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.

13th 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 overexpress ion 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 CO2 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 CO2 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 CO2 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 (Δgpd1) 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 30°C

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 30°C, 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 CO2 production. The Risograph was set at 30°C

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 µm filter to

avoid remaining yeast cells, and collected in three 1.5 mL

Eppendorf tubes. The tubes were kept at -20°C for further

analysis. After defrosting the 1.5mL Eppendorf tubes, about

1mL was taken and filtered again with a 0.22 µm 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 (Δgpd1) 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 Δgpd1,

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 Δgpd1

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 CO2. 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, Δgpd1 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 Δgpd1 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 aren’t as significant as for glycerol.

Nevertheless, the data show that after three hours, more

glucose is fermented into ethanol and CO2 by OE-GPD1.

Furthermore, Δgpd1 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 Δgpd1.

For the glycerol concentrations, four measurements were

taken at 0, 60, 120 and 180 minutes for the wild-type,

Δgpd1 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 Δgpd1 yeast cells. This graph also grounds the statement

again that Δgpd1 and OE-GPD1 produce respectively less

and more glycerol compared to the wild-type yeast.

0

1

2

3

Wild-type OE-GPD1 Δ gpd1

Rat

io o

f co

nce

ntr

atio

ns

rela

tive

to

w

ild t

ype

at 1

80

min

[-]

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Wild-type OE-GPD1 Δ gpd1

Co

nce

ntr

atio

n [

mm

ole

/ g

yeas

t]

Fig. 1 Intracellular glycerol concentrations and standard deviation of Saccharomyces cerevisiae S288C for OE-GPD1, Δgpd1 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

Δgpd1 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 Δgpd1 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 CO2 with Risograph analysis

We measured CO2 production of our three strains during

high sugar bread dough fermentation, as a proxy for

fermentation rate and capacity. For the wild-type, Δgpd1,

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 CO2 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 Δgpd1 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, CO2 production

rate starts to rise almost linear after its lag phase, with an

average increase in production rate of 0.1 mL CO2/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 Δgpd1,

there is no fermentation in the beginning. The Δgpd1 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 Δgpd1 starts fermenting

glucose. OE-GPD1 does not only start its fermentation

earlier, it also has a stronger slope. The CO2 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 CO2 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 Δgpd1 graph shows again that there is no

significant CO2 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.

0

1

2

3

4

5

6

7

8

9

10

11

0 60 120 180

Co

nce

ntr

atio

n g

lyce

rol [

mm

ole

/g y

east

]

Time [min]

Fig. 3 The concentration of glycerol in the bread dough during the

fermentation process of Saccharomyces cerevisiae S288C for OE-

GPD1 (■), Δgpd1 (×) and wild-type (▲). Glycerol levels rise faster and

are also higher for the OE-GPD1 as for the wild-type.

0

5

10

15

20

25

0 60 120 180Co

nce

ntr

atio

n e

than

ol [

mm

ole

/g y

east

]

Time [min]

Fig. 4 The concentration of ethanol in the bread dough during the

fermentation process of Saccharomyces cerevisiae S288C, OE-GPD1

(■),Δgpd1 (×) 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 (Δgpd1), 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

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 60 120 180

Rat

e [m

L C

O2/

min

]

Time [min]

A

B

C

Fig. 5 The evolution of the production rate of CO2 (mL) per minute of Saccharomyces cerevisiae S288C, OE-

GDP1 (A), wild-type (B) and Δgpd1 (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, Δgpd1 starts to show a fermentation behaviour near the end.

