APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2004, p.

33923400
0099-2240/04/$08.000 DOI: 10.1128/AEM.70.6.33923400.2004
Copyright 2004, American Society for Microbiology. All Rights Reserved.

Vol. 70, No. 6

Biomass Content Governs Fermentation Rate in Nitrogen-Deficient

Wine Musts
Cristian Varela, Francisco Pizarro, and Eduardo Agosin*
Departamento de Ingeniera Qumica y Bioprocesos, Escuela de Ingeniera, Pontificia Universidad Cato
lica
de Chile, Santiago, Chile
Received 17 December 2003/Accepted 5 March 2004

Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most
prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen
diminishes a yeasts metabolic activity, as well as the biomass yield, although it has not been clear which of
these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions
with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We
quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell
viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher
initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar
uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in
spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a
problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter
the chemistry of the must.
brane is the primary region for controlling sugar uptake and
the subsequent ethanol production (34, 39).
Currently, the most common method for dealing with nitrogen-deficient fermentations is adding supplementary nitrogen
(usually ammonium phosphate). The timing of the addition is
key for ensuring a successful fermentation (3, 32). Early addition affects both the fermentation rate and the biomass yield.
Late addition has a minimal effect on biomass formation, however, and only increases the fermentation rate (3). Unfortunately, it is not possible to distinguish clearly between the effect
of nitrogen on the fermentation rate and the effect of nitrogen
on the biomass yield as the two effects are interdependent.
We propose that the rate of fermentation is indeed a twocomponent function comprising an intracellular component (a
property of the cell metabolism) and a cellular component
(which is dependent on the mass of cells actively fermenting).
In this work, we used metabolic flux balancing and biomass
concentration experiments to isolate the two components and
to quantitatively evaluate how these components affect the rate
of fermentation. The metabolic phenotype is best represented
by the flux distribution throughout the network of intracellular
pathways (26, 38, 40). Adding biomass with cells at the same
metabolic state allowed us to evaluate the effects on the fermentation rate. We then assessed the relative significance of
the two components in nitrogen-deficient sluggish fermentations.

Sluggish and stuck fermentations are common in the wine

industry. Factors that affect the yeast growth rate and that lead
to problematic fermentations include limited nutrient contents, ethanol toxicity, fatty acid toxicity, temperature extremes
(5), the ratio of nitrogen sources to carbon sources in the
medium (21), and the initial quantity and quality of the amino
acids (29). Sluggish cultures may prolong the process time
from days to weeks. In stuck fermentations, high levels of
residual sugar are left following the arrest of fermentation.
Several difficulties occur at the cellular level; these include
diminished sugar transport capacity, the inability of yeasts to
produce compatible solutes in response to high osmolality (20,
28), and cell membrane integrity issues in the presence of high
concentrations of ethanol (8). As such mechanisms may act
simultaneously in cells and to various extents, it is difficult to
isolate and reproduce the mechanisms in stuck and sluggish
fermentations.
Quantitatively, nitrogen is the second most abundant nutrient in wine fermentations. It is essential for yeast metabolism
and growth. Consequently, a lack of nitrogen triggers sluggish
fermentations (1, 8, 13). In previous studies workers found
differences in nitrogen-related curves (e.g., curves of biomass
versus assimilable nitrogen) which indicated that the fermentation rate and biomass yield functions are distinct (9, 10; W.
Agenbach, Proc. S. Afr. Soc. Enol. Vitic., p. 6688, 1977). In
other studies the workers associated nitrogen deficiency with a
high sugar transporter turnover rate, which resulted in a loss of
sugar uptake capacity in the cells (5, 32). The cellular mem-

MATERIALS AND METHODS

Yeast strain and growth conditions. Saccharomyces cerevisiae Prise de Mousse
EC1118 (Lalvin, Zug, Switzerland), a strain commercially available worldwide
for the wine industry, was used throughout this study. Initial seed cultures were
grown in YPD medium at 28C in aerobic conditions. Defined artificial must,
which simulated standard grape juice, was used in bioreactor fermentations.

* Corresponding author. Mailing address: Pontificia Universidad

Cato
lica de Chile, Casilla 306 Correo 22, Santiago, Chile. Phone: 562
354 49 27. Fax: 562 354 58 03. E-mail: agosin@ing.puc.cl.
3392

