Experiment 1: The glycolytic pathway – Fermentation by a yeast suspension

Objective

1. To determine the rate of yeast fermentation of different sugars via production of carbon
dioxide and concentration of ethanol produced.
2. To investigate alcohol fermentation in yeast under different conditions.

Introduction

Cells obtain the energy stored in sugars by breaking the molecules apart in a series of enzyme-
mediated reactions. Energy is generated most efficiently in the presence of oxygen via the
process aerobic respiration. Under condition where oxygen is absent, some cells are still able
to break down glucose via the process of anaerobic respiration which is known as fermentation.
To produce ATP from glucose, whether by fermentation or cellular respiration, cells must first
partially broken down by glycolysis and yeast synthesizes ATP through both of this major
biochemical pathways (Jones, 2006). During both respiration and fermentation, yeast cells
break down glucose molecules within the cell to release energy by a process
called glycolysis, and some of this energy is captured and stored in the ATP’s high-energy
phosphate bonds. The breakdown of glucose also releases carbon atoms, which become
available for biosynthetic reactions, enabling the yeast to grow and reproduce by budding. The
rest of the carbon ends up in the by-products of these reactions, such as carbon dioxide, ethanol,
and other more minor compounds. Other fermentable sugars, such as maltose, are first
converted to glucose before entering these metabolic pathways (Aquilla, 2019).

Glycolysis requires eleven enzymes which degrade glucose to lactic acid. Alcoholic
fermentation follows the same enzymatic pathway for the first ten steps. The last enzyme of
glycolysis, lactate dehydrogenase, is replaced by two enzymes in alcoholic fermentation. These
two enzymes, pyruvate decarboxylase and alcoholic dehydrogenase, convert pyruvic acid into
carbon dioxide and ethanol in alcoholic fermentation (Morton, 2019). Through the glycolysis
process, glucose or some other carbohydrates are converted through a series of enzymatic
reactions into pyruvate. In cellular respiration, two hydrogen atoms are removed from glucose
and transferred to a coenzyme called nicotinamide adenine dinucleotide (NAD+), reducing this
compound to NADH. The transfer of electrons from NADH to the electron transport chain
releases energy, and this energy can be used to synthesize ATP (Jones, 2006).

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 Under anaerobic condition, there are two common types of fermentation which are alcoholic
fermentation and lactic acid fermentation. Animal, including human being, certain fungi, and
some bacteria convert pyruvate produced in glycolysis to lactate or lactic acid. Plants and some
fungi, yeast in particular, convert pyruvate to ethanol and carbon dioxide. Yeast cells obtain
energy under anaerobic condition by the process of alcoholic fermentation. Alcoholic
fermentation begins with glycolysis, a series of reactions breaking glucose into two molecules
of pyruvate with a net yield of 2 ATP and 2 NADH molecules. In anaerobic environment, the
pyruvate is converted to ethanol and carbon dioxide. In this process, the 2 NADH molecules
are oxidized, replenishing the NAD+ used in glycolysis. Cellular respiration is much more
efficient than fermentation in producing ATP whereby Cellular respiration can produce a
maximum of 38 ATP molecules, while fermentation produces only 2 ATP molecules
(Heckman, 2019).

Results
Table 1: CO2 Evolution

Time of Tube no.

incubation 1 2 3 4 5
0’ - - - - -
15’ + ++ + ++ +
30’ + +++ + +++ +
45’ ++++ +++ ++ +++ ++
60’ ++++ ++++ ++ ++++ ++

0: No air bubble + +: Less air bubbles + + + +: Most air bubbles

+: Least air bubbles + + +: More air bubbles

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 Table 2: Ethanol standard curve

Tube no. S1 S2 S3 S4 S5
[ethanol] mM 0 0.25 0.5 0.75 1.0
Volume of 1mM ethanol (L) 0 25 50 75 100
Volume of water (L) 100 75 50 25 0
Absorbance at 340nm 0 0.073 0.151 0.216 0.279

Table 3: Ethanol concentration in samples

[Ethanol] in
Time of [Ethanol] [Ethanol]
Tube Absorbance protein free
Sampling allowing for produced
no. at 340nm samples
(min) dilution (mM) (mM)
(mM)
15 0.894 3.175 98.425 90.21
1
30 0.896 3.183 98.673 90.458
15 0.561 1.988 61.628 53.413
2
30 0.578 2.049 63.519 55.304
15 0.152 0.529 16.399 8.184
3
30 0.176 0.615 19.065 10.85
15 0.711 2.523 78.213 69.998
4
30 0.784 2.783 86.273 78.058
0 0.078 0.265 8.215 0.000
5 15 0.116 0.401 12.431 4.216
30 0.119 0.412 12.772 4.557

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 Figure 1: A graph of Absorbance at 340 nm against Concentration of
ethanol (mM)

Calculation

Based on Table 2, formula M1V1 = M2V2 is used to find the volume of water needed to prepare
the different concentration of ethanol solution. M1 and V1 represents the concentration (mM)
and volume of ethanol respectively. M2 and V2 represents the concentration (mM) and volume
of water respectively. To find the volume of water, the equation V2 = (M1/M2) x 100 μL.

