BY Angela Lee Linderholm and
Linda F. Bisson
Department of Viticulture & Enology
University of California, Davis
Formation of hydrogen sulfide (H2S)
is a major problem in winemaking because, if untreated, it can leave
a wine with an undesirable rotten-egg sensory characteristic. The
fact that Saccharomyces cerevisiae is responsible for the
sulfide defect in wine has been well-established.2,3,4,5
Production of hydrogen sulfide by Saccharomyces cerevisiae
strains ranges from 0 mg/L to 290 mg/L,
well above the human detection threshold of 11 ng/L.1
Sources of hydrogen sulfide
Hydrogen sulfide can come from different sources (Figure I). If
sulfur is used in the vineyard too close to harvest, the reductive
conditions that are created during fermentation can chemically convert
this sulfur to hydrogen sulfide. Volatile sulfur compounds can also
be formed during degradation of the sulfur containing amino acids,
methionine, and cysteine, which usually are in low concentration
in most juices barring supplementation.
Some strains have been shown to reduce sulfite (used in wine as
an antimicrobial or antioxidant) to hydrogen sulfide, but this trait
appears to be rare. The majority of H2S
produced occurs during the process by which Saccharomyces
makes sulfur-containing amino acids.
Saccharomyces cerevisiae is able to reduce sulfate from its
environment to sulfide and then incorporate it into amino acids
through several more enzymatic steps (Figure II). Sulfide that is
not incorporated into these amino acids is converted to H2S in a
pH-driven reaction. Because winemaking conditions are typically
at low pH, the abundance of hydrogen molecules favors the reaction
to create the volatile gas, H2S.
Hydrogen sulfide can be formed at different times during wine production.
Volatile sulfide formation can occur during the active phase of
fermentation or as the wine ages on yeast lees post-fermentation.
In the first case, it is thought that the sulfide is derived from
either of the sources listed above.
Post-fermentation, sulfide is believed to arise either from degradation
of sulfur-containing compounds in the yeast lees or from the re-release
of chemically-entrapped sulfide during ageing (Figure III). In the
latter situation, the entire sulfide formed during fermentation
was not released as hydrogen sulfide gas, but a fraction was bound
to other compounds or trapped in a non-volatile form in the wine.
As ageing changes the reductive environment of a wine, this sulfur
can be released from its bound or altered form, creating H2S, a
reaction favored by the low pH of the wine. Thus, a sulfide problem,
thought to be cured, can return.
Factors affecting sulfide formation
Hydrogen sulfide production varies depending upon the growth conditions
of the yeast. Environmental and nutritional factors such as levels
of elemental sulfur,5
presence of sulfur dioxide,6
organic compounds containing sulfur,2
and vitamin deficiency7
have been associated with the production of volatile sulfur compounds.
However, the effect of these variables is dependent upon media composition
and growth conditions. It has also been observed that production
levels vary dramatically across strains in response to these conditions.2,8
This variation suggests that differences in internal enzyme regulation
and activity impact H2S production.
The existence of such dramatic variation in sulfide production across
yeast strains has made it difficult to devise generalized recommendations
guaranteed to prevent the appearance of H2S
The sulfate reduction pathway that generates hydrogen sulfide also
generates many toxic intermediates and is biologically expensive
in terms of consumption of cofactors and cellular energy stores.
But, at the same time, the end products of this pathway are essential
for ethanol tolerance and maintenance of the redox status of the
cell, two critical issues challenging the yeast during wine fermentations.
Thus, strategies that simply knock out the activity of this pathway
are not viable for the construction of strains with reduced sulfide
production. Instead, it is important to understand the basis of
the naturally occurring low sulfide production trait so that it
can be exploited to generate strains with reduced tendency towards
Our goal is to determine the biological basis of variation in sulfide
production in yeast strains under winemaking conditions, and to
define the interaction between strain and environment so that sulfide
formation can be more effectively controlled during wine production.
Although it is true that copper treatments can eliminate sulfide
from fermentation and wine via precipitation, the residual copper
must then be considered. It is far more desirable to not have the
sulfide formed in the first place.
