A.  Learning Objectives

In this lab, students will:

• use a respiration chamber to assess the rate of cellular respiration of germinating peas.

• design and conduct an experiment to test the role of enzymes in cellular respiration.

• determine the fermentation rate of different sugars by yeast.

B. Textbook Correlation: 

Please review Sections 7.1 and 7.2 of Chapter 7: Cellular Respiration, Fermentation, and Secondary Metabolism when preparing for the lab.

C.  Introduction

Write a three paragraph introduction to carbohydrate metabolism

In paragraph 1, discuss the process of cellular respiration.  In this introduction, discuss the three major stages of respiration, list where each stage occurs in the eukaryotic cell, and cite the energy intermediates that capture the energy.  Also, discuss the role of the electron transport chain in harvesting energy.  In this explanation, mention the roles of NADH, FADH₂, O₂, and the major complexes of the chain.  Also discuss the role of the proton gradient in driving the production of ATP by the enzyme ATP synthase.  Lastly, discuss how proteins and lipids also drive the reactions of respiration.  Include an image or animation of the electron transport chain and also include an image of how these macromolecules fit into the process.

In paragraph 2, discuss the production of ATP in the absence of oxygen.  Specifically focus on the function of the reactions of fermentation in allowing cells to continue to produce ATP using glycolysis.  Next, discuss the lactic acid and ethanol fermentation pathways and the cells that carry out these reactions.  Finally, cite some of the major benefits humans have derived from the exploitation of this pathway.  Includes images or animations of both ethanol and lactic acid fermentation.

In paragraph 3, discuss the role of enzymes in these energy harvesting pathways.  Discuss the roles of two classes of enzymes, kinases and dehydorgenases.  Also in your write-up, include a summary of how enzymes are controlled or regulated in the cell, using phosphofructokinase as a specific example.

In these experiments, you will first learn how to measure the respiration rate of germinated peas using a respiration chamber. Then you will be able to design and conduct an experiment to investigate the role of enzymes on pea respiration rate. Although glucose is the preferred carbohydrate for glycolysis and fermentation, you will perform an experiment to measure the fermentation rate of sugars other than glucose by yeast.

D. Respiration

When a seed germinates into a seedling, energy is required.  To meet this energy requirement, seeds store carbohydrates as a source of energy.  These carbohydrates are catabolized and the energy released drives the production of ATP.  

In this experiment you are going to measure the respiration rate of germinating peas by measuring the amount of CO₂ they produce. Refer back to the cellular respiration equation—as glucose is broken down, carbon dioxide is produced. Each group will perform the experiment on germinating peas and non-germinating peas. 

Hypothesis:  Which set of peas will demonstrate the highest rate of cellular respiration?  Explain.

Procedure:

Part I:  Assembly and Incubation

1. On your bench, there are two bottles labeled germinated and ungerminated.   Fit the rubber stopper with the attached glass tubes into the respiration bottles.

2. Submerge the rubber tubing into the water-filled test tubes at your bench.

3.  Insert the rubber stoppers and take note of the time. 

4.  Incubate for 60 minutes.

Part II:  Measuring the rate of respiration

As stated earlier, we are measuring the rate of respiration as a function of CO₂ gas production.  It is difficult to measure the volume of gas produced because it is colorless and odorless.  To overcome this obstacle, we will bubble CO₂ though water, which produces carbonic acid (H₂CO₃).  We will then use a pH indicator to directly measure the amount of H₂CO₃ in our jars.  In this manner, we will indirectly measure the amount of CO₂ produced in these reactions. 

5.  Replace the water in each test tube with phenol red.  Re-submerge the rubber tubing below the surface of the liquid. 

6.  Remove the rubber stopper from the top of the jar and slowly pour water into the jar.  The carbonic acid will be displaced and move through the rubber tubing into the tube filled with phenol red.  A yellow color is an indication of acidic conditions.  Please obtain video of this step and narrate what is happening in each tube.

E.  Effect of enzymes on the rate of respiration:

The chemical reactions of respiration are controlled by enzymes.  Therefore, the respiration rate in germinating peas can be influenced by environmental factors like temperature. In this exercise you will design and conduct an experiment to test the impact of enzymes on the rate of respiration.  We have provided the materials necessary:  another jar of peas, a beaker of water with boiling chips, a hot plate, and another experimental apparatus.  Design your experiment before attending the lab.

1. Hypothesis:

2. Experimental design:

3. Results (Video of bubbling of water)

F.  Fermentation

During fermentation, yeast produce ethanol and CO₂. While glucose is the preferred substrate, other sugars can also be fermented by yeast. In this experiment you will determine yeast’s relative fermentation rates of different sugars. The rate of fermentation can be measured by capturing and measuring the CO₂ as it is produced by the yeast.

Procedure

1.  On your bench, there are four beakers each containing a 15 mL sugar solution: 

            Beaker 1:  Glucose

            Beaker 2:  Glucose

            Beaker 3:  Fructose

            Beaker 4:  Lactose 

2. To beakers 1, 3, and 4 add a 0.5g piece of fresh cake yeast.  Stir with a clean glass rod.  Beaker 2 will not have yeast.

3. Add the entire content of each beaker into separate fermentation tubes called Swan tube due to the curvature of the neck.  Cover the opening of the tube with your thumb and invert each tube so the long tail is filled with solution.

4. Incubate the tubes at 37°C for one hour. 

5. Record the size of the CO₂ bubble in mm every 30 minutes.  Accompany each measurement with a photo of each tube.

Presentation:

You will record two presentations for this lab following the existing format (Introduction, Experimental Design/Execution, Results, Conclusions)

     1.  Cellular Respiration (Sections D and E).

     2.  Fermentation (Section F).