Burping Yeast: An Investigation of Cellular Respiration Student Materials Introduction... 2 Lab Protocol... 4 Data Collection Worksheet... 6 Pre-Lab Questions... 7 Post-Lab Questions and Analysis... 8 Last updated: 10/11/2018 Copyright MassBioEd 2013 1
Burping Yeast: An Investigation of Cellular Respiration Introduction All living things have a number of characteristics in common. Whether a single-celled bacterium or a human, all living things grow, develop, sense their environment, and work to maintain stable internal conditions. Each of these activities requires energy, which brings us to another shared characteristic of all life: all living things convert the energy stored in food into cellular energy. Of course, what constitutes food for a plant or bacteria is a bit different from what you consider food. But no matter what form the food takes, it almost always comes down to the same basic building block the simple sugar glucose (C 6H 12O 6). The process of converting energy stored in the chemical bonds of glucose into the chemical bonds of the molecule ATP is called cellular respiration. During cellular respiration, glucose is broken down by a series of chemical reactions. As the chemical bonds of glucose are broken, the energy and electrons released can be used to produce ATP, or adenosine triphosphate. ATP, like glucose, is an energy storage molecule, but unlike glucose, cells can use ATP directly. ATP is used by cells to do everything from replicating DNA to transporting some molecules across the cell s membrane. In humans, it is ATP that makes our hearts beat, our muscles contract, and our lungs fill with air in short, ATP provides the energy that makes cells go no ATP, no life. Most cellular respiration occurs in the presence of oxygen and is called aerobic ( with oxygen ) cellular respiration. When oxygen is not available some cells (yeast and overworked muscles cells, for example), can undergo an anaerobic ( without oxygen ) process called fermentation. Both pathways begin with a set of reactions known as glycolysis, which transforms glucose to the 3-carbon molecule pyruvate. In the aerobic pathway, the pyruvate is further broken down in the reactions of the Krebs cycle. For most biochemical reactions (even spontaneous reactions) to occur in a time frame relevant in a cell, an energy barrier called the energy of activation must be overcome. Enzymes are specialized proteins that speed up chemical reactions by lowering the activation energy required. Enzymes are affected by the conditions of their environment, including ph and temperature. Each enzyme is specific for a single reaction and is effective within a narrow range of conditions. If conditions vary too much from this range, then the enzyme cannot work. Where do the carbon atoms from pyruvate end up? You know the answer to this question! Take a deep breath and hold it. Now blow out. What are you exhaling? Electrons released from the breakdown of glucose (and pyruvate) following the Krebs cycle are carried to the electron transport chain. What molecule accepts the electrons from glucose? You know the answer to this question, too! Take a deep breath. What did you just inhale? When oxygen is absent, where do the electrons released from the breakdown of glucose and pyruvate go? They are given back to pyruvate, generating ethanol and CO 2 (in the case of yeast) or lactic acid (in the case of muscle cells). Why does respiration or fermentation by yeast cause bread dough to rise? Copyright MassBioEd 2013 2
Both aerobic respiration and fermentation produce ATP. The aerobic pathway (via glycolysis, the Krebs cycle, and electron transport chain) produces much more ATP than fermentation per molecule of glucose more than 30 ATPs for the aerobic pathway and just 2 ATPs (from glycolysis) for fermentation. Figure 1 shows the major sets of reactions involved in fermentation and aerobic respiration. Figure 1. Overview of the ATP production during cellular respiration. The aerobic cellular respiration pathway (glycolysis + Krebs cycle + electron transport chain) generates 25-32 molecules of ATP per molecule of glucose. By contrast, fermentation generates just 2 ATPs per molecule of glucose. The overall equations for both aerobic cellular respiration and alcoholic fermentation (the type of fermentation carried out by yeast) are below. Aerobic Cellular Respiration: Alcoholic Fermentation: C 6H 12O 6 + 6O 2 6CO 2 + 6H 2O + energy Glucose + oxygen carbon dioxide + water C 6H 12O 6 