Lab 2 The glycolytic pathway – Stoichiometry of glycolysis in yeast

Objectives

- To determine the efficiency of fermentation versus respiration by analyzing ethanol

production and glucose catabolism.
- To examine the effect of two different inhibitors on fermentation.

Introduction

Glucose is the most important energy-producing molecule of organisms. There are three
different glucose metabolism processes which are glycolysis under anaerobic conditions,
complete oxidation under aerobic conditions, and the pentose phosphate pathway. Glycolysis
is the metabolic pathway that converts glucose into pyruvate in cytoplasm, which produces
adenosine triphosphate (ATP). The whole pathway of glycolysis, containing 10 steps of
chemical reactions with each catalyzed by a specific enzyme (Li et al., 2015). Glycolysis is
used by all organisms for energy generation. The final product of glycolysis of yeast is
pyruvate in aerobic condition and ethanol in anaerobic conditions. Pyruvate enters the Krebs
cycle for further energy production (Kumari, 2018).

Yeast are also known as Saccharomyces cerevisia. They can convert sugars to ethanol and
carbon dioxide at both anaerobic and aerobic conditions. It is possible for them to rapidly
switch between respiratory and fermentative sugar metabolism in response to changes in the
availability of oxygen and fermentable sugars. This metabolic flexibility is likely to have
arisen during evolution in environments where there are strong temporal and/or spatial
fluctuations in sugar and oxygen levels (Van den Brink et al., 2008). When oxygen is absent,
acetaldehyde is the final electron acceptor and gets converted into ethanol under purely
fermentative growth. Under aerobic conditions, respiration is possible with oxygen as the
final electron acceptor, but S. cerevisiae still exhibits alcoholic fermentation until the
sugar/glucose is depleted from the medium (Hagman and Piškur, 2015). Metabolic flexibility
of S. cerevisiae became an important characteristic for its multiple industrial applications.

Fermentation process of yeast will also be affected by several factors. These include the
mode of substrate feeding, nutrient supplementation, temperature, osmotic pressure, oxygen,
intracellular ethanol accumulation, and present of inhibitors such as fluoride ions and
bisulphate ions (D'Amore, 1992). Therefore having an optimum conditions are very important
for the successful fermentation process of yeast.
Results

Table 1 Ethanol Standard Curve

Tube no. S1 S2 S3 S4 S5
[ethanol] Mm 0 0.25 0.5 0.75 1.0
Volume of 1mM 0 25 50 75 100
ethanol (μL)
Volume of water 100 75 50 25 0
(μL)
Absorbance at 0.000 0.010 0.027 0.043 0.060
340nm

Table 2 Ethanol Concentrations in Sample

Tube no. 1 2 3 4 5
Absorbance at 0.016 0.026 0.027 0.023 0.020
340nm
[Ethanol] in 0.304 0.467 0.484 0.418 0.369
diluted samples
(mM)
[Ethanol] in 15.20 23.350 24.20 20.90 18.45
incubation
mixtures (mM)
[Ethanol] 0.000 8.15 9.00 5.70 3.25
produced in
tubes 2-5 (mM)

Table 3 Glucose Standard Curve

Tube no. S1 S2 S3 S4 S5 S6

[Glucose] Mm 0 0.2 0.4 0.6 0.8 1.0

 Volume of 0 40 80 120 160 200
1mM glucose
(μL)

Volume of 200 160 120 80 40 0

water (μL)

Absorbance at 0.00 0.145 0.228 0.297 0.403 0.481

510nm

Table 4 Glucose Concentrations in the Sample

Tube no. 1 2 3 4 5

Absorbance at 0.502 0.463 0.358 0.179 0.295

510nm

[Glucose] in 0.978 0.940 0.713 0.328 0.578

diluted samples
(mM)

[Glucose] in 48.90 47.00 35.65 16.40 28.90

incubation
mixtures (mM)

[Glucose] 0.00 4.00 15.35 34.60 22.10

catalized in
tubes 2-5 (mM)

Table 5 Stoichiometry

Tube no. 1 2 3 4 5
Moles ethanol 0 2.03 0.58 0.16 0.15
produced per mole
glucose
catabolized
 Standard Curve of Absorbance at 340nm against Concentration of
Ethanol
0.07
0.06
Absorbance at 340 nM

y = 0.0612x - 0.0026
0.05
0.04
0.03
0.02
0.01
0
-0.01 0 0.2 0.4 0.6 0.8 1 1.2
[Ethanol] mM

Figure 1 A graph of standard curve of absorbance at 340nm against concentration of ethanol.

