IB Biology Internal Assessment

Research question: What is the effect of changing the pH of sucrose solution (pH 2, 4, 7, 9) on the
mean rate of aerobic respiration of Saccharomyces cerevisiae, as measured by volume of carbon
dioxide gas produced (in cm3) through the downwards displacement of water by the end of 3 minutes?

Background information:
Fungi are eukaryotic organisms which exist as molds, yeasts or a combination of the two forms (McGinnis and
Tyring). Commonly found in soil, plant surfaces and especially abundant in sugary mediums, yeasts are fungi
which comprise solitary cells that reproduce asexually by budding (Encyclopedia Britannica).

As fungi, yeasts act as decomposers and are central to the cycling of materials and energy in the ecosystem.
Fungi contribute to the mineralisation of organic materials and the dissipation of carbon and energy within the
soil ecosystem. They are involved in nutrient cycles such as the nitrogen cycle and sulphur cycle, and also
have the ability to solubilise insoluble phosphates (Botha). Therefore, through the metabolic activity of fungi,
elements which are essential to biological systems are released from decaying matter and made available to
living organism. Fungi such as yeasts are critical to ecosystems, such that decomposers, (fungi and bacteria),
alongside primary composers are the most basic components of a functioning ecosystem (Klironomos).

Saccharomyces cerevisiae is a type of budding yeast, selected strains of which are often used in baking and
brewing industries due to its ability to ferment sugar into carbon dioxide and ethanol (Encyclopedia Britannica).
Oxidation of sugars and other organic materials provides yeasts with a source of energy, and yeasts are
differentiated by their ability to use different carbon sources such as simple sugars (McGinnis and Tyring).
Therefore, glucose is the starting substrate of respiration, a process through which energy is obtained. In the
presence of oxygen, respiration is aerobic, the chemical equation for which is as shown: C6H12O6 + 6O2 →
6CO2 + 6H2O + 36ATP (Allott and Mindorff).

The first step of aerobic respiration is glycolysis (see Fig. 1) which occurs in the
cytoplasm of the cell and is facilitated by various enzymes. Two adenosine
triphosphate (ATP) molecules are used to initiate glycolysis. Glucose molecules
are phosphorylated meaning the phosphates from the ATP molecules are
added to the glucose, forming fructose-1,6-biphosphate. This is a less stable
molecule that splits into two 3-carbon sugars through lysis, producing two
sugars called glyceraldehyde-3-phosphate (G3P) which are oxidised to form
NADH, a reduced molecule of NAD+. Meanwhile, released energy is used to
add an inorganic phosphate to the remaining 3-carbon compound, resulting in a
compound with two phosphate groups which are later removed by enzymes to
be added to adenosine diphosphate (ADP) to form ATP. The end result is four
ATP molecules, hence a
net gain of two ATP
molecules, and four
molecules of pyruvate (Allott and Mindorff).

Aerobic respiration occurs in the mitochondria. If oxygen

is present, pyruvate from glycolysis enters the matrix of
the mitochondria (see Fig. 2) through active transport
and the pyruvate is decarboxylated. This reaction
involves the loss of a carbon, giving the waste gas carbon dioxide, to form the 2-carbon acetyl group which is
oxidised with the formation of reduced NAD+. The acetyl group combines with coenzyme A (CoA), forming
acetyl CoA. This process is known as the link reaction and it is controlled by a system of enzymes (see Fig. 3)
(Allott and Mindorff).

Acetyl CoA enters the Krebs cycle, a cycle of enzymatic reactions that
releases energy (see Fig. 4), where it combines with a 4-carbon
compound called
oxaloacetate,
giving a 6-carbon
compound called
citrate. Citrate is
oxidised and
decarboxylated to a
5-carbon compound, releasing carbon dioxide from the cell.
Meanwhile, NAD+ is reduced to NADH. The 5-carbon
compound is then oxidised to a 4-carbon compound and
once again NAD+ is reduced to NADH. The 4-carbon
compound undergoes certain changes resulting in the
production of another NADH and the coenzyme FAD is
reduced to FADH2. ATP is also reduced to ADP, also giving
an inorganic phosphate. Oxaloacetate is reformed and the
cycle repeats (Allott and Mindorff).

