Alcoholic Fermentation in Yeast A Bioengineering Design Challenge1

I. Introduction
Yeasts are single cell fungi. People use yeast to make bread, wine and beer. For your experiment,
you will use the little brown grains of yeast you can buy if you want to make bread.
Alcoholic fermentation is the main process that yeast cells use to produce ATP. The process of
alcoholic fermentation is summarized in these chemical equations.

Notice that during alcoholic fermentation:

The sugar glucose is broken down to the alcohol ethanol and carbon dioxide.
ATP is synthesized from ADP and P.
Energy released by the first reaction provides the energy needed for the second reaction.
1. For the first chemical equation shown above, label each chemical formula with the name of the
molecule.
2. Why do cells need ATP?

Alcoholic fermentation is a complex process that includes twelve different chemical reactions. Each
of the twelve chemical reactions in alcoholic fermentation of glucose requires an enzyme.
3. What are enzymes? What does it mean to say that "a chemical reaction requires an enzyme"?

II. Testing for Alcoholic Fermentation in Yeast Cells

One way to test for alcoholic fermentation in yeast cells is to evaluate whether CO2 is produced by
yeast cells in water with sugar vs. water without sugar.
4. If yeast cells carry out alcoholic fermentation, would you expect CO2 to be produced by:
yeast cells in sugar water?
yes___
no ___
yeast cells in plain water (without sugar)?
yes___
no ___
Explain your reasoning.

By Drs. Ingrid Waldron and Jennifer Doherty, University of Pennsylvania, Biology Department, 2016. Teachers are encouraged to copy this
Student Handout for classroom use. A Word file with the Student Handout and Teacher Preparation Notes with instructional suggestions and
background information are available at http://serendip.brynmawr.edu/sci_edu/waldron/#fermentation .

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CO2 can be produced by a simple chemical reaction without any living cells or enzymes and
without producing ATP (e.g., baking soda and vinegar react to produce CO2). This raises the
possibility that dead yeast cells in sugar water produce CO2 as a result of a simple chemical
reaction without carrying out alcoholic fermentation. To evaluate this possibility, you will test
for CO2 production by dead yeast cells that have been boiled so the enzymes needed for
alcoholic fermentation have been denatured and cannot catalyze the chemical reactions
required for alcoholic fermentation.
5. Can dead yeast cells that were boiled carry out alcoholic fermentation of sugar to produce
CO2?
yes___
no ___
Explain why or why not; include enzymes in your explanation.

6. In your experiment to test whether alcoholic fermentation occurs in yeast cells, you will test
whether CO2 is produced under each of the conditions listed in first column of this table.
Explain how each of these tests will help you evaluate whether yeast cells carry out alcoholic
fermentation. Identify the two tests that are controls and explain why each is useful.
How testing whether these yeast cells can produce CO2
will help you know whether alcoholic fermentation occurs in yeast cells

Living yeast cells

in sugar water

Living yeast cells

in plain water

Dead yeast cells

in sugar water

The sugar you will use in your experiment is sucrose, the common sugar that people use for
baking and cooking. Sucrose is a disaccharide that yeast cells convert to glucose for alcoholic
fermentation.
When the yeast cells produce CO2, this gas is trapped in bubbles in a layer of foam. Your
teacher will pass around a cup with a foam layer so you will know what it looks like.

In your experiment to test for alcoholic fermentation in yeast cells:

Experimenter 1 will test for CO2 production by living yeast cells in plain water.
Experimenter 2 will test for CO2 production by living yeast cells in sugar water.
Experimenter 3 will test for CO2 production by dead yeast cells in sugar water.
Experimenter 4 will be in charge of timing and measuring temperature.
Use the following procedure for your experiment:
Experimenter 1

Experimenter 2

- Label your cups.

- Add 80 mL of 35C water.

Experimenter 3
- Label your cup.
- Add 80 mL of 35C
dead yeast
suspension.

- Tear a piece of scrap paper into quarters; each experimenter should

take a quarter-piece of paper for the next step.
Experimenter 1:
- Weigh 4 g of yeast
and put this on your
piece of paper.

Experimenter 2:
- Weigh 4 g of yeast and
0.5 g of sucrose and put
these on your piece of
paper.

Experimenter 4

Experimenter 3:
- Weigh 0.5 g of
sucrose and put this
on your piece of
paper.

- Bend your paper and pour the yeast and/or sucrose into your cup.
- Stir vigorously with a plastic spoon for 1 minute. Smash any clumps of
yeast and if necessary use your second spoon to scrape off any yeast
that is stuck to the first spoon.
- Put your cups in the container for the warm water bath.

- Add 35C water to the

container for the warm
water bath, just to the level
of the liquid in the cups. Do
not add more water or the
cups will tip over!
- Put the thermometer in
the water in the bath.
- Time 1 minute of stirring
for Experimenters 1-3.
- Measure the temperature
of the bath. Record this
starting temperature in
question 7 on the next page.

