BIO 3A Lab: Cellular Respiration (02/09) Page 1 of 4 Biology 3A LABORATORY Cellular Respiration and Fermentation Objectives • To study processes of anaerobic and aerobic respiration • To determine the amount of oxygen consumed during aerobic respiration • To determine the amount of carbon dioxide produced during aerobic respiration • To study the effect of substrate difference on anaerobic respiration in yeast • To investigate the process of fermentation used in food making PLEASE BRING A USB THUMBDRIVE TO LAB TODAY Introduction All living organisms require energy in order to sustain the many processes involved in life. The energy for these processes is provided by cellular respiration, a catabolic process that releases energy (exergonic), most often as ATP. It is essential that the chemical reactions involved in cellular respiration occur at a rapid rate and within optimum conditions. Enzymes are critical in this process. Aerobic respiration in germinating peas Cellular respiration involves glycolysis, the Krebs cycle and the electron transport chain. As you may recall from lecture, glycolysis is essentially an anaerobic process since it is not dependent upon the presence of oxygen. The fate of pyruvate, the end product of glycolysis, is dependent on the presence of oxygen. If oxygen is not present, the two pyruvates (from the complete oxidation of one glucose molecule) will remain in the cytosol and undergo the anaerobic process called fermentation. There is no “extra” energy yield from fermentation. If oxygen is present, the pyruvates will be shuttled to a mitochondrion, altered and enter into a series of reactions involving the Krebs cycle and the Electron Transport Chain (ETC). Both of these processes are dependent on the presence of oxygen and are aerobic in nature. The Krebs cycle only produces 1 ATP molecule directly per cycle. However, it is indirectly responsible for the greatest ATP production by generating coenzymes, both NADH and FADH2. When these coenzymes are reoxidized in the electron transport chain, many molecules of ATP are generated (a theoretical 36 – 38 ATP per glucose). Many living organisms undergoing aerobic respiration will use oxygen and produce carbon dioxide. In this lab you will indirectly determine metabolic rate during aerobic respiration in germinating peas placed in a manometer (a closed chamber) by monitoring the amount of CO2 produced. Procedure A: Setup: 1. Obtain 10 – 12 four- to six-day old germinating peas, determine the mass and record the mass on Table 3. 2. Obtain one Pasco Xplorer GLX data logger, CO2 probe and the CO2 measurement container. 3. To the container, add a 2 cm ball of absorbent cotton to the bottom. 4. Add normal peas to the container. 5. Wrap the container with aluminum foil to inhibit photosynthesis. 6. Place the CO2 probe onto the container. BIO 3A Lab: Cellular Respiration (02/09) Page 2 of 4 Measurement: 1. Connect the CO2 probe to the Xplorer GLX WITHOUT tipping the container upside down (do not let the wet peas hit the measuring probe) to one of the four PASPORT sensor ports (They look like serial cable ports at the top end of the screen). 2. Turn on the GLX (small green button on the bottom left hand side of the handle) 3. If the Graph is not already displayed on the screen press the Home Screen ( ) and F1 ( ) at the same time to go to the Graph. 4. If you need to start a new graph, Press F4 then press #7 to start a new graph ready to display data from the PASPORT sensor. 5. Equilibrate the sealed peas in the container for 5 minutes on the table. Do not handle the containers with your warm hands to assure accurate results. 6. After five minutes, Press to start data recording for 10 minutes. 7. To stop data recording, press again. 8. Press the Home Screen ( ) button for the menu display. 9. Use the cursor and select Graph menu and press activate ( ). 10. Return the peas to the original container and discard the used cotton ball. Clean out the container if need be. Do not remove the aluminum foil. 11. Obtain a new 2 cm cotton ball and repeat the procedure with the freeze/thawed peas. To Download your data to your USB device: 1. Attach your USB drive to the USB port on the right side of the display to save your file to your USB drive. 2. Select Data Files, press ( ). 3. Select the file and press F1. Next to the name, it should say [Open]. 4. Press Home ( ). 5. Cursor down to Table and press activate ( ). 6. Press F4 and cursor down to Export All Data and press ( ). 7. Your data should be downloaded to you drive. 8. Open Excel and select the file (Export file) from your drive. The Text Import Wizard will pop-up. 9. Click Next. Make sure that TAB is selected (that’s the default) on the Tab Delimiters and click next. 10. Using Excel, select scatter plot for the entire 10 minute duration. Add a trendline with the equation and r2 value. 11. Return the peas to the original container and discard the used cotton ball. Clean out the container for the next group. CO2 production during aerobic respiration From the acid, bases and buffers lab, you should recall that CO2 can combine with water to form carbonic acid, which dissociate as follows: CO2 + H2O  H2CO3  H+ + HCO3-  H+ + H+ + CO32- (3)BIO 3A Lab: Cellular Respiration (02/09) Page 3 of 4 In this exercise, you will indirectly determine the amount of CO2 produced during cellular respiration in a plant and an aquatic animal. You will use phenolphthalein to detect changes in pH resulting from CO2 production (H2CO3). Recall that phenolphthalein is red in basic solutions and colorless in acidic solutions. Since we are not directly measuring CO2 production, calculate a relative measure of respiration by measuring the volume of NaOH required to neutralize carbonic acid. Procedure B Setup & Volume determination 1. Place 75 ml of the dechlorinated water in each of the three labeled 150 ml beakers(“control”, “fish”, and “plant”). The solution has been