Fermentation Under Different Temperatures

October 2023
Introduction
Living organisms require energy to survive, however, their way of attaining it can vary.
Some organisms go through cellular respiration and glycolysis, and others go through the process
of fermentation. The different ways of attaining energy determine certain characteristics of the
organism, for example, bacteria and fungi obtain energy by fermentation, break down
carbohydrates with the help of enzymes in the absence of oxygen, and, consequently, produce a
large quantity of CO2 depending on the temperature of the environment.
Fermentation is a biological process to obtain energy that bacteria and fungi cells
undergo, this process is a type of anaerobic cellular respiration, meaning that the process occurs
in the absence of oxygen. It begins with glycolysis, breaking down glucose into two pyruvate
molecules that produce two ATP and two NADH. It starts off with a glucose molecule and two
ATP molecules power the process to break down and rearrange the glucose molecule into two
three-carbon and one phosphate molecules. Then, two phosphates join with the molecule turning
it into a three-carbon two phosphate, while two NAD+ gain energy to attach with hydrogen ions,
creating two NADH and 2H+. Finally, four ADPs come to rearrange the molecule and utilize the
phosphates to make four ATP molecules, leaving the molecule as two pyruvates.
Once glycolysis happens, the fermentation can go either two ways, to produce lactic acid
or ethanol in an alcoholic fermentation. For the lactic acid, the pyruvate molecules help
regenerate the NAD+, going through lactate dehydrogenase, creating the lactate molecule. On
the other hand, alcoholic fermentation has two pyruvate molecules that lose 2 CO2, turning into
two Acetaldehyde molecules. These molecules go through a chemical reaction, producing the
final two ethanol molecules.
The fermentation allows the glucose to be continuously broken down due to the recycling
of NADH to NAD+. An example of this is in muscle cells, with an excess of exercise and lack of
oxygen, the cells start to ferment causing fatigue and producing a waste product called lactic
acid, which is used to break down carbohydrates.
The fermentation process helps organisms to attain energy due to the arrangement and
movement of the molecules. This causes the release of energy from the bonds of the molecules,
helping ADP and NAD+ to join with a phosphate group or hydrogen ion.
Yeast plays a crucial role in fermentation, it's used to start the process, and it modifies the
carbohydrates into several different metabolic products. The yeast turns sugar into alcohol and
carbon dioxide, which gives origin to the substance’s taste, texture, and aroma. However, when
utilizing yeasts, it needs to be careful due to its sensitivity to temperature. The activity of yeast
relies on temperature to make it more or less efficient when going through the process, with a
higher activity caused by the right temperature, carbon dioxide production increases achieving
maximum efficiency.
Temperature is a very sensitive variable in fermentation, depending on the temperature,
the process can either be very successful or cause death to the cells. If the temperature is too hot,
around 40 degrees Celsius, the yeast cells can be overworked, which could lead to cell death. If
it’s too cold, around 0-10 degrees, the yeast cells become lethargic, bringing a slow fermentation,
with little energy being produced. However, if the temperature is right in the middle, around
20-30 degrees, the yeast cells thrive and are able to efficiently go through the fermentation
process.
CO2 production throughout fermentation can vary depending on the temperature of the
environment. With a higher efficiency of fermentation, the CO2 production increases due to the
higher rates of breaking down of carbohydrates. Throughout the fermentation process, CO2 is
produced when there is oxidation in the carbon molecules. The production of CO2 during
fermentation starts with glycolysis, then it goes through Pyruvate Decarboxylation, which is
when a pyruvate acid, a product of glycolysis, is converted into acetaldehyde after going through
decarboxylation, the result of this is the production of CO2.
The most commonly used yeast is Saccharomyces cerevisiae which is a fungi that
converts sugars into CO2 and ethanol. This type of yeast is utilized in wine and beer making,
baking, and brewing. Saccharomyces cerevisiae is good for making bread or beer due to the
quick production of ethanol and CO2 helping the bread to be fluffy and full of air pockets, and
beer to become alcoholic.
The fermentation process is crucial to several biological systems for cells to obtain
energy by breaking down carbohydrates. Fermentation is a sensitive process, where several
different factors can affect it, especially temperature. Depending on the temperature of where the
process takes place, can drastically change the production rate of CO2, and the efficiency of the
fermentation. In conclusion, this process is significant for many biological processes such as
muscle cells, yeast fermentation, methanogenesis, and bacterial fermentation.
Testable Question:
How does temperature affect the production of CO2 during the process of fermentation?

