Spectrophotometric Analysis of Ethanol and Glucose Concentrations in Yeast Culture Media

Fermentative growth on glucose is one of the most widely studied conditions of yeast growth in the laboratory. The production of ethanol from sugars is relevant to the wine, beer, and bread industries and to production of biofuels. Assaying the levels of glucose and ethanol in yeast growth medium allows the experimenter to determine the consumption of the carbon source glucose and the production of ethanol. This protocol describes enzyme-coupled assays for determination of glucose and ethanol concentrations in a sample of cell-free culture medium. Enzymes convert glucose or ethanol into other compounds through chemical reactions that reduce NAD(P)⁺ to NAD(P)H, and the production of NAD(P)H is measured using a spectrophotometer. The methods presented are highly sensitive, with a detection limit of ∼0.4 mg/L of glucose and 50 mg/L of ethanol, and also have the advantage of high specificity. For example, glucose and fructose have identical chemical formulas and thus cannot be distinguished by a mass spectrometer, but the enzyme assay presented here is specific for glucose. The glucose assay can be coupled to other assays to determine the quantity of additional carbohydrates such as fructose, trehalose, and glycogen.