## 2 Materials and Methods
### Physiological Characterization of Non-Saccharomyces Yeasts  
Candidate non-_Saccharomyces_ yeasts for downstream characterization experiments were selected based on results obtained from sequencing analysis and the absence of toxin production based on previously published studies. For example, strains with highest statistical confidence based on BLAST results, de novo sequences alignment, and phylogenetic analysis were selected as candidate strains. Candidate strains _Hanseniispora uvarum_ PSWCC70, _H. oomutiae_ PSWCC64, _Pichia kluyveri_ PSWCC62, _P. kudriuzvevii_ PSWCC102, and _Aureobasium pullulans_ PSWCC82 isolated from spontaneously fermenting Chambourcin must were chosen for downstream analysis. _Saccharomyces cerevisiae_ BY4742 was used as a laboratory control.  
Candidate non-_Saccharomyces_ yeast strains were grown under varying concentrations of sulfite and ethanol using a microplate-based method described by [PERSON] et al. (2014) and [PERSON] et al. (2015) with minor modifications related to the concentration of substrate [44; 45]. For characterization of sulfite tolerance, yeast strains were grown in liquid YPD media with final concentrations of 0, 40, 60, 80, 100 mg/L sodium metabisulfite (VWR International, Radnor, PA) at pH = 3.0. Sulfites are most effective as an antimicrobial agent in acidic conditions due to the effect from the molecular \(SO_{2}\) penetrating the cell wall and disrupting the enzymatic. For characterization of ethanol tolerance, yeast strains were grown in liquid YPD media (pH = 6.5) containing 0, 8, 10, 12, and 14% (\(v/v\)) ethanol (Decon Labs, King of Prussia, PA, USA). Varying ethanol concentrations were chosen to represent 12-14% ethanol typically present in red wines.  
Five candidate strains and one control strain BY4742 were streaked on YPD agar (1.5% Bacto Agar) (Difco, Sparks, MD, USA) to obtain single colonies which were transferred to 5 mL of liquid YPD media and allowed to grow for 24 h at 25 \({}^{\circ}\)C with shaking (200 rpm). Cell pellets were collected by centrifugation at 4000\(\times\)\(g\) for 5 min and washed twice with sterile phosphate-buffered saline (PBS) and resuspended in fresh liquid YPD media to obtain a starting optical density (OD) of 0.2 at 600 nm. A starting OD\({}_{600}\) of 0.2 is approximately equal to a cell density of 10\({}^{6}\)\(\sim\)10\({}^{7}\) cfu/mL. The resulting yeast culture (20 \(\upmu\)L) was added to 180 \(\upmu\)L liquid YPD media supplemented with varying concentrations of sodium metabisulfite or ethanol described above. The microplate-based assay was conducted at 25 \({}^{\circ}\)C and OD\({}_{600}\) was measured every 30 min after orbital shaking for 10 s using the continuous measurement mode on a microplate reader for 48 h (BioTek Instruments, Winooski, VT, USA). To determine ethanol and sulfite tolerance of these isolates, the ratio (%) between growth of the isolate in YPD media with and without sodium metabisulfite or ethanol at the end of incubation time (t = 48 h) was calculated using the following equation:  
\[\text{Growth ratio }(\%)=\frac{\text{Growth in substract }(\text{OD}_{600})}{\text{Growth in YPD }(\text{OD}_{600})}\ \times 100\%\]  
Isolates with a percentage growth ratio of larger than 10% were considered tolerant [45]. Three biological experiments containing triplicate samples for each experimental condition were used for sulfite and ethanol tolerance experiments.