Factors Affecting Cellular Respiration

Nicole Anne Alcantara, Nicolas Medenilla III*, Maria Patricia Obordo

Biology Department, De La Salle University, 2401 Taft Ave., Manila 0922, Philippines

*Corresponding Author: nicolas_medenillaiii@dlsu.edu.ph

ABSTRACT

Cellular respiration in eukaryotic cells occurs as sugars from food are being converted
into energy (ATP) and other byproducts to be used in the cell’s processes. In this
experiment, the rate at which germinating, dry and boiled peas are consuming oxygen
was tested. With this, the experimenters were able to determine at which state and
environmental condition the variables are able to thrive better which is being in an
environment rich in oxygen rather than acidic (pyrogallic acid). Dry peas, on the other
hand, will only restore its function if soaked in water to regain its moisture. Also boiled
peas have been denatured, thus making it impossible to germinate. Cellular respiration as
exhibited by fermentation of yeast suspension was also tested. This resulted in the yeast
generating more oxygen bubbles under the condition that yeast will be mixed with 10%
glucose and sucrose (tube B and D), in separate tubes. Phenol red test also indicated the
aforementioned tubes to be acidic, as a result of fermentation, which yielded a yellow
color. The enzyme-substrate reaction was also tested through putting potatoes and turnips
(with guaiacol) into the same conditions being H₂O₂ as its substrate and its innate
enzymes as an active site. Banana bits were placed in different conditions such as the
change in temperature and pH. The substrate used in this experiment is hydrogen
peroxide (H₂O₂). The results of this experiment is that the pH level is within normal
range and the temperature is normal and considerably high to utilize the reaction. All
conducted experiments therefore show that extrinsic and intrinsic factors in which plants
are placed greatly affect the cellular respiration.

Keywords: cellular respiration, fermentation, enzyme, substrate

when chemical energy is converted from the

INTRODUCTION
Cellular respiration is referred to as the
chemical process or the biological mechanism in
which a conversion of the sun’s energy is
exhibited by the cell. The energy is then stored
in foodstuff molecules that lead to making
cellular activity possible. (Reid, 1940)
Adenosine triphosphate (ATP) is the product
molecules of oxygen. Cellular respiration takes
place in the mitochondria and has four steps. Its
first step is glycolysis, wherein the 6-carbon
molecule separates into two pyruvate molecules.
(Schwartz and Brown, 2013) Pyruvate is the
second step in cellular respiration; pyruvate
molecules are produced after glycolysis takes
place, these molecules are then transported into
the middle part of the mitochondria, named the
mitochondrial matrix. (Millerd, Bonner,
Axelrod, & Bandurski, 1951). The Krebs cycle is
also known as the citric acid cycle; the
irreversible oxidation of carbohydrates, lipids,
and proteins are involved in this step. In
addition, carbon dioxide is liberated and
generates NADH. (Buchanan & Arnon, 1990).
Oxidative phosphorylation is the last pathway in
bringing out ATP. (Smeitink, Van den Heuvel,
& DiMauro, 2001). Oxidative phosphorylation
indicates the formation of ATP due to the
transfer of electrons from NADH or FADH2 to
O2 by electron carriers.
The objectives of this research are to
determine and become familiar with the
compounds involved in cellular respiration, from
start to finish; to rule out how the nature of the
substrate can affect the rate of fermentation; and
to become acquainted on how respiratory
enzymes work.
The experimental hypothesis is that a high
amount of oxygen and CO2 are needed to
activate and increase the rate of respiration. In
order to show the effectiveness of providing and
preventing the exchange of oxygen and CO2,
germinating seeds are commonly used to
showcase this. Factors such as heat, acids, and
mixtures are used on plants to test a variety of
effects on the process of cellular respiration.
MATERIALS AND METHODS
Procedure 1
The experimenters used two flasks, one
containing pyrogallic acid which limits and
absorbs all oxygen in the container (flask A) and
the other (flask B) contains distilled water. Both
containers have a pouch of germinating seeds
suspended above the solutions. In this procedure,
the experimenters seek to determine in which
condition seeds germinates, in an oxygen-rich or
oxygen-deprived environment. The flask in
which the seeds begin to germinate will prove its
oxygen consumption.
Procedure 2
In this procedure, germinating and dry peas were
placed in test tubes to determine their rate of
respiration. Each tube contains cotton soaked in
potassium hydroxide which will solidify in the
presence of CO₂ (waste product of respiration).
A respirometer was used to quantify the amount
of CO₂ produced as the peas took in oxygen for
respiration.
Procedure 3
To support the results of procedure 2,
germinating and boiled seeds were placed into
separate test tubes containing a certain amount
of hydrogen carbonate indicator solution. The
seeds are suspended above the solution using a
gauze and the test tubes are sealed off. In the
presence of the waste product of respiration,
CO₂, the solution underneath will change in
color, thus, this data will be used in analyzing
the results.
Procedure 4
This experiment seeks to determine how gas
evolves in aerobic respiration. A series of
beakers were aligned, U-shaped test tube A
contains potassium hydroxide solution which
absorbs carbon dioxide from the air, beaker B
and D contains lime water solution and lastly
beaker C contains the germinating seeds. These
containers are connected by a glass apparatus
ending in beaker D with a suction pump. As air
is being pumped, the experimenters observed the
change in color of the lime water in beakers B
and D, if any.
Procedure 5
Five fermentation tubes, two 50 ml of 10%
glucose and sucrose solution, two 25 ml of 10%
of the same solutions, two 25ml yeast suspension
and one 50 ml yeast suspension was prepared to
test the effect of substrate on fermentation. The
contents of each fermentation tube are as
follows:
A: 50 ml of 10% glucose solution
B: 25 ml of 10% glucose solution + 25ml yeast
suspension
C: 50 ml of 10% sucrose solution
D: 25 ml of 10% sucrose solution + 25ml yeast
suspension
E: 50ml yeast suspension
Air bubbles should not be present in the tube. All
samples will be fermented for 2 hours while
tabulating the measurement of the amount of gas
bubbles produced. After which, a phenol red test
will be conducted to determine the acidity of the
tubes.
The first procedure tested in which environment
germinating seeds would thrive, oxygen-rich or
oxygen-deprived. Pyrogallic acid solution, a
mixture of 10g sodium hydroxide, 100ml
distilled water and 10g pyrogallic acid, is seen in
Figure 1 (left flask). This is known to efficiently
absorb the oxygen content of a closed
environment (Britannica, 1998). This test flask
containing the said solution enabled the
experimenters to set this as an oxygen-deprived
environment. This flask showed no germination
after days of observation.

