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The Effect of the Substrate on the Rate of Cellular Respiration of Yeast
(Saccharomyces Cerevisiae)1
Sophia Marie A. Malagday
BIO 11.1 21L
October 29, 2018
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A Scientific paper submitted in partial fulfillment of the requirements in BIO 11.1
(Investigations in College Biology) laboratory under Prof. John Vincent R. Pleto, 1st Semester,
2018-2019
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ABSTRACT
The effect of the nature of substrates on the rate of cellular respiration of
yeast (Saccharomyces cerevisiae) was determined using the Smith
fermentation tube method. In five tubes, a solution of 15mL distilled water
and 15mL 10% yeast suspension was mixed with the following solutions
(all at 10% concentration): (1) – albumin, (2) oil, (3) – glucose, (4) –
sucrose, (5) – distilled water. The opening of the tube is then plugged with
cotton to enforce anaerobic respiration. The height of the carbon dioxide
formed was measured every five minutes for thirty minutes. The
experiment had two replicates. Then, the volume of the carbon dioxide gas
produced was computed and used to determine the average rate of carbon
dioxide produced in each substrate. Glucose had the highest rate of carbon
dioxide production with an average height of 2.2 cm and a standard
deviation of 1.48. This is followed by sucrose, with an average height of
1.57 cm and a standard deviation of 0.94, and then oil, which had an
average height of 0.07 cm and a standard deviation of 0.11. No carbon
dioxide gas was observed in both tubes containing albumin and distilled
water as the substrates. The results confirmed the prediction of the
hypothesis and was coinciding with the expected outcome. Results showed
that the simpler the substrate, the higher the rate of carbon dioxide
production, and thus, the faster the rate of respiration.
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.
INTRODUCTION
The cell contains many different substances that it effectively utilizes in order to
ensure maintenance and survival. Some of the substances within the cell are organic
substances. Organic substances are known to possess potential energy due to the way the
electrons are arranged in the bonds between atoms and the cell makes use of these
organic substances by breaking them down into smaller molecules or simpler waste
products through enzyme activity, in order to obtain the energy it needs to perform
functional activities that will sustain the cell (Urry, Cain, Wasserman, Minorsky, &
Reece, 2017). When organic substances, also known as substrates, are broken down by
certain enzymes into simpler products, energy is released. This process is known as
cellular respiration (Adajar et. al, 2018). Cellular respiration includes two types: aerobic
respiration and anaerobic respiration. Aerobic respiration breaks down the substrate with
the aid of water, oxygen, and enzymes, while anaerobic respiration breaks down the
substrate, albeit incompletely, without the use of oxygen. Products of anaerobic
respiration can further be broken down to obtain more energy. This is to say that the
simpler the substrate, the faster it is to break down, the higher the rate of cellular
respiration.
Yeast (Saccharomyces cerevisiae) is a fungus that is single-celled or grow as
solitary cells that reproduce through budding (McGinnis & Tyring, 1996). Yeast respires
both aerobically and anaerobically and may simultaneously respire through both
processes. The one which determines which process predominates is the amount of
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oxygen present in the system (Adajar et. al, 2018). In fermentation, yeast utilizes the
anaerobic process in order to produce the energy it needs.
In this experiment, the effect of the substrate on the cellular respiration of yeast
will be observed through the Smith Fermentation Tube Method. The substrates to be
tested are Albumin (protein), oil (lipid), glucose (monosaccharide), sucrose
(disaccharide), and distilled water. Volume of CO2 produced, partial rate of CO2
production, and average rate of CO2 production will be used for comparison of the
different substrates.
The hypothesis is that if the substrate affects the rate of cellular respiration in
yeast, then the simpler the substrate, the faster the rate of cellular respiration.
This study aims to identify the effect of the substrates used in the production of
carbon dioxide gas of yeast brought about by cellular respiration on the rate of respiration
of yeast.
