Cooling–Heating Phase Behavior of Hypersaline Culture Media Studied by DSC and Cryomicroscopy

Hypersaline culture media used for cultivation of Dunaliella salina represent complex multicomponent aqueous systems whose cooling–heating phase behavior remains insufficiently characterized. In this study, the thermal transitions of two biologically relevant hypersaline media (Artari and Ramaraj) were investigated using differential scanning calorimetry (DSC) and cryomicroscopy. The media were examined at NaCl concentrations of 1.5, 2.0, and 4.0 M, corresponding to moderate to highly concentrated brine conditions comparable to natural salt lakes and evaporative basins. DSC analysis revealed pronounced salinity-dependent suppression of ice crystallization and modification of melting transitions relative to classical NaCl–water systems. Increased NaCl concentration reduced recrystallization during heating and shifted peak temperatures, indicating kinetic and compositional effects in the unfrozen fraction. Rapid cooling promoted formation of partially amorphous phases, consistent with limited vitrification in highly concentrated media. Cryomicroscopy directly confirmed changes in ice morphology, nucleation density, and crystal growth dynamics under varying salinity and thermal histories. The combined calorimetric and microscopic approach demonstrates that complete hypersaline cultivation media exhibit phase behavior that cannot be fully extrapolated from simplified binary systems. These findings provide new insight into the physicochemical stability of multicomponent brines during cooling and highlight the critical role of salinity and thermal history in controlling crystallization pathways in hypersaline aqueous environments.