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Ethanol Fuel

Fermenting Fuel

Grade Level: High School

Ethanol is a renewable source of fuel for vehicles, which is widely produced from corn. Ethanol production is reliant on the anaerobic fermentation of corn sugars by yeast. Scientists and industry professionals are constantly working to make the fermentation procedure more efficient. Different enzymes are added to the corn in order to break the starch into simple sugars that the yeast can process into ethanol. This lab allows students to experiment with different variables in the fermentation process to determine their effect. Students can then use their findings to develop a fermentation procedure that they may use on a larger sample for the distillation lab, Corn Mash and Distillation.

Variables teachers and/or students can select to test:

  • Feedstock: Suggest using ground corn if students are designing procedure for distillation
  • Enzymes: none, alone, or in combination
  • Effect of pH on yeast and enzymes
  • Amount of yeast solution added
  • Starting yeast in a 2% glucose solution

Students will design and conduct their own investigations to answer one or more of the following driving questions or develop their own. (Teacher may choose the questions, and it is recommended that all students test enzymes separately, mixture and control as one investigation.

  • What is the effect of each enzyme or combination on glucose concentration and rate of fermentation?
  • How much of an effect does starting yeast in glucose solution have on fermentation rate?
  • What is the optimal amount of yeast solution per gram of feedstock to produce the most ethanol in the allotted time frame?
  • Do the enzymes work more effectively at releasing glucose at a certain pH?
  • How does baker’s yeast compare to brewer’s yeast in ability to produce ethanol from corn?

Students will measure the amount of CO2 produced in the time frame that the teacher selects. This can be one class period or overnight. If allowed to run overnight, time-lapse video of the fermentation allows students to plot data points from the times they were not able to directly observe the fermentation.

Teaching the Lesson

Science Performance Expectation

  • HS-LS2-3 Construct and revise an explanation based on evidence for the cycling of matter and flow of energy in aerobic and anaerobic conditions. Clarification Statement: Emphasis is on conceptual understanding of the role of aerobic and anaerobic respiration in different environments.

Science Disciplinary Core Ideas

  • LS2: Ecosystems: Interactions, Energy, and Dynamics
  • LS2.B: Cycles of Matter and Energy Transfer in Ecosystems Photosynthesis and cellular respiration (including anaerobic processes) provide most of the energy for life processes.

Science Practices: High School (9-12)

  • Constructing explanations and designing solutions in 9–12 builds on K–8 experiences and progresses to explanations and designs that are supported by multiple and independent student-generated sources of evidence consistent with scientific ideas, principles, and theories.
    • Make a quantitative and/or qualitative claim regarding the relationship between dependent and independent variables.
    • Construct and revise an explanation based on valid and reliable evidence obtained from a variety of sources (including students’ own investigations, models, theories, simulations, peer review) and the assumption that theories and laws that describe the natural world operate today as they did in the past and will continue to do so in the future.
    • Apply scientific ideas, principles, and/or evidence to provide an explanation of phenomena and solve design problems, taking into account possible unanticipated effects.
    • Apply scientific reasoning, theory, and/or models to link evidence to the claims to assess the extent to which the reasoning and data support the explanation or conclusion.
    • Design, evaluate, and/or refine a solution to a complex real-world problem, based on scientific knowledge, student-generated sources of evidence, prioritized criteria, and trade off considerations.

Cross Cutting Concept

  • Energy drives the cycling of matter within and between systems.
  • The student will differentiate between aerobic respiration and anaerobic fermentation.
  • The student will design and conduct an investigation to determine the effect of enzymes on fermentation and/or glucose release.
  • The student will collect and analyze data to determine the rate of fermentation.
  • Using data students will design a recipe for producing ethanol in a larger scale lab for distillation.

Materials for Preparing Samples:

  • Ground corn (enough for 1 g per sample)
  • Balance
  • Rubber stoppers
  • Beaker (500 ml)
  • Hot Plate
  • Test tube holder
  • Stirring rod

Materials for Fermentation:

  • Fermenting Fuel Student Handout
  • 5 test tubes of prepared samples (per group)
  • Test tube rack (per group)
  • Rubber stoppers with tubing connectors and tubing to collect CO2 produced (per group)
  • Tub for water displacement (per group)
  • 3D printed manifold with 5, 50 ml centrifuge tubes (per group)
  • Distilled water
  • Food coloring for more visibility (recommended especially if time-lapse is being used)
  • Disposable pipettes
  • Dry activated yeast (may want to provide different varieties if students want to test)
  • Amylase solution (1 tsp/500 ml water or 1g/125 ml)
  • Glucoamylase solution (1 tsp./500 ml water or 1g/125 ml)
  • Buffer solution (pH5; pH4 optional if pH is being tested)
  • Other materials students may need (beaker for warm water, etc.)
  • This lab contains materials that students might consider edible. Remind students that while working in a lab, all materials should be considered dangerous and are not be tasted. There should be no eating or drinking in the laboratory.
  • Students should wear appropriate eye protection.

