Biology, Answering the Big Questions of Life/Fermentation

Goal
The goal of the Fermentation lab is to introduce the students to the idea of sugar catabolism. They realize that they have seen this occurring in their everyday life but did not know the overall details.

Point out the fact that yeast can breakdown sugar both aerobically and anaerobically, but if they do it anaerobically they yield only 2 ATP energy from each glucose, while they yield 36 or 38 ATP whey they use oxygen.

The lab is designed to clearly show the beginning and end points of the reaction. The students add sugar to water and then add the yeast. In the end, they smell the alcohol and test the CO2.

This lab can be done only qualitatively or quantitatively. Chose which is most appropriate for your students.

Yeast Fermentation Lab
The Fermentation lab is fun and you can easily change the difficulty.

For the simplest case, make it a demonstration. Place the balloon on top of the flask, and let it sit on the teacher's desk while you lecture. The balloon will slowly enlarge.

For more complex protocols allow the student to distill the alcohol produced, and test that CO2 is present by using Lime Water.

Remember that the time required for this lab will vary due to temperature and the quality of the yeast. If students do not get results, let the reaction continue 20 more minutes and try again.

Observing a Yeast sample
If you make a microscope slide of the yeast, you will see sand like dots at 40X, small clumps of round things at 100X, and clumps of nucleated cells at 400X

If you do not dye the cells with a dye that kills the cells like iodine, then the cells will continue to grow, and the students can observe bubbles forming under the glass even without a microscope. Have them draw the slide as soon as they make it, and then draw it again after 15 minutes. The bubbles should be larger and more pronounced. Ask them what is happening in the slide.

Burning Ethanol
1. Your instructor will show you that ethanol still contains energy by burning a sample of 95% ethanol.

A. Pour some 95% ethanol on a watch glass and carefully light.

B. Turn off the light to see it easier.

C. Be careful. I burned my hand doing this experiment today!

D. Tell the students that although ethanol is the end product of fermentation, it still holds energy in the molecules. The way that cells get that energy out is Aerobic respiration.

E. Cover the watchglass with a beaker and watch as the fire goes out. Tell the students that the burning of pyruvate requires oxygen, just like the flame does.

CO2 Demonstration
2. Look at the demonstration flask, and see if you can observe the presence of CO2 gas in the mixture.

A. Cap an Erlenmeyer flask with a stopper containing one hole. Place a glass tube through the hole.

B. Attach this to plastic tubing that goes into another beaker filled with lime water.

C. As fermentation occurs, CO2 will be produced. It will go through the tube and bubble out through the lime water. Hopefully, you will see a white precipitate.

D. Using Lime water CaOH2 to precipitate CO2 is a classic chemical test. The white powder is chalk. The wikipedia entry on CO2 says:

"The gas may be bubbled into calcium hydroxide solution (lime water). The lime water will turn milky if CO2 is introduced because of the formation of calcium carbonate."

The equation is:

Limewater + Carbon Dioxide --> Chalk + Water :

Ca(OH)2(aq) + CO2(g) --> CaCO3(s) + H2O(l)

For more info, see the Wikipedia article on [lime water]

Measuring the CO2 content.
Use round balloons for this experiment. Then have the students wrap a string around the widest part of the balloon. Lay down the string and measure the length. This tells you the circumference of the circle.

Calculate the radius to the center of the circle using the equation:

$$C/2 \pi  = r$$

Once you have that, you can figure out the volume of the balloon using the equation:

$$V=4/3 \pi r^3$$

The easiest thing is to compare volumes directly, but it should be able to figure out the amount of CO2 produced.

Assuming that it is pure CO2, and assuming standard temperature and pressure, you can figure out the moles of CO2 generated by this reaction by using the ideal gas law.

The Ideal Gas Law

P is pressure, V is volume, n is moles, T is temperature, and R is the gas constant, which has various values depending on the unit of pressure:

$$\qquad PV=nRT$$

To get the moles of CO2 released use the equation:

$$\qquad n= PV/RT$$

Two moles will have been released for each glucose molecule broken down by glycolysis.

glucose --> 2 Ethanol + 2 CO2

so multiply the number of moles by two to get the number of moles glucose burned in your fermentation reaction.

Variations
1. Repeat the experiment with different carbohydrate sources to find which sugars are utilized best.

2. Measure the volume of the balloon at different time points, or pour the reactants into test tubes and take a measurement of both the sugar and the gas volume at different time points.

(Be sure to ask the students what sources of error they may have in their experiments.)

Possible problems
If you want to go the quantitative route, please note these possible sources of error.

The corn syrup will stick to the graduated cylinder, and if the students are impatient, they may not get all of the sugar out. I have seen almost 5 mls of sugar left in the flask after they have "emptied" it. If you are using the amount of sugar measured in your calculations, it is most likely off.

If you want to calculate the amount of CO2 made, use the mMol/L measurement of the sugar found on the test strips so that you can compare how much sugar was in the original amount.