Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

PROTOTYPE MODEL OF THE DEMONSTRATION OF ALCOHOLIC

FERMENTATION

Garcia Alfred Goran1*, Getuiza Irah Mae1, Guevarra Gwyneth Ysabelle1, Ibarra Jelean
Ira1, Javier Kate Angela1, Lim Chelsea1, Lopez Hillary Joyce1, And Lucas Ruinna1
1
Senior High School & Group Four of Grade 12-Health Allied 2, University of Santo Tomas, Manila, Philippines
Corresponding author e-mail: [email protected]

ABSTRACT
Anabolic Metabolic Process or simply, Anaerobic Respiration is the counterpart of Aerobic
Respiration and it takes place when there is no oxygen. There are two types: Lactic Acid Formation and
Alcohol Fermentation, in which the two differ with the products of their process and elements involved.
The experiment shows the fermentation of sugar through the yeast and application of anaerobic in a simple
way. The set up consists of two bottles: the fermentation bottle, consisting of the water, sugar, yeast mixture
and the air exclusion bottle which consist of the water, vegetable oil mixture. When one is sealed off with
a clay and the latter open to air. They are connected with a straw, that allows gas to move into the
fermentation bottle. Upon final observation, the fermentation bottle’s color became vivid (golden yellow
liquid) and less turbid as the components of the mixture settled at the bottom of the container. Less bubbles
are observed in comparison with the initial observation. This can be explained as the release of 𝐶𝑂# . As
for the Air Exclusion Bottle, the only change present was the presence of bubbles in the oil bed. This can
be explained as the transfer of 𝐶𝑂# from the Fermentation bottle, reached this container. In conclusion, the
experiment showed Alcoholic / Ethanol Fermentation and its emphasis on the effect of the release of 𝐶𝑂# to
the Fermentation Process. The process starts when the sugar breaks down the yeast cells and releases 𝐶𝑂# as
its byproduct, once the energy from the yeast cells are broken down it settles at the bottom of the container,
thus the final observation presents and the final stage of the fermentation process in the experiment.

Keywords: Carbon Dioxide, Ethanol Fermentation, Yeast

INTRODUCTION
Anabolic metabolic process in the cell occurs when there is no oxygen process. According to
Biology Dictionary (2017), this process starts with the glycolysis in order to break down the glucose into
two molecules of pyruvic acid. To follow, pyruvic acid is broken down further into acetaldehyde, which it
is used to produce ethanol, and carbon dioxide. Lactic acid is formed due to the absence of oxygen which
acts as the acceptor of hydrogen. Fermentation happens once the NADH does not enter the hydrogen and
electron transport chain.[1] Yaneff (2017) stated that as an application in real-life, Alcohol Fermentation,
also known as Ethanol Fermentation, is the anaerobic pathway carried out by yeasts in which simple sugars
are converted to ethanol and carbon dioxide. The process of fermentation involves Brewer’s Yeast or
Baker’s Yeast (Saccharomyces Cerevisiae) which is a species of yeast wherein Saccharo means sugar and
myces means yeast or fungi, which allows the breakdown of sugar even in the absence of oxygen and results
in byproducts that humans benefit from.[2] But fermentation does not only applied in the making of beer,
Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

wine, and alcohol because according to Kimbrough (2000), other examples just like in the conversion of
milk to cheese and the making of bread also shows the fermentation of carbohydrates by the use of yeast.[3]
With this experiment, sugar or water mixture will be fermented just by using an ordinary baker’s
yeast. Through this, the students aims to achieve the follow objectives: (a) to make use of yeast and sugar
to effect a fermentation in a simple way, (b) to compare the fermentation bottle and air exclusion bottle in
terms of its color, turbidity, transparency, presence of bubbles, and odor before and after the experiment,
(c) to classify the differences and similarities between aerobic and anaerobic fermentation if the substrate
used is glucose, (d) to distinguish the significance of oil in an aerobic respiration and (e) to determine other
methods on the production of gas by the fermentation of yeast.
Just by the use of simple products at home simple fermentation can be done. Based from
Thaweboon and Tri (2011), the use of these various dietary sugars is essential in producing organic acids
which are vital in the fermentation process. This acids act as catalyst in fermented products. All test
microorganisms could ferment sucrose, glucose, fructose and trehalulose to pH lower than 5.5. [4] Buehler
(2014) states that sugar can speed up the rate of fermentation up to three percent. This is caused by the
process when the sugar starts to break or dehydrate the yeast cells which is called the crenation. [5] Hewitson,
J. And Hill, C. (n.d.) States that Energy is required in yeast in its growth and development, during
fermentation, sugar delivers energy for the yeast. Yeast uses oxygen to produce energy from sugar. This is
called respiration. The greater quantity of sugar, the more active the yeast will be. [6] On the other hand,
shortage in oxygen will result the yeast to produce alcohol and carbon dioceses as byproducts.
Through this experiment the people will have an overview or projection of the Anaerobic
Respiration.

