Group 41 Glycerol in Bread Dough Fermentation 1

The effect of yeast glycerol production level on bread dough

fermentation capacity
Neckebroeck B., Nootens S., Samlali K., Vandenkerckhove J.
13
th
of May 2014

Abstract
Glycerol production in yeast is known to be a major response to high osmotic stress situations such as in solid state bread dough
fermentation. Moreover, it has recently been argued that GPD1, the Saccharomyces cerevisiae gene encoding glycerol-3-
phosphate dehydrogenase, is the rate limiting gene in glycerol metabolism. We investigated the role of GPD1 on glycerol/ethanol
productions and on bread dough fermentation capacity. Our results showed that an overexpression of GPD1, increasing the
glycerol production, leads to a faster adaptation to osmotic stress in dough and a better fermentation capacity. On the other hand,
deletion of GPD1 leads to a long lag phase and a delayed fermentation capacity. This demonstrates that proper response to
osmotic stress through glycerol production is important for proper dough fermentation. Glycerol production is a necessity for
fermentation.

1. Introduction
Glycerol, 1,2,3-propanotriol, is a by-product of the
alcoholic fermentation process by yeast. The whole
fermentation metabolic pathway is divided into different
branches. The production of ethanol (EtOH) and CO
2
is the
primary branch while glycerol is a secondary metabolite.
The production of these two metabolites is anti-correlated.
With a rise of glycerol levels, the primary metabolites
ethanol and CO
2
will be less produced (Michnick et al.,
1997). Glycerol has two different roles in the fermentation
process. First, the production of glycerol will consume the
NADH produced during the glycolysis and by doing this, it
will maintain the cytosolic redox balance (Wang et al.,
2001). The second role of glycerol production is to handle
high osmotic stress situations. To prevent water loss during
high osmotic stress, microorganisms produce compatible
solutes like ions, amino acids or polyols and accumulate
them. Glycerol is one of those solutes (Brown, 1976 ;
Blomberg and Adler, 1992 ; as cited by Aslankoohi et al.,
2013). The low extracellular free water causes an increased
rate of glycerol production (Blomberg and Adler, 1989).

Bread dough is a solid state fermentation (SSF) medium.
SSF differs from liquid fermentation due to a lower amount
of free extracellular water available. This is a high osmotic
stress condition for the yeast, which increases the rate of
glycerol production as stated earlier (Aslankoohi et al.,
2013). Microarray analysis shows there is a strong up-
regulation of the genes involved in the High-Osmolarity
Glycerol response (HOG) pathway during high osmolarity
(Tanaka et al., 2006). The HOG pathway is a signal
transducing pathway. Also many other signalling pathways
are activated by the osmotic stress, adjusting osmosensing
proteins like MAP kinase. These pathways are still not all
identified and interact closely to finally activate important
genes in the metabolic pathway to glycerol (Rep et al.,
1999). One of these important genes is the GPD1 gene.
After the activation of the GPD1 gene, contributing to
glycerol production, a two-step process begins. In the first
step of glycerol metabolism, dihydroxyacetonephosphate
(DHAP) is converted into glycerol-3-phosphate (G3P).
Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes this
reaction. It consists of two isoenzymes, gpd1p and gpd2p,
which are respectively osmotic-induced and constitutive
enzymes. From these, gpd1p, a cytoplasmic NAD
+
-
dependent enzyme, is the key enzyme and the result of the
expression of the GPD1 and/or GPD2 gene (Wang et al.,
2001). Secondly, glucose-3-phosphate is further
transformed to glycerol by dephosphorylation due to the
enzym glycerol-3-phosphatase (gpp). This also consists of
two isoenzymes, gpp1p and gpp2p, respectively constitutive
and osmotically-induced. The production of gpp2p enzyme
is also dependent on the activation of its gene by the
osmotic signal in a bread dough or other hyperosmotic
environments (Wang et al., 2001).

Previous studies show that GPD1 is the rate limiting gene in
glycerol metabolism and overexpression of this gene leads
to an increase in production of glycerol. Most of the GPDH
protein activity comes from the isoenzym gpd1p (Michnick
et al., 1997). This isoenzym is expressed by the GPD1 gene.
As a result of the overproduction of gpd1p enzyme, a
threefold and higher increase in glycerol levels can be
obtained. This increase of glycerol leads to decreased
fermentation rate and a lower ethanol and CO
2
levels which
is not favourable for the leavening of dough. On the other
hand, this increase also facilitates the adaptation to high
osmotic stress (Michnick et al., 1997).

