Research Question: How does the use of different simple sugar substrates

(glucose, fructose, sucrose, maltose, and lactose) (g or ml) affect the rate of
oxygen uptake (ml/minute) in Saccharomyces cerevisiae within 60 minutes?

By Ratu Salsabila Astrakusuma

1: Introduction
Carbohydrates are sugars (saccharides) naturally found in the food we eat like fruits, vegetables, dairy,
legumes, grains, as well as in manufactured products such as sports drinks and energy bars that provide us
with the energy to live (Mueller and Hingst, 2013). As a part of my effort to understand how I can improve
my health, proper nutrition and exercise have become topics of interest. Publications that advice to “avoid
simple sugars and consume more complex sugars,” are often encountered, claiming that complex sugars pack
more energy and digest slower, making them more filling and a good option for weight control (Cherney,
2018). Complex sugars are also ideal for people with type 2 diabetes, as they help manage blood sugar spikes
after meals (Cherney, 2018). However, while staying within the 40-60 grams per day range, we can use
simple sugars to our advantage, fueling our normal levels of exercise and daily activities (Baur, 2011).
Balancing that with a diet of 45-65% carbohydrate consumption while meeting daily calorie needs allows the
storage of energy enough for 3 hours of moderate-intensity exercise (Mueller and Hingst, 2013). Examples of
simple sugars include glucose, fructose, sucrose, maltose, and lactose, which are easily and rapidly converted
into energy after consummation. These sugars are found in all kinds of healthy fruits and dairy products like
milk.
Heavy or intense exercise for over 60 minutes without adequate carbohydrate consumption can increase the
risk of glycogen depletion, which causes a shortage of energy being sent to the brain, leading to symptoms
like dizziness, cramping, and extreme fatigue (Mueller and Hingst, 2013). Thus, to understand the appropriate
amount of simple sugar needed for our daily energy needs, we need to understand how the use of different
simple sugar substrates affect the rate of cellular respiration in organisms, and whether their effects are
significantly different. In class, we learned about how an organism’s respiration rate can be determined by
using a respirometer that measures the rate of exchange of O 2 and CO2, making respirometry the ideal method
for this investigation (Cornell, 2016). However, it is important for the sugars used to be pure and in the form
that is easily accessible, like fructose as syrup, lactose in milk, and sucrose as table sugar, so the results of this
investigation are applicable in real life.

2: Investigation
2.1 : Hypothesis

H0: The rate of respiration in Saccharomyces cerevisiae is not significantly affected by changes in the type of
simple sugar substrate.
H1: The rate of respiration in Saccharomyces cerevisiae is significantly affected by changes in the type of
simple sugar substrate, with monosaccharaides resulting in higher rates compared to disaccharides.

2.2 : Background Knowledge

Cell respiration is the controlled release of energy by converting organic compounds into adenosine triphosphate,
otherwise known as ATP (Bailey, 2019). Cell respiration begins with the breakdown of those organic compounds
into glucose, which then is converted through glycolysis into pyruvate, followed by one of two pathways: anaerobic
or aerobic respiration, depending on the availability of oxygen. Unlike anaerobic respiration, aerobic respiration
occurs in the mitochondria and requires oxygen, as well as yielding much more energy (ATP). The simplified
energy conversion is as follows (Millar, 2011):

1
 C6H12O6 + 6O2  6CO2 +
6H2O + energy (38ATP)

Figure 1.1 Aerobic Respiration Overview (Millar, 2011)

Figure 1.2 Hydrolysis of Sucrose (Shipman et al., 1993)

However, the complete process of aerobic respiration is in fact a complex metabolic pathway, comprising of at least
30 separate steps that can be summarized into 3 main processes: glycolysis (the breakdown of glucose by enzymes,
releasing energy and pyruvic acid), Krebs cycle, and the respiratory chain (Millar, 2011). The organic compounds
used for cell respiration are usually sugars or carbohydrates, though sometimes fats and proteins are also used.
Most carbohydrates enter cellular respiration during hydrolysis, when enzymes and water break down complex
carbohydrates into simple sugar monomers (Berg et al., 2002). Sugar monomers, also called monosaccharaides
2
 (glucose, fructose, and galactose) are small enough to be absorbed directly into the bloodstream. Meanwhile, bigger
carbohydrates which include disaccharides like sucrose (glucose and fructose), maltose (2 glucose molecules), and
lactose (galactose and glucose), and complex sugars (polysaccharides) like starch (multiple glucose molecules
linked together) are broken down into monomers (Figure 1.2) before being converted into ATP or stored as
glycogen (FDA, 2019). This is why, in the initial hypothesis (H 1), monosaccharaides are expected to result in faster
cellular respiration, as disaccharides need to be broken down before being converted and used as glucose.

