This lesson explains the processes of cellular respiration and fermentation and how it applies to the production and processing of honey. Grades 9-12

adenosine triphosphate (ATP): a compound that has three phosphate groups and is used by cells to store energy

carbohydrate: an organic compound that is the main source of energy for the body; composed of carbon, oxygen, and hydrogen atoms

cellular respiration: process by which cells release energy from glucose and convert it to ATP

fermentation: a food-based reaction caused by the action of enzymes that breaks compounds into simpler substances; used for food preservation and preparation

invertase: an enzyme produced by yeast that catalyzes the hydrolysis of sucrose, forming invert sugar

Prior to this lesson high school students should have some background knowledge of the processes of cellular respiration and fermentation. They should also know that carbohydrates give living organisms the energy to carry out their daily activities. This lesson plan will build on students’ existing knowledge. 

Key STEM Ideas

All living organisms rely on cellular respiration and/or fermentation to transform glucose into usable energy (ATP). Living organisms often compete with one another for the same glucose source. 

Cellular respiration requires the presence of oxygen and yields more usable energy while fermentation occurs in the absence of oxygen and produces much less usable energy.

The byproducts of fermentation (including alcohol, carbon dioxide, and lactic acid) can be beneficial or harmful depending on the intended or unintended nature of the fermentation.

Connections to Agriculture

Honey bees are valuable managed pollinators of many agricultural crops and produce honey, an energy-rich food source and natural sweetener. Bees gather nectar from flowering plants and return it to their hive where they add enzymes from their honey stomachs. The added enzyme, invertase, breaks down sucrose, a disaccharide into its component monosaccharides, glucose and fructose. Glucose will be used by the bee in the cellular respiration process to produce ATP for energy. The bee places the honey mixture in the honeycomb and fans the mixture with their wings. This process helps to dehydrate (remove water) the mixture to ideally contain 16-18.5% moisture. The bee then caps the cell and the honey is effectively stored inside.

If the bees are unable to remove the excess moisture from the honey and the moisture content is greater than 18.6%, fermentation is more likely to occur. Fermentation is the process of changing carbohydrates to ethanol and carbon dioxide, the same process used in brewing alcoholic beverages. If the moisture content of the honey is too high or if cold weather sets in, bees will not cap the honeycomb cells. The cells will then sit open and will likely eventually ferment. 

^{Honeycomb showing capped and uncapped cells. Source: National Agriculture in the Classroom}

Producing a high quality honey crop requires that beekeepers protect it from fermentation. Beekeepers can reduce the moisture content of honey by placing it in a ‘hot room’ and/or with a dehydrator to decrease the water content.

When the moisture content is too high, uncontrolled fermentation can take place either in the hive or after bottling the harvested honey. This can add unwanted byproducts to the honey ruining its flavor and making it inedible as honey. If honey starts to ferment in the hive, the honeycomb cells are filled with bubbles and an odor of yeast can be smelled. Sometimes foam oozes out and collects under the frames. In a controlled circumstance fermentation of honey can be purposefully utilized to produce an alcoholic drink known as mead.

Protecting honey from fermentation is important to both humans and bees. As a food source, fermented honey is not valuable to humans for culinary use. Fermented honey can also be detrimental to the health of bees. Bees instinctively produce honey in the summer months to be a stored food source for the winters. Honey with high levels of alcohol can be poisonous to bees.

In a general sense, controlled fermentation of glucose can be used to produce products such as bread, yogurt, and biofuels such as ethanol. Understanding the process of fermentation along with its correct application allows us to protect important bee pollinators and produce more of the quality food and fuel the world demands.

1. Display the following pictures for your students to see. Instruct students to compare the pictures and ask the following questions:
   • What are these? (honeycomb from a beehive)
   • What do you notice that is different in these two pictures? (The picture on the right has bubbles in it)
   • What are some reasons that could cause the honey in this comb to bubble? (allow students to offer ideas)
   • Think about the scientific processes you are aware of. What creates bubbles? (fermentation)
   • What do you think could cause this honey to ferment? 

Image Sources:

• Left: Honey in Comb. Source: National Agriculture in the Classroom
• Right: Fermented Honey in Comb: Randy Burlew: http://honeybeesuite.com

Activity 1: How does the concentration of sugar affect yeast's ability to consume sugar and produce CO₂ as a waste product?