- Time 10 minutes.
- Make observations and record what you observe in the second and
- At 8 minutes, measure and
third columns of the table in question 7 on the next page. Do not bump
record the ending
the cups!
temperature of the warm
water bath.
- At the end of 10 minutes, measure the depth of the foam layer at the - At 9 minutes give a
edge of the cup and record your results in the last column in the table
warning.
in question 7. If the foam layer is not even, measure the depth at the
- At 10 minutes announce
thinnest and thickest points and record both measurements and the
that it is time to measure
average.
the depth of the foam layer
in the cups.

- Empty and clean the cups and bath. Clean up your workspace.

- Report the depth of the

foam layer in each cup and
the starting and ending
temperatures to your
teacher.

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7. Record your observations in this table.
Starting temperature _______ C
Any bubbles
right at the beginning?

Ending temperature _______ C

Observations
1-9 min.

Depth of Foam
Layer (mm) at
10 min.

Living yeast in
plain water (1)
Living yeast in
sugar water (2)
Dead yeast in
sugar water (3)
8. Compare your results in question 7 with your answers to questions 4-6. Do your results support
the conclusion that alcoholic fermentation occurs in yeast? Explain your reasoning.

9a. Your teacher will provide the results for all the student groups in your class. Do the class results
support your conclusions in question 8? If not, briefly summarize your conclusions based on the
class results.

9b. Were the results similar for all the cups with living yeast in sugar water? If not, what could be
the reasons for any differences in results?

10. If you were going to repeat your experiment, how could you improve your methods to get more
reliable and valid results?

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III. Bioengineering Design Challenge
When bakers make bread, they include flour with the yeast, sugar and water. The gluten protein in
the flour gives elasticity to the dough and traps the CO2 bubbles produced by the yeast so the bread
dough rises and the bread becomes fluffy. The fluffiness of the bread can be influenced by the
relative amounts of yeast, sugar, water and flour, as well as other ingredients in the dough. The
fluffiness of the bread can also be influenced by the temperature of the dough as it rises and how
long the dough rises.
.

Design Challenge. Jim Baker wants to make his bread as fluffy as possible without spending too
much time waiting for the dough to rise. He has asked your class to find the amount of sucrose and
temperature that produces the most CO2 in 10 minutes. He does not want his bread to be too
sweet, so he doesn't want to use any more sucrose than needed for maximum CO2 production. To
maintain good flavor and texture of the bread, he wants to keep the amount of yeast the same as in
your experiment in Part II.
Scientific Background. CO2 is produced by alcoholic fermentation, which includes multiple chemical
reactions, each catalyzed by a different enzyme. Fortunately, you do not need to think about all of
these chemical reactions and enzymes to predict the effects of changes in the amount of sucrose or
temperature on the rate of CO2 production; you can get sufficiently accurate predictions by thinking
about the expected effect for a single reaction catalyzed by a single enzyme.
11a. What happens to the rate of a chemical reaction catalyzed by an
enzyme when the concentration of substrate increases? Give molecular
explanations for the relationships between substrate concentration and
rate of reaction. In the graph, draw a curve to show the expected
change in the rate of CO2 production as the amount of sucrose
increases.

11b. What happens to the rate of a chemical reaction catalyzed by an

enzyme when temperature increases? Give molecular explanations for
the relationships between temperature and rate of reaction. In the
graph, draw a curve to show the expected change in the rate of CO2
production as the temperature increases.

Proposing and Testing Your Design Solution

12. Reread the Design Challenge above and list here the criteria for a good solution for this Design
Challenge.

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13a. Think about the best Design Solution for this Design Challenge. Based on your answers to
questions 11 and 12 and the experiment in Part I, how much sucrose do you think will give the best
Design Solution? What range of grams of sucrose in 80 mL H2O do you think should be tested to
identify the best Design Solution? Explain your reasoning.

13b. What range of temperatures do you think should be tested to identify the best Design
Solution? Explain your reasoning.

14. Based on your answers to question 13, what combinations of sucrose concentrations and
temperatures should be tested to identify the best Design Solution?

Your teacher will lead a discussion of the proposed experiments and develop a Class Investigation
Plan. For practical reasons, each student group will test three different amounts of sucrose at one
temperature and different groups will test at different temperatures. Record the assigned
temperature and the assigned amounts of sucrose for your group in question 15.
Use the following procedure for your experiment.
Experimenters 1-3
- Label your cups.
- Add 80 mL of water at your assigned temperature.
- Weigh 4 g of yeast and your assigned amount of
sucrose onto a quarter-sheet of scrap paper.

