Sulphur dioxide is a molecule commonly known as SO2. It is used in many dried fruits (figs, raisins apricots, etc) as an antimicrobial agent and has been used in winemaking by the Romans, when they discovered that burning sulphur candles inside empty wine vessels keeps them fresh and free from vinegar smell.

Sulphur dioxide is used during several steps of the winemaking process. It is added to prevent the unwanted developments of microorganisms, as an anti-oxidant, as an antioxidasic to inhibit polyphenol oxidases (laccase and tyrosinase) and as a dissolvent.

However, sulphites can have a negative impact on wine sensory properties, can delay the onset of malolactic fermentation, and can cause some health concerns in case of high concentrations in the final wine. That’s why SO₂ levels in wine are regulated. On wine bottles, "contains sulphites" must be displayed on the label when found above 10 mg/L. Consequently, it is important in the winemaking process to control and manage the SO2 content of wine in order to maintain the lowest possible concentration while preserving its interesting properties.

SO₂ can be added in wines in several forms such as liquid gas, SO₂ solution, potassium metabisulphite powder or effervescent tablets .

SO₂ is not only and exogenous compounds, as it can also be produced by yeast as it will be discussed in this document.

Sulphur dioxide can be found in many forms in wines, and it will have an impact on the final concentration found in the product. It is important to understand the nature of the form that it takes in the wine and the impact it has.

• Free SO₂ : the active and most efficient form of the sulphites found in wines. This is the form that will be active as an antimicrobial agent, as well as an antioxidant. It is called free because it is not bound or attached to any other compounds.
• Bound SO₂ : When the SO₂ is added to wine or must, a portion will be bound by sugars and by aldehydes (such as acetaldehyde) and by ketones. This form of SO₂ is not active.
• Total SO₂ : Free + Bound SO₂.
• Molecular or Active SO₂ : the molecular SO₂ is the most active and efficient form of the free SO₂. This form of SO₂ is more precise than the free SO₂ in the degree of protection that it offers to the wine. It’s calculated with a formula taking in account pH, temperature, the % of alcohol and the free SO₂. The pH of the wine is one of the main factor intervening in the balance molecular, free and total SO₂. Generally, a concentration between 0,35 mg/L and 0,60 mg/L of molecular SO₂ will allow for a proper protection of the wine.

Saccharomyces cerevisiae wine yeast, whether selected or spontaneous, will produce SO₂. Wine yeasts are able to produce from a few mg/L of sulphites to more than 90 mg/L, depending on the fermentation conditions and the yeast strain. It was reported by Delteil (1992) that 30% of indigenous wine yeast from Côte Rôtie (France) were strong SO₂ producers. Sulphur dioxide is an intermediate metabolite in the sulfate assimilation pathway (figure 2) leading to sulphur amino acid synthesis. Under certain conditions, it may be synthesized in excess then excreted into the medium. Furthermore, sulphites are precursors for the synthesis of sulphide, a highly undesirable by-product. Although the sulfate assimilation pathway has been widely studied, little is known about the parameters that influence sulphite production, and the molecular basis responsible for the differences between yeast strains has not yet been completely identified.

The best strategies to avoid such situation is 1) to select a wine yeast that will produce very little SO₂, 2) to know if your selected yeast has a high demand for nitrogen during fermentation and 3) to properly manage alcoholic fermentation.

The parameters influencing the production of sulphur compounds by yeast are :

1. Temperature : it has been shown that at low temperature (16 °C), sulphur production is greater than at 28 °C (Figure 3).
2. The wine yeast used : we know that the production of SO₂ by wine yeast is genetically and environmentaly determined. All wine yeast, selected or spontaneous, will produce various concentration of SO₂. (Figure 4).

Many wine yeast were characterized based on their SO₂ production in a synthetic media. Figure 4 (on next page) illustrate the range of concentration produced by the different wine yeast from the lowest at 5 mg/L to the highest at 90 mg/L. The concentration of SO₂ produced are those that are intrinsically produced by the wine yeast since there were no sulphur addition to the synthetic must. When selecting a yeast for winema-king, based on the condition of the must, the level of SO₂ added, then this factor can be taken into consideration based on the winemaking itine-rary chosen and the wine style desired.

For example, if malolactic fermentation is desired, and knowing the sensitivity of wine bacteria to SO₂, then a wine yeast producing lower concentration of sulphite can be selected. Recent wine yeast selection has also been focused on finding a microorganism able to produce less or no SO₂. During a collaborative work between Lallemand, Institut Coopératif du Vin (ICV France) and the SupAgro INRA (France), a natural wine yeast, Lalvin ICV oKay® was obtained with a directed breeding strategy approach that produces very low levels of SO₂, H₂S and acetaldehyde (SO₂ binding compound). This wine yeast has shown in all situations, a lower production of SO₂, as seen in Figure 5. In the different wines, Lalvin ICV oKay® was in some instances, not producing any SO₂, as shown when there is no red column for this specific yeast. Oenological and sensory properties of the Lalvin ICV oKay® have been shown to be very positive to produce quality wines. Moreover, since this wine yeast produces little or no SO₂ during alcoholic fermentation, malolactic fermentation is compatible when needed.