The most common sugar found in nature is glucose, which plays a key role in cellular respiration and
photosynthesis, as well as contributing to blood sugar levels (Shipman et al., 1993). Glucose is the organic
compound other compounds get converted into when entering the cellular respiration process, as its structure
allows for it to be very compactly stored as
glycogen (Berg et al., 2002). Its compact
structure allows large amounts energy to be
stored in a small volume with little effect on
cellular osmolarity (Winchester, 2011). On the
other hand, the monosaccharide fructose, along
with galactose, has no contribution to blood
sugar levels but is broken down by the liver into
glucose to be used or to be stored as glycogen
Figure 1.3 Carbohydrate Metabolism Overview (Eschopp, 2017) (Figure 1.3) (Groves, 2018). Additionally, when
sucrose is broken down, glucose is instantly used
while fructose enters glycolysis: addition of a phosphate group turns it into fructose-6-phosphate, which is then
metabolized into glucose-6-phosphate and then into usable glucose through gluconeogenesis (Berg et al., 2002).

Eukaryotic organisms, like Saccharomyces cerevisiae, use their mitochondria as the main site for most cell
respiration processes. ‘Saccharomyces’ is Latin for ‘sugar fungus’, clarifying that this genus is usually found in
sugar-rich environments (Gerke et al., 2006). Saccharomyces cerevisiae is characterized by its role in bread-
making, wine and beer (‘cerevisiae’ is Latin for ‘of beer’), among others
(Marques et al., 2015).

S. cerevisiae was the first eukaryotic cell that had its complete genome
sequenced (Barrell et al., 1996) and is often used for scientific
investigations as a model for eukaryotic cells, as they are easily accessible
and highly tolerant towards changes in the environment, including pH,
osmolarity and temperature changes, as well as being ‘able to survive as a
generalist at low abundance in a vast ranges of habitats’ (Goddard and S. cerevisiae, electron micrograph
(Murtey and Ramasamy, 2016)
Greig, 2015). Additionally, like others in its genus, S. cerevisiae can
consume different substrates as sources of carbon (Samani et al., 2015),
making it the appropriate organism to be used for this experiment.

2.3 : Variables

Variable Unit How it is measured

Monosaccharaides: glucose (s) and The state of matter of the sugars varies according to
Independent
fructose (l) and Disaccharides: maltose Grams (g) how they are naturally found, so that the experiment
Variable
(l), sucrose (s), lactose (aq) results would be applicable to real life.
The rate of respiration is measured by respirometry,
Dependent Rate of oxygen uptake (ml minute-1)
ml minute-1 where for every minute, the cumulative oxygen uptake
Variable within 60 minutes (±0.1ml)
(ml) is recorded for 60 minutes.
Variable Unit How results can be affected How it is measured
Controlled The amount of sugar Grams (g) Using the same amount of sugar Before each experiment, the
Variable used is 2.5g (±0.01g) decreases the possibility of factors sugar is weighed on an electric
3
 other than the independent scale (0.00g) (±0.01g). Solid
variable to affect the results, and sugars are weighed to equal 2.5g,
allows for the yeast to use the while the liquid sugars used was
same amount of substrate to use calculated from ml to equal 2.5g
for respiration. of solid sugars. (Appendix A)
Before each experiment executed,
The amount of Using the same amount of yeast
the yeast is weighed on an
Saccharomyces for each test ensures that the
Grams (g) electric scale (0.00g) (±0.01g).
cerevisiae used is biological process of cell
All the yeast used also came from
2.5g (±0.01g) respiration remains constant.
the same packet and storage.
A difference in water volume may The water is measured using a
The volume of result in unmeasured varying sugar volumetric 10ml (±0.05ml)
Milliliters
distilled water used is concentrations to be used in each pipette. The preliminary
(ml)
30ml (±0.05ml) experiment, hence would make the experiment identified this volume
results unreliable. to be appropriate.
S. cerevisiae requires lukewarm
Before each experiment, the
(36.5 to 40.5°C) water for the
The distilled water temperature of the distilled water
Degrees yeast to activate, which is close to
temperature is kept at is checked, using a thermometer
Celsius (°C) that of human body temperature
37-40°C (±0.01°C) (110°C) (±0.01°C), to ensure that
(37°C) (Joachim and Schloss,
it is within this range.
2013).
KOH (strong alkali) absorbs the Before each experiment, the
The amount of carbon dioxide (CO2) produced by KOH is weighed on an electric
potassium hydroxide cell respiration. If different masses
scale (0.00g) (±0.01g). The use of
Grams (g)
(KOH) is kept at 5g of KOH is used, the rate of CO 2 KOH allows for only oxygen
(±0.01g) absorption may be inconsistent for
uptake to be the gas that is
each experiment. measured in the experiment.
The preliminary experiment
The duration of Keeping the duration of identified this duration as the
Minutes
experiment is kept at experiment constant allows the appropriate duration for each
(min)
60 minutes (1 hour) results to be comparable. experiment. The data is recorded
in one-minute intervals.

