Análise do efeito do agente crioprotetor dimetilsulfóxido (DMSO) visando melhorar a criopreservação de microalgas

Abstract

Cryopreservation is a technique that adds ease of maintenance of collections and genetic preservation of strains, but it has a high failure rate and problems in the rehabilitation of strains, using cryopreservers (CPA) such as alcohols, sorbitol and dimethylsulfoxide (DMSO) to inhibit the formation of ice crystals inside the cells increasing the success of the protocol, even considering the toxicity of these substances. The objective of the present work was to produce an adapted protocol for the freezing of species of the genus Kirchneriella using DMSO based on the physiological response of the cells. For this, Kirchneriella lunaris was the organism chosen and the impact of different concentrations (v/v) of DMSO and exposure times on absorbance (570, 684 and 750 nm), cell density (cells/mL) and photosynthetic yield was verified. maximum. Initially, the growth curve was performed to determine the point of highest biomass concentration and cultivation time. After determining the optimal time, experiments were started to validate possible protocols, through a toxicity test in 96-well plates, monitored daily by absorbance to define the DMSO amplitude, with concentrations ranging from 0-20% of the chosen CPA. We proceeded to evaluate the growth and experiment of photosynthesis (PhytoPAM) under the effect of DMSO. The results showed that the best protocol is in the range of 5-10% (v/v) of DMSO, concentrations in which the microalgae can withstand the toxicity of the cryopreserver and can resume growth after thawing. It is concluded that the cryopreservation protocol using CPA DMSO 10% obtained better results in the freezing of strains of Kirchneriella lunaris (CCMA-UFSCar 087, 123 and 443) and in the resumption of growth. This result can be extended to other species of the genus Kirchneriella, but in this case a more in-depth study should be carried out, as possible limitations must be considered due to morphological, physiological and genetic differences that are found in different species and directly alter the cryoprotectant efficiency. DMSO during cryopreservation.