22.4
Sulfur Metabolism

22.4.1 Introduction

Yeasts require sulfur for growth due to its role in sulfur‐containing amino acids, peptides, and proteins [1–3]. Unlike nitrogen, sulfur availability is rarely limiting to fermentations, and assimilable sources include inorganic species such as elemental sulfur, sulfate, and sulfite, and organic sources such as amino acids and peptides. However, grapes generally contain low amounts of organic sulfur compounds [4] such as cysteine and methionine, and the principal source of sulfur available to yeasts during winemaking is sulfate (SO42−) [2], in which sulfur is in its highest oxidation state (+6). Sulfate is naturally abundant in grape juices or musts and can be found at concentrations up to several hundred mg/L1 [5,6]. Several critical sulfur compounds affecting wine aroma and quality are produced as yeast secondary metabolites during fermentation (Chapter 10), and of primary interest in this regard is the formation of H2S as a byproduct of sulfur amino acid metabolism. Fortunately, while total H2S production for the duration of fermentation can be in the order of several hundred μg/L, much of this is entrained in CO2 and lost due to volatilization such that only a fraction typically remains in the finished wine (e.g., 1–20 μg/L). Nonetheless, formation of H2S is a malodorous nuisance to the winemaker during fermentation, and may lead to more challenging problems through the production of related volatile sulfur compounds that are not as easily purged from wine. Knowledge of formation of sulfur compounds by yeast is therefore indispensible and begins with an understanding of the origins of H2S.

22.4.2 Sulfide production and assimilation

Biosynthesis of S‐containing amino acids requires formation of sulfide (S2−), the most reduced form of sulfur (oxidation state –2). To form sulfide from sulfate (the major sulfur source), yeast employ the sulfate reduction sequence (SRS) pathway (shown in Figure 22.4.1), which uses several enzymes for the uptake and activation of sulfate, followed by its reduction to sulfite and then sulfide. Activation of sulfate by adenylation markedly increases its reduction potential, making its reduction to sulfite more thermodynamically favorable [7]. Following the reduction sequence, additional enzymatic steps see the incorporation of sulfide into cysteine and methionine via coupling with O‐acetylhomoserine (O‐AHS) [1,8]. Notably, while sulfite (SO32−) is produced as an intermediate in the SRS pathway, extracellular sulfite (i.e., from the addition of bisulfite during winemaking) can also be utilized after diffusing into the cell (as SO2, see below). This sulfur assimilation pathway can be summarized as follows:

  • Extracellular sulfate is transported into the cell by sulfate permease.
  • Sulfate is adenylated by ATP sulfurylase (1), forming adenosine‐5′‐phosphosulfate (APS) and pyrophosphate (PPi).
  • APS is phosphorylated to 3′‐phosphoadenosyl‐5′‐phosphosulfate (PAPS) by APS kinase (2).
  • PAPS is reduced to sulfite by PAPS reductase (3) and NADPH.
  • If not excreted, sulfite is reduced to sulfide by sulfite reductase (4) and NADPH prior to its assimilation into S‐containing amino acids or dissimilation as waste product H2S.
  • Further steps by sulfhydrylase, synthase, lyase, and methyltransferase enzymes produce homocysteine, which leads to cysteine and methionine.
Image described by caption.

Figure 22.4.1 Representation of the pathway for assimilation of sulfate into sulfur amino acids by yeast. After uptake, sulfate is adenylated with adenosine‐5′‐triphosphate (ATP) by the action of ATP sulfurylase (1), forming adenosine‐5′‐phosphosulfate (APS) and pyrophosphate (PPi); APS is phosphorylated to 3′‐phosphoadenosyl‐5′‐phosphosulfate (PAPS) by APS kinase (2); PAPS is reduced to sulfite by PAPS reductase (3) and NADPH, releasing phosphoadenosine phosphate (PAP); sulfite can be excreted, or reduced to sulfide by sulfite reductase (4) and NADPH; sulfide can be assimilated into S‐containing amino acids or dissimilated as waste product H2S

Generally, factors that result in accumulation of sulfide but do not facilitate its incorporation into S‐containing amino acids will result in greater accumulation of H2S in the cell and diffusion into the fermenting must. The major sources of variation in H2S formation (yeast genetics, nitrogen availability, etc.) are discussed in more detail below.

