Polytechnic University of the Philippines

Sta. Mesa Manila

College of Science
Department of Biology

BIOL 30085
General Physiology
Laboratory Activity no. 2

ANAEROBIC REACTION IN FERMENTATION

Artap, Nina Kiana D. | Baao, Louisse May M. | Jurado, Trisha Mei R. | Sase, Paul Andre T. |
Taquiqui, Arvin T. | Tolentino, Mark Lauren I.
______________________________________________________________________________

Abstract:
Fermentation is not the only process to acquire energy in the absence of oxygen
or anaerobically. Some organisms make use of inorganic molecules as a final electron
acceptor for an electron transport chain. This process is called anaerobic cellular
respiration and is commonly performed by bacterial organisms. The experiment
demonstrates the process of fermentation within yeast and sugar and how anaerobic
reactions occur within the use of limewater. To demonstrate the process of fermentation
in yeast and sugar, the proponents of the study made use of five plastic bottles capped
with balloons containing tap water with 1 tsp. of white sugar, boiled water containing 2
tsp. of white sugar and 2 ¼ tsp. of yeast, warm water containing 2 ¼ tsp. of yeast, warm
water containing 3 tsp. of white sugar and 2¼ tsp. of yeast, and tap water containing 1
tbsp. of flour and 2 ¼ tsp. of yeast, respectively. In this experiment, proponents concluded
that the inflation of the balloons are dependent on the content of the bottle. To
demonstrate the capability of yeast to respire without air, the proponents used yeast with
Limewater and observed after two (2) ½ hours, respectively, which was divided into two-
part observation, hence one (1) hour and fifteen (15) minutes each. The utilized oil
prevented the diffusion of air into the yeast in the glucose solution that ensured the
complete anaerobic respiration. The final part of the activity involved the production of
yogurt after twenty-four (24) hours of refrigeration. Lactic acid was produced by a
bacteria called Lactobacillus bulgaricus that gives a yogurt its distinct flavor, another
bacteria found in the yogurt was Streptococcus thermophilus that reduced the firmness
and improved viscosity, and water retention. The sour taste of yogurt caused by the lower
pH that coagulated and precipitated, formed the solid curd that made up the yogurt.

Keywords: anaerobic respiration, fermentation, yeast, anaerobic cellular respiration

______________________________________________________________________________

1. Introduction foods and beverages (Shurtleff & Aoyagi,

Fermentation already exist centuries 2020). At first, they used fermentation mainly
ago as people in Neolithic times had been to make bread rise and for beers. After
practicing this process to create variety of thousand more years, Chinese used

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
fermentation to derive food products such as Today, there are machineries and new
soybean and tea into medicine that could treat techniques done by various companies to
several illnesses primarily on skin diseases. produce products like antibiotics and sell it on
After few more centuries, Theodore Schwann the market. In this activity, students would like
concluded that fermentation is a product of to fully understand fermentation by
living things which was then supported by demonstrating the reaction of yeast using
Louis Pasteur who said that fermentation is limewater and making a lab-made yogurt.
caused by yeast. There were no written Throughout the activity, the students observed
documents who discovered and invented experimentation and recorded the data for
fermentation but throughout the years, further analyzation.
promising products were made including
breads, beers, several condiments, dairy
products, and other delicacies (Coyle, 2019). 2. Methodology

Few more years later in 1907, a German 2.2 Fermentation of Yeast and Sugar
chemist was awarded a Nobel Prize Award in In this activity, 5 plastic bottles, white

his discovery on cell-free fermentation, yeast sugar, yeast 5 balloons, a plastic funnel,
enzymes (Famous Scientists, 2017). After graduated cylinder and tap water were prepared
almost two decades, scientists Hans von Euler- for the experiment.
Chelpin and Arthur Harden were awarded the First, the plastic bottles were labeled 1-