The electron transport chain is the first step of cellular

respiration which requires oxygen, occurring on the inner
mitochondrial membrane and on the membranes of the cristae.
Electron carriers are embedded in these membranes. These are
molecules capable of accepting one or more electrons and
transferring them to other molecules, releasing energy that is
used to produce ATP. These electrons are supplied by NADH
and FADH2 which are oxidised to NAD+ and FAD respectively.
As each carrier molecule has a different electronegativity,
electrons pass from one carrier to another as the receiving
molecule has a higher attraction for electrons. FADH2 enters the
chain at a lower free energy level than NADH, resulting in the
production of two ATP molecules whereas NADH allows the
production of three ATP molecules. Oxygen is the final electron acceptor due to its high electronegativity. As
two electrons combine with the oxygen so do two hydrogen ions, forming water (Allott and Mindorff).

The electron transport chain, allowing for the addition of the phosphate and energy to ADP to form ATP. This
process is called chemiosmosis, where electron carrier proteins use energy to pump protons (as hydrogen
ions) across the inner membrane of the mitochondria into the intermembrane space. A proton concentration
gradient is formed in the mitochondrial matrix due to the accumulation of protons in the intermembrane space.
The inner mitochondrial membranes are embedded with ATP synthase, an enzyme which uses the energy of
the protons which move down their concentration gradient from the intermembrane space to the mitochondrial

2
matrix, allowing the phosphorylation of ADP. Therefore, ATP is produced by oxidative phosphorylation. Overall,
there is a net gain of 36 ATP by aerobic respiration (Allott and Mindorff).

The rate of aerobic respiration can be influenced by various factors, including pH conditions. The optimal pH
range for yeast growth varies from pH 4 to 6 (Narendranath and Power). Though there is limited literature
regarding the physiological basis for the preferred pH range for respiration is based on the optimal pH
conditions of the involved enzymes. It is also important to maintain an intracellular pH during yeast growth and
each enzyme works best at an optimum pH that is acidic due to the acidophilic nature of S. cerevisiae. As the
extracellular pH deviates further from the optimum intracellular pH, the yeast cell must invest more energy in
pumping hydrogen ions in and out of the cell to maintain the intracellular pH. It may become difficult to maintain
the intracellular pH if the extracellular pH deviates too much from the intracellular pH, preventing the normal
function of enzymes (Narendranath and Power). Enzymes denature as the intracellular pH deviates too far
from the optimum as the hydrogen ions (if the pH is too acidic) or hydroxide ions (if the pH is too alkaline)
cause a conformational change in the active sites of enzymes, preventing substrate molecules from binding to
it. Enzymes play a vital regulatory role throughout aerobic respiration. Through the conformational change of
the active sites of the enzymes due to extreme pH conditions, the rate of aerobic respiration will decrease
(Allott and Mindorff).

In nature, the extracellular pH of yeast can be affected by acid rain, precipitation which has a pH of roughly 5.2
or lower. This is mainly caused by the emission of sulphur dioxide and nitrogen oxides from human activities,
particularly the combustion of fossil fuels (Butler and Likens). The mean pH of rainwater in Singapore is 4.2
(Hu et al.).

Hypothesis:
The rate of respiration of S. cerevisiae will be around pH 5 (pH 4-6)
due to it being an acidophilic organism. This pH is the optimum for
the enzymes involved in facilitating aerobic respiration. When the
extracellular pH deviates from this range, to pH 2 or 9, then it is
expected that the enzymes will denature. In pH 2, the gain of
hydrogen ions will distort the tertiary structure of the enzymes
(specifically its active site), and the gain of hydroxide ions in pH 9
will have the same effect. This will prevent the binding of substrate
molecules to the active site, and hence the enzyme can no longer
catalyse the reaction by lowering its activation energy which occurs
with the binding of the substrate to the active site. Therefore, less
carbon dioxide gas should be collected at the extreme extracellular
pH conditions (pH 2 and 9) and the peak of gas production should be in the range of pH 4-6 (see Fig. 6).

