Customized cryopreservation protocols for chlorophytes based on cell morphology

The development of efficient algae cryopreservation methods is pivotal to the establishment of long-term culture collections as well as algae breeding and genetic modification programs. However, the unpredictable responses of distinct algal species to cryopreservation agents and protocols have hampered the standardization of universal methods so far. The results presented in this study indicate that intrinsic biological factors (i.e.: cell morphology and phylogenetic origin) play a role in the definition of which culture age and cryoprotectant agent type and concentration should be used to achieve successful cryopreservation. Through the use of Central Composite Rotatable Design (CCRD) it was possible to define optimized protocols for cryopreserving three major morphotypic groups found among chlorophytes (i.e.: coenobium, coccoid or palmella forming strains). These optimized protocols were then validated upon fifteen strains from Sphaeropleales, Chlamydomonadales and Chlorelalles clades. Analysis reveals that the use of DMSO as the sole cryoprotectant provided the highest postfreezing cell viability recovery rates for 80% of the coccoid strains tested, while the combination of glycerol and PEG400 allowed efficient cryopreservation of 100% of the coenobium and palmella forming strains evaluated. In addition, results suggest that coenobium-forming strains have higher resistance to freeze/thawing when frozen at mid-log phase of growth, while coccoid and plamelloid strains present higher survival rates when cryopreserved at late-log phase. Such information is of valuable practical use since a simple visual inspection of algae strain's predominant morphotype can guide the choice of the most suitable cryopreservation protocol.