Cryopreservation-recalcitrance in microalgae: novel approaches to identify and avoid cryo-injury

Standard two-step freezing protocols are unsatisfactory for Euglena gracilis and many other microalgae, particularly those with larger cell sizes, complex morphologies and/or those susceptible to environmental stress. Using techniques that allow mechanisms of injury and sites of damage to be identified (e.g. monitoring oxygen evolving capacity, detection of (OH)-O-., microscopic visualisation of intracellular ice and structural/ultrastructural damage), it is possible to improve conventional cryopreservation methodologies. In E. gracilis this has resulted in the development of protocols which increased post-thaw viability levels from 0 to 20%. Alternative cryoprotection strategies tested included vitrification and encapsulation/dehydration. Vitrification was unsuccessful due to the high toxicity of the solutions. Encapsulation/dehydration, with or without two-step cooling were suitable for cryopreservation of E. gracilis, the latter resulted in the highest levels of post-thaw viability (40%) and viability was maintained after 12 months storage.