Cryopreservation of Chlorella vulgaris Using Different Cryoprotectant Agents

The objective of this study is to evaluate the cryopreservation of Chlorella vulgaris using different substances. The C. vulgaris was cultured in medium MH, the microalgae were grown under a 12-12 h light- dark photoperiod, illumination with 40 W led lamps, and a controlled temperature of 28±1 ºC. C.vulgaris was cultured for 15 days and the culture was aliquoted into 3-mL cryogenic tubes. The 3-mL aliquot was centrifuged, the supernatant was discarded, and the pellet was resuspended in different cryoprotectant solutions, T1-PVS1, T2-PVS2, T3-PVS2 (1% phloroglucinol), T4 (2 M glycerol), and T5 (5% methanol). The samples were rapidly frozen in liquid nitrogen (-196 °C) and analyzed after 15, 150, and 300 days of freezing. Cell viability was determined in cultures grown for 20 days. The only effective treatment was T5, which promoted the growth of thawed cultures in both solid and liquid media. After 15 days of freezing in liquid nitrogen and 20 days of culture growth, the number of viable and nonviable cells was 3.42±0.72 × 107 and 0.06±0.009 × 107, respectively, and viability was 98.2%. Similar values were obtained after 150 and 300 days of freezing- 2.17±0.15 × 107 and 2.35±0.18 × 107 viable cells, 0.05±0.02 × 107 and 0.10±0.02 × 107 nonviable cells, and viability of 97.6% and 95.8%, respectively. The cryopreservation protocol for microalgae C. vulgaris using 5% methanol was effective; therefore, it is possible to maintain this strain under axenic conditions in liquid nitrogen for long periods.