S t u c k and S l u g g i s h F e r m e n t a t i o n s

L I N D A F. B I S S O N *
Premature arrest of fermentation constitutes one of the most challenging problems in wine production. The
causes of stuck and sluggish fermentations are numerous, troublesome to diagnose, and difficult to rectify. It
has become well-established that fermentation rate decreases due to a targeted loss of hexose transport
capacity. Factors which have been correlated with incomplete fermentations also regulate transporter
expression, turnover and function. Several causes of slow and incomplete fermentations, including ethanol
toxicity, have been well-characterized under enological conditions as described herein. Other potential factors
that may impact cell growth, viability, and fermentative activity have not been sufficiently evaluated. The role
of these factors in problematic enological fermentations is discussed.
KEY WORDS: fermentation, stuck fermentation, ethanol toxicity, hexose transport capacity

though viable biomass remains high. Numerous factors

have been identified t h a t impact cell viability and yield
[2,16,73]. Conditions which affect fermentation rate of
individual cells have also been identified [16,73] and
partially overlap with those factors impacting viability
and biomass yield.

Slow and incomplete alcoholic fermentations are a

chronic problem for the wine industry. Incomplete or
"stuck" enological fermentations are defined as those
leaving a higher t h a n desired residual sugar content in
the wine at the end of the alcoholic fermentation. A
residual sugar level of less t h a n 0.4% (4 g/L, glucose +
fructose) is often considered by winemakers to indicate
a complete or "dry" fermentation, but final sugar concentrations are typically below 0.2% at the end of yeast
fermentation. A slow or sluggish fermentation is one
t h a t requires a longer t h a n average time to reach a low
residual sugar content. A routine fermentation will
usually go dry within 7 to 10 days (depending upon
temperature), while a sluggish fermentation will take
considerably longer, perhaps even months, to complete.
Sluggish consumption of sugar by the yeast is commonly an indicator of adverse environmental or physiological conditions, and such fermentations may eventually become stuck at a high residual sugar concentration. Stuck fermentations are a serious problem because wines with a high post-fermentation sugar content are very susceptible to microbial spoilage and cannot be b o t t l e d u n t i l it is k n o w n t h a t t h e y are
microbially stable. Slow and incomplete fermentations
may require special antioxidation operations because of
the loss of the protective carbon dioxide blanket of an
active fermentation. In addition, such wines tie up fermentor space and may be stylistically unacceptable.

The circumstances yielding stuck and sluggish fermentations can frequently be alleviated thus allowing
the fermentation to go to dryness. However, early and
accurate diagnosis of the cause of fermentation arrest
are critical in order to eliminate correctly the specific
stress affecting the yeast in a timely fashion [16,73].
Once cells lose viability or p e r m a n e n t l y adapt to the
adverse conditions of the environment by reducing rate
of sugar consumption, it is very difficult to restore the
rate of fermentation. Currently, a slow or incomplete
fermentation is not recognizable until the rate of sugar
consumption has been observed to decrease. By the
time the rate has dramatically slowed, it is often too
late to modify the adverse conditions or prevent the
stress-induced fermentation arrest of the yeast. Continued incubation of the cells under conditions of stress
generally leads to loss of viability and death of the
culture. We have noted t h a t re-initiation of fermentations t h a t contain a large population of nonviable cells
is particularly challenging [16].
A major impediment to the rapid identification,
correct diagnosis, and t r e a t m e n t of a stuck or sluggish
fermentation is a lack of basic information on the biology of yeast during grape juice fermentation. A significant amount of information is known about the metabolism, physiology, cell biology, and adaptation to stress
in Saccharomyces cerevisiae under laboratory conditions, but this organism has been less thoroughly characterized in the n a t u r a l environment of grape juice.
Unfortunately, laboratory conditions do not mimic the
n a t u r a l environments of yeast. Therefore, the application of this wealth of information is somewhat limited.
There has been a long series of important studies on
control of fermentations in grape juice [see 5 and references therein], but these studies, while helpful in m a n y
aspects, were by necessity empirical in n a t u r e and
made without benefit of our current knowledge of the
intermediary metabolism and molecular genetics of

F e r m e n t a t i o n rate is a function of both the total

viable biomass and rate of sugar utilization of the individual cells [91]. Grape juice factors which limit growth
or lead to cell death will reduce viable biomass and
cause a decrease in sugar utilization t h a t can result in a
stuck fermentation. A sluggish fermentation may occur
if the rate of fermentation per cell decreases even
*Professor and Maynard A. Amerine Endowed Chair, Department of Viticulture and Enology,
University of California, Davis, CA 95616-8749 [email: <lfbisson@ucdavis.edu>; Fax: (530) 7520382].
Acknowledgements: I would like to thank Ralph Kunkee, Rich Morenzoni, Roger Boulton,
Nancy Irelan, Christian Butzke, David Block, David Mills, and Laura Lange for critical reading of
the manuscript and for sharing unpublished observations. I would also like to thank the reviewers
of this manuscript, Drs. Gafner and Sponholz, for their very helpful comments. Finally, I would
like to thank the reviewers of an AVF grant on this topic who read an earlier version of this review.
Their critical suggestions greatly improved the final version.
Manuscript submitted for publication 9 July 1998; revised 18 December 1998.
Copyright 1999 by the American Society for Enology and Viticulture. All rights reserved.

107
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yeast. However, with the completion of the genome

sequencing project for Saccharomyces and the development of new technologies for the analysis of whole
genome and protein expression [38,124,129,136], it is
now possible to thoroughly investigate yeast biology
under enological conditions.

