A Modified Cryopreservation Method of Psychrophilic Chlorophyta Pyramimonas sp. from Antarctica

Abstract

Polar psychrophiles which thrive under extreme conditions such as cold temperature, high salinity, and high dose ultraviolet light, emerge as novel targets for biotechnology. To prevent genetic drift and the possibility of contamination by subculturing, cryopreservation was employed for two psychrophilic microalgae, Porosira sp. (KOPRI AnM0008) and Pyramimonas sp. (KOPRI AnM0046), which have anti-freeze activities. Five cryoprotectants (dimethyl sulphoxide, ethylene glycol, glycerol, methanol and propylene glycol) showed toxicity at 20-30% (v/v). The optimal cryoprotectant concentration and equilibration time were less than 20% and 10 min, respectively. Cryopreservation was carried out in the presence of cryoprotectants either by direct freezing in liquid nitrogen (<TEX>$LN_2$</TEX>) or controlled freezing using a controlled rate freezer followed by storage in the <TEX>$LN_2$</TEX> tank. As a result, Pyramimonas sp. (KOPRI AnM0046), a psychrophilic chlorophyta was revived. Cryopreserved Porosira sp. was not revived from either freezing protocols probably due to the silicic cell wall and its relatively large cell size. In the case of Pyramimonas sp. (KOPRI AnM0046), the controlled freezing method showed higher revival yield than the direct freezing method.