Question

Create graphs for Figures 1-4 (circled on pages 111 & 114) based on the data given in Tables 2 & 4.

Lab # 8 Cellular Respiration and Fermentation I. Goals and Objectives At the completion of this laboratory exercise, students will be able to: A Differentiate between the intermediates and products of fermentation versus acrobic cellular respiration in yeast. Relate rates of fermentation with sugar availability in yeast. Utilize a reduction-oxidation dye to determine the effect of varying yeast concentration on aerobic cellular respiration. B. C. II. Introduction This lab investigates fermentation and cellular respiration, cellular processes that convert the energy of glucose into a form useable by cells, adenosine triphosphate (ATP). The energy in ATP can then be used to perform cellular work. Fermentation can occur in some cell types under anaerobic (without oxygen) conditions and aerobic cellular respiration requires the presence of oxygen. Fermentation and aerobic cellular respiration both consist of oxidation-reduction reactions (redox reactions). Redox reactions involve the transfer of electrons. Oxidation is the loss of electrons and reduction is the gain of elections (helpful acronym - OIL RIG). In cellular respiration, electrons from glucose are transferred to NAD+ resulting in the oxidation of glucose and reduction of NAD+ to NADH. In aerobic cellular respiration, NADH transfers electrons to the electron transport chain and ultimately oxygen is reduced. This process releases energy that can be used to synthesize ATP CsH120s+606H20 +6C02 +ATP Aerobic cellular respiration consists of three metabolic stages: glycolysis, citric acid (Kreb 's) cycle, and the electron transport chain/chemiosmosis/oxidative phosphorylation. With the exception of glycolysis, the stages of aerobic cellular respiration occur within the mitochondria (Figure 1). Fermentation occurs in the cytoplasm and only involves glycolysis. Depending on the cell type, one of two types of fermentation may occur, alcoholic or lactic acid fermentation, which produce a net gain of2 ATP, far fewer than aerobic cellular respiration. Alcoholic fermentation can occur in yeast and begins with glycolysis, a series of reactions that breaks glucose into two molecules of pyruvate with a net yield of 2 ATP and 2 NADH. The pyruvate (3 carbons) may then be further broken down into ethyl alcohol (2 carbons) and CO. In this process, the 2 NADH molecules are oxidized which return to replenish the NAD+ used in glycolysis (Fig. I). Cytosol PYRUVATE No Os Ethamol coz Figure 1. Stages of cellular respiration and fermentation. III. Experiments Experiment 1. Alcoholic Fermentation In this lab you will examine alcoholic fermentation in a yeast cell, Saccharomyces cerevisiae, or "baker's yeast". Yeast cells can perform cellular respiration but often switch to alcoholic fermentation (facultative anaerobes). The carbon dioxide (CO2) produced in the reaction forms bubbles out of the solution which can be used as an indication of the relative rate of fermentation taking place. Fig. 2 shows the respirometers you will use to collect CO2 (1) Hypothesize about the effect of different concentrations of yeast on the rate of fermentation: а. Но: b. Ha: (2) Predict the results of the experiment based on your hypothesis (use an if/then 108 Note: The respirometer being used in lab maybe designed differently than the one shown in Fig. 2. Follow the IA's instructions regarding the setup with your instrument. For example, the binder clip at the top may not be used if a pipette is sealed through the center of a stopper on the flask. Be careful not to move the pipette in the stopper because the seal with the stopper will loosen. This will cause loss of pressure in the chamber and the data will not be accurate. Carbon dioxide is formed here Figure 2. Respirometer used for yeast fermentation Protocol 1. Label six test tubes (fermentation tubes) so that you can identify them. Add warm solutions as in Table 1 to appropriate tubes. Be sure to mix the yeast suspension well. 2. To each test tube, add a 0.5 ml graduated pipette with attached piece of plastic tubing inserted through a rubber stopper. The yeast suspension should be forced into the pipette. Record this mark as the initial reading 109 Table 1. Contents of fermentation solutions (volumes in ml) Tube # DI Water (ml) Yeast Suspension (ml) Glucose Solution (ml 1.5 0.5 0.5 1.0 1.5 4.0 3.5 1.5 1.5 1.5 1.5 2.5 (If the above step does not generate enough pressure to force the suspension into the tube sufficiently, attach a pipette pump to the free end of the tubing of the first pipette. Use a syringe to suck up the fermentation solution up past the 0 ml line). 