Lab 6 Cellular Respiration

Lab 6: Cellular Respiration Metabolism is the sum of all chemical reactions in a living organism. These reactions can be catabolic or anabolic. Anabolic reactions use up energy to actually build complex biomolecules (think of anabolic steroids building muscle mass). The energy for anabolic reactions usually comes from ATP, which is produced during catabolic reactions. Catabolic reactions break down complex biomolecules, such as carbohydrates and lipids and release the energy stored within. 1. Is cellular respiration anabolic or catabolic? Explain. Cellular respiration is a catabolic reaction, because this process breaks down carbohydrates to release energy. 2. Is photosynthesis anabolic or catabolic? Explain. Photosynthesis is anabolic, because this process uses energy to build carbohydrates. Enzymes are proteins that facilitate chemical reactions in living systems by acting as catalysts in biochemical reactions. Enzymes speed the rate of the reaction by either bringing the reactants into close proximity or by binding to a single reactant and splitting it into smaller parts. Enzymes have a property known as specificity, which simply means that each enzyme catalyses a specific biochemical reaction. Enzymes are indispensable molecules of life. Enzymes are functional within a given range of temperatures and pH values for that enzyme. Part 1: Starch Synthesis and Storage Using energy from the sun, photosynthetic organisms convert carbon dioxide and water molecules into glucose. Plants don’t store this chemical energy as glucose. Using enzymes, plants link the glucose molecules together and store them as the polysaccharide starch. Potatoes are the primary starch storage site for the potato plant. Iodine reacts with starch to form a bluish color. You can see the starch stored in a potato cell by staining the cells with iodine. Materials Microscope slide Coverslip Iodine Potato cells Procedure 1. Cut a thin slice or scrape a few cells from the surface of a potato. 2. Make a wet mount of the potato cells and stain them using a drop of iodine. 3. Observe your cells under high power. 4. Draw a few cells and label the following structures: A. cell wall B. plastid with starch grains (stains purple) 1. Where did the starch in the potato cells come from? Enzymes inside of plant plastids link glucose molecules together to form a polysaccharide starch. The glucose came from photosynthesis. Tap water Part 2: Starch Digestion When you eat starchy foods like bread or potatoes, your body must digest the complex carbohydrate into monosaccharides, such as glucose or fructose, before it can be absorbed by the body. Alpha amylase, an enzyme found in your saliva, catalyzes the first step in starch digestion. Just for the fun of it, snag an unsalted saltine cracker. Begin chewing the cracker, but do not swallow it. Keep chewing it, and allow the amylase from your saliva to break the starch into maltose. 1. Can you taste the difference in the cracker after chewing for a very long time? What happened? Yes. The cracker tasted sweeter after chewing the cracker for several minutes. The enzyme amylase, in my saliva, broke down the starch in the cracker into glucose. The glucose tasted sweet whereas the starch did not. In this lab, you will assess the effect of amylase on a carefully prepared starch solution. Materials 2 Ehrlenmeyer Flask, 250 ml Glass stirring rod Hot pads Wax pencil Amylase Corn starch 2 glucose test strips Hot plate Procedure 1. Label your two flasks A and B. 2. Add 100 ml of water and 10 g of corn starch to each flask. 3. Using the hot plate, gently heat this mixture, stirring continuously with a glass stir rod. 4. When the mixture thickens, remove it from the heat and cool it under running water to lukewarm. 5. Shake the Amylase solution thoroughly to mix the enzyme. 6. Add 2 ml of Amylase to flask A. Do not add enzyme to flask B. 7. Allow both flasks to sit for 15 minutes and note any change in the viscosity of the two starch solutions. 8. To see if starch digestion occurred, place a small amount of solution onto each glucose test strip. Compare the color on the test strip with the known standards. Viscosity (thickness) Beaker A: Amylase treatment Beaker B: No enzyme treatment Glucose test (+ or -) Low viscosity (+) High viscosity (-) 1. Is there any difference in viscosity between the two beakers? If so, explain why there is a difference. Yes, there is a difference in viscosity between the two beakers after the treatment. The initial solution was thick due to the tangle of long insoluble polysaccharide starch chains. The enzyme amylase was added to beaker A. Amylase broke down the long chains of the polysaccharide starch (from the corn starch) into many monosaccharide glucose molecules. Glucose (sugar) is soluble in water. Therefore, the glucose dissolved in water and the solution turned liquid. 