Anecdotal information from wine producers experimenting with screwcap
closures suggests that they are experiencing more problems from
sulfide formation in the bottle than in the same wine with cork
or synthetic closures. Although the exact explanation of this phenomenon
will require some experimentation, it is possible that sulfide formed
during or after fermentation may indeed be trapped in a non-volatile
chemical form resistant to copper precipitation that reverts back
to H2S over time under the reductive conditions established with
screwcap closures. Similar comments have been made about de-alcoholized
wine and the re-appearance of a sulfide problem.
Although several studies have predicted the existence of such a
sulfide reservoir in wine, the nature of the bound compound remains
unknown in spite of significant effort to identify it. This is further
impetus to discover and develop wine yeast strains that will not
Understanding role of strain
variation in sulfide production
The specific aim of our research program is to understand hydrogen
sulfide production in Saccharomyces cerevisiae at both the
genetic and physiological level, and to define the genetic changes
that have arisen naturally in native wine strain populations that
result in reduced sulfide formation by these strains. This is no
small task, as the genetic variation seen indicates a complex inheritance,
meaning multiple genes are likely involved.
As a first step, we have characterized sulfide production in a large
group of strains of S. cerevisiae, including native and laboratory
strain isolates and commercial wine and beer strains.8
We have found that some rare strains are consistently high or consistently
low producers of sulfide regardless of the medium in which they
However, most strains show a strong influence of growth conditions
and medium composition on sulfide formation, and produce varying
levels of sulfide in response to those changing conditions. The
levels of sulfide produced may vary over 2,000 fold for a given
strain in response to changing environmental conditions. Other strains
show very little difference in sulfide levels produced under the
same conditions. The highest level of production observed in commercial
or native isolates is roughly 300 mg/L of sulfide.
Regardless of the condition, high temperature, low temperature,
high nitrogen, low nitrogen, vitamin deficiency, mineral excess
or deficiency, strains can be found that will show varying sulfide
production levels in response to the specific environmental condition
evaluated. The magnitude of the response also varies, with some
low level-producing strains suddenly showing a very high level of
production if something in their environment dictates.
Predicting strain behavior
We have found it difficult, if not impossible, to predict strain
behavior with respect to the levels of sulfide formed on the sole
basis of physiological studies and medium composition.
Several different methods have been proposed to evaluate the H2S-producing
behavior of yeast strains. Since sulfide forms a dark-colored precipitate
with heavy metal ions, colony color analyses have been developed
that correlate the amount of dark pigment produced with the level
of sulfide being formed under the given growth conditions.
such medium is called BiGGY agar. BiGGY (bismuth glucose
glycine yeast agar), is a differential medium that evaluates relative
production of sulfide. Sulfide generated by the yeast colony forms
a black precipitate and the intensity of color is dependent on the
amount of sulfide produced. Some examples of the different colony
colors that can be observed are shown in Figure IV.
Copper-based media can also be used in a similar way. Finally,
the sulfide formed during growth in liquid media can be quantified
in the gaseous phase released during fermentation. Since H2S is
volatile, it is driven off in the carbon dioxide stream produced
during fermentation of sugar. The sulfide in this stream can be
trapped and quantified. This can be done using a liquid trap-based
method 4 or by allowing the gas stream to pass
over a column containing lead that will bind to the sulfide. 3
Finally, sulfide from the headspace can be analyzed using gas chromato-graphy.
We have used all of the above methods to characterize sulfide formation
in wine strains. We find that the convenience of the lead trap method
allows screening of large populations of organisms and that other
more quantitative methods can then be used on selected strains.
Interestingly, there is not good agreement between the plate-based
colored colony and gas stream data. Strains that yield dark colonies
often do not give significant sulfide production in liquid media.
Early reports in literature suggested that BiGGY-agar assesses sulfite
reductase activity under conditions of the plate and that the color
of colonies on BiGGY was correlated with levels of H2S
Brown color colonies produced a detectable amount of H2S
by aroma while white colonies produced no detectable sulfide by
aroma. In our research, this was not consistently accurate.
