Materials Included In Kit

Additional Materials Required

Prelab Preparation

Pond Water

Pond water will produce the best results when conducting this lab. If a pond is nearby, simply collect 75 mL of pond water per group. You will need some type of container; a 2-gallon clean milk jug will work well.

If gathering pond water is not an option, making your own pond water is an excellent alternative. This pond water will be nutrient-rich and support the oil-eating bacteria during the cleaning process.

To prepare the pond water, follow the instructions below at least two days in advance.

1. Fill two beakers each with 1,000 mL of tap water and bring to a boil.
2. Remove from heat and allow the tap water to cool to room temperature.
3. While water is cooling, cut half of the timothy hay (about 10 grams) into small pieces, ½" long, and place into a 1,000-mL Erlenmeyer flask.
4. Repeat step 3 with remaining 10 g of timothy hay in a separate 1,000-mL Erlenmeyer flask.
5. Pour cooled, boiled water into each Erlenmeyer flask until the hay and water reach 1,000 mL (about 850 mL of water).
6. Loosely cover the Erlenmeyer flasks, allowing the mixture to “breathe.”
7. Allow the Erlenmeyer flasks to stand undisturbed for 2 days in a warm place, 60–90 °F. When a scum (thin film) forms on the surface of the water, proceed with the oil-eating bacteria preparation.
8. Strain the water to remove any hay pieces before distributing to student groups. The procedure should yield 1,500+ mL of pond water. Each group should receive approximately 75 mL.

Oil-Eating Bacteria

At least two hours prior to the laboratory work, soak the oil-eating bacteria powder in tap water. Mix 5 g of the bacteria powder mixture in 375 mL of lukewarm water. Stir the mixture occasionally during this incubation period. Dispense 25 mL of the active suspension for each lab group into a plastic medicine cup prior to the laboratory work.

Control Beach

On the day of the experiment, create a control beach for students to compare their results. Fill one chamber of partitioned Petri dish approximately half full with 20–25 g of sand. Place one drop of oil near the outer edge to observe absorption. Then add 3 mL of pond water with pipet to saturate beach material. Place 5 more drops of oil in the center of the sand. Repeat same procedure with fine gravel in chamber 2 and coarse gravel in chamber 3. Cover with lid and place in warm location. You may need to add pond water during the lab to retain moisture.

Safety Precautions

Although the oil-eating bacteria used in this laboratory are naturally occurring and non-pathogenic, care should be taken when handling the bacteria. Follow all normal laboratory safety procedures. Instruct your students not to touch their face or mouth with their hands when using the bacteria and be sure to wash their work area and hands thoroughly when lab work is completed. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Follow Flinn Suggested Disposal Method for Type I Biological Materials. This method involves autoclaving or chemical sterilization.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs. The activity will require about 30 minutes a class period to set up and about 10–15 minutes for follow-up observations each day over the duration of the experiment. The recommended length of time for the experiment is seven days.

• The bacterial culture provided is a mixture of microbes that contains over 140 billion bacteria cells per ounce in a bentonite clay carrier. The bentonite helps absorb oil into one place (as evidenced by the clumping in the experiment) and the microbes biodegrade (“eat”) the oil.
• Water and oxygen must be present for the microbes to flourish. Oxygen is a key variable since they are all aerobic bacteria.
• In addition, the microbes work best in a near neutral pH range and at warm temperatures up to 110 °F. Best results are noticed in plates that have these “ideal” conditions.
• The oil-eating bacteria slowly “eats” the oil so the total disappearance of the oil is slow. The oil will probably not be completely gone after one week and may be perceived as a failure by students. This is a key part of the lesson—real oil spills are hard to clean up and bioremediation takes a long time. The changes in appearance noted in the answers indicate what students are likely to observe. Observation and results will vary from group to group.
• Possible extensions to this activity might include using oil-absorbing powders on oil spills. The oil-eating bacteria can be exposed to different conditions (e.g., sunlight/dark, temperature, concentration, type of water) to see what affects the rate of bioremediation. Have students share observations with classmates, research oil spill control methods further and hold an oil-spill control seminar. Discuss the advantages and disadvantages of various methods for controlling an oil spill.