Standard Curve of Absorbance at 510nm against Concentration of

Glucose
0.6
Absorbance at 510 nm

0.5
y = 0.464x + 0.027
0.4
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2
[Glucose] mM

Figure 2 A graph of standard curve of absorbance at 510nm against concentration of glucose.

Calculations

Table 1

M1V1 = M2V2

M1 = Initial molarity of ethanol M2 = Final molarity of ethanol

V1 = Initial volume of ethanol V2 = Final volume of ethanol

Volume of water = final volume of solution – volume of ethanol

S1

M1 = 1mM M2 = 0mM V2 = 100μL

𝑀2 0
Volume of ethanol, V1 = 𝑀1 × V2 = 1 × 100 = 0μL

Volume of water = 100μL - 0μL = 100μL

S2

M1 = 1mM M2 = 0.25mM V2 = 100μL

𝑀2 0.25
Volume of ethanol, V1 = 𝑀1 × V2 = × 100 = 25μL
1

Volume of water = 100μL - 25μL = 75μL

S3

M1 = 1mM M2 = 0.5mM V2 = 100μL

𝑀2 0.5
Volume of ethanol, V1 = 𝑀1 × V2 = × 100 = 50μL
1

Volume of water = 100μL - 50μL = 50μL

S4

M1 = 1mM M2 = 0.75mM V2 = 100μL

𝑀2 0.75
Volume of ethanol, V1 = 𝑀1 × V2 = × 100 = 75μL
1

Volume of water = 100μL - 75μL = 25μL

S5

M1 = 1mM M2 = 1.0mM V2 = 100μL

𝑀2 1.0
Volume of ethanol, V1 = 𝑀1 × V2 = × 100 = 100μL
1

Volume of water = 100μL - 100μL = 0μL

Table 2

1. Based on the equation y = 0.0612x - 0.0026 shown in ethanol standard curve in Figure 1,
[Ethanol] in diluted samples is calculated by applying y = absorbance at 340nm and
unknown x = [Ethanol] in diluted samples.
2. [Ethanol] in incubation mixtures is calculated by multiplying value of [Ethanol] in diluted
samples with a dilution factor of 50x.
3. [Ethanol] produced in tubes 2-5 is calculated by subtracting the value of [Ethanol] in
incubation mixtures in tubes 2-5 with the control value in tube 1.

Tube 1

1. y = 0.016
0.016 = 0.0612x – 0.0026
x = 0.304mM
2. 0.304mM × 50 = 15.2mM
3. 0.00 mM

Tube 2

1. y = 0.026
0.026 = 0.0612x – 0.0026
x = 0.467mM
2. 0.467mM × 50 = 23.35mM
3. 23.35mM – 15.2mM = 8.15mM

Tube 3

1. y = 0.027
0.027 = 0.0612x – 0.0026
x = 0.484mM
2. 0.484mM × 50 = 24.2mM
3. 24.2mM – 15.2mM = 9mM
Tube 4

1. y = 0.023
0.023 = 0.0612x – 0.0026
x = 0.418mM
2. 0.418mM × 50 = 20.9mM
3. 20.9mM – 15.2mM = 5.7mM

Tube 5

1. y = 0.020
0.020 = 0.0612x – 0.0026
x = 0.369mM
2. 0.369mM × 50 = 18.45mM
3. 18.45mM – 15.2mM = 3.25mM

