This document provides background information and procedures for a lab experiment on alcoholic fermentation in yeast. The objectives are to understand alcoholic fermentation and how varying experimental conditions, like sugar source or yeast concentration, can affect the rate of fermentation as measured by CO2 production. Students will set up fermentation reactions with different sugar solutions, measure CO2 inflation of balloons over an hour, and analyze the results to determine the independent variable (sugar source) and dependent variable (CO2 production).

1. Lab Topic 6
Alcoholic Fermentation
Written by Nickie Cauthen, Clayton State University Morrow,
GA
Objectives
1. Define the terms in bold type.
2. Understand the process of alcoholic fermentation.
3. Determine the independent and dependent variables in the
experiment.
4. Understand why CO2 is measured in alcoholic fermentation
5. Understand why and how the rate of alcoholic fermentation
can be altered.
6. Interpret the data generated in the experiment.
Introduction
Organisms are open systems allowing them to take in
components from the environment and convert them to a form of
energy that can be used by the cell.
Many organisms undergo a process called cellular respiration to
produce energy in the form of adenosine triphosphate, or ATP.
ATP is then used to power most cellular activities. The
following general formula describes this process:
C6H12O6 + 6O2 6CO2 + 6H2O + ATP
Glucose is broken down in a series of chemical reactions that
release small, manageable amounts of energy. This allows the
cells to capture the energy more efficiently than if the glucose
were broken down in one single reaction. Glucose is broken
down in a series of chemical reactions that begin in the
cytoplasm of the cell.

2. The reactants are then transferred to the mitochondria where the
process is continued. The process that occurs in the cytoplasm
of the cell is called glycolysis and does not require oxygen,
meaning it is an anaerobic process. This portion of the process
makes 2 net ATP molecules per molecule of glucose. In the
mitochondria of the eukaryotic cell, oxygen is employed to
more efficiently harness the energy stored in the glucose
molecules. The use of oxygen, an aerobic process, allows the
cell to make a total of 34-38 ATP molecules per molecule of
glucose.
When oxygen is available, cells tend to produce ATP in the
mitochondria since this process produces more ATP than
glycolysis alone. Some organisms, like yeast and the muscle
cells of animals, have the ability to continue to produce ATP
when oxygen is no longer available by a process called
fermentation. During this process in yeast, the glucose is
broken down by glycolysis in the cytoplasm and 2 net ATP, 2
NADH, and 2 molecules of pyruvate are produced. Since
oxygen is not present, pyruvate, the product that results at the
end of glycolysis, is converted to ethanol and CO2. The NADH
is converted to NAD+ and H+ and the ATP is used for cellular
work. The general formula below shows the conversion of
glucose to 2 molecules of pyruvate, 2 molecules of NADH, and
2 net ATP in glycolysis. In yeast cells when oxygen is absent,
fermentation occurs to convert the 2 molecules of pyruvate
(produced in glycolysis) to 2 molecules of ethanol and 2
molecules of CO2 and to convert the 2 molecules NADH (also
produced in glycolysis) to 2 molecules of NAD+ and 2H+.
C6H12O6 2 C3H4O3 + 2 net ATP + 2 NADH 2 C3H5OH +
2CO2 + 2 NAD+ + 2 H+
(glucose) (pyruvate) (ethanol)
GlycolysisFermentation

3. Fermentation allows NADH that is produced during glycolysis
to be recycled back to NAD+, providing NAD+ for the
additional rounds of glycolysis. This process, called alcoholic
fermentation, allows a cell to continue to produce ATP and meet
its energy needs when oxygen is no longer available.
In today’s lab we will investigate alcoholic fermentation in
yeast. Certain types of yeast are economically important and
pay roles in the food and beverage industries. For example,
yeast uses alcoholic fermentation to convert sugars found in
barley to ethanol to make beer. The CO2 that is produced when
the yeast converts glucose to ATP makes yeast breads rise. You
will be studying variables that affect the rate of alcoholic
fermentation in yeast.
Exercise 6.1
Alcoholic Fermentation
In this laboratory exercise you will set up conditions that are
favorable for alcoholic fermentation in yeast and test the effect
of the concentration of yeast on the rate of alcoholic
fermentation. You will measure the rate of alcoholic
fermentation by collecting the CO2 that is produced by the
yeast as a byproduct of alcoholic fermentation. The more CO2
that is produced the faster the rate of alcoholic fermentation.
Hypothesis: Write a hypothesis that describes the effect of
using different sugar sources on alcoholic fermentation. Write
this hypothesis on your report sheet (number 1).
Procedure:
1. After running water from a faucet for 1-2 minutes, fill the pot
or baking dish with 2 inches of hot tap water. The water should
be approximately 50°C. Use a thermometer to monitor the