2.4 : Preliminary Experiment

A preliminary experiment was carried out to assess if alterations to the original methodology were necessary.
The method used for the preliminary experiment was identical to that in section 3.3, save for the fact that the
amount of yeast and volume of water used in the preliminary experiment was 25g (±0.01g) of yeast and 10ml
of distilled water as opposed to 2.5g (±0.01g) of yeast and 30ml (±0.05ml) of distilled water. The reason to this
was because an insufficient volume of water was used and the yeast mixture initially overflew the
respirometer. Duration was also considered during the preliminary experiment, starting with 45 minutes per
experiment as the initial duration, before extending it to 60 minutes.

3: Procedure
3.1 : Apparatus

1. 2.50g (±0.01g) Saccharomyces cerevisiae, tube, 100ml Erlen Meyer (±1ml), 100ml Erlen
sourced from the same packet of instant yeast. Meyer rubber stopper with 2 holes).
2. 2.50g (±0.01g) of simple sugar. The 5 types of 5. 0.2ml (±0.01ml) rubbing alcohol (70%
sugars to be prepared are: Glucose (s), Fructose Alcohol; ethanol).
(l), Sucrose (s), Maltose (l), Lactose (l). All 6. One small bottle of green food coloring.
liquid (ml) materials are converted to grams. 7. 30cm sewing thread.
3. Distilled water at 37-40°C (±0.01°C). 8. Water bath (ensure that the water bath is able to
4. Custom Respirometer (S-tube, 5cm cylindrical reach at least 50°C in temperature).
4
9. Stirring rod. 15. 5ml micro-centrifuge tubes.
10. 0.00g Digital weighing scale (±0.01g). 16. Graph paper with 3mm increments as a ruler
11. 100ml beaker (±1ml). for oxygen uptake (±0.1ml).
12. Volumetric 10ml pipette (±0.05ml). 17. Pressure cooker and stove (autoclave
13. 1ml syringe (±0.01ml). substitute).
14. 5.00g (±0.01g) Potassium Hydroxide (KOH). 18. Thermometer (110°C) (±0.01°C).
19. Marker, transparent adhesive tape, and scissors.

3.2 : Photograph of set-up

Photographs taken by myself using an Oppo F7 phone, on 11/8/2019, displaying the respirometer set up.

3.3 : Methodology
Preparing the Potassium Hydroxide (KOH):
1. Tie the end of a 30cm thread around the head of the 5ml micro-centrifuge tube. Make sure that the micro-
centrifuge hangs from the thread properly to minimize the risk of spillage.
2. Using a 0.00 (±0.01g) digital scale, measure 5.00g (±0.01g) of potassium hydroxide (KOH) and place them in
the micro-centrifuge tube, keep the lid of the micro centrifuge tube open for the potassium hydroxide to
absorb the CO2 later on during the experiment.
Preparation of experiment:
1. Attach the S-tube and the cylindrical tube into the 2-holed rubber stopper.
2. Mark a straight line and make a mark for every 3mm on the graph paper using a pen or marker. For every
3mm the green alcohol (indicator) rises, 0.2ml (±0.1ml) of oxygen is consumed for respiration. Attach the
graph paper with on the S-tube to be used as a ruler.
3. Use one drop of green food coloring to give color to alcohol and insert 0.2ml (±0.01ml) colored alcohol into
the S-tube using a 1ml syringe (±0.01ml) until it reaches the bottom of the tube. Ensure that the marked line
for 0.0ml is the same height as the top of the colored alcohol to make sure that it starts at 0.0ml (±0.01ml).

Preparing the distilled water:

1. Add 100ml (±1ml) of distilled water into a 100ml beaker (±1ml).
2. Using a water bath, heat up the distilled water to 50°C (±0.01°C), using a thermometer to track the
temperature, so that it could cool down and kept at 37-40°C (±0.01°C) during the experiment. Reheat in
between experiments when the temperature of water goes below 37°C.
Procedure:
1. Using a 0.00 (±0.01g) digital scale, prepare 2.5g (±0.01g) of sugar and pour into the 100ml Erlen Meyer
(±1ml).
2. Insert 30ml (±0.05ml) of distilled water using a 10ml volumetric pipette (±0.05ml) into the Erlen Meyer and
stir until fully mixed using a stirring rod. Note that water measurements are not done for the lactose sugar

5
 experiment as the process using lactose uses the liquid available in milk (read 3.4 Justifications).
3. Using a 0.00 (±0.01g) digital scale, measure 2.5g (±0.01g) of S. cerevisiae and add it into the before stirring
the mixture until homogenous using a stirring rod. Cover the hole of the cylindrical tube to constrain the
airflow.
4. Start recording the cumulative value of oxygen uptake per 0.2ml (±0.1ml) using the ruler attached on the S-
tube that had been previously prepared, note down the value every 1 minute for 60 minutes.
5. Repeat 3 times for each sugar type.