Variables
Independent variable: The extracellular pH (pH 2, 4, 7, 9) of S. cerevisiae. This will be changed by adding 40
cm3 of the respective pH buffer to 10 cm3 of sucrose solution. There will also be a control group where no pH
buffer is added to the sucrose solution.
Dependent variable: The rate of aerobic respiration of yeast in the respective pH solutions of sucrose and in
the control group. This will be measured by measuring the carbon dioxide gas produced by the aerobic
respiration of yeast through the downwards displacement of water. The volume of gas produced in cm3 can be
divided by the number of minutes that the yeast was allowed to respire, to give the volume of gas produced per
minute.
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Table 1: List Of Controlled Variables, Implications To The Investigation And How It Is Controlled
Variable Why it needs to be controlled How it will be controlled

Temperature The temperature affects the rate of respiration as it influences The measuring cylinders
of sucrose the activity of the enzymes involved in this process. At higher where the sucrose solution
and pH buffer temperature, the enzyme activity increases as substrate and the pH buffer are
solutions molecules gain kinetic energy, increasing the chance of them measured will be kept in a
binding to the active site of the enzymes, catalysing aerobic hot water bath. A
respiration of yeast. At lower temperatures, the enzyme thermometer will be used to
activity is lower as the substrate molecules lose kinetic measure the respective
energy and the frequency of the substrate binding to the solutions and they will be
active site decreases. Beyond a certain temperature, the removed from the bath and
three-dimensional conformation of the active site of the mixed together upon
enzymes changes, preventing the substrate from binding to reaching 40 ºC.
the active site of the enzyme. (Allott and Mindorff) In this
case, enzymes denature and products can not be formed,
inhibiting aerobic respiration.

Concentration This must be kept constant because if there is a greater This will be controlled by
of sucrose for number of sucrose molecules per unit volume in the using a fixed concentration
sucrose environment of the yeast respiration, then the rate of aerobic of sucrose solution of 1.0 M
solutions respiration will be higher. This is because glucose is the for all of the trials.
beginning substrate molecule in respiration and hence by
increasing the concentration of substrate molecules, the rate
of respiration increases, and vice versa if the concentration is
lower.

Mass of yeast This must be kept constant because if there is a greater mass A digital weighing scale will
added used of yeast left to respire then there will be more carbon dioxide be used to measure 2.00
for each trial gas produced due to a larger number of organisms grams of yeast which will
performing aerobic respiration. be added to the solutions of
pH buffer and sucrose.

Ratio of This must be controlled because by adding a different volume This will be kept constant
sucrose of pH buffer with the sucrose solution, or vice versa, the by adding 10 cm3 of
concentration concentration of hydrogen ions (if pH < 7) or hydroxide ions sucrose solution together
to pH buffer (if pH > 7) per unit volume of the solution may vary. This will with 40 cm3 of pH buffer for
solution in affect the activity of the enzymes and can affect to what each trial, except the
each trial degree their tertiary structure is distorted by these ions. If the control group which will
concentration of ions is lower, then the tertiary structure will only contain 10 cm3
be distorted to a lesser extent causing the rate of aerobic sucrose solution to see the
respiration to be higher, vice versa when the concentration is effect of the absence of pH
higher. buffer solution.

Time that the If more time is allowed, then there will be a greater volume of A stopwatch will be used to
yeast is carbon dioxide gas collected as the yeast will be left to measure a duration of 3
allowed to respire for a longer duration of time. Similarly, if less time is minutes for every trial.
respire for allowed, a lower volume of gas will be produced.

Pre-trials: To test which concentration of sucrose solution would be optimal for the aerobic respiration of S.
cerevisiae, the yeast was added to sucrose solutions of concentrations 0.6 M, 0.8 M and 1.0 M respectively.
The volume of carbon dioxide gas collected via the downwards displacement of water was indicative of the rate

4
of aerobic respiration. The mean volume of gas collected was highest at 1.0 M sucrose concentration, and
therefore this concentration was used in the investigation.
Table 2: Pre-trial data table investigating the ideal sucrose concentration (0.6, 0.8, 1.0 M) for the aerobic
respiration of Saccharomyces cerevisiae, as measured by the volume of carbon dioxide gas collected by the
downwards displacement of water in 3 minutes
Extracellular sucrose Volume of carbon dioxide gas collected by the downwards displacement of water / ±
concentration of S. 0.5 cm3
cerevisiae (M)
Trial 1 Trial 2 Mean

0.6 13.0 12.0 12+13

= 12. 5
2

0.8 15.0 17.0 16.0

1.0 20.0 15.0 17.5

Materials
Table 3: Materials list
Materials Quantity Uncertainty Materials Quantity Uncertainty