On the other side, reducing the glycerol production can
affect the fermentation capacity of the bread dough as well.
One way to do this, is to use a GPD1 deletion mutant. (Jain
et al., 2011). In this case, the yeast will use alternative
pathways to reduce the NADH produced during
fermentation. As stated earlier, gpd1p is a limiting enzyme
for glycerol production. However, in the absence of GPD1,
the gpd2p enzyme expressed by the GPD2 gene, can
maintain a lower production level of glycerol (Michnick et
al., 1997).
The aim of this study is to gain an insight in the effect of
glycerol production level on yeast's bread dough

Group 41 Glycerol in Bread Dough Fermentation 2
fermentation capacities. To this end, we use mutants with
deletion and overexpression of GPD1 with respectively
lower and higher glycerol production compared to the wild
type. Our results show that an overexpression of GPD1,
increasing the glycerol production, leads to a faster
adaptation to osmotic stress in dough and a better
fermentation capacity. On the other hand, deletion of GPD1
leads to a long lag phase and a delayed fermentation
capacity.
2. Materials and Methods
2.1 Strains
Saccharomyces cerevisae S288C was used to determinate
the effect of glycerol production during bread dough
fermentation. Three different mutants were used: wild-type,
GPD1 deletion mutant (gpd1) and GPD1 overexpression
mutant (OE-GPD1). The overexpression mutant was made
by introducing high-copy pVT100U-GPD1 vectors into the
yeast wild-type. The deletion mutant was made by PCR
deletion and homologous recombination. For each mutant,
two repeats were made.
2.2 Culture conditions and harvesting
Yeast cultures were prepared in the course of three days.
During the first day, yeast cells were grown overnight in 3
mL YPS medium (Yeast extract- Peptone- 2% Sucrose). On
the second day, 1 mL of stationary phase cells were
inoculated in baffled Erlenmeyer flasks containing 500 mL
YPS and were grown overnight to reach an early stationary
phase. The flasks were placed in an incubator (New
Brunswick Scientific Incubator Shaker, Innova 40) at 30C
and 250rpm. And finally, on the third day, the culture was
harvested by centrifugation (EBA 21 centrifugeuse Hettich
2800 g) at 2800rpm (870g) for 3 min. The supernatant was
discarded and the pellets were collected and weighed in 50
mL Falcon tubes. They were washed with PBS to prevent
the release of glycerol before introduction into bread dough.
2.3 Intracellular glycerol levels measurement
To measure intracellular glycerol level of yeast cells, all
intracellular molecules containing more than one alcohol
function (polyols) were first extracted. This measurement
was performed within two biological replicates. The cell
suspensions were washed twice with iso-osmotic buffer.
Then the cell pellets were added for 5 min to 3 mL boiling
0.1 M-TRIS/HCL buffer at pH 7,7 composed of 2mM-
EDTA. This mixture was centrifuged at 15 000g for 10 min
in order to remove the cell debris. Finally, the concentration
of the glycerol of the extract was measured using HPLC
method.
2.4 Bread dough production and fermentation
Bread dough was prepared by using the following
ingredients: 18.0 g sucrose, 100.0 g biscuit flour, 1.5 g
sodium chloride, 45 mL demineralised water and 5.3 g yeast
mixed in a 100-g pin bowl mixer (National Manufacturing,
Lincoln, NE, USA), for 4 min. Each dough sample was
divided into five pieces. Three pieces were fermented in a
fermentation cabinet at 30C, with a relative humidity of
95% during resp. 60, 120 and 180 min. Another piece was
used for immediate glycerol extraction as a zero time
sample. The remaining piece was used in the Risograph
instrument (National Manufacturing, Lincoln, NE, USA) to
measure CO
2
production. The Risograph was set at 30C
with continuous measurement at 1-min intervals. For each
sample, the experiment was done in two biological
replicates.
2.5 Glycerol extraction and analysis
The glycerol was extracted from the dough by adding 40mL
demineralised water to a 20g piece of dough from the
fermentation cabinet and the 0-time sample. After
blending two times for 10 sec in a blender to dissolve the
glycerol, the mixture was transferred in 1.5 mL Eppendorf
tubes and centrifuged at 11.000 rpm (Eppendorf Micro
centrifuge 5415D) for 3 min. Pellets were thrown away
while supernatant was passed through a 0.22 m filter to
avoid remaining yeast cells, and collected in three 1.5 mL
Eppendorf tubes. The tubes were kept at -20C for further
analysis. After defrosting the 1.5mL Eppendorf tubes, about
1mL was taken and filtered again with a 0.22 m filter into
a high-performance liquid chromatograph (HPLC) vial. The
concentrations of the different compounds extracted from
the dough were measured using ion-exclusion HPLC. The
vials were put in a LC-20AT modular HPLC system
(Shimadzu, Kyoto, Japan) equipped with a Rezex ROA-
Organic acid guard column and analytical column
(Phenomenex, Torrance, CA, USA). Detection was done by
a RID-10A refractive index detector (Shimadzu). The
mobile phase consisted of 2.5 mM sulphuric acid.
3. Results
3.1 Intracellular glycerol level as a control of the
working OE-GPD1
In this experiment, the overexpression mutant of the GPD1
gene (OE-GPD1) is used for observing the effect of higher
glycerol production on the fermentation process. This is
why, as a first experiment, intracellular glycerol levels are
measured as a control in order to see whether or not changes
in the genome lead to changes in the phenotype. The
deletion mutant (gpd1) is used for obtaining a lower
glycerol level. This is also checked by measuring
intracellular levels. OE-GPD1 indeed shows a higher
intracellular glycerol level (Fig. 1). Compared to the wild-
type a threefold increase can be observed. In case of gpd1,
the measurement of the intracellular glycerol shows a