VOL. 70, 2004

EFFECTS OF LOW NITROGEN ON FERMENTATION RATE

Modified MS300 medium (33) contained (per liter) 120 g of glucose, 120 g of
fructose, 6 g of citric acid, 6 g of DL-malic acid, 750 mg of KH2PO4, 500 mg of
KH2SO4, 250 mg of MgSO4 7H2O, 155 mg of CaCl2 2 H2O, 200 mg of NaCl,
4 mg of MnSO4 H2O, 4 mg of ZnSO4, 1 mg of CuSO4 5H2O, 1 mg of KI, 0.4
mg of CoCl2 6H2O, 1 mg of H3BO3, 1 mg of NaMoO4 2H2O, 20 mg of
myo-inositol, 2 mg of nicotinic acid, 1.5 mg of calcium panthothenate, 0.25 mg of
thiamine hydrochloride, 0.25 mg of pyridoxine hydrochloride, and 0.003 mg of
biotin. The nitrogen concentration for normal fermentations was 380 mg liter1,
and the nitrogen concentration for sluggish fermentations was 65 mg liter1. The
nitrogen sources used were 18.6% (wt/wt) ammoniacal nitrogen (NH4Cl), 20.5%
(wt/wt) L-proline, 16.9% (wt/wt) L-glutamine, 1.25% (wt/wt) L-arginine, 6% (wt/
wt) L-tryptophan, 4.9% (wt/wt) L-alanine, 4% (wt/wt) L-glutamic acid, 2.6%
(wt/wt) L-serine, 2.6% (wt/wt) L-threonine, 1.6% (wt/wt) L-leucine, 1.5% (wt/wt)
L-aspartic acid, 1.5% (wt/wt) L-valine, 1.3% (wt/wt) L-phenylalanine, 1.1% (wt/
wt) L-isoleucine, 1.1% (wt/wt) L-histidine, 1.1% (wt/wt) L-methionine, 0.6% (wt/
wt) L-tyrosine, 0.6% (wt/wt) L-glycine, 0.6% (wt/wt) L-lysine, and 0.4% (wt/wt).
The following anaerobic factors were added to the medium: 15 mg of ergosterol
per liter, 5 mg of sodium oleate per liter, and 0.5 ml of Tween 80 per liter. Since
proline is not assimilable by yeast under anaerobic growth conditions (18), the
ammonium salt and -amino acids (all amino acids except proline) were considered assimilable sources of nitrogen. Therefore, for normal and sluggish
fermentations, the concentrations of assimilable nitrogen were 300 and 50 mg
liter1, respectively.
Cultivation conditions. The bioreactors, a 2-liter Bioflo IIc bioreactor (New
Brunswick Scientific Co., Edison, N.J.) with a 1.5-liter working volume, a 3-liter
Bioflo IIc bioreactor with a 2.5-liter working volume, and a 50-liter Bioengineering bioreactor (Bioengineering, Wald, Switzerland) with a 35-liter working volume, were inoculated to obtain an initial density of 106 cells ml1. Cells were
washed with 0.9% NaCl to eliminate any remaining nitrogen from rich media
prior to inoculation. The temperature was maintained at 28C, and the pH was
maintained at 3.5. Nitrogen was sparged through the medium for 30 min at a rate
of 250 ml min1 before inoculation to eliminate any oxygen in the medium. The
medium was agitated at 100 rpm to keep the cells in suspension. Carbon dioxide
production in addition to nitrogen sparging and agitation ensured that that the
conditions were anaerobic throughout the experiment.
Two batch cultures were compared in this work. In a normal fermentation, 300
mg of assimilable nitrogen (ammonia and amino acids) per liter was used, while
a second culture contained 50 mg of assimilable nitrogen per liter to force
sluggish fermentation. Three independent experiments were carried out for each
type of batch culture.
Cell concentration experiments. At the end of the exponential phase of one of
the sluggish fermentations, an 11.5-liter sample containing 1.2 g (dry weight) of
cells per liter was removed from the 35-liter batch culture. A 7.5-liter aliquot was
centrifuged (3,000 g at room temperature), and the pellet was resuspended in
1.5 liters of supernatant. Hence, the cell concentration was increased fivefold
without altering the chemical composition of the must. In a similar fashion,
biomass from the remaining 4 liters was centrifuged and resuspended in 2 liters
of supernatant to obtain a twofold increase in the cell concentration. After
resuspension, the concentrated cultures were loaded into sterile bioreactors and
sparged with nitrogen before the two fermentations were allowed to continue.
Analytical techniques. Carbon dioxide evolution in the bioreactor was determined with a Gallus 1000 volumetric flux transductor (Schlumberger, Buenos
Aires, Argentina). Culture samples were taken periodically to establish the
fermentation status. These samples were analyzed to determine the dry cell
weight, the cell number, and the concentrations of glucose, fructose, intra- and
extracellular organic acids and amino acids, ammonia, and free amino acid
nitrogen. Dry cell weight was estimated by filtering cells and washing them twice
with distilled water and then drying the preparation to a constant weight at 85C.
Cell numbers were estimated with a Neubauer chamber (Brand Biotech).
Glucose, fructose, and trehalose concentrations were measured by high-performance liquid chromatography (HPLC) by using a Waters high-performance
carbohydrate cartridge as described by the manufacturer (Waters Corporation).
Organic acids, glycerol, and ethanol concentrations were measured by HPLC by
using a Bio-Rad HPX-87H column (40). Amino acids were derivatized with
Waters AccQ Fluor reagent, and then the concentrations were measured by
HPLC by using an AccQ Tag amino acid analysis column in accordance with the
instructions of the manufacturer (Waters Corporation). The ammonia concentration was measured enzymatically by using glutamate dehydrogenase (Sigma
Chemical Co., St. Louis, Mo.). The concentration of free amino acid nitrogen
was determined by using the -phthaldehyde/N-acetyl-L-cysteine spectrophotometric assay (NOPA) procedure (12). Since the NOPA reagent reacts only with
primary amino groups, the nitrogen in other groups in a molecule is unaffected.
In arginine, for example, just one nitrogen molecule reacts.