4. Fold the tubing over and clamp it shut with the binder clip so the solution does not run out. 5. Record the initial reading on the pipette and record in Table 2. Record the new reading every 2 min in the "CO2 evolved" column for 10 mins. If you did not bring the solution to the 0 ml line, record the change in CO2 from the "initial reading" of the pipette. The change in the volume of the suspension should be directly related to the amount of CO; produced. 110 Table 2. Total CO2 evolved from different concentrations of yeast. Time (min) Tube 1 Tube 2 Tube 3Tube4 Tube 5 Tube 6 CO2 evolved evolved evolved evolved evolved evolved CO2 CO2 CO2 CO2 Initial Reading 4 10 9% 12 (3) For the data in the table above (Table 2), calculate the change in CO2 evolved over time for each tube. To do this, subtract the reading at time 0 from the reading at a given time point. (Reading at time x - reading at time 0). Why does this make it easier to compare the tubes? (4) Which of the tubes serve as the negative controls? Why? (5) Why were differing amounts of water added to the different respirometer tubes? (6) Which tube had the highest rate of fermentation? Due next lab... Prepare a graph of the evolution of carbon dioxide over time for all tubes. This is Figure 1. Graph the relationship between carbon dioxide and yeast concentration at a single time point. This is Figure 2. Experiment 2. Cellular Respiration In this lab exercise, you will investigate acrobic cellular respiration in baker's yeast. At one step in the Krebs cycle, succinate is converted to fumerate in a redox reaction (Fig. 3). We will use this step in the Krebs cycle to study the rate of aerobic cellular respiration under different conditions. We will add a substance called DPIP (di-chlorophenol-indophenol). DPIP is an electron acceptor that intercepts electrons released from succinate. This changes DPIP from an oxidized to a reduced state. DPIP is blue in its oxidized state, but changes to colorless as it is reduced. e- DPIP: BlueClear (oxidized) (Reduced) (7) Hypothesize about the effect of an increased amount of substrate on the rate of cellular respiration а. Но: b. Ha: (8) Prediction: Predict the results of the experiment based on your hypothesis. (9) What are we measuring with the spectrophotometer? Figure 3. Succinate is converted to fumarate in Krebs cycle. Glucose Glycolysis Pyruvate Fumarate A step in Krebs cycle FADH2 Krebs 2H'/e- FAD Succinate 112 Table 4 Tube 0 min5 min 1 min 1.5 min 2 min 2.5 min 3.0 min 3.5 min 4.0 min 9901 05 ol 07 10111 91 123 220 1.200.177 .1 40 13 1.151 40 837 455 51 6063 .0u -125 1109 o 02 054 1.077 1071 1.0 (12) Does the data support your hypothesis? (13) In what tube did absorption decrease the fastest? What is a possible explanation of this finding? For Next Lab... Prepare a graph of the change in absorbance versus time for all tubes. This is Figure 3. Prepare a graph of absorbance as a function of succinate concentration for a particular time point. This is Figure 4. 114

Answer 1

2.1 Tube 1 Tube 2 Tube 3 Tube 4 -Tube 5 Tube 6 1.9 1.8 1.7 1.6 1.5 10 Time in minutes

Figure 1

6 minute 1.91 1.9 S 1.89 Σ 1.88 1.87 1.86 1.85 6 minute 1.84 焦.HA ะ 1.83 1.82 0 0.5 0.5 1 15 4 Yeast Concentration

Figure 2

1.3 1.2 E 1.1 Tube 2 Tube 3 -Tube 4 Tube 5 -Tube 6 0.9 0.6 0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 Time in minutes

Figure 3

6 minute 1.91 1.9 S 1.89 Σ 1.88 1.87 1.86 1.85 6 minute 1.84 焦.HA ะ 1.83 1.82 0 0.5 0.5 1 15 4 Yeast Concentration

Figure 4