The reactions within cells which result in the synthesis of ATP using energy stored in glucose are referred to as cellular respiration. Aerobic respiration requires oxygen as the final electron acceptor. Fermentation does not require oxygen.
The equation for aerobic respiration is below.
C6H12O6 + 6O2
→ 6CO2 + 6 H2O + 36 or 38 ATP
In aerobic respiration (equation above) glucose is completely broken down to CO2 + H2O
but during fermentation, it is only partially broken down. Much of the
energy originally available in glucose remains in the products
produced. Plant and fungal cells produce alcohol as a result of
fermentation and animal cells produce lactic acid. The equation for
alcohol fermentation is below.
C6H12O6 → 2CO2 + 2C2H5OH + 2 ATP
Notice from the above equations that aerobic respiration produces much more ATP per glucose molecule than fermentation.
We
will investigate fermentation by measuring the amount of carbon dioxide
produced by yeast. The rate of cellular respiration is proportional to
the amount of CO2 produced (see the equation for fermentation above).
In
this experiment, we will measure the rate of cellular respiration using
either distilled water or one of four different food sources. Create a
hypothesis regarding the rate of cellular respiration for each of the
different food sources listed in the step below.
1) Hypothesis- The words "gas bubble" should be used in your hypothesis.
Fill
each of five small test tubes one-half full with the solutions listed
below. Each tube should be filled to exactly the same level.
Tube 1 - glucose (a monosaccharide)
Tube 2 - fructose (a monosaccharide)
Tube 3 - sucrose (a disaccharide)
Tube 4 - starch
Tube 5 - distilled water
Use
a dropper to finish filling tube 1 with a thoroughly-mixed yeast
suspension. Be sure to mix the yeast suspension immediately before
adding it to the tubes. The tube should be filled as full as possible
while holding it over a sink. Carefully invert a larger tube and place
it over the smaller tube containing the yeast suspension and glucose.
Push the smaller tube all the way into the larger tube using your
finger or a pencil and then invert both tubes so that the opening of
the larger tube is up. Repeat this procedure for the other four
tubes.
Below: Tubes containing yeast and a sugar solution are inverted so that CO2 produced by the yeast can collect.
 | Click on the image to view an enlargement. Press "back" to return here. |
Place the five test tubes in a 37 degree incubator and record the time.
2) Time at the start of incubation: _________
The
tubes should be checked approximately every 5 minutes to observed the
size of the gas bubble that accumulates in the small tube.
The
experiment should be stopped when the gas bubble in any of the tubes is
approximately one half the length of the tube. Record the time when the
experiment is terminated.
3) Time when yeast is removed from the incubator: _________
Proceed to the Aerobic Respiration part of this exercise while waiting for this experiment to complete.
 | The level of the liquid can be seen through the sides of the test tubes. Click on the image to view an enlargement. Press "back" to return here. |
After
the tubes are removed from the incubator, hold each tube over a sink
and quickly invert them as shown below. Use your finger or a pencil to
keep the small tube in place while inverting so that the liquid inside
the small tube remains in the small tube. Lift the larger tube off of
the smaller tube and set the smaller tube in a test tube rack. Repeat
this procedure with the other tubes.
The
size of the gas bubble produced by the yeast can be measuring the
amount of liquid remaining in the tube and subtracting it from the
total volume of the tube. Measure the amount of liquid in each of the
tubes with a 10 ml graduated cylinder and record that value in the
table below.
Measure the total volume of one
of the small tubes with a 10 ml graduated cylinder. With this number
you can calculate the volume of gas produced. by each tube. Perform
these calculation and enter the values in the table below.
4) Total volume of a smaller tube: _______
| Tube | Contents | Milliliters of
liquid remaining | Milliliters of
CO2 produced | Milliliters of CO2
produced per minute |
| 1 | glucose | | | |
| 2 | fructose | | | |
| 3 | sucrose | | | |
| 4 | starch | | | |
| 5 | distilled water | | | |
5)
Which food source produced in the highest rate of cellular respiration?
Which food source produced the slowest rate? Explain your results for
tube 5 (distilled water).
Below: The level of liquid in
each of the tubes below is indicated with a blue line. Notice that each
of the sugars (glucose, fructose, and sucrose) produced approximately
the same amount of CO2. Sucrose is expected to produce CO2
at a slower rate because it is a disaccharide and must first be
converted to glucose by the cell. Fructose is easily converted to
glucose by yeast cells. Yeast cells in water produced little CO2 because they do not have a source of sugar.
We will measure the rate of aerobic cellular respiration in beans by measuring the volume of O2
consumed using the apparatus shown below. The apparatus consists of
three test tubes with stoppers and a graduated pipette inserted into
each stopper. A colored liquid is placed in each of the pipettes. When
the volume of gas in the test tube changes, the liquid in the pipette
will move.
 | The assembled respirometer apparatus. Click on the photograph to view an enlargement. |
Oxygen consumption cannot be measured simply by putting beans in the test tubes because beans are also producing CO2. Any change in gas volume will be due to both O2 consumption and CO2 production. In order to minimize the confounding effect of CO2, KOH will be added to the tubes. It reacts with CO2 to form solid potassium carbonate. The solid will not have a measurable increase in the volume inside the tubes.