2. Which beaker represents the control treatment in this experiment? Is this a positive or negative control? Explain. Beaker B is a negative control. This is because we were expecting to see no effect and we did not see an effect in this treatment. The No Amylase Treatment was used as a comparison with the Amylase Treatment, which helped me recognize the effect of amylase on starch. 3. Design an experiment that would study the influence of a factor like pH or temperature on enzyme activity. To test the influence of temperature on enzyme activity I would add the enzyme amylase to the starch solution at several different temperatures: 1. 2. 3. 4. 5. 6. Label 6 flasks a, b, c, d, e, and f. Add 100 ml of water and 1.0 grams of corn starch to each flask Heat all flasks till thickens Remove flask (a) from heat and bring to room temperature. Remove flask (b) from heat and put on ice Leave c, d, e, and f on the heat so that c is at 35 degrees C, d is at 45 degrees C, e is at 55 degrees C, and f is at 65 degrees C. 7. Add 2 ml of amylase to each flask. 8. Allow thickened cornstarch in each flask to sit for 15 minutes at their respective temperatures. 9. Test for starch digestion. Part 3: Aerobic Respiration Carbon dioxide is a byproduct of aerobic cellular respiration. Measuring carbon dioxide production is an indirect way of measuring whether or not cellular respiration is occurring. Your task in this lab is to determine whether or not various sets of bean seeds are going through cellular respiration. Materials Flasks Rubber stoppers Germinated bean seeds “Respiration bottle” setups Germinated + boiled bean seeds Phenol red Dry ungerminated bean seed Procedure 1. Fill one flask about 1/3 full of the bean seeds labeled “Germ”. These bean seeds were soaked overnight and then drained and covered in a wet paper towel for 2 days to allow the seeds to begin germinating. 2. Fill the other flask about 1/3 full of the bean seeds labeled “Germ-Boil”. These bean seeds were soaked as for “Germ”, but were then boiled for 3-4 minutes and cooled to room temperature on the day of the lab. 3. Your instructor will set up the control flask containing ungerminated seeds. 4. Place rubber stoppers over each flask and allow flasks to sit for approximately one hour. 5. After one hour, replace the rubber stopper with a second stopper containing a funnel and rubber tubing attached to a glass tube. Place the glass tube into a test tube about ½ full with water. 6. Add several drops of phenol red solution to the test tube. Phenol red, a pH indicator, is red when pH>7 (basic) and yellow when pH<7 (acidic). 7. Put several hundred ml of tap water into a beaker and slowly pour the water into the flask via the funnel at the top. This will force the gases in the bottle to bubble into the test tube containing the phenol red solution. 8. Record the color of the phenol red solution as gases present in the flask bubble through the solution. 9. Clean out the test tube, then repeat steps 5-8 for the second flask. Record your observations in the table below. A. Remember, the CO produced during cellular respiration combines with water to form carbonic acid. 2 The carbonic acid dissociates into hydrogen and bicarbonate ions, and the hydrogen ions decrease the pH. CO 2 + HO 2 (carbon dioxide + water) Bean seeds Germinating- unboiled Germinating- boiled Ungerminated- control ↔ H CO 2 (carbonic acid) Indicator color yellow red red 3 HCO3- + H+ ↔ (bicarbinate ion + hydrogen ion) Results (CO present2 or absent) present absent absent Lab Questions 1. Which set of seeds was undergoing cellular respiration? How do you know? The unboiled germinated seeds underwent cellular respiration. The unboiled germinated seeds released carbon dioxide which mixed with water to form carbonic acid which then dissociated into hydrogen ions and bicarbonate ions. The phenol red turned yellow in this acidic condition. 2. What might have happened to the enzymes in the germinated and boiled seed treatment? Boiling might have denatured the enzymes needed for cellular respiration. 