Metabolic Basis of Stuck and

Sluggish Fermentations
The metabolic basis of stuck and sluggish fermentations has been fairly well established. The decrease in
rate of sugar consumption is correlated with a decrease
in sugar uptake capacity [16,74,85,125,128] while the
rest of the glycolytic pathway remains intact and fully
active. Analysis of an isogenic set of industrial strains
of Saccharomyces differing only in ploidy, demonstrated that plasma membrane transport capacity, and
not level of internal enzymes, dictates the overall CO 2
production rate [126]. Given the potential toxicity of
intracellular free glucose which would occur if a downstream metabolic step of glycolysis were decreased instead [12,16,144], the targeted loss of transport capacity represents an important survival mechanism for the
cells. Glucose and fructose consumption are reduced in
response to various environmental or cellular conditions of stress. Nutrient limitation (macronutrient and
micronutrient), low pH, lack of oxygen, lack of adequate
agitation, temperature extremes, presence of toxic substances, presence of other microorganisms, imbalance
of cations, and poor strain tolerances (particularly to
ethanol or acetaldehyde), which have all been associated with stuck and sluggish fermentations [for review
see 2,16,47,73], impact glucose and fructose transporter expression and activity [12].
Yeast balance the rate of entry of sugar into the cell
with the rate of catabolism [10]. The overall rate of flux
through glycolysis is equivalent to the rate of flux
through the transport step [69,85,97,126,128]. A few
years ago this was thought to be a consequence of
transport being inherently slow as compared to phosphorylation and subsequent metabolism. However, it is
now generally accepted that transport processes are
not simply slow in a biochemical sense but are exquisitely regulated so as to match, but not exceed, the
metabolic needs of the cell [12,14,74,144].
The process of sugar uptake in Saccharomyces has
been extensively investigated in the last decade [for
review see 12,14,16,67,74], and has been examined under enological conditions [69,85,86,87,125,126,
128,131,132]. Saccharomyces possesses a multigene
family of t r a n s p o r t e r s , called the H X T genes
[12,14,67,68]. The "HXT" designation stands for hexose
transporter [68]. There are 18 members of the H X T
family (HXT1 through HXT17 and GAL2), which display a high identity in coding sequence (65% - 99%
identical), share common functional motifs and predicted secondary structure [12,14,67,74]. These proteins share the same structural features (12 membrane
spanning domains) and conserved residues of members
of the Major Facilitator Superfamily [14,74,94]. This

family includes transporters from animals and plants

in addition to fungi and bacteria, reflecting a high degree of conservation of the structure and function of
proteins that translocate sugars across lipid bilayers.
Members of this family have been unequivocally shown
to translocate glucose across membranes with catabolically relevant kinetics [12,14,114,115]. In addition, the
members of the H X T family display differences in regulation at both the transcriptional and posttranslational
levels [12,14,64,68,76,102,103,114,115,155,156]. HXT1
and HXT3 encode low affinity transporters (Km50- 100
mM) while HXT6, HXT7, and GAL2 are high affinity
glucose transporters ( K = 1-2 mM) [114]. HXT2 and
HXT4 have been shown to possess a moderate low
affinity ( K = 10 mM) [114]. The HXT2 transporter,
Hxt2p, apparently can exist in either a low and high
affinity state, suggesting that environmental conditions can modify the kinetics of this protein [114]. A
kinetic analysis of the remainder of the members of the
H X T family has not been reported. Deletion of the
HXT2 gene has been shown to result in a sluggish
fermentation confirming the critical role of transport in
glucose utilization [69]. Related genes, SNF3 and
RGT2, have been shown to encode regulatory proteins
involved in the detection of glucose and are not predicted to play a direct role in transport for catabolism
[12,14,28,77,100,149]. Snf3p functions as a low glucose
sensor while the related protein, Rgt2p is a sensor of
high glucose concentrations [28,77,100,149]. Both of
these proteins are members of the Major Facilitator
Superfamily [12,14,74] and regulate expression of
members of the H X T family [28,77,100,149]. Other
transporter-like proteins have been identified [94],
with as yet unknown functions.
One of the key factors regulating H X T gene expression is glucose itself [12,102,155,156]. Transporter
genes are regulated by both glucose induction and glucose repression at the transcriptional level. Genes
regulated by glucose induction are not expressed in the
absence of glucose in the medium whereas glucose repressed genes are not expressed at high glucose concentrations, becoming derepressed upon glucose exhaustion. Some genes, like HXT1, are induced by high levels
of glucose, while others, HXT2 and HXT4, are repressed by high glucose concentrations. Thus, HXT1
will not be expressed if external glucose levels are too
low, while the moderate affinity transporters, HXT2
and HXT4 will not be expressed on high concentrations
of glucose. The HXT2 gene represents a special case as
this gene is both glucose repressed and glucose induced
[102,156]. This pattern of regulation allows for HXT2
expression only under conditions of low glucose. Glucose repression of these genes is mediated by the general glucose repression pathway [156]. Glucose induction involves the removal of a negative transcriptional
factor, called Rgtlp, which is mediated by SNF3 under
low glucose and RGT2 under high glucose conditions
[100,101]. Other factors such as permissive growth conditions, also regulate H X T gene expression [76,155,
156]. We have observed that the level of nitrogen in the
medium impacts HXT1 expression and can override

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glucose control [N. M. Fong and L. F. Bisson, unpublished observations]. Further, HXT2 is expressed during enological fermentations under conditions of a normally repressing sugar concentration [P. Vagnoli and
L. F. Bisson, unpublished observations].
Other yeasts t h a t have been evaluated have far
fewer transporter genes, frequently no more t h a n three
[14]. Why, then, are there so m a n y transporters in
Saccharomyces? Several explanations have been proposed for the existence of a multigene family of transporters in Saccharomyces. The transport of glucose and
fructose in this yeast occurs by facilitated diffusion,
meaning that no expenditure of energy is involved, the
process is not concentrative, and accumulated free
sugar can exit via the same t r a n s p o r t e r molecules
[26,27,49,54,134]. Facilitated diffusion systems are optimally operational only over a narrow substrate concentration range. This is a consequence of two phenomena: the inherent kinetics of the transport process and
substrate inhibition [14,86,87,93].
Translocation of a sugar molecule into the cell requires first binding and then recognition of the sugar by
the transporter. As a result the transporter must possess a mechanism for substrate identification. Transporters that operate at low substrate concentrations
(the high affinity transporters) are thought to have an
open structure with more t h a n one substrate recognition or binding site. These proteins are capable of recognizing their substrate from more t h a n one position,
meaning t h a t the sugar molecule can associate with the
t r a n s p o r t e r regardless of the orientation of its approach. For example, the molecule could be recognized
w h e t h e r it approached from the carbon 6 or 1 position
via a specific binding event to different regions of the
transporter. Once the sugar substrate is bound and
recognized as correct there is a conformational change,
and the transporter adopts an inward or cytoplasmic
facing position with subsequent release of the sugar
into the cell. This controlled movement of the protein
requires a certain amount of plasma m e m b r a n e fluidity. The m e m b r a n e lipid composition must allow the
conformational change of the t r a n s p o r t e r protein, but
also restrict it so t h a t the empty carrier can readily
undergo a second conformational change to again face
the outside of the cell. Thus, proper m e m b r a n e fluidity
is critical to maintenance of fermentation rate. It has
been assumed that hexose t r a n s p o r t e r activity is immune to the disruptive effects of ethanol because transport activity is maintained in the presence of ethanol,
but this assumption bears further scientific scrutiny.
Some members of the H X T family m a y be designed to
work in an ethanol environment, while others might
not.
At high s u b s t r a t e concentrations, t r a n s p o r t e r s
with multiple substrate recognition sites are subject to
substrate inhibition. Substrate inhibition occurs when
multiple s u b s t r a t e molecules a t t e m p t to simultaneously bind to the transporter [93]. In effect this jams
the transporter which prevents the requisite conformational change and therefore no sugar is translocated

