Lab 9: Fermentation in Yeast

Objectives:

At the end of lab you should be able to:

1. Understand the metabolic process of fermentation.

2. Make a recommendation about which sugars yeast can use during the process of fermentation.

3. Graphically present data taken from measurements of yeast fermentation.

4. Summarize the lab experiment in the form of a written lab report.

Key Terms:

fermentation (anaerobic respiration)

aerobic respiration
enzymes
catalysts
substrate
reaction product
metabolic pathways
glycolysis
glucose → pyruvate
fructose
sucrose
lactose
alcohol fermentation
pyruvate → ethanol + carbon dioxide
substrate specificity of enzyme
complementary binding of enzyme and substrate
rate of reaction
temperature optimum
fermentation tubes

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 Lab 9: Fermentation in Yeast
BACKGROUND INFORMATION

Yeast (Saccharomyces cerevisiae) are single-celled fungi that are found in many different
environments in nature. Yeast cells are different from animal cells in that they can survive with
or without oxygen. The reactions of fermentation are defined as metabolic reactions that occur
in the absence of oxygen (anaerobic respiration).
Alcohol fermentation is one of the best-known metabolic reactions of yeast cells. During
alcohol fermentation, enzymes present in yeast are able to convert sugars into ethanol (ethyl
alcohol), releasing carbon dioxide as a byproduct. Yeasts are used by the beer and wine industry
to ferment sugars found in fruit and grains into alcohol. In baking, carbon dioxide given off by
the fermenting yeast causes bread to rise. Fermentation reactions provide the yeast cells with a
small amount of energy for reproduction and other cellular activities. Eventually, however, the
alcohol concentration becomes toxic and the yeast cells die.
Animals and several microorganisms can carry out another type of fermentation known
as lactic acid fermentation, where sugars are converted to lactate and carbon dioxide is not
produced. Human muscle cells make some energy by lactic acid fermentation when oxygen is
scarce. Lactic acid fermentation by certain types of fungi and bacteria is used in the dairy
industry to produce yogurts and some cheeses.
In the presence of oxygen, yeasts can use a different type of metabolism called aerobic
respiration. During aerobic respiration, yeast cells form acidic by-products instead of alcohol
and can completely break down sugars to carbon dioxide. Aerobic respiration yields more energy
for yeast and will always be used if oxygen is present.

Metabolic Pathways and Enzymes

Like all living cells, yeasts contain many different enzymes. Enzymes are biological
catalysts. Catalysts increase the rate of reactions without being consumed themselves. Enzymes
are also specific, in that each enzyme has a very particular three-dimensional shape, which
allows it to recognize and bind to only a few different molecules. Living cells can have hundreds
of chemical reactions occur in them because cells contain hundreds of different enzymes, each
catalyzing a different reaction.
In enzyme-catalyzed reactions, a specific enzyme molecule binds to a specific substrate
molecule and catalyzes a reaction of that substrate. At the end of the reaction, the enzyme
releases the product, in order to be able to catalyze another reaction. Enzyme reactions are
written:
enzyme + substrate → product + enzyme

In living cells, the hundreds of reactions of metabolism are organized into many
sequences of reactions, each leading to a different product. Each sequence of reactions is called
a metabolic pathway. In any one metabolic pathway, each reaction is dependent on the one that
comes before it.
The metabolic pathway of glycolysis is an example of the very organized reaction
sequences that are typical of all living cells. When you look at the reactions of glycolysis, you
will notice that only small changes (perhaps only three or four atoms) are made in the substrate

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of any one reaction. [Please refer to Figure 9.8 on pages 170-171 of your textbook for a summary
of the reactions involved in glycolysis.]
By changing molecules only bit by bit, cells can trap the maximum amount of energy
from the breakdown reactions. (If the sugar were reacted quickly, as in burning, most of the
energy in the molecule would be released as heat.) Without enzymes and metabolic pathways,
the type of life that exists on earth would not be possible.