Based on the equation y = 0.2804x + 0.0036 shown in ethanol standard curve, [Ethanol] in
free protein samples is calculated by applying y = absorbance at 340nm which is shown in the
Table 2 and unknown x = [Ethanol] in free protein samples. [Ethanol] allowing for dilution is
calculated by multiplying value of [Ethanol] in free protein samples with a dilution factor of
31x.

100 𝜇𝐿 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
Dilution factor =
100 𝜇𝐿 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒+2𝑚𝐿 𝑇𝐶𝐴+1𝑚𝐿 𝑁𝑎𝑂𝐻

0.1𝑚𝐿
=
3.1𝑚𝐿
= 31x
[Ethanol] produced is calculated by:

[Ethanol] allowing for dilution at particular time – [Ethanol] allowing for dilution at Zero time

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Tube 1

 15 mins, absorbance = 0.894nm

1) y = 0.2804x + 0.0036

0.894= 0.2804x + 0.0036

x = 3.175mM

2) Ethanol allowing for dilution(mM) = 3.175 x 31 = 98.425mM

3) [Ethanol] produced (mM) = 98.425 – 8.215 = 90.21mM

 30 mins, absorbance = 0.896nm

1) y = 0.2804x + 0.0036

0.896 =0.2804x + 0.0036

x = 3.183mM

2) Ethanol allowing for dilution(mM) = 3.183 x 31 = 98.673mM

3) [Ethanol] produced (mM) = 98.673 – 8.215 = 90.458mM

Tube 2

 15 mins, absorbance =0.561nm

1) y = 0.2804x + 0.0036

0.561= 0.2804x + 0.0036

x =1.988mM

2) Ethanol allowing for dilution(mM) = 1.988 x 31 = 61.628mM

3) [Ethanol] produced (mM) = 61.628 – 8.215 = 53.413mM

 30 mins, absorbance = 0.578

1) y = 0.2804x + 0.0036

0.578 = 0.2804x + 0.0036

x = 2.049mM

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2) Ethanol allowing for dilution = 2.049 x 31 = 63.519mM

3) [Ethanol] produced (mM) = 63.519 – 8.215 = 55.304 mM

Tube 3

 15 mins, when absorbance, y =0.152nm

1) y = 0.2804x + 0.0036

0.152= 0.2804x + 0.0036

x = 0.529mM

2) Ethanol allowing for dilution(mM) = 0.529 x 31 = 16.399mM

3) [Ethanol] produced (mM) = 16.399 – 8.215 = 8.184 mM

 30 mins when absorbance, y =0.176nm

1) y = 0.2804x + 0.0036

0.176 = 0.2804x + 0.0036

x = 0.615mM

2) Ethanol allowing for dilution(mM) = 0.615 x 31 = 19.065mM

3) [Ethanol] produced (mM) = 19.065 – 8.215 = 10.85 mM

Tube 4

 15 mins, when absorbance, y =0.711nm

1) y = 0.2804x + 0.0036

0.711 = 0.2804x + 0.0036

x = 2.523mM

2) Ethanol allowing for dilution(mM) = 2.523 x 31 = 78.213mM

3) [Ethanol] produced (mM) = 78.213 – 8.215 = 69.998mM

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 30 mins when absorbance, y = 0.784nm
1) y = 0.2804x + 0.0036

0.784 = 0.2804x + 0.0036

x = 2.783mM

2) Ethanol allowing for dilution(mM) = 2.783 x 31 = 86.273mM

3) [Ethanol] produced (mM) = 86.273 – 8.215 = 78.058mM

Tube 5

 0 mins (zero time), when absorbance, y =0.078nm

1) y = 0.2804x + 0.0036

0.078 = 0.2804x + 0.0036

x =0.265mM

2) Ethanol allowing for dilution(mM) = 0.265 x 31 = 8.215mM (control reading)

