Cryo-injury in algae and the implications this has
to the conservation of micro-algae

• Authors: John G. Day , Roland A. Fleck
• Languages of publication: EN

Abstracts

EN

As with all products and processes exploiting
biological materials algal biotechnology has an absolute
requirement for stable, function-fulfilling master stockcultures.
Conventionally microalgae and cyanobacteria
are maintained by serial transfer with inocula (2.5-25%
v/v) being transferred to fresh medium and the culture
being held under controlled environmental conditions.
This method is satisfactory for many algal taxa, but by its
nature cannot provide an absolute guarantee of phenotypic
or genotypic stability. Cryopreservation at ultra-low
temperatures (> -130oC) is the only methodology that can
provide this level of security to master stock-cultures;
however, many algae are recalcitrant to cryopreservation
with low or no survival. This review explores the reasons of
this cryo-recalcitrance on the application of conventional
colligative, two-step cryopreservation protocols and
points towards the options available to enhance postcryopreservation
viability.

Keywords

EN

Algal biotechnology; biofuels; biologicalresource; cryopreservation; cryoinjury; cryoprotectant

• Publisher: De Gruyter Open
• Journal: Microalgae Biotechnology
• Year: 2015
• Volume: 1
• Issue: 1

Dates

received

15 - 4 - 2014

online

26 - 3 - 2015

accepted

6 - 10 - 2014

Contributors

author

John G. Day

• Culture Collection of Algae and
  Protozoa, Scottish Association for Marine Science, Scottish Marine
  Institute, Oban, Argyll, PA37 1QA, UK

author

Roland A. Fleck

• Centre for Ultrastructural Imaging, King’s College
  London, New Hunt’s House Guy’s Campus, London, SE1 9RT, UK