3. Where does cellular respiration occur inside the cells of the germinating seed? mitochondria 4. In order to obtain all the energy possible from a molecule of glucose (≈36 ATPs), what substance must be available? oxygen 5. Was the control in this experiment a positive or negative control? How could you design THE OPPOSITE TYPE OF CONTROL for this experiment? The control for this experiment was the ungerminated seeds. This is a negative control. The opposite control would be a positive control. (1) We could set up a flask with water and phenol red and blow carbon dioxide into it to produce an acidic condition and turn the phenol red yellow. We could use this positive control to compare with our experimental treatment. (2) If we wanted to see what the effect of boiling would have on the beans, we would have designated the unboiled germinated beans as a positive control to compare with our boiled germinated treatment. Part 4: Anaerobic Respiration During anaerobic respiration, only 2 molecules of ATP can be generated for one molecule of glucose. Pyruvate is a waste product produced during glycolysis, and unless pyruvate is metabolized, it will prevent anaerobic respiration from proceeding. There are two ways that pyruvate can be metabolized. In yeasts and certain other microbes, pyruvate is turned into ethyl alcohol (ethanol). In animals and some bacteria, pyruvate is turned into lactic acid. 1. Clearly draw the anaerobic cellular respiration pathway in yeast. “Wine Making” Experiment Working in your group, you will design an experiment to test a hypothesis regarding the rate of fermentation. Some of the experimental variables you might manipulate in the fermentation process are: • Sugar type (Possibilities: sucrose, lactose, glucose, and dextrin) • Yeast quantity (use 1 gram as your control value…) • Temperature (18˚C-room temperature, 37˚C, 55˚C, or 0˚C ice bath) 1. What is the question your group is asking for this experiment? Will more yeast increase the amount of fermentation? 2. Describe your hypothesis: If the amount of yeast is increased, then the amount of carbon dioxide will also increase. Experimental Design To test your hypothesis, set up three or four fermentation tubes. Be sure to include a control in your experiment. General experimental design- modify as necessary. 1. Fill each test tube about half full with water. 2. Add 1 gram of yeast 3. Add 1 gram of a sugar. 4. Place a balloon over the top of each tube. 5. Place the tubes in a test tube holder at the appropriate temperature. 6. After 30 minutes observe tubes and measure the circumference of the balloon. Describe your experimental design: 1. Fill each test tube about half full with water. 2. Add 1 gram of a sugar (glucose) to each test tube. 3. Add 1 gram of yeast to the control. 4. Add 0.5, 1.5, and 2 grams of yeast to the other test tubes. Mix 5. Place a balloon, secured in place wi5th a rubber band, over the top of each tube. 6. Place the tubes in a test tube holder in the 37˚C bath. 7. After 30 minutes observe tubes and measure the circumference of the balloon. Tube Contents Variable Circumference of Balloon (in 1 1 g of glucose, water 0.5 g of yeast 5 cm 2 1 g of glucose, water 1.5 g of yeast 9 cm 3 1 g of glucose, water 2 g of yeast 9 cm 4 (Control) 1 g of glucose, water 1g of yeast 7 cm 1. What was the control in your experiment? Was it a positive control or a negative control? Explain. The control in this experiment was the treatment with 1 gram of yeast. This is a positive control, because I expected to see an effect and I did see an effect in this treatment. 2. Did the results support your hypothesis? If not, why do you think your results did not support your hypothesis? What would your new hypothesis be? Yes and no. The increase in yeast caused the increase in carbon dioxide from 0.5 to 1.5 grams, but additional yeast after that did not cause an increase in carbon dioxide. It could be possible that an increased amount of yeast will need more than 1 gram of sugar to ferment into more carbon dioxide. 3. What further experiment(s) would you perform to support or refine your hypothesis? In the above experiment, the sugar was set at 1 gram for all of the treatments. To refine my experiment I would increase the amount of sugar to 1.5 grams of sugar to see if this would change my results. In an experiment, it is important to change one variable at a time: sugar, or temperature, or yeast. If your hypothesis has to do with temperature, you need to stick to varying this variable without adding a second variable. A second variable would complicate matters and you would not be able to figure out which variable caused the results. cm)