LAB 12:
Fermentation and Respiration
I. Objectives:
Upon completion of this topic you should know:
o The role of glucose and ATP in the powering of cellular reactions
o The role of fermentation in metabolism
o What the products of fermentation are in yeast
o How different sugars, temperature, and pH affect the rate of fermentation
o How aerobic respiration differs from fermentation
II. Safety Considerations:
o Parts A to C (Yeast Fermentation) note that the 70oC water bath is hot. Also be careful
not to leave spilled ice on the floor for others to slip on.
o In Part D, the 15% KOH is caustic, so avoid contact with it.
III. Introduction:
All cells must acquire and then use energy to carry out their necessary functions (moving,
growing, dividing, etc). Cells acquire their energy by either using sunlight to make the highenergy molecule glucose, or by consuming organisms that have glucose (or other high-energy
molecules). Regardless of how the glucose iss obtained, all organisms (even plants) must then
break down glucose in order to harness the stored energy and do something useful. It may be
surprising that no cellular process is powered directly by the breakdown of glucose. Glucose
stores so much energy that if it were all released at once, it could damage the cell. In much the
same way your car burns its gasoline a small amount at a time (instead of in one big fiery
explosion), cells harvest the chemical energy of glucose a small amount at a time over several
steps. Collectively, the set of chemical reactions used to harvest the chemical energy of glucose
as it is broken down into carbon dioxide (CO2) and water is called cellular respiration.
Through cellular respiration, the chemical energy in glucose is used to create a large number of
high-energy molecules of adenosine triphosphate--ATP.
ATP is an extremely abundant molecule in the cell and is used directly to power a large number
of energy-consuming activities of the cell. Because of this, it is sometimes referred to as the
“universal energy currency” of cells. ATP is a relatively simple molecule that is formed by
putting a third phosphate group onto a molecule of adenosine diphosphate (ADP). The addition
of the third phosphate requires energy, which is then stored in the bond once the bond is formed
(see Figure 1). Enzymes needing energy to power a reaction break the phosphate bond of ATP
to liberate and use the chemical energy it stores.
1
phosphate (P)
P
energy
+
Adenosine
P
Adenosine
P
Adenosine diphosphate (ADP)
P
P
P
Adenosine triphosphate (ATP)
Figure 1. ATP Synthesis
ATP synthesis requires energy, while its breakdown to ADP and phosphate
gives off energy. The reaction is reversible.
The purpose of cellular respiration is to harvest the chemical energy stored in glucose to
synthesize ATP from ADP and phosphate. For most cells, cellular respiration can be divided
into two general steps; glycolysis and oxidative phosphorylation. Glycolysis is the set of
chemical reactions that starts the breakdown of glucose. These reactions take place in the
cytosol and do not require oxygen (O2) and are therefore anaerobic. If oxygen is available cells
are able to use the pyruvate from glycolysis in the oxidative phosphorylation phase of aerobic
respiration. If oxygen is not available, cells are only able to carry out fermentation which yields
far less ATP than does aerobic respiration. See Figure 2 below (this is figure 9.19 from your
text).
Figure 2. Energy conversion in cells under aerobic vs. anaerobic conditions. (From
Campbell et al., 2009)
If oxygen is available, the pyruvate produced in glycolysis will be used in oxidative
phosphorylation in the next set of chemical reactions. These reactions occur in the
mitochondria of the cell and continue the process of glucose breakdown. Oxidative
2
phosphorylation produces much more ATP than glycolysis, but it requires O2. Because it
requires O2, oxidative phosphorylation is called aerobic (“with air”) respiration. Figure 3
illustrates the steps of cellular respiration.
Figure 3. Overview of Cellular Respiration. (From Campbell et al., 2009)
Some cells can capture energy from glucose in oxygen-deficient environments by carrying out
only glycolysis. Since NAD+ is used in glycolysis and converted to NADH, in order to continue
carrying out glycolysis NAD+ must be regenerated. In yeast, NAD+ is regenerated in a two-step
process—alcoholic fermentation. In the first step, CO2 is released as pyruvate is converted to
acetaldehyde. In the second step, acetaldehyde is reduced to ethanol by NADH, which
regenerates NAD+. When oxygen is not present in our cells, our cells carry out fermentation
also, but in this case, lactic acid is produced in the regeneration of ATP and no CO2 is released
(Figure 4).
