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Ethanol Fuel

DDG Nutrient Testing

Grade Level: High School

Corn is a vital part of our society. It has many uses, such as feeding people and producing ethanol. This lab can be used in two different ways. It can be used as a stand-alone lab if you do not have the time or resources to conduct a full ethanol distillation lab. However, you can also use this lab to enhance the Ethanol- Corn Mash and Distillation and to observe the different levels of nutrient availability before fermentation, after fermentation, and again after distillation. We will focus on what is happening to the nutrients, along with discussing macromolecules and the importance they provide to organisms.

The first part of the lab is creating a corn mash that will ferment and form methanol and ethanol. Discussions about ethanol can begin here. The main focus will be on what the yeast is doing to the corn, as well as the purpose of the enzymes that are present in the corn mash. The next part of the lab will be to compare the nutrient availability with a corn slurry (this can be made at the start of the second lab day), and the corn mash. This lab focuses on the liquid feed. Lugol’s Iodine provides a black color change when present in starch. The Buiret Test Solution provides a purple color change when present in protein. The last indicator provides an orangish-red color change when heated and present with glucose. These changes provide an important laboratory practice in observations.

Teaching the Lesson

Kansas College and Career Ready Standards


Students who demonstrate understanding can

  • HS-LS1-6. Construct and revise an explanation based on evidence for how carbon, hydrogen, and oxygen from sugar molecules may combine with other elements to form amino acids and/or other large carbon-based molecules.
  • HS-ETS1-3. Evaluate a solution to a complex real-world problem based on prioritized criteria and trade-offs that account for a range of constraints, including cost, safety, reliability, and aesthetics as well as possible social, cultural, and environmental impacts.


  • W.9-10.1 Write arguments to support claims in an analysis of substantive topics or texts, using valid reasoning and relevant and sufficient evidence.
  • W.9-10.2 Write informative/ explanatory texts to examine a topic and convey ideas, concepts, and information through the selection, organization, and analysis of relevant content.
  • W.9-10.4 Produce clear and coherent writing in which the development, organization, and style are appropriate to task, purpose, and audience

Learning Objectives

  • Students will understand reactions and how indicators can be used to identify materials in a solution.
  • Students will understand the hydrocarbon structure of the nutrients found in the solutions.
  • Students will gain a better understanding of laboratory procedures.
  • Students will understand the use of macromolecules in growth and development of organisms.


For Preparation of Corn Mash:

  • Preparation of Enzymes
  • 2, 500ml beakers
  • 500 ml distilled water
  • Liquid Amylase Enzyme Concentrate
  • Liquid Glucoamylase Enzyme Concentrate

Preparing the Mash

  • Hot plate
  • Beakers (100, 500, 600 ml)
  • Graduated cylinders (1, 1000 ml)
  • Digital thermometer
  • Balance
  • Pipettes
  • Distilled water
  • 50g Ground Corn
  • Buffer Solution pH 5
  • Prepared yeast solution
  • Prepared Amylase Solution
  • Prepared Gluco-Amylase solution
  • Yeast

For Workstations

Safety Considerations

All glassware has a possibility of breaking. Protective eyewear should be worn at all times. The indicators being used can cause skin irritations and have the possibility of staining clothes.

Biuret Reagent, Lugol’s Solution, and Benedict’s Solution are all possible skin irritants. Please review all Safety Data Sheets for reagents used in lab for further safety information.

Procedures for Instruction

Note: The following procedures are what you will use if you are doing this as a stand-alone lab. You will need to make up the mash and slurry prior to starting the lab. You also have the option to perform this lab as part of the sequence in ethanol distillation. Kansas Corn: Ethanol – Corn Mash and Distillation (available online at is the recommended unit to perform first in the sequence. The DDG Nutrient Test can be done before and after fermentation, as well as after distillation is complete, to compare the levels of nutrients in the different stages of production.

Length of Time for Preparation: 1 day

  • Day 1: Set up workstations for lab

Length of Time for Classroom Teaching: 1-2 days classroom work

  • Day 1: Prepare corn mash for fermentation, students can fill in the Introduction, Question, Hypothesis, and Procedure on Student Lab Report Sheet.
  • Day 2: Test nutrient availability of liquid

Preparation Procedure/Instruction

Preparation of yeast solution (1-2 hours before the lab)

  1. Vigorously shake your distilled water to oxygenate it fully.
  2. Measure and mix together 300 ml of distilled water and 0.6 g of glucose and add it to a 500 ml beaker to make a 2% glucose solution.
  3. Measure 7 grams of dried fresh yeast and add it to your 2% glucose solution. Gently stir and cover beaker with a watch glass or plastic wrap.
  4. Allow your 2% glucose/yeast solution to culture for 2-4 hours. This will “wake up” your yeast, feed them, and start them metabolizing.

