Fermentation in the Yeast Saccharomyces cerevisiae
BY DR. SUSAN PETRO
Introduction
Yeast are simple unicellular organisms belonging to the Kingdom Fungi. They
are relatives of molds, mildews and mushrooms. Like all organisms, yeast need energy to
do the work of living (synthesis, transport, reproduction, etc.) They obtain this energy
through a process called cellular respiration, which involves the oxidation of organic
molecules. Some of the energy produced by this oxidation is stored in the chemical
bonds of adenosine triphosphate (ATP) - the energy currency of the cell. There are two
types of cellular respiration - aerobic and anaerobic.
Aerobic respiration
In most cells, including yeast, respiration begins in the cytoplasm with the
oxidation of glucose to pyruvate by a ten-step process called glycolysis. This process
requires a ‘push’ of two ATPs to get started, but once rolling it produces four ATPs
directly by a process called substrate level phosphorylation. Additional energy is stored
by adding high-energy electrons to the electron carrier NAD+ (nicotinamide adenine
dinucleotide) converting it to NADH + H+. This process is called reduction (the positive
charge is being reduced by the addition of negatively charged electrons). Glycolysis
occurs with or without the presence of oxygen.
If oxygen is present, most organisms (including yeast) continue respiration by
oxidizing the two pyruvates produced by glycolysis to CO2 via the formation of acetyl
CoA and then the Kreb’s citric acid cycle. Two additional molecules of ATP are
produced directly by the Kreb’s cycle, again by substrate level phosphorylation. The
formation of acetyl CoA and the Krebs cycle store additional energy in NADH + H+ and
FADH2 (flavin adenine dinucleotide). Again this energy is stored by reduction, the
process of adding high-energy electrons to the NAD+ and FAD converting them to
NADH + H+ and FADH2. NADH + H+ and FADH2 later transfer their high-energy
electrons to the series of complexes comprising the electron transport chain. As the
electrons pass down the electron transport chain the energy they lose is trapped as usable
energy in ATP through the mechanism of chemiosmosis. Oxygen is the final electronacceptor in the electron transport chain, which is why the Kreb’s cycle and electron
transport chain cannot proceed if oxygen is absent. The entire process of aerobic
respiration consisting of glycolysis, acetyl CoA formation, the Krebs citric acid cycle, the
electron transport chain and chemiosmosis produce 30 - 32 ATPs for each molecule of
glucose depending on the type of cell. In heart and liver cells for example 32 ATPs are
produced while in brain cells 30 ATPs are produced.
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Anaerobic respiration
Yeast are classified as facultative anaerobes, which means they are capable of
both aerobic and anaerobic respiration. When oxygen is unavailable, yeast carry out
fermentation, a type of anaerobic respiration. The difference between aerobic and
anaerobic respiration lies in how the NADH + H+ produced in glycolysis is converted
back to NAD+. In aerobic respiration the hydrogens (electrons) from NADH +H+ are
passed to oxygen in the electron transport chain yielding approximately 2.5 ATPs per
NADH + H+ while in fermentation the hydrogens (electrons) are passed on to
acetaldehyde to form ethanol yielding no ATPs per NADH + H+ as follows:
Step 1 - Pyruvate (from glycolysis) → Acetaldehyde + CO2
Step 2 - Acetaldehyde
NADH + H+
Ethanol
NAD+
Humans have made use of the byproducts of fermentation for centuries - the CO2
to make bread rise and the ethanol in beer and wine. From the yeast's viewpoint both
CO2 and ethanol are waste products and in fact ethanol is toxic, killing the yeast
organisms when it reaches a concentration between 14-18%. This is why the percentage
of alcohol in wine and beer doesn't exceed approximately 16%. In order to produce
beverages with higher concentrations of alcohol (liquors), the fermented products must be
distilled.
Fermentation produces only 2 ATPs per glucose molecule (via glycolysis).
Aerobic respiration produces 30 - 32 ATPs from a molecule of glucose. Thus the ability
of yeast to live in the absence of oxygen comes at a price - fermentation produces 19-fold
fewer ATPs per glucose molecule than does aerobic respiration.
