Yeast and Anaerobic Fermentation
Yeast are eukaryotic unicellular fungi. Like many other eukaryotes, they typically use aerobic cellular
respiration to break down glucose and generate the ATP needed for their vital functions. Aerobic
respiration includes the processes of glycolysis, the Krebs cycle, the electron transport chain, and
chemiosmosis and produces 36-38 molecules of ATP.
The chemical reaction for aerobic respiration of glucose is C6H12O6 + 6 O2 → 6 CO2 + 6 H2O.
Yeast also have the ability to switch to fermentation when in an anaerobic, or oxygen-free atmosphere.
This gives the organisms a chance to continue to produce ATP, although at a much lower level than
The chemical reaction for fermentation of glucose is C6H12O6 → 2 CO2 + 2 C2H5OH
In addition to producing ATP for the yeast cells, this reaction also produces two waste products: carbon
dioxide and ethyl alcohol. The carbon dioxide gas can measured quantitatively and used to approximate
the fermentation rate of the yeast organisms.
- As eukaryotes, what cellular structures would you expect to be present in yeast?
- How do the waste products of yeast fermentation differ from those of fermentation in animals?
- If the yeast remained in an anaerobic environment for an extended period of time and
fermentation continued, what could eventually happen to them?
Biology Teaching Resources http://www.aurumscience.com Page 1
Procedure 1: Qualitative Observation of Cellular Respiration in Yeast
To observe the transition of yeast from aerobic respiration to anaerobic respiration.
Janus Green B is an indicator solution that changes color based on whether or not oxygen is present. In
the presence of oxygen, it produces a blue color. In the absence of oxygen, it produces a pink color.
What color change do you predict if Janus Green B is added a mixture of yeast and glucose in a test tube?
What color change do you predict if the yeast suspension is sealed so oxygen cannot enter?
Dry yeast (3g)
Janus Green B indicator solution
1. Mix the dry yeast with 30mL of distilled water at a temperature of 40ºC.
2. Make a 5% glucose solution.
To make a % solution, add the grams of solute to the beaker, then fill with water up to 100mL.
Example: To make a 1% glucose solution, add 1 gram of glucose then fill to 100mL with water.
3. Add 10mL of the 5% glucose solution to the test tube.
4. Add two drops of the Janus Green B indicator solution to the test tube.
5. Add 10mL of yeast solution to the test tube.
- What color change do you observe at this step? _________________________
6. Using a pipette, place enough oil over the top of the glucose / yeast solution in the test tube to
completely cover it.
- What color change do you observe at this step? __________________________
Based on your observations, describe what type of respiration is occurring in the test tube before and after
the layer of oil is added.
Procedure 2: Microscopic Examination of Yeast
To observe the cellular structure of yeast organisms.
Test tube of yeast, glucose, and Janus Green B.
Blank slide and cover slip
1. Using a pipette, take a drop of the yeast/glucose/Janus B solution. Avoid sampling the oil on top.
2. Place a drop of the yeast mixture on a clean glass microscope slide.
3. Place a cover slip on top of the drop, keeping air bubbles out.
4. Examine the yeast slide under a microscope at 100X power.
Make a sketch of the structure of one of the yeast cells under high power. Label any structures you can
see, paying particular attention to the mitochondria, which are specifically stained by the Janus Green B.
Microscope Power: __________
Janus Green B is an indicator that specifically reacts with oxygen. Given this information, why would it
be an effective stain for the mitochondria organelles?
Procedure 3: Quantitative Measurement of Carbon dioxide in Yeast Fermentation
1. To study the yeast fermentation rate of three sugars: glucose, sucrose, and starch.
2. To study the effect of an enzyme activator (Magnesium sulfate, MgSO4) and an enzyme inhibitor
(Sodium fluoride, NaF) on yeast fermentation rate.
