## 3 Results
### Physiological Characterization of Non-Saccharomyces Ycasts
opuntiae_ PSWCC64 and _A. pullulans_ PSWCC82 (60%), and _H. uvarum_ PSWCC70 and _P. kluyveri_ PSWCC62 (40%). All non-_Saccharomyces_ strains produced similar levels of ethanol measured as \({}^{\circ}\)g EtOH/g Sugars\({}^{\circ}\)except _A. pullulans_ PSWCC82. Interestingly, the conversion efficiency of _H. uvarum_ PSWCC70 (0.54 (\(\pm\)0.03)) was not significantly different when compared to _S. cerevisiae_ BY4742 (0.51 (\(\pm\)0.05)) (Figure 4B). Figure 4: Composition of major sugars, sugar consumed, and ethanol yield in sterile juice compared to inoculated fermentations with candidate non-_Saccharomyces_ yeast strains measured by UHPLC analysis. (**A**) Glucose (blue) and fructose (orange) represent major sugars in grape juice and wine. (**B**) Percent sugar consumed and ethanol yield during fermentation. Circles represent ethanol yield measured at the end of fermentation. Different types of yeasts used in this study are represented on the x-axis. Data presented as mean ratio \(\pm\) SEM with a common superscript indicating significance (\(p<0.05\)). Strains used in this study are represented on the x-axis, _S. cerevisiae_ BY4742 was used as a control. Ethanol and glycerol are two major primary metabolites produced during alcoholic fermentation of yeast and are important indicators of fermentation. The amount of ethanol produced by _P. kudriazevii_ PSWCC102 (74.71 g/L) was not statistically different compared to _S. cerevisiae_ BY4742 (74.29 g/L), but was significantly higher than ethanol levels produced by _H. uwarum_ PSWCC70 (37.84 g/L), _H. oputiae_ PSWCC64 (52.90 g/L), _P. kuyveri_ PSWCC62 (2.75 g/L), and _A. pullulans_ PSWCC82 (32.11 g/L). However, there was no significant difference between glycerol production by _S. cerevisiae_ BY4742, _P. kluyveri_ PSWCC62, _P. kudriazevii_ PSWCC102, and _A. pullulans_ PSWCC82 (5.95-7.54 g/L) (Figure 5A). One aspect of wine quality relative to fermentation-derived nonvolatile metabolites are the types and levels of organic acids. Organic acids contribute to sourness, bitterness, and tartness of final wines [49]. Although present at low levels compared to ethanol, five organic acids were chosen for analysis of common acids present in inoculated fermentations. Specifically, _P. kudriazevii_ PSWCC102 produced 1.90 \(\pm\) 0.69 g/L of malic acid and 0.81\(\pm\)0.06 g/L of acetic acid with no significant difference compared to _S. cerevisiae_ BY4742. Concentration of tartaric acid decreased after fermentation by most of the yeast strains (0.29\(\sim\)0.54 g/L) and was significantly different compared to sterile juice (0.69 \(\pm\) 0.02 g/L). The control strain, BY4742 produced the highest amount of citric acid (0.20 \(\pm\) 0.003 g/L) followed by _A. pullulans_ PSWCC82 (0.17 \(\pm\) 0.005 g/L). _Hansenispora oputiae_ PSWCC64 produced the highest amount of succinic acid (0.17 \(\pm\) 0.02 g/L) followed by _S. cerevisiae_ BY4742 with no significant difference (Figure 5B). Partial least squares-discriminant analysis (PLS-DA) was used to identify how nine core nonvolatile compounds present in inoculated fermentations drive differentiation of candidate non-_Saccharomyces_ yeast strains compared to _S. cerevisiae_. In PLS-DA score plot the first two components explained 76.5% of variance which separated experimental control (sterile juice) and inoculated fermentations (Figure 6A). Uninoculated juice was included in this analysis as a benchmark for comparison to ensure that the model system using Chambourcin sterile juice is reliable and reproducible to characterize candidate yeast strains for use in winemaking. The main compounds separating the samples analyzed in component 1 are glucose (C3) and fructose (C5) (negative loading), and malic acid (C4), succinic acid (C6), and ethanol (C9) (negative loading). In the case of PC2, tartaric acid (C2) was the major compound responsible for the separation between inoculated fermentations (Figure 6B).