Purpose - Part 1
In this exercise the effect of the following compounds on fermentation rate in
bakers’ yeast (Saccharomyces cerevisiae) as measured by CO2 production will be
observed. The genus name of this yeast means sugar fungus and the species epithet is
Latin for beer as this species is also used in brewing. It is the official state microbe of
Oregon. Glucose will be used as the substrate except in the lactose tube. In the lactose
tube lactose will be the substrate.
• Lactose - a disaccharide composed of glucose and galactose
• MgCl2 - provides Mg++, a cofactor in three of the enzymes of glycolysis:
phosphofructokinase (step 3), enolase (step 9) and pyruvate kinase (step 10)
• NaF - an inhibitor of the enzyme enolase, the catalyst for step 9 of glycolysis
• In addition, the effect of pH on fermentation rate will be observed.
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Each table is assigned one of the variables to determine its effect on
fermentation rate in the yeast, Saccharomyces cerevisiae.
Lab Table 1 – Is yeast necessary for the production of CO2?
Procedure – Part 1
Measure 50 ml of pH 4 buffer and place in a beaker.
Add 1.25 grams of yeast to the pH 4 buffer in the beaker.
Now add 2.5 ml of 5.0% glucose solution to the beaker.
Mix well with a glass stir rod and add the mixture to a fermentation tube.
Determine what would go in the control fermentation tube for this
experiment.
Prepare a control tube.
Follow post-mixing directions below.
Lab Table 2 – What is the effect of pH on the production of CO2 by Saccharomyces
cerevisiae?
Procedure – Part 1
Measure 50 ml of pH 4 buffer and place in a beaker.
Add 1.25 grams of yeast to the pH 4 buffer in the beaker.
Now add 2.5 ml of 5.0% glucose solution to the beaker
Mix well and add the mixture to a fermentation tube.
Repeat directions for pH 2 buffer and pH 6 buffer.
Follow post-mixing directions below.
Lab Table 3 – What is the effect of MgCl2 on the production of CO2 by
Saccharomyces cerevisiae?
Procedure – Part 1
Measure 50 ml of pH 4 buffer and place in a beaker.
Add 1.25 grams of yeast to the pH 4 buffer in the beaker.
Now add 2.5 ml of 5.0% glucose solution to the beaker.
Add 5.0ml of 0.1M MgCl2 to the beaker.
Mix well and add the mixture to a fermentation tube.
Determine what would go in the control fermentation tube for this
experiment.
Prepare a control tube.
Follow post-mixing directions below.
Lab Table 4 – What is the effect of NaF on the production of CO2 by
Saccharomyces cerevisiae?
Procedure – Part 1
Measure 50 ml of pH 4 buffer and place in a beaker.
Add 1.25 grams of yeast to the pH 4 buffer in the beaker.
Now add 2.5 ml of 5.0% glucose solution to the beaker.
Finally add 5.0ml of 0.1M NaF to the beaker.
Mix well and add the mixture to a fermentation tube.
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Determine what would go in the control fermentation tube for this
experiment.
Prepare a control tube.
Follow post-mixing directions below.
Lab Table 5 – What is the effect of using lactose as a substrate instead of glucose on
the production of CO2 by Saccharomyces cerevisiae?
Procedure – Part 1
Measure 50 ml of pH 4 buffer and place in a beaker.
Add 1.25 grams of yeast to the pH 4 buffer in the beaker.
Now add 2.5 ml of 5.0% lactose solution to the beaker.
Mix well and add the mixture to a fermentation tube.
Determine what would go in the control fermentation tube for this
experiment.
Prepare a control tube.
Follow post-mixing directions below.
Post-mixing directions for all tables.
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Make certain that you tip the fermentation tube when you fill it so the blind
end is completely filled with fluid.
Insert a foam plug in the outlet of each tube.
Put the tubes on the tray on your lab bench and tape the bases of the tubes to
the tray so they don’t accidently tip over.
Place the tray with its fermentation tubes into the 29ºC incubator.
Every 10 minutes for 60 minutes measure the height in millimeters of the
column of accumulated CO2. Your lab rulers are calibrated in centimeters i.e.
the 1 demarcation is 1 centimeter. The ten lines between zero and 1 are
millimeters.