CO2 + 2KOH → K2CO3 + H2O
Temperature
will also affect the measurement of gas consumption because gasses
expand when they warm and contract when they cool. This effect will be
minimized by keeping the respirometer tubes immersed in water at room
temperature. Water temperature changes slowly, so the water will
minimize temperature fluctuations inside the tubes.
Create a hypothesis regarding the rate of cellular respiration in each of the tubes containing bean seeds.
6) Hypothesis The words "dye" or "colored liquid" should be used in your hypothesis.
Obtain
the materials for assembling the respirometer apparatus. A tank
containing water at room temperature will be needed to hold the three
respirometer tubes. Three large test tubes with stoppers and graduated
pipettes will also be needed.
Push a small wad of cotton to the
bottom of each respirometer tube. The cotton should occupy
approximately 2 cm of space on the bottom of the tube. Use a dropper to
add 15% KOH solution to the cotton in each tube. Use enough KOH to
saturate the cotton but not enough to pour out of the test tube. Use
the same amount of KOH in each tube. Be careful not to let KOH come in
contact with the sides of the test tube because it will kill the bean
seeds.
Push a small wad of dry cotton on top of the KOH-saturated
cotton in each tube. This will prevent KOH from coming in contact with
the bean seeds and killing them.
Fill one of the tubes half full with germinated bean seeds. Count the number of bean seeds used.
7) Number of germinated bean seeds used: ___________
Add
the same number of nongerminated soybeans to a second tube. The third
tube will remain empty to measure the effect of temperature changes.
Assemble each apparatus and place the tubes in the water tank.
Be sure that the rubber stopper is inserted tightly into the test tube. The valve in the stopper of each tube should be kept open so that air can move through the stopper
and into or out of the tube. After the respirometer tube is inserted
into the water tank, use a dropper to push a drop of a colored liquid
into the tip of each of the graduated pipettes. Try to force the marker
into the region past the tip where its position can be read using the
calibrations on the pipette.
 | The valve shown at the right should be open while you add the marker dye. |
Before
adding the marker dye, double-check to see that the valve on top of the
stopper is open and that air can pass through. Sometimes the rubber
tube that passes through the valve remains squeezed shut even though
the valve is open. Be sure that you can see an opening through the
tube.
Squeeze the plastic bulb to push the liquid marker
dye into the glass pipette as shown below. The glass pipette is marked
with milliliter marks but the narrow tip does not have any marks. The
dye should be pushed far enough into the pipette that it is in the area
where there are milliliter marks. This will enable you to read how far
the dye moves from the beginning of the experiment to the end.
 | Notice that the dye has been pushed several centimeters into the glass pipette. |
8)
Record the time that the respirometer is set up and the dye is in
place.__________ You will be ready to begin the experiment after the
tubes have been in the water for 10 minutes.
Before
beginning the experiment, be sure that the dye in the pipette is in a
region of the pipette that has calibration marks. This is important
because you will measure the amount of movement of the dye. Also,
check the rubber stoppers to be sure that they are firmly inserted into
the test tubes.
After the respirometer has been idle for 5 minutes, close the valve in the top of each of the tubes.
After the valves are closed, any air movement into the tubes will cause
the dye in the pipette to move inward. After the valves are closed,
record the position of the dye and continue to record its position
every 10 minutes for a total of thirty minutes.
When you are finished, do not disassemble the apparatus. It will be needed in the temperature experiment below.
| Tube | Initial reading
(after valves are closed) | Reading after
10 min. | Reading after
20 min. | Final reading
(after 30 min.) | Change in volume (ml)
(initial - final) | Correction for
temperature change |
| Germinated beans | | | | | | |
| Nongerminated beans | | | | | | |
| No beans | | | | | | XXXXXX |
Effect of Temperature
In
the experiment below, you will use the respirometer that does not
contain bean seeds from the apparatus above and expose it to hot and
cold temperatures. Create a hypothesis regarding the movement of the
liquid in the respirometer tube.
9) Hypothesis:
After
you take your last reading, remove the respirometer that does not
contain any bean seeds and place it in a beaker of cold water to
observe movement of the fluid in the pipette. Next, put the tube in a
beaker of warm water. What happened in each case? Record your
observations below.
| |
Observation |
| Respirometer in cold water | |
| Respirometer in warm water | |
Questions
10) What is the function of using a respirometer without any bean seeds in this experiment?
11)
What two gasses are involved in aerobic respiration? How should they
have affected the dye in the tube that contained germinated bean seeds?
To answer this question, review the equation for cellular respiration
and review the discussion of KOH above.
12) Explain how
temperature fluctuations affect the apparatus and how these were
corrected.Your answer to this question should mention the use of
subtraction to correct for temperature change.