3393

Viability was measured by using a LIVE/DEAD yeast viability kit as described

by the manufacturer (Molecular Probes, Eugene, Oreg.) and a fluorescence
microscope (Leitz Laborlux S) equipped with an XF56 filter (Omega Optical).
Metabolically active cells contained small red fluorescent intravacuolar structures, while dead cells emitted diffuse, green-yellow fluorescence.
Intracellular metabolites were extracted with boiling buffered ethanol (14),
and then the concentrations were determined by HPLC. Glycogen and trehalose
were extracted with 0.25 M Na2CO3 prior to enzymatic hydrolysis to glucose (27).
DNA concentrations were determined by the Burton method (15). RNA concentrations were determined by the Schmidt-Tannhauser procedure (4). Protein
concentrations were determined by the biuret method (41). The total cellular
carbohydrate concentrations were determined by the phenol method (15). Lipid
concentrations were determined gravimetrically after chloroform and methanol
extraction (11).
Stoichiometric model. The stoichiometric model used to represent the metabolic network of S. cerevisiae was adapted from the model of Nissen et al. (26).
A few modifications were necessary to account for fructose uptake, transport
reactions, and amino acid biosynthesis pathways. The pathway network consists
of glycolysis, the pentose phosphate pathway, the pyruvate carboxylase reaction,
the synthesis of ethanol, glycerol, and acetate, the tricarboxylic acid cycle, synthesis and transport reactions for the amino acids arginine, glutamine, tryptophan, alanine, glutamate, serine, threonine, leucine, aspartate, valine, phenylalanine, and isoleucine (16, 42, 43), transport reactions for incorporation and
secretion of various metabolites, and the synthesis pathways for macromolecular
components (26).
Metabolic flux analysis: consistency analysis. The flow rates of material (i.e.,
the fluxes through the pathways of the bioreaction network) are estimated from
measurements of substrate uptake and product formation rates. Measurements
and fluxes are linked through metabolite (stoichiometric) balances (38). Intracellular metabolite pools are assumed to be at a steady state; however, it is
necessary to establish the macromolecular composition in order to quantify the
drain of metabolites in biomass synthesis (19).
An analysis of metabolite balancing was carried out to determine the flux
distribution in each culture at three equivalent stages during fermentation. To
compare normal and sluggish flux distributions, the metabolic fluxes for the
cultures were compared at the same alcohol concentration. The substrate uptake
rates and product and macromolecular component formation rates were calculated for batch cultures as previously described (37) and were used to estimate
the metabolic fluxes. The relative standard deviations considered for the measured rates, based on the corresponding measurement variances, were as follows:
carbon dioxide evolution rate, ammonia, and macromolecular components, 10%;
and glucose, organic acids, and amino acids, 5%. The stoichiometric matrix
derived from the model has a condition number of 100 and indicates that the
model is numerically robust (38).
Prior to any flux analysis, we verified that the balance for biomass macromolecular components accounted for 95 to 100% of the cell weight in all of the
samples analyzed and, after this, that carbon balances accounted for 95 to 100%
in the three flux distributions obtained.

RESULTS
Two fermentations were performed under winemaking conditions. The cultures differed only in the initial nitrogen content. The first culture contained 300 mg of assimilable nitrogen
per liter, which is sufficient to avoid sluggish fermentations (6).
The second culture, on the other hand, contained 50 mg of
assimilable nitrogen per liter, a concentration which is sufficiently low to cause a sluggish fermentation. Each type of
fermentation was performed in triplicate.
Normal fermentations. In normal fermentations, the average time to reach dryness (4 g of sugar liter1) was 6 days
(170 h) (Fig. 1A). Glucose was consumed in preference to
fructose (23). This was due to differences in the various transporters affinities for these sugars (7, 30). The biomass growth
curve revealed an exponential phase and a stationary phase
that began at 48 h, at which point the biomass concentration
was 5.8 g liter1. Ethanol synthesis occurred mainly in the
stationary phase and resulted in a final ethanol concentration

3394

VARELA ET AL.

APPL. ENVIRON. MICROBIOL.

FIG. 1. (A) Normal fermentation profile (300 mg of N liter1).

(B) Sluggish fermentation profile (50 mg of N liter1). Symbols: F,
glucose consumption; , fructose consumption; , ethanol production;
, biomass production.

of 12.7% (vol/vol) (Table 1). These values are typical of experiments reported for normal fermentations for several commercial strains grown under similar conditions (31).
Assimilable nitrogen was depleted after 24 h of culture, and
this coincided with the highest specific growth rate (0.2 h1).
Even though all assimilable nitrogen was consumed, the cell
viability remained greater than 97% until all of the sugar was
consumed.
Besides ethanol, the yeast produced a number of other products during the fermentation. The final concentration of glycerol, quantitatively the second most important product of wine
fermentation, was 7.8 g liter1, and glycerol was produced
mainly during the exponential growth phase, in response to an