References

• [1] Snell J.J.S., General Introduction To Maintenance Methods. In:Kirsop B.E., Doyle A. (Eds.), Maintenance of Micro-organismsand Cultured Cells, A Manual of Laboratory Methods, AcademicPress Ltd., London, UK, 1991
• [2] Grout B., Morris J., McLellan, M., Cryopreservation and themaintenance of cell lines. Tibtech, 1990, 293-297
• [3] Day J.G., Cryo-conservation of microalgae and cyanobacteria,CryoLetters Suppl., 1998, 1, 7-14
• [4] Pringsheim E.G., Pure Cultures of Algae, Cambridge UniversityPress, Cambridge, UK, 1946
• [5] Lorenz M., Friedl T., Day J.G., Perpetual Maintenance of ActivelyMetabolizing Microalgal Cultures. In: Andersen R.A. (Ed.), AlgalCulturing Techniques, Academic Press, New York, 2005
• [6] Day J.G., Brand J.J., Cryopreservation Methods for MaintainingCultures, In: Andersen R.A. (Ed.), Algal Culturing Techniques,Academic Press, New York, 2005
• [7] Day J.G., Watanabe M.M., Morris G.J., Fleck R.A., McLellanM.R., Long-term viability of preserved eukaryotic algae, J. Appl.Phycol., 1997, 9, 121-127[Crossref]
• [8] Hédoin H., Pearson J., Day J.G., Philip D., Young A.J., Hall T.J.,Porphyridium cruentum A-408 and Planktothrix A-404 retaintheir capacity to produce biotechnologically exploitablemetabolites after cryopreservation, J. Appl. Phycol., 2006, 18,1-7
• [9] Hipkin R., Day J.G., Rad Menéndez C., Mock T., The firstevidence of genotypic stability in a cryopreserved transgenicdiatom, J. Appl. Phycol., 2014, 26, 65-71[Crossref]
• [10] Morris G.J., The Cryopreservation of Chlorella 1. Interactionsof Cooling, Protective Additive and Warming Rate, Arch.Microbiol., 1976a, 107, 57-62
• [11] Watanabe M.M., Shimizu A., Satake K. NIES-Microbial CultureCollection at the National Institute of Environmental Studies:Cryopreservation and database of culture strains of microalgae.In: Watanabe M.M. (Ed.) Proceedings of Symposium on CultureCollection of Algae. NIES, Tsukuba, Japan, 1992
• [12] Bodas K., Brennig C., Diller K.R., Brand J.J., Cryopreservation ofblue-green and eukaryotic algae in the culture collection at theUniversity of Texas at Austin, Cryo-Letters, 1995, 16, 267-274
• [13] Wood S.A., Rhodes L.L., Adams S.L., Smith J.F., Tervit H.R., CaryS.C., Maintenance of cyanotoxin production by cryopreservedcyanobacteria in the New Zealand culture collection,NewZealand J. Mar. Freshwater Res., 2008, 42, 277–283[Crossref]
• [14] Taylor R., Fletcher R.L., Cryopreservation of eukaryotic algae - areview of methodologies, J. Appl. Phycol., 1998, 10, 481-501[Crossref]
• [15] Franks, F. Biophysics and Biochemistry at Low Temperatures,Cambridge University Press, Cambridge, UK, 1985
• [16] Canavate J.P., Lubian L.M., Effects of culture age on cryopreservationof marine microalgae, Eur. J. Phycol., 1997, 32, 87-90[Crossref]
• [17] Morris G.J., Cryopreservation: An Introduction to Cryopreservationin Culture Collections Institute of Terrestrial Ecology,Cambridge. UK, 1981
• [18] Morris G.J., Clarke A., The cryopreservation of Chlorella 4.Accumulation of lipid as a protective factor, Arch. Microbiol.,1978, 119, 153-156
• [19] Morris G.J., The Cryopreservation of Chlorella 2. Effect ofGrowth Temperature on Freezing Tolerance, Arch. Microbiol.,1976b, 107, 309-312
• [20] Ben-Amotz A., Gilboa, A., Cryopreservation of marineunicellular algae. II. Induction of freezing tolerance, Mar. Ecol.Prog. Ser., 1980, 2, 221-224
• [21] Morris, G.J., McGrath J.J., Intracellular ice nucleation and gasbubble formation in Spirogyra, CryoLetters, 1981, 2, 341–352
• [22] Box J.D., Cryopreservation of blue-green alga Microcystisaeruginosa, Br. Phycol. J., 1988, 23, 385-386
• [23] Thomashow M.F., Plant cold acclimation: Freezing ToleranceGenes and Regulatory Mechanisms, Annu. Rev. Plant Physiol.Plant Mol. Biol., 1999, 50, 571-599[Crossref]
• [24] Agarwal P.K., Agarwal P., Reddy M.K., Sopory S.K., Role of DREBtranscription factors in abiotic and biotic stress tolerance inplants, Plant Cell Rep., 2006, 25, 1263-1274[Crossref]
• [25] Webb M.S., Gilmour S.J., Thomashow M.F., Steponkus P.L.,Effects of COR6.6 and COR15am Polypeptides Encoded byCOR (Cold-Regulated) Genes of Arabidopsis thaliana onDehydration-Induced Phase Transitions of PhospholipidMembranes, Plant Physiol., 1996, 111, 301-312
• [26] Gray G.R., Chauvin L.P., Sarhan F., Huner N.P.A., ColdAcclimation and Freezing Tolerance (A Complex Interaction ofLight and Temperature), Plant Physiol., 1997, 114, 467–474