MATERIALS AND METHODS

In order to perform the experiment the following materials are needed: (a) two (2) 500mL
clean and dry plastic bottles (as fermentation bottle and air exclusion bottle), (b) 85mL distilled
water, (c) 2 tablespoons of sugar (sucrose), (d) 30mL vegetable oil, (e) bendable plastic drinking
straws, (f) modeling clay or tape, and (f) 1 teaspoon of yeast. In doing the experiment, proper steps
and process must be observed to ensure that the set-up will be a success. First, the researchers should label
the bottles, one must be labeled as fermentation bottle or FB and the other is labeled as the air exclusion
bottle or AEB. Second, they must put 85 mL of distilled H2O or water to the fermentation bottle. And add
2tbsps of sugar and gently swirl until the sugar is completely dissolved (refer to Figure 1). Third, they must
add 1tsp of yeast into the fermentation bottle, which contains the sugar and water. Afterwards, they must
swirl it for about 30 seconds. Furthermore, they must make sure that the yeast is not completely dissolved,
light swirling is advised (refer to Figure 2). Fourth, they must put 150 mL of distilled H2O into the air
exclusion bottle. Afterwards, 30mL vegetable oil must be added until there is a layer of oil on top of the
water that is approximately 0.5 - 1 inch (1 - 3 cm) thick. Fifth, the bottles must be placed next to each other
and assembled with the bendable drinking straws (refer to Figure 3). Another is that they must ensure that
the end of the straw is inserted into the fermentation bottle is only 7-8 cm from the top of the bottle above
the level of the yeast, sugar and water mixture. The straw should be inserted into the air exclusion bottle
such that it is well below the oil level, submerged into the water layer, although not touching the bottom of
the bottle. Sixth, the straws must be adjusted and trimmed if needed to get the right measurement of straw.
Seventh, the straws must be joined by sealing it with modelling clay, and the seal must be airtight.
Furthermore, the straws must be placed into the bottles, and the modelling clay or tape must be used to seal
the straw into the fermentation bottle. The researchers should make sure that it is an airtight seal (refer to
Figure 4). While the other bottle that contains water and oil will be left open to the air. Lastly, the researchers
must place the fermentation set-up somewhere where it won’t be disturbed and it will neither be exposed
Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

to extreme heat or extreme cold. Fermentation needs to proceed for 5 - 7 days. After 7 days, check the set-
up again and observe for changes or difference of the set-up from the previous week (refer to Figure 5).

RESULTS AND DISCUSSION

Results
Table 1. Characteristics of the Fermentation Bottle on the initial and final observation.
Initial Observation Final Observation
Color Pale Yellow/Yellowish White Golden Yellow Liquid
Turbidity More turbid Less turbid (components of the
bottle has settled on the bottom
of the bottle)
Transparency Hazy, Opaque Clearer
Presence of Bubbles Presence of bubbles is observed Less bubbles are observed
Odor Smells like alcohol or wine Smells like alcohol or wine (no
change)

Before the fermentation, the liquid inside the fermentation bottle is hazy and opaque and has pale
yellow color. After the fermentation, the liquid was clearer, and the color turned into a pale golden yellow.
Settlement of the particles in the mixture was also observed after fermentation. The components inside the
bottle had contained more bubbles inside on the initial observation compared to the final observation.
However, the odor that has a smell of alcohol or wine, remained the same from before to after fermentation.
(Table 1)

