APBiology Unit 2, Chapter 8

Research Question

What factors affect the rate of cellular respiration in multicellular organisms?

Background

Living systems require free energy and matter to maintain order, to grow, and to reproduce. Energy deficiencies are not only detrimental to individual organisms, but they cause disruptions at the population and ecosystem levels as well. Organisms employ various strategies that have been conserved through evolution to capture, use, and store free energy. Autotrophic organisms capture free energy from the environment through photosynthesis and chemosynthesis, whereas heterotrophic organisms harvest free energy from carbon compounds produced by other organisms. The process of cellular respiration harvests the energy in carbon compounds to produce ATP that powers most of the vital cellular processes. In eukaryotes, respiration occurs in the mitochondria within cells. If sufficient oxygen is available, glucose may be oxidized completely in a series of enzyme-mediated steps, as summarized by the following reaction:

C6H12O6 + 6O2(g) → 6CO2(g) + 6H2O + energy More specifically,

C6H12O6 + 6O2 → 6CO2 + 6H2O + 686 kilocalories of energy per mole of glucose oxidized

The chemical oxidation of glucose has important implications to the measurement of respiration. From the equation, if glucose is the energy source, then for every molecule of oxygen consumed, one molecule of carbon dioxide is produced. Suppose you wanted to measure the overall rate of cellular respiration. • What specific things could you measure? • Which of these might be easier or harder to measure? In these procedures, you will learn how to calculate the rate of cellular respiration by using a respirometer system, gas pressure sensors, or O2 and CO2 gas sensors with computer

interface. These measure relative volume (changes in pressure or type of gas) as oxygen is consumed by germinating plant seeds. As oxygen gas is consumed during respiration, it is normally replaced by CO2 gas at a ratio of one molecule of CO2 for each molecule of O2 (remember the formula for respiration!). Thus, you would expect no change in gas volume to result from this experiment. However, in the following gas pressure procedures the CO2 produced is removed by potassium hydroxide (KOH). KOH reacts with CO2 to form the solid potassium carbonate (K2CO3) through the following reaction:

CO2 + 2KOH → K2CO3 + H2O Thus, as O2 is consumed, the overall gas volume in the respirometer decreases. The change in volume can be used to determine the rate of cellular respiration. Because respirometers are sensitive to changes in gas volume, they are also sensitive to changes in temperature and air pressure; thus, you need to use a control respirometer. What would be a good control for this procedure? Talk with another student for a minute, and come up with at least one possible control you could use. As you work through Procedures, think about this question: What factors can affect the rate of cellular respiration? In Designing and Conducting Your Investigation, you will design and conduct an experiment(s) to investigate at least one of your responses to this question or some other question you have. Your exploration will likely generate even more questions about cellular respiration. The investigation also provides an opportunity for you to apply and review concepts that you have studied previously, including the relationship between cell structure and function (mitochondria); enzymatic activity; strategies for capture, storage, and use of free energy; diffusion of gases across cell membranes; and the physical laws pertaining to the properties and behaviors of gases.

■■Learning Objectives • To learn how a respirometer system can be used to measure respiration rates in plant

seeds or small invertebrates, such as insects or earthworms • To design and conduct an experiment to explore the effect of certain factors, including

environmental variables, on the rate of cellular respiration • To connect and apply concepts, including the relationship between cell structure and

function (mitochondria); strategies for capture, storage, and use of free energy; diffusion of gases across cell membranes; and the physical laws pertaining to the properties and behaviors of gases

■■Getting Started Answer the following questions before you begin; 1. Why is it necessary to correct the readings of the respirometers containing seeds with the

readings taken from respirometers containing only glass beads? Your answer should refer to the concepts derived from the general gas law:

PV = nRT

Where P = pressure of the gas V = volume of the gas n = number of moles of the gas R = the gas constant (its value is fixed) T = temperature of the gas 2. What happens to the volume of the gas being measured (O2 consumption or CO2

production) when the temperature or pressure changes during the experiment? If pressure and temperature remain constant, will the volume of gas in the respirometers increase or decrease? Explain.

Hint: Several tutorials and animations explaining the general gas law are available online (e.g., http://www.nclark.net/GasLaws). 3. Imagine that you are given 25 germinating pea seeds that have been placed in boiling

water for five minutes. You place these seeds in a respirometer and collect data. Predict the rate of oxygen consumption (i.e., cellular respiration) for these seeds and explain your reasons.

4. Imagine that you are asked to measure the rate of respiration for a 25 g reptile and a 25 g mammal at 10°C. Predict how the results would compare, and justify your prediction. Produce a graph to illustrate your prediction.