imbalance in reduction equivalents triggered by protein synthesis (35). Other significant compounds produced by the yeast
were succinic and acetic acids, whose final concentrations were
1.8 and 1.0 g liter1, respectively.
Sluggish fermentations. The average batch time in sluggish
fermentations was 29 days (700 h), and these fermentations left
16 g of residual sugar liter1 (Fig. 1B). As in the normal
fermentations, glucose consumption was preferred over fructose consumption. The biomass entered the stationary phase
after 72 h, when the biomass concentration was 1.5 g liter1.
The ethanol concentration, meanwhile, reached 9.5% (vol/vol)
(Table 1).
Assimilable nitrogen was completely depleted from the medium in the first 35 h (data not shown). Nonetheless, the yeast
continued to consume sugar for nearly 27 days in a nitrogenfree medium. Moreover, the cell viability remained high (97%)
until the end of the fermentation (data not shown). The final
concentrations of glycerol, succinic acid, and acetic acid were
7.2, 1.0, and 1.0 g liter1, respectively.
Cellular composition. The macromolecular composition of
yeast was determined for both types of cultures. The relative
sugar consumption was used as the basis for comparison as the
time scales were different (2). Insufficient biomass made determination of macromolecular composition unreliable before
20 h of culture.
The protein content decreased from 70 to 45% of the cell
weight for normal conditions and from 30 to 24% of the cell
weight for conditions that led to sluggish fermentations (Fig.
2A). The RNA content also decreased throughout the fermentations (from 9 to 4% and from 24 to 7%, respectively) (data
not shown). Conversely, the levels of the total carbohydrates
increased from 20 to 50% in normal cultures and from 26 to
63% in sluggish fermentations (Fig. 2B). The decreases in the
protein and RNA contents were due to the reduced requirements for these components when the cells entered the stationary phase and to increased cell weight resulting from the
accumulation of carbohydrates (35). An interesting finding was
the high initial RNA level in sluggish fermentations, which
could have been the result of metabolic adjustments in response to lower nitrogen availability. In both normal and sluggish fermentations, the sum of the total carbohydrate, protein,
and RNA contents accounted for 96% of the dry cell weight on
average. This proportion was constant throughout the fermentations, and the remaining cell weight comprised less than 1%
DNA and around 3% lipids.
Glycogen and trehalose accumulated when nitrogen was exhausted upon entry into the stationary phase for a normal

TABLE 1. Final fermentation values

Culturea

Fermentation
time (h)b

Cell dry wt
(g liter1)

Sugar fermentation
rate (g liter1
day1)

Ethanol
concn (%,
vol/vol)

Glycerol concn
(g liter1 )

Succinic acid
concn (g
liter1)

Acetic acid
concn (g
liter1)

Normal (300 mg of N liter1)

Sluggish (50 mg of N liter1)
Concentrated twofold
Concentrated fivefold

170 12
700 10
330
120

5.8 0.1
1.5 0.1
3.2
8.0

33.5 2.6
7.6 0.3
17.1
47.7

12.7 0.9
9.5 0.4
13.4
11.2

7.8 0.6
7.2 0.3
8.1
9.4

1.8 0.2
1.0 0.1
1.4
1.4

1.0 0.1
1.0 0.1
0.6
0.8

a
Data for the normal and sluggish fermentations were obtained from three independent experiments. Musts for the concentration experiments were obtained from
a single sluggish fermentation.
b
Time for 93% of the sugar consumption for sluggish fermentations.

VOL. 70, 2004

EFFECTS OF LOW NITROGEN ON FERMENTATION RATE

FIG. 2. Macromolecular composition for normal cultures (solid

symbols), sluggish cultures (open symbols), and concentrated cultures
(gray squares). (A) Protein. (B) Total carbohydrates. (C) Trehalose
(circles), glycogen (triangles), and ethanol (dotted line).

fermentation and throughout the stationary phase for sluggish

fermentations (Fig. 2C). Glycogen accounted for 1.6% of the
dry cell weight in normal fermentations and 3% of the dry cell
weight in sluggish fermentations. Trehalose, however, accounted for 18% of the dry cell weight at the end of the
stationary phase, and the level was highly correlated with the
increase in the amount of ethanol in both cultures (Fig. 2C).
Regardless of the differences in the initial nitrogen concentra-

3395

tions, we found that the amounts and profiles of accumulated

trehalose were similar in all types of cultures.
Metabolic fluxes. Metabolic carbon flux distributions were
obtained for normal and sluggish fermentations in order to
study the effect of assimilable nitrogen on the metabolism of
the yeast. Three fermentation points were compared at specific
concentrations of alcohol: in the exponential phase, at the
onset of the stationary phase, and during the stationary phase.
We decided to examine alcohol content rather than relative
sugar consumption because although the amount of alcohol is
related to sugar consumption and reflects the internal state of
the cell, it also reveals environmental conditions caused by
alcohol-induced stress. The flux distributions in the network at
ethanol concentrations of 1.2, 6.4, and 9.5% (vol/vol) are
shown in Fig. 3. The fluxes were expressed as specific fluxes (in
millimoles of C per gram [dry weight] of cells per hour) to aid
comparisons of fermentations with different biomass concentrations.
During the exponential phase, 65% of the carbon consumed
came from glucose in both types of cultures. At the onset of the
stationary phase carbon was consumed equally from fructose
and glucose. In the late stationary phase fructose supplied 60%
of the carbon in normal fermentations and 75% of the carbon
in sluggish fermentations. In each phase, most of the carbon
was used in energy production (ethanol synthesis). The carbon
fraction used for ethanol production increased from the exponential phase to the late stationary phase, from 53 to 63% in a
normal fermentation and from 59 to 67% in a sluggish fermentation (Fig. 3).
The decline in the flux towards glycerol (see Appendix,
equation 13) was consistent with the decrease in reduction
equivalents following the growth phase (35). In all three phases
less than 4% of the carbon was used for the production of
other metabolites (mainly succinic and acetic acids). The fraction of carbon directed to the pentose phosphate pathway was
growth rate dependent. Thus, when the requirement for intermediates from this pathway diminished (at the start of the
stationary phase), the flux through the pathway decreased
(equation 14). The flux of carbon in the tricarboxylic acid cycle
was kept very low under all conditions, mainly to supply the
necessary metabolites for biosynthesis.
Normal fermentations had larger fluxes than sluggish fermentations at the three points compared. Only small differences in flux distributions were observed between the two types
of cultures when the fluxes were normalized with respect to
total sugar entry. Therefore, the flux distribution was largely
unaffected by the initial nitrogen concentration within the metabolic network. In fact, the main difference between normal
and sluggish fermentations was the kinetics of carbon entry
into the cells.
Cell concentration experiments. In a new approach, we isolated the effect of biomass from the effect of metabolic activity
in low-assimilable-nitrogen musts by increasing the concentration of cells in the same metabolic state. At the beginning of
the stationary phase of a sluggish fermentation, two samples
were extracted, and the biomass was concentrated twofold for
one sample and fivefold for the other sample. The two resulting fermentations were then continued with larger amounts of
biomass without altering other fermentation parameters, and