3.4 : Justification
The presented independent variable values were used for specific reasons. Glucose is the main
substrate used for cell respiration and thus functioned as the control sample. Sucrose represented
table sugar often used in households and manufactured foods, fructose represented the sweetest
type of sugar often found in fruits along with its use in kitchens as liquid sugar, maltose
represented sugar used in bakeries and confectionery production, and lactose represented sugar
found in milk and dairy products (IFT, 2000).
The dependent variable (rate of oxygen uptake (ml minute -1) within 60 minutes (±0.1ml)) was chosen
because aerobic respiration is dependent and linear to the rate of oxygen uptake (Wagner et al.,
2011). A simple respirometer designed to measure oxygen uptake consisting of a sealed container
with the S. cerevisiae together with a substance to absorb the carbon dioxide given off during
respiration, such as potassium hydroxide (KOH), was the appropriate set up for this experiment.
Therefore, the rate of oxygen uptake would be a representation of the rate of respiration. The
respirometer model used was inspired by Timothy Hagge, as it allows for the mixture of yeast
mixed with sugar and water as the organism to be used (Hagge, 2015).
3 repeats were carried out for each sugar substrates to ensure the reliability of result data. Lactose was
sourced from full-cream whole milk, and according to calculations, 29.6ml (rounded to 30ml) of
milk is required to obtain 2.5g of lactose, as the milk used contained 20g of lactose for every cup
(236.5ml) of whole milk (UVA, 2012). Therefore, water was not added for the experiments using
lactose, and those experiments instead used 30ml of milk. Like the distilled water, the milk is
heated up to 50°C (±0.01°C), using a thermometer to track the temperature, so that it could cool
down and kept at 37-40°C (±0.01°C) during the experiment. This temperature was chosen because
S. cerevisiae requires lukewarm (36.5 to 40.5°C) water for the yeast to activate, which is close to
that of human body temperature (37°C) (Joachim and Schloss, 2013). Finally, liquid fructose and
maltose (ml) was converted into grams to ensure that the mass of the sugar is constant.

3.5 : Risk Assessment

Safety Issues: Potassium hydroxide, or KOH, is a corrosive strong alkali that can be poisonous and is
especially dangerous when in its stable solid form. As safety precautions, wearing a proper lab
coat, goggles, and a respirator is appropriate. Glassware is also used in this experiment and may
lead to injuries if they break, therefore proper handling of these glassware is necessary (McIntosh,
2010). They should be inspected before use; cracked glassware should be disposed of, and
contaminated glassware should be cleaned. Lastly, as syringes are used in this experiment, it is
important to make sure that it is being used carefully and that it is disposed immediately pin a
sharps disposal container (FDA, 2018).
Ethical Issues: There were no ethical issues to be taken into account.
Environmental Issue: There were no environmental issues to be taken into account.

6
 4: Raw Data
Raw Data Table 1: Distance of movement (mm) (±0.1mm) by colored alcohol in the respirometer per
minute of 60 minutes using different sugar substrates.
Distance of Movement by Colored Alcohol (mm) (±0.1mm)
Glucose (mm) (±0.1mm)Fructose (mm) (±0.1mm)Maltose (mm) (±0.1mm)Sucrose (mm) (±0.1mm)Lactose (mm) (±0.1mm)
G1 G2 G3 F1 F2 F3 M1 M2 M3 S1 S2 S3 L1 L2 L3
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 18 19.5 18 1.5 3 1.5 18 19.5 19.5 3 3 1.5 1.5 1.5 1.5
2 36 39 37.5 3 6 4.5 39 39 37.5 3 3 3 1.5 3 1.5
3 54 58.5 57 6 9 7.5 60 58.5 55.5 4.5 3 4.5 3 3 1.5
4 72 75 76.5 7.5 12 10.5 81 78 76.5 4.5 4.5 6 3 4.5 3
5 91.5 91.5 93 10.5 15 15 102 97.5 97.5 6 4.5 7.5 4.5 4.5 4.5
6 111 108 109.5 13.5 19.5 19.5 124.5 117 118.5 9 6 9 4.5 6 4.5
7 133.5 124.5 126 16.5 24 24 147 136.5 139.5 9 7.5 10.5 4.5 6 6
8 156 145.5 142.5 19.5 28.5 28.5 169.5 156 160.5 10.5 9 12 6 6 6
9 178.5 166.5 159 24 33 33 192 175.5 181.5 13.5 10.5 13.5 6 6 6
10 201 190.5 178.5 28.5 36 37.5 214.5 195 202.5 16.5 13.5 15 6 7.5 7.5
11 223.5 214.5 198 33 39 42 237 214.5 223.5 19.5 13.5 16.5 7.5 7.5 9
12 243 238.5 217.5 37.5 42 46.5 261 234 243 22.5 16.5 19.5 7.5 9 10.5
13 262.5 264 237 42 45 49.5 285 255 262.5 24 19.5 22.5 9 9 10.5
14 282 289.5 256.5 45 49.5 52.5 309 276 283.5 28.5 24 25.5 9 10.5 10.5
15 301.5 315 282 48 54 55.5 333 297 304.5 33 28.5 28.5 9 10.5 12
16 321 337.5 307.5 51 55.5 58.5 357 318 325.5 36 33 31.5 9 12 12
17 340.5 360 333 54 57 61.5 384 339 348 39 37.5 34.5 10.5 12 12
18 360 378 358.5 57 60 63 411 360 370.5 42 42 37.5 12 12 13.5
19 384 396 384 61.5 63 64.5 438 384 393 45 46.5 40.5 12 13.5 13.5
20 408 417 409.5 66 66 67.5 465 408 415.5 49.5 46.5 43.5 13.5 15 13.5
21 432 438 435 70.5 69 70.5 493.5 432 438 54 51 49.5 13.5 15 15
22 456 459 457.5 76.5 72 73.5 522 456 460.5 58.5 55.5 55.5 13.5 15 16.5
23 480 486 480 82.5 75 78 550.5 480 486 64.5 61.5 61.5 13.5 15 16.5
24 504 513 502.5 88.5 79.5 82.5 579 504 511.5 70.5 67.5 67.5 15 16.5 18
25 528 540 526.5 94.5 84 87 607.5 528 540 76.5 73.5 73.5 15 16.5 18
26 552 567 550.5 100.5 90 91.5 636 552 570 82.5 79.5 81 15 16.5 19.5
27 576 591 574.5 106.5 97.5 96 664.5 576 600 88.5 85.5 88.5 15 18 21
Time (minutes)

