**2. Results**

In order to evaluate the e ffect of SO2 addition on the yeas<sup>t</sup> population structure, triplicate uninoculated fermentations were established in Chardonnay grape juice across two consecutive vintages. The e ffect of these di fferent SO2 concentrations on wine volatile composition was also evaluated.

#### *2.1. SO2 Addition A*ff*ects Yeast Population Structure*

In vintage 2018, the grape juice was treated with one of five di fferent concentrations of total SO2 (0, 40, 60, 80 and 100 mg/L). In addition to its antimicrobial e ffect, SO2 is also a powerful antioxidant [17]. To di fferentiate between the antimicrobial and antioxidant e ffects of the SO2 addition, an alternate antioxidant, glutathione (GSH, 250 mg/L), was also assessed for its e ffects on the yeas<sup>t</sup> community structure.

The progress of each ferment was tracked via sugar consumption (Figure S1), with samples taken immediately after SO2 or GSH addition (T1), at 90% of sugar remaining (T2), 50% sugar remaining (T3) and 10% sugar remaining (T4), for meta-barcoding analysis using the fungal Internal Transcribed Spacer (ITS) region [18,19]. The addition of GSH did not a ffect the duration of fermentation, however, SO2 had a significant impact on the length of time required for the fermentation to reach completion, with two of the 100 mg/mL treatments requiring five to seven days longer than the control ferments (26 day fermentation) and one of the 100 mg/mL treatments becoming stuck with 13 g/<sup>L</sup> of residual sugar.

Across the 18 samples from 2018 (6 treatments in triplicate), Operational Taxonomic Units (OTUs) that could be assigned to a total of 26 fungal genera were detected that exceeded 0.01% of the total abundance in at least one sample (Figure 1; full results in Table S1). Triplicate samples were shown to be highly concordant for each combination of SO2 concentration and timepoint (Figure S2). The highest level of fungal diversity was observed at the T1 timepoint, while *Hanseniaspora*, *Metschnikowia*, *Saccharomyces* and *Torulaspora* dominated the fermentations from T2 through T4, accounting over 95% of the total ITS reads (Figure 1).

As seen for the fermentation kinetics, the GSH addition did not a ffect the overall population structure relative to the control samples, however, the addition of SO2 had a significant, but di fferential effect on the four main genera observed across the samples (Figure 1). *Metschnikowia* displayed the highest sensitivity to SO2, with 40 mg/<sup>L</sup> completely inhibiting the detection of this genus by the T2 timepoint. *Torulaspora* was shown to have a higher abundance at 40 mg/<sup>L</sup> relative to 0 mg/L, however, this genus was progressively inhibited by higher concentrations of SO2 in a gradient from 60 through to 100 mg/L, at which point it was completely inhibited at timepoint T2. *Hanseniaspora* and *Saccharomyces*

were both shown to be tolerant across all the tested SO2 concentrations, with *Hanseniaspora* increasing in its total proportion relative to the other genera as the concentration of SO2 was increased.

**Figure 1.** Genus-level metabarcoding analysis of community response to SO2 addition. Vintage 2018 Chardonnay juice was treated with increasing concentrations of total SO2 (mg/L) or glutathione (GSH, 250 mg/L) as an alternate antioxidant. Ferment samples were taken at four timepoints (T1, at crush; T2, 10% sugar utilization; T3, 50% sugar utilization; T4, 90% sugar utilization) and subjected to ITS metabarcoding. Only genera that exceeded 0.1% abundance in at least one sample are shown.

As fungal ITS sequencing generally affords the ability to define OTUs to the species level, the genus level counts were partitioned into species-level units to determine the effect of SO2 concentration on the abundance of individual species. There were 29 species that exceeded 0.1% of the total abundance in any sample, with the genus *Hanseniaspora* displaying the highest number of individual species (*n* = 4). While the addition of SO2 was shown to increase the overall abundance of *Hanseniaspora* at the genus level, there was a far more complex response profile when species designations were taken into account (Figure 2). Rather than a general increase in all species of *Hanseniaspora*, two species, *H. uvarum* and *H. opuntiae*, were the dominant species when SO2 was absent (GSH and SO2 0 mg/<sup>L</sup> treatments). However, the addition of 40 mg/<sup>L</sup> of SO2 resulted in a drastic shift in the species composition such that *H. osmophila* was the sole representative of this genus at 40 mg/<sup>L</sup> of SO2. The relative abundance of this species increased substantially as SO2 levels were raised, producing the overall increase in *Hanseniaspora* that was observed at the genus level.

**Figure 2.** Species-level meta-barcoding analysis of community response to the SO2 addition. Vintage 2018 Chardonnay juice was treated with increasing concentrations of total SO2 (mg/L) or glutathione (GSH, 250 mg/L) as an alternate antioxidant. Ferment samples were taken at four timepoints (T1, at crush; T2, 10% sugar utilization; T3, 50% sugar utilization; T4, 90% sugar utilization) and subjected to ITS metabarcoding. Only species that exceeded 0.1% abundance in at least one sample are shown. For those OTU where a species-level designation was not possible, the genus-level taxonomic classification of the OTU was used. Only members of the genus *Hanseniaspora* are colored.