Group 41 Glycerol in Bread Dough Fermentation 3
fourfold decrease. This confirms the fact that the mutant
yeasts used for the experiments are indeed producing more
glycerol in the case of the OE-GPD1 and less for the gpd1
mutant relative to the wild type strain. Therefore, the effect
of yeast glycerol production on performance of bread dough
fermentation capacity can be examined with those three
strains.
3.2 Measurement of extracellular glycerol and
ethanol levels by HPLC analysis
During the fermentation process, glucose is converted into
ethanol and CO
2
. But a secondary pathway also turns some
glucose into glycerol during bread dough fermentation to
combat the external osmotic pressure on the yeast. To
investigate if the deletion and overexpression of the GDP1
gene leads to a significant variation of the glycerol level in
the dough, glycerol and ethanol levels were measured with a
HPLC instrument. Data was collected from two biological
replicates of wild-type, gpd1 and OE-GPD1 of
Saccharomyces cerevisiae S288C. The data for both
replicates showed very similar profiles and data was
analysed by means of the average value. Four measurements
were taken at 0, 60, 120 and 180 minutes after the start of
the fermentation for the three different mutants.

The concentrations of glycerol and ethanol are given
relative to the concentration for the wild type yeast after
three hours of fermentation (Fig. 2). It is clear that the
dough with the gpd1 contains less glycerol compared to
the wild-type dough and the bread dough fermented by OE-
GPD1 strain contains more glycerol compared to the one
fermented by the wild-type yeast. If the conclusions of the
previous paragraph are reviewed, it becomes clear that the
glycerol concentrations inside the cell and in de dough are
correlated. If the yeast produces more glycerol inside the
cytoplasm, it also transports more glycerol outside the cell
into the bread dough. This is a second proof that the used
mutants were indeed suitable for this experiment on the
effects of glycerol production.

For ethanol levels, the difference between OE-GPD1 and
the wild-type yeast arent as significant as for glycerol.
Nevertheless, the data show that after three hours, more
glucose is fermented into ethanol and CO
2
by OE-GPD1.
Furthermore, gpd1 shows an interesting characteristic. Not
only is the concentration of glycerol much lower compared
to the wild-type yeast, also the concentration of ethanol is
very low. This concentration is only about 5% of the wild-
type ethanol concentration. This means that the deletion of
the GPD1 gene not only leads to a lower glycerol
production and concentration but also effects the
fermentation of glucose into ethanol as a primary pathway.
Moreover, it is possible that fermentation has not started yet
due to the low end-point ethanol concentration for gpd1.