28 603 615 598.5 112.5 105 100.5 693 600 631.5 94.5 91.5 94.5 15 18 22.5
29 630 639 622.5 120 112.5 108 721.5 633 663 100.5 97.5 100.5 16.5 18 24
30 657 663 652.5 127.5 120 115.5 750 666 694.5 106.5 103.5 106.5 16.5 19.5 24
31 684 687 682.5 135 127.5 123 778.5 699 726 111 109.5 112.5 18 19.5 25.5
32 711 711 708 142.5 136.5 132 807 732 757.5 115.5 115.5 118.5 19.5 19.5 27
33 738 736.5 733.5 148.5 147 141 837 765 789 121.5 121.5 123 21 21 27
34 750 760.5 759 154.5 157.5 153 868.5 798 822 127.5 126 127.5 21 22.5 27
35 762 784.5 784.5 160.5 165 162 900 831 855 133.5 130.5 132 22.5 24 28.5
36 769.5 796.5 804 166.5 171 168 931.5 864 888 141 136.5 136.5 22.5 25.5 28.5
37 777 804 814.5 171 175.5 174 960 897 921 148.5 142.5 141 24 27 30
38 784.5 811.5 825 175.5 180 180 988.5 930 954 156 148.5 145.5 25.5 28.5 30
39 792 817.5 828 180 184.5 186 1012.5 963 987 160.5 154.5 150 27 30 30
40 795 823.5 831 184.5 187.5 192 1036.5 996 1021.5 162 159 154.5 28.5 30 31.5
41 798 826.5 834 189 190.5 196.5 1056 1029 1053 163.5 163.5 159 30 31.5 31.5
42 801 829.5 837 195 193.5 199.5 1075.5 1062 1081.5 165 168 163.5 30 33 33
43 804 831 840 201 196.5 202.5 1095 1096.5 1108.5 165 169.5 165 31.5 34.5 34.5
44 807 832.5 844.5 207 199.5 205.5 1114.5 1131 1135.5 165 169.5 166.5 33 34.5 34.5
45 810 834 849 213 205.5 208.5 1134 1158 1158 166.5 169.5 166.5 33 36 36
46 813 837 853.5 219 211.5 213 1153.5 1185 1180.5 166.5 171 168 34.5 37.5 36
47 814.5 840 858 226.5 219 217.5 1170 1212 1203 168 171 169.5 34.5 37.5 37.5
48 816 843 862.5 234 226.5 222 1186.5 1239 1222.5 168 171 169.5 36 39 39
49 817.5 846 867 241.5 234 228 1203 1266 1242 169.5 171 169.5 36 39 40.5
50 817.5 847.5 870 249 243 234 1219.5 1293 1260 169.5 171 169.5 37.5 40.5 40.5
51 817.5 849 873 255 252 244.5 1230 1305 1278 171 172.5 171 39 42 40.5
52 819 850.5 876 261 259.5 255 1240.5 1317 1294.5 171 172.5 171 40.5 43.5 42
53 820.5 852 880.5 265.5 267 265.5 1251 1323 1308 171 172.5 171 42 45 43.5
54 822 853.5 885 270 274.5 273 1261.5 1329 1320 171 174 171 42 45 45
55 823.5 855 889.5 274.5 282 280.5 1264.5 1335 1330.5 171 174 172.5 43.5 46.5 45
56 825 856.5 894 280.5 289.5 288 1267.5 1341 1339.5 171 174 172.5 45 46.5 46.5
57 828 858 900 288 297 295.5 1270.5 1347 1345.5 172.5 175.5 172.5 45 48 48
58 828 859.5 906 295.5 304.5 303 1273.5 1353 1351.5 172.5 175.5 172.5 46.5 48 49.5
59 829.5 861 912 303 312 310.5 1276.5 1362 1357.5 172.5 175.5 174 46.5 49.5 51
60 832.5 862.5 918 306 319.5 318 1279.5 1371 1363.5 174 175.5 174 48 51 51