Glycolysis and Fermentation by Yeast

Virtually all living cells (including yeast) get energy by breaking down glucose. The
glycolysis pathway is the starting point for glucose breakdown, so essentially all cell metabolism
begins with and depends upon glycolysis. This metabolic pathway is believed to be one of the
first pathways that evolved in living cells.
The reactions of glycolysis harvest chemical energy by converting glucose into pyruvate.
But pyruvate is not stable and must undergo further reactions. A cell that is using oxygen will
take the end product of glycolysis, pyruvate, and start to break it down further, using the
metabolic pathways of the citric acid cycle (or Krebs cycle) followed by oxidative
phosphorylation. But a cell that cannot use oxygen, for whatever reason, will ferment pyruvate
into some product. In the case of yeast growing without oxygen, after glycolysis, alcohol
fermentation will convert pyruvate into alcohol.

Measuring Rates of Enzyme Catalyzed Reactions

In order to study reactions of metabolism, including fermentation, it is necessary to be

able to make some type of measurement of the reaction. When there is a way to make a
measurement, preferably a quantitative one, it becomes much easier to study the cell or the
enzyme activity and to compare conditions that are important to the lives of cells.
Reaction rates of enzyme catalyzed reactions are very often the method of measuring
activity of cells and their metabolism. The rate of an enzyme catalyzed reaction is measured by
either the amount of substrate consumed per unit time or the amount of product formed per
unit time. In studying fermentation in this laboratory, you will measure the amount of one of the
products of glycolysis, carbon dioxide, formed in a given amount of time.

Substrate Specificity of Yeast Cell Enzymes

Because yeasts occur in many places in nature, they must contain many enzymes that are
capable of using a wide variety of substrates. The ability of yeasts to ferment a number of
different carbohydrates means that the cell must have at least one different enzyme for each
individual carbohydrate that it uses. These specific enzymes will chemically change the
carbohydrate into some other type of molecule that the yeast can use for energy.
However, there is a limit to the number of different enzymes that a cell can produce.
Therefore, there is also a limit, set by the presence or absence of specific enzymes, to the number
of substrates that an organism can break down. In this way the substrate specificity of the
enzymes of an organism will determine the number of substrates that the entire organism can use
for its nutrition.

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 In these laboratory exercises, you will compare the rates at which yeast cells can ferment
different sugars during the time of the lab period. The sugars tested will include sucrose,
glucose, fructose, and lactose. You will be able to observe that not every sugar can be used by
the yeast. (Please refer to Appendix A at the end of this handout for more information about
sugar structures.)

Temperature Optima of Yeast Cell Enzymes

Each enzyme has a temperature at which it reacts with its substrate at its maximum rate.
This temperature is called the temperature optimum. Because an organism is a collection of
enzymes, the organism itself will have an optimum growth temperature. This optimum growth
temperature will be closely related to the temperature optima of the various enzymes of the
organism.
At temperatures below the optimum temperature, the reactions will be slower because all
molecules have less energy and move more slowly at lower temperatures. At temperatures above
the optimum temperature, the reactions also will be slower, but for a different reason. Above the
optimum temperature, the three-dimensional structure of the enzyme protein is destroyed.
Although the optimum temperature for yeast is 35oC, yeast has an unusually high
tolerance to high temperatures for an hour or so (a very useful property for baking bread). In
order to obtain maximum results, you will use much higher temperatures in this laboratory.

TESTING OF SUBSTRATE SPECIFICITY OF YEAST FERMENTATION

For today’s experiment, you are a food chemist for Buberweiser Brewery researching the
production of a new, hoppy pale ale. You are responsible for investigating the ability of yeast to
ferment several different types of sugars during the beverage production.
The traditional way of making beer at Buberweiser is to add yeast to a solution
containing sucrose. Your production team makes the following observations:

1. The yeast in the developing beer produces gas bubbles, which, upon analysis are
found to be carbon dioxide (CO2).
2. Ethanol is also produced as a byproduct.
3. After a time, this fermentation process stops on its own. You can get it started again
by adding sucrose.
4. Each time you add sucrose to a stopped fermentation reaction, the number of yeast
cells increases as the CO2 and ethanol are produced while the amount of sucrose
decreases.

