Lab 8 RESPIRATION: ENERGY CONVERSION OBJECTIVES • Give the overall balanced equations for aerobic respiration and alcoholic fermentation; • Distinguish between the inputs, products, and efficiency of aerobic respiration and those of fermentation; • Identify the structures and list the functions of each part of a mitochondrion. INTRODUCTION The energy needed for living processes is stored in the chemical bonds holding carbohydrate atoms together. However, the cell cannot directly use the chemical bond energy of carbohydrates. Rather, the energy must be converted by a metabolic pathway to form adenosine triphosphate (ATP), the socalled universal energy currency of the cell. The bond energy of carbohydrates is transferred to ATP during phosphorylation, the addition of a phosphate group to adenosine diphosphate (ADP). When the bond holding this new phosphate group is broken during respiration, the energy released is available for a great variety of cellular reactions. Thus, the needs of a cell are linked by the energy (ATP)-releasing exergonic reactions of respiration to the energy (ATP)-requiring endergonic reactions. This last group of reactions is important for maintaining or synthesizing cellular structures, or doing cellular work. There are four categories of respiration: (1) aerobic respiration, an oxygen-dependent pathway common in most organisms; (2) anaerobic respiration, a pathway utilized by some bacteria; (3) alcoholic fermentation, an ethanol-producing process occurring in some yeast; and (4) lactic acid production, a pathway taken by some animal cells that normally rely on aerobic respiration, but which are subjected to oxygen-deficient conditions. (During strenuous activity, your skeletal muscles may switch to lactic acid production for energy.) Aerobic respiration is definitely the most energy-efficient, which means that the amount of energy captured in the forms of ATP relative to the amount available within the bonds of the carbohydrate. For aerobic respiration, the general equation is: C6H12O6 glucose + 6 O2 enzymes oxygen → 6 CO2 + 6 H 2O carbon dioxide water + 36 ATP1 chemical energy If glucose is broken down completely, to CO2 and H2O , about 686,000 calories of energy are released. Each ATP molecule represents about 7,500 calories of usable energy. The 36 ATP represent 270,000 calories of energy (36 x 7,500 calories). Thus, aerobic respiration is about 39% efficient [(270,000/686,000) x 100%]. 1 Depending upon the issue, as many as 38 ATP may be found. !77 By contrast, fermentation and anaerobic respiration yield only 2 ATP. Thus, these processes are only about 2% efficient [(2 x 7,500/686,000 x 100%]. Obviously, breaking down carbohydrates by aerobic respiration gives a bigger payback than the other means. Regardless of the specific respiratory pathway, an organism uses, one series of metabolic steps occurs: glycolysis. The word glycolysis should be a tip-off concerning what happens during this process. The Greek word glykos means "sweet," referring to sugar, while lysis means "loosening." During glycolysis the 6-carbon sugar glucose, C6H12O6, is split into two 3-carbon pyruvate molecules. This universal event occurs within the cytoplasm of all living cells, whether bacteria, protistans, fungi, plants, or animals, including humans. The net energy yield from glycolysis is 2 ATP per molecule of glucose. I. AEROBIC RESPIRATION The fate of pyruvate molecules produced by glycolysis depends upon the organism and environmental conditions. If oxygen is abundant and the organism normally undergoes aerobic respiration, pyruvate is further metabolized by a cyclic (circular) pathway known as Krebs Cycle, which generates a small amount of ATP and releases CO2. For the most part, the Krebs cycle functions to reduce (donate electrons) to special electron carriers. These electron carriers eventually become oxidized (lose electrons) during oxidative phosphorylation, where large amounts of ATP are produced. The Krebs cycle and electron transport phosphorylation occur in the mitochondrion. Why must oxygen by present? Whenever one substance is oxidized (lose electrons), another must be reduced (accept, or gain, those electrons). The final electron acceptor of electron transport phosphorylation is oxygen. Tagging along with the electrons as they pass through the