While white colonies did not produce H2S in our trials, some lighter-staining
colonies did make more than darker staining ones under fermentation
conditions. This is true because grape juices and synthetic juices
are different in composition. These compositional differences create
differences in flux through the sulfate reduction pathway, therefore
differing levels of sulfide production occur. Because of this, actual
fermentations must be conducted in order to accurately gauge a particular
yeast strains H2S production.
To define H2S production in yeast
strains in our work, we perform small-scale 300mL fermentations
in synthetic juice media (called Minimal Must Media.)3,8
This media is modeled after grape juice but allows us to control
individual components such as sugar concentration and nitrogen levels
and growth conditions such as temperature and degree of mixing or
shaking. Fermentations are incubated at 25†C with slow shaking to
insure mixing of nutrients.
Hydrogen sulfide production is monitored with lead acetate tubes
attached to the flasks. Carbon dioxide produced during fermentation
pushes the volatile gases through the tube and the sulfur reacts
with the lead acetate-coated beads to produce a dark gray band.
The height of the gray column reflects the amount of H2S production
Varying media composition and growth conditions indicated that there
is indeed significant variation in strain behavior with respect
to formation of hydrogen sulfide, more variation than previously
thought from earlier studies that investigated fewer strains. Some
strains formed more H2S at lower nitrogen levels, some more at higher
nitrogen levels, and some appeared insensitive to nitrogen content
with respect to sulfide formation.
Effect of nutrient levels
A more detailed analysis revealed that, in most cases, strains do
indeed respond to nitrogen similarly, but the trigger-levels at
which their behavior changes will vary among strains. This means
that as nitrogen is increased or decreased, varying levels of H2S
will be produced. But the nitrogen concentration and the nature
of the nitrogen compound required for a given strain to decrease
sulfide formation may be different than for other strains.
We have studied sulfide production in various strains between the
ranges of 120mg/L to 430mg/L nitrogen equivalents. At the highest
nitrogen concentration, the level of production of hydrogen sulfide
varied from 17% to 70% of that seen at the lowest level for commercial
strains. Some native isolates showed even less production at high
The magnitude of the amount of sulfide released by individual strains
will vary as well as the change in amount with nitrogen addition.
Sulfide production at the lower nitrogen level ranged from undetectable
to 150 mg/L for the strains tested while, at the lower nitrogen
level, production varied from undetectable for only a couple of
strains to 300 mg/L.
Since most previous studies selected a single or a few nitrogen
levels but did not examine yeast strains across a wide range of
nitrogen concentrations, the consistency of response to increasing
nitrogen that we observed has gone unnoticed. Our results indicate
that for some strains, although nitrogen supplementation may decrease
sulfide production, the residual level that is produced may still
be well over the threshold of detection.
These findings suggest that winery protocols for nutrient supplementation
to reduce sulfide formation must take strain variation into account.
Similarly, vitamin limitation will impact strains differently with
respect to the amount of sulfide released. Certain vitamins are
needed in order for reduced sulfide to be incorporated into amino
acids. Deficiency for these vitamins can mimic a nitrogen limitation
and lead to elevated levels of sulfide.
Other researchers have found that the interaction between nitrogen
and vitamin levels is also an important factor in sulfide formation.7
Limiting both nitrogen and vitamin levels can result in even higher
levels of sulfide release than either one alone.
Other growth conditions also impact sulfide formation, but our data
suggests that nitrogen and micronutrient levels play a major role
in defined media studies. Interestingly, we have not seen a strong
effect of sulfite addition in juice or wine on sulfide formation.
However, we do know that in juice, factors other than nutrient levels
are also important.
In one study, Pinot Noir juices were characterized for sulfide formation
and amino acid analyses of the juices were performed.8
A synthetic juice was then created mimicking the amino acid levels
of the Pinot Noir juice that had produced high levels of sulfide.
That high level of sulfide formation was not found in the synthetic
juice, suggesting that nutrition alone is not the sole factor dictating
sulfide formation. Manipulation of nutrient levels may not lead
to elimination of a sulfide problem.
Defining genetic basis
of sulfide production
An alternate approach toward eliminating sulfide formation in wine
strains is to use genetic analyses to identify the genes most impacting
sulfide production with the aim of altering those genes so that
sulfide levels will be reduced.
