Alcoholic Fermentation in Yeast – A Bioengineering Design Challenge1

I. Introduction

1. The ingredients for bread include flour, yeast, sugar and water. What makes the dough rise,
so the bread will be fluffy instead of flat?

2. Examine the little dry grains of yeast that are used to make bread dough. Do you think that
these grains of yeast are alive? Explain why or why not.

One way to test whether these little dry grains of yeast are alive is to test whether they can
carry out alcoholic fermentation.

Alcoholic fermentation is the main process that

produces ATP in yeast cells. This figure shows that
during alcoholic fermentation:
 The sugar glucose is broken down to the
alcohol ethanol plus carbon dioxide.
 ATP is synthesized from ADP and P.
 Energy released by the first reaction The pair of curved arrows represents coupled
provides the energy needed for the second chemical reactions; the top reaction provides
reaction. energy for the bottom reaction.

3a. Why is this type of fermentation called alcoholic fermentation?

3b. Why do cells need ATP?

3c. How can alcoholic fermentation result in the production of gas bubbles?

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.

4. What are enzymes? What does it mean to say that "a chemical reaction requires an
enzyme"?

1By Drs. Ingrid Waldron and Jennifer Doherty, University of Pennsylvania, Biology Department, © 2018. 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://serendipstudio.org/sci_edu/waldron/#fermentation .
 II. Do the dry grains of yeast contain living cells that can carry out alcoholic fermentation?
To test for alcoholic fermentation, you will put the little dry grains of yeast in sugar water and
observe whether or not bubbles are produced. You will use controls to check whether any bubbles
produced by yeast in sugar water are the result of alcoholic fermentation. These controls will test
whether grains of yeast can produce bubbles by a simple physical process or a simple chemical
reaction like the reaction between vinegar and baking soda.

For one control, you will use definitely dead yeast cells that have been boiled so enzymes in the
grains of yeast have been denatured and are inactive. You will compare bubble production by these
definitely dead yeast cells vs. bubble production by the possibly living yeast cells in grains of yeast
that have not been boiled.

5a. Can the definitely dead yeast cells carry out alcoholic fermentation of sugar to produce CO2?
yes___ no ___

5b. Explain why or why not; include enzymes in your explanation.

5c. You will put possibly living yeast cells in sugar water in one cup and definitely dead yeast cells in
sugar water in another cup. How would you interpret each of these possible combinations of
results?
Possibly Living Definitely Dead
Interpretation
Yeast Cells Yeast Cells

Produce bubbles of gas No bubbles

Produce bubbles of gas Produce bubbles of gas

6a. As another control, you will compare bubble production by the possibly living yeast cells in
sugar water vs. plain water without sugar. Can living yeast cells in plain water carry out
alcoholic fermentation? yes ___ no ___

6b. Explain why or why not.

6c. What combination of results would provide evidence that the little dry grains of yeast
contain living yeast cells that can carry out alcoholic fermentation?

The sugar you will use is sucrose, the common sugar that people put in their coffee and use for
baking. 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.
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 In your experiment to test for alcoholic fermentation in yeast cells:
 Experimenter 1 will test for CO2 production by possibly living yeast cells in plain water.
 Experimenter 2 will test for CO2 production by possibly living yeast cells in sugar water.
 Experimenter 3 will test for CO2 production by definitely 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 Experimenter 3 Experimenter 4
- Label your cup.
- Label your cups. - Add 80 mL of 35°C
- Add 80 mL of 35°C water. dead yeast
suspension.

- Tear a piece of scrap paper into quarters; each experimenter should - Add 35°C water to the
take a quarter-piece of paper for the next step. container for the warm
water bath, just to the level
Experimenter 1: Experimenter 2: Experimenter 3: of the liquid in the cups. Do
- Weigh 4 g of yeast - Weigh 4 g of yeast and - Weigh 0.5 g of not add more water or the
and put this on your 0.5 g of sucrose and put sucrose and put this cups will tip over!
piece of paper. these on your piece of on your piece of - Put the thermometer in
paper. paper. the water in the bath.

