Fermentation Laboratory
Introduction:
Aerobic cellular respiration is a sequence of three metabolic stages: glycolysis in the
cytosol, and the transition reaction, Krebs cycle and the electron transport chain in
mitochondria. Fermentation involves glycolysis but does not involve the transition
reaction, Krebs cycle or the electron transport chain which are inhibited at low oxygen
levels. Two common types of fermentation are ethyl alcoholic fermentation and lactate
fermentation. Animals, certain fungi, and some bacteria convert pyruvate produced in
glycolysis to lactate (lactic acid). Plants and some fungi, yeast in particular, convert
pyruvate to ethyl alcohol and carbon dioxide. Aerobic cellular respiration is much more
efficient than fermentation in producing ATP. Aerobic cellular respiration of a glucose
molecule can produce a maximum of 38 ATP molecules while fermentation produces
only 2 ATP molecules.
Yeast are unicellular members of the Kingdom Fungi. Fungi are heterotrophs
which are unable to produce their own nutrient molecules and therefore must obtain their
nutrients from the environment. Fungi, like all other organisms, need nutrient molecules
to supply their energy needs. They rely on sugar found in their environment to provide
them with this energy so that they can perform chemical, transport, and mechanical work
as well as grow and reproduce.
Yeast, like bacteria, grow in or on their food source. They produce and release
digestive enzymes into their environment where the sugar molecules are located. Outside
the yeast cell, the sugar molecules are broken down by the digestive enzymes into smaller
molecules that can be absorbed by the yeast. If oxygen not available within the yeast cell,
the sugar molecules are processed by an anaerobic cellular respiration pathway which
releases the sugar’s stored energy, some of which is captured in ATP molecules for cell
use. The pathway also produces two waste products, carbon dioxide and ethyl alcohol
(ethanol) which are released from the cell. The anaerobic cellular respiration pathway
used by yeast to release nutrient energy for cell use is called ethyl alcohol fermentation.
The chemical reaction for ethyl alcohol fermentation of glucose is:
Reactant
C6H12O6
Products
2CH3CH2OH + 2CO2 + 2 ATP
Glucose
2 ethyl alcohol + 2 carbon dioxide + 2 ATP
Although the 2 ATP produced per glucose molecule that undergoes anaerobic cellular
respiration in the yeast cell represents only two percent of the total energy of the glucose
molecule, this chemical reaction is necessary to release energy for the yeast’s survival. Most of
the energy of the glucose molecule remains in the resulting pair of ethyl alcohol molecules.
Although unusable by the yeast cell, the ethyl alcohol and carbon dioxide molecules are
advantageous to us. Ethyl alcohol has widespread use as a solvent of substances intended
for human contact or consumption, including scents, flavorings, colorings, and
medicines. In chemistry, it is both an essential solvent and a feedstock for the synthesis of
other products. The intoxicating effects of ethyl alcohol consumption in the form of
alcoholic beverages have been known since ancient times. Ethyl alcohol also has a long
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history as a fuel for heat and light as well as a fuel for internal combustion engines. When
yeast is added to flour, the resulting carbon dioxide is the factor responsible for making
bread rise.
In this laboratory investigation, you will investigate alcoholic fermentation in a
yeast, Saccharomyces cerevisiae, also known as baker’s yeast. When oxygen is low,
some fungi, including yeast, and most plants, switch from aerobic cellular respiration to
ethyl alcoholic fermentation. In bread making, starch in the flour is converted to glucose
and fructose, which then serve as the starting compounds for fermentation. The resulting
carbon dioxide is trapped in the dough, causing it to rise. Ethyl alcohol is also produced
in bread making but evaporates during baking. In this laboratory experiment, the carbon
dioxide (CO2) produced, being a gas, bubbles out of the solution and can be used as an
indication of the relative rate of fermentation taking place. The rate of fermentation, a
series of enzymatic reactions, can be affected by several factors, for example,
concentration of yeast, concentration of glucose, or temperature. In this investigation you
will investigate the effects of yeast concentration. In your independent study you may
choose to investigate other independent variables.