2CO 2 + 2C 2H 5OH + energy Glucose carbon dioxide + ethyl alcohol In the lab today, you will investigate the breakdown of glucose by baker s yeast (Saccharomyces cerevisiae). Like all eukaryotes, baker s yeast cells contain a nucleus and other membrane-bound organelles including mitochondria, the site of aerobic cellular respiration in eukaryotes. But baker s yeast is special in that it is a facultative anaerobe, which means that under some conditions, such as low oxygen concentration and/or high glucose concentration, the cells can switch from aerobic respiration to fermentation. Notice from the equations that as the cells carry out both forms of cellular respiration, they break down glucose and release carbon dioxide gas. In this lab you will compare rates of under different conditions by counting the number of carbon dioxide bubbles released by the yeast over 10 minutes. Copyright MassBioEd 2013 3
Burping Yeast Lab Protocol Materials: Check your workstations to make sure all supplies are present before beginning the lab. Student Workstation: 3 600-mL beakers (or 1-quart containers) 3 test tubes 1 test tube rack 5 transfer pipettes 3 metal hex nuts 3 2.0-mL microcentrifuge tubes microcentrifuge tube rack 1 p1000 micropipette and pipette tips 1 thermometer timer calculator permanent marker (such as Sharpie) 0.5 M glucose solution 10% yeast solution Common Workstation: 5-6 C water 40 C water 20-22 C water Protocol 1. Using the p1000 micropipettor, add 1000 L of yeast solution and 500 L of glucose solution to each of three microcentrifuge tubes. Caution! To avoid contamination, be sure to use a new tip for each solution. Cap the tubes and gently mix the solution by tapping the bottom of the tube with your finger. Set up your respirometers: Important! Each student in the group should set up a respirometer, so there will be three total per group. 2. When preparing the respirometer, it is very important that you get all of the yeast-glucose solution into the bulb of the transfer pipette. Follow the steps below to deposit the solution into the bulb of a transfer pipette. a) Obtain a clean transfer pipette. b) Completely depress the bulb of the pipette. c) Slowly draw up the entire 1.5 ml of yeast-glucose solution from one tube into the pipette. d) Flip the pipette bulb side down. e) Release the bulb to draw the solution into the bulb. f) Slide a metal hex nut over the shaft of the transfer pipette loaded with the yeast-glucose as shown in Figure 2 and place the respirometer (still inverted) into a test tube rack. Use an additional hex nut if needed. Figure 2. Respirometer. Carefully follow steps 2a 2f to ensure that all of your yeast-glucose solution is loaded into the bulb of the transfer pipette. Copyright MassBioEd 2013 4
Prepare all three respirometers before going on to step 3 Set up the experimental conditions and control: 3. Obtain 3 600-mL beakers and label them each with the permanent marker A, B, and C. Fill each one up, almost to the top, as outlined below: Beaker A: fill with water from source A (coolest temperature water) Beaker B: fill with water from source B (medium temperature water) Beaker C: fill with water from source C (warmest temperature water) 4. Fill a test tube with water from one of each of the three beakers (A, B, and C) and place it in the corresponding beaker. Example: Fill a test tube with water from Beaker A and then place it in Beaker A. Repeat for the remaining two beakers. 5. Slowly lower a respirometer (still inverted) into each of the test tubes in the three beaker water baths, as shown in Figure 3. Be sure that the tip of the respirometer is submerged. 6. Set your timer for three minutes. Do NOT record any data until the timer goes off. This time allows yeast-glucose solution in the respirometer to reach the same temperature as the surrounding water. While waiting for the three minutes to elapse, assign each of your group members to a beaker for data collection (Beaker A, B, or C) and place a thermometer into each beaker. 7. Once the timer goes off, record the starting water temperature in your beaker in the appropriate place in data collection table (next page). Important! Be sure to record this and all data you collect in the column that corresponds to your assigned beaker. Collect Data 8. Set the timer for one minute. Figure 3: Complete set-up. The respirometer has been placed into a test tube containing water from its associated beaker, and then the test tube has been placed into the beaker. 