Demonstrating Cellular Respiration and Fermentation

Cellular respiration and fermentation are 2 of the most challenging concepts for introductory biology students, who may become so consumed by memorizing steps of the Krebs cycle and glycolysis that they lose sight of the big picture. The following demonstrations place aerobic cell respiration and fermentation firmly in grasp. First, students observe respiration in germinating seeds by detecting the carbon dioxide produced. Next, they observe the carbon dioxide gas produced by yeast fermentation.

Demonstrating Aerobic Cellular Respiration with Germinating Seeds

Have students observe the respiration of germinating seeds, using bromthymol blue as a pH indicator. You can keep this demonstration simple by observing pH changes in solution as seeds respire. Alternatively, you might add variables to the demonstration (e.g., compare the respiration rates of various plant seeds with the same total mass). Additionally, you can test whether photosynthesis is occurring by placing duplicate samples in the dark.

Materials

• Hydrated Seeds
• Sealable Bag
• Bromthymol Blue, 0.04%
• Sodium Hydroxide, 1%
• Test Tubes
• Rubber Stoppers
• 250-mL Flaask
• Test Tube Rack
• Distilled Water or Springwater
• Paper Towel

Safety tip

Note:Wear gloves and safety goggles while handling the bromthymol blue solution and sodium hydroxide.

Procedure

1. Two days before the demonstration, rehydrate the seeds (we recommend wheat, barley, peas, or mung beans). Place the seeds in a cup or beaker. Use dechlorinated water to cover the seeds to a depth at least 3 times their height in the container to compensate for the expansion of the seeds as they swell. Allow the seeds to soak overnight.
2. Pour off the remaining water and fold the seeds into a wet paper towel. Place the towel in a sealable bag. Close the bag and store the seeds in a dark place over a second night.
3. Prepare the bromthymol blue solution by adding 1.5 mL (about 30 drops) of 0.04% bromthymol blue to 80 mL of springwater or distilled water. The prepared bromthymol blue solution should be green, but the shade of green may vary depending on the pH of your water source. A color change will easily be observed if the solution given to students is slightly basic. To create a slightly basic solution, add the sodium hydroxide dropwise to the bromthymol blue until the color changes from green to a deep blue (10 to 20 drops).
4. Explain the use of bromthymol blue as a pH indicator to the class. The chemical bromthymol blue is an indicator that appears blue in an alkaline (base) solution and yellow in an acidic solution. Carbon dioxide that is added to the solution surrounding the seeds combines with water to form carbonic acid (H2CO3), turning the bromthymol blue to a yellow-green color. Remove the carbon dioxide from solution and the bromthymol blue will turn a deeper blue.
5. Fill a test tube ¾ full with rehydrated seeds. Pour the bromthymol blue solution over the seeds until the tube is completely full. Seal the tube with a rubber stopper.
6. Prepare a control tube with only bromthymol blue solution. Fill the tube until it is completely full, and seal it with a rubber stopper.
7. Store the samples until the end of the class period or overnight and have students make observations regarding the color of the solution or any other changes they observe. (You may set up duplicate samples in the dark to rule out any possible effects of photosynthesis on the experiment.)

Class discussion

Have students consider the seeds’ respiration. Oxygen is present in the test tube, and the carbon dioxide gas is a product of the process of aerobic cellular respiration. (To a much lesser extent and for a limited time, germinating seeds may also perform anaerobic respiration, which also produces carbon dioxide.) Discuss the purpose of the control tube with only bromthymol blue solution.

Students may think that the seeds are photosynthesizing. If chlorophyll and light are present, the seeds may be photosynthesizing. Students should know that plants’ photosynthesis uses carbon dioxide and produces oxygen. While additional oxygen would not significantly change the pH, removal of carbon dioxide from solution would make the solution more basic and turn the bromthymol blue a deeper blue color.

Demonstrating Fermentation with Yeast

Students observe the carbon dioxide gas produced as yeast ferments sugar. You can simply demonstrate yeast fermentation of a 10% glucose solution, or you can introduce variables and have students compare the degree of fermentation (as indicated by the volume of carbon dioxide generated) occurring under each condition. Suggested conditions to vary include the concentration of glucose, the sugar type, the temperature of incubation, and the amount of light.

Materials

• Sugar(s): Glucose, Sucrose, Lactose, and/or Sucralose (artificial sweetener)
• Packet of Activated Dry Yeast
• Water (distilled or spring)
• Fermentation Tube(s) (a plastic graduated test tube and a larger glass test tube)
• Beaker or Test Tube Rack
• 2 100-mL Flasks
• 100-mL Graduated Cylinder
• Balance
• Hot Plate
  

Proceedure

1. Prepare the 10% sugar solution(s) in distilled water. Yeast can ferment glucose and sucrose but not lactose or sucralose (artificial sweetener).
2. Prepare the yeast suspension immediately before class.
   1. Warm 70 mL springwater or distilled water to approximately 37° C and add 7 g of yeast (1 packet). If you are using a 15-mL conical tube for the small tube, this volume will be sufficient for 8 setups. Scale up or down depending on the desired volume.
   2. Activate the yeast by swirling it for 1 to 2 min. Make sure the yeast distributes evenly in suspension. The presence of clumps leads to inconsistent results. Start a fresh yeast suspension for each class period as the rate of fermentation diminishes over time.
3. Dilute the yeast 1:1 with the sugar solution so that the final concentration of sugar is 5%.
4. Transfer yeast into your fermentation tube by filling the graduated tube with the yeast suspension. Invert the large tube and slide it over the graduated tube with the yeast suspension. Firmly holding the tubes together, invert the apparatus so that the large tube is upright as seen in the figure. A small amount of yeast suspension will flow out of the graduated tube, causing a small air bubble to form in its tip.
5. Have students record their observations of the yeast and the amount of carbon dioxide collected in the graduated tube at regular intervals for 15 min. (They will need to subtract the volume of the original air bubble to get final measurements.) If you are using the activity as an experiment, assign each group a different treatment to observe.

Class discussion

The volume of carbon dioxide released is proportional to the quantity of sugar fermented and is therefore a measure of fermentation. Fermentation results from the action of enzymes. In yeast cells, enzymes convert sugars into carbon dioxide and ethanol. A yeast can ferment a particular type of sugar only if the yeast cell contains the proper enzyme(s) to break down that molecule. Baker’s yeast contains the enzymes to break down glucose and sucrose but not lactose or sucralose.

This kit highlights physiological differences between aerobic cellular respiration and fermentation. Students learn about the production of ATP in glycolysis, Krebs cycle, and electron transport system, and then compare these to the ATP production of both lactic acid fermentation and alcohol fermentation. They observe a demonstration of carbon dioxide production by the aerobic respiration of wheat seeds and the anaerobic respiration of yeast. Next, students apply scientific methodology to investigate yeast alcohol fermentation in the presence of different sugars. They answer questions, perform calculations, and interpret results to evaluate their hypothesis and demonstrate their learning.

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