Procedure 6

Enzymes involved in oxidation reactions in

varying conditions were put to a test in this
procedure. In the first condition, potato samples
were placed in one test tube containing hydrogen
peroxide and red dye to determine how much
oxygen bubbles were produced as a product of Figure 1. Flasks Containing Pyrogallic Acid
oxidation. The same conditions, in six test tubes, Solution (left) and Distilled Water (right).
were made with the turnip sample (peroxidase
enzyme) but guaiacol was added to act as an Figure 2 shows germination in the second flask
indicator of how much oxygen was created from containing 100ml distilled water (right flask in
the reaction. After which, a pH level of 10 was Figure 1). This then represents an environment
introduced to each test tube to see the effects of rich in oxygen which allowed the seeds to
pH level to the reaction. Results were tabulated propagate given the limited amount of oxygen
for each time limit starting from 0 minutes to 5 caused by sealing the flask. Roots evidently start
minutes in one minute increments. The next to grow which indicates that the seeds were able
procedure for this part of the experiment was to to perform their cellular functions.
test the different pH levels. Banana bits (enzyme
carrying sample) were placed in three different
test tubes containing the same amount of H₂O₂
but each has a different pH level ranging from
acidic (A), normal (B) and basic (C). The other
condition the samples were placed in were the
different water temperatures ranging from
normal room temperature (D), ice bath
approximately 0oC (E) and boiling 100oC (F).
The amount of bubbles were observed and
measured which indicates the product of
enzyme-substrate reaction. Figure 2. Right Flask Germination.