 15 mins, when absorbance, y =0.116nm

1) y = 0.2804x + 0.0036

0.116 = 0.2804x + 0.0036

x = 0.401 mM

2) Ethanol allowing for dilution(mM) = 0.401 x 31 = 12.431mM

3) [Ethanol] produced (mM) = 12.431 – 8.215 = 4.216 mM

 30 mins when absorbance, y =0.119nm

1) y = 0.2804x + 0.0036

0.119 = 0.2804x + 0.0036

x = 0.412mM

2) Ethanol allowing for dilution(mM) = 0.412 x 31 = 12.772mM

3) [Ethanol] produced (mM) = 12.772 – 8.215 = 4.557 mM

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Discussion

Yeast undergoes fermentation and produces ethanol and carbon dioxide. In this experiment,
we are able to study the effectiveness of different sugars such as glucose, fructose, lactose and
sucrose in carbon dioxide evolution and alcoholic fermentation. First, there are 5 tubes,
labelling as 1, 2, 3, 4 and 5. Tube 1 represents glucose, tube 2 represents fructose, tube 3
represents lactose, tube 4 represents sucrose and tube 5 represents water which acts as a control.

Table 1 shows that the results of carbon dioxide evolution in different incubation tubes. Yeast
induces a rapid conversion of sugars to ethanol and carbon dioxide at both anaerobic and
aerobic conditions (Haqman. and Piškur, 2015). The carbon dioxide produced is trapped in the
Durham tube. The longer the time for yeast fermentation, the more the carbon dioxide produced.
Therefore, the longer the time of incubation, the more the gas bubbles trapped in the Durham
tube. Different sugars will release different amount of carbon dioxide.

Based on the results in table 1, at 0’ minute, there is no air bubbles have been trapped in the
Durham tube in every single incubation tubes. This indicates that there is no yeast fermentation
process at this time. Therefore, carbon dioxide is not produced. At 15’ minutes, air bubbles are
present in the Durham tube in all tubes. It is observed that tube 1 and tube 4 which are glucose
and sucrose respectively have higher amount of carbon dioxide produced compared to tube 2
and 3 which are fructose and lactose. There is little gas bubbles trapped in tube 5. At 30’
minutes, tube 1 and 4 have higher amount of carbon dioxide than tube 2 and 3. Little air bubble
is observed and trapped in Durham tube in tube 5. At 45’ minutes, it is observed that glucose
in tube 1 has reached the maximum amount of carbon dioxide released. Fructose in tube 2 and
sucrose in tube 4 have the same amount of carbon dioxide released at this time. Meanwhile,
lactose in tube 3 produce a small amount of carbon dioxide. Little air bubble is trapped in
Durham tube in tube 5. At 60’ minutes, glucose in tube 1, fructose in tube 2 and sucrose in tube
4 have reached the maximum amount of carbon dioxide released. Lactose produce smaller
amount of carbon dioxide if compared to other sugars. Water in tube 5, acts as a control, little
air bubble is produced.

Table 2 shows that the absorbance value at 340nm increases as the concentration of ethanol
increases. Figure 1 shows the standard curve of absorbance at 340nm against the ethanol
concentration. With the equation given from the standard curve which is y = 0.2804x + 0.0036,
the absorbance values at 340nm of each incubation tube at different incubation time can be

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calculated. With these values, ethanol concentration produced from yeast fermentation can be
obtained.

Based on table 3, we could conclude that as the longer the time of yeast fermentation, the
higher the concentration of ethanol produced. Glucose in tube 1 has the highest concentration
of ethanol produced, followed by sucrose in tube 4, fructose in tube 2, lactose in tube 3 and
water in tube 5.

Glucose is simple sugar which has higher rate of carbon dioxide and ethanol production than
complex sugars. Hence, glucose can be directly used in glycolysis reaction. Glucose will
ferment the most as less energy is required to break down into pyruvate and
acetaldehyde. Sucrose which is disaccharide would ferment more slowly because it would first
undergo hydrolysis. Before fermentation starts, sucrose has to be hydrolysed into glucose and
fructose first with the enzyme invertase. This is the enzyme found in yeast. Given that glucose
and fructose have same molecular formula and both of them are monosaccharides. In fact,
almost all the monosaccharides can produce carbon dioxide and ethanol. Also, when 6-Carbon
sugar attached to a 5-Carbon sugar, it would limit production of carbon dioxide and ethanol.
Therefore, sucrose produce lower amount of carbon dioxide and ethanol than glucose.