into the cell. Under these conditions, the overall rate of

sugar uptake decreases [86,87,93]. At high sugar concentration transporters with a relatively closed structure are required. These transporters are thought to
have a single substrate binding site and to be less
susceptible to substrate inhibition. Because the transporter is less accessible to the substrate, these transporters display a high K m (the low affinity transporters). As a consequence they are not effective as substrate concentration drops below their K m value. Thus,
one reason for the large hexose t r a n s p o r t e r family is
the need to shift from situations of very high substrate
concentration to low, while maintaining flux rates into
the cell. The strategy of coupling H X T gene expression
to external glucose concentrations assures t h a t transporters with a proper affinity and sensitivity to substrate inhibition will be expressed at the appropriate
time during fermentation.
A second reason for the existence of multiple protein species catalyzing the same catabolic step concerns
the speed of adaptation to changing conditions t h a t
such an a r r a n g e m e n t allows. We have found t h a t multiple transporters are expressed simultaneously by the
cells, and transport should be viewed as the summation
of these independent activities, which we have termed
the "consortium hypothesis" [12]. If it becomes necessary to partially reduce the uptake capacity, this is
more easily achieved by eliminating one transporter
protein species in its entirety r a t h e r t h a n attempting to
partially down regulate a single species. For example, if
three transporters are all simultaneously expressed
and contribute equally to uptake and the cell needs to
reduce uptake capacity by a third, it is easier to eliminate one of the species entirely t h a n to fractionally
reduce the activity of each protein.
In Saccharomyces transport is the main site of control of the rate of sugar fermentation [10]. When environmental conditions become adverse, fermentation
rate is decreased due to the specific degradation of the
H X T t r a n s p o r t e r s [16,18,75,85,125,128]. We have
shown t h a t this degradation occurs in the vacuole and
t h a t the H X T proteins are internalized by a unique
endocytotic mechanism as has been observed for the
galactose and maltose transporters [116; J. S. Mazer
and L. F. Bisson, unpublished observations]. Blocking
proteolysis of the t r a n s p o r t e r results in cell death.
Thus, continued glucose catabolism under adverse conditions is itself toxic to the cells. While this explains the
physiological response of t r a n s p o r t e r degradation, it
means t h a t it will not be feasible to prevent stuck
fermentations by simply blocking transporter turnover.
Thus, it is instead necessary to identify and correct the
exact cause of the adverse condition resulting in transporter loss.
It is generally concluded from competition studies
t h a t fructose and glucose share the same transporters,
meaning t h a t fructose competitively inhibits glucose
uptake [26,27,54,134]. The K for fructose is generally
2.5 to 5 fold greater t h a n the K for glucose, but the V
of transport of fructose is generally higher t h a n that for

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glucose [24,26,27,54,134]. The lower affinity for fructose is thought to be due to the fact that glucose and
other sugars are transported in the pyranose rather
than the furanose form [27,54,74]. Roughly 30% of the
fructose present in solution is in the furanose form,
meaning that the actual transport-competent concentration of fructose is below the total concentration [27].
From the difference in kinetics, it is predicted that
glucose will be consumed at a faster rate changing the
environmental ratio of glucose to fructose from 1:1. As
an adaptation to this alteration in relative substrate
concentrations, the hexokinase isozyme expressed
switches from hexokinase II, which displays an equal
reaction rate for both sugars, to hexokinase I which
displays a faster reaction rate against fructose, (glucose
to fructose ratio of 1:3) [reviewed in 51]. This switch
allows the cell to compensate for the change in external
glucose to fructose ratio and maintain glycolytic flux. It
has recently been suggested that low-level expression
of Hexokinase I is associated with strains prone to
sluggish fermentations [132]. The substrate specificity
of all of the members of the H X T transporter family has
not been evaluated in detail. It is certainly possible that
a similar change to transporters with a higher affinity
towards fructose would have to occur to match the
change in hexokinase species in order to increase the
rate of flux of fructose into the cell. Some of the HXTs
may in fact be "fructophilic" in order to maintain metabolic rates.
The central role of transporter activity in the regulation of fermentation rate is thus well established.
Loss of transporter activity has been definitively associated with stuck and sluggish enological fermentations
in several laboratories [16,18,69,85,125,128]. This loss
of activity appears to represent an important survival
mechanism preventing the uptake of potentially toxic
sugar levels. More detailed information is needed on
the factors regulating expression of each of the 18 H X T
genes under enological conditions to more clearly define the role of these genes in maintenance of fermentation rate.

Factors Affecting
F e r m e n t a t i o n Rate
Several factors have been shown to impact fermentation rate and lead to sluggish or stuck fermentations:
nutrient limitation, ethanol toxicity, organic and fatty
acid toxicity, presence of killer factors or other
microbially-produced toxins, cation imbalance, temperature extremes, pesticide, and fungicide residues,
microbial competition and poor enological practices (excessive SO 2 use; excessive must clarification; lack of
agitation; lack of a t t e n t i o n to t e m p e r a t u r e )
[2,16,47,73]. These factors can interact synergistically,
and in combination may be far more inhibitory than
any factor individually. For example, the minimum and
maximum temperatures of growth are altered by the
presence of ethanol, organic acids and fatty acids
[79,121,123]. The minimum temperature supporting
growth of Saccharomyces is quite ethanol sensitive,

