## 2 Materials and Methods
### Microbiological Analyses and Yeast Molecular Identification  
Samples from all fermenters were taken every 48 h for enumeration of the yeast population, while after 48 h of yeast inoculation, at the middle and at the end of fermentation for molecular typing. From each fermenter, 1 mL sample per time point was taken to study the microbial growth. A sample taken from a fermenter without yeast inoculation was used as negative control during the whole fermentation procedure. Each sample was serially diluted in sterile saline. The measurements of the _Saccharomyces_ yeast and indigenous non-_Saccharomyces_ yeast population levels were accomplished by plating serial dilutions on Wallerstein Laboratory Nutrient agar (WLN) or Lysine medium agar, respectively. All samples that were grown in WLN medium were plated in duplicate, with one plate being incubated at 37 \({}^{\circ}\)C for 24 h, and the other being incubated at 28 \({}^{\circ}\)C for 48 h, as _S. pustorianus_ cells are thermosensitive and cannot grow significantly at temperatures that are above 30 \({}^{\circ}\)C. The incubation of Lysine agar plates was performed at 28 \({}^{\circ}\)C for 48-72 h. The cell \begin{table}
\begin{tabular}{c c} \hline \hline  & **Yeast Strains in Pure Cultures** \\ \hline Code & Yeast strains \\ Sp2 & _S. pastorianus_ Sp2 \\ Sc1 & _S. cerevisiae_ CK S102 \\ Sc2 & _S. cerevisiae_ SH 12 \\  & Yeast strains in mixed cultures \\ Code & Yeast strains \\ Sp2 70\%–Sc1 30\% & _S. pastorianus_ Sp2 70\%–_S. cerevisiae_ CK S102 30\% (_w/w_) \\ Sp2 80\%–Sc1 20\% & _S. pastorianus_ Sp2 80\%–_S. cerevisiae_ CK S102 20\% (_w/w_) \\ Sp2 90\%–Sc1 10\% & _S. pastorianus_ Sp2 90\%–_S. cerevisiae_ CK S102 10\% (_w/w_) \\ Sp2 95\%–Sc1 5\% & _S. pastorianus_ Sp2 95\%–_S. cerevisiae_ CK S102 5\% (_w/w_) \\ Sp2 99\%–Sc1 1\% & _S. pastorianus_ Sp2 99\%–_S. cerevisiae_ CK S102 1\% (_w/w_) \\ Sp2 70\%–Sc2 30\% & _S. pastorianus_ Sp2 70\%–_S. cerevisiae_ SH 12 30\% (_w/w_) \\ Sp2 80\%–Sc2 20\% & _S. pastorianus_ Sp2 80\%–_S. cerevisiae_ SH 12 20\% (_w/w_) \\ Sp2 90\%–Sc2 10\% & _S. pastorianus_ Sp2 90\%–_S. cerevisiae_ SH 12 10\% (_w/w_) \\ Sp2 95\%–Sc2 5\% & _S. pastorianus_ Sp2 95\%–_S. cerevisiae_ SH 12 5\% (_w/w_) \\ Sp2 99\%–Sc2 1\% & _S. pastorianus_ Sp2 99\%–_S. cerevisiae_ SH 12 1\% (_w/w_) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Saccharomyces_ yeast species in pure and mixed cultures in different inoculation ratios. growth of total and wild yeast populations was measured in terms of colony-forming units (CFU/mL). After the colony enumeration of each plate, the WLN plates that were incubated at 28 \({}^{\circ}\)C were stored at 4 \({}^{\circ}\)C in order for them to be used for a yeast genetic fingerprint analysis. The DNA fingerprinting took place after 48 h, at the middle and at the end of the fermentation process in order to confirm that the dominated strains of the _Saccharomyces_ species were the inoculated ones and not the indigenous ones. _Saccharomyces_ strains that were responsible for the fermentation in each fermenter were identified by inter-delta sequence profile analysis using \(\delta\)12 (5\({}^{\prime}\)-TCAACAATGGAAATCCCAAC-3\({}^{\prime}\)) and \(\delta\)2 (5\({}^{\prime}\)-GTGGATTTTTATTCCAACA-3\({}^{\prime}\)) primers [7; 13]. A single colony was picked from the WLN plate of interest (the one that was incubated at 28 \({}^{\circ}\)C and stored at 4 \({}^{\circ}\)C), and the colony cells were resuspended in 20 \(\upmu\)L of 0.02 N NaOH. Then, the cells were lysed through heat, after 10 min of incubation at 98 \({}^{\circ}\)C in their resuspension solution, and 3 \(\upmu\)L of lysed cells were used as template for each PCR reaction. A total of 16 colonies per fermenter and per time point, corresponding to at least half of the colonies that were enumerated, were tested through PCR reaction with \(\delta\)12/\(\delta\)2 primer set, as well as 16 colonies from the negative control fermenter.