Next, fructose is utilised slower than glucose when two sugars are fermented separately. This
is due to fructose’s 5-carbon structure is greatly different from glucose 6-carbon structure. The
yeast strain has a higher affinity for glucose than fructose (D’more et al. 1989). Also, fructose
cannot be directly used in glycolysis as it needs to transform into intermediate first. Therefore,
fructose produce lower amount of carbon dioxide and ethanol than glucose and sucrose.
Meanwhile, lactose produce the lowest ethanol concentration and carbon dioxide amount in
fermentation. This is because yeast cell cannot metabolize lactose. Lactose require enzyme
lactase, which is not produced by the yeast (Latremouille, 2016).

There are few precautionary steps in this experiment. First, no air bubble is present in the
Durham tube before inserting the tube into incubation mixture. The presence of air bubbles will
give rise to inaccurate results. Next, the incubation time for each incubation tube is noted
carefully to prevent further fermentation. Also, it is important not to touch optical surfaces of
cuvette. This is to avoid measurement errors when using spectrophotometer.

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Questions

1. Which substrates were the most effective for alcohol fermentation by the yeast?
Explain your reasoning.
Based on the result from table 3, we concluded that glucose and sucrose were the most
effective for alcohol fermentation by yeast as they produce higher amount of ethanol than
other sugars. Glucose is a monosaccharide and it is in the simplest form. Therefore, it can
be used directly in glycolysis process. Hence, a high amount of alcohol can be produced by
using glucose in yeast fermentation. Next, sucrose is disaccharide, which is made up of
glucose and fructose. Sucrose fermentation undergoes extracellular hydrolysis, mediated
by the periplasmic invertase, producing glucose and fructose that are transported into the
cells and metabolized (Batista et al. 2004). In fact, most of the monosaccharides can
produce ethanol. Glucose and fructose are utilized simultaneously. Therefore, high amount
of ethanol is produced by using sucrose.

2. Which substrate was the least effective and why?

Based on the results, we could conclude that lactose was the least effective in yeast
fermentation. This is because yeast does not have a lactose metabolization system
(Domingues et al. 2010). Yeast does not have enzyme lactase. Lactase content of yeast
extracts was negligible or zero. Therefore, yeast cannot break down lactose into glucose
and galactose. Hence, there is little amount of ethanol produced.

Conclusion

The objective of studying the effects of different sugars in yeast fermentation is achieved
successfully. In the results, we could concluded that glucose is the most effective substrate in
yeast fermentation, followed by sucrose, fructose and lactose. Glucose produces the highest
amount of carbon dioxide and ethanol if compared to other sugars as it is a simplest form of
sugar. Lactose is the least effective substrate in yeast fermentation as yeast does not have
enzyme lactase to break down lactose. Therefore, it produces the least amount of carbon
dioxide and ethanol if compared to other sugars.

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References

Aquilla, T., 2019. The Biochemistry of Yeast - Aerobic Fermentation [Online]. Available at:
https://www.morebeer.com/articles/how_yeast_use_oxygen [Accessed 10 Jun. 2019].

Batista, A., Miletti, L. and Stambuk, B., 2004. Sucrose fermentation by sacccharomyces
cerevisiae lacking hexose transport. Journal of Molecular Microbiology and
Biotechnology, 8(1), 26-33.

D'Amore, T., Russell, I. and Stewart, G.G., 1989. Sugar utilization by yeast during
fermentation. Journal of Industrial Microbiology, 4(4), 315-323.

Domingues, L., Guimarães, P. and Oliveira, C., 2010. Metabolic engineering of saccharomyces
cerevisiaefor lactose/whey fermentation. Bioengineered Bugs, 1(3), 164-171.

Hagman, A. and Piškur, J., 2015. A study on the fundamental mechanism and the evolutionary
driving forces behind aerobic fermentation in yeast. Plos One, 10(1), 116-124.

Heckman, J., 2019. Glycolysis (Saccharomyces cerevisiae) - WikiPathways [Online].

Available at: https://www.wikipathways.org/index.php/Pathway:WP253 [Accessed 10
Jun. 2019].

Jones, A., 2006. Glycolysis revisited. Biochemical Education, 13(1), pp.35-36.

Latremouille, G., 2016. The Effect of Sucrose Concentration on the Percentage Change in
Carbon Dioxide during Ethanol (Yeast) Fermentation. [Online]. Available at:
https://www.researchgate.net/publication/316940265_The_Effect_of_Sucrose_Concent
ration_on_the_Percentage_Change_in_Carbon_Dioxide_during_Ethanol_Yeast_Ferme
ntation/citation/download [Accessed 2 Jun. 2019].

Morton, J., 2019. Glycolysis and Alcoholic Fermentation [Online]. Available at:
https://www.icr.org/article/glycolysis-alcoholic-fermentation/ [Accessed 10 Jun. 2019].

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