For the glycerol concentrations, four measurements were
taken at 0, 60, 120 and 180 minutes for the wild-type,
gpd1 and OE-GPD1 strains (Fig. 3). For the deletion
mutant, only the end point concentration is shown.
Concentration rises faster in the dough with OE-GPD1 than
for dough with wild-type yeast. This means there is more
glycerol produced per minute which is partly the result of a
higher fermentation rate. The concentration after three
hours in the dough in which OE-GPD1 was used is 2.5
times higher compared to the concentration for wild type
yeast (See also Fig. 2). Very little glycerol is produced by
the gpd1 yeast cells. This graph also grounds the statement
again that gpd1 and OE-GPD1 produce respectively less
and more glycerol compared to the wild-type yeast.
0
1
2
3
Wild-type OE-GPD1 gpd1
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Fig. 1 Intracellular glycerol concentrations and standard deviation of
Saccharomyces cerevisiae S288C for OE-GPD1, gpd1 and wild-type..
OE-GPD1 produces four times more glycerol compared to the wild-
type. The deletion of the GPD1 gene delivers a fourfold decrease in
glycerol levels. This confirms the mutants work as intended.
Fig. 2 Extracellular glycerol and ethanol concentration in bread dough
produced by Saccharomyces cerevisiae S288C, for OE-GPD1 and
gpd1 compared to the wild-type at end point (180min). Glycerol (),
Ethanol (+). Glycerol levels are as predicted by the intracellular levels.
Ethanol levels of the wild-type and OE-GPD1 are almost equal. The
ethanol levels of the gpd1 show that the fermentation capacity is
drastically affected by glycerol levels.


Group 41 Glycerol in Bread Dough Fermentation 4
The ethanol level and the glycerol level at 0 minutes are
similar (Fig. 3, Fig. 4). After this point, the values of
ethanol increase faster than those of glycerol. As stated
earlier, ethanol is a product of the primary metabolic
pathway. Glycerol is only produced in a secondary
metabolic pathway. This explains the different increasing
rates for ethanol and glycerol.
3.3 Determination of fermentation capacity by
measuring CO
2
with Risograph analysis
We measured CO
2
production of our three strains during
high sugar bread dough fermentation, as a proxy for
fermentation rate and capacity. For the wild-type, gpd1,
OE-GPD1 strains both biological replicates show very
similar profiles. The average of the 2 replicates is displayed
(Fig 5, Fig. 6). The rate of CO
2
production at 1 min time
intervals is provided as function of time during three hours
(Fig. 4). All the strains show a lag phase. In this period, the
yeast is not yet fermenting. This lag phase corresponds to
the latency while the yeast cells adapt to their high-osmotic
environment in sweet dough. This results in the induction of
signalling pathways like the HOG pathway. This results in
glycerol production which helps the cells to overcome the
osmotic stress as an osmolyte and redox balancer. The yeast
cells start to ferment. For the OE-GPD1 mutant, the lag
phase is significantly shorter than the gpd1 or wild-type
strain. The wild-type shows a relatively long lag phase.

After the lag phase, active fermentation starts and is
optimized during time. For the wild-type, CO
2
production
rate starts to rise almost linear after its lag phase, with an
average increase in production rate of 0.1 mL CO
2
/min per
hour (Fig. 5). A higher production of glycerol is obtained
and the yeast cell will resist the high-osmotic stress by
accumulating the glycerol in its cytosol. In case of gpd1,
there is no fermentation in the beginning. The gpd1 only
starts its fermentation near the end of the experiment.
Slowly, during the last hour of the experiment, the rate
starts to increase, which shows that gpd1 starts fermenting
glucose. OE-GPD1 does not only start its fermentation
earlier, it also has a stronger slope. The CO
2
production rate
rises faster than the wild-type. This means that the
fermentation process is much more efficient. This strain
reaches to maximum fermentation rate faster than the two
others. This implies that the overexpression is adapting
faster to a high osmolarity environment, in this case, sweet
dough with 18% sugar.

For the total CO
2
production, we can conclude the same
(Fig. 6). The slope of the OE-GPD1 is much steeper than
the slope of the wild-type graph. This indicates its higher
fermentation capacity. The increase has a fast non-linear
behaviour, which shows that the OE-GPD1 mutant strain
adapts faster to high osmotic environments than the wild-
type. For fermentation in high sugar bread dough (in this
case 18% sugar), figure 6 confirms that OE-GPD1 is indeed
beneficial. The gpd1 graph shows again that there is no
significant CO
2
production in the beginning of the
experiment. However, in the last hour, fermentation starts
with a very low rate. The final fermentation rates confirm
that OE-GPD1 is beneficial for short term fermentation
such as bread dough fermentation.

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Time [min]
Fig. 3 The concentration of glycerol in the bread dough during the
fermentation process of Saccharomyces cerevisiae S288C for OE-
GPD1 (), gpd1 () and wild-type (). Glycerol levels rise faster and
are also higher for the OE-GPD1 as for the wild-type.
0
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Time [min]
Fig. 4 The concentration of ethanol in the bread dough during the
fermentation process of Saccharomyces cerevisiae S288C, OE-GPD1
(),gpd1 () and wild-type (). Ethanol levels of wild-type and OE-
GPD1 do not differ a lot during fermentation pointing out that both
strains have an equal activity in the primary metabolic pathway.

