Fungal Diversity and Dynamics during Grape Wine Fermentations with Different Sulfite Levels and Yeast Inoculations

Results and Discussion

Composition analysis of juice/wine. The composition analysis of the Noble juice showed 18.2% soluble solids, 0.3% TA, and pH 3.16, whereas the Vignoles juice had 24.2% soluble solids, 1.03% TA, and a pH of 3.02. In general, Vitis vinifera grapes for commercial wine production have 20 to 23% soluble solids, a TA of 0.6 to 0.7%, and a pH < 3.3 to 3.5. The Noble and Vignoles juices had composition values that fall outside of this range but are typical for these varieties when grown in Arkansas. Muscadine grapes often contain lower TA than other winegrapes (Barchenger et al. 2015, Zhang et al. 2017), whereas Vignoles had higher soluble solids and TA (Howell et al. 1991, Wilker et al. 2004). Dry table wine contains 85 to 89% (w/w) water and 9 to 13% ethanol, with the remainder consisting of glycerol, acids, residual sugars, polyphenols, polysaccharides, minerals, and volatile compounds (Waterhouse et al. 2016). Wines typically have glycerol concentrations of 7 to 10 g/L (Waterhouse et al. 2016), and the glycerol levels of all Noble and Vignoles wines were near the typical range of 5 to 6 g/L for Noble and 4 to 8 g/L for Vignoles and did not differ greatly during and after fermentation. The Vignoles juice had a higher level of initial total sugars (263 g/L) than the Noble juice (194 g/L), which resulted in higher ethanol levels in the wine.

The three-way interaction between sulfite levels, yeast inoculations, and fermentation time was significant for both varieties (Figures 2 and 3). The type of yeast inoculation had a larger effect than sulfite level on fermentation performance (sugar conversion to ethanol). Increasing sulfite levels did not impact fermentation performance of S. cerevisiae but did impact fermentation performance of uninoculated juice and juice inoculated with T. delbrueckii. Although there was a decrease in total sugars and an increase in ethanol for both varieties, the effect of sulfite levels on fermentation performance was larger for Vignoles than for Noble.

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Figure 2

Effects of sulfite levels (SO₂: 0, 10, and 20 mg/L), yeast inoculation type (uninoculated, Saccharomyces cerevisiae, and Torulaspora delbrueckii), and fermentation time (0, 14, and 21 days) on total sugars, ethanol, and total organic acids in Arkansas-grown Noble juice/wine. Each standard error bar was constructed using 1 standard error from the mean. Means with different letters for each attribute are significantly different (p < 0.05) according to Tukey’s honest significant difference test. Total sugars were calculated as the sum of glucose and fructose. Total organic acids were calculated as the sum of citric, tartaric, malic, succinic, lactic, and acetic acids.

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Figure 3

Effects of sulfite levels (SO₂ 0, 10, and 20 mg/L), yeast inoculation types (uninoculated, Saccharomyces cerevisiae, and Torulaspora delbrueckii), and fermentation time (0, 14, and 21 days) on total sugars, ethanol, and total organic acids in Arkansas-grown Vignoles juice/wine. Each standard error bar was constructed using 1 standard error from the mean. Means with different letters for each attribute are significantly different (p < 0.05) according to Tukey’s honest significant difference test. Total sugars were calculated as the sum of glucose and fructose. Total organic acids were calculated as the sum of citric, tartaric, malic, succinic, lactic, and acetic acids.

The levels of sugars, glycerol, ethanol, and organic acids in wines after fermentation at day 21 are presented in Table 1. Total sugars are presented as the sum of glucose and fructose, and these residual sugars varied by treatment. For both grape varieties, regardless of sulfite level, uninoculated juice and juice inoculated with T. delbrueckii had higher total residual sugars (~62 and ~51 g/L for uninoculated and T. delbrueckii-inoculated juice, respectively) than juice inoculated with S. cerevisiae (~2 g/L), resulting in lower ethanol levels (~109 and ~117 g/L for uninoculated and T. delbrueckii-inoculated juice, respectively, compared to ~145 g/L for S. cerevisiae-inoculated juice). The glycerol levels of wine at 21 days of fermentation for both grape varieties were low (~4 to 8 g/L), and total organic acids ranged from 11 to 17 g/L. Total organic acids consisted of the sum of citric, tartaric, malic, succinic, lactic, and acetic acids, and these individual acids varied by treatment. The fermentation performance of Vignoles and Noble juice/wine will be described in the following sections.

Noble juice/wine. Sugars of Noble. The initial total sugar levels of the Noble juice prior to fermentation (day 0) were 191 to 198 g/L (Figure 2). The sulfite levels affected total sugars in the uninoculated juice/wine at days 14 and 21. On both days, uninoculated juice with 0 mg/L SO₂ had lower total sugars than uninoculated juice with 20 mg/L SO₂. The uninoculated juice at day 14 had total sugar levels of 67 to 103 g/L (70, 67, and 103 g/L for uninoculated juice at 0, 10, and 20 mg/L sulfite, respectively). After 21 days of fermentation, uninoculated juice contained a total sugar concentration of 49 to 71 g/L, indicating that fermentation was incomplete or “stuck”. Total sugar levels of wine at day 21 in uninoculated juice with 0, 10, and 20 mg/L sulfite were 49, 54, and 71 g/L, respectively. Table 1 shows that in uninoculated juice at day 21 regardless of SO₂ level, the glucose and fructose levels of these residual sugars were about equal.

Most of the sugars were fermented in S. cerevisiae-inoculated juice after 14 days of fermentation (total sugars < 2 g/L). The total sugar content of S. cerevisiae-inoculated juice/wine at day 21 was less than 0.4 g/L regardless of SO₂ level. At day 21, S. cerevisiae-inoculated juice had no fructose and very little glucose (<0.35 g/L) regardless of SO₂ level (Table 1).