3396

VARELA ET AL.

APPL. ENVIRON. MICROBIOL.

FIG. 3. Metabolic fluxes in normal fermentations (black numbers) and sluggish fermentations (white numbers on a black background) at the
following three alcohol concentrations (from top to bottom in each case): 1.2, 6.4, and 9.5% (vol/vol). The values are expressed in millimoles of
C per gram (dry weight) of cells per hour. The flux used for maintenance is expressed as a percentage of the total ATP produced. CARB,
carbohydrates; GLC, glucose; FRUC, fructose; F6P, fructose 6-phosphate; R5P, ribose 5-phosphate; DHAP, dihydroxyacetone phosphate; G3P,
glyceraldehyde 3-phosphate; E4P, erythrose 4-phosphate; GLY3P, glycerol 3-phosphate; 3PG, 3-phosphoglycerate; GLYC, glycerol; SER, serine;
PEP, phosphoenolpyruvate; AC, acetate; ACCOA, acetyl coenzyme A; LIP, lipids; OAA, oxaloacetate; PYR, pyruvate; ADE, acetaldehyde;
ETOH, ethanol; ASP, aspartate; ISOCIT, isocitrate; GLN, glutamine; FUM, fumarate; AKG, -ketoglutarate; GLU, glutamic acid; PROT,
protein; SUC, succinate; SUCEX, extracellular succinate; mATP, maintenance ATP.

the results were compared with the results of three sluggish

fermentations.
After concentration, the amounts of biomass obtained in the
stationary phase in the two experiments were 3.2 g liter1 for
the culture concentrated twofold and 8.0 g liter1 for the
culture concentrated fivefold. The fermentation times were 53
and 86% shorter, respectively, than the average time taken for
the sluggish fermentations to consume 93% of the sugar (Table
1). The rate of sugar fermentation increased linearly with the
biomass concentration (two- and sixfold, respectively).
At the end of the fermentations of both concentrated cultures there was no residual sugar, which was reflected in significantly higher final concentrations of ethanol (Table 1). The
profiles of proteins, total carbohydrates (Fig. 2A and B), trehalose, and glycogen (data not shown) were still similar to the
profiles for a sluggish fermentation.

DISCUSSION
Nitrogen deficiency, the most common cause of sluggish
fermentations, indirectly affects the fermentation rate through
intracellular activity and biomass yield. To gain further insight
into the phenomenon of problematic cultures caused by low
levels of assimilable nitrogen, we studied fermentations using
an artificial must under winemaking conditions (anaerobiosis,
low pH, high initial sugar content).
Depletion of assimilable nitrogen occurred halfway through
the exponential phase of both normal and sluggish fermentations, irrespective of the initial amount of nitrogen available,
and this confirmed that nitrogen is the growth-limiting substrate. In the sluggish fermentations even the time required to
consume lower levels of initial nitrogen was less than that for
normal cultures. The biomass yield was significantly lower in