electron transport process are protons (H+). When the electrons and protons are captured by oxygen, water (H2O) is formed: 2 H+ + 2 e- + O2 → H 2O In the following experiments, we examine aerobic respiration in two sets of pea seeds. A. Carbon Dioxide Production Seeds contain stored food material in the form of some carbohydrate. When a seed germinates, the carbohydrate is broken down, liberating energy (ATP) needed for growth of the enclosed embryo into a seedling. Dry pea seeds have been soaked in water to start the germination process. Another set was not soaked. This experiment will compare carbon dioxide production between germinating pea seeds, germinating pea seeds that have been boiled, and ungerminated (dry) pea seeds. This experiment investigates the hypothesis that germinated and boiled seeds do not produce carbon dioxide through aerobic respiration. Procedure 1. Place about 250 mL of tap water in a 600 mL beaker, put the beaker on a hot plate, and bring the water to a boil. 2. Obtain three respiration tube apparatus setups (See Figure 8-1) labeled "Germ" for germinating pea seeds, "Germ-Boil" for those you will boil, and "Ungerm" for ungerminated (dry) seeds. !78 3. Obtain and put 40 germinating pea seeds in each of two appropriately labeled respiration tubes to fill them approximately halfway. Add 40 ungerminated seeds to the third tube. 4. Dump the germinating peas from the "Germ-Boil" tube into the boiling water bath; continue to boil for 5 minutes. After 5 minutes, turn off the heat, put on a heat-resistant glove, and remove the water bath. Pour the water off into the sink and cool the boiled peas by pouring cold water into the beaker. Allow 5 minutes for the peas to cool, then, pour off the water. Now replace the peas into the "Germ-Boil" respiration tube. 5. Fit the rubber stopper with attached glass tubes into the respiration tubes. Add enough water to the smaller test tube to cover the end of the glass tubing that comes out of the respiration tube. (This keeps gases from escaping from the respiration tube.) Glass tubing Thistle tube Peas Fluid-filled test tube Figure 8-1 Respiration tube apparatus. 6. Set the three tubes aside for the next hour and perform the other experiments in this lab. 7. After an hour, dump the water in each small test tube into the sink and replace it with 10mL of Phenol Red solution. Phenol Red solution, which should appear pinkish in the stock bottle, will be used to test for the presence of carbon dioxide (CO2) within the respiration bottle. If CO2 is bubbled through water, carbonic acid (H2CO3) forms, as shown by the following equation: CO2 + H2O → H2CO3 Phenol Red solution is mostly water. When the Phenol Red solution is basic (pH > 7), it is pink; when it is acidic (pH < 7), the solution is yellow. The Phenol Red solution in the stock bottle is (color) Therefore, the stock solution is (acidic/basic) 8. Put several hundred ml of tap water in the 600 mL beaker. !79 9. Remove the stopper plugging the top of the thistle tube and slowly pour water from the beaker into each thistle tube. The water will force out gases present within the bottles. If CO2 is present, the Phenol Red will become yellow. 10. Record your observations in Table 8-1. Table 8-1 CO2 Evolution by Pea Seeds Pea Seeds Phenol Red Color CO2 Present or Absent Germinatingunboiled Germinatingboiled Ungerminated 11. After you have completed this section of the lab, wash your dirty glassware with soap, rinse with tap water and dry with paper towels. INVERT test tubes in the test tube rack for proper drainage. Finally, tidy up your work area making certain that all materials used for this exercise are there for the next class. Questions 1. Which set(s) of seeds underwent respiration? 2. What happened during boiling that caused the results you found? (Hint: think enzymes). 