Abstract
The development of cryopreservation methods for microalgae opens great prospects for marine biotechnology and aims to establish a bank of cryopreserved cultures. Eight of ten marine microalgae species used in this study (the diatoms, green, red, and golden algae), including five previously untested species, were successfully recovered after freezing to ultra-low temperatures (−196 °C) using penetrating (dimethyl sulfoxide, glycerol, and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants. We found that ethylene glycol in combination with trehalose possessed the most effective cryoprotective activity among the algae cryoprotectants tested. However, the chief factor for the successful preservation of microalgal cells during freeze–thawing was shown to be the cooling rate. Cooling was performed in two ways: step or fast droplet freezing. The droplet freezing described here was effective only for cryopreserving green algae, whereas step freezing was optimal for all other algal species. Three diatoms of the genus Attheya were successfully cryopreserved for the first time, but none of the tested protocols had a positive result for the diatoms belonging to Pseudo-nitzschia. The failure may be explained rather by peculiarities in the cell wall composition (higher content of silica and fewer organic components) than by the specific (long and thin) shape of these cells. The pigment content in all of the studied species tended to decrease after thawing as compared with unfrozen cells and increase significantly during cell recovery. Cryosensitivity of marine algae depended on the differences in natural intrinsic characteristics rather than their taxonomic position.
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Abreu L, Borges L, Marangoni J, Abreu PC (2012) Cryopreservation of some useful microalgae species for biotechnological exploitation. J Appl Phycol 24:1579–1588
Aizdaicher NA (2008) Collection of marine microalgae at the A. V. Zhirmunsky Institute of Marine Biology. Russ J Mar Biol 34:139–142
Akarasanon K, Damrongphol P, Poolsanguan W (2004) Long-term cryopreservation of spermatophore of the giant freshwater prawn, Macrobrachium rosenbergii (de Man). Aquac Res 35:1415–1420
Baust JM (2002) Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell Preserv Tech 1:17–31
Ben-Amotz A, Avron M (1973) The role of glycerol in the osmotic regulation of the halophilic alga Dunaliella parva. Plant Physiol 51:875–878
Ben-Amotz A, Gilboa A (1980) Cryopreservation of marine unicellular algae. I. A survey of algae with regard to size, culture age, photosynthetic activity and chlorophyll-to-cell ratio. Mar Ecol Prog Ser 2:157–161
Benhra A, Radetski CM, Ferard JF (1997) Cryoalgotox: use of cryopreserved alga in a semistatic microplate test. Environ Toxicol Chem 16:505–508
Benson EE (2008) Cryopreservation of phytodiversity: a critical appraisal of theory practice. Crit Rev Plant Sci 27:141–219
Bhosale P (2004) Environmental and cultural stimulants in the production of carotenoids from microorganisms. Appl Microbiol Biotechnol 63:351–361
Borowitzka MA (1995) Microalgae as sources of pharmaceuticals and other biologically active compounds. J Appl Phycol 7:3–15
Borowitzka LJ, Brown A (1974) The salt relations of marine and halophilic species of the unicellular green alga, Dunaliella. Arch Microbiol 96:37–52
Brown MR, McCausland MA, Kowalski K (1998) The nutritional value of four Australian microalgal strains fed to Pacific oyster Crassostrea gigas spat. Aquaculture 165:281–293
Cañavate JP, Lubińn LM (1995) Some aspects on the cryopreservation of microalgae used as food for marine species. Aquaculture 136:277–290
Crawford RM, Gardner C, Medlin LK (1994) The genus Attheya. I. A description of four new taxa, and the transfer of Gonioceros septentrionalis and G. armatas. Diatomol Res 9:27–51
Day JG (1998) Cryoconservation of microalgae and cyanobacteria. Cryo-Letters 1:7–14
Day JG, Brand JJ (2005) Cryopreservation methods for maintaining microalgal cultures. In: Andersen RA (ed) Algal culturing techniques. Elsevier, Amsterdam, pp 165–187
Day JG, Harding K, Nadarajan J, Benson EE (2008) Cryopreservation: conservation of bio-resources at ultra-low temperatures. In: Walker JM, Rapley R (eds) Molecular biomethods handbook, 2nd edn. Humana, Totowa, pp 917–947