and may be elevated to as high as 27C, depending

upon conditions [123]. Tolerance to both ethanol and
temperature is very strain dependent. In addition, cells
grown at high temperature are susceptible to inhibition
by oxygen exposure under non-growing conditions [33].
Thus, correct diagnosis of the cause of a stuck fermentation necessitates consideration of possible synergistic
effects in addition to a single individual cause.
N u t r i e n t l i m i t a t i o n : The best characterized of
the conditions leading to stuck and sluggish fermentations is nutrient limitation [2,16,47,53,56,58,73]. Nutritional requirements can be classified as those needed
for proliferation and those required for maintenance of
metabolically active non-proliferative or stationary
phase. These two categories are not mutually exclusive
as the same nutrients may be required for both phases.
Stationary phase nutrition has not been as well studied
as growth nutrition. Nutrients required for periods of
non-growth include an energy source, ample amino
acids for synthesis of actively degraded proteins, and
compounds required to minimize the inhibitory effects
of ethanol. The latter class of compounds have been
termed "survival factors" [72,73], and include long
chain saturated and unsaturated fatty acids and ergosterol. It is important to note that in addition to disruptive effects of plasma membrane activity, ethanol can
also disrupt non-membrane cytoplasmic functions
through the dielectric disorder of the aqueous phase of
proteins [59,60,61]. Several compounds have been suggested to be important in protecting internal processes
from ethanol: trehalose, proline and glycine
[82,83,147]. Trehalose is made from glucose and proline
is plentiful in grape juice, so these two compounds
would be predicted to be in excess during grape juice
fermentation. In contrast, glycine levels are quite low.
Thomas et al. [147] reported a synergistic effect of
proline and glycine in fermentation of high gravity
media. In light of these findings, the impact of limiting
glycine on grape must fermentation, particularly of
musts with a high sugar content as would occur with
late harvest or Botrytis infection, warrants investigation.

The two macronutrients most frequently implicated as causes of stuck fermentations when present in
insufficient quantities are nitrogen and phosphate
[2,16,47,53,73]. Lack of micronutrient vitamins and
minerals have also been shown to limit fermentation
rate, as has a deficiency of oxygen. Depending upon the
nature and severity of the limitation, starvation for a
required nutrient can reduce the rate of cell growth,
limit maximal biomass production, decrease viability
and affect the fermentation rate of individual cells.
In contrast to many other proteins, the sugar transporters maintain high rates of turnover in stationary
phase cells [14,15,85,125,128]. Thus, a supply of nitrogen must be available to allow continued resynthesis of
these proteins. Since a high ethanol concentration inhibits the translocation of amino acids and other nitrogen sources [146], the nitrogen must be available early
in fermentation and stored in the vacuole for later use

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STUCK and SLUGGISH FERMENTATION- 111

[16]. It has recently been shown t h a t supplementation

of stationary phase fermentations with specific amino
acids prolongs maximal fermentative activity [82]. Previous work suggests that ammonium does not have this
effect [73]. Amino acid additions may enhance the ability to synthesize rapidly degraded proteins such as the
glucose transporters. Some amino acids were far more
effective t h a n others and t h a n a mixture of amino acids
[82]. The effectiveness of the amino acids appeared
unrelated to their utility as nitrogen sources supporting growth. One of the most effective amino acids was
glycine [82], a very poor nitrogen source for Saccharomyces. This is consistent, however, with reports of the
stimulatory value of glycine supplementation in very
high gravity fermentations [147]. The nitrogen requirement for maintenance of stationary phase fermentation
was shown to differ dramatically by strain, much more
so t h a n nitrogen requirements during growth [81]. It
has also recently been shown that high ammonium
content of juice may inhibit efficient utilization and
biomass formation of wine strains under enological conditions [127]. Thus, nitrogen-containing compounds
must be in balance for optimal utilization and growth.
Numerous studies have underscored the importance of
proper nitrogen nutrition for completion of fermentation under enological conditions [1,4,11,53,58,90,91,
119,120,130].
Phosphate limitation has also been shown to impact cell growth and biomass yield as well as directly
affecting fermentation rate [16,51,73]. Ample phosphate must be present to maintain cellular pools of Pi,
ADP and ATP to drive glycolysis. Imbalances in the
AMP/ATP or ADP/ATP ratios have been suggested to
play a key role in regulation of glycolytic flux [51].
Hexose transport might also be responsive to this ratio
either directly or via the inhibition of glycolysis. Mutational loss of one of the irreversible steps of glycolysis,
hexose phosphorylation, phosphofructokinase or pyruvate kinase, results in greatly diminished glucose uptake [12].
Deficiencies and imbalances in minerals and cations can r e s u l t in r e d u c e d f e r m e n t a t i o n r a t e s
[13,39,153]. In fact, t r e a t m e n t of grape juice with metal
chelating resins has been proposed as a means to prevent microbial spoilage and fermentation [44]. Minerals serve as cofactors for glycolytic and other enzymatic
reactions and limitations of minerals such as zinc and
magnesium directly affect sugar catabolism. Calcium
limitation increases ethanol sensitivity [92]. High manganese depresses uptake of magnesium and vice versa
[13], so a disparity in these ions will lead to a deficiency
situation. We have also recently shown that an imbalance of pH and potassium ions can lead to a stuck
fermentation in model juice-like media, and such irabalances may be present in grapes from vines that
display poor potassium uptake from soil [70].
Saccharomyces is capable of making all essential
vitamins except biotin, but research has shown that the
presence of other vitamins is highly stimulatory to
growth and fermentation [47,73,90,99]. It has recently