• [27] Leeson E.A., Cann J.P., Snell J.J., Maintenance of algae andprotozoa. In: Kirsop B.E., Snell J.J. (Eds.), Maintenance ofMicroorganisms, Academic Press, London UK, 1984
• [28] Terumoto I., Frost and drought injury in Lake-Balls. LowTemperature Science, Biological Science, 1959, B17, 1-7
• [29] Nagao M., Matsui K., Uemura M., Klebsormidium flaccidum,a charophycean green alga, exhibits cold acclimation that isclosely associated with compatible solute accumulation andultrastructural changes, Plant Cell Environ., 2008, 31, 872-885[Crossref]
• [30] Fleck R.A., The Assessment of Cell Damage and Recovery inCryopreserve Freshwater Protists, PhD thesis, Univ. AbertayDundee, Dundee, 1998
• [31] Benson E.E. Free Radical Damage in Stored Plant Germplasm,IBPGR, Rome, 1990.
• [32] Benson E.E., Lynch P.T., Jones J., The detection of lipidperoxidation products in cryoprotected and frozen rice cellsconsequences for post-thaw survival, Plant Sci., 1992, 85,107-114
• [33] Fleck R.A., Benson E.E., Bremner D.H., Day J.G., Studies offree radical-mediated cryoinjury in the unicellular green algaEuglena gracilis using a non-destructive hydroxyl radicalassay: A new approach for developing protistan cryopreservationstrategies, Free Radical Res., 2000, 32, 157-170
• [34] Farrant J. General Observations on Cell Preservation, In:Ashwood-Smith M.J., Farrant J. (Eds.), Low TemperaturePreservation in Medicine and Biology, Pitman Medical Ltd.,Kent, UK, 1980
• [35] Santarius K.A., Freezing of isolated thylakoid membranes incomplex media. X. Interactions among various low molecularweight cryoprotectants, Cryobiology, 1996, 33, 118-126[Crossref]
• [36] Fleck R.A., Benson E. E., Bremner D.H., Day J.G., A comparativestudy of antioxidant protection in cryopreserved unicellularalgae Euglena gracilis and Haematococcus pluvialis,CryoLetters, 2003, 24, 213-228
• [37] Heesch S., Day J.G., Yamagishi T., Kawai H., KüpperF.C., Cryopreservation of the model alga Ectocarpus(Phaeophyceae), CryoLetters, 2012, 33, 327-336
• [38] Gäbler-Schwarz S., Rad Menéndez C., Achilles-Day U.E.M.,Campbell C.N., Day J.G., Cryopreservation of Phaeocystisantarctica, CryoLetters, 2013, 34, 561-570
• [39] Taylor M.J., Physico-chemical principles in low temperaturebiology. In: Grout B.W.W., Morris G.J., (Eds.), The Effects of LowTemperatures on Biological Systems, Edward Arnold, London,London, 1987.
• [40] Mazur P., Cryobiology: The Freezing of Biological Systems,Science, 1970, 168, 939-949
• [41] Levitt J. Responses of Plants to Environmental Stress, AcademicPress, New York, USA, 1980
• [42] Fleck R.A., Day, J.G., Clarke, K.J., Benson, E.E., Elucidation ofthe metabolic and structural basis for the cryopreservationrecalcitrance of Vaucheria sessilis, CryoLetters, 1999, 20,271-282
• [43] Steponkus P.L., Langis R., Fujikawa, S., Cryopreservation ofplant tissues by vitrification. In: Steponkus P.L (Ed.), Advancesin Low-Temperature Biology, Jai Press Ltd., USA, 1992
• [44] Polge C., Smith A.U., Parkes, A.S., Revival of spermatazoa aftervitrification and dehydration at low temperatures, Nature,1949, 164, 666
• [45] Mazur P., Causes of injury and frozen and thawed cells. FederalProceedings, 1965, 24, S175–S182
• [46] Ashwood-Smith M.J., Farrant J., Low Temperature Preservationin Medicine and Biology, Pitman Medical Ltd., Kent, UK, 1980
• [47] Farrant, J., Ashwood-Smith M.J., Practical Aspect, In: Ashwood-Smith M.J., Farrant J. (Eds.), Low Temperature Preservation inMedicine and Biology, Pitman Medical Ltd., Kent, UK, 1980
• [48] Morris G.J., Coulson G.E., Engels M., A cryomicroscopic studyof Cylindrocystis brebissonii and two species of MicrasteriasConjugatophyceae Chlorophyta during freezing and thawing, J.Exp. Bot., 1986, 37, 842-856[Crossref]
• [49] Fleck R.A., Day J.G., Rana K.J., Benson, E.E., Visualisation ofcryoinjury and freeze events in the coenocytic alga Vaucheriasessilis using cryomicroscopy. CryoLetters, 1997, 18, 343-355
• [50] Roberts S., Grout B.W.W., Morris G.J., Consecutive observationsof a frozen cell sample by cryogenic light microscopy andcryogenic scanning electron microscopy. Cryo-Letters, 1987, 8,122-129
• [51] Fleck R.A., Pickup R.W., Day J.G., Benson E.E., The use offlow-cytometry and cryomicroscopy to characterise cryopreservation-induced injuries in Euglena gracilis, Cryobiology,2006, 52, 261-268.[Crossref]