The study was conducted at the Institute of Biological Sciences, Wing C,
University of the Philippines, Los Baños, Laguna, Philippines, on October 4, 2018.
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MATERIALS AND METHODS
In determining the effect of the substrate on the cellular respiration of yeast
(Saccharomyces cerevisiae), the Smith Fermentation Tube Method was used.
Five Smith Fermentation Tubes were obtained and five of the respective tubes
were filled with 15mL of their respective solutions (all at 10% concentration): (1) –
albumin (protein), (2) – oil (lipid), (3) – glucose (monosaccharide), (4) – sucrose
(disaccharide), (5) – distilled water. 15 mL distilled water and 15mL 10% suspension
were then prepared for and added to each solution. The mixtures were then shaken gently
to ensure there is no air trapped at the closed end of the tube.
The openings of the Smith fermentation tube were plugged with cotton to remove
the source of oxygen and ensure that the yeast in the mixture undergoes anaerobic
respiration. The tubes were then kept upright and set aside. Two replicates were
performed for this experiment.
The yeast performs respiration as soon as it comes in contact with the substrate
and oxygen is removed from the system. It is immediately timed, and the height of the
space occupied by CO2 is measured every five minutes for thirty minutes. This is done
for each of the tubes containing the different solutions. Afterwards, the radius of the
vertical arm of each Smith fermentation tube is measured, and the volume of CO2
produced, partial rate of CO2 production, and average rate of CO2 production for each
substrate were determined using the following formulas:
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𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑪𝑶𝟐 𝒑𝒓𝒐𝒅𝒖𝒄𝒆𝒅 = 𝜋𝑟 2 ℎ
where: r = radius of the vertical arm
h = the height of the space occupied by 𝐶𝑂2
𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒓𝒂𝒕𝒆 𝒐𝒇 𝑪𝑶𝟐 𝒑𝒓𝒐𝒅𝒖𝒄𝒕𝒊𝒐𝒏 =
𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐶𝑂2 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑
𝑡𝑜𝑡𝑎𝑙 𝑡𝑖𝑚𝑒
𝑷𝒂𝒓𝒕𝒊𝒂𝒍 𝒓𝒂𝒕𝒆 𝒐𝒇 𝑪𝑶𝟐 𝒑𝒓𝒐𝒅𝒖𝒄𝒕𝒊𝒐𝒏 =
𝑉𝒊 − 𝑉𝒊−𝟏
𝑡𝑖 − 𝑡𝑖−1
Where: 𝑉𝒊 = volume of 𝐶𝑂2 at a given time
𝑉𝒊−𝟏 = volume of 𝐶𝑂2 immediately before 𝑉𝒊
𝑡𝑖 = time when 𝑉𝒊 was measured
𝑡𝑖−1 = time immediately before 𝑡𝑖
RESULTS AND DISCUSSION
The rate of the production of carbon dioxide can be used to measure the rate at
which yeast performs anaerobic respiration. Carbon dioxide is one of the waste products
of yeast as it undergoes fermentation, also anaerobic respiration, with glucose usually as
the substrate. As the substrate, glucose, is incompletely broken down in anaerobic
respiration, it releases less energy compared to aerobic respiration. Carbon dioxide is then
released as a waste product of the anaerobic process of obtaining energy of yeast. It can
be said, then, that carbon dioxide can be used as an indicator of the rate of the anaerobic
process or how fast the substrate is being broken down. The faster the rate of anaerobic
respiration, the more carbon dioxide is produced and released.
The height of the carbon dioxide gas formed in the arms of each Smith
fermentation tube were measured every 15 minutes for 30 minutes for both replicates.
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The average height per time was computed and tabulated. The standard deviation was
determined.
Table 1. The average height of Carbon dioxide gas formed on the solution of yeast and
distilled water with different substrates – albumin, oil, glucose, sucrose, and distilled
water.