5. Imagine that you are asked to repeat the reptile/mammal comparison of oxygen consumption, but at a temperature of 22°C. Predict how these results would differ from the measurements made at 10°C, and explain your prediction in terms of the metabolism of the animals. Produce a graph to illustrate your prediction.

6. What difficulties would there be if you used a living green plant in this investigation instead of germinating seeds?

Procedure 1 - Water Respirometers

1. Both a room-temperature bath (approximately 25°C) and a 10°C bath should be set up immediately to allow for time to adjust the temperature of each. Add ice to attain 10°C.

2. Respirometer 1: Obtain a 100 mL graduated cylinder and fill it with 50 mL of H2O 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.

Pea Volume _______________ mL 3. Respirometer 2: Refill the graduated cylinder with 50 mL of H2O. Drop 25 dried peas (not

germinating) into the graduated cylinder and then add enough glass 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 H2O. Determine how many glass 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, soak the cotton with 15% KOH. (Completely damp but not dripping wet) 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 1). It is important that the amounts of cotton and KOH be the same for each respirometer.

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.

8. Keep the tips of the pipettes out of the water during the seven minute equilibration period. You may make a sling of masking tape attached to each side of each of the water if necessary. Vials 1, 2, and 3 should rest in the room-

Figure 1:

Assembled

Respirometers

Figure 2:

Respirometers in the

Water Bath

temperature water bath (approximately 25°C) and vials 4, 5, and 6 should rest in the 10°C water bath (Figure 2).

9. After the equilibration period of seven minutes, place a small drop of food coloring in the tip of each pipette, then immerse all six respirometers entirely in their water baths. Water will enter the pipettes for a short distance and then stop. The food coloring is to assist in tracking the movement of the water into the pipette. If the water continues to move into a pipette, check 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). Check 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 6.1. If the water does not descend into the pipette to the calibration markings you will have to measure the distance manually.

Table 1: Measurement of 02 Consumption Using Volumetric Methods

Temp (˚C)

Time (min)

Beads alone Germinating Peas Dry Peas and Beads

Reading at end of interval

Change from initial* (correction factor)

Reading at end of interval

Change from initial*

Corrected change ‡

Reading at end of interval

Change from initial*

Corrected change‡

Initial (0)

0 – 5

5 – 10

10 – 15

15 – 20

Initial (0)

0 -5

5 – 10

10 – 15

15 - 20

* Change = (initial reading at time 0) – (reading at end of interval) ‡ Corrected change = (change – correction factor)

5. From the slope of the four lines on the graph, determine the rate of O2 consumption of germinating and dry peas during the experiments at room temperature and at 10°C. Recall that rate =

x

y

Table 6.2: Measurement of 02 Consumption Using Volumetric Methods Condition Calculations Rate in ml O2/minute Germinating @ 10C Germinating @ room temperature

Dry Peas @ 10C Dry Peas @ room temperature

PROCEDURE 2 – GAS PRESSURE SENSORS

MATERIALS

computer glass beads Vernier computer interface ice Logger Pro non-absorbent cotton 2 Vernier Gas Pressure Sensors thermometer 15% KOH in a dropper bottle test tube rack 15 germinating peas timer with a second hand 15 non-germinating peas three 18 150 mm test chambers 100 mL graduated cylinder two 1-hole rubber stopper assemblies absorbent cotton two 1 L beakers forceps ring stand 2 utility clamps

1. Connect the plastic tubing to the valve on the Gas

Pressure Sensor. 2. Connect the Gas Pressure Sensor to the computer

interface. Prepare the computer for data collection by opening the file “11C Cell Resp (Pressure)” from the Biology with Computers folder of Logger Pro.

To test whether germinating peas undergo cell respiration, you will need to

set up two water baths. prepare a respirometer for the germinating peas.

prepare a second, control respirometer containing glass beads.

3. Set up two water baths, one at about 25°C and one at about 10°C. Obtain two large beakers and ¾ fill each with water. Add ice to attain the 10°C water bath.

4. To be sure the volumes of air in all respirometers are equal; you will need to measure the volume of the fifteen peas that will be in the experimental respirometer. The control respirometer must have an equal volume of glass beads (or other non-oxygen consuming material) to make the air volume equal to the respirometer with germinating peas. Similarly, glass beads will be used to account for any volume difference between the germinating and non-germinating peas.

Figure 3:

Respirometer in the

Water Bath

Respiration API Lab 6 APUnit 2 2015.docx Page 7 of 11

5. Obtain three glass tubes and label them “T1”, T2”, and “T3”. . 6. Place a 2 cm wad of absorbent cotton in the bottom of each test tube. Using a

dropper pipette, carefully add a sufficient amount of KOH to the cotton to soak it. (Note: Do not allow any of the KOH to touch the sides of the test tube. The sides should be completely dry, or the KOH may damage the peas.) CAUTION: Potassium hydroxide solution is caustic. Avoid spilling it. See figure 4.