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.

Prescribed preparation of corn mash

  1. Add about 500 ml water to a 1 liter beaker and heat to boiling on a hot plate.
  2. Weigh out 50 g of ground corn. Add ground corn to the 500 ml Erlenmeyer flask and stir.
  3. Add 10.0 ml of amylase solution.
  4. Add 200 ml distilled water to the ground corn and stir.
  5. Use a ring stand and utility clamp to insert the flask into the boiling water as shown in the image below.
  6. Boil for 10 minutes, the temperature in the flask will be 90-95°C
  7. After boiling is completed, remove the flask from the hot water bath and allow it to cool to 50°C or below.
  8. Stir the resulting mixture and add it to the cooled corn mash. Stir the mixture occasionally with a stirring rod throughout the next 10 minutes.
  9. At the end of the 10-minute period, measure 20 ml of the pH 5 buffer. Shake the buffer solution and add it to the corn mash to maintain a slightly acidic pH.
  10. Shake the glucoamylase solution, then measure 5 ml of glucoamylase solution. Add it to the corn mash.
  11. Add 5.0 g of yeast to the corn mash and stir the entire mixture well.

Corn Slurry Preparation and Workstation Set-up.

  • You have the option to make the corn slurry ahead of time, or you can let the students make the slurry in class. For one group, grind 5 g of corn and mix it with 20 ml of distilled water
  • Filter the slurry, keeping the liquid food sample.
  • For each work station:
    • Provide each group with a small beaker of 5-10 ml of the corn slurry and a separate beaker with 5-10 ml of the fermented corn mash.
    • Provide each group with 2 separate pipets.
  • The indicators can be set out to be used when needed, or about 3 ml of each can be provided for each group.
  • Each group will need 6 test tubes and a test tube rack.

Background Information

In the United States, commercial production of fuel ethanol involves breaking down the starch present in corn into simple sugars, also called “glucose”. These sugars are than fed to yeast, which begins the process of fermentation. The main product is used as ethanol – it is primarily found as a fuel additive in the gasoline used in our vehicles. The ethanol industry utilizes many different parts of the production process. Some of the other products produced include animal feed, corn oil, and carbon dioxide. The product used in animal feed is known as distillers grains. There are two types of these grains – wet distillers grains and dry distillers grains. Wet distillers grains have a high water content and low shelf life. This results in transportation to local farmers near the ethanol plant for use in feed for livestock. Dry distillers grains have been dried to have a low water content. They have a longer shelf life and can provide feed for farmers at locations further away.


Classroom Discussion

Introduce the topic and assess students for prior understanding. Let students discuss their ideas, and guide the discussion without telling them if they are right or wrong.

Making Fermented Corn Mash and Forming Predictions:

  • Our job is to try and figure out what product will provide us with the highest nutrient availability. We will be comparing a corn slurry and a corn mash after fermentation.
    • What is fermentation?
    • What role does the yeast play in the fermentation process?
    • What is the purpose of the enzymes?
    • Which product do you believe will have more nutrients? Why do you think this?
    • What purposes does the fermented corn mash provide? What can be made?
    • What are some of the products produced?
  • As students are finishing the questions above allow them time to review the procedure. They can begin filling in the DDG Lab Report Sheet.
    • They should start with the introduction. This should give examples of the lab to be conducted, provide objectives of the lab, and should discuss the importance of the lab.
    • The next step will be developing a question for them to answer.
      • In our lab the questions to be answered is:
        • Does fermenting corn provide a higher nutrient availability?
      • Some possible questions for them to find as a secondary lab to this one:
        • Does the type of yeast affect the nutrient availability?
        • Does the amount of yeast affect the nutrient availability?
        • Does the type of enzyme affect the nutrient availability?
    • Students should develop a hypothesis as to what they think the outcome will be.
    • Have students write in the procedure for the lab that they will be conducting the next day.

Procedure for Lab

Carbohydrate Indicator Test (Glucose)

  1. Label 1 test tube Corn Slurry and a 2nd test tube Corn Mash
  2. Add 2 ml of corn slurry to a test tube and mix 2 ml of Benedict’s Solution.
  3. Cover with Parafilm and mix.
  4. Place test tube in a boiling water bath for 2 minutes.
  5. Record color change in the table on the DDG Student Lab Report Sheet
  6. Repeat process in a new test tube with the fermented corn mash.