For juice inoculated with T. delbrueckii, total sugars had dropped to 64 to 67 g/L at day 14. At day 21, total sugars in T. delbrueckii-inoculated juice/wine had further dropped to 40 to 42 g/L, indicating that the fermentation was incomplete. Total sugar levels of T. delbrueckii-inoculated juice/wine at day 21 at sulfite levels of 0, 10, and 20 mg/L were 40, 42, and 42 g/L, respectively. The lower growth rates of T. delbrueckii species compared to S. cerevisiae, as well as their inability to consume all available sugars, have previously been demonstrated (Bely et al. 2008). Regardless of SO₂ level, T. delbrueckii-inoculated juice at day 21 had more fructose (28 to 29 g/L) remaining than glucose (12 to 13 g/L) (Table 1).

Ethanol of Noble. The sulfite levels showed an effect on ethanol in uninoculated juice/wine at days 14 and 21 (Figure 2). At day 14, juice with 0 and 10 mg/L SO₂ had higher ethanol levels than juice with 20 mg/L SO₂ (SO₂ 0 mg/L: 80 g/L, SO₂ 10 mg/L: 82 g/L, and SO₂ 20 mg/L: 58 g/L). At day 21, juice with 0 mg/L SO₂ had higher ethanol levels than juice with 20 mg/L. Ethanol levels at day 21 in uninoculated juice at 0, 10, and 20 mg/L sulfite were 102, 100, and 87 g/L, respectively.

For S. cerevisiae-inoculated juice, ethanol increased drastically from day 0 to day 14 (124 to 128 g/L) with similar levels at day 21 (131 to 133 g/L) and no significant difference between sulfite levels. On day 14, S. cerevisiae-inoculated juice contained more ethanol (124 to 128 g/L) than T. delbrueckii-inoculated juice (78 to 83 g/L) and uninoculated (58 to 82 g/L) juice. Ethanol levels at day 21 in S. cerevisiae-inoculated juice at 0, 10, and 20 mg/L sulfite were 133, 131, and 131 g/L, respectively. The larger increase in ethanol and decrease in total sugars during fermentation confirmed that S. cerevisiae had better conversion efficiency.

Ethanol concentration increased progressively in juice inoculated with T. delbrueckii, reaching 78 to 83 g/L at day 14 and 102 to 112 g/L at day 21; there was no significant difference in fermentation performance between sulfite levels. Ethanol levels at day 21 for the T. delbrueckii-inoculated juice at 0, 10, and 20 mg/L sulfite were 109, 112, and 102 g/L, respectively.

Organic acids of Noble. Total organic acid levels of the Noble juice increased during fermentation (Figure 2). At day 0, total organic acid levels were 7 to 8 g/L. From day 0 to day 14, total organic acids increased in all three inoculation treatments of the juices. The largest increase was observed for uninoculated juice (11 to 13 g/L), followed by juice inoculated with T. delbrueckii (11 to 12 g/L), and then S. cerevisiae (10 to 11 g/L). In uninoculated juice, the lower the sulfite levels, the higher the total organic acids produced at day 14. In uninoculated juice at day 21, there were no differences among the total organic acid levels of the three sulfite levels (SO₂ 0 mg/L: 13 g/L, SO₂ 10 mg/L: 14 g/L, and SO₂ 20 mg/L: 13 g/L). A similar pattern was found in juice inoculated with S. cerevisiae. However, at day 21 for juice inoculated with T. delbrueckii, total organic acid levels were higher in juice with 10 and 20 mg/L sulfite (13 g/L) than in juice without sulfite (12 g/L). Bokulich et al. (2015) found that low levels of SO₂ in uninoculated fermentations led to slower fermentations with higher levels of lactic and acetic acid bacteria. Thus, total organic acids can vary depending on the presence or absence of lactic and acetic acid bacteria, but these bacteria were not evaluated in our study. In all inoculated juices at day 21, regardless of SO₂ level, tartaric acid was the most prominent acid (3.6 to 4.9 g/L); in the T. delbrueckii-inoculated juices, succinic acid levels were also high (3.1 to 3.3 g/L) (Table 1).

Vignoles juice/wine. Sugars of Vignoles. At day 0, initial total sugar levels of Vignoles were 250 to 278 g/L (Figure 3).

Uninoculated juice had total sugar levels of 58 to 128 g/L at day 14. There were differences in total sugars between sulfite levels, with lower total sugars at higher sulfite levels for uninoculated juice (SO₂ 0 mg/L: 128 g/L, SO₂ 10 mg/L: 98 g/L, and SO₂ 20 mg/L: 58 g/L). Total sugars of wine from uninoculated juice at day 21 with 0, 10, and 20 mg/L sulfite were 81, 69, and 50 g/L, respectively, indicating that fermentation was incomplete. Similarly, Bokulich et al. (2015) also found lower sugar levels with 20 mg/L sulfite compared to 15 and 0 mg/L sulfite in uninoculated fermentations of Chardonnay at day 21. In uninoculated juice at day 21, regardless of SO₂ level, fructose levels (43 to 60 g/L) were higher than glucose levels (7 to 21 g/L) (Table 1).

Most of the sugars were fermented in the S. cerevisiae-inoculated juice after 14 days of fermentation, with total sugar levels of 2 to 10 g/L (Figure 3). At day 21, sugars were almost completely fermented (less than 7 g/L) for juice inoculated with S. cerevisiae (SO₂ 0 mg/L: 7 g/L, SO₂ 10 mg/L: 3 g/L, and SO₂ 20 mg/L: 2 g/L). There were no differences in total sugar levels at days 14 and 21 between sulfite levels in juice inoculated with S. cerevisiae. In S. cerevisiae-inoculated juice at day 21, regardless of SO₂ level, glucose levels were very low (0 to 0.2 g/L), as were fructose levels (2 to 6 g/L) (Table 1).