Fermentation is an anaerobic process (without oxygen), carried out by yeast, bacteria, and even muscle cells. This is an alternative pathway for organisms to release energy from food when oxygen is not available. Yeast and muscle cells can carry out both aerobic respiration and anaerobic fermentation, providing an energy source when oxygen is scarce. The first step in both aerobic and anaerobic respiration is the breaking of a glucose molecule, called “glycolysis”. This yields two new ATP molecules (cellular fuel) per molecule of glucose broken. If oxygen is not available, the cell needs to recycle the molecules needed in order to break more glucose. In doing this, it converts the broken pieces of glucose (pyruvate) into different molecules. Yeast convert the pyruvate into ethanol, which is a renewable fuel source. This process also produces two carbon dioxide molecules for each glucose molecule fermented.  Students can determine the rate and amount of fermentation by measuring the amount of carbon dioxide it produces, as shown in the equation below.

In this activity, students will be using yeast, which needs glucose to break for fuel. Glucose and fructose are both simple sugars, but the ethanol industry feeds yeast corn, in which most of the sugars are contained in long chain molecules, or polymers called “starch”. The differences in these food sources are shown in the image below.

Several enzymes have the purpose of breaking starch up into smaller pieces, which are more useful for the yeast. Amylase is an enzyme that breaks starch into smaller pieces, while glucoamylase is more specialized in removing individual glucose molecules from the ends of the starch. Being proteins, enzymes have specific conditions in which they function more effectively, such as pH and temperature. Students may research or experiment to find the most efficient range for the enzymes used.

Procedures for Instruction

Length of Time for Preparation: 1 hour

Length of Time for Classroom Teaching:

  • Day 1: 1 hour (Preparing samples and designing experiment)
  • Day 2: 1 class period (running experiment, 1 hour or block)
  • Day 3: 1 hour (reflections, conclusions, discussion, and assessment)

Introducing Lab:

  1. Assign lab groups and present students with the basic procedure for preparing samples and fer­menting, and the variables that they may alter in an attempt to improve the procedure.
  2. Make sure each student receives a copy of the Fermenting Fuel Student Handout (pg. S1-S7, or available online at so they can reference background information, instructions, and enter data as well as answer reflection and conclusion questions. (Sample data and key are included on pg. T11-T13.)
  3. Allow students to design the experiment with up to five samples, reminding them that there must be a comparison sample that is only one variable different from another sample.
  4. The design should be written so that each sample has a recipe on the student handout.

Preparing Samples: (May be student or teacher prepared)

  1. Add approximately 250 ml of water to a 500 ml beaker and heat to boiling.
  2. Add 1 g of ground corn to each sample test tube and add 5 ml of distilled water.
  3. Mix the corn and water mixture with a stirring rod and add a test tube holder.


  1. Place test tubes in boiling water for 10 minutes, as shown in the image above, to break down corn. (Note: This time can be shortened or step skipped if students want to test the importance of heating time.)
  2. Place a stopper in the samples and allow to them cool. The samples may be placed in cold water bath or allowed to cool overnight. Do not add enzymes until sample has cooled.

Fermentation Procedure:

  1. Fill tub with enough water to cover the 35 ml mark on the tubes.
  2. Tip the manifold over on its back and tap the tubes to remove bubbles.
  3. Students will add the ingredients to each sample as they have prescribed in their procedure.
  4. Mix each sample with a stirring rod, rinsing between samples, and place in test tube rack.
  5. Stopper each sample with a one-hole stopper and insert tubing into opening in manifold below the gas collection tube for that sample. Repeat for each sample.
  6. Tip manifold up so that gas is now being collected.
  7. If time lapse recording is to be used, set up camera.
  8. Measure and record volume of CO2 collected every 5 minutes.
  9. Graph data and analyze results.
  10. Teacher may allow students to design another experiment or procedure for fermentation to use in the follow up ethanol lab.



3D Printed manifold for gas collection.

The .stl file for this manifold






Manifold with centrifuge tubes inserted. The manifold will be submerged in water in this position to fill with water.







Filled manifold with tubing from samples inserted. Ready to collect any CO2 gas produced.




Student set up testing rate of fermentation with different varieties of yeast.







Student set up above with iPad included for time lapse recording of data.

Fermenting Fuel Time Lapse  Video recorded during this experiment.







Student set up to test the effect of a warm water bath (38C-40C) on the rate fermentation.






Multiple documents are available for the teachers and students to use during this lab. Use the Fermenting Fuel Student Handout to guide students through the lab, but to also conclude and reflect on what is was learned. Sample data, assessments, and answer keys are provided for teacher guidance.

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Any educator electing to perform demonstrations is expected to follow NSTA Minimum Safety Practices and Regulations for Demonstrations, Experiments, and Workshops, which are available at, as well as all school policies and rules and all state and federal laws, regulations, codes and professional standards. Educators are responsible for abiding appropriate legal standards and better professional practices under a duty of care to make laboratories and demonstrations in and out of the classroom as safe as possible. If in doubt, do not perform the demonstrations.

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