Evaluation and optimization of a methodology for the long-term cryogenic storage of Tetradesmus obliquus at − 80 °C

  • Jose A. Garrido-CardenasEmail author
  • Xiaoyu Han
  • Diego L. Alonso
  • Federico García-Maroto
Methods and protocols


Cryopreservation is a common methodology for long-term microalgae storage. Current cryopreservation methods are based on using diverse cryoprotectants and two-step cooling protocols, followed by sample storage at the temperature of liquid nitrogen (− 196 °C). However, the use of this methodology requires a continuous liquid N2 supply as well as facilities with dedicated equipment, which is not affordable for every laboratory. In our work, we report on the successful development of a simple and cost-effective method for the long-term cryogenic storage of Tetradesmus obliquus at temperatures (− 80 °C) used in commonly available deep freezers that are more readily accessible to laboratories. Two procedures were evaluated that were originally devised for other microalgae; this was followed by the optimization of critical parameters such as the sample’s microalgal concentration and the cryoprotectant reagent’s incubation time. Cell viability was monitored using the survival rates obtained by direct agar plating and the growth recovery times in liquid cultures. Viability-related variables were recorded following different storage times of up to 3 years. The main operational factors involved in the process (cell concentration, incubation time, and storage time) were statistically analyzed with regard to their influence on the survival rate. The statistical analysis showed interdependence (a two-factor interaction) between the cellular concentration and the cryoprotectant’s incubation time, on the one hand, and between the incubation time and the storage time on the other. Survival rates above 70% were obtained under optimized conditions after 3 months of storage, along with 20–35% viabilities after 3 years. These results open up the possibility of extending this method to other Scenedesmaceae, or even other microalgal species, and for its use in resource-limited laboratories.


Tetradesmus Scenedesmus Cryogenization Microalgal preservation 



This study was funded by the Ministerio de Economía, Industria y Competitividad, Gobierno de España (grant number AGL2016-74866-C3-2-R).

Compliance with ethical standards

This article does not contain any studies with human participants performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_9650_MOESM1_ESM.pdf (559 kb)
ESM 1 (PDF 558 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biology and GeologyUniversity of AlmeriaAlmeríaSpain
  2. 2.Natural Products Biotechnology Group, Centro de Investigación en Agrosistemas Intensivos Mediterráneos y Biotecnología Agroalimentaria (CIAMBITAL), and Campus de Excelencia Internacional del Mar (CEI-Mar)University of AlmeriaAlmeríaSpain
  3. 3.Department of Chemistry and PhysicsUniversity of AlmeriaAlmeríaSpain

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