Complex Carbohydrate Indicator Test (Starch)

  1. Label 1 test tube Corn Slurry and a 2nd test tube Corn Mash
  2. Add 1 ml of corn slurry in a test tube and add 1 drop of the Lugol’s Solution.
  3. Cover with Parafilm and mix.
  4. Do not heat solution.
  5. Record color change in table on the DDG Student Lab Report Sheet
  6. Repeat process with the corn mash.

Protein Indicator Test (Do not heat; heating will cause the proteins to breakdown, resulting in a negative test).

  1. Label 1 test tube Corn Slurry and a 2nd test tube Corn Mash
  2. Add 1 ml of corn slurry and add 2 ml of Biuret Reagent to a test tube.
  3. Cover with Parafilm and gently mix.
  4. Wait 30 seconds for a color change.
  5. Record color change in the table on the DDG Student Lab Report Sheet
  6. Repeat process with the fermented corn mash.

Teacher Resources

Nutrient Test Results

Benedict’s Solution will detect the presence of glucose, and it will provide an orange to red precipitate (form a solid) after heating. The Lugol’s Solution (Iodine) will turn black when starch is present. The Buiret Reagent will turn purple when protein is present.

Additional Resources

Lab Analysis

Have the students answer the following questions on the DDG Student Lab Report Sheet:

  • Were your predictions correct?
    • Students will look back at their pre-lab predictions and use the data collected from the lab to show if they were correct or not.
  • What do the results tell you about what happened during the fermentation process?
    • They should be making comparisons of the results from the different stages of production. If used as a stand-alone lab, they will have two comparisons – corn slurry and then after fermentation.
  • Where are the nutrients located in the slurry?
    • At this stage, you may want to have students test this by taking samples from varying locations in the slurry and performing the lab again.
    • This can be done in groups. Each group can compare the results they obtain with the results of other groups to help them determine the answer.
  • Where are the nutrients located after fermentation?
    • At this stage, you may want to have students test this by taking samples from varying locations in the slurry and performing the lab again.
    • This can be done in groups and each group can compare the results they obtain with the results of other groups to help them determine the answer.
  • Why would this not be the last step in identifying nutrient availability?
    • What are some other options we have? (Note: You can suggest possible research topics for students.)
    • This can be a good place to research and discuss what happens in ethanol plants and why they provide distillers grains.
  • This point can also be used to describe the varying sizes and shapes of the molecules present. They can draw and label glucose, as well as make a short chain of starch and a short protein. After they complete this task, they can review why these nutrients are important and describe the role these nutrients have in growth and development of livestock.

Reflection and Conclusion

Students should have made observations about the results they found in the test. Students should be able to identify where these distillers grains are produced and the importance it has for the ethanol industry. The following include some sample questions to pose for the students after the tests are completed:

  1. What does the results tell us about the availability of nutrients before fermentation? After fermentation?
  2. Which product would be better to use as a food source? Why?
  3. Is there a nutrient missing that is needed for life functions?
  4. Why would an ethanol plant want to produce distillers grains?

Students should understand that the production of ethanol not only makes ethanol that we use in gasoline, but it also provides farmers with resources to help cattle and other livestock grow by using distillers grains as a food source.

Science and Agriculture Careers

To wrap-up this topic, students can research some of the following careers. Students can find out what role each of the careers plays in studying nutrients and their functions in agriculture. Students can complete this part of the write up on the DDG Student Lab Report Sheet.

Agricultural Inspector
Agricultural Specialist
Food Specialist
Soil and Plant Scientist

To learn more about agriculture careers visit You can also find career profiles at Careers in Corn.



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.

About Kansas Corn STEM

Investing in Kansas teachers and students is a priority for the Kansas Corn Commission. We are committed to providing materials and training to support STEM education while fostering an understanding of how corn farming and agriculture fit into our daily lives. Professional development workshops are offered to teachers seeking to expand their knowledge and inquiry-based teaching skills. Workshop participants receive free lab supplies needed for the lessons.

Workshop Info

This lesson is the work product of the Kansas Corn Commission. Our lessons are written in collaboration with Kansas teachers for use in the classroom. Teachers may copy and share this curriculum. Use of this product for commercial or promotional use is prohibited without express permission of Kansas Corn.

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