0

10

20

30

40

50

60

70

80

90

0 60 120 180

Tota

l pro

du

ctio

n C

O2 [

mL]

Time [min]

A

B

C

Fig. 6 The evolution of the total production of CO2 (mL) of Saccharomyces cerevisiae S288C, OE-GPD1 (A), wild-type (B) and Δgpd1 (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); (Tamás 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 CO2

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 Δgpd1 mutant has

a much longer lag phase relative to the wild-type. Only after

two hours, the CO2 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 Δgpd1

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 Δgpd1 mutant. After this point, all three

curves showing the evolution of total CO2 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 Δgpd1

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 Δgpd1. The OE-GPD1

produces more CO2 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 CO2 can also be a by-product of the TCA

cycle. Looking at the Δgpd1 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

CO2 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 CO2 production rate would be to use a Δgpd1 mutant in

bread dough with a low osmolarity level. Concerning the

Δgpd1 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 CO2 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 CO2 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 Δgpd1 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 Δgpd1. 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

CO2 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 (Δgpd1), a long lag phase

contributes to a late induction of fermentation, shown by the

slow increasing CO2 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. Again,

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 Δgpd1, 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 CO2 production

rate rises faster for the OE-GPD1 than the wild-type,

contributing to a logarithmic curve instead of a linear for the

total CO2 production during fermentation. Both higher glycerol

and CO2 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

1. Aslankoohi, E., Zhu, B., Rezaei, M.N., Voordeckers, K., Maeyer, D.D., Marchal, K., Dornez, E., Courtin, C.M., Verstrepen, K.J., 2013.

Dynamics of the Saccharomyces cerevisiae transcriptome during bread dough fermentation. Appl. Environ. Microbiol. 79, 7325–7333.

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, 169–176.

3. Blomberg, A., Adler, L., 1989. Roles of glycerol and glycerol-3-phosphate dehydrogenase (NAD+) in acquired osmotolerance of Saccharomyces cerevisiae. J. Bacteriol. 171, 1087–1092.

4. Blomberg, A., Adler, L., 1992. Physiology of osmotolerance in fungi. Adv. Microb. Physiol. 33, 145–212.

5. Brown, A.D., 1976. Microbial water stress. Bacteriol. Rev. 40, 803–846. 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, 1427–1435. 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, 1360–1371.

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, 783–793.

doi:10.1002/(SICI)1097-0061(199707)13:9<783::AID-YEA128>3.0.CO;2-W

10. Overkamp, K.M., Bakker, B.M., Kötter, 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, 2814–2821. 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, 715–727.

13. Tamás, 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, 1087–1104. 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 baker’s yeast during initial stages of model dough-fermentation. Food Microbiol. 23, 717–728. 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, 199–224.

16. Wang, Z., Zhuge, J., Fang, H., Prior, B.A., 2001. Glycerol production by microbial fermentation: A review. Biotechnol. Adv. 19, 201–223. doi:10.1016/S0734-9750(01)00060-X

Faculteit Bio-ingenieurswetenschappen, Kasteelpark Arenberg 20, 3001 Heverlee, België

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,

Δgpd1 and wild-type relative to wild-type. Relative to wild-type, there

is more glycerol for OE-GPD1 and less for Δgpd1. The ethanol levels for OE-

GPD1 and wild-type aren’t significantly different but for Δgpd1 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, Δgpd1 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 Δgpd1. For OE-GPD1 this is partly

due to a higher fermentation rate.

Fig. 3 Evolution of the production rate of CO2 (mL) per minute of

Saccharomyces cerevisiae S288C, OE-GDP1, wild-type and Δgpd1. The

strains begin fermentation as soon as the lag phase ends and CO2 production rate

rises. The shorter lag phase for OE-GPD1 means this strain adepts faster to the high

osmotic stress than the other strains. Δgpd1 has a long lag phase and is only starting

fermentation near the end of the experiment. Only after this period Δgpd1 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 Δgpd1. The

slope of OE-GPD1 graph is steeper than the slope of wild-type, which is steeper than

the slope of Δgpd1. This means OE-GPD1 has the highest fermentation capacity and

Δgpd1 the lowest. The final rates show that OE-GPD1 in beneficial for a short term

fermentation like bread dough fermentation.

0

0,1

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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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Wild-type OE-GPD1 Δ gpd1

Rat

io o

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t 1

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OE-GPD1

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OE-GPD1

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Δgpd1

Glycerol,

EtOH and CO2

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.