At day 14, total sugar levels of T. delbrueckii-inoculated juice at 0, 10, and 20 mg/L sulfite had decreased (115, 87, and 50 g/L, respectively) (Figure 3). There were differences in total sugars between the three sulfite levels, with lower total sugars at higher sulfite levels at days 14 and 21. After 21 days of fermentation, total sugar levels of wine from T. delbrueckii-inoculated juice with 0, 10, and 20 mg/L sulfite were 81, 58, and 42 g/L, respectively, indicating that the fermentation was incomplete. In T. delbrueckii-inoculated juice at day 21, regardless of SO₂ level, fructose levels (36 to 56 g/L) were higher than glucose levels (5 to 25 g/L) (Table 1).

Ethanol of Vignoles. At day 14, ethanol concentration had increased for all three yeast inoculations, with S. cerevisiae (153 to 162 g/L) having better fermentation performance than T. delbrueckii (97 to 128 g/L) and uninoculated (79 to 119 g/L) juices (Figure 3).

At day 14, ethanol levels in uninoculated juice differed for the three levels of sulfites (SO₂ 0 mg/L: 79 g/L, SO₂ 10 mg/L: 95 g/L, and SO₂ 20 mg/L: 119 g/L). At day 21 for uninoculated juice, higher levels of ethanol were observed at the highest level of sulfites (SO₂ 0 mg/L: 115 g/L, SO₂ 10 mg/L: 112 g/L, SO₂ 20 mg/L: 137 g/L).

For S. cerevisiae-inoculated juice, there were no differences in ethanol levels at day 14 between the three sulfite levels (SO₂ 0 mg/L: 162 g/L, SO₂ 10 mg/L: 153 g/L, SO₂ 20 mg/L: 153 g/L). However, there was a difference at day 21, with lower ethanol levels observed with the lowest addition of sulfites (SO₂ 0 mg/L: 163 g/L, SO₂ 10 mg/L: 150 g/L, SO₂ 20 mg/L: 164 g/L). As for Noble juice, a larger increase in ethanol and decrease in total sugar levels during fermentation confirmed that S. cerevisiae had the best conversion efficiency in Vignoles juice.

Juice inoculated with T. delbrueckii had its highest level of ethanol when inoculated with the highest level of sulfite at day 14 (SO₂ 0 mg/L: 98 g/L, SO₂ 10 mg/L: 97 g/mL, and SO₂ 20 mg/L: 128 g/L) and day 21 (SO₂ 0 mg/L: 115 g/L, SO₂ 10 mg/L: 120 g/L, SO₂ 20 mg/L: 142 g/L).

Organic acids of Vignoles. The level of total organic acids in Vignoles juice at day 0 was 14 to 18 g/L. The fermentation pattern of Vignoles was different from that of Noble as there was not an increase of total organic acids during fermentation. In general, regardless of yeast inoculation treatment, total organic acid levels were slightly higher in juices/wines without sulfites compared to those with sulfites during fermentation, but this difference was not always significant. This can be explained by the fact that the absence of sulfites allows the growth of lactic and acetic acid bacteria, which affect organic acid levels (Bokulich et al. 2015). Total organic acid levels differed more with sulfite level in the Vignoles fermentation than in the Noble fermentation. In all inoculation treatments of juice at day 21, regardless of SO₂ level, malic acid was the most prominent acid (5.2 to 6.4 g/L). The second-highest acids varied by inoculation treatment and was also impacted by SO₂ level (Table 1).

Sequence analysis of juice/wine. The fungal diversity analysis of the Noble and Vignoles juices/wines generated 529 and 418 Operational Taxonomic Units, respectively. About four samples from each variety were removed from the analysis due to either a low number of sequence reads (<400 sequences) or dissimilarities compared to replicates. The fungal taxonomic composition of the Noble juice/wine included seven phyla (relative abundance of the fungal communities: Ascomycota, 92.2%; Basidiomycota, 2.3%; and Chytridiomycota, Glomeromycota, Mortierellomycota, Olpidiomycita, and Rozellomycota combined, 0.1%) and 129 genera. The fungal taxonomic composition of the Vignoles juice/wine included six phyla (relative abundance of the fungal communities: Ascomycota, 85.8%; Basidiomycota, 4.6%; and Chytridiomycota, Glomeromycota, Mortierellomycota, and Olpidiomycita combined, 0.4%) and 115 genera. Unknown sequences (Fungi _unclassified) represented 5.4% and 9.1% in Noble and Vignoles, respectively, meaning these sequences were not assigned to any fungi during the taxonomic assignment procedure (RDP Classifier against the UNITE fungal ITS reference data set). Morrison-Whittle et al. (2018) also found Ascomycota to be a major phylum in juice and spontaneous wine fermentations in New Zealand (92.1% of all sequences), followed by Basidiomycota (0.4%), and unknown sequences (7.5%) but did not identify more phyla. The percentage of unknown sequences were similar to those found in Vignoles and Noble. The data at the genus and phylum levels will be further discussed for the two grape varieties, and data for the phylum level is shown in the supplemental figures.

Indigenous fungal communities of juice from the two grape varieties. The indigenous fungal communities of the Noble and Vignoles grape varieties were identified from the juice of grapes prior to fermentation. The fungal genera with a relative abundance higher than 1% in each variety are presented in Table 2. Grapes from both varieties were initially dominated by unclassified taxa: Nectriaceae_unclassified (40.7 and 45.2%) and Fungi_unclassified (15.9 and 17.6%) for Noble and Vignoles, respectively. Identifiable genera were represented in smaller relative abundance but were distinct between the two varieties. Podosphaera was present in both grape varieties but in higher abundance in Vignoles (9.5%) than in Noble (5.5%). Candida was also present in both grape varieties, with a higher abundance in Noble (6.3%) than in Vignoles (3.4%). Noble juice harbored abundant numbers of Uwebraunia (5.2%) and Zygoascus (1.8%). These two genera were either not present or present at a low relative abundance in Vignoles (Uwebraunia, 0.4% and Zygoascus, not detected). In contrast, Vignoles juice harbored a higher relative abundance of Filobasidium (2.4%) compared to Noble (0.2%).