been demonstrated that Kloeckera apiculata is quite

efficient at stripping thiamine from grape juice in a
m a t t e r of hours, thus leading to a deficient situation for
Saccharomyces [9]. The presence of acetic acid has been
reported to reduce the ability of Saccharomyces to
transport and retain thiamine [57]. Thus, one likely
cause of stuck fermentations arising from uninoculated
juices is the depletion of thiamine by wild yeasts with
the production of acids that inhibit transport of the
residual vitamin by Saccharomyces. Sulfur dioxide reacts directly with thiamine, reducing the level of this
vitamin. Excessive use of SO 2 may also lead to a deftciency situation, especially if it follows cold settling.
This is another example of the synergistic interaction of
factors that can lead to poor fermentation performance.
Depletion of thiamine may also pose a problem for
inoculated fermentations as well [73], especially under
conditions favoring the growth of Kloeckera.
It is important to note that the nutritional requirements of yeast during grape juice fermentation may be
influenced by inhibitory substances present in the medium. Plants produce several inhibitory compounds,
the phytoalexins, in response to fungal invasion
[78,138]. One class of these compounds is derived from
the amino acid phenylalanine [78,138]. It is not surprising, therefore, that one of the groups of mycotoxins
produced by the molds during plant infection are inhibitors of phenylalanine biosynthesis and mobilization [66]. Saccharomyces has been reported to be insensitive to these mycotoxins [80], but that analysis was
conducted on rich media where amino acids were
plentiful. It is possible t h a t Saccharomyces may be
impacted by these mycotoxins if phenylalanine levels in
the must are low. Indeed, a correlation between fermentation arrest and phenylalanine deficiency has
been observed [16; R. B. Boulton, personal communication], which was not able to be replicated in defined
media studies. It is possible that the phenylalanine
requirement arose because of the presence of mold toxins in the juice.
Alternatively, phenylalanine limitation may impact the cells ability to communicate at high density
(called quorum sensing [see 50]). We have observed
t h a t cells of Saccharomyces at high densities produce
relatively large amounts of phenethyl alcohol, which is
derived from phenylalanine. It is possible that this
compound functions in quorum communication and the
inability to produce it may lead to senescence of the
culture. Very high levels of phenethyl alcohol (1.5 g/L)
have been shown to inhibit a variety of cellular functions in Saccharomyces [8,17]. While the inhibitory concentrations are well above those found in wine (trace to
50 mg/L) [5], it is possible t h a t lower concentrations
play a role in the correct entry into the non-proliferative state (see discussion below).
E t h a n o l t o x i c i t y a n d t h e r o l e of s u r v i v a l factors: Another major cause of stuck fermentations is
ethanol toxicity [23,31,59,145,146]. Ethanol appears to
impact plasma membrane fluidity in a complex fashion.
Ethanol decreases polarity in aqueous surroundings

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but increases it in hydrophobic environments [25]. The

effect of ethanol on plasma membrane functions, and
whether a decrease or increase in fluidity is observed,
depends upon the conditions of the assay and the membrane component evaluated [3,23,25,59]. The cell responds to ethanol by producing a membrane rich in
u n s a t u r a t e d f a t t y acids and ergosterol [3,25,35,
59,84,145,148,150]. Unsaturated fatty acid content increases approximately two-fold, while ergosterol levels
increase 18-fold [25]. The content of saturated long
chain fatty acids also increases [25]. In addition to an
increase in unsaturated fatty acids and sterols, the
protein content is reduced ethanol-tolerant membranes. The changes in plasma membrane composition
have been proposed to alter the plasma membrane
fluidity to compensate for the disruptive effects of ethanol on fluidity. However, this conclusion has recently
been questioned [59,60,61]. Ethanol is a polar molecule
and is not found in high concentrations in nonpolar
environments as would occur inside of the lipid bilayer.
Instead, it has been suggested that ethanol impacts
membrane function by polar, dielectric, and hydrogen
bond interactions with the polar head groups of the
phospholipids and integral membrane proteins rather
than changing plasma membrane fluidity [59,60,61].
The changes in fatty acid desaturation, decrease in
protein content and increase in ergosterol level are
proposed to occur to counter the disruptive effects of
ethanol on phospholipid head groups and proteins,
r a t h e r t h a n to a l t e r p l a s m a m e m b r a n e fluidity
[59,60,61].
Ethanol sensitivity is a variable property among
strains of Saccharomyces. It is also impacted by the
availability of sterols and long-chain saturated and unsaturated fatty acids in the medium as these compounds cannot be readily synthesized by the yeast under anaerobic conditions [6,25,30,32,72,73,148,150].
Both fatty acid desaturase and squalene oxidase require molecular oxygen as electron acceptor. Grape
must contains unsaturated fatty acids and plant sterols
which can be utilized by yeast. Previous work demonstrated that the major grape sterol, oleanolic acid
[109,110], could not replace the ergosterol requirement
[73,148]. This finding is not surprising since oleanolic
acid would not be predicted to supplant the yeast sterol
based upon analysis of the structural requirements for
the compound [95,96,108]. We evaluated the effect of
other plant sterols found in grape, stigmasterol, B-sitosterol, dihydrobrassica sterol, and campesterol. These
plant sterols could only replace the so-called "bulk" or
structural sterol requirement [95,96,108], but could not
replace the regulatory role of ergosterol in control of
protein and membrane function. They were not as effective as the yeast ergosterol in formation of an ethanol tolerant membrane and resulted in a more sluggish
fermentation, but the fermentation did go to dryness
[N. Peay and L. F. Bisson, unpublished observations]. If
the grape juice is deficient in fatty acids, these components can be harvested from internal membranes such
as those of the mitochondria [reviewed in 16], but this
may result in loss of mitochondrial activity and ability

to respire [16]. Recent work suggests that the "survival

factors" may be critical in maintenance of fermentation
rates in spite of the fact that glucose transporters have
been reported to be relatively ethanol insensitive [K.
Morrisey and L. F. Bisson, unpublished observations].
Further analysis of the factors involved in ethanol sensitivity and tolerance is clearly needed.
Ethanol is believed to be toxic to cells because it
increases the passive proton flux into the cell, placing
stress on the cellular capacity to maintain pH homeostasis [22]. This leakage is thought to occur via the
disruptive effects on ethanol on protein structure
rather than through general affects on plasma membrane fluidity [59,60,61]. This finding is based upon the
observation that passive flux of undissociated acids is
not similarly affected by ethanol and membranes of
high fluidity are not ethanol tolerant [59,60,61]. It has
recently been suggested that the increase in passive
proton flux may not play a major role in ethanol toxicity
[23,118], since it appears that in ethanol challenge
assays (ethanol added to a culture) cell death occurs
prior to the appearance of an altered cytoplasmic pH.
The impact of ethanol on the plasma membrane and
disruption of protein function and accompanying leakage of cytoplasmic components may be more immediately toxic t h a n the pH change of the cytoplasm
[7,62,84,122,146]. Also, ethanol affects translocation of
other ionic species, Ca 2and Mg 2, which may be important in toxicity [39,92,153]. Cultures naturally accumulating ethanol rather than being challenged by it are
able to adapt the protein composition of the membrane
which may be as important of a factor in ethanol tolerance as alteration of the lipid composition [59]. Ethanol
will also affect the function and stability of cytoplasmic
enzymes [59,60], which may also play a role in toxicity.
Ethanol may directly impact transporter function by
affecting substrate recognition, binding or the dynamics of the conformational change that is required for
substrate translocation into the cell.
When considering the toxic effects of ethanol, it is
also important to remember that acetaldehyde, the immediate precursor to ethanol in catabolism, is also
toxic [139,140], and may be in large part responsible for
"ethanol sensitivity" [59,60]. Since alcohol dehydrogenase is a freely reversible enzyme, acetaldehyde will
accumulate in parallel to ethanol and is toxic at much
lower concentrations [59,60,139,140]. Indeed, strain
differences in ethanol tolerance has been associated
with cellular levels of acetaldehyde; those strains with
the lowest cytoplasmic acetaldehyde level being the
most tolerant [59,60].
L o w pH: Saccharomyces is tolerant to low pH fermentations and can readily grow in the juice pH range
of 2.8 to 4.2 [16,47,52,73]. Below pH 2.8, both growth
and fermentation are inhibited [16]. The ethanol, organic and fatty acid tolerances of many strains are
reduced at very low pH values [105], and we have seen
that the potassium concentration is a key factor in pH
tolerance [70]. Saccharomyces excretes protons during
fermentation and may reduce the pH of the medium by

