Microalgal strains have been traditionally maintained as live cultures. The time, cost and risk of maintaining these live cultures, has prompted culture collections to turn to cryopreservation to maintain strains that are less frequently used. The protocols used to this end, have been developed from those used for animal sperm preservation, and are not universally suited for microalgae.
Most microalgal strains preserved are obtained from active cultures and preserved in 0.5 to 1.0 mL containers. This small volume is enough to start cultures in small volumes (1-20 mL). The recovery of these cultures for aquaculture use, requires careful planning, to reach the needed volumes required for animal feeding. The variability on the cell counts in active cultures, increases the uncertainty of the time needed recover and expand the cultures for application as feeds. Higher concentration cultures may allow a faster recovery and expansion of the cultures. One of the variables that can impact the microalgal cryopreservation process, is the cryoprotectant type, equilibration time and the relation between the concentration of cryoprotectant and the cells concentration.
In this work, we explored the effect of cell concentration, equilibration time and ratio of concentration of cryoprotectant to cell numbers. Samples of microalgal cultures where concentrated by centrifugation in arrange of 1- 50X. Triplicate aliquots of each concentration were mixed with a solution of DMSO in culture media, in a ratio of 1:1. The samples were equilibrated for times of 5,10,15,20,25,30,35 and 40 minutes. Samples were inoculated in culture media and their growth monitored for 10 days or 5 days after start of the exponential growth phase.
For each culture concentration, the two treatments with the best growth rate were selected to test freezing rates from 10 - 40 °C. The samples were treated under the conditions selected. Triplicate samples of the selected treatments, without dilution, were placed in 0.5 mL straws, and frozen at each freezing rate to a temperature of -80° C and maintained at this temperature for 5 minutes. The samples were then placed immediately in liquid nitrogen. The samples were stored in dewars under liquid nitrogen vapor for at least 5 days. The samples were thawed, and their growth monitored as described above.
For all experiments, cell counts were performed on several quality control points, including the original culture, the culture after concentration (centrifugation), culture after cryoprotectant addition and equilibration (before freezing in the freezing rate experiment), and samples after inoculation in the culture media (thawed samples in the freezing rate experiment). Cell counts were done every other day thereafter for 10 days, or 5 days after the start of exponential growth. Results show that freezing rate can impact the survival of cryopreserved microalgae, while cryoprotectant stabilization times showed little to no impact.