Metabolism: Cellular Respiration, Fermentation and Photosynthesis Introduction: All organisms require a supply of energy and matter to build themselves and to continue to function. To get that supply of matter and energy living organisms break down complex organic compounds and release the energy in those compounds. Animals, fungi and many bacteria get those complex organic compounds in the food they eat, i.e., food is both our fuel (energy supply) and supply of building blocks. The process by which the food is broken down and energy is released and recaptured to perform work in our cells is respiration or fermentation and each comes in many forms. Plants and some other organisms that are photosynthetic don t eat organic compounds. Instead they take water and carbon dioxide and build their own complex organic compounds. The energy that is required to build those compounds is solar, or light energy and the process by which this occurs is called photosynthesis. After building those organic compounds plants and other photosynthetic organisms still use respiration to release the chemical energy that is stored in the chemical bonds. Aerobic or Cellular Respiration Aerobic respiration is the complete burning: of glucose molecules in the presence of oxygen. The complete formula for this reaction is: C 6 H 12 O 6 + 6 O 2 6 CO 2 + 6 H 2 O (energy is released) By looking carefully at the formula you can see that the reason we need to breathe is to take in oxygen so that glucose (and other foods we eat) can be broken down and energy released. Whenever we break down food, carbon dioxide and water are the end products. As we breathe, oxygen and carbon dioxide are exchanged in our lungs. This occurs in the blood flowing through the alveoli. When we inhale we take in oxygen into our lungs. That oxygen is picked up by hemoglobin and carried to all the cells in the body so that they can perform aerobic respiration. The hemoglobin exchanges the oxygen for carbon dioxide. As we exhale, hemoglobin releases carbon dioxide that it acquired from the cells. During this laboratory we will measure the amount of CO 2 in your breath at a calm state and then again after you have been exercising to raise your metabolic rate. When CO 2 is bubbled through water it dissolves and forms a small amount of carbonic acid (H 2 CO 3 ). This then dissociates to H + + HCO 3, and further to H + + CO 3 2, thereby turning the water acidic. Phenolphthalein is colorless in acidic solutions and pink when basic. By measuring the amount of NaOH (a base) it takes to neutralize the acid (turn the color pink), we can measure the relative amount of CO 2 in your breath. In particular, we can compare the amount of CO 2 while you are in a calm state vs. one where you ve increased the rate of respiration. Fermentation Anaerobic respiration or respiration that occurs without using oxygen takes many different forms including fermentation. Fermentation is the partial burning or breakdown of glucose (or again other food molecules). Fermentation releases much less energy from glucose than aerobic respiration since it is only a partial burn and chemical energy is still in the remaining organic compound. Fermentation itself takes many different forms. The version of fermentation that is best known (and lots of folks favorite) is shown in the chemical reaction below: C 6 H 12 O 6 2 CO 2 + 2 C 2 H 5 OH (alcohol) (releases some energy) Many bacteria and yeast can perform this reaction, i.e., breaking down sugars into alcohol and carbon dioxide. Yeast can also perform aerobic respiration and will only switch to fermentation if there is no oxygen present. In this experiment we will test the ability of yeast to ferment a number of mono, di, and polysaccharides by measuring the amount of carbon dioxide given off in a respirometer. To ensure that the yeast ferment instead of performing aerobic respiration, the availability of oxygen will be limited. 1
Photosynthesis In photosynthesis solar energy is captured and converted to chemical energy. That chemical energy is then used to build sugars from inorganic molecules. Plants basically build themselves from water and thin air. Or more accurately, the CO 2 in the air. This is an extremely important process since for the majority of organisms on this planet this is the ultimate source of food, i.e., we eat the plant or we eat something that ate the plant. Photosynthesis comes in various forms but all plants, algae and one group of bacteria, the cyanobacteria, perform the same version of photosynthesis referred to as oxygenic photosynthesis. The oxygenic refers to the fact that oxygen is one of the by-products of the process. The complete chemical reaction is shown below. 6 CO 2 + 6 H 2 O (+ light energy) C 6 H 12 O 6 + 6 O 2 Look carefully at the reaction for photosynthesis and the reaction for aerobic respiration. You should be able to see that the complete process is chemically the reverse of each other although there is a difference in the energy released and consumed. In aerobic respiration energy is released and recaptured in the form of chemical bond energy and in photosynthesis it is light energy that drives the reaction. Even though the complete processes are the reverse of each other each process takes MANY steps and these are NOT the reverse of each other. In eukaryotes, photosynthesis and aerobic respiration take place in different locations of the cell. Since plants use up carbon dioxide when performing photosynthesis a photosynthesizing plant should be able to use up the carbon dioxide that is released during aerobic respiration. In this lab we will use the ph indicator dye that turns yellow in acidic solutions and red in basic solutions to measure this. Procedures: Both the Photosynthesis and CO 2 Uptake and Anaerobic Respiration Fermentation procedures should be set-up before doing the Aerobic Respiration and CO 2 Production experiment. You can set-up the photosynthesis or Anaerobic Respiration Procedure first. Photosynthesis and CO 2 Uptake Phenol Red is an indicator that turns yellow in an acidic solution and red in a basic one. Breathing into water turns a solution acidic. If a plant uses CO 2 during photosynthesis, it can be assumed that the solution will become basic again. That is what we will test. (NOTE: THE ELODEA SHOULD BE KEPT IN THE DARK OVERNIGHT, PRIOR TO THE EXPERIMENT) 1. Add about 50 ml of the Phenol Red in an Erlenmeyer flask. 