INV~t:;TIGATION 6 CELLULAR RESPIRATION* 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: More specifically, C H 0 60 6 CO 6 H 686 kilocalories of energy 0 6 12 6 + 2 ~ 2 + 2 + mole of glucose oxidized 1be 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 conswned, 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 Procedures, you will learn how to calculate the rate of cellular respiration by using a respirometer system (either microrespirometers or gas pressure sensors with computer interface). These measure relative volume {changes in pressure) as oxygen is consumed by germinating plant seeds. As oxygen gas is consumed during respiration, it is normally "Transitioned from the AP Biology Lab Manual (200J) Investigation 6 S71
replaced by C0 2 gas at a ratio of one molecule of C0 2 for each molecule of 0 2 Thus, you would expect no chahge in gas volume to result from this experiment. However, in the following procedure the C0 2 produced is removed by potassium hydroxide (KOH). KOH reacts with C0 2 to form the solid potassium carbonate (K 2 C0 3 ) through the following reaction: Thus, as 0 2 is consumed, the overall gas volume in the respirometer decreases. 1he 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. vvhat would be a good control for this procedure? 1alk 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 Obiectives 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 ceuular 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 General Safety Precautions You must wear safety goggles or glasses, aprons, and gloves during this investigation(s) because KOH (or the alternative, NaOH in Drano) is caustic. Follow your teacher's instructions when using tl1e hot glue gun to seal microrespirometers. Do not work in the laboratory without your teacher's supervision... S72 Investigation 6
THE INVESTIGATIONS Getting Started Your teacher may assign the following questions to see how much you understand concepts related to respiration before you design and conduct your own investigation: 1. Why is it necessary to correct the readings of the respirometers containing seeds "With the readings taken from respirometers containing only glass bead'l? Your answer should refer to the concepts derived from the general gas law: Where P = pressure of the gas V = volume ofthe gas n = number of moles of the gas R = the gas constant (its value is fi."':ed) T = temperature of the gas PV=nRT 2. vvhat happens to the volume of the gas being measured (0 2 consumption or C0 2 production) when the temperature or pressure changes during the experiment? If pressure and temperature remain constant, will the volume ofgas in the respirometers increase or decrease? Please 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. 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. 6. What difficulties would there be if you used a living green plant in this investigation instead of germinating seeds? lnvestigatioo6 S73
Materials: black eyed peas white beans glass beads graduated cylinder red water Procedure: 3 syringes graduated glass pipette plastic tubing plastic pipette KOH absorbent cotton non-absorbent cotton short bottle cap 1. Using the water displacement method: fill a graduated cylinder with 50 ml of water. Put in 20 germinating beans/peas (Those that have been soaking in water) of your choice. Record the new volume. This is the volume you will"match" in steps 3 and 6. 2. Empty the graduated cylinder, keeping the beans/peas. 3. Place the beans/peas in the syringe. 3. Using the water displacement method described above (#1), this time matching that volume with glass beads. 4. Empty the graduated cylinder, keeping the glass beads. 5. Place the glass beads in the 2nd syringe. 6. Using the water displace method describe above (#1), place 2- non-germinating beans/peas of the same type. You should not reach the same volume as you did in #1. Use enough glass beads to match that volume. 7. Empty the graduated cylinder, keeping the beans/peas and glass bead mixture, placing them in the 3'd syringe. 8. In each of the syringe place a "small wad" of NON-absorbent cotton. 9. On top of this non-absorbent cotton, place a "small wad"' of absorbent cotton. 10. Using the plastic pipette, put in approximately 5 drops of KOH solution (found at my table). 11. Insert the syringe plunger to meet the cotton. 12. Attach 4" strip of plastic tubing to your loaded syringe. 13. Attach the graduated glass pipette to the plastic tubing. You have just made a "respirometer". 14. Draw in a bit of red water (about 1 ml) to your syringe. 15. Using the bottle cap, lay out the syringe and tubing as illustrated. 16. Every 3 minutes record where the red water lies on each graduated pipette in the tables below. Do this for 24 minutes. 17. Using your data, you will graph the rate of cellular respiration: time will be on your x-axis, and 0 2 concentration will be on your y-axis. Graph carefully, as you will be calculating slope from your graphed line. 18. *** If there are changes in the Glass beads then you must subtract that value, at that time, from the volumes at the germinating and non-germinating**** Questions: 1. What are the safety concerns and precautions for this lab? 2. What is the purpose of the KOH? 3. What is the purpose of using both the absorbent and non-absorbent cotton? 4. Why are we using the water displacement method instead of using the same volume measurement on the syringe? 5. Why are we checking the seals of each component of the apparatus? Table 1 Respirometer 1: Germinating Beans/Peas Time Interval Omin 3 min 6min 9min 12 min 15 min 18min 21min 24min (reading- time N/A Table 2 Respirometer 2: Glass Beads Time Interval Omin 3 min 6min 9min 12min 15min 18min 21 min 24min (reading- time N/A Table 3 Respirometer 3: Non-germinating Beans/Peas+ Glass Beads Time Interval Omin 3 min 6min 9min 12 min 15 min 18min 21min 24min (reading - time N/A
Analysis: Graph the rate of 0 2 Consumption on the following graph using different colors- 1 each for the germinating bean/pea, nongerminating bean/pea and the glass beads. Use a line of BEST FIT for each line so that you can calculate slope. TITLE of GRAPH: (Be sure to label the axes as well) 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 5 10 15 20 Analyzing Results? Questions 1. State a hypothesis that relates to what is being tested by this lab exercise. 2. State a hypothesis that specifically relates to the state of seed germination that is being tested by this lab exercise. 3. Calculate the RATE of oxygen consumption for the germinating seeds. Rate can be calculated by determining the SLOPE of the line from your graph above. 4. In this lab exercise, what is the purpose of the... a) Beads b) KOH c) Respirometer 5. Explain why the water moved within the pipet. 6. What additional questions can you expolore about cellular respiration using the same respirameters from this experiment? 7. Do you have any suggestions for improving the design of the respirameters for measuring oxygen concsumption/cellular respiration? 8. What other cosiderations/evaluations can be made? Evaluating Results 1. Was your initial hypothesis about the affect of you factor on the rate of cellular respiration supported? Why or why not 2. What were some challenges you had in performing your experiment? Did you make any incorrect assumptions? 3. Were you able to perform without difficulty the mathematical routines required to analyze your data? If not, what calculations where challenging or required help from your classmates or teacher? What have you learned to diminish these difficulties?