The storage of anhydrobiotic cultures of microalgae and cyanobacteria of A. O. Kovalevsky Institute of Biology of the Southern Seas of RAS

Abstract

Reliable preservation of microalgae cultures and creation of genetic banks of strains is one of the important tasks of modern biology. To date, 792 collections of various cultivated organisms from 76 countries are registered in the catalog of the World Federation for Culture Collections in the WDCM CCINFO database. This is the most extensive consolidated database of culture collections, which includes both well-known large collections and small repositories of research and educational institutions from all over the world. The database contains 47 algological collections and 80 collections of various microorganisms, which also include microalgae and cyanobacteria cultures. Only 30 biological collections are registered in Russia, from which only 13 contain algae strains. The most common technique of microalgae cultures storage is the method of their periodic re-sowing onto liquid media or agar. It is used in 127 collections (99 % of the total number in the catalog). Other methods used are: cryopreservation – in 33 collections (27 %), lyophilization – in 13 (11 %), L-drying – in 5 (4 %), freezing – in 19 (16 %),  and immobilization in alginate beads – in 1 (0.8 %). However, when using these methods, there is a change in morphological and functional features of cells of the cultures stored, as well as their shredding. In addition, cultures maintaining in a viable state is time-consuming and requires expensive equipment. Preservation of microalgae, transferred to the state of anhydrobiosis by dehydration, is simple and cost-effective. Anhydrobiosis is a deep and long-term inhibition of metabolism, reversible under favorable conditions; it is a quite common phenomenon in nature. The only collection in the WDCM CCINFO database that applies the method of transferring cells to a resting state (for soil algae) is the collection of algae cultures of the National University of Kyiv (ACKU WDCM 994). Many years of experiments on the transfer of microalgae to the state of anhydrobiosis allowed us to develop a method of long-term preservation of microalgae without the use of nutrient media. This technique includes cells transfer to the state of anhydrobiosis, their preservation in a dehydrated state, and subsequent removal to an active culture. In order to preserve algological biodiversity, IBSS RAS created a repository of microalgae transferred to the state of anhydrobiosis, which can be converted to active cultures if necessary. The objects of the repository were marine unicellular algae, as well as freshwater and halobic species of lower phototrophs which are perspective for biotechnology and aquaculture. The cultures were obtained as an inoculum from IBSS RAS collection of live cultures of planktonic microalgae. The algae were grown in an accumulative mode under constant lighting. The biomass was collected during cultivation of algologically pure microalgae cultures at the growth retardation or at the stationary stage. Cells were separated from the culture medium by centrifugation or by filtering them on a plankton sieve. Then the algae were dehydrated and maintained in hermetic zipper bags placed in plastic containers of 100 to 500 ml, at a temperature of +18…+21 °C in the dark in a specially equipped room. The main part of the collection is represented by strains from the phyla Chlorophyta, Cyanophyta, Bacillariophyta, and Rodophyta. The list of species, the number of isolates stored, and the information on preservation forms are provided in this article. The technological regulations for maintenance and replenishment of the storage of anhydrobiotic cultures are described. The repository is at the stage of formation. Its future lies in the fund expansion to include marine, freshwater, and halobic species. Optimization of the dehydration method will allow the transfer of microalgae belonging to different systematic phyla to the state of anhydrobiosis.

Keywords: microalgae, anhydrobiosis, viability, dehydration, storage of microalgae and cyanobacteria