De Antoni GL, Perez P, Abraham A, Anon MC (1989) Trehalose, a cryoprotectant for Lactobacillus bulgaricus. Cryobiology 26:149–153
Declerck S, Angelo-Van Coppenolle MG (2000) Cryopreservation of entrapped monoxenically produced spores of an arbuscular mycorrhizal fungus. New Phytol 148:169–176
Diniz-Mendes L, Bernardes E, de Araujo PS, Panek AD, Paschoalin VM (1999) Preservation of frozen yeast cells by trehalose. Biotechnol Bioeng 65:572–578
Eidtmann A, Schauz K (1992) Cryopreservation of protoplasts from sporidia of Ustilago maydis. Mycol Res 96:318–320
Farrant J, Walter CA, Lee H, Morris GJ, Clarke KJ (1977) Structural and functional aspects of biological freezing techniques. J Microsc 111:17–34
Fenwick C, Day JG (1992) Cryopreservation of Tetraselmis suecica cultured under different nutrients regimes. J Appl Phycol 4:105–109
Ford CW, Percival E (1965) The carbohydrates of Phaeodactylum tricornutum. Part I. Preliminary examination of the organism, and characterisation of low molecular weight material and of a glucan. J Chem Soc 1298:7035–7041
Geresh S, Adin I, Yarmolinsky E, Karpasas M (2002) Characterization of the extracellular polysaccharide of Porphyridium sp.: molecular weight determination and rheological properties. Carbohydr Polym 50:183–189
Goncharova SN, Sanina NM, Kostetsky EY (2000) Role of lipids in molecular thermoadaptation mechanisms of seagrass Zostera marina. Biochem Soc Trans 28:887–890
Gouveia L, Coutinho C, Mendonça E, Batista AP, Sousa I, Bandarra NM, Raymundo A (2008) Functional biscuits with PUFA-ω3 from Isochrysis galbana. J Sci Food Agric 88:891–896
Guillard RL (1975) Culture of phytoplankton for feeding marine invertebrates. In: Smith W, Chanley M (eds) Culture of marine invertebrate animals. Springer, NY, pp 29–60
Guillard RR, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:229–239
Hasle GR, Lange CB, Syvertsen EE (1996) A review of Pseudo-nitzschia, with special reference to the Skagerrak, North Atlantic, and adjacent waters. Helgol Meeresun 50:131–175
Heaney-Kieras J, Chapman D (1976) Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum. Carbohydr Res 52:169–177
Hecky RE, Mopper K, Kilham P, Degens ET (1973) The amino acid and sugar composition of diatom cell-walls. Mar Biol 19:323–331
Hubalek Z (2003) Protectants used in the cryopreservation of microorganisms. Cryobiology 46:205–229
Jiménez C, Pick U (1994) Differential stereoisomer compositions of β, β-carotene in thylakoids and in pigment globules in Dunaliella. J Plant Physiol 143:257–263
Kabanova YG (1961) Concerning in vitro culturing of marine planktonic diatoms and peridinea algae. Trio IO AN SSSR 47:203–216
Kana TM, Geider RJ, Critchley C (1997) Regulation of photosynthetic pigments in micro-algae by multiple environmental factors: a dynamic balance hypothesis. New Phytol 137:629–638
Katayama T, Murata AI, Taguchi S (2011) Responses of pigment composition of the marine diatom Thalassiosira weissflogii to silicate availability during dark survival and recovery. Plankon Benthos Res 6:1–11
Kiyosawa K (1993) Permeability of the Chara cell membrane for ethylene glycol, glycerol, meso-erythritol, xylitol and mannitol. Physiol Plant 88:366–371
Kono S, Kuwano K, Ninomiya M, Onishi J, Saga N (1997) Cryopreservation of Enteromorpha intestinalis (Ulvales, Chlorophyta) in liquid nitrogen. Phycologia 36:76–78
Kröger N, Poulsen N (2008) Diatoms—from cell wall biogenesis to nanotechnology. Annu Rev Genet 42:83–107
Kurokura H, Namba K, Ishikawa T (1990) Lesions of spermatozoa by cryopreservation in oyster Crassostrea gigas. Nippon Suisan Gakkaishi 56:1803–1806
Lovelock JE, Bishop MWH (1959) Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183:1394–1395
Martin-Jézéquel V, Hildebrand M, Brzezinski M (2000) Silicon metabolism in diatoms: implications for growth. J Phycol 36:821–840
Masojídek J, Torzillo G, Kopecký J, Koblížek M, Nidiaci L, Komenda J, Lukavská A, Sacchi A (2000) Changes in chlorophyll fluorescence quenching and pigment composition in the green alga Chlorococcum sp. grown under nitrogen deficiency and salinity stress. J Appl Phycol 12:417–426
Mazur P (1960) Physical factors implicated in the death of microorganisms at subzero temperatures. Ann NY Acad Sci 85:610–629
Mazur P (1970) Cryobiology: the freezing of biological systems. Science 168:939–949