Table 3

M1V1 = M2V2

M1 = Initial molarity of glucose M2 = Final molarity of glucose

V1 = Initial volume of glucose V2 = Final volume of glucose

Volume of water = final volume of solution – volume of glucose

S1

M1 = 1mM M2 = 0mM V2 = 200μL

𝑀2 0
Volume of glucose, V1 = 𝑀1 × V2 = 1 × 200 = 0μL

Volume of water = 200μL - 0μL = 200μL

S2

M1 = 1mM M2 = 0.2mM V2 = 200μL

𝑀2 0.2
Volume of glucose, V1 = 𝑀1 × V2 = × 200 = 40μL
1

Volume of water = 200μL - 40μL = 160μL

S3

M1 = 1mM M2 = 0.4mM V2 = 200μL

𝑀2 0.4
Volume of glucose, V1 = 𝑀1 × V2 = × 200 = 80μL
1

Volume of water = 200μL - 80μL = 120μL

S4

M1 = 1mM M2 = 0.6mM V2 = 200μL

𝑀2 0.6
Volume of glucose, V1 = 𝑀1 × V2 = × 200 = 120μL
1

Volume of water = 200μL - 120μL = 80μL

S5

M1 = 1mM M2 = 0.8mM V2 = 200μL

𝑀2 0.8
Volume of glucose, V1 = 𝑀1 × V2 = × 200 = 160μL
1

Volume of water = 200μL - 160μL = 40μL

S6

M1 = 1mM M2 = 1.0mM V2 = 200μL

𝑀2 1.0
Volume of glucose, V1 = 𝑀1 × V2 = × 200 = 200μL
1

Volume of water = 200μL - 200μL = 0μL

Table 4

1. Based on the equation y = 0.464x + 0.027 shown in glucose standard curve in Figure 2,
[Glucose] in diluted samples is calculated by applying y = absorbance at 510nm and
unknown x = [Glucose] in diluted samples.
2. [Glucose] in incubation mixtures is calculated by multiplying value of [Glucose] in diluted
samples with a dilution factor of 50x.
3. [Glucose] catalized in tubes 2-5 is calculated by subtracting the control value in tube 1 with
the values of [Glucose] in incubation mixtures in tubes 2-5.

Tube 1

1. y = 0.502
0.502 = 0.464x + 0.027
x = 1.02mM
2. 1.02mM × 50 = 51.0mM
3. 0.00mM

Tube 2

1. y = 0.463
0.463 = 0.464x + 0.027
x = 0.940mM
2. 0.940mM × 50 = 47.0mM
3. 51.0mM – 47.0mM = 4.0mM

Tube 3

1. y = 0.358
0.358 = 0.464x + 0.027
x = 0.713mM
2. 0.713mM × 50 = 35.65mM
3. 51.0mM – 35.65mM = 15.35mM

Tube 4

1. y = 0.179
0.179 = 0.464x + 0.027
x = 0.328mM
2. 0.328mM × 50 = 16.4mM
3. 51.0mM – 16.4mM = 34.6mM
Tube 5

1. y = 0.295
0.295 = 0.464x + 0.027
x = 0.578mM
2. 0.578mM × 50 = 28.9mM
3. 51.0mM – 28.9mM = 22.1mM

Table 5

Calculate the ratio of moles of ethanol produced to moles of glucose consumed for tubes 2-5.

Tube 1

0.00
Moles ethanol produced per mole glucose catabolized = 0.00 = 0

Tube 2

8.15
Moles ethanol produced per mole glucose catabolized = 4.00 = 2.03

Tube 3

9.00
Moles ethanol produced per mole glucose catabolized = 15.35 = 0.58

Tube 4

5.7
Moles ethanol produced per mole glucose catabolized = 34.60 = 0.16

Tube 5

3.25
Moles ethanol produced per mole glucose catabolized = 22.10 = 0.15
Discussion
The ability of baker's yeast also known as Saccharomyces cerevisiae can rapidly increase
its glycolytic flux upon a switch from respiratory to fermentative sugar metabolism. The
Baker's yeast can rapidly switch between respiratory and fermentative sugar metabolism in
response to changes in the availability of oxygen and fermentable sugars (Van den Brink et al.,
2008). In this experiment, Saccharomyces cerevisiae yeast is aerobically adapted in order to
monitor the efficiency of fermentation versus respiration by analysing ethanol production and
glucose catabolism under different conditions, and also to investigate the effects of two
inhibitors, fluoride ions and bisulphite ions, on rate of fermentation on glucose. There are five
test tubes containing glucose suspension, potassium phosphate and yeast tested with different
variables. Tube 1 acts as control as it is directly kept in ice after yeast is added, tube 2 is used
to study respiration and ethanol production as oxygen is added into the suspension by oxygen
pump, tube 3 is used to study fermentation and ethanol production as it is equipped with
optimum condition for fermentation; and tube 4 and tube 5 are added with inhibitors which are
sodium fluoride and sodium bisulphite respectively. Tube 1 is used as control as the
Saccharomyces cerevisiae, the yeast responsible for fermentation used in this experiment has
the optimum growth temperature is 35°C, so fermentation would not occur in this tube as it is
placed in ice and does not favour fermentation.