Preparation: Depending on time availability, you may want to have sugar solutions and yeast suspension ready prior to the lab. Mixing instructions are included on page 3 of the attached Teacher KEY. Prepare respirometers ahead of time, or show students how to assemble their own respirometers. Designate where used sugar and yeast solutions are to be disposed in your classroom.

1. Give each student 1 copy of the Fermentation of Honey student handout. and review the processes of cellular respiration and fermentation with students from pages 1-2.
2. Divide students up into groups of 3-4. Give each student 1 copy of the Fermentation of Honey student handout.
3. Go through the lab activity procedures found below as well as on their Fermentation of Honey student worksheet (page 3):
   1. Gather the materials needed for this lab.
   2. Measure out 10 ml of the 1% sugar solution and place the solution into a 50 ml beaker.
   3. Measure out 10 ml of the yeast solution and add it to the 50 ml beaker with the sugar solution.
   4. Allow the yeast and sugar mixture to incubate for 5 minutes occasionally swirling the beaker.
   5. Repeat the procedure with the other concentrations of sugar.
   6. Draw 3 ml of the yeast and sugar mixture into the syringe.
   7. Continue drawing the syringe until it has 1 ml of air on top of the sugar-yeast mixture.
   8. Add a drop of water into the bottom of the pipette and attach the pipette to the top of the syringe with plastic tubing. Stand the respirometer upright.
   9. Begin timing when the drop of water reaches 0 on the graduated pipette.
   10. Record the amount of CO₂ produced every 2 minutes in the data table.
   11. Repeat with the other concentrations of sugar.
4. After students record their observations on the data collection table on page 3 of the handout, take some time to share results and discuss any differences in results between lab groups.
5. Allow time for students to answer the three questions in "Part 1" of their handout.
6. Facilitate discussion of answers to the "Part 1" questions and what students observed during the lab activity. Possible questions include:
   • What made the water droplet move up the pipet? (Creation of C0₂ was created as a byproduct of fermentation of glucose by the yeast. This increased the pressure in the pipette and pushed the water up.)
   • What gas was formed during fermentation? (Carbon dioxide)
   • Does a higher sugar concentration necessarily mean more energy can be produced by yeast? (Student answers may vary, but their data should indicate that more sugar isn’t necessarily better for fermentation.)
   • How is measuring the production of CO₂ a measure of fermentation and glucose metabolism? (We know this is a byproduct of respiration and should be observable by the presence of bubbles or moving the water droplet up the pipette.)

Activity 2: Predicting Fermentation in Honey

1. Discuss as a class the process bees go through to ripen nectar into honey using the diagram provided in the Fermentation of Honey student handout.
2. Divide students into their lab groups once again. Using their data from "Part 1," have them graph their data and answer the three questions in "Part 2" where they will predict if fermentation will occur in fully ripened honey.
3. Bring class back together to discuss the answers to "Part 2" of the handout. Share the graphed data as a class and have a discussion about replication.
   • Did all groups collect and record similar data?
   • What are some potential sources of variation or error?
4. For "Part 3" of the handout, have students work on their own to problem-solve the four questions on the worksheet about a beekeeper protecting honey from unwanted fermentation. This section can be used as an assessment of individual understanding or simply by conducting a class discussion of answers to "Part 3."

After conducting these activities, review and summarize the following key concepts:

• Uncontrolled fermentation in honey by wild yeast can result in an unpalatable food product for people, loss of market value for the beekeeper, and an unusable food source for bees.
• If bees eat too much fermented honey the alcohol can be poisonous to them resulting in bee loss and as well as their pollination abilities for our food crops.
• Beekeepers can prevent fermentation from occurring by lowering the water content to below 18.6% with a dehydrator or storing honey in a heated room to promote evaporation of excess moisture.

Sources:

1. http://www.benefits-of-honey.com/honey-bee-facts.html
2. http://positivemed.com/2013/07/31/10-interesting-fun-facts-about-honey-and-honey-bees/
3. http://mentalfloss.com/article/68528/15-honey-facts-worth-buzzing-about
4. Crane, Eva, "A Book of Honey."
5. Simple respirometer image and basic respiration measurement procedures courtesy of http://www.cur.org/assets/1/7/ystferm.pdf

Author Affiliations:

• Burke Morrow: Lincoln East High School, Lincoln, NE
• Erin Ingram: University of Nebraska-Lincoln, IANR Science Literacy Initiative, National Center for Agricultural Literacy

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