Desenvolvimento de métodos otimizados para criopreservação de microalgas verdes (Chlorophyta)

Abstract

In view of the importance of preserving algal genetic resources deposited in reference collections for the support of enhancement programs, it is necessary to develop methodologies for the conservation of microalgae in a metabolically inactive state for long periods. In this context, the objective of this work was to establish optimized cryopreservation protocols for microalgae species with distinct cellular architectures (cenobial colonial, unicellular coccoid and palmeloid colonial), determining the optimal parameters for freezing, including the algal growth phase, and also the type and concentration of cryoprotective agents. Three cryoprotectants were tested: two agents with high cell permeability, Glycerol and Dimethylsulfoxide (DMSO), and a non-permeable agent, Polyethylene Glycol 400 (PEG 400). The optimization of the variables was carried out using a central composite rotational design (CCRD) 22. For most of the single-celled coccoid strains analyzed, the freezing during the deceleration phase of algal growth (6th day of culture) in the presence of 7% DMSO cryoprotectant v/v) resulted in the highest rates of cell viability after cryopreservation (up to 94%). On the other hand, the highest rates of cellular viability of the cenobial algae (62.6%) were obtained by freezing during the exponential algal growth phase (3rd day of cultivation) in the presence of combined glycerol and PEG 400 agents in the concentration of 5% (v / v) for each one. Different behavior was also observed for the analyzed palmeloid strains, with the highest recovery rates (78.8%) being obtained by freezing during the deceleration phase of algal growth (6th day of culture) in the presence of the combination of cryoprotectants, glycerol (5% v/v) + PEG 400 (5% v/v). The results obtained suggest that the morphology presented by the algal stem can be a good indicator for the choice of cryoprotectant to be used for cryopreservation.