The influence of ultra-low temperatures on marine microalgal cells

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.