Effects of salt stress and cooling regimes on the pigment composition of Dunaliella salina cells

Abstract

Background: Microalgae are able to produce a significant amount of biologically significant substances. In connection with the growing popularity of microalgae, it is important to develop effective methods for storing cultures and creating strain banks. This will not only meet the needs of science and biotechnology for viable and sustainable crops, but will also solve the problem of biodiversity conservation. Objectives: study the effect of salt stress and cooling regimes on the pigment composition of microalgae Dunaliella salina cells in order to increase their safety after freezing-thawing. Materials and methods: The objects of the study were the unicellular green microalgae D. salina. Cultivation was carried out according to the standard method on nutrient media with different amounts of NaCl and trace elements. Adaptation to low temperatures was carried out by exposure of samples in the dark at temperature 4°C for 24 hours. Freezing was performed by placing 1 ml of the cell suspension in a 1.8 ml polypropylene cryogenic vial (Nunc, Sigma-Aldrich), cooled at a rate of 1 deg/min using a Mr. Frosty with following regimes: to -10°С, -40°С, -40°С followed by immersion to liquid nitrogen or direct immersion to liquid nitrogen (-196°С). Thawing was carried out in a water bath (37°С) with continuous shaking for 1–2 min. Microscopic examinations were carried out on an LSM-510 Meta laser scanning microscope (Carl Zeiss, Germany) upon excitation by a diode laser with a wavelength of 405 nm and 573 nm using a Nile Red stain. Results: It has been established that the formation of intracellular lipid globules and the synthesis of carotenoids in D. salina cells contribute to an increase in the concentration and number of motile cells after freezing-thawing. It has been shown that during rapid cooling, adaptive mechanisms do not have time to turn on in cells, and complete destruction of carotene-containing lipid globules occurs. Conclusions: Cryopreservation of D. salina cells should be carried out at a rate of 1 deg/min to -40°C, followed by immersion in liquid nitrogen and a mandatory stage of precultivation at 4°C for 24 hours. This approach allows the cells to adapt to a decrease in temperature, which contributes to the best result after freezing-thawing.