2. Using a straw, breath slowly into the phenol red solution until it almost yellow (it may still be very slightly orange). 3. Add two pieces of Elodea which are about the same length into two separate tubes (about 3 inches long). 4. Fill the two test tubes so that the phenol red solution just covers the top of the elodea. 5. Place one tube directly in front of a bright light for about 60 minutes while your fermentation experiment is proceeding. 6. Completely cover the 2 nd tube with aluminum foil and place it in a rack at your desk. 7. At the end of 60 minutes, Compare the Two Tubes (record your observations). Was there a change in color in either tube? Were there any bubbles in either of the tubes? 2
Anaerobic Fermentation: Insert a small tube tightly to the end of the large tube with your finger or a pencil and quickly invert both tubes (below). Practice the following procedure before using the sugar solutions by filling a small tube with water and inverting it into the large tube. The bubble should be about 2-3 mm in height. The smaller the bubble the better. 1. Obtain five large and small test tubes. 2. Label each SMALL tube with a wax marker ½ of the way from the bottom. The exact measurement (i.e. ½) is not important but it is important to line up the five small tubes and ensure that they are marked identically. 3. Carbohydrate Solutions: Fill each small tube to the ½ mark with the appropriate carbohydrate solution. 4. Label the LARGE tubes with your initials and the solutions listed in Table 1 (Do not label with a tube # -- list the actual solution). They will be immersed in a water bath so label the top (open) end of the large tubes. 5. ABSOLUTELY CRITICAL: You must stir the yeast EACH TIME prior to filling the small tube. Use your pipette to squirt the yeast solution in and out several times. 6. Add enough yeast to each tube so that it is nearly full, then swirl it so the yeast and sugar solutions mix. 7. Completely fill the rest of the tube with yeast so that it is overflowing and then invert it in the large tube. If the yeast settled, you may need to tap the small tube to dislodge it. 8. Measure the initial height of the air space in the small tube and record it in Table 1. 9. Place the respirometers in a 37 C water bath to incubate for at least one hour. Record the final height of each gas bubble. Check on the tubes approximately every five minutes. 10. IMPORTANT: Watch the tubes carefully. You may need to suction off some of the excess solution which is expelled from the small tubes. This will prevent them from floating up and losing gas. 11. IMPORTANT: If some of the tubes fill with gas before the hour is up, stop the experiment and measure the gas bubble in all tubes at that point. 3
Aerobic Respiration and CO 2 Production Both procedures must be done by the same person. Sit calmly for a few minutes before doing the first part of the procedure. 1. Add about 2 inches of distilled or deionized water to a 125 or 250 ml Erlenmeyer flask. 2. Add 3-4 drops of phenolphthalein to the flask. 3. Take a normal breath and hold it for 10 seconds. Breathe out slowly through a straw for 10 seconds so that the gas bubbles through the water. 4. Repeat this four more times, for a total of five breaths. 5. It is CRITICAL that you hold your breath the same length of time, breath out at the same rate, and use the same volume of air for each of the five breaths before & after exercising. 6. Add 0.1 M (or 0.5%) NaOH one drop at a time until it turns pink while swirling the flask. It should stay pink for 15-20 seconds or more. If it turns clear again, continue adding one drop at a time until it stays pink for 15-20 seconds. 7. # drops NaOH needed to neutralize the solution: 8. Rinse out the flask and replace the distilled water. 9. Add 3-4 drops of phenolphthalein to the flask. Do some form of exercise to get your heart rate up and then sustain the exercise for a couple of minutes (e.g. walk briskly (or run) outside the room for a few minutes, do jumping jacks, pushups, or whatever exercise you feel comfortable performing). 10. Repeat the procedure 4-6, using the same volume of air as above for each breath. 11. # drops NaOH to neutralize the solution: 12. If your 2nd number is not higher than the 1 st one you will need to redo the experiment. Record all of your data in the tables and on the front board so everyone can get a list of class averages. 4
Biology 160 Name and Lab Section Table 1: Fermentation of Carbohydrates Tube Initial Height (mm) Final Height (mm) Change (mm) Glucose Sucrose Fructose or Lactose Starch H 2 O Table 2: Class Average of Carbohydrate Fermentation Change & CO 2 Respiration Both Partners Initials Glucose Sucrose Fructose or Lactose Starch H 2 O # NaOH Calm # NaOH Rapid Class Average Observations of the two tubes containing Elodea and phenol red (and any differences): Please describe any colors changes that occurred and differences between the two tubes. 5
Questions: 1) What are the gas bubbles in the fermentation experiment? Explain why the gas bubbles were formed by the yeast. 2) Why is alcohol made in sealed containers? What would you expect to happen if the process was open to air? 3) Why must both of the CO 2 breathing procedures be done by the same person? 4) What conclusions can you draw about exercise, respiration, & CO 2? Explain. 5) It is expected that the two tubes with Elodea had different colors at the end of the experiment. What is happening to change the color in one tube and why doesn t it happen in the other? 6