9. When all group members are ready, start the timer and begin counting the number of bubbles that rise from the tip of your assigned respirometers (A, B or C). Important! Be sure to watch carefully it is easy to miss the small bubbles! When the timer goes off, record your data in the data collection table. Once everyone is ready, reset and restart the timer and start your count again. Repeat for 8 more times for a total of 10 1-minute intervals. 10. At the end of the 10-minute period, calculate the total number of bubbles you observed and record the data in the appropriate place in the data collection table. 11. Complete your data table by copying the data recorded by each of your other group members. 12. Record any other observations you make during the experiment. Copyright MassBioEd 2013 5
Burping Yeast: An Investigation of Cellular Respiration Data Collection Worksheet Name: Other group members: Class/period: Date: Assigned Beaker (circle one): A B C Time Number of Bubbles/Minute (After 4 minute equilibration time) Beaker A coolest-temperature Beaker B medium-temperature Beaker C warmest-temperature Starting Temp: C Starting Temp: C Starting Temp: C Minute 1 Minute 2 Minute 3 Minute 4 Minute 5 Minute 6 Minute 7 Minute 8 Minute 9 Minute 10 Total number of bubbles Copyright MassBioEd 2013 6
Burping Yeast: An Investigation of Cellular Respiration Pre-Lab Questions Directions: After reading through the introduction and protocol for the Burping Yeast lab, answer the questions below. 1. Why is cellular respiration important to all living things? 2. What is the difference between glucose and ATP in terms of the cell s ability to use it? 3. Like baker s yeast, human muscle cells can perform fermentation. Explain why muscle cells are more likely to ferment during strenuous exercise than when at rest. 4. What is the role of enzymes in cellular respiration? 5. How will you measure the rate of cellular respiration in this experiment? 6. What is the purpose of the metal nut on the respirometer? 7. Propose a hypothesis about how temperature will affect the yeast s rate of cellular respiration. 8. What are the dependent and independent variables for this experiment? Copyright MassBioEd 2013 7
Burping Yeast: An Investigation of Cellular Respiration Post-Lab Questions and Analysis Directions: After completing the Burping Yeast lab, answer the questions below. 1. Why was counting the number of bubbles an appropriate way to measure the rate of cellular respiration occurring within the yeast? 2. What are some potential limitations to using this method to measure cellular respiration? 3. In Excel, or by hand, generate a bar graph to compare the total number of bubbles counted for each of the three beakers. Remember to label all axes and include an appropriate title. Copyright MassBioEd 2013 8
4. Calculate the average number of bubbles per minute for each beaker and record the information below. Beaker A: Beaker B: Beaker C: 5. In Excel, or by hand, create a second bar graph (like that for #2 above) that includes both the total number of bubbles/10 minutes AND the average number of bubbles/minute for each temperature. Remember to label all axes, include an appropriate title, and label or include a key to indicate which bars represent the total number of bubbles and which represent the average number of bubbles. 6. What did the results indicate about the effect that temperature has on the rate of cellular respiration in baker s yeast? Copyright MassBioEd 2013 9
7. Why did the temperature affect the rate of cellular respiration? 8. Do you think the first organisms on Earth used an aerobic cellular respiration pathway or an anaerobic cellular respiration pathway? Explain your answer. Hint: The first organisms on Earth were not photosynthetic. Where does the oxygen in Earth s atmosphere come from? 9. Bread dough is usually made using flour, water, salt, and sugar. You have learned that it will rise to a fluffy ball when baker s yeast is added and uses the sugar for fermentation. a. Do you think that bread dough made without sugar would rise? Explain your reasoning. b. Bread dough made with wheat flour can double or even triple in volume, but bread dough made with flour from other grains may not rise as much. Form a hypothesis that explains the difference. 10. Under what ph and temperature conditions do you think a human muscle cell carries out cellular respiration? What about a cell from the lining of the human stomach? Copyright MassBioEd 2013 10