General Biology II – Major Life Processes in Plants and Animals
An Investigation of Ethanol Fermentation in Yeast
Adapted From: George E. Glasson and Woodrow L. McKenzie. Investigative Learning in Undergraduate Freshman Biology
Laboratories. JCST Dec 1997/Jan 1998
Steven Spilatro. Yeast on the Rise: Investigative Study of Fermentation in the Introductory Biology Classroom. Tested Studies for
Laboratory Teaching
This lab activity is intended to provide an opportunity for students…
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To examine some parameters that affect ethanol fermentation by yeast
To practice designing experiments that examine a research question
To practice gathering, interpreting and presenting data
To gain experience with oral presentations
Objectives:
This lab aims to contribute to the development of the following components of the Cégep Champlain
St.Lawrence Science Program Graduate Profile:
I. Apply The Experimental Method
II. Take A Systematic Approach To Problem Solving
III. Use The Appropriate Information Technologies
IV. Reason Logically
V. Communicate Effectively
VI. Learn In An Autonomous Manner
VII. Work As Members Of A Team
VIII. Make Connections Between Science, Technology and
Social Progress
X. Become Familiar With the Context in Which Scientific
Concepts are Discovered and Developed
XI. Develop Attitudes Appropriate For Scientific Work
XII. Apply What They Have Learned To New Situations
This lab also contributes to the attainment of the following elements of the 00XU objective:
1.
3.
To analyze the relationship between structure and function in multicelled organisms
To explain the function of conservation, regulation and reproduction in multicelled organisms.
This lab also contributes to the development of the follow performance criteria of the 00XU objective:
• Proper use of concepts and terminology
• Clear description of the main stages in a biological process
• Description of cellular processes of transformation of matter and energy
• Clear description of the factors that influence the transformation of matter and energy
• Use of techniques of observation or experimentation
• Observance of the experimental method and, where applicable, the experimental procedure
• Adherence to safety and environmental protection regulations
Prelab
Bring graph paper to lab
Background Information
from Raven, P.H. and G.B. Johnson 2002. Biology 6th Edition. McGraw-Hill.
Anaerobic Respiration
In the presence of oxygen, cells can respire aerobically, using oxygen to accept the electrons
harvested from food molecules. In the absence of oxygen some organisms can respire anaerobically, using
inorganic molecules to accept the electrons. For example, many bacteria use sulfur, nitrate, or other
inorganic compounds as the electron acceptor in place of oxygen.
Fermentation
In the absence of oxygen, aerobic metabolism cannot occur, and cells must rely exclusively on
glycolysis to produce ATP. Under these conditions, the hydrogen atoms generated by glycolysis are donated
to organic molecules in a process called fermentation.
Eukaryotic cells are capable of only a few types of fermentation. In one type, which occurs in singlecelled fungi called yeast, the molecule that accepts hydrogen from NADH is pyruvate, the end product of
glycolysis itself. Yeast enzymes remove a terminal CO2 group from pyruvate through decarboxylation,
producing a two-carbon molecule called acetaldehyde. The CO2 released causes bread made with yeast to
rise, while bread made without yeast (unleavened bread) does not. The acetaldehyde accepts a hydrogen
Fermentation and Biofuels
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atom from NADH, producing NAD+ and ethanol (ethyl alcohol). This particular type of fermentation is of
great interest to humans, since it is the source of the ethanol in wine and beer. Ethanol and CO2 are byproducts of fermentation.
2 ADP
2 ATP
glycolysis
glucose
2 ethanol
2 NAD+
2 NADH
CO2
pyruvate
2 acetaldehyde
Figure 1. Alcohol fermentation in yeast
The Lab Activity
One of the many industrial applications of fermentation is the production of biofuels. Biofuels such as
ethanol and biodiesel are considered environmentally-friendly alternatives to fossil fuels because they are
renewable and produce fewer emissions and greenhouse gasses. (See the Bio 2 Web page for more
information on biofuel production and use in Canada)
Although the industrial production of biofuels requires many procedures, it is still based on the basic
principles of fermentation. As a class, we will try to determine the optimal conditions for some factors that
affect fermentation.
The first week will be devoted to becoming familiar with the procedure. In addition, each group will be
assigned one of the following factors to test during the second week:
1.
2.
3.
4.
5.
6.
7.
8.
Type of carbohydrate: compare fermentation of glucose, sucrose, lactose, etc.
Concentration of glucose: compare fermentation using glucose concentrations from 0 to 5%
Concentration of salt: compare fermentation in 0% to 10% salt environments
Acid environment: compare fermentation in acidic environments pH 2 – pH 7
Alkaline environment : compare fermentation in basic environments pH 7 to pH 12
Temperature: compare fermentation at temperatures from 4°C to 60°C.