3
Figure 4. Alcohol and Lactic Acid Fermentation (From Campbell et al., 2009)
Introduction for Part A – Yeast Fermentation Of Different Sugars:
In this experiment, we will test the ability of yeast to ferment different sugars. Two of the sugars
(glucose and fructose) are monosaccharides, or simple sugars. The other two sugars (sucrose and
lactose) are disaccharides--they are each made up of two simple sugars. Sucrose is composed of
linked glucose and fructose monosaccharides and lactose is composed of one glucose
monosaccharide and one galactose monosaccharide. Only monosaccharides can be used directly
by yeast in fermentation. This means that sucrose and lactose must be broken down to their
component monosaccharides by the yeast before fermentation can occur.
Because CO2 is released during fermentation of sugars by yeast, it provides a convenient way of
measuring how much fermentation (glycolysis) has taken place. The rate of fermentation can be
measured by placing a small amount of yeast and sugar solution in a fermentation tube. As CO2
is produced, the bubbles collect at the top of the tube. The fermentation rate of the yeast can be
calculated by measuring the volume of CO2 at the top of the tube and dividing it by the amount
of time it took for that volume to form.
In this exercise, you will be testing and comparing the fermentation rates of yeast cells that are
using different sugars. It is important that you label your tubes (with a grease pencil) so that you
4
can identify what sugars are in each tube. After the solutions are mixed in the tubes, you will be
tipping the tubes, placing them in a water bath, and recording the time. As soon as the gas in
ONE of the tubes reaches the halfway mark, you will record the time and remove ALL the tubes,
including those that haven’t yet reached the halfway mark. You will take the tubes back to your
table and IMMEDIATELY measure the volume of gas in each, recording the volumes and time
in the appropriate table.
Materials for Part A, B, and C:
2 - 100 ml beakers
4 fermentation tubes
2 - 10 ml graduated cylinders
1 grease pencil
2 pipettes
Procedure for Part A:
1. Label 4 clean fermentation tubes (1-4). Take one of your beakers to the side of the room and
obtain 50 ml of stock yeast suspension. Be sure to mix the suspension before dispensing.
Using the graduated cylinder in your tray, measure and pour 5 ml of the yeast suspension into
each of your fermentation tubes. You will probably need to use a pipette to accurately bring
the volume of the graduated cylinder to exactly 5 ml.
2. To each of the tubes now add 7 ml of a single sugar. Use the graduated cylinder (rinsing it
and the pipette after each use so as not to cross-contaminate your solutions) to measure and
pour:
7 ml of glucose solution to Tube 1
7 ml of fructose solution to Tube 2
7 ml of sucrose solution to Tube 3
7 ml of lactose solution to Tube 4
3. Tip the fermentation tubes so that the vertical column of each tube fills with the liquid.
4. Place ALL of the tubes in a 37° C (body temperature) water bath and record the time.
5. Monitor the amount of CO2 produced. This may take some time. When ONE of the tubes is
half filled with CO2 (3 ml) record the time, and remove ALL tubes from the water bath.
6. Measure the volume (in ml) of gas in each tube and record it in Table 1 below. Calculate the
fermentation time by subtracting the starting time from the ending time, and record it in the
table. Calculate the fermentation rate of each of the sugars from these data.
7. WASH AND RINSE ALL OF YOUR TUBES (contents can be poured down the sink). Tip
the tubes as you wash them to thoroughly clean them.
Table 1. Results from Part A.
Tube #
Sugar
Start
Time
1
Glucose
2
Fructose
3
Sucrose
4
Lactose
End
Time
Duration
(min)
5
Volume of
CO2 (ml)
Fermentation Rate
(ml CO2/min)
Introduction for Part B – Effect of Temperature on Fermentation
In this experiment you will be investigating the effects of temperature on the fermentation rate of
yeast. As in Part A, CO2 production will be used as a measure of fermentation rate. However,
instead of manipulating the sugar types, only one sugar (glucose) will be used in all the
fermentation tubes. The fermentation tubes will be placed in water baths of different
temperatures (0°C, ~22°C, 37°C, and 70°C) to see how the temperatures affect the fermentation
rates.