VOL. 70, 2004

EFFECTS OF LOW NITROGEN ON FERMENTATION RATE

sluggish cultures, and indeed, every stage of such fermentations occurred later than it occurred in normal cultures.
In low-assimilable-nitrogen sluggish fermentations the size of
the protein fraction was consistently one-half the size in normal
fermentations. Lower protein levels made the relative amounts of
RNA and carbohydrates proportionally higher in sluggish fermentations. The protein, RNA, and carbohydrate profiles were
similar for both culture types even though the quantities of these
macromolecules were different in absolute terms.
Synthesis of trehalose and glycogen occurred mainly at the
onset of the stationary phase in normal fermentations and
mostly in the stationary phase in sluggish fermentations. Total
carbohydrates accumulated throughout the fermentations not
only due to the production of reserve carbohydrates but also
due to the production of other carbohydrates (data not
shown). Nissen et al. (26) described the accumulation of mannan, glucan, and chitin as an effect of the decline in the specific
growth rate of S. cerevisiae.
Trehalose has been reported to accumulate in response to
nutrient depletion (36), as well as in response to other types of
stress, including heat shock and osmotic stress (17, 28). We
found that there was a strong correlation between trehalose
accumulation and ethanol production. Trehalose is directly
related to the survival of a cell exposed to ethanol (24). Wine
yeasts respond to an increase in the ethanol level by accumulating trehalose to mitigate damage to membranes and proteins. Trehalose manages to reduce ion leakage caused by
ethanol (25). It also protects the cell from endocytosis inhibition due to high ethanol levels (22), which contributes to more
efficient nitrogen utilization, and this may explain sustained
cell viability in nitrogen-poor musts. As we found no significant
change in medium osmolality (there was a change from 1,500
to 2,000 mOsm kg1, calculated from the contributions of
sugar, ethanol, and glycerol), in our experiments trehalose was
not accumulated in response to higher osmotic pressure.
The principal difference in the flux distribution between a
normal fermentation and a sluggish fermentation was the kinetics of sugar entry into the network. The specific carbon
uptake rate was always higher in a normal culture (3.6-fold
higher in the exponential phase and 10-fold higher in the late
stationary phase) (Fig. 3). Thus, there was a net difference in
the amount of sugar transported per gram of biomass. This
could have been due either to weaker transporter activity in
sluggish fermentation cells or to fewer transporters per cell.
With regard to the latter possibility, it has been found that
nitrogen deficiency has an impact on the transporter turnover
rate and on the expression of at least one transporter, HXT1
(5). In sluggish fermentations there are lower protein levels
and also a proportionally lower flux compared to the protein
levels and flux in a normal fermentation. Normalized flux distributions are similar, so we wonder whether the lack of nitrogen affects the expression of specific proteins or diminishes the
expression of the entire proteome. Expressing the fluxes in
terms of total protein instead of dry cell weight reduced the
differences observed in the sugar transport rate twofold. This
suggests that there is a combination of specific and general
protein regulation.
Inside the metabolic network, fluxes associated with nitrogen sources represent a fraction of the total flux; the most
noticeable difference is the flux towards protein. Consequently,

3397

in both culture types, differences in nitrogen-related pathways

had no effect on flux distribution inside the network.
The carbon fraction used for ethanol production (energy
production) increased throughout the fermentation in both
cultures, from 53 to 63% for normal cultures and from 59 to
64% for sluggish cultures. As there is no energy requirement
for growth in the stationary phase, this increase in energy was
used entirely for maintenance. In both musts, there were high
energy costs in the late stationary phase in response to ion
diffusion into the cytoplasm (due to ethanol concentration)
and protein turnover (due to nitrogen depletion). In sluggish
fermentations, only the initial maintenance requirements were
higher than those in normal fermentations (Fig. 3). The higher
initial energy cost may have occurred as the cells regulated
their metabolism in response to the lower levels of assimilable
nitrogen available. Afterwards, maintenance requirements
were directly correlated to ethanol stress in both cultures.
Evaluating the impact of increasing biomass upon the rate of
fermentation in cell concentration experiments revealed that
(i) the higher the concentration of biomass, the quicker fermentation was completed, even when cells grown with a severe
shortage of nitrogen were used; and (ii) that the rate of fermentation was a linear function of biomass, while fermentation
time was an exponential function of biomass.
The reduced fermentation times observed in concentrated
cultures established that the main problem with a limited initial nitrogen content is that it curbs biomass formation. Despite
a lower specific instantaneous sugar uptake rate, the concentrated sluggish fermentations concluded rapidly. The lack of
fermentative mass was responsible not only for lower fermentation rates but also for incomplete sugar consumption and a
lower ethanol yield in wine obtained from nitrogen-deficient
musts.
In summary, by isolating the effects of metabolic flux and the
amount of biomass we found that the viable cell concentration
governs the fermentation rates in nitrogen-poor musts. From
an industrial perspective, these findings suggest two alternatives to deal with nitrogen-deficient musts. The first alternative
involves early nitrogen supplementation, as the resulting increase in biomass should ensure a normal fermentation profile.
The second alternative involves addition of viable biomass
from other fermentation tanks (possibly by decantation or filtration) that would also result in the restoration of the normal
fermentation profile. Not only does adding biomass from other
fermentations reduce the time to completion, but it should also
be less likely to affect negatively the resulting wine, as there
should be no significant changes in the concentrations of the
main components present in the must. Nevertheless, possible
effects on aroma and flavor profiles warrant further investigation.
APPENDIX
In the following biochemical reactions used in the yeast
model the subscripts CYT, MIT, and EX indicate cytosolic,
mitochondrial, and extracellular metabolites, respectively;
AICAR refers to 5-phosphoriboxyl-5-aminoimidazole-4-carboxamida; THF refers to tetrahydrofolate.
Glycolysis 1. Glucose 1/6 ATP 3 Glucose 6-phosphate
1/6 ADP 1/6 H

3398

VARELA ET AL.