Variety of yeast: compare fermentation of different varieties of yeast
Ethanol concentration: compare fermentation in 0% - 10% ethanol environments
Note: It may be possible to choose the factor your group will examine. The earlier you choose, the more
likely you will get what you want.
The Lab Assignment
Each team will orally present the results and conclusions of their study to the class.
The experimental protocol used and raw data gathered will be submitted in writing.
Materials
All groups will have access to the following solutions and equipment:
yeast in solution (8% yeast in dH2O) balances and weigh boats
distilled water
alternative varieties of yeast
glucose
2, 1 ml pipettes attached together
test tube rack
controlled temperature water bath
range of pH buffers glucose and other simple carbohydrates
Fermentation and Biofuels
50 ml flask
5 ml syringe
salt
stop watches
10 ml pipette
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WEEK 1 - Designing the Tests – Common Procedure
Fermentation produces two by-products: ethanol and carbon dioxide. Although we are interested in the
production of ethanol, it is much easier to measure the CO2 released and use it as an indicator of
fermentation. Fermentation will take place in a syringe and the gas produced (CO2) will be collected in
1 mL pipettes.
1. Weigh out 1.0 g of glucose and place it in a 50 mL flask.
2. Add 10 mL of distilled water to the flask and gently swirl until the glucose is completely
dissolved.
3. Transfer 10 mL of the yeast suspension (8% yeast in water) into the flask.
4. Allow the yeast suspension to incubate for 5 minutes, with occasional swirling.
5. Use a 5 mL syringe to take up 3 mL of the yeast suspension. Invert the syringe and take
in 1 mL of air above the liquid.
pipette
6. Add a small drop of water to the assembled 1 mL pipettes.
7. Allow the drop to reach the 0 mark and attach the pipettes to the yeast-filled syringe.
(Figure 1)
8. As CO2 is released the water bubble will rise. Record the position of the bubble at 2minute intervals (more frequently if CO2 production is rapid.)
9. Plot the raw data on graph paper, and draw a line through the linear part of the curve.
Using only the linear part of the data, calculate the slope of the line as change in
volume ÷ change in time. This yields the rate of respiration as milliliters of CO2 per
minute.
water droplet
10. Record the rate of respiration in the class data sheet so that a class average can be
calculated.
Table 1. Sample raw data
Time (sec)
0
3
6
7
8
9
11
Reading (ml)
0.18
0.25
0.38
0.48
1
0.55
0,9
0.65
0.84
1 ml air
Rate = 0.54/6
= 0.09 ml/min
3 ml yeast
0,8
0,7
0.54 ml
ml CO2
0,6
0,5
0,4
6 min
syringe
0,3
0,2
Figure 1. Set up for yeast
fermentation experiment
0,1
0
0
2
4
6
8
10
12
14
minutes
Fermentation and Biofuels
Figure 2. Example of how to graph fermentation raw data. The raw data (Table 1, the actual
CO2 volume measurements) is graphed as shown. Note that the rate of fermentation is
calculated from the slope of the line drawn through the linear part of the curve.
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WEEK 1 - Designing the Tests – Group Procedure
Determine how you will perform your assigned experiment. Perform trial runs. You may wish to represent
your data graphically. Does it make sense? Can it be interpreted and explained logically? Modify your
protocol as needed to obtain meaningful results.
WEEK 2 - Experimentation
Perform experiments designed during week one.
Written Assignment
Due March 27, 2008
Write up a detailed description of the procedure you designed.
Provide tables of all data gathered.
Summarize data graphically.
Oral Presentation Assignment
Due March 27, 2008
Present the results of your study to the class in a 10-minute (max!) presentation
Include: What parameter did your team study?
Provide a VERY brief description of your experimental protocol. You only need to describe the
differences between your protocol and the common procedure.
Summarize your results graphically.
Explain the implications of your results to the industrial production of ethanol.
To avoid delays, students will not be allowed to log on to the lab computer. If you wish to use the digital
projector for your oral presentation you may
a. Send to my e-mail address (adera@slc.qc.ca) a clearly labeled PowerPoint file you wish to present by
noon, Thursday, March 27. To facilitate the presentations, I will download all files received into a
single folder.
b. Alternatively, you may access your file from your USB key, providing you have previously verified
that the lab computer is able to recognize your key. USB keys that have not been “recognized” by the
lab computer prior to the presentation time will not be accepted.
Peer Evaluation
Each member of your lab group must complete a Peer Evaluation report and hand it in separately from the
lab report.
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