The fermentation of sugars using yeast: A discovery experiment

Introduction

Enzyme catalysis¹ is an important topic which is often neglected in introductory chemistry courses. In this paper, we present a simple experiment involving the yeast-catalyzed fermentation of sugars. The experiment is easy to carry out, does not require expensive equipment and is suitable for introductory chemistry courses.

The sugars used in this study are sucrose and lactose (disaccharides), and glucose, fructose and galactose (monosaccharides). Lactose, glucose and fructose were obtained from a health food store and the galactose from Carolina Science Supply Company. The sucrose was obtained at the grocery store as white sugar. The question that we wanted to answer was “Do all sugars undergo yeast fermentation at the same rate?”

Sugar fermentation results in the production of ethanol and carbon dioxide. In the case of sucrose, the fermentation reaction is:

\[C_{12}H_{22}O_{11}(aq)+H_2 O\overset{Yeast\:Enzymes}{\longrightarrow}4C_{2}H_{5}OH(aq) + 4CO_{2}(g)\]

Lactose is also C₁₂H₂₂O₁₁ but the atoms are arranged differently. Before the disaccharides sucrose and lactose can undergo fermentation, they have to be broken down into monosaccharides by the hydrolysis reaction shown below:

\[C_{12}H_{22}O_{11} + H_{2}O \longrightarrow 2C_{6}H_{12}O_{6}\]

The hydrolysis of sucrose results in the formation of glucose and fructose, while lactose produces glucose and galactose.

sucrose + water \(\longrightarrow\) glucose + fructose

lactose + water \(\longrightarrow\) glucose + galactose

The enzymes sucrase and lactase are capable of catalyzing the hydrolysis of sucrose and lactose, respectively.

The monosaccharides glucose, fructose and galactose all have the molecular formula C₆H₁₂O₆ and ferment as follows:

\[C_{6}H_{12}O_{6}(aq)\overset{Yeast Enzymes}{\longrightarrow}2C_{2}H_{5}OH(aq) + 2CO_{2}(g)\]

Experiment

In our experiments 20.0 g of the sugar was dissolved in 100 mL of tap water. Next 7.0 g of Red Star^® Quick-Rise Yeast was added to the solution and the mixture was microwaved for 15 seconds at full power in order to fully activate the yeast. (The microwave power is 1.65 kW.) This resulted in a temperature of about 110 ^oF (43 ^oC) which is in the recommended temperature range for activation. The cap was loosened to allow the carbon dioxide to escape. The mass of the reaction mixture was measured as a function of time. The reaction mixture was kept at ambient temperature, and no attempt at temperature control was used. Each package of Red Star Quick-Rise Yeast has a mass of 7.0 g so this amount was selected for convenience. Other brands of baker’s yeast could have been used.

This method of studying chemical reactions has been reported by Lugemwa and Duffy et al.^2,3 We used a balance good to 0.1 g to do the measurements. Although fermentation is an anaerobic process, it is not necessary to exclude oxygen to do these experiments. Lactose and galactose dissolve slowly. Mild heat using a microwave greatly speeds up the process. When using these sugars, allow the sugar solutions to cool to room temperature before adding the yeast and microwaving for an additional 15 seconds.

Fermentation rate of sucrose, lactose alone, and lactose with lactase

Fig. 1 shows plots of mass loss vs time for sucrose, lactose alone and lactose with a dietary supplement lactase tablet added 1.5 hours before starting the experiment. All samples had 20.0 g of the respective sugar and 7.0 g of Red Star Quick-Rise Yeast. Initially the mass loss was recorded every 30 minutes. We continued taking readings until the mass leveled off which was about 600 minutes. If one wanted to speed up the reaction, a larger amount of yeast could be used. The results show that while sucrose readily undergoes mass loss and thus fermentation, lactose does not. Clearly the enzymes in the yeast are unable to cause the lactose to ferment. However, when lactase is present significant fermentation occurs. Lactase causes lactose to split into glucose and galactose. A comparison of the sucrose fermentation curve with the lactose containing lactase curve shows that initially they both ferment at the same rate.

Fig. 1. Comparison of the mass of CO₂ released vs time for the fermentation of sucrose, lactose alone, and lactose with a lactase tablet. Each 20.0 g sample was dissolved in 100 mL of tap water and then 7.0 g of Red Star Quick-Rise Yeast was added.

However, when the reactions go to completion, the lactose, lactase and yeast mixture gives off only about half as much CO₂ as the sucrose and yeast mixture. This suggests that one of the two sugars that result when lactose undergoes hydrolysis does not undergo yeast fermentation. In order to verify this, we compared the rates of fermentation of glucose and galactose using yeast and found that in the presence of yeast glucose readily undergoes fermentation while no fermentation occurs in galactose.

