090 The influence of cryopreservation on marine microalgal cells

Abstract

The development of cryopreservation methods for microalgae opens great prospects for marine biotechnology and aims to establish a bank of cryopreserved cultures. Nine of eleven marine microalgal 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 (Me 2 SO, glycerol, and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants. The chief factor for the successful preservation of microalgal cells during freeze-thawing was shown to be the cooling rate. The survival rate for many algal species did not exceed 15%, but recovery of the initial cell concentration usually took 6–12 days after thawing. The pigment composition and content were evaluated as additional indicators of the algal functional activity. The pigment content tended to decrease after thawing as compared with unfrozen cells and increase during cell recovery. Three diatom species ( Attheya arenicola , A. longicornis , and A. ussurensis ) were successfully cryopreserved for the first time. For these weakly silicified marine diatoms, step freezing created the possibility of gradual cryoprotectant penetration inside the cells and a stepwise release of water out of the cells. None of the tested protocols gave a positive result for the diatoms of the Pseudo-nitzschia genus. The failure in cryopreservation of the Pseudo-nitzschia species may be explained by peculiarities in the cell wall composition: the Pseudo-nitzschia cell wall is composed of more silica and fewer organic components than those of the other tested diatoms, which makes the wall much more rigid and, probably, not permeable enough for penetrating protectants, even if the step freezing method is used. In our experiments with the Pseudo-nitzschia species, we observed that most of the thawed cells were dead but had almost undamaged cell walls. In contrast, the high tolerance to a wide range of cryopreservation conditions of the tiny green algae may be explained by its low water content, small cell size and thin cell wall. Cryosensitivity of marine algal cells was shown to depend on the differences in natural intrinsic characteristics rather than their taxonomic position. The mechanisms by which some algae withstand freezing better than others are not yet clear, but we suggest that the biochemical and physical properties of algal cell walls determine the water flow inside and outside the cell and enable the high flexibility needed for adaptation to different environments. Our results are a step in the development of a cryopreservation technology for some marine microalgae and the search for the nature of their cryoresistance. Source of funding: Grant 1 12-I-P6-07 of the Far Eastern Branch of Russian Academy of Sciences, grant 1 12-04-31974 of the Russian Foundation for Basic Research, grant 1 12-04-13006-12 of the Russian Foundation for Basic Research-Far Eastern Federal University, and grant 1 11 G34.31.0010 of Program at the Far Eastern Federal University. Conflict of interest: None declared. borodandy@gmail.com