Procedure for Part B:
1. Label 4 clean fermentation tubes (1-4). Using the graduated cylinder in your tray, measure
and pour 5 ml of the yeast suspension (obtain more from the side bench if necessary) into
each of your four fermentation tubes. Be sure to mix the suspension before dispensing. You
will probably need to use a pipette to accurately bring the volume of the graduated cylinder
to exactly 5 ml.
2. Using the graduated cylinder, add 7 ml of glucose solution to each of the fermentation tubes.
3. Tip the fermentation tubes so that the vertical column of each tube fills with the liquid.
4. Place tube #1 in the 0° C ice-water bath; tube #2 in the room temperature bath (record the
temperature in Table 2); tube #3 in the 37° C water bath; and tube #4 in the 70° C water bath.
Record the time in Table 2.
5. Monitor the amount of CO2 produced. This may take some time. When ONE of the tubes is
half filled with CO2, note the time, and remove ALL tubes from the water bath.
6. Begin Part D while your tubes are incubating.
7. Measure the volumes (in ml) of gas in each tube and record them in the table below.
Calculate the fermentation time by subtracting the starting time from the ending time, and
record it in the table. Calculate the fermentation rates at each of the temperatures from these
data.
8. BEFORE YOU THROW ANYTHING AWAY!!!...Take tubes #1 (0° C) and #4 (70° C) and
place them both in the 37° C water bath for 5 minutes to let them both equilibrate to that
temperature. After 5 minutes, tip the tubes to mix and remove the air, and leave them in the
bath to incubate. Record the time.
9. Monitor the amount of CO2 produced. This may take some time. When ONE of the tubes is
half filled with CO2, note the time, and remove BOTH tubes from the water bath.
10. Measure the volume (in ml) of gas in each tube and record them it Table 2 below. Calculate
the fermentation time by subtracting the starting time from the ending time, and record it in
the table. Calculate the fermentation rate at each of the temperatures from these data.
11. WASH AND RINSE ALL OF YOUR TUBES (contents can be poured down the sink). Tip
the tubes as you wash them to thoroughly clean them.
6
Table 2. Results from Part B.
Tube
#
Temp.
(° C)
1
2
0
3
37
4
70
1
0  37
4
70  37
Temp.
(° F)
Start
Time
End
Time
Duration
(min)
Volume of
CO2 (ml)
Fermentation Rate
(ml CO2/min)
Introduction for Part C – Effect of pH on Fermentation
In this experiment you will investigate the effect of pH on the fermentation rate of yeast.
Remember, pH is a measure of the H+ (acid) levels of a solution, and lower pH values indicate a
more acidic solution. As in parts A and B, CO2 production will be used as a measure of
fermentation. In this experiment, the sugar type (glucose) and the temperature (37° C) will
remain constant. However, each glucose/yeast suspension will be incubated at a different pH
(3.4, 4.5, 5.5, and 6.5), to see how pH affects the fermentation rate.
Procedure for Part C:
1. Label 4 clean fermentation tubes (1-4). Take your graduated cylinder to the side table
where you will find four small flasks of yeast, each labeled with a different pH (3.5, 4.5,
5.5, and 6.5). Swirl each of the yeast suspensions before you pour them. Pour 5 ml of
the buffered yeast into the tubes (as shown below):
Tube #1 – pH 3.5;
Tube #2 – pH 4.5;
Tube #3 – pH 5.5;
Tube #4 – pH 6.5
Use a pipette to accurately bring the volume of the graduated cylinder to exactly 5 ml.
2. Using the graduated cylinder, add 7 ml of glucose solution to each of the fermentation
tubes.
3. Tip the fermentation tubes so that the vertical column of each tube fills with the liquid.
4. Place ALL four fermentation tubes into the 37° C water bath. Record the time in Table 3.
5. Monitor the amount of CO2 produced. This may take some time. When ONE of the
tubes is half filled with CO2, note the time, and remove ALL tubes from the water bath.