One might think that eliminating the sulfate reduction pathway all
together would be a good strategy to reduce sulfide formation. But
this would then require supplementation of the media with sulfur-containing
amino acids and result in possible formation of volatile sulfur
degradation products from these amino acids by yeast or by bacteria
present in wine. This solution is similar to curing one problem
by creating another. We decided instead to focus our efforts on
determining why some native yeast strain isolates identified from
our studies do not produce sulfide.
The brewing literature suggests that the main driving force in sulfide
release is a diminished ability to incorporate reduced sulfide into
amino acids, caused by relatively lower levels of activity of the
enzymes downstream of sulfite reductase (Figure II).
Published reports demonstrated that enzymes that consume sulfide,
in the sulfate reduction pathway, are responsible for controlling
H2S production in brewing strains.
They demonstrated that by modifying strains to over-express one
of these enzymes, H2S production
was reduced in their strains.10,11
However, only a few brewing strains were evaluated in these studies
and it was not clear that over-expression of these enzymes would
reduce sulfide production in wine strains.
Role of variation in genes
of the sulfate reduction pathway
Given this information, we therefore decided initially to evaluate
the role of the level of expression of these enzymes in H2S
production in wine strains.12
The enzymes we evaluated are the three immediately downstream of
sulfite reductase encoded by MET17, MET6, and CYS4.
We took multiple approaches to determining the role of these enzymes
in sulfide formation in wine strains.
First, we obtained these genes that encode for the activity of these
three enzymes from laboratory and brewing strains. We then used
these genes to look at the effect of increased expression of their
encoded enzyme levels on sulfide formation in wine yeast strains.
Although we were able to show dramatically higher levels of activity
of enzymes encoded by these genes, there was no universal reduction
in H2S production with CYS4, MET6 or MET17.
The second approach was to measure levels of the enzymes encoded
by these genes in wine strains to assess variation in activity.
We did see differences in the level of activity of these genes,
but it was not correlated with sulfide production behavior, suggesting
that biological entrapment of reduced sulfur into amino acids alone
could not explain the variation observed in sulfide production.
Experiments described above examine levels of expression but not
differences in possible regulation or activity of the enzymes themselves.
To evaluate this factor, genes responsible for expression of MET17,
MET6, and CYS4 enzymes were sequenced from 20 different
The function or regulation of a protein can change without a measurable
change in activity. To test this possibility, we sequenced three
genes encoding proteins that function downstream of sulfite reductase
in 20 strains that showed varying levels of sulfide production.
Since all yeast strains alive today have been exposed to DNA alteration,
how do we define the original undamaged DNA sequence?
Mutations or changes in the primary DNA sequence arise naturally
in yeast populations for many reasons. Sometimes a mistake is made
during DNA replication and an incorrect base is inserted into the
sequence that fails to be recognized as an error by the DNA repair
In other cases, the yeast strain has been exposed to environmental
stress or DNA-damaging agents that harm the DNA sequence. The yeast
cell will die if the damage is not repaired. In some circumstances,
the damage can be repaired but not to the original sequence as that
exact information has been irreplaceably lost. The result is a viable
strain, but one that is genetically different from a strain that
was not exposed to DNA damage.
Therefore, the original, undamaged DNA sequence must be defined
by consensus, meaning that the sequence that most unrelated strains
possess is thought to be the undamaged one with rarer changes in
sequence believed to have arisen from either an error in replication
of the sequence or from external damage of the DNA.
The nature of the mutation can also be important. Some mutations
result in dramatic changes to the structure and composition of the
protein or enzyme made from that gene. A mutation can destroy the
sequence such that it is no longer recognized as encoding a protein
or that the protein encoded is so defective that it is no longer
These kinds of mutations lead to a loss of function of the protein
or enzyme, which can be quite damaging to the yeast in its native
environment. Other mutations are more subtle, that is, change one
amino acid in the protein altering the activity or regulation of
the protein but not eliminating its function altogether. These mutations
can often only be found by sequencing the gene itself.
We decided to sequence the MET17, MET6, and CYS4
gene in 20 wine strains, both commercial and native isolates, to
assess the level of naturally-occurring mutations found in members
of this pathway. The 20 strains selected were unrelated to each
other and vary from laboratory wild-type strains.
