I wouldn't teach biochemistry without doing an exercise in fermentation. We all spend so much time teaching metabolism yet there are so few reliable, inexpensive lab exercises that allow students to examine metabolism in action. Both bacteria and mitochondria present interesting examples of accessible metabolism but they both require a good deal of peripheral preparation. In this lab, students examine the substrate specificity of fermentation and analyze the product gas.

Fermentation can be carried out and studied in a number of different containers, even inverted test tubes. In this exercise, fermentation is carried in 60-cc plastic syringes with plastic stopcocks attached to the Luer tips. Students prepare their fermentation mixtures by dissolving 0.6 g of carbohydrate in 30 mL of water in a flask, then suspending 0.6 g of yeast (commercial dry yeast in jars keeps for many years in the refrigerator). Carbohydrates employed are glucose, fructose, galactose, glycerol, sucrose, lactose, maltose, and starch. The fermentation mixtures are poured into the syringes, which are purged of air and placed in a water bath at 42-44 degrees. Students examine them occasionally for gas production. After thirty minutes to an hour, they remove the tubes from the water bath and determine the gas volume.

Glucose, fructose, and sucrose are rapidly fermented, maltose more slowly. Galactose, glycerol, lactose, and starch are not fermented. Gas yields in the best syringes are 25-30 mL.

Two simple tests are used to identify the gas. In the first, about 2 mL of the gas is "injected" from a syringe directly into a screw-capped test tube containing a few milliliters of limewater (saturated calcium hydroxide or calcium oxide). When the tube is capped and shaken, a cloudy precipitate of calcium carbonate forms. In the second test, a 2-mL sample of gas is forced into a 5-mL syringe. Students dip the end of the syringe into a 50-mL beaker containing 10 mL of 1 M NaOH, and draw up about 1 mL of the solution. The NaOH solution is drawn further into the syringe over a minute or so as the carbon dioxide dissolves in the water and reacts with the base.

For a write-up, students are asked first to tabulate all the class's results. They are then asked (1) to describe the reactions by which glycerol would be fermented and to explain why it was not fermented, (2) would they expect sorbitol to be fermented, (3) what glycosidases must yeast possess, or lack, to account for class results, (4) to write balanced equations describing the results of the two tests of the gas, and (5) to calculate, from the gas volumes, the weight percent of ethanol in their best brews given a fermentation volume of 30 mL, a temperature of 40 degrees C, the vapor pressure of water, and the current barometric pressure. It might be worth the effort sometime to complete the exercise with an ethanol assay.

Revised 6/13/07