This procedure tested the rate of respiration in

RESULTS AND DISCUSSION peas and was measured in 0-20 minutes with 5
minute increments. The germinating peas
showed the most amount of oxygen consumed in
millimeters as compared to the dry peas. As time
progressed, the amount of oxygen the
germinating peas consumed also increased.
Unlike dry peas, it almost appears to be constant
at 0.001 mm of consumed oxygen. This is
because the setting in which the dry peas were
placed made it impossible for them to rehydrate,
bringing back their functionality. Dry peas have
been denatured, essentially lowering its moisture
content which then prevents germination
(Rowland, 1981). The rate of respiration in
germinating peas ranged from 0.000mm to
0.350mm (Appendix).
Figure 3. Rate of Respiration in Peas.
The third procedure tested the rate of production
of energy between germinating seeds and boiled
seeds. After leaving the flasks for a few hours,
the thermal flask containing germinating seeds
produced heat while the thermal flask containing
the boiled seeds did not produce any signs of
heat. The germinating seeds were still alive and
respiring compared to the boiled seeds that were
considered dried out. As with the previous
procedures, any signs of the germination process
occur only when the seeds are able to absorb
certain amounts of oxygen, light, and water
within their surroundings.
Figure 4. Thermal flasks containing
Germinating Seeds and Dry Seeds.
As air is being pumped through the connecting
glass tube, the potassium hydroxide in the U-
shaped test tube absorbs the air and is passed to
beaker B containing lime water. There was no
recognizable change in the color to the lime
water. The air then travels to beaker C wherein
the germinating seeds are placed. The seeds have
used the oxygen in the air to perform its various
functions specifically aerobic respiration. Air
then travels to the final beaker with the same
lime water as beaker B and suddenly its color
changes. The cloudy appearance of the lime
water indicates that the initial air that was
pumped into the previous beakers was not the
same as it was in the final container. This is
caused by the aerobic respiration that took place
in the seeds resulting in the production of CO₂,
one of the waste products of the process.
Figure 5. Production of CO₂ in Aerobic
Respiration.
Enzymes generally act as catalysts which enable
the speeding up of certain chemical reactions
which take place in an organism, in this case,
yeast suspension (Shiel, n.d.). Substrates, on the
other hand, are molecules that act upon the
enzyme’s active site to be broken down to its
simplest form for an organism to perform its
specified function (Biology Dictionary, 2017).
Sugars such as sucrose, fructose and glucose are
often converted into energy (ATP) which is
vastly used in the different processes of
organisms like fermentation, which produces
foam. In this experiment, two types of sugars,
namely glucose and sucrose, were used to test
their effects on the fermentation of yeast. The
results in Table 1 have shown the most amount
of gas bubbles produced after two hours is seen
in tube B which contains 25 ml of 10% glucose
solution + 25ml yeast suspension followed by
tube D containing 25 ml of 10% sucrose solution
+ 25ml yeast suspension. This is because
glucose does not need extra energy to break it
down to be converted to ATP needed in
fermentation as compared to sucrose (Anderson,
2019). Tubes A and C did not form any
fermentation bubbles because the substrate used,
both glucose and sucrose, has no enzyme to react
to. As for tube E, there was only minimal foam
that formed in the apparatus because, although
fermentation happened, there was no substrate
introduced to speed up the reaction.
Table 1. Effect of Substrate to Fermentation.
Amount of Gas Bubbles (mm)
Tube
after 2 hours
A 0mm
B 6mm
C 0mm
D 3mm
E 1mm
A phenol red test was conducted to determine
which of the samples became acidic after
fermentation. A change in color would indicate
the change in pH. When the color turns yellow,
it is safe to assume that its pH is less than 6.8
(acidic), orange means that its pH is greater than
6.8 but less than 7.4 and is not to be considered
acidic and lastly red would mean that its pH is
greater than 7.4 (“Microbiology Labs I”, n.d.).
This part of the experiment yielded the results
that tubes containing both substrate and enzyme
are more acidic than the other samples as seen in
Table 2.
Table 2. Fermentation Phenol Red Test.
Tube Color (after adding phenol red)
A Red
B Yellow
C Red
D Yellow
E Orange
Enzymes present in potatoes were used to react
with the substrate hydrogen peroxide (H₂O₂) to
observe its oxidation reaction. The substrate was
broken down into water and oxygen as it reacted
with the potato’s active site. As seen in Figure 6,
red dye was used to have better visualization of
air bubbles, the reaction produced a lot of
oxygen bubbles. This then is a clear indication
that an oxidation reaction has occurred.
Figure 6. Potato Enzyme Oxidation Reaction.
In this part of the experiment, the enzyme,
peroxidase, found in turnips were used to react
with the same substrate as mentioned before,
H₂O₂. However, an indicator was introduced,
without affecting the chemical reaction, which is
guaiacol. This indicator turns brown as oxygen
interacts with it turning guaiacol into
tetraguaiacol. This is used in order to quantify
the results of how fast the reaction occurs over
time (1 being clear and 7 most brown). Table 3
shows that as the time progresses, the
tetraguaiacol turns browner than it was 0
minutes into the reaction. As a pH of 10 was
introduced per time limit, the color in the test
tube turns clear or transparent. This indicates
that a change in pH has an effect on the reaction.
Table 3. Enzymatic Reaction of Peroxidase and
pH Change.
 Figure 7. Effect of pH Level to Enzymatic
Reaction.
Baseline pH 10