The other indigenous fungal genera (present at >1% relative abundance for at least one of the two grape varieties) included Phialemoniopsis, Meyerozyma, Penicillium, Cyberlindnera, Hanseniaspora, and Aspergillus (Table 2). Interestingly, Aspergillus and Penicillium were present at a higher relative abundance in Noble (1.1 and 1.7%, respectively) than in Vignoles grapes (0.5 and 0.6%, respectively). The presence of these two filamentous fungi could be expected to be higher in Vignoles because the grapes are smaller and in tighter clusters than Noble grapes.

A high percentage of Nectriaceae_unclassified was found in both grape varieties (>40%). These results confirmed that a core of microorganisms was shared between varieties and also that distinct microorganisms were found in each grape variety (e.g., Zygoascus in Noble). This was also observed in previous studies that demonstrated the impact of grape variety on indigenous grape microbiota (Bokulich et al. 2014, Agarbati et al. 2019). Agarbati et al. (2019) observed that Aureobasidium pullulans and Hanseniaspora uvarum were the most widespread yeast species at harvest in two Italian grape varieties, but they also identified specific differences in yeast frequency between the two grape varieties. For instance, Pichia spp. were prevalent in the Verdicchio variety, and L. thermotolerans and Zygoascus meyerae were found in the Montepulciano variety. Bokulich et al. (2014) demonstrated that grape mycobiota of Chardonnay, Zinfandel, and Cabernet Sauvignon grapes from different regions of California were dependent on grape variety along with region and climatic factors. For example, Penicillium was significantly more abundant in Chardonnay; Dothideomycetes, Agaricomycetes, Tremellomycetes, Microbotryomycetes, and Saccharomycetaceae were abundant in Cabernet Sauvignon; and Eurotiomycetes (Aspergillus), Leotiomycetes, and Saccharomycetes (notably Candida zemplinina) were abundant in Zinfandel varieties. As a result of either specific genetic features or vineyard management that is specific to certain grape varieties, distinct fungi have been found on different grape varieties. The high abundance of C. zemplinina (Starmerella bacillaris) on Zinfandel grapes could be because Zinfandel berries have thin skins that allow juice and nutrients to be more available for growth of these fermentative yeasts. Because a large proportion of fungi present on grapes are unclassified, more studies combining HTS and cultivation are needed for further identification.

Fungal diversity and successions during fermentation. Regardless of yeast treatment, the Shannon Diversity Indices (Figure 4) showed similar patterns in fungal diversity for both varieties during fermentation. At day 0, there was high diversity, followed by a decrease in diversity at day 14, and then an increase in diversity at day 21. Surprisingly, this pattern was more notable for fermentation of Noble. As expected, uninoculated juice maintained higher fungal diversity compared with juices inoculated with T. delbrueckii and S. cerevisiae.

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Figure 4

Shannon Diversity Indices of Arkansas-grown Noble (A) and Vignoles (B) juice/wine at day 0, 14, and 21 of fermentation. Uni: uninoculated juice, S. cer: Saccharomyces cerevisiae-inoculated juice, and T. del: Torulaspora delbrueckii-inoculated juice.

For Noble, diversity increased only slightly between days 14 and 21, whereas for Vignoles, diversity returned to levels comparable to initial fermentation. This indicated that the indigenous fungi of Vignoles were more resilient to the fermentation processes, even in the presence of yeast inoculations. This observation may drive variety-specific organoleptic properties through secondary metabolic processes. Because the indigenous grape microbiota and diversity of the Noble and Vignoles juices/wines differed, the dynamics of the fungal communities during fermentation were analyzed separately.

Although NMDS plots are typically used to visualize the level of similarity in a data set, the significance of the points in the data set are hard to visualize when there are many factors (such as sulfite levels and yeast inoculations) and several plots in a figure. At each day of fermentation, the fungal communities tended to cluster by type of yeast inoculation (uninoculated, S. cerevisiae, and T. delbrueckii) rather than by sulfite level, more so in Noble than Vignoles (Supplemental Figures 1 and 2).

Noble fungal community dynamics during fermentation. Beginning fermentation of Noble. At day 0 of fermentation, the fungal communities in Noble juice clustered by the type of yeast inoculation (uninoculated, S. cerevisiae, and T. delbrueckii), with S. cerevisiae and uninoculated clustered together and apart from juice inoculated with T. delbrueckii (Supplemental Figure 1).

The fungal profiles at the phylum level were similar among the three types of inoculations and were dominated by the Ascomycota phylum (81.3% for uninoculated, 81% for S. cerevisiae, and 89.2% for T. delbrueckii), followed by the Basidiomycota phylum (5.4% for uninoculated, 5.7% for S. cerevisiae, and 2.9% for T. delbrueckii) (Supplemental Figure 3). Unclassified Fungi represented 13.2, 13.2, and 7.9% of the fungal communities of uninoculated, S. cerevisiae-inoculated, and T. delbrueckii-inoculated juices, respectively. A higher relative abundance of Ascomycota and smaller relative abundance of Fungi_unclassified and Basidiomycota were detected in T. delbrueckii-inoculated juice, compared to S. cerevisiae-inoculated and uninoculated juices. There were no major dissimilarities between sulfite levels (0, 10, and 20 mg/L) for the three yeast inoculations of Noble juices. However, as sulfite levels increased, a small increase of Ascomycota and a small decrease of Fungi_unclassified and Basidiomycota were detected for both inoculations.