1.0 M sucrose solution 250 cm3 - Water trough 1 -

pH buffer solution at pH 2, 4, 200 cm3 - Kettle 1 -

7 and 9 each

Baker’s yeast (S. cerevisiae) 50 g - Rubber bung 1 -

50 cm3 measuring cylinder 2 ± 0.5 cm3 Delivery tube 1 -

25 cm3 measuring cylinder 1 ± 0.5 cm3 Clamp stand 1 -

Thermometer 2 ± 0.5 ºC Clamp 1 -

1000 cm3 beaker 1 - Stopwatch 1 -

Electronic weighing scale 1 ± 0.01 g Small piece of scrap paper 1 -

Glass stirring rod 1 - Soap bottle 1

Plastic spoon 1 -

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Method:
1. Fill a water trough with tap water so that it is mostly full.
2. Position a clamp stand behind the water trough.
3. Attach a clamp to the clamp stand and tighten it so that it does not fall off during the experiment.
4. Fill a 50 cm3 measuring cylinder to the brim with tap water.
5. Place a hand over the opening of the measuring cylinder and invert it into the water trough.
6. Attach the measuring cylinder to the clamp such that some space is left between the opening of the
measuring cylinder and the bottom of the water trough to allow the delivery tube to fit inside.
7. Add tap water in a kettle and turn it on to allow it to heat the water.
8. Pour the hot water in the 1000 cm3 beaker, after it has finished heating, such that it is roughly ¾ full.
9. Add cold tap water to the beaker so that the temperature is not too high (as the sucrose solution and
buffer solution will be warmed using this water and if the temperature is too high then the enzymes of
the yeast may denature when added to the solution). Use a thermometer to monitor the temperature
and add hot or cold water as needed until the temperature is roughly 50 ºC.
10. Measure 10 cm3 of 1.0 M sucrose solution in a 25 cm3 measuring cylinder.
11. Measure 40 cm3 of pH 2 buffer solution in a 50 cm3 measuring cylinder.
12. Place the measuring cylinders in the beaker containing hot water.
13. Place a thermometer in each measuring cylinder and monitor the temperature until it reaches 40 ºC.
14. Place a piece of scrap paper on a digital weighing scale and tare the scale so that the mass of the
paper is not taken when measuring the mass of the Baker’s yeast (do so while the sucrose and pH
buffer solutions are still heating up as the solution may begin to cool in the time taken to measure the
yeast if it is done waterwards).
15. Use a plastic spoon to add gradual amounts of Baker’s yeast on the scrap paper until it reaches 2 g.
16. Pull the measuring cylinder out of the hot water once the solution inside has reached 40 ºC.
17. Transfer the sucrose solution into the 50 cm3 measuring cylinder.
18. Pour the yeast into the measuring cylinder and immediately use a glass stirring rod to stir the solution to
mix in the yeast well.
19. Quickly attach the rubber bung and delivery tube to the measuring cylinder and place the open end of
the delivery tube so that it is in the opening of the 50 cm3 measuring cylinder (so that the bubbles of
carbon dioxide gas produced can enter the measuring cylinder, displacing the water inside.)
20. Start a stopwatch immediately and wait for 3 minutes to pass.
21. Remove the delivery tube from the water trough after exactly 3 minutes and record the volume of gas
collected.
22. Empty the water inside the 50 cm3 measuring cylinders, washing out the yeast using soap to remove
excess yeast. Also wash the stirring rod with soap.
23. Repeat steps 4-6 and 10-22 for 4 additional trials using pH 2 buffer solution.
24. Repeat step 23 for the other pH buffer solutions (for the control group no pH buffer will be added, but
instead only 10 cm3 of 1.0 M sucrose solution to observe the impact of a lack of pH buffer on the rate of
aerobic respiration.)
Safety precautions and environmental factors:
1. pH buffer solution may cause eye or skin damage, hence it is important to wear safety goggles as well
as gloves to prevent this from happening. Any exposed areas must be washed with soap and
contaminated clothing should be washed before reuse.
2. Buffer solution waste water should only be disposed down the sink if its pH level is between 5 and 10,
otherwise it is considered as hazardous chemical waste. This should be stored and disposed as
hazardous waste (UC San Diego).
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 3. Glassware should be handled with care and not kept close to the edge of the work bench to minimise
chance of breakage and injury.
4. Be careful when handling hot water to prevent burns. When moving the beaker containing hot water, do
not directly touch the glass which the water is in contact with. Also do not touch the water kettle directly
as it is heating water.