Group 41 Glycerol in Bread Dough Fermentation 5









4. Discussion
The Saccharomyces cerevisiae S288C cells are exposed to
an osmotic shock due to the tough conditions in the bread
dough. Bread dough is a solid state fermentation medium
(SSF). This consists of a solid matrix with little water
available. When an extracellular lack of water is detected by
the cell, intracellular water is floating out of the yeast cell
due to the osmotic gradient over the cell membrane
(Hohmann et al., 2007). In order to cope with this stress,
yeast will produce glycerol as an osmolyte.
The 18% sugar (sucrose) concentration in the dough reduces
the extracellular water activity and induces the fermentation
process of the yeast. The other purpose of such high sugar
content was to always have a sufficient amount of sugar
available so the fermentation capacity during a three hour
experiment is not negatively affected by a lack of sugars.
The flour also contains all the essential compounds and
nutrients in sufficient quantities, crucial for growth and
fermentation of bread dough during three hours. Together,
these deliver ideal non-limiting conditions for an
experiment on fermentation capacity. To measure the effect
of glycerol on fermentation capacity in these conditions, a
GPD1 deletion mutant (gpd1), a GPD1 overexpression
(OE-GPD1) and a wild-type strain will be used. They show
three different profiles, which will now be discussed.

At the beginning of the experiment, the yeasts show a
transient period. The yeast cells are adapting to their new
environment and its osmotic stress. In such condition, when
the extracellular water activity is low, the yeast cells want
to maintain an osmotic balance relative to the medium.
Therefore, yeast cells produce more glycerol which is an
osmoregulating compound. The production of this osmolyte
is induced by signalling pathways such as the HOG
0
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Time [min]
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Fig. 5 The evolution of the production rate of CO2 (mL) per minute of Saccharomyces cerevisiae S288C, OE-
GDP1 (A), wild-type (B) and gpd1 (C). CO2 levels were continuously measured at 1 min time intervals by a
Risograph. The OE-GDP1 shows a short lag phase. The wild-type shows a lag phase double as long. CO2
production rate for OE-GDP1 rises faster as wild-type, which shows its better ability to ferment in sweet dough.
Near the end, CO2 production rate is lowering. Also, gpd1 starts to show a fermentation behaviour near the end.
0
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Fig. 6 The evolution of the total production of CO2 (mL) of Saccharomyces cerevisiae S288C, OE-GPD1 (A),
wild-type (B) and gpd1 (C).

Group 41 Glycerol in Bread Dough Fermentation 6
pathway, triggered by the osmotic shock. The turgor
pressure is regulated by the accumulation of glycerol within
the cytosol, by closing the Fps1p channel (Luyten et al.,
1995); (Tams et al., 1999). Moreover, another reason why
S. cerevisiae produces glycerol is because of its NADH
consuming process (Van Dijken and Alexander Scheffers,
1986). When glucose passes through the glycolysis, and in
the primary metabolic pathway towards ethanol, several
steps require the reduction of NAD
+
into NADH. There is a
high demand for NAD
+
to obtain a redox balance. Glycerol
production would deliver this NAD
+
.

The duration of this transient period, the lag phase, changes
from strain to strain. Compared to the wild-type strain, the
OE-GPD1 mutant shows a significant shorter lag phase. In
fact, figure 5 already shows an increase of the CO
2

production rate after half an hour. This means that the OE-
GPD1 mutant adapts faster to the osmotic stress condition.
This is logical because the OE-GPD1 produces more
glycerol osmolyte (Fig. 1) contributing to a faster reach of
osmotic equilibrium. On the contrary, the gpd1 mutant has
a much longer lag phase relative to the wild-type. Only after
two hours, the CO
2
production rate is seemingly starting to
rise slowly (Fig. 5) which suggests the fermentation is
starting. As stated earlier, glycerol is a compatible solute
and the lack of it lengthens the lag phase and affects the
growth of the cells and the efficiency of their metabolism.
Compared to the wild-type, four times less glycerol is
produced (Fig. 1). We can declare this due to the deletion of
the GPD1 gene. It has been shown that the GPD1 gene is
the major contributing gene to the glycerol (Michnick et al.,
1997). However, the GPD2 gene is also still responsible for
some glycerol production. This declares why the gpd1
mutant is still capable to adapt to its environment at a very
low pace and survives the high osmotic stress conditions.

