OVERCOMING RECALCITRANCE IN Porphyridium aerugineum GEITLER EMPLOYING ENCAPSULATION-DEHYDRATION CRYOPRESERVATION METHODS

Cultures of the recalcitrant microalga Porphyridium aerugineum were cryopreserved. A two-step, uncontrolled rapid freezing protocol, using methanol as cryoprotectant resulted in 23.8% viable cells. Cultures in the exponential growth phase, grown under low light intensity to prevent vacuole formation in cells, cryopreserved using a passive freezer, showed 22.4% viability. This value was enhanced to 31.5% when a controlled-rate freezer was employed. Optimized cultures in the exponential growth phase, cultivated in medium supplemented or not with vitamin B12, were then tested for freezing using the encapsulation-dehydration protocol. High cell loss was observed early during the sorbitol dehydration steps, but 63.6% of the remaining encapsulated cells were viable after thawing. This study confirmed the potential of encapsulation-dehydration as a method allowing to improve the low viability obtained with two-step freezing protocols. It also showed the importance of monitoring the response of algal cells to bead osmotic and evaporative dehydration pretreatments before freezing.