Table 4: Raw data table showing the correlation between the extracellular pH of Saccharomyces cerevisiae
(pH 2, 4, 7, 9) and the volume of carbon dioxide gas collected by the downwards displacement of water in 3
minutes
Extracellular pH Volume of carbon dioxide gas collected by the downwards displacement of water / ± 0.5 cm3
of S. cerevisiae
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

2.0 (control)* 2.0 3.0 4.0 2.0 2.0

2.0 5.0 3.0 6.0 5.0 8.0

4.0 10.0 9.0 9.0 8.0 5.0

7.0 8.0 7.0 13.0 13.0 10.0

9.0 0.0 0.0 0.0 0.0 0.0

Uncertainty: The uncertainty of the 50 cm measuring cylinder is ± 0.5 cm as it has a precision to 1 cm3 and
3 3

the uncertainty is the precision divided by 2, which gives an uncertainty of ± 0.5 cm3.
*Anomalous data

Qualitative observations:
Foam and bubbles of a light brown colour can be observed to be forming at the top
of the solution of sucrose and pH buffer (see Fig. 8). This shows that S. cerevisiae
is undergoing aerobic respiration as bubbles of carbon dioxide gas are being
produced. The layer of foam becomes thicker from the control group to pH 2 to pH
4 and then to pH 7. For pH 9, the layer is much thinner. This shows that as the
extracellular pH approaches 7, the mean rate of aerobic respiration increases as
the pH is nearer to the optimum pH of the enzymes involved in aerobic
respiration.

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Table 5: Processed data table showing the correlation between the extracellular pH of Saccharomyces
cerevisiae (pH 2, 4, 7, 9) and its rate of aerobic respiration
Extracellular pH Rate of aerobic respiration / cm3 min-1 Mean rate of Standard
of S. cerevisiae aerobic respiration deviation /
Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 / cm3 min-1 cm3 min-1
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑔𝑎𝑠 𝑠𝑢𝑚 𝑜𝑓 𝑟𝑎𝑡𝑒𝑠
2.0 (control)* 𝑡𝑖𝑚𝑒 (𝑚𝑖𝑛𝑠) 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑡𝑟𝑖𝑎𝑙𝑠
2 0.7+1+1.3+0.7+0.7
3
= 0. 666... 5
= 0. 8
≈0.7 1.0 1.3 0.7 0.7 ≈0.9 0.2**

2.0 1.7 1.0 2.0 1.7 2.7 1.8 0.5

4.0 3.3 3.0 3.0 2.7 1.7 2.7 0.6

7.0 2.7 2.3 4.3 4.3 3.3 3.4 0.8

9.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

**Table 6: Sample calculations showing how the standard deviation was found for the control group
Rate of aerobic Difference between the volume of gas collected per (𝑥𝑖 − 𝑥)
2

respiration per trial (𝑥𝑖) trial and the mean volume of gas collected (𝑥𝑖 − 𝑥)

0.7 0.7 - 0.9 = -0.2 0.04

1.0 1.0 - 0.9 = 0.1 0.01

1.3 1.3 - 0.9 = 0.4 0.16

0.7 0.7 - 0.9 = -0.2 0.04

0.7 0.7 - 0.9 = -0.2 0.04

2
Σ(𝑥𝑖 − 𝑥) = 0. 04 + 0. 01 + 0. 16 + 0. 04 + 0. 04 = 0. 29
2
Σ(𝑥𝑖−𝑥) 0.29
Since 𝑛 = 5, 𝑛
= 5
= 0. 058
2
Σ(𝑥𝑖−𝑥)
𝑛
= 0. 058 = 0. 2408... ≈ 0. 2cm3 min-1

The rate of aerobic

respiration of S. cerevisiae
increases as the
extracellular pH increases
towards pH 7, after which
there is a rapid decrease
in the rate.

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Conclusion:
The research question for this investigation was: What is the effect of changing the pH of sucrose solution
(pH 2, 4, 7, 9) on the mean rate of aerobic respiration of Saccharomyces cerevisiae, as measured by
volume of carbon dioxide gas produced (in cm3) through the downwards displacement of water by the
end of 3 minutes?