Table 2 shows the concentration of ethanol produced in the sample. Concentration of

ethanol produced in each tube is analysed by measuring the absorbance with spectroscopy
method at a wavelength of 340 nm. Then, a graph of standard curve of absorbance at 340nm
against concentration of ethanol was plotted then concentration of ethanol in diluted sample in
the incubated mixture was determined by multiplying the concentration obtained from the
linear equation from the plotted standard curve, as shown in Figure 1 above, with dilution factor
of 50. The concentration of ethanol catalysed in each tube is determined by subtracting the
concentration obtained in incubated mixtures from Tube 2 to 4 with the control, Tube 1. From
the results shown above, the concentration of ethanol produced in each tube is the highest in
Tube 3, with a value of 9.0mM as it is provided with optimum condition for fermentation to
occur, followed by Tube 2, at 8.15mM. The result which showed that Tube 3 contains higher
ethanol concentration is due to Pasteur effect, which is an inhibiting effect of oxygen on
the fermentation process has occurred in Tube 2. Pasteur effect occurs when oxygen is added
into an anaerobic respiration process, which shifts to aerobic respiration due to the tendency of
the cells to undergo a process which are easier to obtain energy (Mathews, 2001). The presence
of oxygen suppresses the rate of glycolysis in normal cells because most of the ATP is provided
by oxidative phosphorylation. So as Tube 2 is supplied with oxygen after addition yeast, the
reaction happening in the tube is aerobic respiration, utilizing less glucose, and did not yield
ethanol other than producing water, carbon dioxide and adenosine triphosphate (ATP) as its
product. However, the results show that ethanol is still produced in tube 2. This can be
explained by the possibility that the oxygen supplied to the tube is insufficient, causing
fermentation which occurs at anaerobic condition will still occur. Tube 4 and 5 produced the
least ethanol, at the concentration of 5.70mM and 3.25mM respectively, which is due to the
presence of inhibitors in both tube. Tube 4 and 5 are added with two different inhibitors, sodium
fluoride and sodium bisulphite with different inhibition strength. Fluoride ions is known to be
able to inhibit alcoholic fermentation and glycolysis by inhibiting the enolase activity which
functions in glycolytic pathway as by catalysing the reversible dehydration of 2-phospho-D-
glycerate to yield phosphoenolpyruvate (Qin et al., 2006). The other inhibitor, Sodium
bisulphite will combine with acetaldehyde to prevent alcoholic fermentation by giving a
bisulfite addition compound that is not a substrate for alcohol dehydrogenase (Voet et al., 2016).
Tube 1 do not produce ethanol as fermentation by using S. cerevisiae because it cannot be
commenced in such a low temperature in ice.

Table 3 shows the absorbance reading of six different glucose concentration at 510nm that
had been diluted with water. S6 has the highest absorbance with value of 0.481nm and followed
by S5 with value of 0.403, S4 with value of 0.297, S3 with value of 0.228, S2 with value of
0.145 and S,0 which acts as a control. These values indicates that the higher the amount of
glucose, the higher the absorbance value.

Table 4 shows the concentration of glucose catabolism in the sample. The process of
measuring the concentration of catabolized glucose in each tube was similar as the process of
measuring the concentration of ethanol produced above. However, in measurement of glucose
catabolism, the spectroscopy method used was at a wavelength of 510 nm and the concentration
of glucose catalysed in each tube was determined by subtracting the concentration obtained in
control, Tube 1 with incubated mixtures, Tubes 2 to 4. Based on our result, tube 4 had the
highest concentration followed by tube 3,5 and 2. However, tube 3 supposed to have the
highest concentration and followed by tube 4, tube 5, and tube 2. Tube 3, 4 and 5 had higher
concentration as compared to tube 2, which means fermentation is much less efficient than
cellular respiration in producing energy and this resulted in greater amount of glucose needed
for oxidation. Our group results may have some error due to handling method when measuring
absorbance so it might be fingerprint on the cuvette that cause the results to be inaccurate. In
fermentation, one molecule of glucose only produces 2 ATPs, whereby in the presence of
oxygen, up to 38 ATPs is produced. Besides, fermentation consumes up to 20 times more
glucose per second than cellular respiration (Solomon et al. 2005). Furthermore, tube 4 and 5
had result in lower concentration due to the presence of inhibitors, which were sodium fluoride
in tube 4 and sodium bisulphite in tube 5. These inhibitor had different inhibition strength, in
which tube 5 resulted in lower glucose catabolism than tube 4 (sodium fluoride).
Table 5 shows the correlation of glucose catabolism and ethanol production which is done by
calculating the moles ethanol produced per mole glucose catabolized. So tube 3 again must
show the highest value of ethanol produced per glucose catabolized, followed by Tube 2, Tube
5 and lastly, Tube 4. Tube 3 yielded the highest ethanol per moles of glucose catabolized
because the fermentation happen under a favourable condition of no inhibitors added, under an
optimum temperature for fermentation using S. cerevisiae, thus allowing glucose to be used in
fermentation. On the other hand, tube 2 which undergoes aerobic respiration utilizes less
glucose compared to anaerobic fermentation. Tube 4 and 5 has the lowest moles of ethanol per
moles of glucose catabolized as the fluoride ion and bisulphite ion inhibitors inhibited the
ethanol yield, though some glucose has been catabolized.
In addition, there are some precaution for this experiment. The micropipette must be used
correctly in order to have an accurate result. The micropipette tips must always be change when
using different solutions. The micropipette cannot be laid down when it has liquid in it as it can
get the liquid into the micropipette itself and might contaminate the other samples that need to
be taken by the micropipette. Next, make sure the cuvette are cleaned and placed in the proper
position to get valid absorbance reading and also avoid staining of fingerprint.