Given the three carbohydrates (glucose, sucrose, and starch), which do you believe will have the greatest
and lowest fermentation rate? Justify your hypothesis using your prior knowledge of the chemical
structure of these molecules.
What effect will adding magnesium sulfate and sodium fluoride have on the rate of fermentation? Justify
your hypothesis using your prior knowledge of enzymes and their role in chemical reactions.
Dry yeast (10g) 10 narrow test tubes
Distilled water 10 wide test tubes
0.1M Magnesium sulfate, MgSO4 solution Test tube rack
0.1M Sodium fluoride, NaF solution Warm water bath (40ºC)
Glucose, sucrose, starch Four 250mL beakers
1. Create a liquid suspension of yeast by adding 10g of dry yeast to 200mL of distilled water at a
temperature of 40ºC. Keep your yeast suspension in the warm water bath until you need it.
2. Prepare each of the following solutions:
5% Glucose solution = Add 5.0g glucose to a beaker, fill with distilled water up to 100mL.
5% Sucrose solution = Add 5.0g sucrose to a beaker, fill with distilled water up to 100mL.
5% Starch solution = Add 5.0g starch to a beaker, fill with distilled water up to 100mL.
3. Label ten of more narrow test tubes #1-10.
4. Place the following in each narrow test tube:
Test tube 1: 15mL distilled water
Test tube 2: 5mL glucose, 10mL distilled water
Test tube 3: 5mL glucose, 5mL MgSO4 , 5mL distilled water
Test tube 4: 5mL glucose, 5mL NaF, 5mL distilled water
Test tube 5: 5mL sucrose, 10mL distilled water
Test tube 6: 5mL sucrose, 5mL MgSO4 , 5mL distilled water
Test tube 7: 5mL sucrose, 5mL NaF, 5mL distilled water
Test tube 8: 5mL starch, 10mL distilled water
Test tube 9: 5mL starch, 5mL MgSO4 , 5mL distilled water
Test tube 10: 5mL starch, 5mL NaF, 5mL distilled water
5. Add yeast solution to test tubes #1-10 until each has been filled to the very top.
6. Place a large test tube over each small test tube, then flip upside down.
7. Mark the bottom of the air bubble at the top of the test tube with a wax pencil.
8. Place in the warm water bath. Measure the change in size of the air bubbles at each time interval.
Table 1: Yeast production of carbon dioxide
Contents of Test Tube Carbon Dioxide Production (mm)
10min 20min 30min 40min 50min
Test Tube 1 Distilled water, yeast
Test Tube 2
Test Tube 3
Test Tube 4
Test Tube 5
Test Tube 6
Test Tube 7
Test Tube 8
Test Tube 9
Test Tube 10
Complete one graph to compare the fermentation rate for each of the different carbohydrates. Graph the
carbon dioxide gas production for test tube #2 (glucose), test tube #5 (sucrose), and test tube #8 (starch).
- Why are we specifically choosing these three test tubes as comparison?
- Which variable is independent (manipulated) and should go on the x-axis?
- Which variable is dependent (measured) and should go on the y-axis?
Graph 1: Effect of different carbohydrates on fermentation rate in yeast
Next, compare the effects of sodium fluoride and magnesium sulfate on the yeast. Complete one graph
for glucose, one for sucrose, and one for starch. Each graph should have three lines.
Graph 2: Effect of magnesium sulfate and sodium fluoride on fermentation of glucose
Graph 3: Effect of magnesium sulfate and sodium fluoride on fermentation of sucrose
Graph 4: Effect of magnesium sulfate and sodium fluoride on fermentation of starch
Based on your results, is your hypothesis supported or rejected? How reliable do you believe your data
is? Are there any external variables or errors that could have affected the fermentation rate of the yeast?
How could the experiment have been changed to minimize these variables or errors?
Describe the ideal environment for fermentation to occur at the highest rate, given your results. Include
the factors of temperature, presence of oxygen, food source, and presence of enzyme activators or