The trend from the processed data shows that as the extracellular pH of Saccharomyces cerevisiae increases,
the mean rate of aerobic respiration increases until the pH reaches 7, after which there is a sharp decrease in
the rate. This can be seen in the processed data that when the pH becomes lower 7, the rate of aerobic
respiration decreases (decreasing from 3.4 cm3 min-1 to 2.7 cm3 min-1 at pH 4 and 1.8 cm3 min-1 at pH 2) and as
the pH is increased from 7, the rate decreases rapidly (decreasing from 3.4 cm3 min-1 to 0.0 cm3 min-1 at pH 9).
Additionally, the qualitative observations also show that the rate of aerobic respiration increases towards pH 7,
as shown by the layer of foam forming on top of the solution which gets thicker towards pH 7 and thinner at pH
9. Furthermore, the graph of the processed data reinforces this trend and also shows, through the curved
trendline, that as the extracellular pH is increased towards 7, the rate of aerobic respiration increases at a
decreasing rate. The peak of the curve on the graph indicates an approximation of the optimum pH as 6.5,
beyond which the downwards curving trendline shows the decreasing rate of aerobic respiration. The data
suggests that yeast is tolerant of extremely acidic pH conditions caused by acid rain. At pH 4, roughly the
mean pH of rain in Singapore, it is still able to respire, and even so at pH 2. This is of advantage to ecosystems
as decomposers can play their role, despite natural phenomena such as acid rain.

The carbon dioxide produced by aerobic respiration is a result of decarboxylation, which occurs in the link
reaction and the Krebs cycle. In the link reaction, pyruvate is decarboxylated, and in the Krebs cycle citrate is
decarboxylated while NAD+ is oxidised to NADH, giving a 5-carbon compound which also undergoes oxidative
decarboxylation. Hence, the carbon atom which is removed combines with two oxygen atoms to give carbon
dioxide gas which is released from the cell. The rate at which carbon dioxide is produced is an indication of the
rate of reaction, and is subject to enzyme activity (which is affected by pH) as they control the products which
are carried forward through to the different reactions involved in aerobic respiration.

There are some aspects of the hypothesis which were supported by the collected data. When the extracellular
pH was 2, the rate of aerobic respiration was relatively low which was one of the predictions made in the
hypothesis. This is because pH 2 is highly acidic, potentially causing enzymes involved in aerobic respiration to
denature due to the high concentration of hydrogen ions. The gain of hydrogen ions distorts the
three-dimensional conformation of the active sites of enzymes involved in aerobic respiration. Throughout
aerobic respiration, various enzymes are involved in catalysing different reactions. If these enzymes denature,
they will not be able to facilitate aerobic respiration, leading to a lower output of carbon dioxide gas per unit
time. As pH 2 is highly deviated from the optimum intracellular pH, more energy is spent pumping hydrogen
ions in and out of the cell and the pH may not be maintained, adversely decreasing the rate of aerobic
respiration. This explains the low rate of aerobic respiration at pH 2. When this is increased to pH 4, the rate of
aerobic respiration increases as the concentration of hydroxide ions decreases, distorting the tertiary structure
of the involved enzymes to a lesser extent. Between pH 4 and 7, the enzyme activity is at its highest and
hence this can be inferred as the optimum pH range for enzymes in S. cerevisiae for aerobic respiration.
Through interpolation, it can be seen that pH 6.5 is the optimum pH as it is at the peak of the curve shown in
the graph. This partially supports the hypothesis though the optimal pH range slightly differs from the range
suggested in the hypothesis pH 4-6. Beyond pH 7, the rate of enzyme activity plummets to 0.0 cm3 min -1 at pH
9 due to the concentration of hydroxide ions being too high, distorting the tertiary structure of important
enzymes, significantly reducing the carbon dioxide output per unit time. This supports the hypothesis which
predicted the rate of aerobic respiration would be close to 0.0 cm3 min-1 at pH 9. As this is greatly deviated from
the optimum intracellular pH, once again more energy is expended to pump hydrogen ions in and out of the
cell, however this is not sufficient to maintain the pH, inhibiting enzyme activity.