The fungal profiles at the genus level presented dissimilarities between the T. delbrueckii-inoculated juice and both the uninoculated and the S. cerevisiae-inoculated juices (Table 3 and Supplemental Figure 4). At day 0, the dominant fungi identified in the uninoculated and S. cerevisiae-inoculated juices were similar. For instance, regardless of sulfite level, the predominant fungi were Nectriaceae_unclassified (42.5 and 44.3% for uninoculated and S. cerevisiae-inoculated juices, respectively), followed by Uwebraunia (5.7 and 5.4%, respectively), Candida (5.4 and 5.1%, respectively), and Podosphaera (4.9 and 5.1%, respectively). Juice inoculated with T. delbrueckii was already dominated at day 0 by Torulaspora, representing 37.6% of the fungal communities, followed by Nectriaceae_unclassified (31.7%), Candida (3%), Uwebraunia (2.9%), and Podosphaera (2.5%). Moreover, fungal profiles varied slightly between sulfite levels, with slight differences in relative abundance of the main fungi and variation of fungi of smaller relative abundance.

Middle fermentation of Noble. At day 14 of fermentation, the fungal communities of Noble juice clustered by type of inoculation (uninoculated, S. cerevisiae, and T. delbrueckii), with fungal communities of the three types of inoculation clustered apart from one another. The three sulfite levels for each type of yeast inoculation also clustered together (Supplemental Figure 1).

The fungal profiles at the phylum level were similar among the three types of inoculations (Supplemental Figure 3). Compared to the beginning of fermentation, the relative abundance of Ascomycota increased and represented 96.7, 98.5, and 99.5% of the fungal communities of uninoculated juice/wine, juice/wine inoculated with S. cerevisiae, and juice/wine inoculated with T. delbrueckii, respectively. No significant dissimilarities were observed between the three sulfite levels for the three types of inoculated juice.

However, the fungal profile at the genus level (Table 3 and Supplemental Figure 4) differed between the three inoculation types. Noble juice/wine inoculated with T. delbrueckii contained more than 97.8% Torulaspora spp. No differences were observed between the three sulfite levels. Juice inoculated with S. cerevisiae contained more than 92% of the genus Saccharomyces. Sulfite addition slightly modified the composition of fungal communities, with an increase in Nectriaceae_unclassified (0.95, 3.6, and 6.5% for 0, 10, and 20 mg/L of sulfites, respectively) and a decrease in Saccharomyces (97.1, 90.1, and 88.9%, respectively) observed with increasing sulfite levels.

However, uninoculated Noble juice (representing spontaneous fermentation) was dominated by Hanseniaspora (65.1%), followed by Zygoascus (11.8%) and Saccharomyces (6.1%). Sulfite levels played an important part in this fermentation; the higher the sulfite level, the higher the relative abundance of Zygoascus (0.21 to 32.5% for 0 and 20 mg/L of sulfites, respectively) and Schizosaccharomyces (0.1 to 7.4% for 0 and 20 mg/L of sulfites, respectively), and the smaller the relative abundance of Hanseniaspora (77.8 to 32.5% for 0 and 20 mg/L of sulfites, respectively). During the first stages of spontaneous fermentation, Hanseniaspora spp. are known to be the dominant non-Saccharomyces yeast species, along with Issatchenkia spp. and Candida spp., and to be able to coexist with S. cerevisiae at later stages of fermentation (Fleet 2003, Di Maro et al. 2007, Pinto et al. 2015, Portillo and Mas 2016, De Filippis et al. 2017, Raymond Eder et al. 2018, Morgan et al. 2019b). The Hansenispora genus can represent up to 75% of the total initial microbiota and, during fermentation, can be up to 99% of the total yeast communities (Cioch-Skoneczny et al. 2018). This yeast is generally undesirable in fermentation due to the production of large concentrations of ethyl and amyl acetates, glycerol, and acetic acid that negatively alter wine flavors and aroma (Johnson et al. 2020). However, some Hanseniaspora species, such as H. vineae, when inoculated in mixed fermentation with S. cerevisiae, can positively effect organoleptic characteristics by increasing fruity aromas in wines (Domizio et al. 2011, Medina et al. 2013, Tristezza et al. 2016).

End fermentation of Noble. At day 21 of fermentation, fungal communities clustered by type of yeast inoculation (uninoculated, S. cerevisiae, and T. delbrueckii) (Supplemental Figure 1). The three sulfites levels clustered together for each type of yeast and overlapped for T. delbrueckii-inoculated wine. However, the fungal communities of S. cerevisiae and uninoculated wines that did not receive sulfite treatment (SO₂ 0 mg/L) clustered apart from wines with 10 and 20 mg/L of added sulfites.

The fungal profiles at the phylum level presented few dissimilarities between the three types of yeast inoculation (Supplemental Figure 3). An increase in relative abundance of Basidiomycota and Fungi_unclassified and a decrease of Ascomycota appeared in the three types of inoculation (uninoculated, S. cerevisiae, and T. delbrueckii), especially in uninoculated wine and wine inoculated with S. cerevisiae. The sulfite additions had an impact on fungal communities for uninoculated and S. cerevisiae-inoculated wines. The lower the sulfite level, the higher the relative abundance of Basidiomycota and Fungi_unclassified. Also, when sulfites were not added to juice for yeast treatments, other fungal phyla appeared. These results showed that sulfites inhibited other fungal growth in wines.