7. Prepare the test tube containing germinating peas (T1):

a. Add 50 mL of water to a 100 mL graduated cylinder. b. Place 15 germinating peas into the water. c. Measure the volume of the peas by water displacement. Record that volume in

Table 1. d. Gently remove the peas from the graduated cylinder and blot them dry with

a paper towel.

e. Add a small wad of non-absorbent dry cotton to the bottom of the glass tube to prevent the peas from touching the KOH saturated cotton.

f. Add these germinating peas to the respirometer labeled “T1”.

8. Prepare the glass tube containing non-germinating peas (T2):

a. Refill the graduated cylinder with 50 mL of water. b. Place 15 non-germinating peas into the water.

c. Measure the volume of the peas by water displacement. Record the volume in Table 1.

d. Add a sufficient number of glass beads to the non-germinating peas and water until they displace exactly the same volume of water as the germinating peas.

e. Gently remove the peas and glass beads from the graduated cylinder and dry them with a paper towel.

f. Add a small wad of dry non-absorbent cotton to the bottom of the glass tube to prevent the peas from touching the KOH saturated cotton.

g. Add the non-germinating peas and glass beads to the respirometer labeled “T2”.

9. Prepare the glass tube containing glass beads (T3):

a. Refill the graduated cylinder with 50 mL of water. b. Add a sufficient number of glass beads to the water until they displace exactly the

same volume of water as the germinating peas. c. Remove the glass beads from the graduated cylinder and dry them. d. Add a small wad of dry non-absorbent cotton to the bottom of the glass tube to

prevent the peas from touching the KOH saturated cotton.

e. Add the glass beads to the respirometer labeled “T3”. Part I Germinating peas, room temperature

10. Insert a single-holed rubber-stopper into glass tube T1 and T3. Note: Firmly twist the stopper for an airtight fit. Secure each test tube with a utility clamp and ring-stand.

11. Arrange glass tubes T1 and T3 in the warm water bath using the apparatus shown in Figure 3. Incubate the test tube for 10 minutes in the water bath. Be sure to keep the temperature of the water bath constant. If you need to add more hot or cold water, first remove about as much water as you will be adding, or the beaker may overflow. Record the resulting temperature of the water bath once incubation has finished in Table 2.

Cotton with KOH

Peas

Cotton non-absorbent

Figure 4

Respiration API Lab 6 APUnit 2 2015.docx Page 8 of 11

Note: Be sure the tubes are submerged to an equal depth, just up to the rubber stoppers. The temperature of the air in the tube must be constant for this experiment to work well.

12. When incubation has finished, connect the free-end of the plastic tubing to the connector in the rubber stopper as shown in Figure 5.

13. Click to begin data collection. Maintain the temperature of the water bath during the course of the experiment.

14. Data collection will end after 20 minutes. Monitor the pressure readings displayed in the live readouts on the toolbar. If the pressure exceeds 130 kPa, the pressure inside the tube will be too great and the rubber stopper is likely to pop off. Disconnect the plastic tubing from the Gas Pressure Sensor if the pressure exceeds 130 kPa.

15. The rate of respiration can be measured by examining the slope of the pressure change vs. time plot at the right of the screen. Calculate a linear regression for the pressure change vs. time graph:

a. Click on the Pressure Change vs. Time graph to select it. b. Click the Linear Fit button, , to perform a linear regression. A floating box will

appear with the formula for a best fit line. c. Record the slope of the line, m, in Table 3 as the rate of oxygen consumption by

germinating peas. d. Close the linear regression floating box.

16. Move your data to a stored data run. To do this, choose Store Latest Run from the

Experiment menu.

Part II Non-germinating peas, room temperature

17. Disconnect the plastic tubing connectors from the rubber stoppers. Remove the rubber stopper from each test tube.

18. Repeat Steps 10 – 16, using test tubes T2 and T3.

Part III Germinating peas, cool temperatures

19. Disconnect the plastic tubing connectors from the rubber stoppers. Remove the rubber stopper from each test tube.

20. Repeat Steps 10 – 16, using test tubes T1 and T3 in a cold water bath. 21. Make a printout of the graph with each of the three trials.