Table 2. Characteristics of the Air Exclusion Bottle on the initial and final observation.
Initial Observation Final Observation
Color Water is colorless; Oil is golden Water is colorless; Oil is golden
yellow yellow
Turbidity Not turbid (oil floats on water) Not turbid (oil floats on water)
Transparency Transparent Transparent
Presence of Bubbles No presence of bubbles is Presence of bubbles are observed
observed
Odor Odorless Odorless

On the other hand, the air exclusion bottle remained the same, it has still the same color of the oil
and water. It also remained transparent and odorless just like before fermentation. However, there is a
presence of bubbles in both oil and water after the fermentation. (Table 2)

Discussion
Aerobic respiration and fermentation are both multi-processes that utilize the transfer of energy
stored in the chemical bonds of glucose to bonds in adenosine triphosphate. This energy stored in ATP can
now be used to perform cellular work which is to provide energy for biosynthetic reactions such as active
transport and growth and repair mechanisms. Furthermore, all organisms use both aerobic respiration and
fermentation to produce ATP and fuel various cell processes. The main difference between the two is that
aerobic respiration requires oxygen while fermentation occurs without oxygen. Furthermore, aerobic
respiration yields 38 ATP while fermentation produces 2 ATP only. This is mainly because oxygen aids in
generating ATP, a component the other process lacks. On the other hand, fermentation produces only 2
ATP molecules because yeast lacks enzymes which are essential to metabolize ethanol, storing much of the
energy in the molecules of ethanol because the process cannot produce additional ATP but only recycles
the NAD+ produced in glycolysis (Pfeiffer & Morley, 2014). [7]
Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

As the yeast fermentation begins, the pyruvate underwent decarboxylation to release carbon dioxide
and retain ethanol in the fermentation bottle. The bubbles present in the FB signifies that 𝐶𝑂# was already
released. Since there is a pathway from the fermentation bottle to the air exclusion bottle using straws, the
gas molecules went upward and travelled to the AEB. It helped to show the evidence of 𝐶𝑂# , because
releasing 𝐶𝑂# from the FB indicates that the fermentation already happened. Moreover, 𝐶𝑂# will not be
released outside since the setup was enclosed tightly. As the 𝐶𝑂# reached the AEB, it bubbled up with the
oil and water. The significance of oil is preventing any air to enter the AEB and also preventing water to
freely evaporate throughout the process. When you place water in an open container, moving air would
draw water away from the surface area above the water resulting to evaporation. It will then be replaced by
drier air and the evaporation increases. But when vegetable oil, an impermeable liquid, is poured, a layer
of oil will float on the surface and will prevent water molecules from making contact with air (Milner,
2017). [8] The presence of bubbles to both bottles indicates also the presence of 𝐶𝑂# . Bubbling occurred
because of the intracellular 𝐶𝑂# produced which may be secreted by the pressure through the cell wall of
yeast that affected the pressure homeostasis. Under diminished pressure of the external environment, the
bubbles become visible in centimeter size and would appear vigorously in the products of fermentation.
The pressure then keeps the bubble size at a minimum to lower the chances of having adverse effects on
cell function (Coetsee, et al., 2012). [9]
Presence of bubbles in both oil and water is observed after the fermentation. However, this was
observed after a couple of hours after arranging the set up but became unobservable during post observation.
A study of the impact of changes including fermentation time of plantain pseudo-substrate generated that
variations in fermentation period greatly affects the percentage composition and volume of ethanol,
showing that the best fermentation period for obtaining optimal production of ethanol is after 96 hours
(Amadi & Ifeanacho, 2016). [10]
A similar experiment was done, entitled "Rise to the Occasion: Investigating Requirements for
Yeast Fermentation", which uses almost the same setup in this experiment. One bottle contained the yeast
and sugar mixture with oil on top to prevent oxygen from reaching the yeast. A straw is then secured on the
opening of the bottle then sealed airtight. The straw extends to second bottle filled with limewater. The
opening of the straw should be submerged into the limewater. The objective of the experiment is to detect
the production of carbon dioxide. When the yeast mixture releases carbon dioxide, the limewater should
turn milky white due to chemical reaction, therefore confirming the presence of carbon dioxide. (Science
Buddies, n.d.). [11] Another experiment utilized the same set-up, but with water on the other bottle and no
oil on both bottles. When carbon dioxide is present on the fermentation bottle, bubbles should be seen
coming out from the straw on the bottle filled with water. (Gunther & Rosilas, n.d.) [12]