Looking at figure 5 and 6, it can be assumed that the
fermentation process starts the earliest for the OE-GPD1
mutant, quickly followed by the wild-type and only in the
third hour for the gpd1 mutant. After this point, all three
curves showing the evolution of total CO
2
production
during the experiment are increasing linearly (Fig. 6). This
shows a constant fermentation rate. Nevertheless, the slope
of the OE-GPD1 mutant curve is much steeper than the one
for the wild type, which is again steeper than the gpd1
curve. This illustrates the fact that the OE-GPD1 is faster
and better adapting to the high osmotic stress situation of an
18% sugar medium than the gpd1. The OE-GPD1
produces more CO
2
compared to the wild-type during short
term fermentation. Remarkable however, the ethanol levels
of the OE-GPD1 are more or less the same as the ethanol
levels of the wild-type during the whole experiment (Fig.
4). The rise in CO
2
can also be a by-product of the TCA
cycle. Looking at the gpd1 mutant, it has a very low
fermentation capacity. This can be declared by the redox
balancing effect of glycerol. The fermentation activity is
probably limited due to accumulation of NADH (Jain et al.,
2011). The low production of glycerol does not oxidize
enough NADH to NAD
+
. A lack of NAD
+
is present in the
cytosol and the redox balance is disturbed. In addition, the
deletion of the GPD1 gene leaves only the GPD2 gene
responsible for glycerol production which will only happen
at a very low rate.

In the third hour of the experiment, a slow decreasing of
CO
2
production rate of the OE-GPD1 mutant is observed
(Fig. 5). This can be declared by a metabolic shift towards
the secondary glycerol metabolic pathway. An increase of
glycerol can be observed (Fig. 3). Also, other effects could
explain this behaviour. First, it could be that sugar levels
are lowering due to the high consumption in the first stage
of fermentation. In this case, the high amount of sucrose in
the medium (18% sugar medium) makes this assumption
unlikely. As we discussed previously, another possibility is
a limited fermentation due to the accumulation of NADH
(Jain et al., 2011). A possible control to prove whether or
not NADH accumulation is the main reason of the decrease
of CO
2
production rate would be to use a gpd1 mutant in
bread dough with a low osmolarity level. Concerning the
gpd1 strain, there is no change in behaviour in the last
hour and fermentation capacity rises.

We can predict that if the experiment had lasted longer than
three hours, the rate of CO
2
production of the OE-GPD1
mutant would continue to lower, while the wild-type would
remain stable. For a long fermentation, rather a wild-type
mutant would be used. But for a short fermentation as in
bread dough, an OE-GPD1 mutant is beneficial. To make
use of this property in industry, glycerol could be initially
added to the bread dough to reach high fermentation
capacity much faster. The high CO
2
levels deliver a greater
rise of bread dough compared to dough with wild-type
yeast. Also, higher glycerol levels would give shelf-stable
bread, made by extrusion-formed methods, more ideal
profiles compared to their normal dense and less deformable
products. Glycerol also reduces ultimate firmness after
storage (Barrett et al., 2000). Concerning the gpd1 strain,
it would be possible to experiment with a new deletion
mutant. Other important genes responsible for glycerol
production and transport or the redox balance could be
deleted as a negative control to prove the necessity of
glycerol for fermentation. However a zero glycerol
production would affect cell growth and would not be useful
in a fermentation experiment. Also an analysis of the
transcriptome would be helpful to understand which
pathways are responsible for the specific behaviour of the
OE-GPD1 near the end of the experiment or the low
fermentation rate of the gpd1. Additionally, our
experiment can be compared to experiments done in
different sugar contents. An 18% sucrose in dough was used
which refers to a sweet dough. If dough was used with only
6% sucrose, less glycerol would be produced because there
is a lower osmotic stress. Also more yeast cells would be
viable under these conditions (Blomberg and Adler, 1989).
This could be beneficial for the fermentation. Indeed, more
cells can perform fermentation and it starts earlier because
the period for adapting to the high osmotic medium would
be shorter in these gentler conditions. Also, in an 6% sugar

Group 41 Glycerol in Bread Dough Fermentation 7
medium, the deletion mutant would act as the wild-type
strain does in this experiment. However, fermentation and
CO
2
production could maybe not happen in such high
numbers, affecting the quality of the dough and its volume.
5. Conclusion
At the end of this study, two assumptions can be made.
First, glycerol production is a necessity for the bread dough
fermentation process. Secondly, more glycerol production is
beneficial in case of short term fermentation of sweet bread
dough.

