Student Guide NAME ——___ DATE Carolina” Cell Respiration for AP Biology Background You are probably familiar with photosynthesis, the metabolic process that plants use to harness energy from the sun. But how do plants acquire energy when they germinate underground, out of the reach of sunlight? They metabolize sugars much like humans do, through cellular respiration. All cells need energy. Energy is contained in the molecular structure of organic compounds such as carbohydrates, proteins, and fats. Carbohydrates, also called sugars, are the primary source of cellular energy. When the bonds of a carbohydrate molecule are broken, in a series of small steps and with the help of specific enzymes, energy is released from the bonds. The energy, stored in a molecule called adenosine triphosphate (ATP), can then be used by the cell. ATP is the chief energy source of cells. It stores energy in the structure of its three-phosphate tail. The removal of a phosphate from the ATP molecule releases eneray that powers almost all metabolic processes. When a phosphate is removed, adenosine triphosphate becomes adenosine diphosphate (ADP). Through cell respiration, fermentation, and other metabolic processes, there is a constant cycling between ATP and ADP. —, coy menu @+ ADP ATP Energy eee | Figure 1. Cycling of ATP and ADP In this lab, we will focus on aerobic cellular respiration. The series of reactions that occurs during aerobic cellular respiration is grouped into steps called glycolysis, Acetyl CoA synthesis, Krebs cycle, and electron transport chain. The breakdown of the sugar molecule is summarized by the following equation: C.H,,0, +60, = 6CO, + 6H,O + 36 ATP 01 sea pe up Comme nS CAROLINA = s-1Cell Respiration Kit for AP Biology Student Guide Glycolysis Glycolysis is a 10-step series of reactions that splits a 6-carbon glucose molecule into two 3-carbon molecules called pyruvate. The process occurs in the cytoplasm of a cell. As glucose is broken down, four ATPs and two NADH molecules are produced. Because two ATP molecules are required to start the reaction, there is @ net gain of two ATP 'f oxygen is present, cells may undergo aerobic cellular respiration to produce more energy by further breaking down the derivatives of glucose. All higher organisms and many microorganisms have the necessary enzymes to perform aerobic cellular respiration. Krebs Cycle Process Starting Material Net Energy Output Glycolysis 1 glucose 2. NADH, 2ATP ‘Acetyl: Co synthesis and 2 pyruvate 8 NADH, 2 FADH,, 2 ATP Electron Transport Chain 10 NADH, 2 FADH, 32 ATP Acetyl-CoA synthesis and Krebs Cycle If oxygen is present, the pyruvate made during glycolysis enters the mitochondria of the cell, where itis initially converted into a molecule called acetyl-CoA. As this happens, one molecule of carbon dioxide (CO,) and one molecule of NADH are produced. The acetyl-CoA is then broken down through a series of six reactions referred to as the Krebs cycle or the citric acid cycle. For each acetyl-CoA broken down, two CO, molecules are produced, and some of the energy released from its bonds is stored in ATP. Some of the energy also passes as electrons to the electron carrier molecules NAD+ and FAD, yielding NADH and FADH,. The electrons held in ‘these electron carriers can be used to make additional ATP through the electron transport chain. Electron Transport Chain The NADH and FADH, produced during glycolysis and the Krebs cycle are used to generate more ATP in the electron transport chain. NADH and FADH, molecules are called electron carriers because they transport electrons and associated hydrogen atoms to a series of membrane-embedded proteins called the electron transport chain. Here, the hydrogens and electrons are stripped from the electron carriers, leaving NAD+ from NADH and FADH from FADH,. These molecules constantly cycle between forms within the cel AAs the electrons from the NADH and FADH, are passed between the proteins of the electron transport chain, energy is released and used to shuttle the hydrogen ions into the intermembrane space of the mitochondria. As the ions accumulate, they form a gradient across a membrane. This gradient creates @ type of pressure that causes hydrogen ions to rush through a channel in the membrane formed by a protein called ATP synthase, ATP synthase functions somewhat like a windmill; the rush of hydrogen ions spins a component of ATP synthase, which drives production of ATP. In other words, ATP synthase adds another phosphate group to ADP. In eukaryotic cells, the electrons from each NADH yield