Height of gas formed (cm)
Time elapsed (minutes)
Albumin
Oil
Glucose
Sucrose
Distilled Water
5
0
0
0
0
0
10
0
0
0.8
0.85
0
15
0
0
1.8
1.45
0
20
0
0
2.9
1.95
0
25
0
0.1
3.5
2.35
0
30
0
0.3
4.2
2.8
0
Mean
0
0.07
2.2
1.57
0
Standard Deviation
0
0.11
1.48
0.94
0
In Table 1, the height, in cm, of the space occupied by carbon dioxide that was
formed in each Smith fermentation tube every five minutes for thirty minutes were
tabulated. The results revealed that after 30 minutes, the Smith fermentation tube with
glucose as the substrate had the highest average height of Carbon dioxide gas with a
height of 2.2 cm and a standard deviation of 1.48. This was followed by the Smith
fermentation tube with sucrose as the substrate having an average height of 1.57cm and a
standard deviation of 0.94. Next would be the Smith fermentation tube with oil as the
substrate having an average height of 0.07cm and a standard deviation of 0.11. Some
presence of Carbon dioxide gas formation was observed in the Smith fermentation tube
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with Albumin as the substrate but is considered negligible. The Smith fermentation tube
with distilled water served as the negative control and given that distilled water is an
inorganic compound, respiration will not occur, and Carbon dioxide gas will not form.
The volume of the carbon dioxide produced in each tube was computed using the
formula 𝑽𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑪𝑶𝟐 𝒑𝒓𝒐𝒅𝒖𝒄𝒆𝒅 = 𝜋𝑟 2 ℎ and then the average volume for each
substrate was determined. The average volume of carbon dioxide in the tube with glucose
as the substrate is found to be 11.92 𝑐𝑚3 . The volume of carbon dioxide in the tube with
sucrose as the substrate is found to be 6.55 𝑐𝑚3 . The volume of carbon dioxide in the
tube with oil as the substrate is found to be 0.71𝑐𝑚3 . The tubes with albumin and
distilled water as the substrates, on the other hand, did not produce any carbon dioxide.
Afterwards, the average rate of carbon dioxide production was computed using the
following formula:
𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒓𝒂𝒕𝒆 𝒐𝒇 𝑪𝑶𝟐 𝒑𝒓𝒐𝒅𝒖𝒄𝒕𝒊𝒐𝒏 =
𝑓𝑖𝑛𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐶𝑂2 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑
𝑡𝑜𝑡𝑎𝑙 𝑡𝑖𝑚𝑒
Volume of Carbon dioxide produced by different substrates
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11,92
12
10
8
6,55
6
4
2
0
0,71
0
0
Albumin
Oil
Glucose
Sucrose
Figure 1. Volume of the carbon dioxide produced from each substrate
Distilled water
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In figure 1, the volume of the carbon dioxide gas that was produced for each
substrate was determined. Results showed that among the substrates, glucose produced
the most carbon dioxide gas. The substrate that produced the most carbon dioxide gas
next to glucose is sucrose, a disaccharide. The solution with oil as the substrate comes
after sucrose. And lastly, Albumin (protein) and distilled water, which both did not
Height of carbon dioxiide gas formed (cm)
produce any carbon dioxide gas.
Average Rate of Carbon dioxide gas production of different
substrates
4,5
4,2
4
3,5
3,5
3
2,9
2,5
2
1,95
1,8
1,45
1,5
1
0,85
0,8
0,5
0
2,8
2,35
0
5
0
10
0
15
0
20
0,1
0
25
0,3
0
30
Time (mins.)
Albumin
Oil
Glucose
Sucrose
Distilled Water
Figure 2. A line graph of the height of carbon dioxide gas produced from different
substrates measured every five minutes for thirty minutes.
In figure 2, the average rate of carbon dioxide gas production of the different
substrates was determined. Glucose has been found to have the highest rate of carbon
dioxide production out of all the substrates used in the experiment. This is followed by
the solution with sucrose as the substrate, and after is the solution with oil as the
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substrate. Next is the solutions with Albumin and distilled water as the substrates. It was
initially established that the simpler the molecule is, the faster it is to break down, the
faster the rate of respiration, and therefore, the higher also the production of carbon
dioxide.