same award for elaborately explaining how 5 to make the procedure easier. Then, a 2 ¼
fermentation work (The Nobel Prize, 2020). teaspoon of yeast were added to the 2nd and
This process, with the help of enzymes, 5th bottles using the plastic funnel. Afterwards,
transforms large organic compounds into a teaspoon of white sugar was added to the 1st
smaller compounds. Enzymesbeing bottle, two for the 2nd bottle and three for the
are
produced by several microorganisms like 4th bottle. Then, a tablespoon of flour was
bacteria, molds, and yeasts which undergo in added to the 5th bottle. Next, a cup of tap water
the process of hydrolysis (Shurtleff & Aoyagi, or more specifically 100ml of water was
2020). poured into the bottles; with boiling water for
the 2nd bottle and warm water for the 3rd and
4th bottles.
Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
After mixing the solution, balloons were placed observations concerning the changes that
at the mouth for each water bottle and was happened with the limewater were recorded.
observed for 3hrs with every change recorded 2.3 Yogurt Making
by the hour. With this activity, materials including a
2.2 Anaerobic Reaction of Yeast using big stainless casserole pot, unflavored yogurt, a
Limewater wooden spoon, 2 clean towels, a plastic
The experiment made use of white container with a lid, 2L of fresh full fat milk
sugar, yeast, distilled water, vegetable oil, a and a hot plate/ stove were prepared.
beaker/flask, test tube, a rubber stopper, rubber First, 2L of milk were poured into a casserole
tubes, a container with a lid and calcium oxide pot and was placed on a hot plate or stove
(lime). under high flame. This was left to boil and was
Prior to the experiment, the limewater was stirred occasionally with the wooden spoon.
prepared beforehand because the solution When the milk reached its boiling point, it was
needed to sit overnight. left to boil for another 10 minutes but with the
In preparing the limewater, 1L of flame brought down to a medium heat. The
distilled water and a teaspoon of calcium oxide milk was stirred with the spoon more often
were mixed together in a container with a lid than before. A big, clean and warm towel was
and this mixture was left to sit overnight. placed on a flat surface with the plastic
In doing the actual experiment, 10g of white container placed on top. After 10 minutes, the
sugar, 10g of yeast and 120ml of distilled water milk was removed from the flame and was
were mixed in a flask. Then, 5ml of vegetable placed in the plastic container. The milk was
oil was poured on top of the solution and was left to sit for 20-25 minutes till it cools down.
covered with a rubber stopper with its tip The temperature of the milk was checked by
dipped in the solution. placing a pinky finger in the milk and keeping
Next, a rubber tube was connected to it there for 8 seconds and when a burning
the rubber stopper with its end placed inside a sensation was felt on the finger upon its
beaker containing 150ml limewater. Finally, removal it just means that the milk is ready.
the flask containing the yeast solution was A small amount of milk was mixed to
placed in warm water. This set-up was left the yogurt culture before pouring it to the milk.
undisturbed for at least 2 ½ hours. The Once poured, the solution was mixed once or

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
twice and a clean smaller towel was placed on
top of the mixture before placing the
container’s lid. After sealing it with the lid, the
bigger towel was wrapped over the container
and was left undisturbed for 4 ½ hours. After 4
½ hours, the towel covering the container was
unwrapped and the lid was removed. This was
left for another 30 minutes uncovered and
undisturbed. After half an hour, the yogurt was
transferred to a fridge and was left there for
24hrs uncovered.

3. Results and Discussion Figure 1.2. Bottle 1; tap water containing 1

tsp. of white sugar after 2 hrs.
3.1 Fermentation of yeast and sugar

Figure 1.1. Bottle 1; tap water containing 1 Figure 1.3. Bottle 1; tap water containing 1
tsp. of white sugar after 1 hr. tsp. of white sugar after 3 hrs.

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
Figure 2.1. Bottle 2; boiled water containing 2 Figure 2.3. Bottle 2; boiled water containing 2
tsp. of white sugar and 2 ¼ tsp. of yeast after 1 tsp. of white sugar and 2 ¼ tsp. of yeast after 3
hr. hrs.

Figure 2.2. Bottle 2; boiled water containing 2 Figure 3.1. Bottle 3; warm water containing 2
tsp. of white sugar and 2 ¼ tsp. of yeast after 2 ¼ tsp. of yeast after 1 hr.
hrs.

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
 Figure 3.2. Bottle 3; warm water containing 2 Figure 4.1. Bottle 4; warm water containing 3
¼ tsp. of yeast after 2 hrs. tsp. of white sugar and 2¼ tsp. of yeast after 1
hr.

Figure 3.3. Bottle 3; warm water containing 2 Figure 4.2. Bottle 4; warm water containing 3
¼ tsp. of yeast after 3 hrs. tsp. of white sugar and 2¼ tsp. of yeast after 2
hrs.

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
 Figure 4.3 Bottle 4; warm water containing 3
Figure 5.2 Bottle 5; tap water containing 1
tsp. of white sugar and 2¼ tsp. of yeast after 3
tbsp. of flour and 2 ¼ tsp. of yeast after 2 hrs.
hrs.