For the deletion mutant (gpd1), a long lag phase
contributes to a late induction of fermentation, shown by the
slow increasing CO
2
production rate at the end of the
experiment. The deletion of the GPD1 gene leads to a lack
of glycerol as an osmolyte and as a redox balancer. The
osmotic equilibrium is not reached and there is an
accumulation of NADH. In this mutant, only the GPD2
gene contributes to the glycerol production in a very
inefficient way and is responsible for the delay of
fermentation. The overexpressed mutant (OE-GPD1) shows
a much higher glycerol production compared to the wild-
type. For this strain, the lag phase is very short. Agai n,
fermentation only starts when sufficient glycerol is present
in the cytosol. Thanks to the overexpression of the GPD1
gene, more glycerol is produced at a faster rate. Looking at
the gpd1, it can be concluded that a lack of glycerol is
linked to a low fermentation capacity. The assumption is
made that glycerol is a necessity.

Although the first statement shows the necessity of glycerol
production to some extend in bread dough fermentation, we
may question if an overproduction of glycerol is beneficial or
detrimental for the bread dough fermentation process. The
results show that in short term fermentation of three hours in
sweet bread dough with an 18% sugar content, an
overproduction of glycerol is beneficial. The CO
2
production
rate rises faster for the OE-GPD1 than the wild-type,
contributing to a logarithmic curve instead of a linear for the
total CO
2
production during fermentation. Both higher glycerol
and CO
2
levels will contribute to a better finished bread
product.
6. Acknowledgements
We would like to thank Elham Aslankoohi in first place, for
supporting us in our research. Her knowledge and
experience of the field has helped us stay on track during
this research project. Also we are grateful for Prof. K.
Verstreepen and his research group, including Prof. C.
Courtin and Mohammad Naser Rezaei, who assisted us
during our experiments in the lab. We would like to thank
the Faculty of Bioscience Engineering, particularly
Christine Peeters, coordinator, for giving us the opportunity
to experience a first research project and letting us have a
look into the world of academical research.



Group 41 Glycerol in Bread Dough Fermentation 8
7. References
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Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation. Appl. Environ. Microbiol. 79, 73257333.
doi:10.1128/AEM.02649-13
2. Barrett, A.H., Cardello, A.V., Mair, L., Maguire, P., Lesher, L.L., Richardson, M., Briggs, J., Taub, I.A., 2000. Textural optimization of shelf-
stable bread: Effects of glycerol content and dough-forming technique. Cereal Chem. 77, 169176.
3. Blomberg, A., Adler, L., 1989. Roles of glycerol and glycerol-3-phosphate dehydrogenase (NAD+) in acquired osmotolerance of
Saccharomyces cerevisiae. J. Bacteriol. 171, 10871092.
4. Blomberg, A., Adler, L., 1992. Physiology of osmotolerance in fungi. Adv. Microb. Physiol. 33, 145212.
5. Brown, A.D., 1976. Microbial water stress. Bacteriol. Rev. 40, 803846.
6. Hohmann, S., Krantz, M., Nordlander, B., 2007. Yeast Osmoregulation.
7. Jain, V.K., Divol, B., Prior, B.A., Bauer, F.F., 2011. Elimination of glycerol and replacement with alternative products in ethanol fermentation
by Saccharomyces cerevisiae. J. Ind. Microbiol. Biotechnol. 38, 14271435. doi:10.1007/s10295-010-0928-x
8. Luyten, K., Albertyn, J., Skibbe, W.F., Prior, B.A., Ramos, J., Thevelein, J.M., Hohmann, S., 1995. Fps1, a yeast member of the MIP family of
channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress. EMBO J. 14, 13601371.
9. Michnick, S., Roustan, J.-L., Remize, F., Barre, P., Dequin, S., 1997. Modulation of glycerol and ethanol yields during alcoholic fermentation
in Saccharomyces cerevisiae strains overexpressed or disrupted for GPDI encoding glycerol 3-phosphate dehydrogenase. Yeast 13, 783793.
doi:10.1002/(SICI)1097-0061(199707)13:9<783::AID-YEA128>3.0.CO;2-W
10. Overkamp, K.M., Bakker, B.M., Ktter, P., Luttik, M.A.H., Van Dijken, J.P., Pronk, J.T., 2002. Metabolic engineering of glycerol production
in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 68, 28142821. doi:10.1128/AEM.68.6.2814-2821.2002
11. Rattin, G.E., Faubion, J.M., Walker, C.E., Mense, A.L., 2009. Measuring Yeast CO2 Production with the Risograph. Cereal Foods World. 54,
261-265.
12. Rep, M., Albertyn, J., Thevelein, J.M., Prior, B.A., Hohmann, S., 1999. Different signalling pathways contribute to the control of GPD1 gene
expression by osmotic stress in Saccharomyces cerevisiae. Microbiology 145, 715727.
13. Tams, M.J., Luyten, K., Sutherland, F.C.W., Hernandez, A., Albertyn, J., Valadi, H., Li, H., Prior, B.A., Kilian, S.G., Ramos, J., Gustafsson,
L., Thevelein, J.M., Hohmann, S., 1999. Fps1p controls the accumulation and release of the compatible solute glycerol in yeast
osmoregulation. Mol. Microbiol. 31, 10871104. doi:10.1046/j.1365-2958.1999.01248.x
14. Tanaka, F., Ando, A., Nakamura, T., Takagi, H., Shima, J., 2006. Functional genomic analysis of commercial bakers yeast during initial
stages of model dough-fermentation. Food Microbiol. 23, 717728. doi:10.1016/j.fm.2006.02.003
15. Van Dijken, J.P., Alexander Scheffers, W., 1986. Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol. Rev. 32, 199224.
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doi:10.1016/S0734-9750(01)00060-X