6. Measure the volume (in ml) of gas in each tube and record it in Table 3 below. Calculate
the fermentation time by subtracting the starting time from the ending time, and record it
in the table. Calculate the fermentation rates of each of the pHs from these data.
7. WASH AND RINSE ALL OF YOUR TUBES (contents can be poured down the sink).
Tip the tubes as you wash them to thoroughly clean them. Make sure to remove all
grease pencil marks.
7
Table 3. Results from Part C.
Tube #
1
2
3
4
Yeast.
pH
3.5
4.5
5.5
6.5
Start
Time
End
Time
Duration
(min)
Volume of
CO2 (ml)
Fermentation Rate
(ml CO2/min)
Part D –Respiration (Aerobic Cellular Respiration)
We will utilize germinating bean seeds to examine the process of cellular respiration that is
summarized in the chemical equation:
C6H12O2 + 6 O2  6 CO2 + 6 H2O + 36 ATP (ENERGY)
As the equation above indicates, there are two gases (oxygen and carbon dioxide) involved in
cellular respiration. Note that for every molecule of oxygen that is consumed, one molecule of
carbon dioxide is produced. Because of this relationship, the total volume of gas in a closed
chamber containing bean seeds carrying out respiration would theoretically remain unchanged
(although its composition would change). However, if the CO2 could be removed as fast as it is
evolved, the volume of gas in the chamber would decrease and this decrease would reflect the
volume of oxygen consumed by the seeds. This latter principle is used in the following exercise
to provide a method for measuring aerobic cellular respiration rate. You will add 15% KOH to
the chambers you will set up as described below. The CO2 that is produced in cellular
respiration will react with the KOH to produce solid potassium carbonate (K2CO3) according to
the equation:
CO2 + 2 KOH  K2CO3 + H2O
You will measure the resulting change in volume using your respirometers to determine
respiration rates.
Procedure for Part D (Figures, Tables and Procedures from: Ward’s, 2002):
1. Set up an ice water bath in a large tray and keep the tray filled with ice at all times. Add a
thermometer. Chill the water to less than 10°C and maintain this temperature throughout
the experiment. Place a Styrofoam pad under the ice water bath to insulate it from the
benchtop.
2. Obtain six vials with steel washers on the bottoms. Number the vials 1 through 6 with a
Sharpie or grease pencil.
3. Fill a 100 ml graduated cylinder with 50 ml water. Add 10 germinating peas and take a
reading of the displaced water. This is the volume of the germinating peas. Record the
8
volume in the space below. Decant the water, remove the peas and place them on a paper
towel; pat the peas dry and set aside.
Volume of germinating peas for vial 1______________
4. Refill the graduated cylinder with 50 ml water. Add 10 dry nongerminating peas. Add
glass beads until the water level is the same as that of the germinating peas. Remove the
peas and beads and place them on a paper towel; pat the peas and beads dry and set aside.
5. Refill the graduated cylinder with 50 ml water. Add glass beads until the water level is
the same as that of the germinating peas. Remove the beads and place them on a paper
towel; pat the beads dry and set aside.
6. Repeat steps 3 through 5 with more germinating peas, nongerminating peas and beads,
and beads. Set this set aside for vials 4-6.
Volume of germinating peas for vial 4______________
7. Place an absorbent cotton ball in each of the six vials and push each down to the bottom
using a pipet or pencil tip. Be sure to use the cotton balls and NOT the non-absorbent
rayon.
8. Without getting any liquid on the sides of the vial, use a pipet to add 1 ml 15% potassium
hydroxide (KOH) to the cotton. Add a piece of non-absorbent rayon that is slightly
smaller than that of the cottonball and place it on top of the KOH-soaked cotton.
9. Using the first set of germinating peas, non-germinating peas and glass beads, and glass
beads, place them in vials 1-3, respectively.
10. Repeat this procedure using your second set of germinating peas, non-germinating peas
and glass beads, and glass beads for vials 4-6.