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S T U C K and S L U G G I S H F E R M E N T A T I O N m 113

as much as 0.3 units [16]. As described below, pH will

have a dramatic effect on the types of bacterial species
present and their persistence, which may significantly
impact fermentation progression.

Extremes of temperature: Exposure to temperature extremes can also inhibit f e r m e n t a t i o n r a t e s

[45,135,142]. The plasma m e m b r a n e is the main target
of the inhibitory effects of high or low t e m p e r a t u r e
[reviewed in 142,154]. T e m p e r a t u r e affects m e m b r a n e
fluidity and, therefore, t r a n s p o r t e r p e r f o r m a n c e .
Lower t e m p e r a t u r e s reduce fluidity and restrict transporter conformational change while high t e m p e r a t u r e s
increase fluidity and result in too great of a dissociation
of t r a n s p o r t e r structure during the conformational
change. Since ethanol and t e m p e r a t u r e target the same
cellular function, it is not surprising t h a t their effects
are synergistic. A dramatic drop in temperature, as
would occur at the end of vigorous fermentation with
continued cooling of the tank, should be avoided when
concerned with the prevention of stuck fermentations.
Abrupt changes in t e m p e r a t u r e also affect cytoplasmic enzyme activity and organelle structure and
function [142,154]. Heat shock leads to the induction of
several stress related proteins, which may also be
present upon entry into stationary phase [29]. The ability to respond to sudden t e m p e r a t u r e changes is dependent upon the ability to synthesize the heat shock proteins. If the t e m p e r a t u r e shock occurs under conditions
of nutrient limitation of the yeast, the cells might not be
able to compensate for the change in temperature.

Zymostatic and zymocidal toxins: Several toxic

substances
tion arrest
substances
viewed in

have also been shown to lead to fermenta[2,16,47,73]. Yeast can produce zymocidal
known collectively as killer factors [re157]. Non-Saccharomyces yeasts such as
Hansenula and Kluyveromyces produce killer factors
which are active against Saccharomyces [89]. Saccharomyces strains can also produce glycoprotein killer
factors t h a t are toxic to susceptible strains of Saccharomyces [21,111]. The effect of killer toxins is dependent
upon medium composition, and the relative ratios of
the sensitive to toxin-producing strains [88]. Conditions may impact killer factor production, activity or
sensitivity of the susceptible strains. Bacteria may also
produce substances toxic towards Saccharomyces. The
bacterial toxin syringomycin produced by the soil bacterium Pseudomonas impacts plasma m e m b r a n e K ,
H +, and Ca 2 fluxes in eucaryotic cells, and has been
shown to inhibit yeast [143]. Bacillus and Streptomyces, also common soil organisms, have been shown to
produce metabolites which limit yeast growth under
enological conditions [63]. Both Streptomyces and Bacillus h a v e been found as w i n e r y c o n t a m i n a n t s
[16,47,48]. Streptomyces can grow quite well in filter
matrices that have not been properly sanitized. However, such a situation can be readily detected due to the
distinctive aroma produced by this organism.
Molds present on the fruit at harvest may produce
mycotoxins to which Saccharomyces is susceptible. It
has been suggested t h a t botrytized fruit contains toxic

substances [73,117], but the n a t u r e of the substance

has not been determined in spite of significant research
efforts. Saccharomyces is in general resistant to many
mycotoxins [80], which is one reason this organism is
used in the production of fuel ethanol from mold-infested grains [65]. Saccharomyces was not found to be
sensitive to mycotoxins during wort fermentation either [133] so it is unclear if mycotoxins in general
would pose a problem under enological conditions. We
have conducted vinifications of fruit heavily infested
with mold post-harvest and have not noticed a correlation with slow or incomplete fermentations. However,
as mentioned above, sensitivity to mycotoxins may be
dependent upon the nutritional composition of the medium.
A more likely problem caused by mold infestation of
fruit is the production of compounds toxic to fungi by
the plant when challenged with fungal infection. Plants
produce numerous compounds (the phytoalexins) and
enzymes (the pathogenesis-related proteins) in response to infection which are designed to eliminate the
pathogen [78,138]. This same response might not occur
in post-harvest infection. Toxic phenolic compounds,
amino acid analogs, and enzymes capable of degradation of fungal cell walls (chitinases and glucanases) can
all be produced in response to infection. The phytoalexins are broadly toxic and may even reduce viability of
the plant cells producing them [138]. It is highly likely
t h a t some of these factors will also impact yeast growth
and fermentation since the yeast are members of the
same taxonomic family as the filamentous fungi and
have a similar cell wall architecture.
Organic and medium-chain fatty acids are also inh i b i t o r y to Saccharomyces [20,40,42,45,71,73,
112,113,151,152]. These compounds may be produced
by bacteria and non-Saccharomyces yeast, but they can
also be formed by Saccharomyces [42,113]. Under normal fermentation conditions, the concentrations found
are not inhibitory, but if mixed culture fermentations
(Saccharomyces and non-Saccharomyces yeast and bacteria) are conducted, the risk for the appearance of this
type of inhibition is greater. As mentioned above, these
acids are more toxic at high ethanol and extremes of
t e m p e r a t u r e [152], and may impact vitamin absorption
and retention [57] which may affect fermentation by

Saccharomyces.
Fungicides and pesticides used in the vineyard may
negatively affect yeast viability if present at high
enough residual concentrations at the time of harvest
[16,73]. These compounds may cause an extended lag
phase or might not lead to an immediate problem and
sluggish fermentations not manifest until later in the
fermentation. It has been suggested t h a t high concentrations of heavy metal ions or use of components containing sodium salts may be inhibitory to fermentation
and growth. We have not found these substances to
have an impact at the levels normally found in juices
and musts. However, they are indeed toxic at higher
levels.
Zymostatic toxins are those that inhibit cell growth

Am. J. Enol. Vitic., Vol. 50, No. 1, 1999

114 - - BlSSON

and metabolic activity but are not immediately lethal.