Yeast strain influences both sulfite and sulfide formation, depending on environmental and fermentation conditions. Most strains produce 10–30 mg/L of sulfite (low sulfite producers) although some can yield more than 100 mg/L (high sulfite producers) [3]. Many of these differences can be rationalized by differences in activities of enzymes depicted in Figure 22.4.1, meaning variations (or defects) in sulfate uptake, activation, and reduction can be responsible [1,9]. Greater production of sulfite can be attributed to increased sulfate permease and ATP sulfurylase activity or lack of repression (or feedback inhibition) by methionine or sulfite, and low sulfite reductase activity or affinity for sulfite. Conversely, low sulfite production is associated with increased activity of sulfite reductase and sulfhydrylases in the presence of sulfate and sulfite.

Similarly, genetic variations in the SRS pathway among yeasts can affect the amount of H2S produced or accumulated. Strains can be categorized as non‐producing, or low, medium, and high H2S producers. Variability in production is similar for wild (including non‐Saccharomyces) and commercial strains, and genetic variation can provide targets to microbiologists interested in identifying new yeast strains with low H2S production. Sulfite reductase activity is of particular importance to controlling H2S [10], because if sulfite accumulates2 it can be diverted away from sulfide production. A number of surveys that assess the production of H2S by different yeast strains (commercial or wild) show that lower sulfite reductase activity is correlated with lower H2S at the end of fermentation [11–13] – and presumably higher total SO2 as a consequence, although this is not always measured.

Media composition will also affect the SRS pathway – in particular, it is well known that low yeast assimilable nitrogen (YAN) is correlated with higher H2S production (see Section 22.4.3). H2S formation is also reportedly higher in filtered juices as compared to synthetic media, possibly due to added stressors (e.g., from phenolics) or factors other than nitrogen deficiency (e.g., lack of methionine [11], see Section 22.4.3). The strain‐dependent interaction of the SRS pathway with must nutrient status and fermentation conditions makes predicting H2S production challenging in real juices and musts. As an example, strains that produced high amounts of H2S in synthetic media also produced high amounts in Syrah juice, but strains that produced low levels of H2S in synthetic media did not necessarily produce low H2S in real juice [13].

A major pathway to H2S production involves energetically taxing sulfate reduction to sulfite and ultimately sulfide during fermentation. However, SO2 can readily diffuse into the yeast cell to yield intracellular sulfite (and bisulfite) [14], which is a better precursor to H2S than sulfate3 [15], especially under nitrogen‐limiting conditions [4]. With the onset of nitrogen starvation, enzymes associated with sulfate uptake and initial stages of the SRS will be inhibited, but sulfite reduction to sulfide remains unaffected. Although sulfite reductase has relatively short‐lived activity and cold‐lability when extracted from yeast [16], reductase activity has been suggested to continue for several weeks after fermentation (presumably for as long as viable cells still exist), leading to continued formation of H2S. Caution is therefore required with the addition of bisulfite (or SO2) to wines with active yeast cells or yeast lees, especially in large tanks [2]; fortunately, the presence of lees will consume oxygen and decreases the need for sulfite addition at this time. Late additions are particularly problematic because they will occur after the purging effects on H2S by CO2 evolution have largely ceased (see Section 22.4.4).4

22.4.3 Nitrogen sources and H2S formation

The S‐containing amino acid end products (especially methionine) perform a regulatory role in the SRS pathway, meaning sulfide production is linked to the metabolic demand for protein biosynthesis (and S‐amino acids). In most musts, yeast assimilable nitrogen (YAN) is limiting and sulfate is in excess. This results in an insufficient pool of amino acid precursors required to sequester sulfide, leading to overproduction of H2S, particularly during the growth phase when sulfide formation is generally at its greatest [4,17]. Ammonium supplementation may effectively suppress formation of H2S [18], as can a number of amino acids, particularly those that foster high growth rates (e.g., serine, arginine, glutamine, and asparagine) or act as precursors in the formation of cysteine and methionine (i.e., serine and aspartate) [4] (Figure 22.4.2). Differences in yeast strain are apparent in the study of Jiranek et al. [4], but both strains showed a lack of suppressive effect from proline, threonine, and cysteine, with the last of these three increasing production of H2S. These outcomes can be rationalized based on the following:

  • Proline is not a source of yeast assimilable nitrogen (Chapter 22.3).
  • Threonine, which arises from aspartate via homoserine as an intermediate, inhibits the biosynthesis of homoserine, which is a key precursor to cysteine and methionine [19].
  • Even though it has a regulatory role in the SRS pathway, cysteine can be catabolized to yield pyruvate, ammonium, and H2S when nutrients are deficient [20].
Grouped bar graph of the production of H2S over 6h in aliquots of fermenting grape juice medium, initially containing sulfite and ammonium, supplemented with ammonium or amino acids 1h before ammonium depletion.

