**3. Discussion**

Winemakers are limited in their ability to influence the native microbial population of grape juice, with SO2 addition representing the main available intervention. Previous microbiological studies have shown that species (and strains) of the major wine yeasts can respond differently to the application of SO2, with commercial strains of *S. cerevisiae* displaying diverse but higher tolerance to SO2 [20], while "wild" yeasts display lower tolerances and are therefore thought to be broadly selected against through the

application of moderate amounts of SO2 prior to the start of fermentation [14–16,21]. Through the application of ITS meta-barcoding, this study demonstrated that the addition of over 40 mg/<sup>L</sup> of total SO2 favored the presence of the non-*Saccharomyces* species *H. osmophila* at the expense of other genera such as *Metschnikowia*, *Torulaspora* and *Kazachstania*. *H. osmophila* has previously been shown to be resistant to SO2 concentrations of over 40 mg/<sup>L</sup> [22,23]. While previous research into the effects of SO2 on grape juice consortia did observe the antagonistic effect of SO2 against non-*Saccharomyces* yeasts, the presence of *H. osmophila* was not specifically reported, although *Hanseniaspora* yeasts were observed at levels of SO2 above 40 mg/<sup>L</sup> [16,24,25].

Much is known regarding the molecular basis of SO2 tolerance in *S. cerevisiae*, where the sulfite efflux pump *SSU1* provides the main mode of resistance [26,27], but very little information is available on the main genetic determinants of SO2 resistance in non-*Saccharomyces* species, although *SSU1* has been suggested to have a major role in *Brettanomyces bruxellensis* [28]. However, recent comparative genomic studies of *Hanseniaspora* spp. have shown that there is a clear differentiation of this genus into two well-defined phylogenetic clades, in which one of the differentiating factors is a homolog of *SSU1*, which is absent in the large clade containing *H. uvarum*, but present in the clade containing *H. osmophila* and *H. vineae* [29–31]. While this may explain the different response to SO2 of *H. osmophila* versus *H. uvarum*, there are likely many other factors that impact the response of a specific species, as *T. delbrueckii*, which also possesses an *SSU1* homolog and displayed increased abundance at up to 40 mg/<sup>L</sup> SO2, and is less tolerant at higher SO2 concentrations than *H. osmophila*. Likewise, *H. vineae*, which was present at levels similar to *H. osmophila* in the control ferments and contains an *SSU1* homolog, did not increase in abundance in response to increases in the concentrations of SO2.

Detailed chemical analysis showed that the addition of SO2 resulted in a significant increase in the concentration of key esters and particularly the aroma compound 2-phenylethyl-acetate, which increased over 9-fold, to levels well above the sensory threshold for this compound, even under modest SO2 additions (40 mg/L). Given the microbiological shift that was observed, it is likely that this change in ester production is due to the increasing prevalence of *H. osmophila* in these ferments with higher SO2 levels. This is supported by published data from fermentations established with purified non-*Saccharomyces* strains, in which mixtures of *H. osmophila* and *S. cerevisiae* (90:10 ratio) were shown to produce higher concentrations of acetate esters (with the exception of isoamyl acetate) and concentrations of 2-phenylethyl-acetate almost 10-fold greater than those observed using *S. cerevisiae* alone [32,33].

In summary, uninoculated fermentations can provide desirable complexity, however, the process lacks the ability to introduce specific fermentation characteristics through the use of commercial starter strains with distinct fermentation aroma and/or flavor profiles. The ability to modulate the yeas<sup>t</sup> community structure of an uninoculated ferment, and the resulting chemical composition of the final wine, demonstrated in this study represents an important tool for winemakers to begin to be able to influence the organoleptic profile of uninoculated wines.

#### **4. Materials and Methods**
