Some aspects on the cryopreservation of microalgae used as food for marine species

Abstract

The response of marine microalgae to different cryopreservation methods was described. Of the six species evaluated, only Chaetoceros gracilis depended on faster cooling rates to increase its postthaw viability (7.2% at 0.25 °C min−1, 29.3% at 4 °C min−1) when using 15% dimethyl sulphoxide in 36 p.p.t. salinity seawater. Tetraselmis chuii, Nannochloris atomus and Nannochloropsis gaditana were the most tolerant species to biological freezing, achieving mean viabilities of 97.9%, 80.5% and 61.6% respectively. Rhodomonas baltica and Isochrysis galbana, T-ISO strain, showed the lowest viability (means of 7.3% and 15.1% respectively) after cryopreservation under the same conditions of salinity, cryoprotectant concentration and cooling rates.

Avoidance of undercooling by inducing ice nucleation when reaching the freezing point did not change viability in comparison to all the procedures that did not include seeding in any of the tested species. Five species showed similar viabilities when a single controlled cooling step to −50 °C procedure was compared to a two-step cooling process, in which algae were plunged into liquid nitrogen (LN) after the first step. Isochrysis galbana represented an exception. A mean viability of 25.8% was achieved when cooled to −50 °C, whereas viability decreased to 4.4% when the second cooling step to −196 °C was used.

Replacing the special biological freezing equipment by a −20 °C freezer to perform the first cooling step resulted in a steady cooling rate after the commencement of ice formation. This was due to the fact that samples reached that temperature in a liquid state. Solidification occurred spontaneously at variable times once −20 °C was reached. A cooling rate of −14 °C min−1 during the change from liquid to solid state was achieved when a −80 °C freezer was used to perform the first cooling step. The performance of the first cooling step in both type of freezers resulted in similar viabilities after thawing from liquid nitrogen, in comparison to the use of special equipment for controlling cooling rates in T. chuii (90.8% for −20 °C and 89.7% for −80 °C), N. gaditana (44.6% for −20 °C and 42.6% for −80 °C) and N. atomus (85.9% for −20 °C and 85.6% for −80 °C). Lower viabilities were recorded for R. baltica and Ch. gracilis cooled to −20 °C and in LN (2.2% and 2.9% respectively) but no difference were found with respect to the first technique, when both species were cooled to −80 °C and in LN (6.3% and 19.9% respectively). I. galbana showed no viability when cooled to −80 °C.