Hypothesis:
During the process of fermentation, if the temperature increases, then the production of CO2 will
also increase.

Experimental Procedure:

Part A - Preparing the measuring setup

Materials:
● Graduated cylinder
● Laboratory flask
● Cork with tube
● Thermometer
● Water bath
● Water
● Flexible tube
● Beaker

Steps:
1. A 2 L beaker was filled with water.
2. A 100 mL cylinder was filled to the brim with water.
3. The 100 mL cylinder was sinked upside down in the 2 L beaker, without making bubbles.
4. The cork with the tube's free end was placed inside the underwater cylinder.
5. The corked end of the tube was left on the counter.
6. This was repeated three more times.

Part B - Preparing the fermentation setup

Materials
● Sucrose - 60 grams
 ● Warm distilled water (40oC)
● Beakers
● Yeast - 36 grams
● Fermentation vessels/Flasks - 100mL glass flasks
● Water Bath
● Thermometer
● Distilled water
● Stopwatch/timer

Steps:
1. Three 250 mL flasks were separated and labeled with the temperature that was tested.
2. 5 grams of sucrose were separated into each flask, then 100 mL of warm distilled water
(about 40oC) were placed into each flask; making sure the sugar dissolved in the water.
3. The three flasks were placed in a waterbath with the temperature that was being tested.
4. 3 grams of baker’s yeast powder were added into the flask (with sugar and water).
5. Each flask was quickly sealed with the cork connected to the water displacement
measuring setup and started the timer, giving it a little mix to evenly distribute the
solution.
6. As the fermentation proceeded, the CO2 was generated and collected in the graduated
cylinder, displacing the water. The volume of CO2 produced was measured at the end of
the 15 minutes by reading the graduated cylinder markings.
7. The steps were repeated for each temperature.

Variables:
Independent: The temperature (Celcius), in this experiment, is the only thing that will change to
see how it affects CO2 production.
Dependent: The CO2 production will be measured in milliliters after experimenting.
Controls: Amount of sugar, amount of water, amount of yeast, type of yeast, time waited after
mixing yeast, and way of measuring CO2 produced.
Results

Table 1 - Production of CO2 with different temperatures

Production of CO2 in Fermentation (mL)

Temperatures Trial 1 Trial 2 Trial 3 Trial Average

(Celcius)

22o 26 30 36 30.7

37o 81 80 86 82.3

40o 98 100 85 94.3

60o 104 87 90 93.7

Figure 1 - Production of CO2 in Fermentation with Different Temperatures

Figure 2 - Experiment Set up, Before Collecting Results

Figure 3 - Sucrose, Yeast, and Water Mix After Fermentation

Figure 4 - CO2 Production 25oC (right) and 37oC (left)

Throughout this experiment, it can be seen that the fermentation is more rapid at warm
temperatures, for example 40o celsius was when the yeast was the most active, having a more
ideal environment for the yeast, the average of CO2 produced was 94.7 mL. Comparing it to a
higher temperature, 60o, it can be seen that the environment was too hot for the yeast to be at its
peak, so the average CO2 produced was 93.7 mL. While higher temperatures produce more CO2,
lower temperatures like 22o produce an average of 30.7 mL of CO2, which is low compared to
the higher temperatures. Even with a smaller temperature difference like 37o produced an
average of 82.3 mL.