Sample Data

Part I. The Slick

Each day describe the condition of the water and the oil in each Petri dish. Consider textures, colors, consistency and turbidity. Draw sketches if helpful.

Part IIa. The Beach, Control

Part IIb. The Beach, Oil-Eating Bacteria

Answers to Questions

1. Why was the “OO” Petri dish used in Part I of this experiment? What purpose did it serve?
   

   The “OO” Petri dish served as a control (i.e., a basis for comparison to see if the oil-eating microbes change the slick in “OEB”).
   

2. Describe the sequence of changes that were shown in the “OEB” Petri dish in Part I. Assuming that these changes illustrate biodegradation, write a working definition of this term.
   

   The oil-eating bacteria mixture clumped the oil slick and the microbes started eating the oil as evidenced by the color changes and the disappearance of the slick. Biodegradation is the breakdown of a non-living substance by a living organism (e.g., oil by bacteria).
   

3. Which beach material seemed to “absorb” oil in a small concentrated area versus allowing it to spread quickly? What problem does absorption in a small area cause the oil-eating bacteria?
   

   The fine sand beach seemed to absorb the oil in one location, minimizing spreading. The problem associated with this type of absorption is the creation of an oxygen-starved environment and the oil-eating bacteria would have a difficult time surviving on this surface.
   

4. Which beach material was the easiest to bioremediate? Explain.
   

   The beach with the most exposed surface area, the coarse gravel beach, would allow the most oxygen to reach the bacteria maximizing the ingestion of oil and bioremediation.
   

5. What evidence would support the claim “the bacteria ingested the oil in the Petri dish”?
   

   Several pieces of evidence collected support the claim that the bacteria ingested the oil. For example, the globs of oil in the oil slick and beach settings only occurred in the experimental Petri dishes. Also, the color changed to show cloudiness and white film near the perimeter and between the pieces of gravel. Finally, over time, the oil around the perimeter became thinner, indicating the oil disappearing.
   

6. What factors might explain the difference in biodegradation on each beach? Remember that the oil-eating bacteria need oxygen as well as oil to thrive and grow.
   

   Each beach allowed biodegradation at some level. The fine sand had the least, most likely due to the lack of oxygen between the fine sand particles. The coarse gravel beach had a fair amount of degradation because of the exposed surface area allowing oxygen to reach the bacteria. The fine gravel also had a noticeable amount of bioremediation, again due to oxygen reaching the bacteria.
   

7. What other uses would oil-eating bacteria have besides cleaning up oil spill disasters? Who else might make use of oil-eating bacteria?
   

   Oil-eating bacteria solutions might be useful in cleaning oil off a garage floor. Service stations that change car oil would find the solution helpful. Any industry that ends up with oil-coated machinery might also find uses for the material.

Introduction

A single quart of oil can spread over an acre or more of water. Imagine what 100,000,000+ gallons from a deep water oil rig like the spill that occurred in 2010 in the Gulf of Mexico can do! Despite efforts to eliminate oil spills, the EPA reports thousands of oil spills each year. What methods are available to respond to an oil spill?

Concepts

• Oil-eating bacteria
• Biodegradation/bioremediation

Background

A large oil spill is one of the most dramatic and terrible environmental disasters. Newspaper headlines follow and heart-wrenching photographs of oil-soaked birds and marine mammals capture our attention and stick in our mind. The oil spreads rapidly and can cover a huge area very quickly. The oil slick may eventually extend hundreds of miles and cause huge environmental damage. Response to cleaning up a spill must be fast if its impact on the environment is to be minimized.

Many methods have been tried to clean up and reduce the impact of an oil spill. Usually an oil spill is crude oil (not refined) and contains volatile substances that have low boiling points. These low-boiling point substances tend to evaporate immediately, reducing the spill by 20–30%, but the toxic substances are released into the atmosphere. The remaining oil is thick and sticky and adheres to anything it touches—rocks, sand, marine life and birds.

One method that has been employed at an oil spill is to surround the oil slick with something called a containment boom. Basically, it is a large float that surrounds the slick so that it cannot spread further. As the boom is slowly pulled into a boat and made smaller, oil is skimmed off the top of the water and the slick slowly shrinks. Absorbent materials are used to clump or soak up the oil. Small oil slicks that consist primarily of highly flammable compounds may be set on fire to dispose of the oil. This is rarely done because most oil slicks are not composed of flammable material.