These observations can be summarized in one reaction: yeast + sucrose → CO2 + alcohol

Through observations and previous experimentation, your team knows that a

fermentation tube containing yeast and sucrose will produce CO2, while a tube containing either
sugar alone or yeast alone will produce no CO2.

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 At your latest production team meeting, it is discovered that your sugar supplier is
substantially increasing their prices for sucrose. As a result of this, your boss challenges your
team with the following questions: Since we know sugar cannot be eliminated, can any sugar be
substituted for sucrose in the fermentation process? If yeast can use other sugars, which is the
best alternative for fermentation?

After we have set up our experiment, together as a class, we will discuss our hypothesis for
this experiment.

In order to answer your questions and test your hypothesis, you will set up a series of five
J-shaped fermentation tubes. Each tube will contain a fresh yeast cell suspension plus a different
solution of a sugar. The sugars will be sucrose, glucose, fructose, and lactose. The tube
containing sucrose will act as a positive control since this is the sugar your production team has
had success with in the past. One tube will be a negative control tube which will contain only
yeast cells suspended in water. Please handle the fermentation tubes very carefully.

PROCEDURE FOR MEASURING GAS PRODUCTION BY FERMENTING YEAST:

1. Make sure your group has five numbered fermentation tubes. Study the volume markings and
learn how to read the amount of gas produced in each tube. You will use graduated cylinders to
measure volumes of yeast and substrate added to each tube. Use a clean graduate cylinder to
measure each sugar solution. You may use the same cylinder for all the yeast measurements.

FERMENTATION TUBES

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2. Carefully add 5 ml yeast suspension to each of the five fermentation tubes. (The yeast
clumps so make sure gently mix the suspension before your measure it out.)

3. Add 15 ml of 1 % sucrose to tube 1 (positive control).

4. Add 15 ml of 1 % glucose to tube 2.

5. Add 15 ml of 1 % fructose to tube 3.

6. Add 15 ml of 1 % lactose to tube 4.

7. Add 15 ml of distilled water to tube 5 (negative control).

8. Carefully mix each tube by covering the open end with a piece of Parafilm and your thumb
and gently tipping it back and forth. When you are finished mixing the yeast and sugar solutions,
tip the tube until the straight, closed arm of the tube is filled to the top.

9. Record the temperature of the incubator you have been assigned to use.

10. Remove the Parafilm and place the tubes carefully in the incubator you have been assigned.
Note the time and then allow the tubes to remain in the incubator for 60 minutes in order for
fermentation to proceed.

11. Record the amount of gas in the arm of each tube on the table on the following page.
If the yeast are fermenting the sugar in the tube, bubbles of gas will collect in the side
arm of the tube.

Remember, the small calibration marks on the arm of each tube represent volume in milliliters.
Use these calibrations to determine the amount of gas collected in the side arm.

Note: Do not be confused by the settling of the yeast cells in the tubes, which will cause the
solution in the upper part of the side arm to look clearer. The edge of a gas bubble collecting in
the side arm should be plainly visible, if gas formation is occurring.

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TESTING SUBSTRATE SPECIFICITY OF YEAST FERMENTATION IN-LAB WORKSHEET

Experimental Question:

Hypothesis:

Note: A hypothesis is a possible explanation for something that was observed. It proposes an
answer to a question. It is usually a well-educated statement based on past experience and
available data, and a scientific hypothesis makes predictions that can be tested and falsified.
When forming your hypothesis, it should be stated in an unambiguous way.

Results:

Table 1. Volume of carbon dioxide produced after 60 minutes of incubation at 55 C

Tube # Tube Contents Volume of CO2 (mL)
1 sucrose
2 glucose
3 fructose
4 lactose
5 water

Think about how you might present this data graphically in your lab report. What type of graph
is most appropriate? What is the dependent variable? Independent variable? We will discuss this
during lab.

Discussion of Results:

Was your hypothesis supported or rejected?