Interestingly, all strains analyzed had the identical sequence for
the MET17 gene. This gene is highly conserved in yeast, meaning
that mutations that alter its activity or regulation at the protein
level are deleterious to the strains carrying that alteration. The
CYS4 gene was identical in all strains but one, and the MET6
gene showed variation in four strains.
The sequence analysis confirmed our earlier observations that differences
in the level of activity of the enzymes involved in incorporating
reduced sulfur into amino acids were not well correlated with sulfide
formation in wine strains. The task then is to determine what genes
do dictate sulfide formation behavior.
Identifying all genes that
affect sulfide formation
Our next approach to identify what genes affect H2S production was
more global. Completion of the genome sequence of Saccharomyces
led to identification of all of its genes. This information resulted
in creation of a set of strains carrying mutations in each and every
gene. Some mutations were lethal, but most were not.
We decided to screen the entire set of the yeast-deletion strains,
comprised of 4,827 mutants, for effects on sulfide production. We
did this initially using BiGGY agar to identify all strains that
lead to a lighter or darker colony color than the wild-type parent
The parent yeast strain of the deletion collection was tan on BiGGY
and produced no H2S during fermentation, and mutants ranged from
white to brown. Wine strains on this medium also display colors
ranging from white to brown.
The mutants were classified into six different color groups: white
(4), light tan (257), tan (4,476), light brown (61), brown (28),
and black (1) (Figure V). The light brown and brown colonies found
by the screen were subjected to a second screen in synthetic juice
media MMM and Pinot noir juice, to evaluate production of hydrogen
sulfide. Fourteen of the 89 strains produced detectable amounts
of H2S consistently using the lead trap method.
Five of the 14 mutations are in the methionine synthesis or transport
pathway, with others involved in various pathways such as amino
acid, or nitrogen compound transport or utilization. While the reasons
why some of the mutations affect H2S production are obvious due
to the pathways they are in, some are not. This inconsistency opens
up new ideas as to what is causing some wine strains to produce
In a related experiment, we have examined mRNA expression profiles
of a collection of 30 wine strains of Saccharomyces that
vary in sulfide production. The aim of this study is to identify
genes that consistently change in expression in relation to production
Preliminary analysis of this data set suggests that it is the relative
ratio of levels of expression of the upstream and downstream enzymes
and relative level of activity of the entire pathway that influences
the ability to produce sulfide. Interestingly, the two lowest-producing
strains that we have identified appear to have levels of expression
consistent with a high level of sulfide production, yet they produce
virtually undetectable amounts during fermentation.
One of these strains carries point-mutations in five out of seven
genes that were sequenced in the genome so has clearly been exposed
to significant DNA damage. We are currently analyzing this strain
to define the gene or genes responsible for the loss of sulfide
formation. If we can identify specific genes that are responsible
for H2S production characteristic, we could cross those genes into
more robust yeast strains to produce a commercial wine yeast strain
with low H2S production, and thereby eliminate H2S formation by
Saccharomyces during wine fermentations a goal that
once seemed unrealistic but that now is within sight.
Our research has shown that there is significant strain variation
in sulfide production and that while nitrogen supplementation does
tend to reduce sulfide levels, even with high nitrogen supplementation,
some strains will still produce sulfide at an order of magnitude
over the threshold of detection under these conditions.
It is also difficult to predict strain behavior in response to nutrient
limitation. Some strains do not show a dramatic response while in
others, sulfide levels may vary over 2,000-fold.
Analysis of known mutations has revealed a set of genes that, when
lost, result in high sulfide production or in the absence of detectable
sulfide. These mutants produced up to 700 mg/L of sulfide, higher
than ever observed in commercial strains under any condition.
Now that we know which genes impact sulfide production, we can determine
which ones are varying in native and commercial yeasts and define
the best strain genotype for low sulfide production
and attain our goal of eliminating the appearance of this off-character
during wine production.
This research was supported by a grant from the American Vineyard
Foundation and the California Competitive Grant Program for Research
in Viticulture and Enology, and by funds of the Maynard A. Amerine
Endowment. Angela Lee Linderholm was recipient of the Paul Monk
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