Summary

In this guided-inquiry lab, students will design and test a procedure reacting waste cooking oil in a blue cheese slurry to create a substance that mimics songbird preen oil, which is both antibacterial and hydrophobic. Students will convert the fatty acids in waste oil to methyl ketones, thought to be the principal antibacterial component of preen oil, using the P. roqueforti mold found in blue cheese. Students will expand their knowledge of biomimicry, inherent properties of preen oil, and chemical synthesis by applying the principles of green chemistry. They will also assess their own process through higher-order problem solving and building on their scientific research skills.

Grade Level

High School

NGSS Alignment

This lab will help prepare your students to meet the performance expectations in the following standards:

• HS-PS2-6: Communicate scientific and technical information about why the molecular-level structure is important in the functioning of designed materials.
• HS-ETS1-1: Analyze a major global challenge to specify qualitative and quantitative criteria and constraints for solutions that account for societal needs and wants.
• Scientific and Engineering Practices:
  • Using Mathematics and Computational Thinking
  • Analyzing and Interpreting Data
  • Planning and Carrying Out Investigations
  • Obtaining, Evaluating, and Communicating Information

Objectives

By the end of this lab, students should be able to:

• Apply principles of green chemistry to laboratory techniques in the creation of a preen-oil-like substance.
• Develop a specific procedure to create a desired product and use guidelines to reach goals along the way.
• Calculate and report concentrations by volume percentages and weight/volume percentages.
• Analyze data to see if the desired result was reached.
• Critique the process and steps taken for future revision and improvement.

Chemistry Topics

This lesson supports students’ understanding of:

• Molecules & Bonding
• Organic Chemistry
• Functional Groups
• Mixtures
• Chemical Change
• Molecular Structure
• Green Chemistry
• Biomimicry

Time

Teacher Preparation: 35-40 minutes

Lesson: 90 minutes

Materials

Note: Necessary materials and quantities are broken down by experimental guideline for one lab group. Amounts are approximate, as students will choose their own value within a range.

A) Create a slurry for your waste cooking oil.

• Waste cooking oil (5 mL)
  • try to source from your school cafeteria or local restaurant
  • any vegetable oil will work
• Deionized/distilled water (~35 mL)
• Blue cheese (~5-7 g)
  • look for one that has P. roqueforti as an ingredient
  • this can be found at most grocery stores
• Sodium chloride (~1 g)
  • non-iodized table salt from a grocery store works well, lab-grade sodium chloride is not necessary
• Beaker, 100 mL
• Stir rod
• 10 mL graduated cylinder
• 50 mL graduated cylinder
• Weigh boats or weigh paper
  • if salt is measured first, the same one can be reused for the cheese
• Metal spatula
• Scale
  • a set of shared scales for the class will work

B) Let the oil mix vigorously in the slurry for a length of time.

• Stir plate
• Stir bar
  • if you do not have access to stir plates/stir bars, students can stir their slurry continuously with a stir rod or shake their slurry in a capped vial or centrifuge tube

C) Filter out just the oil; separate from any slurry solids and water.

• Pipette
• Coffee filters (1-3 needed)
• 50 mL beaker

D), E), F) Testing the preen oil properties.

• Deionized/distilled water (0.5 mL)
  • *Deionized water must be used in methyl ketone test
• 3 M NaOH (0.5 mL per group)
  • Note: A freshly made 3 M Na₂CO₃ (washing soda) solution can be substituted as a greener, and less hazardous, base in the iodoform test.
• 2% iodine/2.4% NaI solution (0.75 mL)
  • you can make your own, but these concentrations are found in commercially available antiseptic iodine solution
• 1 mL graduated plastic pipettes (3 needed)
• Test tube (3 needed)
  • test tubes must be large enough to hold ~5 mL (for example: 13 ´ 75 mm)
• Test tube rack (or beaker to hold the 3 test tubes)
• Tap water (2-3 mL)
  • used in testing for hydrophobicity
• 2% methylene blue solution in dropper bottles (2 drops)
• Parafilm cut in ~1” square
  • Used to cover top of one of the test tubes

Safety

• Always wear safety goggles when handling chemicals in the lab.
• Students should wash their hands thoroughly before leaving the lab.
• When students complete the lab, instruct them how to clean up their materials and dispose of any chemicals.
• Gloves should be made available for student use. Gloves must be worn by students while performing the iodoform test.
• Students with a topical allergy to iodine should not physically handle any materials that have been in contact with the iodine solution.
• Sodium Hydroxide (NaOH) can cause severe skin burns and eye damage. Do not breathe vapors, mist or sprays.
• Sodium Carbonate (Na₂CO₃) can cause skin corrosion/irritation.
• 2% iodine/2.4% NaI solution can cause skin and serious eye damage/corrosion.

Teacher Notes

• To learn even more about using this lab in your classroom, read the associated article, A Green Chemistry Guided-Inquiry Lab, published in the November 2020 issue of Chemistry Solutions.