Figure 5.1 Bottle 5; tap water containing 1 Figure 5.3. Bottle 5; tap water containing 1
tbsp. of flour and 2 ¼ tsp. of yeast after 1 hr. tbsp. of flour and 2 ¼ tsp. of yeast after 3 hrs.
Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
 The figures showed the five (5) bottles mixture of yeast and flour) due to the flour
observed after one (1), two (2), and three (3) being composed of starch, which is a
hours duration which contained tap water with polysaccharide that needs to be broken down
1 tsp. of white sugar, boiled water containing 2 into its glucose monomer to act as a substrate
tsp. of white sugar and 2 ¼ tsp. of yeast, warm which, without anything to break down, would
water containing 2 ¼ tsp. of yeast, warm water yield the lowest amount of Carbon dioxide
containing 3 tsp. of white sugar and 2¼ tsp. (CO2) produced (Joe, 2003). The greatest rate
of yeast, and tap water containing 1 tbsp. of of balloon inflation is seen on the fourth bottle,
flour and 2 ¼ tsp. of yeast, respectively. as the ingredients under it (yeast, sugar and
warm water) are the ideal set of substances that
The observation of yeast by Anton van
will allow more production of CO2 due to it
Leeuwenhoek has led to numerous applications
having the greatest number of sugar level that
of the fungus that has, since then, produced
the yeast would consume and would therefore
numerous alcoholic products and a more
generate a greater level of CO2 production.
efficient way of the production of bread. More
scientists have since then discovered
fermentation, but our modern understanding of 3.2 Anaerobic reaction of yeast using
the role of yeast in this process is accredited to limewater
Louis Pasteur, who demonstrated
experimentally that fermented beverages result
from the action of living yeast transforming
glucose into ethanol (Alba-Lois & Segal-
Kischinevzky, 2010).

The experiment has demonstrated this

effect. The first bottle did not have any changes
in the balloons as its component are only sugar
and have nothing to react to. The second, third,
fourth and fifth bottles have their balloons all
Figure 6.1 Anaerobic reaction of Yeast
inflated but with varying sizes, the smallest using Limewater observed after first 1 hour and
inflated balloon present in the 5th bottle (a 15 minutes

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
 3.3 Yogurt making

Figure 6.2 Anaerobic reaction of Yeast using

Limewater observed after second 1 hour and 15
minutes Figure 7. Yogurt made after 24 hrs.
Figures 6 showed the anaerobic
reaction of Yeast using Limewater observed Figure 7 showed the yogurt made after

after two (2) ½ hours, respectively, which was twenty-four (24) hours of refrigeration. It was
divided into two-part observation. Hence one observed that milky and watery substance were
(1) hour and fifteen (15) minutes each, as formed on the upper surface of the yogurt. As
shown on figures 6.1 and 6.2 respectively. described by Sugar (2018, April 18), the thin
layer of clear liquid at the top of the yogurt is
This experimentation demonstrated the
known as whey. Although it consists mainly of
capacity of yeast to respire without air,
water, whey also contains a little bit of protein,
showing the ability of both being an aerobic
potassium, and calcium. Stirring in the whey
and anaerobic fungi. The set up utilized oil to
not only adds nutrition to your yogurt but also
prevent the diffusion of air into the yeast in the
offers a creamier consistency.
glucose solution (Gillam, 2015) that will
ensure complete anaerobic respiration. The Mark (n.d.), explained the formation of

lime water turned cloudy due to the presence of yogurt in his study. According to him, Yogurt
Carbon dioxide (CO ) that have been released is made by the fermentation of lactose by
2