A second set of fermentations were established in the subsequent year (2019) using a finer set of SO2 treatment intervals (0, 10, 20 and 40 mg/L). Consistent with the observations from the 2018 vintage, the SO2 addition affected fermentation kinetics, particularly for the 40 mg/<sup>L</sup> treatments (Figure S1). The 2019 ferments displayed a different overall yeas<sup>t</sup> diversity compared with the 2018 samples, with a lack of OTUs that could be assigned to *Metschnikowia* and prominent contributions from OTUs assigned to *Candida* spp., which increased over the range of SO2 concentration used, and *Kazachstania* spp. that were present at up to 20 mg/<sup>L</sup> of SO2 (Figure 3). However, when the species level contributions were investigated, there were clear similarities between the two vintages in the dynamics of the OTUs assigned to the genus *Hanseniaspora* (Figure 4). As also seen in 2018, *Hanseniaspora* was represented by the greatest number of species designations in 2019. The 2019 ferments also displayed a clear species shift that was associated with the use of SO2, with the proliferation of *H. uvarum*, *H. opuntiae* and *H. vineae* all being inhibited by SO2 in a concentration-dependent manner, while the proportion of *H. osmophila* was shown to be enhanced by the addition of 40 mg/<sup>L</sup> of SO2.

**Figure 3.** Genus-level meta-barcoding analysis of community response to the SO2 addition. Vintage 2019 Chardonnay juice was treated with increasing concentrations of total SO2 (mg/L). Ferment samples were taken at four timepoints (T1, at crush; T2, 10% sugar utilization; T3, 50% sugar utilization; T4, 90% sugar utilization) and subjected to ITS metabarcoding. Only genera that exceeded 0.1% abundance in at least one sample are shown.

In order to compare the 2018 and 2019 data, the metabarcoding time course results were analyzed by Bray–Curtis dissimilarity analysis (Figure 5). The T1 samples from across both vintages were broadly similar, with the ferments characterized by the presence of non-fermentative genera such as *Aureobasidium*, *Cladosporium* and *Epicoccum*. As observed in the abundance plots, ferments progressed towards being dominated by *S. cerevisiae* (Axis 1), however, there was a clear division between samples with 40 mg/<sup>L</sup> or more of added SO2, which deviated along Axis 2 towards *H. osmophila*, while the samples with less than 40 mg/<sup>L</sup> were dominated by the signal from *H. uvarum*. Thus, despite differences in the overall microbial populations of the wild fermentations performed across the two vintages, both displayed consistent alterations in the microbial community due to the amount of SO2 addition.

**Figure 4.** Species-level metabarcoding analysis of community response to the SO2 addition. Vintage 2019 Chardonnay juice was treated with increasing concentrations of total SO2 (mg/L). Ferment samples were taken at four timepoints (T1, at crush; T2, 10% sugar utilization; T3, 50% sugar utilization; T4, 90% sugar utilization) and subjected to ITS metabarcoding. Only species that exceeded 0.1% abundance in at least one sample are shown. For those OTU where a species-level designation was not possible, the genus-level taxonomic classification of the OTU was used. Only members of the genus *Hanseniaspora* are colored.

#### *2.2. SO2 Addition Influences Wine Volatile Composition*

Given the significant effect of SO2 addition on the microbial community structure, it was of interest to understand whether these different SO2 treatments were also associated with changes to the chemical composition of the wine. This was assessed through an analysis of the volatile yeas<sup>t</sup> metabolites known to contribute to the aromatic profile of wine. Of the 39 aroma compounds analyzed, 18 displayed a significant difference (ANOVA, *p* ≤ 0.001) in concentration across one of the SO2 regimes in either 2018 or 2019 (Table S2). Of these, ten analytes displayed more than a 1.5-fold decrease in at least one of the SO2 treatments, while seven displayed an increase of the same magnitude (Figure 6). Three analytes, 2-methylpropanol (decreasing in response to SO2) and 2-phenylethyl acetate and hexyl acetate (increasing), displayed the same effect across the 2018 and 2019 vintages. In all cases, there was a significant effect at 40 mg/<sup>L</sup> SO2. Furthermore, in most situations in which a significant difference in analyte concentration was observed across multiple SO2 regimes, there was a correlation between SO2 concentration and the magnitude of change. The largest change in analyte concentration was observed for the desirable aroma compound 2-phenylethyl-acetate, with the 100 mg/<sup>L</sup> SO2 treatment in 2018 displaying over two orders of magnitude more of this metabolite than the control, and the 2019 40 mg/<sup>L</sup> SO2 treatment having over nine times as much 2-phenylethyl-acetate as the control (Table S2). More generally, higher SO2 concentrations resulted in decreases in short chain acetates and higher alcohols and increases in 6-carbon and 8-carbon esters and acids. There was no effect of SO2 concentration on low molecular weight volatile sulphur compound production.

**Figure 5.** Dissimilarity analysis of ITS-amplicon abundance from vintage 2018 and 2019 fermentations. (**A**) Triplicate samples from each time point were subjected to Bray–Curtis dissimilarity analysis (clustered by PCoA) based upon the top 10 most abundant species and are shaded by treatment condition. (**B**) The weightings of the top 10 most abundant species relative to the plots in part (**A**). Points are shaded by species. For those OTU where a species-level designation was not possible, the genus-level taxonomic classification of the OTU was used.

**Figure 6.** Concentration differences in aroma compounds due to the addition of SO2. (**A**) Analytes with significantly reduced (ANOVA, *p* ≤ 0.001) concentrations in 2018. (**B**) Analytes with significantly increased (ANOVA, *p* ≤ 0.001) concentrations in 2018. (**C**) Analytes with significantly decreased (ANOVA, *p* ≤ 0.001) concentrations in 2019. (**D**) Analytes with significantly increased (ANOVA, *p* ≤ 0.001) concentrations in 2019. Individual bars are shaded according to their significance group and the estimated aroma thresholds (see Materials and Methods) are indicated in red.
