Optimized cryopreservation of Ettlia sp. using short cold acclimation and controlled freezing procedure

Abstract

Despite widespread use as a preservation method, cryopreservation remains recalcitrant for microalgae preservation due to low viability and strain specificity. Herein, a cryopreservation method for Ettlia sp. YC001 was optimized using the following strategies. Cold acclimation at 4 °C for 24 h prior to the freezing procedure enhanced the expression of the cold response pathway and heat-shock proteins, helping to both stabilize the proteins against cold stress and prevent rapid dehydration. Three independent parameters of cryopreservation, the cooling rate, plunge temperature, and thawing temperature, were optimized with various cryoprotectants using a response surface methodology (RSM). Finally, the cellular damage was minimized when using the optimized controlled cooling procedure with an accurate programmable freezer. When applying the optimized conditions, the cell viability of the cryopreserved strain YC001 was >？90% after 1 year. It is believed that the suggested approach can also be applied to other commercial microalgal strains.