4. temperature throughout the experiment. Change the water out
every half hour to keep it warm.
2. Yeast solution - Add 1 packet of baker’s yeast to 1 cup of
warm water. Stir for at least one minute. Place in the warm
water bath.
3. In a separate cup, add 1/3 cup of light corn syrup and 2/3 cup
of warm water. Stir for at least one minute and place in warm
water bath.
4. In another cup, add 3 teaspoons of table sugar to 1 cup of
warm water. Stir for at least one minute and place in warm
water bath.
5. In another cup, add 3 teaspoons of artificial sweetener to 1
cup of warm water. Stir for at least one minute and place in
warm water bath.
6. Label each empty plastic water bottle with a different sugar
source.
7. Add 1/3 cup of the sugar source to each respective bottl e.
8. Add1/3 cup of yeast solution to each bottle. Cap and invert
several times to mix.
9. Identify negative control – Fill a fourth water bottle with the
appropriate substances to make a negative control experiment.
10. Uncap all the water bottles and stretch a balloon over the
top. Make sure the balloon is mostly deflated.
11. Place all water bottles in the warm water bath. This is time
zero (0 minutes) for all samples; record 0 cm for Bottles 1, 2, 3
in the 0 minutes column in Table 6.3 on the report sheet.
12. If fermentation occurs in the water bottle, CO2 will be
released, and it will inflate the balloon. To ensure accurate
results, confirm that the balloon fits tightly and monitor the
temperature of the water bath. Temperatures between 35 and
45°C are sufficient. If the temperature dips, gradually replace
water in the bath with hot water from the tap.
13. At 10-minute intervals measure the inflation of each balloon
by wrapping a piece of string around it, making a mark where
the string overlaps with the beginning of the string and the
holding the string next to a ruler. Record the measurement in

5. cm in Table 6.3 on your report sheet. Do this for each of your
samples for 60 minutes. Measure from the line that you mark
each time a measurement is taken.
Lab 6-1: Background Information and Protocol
Fermentation report sheet-1online.docx
Report Sheet—Lab Topic 6
1. Record your hypothesis and predict the outcome of the
experiment.
Hypothesis:
Table 6.2: Predict the outcome of your experiment
Condition
Predict the results
Bottle 1 (Light Corn Syrup)
Bottle 2 (Table Sugar)
Bottle 3
(Artificial Sweetener)
2. Record and graph the data from your experiment below.

6. Table 6.3
Bottle number
Minutes
0
100
20
30
40
50
60
1
2
3

7. Lab 6-1 : Report sheet
3. Graph your data using Excel and paste it below or attach it in
a separate document. You will NOT get credit if you draw a
graph. You must use a graphical program such as Excel.
4. In the space below. Insert a photo of your different bottles
with the balloons on them at end of the experiment.

8. 5. The independent variable in this experiment is a. intensity
of light.
b. amount of CO2 produced. c. yeast concentration
d. sugar source
6. The dependent variable in this experiment is a. intensity of
light.
b. amount of CO2 produced. c. yeast concentration
d. sugar source
7. Why are you able to evaluate CO2 production as a
measurement of alcoholic fermentation? What does it tell you
about alcoholic fermentation?
8. State the conclusion from this experiment. What
experimental evidence supports your conclusion? Were your
predictions correct? Explain why or why not.

9. 9. What is the role of yeast in alcoholic fermentation?
10. What is the role of glucose in alcoholic fermentation?