• [52] Roberts S., Grout, B.W.W., An ultrastructural investigation intofreezing-induced dehydration of Chlamydomonas reinhardtiicw15 positive, Cryobiology, 1988, 25, 528
• [53] Santarius K.A., Freezing of isolated thylakoid membranes incomplex media IV. Stabilization of cf-1 by ATP and sulphate, J.Plant Physiol., 1978, 126, 409-420
• [54] Heber U., Tyankova L., Santarius K.A.. Stabilisation andinactivation of biological membranes during freezing in thepresence of amino acids, Biochim Biophys Acta, 1971, 241, 578
• [55] Wise R.R., Chilling enhanced photooxidation: The production,action and study of reactive oxygen species produced duringchilling in the light, Photosynth. Res., 1995, 45, 79-97[Crossref]
• [56] Brown R.H., Collins, N., Merrett, M.J., Peroxidase activity inEuglena gracilis, Plant Physiol., 1975, 55, 1123-1124[Crossref]
• [57] Grout B.W.W. Introduction to the in vitro preservation ofplant cells, tissues and organs. In: Grout B. (Ed.), GeneticPreservation of Plant Cells In Vitro, Springer, Berlin, Berlin,1995
• [58] Romo S. Becares E., Preservation of filamentous cyanobacteriacultures (Pseudanabaena galeata Bocher and Geit ler inemaamphibium) (Ag. ex Gom.) Anagn. under low temperatures, J.Microbial Methods, 1992, 16, 85-89
• [59] Stillinger F., A Topographic View of Supercooled Liquids andGlass Formation, Science, 1995, 267, 1935-1939
• [60] Day J.G., Watanabe M.M., Turner M.F., Ex situ conservationof protistan and cyanobacterial biodiversity : CCAP - NIEScollaboration 1991 – 1997, Phycol. Res., 1998, 46 Suppl., 77-83
• [61] Pegg D.E., Wusteman M. C., Boylan S., Fractures incryopreserved elastic arteries: Mechanism and prevention,Cryobiology, 1996, 33, 658-659
• [62] Beaty M.H., Parker B.C., Cryopreservation of eukaryotic algae,Virginia J. Sci., 1992, 43, 403-410
• [63] Buhmann M.T., Day J.G., Kroth P.G., Post-cryopreservationviability of the benthic freshwater diatom Planothidiumfrequentissimum depends on light levels, Cryobiology 2013,67, 23-29[Crossref]
• [64] Müller J., Day J.G., Harding K., Hepperle D., Lorenz M., Friedl T.,Assessing genetic stability of a range of terrestrial microalgaeafter cryopreservation using amplified fragment lengthpolymorphism (aflp), Am. J. Bot., 2007, 94, 799-808[Crossref]
• [65] Day J.G., Benson E.E., Harding K., Knowles B., Idowu M.,Bremner D., et al., Cryopreservation and conservation ofmicroalgae: the development of a pan-european scientific andbiotechnological resource (the COBRA project), CryoLetters,2005, 26, 231-238
• [66] Morris G.J., Cryopreservation of 250 strains of Chlorococcalesby the method of two-step cooling, Eur. J. Phycol., 1978, 13,15-24
• [67] Kono Y., Kashine S., Yoneyama T., Sakamoto Y., Matsui Y.,Shibata, H., Iron chelation by chlorogenic acid as a naturalantioxidant, Biosci. Biotechnol. Biochem., 1998, 62, 22-27[Crossref]
• [68] McAnulty J.F., Haung, X.Q., The efficacy of antioxidantsadministered during low temperature storage of warm ischemickidney tissue slices, Cryobiology, 1997, 34, 406-415[Crossref]
• [69] Day J.G., Fleck R.A., Benson E.E., Cryopreservationrecalcitrancein microalgae: novel approaches to identify andavoid cryo-injury, J. Appl. Phycol., 2000, 12, 369-377[Crossref]
• [70] Harding K., Day J.G., Lorenz M., Timmermann H., Friedl T.,Bremner D.H., et al., Introducing the Concept and Application ofVitrification for the Cryo-conservation of Algae “A Mini Review”,Nova Hedwigia, 2004, 79, 207-226[Crossref]
• [71] Armitage W.J., Survival of corneal endothelium followingexposure to a vitrification solution, Cryobiology, 1989, 26,318-327[Crossref]
• [72] Day J.G., Lorenz M., Wilding T., Friedl T., Harding K., PröscholdT., Brennan D., et al., The use of physical and virtualinfrastructures for the validation of algal cryopreservationmethods in international culture collections, CryoLetters 2007,28, 357-376
• [73] Massie I., Selden C., Hodgson H., Fuller B., Gibbons S.,Morris G.J., GMP Cryopreservation of Large Volumes of Cellsfor Regenerative Medicine: Active Control of the FreezingProcess. Tissue Engineering Part C Methods, 2014 01/2014;DOI:10.1089/ten.TEC.2013.0571[Crossref]
• [74] Müller J., Friedl T., Hepperle D., Lorenz M., Day J.G. Distinctionof isolates among multiple strains of Chlorella vulgaris(Chlorophyta, Trebouxiophyceae) and Testing Conspecificitywith Amplified Fragment Length Polymorphism and ITS rdnasequences, J. Phycol., 2005, 41, 1236-1247[Crossref]

Identifiers

DOI

10.1515/micbi-2015-0001