and exploring Algal Biodiversity. September 2–6, Sammlung von Algenkulturen at Göttingen University (SAG).
Gilboa A, Ben-Amotz A (1979) An improved method for rapid assaying of viability of cryopreserved unicellular algae. Pl. Sci. Lett. 9: 23–25.
Grout BWW (1995) Introduction to the in vitro preservation of plant cells, tissues and organs. In: Grout B (ed.), Genetic Preservation of Plant Cells In Vitro, Springer, Berlin, pp. 1–20.
Hall TJ, James PR, Cambridge G (1993) Development of an in vitro hepatotoxicity assay for assessing the effects of chronic drug exposure. Res. Comm. Chem. Pathol. Pharmacol. 79: 249–256.
Harding K, Day JG, Lorenz M, Timmermann H, Friedl T, Bremner DH, Benson EE (2004) Introducing the concept and application of vitrification for the cryo-conservation of algae “A Mini Review”. Nova Hedwigia 79: 207–226.
Holm-Hansen O (1963) Viability of blue-green algae after freezing. Physiolo. Plant. 16: 530–539.
Jaworski GHM, Wiseman SW, Reynolds CS (1988) Variability in sinking rate of the freshwater diatom Asterionella formosa: the influence of colony morphology. Br. Phycol. J. 23: 167–176.
Kay RA (1991) Microalgae as food and supplement. Crit. Rev. Food Sci. Nutr. 30: 555–573.
Kirsop B, Doyle A (1991) Maintenance of Microorganisms and Cultured Cells. Academic Press Ltd., London, p. 308.
Kyle DJ, Arterburn LM (1998) Single cell oil sources of docosahexaenoic acid: clinical studies. World Rev. Nutr. Diet. 83: 116–131.
Lee JJ, Soldo AT (1992) Protocols in Protozoology. Society of Protozoologists, Kansas, USA.
Lehtimaki J, Moisander P, Sivonen K, Kononen K (1997) Growth, nitrogen fixation, and nodularin production by two Baltic sea cyanobacteria. Appl. Environ. Microbiol. 63: 1647–1656.
Li Q, Sham HL (2002) Discovery and development of antimitotic agents that inhibit tubulin polymerisation for the treatment of cancer. Expert Opinion on Therapeutic Patients 12: 1663–1702.
Lorenz RT, Cysewski GR (2000) Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol. 18: 160–167.
Lukavský J, Elster J (2002) CCALA Trebon 1913–2002. Culture Collections of Algae: increasing accessibility and exploring Algal Biodiversity. In: Abstracts of Culture Collections of Algae: increasing accessibility and exploring Algal Biodiversity, September 2–6 2002, Sammlung von Algenkulturen at Göttingen University (SAG).
Mlouka A, Comte K, Castets AM, Bouchier C, Tandeau de Marsac N (2004) The gas vesicle gene cluster from Microcystis aeruginosa and DNA rearrangements that lead to loss of cell buoyancy. J. Bacteriol. 186: 2355–2365.
Morris GJ (1976) The cryopreservation of Chlorella 1. Interactions of rate of cooling, protective additive and warming rate. Arch. Microbiol. 107: 57–62.
Morris GJ (1978) Cryopreservation of 250 strains of Chlorococcales by the method of two step cooling. Br. Phycol. J. 13:15–24.
Morris GJ (1981) Cryopreservation: An Introduction to Cryopreservation in Culture Collections. Institute of Terrestrial Ecology, Cambridge, UK, p. 27.
Morris GJ, McGrath JJ (1981) Intracellular ice nucleation and gas bubble formation in Spirogyra. CryoLetters 2: 341–52.
Moore RE (1996) Cyclic peptides and depsipeptides from cyanobacteria: a review. J. Ind. Microbiol. 16: 134–143.
Muhling M, Belay A, Whitton B (2005) Screening Arthrospira (Spirulina) strains for heterotrophy. J. Appl. Phycol. 17: 129–135.
Muir JC, Pattenden G, Ye T (2002) Total synthesis of (+) – curacin A, a novel antimiotic metabolite from a cyanobacterium. J. Chem. Soc. Perk. 1: 2243–2250.