5: Processed Data
 The movement of liquid is directly proportional to the uptake of oxygen (O 2) as the liquid is displaced
by the gas. For every 3mm in distance the colored alcohol moves in the respirometer, 0.2ml of
oxygen consumed.

Processed Data Table 1: Comparison of Saccharomyces cerevisiae average oxygen uptake (ml)
within 60 minutes using different sugar substrates (±0.01ml).
Time (Minutes)
0 2 3 4 5 7 10 11 12 13 14 15 17 18 20
21 23 24 25 26 28 31 32 33 34 35 36 38 39 41
42 44 45 46 47 49 52 53 54 55 56 57 59 60
0.0 2.5 3.8 5.0 6.1 8.5 12.7 14.1 15.5 17.0 18.4 20.0 23.0 24.4 27.4
Glucose 29.0 32.1 33.8 35.4 37.1 40.4 45.6 47.3 49.1 50.4 51.8 52.7 53.8 54.2 54.6
54.8 55.2 55.4 55.6 55.8 56.2 56.6 56.7 56.9 57.1 57.2 57.5 57.8 58.1
0.0 0.3 0.5 0.7 0.9 1.4 2.3 2.5 2.8 3.0 3.3 3.5 3.8 4.0 4.4
SUGAR SUBSTRATE

Fructose 4.7 5.2 5.6 5.9 6.3 7.1 8.6 9.1 9.7 10.3 10.8 11.2 11.9 12.2 12.8
13.1 13.6 13.9 14.3 14.7 15.6 17.2 17.7 18.2 18.6 19.1 19.6 20.6 21.0
0.0 2.6 3.9 5.2 6.6 9.4 13.6 15.0 16.4 17.8 19.3 20.8 23.8 25.4 28.6
Maltose 30.3 33.7 35.4 37.2 39.1 42.8 49.0 51.0 53.1 55.3 57.5 59.6 63.8 65.8 69.7
71.5 75.1 76.7 78.2 79.7 82.5 85.6 86.3 86.9 87.3 87.7 88.1 88.8 89.2
0.0 0.2 0.3 0.3 0.4 0.6 1.0 1.1 1.3 1.5 1.7 2.0 2.5 2.7 3.1
Sucrose 3.4 4.2 4.6 5.0 5.4 6.2 7.4 7.8 8.1 8.5 8.8 9.2 10.0 10.3 10.8
11.0 11.1 11.2 11.2 11.3 11.3 11.4 11.4 11.5 11.5 11.5 11.6 11.6 11.6
0.0 0.1 0.2 0.2 0.3 0.4 0.5 0.5 0.6 0.6 0.7 0.7 0.8 0.8 0.9
Lactose 1.0 1.0 1.1 1.1 1.1 1.2 1.4 1.5 1.5 1.6 1.7 1.7 1.9 1.9 2.1
2.1 2.3 2.3 2.4 2.4 2.6 2.8 2.9 2.9 3.0 3.1 3.1 3.3 3.3

Calculation of average oxygen uptake:

∑ OxygenUptake (ml) on each minute of each repeat
Number of repeats
Example calculation for average volume

Average oxygen uptake for glucose at minute 30:

∑ 43.8+ 44.2+43.5 =43.8 ml
3
Data is rounded to 1 decimal places (1.d.p), because the uncertainty of the oxygen intake is ±0.1ml.
All values in processed data table 3 were also rounded to 2.d.p for the same reason.

S. cerevisiae oxygen uptake (ml) within 60 minutes (±0.1ml)

100
Oxygen Intake (ml) (±0.1ml)

80

60

40

20

0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60

Time (minutes)
Glucose Fructose Maltose Sucrose Lactose

Processed Data Graph 1: Comparison of Saccharomyces cerevisiae average oxygen uptake (ml)
every minute within 60 minutes using different sugar substrates (±0.1ml).
 The error bars were set to the maximum error per reading, which was found to be 3.03% (rounded to
3 significant figures (s.f)).
Calculation for maximum percentage error

Maximum Percentage Error=Uncertainty of apparatus used ¿ measure dependent variable ¿

Lowest dependent varia
0.1
Maximum Percentage Error= × 100=3.03 % (rounded¿ 3 significant figures ( s . f ))
3.3
However, the test does not show if there is a significant relationship between the types of sugar substrate
used and S. cerevisiae oxygen uptake (ml) within 60 minutes (±0.1ml). Hence, further data processing
needs to be conducted.

