Optimisation and evaluation of cryopreservation method for Aurantiochytrium limacinum

Marine microorganisms are gaining increased global importance owing to their richness in various bioactive compounds. The thraustochytrid Aurantiochytrium limacinum produces high amounts of the omega-3 fatty acid, DHA, and thus is of biotechnological importance. Aurantiochytrium limacinum cultures are usually maintained by continuous sub-culturing or plating, which is labour and resource-intensive. Having a cryopreservation technique will be useful for the preservation of A. limacinum cultures. Hence, we explored and developed a cryopreservation technique to preserve A. limacinum. Cryopreservation was done at two different temperatures by using a mechanical freezer (- 80 degrees C) and liquid nitrogen (- 196 degrees C). In the mechanical freezer, the cells were preserved in glycerol, while in liquid nitrogen four widely used cryoprotectants namely ethylene glycol, glycerol, methanol and dimethyl sulfoxide were used. The cells stored in a mechanical freezer had a relatively constant growth rate up to 12 months of storage. For liquid nitrogen storage, glycerol, methanol and dimethyl sulfoxide showed promising cryoprotectant activity with viability as high as 80% even after 12 months of storage. However, ethylene glycol did not exhibit any cryoprotective effect evidenced by marginal viability. Post-thaw assessment is crucial for judging the success of cryopreservation protocol and therefore, the study also looked into the functionality of cells on revival. On reviving no significant difference in the fatty acid profile and cell morphology among treatments was observed. The cryopreservation technique thus developed for preserving A. limacinum will help save human efforts and time and also conserve the organism's genetic integrity.