## 3 Results
### Physiological Characterization of Non-Saccharomyces Ycasts  
_Hansenispora uvarum_ PSWCC70_, H. opuntiae_ PSWCC62_, P. kudriuzvezu_ PSWCC102, and _Aurebosidium pullulans_ PSWCC82 were candidate yeasts selected for downstream microbial and chemical analysis. Previous literature suggests _Hansenispora_ spp. and _Pichia_ spp. can utilize fruit sugars for fermentation and have not been shown to produce harmful toxins whereas _Aurebosidium pullulans_ is well known for the production of extracellular enzymes such as pectinases [59]. Therefore, we characterized sulfite and ethanol tolerance of these five strains compared to control _S. cerevisiae_ BY4742. _Hansenispora_ opuntiae_ PSWCC64 showed a tolerance at 80 mg/L sodium metabisulfite, and other candidate strains and _S. cerevisiae_ BY4742 had tolerance at 100 mg/L. _Hansenispora_ uvarum_ PSWCC70 and _H. opuntiae_ PSWCC64 were relatively sensitive to sulfite with significantly decreased growth ratio when sodium metabisulfite concentration was higher than 80 mg/L (Figure 3A). On the other hand, all five candidate strains demonstrated lower tolerance to ethanol compared to BY4742. _Hansenispora_ uvarum_ PSWCC70_, H. opuntiae_ PSWCC64, and _P. kluyveri_ PSWCC62 demonstrate  
Figure 2: Main contributing fungal species during spontaneous fermentation of Chambourcin grape must. **(A)** Relative proportion of identified fungal species (%) from 120 randomly selected colonies (20 isolates/sampling time; 40 isolates from each location) obtained during spontaneous fermentation. **(B)** Fungal species identified on Dichloran Rose Bengal Chloramphenicol (DRBC) agar plates at 0 and 24 h (20 colonies/time point). tolerance at 8% of ethanol while _A. pullulans_ PSWCC82 was not tolerant to any concentration of ethanol tested. Interestingly, _P. kudriuzevii_ PSWCC102 was the only non-_Saccharomyces_ yeast candidate with comparable ethanol tolerance to BY4742 grown in 10% ethanol (Figure 3B). These results suggest that _H. ounutiae_ PSWCC64 and _P. kudriuzevii_ PSWCC102 could adapt to high ethanol environment better than the other candidate non-_Saccharomyces_ yeasts during the alcoholic fermentation process. Figure 3: (**A**) Sulfate and (**B**) ethanol tolerance of candidate non-_Saccharomyces_ isolates compared to laboratory control, _S. cerevisiae_ BY4742. The heights of bar graphs represent tolerance which is defined as a ratio greater than 10% between growth of a strain in media supplemented with and without sulfite or ethanol. Yeast Peptone Dextrose (pH 3.0) was used as a growth medium for sulfite tolerance assay whereas YPD at pH 7.0 was used for ethanol tolerance (see Section 2). Dotted line across the y-axis represents a 10% cutoff. Strains used in this study are represented on the x-axis. Data is presented as mean ratio \(\pm\) SEM with a common superscript indicating significance (\(p<0.05\)). Characterization of Core Nonvolatile Compounds Highlight Fermentiative Potential of Candidate Non-Saccharomyces Ycasts  
Nine nonvolatile compounds that represent fermentative potential of non-_Saccharomyces_ yeasts compared to control strain, _S. cerevisiae_ BY4742 were measured at the end of fermentation (Figure S1). Glucose (89.56 \(\pm\) 0.78 g/L) and fructose (90.75 \(\pm\) 0.56 g/L) are major sugars in Chambourcin juice. We hypothesized that candidate non-_Saccharomyces_ strains can contribute to production of fermentation important metabolites. In support of this, the ability of non-_Saccharomyces_ strains to convert sugars into different wine important fermentation by-products was examined. There was no significant difference in residual sugars measured in inoculated fermentations of _Pichia kudriuzevii_ PSWCC102 (35.99 g/L) compared to _S. cerevisiae_ BY4742 (36.88 g/L) (Figure 4A). Residual sugars are typically expressed as \({}^{\circ}\)Brix and are important to monitor the progress of fermentation. During inoculated fermentations, _S. cerevisiae_ BY4742 and _P. kudriuzevii_ PSWCC102 converted about 80% of total sugars (glucose and fructose) followed by _H.