Question

1. Describe the Pasteur Effect. Do your results demonstrate the Pasteur Effect?

Pasteur effect can be described as the slowing of glycolysis in the presence of oxygen
or described as an inhibiting effect of oxygen on the fermentation process (Krebs, 1972).
Based on our result, Tube 3 undergoes fermentation process and produces more ethanol
compared to Tube 2 which undergoes aerobic respiration process. It means that more
 glucose breakdown without presence of oxygen, therefore it does demonstrate Pasteur
Effect.

2. Explain the mode of action of fluoride and bisulphate on the fermentation pathway.
Predict their likely effect if the yeast treated with these agents had been respiring.

Sodium fluoride and sodium bisulphite are added into Tube 4 and Tube 5 respectively,
both of them inhibit the fermentation pathway. Based on result, bisulphite is a more
powerful inhibitor as compared to fluoride, this is because Tube 4 with fluoride
inhibitor is capable to metabolize higher amount of glucose compare to Tube 5 with
bisulphite inhibitor.

3. What is the predicted yield of ethanol per mole of glucose for each tube? Suggest
plausible reasons for each tube, why the actual yield of moles of ethanol per mole of
glucose is different from that predicted?

The predicted yield of ethanol per mole of glucose is calculated based on stoichiometry
equation. The actual fermentation process will convert one mole of glucose to two
moles of ethanol. Table below shows the predicted yield and actual yield of ethanol per
mole of glucose of each tube.

Yield of Tube
ethanol/mole
1 2 3 4 5
of glucose
Predicted 0 0 2 0.5 1.5
yield
Actual yield 0 2.03 0.58 0.16 0.15

According to table above, it has significant difference between predicted yield and
actual yield. Tube 1 shows no deviation as it serves as a control and does not produce
any ethanol. No ethanol should be produced in Tube 2 as expected because respiration
process undergoes aerobic respiration completely. However, Tube 2 still produced a
 quantity of ethanol mainly because there is insufficient oxygen supply into tube, hence
some yeast cells carry out fermentation and ethanol are formed. For Tube 3, 2 moles of
ethanol are predicted to be produced based on stoichiometry. Actual yield only
produces less than one mole of ethanol mainly because the reaction is not so efficient.
For Tube 4 and 5, there are expected to have a lower yield of ethanol concentration
compare to Tube 3. This is because inhibitors such as sodium fluoride and sodium
bisulphite have been added to Tube 4 and 5 to lower down the production of ethanol.
The actual yield of ethanol per mole of glucose may not be as accurate as predicted due
to some factors. Contaminated cuvette gives inaccurate reading of absorbance in
spectrophotometer during experiment. Inaccurate reading of absorbance causes
elevated in final results.

4. Why is the TCA (tricloroacetic acid) precipitation step, from the previous lab, not
needed this time?

Trichloroacetic acid (TCA) precipitation of proteins is commonly used to concentrate

protein samples or remove contaminants (Koontz, 2014). In this experiment, the TCA
precipitation step is not involved or applied on samples because samples used were
already diluted and this experiment does not need high protein concentration for further
steps.

Conclusion

In conclusion, the objective of this experiment is successfully achieved. Fermentation is more

efficient in producing ethanol compared to cellular respiration. More ethanol will be produced
per one molecule of glucose in this two case. From this experiment, it can be observed that the
two inhibitors added in this experiment which are sodium bisulfide and sodium fluoride lags
the fermentation reaction.
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