5.1: Statistical Test

To establish a statistical difference between the 5 different groups, a one-way analysis of variance
(ANOVA) test was conducted on processed data table 2 using a software that allows for
statistical tests on Microsoft Excel, StatPlus. This test allowed a test for a statistical relationship
across all sugar substrate oxygen uptake.

H0: The rate of respiration in Saccharomyces cerevisiae is not significantly affected by changes in the
type of simple sugar substrate.
H1: The rate of respiration in Saccharomyces cerevisiae is significantly affected by changes in the type
of simple sugar substrate, with monosaccharaides resulting in higher rates compared to disaccharides.

The results of the test can be seen in processed data table 2. The null hypothesis (H0) was tested
through the ANOVA test.

Processed Data Table 2 (Screenshot from Microsoft Excel): A table displaying how the one-way
ANOVA test was carried out.

Note:
Results Format
Results: F [degrees of freedom (d.f) between groups, d.f total] = F value p (probability level)
Results: F (4, 19) = 3.81821 p= 4.037E-53

Based on the low probability value of 4.037E-53 (p≤ 0.05), the null hypothesis (H 0) shown in section 2.1
is rejected and the initial hypothesis (H 1) is accepted. Therefore there is a statistically significant
relationship between the type of sugar substrate used and S. cerevisiae oxygen uptake (ml) within 60
minutes (±0.1ml). Additionally, the F-value (97.6814) is much higher than the F-critical value
(2.40174), which further supports that there is a statistical significant relationship between the
independent and dependent variable.
5: Evaluation

5.1 : Conclusion
As the result of this experiment, it can be concluded that the rate of respiration in Saccharomyces
cerevisiae is significantly affected by changes in the type of sugar substrate. Therefore, the
null hypothesis (H0) shown in section 2.1 is rejected and the initial hypothesis (H1) is accepted.
This is shown in Processed Data Table 2 that has a very low probability value of 4.037E-53 (p≤
0.05). This low probability value indicates a statistically significant relationship between the type
of sugar substrate used and S. cerevisiae oxygen uptake (ml) within 60 minutes (±0.1ml). It is
also shown that the F-value (97.6814) is much higher than the F-critical value (2.40174), further
supporting my hypothesis that the rate of respiration in S. cerevisiae is significantly affected by
changes in the type of simple sugar substrate. However, Processed Data Graph 1 shows that the
extension to the initial hypothesis (H1) is untrue, as the use of monosaccharaides did not show
higher rates of oxygen uptake compared to disaccharides.

As the control of all the sugar substrates used, it was expected that the use of glucose would have
result in the most rapid uptake of oxygen before plateauing, as glucose is being directly
consumed for cell respiration (Bear and Rintoul, 2016). However, as seen in Processed Data
Graph 1, the oxygen uptake by S. cerevisiae using maltose as its sugar substrate resulted in the
highest cumulative oxygen uptake, ending with a cumulative oxygen uptake volume of 89.2ml
(±0.1ml). As seen in Processed Data Graph 1, glucose and maltose resulted in the fastest uptake
of oxygen for the first 30 minutes, with a similar rate of oxygen uptake. However, for the second
half of the experiments, maltose continued to rise consistently, while glucose reached a plateau at
approximately 35 minutes into the experiment. This this can be explained by how maltose is
made of 2 glucose molecules which, through hydrolysis, yields twice the amount of glucose used.
Although the breaking down of maltose was expected to decrease the rate of oxygen uptake, it
resulted in a more consistent rate of oxygen uptake due to the abundance of glucose available
(Rawn et al., 2014).

Lactose resulted in the lowest cumulative oxygen uptake, resulting in only a cumulative oxygen
uptake of 3.3ml (±0.01ml). This may be caused by how S. cerevisiae is unable to utilize all of the
sugars equally well (Pepin, 2015). While glucose and fructose can be metabolized by S. cerevisiae
completely, galactose is not utilized at all, as it may not have the proper enzyme to metabolize
galactose or the proper proteins to transport galactose across its membrane (Timson, 2007). However,
S. cerevisiae does allow for hydrolysis to happen, meaning that the lactose used in this experiment
still resulted in oxygen uptake because lactose can be broken up into the monosaccharaides glucose
and galactose. Although galactose is not utilized, the glucose allows lactose to still be consumed by S.
cerevisiae, despite the result being very low.