Experimenter 4
- Add water at the assigned temperature to
the container for the bath, up to the level of
the liquid in the cups.
- Put in the thermometer.

- Bend your paper and pour the yeast and sucrose

into your cup.
- Stir vigorously with a plastic spoon for 1 minute.
Smash any clumps of yeast.
- Put your cups in the container for the bath.

- Time 1 minute of stirring for

Experimenters 1-3.
- Measure the starting temperature of the
bath and record it in question 15.

- Do not bump the cups! At the end of 10 minutes,

measure the depth of the foam layer and record your
results in question 15. Measure the depth of the
foam layer at the edge of the cup. If the foam layer is
not even, measure the depth at the thinnest and
thickest points and calculate the average.
- Clean up.

- Time 10 minutes. At 8 minutes, measure

and record the ending temperature of the
warm water bath. At 9 minutes give a
warning, and then at 10 minutes announce
that it is time to measure the depth of the
foam layer in the cups.
- Report your results to your teacher.

15. Assigned Temperature ____ C Starting Temperature _____ C Ending Temperature _____ C
Cup 1
Cup 2
Cup 3
Amount of Sucrose (grams in
80 mL of water)
Depth of Foam Layer (mm)
at 10 minutes

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16. Your teacher will provide the information to fill out as many of the columns as possible in this
table. (Each column gives the results from one student group.)
Depth of Foam Layer (mm)
Lowest Assigned
Middle Assigned
Highest Assigned
Temperature
Temperature
Temperature
Amount of Sucrose
(______C)
(______C)
(______C)
(grams in 80 mL of
water)
Test 1 Test 2 Test 3 Test 1 Test 2 Test 3 Test 1 Test 2 Test 3
Lowest amount
(_______ grams)
Medium amount
(_______ grams)
Highest amount
(_______ grams)
Starting temperature
Ending temperature

Lowest Assigned Temperature

Middle Assigned Temperature

Highest Assigned Temperature

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

17. Graph the results for the depth of the foam layer vs. the amount of sucrose at each
temperature. Label the axes and provide a key that distinguishes between the results for tests 1, 2
and 3 in each graph.

Amount of Sucrose

Amount of Sucrose

Amount of Sucrose

18a. Interpret the results shown in these graphs. What effects did the amount of sucrose have on
the amount of CO2 produced?

18b. Were the effects of the amount of sucrose consistent for tests 1-3 at each assigned
temperature? If not, what might account for any observed differences?

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18c. Were the effects of the amount of sucrose consistent at different temperatures?

18d. Are the observed relationships between amount of sucrose and amount of CO2 produced
compatible with your predictions in question 11a? If not, what scientific explanation could account
for the observed results?

19. Graph the depth of the foam layer vs. starting temperature for each amount of sucrose. Label
the axes and provide a key that distinguishes between the results for tests 1, 2 and 3 in each graph.

Starting Temperature

Highest Amount of Sucrose

Depth of Foam Layer (mm)

Middle Amount of Sucrose

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Lowest Amount of Sucrose

Starting Temperature

Starting Temperature

20a. What effects did temperature have on the amount of CO2 produced?

20b. Were the effects of temperature consistent for tests 1-3 at each sucrose level? If not, what
might account for any observed differences?

20c. Were the effects of temperature consistent for different amounts of sucrose?

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20d. Are the observed relationships between temperature and amount of CO2 produced compatible
with your predictions in question 11b? If not, what scientific explanation could account for the
observed results?

21. Summarize your conclusions concerning the optimum Design Solution. What advice would you
give Jim Baker concerning the optimum amount of sucrose and temperature to maximize CO2
production in 10 minutes, but with no more sucrose than needed? Explain how your recommended
Design Solution is supported by the experimental results. Describe any uncertainty you have about
your recommendations.

Improving Your Design Solution

22a. What additional information could help you to better identify the amount of sucrose and
temperature that would give an optimum Design Solution? Explain your reasoning.

22b. Describe the experiments that you think would be most important to do next in order to better
identify the optimum amount of sucrose and temperature.

Your teacher will lead a class discussion of the proposed experiments to develop a Class
Investigation Plan. Then, your teacher will assign each group specific experiments from this plan.
23. Make a data table, carry out your experiment, record your results, and report your results to
your teacher.

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Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

24. Graph the data from all the student groups.

25. Do these results support your proposed design solution in question 21? What different or
additional conclusions are supported by these new results?

26. On a separate sheet of paper prepare a report for Jim Baker. Include the following:
Describe your recommended Design Solution. Relate your Design Solution to the criteria in
the Design Challenge and the evidence that supports your recommendations. Describe any
uncertainty you have about your recommendations.
Would you advise continued testing with the same procedures or would you advise Jim
Baker to begin testing with bread dough? What are the relative advantages and
disadvantages of these two ways of testing different possible Design Solutions?