The bananas were placed into three different

0min 1 1
temperature conditions. Test tube D being
normal room temperature, E has approximately
1min 2 1
0oC and F has a little over 100 oC. In this part, the
experimenters have observed that the enzymatic
2min 3 1
reaction occurring in test tube A created the most
3min 5 1
oxygen bubbles followed by the cooler
temperature, B, and surprisingly no bubbles have
4min 5 1
formed in C (Figure 8). This was a surprising
result because according to Santosh (2018)
5min 7 1
enzymatic reactions are believed to increase in
higher temperatures. Although as mentioned in
the previous part, enzymatic activity is not only
sensitive to pH but as well as temperature
In the previous experiment, only a basic pH of (Worthington et.al, n.d.). A slight change in
10 was introduced to the turnip sample. In this temperature will affect the reaction rate by 10%-
part, the three levels of pH, namely acidic, 20% (Worthington et.al, n.d.). Enzymes,
neutral and basic, were put to a test to determine although thriving in increasing temperature, may
their effects on the enzyme-substrate reaction. also denature or lose their function when the
The enzyme used can be found in bananas and temperature is too high which means there is
the substrate was still hydrogen peroxide also an optimum level in which they thrive to
(H₂O₂). Figure 7 shows that test tube A, located serve their function (Worthington et.al., n.d.).
on the left most part of the rack and has an acidic
pH, shows 3mm worth of oxygen bubbles
formed. On the contrary, both basic and neutral
pH containing tubes, B and C, showed little to
no reaction. Catalysts are sensitive to pH level
(Worthington et.al, n.d.). In cases such as this
experiment, acidic and basic environments put a
halt to the enzymes causing little to no reaction
at all.An optimum pH must be met in order for
the reaction to take place (Worthington et.al.,
n.d.).

Figure 8. Effect of Temperature to Enzymatic

Reaction.

