Introduction

This lab introduces students to fermentation – the biological process that produces ethanol fuel. Using yeast and sugar or corn syrup, students will model the production of bioethanol, observe CO₂ release, and connect the results to real-world biofuel systems. 

Student Objectives

Students will be able to

• Model ethanol fermentation by observing yeast converting sugars into CO₂ and ethanol.
• Collect and graph CO₂ production data from a single setup.
• Connect results to real-world bioethanol systems and their tradeoffs.

Background and Context

Ethanol is one of the most widely used biofuels in the world and is produced from plant-based sources. In Brazil, ethanol production relies heavily on sugarcane, which is particularly efficient because sugarcane contains high concentrations of sucrose that can be easily fermented. In contrast, the United States primarily uses corn as its main feedstock for ethanol production, since corn is widely grown and heavily subsidized. Beyond these first-generation biofuels, researchers are actively developing cellulosic biofuels that come from plant residues, grasses, and other non-food biomass. These so-called second-generation biofuels have the potential to reduce some of the concerns tied to traditional ethanol production by making use of materials that are not part of the human food chain.

The fundamental chemical process underlying ethanol production is fermentation, during which microorganisms such as yeast convert glucose (C₆H₁₂O₆) into ethanol (C₂H₅OH) and carbon dioxide (CO₂). The balanced chemical equation for this reaction is: 

C₆H₁₂O₆ → 2C₂H₅OH + 2CO₂.

In this process, a single molecule of glucose is broken down to form two molecules of ethanol and two molecules of carbon dioxide, releasing energy that the yeast uses to sustain its metabolism.

Despite its benefits as an energy source, the use of ethanol as a biofuel raises important social, environmental, and technological questions. One of the most significant issues is the “food vs. fuel” debate, which highlights the concern that growing crops for fuel might compete with growing food for human consumption, potentially raising food prices and impacting food security. Land use is another major issue, as expanding biofuel production may lead to deforestation, habitat fragmentation, biodiversity loss, and increased water and fertilizer demand. Moreover, through indirect land-use change, it may even undermine some of the greenhouse-gas benefits biofuels are intended to provide.

Materials

• Student Handout

Per group:

• 1 packet of dry yeast
  Note: One packet of dry yeast is equivalent to 2 tsp.
• Feedstocks: 2 tbsp of sugar OR 2 tbsp of corn syrup
• 1 cup of warm water: ~40°C or 100°F (1 cup per trial)
• 1 500 mL clear plastic bottle or glass beaker
• 1 balloon
• Funnel
• Stopwatch or timer
• Flexible measuring tape or string and ruler
• Safety goggles and gloves
• Graph paper or digital graphing tool

Optional for ethanol detection (teacher demonstration; recommended for safety)

• Iodoform test reagents (iodine solution, sodium hydroxide) in small amounts
• Heat source (warm water bath)

Procedure:

1. Students will follow the directions in the Student Handout to set up and conduct the experiment, recording observations and collecting data. They will also make a graph to display their results and answer conclusion questions to analyze and reflect on experiment results and make connections to real-world bioethanol production systems.
2. Optional Extension: Once students are completed with their tests, the teacher may perform an iodoform test on samples. 

• Add a few drops of iodine solution and sodium hydroxide to fermented liquid. 
• Heat gently in a water bath. 
• A yellow precipitate (iodoform) indicates ethanol presence. 

Assessment Rubric

Answer Key

The Student Guide contains the Fermentation and Biofuels Lab – Student questions.

Analysis Questions Answer Key

1. Answers will vary. (Example: The graph rose quickly at first, and then started to flatten. From 0-15 minutes, the slope was steep. After 15 minutes, it increased more slowly, showing a plateau as fermentation slowed.)
2. Answers will vary. (Sample response: First 15 minutes – from 5 cm to 11.5 cm = 6.5 cm over 15 minutes = 0.43 cm/min. Second 15 minutes – from 11.5 cm to 12.9 cm = 1.4 cm over 15 minutes = 0.09 cm/min. Fermentation is fastest early on and slows later as sugar is used up.)
3. Answers will vary. (Sample response: Final diameter 12.9 cm / 2 = Radius 6.45 cm. 4/3 x 3.14 x 6.45³ = approximately 1,124 cm³ (1.12 liters of gas).
4. Answers will vary. (Example: Our data was mostly consistent. Possible errors include: the balloon not being perfectly spherical, stretching the balloon while measuring, small leaks around the bottle neck, and reading diameter at slightly different angles each time.)
5. Answers will vary. (Sample response: Yes, I predicted 12 cm in 30 minutes, and we measured 12.9 cm. It wasn’t exact, but very close.)
6. Answers will vary. (Sample response: I would keep the yeast-sugar mixture at a steady warm temperature (around 40 degrees Celsius) by placing the bottle in a warm water bath. That should speed up the yeast activity from the start, and increase the rate of CO² production.)
7. Answers will vary. (Example: It’s the same core biology – yeast converts sugars to ethanol and CO₂. Industry just does it in large, controlled fermenters with strict temperature control, mixing, and sterile conditions, then distills the ethanol to high purity and handles the CO₂ byproduct.)
8. Answers will vary. (Example: Challenges include getting enough reliable feedstock, land and water use, maintaining sterile conditions at huge volumes, preventing contamination, supplying nutrients, energy-intensive distillation, and transporting materials and fuel, all while keeping costs and emissions low.)
9. Answers will vary. (Example: Benefits include domestically produced fuel, potential lower lifecycle CO₂ than gasoline, and rural economic benefits. Drawbacks include competition with food, land-use change, fertilizer and water demand, biodiversity impacts, and variable yields due to weather.
10. Answers will vary. (Example: It might be better because it uses non-food biomass, can grow on marginal land, and may cut lifecycle emissions. It is challenging because tough plant cell walls require pretreatment and costly enzymes, slower conversions, and sometimes lower, less predictable yields.)