9
There are various aspects of this investigation which do not support the hypothesis. The optimum pH that is
shown through the collected data (pH 6.5) is outside the optimum pH range proposed in the hypothesis (pH
4-6). However, this may be due to the irregular increments that the extracellular pH was increased by in this
investigation which did not allow the effect of this pH range on the rate of aerobic respiration to be fully
analysed. Therefore, due to the gap between pH 4 and 7, the optimum pH for aerobic respiration of S.
cerevisiae can not be fully concluded as there is still a chance that the optimum pH may be lower than pH 6.5
and between pH 4 and 6, which would be in favour of the hypothesis. The processed data table shows pH 7 to
have the highest rate of aerobic respiration. However, this can also be attributed to flaws in the methodology.
During the investigation, due to a shortage of pH 7 buffer solution, distilled water was used instead as it was
expected to have a pH level of 7. However, after carrying out the investigation and using universal indicator to
test the pH of distilled water, the water turned yellow, indicating a pH of 6. This may account for why trial 1 and
2 for pH 7 show a lower rate of aerobic respiration than the following trials. In this regard, the data may be in
support of my hypothesis, however further testing must be done to confirm this. Additionally, the shape of the
obtained graph appears to be negatively skewed, in contrast to the graph proposed in the hypothesis which is
positively skewed to account for the acidophilic nature of S. cerevisiae. Once again, this may be due to flaws in
the methodology, specifically the irregular increments of increasing the extracellular pH.

Anomalous data and error analysis:

The main anomalous data points in this investigation was the control group itself. It was expected that the
control group would provide similar data to pH 7, however, the rate of aerobic respiration was even lower than
that of pH 2. Upon using universal indicator to test the pH level of the sucrose solution, the solution turned red,
indicating pH 2. As the sucrose solution was provided, contamination when preparing the solution may account
for its acidity, which in turn accounts for the very low rate of respiration in the control group, having a mean rate
of 0.9 cm3 min-1. The standard deviation for the control group (0.2 cm3 min-1) is relatively low and therefore this
further suggests that rather than being subject to methodological error, there was an issue with the solution
itself. This may have also altered the pH for other trials, reducing the reliability of the data.

Furthermore, when looking at the standard deviation of the trials, pH 7 has the greatest deviation and also the
greatest error bars. This can be attributed to the usage of distilled water rather than pH 7 buffer solution as
discussed previously. However, due to the larger margins of error, it is not necessarily true that pH 6.5 is the
optimum pH as the graph may become more positively skewed considering that the rate of aerobic respiration
could be as much as 0.8 cm3 min-1 lower. The second largest standard deviation was for pH 4 (0.6 cm3 min-1),
due to a range of possible factors such as ineffective temperature control, not properly mixing the yeast in the
solution of pH buffer and sucrose, human reaction time, parallax error when measuring solutions etc. These
factors can account for a similar standard deviation for pH 2 (0.5 cm3 min-1). The standard deviation was 0.0
cm3 min-1 for pH 9, showing very reliable results, suggesting that this is a denaturing point for the enzymes.

Validity:
This is a 2005 study which was published in the journal,
Applied and Environmental Microbiology, and conducted
by Neelakantam V. Narendranath as well as Ronan
Power. This study looked at the relationship between pH
and medium dissolved solids in terms of growth and
metabolism of Lactobacilli and Saccharomyces
cerevisiae during ethanol production. For this part of the
investigation, four sets of media were made for different
maltodextrin concentrations and the pH was adjusted
using 85% o-phosphoric acid. Suitable dilutions of
overnight cultures of the organisms were performed in
250 mL Erlenmeyer flasks which had 50 mL of the
respective media. After 48 hours, during which the flasks
were incubated at 30 ºC, samples were withdrawn and
filtered using a 0.2 μm filter. Using high performance
liquid chromatography (HPLC), the ethanol production of the filtrate was analysed.
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It is consistently shown for each concentration of dissolved solids that pH 5.5 produces the greatest
concentration of ethanol, followed by pH 5.0, then pH 4.5 and finally pH 4.0 (see Fig. 9). This validates the data
from this experiment as pH will have similar effects of aerobic and anaerobic respiration, due to the enzymes
having similar optimum pH conditions. The study validates this investigation because through interpolation, it
can be seen that trend in the data from this investigation shows similar results. Looking at the graph, the rate of
aerobic respiration does indeed increase from pH 4.0 to 5.5. However, the 2005 investigation only looks at a
small portion of the pH values which was used in my experiment and hence it does not guarantee that my data
is reliable. However, aspects of the methodology can be applied to my investigation. Notably, the media were
incubated at 30 ºC which would be a good temperature for the enzyme activity in S. cerevisiae, without causing
denaturation and providing reliable results. In contrast, in my experiment due to lack of effective temperature
control, the solutions were heated to about 45 ºC which may have caused enzymes to denature. Also, the use
of HPLC reduces chances of human error which were present in my experiment due to human reaction time
when starting the timer and removing the delivery tube.

