Mapping membrane fluidity by fluorescence anisotropy to better understand bacterial toloerance to osmotic and cold stresses encountered during freezing

Lactic acid bacteria are principally preserved by freezing before their industrial use. However, freezing induces cold and osmotic stresses that, in turn, cause significant cell injury and death. This work is aimed at analyzing the individual and combined effects of both stresses on the cell physiological and biophysical responses of Lactobacillus delbrueckii subsp. bulgaricus to better understand their relative contribution to the global freeze-injury experienced by cells. By comparing two Lb. bulgaricus strains presenting contrasted freeze-resistances and investigating membrane fluidity at the cellular level by Synchrotron deep UV fluorescence microscopy, potential features associated with bacterial tolerance to such stresses were identified. In particular, we identified a stronger deleterious influence of the osmotic stress compared to the cold stress. Furthermore, cells exhibiting a more fluid membrane when lowering the environmental temperature appeared to be associated with a higher resistance to osmotic stress. Intra and inter cellular heterogeneity in membrane fluidity within both bacterial populations also came out as a relevant parameter to be assessed. They were put into perspective of stress tolerance, and their variations will be discussed