Cellular Respiration
Introduction
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.
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:
Fill each of five small test tubes two-thirds 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 remainingMilliliters of
CO2 producedMilliliters of CO2
produced per minute1 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.
Aerobic Respiration
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:
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. A 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. If necessary, this can be accomplished by using a plastic pipette that has been cut so that it fits over the tip of the glass graduated pipette. The photograph below shows a plastic pipette that has been cut for this purpose.
Squeeze the plastic bulb to push the liquid marker dye into the glass pipette as shown below.
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.
After the respirometer has been idle for 10 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 changeGerminated 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? 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.