three ATP ‘molecules, and each FADH, yields two ATP molecules. The electron transport chain produces 16 ATP molecules for each pyruvate molecule. Remember that there are two pyruvate molecules from every glucose molecule that enters the system. Thus, from aerobic respiration of each glucose molecule, approximately 36 ATP molecules are made, ‘Oxygen comes into play only during the final step of the electron transport chain. Remember that electrons are passed from protein to protein. When the electrons get to the end of the chain, they do not simply “fall off.” The electrons, along with the hydrogens, are passed to oxygen, resulting in the formation of water (H,0). For this reason, oxygen (O,) is called the terminal electron carrier. As oxygen picks up electrons CAROLINA = s-2 20 tm ge Sep Ceres US,Cell Respiration Kit for AP Biology Student Guide 2e- fromNADH |@YO ».7 Gt at Oo HO ATE Synthase from FADH, @ (6 bp sp Figure 2. Electron transport chain and ATP synthase at the end of the electron transport chain, it enables more electrons to be passed through. If oxygen is not available to pick up electrons, no more electrons can enter the system from NADH and ADH, Thus, in the absence of oxygen, the electron transport and Krebs cycle processes cease to function. Using a Respirometer ‘You will use a respirometer to measure the rate of respiration of germinating and dormant pea seeds. The respirometer is composed of a vial that contains the peas and a volume of air; the mouth of the vial sealed with a rubber stopper with a pipet inserted into the hole. During the experiment, the respirometer is submerged in water. if the peas respire, they will use oxygen and release carbon dioxide. Because 1 mole of carbon dioxide is released for each mole of oxygen consumed, there is no change in the volume of gas in the respirometer. (Avogadro's Law: At constant temperature and pressure, 1 mole of any gas has the same volume as 1 mole of any other gas.) You will alter this equilibrium by placing a solution of potassium hydroxide (KOH) in the vial. Potassium hydroxide reacts with carbon dioxide to form potassium carbonate, which is a solid. The following reaction occurs: CO, + 2KOH = K,CO, + H,0 Because the carbon dioxide produced is removed by reaction with potassium hydroxide, as oxygen is used by cellular respiration, the volume of gas in the respirometer will decrease. As the volume of gas decreases, water moves into the submerged pipet. You will use this decrease of volume, as read from the scale printed on the pipet, as a measure of the rate of respiration. Pre-laboratory Questions 1. During respiration, a seed metabolizes sugars. What is the source of the sugar metabolized by the seed? 2. What variables do you think affect the respiration rate of seeds? Brainstorm within your group to generate a list. 01 uh Ba Spy as WU CAROLINA = 5-3Cell Respiration Kit for AP Biology Student Guide Guided Activity Materials tray of room-temperature water 3 rubber stoppers. 25 germinating pea seeds 15% KOH solution (shared) 25 dry pea seeds dropping pipet beads forceps 3 respirometers paper 50-mL. graduated tube stopwatch, timer, or clock with a second hand 3 absorbent cotton balls thermometer nonabsorbent cotton Procedure 1, Set up the water baths. The water bath will buffer the respirometers against temperature change during the experiment. a. Place a sheet of paper in the bottom of the water bath. This makes the graduated pipet easier to read. b. Place a thermometer in each tray. Observe the thermometer to make sure the temperature of the water bath is stable, 2. Prepare the peas and beads. a. For Respirometer 1, put 25 mL of water in your 50-mL. graduated tube. Drop in 25 germinating peas. Determine the volume of water that has been displaced, which is equivalent to the volume of peas. Record the volume of the germinating peas. Remove these peas and place them on a paper towel. b. For Respirometer 2, refill the graduated tube to 25 mL by adding water. Drop 25 dry, dormant peas into the tube. Next, add enough beads to equal the volume of the germinating peas. Remove the dormant peas and beads, and place them on a paper towel. For Respirometer 3, refill the graduated tube to 25 mL with water. Add enough beads to equal the volume of the germinating peas. Remove these beads and place them on a paper towel. 