by the consumption of the glucose solution of bacterial enzyme in the process of anaerobic
the yeast. This gas will in turn react with the respiration. Lactose is broken down by lactase
calcium hydroxide and will then form calcium enzyme produced by bacteria which produces
carbonate that is insoluble in water and will lactic acid and acetaldehyde which gives the
produce a milky white precipitate. yogurt its distinct flavor. These chemicals
lower the pH of the milk causing it to have a
Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
sour, tart taste. The lower pH also affects the set of subtance or components that are in the
casein or milk protein, causing it to coagulate bottle.
and precipitate, forming the solid curd that
The experiment concerning the aerobic
makes up yogurt.
and anaerobic reaction of yeast shows that it
Bacteria found in yogurt includes, S. demonstrated the capacity of yeast to respire
thermophilus as the starter culture for yogurt without air, showing the ability of both being
production which reduces the firmness and an aerobic and anaerobic fungi. The utilized oil
improves viscosity, water retention, and the prevented the diffusion of air into the yeast in
mouthfeel of yogurt and L. bulgaricus which the glucose solution that ensured the complete
produces lactic acid used to preserve milk. anaerobic respiration.
(Botello, 2016, November 18).
Yogurt was a fermentation of lactose by
bacterial enzyme in the process of anaerobic
4. Conclusion
respiration. Lactic acid was produced by a
In this activity, different experiment bacterium called L. bulgaricus that gives a
and methods were conducted and used to yogurt its distinct flavor, another bacteria
observed the role of yeast on fermentation and found in the yogurt was S. thermophilus that
identifying the aerobic and anaerobic reaction reduced the firmness and improved viscosity,
of yeast using Limewater as well as describing and water retention. The sour taste of yogurt
the fermenttion of yogurt. caused by the lower pH that coagulated and

In observation of yeast, the experiment precipitated, formed the solid curd that made
shows that every bottle that has yeast their up the yogurt.
balloon inflated with varying sizes. The biggest
inflated balloon was bottle 4 due to its ideal set
of substance that allowed more production of
CO₂ that has the greatest number of sugar level
that the yeast consumed while the bottle 1 did
not inflate because it has nothing to react with.
In this experiment, it simply shown that the
inflation of the balloon was dependent on the

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP
References:
Aerobic and Anaerobic Respiration. Retrieved January 12, 2020, from
https://www.bbc.co.uk/bitesize/guides/zm6rd2p/revision/3
Alba-Lois, L. & Segal-Kischinevzky, C. (2010) Beer & Wine Makers. Nature Education 3(9):17
Anonymous. (n.d.). Tests for Gases. Retrieved January 12, 2020, from
https://chemstuff.co.uk/analytical-chemistry/tests-for-gases/
Blow Up a Balloon with Yeast. Retrieved January 11, 2020, from https://sciencebob.com/blow
-up-a-balloon-with-yeast/
Botello, Evangelina. (2016, November 18). Yogurt Fermentation Lab. Retrieved from
https://www.coursehero.com/file/18914481/Yogurt-Fermentation-Lab/
Corriher, S. (n.d.) Yeast's Crucial Role in Breadbaking. Retrieved January 12, 2020, from
https://www.finecooking.com/article/yeasts-crucial-roles-in-breadbaking
Coyle, D. (2019, January 15). Health Line. Retrieved from What Is Fermentation? The Lowdown
on Fermented Foods: https://www.healthline.com/nutrition/fermentation
Famous Scientists. (2017, April 15). Retrieved from Eduard Buchner:
https://www.famousscientists.org/eduard-buchner/
Jessup, N. (2014, November 9). Yeast and Sugar. Retrieved January 11, 2020, from
https://foodchemblog.com/yeast-and-sugar/
Joe, B. B. (2003). Effects of Various Carbohydrates on Fermentation. Retrieved January 12,
2020, from http://www.utsc.utoronto.ca/~pnda/SR/BGYA01/Fermentation.pdf
Mark, Neil. (n.d.). Flavored Yogurt. Retrieved from
https://www.scribd.com/doc/254690658/Yogurt-Lab-Report
Rembac, J. (2010, September 1). Growing Yeast: Sugar Fermentation. Retrieved from
https://www.education.com/science-fair/article/biology_foamy/
Shurtleff, W., & Aoyagi, A. (2020). Soy Info Center. Retrieved from A Brief History of
Fermentation, East and West: https://www.soyinfocenter.com/HSS/fermentation.php
Sugar, Jenny. (2018, April 18). Why the Liquid on Top of Your Yogurt Matters. Retrieved from
https://www.popsugar.com/fitness/What-Liquid-Top-Yogurt-7660510
The Accidental Science of Cooking. (n.d.). Yeast Air Balloons. Retrieved January 12, 2020,
from https://www.exploratorium.edu/cooking/bread/activity-yeast.html
The Nobel Prize. (2020). Retrieved from Hans von Euler-Chelpin:
https://www.nobelprize.org/prizes/chemistry/1929/euler-chelpin/facts/

Contributions:
Abstract/ Keywords/Title: TOLENTINO
Introduction BAAO
Methodology: JURADO
Results and Discussion: TAQUIQUI, SASE
Conclusion/References: ARTAP