Effect of the antifreeze protein from the arctic yeast Leucosporidium sp. AY30 on cryopreservation of the marine diatom Phaeodactylum tricornutum.

Abstract

Antifreeze proteins are a group of proteins that allow organisms to survive in subzero environments. These proteins possess thermal hysteresis and ice recrystallization inhibition activities. In the present study, we demonstrated the efficiency of a recombinant antifreeze protein from the Arctic yeast Leucosporidium sp. AY30, LeIBP, in cryopreservation of the marine diatom Phaeodactylum tricornutum, which is one of the classical model diatoms and has most widely been studied with regard to its ecology, physiology, biochemistry, and molecular biology. P. tricornutum cells were frozen by either a fast or two-step freezing method in freezing medium containing 10 % dimethyl sulfoxide, glycerol, propylene glycol, and ethylene glycol, respectively, with or without LeIBP supplement. When cells were frozen using the two-step freezing method, cell survival was significantly increased and statistically the same as that of unfrozen native cells in the presence of 0.1 mg/ml LeIBP in 10 % propylene glycol or 10 % ethylene glycol at day 11 of post-thaw culture. In the presence of LeIBP, the concentration of chlorophyll a was dramatically increased to 14-, 48-, 1.6-, and 8.8-fold when cells were frozen in freezing medium containing dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. Scanning electron microscopy observations demonstrated that the cells were also successfully preserved and epitheca or hypotheca were not deformed. These results demonstrate that LeIBP was successfully applied to improve cryopreservation of the marine diatom P. tricornutum.