11. Insert the graduated pipet into the wide end of a stopper so that the tapered end of the
pipet is furthest from the stopper and so that the pipet extends just beyond the bottom of
the stopper.
12. Firmly insert the stopper into the vial. The seal that has been created between the stopper
and the vial should be sufficient enough to prevent the pipet from easily moving up and
down in the stopper. Place a washer over the pipet tip and guide it down the pipet until it
rests on the stopper. Repeat this entire step for the remaining five vials. The first set of
respirometers should look like those shown in Figure 3 below.
9
10
13. Place vials 1-3 in the room-temperature waterbath with the pipet tips resting on the edge
of the tray as shown in Figure 4. Place vials 4-6 in the chilled waterbath in the same
manner. Allow all respirometers to equilibrate for 10 minutes.
14. Add one drop of food coloring to the exposed tip of each respirometer and wait one
minute. Turn each of the respirometers so that the graduation marks on the pipets are
facing up. Carefully immerse all six respirometers in their waterbaths. Do not touch the
respirometers once the experiment has started! Let the respirometer equilibrate for
another 5 minutes before proceeding to step 15.
NOTE: It is normal for a small amount of water to enter the pipets when they are first immersed
and for a small amount of food coloring to enter the water. However, if a pipet begins
to fill with water, that respirometer has a leak that should be repaired immediately in the
following manner: Remove the vial from the water and remove the stopper assembly. Blot the
end of the pipet on a paper towel to remove all liquid. Reassemble the respirometer in the same
manner as in Steps 11 and 12 of this procedure. Be sure to firmly insert the stopper to prevent
leaks. Submerge the vial portion of the respirometer and add one drop of food coloring to the tip.
Carefully submerge the tip of the respirometer in the same manner as previously mentioned.
15. Read all of the respirometers to the nearest 0.01 ml and take the temperature of each
water bath. Record the initial readings and the temperature of each water bath in Table 1
in the Analysis section of the lab.
16. Take additional readings every five minutes for 30 minutes and record the readings and
temperature in Table 1.
17. When all of the readings have been taken, calculate the difference and corrected
difference for each result and record each value in Table 1.
Difference = (initial reading at time 0) – (reading at time X)
Corrected difference = (initial pea reading at time 0 – pea seed reading at time X) – (initial bead
reading at time 0 – bead reading a time X)
NOTE: The corrected difference is being used because this procedure is very sensitive and may
be influenced by factors such as an increase in ambient temperature or varying barometric
pressure from passing weather.
18. On the graph paper provided, graph your results from the corrected difference column in
Table 1 for the germinating peas and dry peas, in both the room temperature and chilled
water baths. Plot the time in minutes.
11
Table 1. Respiration in bean seeds.
References:
Campbell NA, Reece JB, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB.
(2009) Biology. 8th ed. San Francisco (CA): Pearson, Benjamin Cummings.
Ward’s AP Biology Lab 5. 2002 WARD’S Natural Science Establishment, Inc
Record the answers to the following questions in your lab notebook:
1. Which sugar was fermented by the yeast at the highest rate?
2. What do the fermentation rates of sucrose and lactose suggest about yeast’s ability to break
down specific disaccharides?
3. Describe the relationship between temperature and fermentation rate in yeast. Is it linear?
4. At what temperature was the yeast fermentation rate the highest? Why might yeast have
adapted to ferment best at this temperature?
12
5. What happened to the yeast fermentation in the tubes placed in the 0° C and 70° C water
baths after they were both allowed to incubate in the 37° C water bath?
Provide an explanation for the results described in question 5 above.
6. Describe the relationship between pH and fermentation rate in yeast.
7. Which used the most oxygen, germinating seeds or non-germinating seeds? What is a
reasonable explanation for this?
8. How did the respiration rate compare between germinating seeds in the ice bath and those at
room temperature?
9. How can you account for any differences that you observe?
10. How do you think the respiration rate of a mouse would compare with bean seeds under each
of these conditions? Why do you conclude this?
11. Were respiration and fermentation affected by temperature in the same way? What is a
reasonable explanation for this?
13