3. Prepare samples. a. Place an absorbent cotton ball in the bottom of each respirometer vial. b, Use a dropping pipet to saturate the cotton with 2 mL of 15% KOH solution. (Caution: Avoid skin contact with KOH. Be certain 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, nonabsorbent cotton on top of the KOH-soaked absorbent cotton. The nonabsorbent cotton will prevent the KOH solution from contacting the peas. It is important that the amount of cotton and KOH solution be the same for all three respirometers. d. Place 25 germinating peas in the vial of Respirometer 1. fe. Place 25 dry peas and beads in the vial of Respirometer 2. 4. Place the equivalent volume of beads in the vial of Respirometer 3. 4g. Insert a stopper fitted with a calibrated pipet into each respirometer the respirometers leak during the experiment, you will have to start over. comnts CAROLINA s-4 The stopper must fit tightly. ICell Respiration Kit for AP Biology Student Guide h, Optional: If your instructor asks that you add food coloring to the water in the pipet, now is the time. Hang a drop of food coloring from the bottle and allow capillary action to pull the drop into the pipet. Place a paper towel beneath the tip to catch any excess food coloring. 4, Place the set of respirometers (1, 2, and 3) in the water bath with their pipet tips resting on the lip of the tray, as shown in Figure 3. Wait 5 minutes before proceeding. This is to allow time for the respirometers to reach thermal equilibrium wit the water. If any of the respirometers begins to fill with water, you have a leak and must start over. 5. After the equilibration period, immerse all the respirometers (including the pipet tips) in the water bath. Position the respirometers so that you can read the scale on each of the pipets. (The paper in the tray underneath the pipets makes them easier to read.) Do not put anything into or take anything out of the water bath until you have taken all the necessary readings. Figure 3. Respirometers in the water bath 6. Allow the respirometers to equilibrate for another 5 minutes. 7. Observe the initial volume reading on the scale to the nearest 0,01 mL. Record the data in Table 1 for Time 0. Also, observe and record the temperature of the water. Repeat your observations for all three samples, Every 5 minutes for 20 minutes, record the temperature of the water and take readings of the volume of air in each of the three pipets. Record your data in Table 1 and then perform the calculations necessary to complete the table. Table 1: Respiration of Peas at Room Temperature Respirometer 1 Respirometer 2 Respirometer 3 Germinating Peas Dry Peas + Beads Beads Only 24 0 - - - 24 5 24) 10 24) 15 23] 20 AV= Vat Time 0- Vat time of current reading Corrected AV = AV (for Respirometer 1 or Respirometer 2) - AV of Respirometer 3 ‘an nea ip omni CARQUNA S-5Cell Respiration Kit for AP Biology Student Guide NAME Date Laboratory Questions 1. Write a hypothesis that this experiment is designed to test 2. Graph the data for respirometers 1 and 2 from your table. Plot the independent variable on the x-axis and the dependent variable on the y-axis, Label each plotted line. The independent variable is The dependent variable is Title: Pn eames A CARQUINA = S-6Cell Respiration Kit for AP Biology Student Guide 3. In this experiment, you measured the change in volume of the gas inside the respirometers. The ideal gas law describes the state of gas under given conditions pV=nRT where p= pressure of the gas V= volume of the gas moles (number of molecules) of gas iniversal gas constant [8314 joules/{kmole)(K)] jemperature of the gas in K Because you are measuring changes in volume, the gas law is restated to solve for volume: RT v = DRT P Using the ideal gas law and your experience in this lab, give the variables that had to be controlled in order for your data to be valid. State the controls used for each variable, and any means used to correct for the influence of the variable(s). 4. Which of the respirometers (1, 2, or 3) serves as a control? Explain your answer. 5. Assuming your control measures worked, tell what variable(s) changed to bring about the observed change in volume (Corrected AV in Table 1 and Table 2)? Consider the ideal gas law. 6. Using your graph and data tables, summarize your findings, comparing results from respirometers 1 and 2. Speculate as to the cause(s) of any difference between the treatments. 