Extending cryoprotectant strategies from Phaeocystis antarctica to Fragilariopsis kerguelensis

Steffi.Gaebler [ at ] awi.de


The diatom Fragilariopsis kerguelensis and the prymnesiophyte Phaeocystis antarctica play an important role in the phytoplankton community in the Southern Ocean and contribute to a great extent to the global primary production. At the Alfred-Wegener-Institute in Bremerhaven Germany both species are kept by means of serial subculturing for research purposes. Because of several disadvantages concerning serial subculturing such as high costs, time, genetic drift and possible contamination, several cryoprotective strategies were tested in this study to cryopreserve these algal species. Cryopreservation is the storage of organic material in liquid Nitrogen (LN2) at -196°C. Most common fluids such as algal medium and other intracellular fluids in algae form ice when being frozen, which harms the cells during cryopreservation. Therefore special strategies must be applied to ensure that after having thawed the samples the algae will recover and grow. These strategies include the treatment with cryoprotective agents (CPA) before freezing, the freezing and thawing method and the post-cryopreservation treatment. In this study a combination of penetrating (p) and non-penetrating (np) CPA were tested as well as the single application of npCPA when plunging the samples directly into LN2. After thawing, both algal species showed viability for each of the CPA treatments proved by vital staining. In some cases there was a statistical significant difference in viability between samples treated with only npCPA and those treated with npCPA+pCPA. It was revealed for the colony-forming P. antarctica samples and for F kerguelensis samples that directly after thawing the differences in mean viability levels were significant between the methyl cellulose (MC) and the fish gelatine (FG) treated samples, the two npCPA. The highest percent numbers of living cells of F. kerguelensis were reached when using MC as npCPA. For the colony-forming P. antarctica samples the combination of a pCPA with MC led to the highest mean viability values. Whereas for the attached aggregates (AA) P. antarctica samples, those treated with a combination of methanol and FG (MeOH+FG) resulted in the highest mean viability values. Two weeks after thawing, in almost all samples viable cells were observed in the vital staining analysis. However four weeks after thawing, no algae growth was recorded. Instead, growth of bacteria especially in all FG treated samples was observed. It was presumed that the main factor, having an impact on recovery and growth, was the behaviour of algae being dependent on seasons. Results of other studies and a self-implemented preliminary study showed good growth despite bacteria when performed at another time than the Antarctic winter season.

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Sedlacek, T. (2013): Extending cryoprotectant strategies from Phaeocystis antarctica to Fragilariopsis kerguelensis , Bachelor thesis, Hochschule Bremerhaven & AWI.

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