Fig. 2. Comparison of the mass of CO₂ released vs time for the fermentation of sucrose, glucose and fructose. Each 20 g sugar sample was dissolved in 100 mL of water and then 7.0 g of yeast was added.

Fermentation rate of sucrose, glucose and fructose

Next we decided to compare the rate of fermentation of sucrose with that glucose and fructose, the two compounds that make up sucrose. We hypothesized that the disaccharide would ferment more slowly because it would first have to undergo hydrolysis. In fact, though, Fig. 2 shows that the three sugars give off CO₂ at about the same rate. Our hypothesis was wrong. Although there is some divergence of the three curves at longer times, the sucrose curve is always as high as or higher than the glucose and fructose curves. The observation that the total amount of CO₂ released at the end is not the same for the three sugars may be due to the purity of the fructose and glucose samples not being as high as that of the sucrose.

Fermentation rate and sugar concentration

Next, we decided to investigate how the rate of fermentation depends on the concentration of the sugar. Fig. 3 shows the yeast fermentation curves for 10.0 g and 20.0 g of glucose. It can be seen that the initial rate of CO₂ mass loss is the same for the 10.0 and 20.0 g samples. Of course the total amount of CO₂ given off by the 20.0 g sample is twice as much as that for the 10.0 g sample as is expected. Later, we repeated this experiment using sucrose in place of glucose and obtained the same result.

Fig. 3. Comparison of the mass of CO₂ released vs time for the fermentation of 20.0 g of glucose and 10.0 g of glucose. Each sugar sample was dissolved in 100 mL of water and then 7.0 g of yeast was added.

Fermentation rate and yeast concentration

After seeing that the rate of yeast fermentation does not depend on the concentration of sugar under the conditions of our experiments, we decided to see if it depends on the concentration of the yeast. We took two 20.0 g samples of glucose and added 7.0 g of yeast to one and 3.5 g to the other. The results are shown in Fig. 4. It can clearly be seen that the rate of CO₂ release does depend on the concentration of the yeast. The slope of the sample with 7.0 g of yeast is about twice as large as that with 3.5 g of yeast. We repeated the experiment with sucrose and fructose in place of glucose and obtained similar results.

Fig. 4. Comparison of the mass of CO₂ released vs time for the fermentation of two 20.0 g samples of glucose dissolved in 100 mL of water. One had 7.0 g of yeast and the other had 3.5 g of yeast.

Discussion

In hindsight, the observation that the rate of fermentation is dependent on the concentration of yeast but independent of the concentration of sugar is not surprising. Enzyme saturation can be explained to students in very simple terms. A molecule such as glucose is rather small compared to a typical enzyme. Enzymes are proteins with large molar masses that are typically greater than 100,000 g/mol.¹ Clearly, there are many more glucose molecules in the reaction mixture than enzyme molecules. The large molecular ratio of sugar to enzyme clearly means that every enzyme site is occupied by a sugar molecule. Thus, doubling or halving the sugar concentration cannot make a significant difference in the initial rate of the reaction. On the other hand, doubling the concentration of the enzyme should double the rate of reaction since you are doubling the number of enzyme sites.

The experiments described here are easy to perform and require only a balance good to 0.1 g and a timer. The results of these experiments can be discussed at various levels of sophistication and are consistent with enzyme kinetics as described by the Michaelis-Menten model.¹ The experiments can be extended to look at the effect of temperature on the rate of reaction. For enzyme reactions such as this, the reaction does not take place if the temperature is too high because the enzymes get denatured. The effect of pH and salt concentration can also be investigated.

References

1. Jeremy M. Berg, John L. Tymoczko and Lubert Stryer, Biochemistry, 6th edition, W.H. Freeman and Company, 2007, pages 205-237.
2. Fugentius Lugemwa, Decomposition of Hydrogen Peroxide, Chemical Educator, April 2013, pages 85-87.
3. Daniel Q. Duffy, Stephanie A. Shaw, William D. Bare, Kenneth A. Goldsby, More Chemistry in a Soda Bottle, A Conservation of Mass Activity, Journal of Chemical Education, August 1995, pages 734-736.
4. Jessica L Epstein, Matthew Vieira, Binod Aryal, Nicolas Vera and Melissa Solis, Developing Biofuel in the Teaching Laboratory: Ethanol from Various Sources, Journal of Chemical Education, April 2010, pages 708–710.