Remove your tray of fermentation tubes from the incubator to do the
measurements so the incubator doesn’t cool down. Putting each fermentation
tube on a flat surface gently swirl the contents to release any gas trapped in the
base by the settling yeast organisms.
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•
Measure from the bottom of the gas
produced (which may be just a few
bubbles at first) to the top of the
tube. Do not confuse the
demarcation line between the
settled yeast organisms and the
fluid above them with the
demarcation line between the fluid
and the gas above it. Line up the
zero on the ruler with the bottom of
the meniscus of the fluid. To read
the measurements it might be helpful
to then place an index card on the top
of the blind end of the fermentation
tube parallel to the table top so it hits
the ruler at the measurement. See
illustration right.
•
Transfer all the class data from the whiteboard to Results Tables 1 and 2
Results Table 1
Time
(min)
CO2
Production
(mm)
CO2
Production
(mm)
CO2
Production
(mm)
CO2
Production
(mm)
CO2
Production
(mm)
CO2
Production
(mm)
CO2
Production
(mm)
CO2
Production
(mm)
Tube with
yeast
Control
Tube
MgCl2
MgCl2
control
NaF
NaF
control
Lactose
Lactose
control
0
10
20
30
40
50
60
Results Table 2
Time
(min)
0
10
20
30
40
50
60
CO2 Production (mm)
CO2 Production (mm)
CO2 Production (mm)
pH 2
pH 4
pH 6
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Part 2
In this part of the exercise the effect of temperature on fermentation in the yeast
Saccharomyces cerevisiae will be observed.
Procedure - Part 2
• There is an index card on each table with the temperature at which your final
fermentation tube will be incubated.
• Check the thermometer inside the refrigerator or incubator to be sure that the
temperature is what it says outside. If not then record the actual temperature.
• Set up the final tube according to Mixing Table 2 below. The pre-measured
buffers to make the yeast solution are in the flasks in the incubators or
refrigerator at your assigned temperature.
• Take 2 grams of yeast and your fermentation tube with the glucose in it to the
incubator/ refrigerator at your assigned temperature.
• If you were assigned room temperature your buffer is on your table and you must
record what the room temperature is.
• Add the 2g of yeast to the buffer flask, mix and add to the fermentation tube. As
previously, make sure the blind end of the tube is full of the yeast suspension.
• Work fast so the temperature does not alter too much.
Mixing Table 2
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Tube
5.0% Glucose
1
2.5ml
pH buffer 4 in incubator/frig/on lab bench
depending on which temp your table was
assigned
50ml
Insert a foam plug and return the fermentation tube to the incubator/refrigerator.
Every 10 minutes for 60 minutes measure the height in millimeters of the column
of accumulated CO2.
Write your results on a chart on the whiteboard.
Transfer all the class data from the whiteboard to Results Table 2.
Results Table 2
Time (min)
CO2 Production
(mm) at
4°C
CO2 Production
(mm) at
Room temp
____°C
0
10
20
30
40
50
60
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CO2 Production
(mm) at
CO2 Production
(mm) at
CO2 Production
(mm) at
37°C
55°C
70°C
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At the end of the experiment thoroughly rinse out your fermentation tubes. Do not
use soap.
Rinse out and save the foam plugs.
Write up
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Write up this exercise as a laboratory report.
This would of course include graphing of your data and evaluation of your slopes
(the b in the equation y = bx + a) to determine the effect of the various
treatments on the rate of the reaction.
Make six separate multi-line linear regression graphs one for each of the following:
1. yeast tube and its control
2. MgCl2 tube and its control
3. NaF tube and its control
4. lactose tube and its control.
5. one for the three pHs (there is no control tube for pH as you can’t have a solution
with no pH)
6. one for the five temperatures
You will then do a seventh graph
7. A derivative graph of fermentation rates for the various temperatures.
(Hint: you did two derivative graphs for the turnip peroxidase lab.)
After observing the effect of NaF on fermentation rate can you think of a reason why
many toothpastes have fluoride in them? (Put in your discussion on NaF)
Latest Revision – April 23, 2014
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