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LAB REPORT GUIDELINES:

A formal lab report is due at the beginning of your lab session on the week of November 11.

The lab report will be worth a maximum of 50 points and is worth 5% of your course grade.

Five points will be deducted from your lab report for each day it is late. If you are more than 15
minutes late to lab on the day your lab report is due, 2.5 points will be deducted from your score.

You MUST write your own lab report. This is NOT a group lab report and plagiarism will be
penalized.

Your report must have the following:

Four sections (with headings): Introduction, Methods, Results, and Discussion (see below)

-Give your paper a title.

-Do not just answer the questions given below. Write your report using paragraphs. Make sure
your sentences are complete and well composed. Do not use bullet points or numbered lists
anywhere in the lab report.
-There is no required length for your lab report. Scientific writing should be clear and concise.
-Your report must be typed and double-spaced.
-Write the report in third person (try not to use I or we).
-Make sure your word tense (past vs. present or singular vs. plural) is consistent and appropriate
throughout the lab report.
-Make sure you provide results in the correct units.
-Proofread your report! Make sure it is free of spelling, punctuation and syntax errors.

Introduction (8 points)
Give a short background on the topic of the experiment.
-What is fermentation and how does it work?
-Why is fermentation important?
-What is the purpose of this experiment?
-What is the general question of interest for your experiment?
-What is the specific hypothesis for the experiment you conducted?

Methods (7 points)
In this section, you need to write out how you conducted the experiment in such a
way that someone could follow it and use it to replicate the experiment.
-Write this section in past tense.
-You must include a description of your experiment- chemicals used, volumes,
controls, etc.
-Remember to write your experiment so that someone could replicate it by
following your report.
-All values in the text must have units (i.e., mL or °C)
-This section should be written in paragraph form with complete sentences, NOT
as a numbered list.

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Results (15 points)
The results section reports the data only.
-You must have a short paragraph of text in your report summarizing the results
generated in lab. The results text describes trends in the data or the outcomes of
the experiment.
-Make sure you do not make interpretations of your data in the results section.
-You need to report the data from your experiment. Summarize the results in a
table. The table must be numbered with a caption above the table.
-You must also report the results from your experiment in the form of a graph.
The graph must have a number and a caption below. The axes must be labeled
with the appropriate variables and units. You may either use Excel or Word to
make your graph or draw the graph by hand.
-Remember, results are NOT interpreted in the results section.

Discussion (15 points)

The discussion section is where you interpret your results and draw your
conclusions based on the data your group collected.
-Discuss all of your results from the experiment, including the controls.
-Was your hypothesis supported or rejected? Do you now have any additional
information to know why this may be the case? Offer an explanation.
-Was there any error in how you completed the experiment that might help to
explain your results? If so, how did this error affect your experiment and how do
you address this in a future experiment?

-In the discussion section of the lab report, outline your recommendations to your
boss based on your data by answering the two questions below:

1. Based on the results of your experiment, what sugar do you recommend that
Buberweiser use in their fermentation process and why?

2. What questions need further testing by the company and why?

Quality of Writing (5 points)

See guidelines above.

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 Appendix A: Sugar Structures
Before we can make predictions about how different sugars might or might not be
utilized by yeast in fermentation reactions, we need to think about the structure of different
sugars. All sugars are carbohydrates. All carbohydrates are the equivalent of a carbon molecule
bonded to a water molecule (CH2O)n, where n stands for the number of CH2O units bonded
together. Sugars are often classified and given a generic name based on the number of carbon
atoms they have. We will be working with six carbon sugars, n = 6, or hexoses.

Sugars that have the same number of carbons are not necessarily identical. Sugars also
differ by the arrangement of the carbon atoms and the bonds between atoms. When molecules
have the same molecular formula, but differ in arrangement, they are called isomers. Each
isomer is usually given a specific name.

Simple sugars are called monosaccharides. When two monosaccharides are bonded
together, they are called disaccharides; when two or more are bonded, they are called
polysaccharides. While some of the sugars pictured below look pretty similar, they are not all
biologically equivalent.

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