Fructose and sucrose resulted in a much lower oxygen uptake in comparison to maltose and glucose,
resulting in a cumulative uptake of 21.0ml (±0.01ml) for fructose, and 11.6ml (±0.01ml) for sucrose.
Sucrose was expected to result in a much lower oxygen uptake compared to fructose due to sucrose
requiring hydrolysis before it can enter cellular respiration. Additionally, fructose was expected to
result in the second fastest cumulative oxygen uptake due to being a monosaccharaide. However, such
a result can be explained by how the conversion of fructose into glucose may delay the occurrence of
the respiratory chain, which would also explain why maltose resulted in a higher rate of oxygen
uptake compared to fructose (Mocke, 2013). In addition, S. cerevisiae is a glucophilic yeast,
indicating its preference for consuming glucose over fructose, which may also play a role to why the
oxygen uptake is much slower in experiments using sucrose and fructose than used glucose and

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 maltose (Mocke, 2013).

5.2 : Strengths
The experiment was done in an isolated system, meaning that the external environment outside of the
respirometer was not likely to affect the S. cerevisiae respiration process. In addition, using a simple eukaryotic
organism (Saccharomyces cerevisiae or baker’s yeast) as a model for a biological pathway observation will allow
the results to be applicable when observing other eukaryotic organisms, especially humans, as S. cerevisiae
would have consistent metabolic activity but is capable of carrying out similar cellular respiratory processes as
other eukaryotic organisms. The apparatus used during the experiment were also sterilized using a pressure
cooker as an autoclave substitute before the experiment, reducing the chance of contamination by other
organisms. The respirometer was consistently used without breaking down and was cleaned, dried, and was
sterilized again before further use, ensuring the precision of the methodology. The experiment also had low
systematic error due to the relatively low uncertainty of the apparatus used in the experiment, since the
uncertainty of the mass of each sugar substrate and S. cerevisiae used was only ±0.01𝑔, which is a small
percentage error relative to the volume of the results being measured that ranged from 3.3ml to 89.2ml, which had
the uncertainty of only ±0.1ml. Furthermore, the small error bars (3.03%) on the graph also indicate a low chance
of error, increasing the certainty and precision of the data gained through the experiment.

5.3 : Weaknesses
Despite its applicability to real-life situations, the use of sugar substrates in different states of matter allowed for
unknown factors (possibly things like viscosity, the different bond strengths between the molecules, etc.) to affect
the results of the experiment. The use of lactose in the form of milk also allowed for many unknown factors to
play a role to why the use of lactose resulted in very low values in comparison to other sugar substrates outside of
that had been discussed. Additionally, the scale of the sample sized used was quite small, as only one source
(brand, form of sugar, and packet) of each sugar substrate was used. This limits the extent to which the conclusion
supports the hypothesis at a larger scale. This could be improved by the use of multiple sugar source of the same
sugar type, along with using a consistent state of matter for each sugar (using all liquid sugar or all solid sugar) to
allow for more evidence supporting whether the type of sugar alone plays a significant role in the rate of
respiration.

The data gained from the experiment is also strictly focused on the short-term effects (60 minutes or 1 (one) hour)
of different simple sugar substrates on the rate of respiration by S. cerevisiae. This limits the extent to which it
examines how the rate of cellular respiration varies with the type of sugar substrate used, as it does not represent
the effect of cellular respiration using different types of simple sugar substrates for daily energy supplementation.
This can be rectified by conducting the experiment over a period of time longer than 12 hours. Such improvement
to the methodology could increase the reliability of results, as it allows us to see the duration in which each sugars
increase the rate of respiration before plateauing, given that, in this experiment, maltose and fructose had not
plateaued and still had an increase in rate even after other sugars have already plateaued.

This investigation can also be further improved by more repetitions of each experiment, as the experiments for
each sugar substrate type was only repeated 3 times. More repetitions would allow for an investigation for a more
accurate and precise comparison of cellular respiration rates between each sugar substrates. Furthermore, the
measurement of carbon dioxide (CO2) production could be taken into account to measure whether the oxygen
uptake is directly proportional and for their ratio to be measurable as well.

5.4 Further Research Questions

An extension to this investigation would be a further study into how the use of polysaccharides affect the rate of
respiration of Saccharomyces cerevisiae, as starch is a major part of the human diet. This would allow for more
information on how more complex sugars are metabolized in the organism. Another extension would be how the

12
use of different sugar substrates affect the rate of oxygen uptake (ml/minute) in S. cerevisiae, or an alternative
organism with varying metabolic pathways over a period of 2 or more hours, given that the results from this
experiment concluded in some sugars not reaching plateau just yet, implying that the entire process of cell
respiration is yet to be completed by S. cerevisiae. Therefore, it would be interesting to uncover if this trend still
continues after a much longer time period than 60 minutes. Such investigation would allow deeper understanding
of the durations in which each sugar substrate type is effective to be consumed.

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