Materials: (per group)
4 respirometers consisting of:
large test tube
1-mL graduated pipette
aquarium tubing
250 mL flask
600 mL beaker
binder clips
pipette pump
3 5-mL graduated pipettes
yeast solution
glucose solution
deionized water
marking pencil
timer
Prelab:
Hypothesis
Hypothesize about the effect of different concentrations of yeast on
the rate of fermentation.
Lab Protocol
Activity I
1. Obtain four 250 mL flasks and fill with approximately 200 mL of tap water. Label the
flasks 1, 2, 3, and 4.
2. To stabilize the respirometers, place the water filled flasks into separate beakers.
3. Obtain four large test tubes (fermentation tubes) and label them 1, 2, 3, and 4. Place each
test tube into a separate flask.
4. Using pipette pumps with graduated pipettes, add solutions as follows to the
appropriate tubes. (Do not cross-contaminate the pipettes!)
Table 1. Contents of Fermentation Tubes (volumes in mL)
Tube DI water
Yeast Suspension Glucose Solution
1
4
0
3
2
6
1
0
3
3
1
3
4
1
3
3
2
5. Attach a piece of aquarium tubing to the large end of each of four 1-mL graduated
pipettes. Then place a pipette with attached tubing into each test tube containing
the fermentation solutions.
6. Attach the pipette pump to the free end of the tubing on the first pipette. Use the pipette
pump to draw the fermentation solution up into the pipette. Fill the pipette past the
calibrated portion, but do not draw the solution into the tubing. Fold the tubing over
and clamp it shut with the binder clip so the solution does not run out of the pipette.
Open the clip slightly, and allow the solution to drain down to the 0-mL calibration line
(or slightly below). Quickly do the same for the other three pipettes. *NOTE: if you can
not get the solution to the 0-mL mark, then just do your best, mark the starting point
with a wax pencil and note your initial reading in Table 2.
7. Record your initial readings for each pipette. This will be the initial time (I).
8. Two minutes after the initial readings for each pipette, record the actual readings (A) in
mL for each pipette in the “Actual (A)” column. Subtract I from A to determine the
total amount of CO2 produced (I-A). Record this value in the “C02 Evolved (I-A)”
column. From this point on, you will subtract the initial reading from each actual
reading to determine the total amount of CO2, evolved.
9. Continue taking readings every 2 minutes for each of the solutions for 20 minutes.
Remember, take the actual reading from the pipette and subtract the initial reading to get
the total amount of C02 evolved into each pipette.
10. Complete Data Table 1.
Table 2. Total CO2 Evolved by Different Concentrations of Yeast
Tube 1
Time
(min)
Actual
(A)
CO2
Evolved
(A-I)
Tube 2
Actual
(A)
CO2
Evolved
(A-I)
Initial
Reading
(I)
2
4
6
8
3
Tube 3
Actual
(A)
CO2
Evolved
(A-I)
Tube 4
Actual
(A)
CO2
Evolved
(A-I)
10
12
14
16
18
20
Data Table 1: CO2 Evolved by Different Concentrations of Yeast
Fermentation Ratio of Yeast to Glucose Total CO2 Produced
Tube #
(mL:mL)
(20 min)
1
2
3
4
Analysis:
1. Was your hypothesis supported by the collected data? Give evidence for your answer.
Activity II:
1. Using Activity I as a model, design an experiment testing a variable that you hypothesize
could affect the rate of ethyl alcohol fermentation in yeast. Possible variables include, but
are not limited to temperature, different nutrient molecules, light, etc… Be creative and
most importantly, use good scientific methodology.
2. Form a hypothesize concerning your experiment.
3. Run the experiment for at least 20 minutes, collecting and recording data in Table 3.
4. Produce a group laboratory report complete with separate data table(s) and graph.
Tube
1
2
3
4
Table 3.
Tube 1
Time
(min)
Actual
(A)
CO2
Evolved
(A-I)
Tube 2
Actual
(A)
CO2
Evolved
(A-I)
4
Tube 3
Actual
(A)
CO2
Evolved
(A-I)
Tube 4
Actual
(A)
CO2
Evolved
(A-I)
Initial
Reading
(I)
2
4
6
8
10
12
14
16
17
18
19
20
5