Cryopreservation of four valuable strains of microalgae, including viability and characteristics during 15 years of cryostorage

Abstract

Recent developments in the technology of cryopreservation have permitted the long-term storage of many strains of microalgae with reliable rates of survival. However, many strains still cannot be recovered from storage in liquid nitrogen. Here, we investigated the effects of various cryoprotectants in achieving comparatively high survival rate around 50%. The strains tested included two freshwater algae, Chlorella vulgaris C-27 and M-207A7, and two marine algae, Nannochloropsis oculata ST-4 and Tetraselmis tetrathele T-501. Cells of these strains were suspended in various cryoprotective solutions and slowly cooled to −40°C prior to immersion in liquid nitrogen. Little or no cryoprotection was seen with dimethyl sulfoxide (DMSO) alone or in combination with sorbitol or proline; with glycerol alone; or with ethylene glycol (EG) alone. However, survival rates of approximately 50% were observed using a cryoprotectant mixture of 5% DMSO, 5% EG, and 5% proline. Viability persisted during a storage period of 15 years. Similarly, chlorophyll content was not significantly changed during this 15-year interval. Thus, the present study demonstrates the advantage of cryopreservation using liquid nitrogen. We expect that this method will contribute to both basic and applied biology through the establishment of cryopreserved microalgal culture collections.