Figure 5

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Figure 1 Figure 6

DATA - GAS PRESSURE SENSORS

Table 1- Pressure Sensor

Peas Volume (mL)

Germinating

Non-germinating

Table 3 - Pressure Sensor

Peas Rate of Respiration (kPa/min)

Germinated, room temperature

Non-germinated, room temperature

Germinated, cool temperature

PROCEDURE 3 – OXYGEN / CO2 SENSOR

MATERIALS

Computer 250 mL respiration chamber Computer interface ice cubes Logger Pro software 1 large beaker O2 Gas Sensor thermometer 15 germinating peas two 100 mL beakers 15 non-germinating peas

PROCEDURE

1. Connect the O2/ CO2 Gas Sensor to the computer interface. 2. Prepare the computer for data collection by opening the file “11A Cell Resp O2” or

“11B Cell Resp CO2” from the Biology with Computers folder of Logger Pro. 3. Obtain 15 germinating peas and blot them dry between two pieces of paper towel.

Use the thermometer to measure the room temperature. Record the temperature in Table 1.

4. Place the germinating peas into the respiration chamber. 5. Place the Gas Sensor into the bottle as shown in Figure 6. Gently push the sensor

down into the bottle until it stops. The sensor is designed to seal the bottle without the need for unnecessary force.

6. Wait two minutes, then begin collecting data by clicking . Data will be collected for 10 minutes.

7. When data collection has finished, remove the Gas Sensor from the respiration chamber. Place the peas in a 100 mL beaker filled with cold water and an ice cube.

8. Fill the respiration chamber with water and then empty it. Thoroughly dry the inside of the respiration chamber with a paper towel.

9. Determine the rate of respiration:

Table 2 - Pressure Sensor

Water bath Temperature (°C)

warm

cool

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a. Click the Linear Fit button, , to perform a linear regression. A floating box will appear with the formula for a best fit line.

b. Record the slope of the line, m, as the rate of respiration for germinating peas at room temperature in Table 2.

c. Close the linear regression floating box.

10. Move your data to a stored run. To do this, choose Store Latest Run from the Experiment menu.

11. Obtain 15 non-germinating peas and place them in the respiration chamber 12. Repeat Steps 5 – 10 for the non-germinating peas.

Part II Germinating peas, cool temperatures

13. Place the respiration chamber in a 1 L beaker. Cover the outside of the chamber with ice.

14. Use the thermometer to measure the water temperature of the 100 mL beaker containing the germinating peas. Record the water temperature in Table 1.

15. Remove the peas from the cold water and blot them dry between two paper towels. 16. Repeat Steps 5 – 9 to collect data with the germinating peas at a cold temperature. 17. To print a graph showing all three data runs:

a. Label all three curves by choosing Text Annotation from the Insert menu, and typing “Room Temp Germinated” (or “Room Temp Non-germinated”, or “Cold Germinated”) in the edit box. Then drag each box to a position near its respective curve. Adjust the position of the arrow head.

b. Print a copy of the graph, with all three data sets and the regression lines displayed.

DATA

Table 1- ___ Gas Sensor

Condition Temperature (°C)

room

cold water

Table 2 - ___ Gas Sensor

Peas Rate of Respiration (%/min)

Germinating, room temperature

Non-germinating, room temperature

Germinating, cool temperature

Respiration API Lab 6 APUnit 2 2015.docx Page 11 of 11

QUESTIONS

1. Do you have evidence that cell respiration occurred in peas? Explain.

2. What is the effect of germination on the rate of cell respiration in peas?

3. What is the effect of temperature on the rate of cell respiration in peas?

4. What was the role of the control respirometer in each series of experiments?

5. Why do germinating peas undergo cell respiration?

ANALYSIS OF RESULTS

1. In this activity, you are investigating both the effect of germination versus non-

germination and warm temperature versus cold temperature on respiration rate. Identify the hypotheses being tested in this activity. If… Then … Because … 2. This activity uses a number of controls. Identify at least three of the controls, and

describe the purpose of each control. 3. Describe and explain the relationship between the amount of O2 consumed and time. 4. Why is it necessary to correct the readings from the peas with the readings from the

beads in procedure 1 and 2? 5. Explain the effect of germination (versus non-germination) on pea seed respiration. 6. To the right is a sample graph of possible data

obtained for oxygen consumption by germinating peas up to about 8°C. Draw in predicted results through 45°C. Explain your prediction.

7. What is the purpose of KOH in procedure 1 and 2?

8. Why did the vial have to be completely sealed around the stopper?

9. If you used the same experimental design to compare the rates of respiration of a 25 g. reptile and a 25 g. mammal, at 10°C, what results would you expect? Explain your reasoning. Illustrate with a graph or relate to a previous graphed prediction.

10. If respiration in a small mammal were studied at both room temperature (2 1°C) and 10°C, what results would you predict? Explain your reasoning. Illustrate with a graph or relate to a previous graphed prediction.

11. Explain why water moved into the respirometers’ pipettes in procedure 1.