- Bend your paper and pour the yeast and/or sucrose into your cup. - Time 1 minute of stirring
- Stir vigorously with a plastic spoon for 1 minute. Smash any clumps of for Experimenters 1-3.
yeast and if necessary use your second spoon to scrape off any yeast - Measure the temperature
that is stuck to the first spoon. of the bath. Record this
- Put your cups in the warm water bath. 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 9 minutes give a
- At the end of 10 minutes, measure the depth of the foam layer at the
warning.
edge of the cup and record your results in the last column in the table
- At 10 minutes announce
in question 7. If the foam layer is not even, measure the depth at the
that it is time to measure
thinnest and thickest points and record both measurements and the
the depth of the foam layer
average.
in the cups.

- Report the depth of the

foam layer in each cup and
- Empty and clean the cups and bath. Clean up your workspace. the starting and ending
temperatures to your
teacher.

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 7. Record your observations in this table.

Starting temperature _______° C Ending temperature _______° C

Depth of Foam
Any bubbles Observations
Layer (mm) at
right at the beginning? 1-9 min.
10 min.
Possibly living
yeast in
plain water (1)
Possibly living
yeast in
sugar water (2)
Definitely dead
yeast in
sugar water (3)

8. Compare your results in question 7 with your answers to questions 5c and 6c. Do your results
support the conclusion that the “possibly living” yeast contained living yeast cells that could
carry out alcoholic fermentation? yes ___ no ___ 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 “possibly 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 11a and 12 and the experiment in Part II, how much sucrose do you think will give the
best Design Solution?

13b. 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.

14a. Based on your answers to questions 11b and 12 and the experiment in part II, what
temperature do you think will give the best Design Solution?

14b. What range of temperatures do you think should be tested to identify the best Design
Solution? Explain your reasoning.

15. Your teacher will lead a discussion to develop a Class Investigation Plan. Suppose that one
student suggests that each student group should decide which combinations of sucrose levels and
temperature they will test. A second student argues that the different student groups should agree
on the most important sucrose levels and temperatures to test and then cooperate to test each of
these sucrose levels at each temperature. Which proposal will provide the most useful information
for figuring out the best Design Solution? Explain your reasoning.

 Record your group’s assigned temperature and assigned amounts of sucrose in question 16.
 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.
- 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 bath.
- 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.
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.
- Time 1 minute of stirring for
Experimenters 1-3.
- Measure the starting temperature of the
bath and record it in question 15.
- 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.
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 16. 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

17. 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)
Amount of Lowest Assigned Middle Assigned Highest Assigned
Sucrose Temperature (______°C) Temperature (______°C) Temperature (______°C)
(grams in 80 Test Test Test Test Test Test Test Test Test
Average Average Average
mL of water) 1 2 3 1 2 3 1 2 3
Lowest
amount
(_______ g)
Medium
amount
(_______ g)
Highest
amount
(_______ g)
Starting
temperature
Ending
temperature

18. 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.
Lowest Assigned Temperature Middle Assigned Temperature Highest Assigned Temperature
Depth of Foam Layer (mm)
Depth of Foam Layer (mm)
Depth of Foam Layer (mm)

Amount of Sucrose Amount of Sucrose Amount of Sucrose

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 19a. Interpret the results shown in the graphs in question 18. What effects did the amount of
sucrose have on the amount of CO2 produced?

19b. Compare the effects of the amount of sucrose for tests 1-3 at each assigned temperature.
Were the effects of the amount of sucrose consistent for these replicate tests? If not, what
might account for any observed differences?

19c. Were the effects of the amount of sucrose consistent at different temperatures?

19d. 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?

20. Graph the average depth of the foam layer vs. starting temperature for each amount of
sucrose. Label the axes.
Lowest Amount of Sucrose Middle Amount of Sucrose Highest Amount of Sucrose
Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Depth of Foam Layer (mm)

Starting Temperature Starting Temperature Starting Temperature

21a. What effects did temperature have on the amount of CO2 produced?

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21b. Were the effects of temperature consistent for different amounts of sucrose? If no, what
could account for the variation?

21c. 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?

22a. 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?

22b. Explain how your recommended Design Solution is supported by the experimental results.

22c. Describe any uncertainty you have about your recommendations.

23a. Describe any additional testing you would recommend to improve your design solution
and/or increase your confidence in your proposed design solution.

23b. 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?