Background:

• This laboratory activity is focused on the implementation of biomimicry via guided inquiry. This activity was developed from scratch, so there is no reference available.
• Useful links regarding the scientific background such as the cheese-making process, properties of P. Roqueforti, the location and role of the preen oil gland, and antibacterial properties of the preen oil can be found below:
  • Biomimicry:
    • What is Biomimicry?
    • Ask Nature
  • Penicillium roqueforti/Blue Cheese
    • University of Guelph: Food Science
  • Rabie, A.M. (1989). Acceleration of blue cheese ripening by cheese slurry and extracellular enzymes of Penicillium roqueforti. Lait, 69, 305-314. DOI: 10.1051/lait:1989424
  • New Brunswick Museum: Moulds—Penicillium
• Preen Oil/Uropygial Gland
  • The Beauty of Birds: The Bird’s Oil Gland
  • O’Reilly S, Summons R, Mayr G, Vinther J. 2017 Preservation of uropygial gland lipids in a 48-million-year-old bird. Proc. R. Soc. B 284: 20171050.

• This lab works best with groups of 3-4 students. If you choose, students can complete a group Pre-Lab Questions and Data Sheet, then individual Post-Lab Questions.
• To adapt for larger groups, more structure could be added to the activity by assigning each group a slurry ratio or length of reaction time. The results could be compiled and compared for the whole class to achieve a thorough data set.
• For smaller groups, the suggested slurry ratios could be withheld from students, who could experiment with amounts like in a more realistic, longer-term research project. This would demand at least 2 or 3 lab periods instead of a one time, 90-minute, lesson.
• The type of waste cooking oil or percent of P. roqueforti in the blue cheese could be varied per group and results compared among groups of students.
• The approximate time required for each section of the lab, as well as sample class schedules and how the lab was adapted for each is show below:

• Class Schedule Adaptations:

• Pre-Lab Tips: Use these tips as your students prepare to complete the lab work.
  • If time allows, or as a pre-lab homework assignment, have students write out a step-by-step procedure that they will then use to guide them through the hands-on experiment.
  • An example of how the materials and processes used are connected to the final properties and product is provided. This can be shared with students to help them make the necessary connections: Blue cheese and waste cooking oil are reacted to form methyl ketones which are acting as the antibacterial compounds in songbird preen oil.
• Suggested visuals to accompany pre-lab:
  • a bird collecting oil from its uropygial gland and preening (Section II-Student Handout)
  • mosquito proboscis vs. mosquito-like needle (Prelab Questions)
  • gecko feet vs. microscale gecko tape (Prelab Questions)

Image shows Blue Cheese Slurry on stir plate

• Procedure Tips: Use these tips to guide your students through the lab.
  • Creating a slurry: dissolve the salt in the water first, then add the cheese and oil
  • Filtering: let the slurry settle into an oil layer and a water layer, then decant or pipette to only remove the top/oil-containing layer; wring only the oil layer through a coffee filter until the resultant oil is translucent, this may take more than one coffee filtration (think cheese-making and separating curds from whey).
  • Students only need to recover enough filtered biomimetic preen oil to test; yield is not being calculated or judged. This means only ~6-10 drops are needed.
  • Stirring slurry: encourage students to use their “down time” to prepare for the next steps of the procedure and/or add to their data sheet.
  • Hydrophobic test: the easiest test is to add a couple drops of oil to a test tube, add an equivalent amount of water; if the oil and water separate into distinct layers, the oil is hydrophobic.

Image shows example of iodoform test results.

• Iodoform test: a yellow precipitate (iodoform) confirms methyl ketones are present; again, freshly made 3 M Na₂CO₃ (washing soda) can be substituted for the 3 M NaOH.
• Image shows an example of test results: Left test tube: successful iodoform test resulting in a pale-yellow precipitate, indicating methyl ketones are present. Right test tube: iodoform test performed on waste cooking oil feedstock; negative control, should not contain methyl ketones and did not produce a yellow precipitate.
• Methylene blue test: methylene blue data needs to be recorded over a period of time for results to be seen; if students record daily methylene blue data on a 1-10 scale, where 1 is clear/no blue and 10 is original deep blue, then the data can be plotted for further analysis.

Image shows samples of Day 5 of a methylene blue test.

• Samples on the Day 5 of a methylene blue test—note the range of blue colors. The far right test tube is still original deep blue, and therefore contains the most antibacterial biomimetic oil.
• Extension suggestions: Students can further expand their knowledge gained in this experiment and make interdisciplinary connections.
  • Write: What natural product or process would you biomimick? What properties are important to maintain?
  • Create: Design a product label for biomimicked preen oil that conveys its uses and properties to consumers.
  • Analyze: Try your experimental procedure again using a different type of feedstock waste oil. Compare and contrast your results.
  • Connect: Penicillins, a family of antibiotic medicines, are derived from the same family of mold as Penicillium roqueforti in blue cheese. Research a penicillin-based medicine and compare and contrast it to the properties and uses of P. roqueforti in preen oil creation.
• An Answer Key document, including expected results is available to download.
• Beyond Chemistry Class:
  • High school biology students could also benefit from this experiment with a heavier focus on the enzymatic functions of the P. roqueforti fungus.
  • The real-world concepts connecting across scientific disciplines and the easily accessible materials make this activity adaptable for a homeschool setting as well.