RELATIONSHIP BETWEEN COOLING RATES, CRYOPROTECTANT CONCENTRATIONS ANDSALINITIES IN THE CRYOPRESERVATION OF MARINE MICROALGAE

The viability of the marine microalgae Rhodomonas baltica Karsten, Iso chrysis affinis galbana (Strain T-ISO) Parke, Chaetoceros gracilis Sch utt, Tetraselmis chuii Butcher, Nannochloropsis gaditana Lubian and Na nnochloris atomus Butcher, cryopreserved employing different cooling r ates, either with or without the addition of the cryoprotective compou nds dimethyl sulfoxide (DMSO) and methanol was studied at two exposure salinities. A viability index, which considered both cell recovery an d the growth capacity of microalgae after thawing, was developed. The growth of thawed algae was compared to that obtained for unfrozen alga e 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) )) x 100, where C-0 and C-7 are, respectively, the initial and final c ell densities measured in the cultures after thawing from - 196 degree s 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 functi on of C-0 in the unfrozen controls. R. baltica was the only species th at showed an improved viability when salinity was reduced from 36 part s per thousand, (average viability 13.7% for 15% DMSO) to 20 parts per thousand (average viability 36.2% for DMSO). The other five species d isplayed better viability only at the higher salinity at all tested co oling rates and cryoprotectant levels. T. chuii, Nannochloropsis gadit ana, and Nannochloris atomus Butcher could be cryopreserved without cr yoprotectant. However, their respective viabilities (32.7, 30.8 and 65 .8%) at 36 parts per thousand S were progressively improved on additio n of 5% DMSO (70.9, 48.2 and 93.5, respectively) and 15% DMSO (91.9, 5 7.0 and 94.2%, respectively). Similar improvements were found for Nann ochloropsis gaditana and Nannochloris atomus when cryopreserved using methanol concentrations of 1% (average viabilities of 46.9 and 91.8, r espectively) and 5% (average viablities of 48.7 and 95.3, respectively ). Methanol was completely ineffective in cryopreserving the other fou r species and caused a lethal effect on T, chuii during freezing. C. g racilis could be cryopreserved with 5% DMSO only at 36 parts per thous and, S. This resulted in a similar viability (11.7%) to that obtained using 15% DMSO in 20 parts per thousand S (13.7%). Keeping cryoprotect ant concentration at 15% DMSO and raising salinity to 36 parts per tho usand 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% D MSO was used in full-strength seawater (36 parts per thousand S). With in the range of cooling rates tested (0.25 to 16 C-o min(-1)), cryopre served microalgae showed higher viabilities at faster rates in the abs ence 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 coo ling rates in the presence of 15% DMSO.