## 3 Results and Discussion
### Adaptive Evolution-Mediated Changes in Glycerol Concentrations  
Because [PERSON] et al. [10] used the ALE-KCI strategy to remodel wine yeast strain metabolisms toward decreased ethanol production and enhanced glycerol biosynthesis [10], the next question we asked was whether KCl-evolved strains redirected their carbon flux towards glycerol overproduction. Therefore, we performed a laboratory-scale fermentation in grape juice containing 18% or 30% sugars. At 21 days of fermentation, when sugars (glucose and fructose) in the media were exhausted, concentrations of glycerol in the media were determined. The highest increase in glycerol production was exhibited by one of the KCl-evolved derivatives of strain 1 (1_KCL_a strain), while KCl-evolved derivatives of strain 3 (3_KCL_a and 2_KCL_b) showed the lowest rises in glycerol synthesis, in comparison to the corresponding ancestral strains (Figure 3A). When 18% grape juice was used as fermentation media, concentrations of glycerol were 5.97 (\(\pm\) 0.62), 7.8 (\(\pm\) 0.82), 8.96 (\(\pm\) 1.32), and 6.86 (\(\pm\) 1.21) g/L for strains 1, 1_YPD, 1_KCL_a, and 1_KCL_b, respectively. In turn, for strains 3, 3_YPD, 3_KCL_a, and 3_KCL_b, concentrations of glycerol were 7.02 (\(\pm\) 0.78), 7.04(\(\pm\) 1.25), 8.89 (\(\pm\) 1.03), and 8.65 (\(\pm\) 0.93) g/L, respectively. In the case of strain 2, glycerol concentrations reached 5.68 (\(\pm\) 1.02), 7.24 (\(\pm\) 0.98), 8.24 (\(\pm\) 0.87), and 7.42 (\(\pm\) 1.24) g/L for strains 2, 2_YPD, 2_KCL_a, and 2_KCL_b, respectively (Figure 3A). Similarly, when fermentation was conducted in grape juice containing 30% sugars, the highest increase in glycerol production was observed for the strain 1_KCL_a, a derivative of strain 1, when compared to its ancestral strain. The lowest rises in glycerol content were observed when 30% grape juice was inoculated with KCl-evolved derivatives of strain 3. At the end of fermentation, the glycerol concentration reached 3.2 (\(\pm\)0.92), 5.43 (\(\pm\)0.99), 7.2 (\(\pm\)0.52), and 4.6 (\(\pm\)0.82) g/L for strains 1, 1_YPD, 1_KCL_a, and 1_KCL_b, respectively; 4.31 (\(\pm\)0.86), 5.2 (\(\pm\)0.99), 5.32 (\(\pm\)1.2), and 4.9 (\(\pm\)0.91) g/L for strains 2, 2, 2_YPD, 2_KCL_a, and 2_KCL_b, respectively; and 4.76 (\(\pm\)0.89), 4.46 (\(\pm\)1.2), 5.26 (\(\pm\)0.99), and 5.81 (\(\pm\)1.2) g/L, respectively (Figure 3A). Our results showing adaptive evolution-mediated increases in glycerol production are consistent with data obtained by [PERSON] et al. [10], who confirmed that the KCl-ALE approach is an effective strategy to remodel yeast metabolism towards enhanced glycerol production and decreased ethanol biosynthesis [10]. However, it should be mentioned that the authors [10] evolved their yeast strain for 450 generations (in contrast to 250 generations in our study), and they implemented a breeding approach in addition to KCl-ALE [10]. It is widely known that intracellular glycerol is a key cellular osmoprotectant [40]. However, it is also accepted that the glycerol content is regulated by several mechanisms, such as metabolic reprogramming of carbon fluxes, control of glycerol export and import, as well as an adjustment of glycerol biosynthesis-related gene expression [17]. In general, the intracellular concentration of glycerol increases in response to high osmotic stress via the high osmolarity glycerol (HOG) pathway. Its central kinase, Hog1p, is activated by phosphorylation and activates, i.e., the expression of genes responsible for glycerol uptake and synthesis [17; 41]. Gene expression can be adapted evolutionarily to stress, e.g., salt stress. [PERSON] et al. [16] showed that yeast cells subjected to NaCl treatment for 300 generations achieved evolutionary adaptation to saline, which, in turn, was associated with both a genome size increase and modest expression changes in several genes, including stress-responsive ones, for example, _Msn4_ (multicopy suppressor of SNF1 mutation), encoding a stress-inducible transcriptional activator [16].