CONCLUSION

The experiment performed clearly portrayed the Anaerobic respiration specifically alcoholic
fermentation, with the objective to use yeast and sugar to create fermentation and to compare the
fermentation bottle and air exclusion bottle without the presence of oxygen. The set-up was able to
successfully exhibit the properties of alcoholic fermentation.
Fermentation is the process of chemical breakdown of substances, often through the use of simple
products at home this process could be demonstrated. In the experiment, alcoholic fermentation was
executed with the aid of yeast that served as the catalyst for the reaction to occur. When the yeast
fermentation process began, 𝐶𝑂# or carbon dioxide is generated in the other container which contained the
water and oil through the tiny bubbles that it released. In the set-up, the gas moves through the straw
however, the presence of oil prevents the air from entering the system.
For the future researchers, it is recommended that they ensure that the methods are strictly followed
so that the experiment would clearly demonstrate alcoholic fermentation. One factor that greatly affects the
success of the experiment is on how they sealed the fermented bottle. Since the experiment is all about
Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

anaerobic respiration, there should be no presence of oxygen inside the fermented bottle. Another factor is
the environment where the experiment will be placed for one week. If the temperature is too high or too
low, the fermentation will not occur.

DOCUMENTATION

Figure 1.

Figure 2.
Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

Figure 3.

Figure 4.
Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

Figure 5. The state of the set-up after 7 days.

REFERENCES
[1] Biology Dictionary (2017). Anaerobic Respiration - Definition, Types, Examples. Retrieved from
https://biologydictionary.net/anaerobic-respiration/

[2] Yaneff, J. (2017). Nutritional Yeast Nutrition Facts, Health Benefits & Recipes. Retrieved from
https://www.doctorshealthpress.com/food-and-nutrition-articles/nutritional-yeast-nutrition/

[3] Kimbrough, D. (2000). Fermentation Distillation. Retrieved from https://www.scribd.com/document/

236567842/8-Fermentation-Distillation

[4] Thaweboon, B. & Tri D. (2011). Fermentation of various sugars and sugar substitutes by oral
microorganism. Retrieved from http://www.apjtb.com/zz/2011s2/24.pdf

[5] Buehler, E. (2014). Yeast and Sugar. Retrieved from http://foodchemblog.com/yeast-and-sugar/

[6] Hewitson, J. & Hill, C. (n.d.). How does sugar affect yeast growth? Retrieved from
http://www.saps.org.uk/saps-associates/browse-q-and-a/169-q-a-a-how-does-sugar-affect-yeast- growth

[7] Pfeiffer, T., & Morley, A. (2014). An evolutionary perspective on the Crabtree effect [Abstract].
Frontiers in Molecular Biosciences, 1. doi:10.3389/fmolb.2014.00017

[8] Milner, W. (2017, April 24). How to Stop Water from Evaporating. Retrieved from https://sciencing.
com/stop-water-evaporating-8719492.html

[9] Coetsee, E., Dithebe, K., Kock, J. L., Pohl, C. H., Swart, C. W., Swart, H. C. & Wyk, P. W., (2012).
Gas bubble formation in the cytoplasm of a fermenting yeast. FEMS Yeast Research, 12(7), 867-869.
doi:10.1111/j.1567-1364.12004.x
Laboratory Exercise # 4 Anaerobic Respiration Fermentation (Enabling PeTa)

[10] Amadi, P., & Ifeanacho, M. (2016). Impact of changes in fermentation time, volume of yeast, and mass
of plantain pseudo-stem substrate on the simultaneous saccharification and fermentation potentials of
African land snail digestive juice and yeast. Journal of Genetic Engineering and Biotechnology, 14(2), 289
297.gdoi:10.1016/j.jgeb.2016.09.002

[11] Rise to the Occasion: Investigating Requirements for Yeast Fermentation. (n.d.). Retrieved from
https://www.sciencebuddies.org/

[12] Gunther H., & Rosilas, M. (n.d.). Comparison of alcohol fermentation performance with different
glucose content. Retrieved from https://www.ncbi.nlm.nih.gov/m/pubmed/24122709/