Evaluation:
Though there were many sources of error in this experiment, one strength is that there were five trials carried
out, which reduces the effect of anomalous data, producing a more reliable mean value.

Table 7: Sources of error in the investigation, its impact on the results and suggested improvements
Source of error Impact on results Suggested improvements

There was ineffective temperature
control as multiple trials were
being carried out at once and the
solutions were being heated too
quickly to monitor due to the high
temperature of the water bath
(~50-60 ºC). Solutions may have
been heated too much causing
enzymes to denature.
Some solutions may have been
heated more than others so they
may have had a higher rate of
aerobic respiration. If heated too
much, enzymes may begin to
denature, reducing the rate of
respiration by causing
conformational changes in the
active site of the enzyme.
One trial should be conducted at a
time and As soon as the
temperature reaches 30 ºC, the
solutions should be removed from
the water bath. The temperature of
the water bath should be at
roughly 40 ºC by adding cold
water to the hot kettle water. The
temperatures of the solutions will
increase more slowly, becoming
easier to monitor.

Not all the yeast was mixed in the
solution as there was some stuck
to the side of the measuring
cylinder as shown in the
qualitative observations.
A lower volume of carbon dioxide
will be produced if there is less
yeast in the solution, making it
appear as though there is a lower
rate of aerobic respiration.
Yeast should be added to a dry
measuring cylinder, only after
which sucrose solution or pH
buffer can be added, preventing
yeast from sticking to the sides.

The delivery tube became clogged
by yeast which had entered the
tube during trials.
This makes it harder for the
carbon dioxide bubbles to leave
the delivery tube, causing the rate
of aerobic respiration seems lower
than the actual value due to
restricted carbon dioxide output.
There should be less overall
solution so that the foam does not
reach the delivery tube. The
volume of the pH buffer solution
could be reduced from 40 cm3 to
30 cm3.

Human error due to the time taken
to start the stopwatch and remove
the delivery tube from the water
trough at the end of three minutes.
Reduces accuracy of results as
the time allowed for aerobic
respiration to take place varies.
The rate of aerobic respiration
may seem higher/lower than
reality if the time allowed is
longer/shorter.
A data logger can be used in
conjunction with a carbon dioxide
sensor to obtain results which
would eliminate human error.
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 The extracellular pH was
increased at irregular increments
(from pH 2 to 4 to 7 to 9).
Does not allow the hypothesis to
be fully tested as it was predicted
that the optimum pH would be
between 4 and 6 but the
investigation did not involve a pH
between these values due to the
gap between pH 4 and 7.
The pH should be increased in
increments of 1 (pH 2, 3, 4, 5, 6, 7,
8, 9) to get a better understanding
of the trend and to see where the
optimum pH lies.

The yeast used was often taken Yeast is perishable when exposed Either only use yeast from an
from a packet which was not to air and moisture (Red Star), unopened packet or once a packet
closed properly and left overnight, causing the rate of aerobic is opened the yeast must be
being exposed to air and moisture. respiration to seem lower when frozen or refrigerated in an airtight
using yeast from an opened pack. container.
Extensions: Temperature control is a major source of error in this experiment. To better understand the effect
of temperature on the rate of aerobic respiration of S. cerevisiae, once again through the downwards
displacement of water. 20 cm3 of 1.0 M sucrose solution can be added to a boiling tube. Different temperatures
can be produced using a hot water bath and a thermometer to monitor temperature. Yeast can then be added
to the solutions and carbon dioxide gas can be collected for 3 minutes through the downwards displacement of
water. From this the rate of aerobic respiration can be calculated.

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