Moore RE (1996) Cyclic peptides and depsipeptides from cyanobacteria: a review. J Ind Microbiol 16:134–143
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 (1981) Cryopreservation: an introduction to cryopreservation in culture collections. Institute of Terrestrial Ecology, Cambridge
Morris GJ (1986) A cryomicroscopic study of Cylindrocystis brebissonii De Bary and two species of Micrasterias Ralfs (Conjugatophyceae, Chlorophyta) during freezing and thawing. J Exp Bot 37:842–856
Nakanishi K, Deuchi K, Kuwano K (2012) Cryopreservation of four valuable strains of microalgae, including viability and characteristics during 15 years of cryostorage. J Appl Phycol 24:1381–1385
Nash T (1966) Chemical constitution and physical properties of compounds able to protect living cells against damage due to freezing and thawing. In: Meryman HT (ed) Cryobiology. Academic, London, pp 179–211
Okauchi M, Kawamuru K, Mizukami Y (1997) Nutritive value of Tahiti Isochrysis. Isochrysis sp for larval greasy back shrimp Metapenaeus ensis. Bull Natl Res Inst Aquac (Japan) 26:1–11
Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1/1:2. doi:10.1186/1746-1448-1-2
Polge C, Smith AU, Parkes AS (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666
Ponomarenko LP, Stonik IV, Aizdaicher NA, Orlova TY, Popovskaya GI, Pomazkina GV, Stonik VA (2004) Sterols of marine microalgae Pyramimonas cf. cordata (Prasinophyta), Attheya ussurensis sp. nov. (Bacillariophyta) and a spring diatom bloom from Lake Baikal. Comp Biochem Physiol B 138:65–70
Rhodes L, Smith J, Tervit R, Roberts R, Adamson J, Adams S, Decker M (2006) Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae. Cryobiology 52:152–156
Roberts R (2001) A review of settlement cues for larval abalone (Haliotis sp.). J Shellfish Res 20:571–586
Sakurada M, Tsuzuki Y, Morgavi DP, Tomita Y, Onodera R (1995) Simple method for cryopreservation of an anaerobic rumen fungus using ethylene glycol and rumen fluid. FEMS Microbiol Lett 127:171–174
Sanina NM, Kostetsky EY, Goncharova SN (2000) Thermotropic behaviour of membrane lipids from brown marine alga Laminaria japonica. Biochem Soc Trans 28:894–897
Sarokin DJ, Carpenter EJ (1982) Ultrastructure and taxonomic observations on marine isolates of the genus Nannochloris (Chlorophyceae). Bot Mar 25:483–491
Schnepf E, Drebes G (1977) The structure of the frustule of Attheya decora West (Bacillariophyceae, Biddulphiineae) with special reference to the organic compounds. Br Phycol J 12:145–154
Searcy-Bernal R, Velez-Espino LA, Anguiano-Beltran C (2001) Effect of biofilm density on grazing and growth rates of Haliotis fulgens post larvae. J Shellfish Res 20:587–591
Simione FP Jr, Daggett PM (1977) Recovery of a marine dinoflagellate following controlled and uncontrolled freezing. Cryobiology 14:362–366
Sournia A (1974) Circadian periodicities in natural populations of marine phytoplankton. Adv Mar Biol 12:325–389
Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96
Taylor R, Fletcher RL (1999) Cryopreservation of eukaryotic algae—a review of methodologies. J Appl Phycol 10:481–501
Tesson B, Genet MJ, Fernandez V, Degand S, Rouxhet PG, Martin-Jezequel V (2009) Surface chemical composition of diatoms. ChemBioChem 10:2011–2024
Walker T, Purton S, Becker D, Collet C (2005) Microalgae as bioreactors. Plant Cell Rep 24:629–641
Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J Plant Physiol 144:307–313
Wetherbee R (2002) The diatom glasshouse. Science 298:547
Youn JY, Hur SB (2009) Cryopreserved marine microalgae grown using different freezing methods. Algae 24:257–265
Funding
This work was supported by the Far Eastern Branch of Russian Academy of Sciences (grant no. 12-I-P6-07), the Russian Foundation for Basic Research (grant no. 12-04-31974), the Russian Foundation for Basic Research-Far Eastern Federal University (grant no. 12-04-13006-12/13), and Program at the Far Eastern Federal University (grant no. 11 G34.31.0010).
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Boroda, A.V., Aizdaicher, N.A. & Odintsova, N.A. The influence of ultra-low temperatures on marine microalgal cells. J Appl Phycol 26, 387–397 (2014). https://doi.org/10.1007/s10811-013-0093-5
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DOI: https://doi.org/10.1007/s10811-013-0093-5