The fungal profile at the genus level (Table 3 and Supplemental Figure 4) presented some variation between days 14 and 21. Overall, day 21 showed a decrease in the predominant fungi of day 14 (uninoculated: decrease of Hanseniaspora from 65.1 to 49.3%, S. cerevisiae-inoculated: decrease of Saccharomyces from 92 to 57.5%, T. delbrueckii-inoculated: decrease of Torulaspora from 97.8 to 90.3%) and an increase in the relative abundance of Nectriaceae_unclassified (from day 14 to day 21: 5.2 to 21.8% for uninoculated, 3.7 to 25.5% for S. cerevisiae, and 0.75 to 5.4% for T. delbrueckii).

Wines inoculated with T. delbrueckii did not show significant dissimilarities in fungal profiles between the three sulfite levels. However, wines inoculated with S. cerevisiae showed an increase in other fungi, such as Candida (0.5 to 1.9%) and Podosphaera (0.8 to 1.8%). Important dissimilarities in fungal profiles appeared between wines inoculated with S. cerevisiae at different sulfite levels. Compared to S. cerevisiae-inoculated wines with sulfites, wine with no added sulfites had a lower relative abundance of Saccharomyces (SO₂ 0 mg/L: 10%, SO₂ 10 mg/L: 83.6%, and SO₂ 20 mg/L: 78.8%) and a higher relative abundance of Nectriaceae_unclassified (SO₂ 0 mg/L: 54%, SO₂ 10 mg/L: 10%, and SO₂ 20 mg/L: 12.8%), Phialemoniopsis (SO₂ 0 mg/L: 1.26%, SO₂ 10 mg/L: 0.36%, and SO₂ 20 mg/L: 0.31%), and Sarocladium (SO₂ 0 mg/L: 1.22%, SO₂ 10 mg/L: 0.15%, and SO₂ 20 mg/L: 0.11%).

Uninoculated wines also presented dissimilarities in fungal profiles depending on the sulfite levels added to the juice. For instance, in uninoculated wines with no addition of sulfites, Nectriaceae_unclassified exhibited a higher relative abundance (SO₂ 0 mg/L: 40.5%, SO₂ 10 mg/L: 12.2%, and SO₂ 20 mg/L: 12.5%), and Saccharomyces genus exhibited a lower relative abundance (SO₂ 0 mg/L: 0.11%, SO₂ 10 mg/L: 3.9%, and SO₂ 20 mg/L: 12.7%).

Higher levels of sulfites promoted Saccharomyces growth in both uninoculated and S. cerevisiae-inoculated wines. Wine inoculated with T. delbrueckii maintained a high relative abundance of Torulaspora throughout fermentation (day 0 to day 21), with a higher relative abundance than that of Saccharomyces in wine inoculated with S. cerevisiae. This confirmed that T. delbrueckii, a yeast naturally found in vineyards, can be a good candidate for producing wines with specific terroir flavors (Azzolini et al. 2012, 2015, Cordero-Bueso et al. 2013, Roudil et al. 2020). However, the genus Torulaspora was not detected in uninoculated wines. It would be interesting to co-inoculate Noble juice with both S. cerevisiae and T. delbrueckii to observe the dynamics of these two yeasts and indigenous grape microbiota using HTS.

Table 1 shows the main fungal genera of Noble wine at 21 days of fermentation. The main fungi detected in uninoculated juice at 0 mg/L SO₂ and at 10 and 20 mg/L SO₂ were Nectriaceae_unclassified and Hanseniaspora, respectively. The main fungi detected in S. cerevisiae-inoculated juice at 0 mg/L SO₂ and at 10 and 20 mg/L SO₂ were Nectriaceae_unclassified and Saccharomyces, respectively. The main fungal genera of T. delbrueckii-inoculated juice at all SO₂ levels was Torulaspora.

Vignoles fungal community dynamics during fermentation. Beginning fermentation of Vignoles. At day 0 of fermentation, fungal communities of Vignoles juice clustered by type of inoculation (uninoculated, S. cerevisiae, and T. delbrueckii) (Supplemental Figure 2).

The fungal profiles at the phylum level varied between the three types of inoculations and the three sulfite levels (Supplemental Figure 5). Overall, as in Noble juice, fungal communities of the three inoculation types in Vignoles juice were dominated by the Ascomycota phylum (76.6, 76.1, and 82.8% for uninoculated, S. cerevisiae, and T. delbrueckii, respectively), followed by the Basidiomycota phylum (6.1, 7.3, and 6%, for uninoculated, S. cerevisiae, and T. delbrueckii, respectively). Unclassified fungi represented 16.2, 16, and 11% of the fungal communities of uninoculated, S. cerevisiae-inoculated, and T. delbrueckii-inoculated Vignoles wines, respectively. A higher relative abundance of Ascomycota was observed for all three types of inoculation when sulfites were added to the juices.

The fungal profiles at the genus level (Table 4 and Supplemental Figure 6) presented dissimilarities between the three types of inoculated Vignoles juices and varied with sulfite level for each type of inoculated juice. A day 0, the five most abundant fungi identified in uninoculated Vignoles juice, regardless of sulfite level, were Nectriaceae_unclassified (48.3%), Podosphaera (8%), Candida (4.7%), Meyerozyma (2%), and Penicillium (2%). Differences in sulfite level slightly affected the relative abundance of fungal communities, mainly those present at lower abundance in uninoculated juice. The Vignoles juice had a high soluble solids level (24.8%) that could have impacted the initial microbial communities.