Figure 22.4.2 Relative production of H2S (log scale, normalized to Strain A, no addition treatment) over 6 h in aliquots of fermenting chemically defined grape juice medium, initially containing sulfite (260 μM) and ammonium (8.3 mM), supplemented with ammonium or amino acids (total equivalent to 14.3 mM of nitrogen) 1 h before the predicted depletion of initial ammonium. Selected data from Reference [4]

Deficiencies of other nutrients involved in production of amino acid precursors (e.g., B group vitamins, pyroxidine and pantothenic acid), while less common,5 can limit the production of methionine and lead to accumulation of H2S [3,15].

Yeast catabolism of cysteine can potentially contribute to H2S formation through β‐lyase activity (Chapter 23.2), although cysteine is an unlikely source of H2S under normal winemaking conditions due to its low abundance. However, degradation of proteins and glutathione (GSH, Chapter 10) may provide a pool of cysteine (or cystine, the oxidized dimeric form of cysteine), and lead to H2S production [2,18,21]. Processes that minimize the presence or degradation of proteins in must (i.e., clarification, pre‐fermentation bentonite treatment, avoidance of proteolytic activity) have been shown to reduce the amount of H2S formed. Analogously, addition of GSH to a must or during yeast rehydration can lead to an increase in H2S production. Interestingly, the uptake of GSH by yeast during rehydration seemed to be important, rather than uptake once fermentation starts [21].

22.4.4 Timing of formation and residual H2S

Lack of must nutrients or their exhaustion during fermentation are commonly recognized as factors that lead to H2S production. Despite the widespread practice of supplementing musts with assimilable nitrogen in the form of DAP, often prior to the onset of fermentation, there can be mixed results in the formation of H2S. In some instances addition of DAP may increase overall H2S production or residual concentration, depending on yeast strain and juice/must composition [17,22] (Figure 22.4.3). Because H2S is highly volatile, the timing of its production, rather than the total amount produced, is important in determining how much might remain in the finished wine [23]. Factors affecting both total H2S production and final H2S concentration can be generalized as follows:

  • Production is low to moderate during early stages of yeast growth and may continue throughout fermentation. Typically these conditions do not respond to nitrogen or nutrient supplementation, and may leave residual H2S in wine.
  • Production is at a maximum during the early–mid phase when yeast is actively growing and YAN becomes depleted – concurrent CO2 evolution at this stage results in volatilization of most of this H2S, although low fermentation vigor can leave residual H2S in wine. DAP and nutrient supplementation can be beneficial for nitrogen‐responsive strains in low YAN conditions.
  • Production is usually low late in fermentation when yeast growth has ceased. However, there is also minimal purging due to decreased CO2 evolution, which can result in increased risk of residual H2S in wine. DAP addition tends to have no effect at this time.
Scatterplot combined with bar graph of variable influence of yeast strain and nitrogen supplementation on the total production and final wine concentrations of H2S in Shiraz and Chardonnay fermentations.

Figure 22.4.3 Variable influence of yeast strain and nitrogen supplementation (total YAN indicated, that is, 250/400 mg/L and 260/410 mg/L)) on the total production and final wine concentrations of H2S in Shiraz (30 kg) and Chardonnay (200 mL) fermentations (note the vastly different y‐scales for total and residual H2S). Unsupplemented control treatments had 100 and 110 mg/L of YAN for Shiraz and Chardonnay fermentations, respectively. Data derived from References [17] and [22]

In summary, higher total amounts of H2S produced during fermentation do not necessarily equate to higher amounts in finished wine, since production after the midpoint of fermentation may be of greatest importance to residual H2S in wine.

References

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  2. 2. Rauhut, D. (2009) Usage and formation of sulphur compounds, in Biology of microorganisms on grapes, in must and in wine (eds König, H., Unden, G., Fröhlich, J.), Springer‐Verlag, Berlin and Heidelberg, pp. 181–207.
  3. 3. Eschenbruch, R. (1974) Sulfite and sulfide formation during winemaking – a review. American Journal of Enology and Viticulture, 25 (3), 157–161.
  4. 4. Jiranek, V., Langridge, P., Henschke, P.A. (1995) Regulation of hydrogen sulfide liberation in wine‐producing Saccharomyces cerevisiae strains by assimilable nitrogen. Applied and Environmental Microbiology, 61 (2), 461–467.
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  8. 8. Thomas, D. and Surdin‐Kerjan, Y. (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 61 (4), 503–532.
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  12. 12. Mendes‐Ferreira, A., Mendes‐Faia, A., Leão, C. (2002) Survey of hydrogen sulphide production by wine yeasts. Journal of Food Protection, 65 (6), 1033–1037.
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Notes