Several pathways are followed in the metabolism of substances in the cell.
Glycolysis is the pathway that is followed in the degradation of sugars or carbohydrates.
In the glycolytic pathway, glucose, which is considered as the simplest sugar, is degraded
and converted to pyruvate. Since it is immediately converted to pyruvate, degradation
proceeds immediately (Da Poian, A. T., El-Bacha, T. & Luz, M. R.M.P., 2010). Glucose,
known as a monosaccharide, is the simplest organic substance amongst the substrates
used. When yeast is subjected to conditions that will facilitate anaerobic respiration,
Glucose is easily broken down and converted to other substances in order to obtain ATP
(Adenosine Triphosphate). Distilled water is not an organic molecule and therefore
cannot be used to produce energy. It is only logical, then, that glucose would have the
highest rate of carbon dioxide gas production amongst the substrates used.
Sucrose, on the other hand, is a disaccharide. It is composed of a fructose and a
glucose molecule linked together by α – 1-1 glycosidic bonds (Khare, 2007). As sucrose
is a sugar (carbohydrate), it will also undergo the glycolytic pathway in order to be
degraded. Sucrose is broken down into glucose and fructose before it proceeds to
glycolysis (SRI International,2010). Having two sugars linked together, it is a more
complex molecule as compared to glucose. Sucrose is therefore harder to break down,
which explains the lower rate of carbon dioxide production.
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Oil is a lipid and is known to be insoluble in aqueous solutions. Lipids are
composed of fatty acids, which are mono carboxylic acid with long chains of
hydrocarbon molecules (Khare, 2007). Lipids undergo β - oxidation before they are
degraded. β - oxidation proceeds only when all the carbohydrates present have already
been broken down (Tornheim, K., & Ruderman, N.B., 2017). Lipids, being composed of
more complex molecules, are harder to break down and catalyze as compared to
saccharides like glucose and sucrose, which then explains its rate of carbon dioxide gas
production. Its insolubility might have also played a role in its rate of carbon dioxide
production.
Albumin is a type of protein found in the human blood plasma (Zunszain,
Ghuman, Komatsu, Tsuchida, & Curry, 2003). Proteins have amino acids as its building
blocks with carboxyl moieties attached to it and may also have a side chain composed of
different molecules (Khare, 2007). In the synthesis of ATP, proteins are usually not
broken down or utilized. According to Tornheim and Ruderman, although proteins are
usually converted and turned over to other molecules, gross proteolysis only occurs in the
event of a prolonged starvation or when disease is present. This means that in order for
protein to be broken down, all energy reserves must first be depleted. Considering this, it
is only logical that Albumin has the lowest rate of carbon dioxide production.
The cell usually utilizes macromolecules in the synthesis or ATP. In this sense,
yeast will perform cellular respiration only when macromolecules are present as these are
the storages of energy.
Based on the data gathered, it can be said that the hypothesis “If the substrate
affects the rate of cellular respiration in yeast, then the simpler the substrate, the faster the
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rate of cellular respiration” was satisfied. The expected outcomes were observed from the
experiment.
SUMMARY AND CONCLUSION
The effect of the substrate on the cellular respiration of yeast (Saccharomyces
cerevisiae) was determined using the Smith fermentation tube method. A solution of
15mL distilled water and 15mL 10% yeast suspensions was prepared and poured in five
fermentation tubes. Afterwards, 15mL of the following solutions (all at 10%
concentration) was added: 1 – albumin (protein), 2 – oil (lipid), 3 – glucose
(monosaccharide), 4 – sucrose (disaccharide), 6 – distilled water (negative control). The
height of the space occupied by the carbon dioxide gas that was produced as yeast
underwent cellular respiration in the different substrates was measured and then recorded
every five minutes for thirty minutes.