CONCLUSION

Cellular respiration remains vital as this involves

the utilization of oxygen formed in
photosynthesis. Germination was not evident in
the first procedure as the seeds were placed in an
oxygen-deprived environment with pyrogalic
acid, distilled water, and sodium hydroxide. On
the other hand, the other flask was placed in an
environment where oxygen was easier to go
through. The first procedure showed a
connection with roots; they begin to grow once
the seeds have accomplished their cellular
functions. The amount of oxygen consumed by
the germinating peas is higher compared to the
dry peas; the environment where the dry peas
were exposed was unable to let them rehydrate
and as a result, their natural qualities were
altered. In terms of the rate of respiration in
peas, it was highlighted that the flask containing
germinating seeds produced heat, and the seeds
were able to undergo respiration unlike the
boiled seeds. This posits that germination only
took place when the seeds were able to absorb
oxygen, light, and water within their
environment. In the procedure regarding the
production of carbon dioxide in aerobic
respiration, the line water appeared to be cloudy;
the seeds were subjected to aerobic respiration as
it remains vital in producing carbon dioxide.
Enzymes function to hasten chemical reactions
and in its experiment, glucose and sucrose were
utilized to determine their effects on yeast
fermentation. Sucrose required more energy in
breaking down to be able to convert to ATP
unlike glucose. Two tubes (Tube A and Tube C)
did not exhibit fermentation bubbles due to the
absence of enzymes. This indicates that the
bacteria was not able to be fermented due to the
source of sugar. Two other tubes (Tube B and
Tube D) did exhibit fermentation bubbles. This
indicates that there was a drop in the pH due to
the production of the acid via fermentation of the
sugar.
In the following enzyme experiments, potatoes
and turnips were used. The tests performed show
that, when their respective enzymes are
activated, potatoes and turnips are able to obtain
an oxidation reaction. Factors such as pH and
temperature have an effect on how enzymes
function. The effect of pH on enzymes depends
on the increase or decrease of their levels.
Temperature will affect the stability of enzymes,
whether they are able to break down components
or slow down molecules until they become
crystallized. In order to create certain products, it
is important to note that the process of
fermentation is affected by numerous factors like
temperature, pH, composition, and the source of
carbon and nitrogen, along with the ability to
activate the enzymes release of carbon dioxide
and lactic acid.
Plants, like any other organisms are delicate.
External and internal factors of plants, in
general, greatly affects its functions. It is
important to keep these factors at their optimum
level to achieve expected results. As for cellular
respiration in particular, factors such as pH,
temperature as well as its intrinsic characteristics
must be well monitored because a slight shift in
these factors can affect its functionality.
AUTHOR CONTRIBUTIONS
Nicole Anne Alcantara: Data curation (lead);
Formal analysis (lead); Investigation (equal);
Methodology (equal); Project administration (equal);
Resources (supporting);
Visualization (lead); Writing review & editing
(equal).
Nicolas Medenilla III: Conceptualization
(supporting); Formal analysis
(supporting);Methodology (equal); Project
administration (equal); Resources (equal);
Supervision (lead); Writing-original draft
(supporting); Writing-review & editing (supporting).
Maria Patricia Obordo: Formal analysis
(supporting); Investigation (equal); Methodology
(supporting); Resources (supporting); Writing
-original draft (equal); Writing review & editing
(equal).
REFERENCES
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Fermentation: Carbon Dioxide Production Lab
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fermentation-carbon-dioxide-production/
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Unpublished raw data.
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Experiment to show that Co2 is released during
germination of seeds [Digital image]. (2019).
Retrieved from https://www.youtube.com/watch?
v=q6QfMMXlzvE
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from https://www.youtube.com/watch?
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A., PhD. (2019). Introduction of Enzymes [PDF].
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Haan, J. (2020). [Banana enzyme and substrate

reaction to pH and temperature]. Retrieved from
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Pyrogallol. (n.d.). Retrieved January 10, 2021, from

https://www.britannica.com/science/pyrogallol

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Moisture Content on Germination, Stand and Yield
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Appendix

Temp Time Beads Alone G. Peas Dry Peas

 (oC) (min) Reading of Difference Reading of Difference Reading of time Difference
time x time x x

22 oC 0 0.900 x 0.900 x 0.831 x

5 0.900 0.000 0.843 0.057 0.832 0.001

10 0.900 0.000 0.722 0.178 0.832 0.001

15 0.900 0.000 0.639 0.261 0.830 0.001

20 0.900 0.000 0.550 0.350 0.819 0.012

Rate of Respiration in Peas.

RUBRIC FOR COLLABORATION AND COOPERATION

(to be graded by each groupmate)

Evaluator: Patricia Obordo

Name: Nicole Alcantara
Scores: Task and Participation (4), Share responsibility and dependability (4), Discussing, listening and interacting
(4), Teamwork (4) = 16 pts.
Name: Nicolas Medenilla
Scores: Task and Participation (4), Share responsibility and dependability (3), Discussing, listening and interacting
(4), Teamwork (4) = 15pts.
Evaluator: Nicolas B. Medenilla III
Name:Nicole Alcantara
Scores: Task and Participation (4), Share responsibility and dependability (4), Discussing, listening and interacting
(4), Teamwork (4) = 16pts.
Name: Patricia Obordo
Scores: Task and Participation (4), Share responsibility and dependability (4), Discussing, listening and interacting
(4), Teamwork (4) = 16pts.
Evaluator:p Nicole Anne G. Alcantara
Name: Nicolas Medenilla
Scores: Task and Participation (2), Share responsibility and dependability (2), Discussing, listening and interacting
(1), Teamwork (1) = 6pts.
Name: Patricia Obordo
Scores: Task and Participation (3), Share responsibility and dependability (3), Discussing, listening and interacting
(2), Teamwork (2) = 10pts.