Müller J, Friedl T, Hepperle D, Lorenz M, Day JG (2005) Distinction of isolates among multiple strains of Chlorella vulgaris (Chlorophyta, Trebouxiophyceae) and Testing Conspecificity with Amplified Fragment Length Polymorphism and ITS RDNA sequences. J. Phycol. 41: (in press).
Oh-Hama T, Miyachi S (1988) Chlorella. In: Borowitzka MA, Borowitzka LJ (eds.), Microalgal Biotechnology. Cambridge Univ. Press Cambridge, UK, pp. 3–26.
Osorio H, Laranjeriro N, Santos LMA, Santos MF (2004) First attempts at cryopreservation of ACOI strains and use of image analysis to assess viability. Nova Hedwigia 79: 227–236.
Panda D, DeLuca K, Williams D, Jordan MA, Wilson SL (1998) Antiproliferative mechanism of action of cryptophycin-52: Kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends. Proc. Natl. Acad. Sci. 95: 9313–9318.
Pearson BM, Jackman PJH, Painting KA, Morris GJ (1990) Stability of genetically manipulated yeasts under different cryopreservation regimes. CryoLetters 11: 205–210.
Phillip D, Hobe S, Molnar P, Paulsen H, Hashimoto H, Young AJ (2002) The binding of xanthophylls to the bulk light-harvesting complex of photosystem II in higher plants: a specific requirement for carotenoids with a 3-OH-β-end group. J. Biol. Chem. 277: 25160–25169.
Richmond A (1988) Spirulina. In: Borowitzka MA, Borowitzka LJ (eds.), Microalgal Biotechnology. Cambridge Univ. Press Cambridge, UK, pp. 85–121.
Rippka R, Iteman I, Coursin T, Comte K, Singer A, Araoz R, Laurent T, Herdman M, Tandeau de Marsac N (2002) Recent progress in the Pasteur Culture Collection of Cyanobacteria. In: Abstracts of Culture Collections of Algae: increasing accessibility and exploring Algal Biodiversity. September 2–6 2002, Sammlung von Algenkulturen at Göttingen University (SAG).
Roberts S, Grout BWW, Morris GJ (1987) Consecutive observations of a frozen cell sample by cryogenic light microscopy and cryogenic scanning electron microscopy. CryoLetters 8: 122–129.
Ryan MJ, Jeffries P, Bridge PD, Smith D (2001) Developing cryopreservation protocols to secure fungal gene function. CryoLetters 22: 115–124.
Ryan MJ, Smith D, Bridge PD, Jeffries P (2003) The relationship between fungal preservation method and secondary metabolite production in Metarhizium anisopliae and Fusarium oxysporum. World J. Microbiol. Biotechnol. 19: 839–844.
Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Chroocoococcales). Bact. Rev. 35: 171–205.
Tompkins J, DeVille MM, Day JG, Turner MF (1995) Culture Collection of Algae and Protozoa Catalogue of strains (6th ed.), Culture Collection of Algae and Protozoa, Ambleside, p. 208.
Walsh JR, Diller KR, Brand JJ (2004) Measurement and simulation of water and methanol transport in algal cells. J. Biomech. Engin. 126: 167–179.
Warren A, Day JG, Brown S (2002) Cultivation of Protozoa and Algae. In: Hurst CJ, Crawford RL, Knudsen GR, McInerney MJ, Stezenbach LD (eds.), Manual of Environmental Microbiology (2nd ed.), ASM Press, Washington D.C., pp. 71–83.
Watanabe MM, Shimizu A, Satake K (1992) NIES-Microbial Culture Collection at the National Institute of Environmental Studies: Cryopreservation and database of culture strains of microalgae. In: Watanabe MM (ed.), Proceedings of Symposium on Culture Collection of Algae. NIES, Tsukuba, Japan, pp. 33–41.
Whitelam G, Codd GA (1983) Photoinhibition of photosynthesis and in vivo chlorophyll fluorescence in the green alga Ankistrodesmus braunii. Plant Cell Physiol. 25: 465–471.
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Hédoin, H., Pearson, J., Day, J.G. et al. Porphyridium cruentum A-408 and Planktothrix A-404 retain their capacity to produce biotechnologically exploitable metabolites after cryopreservation. J Appl Phycol 18, 1–7 (2006). https://doi.org/10.1007/s10811-005-9007-5
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DOI: https://doi.org/10.1007/s10811-005-9007-5