If such compounds are present, the fermentation can
usually be re-initiated by blending with other juices or
wines, assuming such a practice does not lead to further toxin production. Sometimes it is necessary to first
remove the initial biomass prior to attempts to reinitiate fermentation or to treat the stuck wine with
yeast ghosts [73]. However, unless it is clear that a
toxin is involved and the nature of the toxin is known,
this practice may simply result in a larger volume of
stuck wine.
M i c r o b i a l i n c o m p a t i b i l i t y : Enological fermentations containing initial high populations of non-Saccharomyces yeast and bacteria are at high risk for the
development of stuck and sluggish f e r m e n t a t i o n s
[16,40,47,48,73]. This is due in part to the competition
for nutrients and production of toxic substances as
described above. However, other factors such as ~high
total cell density may also be important in the reduction of fermentation rate. We and others have found
that mixed culture fermentations (Saccharomyces and
bacteria) require higher than normal vitamin supplementation, and that the arrested fermentation may not
be re-startable until the existing biomass is removed
via racking or centrifugation [T. Rynders and L. F.
Bisson, unpublished observations]. It is also important
to note that there are incompatible pairings of wine
yeast and malolactic (ML) bacteria. Some Saccharomyces strains are very susceptible to inhibition by the ML
bacteria, while other strains are not. Yeasts inhibited
by one bacterium are not necessarily inhibited by all
strains of ML bacteria. To avoid this type of fermentation problem, the compatibility properties of the ML
bacteria and yeast strains that the winemaker desires
to use should be evaluated. It has recently been shown
that a novel bacterium, Lactobacillus kunkeei, referred
to as the "ferocious Lactobacillus", frequently causes
stuck fermentations regardless of the yeast strain(s)
present [41,43].
Other factors impact the appearance and persistence of the non-Saccharomyces flora. Many bacterial
species are unable to grow at the lower pH range found
in wine, below 3.4, but will grow above this pH [16,73].
Wild yeasts such as Kloeckera apiculata (perfect form:
Hanseniaspora uvarum), are as pH tolerant as Saccharomyces, but are more tolerant of low temperatures
[47,52]. We and others [47] have found that holding of
must or juice at a low temperature (cold maceration/
extraction for red musts or cold settling for white
juices) allows the build up of Kloeckera populations.
Fermentation at low temperature gives these organisms a competitive advantage over Saccharomyces.
Kloeckera is far more ethanol tolerant at low temperature than at high temperatures. As mentioned above,
these practices can lead to vitamin depletion of the
juice or must [9].
E n o l o g i c a l p r a c t i c e s : Several enological practices can also be the cause of fermentation arrest
[2,16,73]. Excessive clarification of musts reduces fermentation rate. This appears to be due to multiple

factors: the loss of nutrients found in particulate matter

such as u n s a t u r a t e d fatty acids and sterols [34,35,46,
55,73], the reduction of vitamins and mineral content
via the removal of microbes that have sequestered
these components [9], and a decrease in the natural
agitation ability of the must [16]. It has also been suggested that must solids serve as nucleating sites for the
release of CO 2, and may provide a solid surface upon
which the yeast can form a biofilm. Excessive use of
sulfur dioxide can also lead to poor fermentation performance, depending upon the pH of the medium and
bound and free c o n c e n t r a t i o n s of the compound
[16,141]. Winery practices such as the addition ofnutrients and aeration may positively impact fermentation
performance and reduce the incidence of stuck and
sluggish fermentations [2,16,73]. Finally, the type of
temperature control employed may be a major contributing factor to fermentation problems. Too large or too
small of a temperature differential between the coolant
and the desired temperature of the must can result in a
sluggish fermentation and even in death of the yeast
[16]. The temperature of the must may not be uniform
and may be either too low near the sides of the tanks or
alternately too high in the center, depending upon heat
t r a n s f e r capability of the s y s t e m e m p l o y e d [16].
"Warm" fermentations above 30C should be avoided,
as this could result in the rapid generation of an inhibitory temperature depending upon the fermentation
rate of the strain and the heat transfer capacity of the
system [discussed in 16]. Too much cooling at the later
stages of fermentation can also cause fermentation arrest. Depending upon the concentration of ethanol and
other components such as fatty and organic acids, the
minimum temperature sustaining fermentation will be
elevated and the maximum decreased. Late in fermentation, the temperature should be held around 22C to
25C (70F - 78F), depending upon the yeast strain.
Yeast perform better with gradual t e m p e r a t u r e
changes as that allows the cells to adapt properly to the
new condition. Temperature swings in excess of 5C
(9F - 10F) should be avoided.
O t h e r f a c t o r s : Several additional factors that may
impact fermentation rate have been less well characterized. It has been suggested that a high concentration
of fructose relative to glucose is inhibitory to yeast
[131,132]. Fructose is not as reactive of a sugar as
glucose, so the basis of this inhibition is unclear. The
high residual concentrations of fructose may be a symptom rather than a cause of stuck and sluggish fermentations. Schfitz and Gafner [132] have suggested that
poor utilization of fructose and a tendency to stick
during fermentation are associated with a deficiency in
level of hexokinase I activity. However, it has been
shown that addition of fructose, and the accompanying
change in the glucose to fructose ratio (GFR) can inhibit
an ongoing fermentation [131] while addition of glucose
to alter the GFR to less than 0.1 can stimulate fermentation activity [131]. It is not clear if this is due to
substrate inhibition of the transport process or not.
Initial high sugar concentrations can lead to a sluggish
initiation of fermentation depending upon the affinity

Am. J. Enol. Vitic., Vol. 50, No. 1, 1999

STUCK and SLUGGISH FERMENTATION- 115

of the transporters expressed in the inoculum.

