Cellular Respiration

Cellular respiration is the release of energy from organic compounds by metabolic chemical oxidation in the mitochondria within each cell. Cellular respiration involves a series of enzyme- mediated reactions.

The equation below shows the complete oxidation of glucose. Oxygen is required for this energy releasing process to occur.

C6H1206 + 602 CO2 + 6H20 + 686 kilocalories of energy/mole of glucose oxidized

By studying the equation above, you will notice there are three ways cellular respiration could be measured. One could measure the:

1. Consumption of 02 (How many moles of 02 are consumed in cellular respiration?)
2. Production of CO2. (How many moles of CO2 are produced in cellular respiration?)
3. Release of energy during cellular respiration.

In this experiment, the relative volume of 02 consumed by germinating and nongerminating (dry) peas at two different temperatures will be measured.

Background Information

A number of physical laws relating to gases are important to the understanding of how the apparatus that you will use in this exercise works. The laws are summarized in the general gas law that states:

PV = nRT

where
P is the pressure of the gas,
V is the volume of the gas,
n is the number of molecules of gas,
R is the gas constant (its value is fixed), and T is the temperature of the gas (in o K).

This law implies the following important concepts about gases:

1. If temperature and pressure are kept constant, then the volume of the gas is directly proportional to the number of molecules of the gas.
2. If the temperature and volume remain constant, then the pressure of the gas changes in direct proportion to the number of molecules of gas present.
3. If the number of gas molecules and the temperature remain constant, then the pressure is inversely proportional to the volume.
4. If the temperature changes and the number of gas molecules is kept constant, then either pressure or volume (or both) will change in direct proportion to the temperature.

It is also important to remember that gases and fluids flow from regions of high pressure to regions of low pressure.

In this experiment, the CO2 produced during cellular respiration will be removed by potassium hydroxide (KOH) and will form solid potassium carbonate (K2CO3) according to the following reaction:

CO2+ 2KOH K2CO3+ H20

Since the CO2 is being removed, the change in the volume of gas in the respirometer will be directly related to the amount of oxygen consumed.

In the experimental apparatus (Figures 5.1 and 5.2), if water temperature and volume remain constant, the water will move toward the region of lower pressure. During respiration, oxygen will be consumed. Its volume will be reduced, because the CO2 produced is being converted to a solid. The net result is a decrease in gas volume within the tube, and a related decrease in pressure in the tube. The vial with glass beads alone will permit detection of any changes in volume due to atmospheric pressure changes or temperature changes.

The amount of O2 consumed will be measured over a period of time. Six respirometers should be set up as follows:

Respirometer Temperature Contents
1 Room Germinating Seeds
2 Room Dry Seeds + Beads
3 Room Beads
4 10oC Germinating Seeds
5 10oC Dry Seeds + Beads
6 10oC Beads

1. Both a room temperature bath (approximately 25oC) and a 10oC bath should be set up immediately to allow for time to adjust the temperature of each. Add ice to attain 10oC
2. Respirometer 1: Obtain a 100 mL graduated cylinder and fill it with 50 mL of H20. Drop in 25 germinating peas and determine the amount of water that was displaced (which is equivalent to the volume of peas). Record the volume of 25 germinating peas. Remove these peas and place them on a paper towel. They will be used in respirometer 1.
3. Respirometer 2: Refill the graduated cylinder with 50 mL of H20. Drop 25 dried peas (not germinating) into the graduated cylinder and then add enough beads to attain a volume equivalent to that of the expanded germinating peas. Remove these peas and beads and place them on a paper towel. They will be used in respirometer 2.
4. Respirometer 3: Refill the graduated cylinder with 50 mL of H20. Determine how many beads would be required to attain a volume equivalent to that of the germinating peas. Remove these beads and place them on a paper towel. They will be used in respirometer 3.
5. Repeat the procedures above to prepare a second set of germinating peas, dry peas plus beads, and beads for use in respirometers 4, 5, and 6, respectively.
6. To assemble the six respirometers, obtain six vials, each with an attached stopper and pipette. Place a small wad of absorbent cotton in the bottom of each vial and, using a dropper, saturate the cotton with 15% KOH. Make sure that the respirometer vials are dry on the inside. Do not get KOH on the sides of the respirometer. Place a small wad of dry cotton on top of the KOH soaked absorbent cotton (Figure 5.1). It is important that the amounts of cotton and KOH be the same for each respirometer.

Figure 1: Assembled Respirometers
7. Place the first set of germinating peas, dry peas + beads, and beads in vials 1, 2, and 3, respectively. Place the second set of germinating peas, dry peas plus beads, and beads in vials 4, 5, and 6, respectively. Insert the stopper fitted with the calibrated pipette. Place a weighted collar on each end of the vial (Figure 5.2).
8. Make a sling of masking tape attached to each side of each of the water baths to hold the pipettes out of the water during an equilibration period of seven minutes. Vials 1, 2, and 3 should rest in the room temperature water bath (approximately 25oC) and vials 4, 5, and 6 should rest in the 10oC water bath (Figure 5.2).





Figure 2: Respirometers in the Water Bath

9. After the equilibration period of seven minutes, immerse all six respirometers entirely in their water baths. Water will enter the pipettes for a short distance and then stop. If the water continues to move into a pipette, cheek for leaks in the respirometer. Work swiftly and arrange the pipettes so that they can be read through the water at the beginning of the experiment. They should not be shifted during the experiment. Hands should be kept out of the water bath after the experiment has started. Make sure that a constant temperature is maintained.
10. Allow the respirometers to equilibrate for three more minutes and then record, to the nearest 0.01 mL, the initial position of water in each pipette (time 0). Cheek the temperature in both baths and record in Table 1. Every 5 minutes for 20 minutes, take readings of the water's position in each pipette, and record the data in Table 1.

Table 1: Measurement of 02 Consumption by Soaked and Dry Pea Seeds at Room Temperature (25oC) and 10oC Using Volumetric Methods



Temp (oC)

Time (min)		 Beads Alone Germinating Peas Dry Peas and Beads
Reading at time X Diff.* Reading at time X Diff.* Corrected diff.** Reading at time X Diff.* Corrected diff.**
Initial - 0                
0 - 5                
5 - 10                
10 - 15                
15 - 20                
Initial - 0                
0 - 5                
5 - 10                
10 - 15                
15 - 20                

* Difference = (initial reading at time 0) - (reading at time X)
** Corrected difference = (initial pea seed reading at time 0 - pea seed reading at time X) - (initial bead reading at time 0 - bead reading at time X)

11. Graph the results from the corrected difference column for the germinating peas and the dry peas at both temperatures.

Questions
1. This experiment uses a number of controls. Identify at least three of the controls, and describe the purpose of each.
2. Describe and explain the relationship between the amount of O2 consumed and time.
3. From the slope of the four lines on the graph, determine the rate of O2 consumption (in mL O2 /min) of germinating and dry peas during the experiments at both temperatures.
4. Why is it necessary to correct the readings from the peas with the readings from the beads?
5. Explain the effect of germination (versus nongermination) on pea seed respiration.
6. What is the purpose of KOH in this experiment?
7. Why did the vial have to be completely sealed around the stopper?
8. If you used the same experimental design to compare the rates of respiration of a 25 g mammal and a 25 g reptile, at 10oC, what results would you expect? Explain.
9. If respiration in a small mammal were studied at both 21oC and 10oC, what results would you expect? Explain.
10. Explain why water moved in the respirometers.