Kansas College and Career Ready Standards

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.

Learning Objectives

• 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

• Fermenting Fuel PowerPoint
• Experiment Worksheet

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)
• Prepared amylase solution
• Prepared glucoamylase solution
• Buffer solution (pH5; pH4 optional if pH is being tested)
• Other materials students may need (beaker for warm water, etc.)

Safety Considerations

• 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.

Background Information

The chemical reaction that powers most vehicles is a combustion reaction that uses gasoline, diesel, or ethanol as fuel. Each of these fuels has a carbon chain and burning these fuels releases carbon dioxide. Carbon dioxide is a greenhouse gas. Its levels are rising in the atmosphere as a result of burning fossil fuels. This increase is thought to be a major component of climate change.

The difference between fossil fuels, such as diesel and gasoline, and biofuels, such as ethanol and biodiesel, is the source of the carbon. When fossil fuels are refined from crude oil that is pumped from deep underground, the carbon in biofuels comes from carbon dioxide, which is fixed from the atmosphere during photosynthesis. The carbon dioxide produced from burning fossil fuels is “old” carbon, not having been in the atmosphere for millions of years, whereas the carbon dioxide produced from burning biofuels is “new” carbon that was in the atmosphere during the lifetime of the plant used to produce the fuel. Burning biofuels requires the removal of a carbon dioxide molecule from the atmosphere for every molecule produced during combustion of the fuels. Producing and burning fossil fuels produces no increase in atmospheric carbon dioxide.

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, allowing 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 2 new ATP molecules (cellular fuel) per molecule of glucose broken. If oxygen is not available, the cell needs to recycle the molecules needed to break more glucose. In doing this it converts the broken pieces of glucose (pyruvate) into different molecules. Most bacteria, and our muscle cells, convert pyruvate into lactic acid, which is important in the production of cheese and yogurt, while yeast and some other bacteria convert the pyruvate into ethanol, which is a renewable fuel source.

As shown in the equation below, 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 produced.

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.

There are several enzymes whose purpose is to break 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. 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)

Preparation of Enzymes (Prepare before the start of the lab)

1. Mix 2.0 mL of amylase concentrate with 98 mL of distilled water to produce a 2% solution. Stir thoroughly.
2. Mix 2.0 mL of glucoamylase concentrate with 98 mL of distilled water to produce a 2% solution. Stir thoroughly.

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 kansascornstem.com) 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. Label 5 test tubes.
3. Add 1 g of ground corn to each sample test tube.
4. Add amylase solution to the test tubes prescribed in the procedure.
5. Add 5-10 ml of distilled water, making sure final levels are equal.
6. Mix the corn and water mixture with a stirring rod and add a test tube holder.
7. 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.)
8. 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.

Procedure for Lab

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.

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.

Teacher Resources

Additional Resources

• Purdue University How Fuel Ethanol is Made From Corn
  This downloadable PDF is a great resource for introductory reading prior to starting the fermentation lab.
• U.S. Department of Energy Alternative Fuels Data Center  Ethanol Production and Distribution Great diagram of the ethanol distribution system.
• Renewable Fuels Association How Ethanol is Made?
  Wet and dry mill processes of ethanol production are explained. Great accompanying graphics.
• Renewable Fuels Association Where is Ethanol Made? 
  This site shares the location of the 205, and counting, ethanol plants in the United States with a link that allows you to choose any plant in the nation and go to their specific website to learn more about them.
• U.S. Energy Information Administration U.S. Fuel Ethanol Production Capacity Increased by 3% in 2019 This resource allows for the analysis of ethanol capacity increases over the last 10 year.

Reflection and Conclusion

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.

Assessment

Fermenting Fuel Student Assessment KEY

Science and Agriculture Careers

To learn more about agriculture careers visit https://agexplorer.ffa.org/.

Disclaimer

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 https://static.nsta.org/pdfs/MinimumSafetyPracticesAndRegulations.pdf, 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.