Thehigh viscosity of some grape musts may inhibit
fermentation. Musts from late harvest grapes with a
high pectin content can display a very sluggish initiation of fermentation [L. F. Bisson, unpublished observations]. It has recently been suggested that high viscosity media may prevent loss of CO 2, elevating the
carbon dioxide pressure of the medium to an inhibitory
level [106,147]. High viscosity solutions generally decrease mixing and diffusion rates, which may result in
slow growth and diminished fermentation rates.
An area that has been largely ignored is the impact
of grape must enzymes on the progression of fermentation. Polyphenol oxidase levels vary dramatically with
the varietal and the season [16]. This enzyme competes
directly with Saccharomyces for available dissolved
oxygen [16]. It has been suggested that the real reason
that SO 2 is able to stimulate fermentation by Saccharomyces lies in the inhibition of the competing polyphenol
oxidase, making oxygen more readily available for Saccharomyces [16] and not in the inhibition of wild (nonSaccharomyces) yeast and bacteria.
The ability to accumulate the disaccharide trehalose has also been shown to be important during fermentation [83]. Viability has been correlated with cellular trehalose levels, perhaps protecting yeast against
both osmotic stress and ethanol toxicity. Inability to
accumulate trehalose as a factor in sluggish and stuck
fermentations has not been evaluated. Proline and glycine have also been d e m o n s t r a t e d to have an
osmoprotective effect [147], and deficiencies in these
amino acids may likewise impact fermentation rates
and cell viability.
It has occasionally been noted by winemakers that
certain vineyard locations yield fruit highly susceptible
to sluggish fermentations, even when nutrient additions are made to the must. This effect may reflect a
particularly high disease pressure of those vines and
the resultant phytoalexin content of the juice, or alternately may suggest that these grapes support high wild
flora populations. We have recently shown that phenolic compounds found in grape can stimulate or inhibit
fermentation rates, depending upon the concentration
and the specific compound [R. Hood and L. F. Bisson,
unpublished observations]. The biological effects of
grape phenolics on yeast have not been well-studied.
Cantarelli reported both inhibitory and stimulatory effects on growth and fermentation rates [19]. Sluggish
fermentations may arise due to a deficiency of stimulatory phenolics or to an excess of inhibitory ones. Differences in phenolic composition may be one reason that
fruit from certain vineyard sites seems prone to fermentation problems.
Finally, an area meriting further studies concerns
the analysis of cell-cell communication during fermentation. It has been demonstrated that several compounds produced by yeast can signal developmental
changes in other yeast in the vicinity. Ammonia pulses
produced by neighboring colonies have been demon-

strated to limit colony growth towards each other [104].

Carbon dioxide accumulation in a dense population
induces sporulation of surrounding cells so that sexual
reproduction might occur [98]. Higher alcohols (fusel
oils) have been shown to bring about pseudohyphae
formation in Saccharomyces at higher concentrations
than typically found in wine [36]. Similar types of signaling may be occurring during fermentation, regulating growth and fermentative activity of the population.
It has recently been reported in Schizosaccharomyces
pombe that the multidrug resistance protein encoded
by the fnxl gene plays a role in entry of cells into
stationary or non-proliferative phase in response to
nitrogen limitation [37]. Cells lacking fnxl fail to respond properly to nitrogen limitation, leading to loss of
viability [37]. It was suggested that the fnxl protein
releases a signaling molecule that is involved in cell-tocell communication of the deficiency, and that cells do
not enter a quiescent phase without receiving both an
internal and external nitrogen starvation signal [37].
Failure to properly enter the quiescent state leads to
cell death. This observation has important implications, especially for reinitiation of stuck and sluggish
fermentations. Saccharomyces possess a gene with significant homology to fnxl [37], and may produce a
similar starvation factor affecting neighboring cells. If
such a factor exists, it may explain the difficulty in
restarting stuck fermentations, as the newly introduced yeast would also respond to these compounds.
Reinitiation of nutrient-limited fermentations may require the removal or dilution of these quiescence factors.
Settled (but not flocculant), populations of Saccharomyces are not as fermentatively active as dispersed
populations and inactive populations quickly settle.
Cell-to-cell communication in high density settled
populations may differ from interactions in dispersed
cultures. Saccharomyces has been shown to undergo
apoptosis or programmed cell death typical of higher
eucaryotes [137]. The senescence factors appear to be
unrelated to those found in mammalian cells and conditions leading to early senescence have not been adequately described [137]. Apoptosis is a phenomenon
associated with communal cells. Induction of death in a
segment of the population would provide nutrients for
the remaining viable cells increasing their chance of
survival. Further analysis of yeast apoptosis may provide important information useful in the re-initiation of
stuck fermentations. We have noticed that re-starting
stuck fermentations may require removal of the existing biomass. Fermentations at full cell density that
arrest may be difficult to restart due to the fact that the
maximal tolerable culture density has been exceeded.
It is highly likely that Saccharomyces will possess some
type of cell density or "quorum" sensing ability as has
been described in other microorganisms [reviewed in
50].

Conclusions
Many factors have been shown to result in slow or
incomplete enological fermentations. However, other

Am. J. Enol. Vitic., Vol. 50, No. 1, 1999

1 16 - - BISSON

causes of fermentation arrest that have not been evaluated merit investigation. While the precise cause of a
stuck and sluggish fermentation might not be currently
discernible, the type of fermentation arrest can be quite
useful at limiting the number of possibilities. Careful
attention to numbers of viable Saccharomyces cells and
judicious use of cooling and intelligent temperature
maintenance in addition to prudent nutrient supplementation should greatly reduce the incidence of slow
and incomplete fermentations. Three general areas
merit further research: non-proliferative phase nutrition, the impact of plant phenolic compounds and phytoalexins on growth and fermentation rates, and determination of the factors involved in quorum and senescence communication among yeast populations. The
completion of the yeast genome sequencing project and
advent of technologies for the analysis of whole genome
and proteome expression profiles should catalyze an
explosive increase in our knowledge of the biology of
yeast during normal and problematic fermentations
leading to better diagnostic tools for the winemaker
and facilitate rectification of slow and incomplete fermentations.

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