Faculteit Bio-ingenieurswetenschappen, Kasteelpark Arenberg 20, 3001 Heverlee, Belgi
telefoon: +32 (0)16 32 16 19 fax: +32 (0)16 32 19 99 www.biw.kuleuven.be
Effect of yeast glycerol production level on bread
dough fermentation capacity
Neckebroeck B., Nootens S., Samlali K., Vandenkerckhove J.
RISOGRAPH




















Fig. 1 Glycerol ( ), Ethanol (+) end point concentrations in the
bread dough of Saccharomyces cerevisiae S288C, OE-GPD1,
gpd1 and wild-type relative to wild-type. Relative to wild-type, there
is more glycerol for OE-GPD1 and less for gpd1. The ethanol levels for OE-
GPD1 and wild-type arent significantly different but for gpd1 the ethanol level,
like the glycerol level, is definitely lower than wild-type. The changes in genome
lead to different glycerol concentrations.





















Fig. 2 Evolution of glycerol and ethanol concentrations in bread
dough during fermentation of Saccharomyces cerevisiae
S288C, OE-GPD1, gpd1 and wild-type. The levels of ethanol
increase faster for all three strains as it is a product of the primary metabolic
pathway. The glycerol and ethanol levels are higher for OE-GPD1 than for wild-
type, which are higher than the levels for gpd1. For OE-GPD1 this is partly
due to a higher fermentation rate.




















Fig. 3 Evolution of the production rate of CO
2
(mL) per minute of
Saccharomyces cerevisiae S288C, OE-GDP1, wild-type and gpd1. The
strains begin fermentation as soon as the lag phase ends and CO
2
production rate
rises. The shorter lag phase for OE-GPD1 means this strain adepts faster to the high
osmotic stress than the other strains. gpd1 has a long lag phase and is only starting
fermentation near the end of the experiment. Only after this period gpd1 can handle
the osmotic stress.





















Fig. 4 The evolution of the total production of CO2 (mL) of
Saccharomyces cerevisiae S288C, OE-GDP1, wild-type and gpd1. The
slope of OE-GPD1 graph is steeper than the slope of wild-type, which is steeper than
the slope of gpd1. This means OE-GPD1 has the highest fermentation capacity and
gpd1 the lowest. The final rates show that OE-GPD1 in beneficial for a short term
fermentation like bread dough fermentation.

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Conclusion
Glycerol is a necessity for the bread dough fermentation process.
In case of a short fermentation in sweet bread dough, a higher glycerol production is beneficial.
We would like to thank Elham Aslankoohi, Prof. K. Verstrepen, Prof. C. Courtin and Christine Peeters.
Lag phase
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Glycerol,
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levels were
inspected
Abstract
Glycerol production in yeast is known to be a major response to high osmotic stress situations such as in solid state bread dough fermentation. Moreover, it has recently been argued that
GPD1, the Saccharomyces cerevisiae gene encoding glycerol-3-phosphate dehydrogenase, is the rate limiting gene in glycerol metabolism. We investigated the role of GPD1 on
glycerol/ethanol productions and on bread dough fermentation capacity. Our results showed that an overexpression of GPD1, increasing the glycerol production, leads to a faster adaptation to
osmotic stress in dough and a better fermentation capacity. On the other hand, deletion of GPD1 leads to a long lag phase and a delayed fermentation capacity. This demonstrates that proper
response to osmotic stress through glycerol production is important for proper dough fermentation.