2. Fructose 1/6 ATP 3 Fructose 6-phosphate 1/6 ADP

1/6 H
3. Glucose 6-phosphate 3 Fructose 6-phosphate
4. Fructose 6-phosphate 1/6 ATP 3 3/6 Glyceraldehyde
3-phosphate 3/6 Dihydroxyacetone phosphate 1/6 ADP
1/6 H
5. Dihydroxyacetone phosphate 3 Glyceraldehyde 3-phosphate
6. Glyceraldehyde 3-phosphate 1/3 NADCYT 1/3 ADP
1/3 P 1/3 H2O 3 3-Phosphoglycerate 1/3 ATP 1/3
NADHCYT 2/3 H
7. 3-Phosphoglycerate 3 Phosphoenolpyruvate 1/3 H2O
8. Phosphoenolpyruvate 1/3 ADP 1/3 H 3 Pyruvate
1/3 ATP
Ethanol, glycerol, and acetate synthesis
9. Pyruvate 1/3 H 3 2/3 Acetaldehyde 1/3 CO2
10. Acetaldehyde 1/2 NADHCYT 1/2 H 3 Ethanol
1/2 NADCYT
11. Acetaldehyde 1/2 NADPCYT 1/2 H2O 3 Acetate
1/2 NADPHCYT H
12. Dihydroxyacetone phosphate 1/3 NADHCYT 1/3
H 3 Glycerol 3-phosphate 1/3 NADCYT
13. Glycerol 3-phosphate 1/3 H 3 Glycerol 1/3 P
Pentose phosphate pathway
14. Glucose 6-phosphate 2/6 NADPCYT 1/6 H2O 3
5/6 Ribose 5-phosphate 2/6 NADPHCYT 2/6 H 1/6
CO2
15. Ribose 5-phosphate 3 2/5 Erythrose 4-phosphate 3/5
Fructose 6-phosphate
16. 5/9 Ribose 5-phosphate 4/9 Erythrose 4-phosphate 3
6/9 Fructose 6-phosphate 3/9 Glyceraldehyde 3-phosphate
Tricarboxylic acid cycle
17. 3/7 Pyruvate 4/7 OxaloacetateMIT 1/7 NADMIT
1/7 H2O 3 6/7 Isocitrate 1/7 CO2 1/7 NADHMIT 1/7
H
18. Isocitrate 1/6 NADMIT 3 5/6 -Ketoglutarate 1/6
CO2 1/6 NADHMIT
19. -Ketoglutarate 1/5 NADMIT 1/5 ADP 1/5 P
1/5 H2O 3 4/5 Succinate 1/5 ATP 1/5 CO2 1/5 NADHMIT
1/5 H
20. Succinate 1/4 Flavin adenine dinucleotide 3 Fumarate 1/4 Reduced flavin adenine dinucleotide 1/4 H
21. Fumarate 1/4 H2O 1/4 NADMIT 3 OxaloacetateMIT
1/4 NADHMIT 1/4 H
22. Acetate 1/2 Coenzyme A ATP H 3 Acetyl
coenzyme A ADP P 1/2 H2O
23. Isocitrate 1/6 NADPMIT 3 5/6 -Ketoglutarate
1/6 CO2 1/6 NADPHMIT
Anaplerotic reaction: pyruvate carboxylase
24. 3/4 Pyruvate 1/4 CO2 1/4 ATP 3 OxaloacetateCYT
1/4 ADP 1/4 P 1/4 H
Carbohydrate synthesis
25. Glucose 6-phosphate 1/6 ATP 3 Carbohydrates 1/6
ADP 2/6 P
Nitrogen metabolism and amino acid biosynthesis
26. -Ketoglutarate 1/5 NH
4 1/5 NADPHCYT 1/5
H 3 Glutamate 1/5 H2O 1/5 NADPCYT
27. Glutamate 1/5 NH
4 1/5 ATP 3 Glutamine 1/5
ADP 1/5 P 1/5 H2O

APPL. ENVIRON. MICROBIOL.

28. 4/9 OxaloacetateCYT 5/9 Glutamate 3 4/9 Aspartate

5/9 -Ketoglutarate
29. 3/8 3-Phosphoglycerate 5/8 Glutamate 1/8
NADCYT 3 3/8 Serine 5/8 -Ketoglutarate 3/8 P 1/8
NADHCYT
30. 10/15 Glutamate 4/15 Aspartate 1/15 NH
4 1/15
CO2 5/15 ATP 1/15 NADHCYT 3 6/15 Arginine 4/15
Fumarate 5/15 -Ketoglutarate 5/15 ADP 5/15 P 1/15
NADCYT 3/15 H2O 4/15 H
31. 3/8 Pyruvate 5/8 Glutamate 3 3/8 Alanine 5/8
-Ketoglutarate
32. Aspartate 2/4 ATP 2/4 NADPHCYT 2/4 H 3
Threonine 1/4 H2O 2/4 ADP 2/4 P 2/4 NADPCYT
33. 6/13 Pyruvate 2/13 Acetyl coenzyme A 5/13 Glutamate 3 6/13 Leucine 5/13 -Ketoglutarate 2/13 CO2
1/13 H2O 1/13 Coenzyme A
34. 6/5 Pyruvate Glutamate 1/5 NADHCYT 1/5 H 3
Valine -Ketoglutarate 1/5 CO2 1/5 NADCYT 1/5
H2O
35. 6/5 Phosphoenolpyruvate 4/5 Erythrose 4-phosphate
Glutamate 1/5 NADHCYT 1/5 H 1/5 ATP 3 9/5
Phenylalanine -Ketoglutarate 1/5 CO2 1/5 ADP 1/5
P 1/5 NADCYT H2O
36. 4/6 Threonine 3/6 Pyruvate 5/6 Glutamate 1/6
NADHCYT 1/6 H 3 Isoleucine 5/6 -Ketoglutarate