For juice inoculated with S. cerevisiae, a clear distinction between fungal profiles appeared at day 0 and was dependent on sulfite level. Juice inoculated with S. cerevisiae without sulfites was dominated by Nectriaceae_unclassified (42.3%), followed by Sporidiobolaceae_unclassified (6.6%), Tremellales_unclassified (2.6%), Phialemoniopsis (2%), and Saccharomycetales_unclassified (1.7%). With the addition of sulfites (SO₂ 10 mg/L and SO₂ 20 mg/L), the presence of Saccharomyces was apparent (SO₂ 0 mg/L: 0.13%, SO₂ 10 mg/L: 48.2%, and SO₂ 20 mg/L: 29.3%). The addition of sulfites promoted a higher relative abundance of Saccharomyces. Intriguingly, the relative abundance of Saccharomyces was higher at a sulfite level of 10 mg/L compared to 20 mg/L. The relative abundance of other fungi also varied between no sulfite addition and the two levels of added sulfites, such as a higher relative abundance of Podosphaera (SO₂ 0 mg/L: 1.3%, SO₂ 10 mg/L: 3.5%, and SO₂ 20 mg/L: 4.9%) and Candida (SO₂ 0 mg/L: 0.5%, SO₂ 10 mg/L: 2.5%, and SO₂ 20 mg/L: 3.8%) and a lower relative abundance of Phialemoniopsis (SO₂ 0 mg/L: 2%, SO₂ 10 mg/L: 1%, and SO₂ 20 mg/L: 1.2%), Nectriaceae_unclassified (SO₂ 0 mg/L: 42.3%, SO₂ 10 mg/L: 23.7%, and SO₂ 20 mg/L: 37.6%), and Sporidiobolaceae_unclassified (SO₂ 0 mg/L: 6.6%, SO₂ 10 mg/L: 0.6%, and SO₂ 20 mg/L: 0.3%).

Juice inoculated with T. delbrueckii, regardless of sulfite level, was dominated by Torulaspora (40.3%), Nectriaceae_ unclassified (28.1%), Podosphaera (3.1%), Candida (2.4%), and Sporidiobolaceae_unclassified (1.5%). The addition of sulfites did alter the fungal communities of juice inoculated with T. delbrueckii. For instance, sulfite addition caused a decrease of the genus Torulaspora (SO₂ 0 mg/L: 59.7%, SO₂ 10 mg/L: 42.6%, and SO₂ 20 mg/L: 18.7%) and increases of Nectriaceae_unclassified (SO₂ 0 mg/L: 7.8%, SO₂ 10 mg/L: 30.8%, and SO₂ 20 mg/L: 45.6%) and Candida (SO₂ 0 mg/L: 0.7%, SO₂ 10 mg/L: 3.4%, and SO₂ 20 mg/L: 3%). Overall, for Vignoles juice, a clear pattern of fungal profiles appeared distinct to each inoculation (uninoculated, S. cerevisiae, and T. delbrueckii) and to sulfite level.

Middle fermentation of Vignoles. At day 14 of fermentation, fungal communities for each type of inoculation (uninoculated, S. cerevisiae, and T. delbrueckii) clustered apart except for the clusters of Vignoles that received 20 mg/L of sulfites (Supplemental Figure 2).

Fungal profiles at the phylum level were similar among the three types of inoculations (Supplemental Figure 5). Compared to the relative abundance at the beginning of fermentation, the relative abundance of Ascomycota increased and represented 95.7, 96.7, and 98.7% of the fungal communities of uninoculated, S. cerevisiae-, and T. delbrueckii-inoculated juices, respectively. The relative abundance of Basidiomycota had decreased at day 14 and was higher in uninoculated juice (SO₂ 0 mg/L: 2%, SO₂ 10 mg/L: 0.9%, and SO₂ 20 mg/L: 0.4%). The relative abundance of Fungi_unclassified had also decreased at day 14 for the three type of inoculations (SO₂ 0 mg/L: 2.2%, SO₂ 10 mg/L: 2.3%, and SO₂ 20 mg/L: 0.9%). No significant dissimilarities were observed among the three sulfite levels for the three types of inoculated juice.

However, the fungal profile at the genus level (Table 4 and Supplemental Figure 6) presented strong dissimilarities among the three types of inoculated juice. Overall, for all three types of inoculated juice, Nectriaceae_unclassified decreased and Saccharomyces increased.

Vignoles juice inoculated with S. cerevisiae was dominated by the genus Saccharomyces (85.8%), followed by Nectriaceae_unclassified (6.7%). The sulfite additions did not affect fungal profiles. However, sulfite levels modified the relative abundance of fungi in uninoculated juice and juice inoculated with T. delbrueckii. For instance, uninoculated juice with no added sulfite and with 10 mg/L SO₂ presented a different fungal profile from uninoculated juice with 20 mg/L SO₂. Hanseniaspora was detected at a higher relative abundance in uninoculated juice with no added sulfite and with 10 mg/L SO₂ than in juice with more sulfite (SO₂ 0 mg/L: 56.8%, SO₂ 10 mg/L: 45.8%, and SO₂ 20 mg/L: 0.02%). This was also true for Candida (SO₂ 0 mg/L: 1.6%, SO₂ 10 mg/L: 1.1%, and SO₂ 20 mg/L: 0.6%). In contrast, a higher relative abundance of Saccharomyces was observed in uninoculated juice with 20 mg/L of sulfites compared to juice with no added sulfite or with 10 mg/L SO₂ (SO₂ 0 mg/L: 23%, SO₂ 10 mg/L: 35.5%, and SO₂ 20 mg/L: 93.2%). The increase in relative abundance of Hanseniaspora in low-sulfite additions or no-sulfite fermentation has been previously described (Morgan et al. 2019b).

Juice inoculated with T. delbrueckii was dominated by the genus Torulaspora when no sulfite was added and when 10 mg/L SO₂ was added (SO₂ 0 mg/L: 96.3%, SO₂ 10 mg/L: 95.8%, and SO₂ 20 mg/L: 16.1%). When a higher level of sulfite (20 mg/L) was added, the genus Saccharomyces dominated (SO₂ 0 mg/L: 0.8%, SO₂ 10 mg/L: 0.5%, and SO₂ 20 mg/L: 74.1%), followed by Torulaspora (16.1%).

