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

Standard two-step freezing protocols are unsatisfactory for Euglena gracili s and many other microalgae, particularly those with larger cell sizes, com plex morphologies and/or those susceptible to environmental stress. Using t echniques that allow mechanisms of injury and sites of damage to be identif ied (e.g. monitoring oxygen evolving capacity, detection of (OH)-O-., micro scopic visualisation of intracellular ice and structural/ultrastructural da mage), it is possible to improve conventional cryopreservation methodologie s. In E. gracilis this has resulted in the development of protocols which i ncreased post-thaw viability levels from 0 to 20%. Alternative cryoprotecti on strategies tested included vitrification and encapsulation/dehydration. Vitrification was unsuccessful due to the high toxicity of the solutions. E ncapsulation/dehydration, with or without two-step cooling were suitable fo r cryopreservation of E. gracilis, the latter resulted in the highest level s of post-thaw viability (40%) and viability was maintained after 12 months storage.