Alcohol Fermentation

Introduction

Use colorful indicators to demonstrate the products of fermentation.

Background

Interest in fermentation, the breakdown of sugar to alcohol, dates far back in human history. Until 1860, it was believed to be a purely chemical process having nothing to do with living organisms. Then Louis Pasteur showed that fermentation involves a living process carried out by yeast and bacteria. Glucose is fermented according to the following net equation.

C₆H₁₂O₆ → 2C₂H₅OH + 2CO₂

Many different intermediate products can be formed in various concentrations during this reaction, depending upon the conditions. In this demonstration, the production of ethyl alcohol (C₂H₅OH) and carbon dioxide (CO₂) will be illustrated. The color indicator, resazurin, will be used to highlight the conditions present in the fermentation chamber.

Materials

Safety Precautions

Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information. Wash hands thoroughly with soap and water before leaving the laboratory.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Small amounts of iodoform may be disposed of following Flinn Suggested Disposal Method #26a. Follow Flinn Suggested Disposal Method for Type I biological materials discussed in the Biological Waste Disposal Section of your current Flinn Scientific Catalog/Reference Manual.

Procedure

1. Fill a test tube ¾ full with 0.01% phenol red solution and place it in a test tube rack.
2. Place 150 mL of distilled water into a 250-mL Erlenmeyer flask. (A larger flask can be used with proportional increases in the other ingredients.)
3. Add 15 g of glucose (or dextrose) to the flask and swirl the contents until all the glucose dissolves.
4. Add 3 g of active dry yeast to the flask and swirl until the yeast is uniformly mixed in the solution.
5. Add three drops of resazurin solution to the solution in the flask.
6. Use a one-holed stopper, a glass tube and flexible plastic or latex tubing to complete the fermentation setup as shown in Figure 1. The plastic or latex tubing will be below the surface of the solution in the test tube.
7. Note the initial color of the solution in both the flask (purple) and the test tube (red).
8. Let the fermentation apparatus sit undisturbed for an extended period of time (even overnight if necessary).
9. Record the color changes of the solutions in both the flask and test tube.
10. Save the solution in the flask for the optional test for alcohol that follows.

Optional: Iodoform Test for Alcohol

a. When the yeast activity has ceased in the flask (bubbling stops), decant the supernatant fluid into a clean flask.

b. Connect the new flask to a condenser and heat the liquid to distill the ethyl alcohol.

c. Catch the first portion of the distillate and test for alcohol as follows:

1. Place 2–3 mL of distillate onto a watch glass.
2. Add several drops of sodium hydroxide solution to the distillate.
3. Add a few drops of Lugol’s iodine solution to the distillate.
4. Heat the watch glass gently on a hot plate and note the formation of iodoform crystals.
5. Examine the distinctive yellow crystals under a microscope.
Note: Iodoform is toxic and has a disagreeable, pungent odor. Do not handle the solid. Work in a fume hood or well-ventilated area.

Teacher Tips

• This kit contains enough chemicals to perform the demonstration seven times.
  Yeast, active dry, 21 g
  Glucose (dextrose), 150 g
  Phenol red solution, 0.01%, 100 mL
  Resazurin solution, 0.1%, 20 mL

• This demonstration can be set up for every class and each setup will be at a different stage throughout the school day. Alternatively, one demonstration can be set up and monitored by various classes throughout the day.
• The reactions in this demonstration are temperature sensitive. If the surroundings are warm, the gas production will occur within minutes. The color changes can occur within one class period or may take several hours.
• Use distilled water for the setup. Some tap waters may contain chemicals that will kill or inhibit the yeast cells.

Discussion

Resazurin solution is commonly used to show the depletion of oxygen in a solution by living microbes. It is often used to test for the relative number of microbes present in milk. When microbes are present, the resazurin goes through a series of color changes indicating the oxygen condition of the solution. The yeast suspension quickly consumes the available oxygen and the anerobic condition for alcohol fermentation results. The solution in the flask changes color from an initial deep purple to pink, peach, orange and finally colorless, indicating the anaerobic conditions in the flask. These changes occur easily within one class period.

As carbon dioxide is produced in the fermentation process, it bubbles through the tube and into the phenol red solution in the test tube. As the CO₂ bubbles through the solution, the pH of the solution gradually decreases. When the pH gets below a neutral 7.0, the phenol red turns to a yellow color. This bubbling and change in the phenol red is a clear indication of the carbon dioxide produced during fermentation. The optional iodoform test provides a quantitative test for the other product resulting from yeast fermentation—ethyl alcohol.