A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION
Identifieur interne : 000229 ( France/Analysis ); précédent : 000228; suivant : 000230A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION
Auteurs : Anne Mortain-Bertrand [France] ; Freddy Etchart [France] ; Marie-Thérèse De Boucaud [France]Source :
- Journal of Phycology [ 0022-3646 ] ; 1996-04.
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Abstract
The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold‐adapted, against 10 and 42% when not cold‐adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold‐adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.
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DOI: 10.1111/j.0022-3646.1996.00346.x
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ISTEX:108FF252280C0740A7023A0B5CB22E5AACA1E575Le document en format XML
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type="university">Université Bordeaux I</orgName></affiliation></author><author><name sortKey="Boucaud, Marie Herese De" sort="Boucaud, Marie Herese De" uniqKey="Boucaud M" first="Marie-Thérèse De" last="Boucaud">Marie-Thérèse De Boucaud</name><affiliation wicri:level="4"><country xml:lang="fr">France</country><wicri:regionArea>Laboratoire de Physiologie Cellulaire Végétale, Université Bordeaux I, Avenue des Facultés, 33405 Talence Cedex</wicri:regionArea><placeName><region type="region" nuts="2">Nouvelle-Aquitaine</region><region type="old region" nuts="2">Aquitaine</region><settlement type="city">Talence</settlement></placeName><orgName type="university">Université Bordeaux I</orgName></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Phycology</title><idno type="ISSN">0022-3646</idno><idno type="eISSN">1529-8817</idno><imprint><publisher>Blackwell Publishing Ltd</publisher><pubPlace>Oxford, UK</pubPlace><date type="published" when="1996-04">1996-04</date><biblScope unit="volume">32</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="346">346</biblScope><biblScope unit="page" to="352">352</biblScope></imprint><idno type="ISSN">0022-3646</idno></series><idno type="istex">108FF252280C0740A7023A0B5CB22E5AACA1E575</idno><idno type="DOI">10.1111/j.0022-3646.1996.00346.x</idno><idno type="ArticleID">JPY346</idno></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-3646</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Chlorophyta</term><term>Dunaliella salina</term><term>carotenogenesis</term><term>cold acclimation</term><term>cryoconservation</term><term>cryoprotectant</term><term>glycerol</term><term>microalga.</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold‐adapted, against 10 and 42% when not cold‐adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold‐adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.</div></front></TEI><affiliations><list><country><li>France</li></country><region><li>Aquitaine</li><li>Nouvelle-Aquitaine</li></region><settlement><li>Talence</li></settlement><orgName><li>Université Bordeaux I</li></orgName></list><tree><country name="France"><region name="Nouvelle-Aquitaine"><name sortKey="Mortain Ertrand, Anne" sort="Mortain Ertrand, Anne" uniqKey="Mortain Ertrand A" first="Anne" last="Mortain-Bertrand">Anne Mortain-Bertrand</name></region><name sortKey="Boucaud, Marie Herese De" sort="Boucaud, Marie Herese De" uniqKey="Boucaud M" first="Marie-Thérèse De" last="Boucaud">Marie-Thérèse De Boucaud</name><name sortKey="Etchart, Freddy" sort="Etchart, Freddy" uniqKey="Etchart F" first="Freddy" last="Etchart">Freddy Etchart</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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