Discussion
In this experiment, the yeast activation and production of CO2 is strongly influenced by
the temperature of the environment. For example, at the lowest temperature tested; 22o celsius,
the average production of CO2 was 30.7 mL, while the highest temperature tested was 60o
celsius, having a 93.7 mL average for the CO2 production. However, the most efficient result was
at 40o celsius, producing an average of 94.3 mL. This occurred in this experiment because of the
high sensitivity to temperature that yeast has, a small change in temperature can change the
activity of it, turning it overworked or lethargic. With a yeast that is at the target temperature, it
will be able to break down more sugar molecules, producing more by-products; CO2 and ethanol.
When the yeast is in a warmer environment, it helps the enzymes become active so they have
enough energy to react and break the carbohydrates down.
Comparing this experiment with a study performed by Slaa, J., Gnode, M., & Else, H.
from the Vrije Universiteit Amsterdam, the results are similar, however their optimal temperature
was 35oC, while this experiment it was 40oC. Despite them measuring the CO2 produced in
grams instead of milliliters, it can be concluded that once the temperature hits 40oC, the yeast
enzymes become denatured or less active due to the lower production of CO2. Their lowest
temperature tested was 20oC and it produced an average of 1.88 g (x10 g) of CO2. The highest
temperature tested was 40oC, and that produced an average of 2.07 g of CO2. However the
temperature with the highest amount of CO2 produced was 35oC, having an average of 5.17 g of
CO2. It can be seen how much the CO2 production has been reduced from 5oC of difference even
though the units aren’t the same, it can be inferred of how much difference there was.
This experiment that was conducted most likely didn’t have these same results as the
Vrije Universiteit Amsterdam due to a few errors in temperature. The temperature for 60 and
40oC was not accurate due to the high production of CO2 in both tests and in all trials. Moreover,
the system to collect the CO2 was most likely leaking or altered due to the excess CO2 collection
(if the temperature was correct). Despite the few errors, the overall results can still be seen; the
lower production of CO2 in lower and higher temperatures, with an efficient optimal temperature
which is around 35-40oC.

Conclusion
In conclusion, the experiment conducted proved the hypothesis partially correct, stating
how during the process of fermentation, once the temperature increases, then the production of
CO2 will also increase. This was correct however the efficiency and exact number of production
varies depending on the right temperature of yeast activation. The results found were that the
yeast produces the most CO2 when the temperature is at 40oC, producing an average of 94.3 mL
of CO2. The hypothesis stated that the CO2 production would increase as the temperature rises,
however once the temperature passed a certain number (around 60oC), the yeast stopped being as
efficient as it was around 40oC, it produced an average of 93.7 mL of CO2, instead of 94.3 mL
that was produced at 40oC. This occurred due to the sensitivity of the yeast, due to the active
fungus that make up the yeast. The slightest changes of temperature and environmental
conditions can strongly affect the efficiency of the fermentation process.
According to an experiment conducted by Slaa, J., Gnode, M., & Else, H. in 2009, it can
be concluded that optimal temperature for fermentation is 35oC, once it passes that, the enzymes
in the yeast become denatured or lose their efficiency, producing less CO2. However overall,
their experiment proved how the production of CO2 with yeast fluctuates, producing less at a
lower and higher temperature, then a higher amount of CO2 with an optimal temperature, which
is from 35-40oC.

Bibliography

“Cell Processes: Fermentation.” Texas Gateway,

https://www.texasgateway.org/resource/cell-processes-fermentation. Accessed 26

February 2024.

Faulkner, F. “Fermentation.” Wikipedia, https://en.wikipedia.org/wiki/Fermentation. Accessed 26

February 2024.

“Fermentation | Definition, Process, & Facts.” Britannica,

https://www.britannica.com/science/fermentation. Accessed 26 February 2024.

“How Does Temperature Affect Fermentation?” Atlas Scientific, 20 June 2023,

https://atlas-scientific.com/blog/how-does-temperature-affect-fermentation/. Accessed 26

February 2024.

“Saccharomyces cerevisiae.” Wikipedia,

https://en.wikipedia.org/wiki/Saccharomyces_cerevisiae. Accessed 26 February 2024.

“Yeast and fermentation: the optimal temperature.” NVON,

https://newsroom.nvon.nl/files/default/LA-2.10.1-Fermentation_article.pdf. Accessed 26

February 2024.