At day 14 of fermentation, each inoculation presented a dominant yeast: Hanseniaspora and Saccharomyces in uninoculated juice, Saccharomyces in juice inoculated with S. cerevisiae, and Torulaspora (or Saccharomyces when increased levels of sulfites were added) in juice inoculated with T. delbrueckii. The increase in sulfite levels had a significant impact on the fungal profiles of the three types of inoculated juice.

End fermentation of Vignoles. At day 21 of fermentation, fungal communities clustered by the type of yeast inoculation, with the fungal communities of uninoculated and S. cerevisiae-inoculated juices closer to each other than to T. delbrueckii-inoculated juice (Supplemental Figure 2).

The fungal profiles at the phylum level presented slight dissimilarities between the three types of inoculation and the three sulfite levels (Supplemental Figure 5). An increase of Basidiomycota (uninoculated: 5.5%, S. cerevisiae: 9.5%, and T. delbrueckii: 4%) and Fungi_unclassified (uninoculated: 9.6%, S. cerevisiae: 17.5%, and T. delbrueckii: 6.7%) appeared in all three types of yeast inoculation, and the increase was especially greater in uninoculated wines and wines inoculated with S. cerevisiae.

The fungal profile at the genus level (Table 4 and Supplemental Figure 6) presented some variation compared to day 14. Overall, from day 14 to day 21, an increase of Nectriaceae_unclassified (uninoculated: 31.8%, S. cerevisiae: 40.2%, and T. delbrueckii: 34.5%) and a decrease of Saccharomyces (uninoculated: 14.7%, S. cerevisiae: 19.3%, and T. delbrueckii: 1.6%) for the three types of inoculations and sulfite additions was observed. Fungi of smaller relative abundance appeared at day 21, including Aspergillus, Lachancea, and Zygoascus.

The emergence of these new fungi at day 21 may be explained by the fact that yeasts present at high relative abundance throughout fermentation died and autolyzed, releasing nutrients (amino acids and vitamins) and allowing the proliferation of other yeast species (such as Necteriaceae_unclassified and Fungi_unclassified in this study) that were previously outcompeted for growth (Fleet 2003). The initial sugar level affects the microbiota, which could influence differences between the initial microbiota of Vignoles versus Noble juice. The higher soluble solids of Vignoles juice resulted in a higher ethanol level that could also have affected microbiota by selecting for microbial communities capable of surviving at higher ethanol levels.

The primary fungi detected in uninoculated juice without sulfite addition and with addition of 10 mg/L or 20 mg/L SO₂ were Hanseniaspora and Nectriaceae_unclassified, respectively. The main fungi identified in S. cerevisiae-inoculated juice without sulfite addition and with addition of 10 mg/L or 20 mg/L SO₂ were Saccharomyces and Nectriaceae_unclassified, respectively. The main fungi detected in T. delbrueckii-inoculated juice at 0 mg/L SO₂ and at 10 or 20 mg/L SO₂ were Torulaspora and Nectriaceae_unclassified, respectively (Table 1).

Overall impact of sulfite additions and yeast inoculations. The highest level of sulfites significantly affected fermentation dynamics. For uninoculated juice, Hanseniaspora was strongly inhibited. Intriguingly, Hanseniaspora was replaced by Saccharomyces for Vignoles and by Zygoascus and Schizosaccharomyces for Noble. For Vignoles, the high level of sulfites promoted Saccharomyces but inhibited other fungi. Even in T. delbrueckii-inoculated juice at higher sulfite levels, Saccharomyces growth was promoted over Torulaspora. This can be a beneficial property in terms of Saccharomyces-driven wine production, but it is important to note that the initial inoculation might be reduced if too much sulfite is added at the beginning of fermentation. This should be taken into consideration when using commercial yeast strains of non-Saccharomyces yeasts in mixed-culture fermentations with S. cerevisiae strains. As mentioned by the manufacturers, these commercially available non-Saccharomyces yeasts are sensitive to SO₂ levels and need to be first inoculated without sulfite additions before a second inoculation with selected S. cerevisiae strains. At lower sulfite concentrations and without the presence of S. cerevisiae, non-Saccharomyces yeasts can grow and produce beneficial chemical compounds that can enhance wine complexity or inhibit spoilage microorganisms (Roudil et al. 2020). To further complete fermentation, S. cerevisiae strains are later added to the fermentation.

Nectriaceae_unclassified growth in Vignoles juice was stimulated at day 21 when higher levels of sulfites were added, whereas for Noble juice, their growth was inhibited. Uninoculated Noble and Vignoles juices were dominated by the Hanseniaspora and Saccharomyces genera. However, the relative abundance of these two genera varied by sulfite level and shifted in opposite directions for the two grape varieties. While the relative abundance of Saccharomyces increased with higher sulfite levels in uninoculated Noble juice, it decreased for uninoculated Vignoles juice. Moreover, a third genus, Zygoascus, was identified at a high relative abundance only in uninoculated Noble juice with sulfite additions; it was not identified in uninoculated Vignoles juice, possibly because the two grape varieties had different compositions (e.g., more total sugars in Vignoles juice). In Noble juice inoculated with S. cerevisiae, Saccharomyces growth and dominance took a few days, but its growth was detected at day 0 during fermentation in Vignoles juice. However, the Saccharomyces genus retained a higher relative abundance in Noble juice than in Vignoles juice at day 21. Both juices inoculated with T. delbrueckii showed a dominant Torulaspora relative abundance from day 0 to day 21, with higher relative abundance in Noble juice at day 21 compared to Vignoles juice, in which a decrease in Torulaspora relative abundance was observed at day 21. These results confirmed that grape variety affected indigenous juice/wine mycobiota and the performance of commercial yeasts.