7. From your graph, calculate the rate of oxygen consumption for each treatment: Ay _ 0.46 mL ir Example: Rate = = S527 = 0.023 ml/min 2. germinating seeds at room temperatur mUmin b, dormant seeds at room temperature mUmin 20 ai Bt Comp A CAROLINA == s-7Cell Respiration Kit for AP Biology Student Guide Inquiry Activity On the basis of what you learned in the Guided Activity, develop 2 question to test about rates of respiration. In developing an experimental question, consider the materials and equipment available to you. Consult your instructor for the availability of additional supplies. if you need water baths prepared ahead of time, inform your instructor. Materials water tray nonabsorbent cotton germinating pea seeds dropping pipet dry pea seeds forceps beads white paper respirometers stopwatch, timer, or clock with a second hand 50-mL graduated tube ‘thermometer (for measuring peas) 15% KOH solution (shared) absorbent cotton balls Recommended: barley seeds alfalfa seeds mung beans Procedure 1. Inyour group, collaborate to come up with a testable question about the respiration rate of seeds. If you have trouble, ask your teacher for guidance. 2, Design an experiment to test your question. Consider the following as you frame your experiment: * Question - What are you testing in your experiment? What are you trying to find out? ‘© Hypothesis - What do you think will happen? Why do you think so? What do you already know that helps support your hypothesis? * Materials - What materials, tools, or instruments are you going to use to find the answer to the question? + Procedure - What are you going to do? How are you going to do it? What are you measuring? How ‘can you make sure the data you collect are accurate? What are the independent and dependent variables in this experiment? What is/are your controls)? What safety practices do you need to use? * Data Collection - What data will you record, and how will you collect and present it? Show and explain any data tables and graphs that you plan to use. 3. Have your teacher approve your experimental plan before you begin the experiment. 4, After you perform the experiment, analyze your data: * Data Analysis - What happened? Did you observe anything that surprised you? Show and explain any tables and graphs that support your data. * Conclusion - What conclusions can you draw from the results of your experiment? How does this compare with your initial hypothesis? Identify some possible sources of error in your experiment. If given the opportunity, how might you conduct the experiment differently? 5. Be prepared to present the findings of your experiment to the class according to your instructor's specification. 201 Gon ab py Cpe US CARGLINA 5-8Cell Respiration Kit for AP Biology Student Guide Experimental Design Template Part : To be completed and approved before be 1g the investigation What question will you explore? On the basis of your previous laboratory exercise, background knowledge, and research, what is the hypothesis that you will test? What will be the independent and dependent variables? What will be the control group(s)?__— What equipment and materials will you need (list items and quantity)? What procedure (step-by-step) will you follow? What safety steps will you follow (equipment and procedures)? How will you collect data? How will you analyze data? ‘Teacher approval to begin your investigation: 00 ee Ble sp ome CAROLINA s-9ell Respiration Kit for AP Biology Student Guide Part B: To be completed during or after your investigation ‘What changes or modifications have you made to the investigation? Attach any data collection or analysis as instructed by your teacher. What results did you see in the experiment? ‘Was the hypothesis accepted or rejected? What conclusions can you draw on the basis of the data and analysis? What sources of error may have existed, and how might the experiment have been conducted differently? What additional questions arose from the experiment? (ate Bo See Compe US CAROLINA © s-10Cell Respiration Kit for AP Biology Student Guide NAME Date Big Idea Assessments 1. Suppose a group performs this experiment but does not add any KOH to their respirometers. If the group reports respiration values for germinating peas similar to the data provided by your group, why might you suspect that the group falsified its data? Remember the equation for respiration: C,H),0, + 60, ~ 6CO, + 6H, +36 ATP 2. During metabolism, bonds between molecules are continuously created and destroyed. The generation of ATP during respiration requires the breakdown of sugar molecules, but not all the energy released from the breaking of the bonds is captured by the cells. Some energy is lost in the form of heat. Design a simple apparatus and experiment that would allow you to measure the respiration rate of germinated and non- germinated peas by temperature. Use the gas law (pV = nR7) to help you. rt sa CAROLINA = s-11