# Response to Sulfur Dioxide Addition by Two Commercial _Saccharomyces cerevisiae_ Strains
## 1 Introduction  
Sulfur dioxide (SO\({}_{2}\)) has been used as an antimicrobial and antioxidant in winemaking for hundreds of years. It is added at different times throughout the winemaking process, including prior to inoculation with commercial yeast strains, in order to suppress any potential spoilage microorganisms from the grape must. Commercial yeasts are usually strains of _Saccharomyces cerevisiae_, and are expected to be more tolerant to SO\({}_{2}\) than noncommercial yeasts and bacteria. However, different yeast strains may respond differently to SO\({}_{2}\) addition, and it is important for winemakers to know which yeasts respond in favourable ways so they may select appropriate strains to use as inoculants. Wine yeasts have four main methods of surviving in the presence of SO\({}_{2}\): (1) Entering a viable but nonculturable (VBNC) state; (2) expelling SO\({}_{2}\) from the cell via specialized sulfite efflux pumps; (3) reduction of SO\({}_{2}\) via incorporation into the sulfur amino acid biosynthesis (SAAB) pathway; and (4) production of acetaldehyde [1]. If the level of SO\({}_{2}\) added to the must is too high, wine yeasts, including both spoilage yeasts such as _Brettanomyces bruxellensis_ and fermentative yeasts such as _S. cerevisiae_, may enter a VBNC state where they are still metabolically active but are not detected by culture-dependent methods [2; 3; 4]. Such a response is rare for commercial yeasts, because they are selected for their ability to withstand stressful environments, including musts or wines containing SO\({}_{2}\)[5]. Additionally, the concentration of SO\({}_{2}\) in fermentations is often not high enough to induce such a state in all but the most vulnerable wine microorganisms [6; 7; 8]. Sulfite efflux is a desirable responseto SO\({}_{2}\) by commercial wine yeasts because the SO\({}_{2}\) is not converted into an undesirable form nor is it bound and rendered inactive, so it is able to resume its antimicrobial activity on more susceptible microorganisms [9]. Sulfite efflux is conducted via the specialized Ssu1p sulfite efflux pump, and is encoded by the _SSUI1_ gene and regulated by the _FZF1_ transcription factor [9; 10; 11; 12]. Expression of _SSUI1_, or its more resistant allele _SSUI1_-_R_, can be induced in the presence of SO\({}_{2}\), and its expression is often constitutively higher in more SO\({}_{2}\)-resistant strains [11; 13]. When SO\({}_{2}\) enters the cell, the higher internal pH converts molecular SO\({}_{2}\) to bisulfite (HSO\({}_{3}\)\({}^{-}\)), which can be incorporated into the SAAB pathway. Once it is in this pathway, bisulfite is reduced to sulfide (S\({}^{2-}\)) and then either used to produce sulfur-containing amino acids or exported from the cell as hydrogen sulfide (H\({}_{2}\)S). This response to SO\({}_{2}\) is undesirable for winemakers and wine consumers alike, because H\({}_{2}\)S has a low detection threshold and can lend a rotten egg or cooked cabbage aroma to the wine [14]. Finally, acetaldehyde is produced by yeasts as an intermediate in many metabolic pathways, including alcoholic fermentation. Acetaldehyde has an extremely high affinity for SO\({}_{2}\), with one mole of acetaldehyde able to bind approximately one mole of SO\({}_{2}\)[1]. Bound SO\({}_{2}\) is no longer active as an antimicrobial agent, so this is an effective method of sulfite resistance. However, high levels of acetaldehyde in wine can give the wine a bruised apple or sherry-like aroma [15]. Like the sulfite efflux pump, highly resistant yeast strains tend to have higher constitutive production of acetaldehyde, even in the absence of SO\({}_{2}\)[16; 17]. Previous studies have been instrumental in determining the mechanisms by which _S. cerevisiae_ strains respond to the presence of SO\({}_{2}\)[11; 13; 17; 18; 19]. However, most of these studies have focused on one of these mechanisms at a time, and few have investigated the ways that these mechanisms may work together to provide sulfite protection [20].