1/6 NH
4 1/6 CO2 1/6 H2O 1/6 NAD CYT
Synthesis of DNA, RNA, proteins, and lipids
37. 5/23 Ribose 5-phosphate 10/23 Glutamine 3/23
Serine 4/23 Aspartate 1/23 CO2 6/23 ATP 1/23
NADPCYT 3 9/23 AICAR 10/23 Glutamate 4/23 Fumarate 1/23 NADPHCYT 6/23 ADP 6/23 P 5/23 H
2/23 H2O
38. 0.4579 AICAR 0.4371 Glutamine 0.0842 THF
0.3313 Aspartate 0.2544 Ribose 5-phosphate 0.4625 ATP
0.0509 NADPHCYT 0.1540 NADCYT 0.3301 H2O 3
DNA 0.4371 Glutamate 0.1278 Fumarate 0.0509
NADPCYT 0.1540 NADHCYT 0.5166 H 0.4625 ADP
0.4625 P
39. 0.5112 AICAR 0.5271 Glutamine 0.0568 THF
0.2993 Aspartate 0.2400 Ribose 5-phosphate 0.4890 ATP
0.0568 NADPCYT 0.1348 NADCYT 0.3427 H2O 3
RNA 0.5271 Glutamate 0.1073 Fumarate 0.1348
NADHCYT 0.0568 NADPHCYT 0.7080 H 0.4890 ADP
0.4890 P
40. 0.0263 Ribose 5-phosphate 0.7189 Glutamate
0.1311 Glutamine 0.2319 Aspartate 0.3944 Pyruvate
0.0869 Serine 0.0597 Erythrose 4-phosphate 0.0895 Phosphoenolpyruvate 1.0084 ATP 0.0522 NADPHMIT
0.0742 NADMIT 0.0201 NADHCYT 0.0883 NADPHCYT
0.0083 SO2
0.7685 H2O 3 Proteins 0.5596 -Keto4
glutarate 0.0518 Fumarate 0.0044 Glyceraldehyde 3-phosphate 0.1155 CO2 0.0110 NH
4 0.0124 THF 0.0742
NADHMIT 1.0617 H 0.0522 NADPMIT 0.0201
NADCYT 0.0883 NADPCYT 1.0084 ADP 1.0084 P
41. 0.8326 Acetyl coenzyme A 0.0662 Glyceraldehyde
3-phosphate 0.1012 Serine 0.4000 ATP 0.7111
NADPHCYT 0.0259 H 3 Lipids 0.0258 H2O 0.4000
ADP 0.4000 P 0.4163 Coenzyme A 0.7111 NADPCYT

VOL. 70, 2004

EFFECTS OF LOW NITROGEN ON FERMENTATION RATE

Oxaloacetate shuttle
42. OxaloacetateCYT 1/4 ATP 3 OxaloacetateMIT 1/4
ADP 1/4 P
Mitochondrial synthesis of ethanol
43. Acetaldehyde 1/2 NADHMIT 1/2 H 3 Ethanol
1/2 NADMIT
Transport reactions
44. NHEX
ATP 3 NH
4
4 ADP P
45. Acetate 3 AcetateEX
46. Ethanol 3 EthanolEX
47. Glycerol 3 GlycerolEX
48. Succinate 3 SuccinateEX
49. AspartateEX 1/4 ATP 3 Aspartate 1/4 ADP 1/4
P
50. GlutamateEX 1/5 ATP 3 Glutamate 1/5 ADP 1/5
P
51. GlutamineEX 1/5 ATP 3 Glutamine 1/5 ADP 1/5
P
52. SerineEX 1/3 ATP 3 Serine 1/3 ADP 1/3 P
53. ArginineEX 1/6 ATP 3 Arginine 1/6 ADP 1/6 P
54. TryptophanEX 1/11 ATP 3 Tryptophan 1/11 ADP
1/11 P
55. AlanineEX 1/3 ATP 3 Alanine 1/3 ADP 1/3 P
56. ThreonineEX 1/4 ATP 3 Threonine 1/4 ADP 1/4
P
57. LeucineEX 1/6 ATP 3 Leucine 1/6 ADP 1/6 P
58. ValineEX 1/5 ATP 3 Valine 1/5 ADP 1/5 P
59. PhenylalanineEX 1/9 ATP 3 Phenylalanine 1/9
ADP 1/9 P
60. IsoleucineEX 1/6 ATP 3 Isoleucine 1/6 ADP 1/6
P
ATP dissipation reaction
61. ATP 3 ADP
ACKNOWLEDGMENTS
We thank Alex Crawford for a thorough revision of the manuscript.
This work was supported by Fondo Nacional para el Desarollo
Cientfico y Tecnolo
gico de Chile (FONDECYT) grant 2010087 and
by Beca de Apoyo a la Realizacio
n de Tesis Doctoral (CONICYT).
Cristian Varela and Francisco Pizarro were supported by a doctoral
fellowship from Consejo Nacional de Investigacio
n Cientfica y Tecnolo
gica de Chile (CONICYT).
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