Another method used to clean up oil spills involves using detergent. When the oil slick is sprayed with a detergent solution, the oil breaks up into clumps which then sink to the bottom. Although these clumps can be hazardous, the problems of the clumps are often easier to deal with than the slick. Many other methods to clean up oil have also been tried. Recently, oil-eating bacteria have been “designed,” cultured and spread on oil-infested surfaces. Bacteria, a living organism, has requirements for survival. At a minimum they require water, oxygen and food. The water and oxygen are obtained from the environment where the oil spill has occurred. Water for the bacteria is needed for chemical reactions to occur. One such reaction is respiration, oxygen taken in from the atmosphere allows a series of reactions to break down carbon-based molecules and release energy. Heterotrophic bacteria consume carbon-based material from the environment, which in the case of an oil spill, would be the oil! The bacteria use oxygen and water to break down the oil into carbon dioxide, water and soluble fatty acids that serve as food fish. As the bacteria reproduce, they eat more and more oil continuing to diminish the slick. When the slick is gone, their food source is gone and the bacteria die, leaving literally nothing behind.

Materials

Safety Precautions

Although the oil-eating bacteria are naturally occurring and non-pathogenic, care should be taken when handling the bacteria. Follow all normal laboratory safety procedures. Do not touch your face or mouth with your hands when using the bacteria and be sure to thoroughly wash your hands and lab bench when lab work is completed. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron.

Procedure

Part I. The Slick

1. Label two Petri dishes with your name and class period. Label one Petri dish “OO” (oil only) and the other “OEB” (oil-eating bacteria). Fill each Petri dish with 30 mL of pond water.
2. Using a pipet, add 3 drops of refined motor oil slowly to the top of the water in each Petri dish to create a mini-oil slick on each.
3. Look carefully at the “oil slick” in each dish and describe the starting condition of the oil and water in the “OO” dish on the Part I Oil-Eating Bacteria Worksheet.
4. Stir the oil-eating bacteria suspension if it has settled. Using a pipet, add 5 mL of the oil-eating bacteria suspension to the Petri dish labeled “OEB.”
5. After adding the bacteria, observe the dish carefully and describe its appearance on the Oil-Eating Bacteria Worksheet, Part I.
6. Place the dishes, as directed by your instructor, where they can sit undisturbed in a warm location for the duration of the experiment.
7. Record daily observations of both dishes over the next week in Part I of the Oil-Eating Bacteria Worksheet. Record the word “weekend” in the observation space provided for Saturday and Sunday.

Parts IIa and b. The Beaches

1. Label the partitioned Petri dish with your name and class period. Create mini-beaches in the three chambers of the Petri dish. Use fine sand in chamber 1, fine gravel in 2 and coarse gravel in 3. Fill each chamber half full of test beach material.
2. Using a pipet, carefully add a drop of oil to each chamber near the outer edge of the Petri dish so that it can be observed through the side of the Petri dish. Which “beach” absorbs the oil the best?
3. Add 3 mL of pond water to each chamber.
4. Add 5 more drops of oil to the center of each beach.
5. Stir the oil-eating bacteria suspension if it has settled. Using a pipet, add 5 mL of the oil-eating bacteria suspension to each of the mini-beaches. Add the bacteria suspension drop by drop over each test beach.
6. Look carefully at the “oil slick” on each beach and describe the starting condition of each beach in Part IIb of the Oil-Eating Bacteria Worksheet.
7. Place the cover on the Petri dish and store it in a warm spot in the laboratory where it can sit undisturbed for the next week. Your instructor will identify the location.
8. Record daily observation of your instructor's beach (control) over the next week in Part IIa of the Oil-Eating Bacteria worksheet. Record the word “weekend” in the observation space provided for Saturday and Sunday.
9. Record daily observations of your beach over the next week in Part IIb of the Oil-Eating Bacteria Worksheet. Record the word “weekend” in the observation space provided for Saturday and Sunday.
10. Disinfect work areas as directed by your instructor and wash your hands thoroughly before leaving the laboratory each day.
11. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10451_Student1.pdf