Relationship between cooling rates,cryoprotectant concentrations and salinities in the cryopreservation of marine microalgae

The viability of the marine microalgae Rhodomonas baltica Karsten, Isochrysis affinis galbana (Strain T-ISO) Parke, Chaetoceros gracilis Schütt, Tetraselmis chuii Butcher, Nannochloropsis gaditana Lubian and Nannochloris atomus Butcher, cryopreservec employing different cooling rates, either with or without the addition of the cryoprotective compounds dimethyl sulfoxide (DMSO) and methanol was studied at two exposure salinities. A viability index, which considered both cell recovery and the growth capacity of microalgae after thawing, was developed. The growth of thawed algae was compared to that obtained for unfrozen algae grown in liquid medium under the same conditions. Viability (V) was calculated according to the equation: V=(C ₀/C _i)x(C ₇/(a·C ₀ ^b ))x100, where C ₀ and C ₇ are, respectively, the initial and final cell densities measured in the cultures after thawing from-196°C, C _i is the maximum initial cell density (complete cell recovery), and a, b are the regression coefficients obtained for C ₇ as a function of C ₀ in the unfrozen controls. R. baltica was the only species that showed an improved viability when salinity was reduced from 36 (average viability 13.7% for 15% DMSO) to 20 (average viability 36.2% for 15% DMSO). The other five species displayed better viability only at the higher salinity at all tested cooling rates and cryoprotectant levels. T. chuii, Nannochloropsis gaditana, and Nannochloris atomus Butcher could be cryopreserved without cryoprotectant. However, their respective viabilities (32.7, 30.8 and 65.8%) at 36 S were progressively improved on addition of 5% DMSO (70.9, 48.2 and 93.5, respectively) and 15% DMSO (91.9, 57.0 and 94.2%, respectively). Similar improvements were found for Nannochloropsis gaditana and Nannochloris atomus when cryopreserved using methanol concentrations of 1% (average viabilities of 46.9 and 91.8, respectively) and 5% (average viablities of 48.7 and 95.3, respectively). Methanol was completely ineffective in cryopreserving the other four species and caused a lethal effect on T. chuii during freezing. C. gracilis could be cryopreserved with 5% DMSO only at 36 S. This resulted in a similar viability (11.7%) to that obtained using 15% DMSO in 20 S (13.7%). Keeping cryoprotectant concentration at 15% DMSO and raising salinity to 36 significantly improved the mean viability of C. gracilis to 21.6%. A low mean viability of 2.1% was obtained for I. galbana when 15% DMSO was used in full-strength seawater (36 S). Within the range of cooling rates tested (0.25 to 16 C° min^-1), cryopreserved microalgae showed higher viabilities at faster rates in the absence of cryoprotectants at both salinities. Generally, the dependence on cooling rate decreased proportionally to the concentration of DMSO or methanol, as demonstrated by the lack of significance for the slope of the regressions. Only C. gracilis appeared to depend on faster cooling rates in the presence of 15% DMSO.