3. Write a conclusion, accepting or rejecting the hypothesis stated at the onset of this experiment (towards th bottom of pg 78). II. FERMENTATION Despite relatively low energy yield, fermentation provides sufficient energy for certain organisms to survive. Alcoholic fermentation by yeast is the basis for the brewing industry. It's been said that yeast and alcoholic fermentation have made Milwaukee famous. The chemical equation for this process is: C6H12O6 Glucose → 2 CH3CH2OH Ethanol + 2 CO2 Carbon dioxide !80 + 2 ATP Energy Starch (amylose), a common storage carbohydrate in plants, is a polymer consisting of a chain of repeating glucose (C6H12O6) units. The polymer has the chemical formula (C6H12O6)n2, where n represents a large number. Starch is broken down by the enzyme amylase into individual glucose units. To summarize: (C6H12O6)n Starch Amylase → C6H12O6 + Glucose C6H12O6 + Glucose C6H12O6 + … Glucose This experiment demonstrates the action of yeast cells on carbohydrates. Procedure 1. Using a marking pen, number three 50 mL beakers. DO NOT WRITE DIRECTLY ON THE BEAKERS. 2. With a CLEAN 25 mL graduated cylinder, measure out and pour approximately 15 mL of the following solutions: NOTE: Wash the graduated cylinder between Solutions. Beaker 1: 15 mL of 10% Glucose Beaker 2: 15 mL of 0.5% Starch Beaker 3: 15 mL of 0.5% Starch and 5 mL of 1% Amylase 3. To each beaker, add a ¼ teaspoon of yeast. Stir with SEPARATE stirring rods. 4. When each is thoroughly mixed, pour the contents into three correspondingly numbered fermentation tubes (see Figure 8-4). Cover the opening of the fermentation tube with your thumb and invert each fermentation tube so that the "tail" portion is filled with the solution. d Tail h Figure 8-4 Fermentation tube A number of carbohydrates share this same chemical formula but differ slightly in the arrangement of their atoms. These carbohydrates are called structural isomers. !81 2 5. Place the tubes in a 37°C incubator. RECOGNITION. INITIAL YOUR TUBES FOR PROPER 6. At intervals of 20, 40, and 60 minutes after the start of the experiment, remove the tubes and, using a metric ruler, measure the distance from the tip of the tail to the fluid level. Record the results in Table 8-3. Calculate the volume of gas evolved using the formula at the bottom of Table 8-3. (If time is short, do your calculations later). Table 8-3 Evolution of Gas by Yeast Cells Volume of CO2 Produced in mL Tube Solution 1 10% Glucose + Yeast 2 0.5% Starch + Yeast 3 0.5% Starch + 0.5% Amylase + Yeast 20 min 40 min 60 min Volume* of gas evolved (mL) * If your tubes are not vulumetric: Calculate the volume of gas evolved by using the following equation: * V = Πr2h, where Π = 3.14, r = radius of tail of fermentation tube (r = ½ d), h = distance from top of tail to level of solution. 7. After completing the experiment, wash all dirty glassware with soap and a scrub, rinse with tap water and dry with paper towel. IT IS IMPERATIVE THAT YOU WASH THE FERMENTATION TUBES IMMEDIATELY AFTER YOU HAVE COMPLETED THIS LAB ACTIVITY. THE REAGENTS USED IN THIS ACTIVITY WILL ADHERE TO THE TUBES. Finally, tidy up your work area, making certain all equipment used in this exercise is ready for the next class. Questions 1. What gas accumulates in the tail portion of the fermentation tube? 2. Which nutrient was preferred by the yeast cells in this experiment? 3. Which nutrient might be preferred if the experiment were to go for an entire day?Think of the amount of calories in each nutient. 2. What are the products of fermentation? !82 Post Lab Questions 1. The pea seed experiment was to determine the effect of boiling on pea seed respiration. Explain the role of the following components in the experiment on carbon dioxide production: 1. germinating pea seeds 2. ungerminated (dry) pea seeds 3. germinating, boiled pea seeds 4. phenol red solution 2. Sucrose (table sugar) is a disaccharide composed of glucose and fructose. Glycogen is a polysaccharide composed of many glucose subunits. Rank the tubes in order of least to most amount of carbon dioxide produced in one hour. 1. Tube 1: glucose plus yeast 2. Tube 2: sucrose plus yeast 3. Tube 3: glycogen plus yeast 3. Bread is made by mixing flour, water, sugar, and yeast to form a dense dough. Why does the dough rise? What gas is responsible for the holes in bread? 4. Examine the electron micrograph of the mitochondrion on the right. 1. What portions of aerobic respiration occur in the liquid portion of region b? 2. What is produced as hydrogen ions cross through the inner membrane into the matrix. 3. What portion of cellular respiration takes place in the cytoplasm outside of this organelle? 5. Oxygen is used during aerobic respiration. What biological process is the source of the oxygen? !83
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