Alcoholic Fermentation

This is a classic demonstration of the specificity and product analysis of sugar fermentation. I like it because it is one of the few, and easier, laboratory demonstrations of metabolism in action. (I do this a bit more intensively in the lab for CHM 341.) In this exercise, students compare fermentation of seven carbohydrates (glucose, fructose, galactose, sucrose, maltose, lactose, and starch), analyze the gas evolved, and answer some questions that help them interpret their results.

The exercise requires some apparatus for observing and collecting the evolved gas. I am fortunate to have on hand a number of fermentation tubes. Another possible arrangement that is inexpensive but satisfactory is a test tube inverted in a larger tube, for example, a 12 x 75-mm tube in a 18 x 150-mm tube or, for larger volumes, an 18 x 150-mm tube in a 100-mL graduated cylinder. There are then the problems of inverting the inner tube without letting any air in and recovering the evolved gas at the end. A third possibility would be a stoppered flask with a gas delivery tube (preferably short and narrow) to permit the gas to be collected in a test tube inverted in a trough.

The fermentation mixture is made by combining equal volumes of a 4% carbohydrate solution and a 4% suspension of yeast, all in water. Incubation in a warm environment (up to 44 degrees is fine) gives 5-15 mL of gas within an hour. I don't have an incubator in the lab, but I have a laboratory oven that can be made to maintain around 42 degrees. I've also found that a mechanical room in the basement has a temperature of about 43 degrees, and have appropriated a corner for the incubations.

Students always find that glucose, fructose, and sucrose ferment well. Galactose, lactose, and starch do not. Typically, we find that maltose ferments as well as glucose. I've read that some strains of yeast have higher maltase activities than others; I've been using Fleischmann's yeast in a 4-oz. jar (for the "Bread Machine"). It's a few years outdated but still works fine if stored dry and cold.

From tubes that fermented well, I had students remove 1 mL of the gas with a syringe and inject it into a small test tube with 2 mL of limewater (saturated calcium hydroxide). Corking and shaking gives calcium carbonate. I haven't had them try to detect ethanol.

When they are done, they are asked (1) how yeast get fructose into glycolysis, by analogy to muscle, (2) what is the structural difference between glucose and galactose and what reactions would be necessary to ferment the latter, (3) what monosaccharides would be produced by hydrolyzing lactose, maltose, sucrose, and starch and which of these reactions yeast seem able to carry out, and (4) to write the equation for the reaction that occurs between the gas and calcium hydroxide.