346 M. Ugliano and P.A. Henschke
Gray 1979). Cysteine is a powerful source of H 2 S when added to a yeast culture,
irrespective of nitrogen limitation, although the response is yeast strain dependent
(Duan et al. 2004; Eschenbruch and Bonish 1976; Jiranek et al. 1995; Moreira
et al. 2002; Rankine 1963). Under nitrogenlimitation, cysteine is accumulated
by various specific and general permeases (Yct1p, Gap1p, Mup1p), depending on
amino acid composition of the medium (Kaur and Bachhawat 2007), and degraded
to H 2 S, pyruvate and ammonia by cysteine desulfhydrase (Tokuyama et al. 1973)
(Fig 8D.8). The concentration of free cysteine in grape juice is typically insuf-
ficient (<20 mg/L; Amerine et al. 1980) to explain the prolonged formation of
large amounts of H 2 S often observed during fermentation. However, cysteine can
be derived from other sulfur containing compounds. Hydrolytic release from grape
proteins by yeast proteases has been postulated (Vos and Gray 1979) or intracellular
cysteine can be derived from the major sulfur reserve compound, glutathione, by
enzymatic hydrolysis induced by nitrogen or sulfur limitation, which also yields
glutamate and glycine (Mehdi and Penninckx 1997). Under nitrogen limitation,
cysteine can be further degraded as a source of nitrogen by cysteine desulfhydrase
(Hallinan et al. 1999). Cysteine might alsoserve as precursor for dimethyl sulfide
and 2-mercaptoethanol (Moreira et al. 2002; Rib ́ereau-Gayon et al. 2000b). The
roles of these various mechanisms as sources of H 2 S in wine fermentation are yet to
be demonstrated conclusively.
Methionine is an important though limited source of organic S and N in grape
must and serves several roles in yeast metabolism (Henschke and Jiranek 1991;
Thomas and Surdin-Kerjan 1997) (Fig 8D.8). Its depletion during the early stages
of growth activates the sulfate reductive assimilation pathway to allow biosynthesis
of the S-amino acids, cysteine and methionine. Upon limitation of assimilable nitro-
gen, insufficientO-acetyl-L-homoserine is apparently formed to efficiently sequester
the H 2 S formed from sulfate reduction, leading to its diffusion out of the cell. Sup-
plementation of nitrogen-limited or starved cells with methionine suppresses H 2 S
liberation from the culture of many but not all wine yeast studied (Duan et al. 2004;
Jiranek et al. 1995a,b; Moreira et al. 2002; Spiropoulos et al. 2000) and hence
methionine supplementation does not appear to offer a satisfactory option for con-
trolling H 2 S production. Furthermore, in some wine strains ammonium supplemen-
tation is ineffective when methionine is low.
Nitrogen availability is widely accepted to represent a critical factor in regu-
lating the formation of H 2 S during fermentation. Because sulfide is a metabolic
intermediate in the synthesis of organic sulfur compounds, shortage of nitrogen
results in H 2 S accumulation, due to restricted synthesis ofO-acetyl-L-homoserine,
the sulfide acceptor in the sulfate reduction pathway. Furthermore, sulfite reductase
activity is only slowly down-regulated allowing H 2 S production to continue (Jiranek
et al. 1996; Stratford and Rose 1985b). Supplementation of grape juice with prefer-
ential nitrogen sources, such as ammonium salts, is therefore frequently carried out
in the winery to control H 2 S formation due to nitrogen starvation. Other nutrients,
particularly vitamins such as biotin, pantothenic acid, and pyridoxine, affect H 2 S
formation. Supplementation with pantothenic acid has been shown to reduce the
ability of some strains to form H 2 S (Edwards and Bohlscheid 2007; Tokuyama