The data were tabulated and analyzed. Results showed that after thirty minutes,
the Smith fermentation tube containing glucose as the substrate, having an average height
of 2.2 cm and a standard deviation of 1.48, had the highest height of carbon dioxide gas
formed amongst all the substrates used. The Smith fermentation tube with Sucrose as the
substrate follows with an average height of carbon dioxide gas formed of 1.57 cm and a
standard deviation of 0.94. Next is the Smith fermentation tube with oil as the substrate
with an average height of carbon dioxide gas formed of 0.07 cm and a standard deviation
of 0.11. No carbon dioxide gas formation was observed from the Smith fermentation
tubes containing albumin and distilled water. Having no presence of carbon dioxide gas is
an indication that cellular respiration did not occur.
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The volume for each of the substrates was computed and the average rate of
carbon dioxide gas production was determined. It revealed that glucose, being a
monosaccharide and the simplest organic substance amongst the substrates used, had the
fastest rate of carbon dioxide production. This is followed by sucrose, a disaccharide and
more complex substance compared to glucose. After sucrose comes oil, which is a lipid
and is a more complex substance compared to saccharides such as glucose and sucrose.
Albumin, being a protein and the most complex organic substance amongst all substrates,
had the slowest rate of carbon dioxide production. Distilled water, which served as the
negative control, is not an organic substance and would, therefore, not produce any
carbon dioxide gas.
The results of the experiment are coinciding with the hypothesis previously stated.
It can therefore be concluded that, organic substances that are constituted by simpler
molecules are easier to break down and therefore, would have a faster rate of cellular
respiration as compared to substances composed of more complex molecules. Substances
that are inorganic cannot be used as a substrate to produce energy, therefore, cellular
respiration would not occur.
Should the study be replicated, it is recommended that the observations and
measurements be more accurate in order to avoid any errors. It is also recommended to
use different organic molecules as substrates in order to further investigate the effect of
the substrate on the cellular respiration of yeast.
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LITERATURE CITED
Adajar, Alcabedos, Dela Vina, Duka, Gonzales, Lado, … Rodriguez. (2018). Designing
experiments. In Bio 11.1 laboratory manual: Investigations in college
Biology (1st ed., pp. 74-75).
Cortes, R. (2015). The effect of nature of substrate on the rate of respiration on Yeast
(Saccharomyces cereviciae). Retrieved from
https://www.academia.edu/18073312/The_Effects_of_Nature_of_Substrate_on_t
he_Rate_of_Cellular_Respiration_on_Yeast
Crispino, M. (n.d.). Cellular respiration of Yeast. Retrieved from
https://www.academia.edu/4268545/Cellular_respiration_of_Yeast_Scientific_Pa
per
Khare, S. K. (2007). Structures and functions of biomolecules. Retrieved from
Researchgate website:
https://www.researchgate.net/publication/237577555_Structures_and_Functions_
of_Biomolecules
McGinnis, M. R., & Tyring, S. K. (1996). Introduction to Mycology. In Medical
microbiology(4th ed.). Retrieved from
https://www.ncbi.nlm.nih.gov/books/NBK8125/
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SRI International. (2010). MetaCyc pathway: Sucrose degradation V (sucrose αglucosidase). Retrieved from https://biocyc.org/META/NEWIMAGE?type=PATHWAY&object=PWY66-373
Tornheim, K., & Ruderman, N.B. (2017). Chapter 2 Intermediary Metabolism of
Carbohydrate , Protein , and Fat.
Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Reece, J. B. (2017).
Cellular respiration and fermentation. In Campbell Biology [Adobe Digital
Editions] (11th ed., pp. 165-168).
Zunszain, P. A., Ghuman, J., Komatsu, T., Tsuchida, E., & Curry, S. (2003). Crystal
structural analysis of human serum albumin complexed with hemin and fatty
acid. BMC Structural Biology, 3(6), 1-2. Retrieved from
http://www.biomedcentral.com/1472-6807/3/6