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A Student's Experiment:

Lessons, Labs, and Lectures: The Biodegradation of Oil by Aerobic Bacteria

Jon Williams. Reprinted with permission from "Carolina Genes." North Carolina Biotechnology Center, Research Triangle Park, NC (Spring, 1994).

At the time this was written, Jon Williams was in the ninth grade at Enloe High School in Raleigh, North Carolina. As a seventh-grader, he presented an award-winning project demonstrating biodegradation of oil at the North Carolina Student Academy of Science competition. Jon had used inexpensive, readily available materials in a simple but effective procedure.

Introduction

Oil released into the environment is a well-recognized problem in today's world. Oil spills affect many species of plants and animals in the environment, as well as humans. The search for effective and efficient methods of oil removal from contaminated sites has intensified in recent years, in part due to the enormous publicity of the Exxon Valdez spill. One promising method that has been researched is the biological degradation of oil by bacteria. The bacteria metabolize the oil in much the same way humans convert food into energy. Like food, oil is a compound rich in carbon. The following experiment can be used to demonstrate that some types of bacteria can degrade oil. Also, other variables can be selected and incorporated into the lesson plan to allow additional experimentation.

Materials

  • eight 1-pint Mason jars
  • aquarium pumps and tubing
  • inorganic nutrients: ammonium phosphate, magnesium sulfate, potassium phosphate, and non-iodinated sodium chloride
  • lightweight machine oil (not motor oil; available at hardware stores)
  • pipettes that can deliver drops or reproducible small volumes
  • distilled water
  • brown paper bags
  • laboratory balance
  • soil sample (preferably collected from an oil-contaminated site); will contain soil bacteria

Procedure

  1. This experiment includes 4 treatments, each done in duplicate. Therefore, label the jars 1A, 1B, 2A, 2B, etc., up to 4B. The jars with the same numerical markings (for example 1A and 1B) will contain identical treatments. The jars should be clean.

  2. Put 150 ml distilled water and 2 grams of machine oil into each of the 8 jars.

  3. The first set of 2 jars (1A and 1B) will contain only distilled water and oil and will serve as controls. Set them aside.

  4. Add into the second set of jars the following mixture of inorganic nutrients: 0.25 g ammonium phosphate; 0.05 g magnesium sulfate; 0.25 g potassium phosphate; 1.25 g non-iodinated sodium chloride. The inorganic nutrients provide nitrogen and minerals to the organisms.

  5. Add into the third set of jars the soil sample. The best place to collect soil is from an area already contaminated with oil. That way it is likely that the surviving bacteria in the sample may be representative of oil-degrading species. One suggestion is to collect bacteria from where engine oil has repeatedly leaked on the ground for several years (service stations, dirt parking lots, etc.). If you don't have access to such a place, soil taken from an organically rich area may be substituted, since oil-degrading bacteria are generally present in most soils. Approximately 5 g of the soil from your chosen area should be added to each of the two jars for this treatment.

  6. Into the fourth and final set of two jars, add both the inorganic nutrients and the soil sample as described in steps 4 and 5.

  7. Cover the top of the jars loosely with inverted Petri dish halves in order to reduce evaporation of the water and oil in the treatments (aluminum foil can be substituted). The dish halves should have a hole drilled in them to allow a piece of tubing to reach into the water and bubble up air from a small aquarium pump. (If you use 4-way splitters, only 2 pumps will be needed for the 8 jars.)

  8. Results may be recorded from each jar every 3 days or weekly for up to 30 days. Take results by performing a "greasy spot" test. To perform this test, cut a brown paper bag into a 16 x 16-inch square. Then, with a ruler and pencil, divide the large square into eight 2 x 2-inch squares. In the corner of each small square, put the number of each different treatment jar (1A, 1B, etc.), one per square. This way, there should be one 2 x 2-inch square for each jar.

    Using a pipette or dropper, draw a small quantity of liquid from just under the top of the water level of one jar. Deposit three drops of this liquid onto the center of the correct square of paper (if the sample was from jar 1A, place the three drops on the square for 1A, etc.). Take samples from the same place in each jar each time (just under the surface of the water. After a few hours, the water will evaporate, leaving a greasy spot in each small square. Circle the circumference of each greasy spot with a pencil and measure and record the diameter of each spot. Average the results from jars containing identical treatments. During the course of the experiment, the spots from the treatments containing the bacteria and inorganic nutrients should be smaller since the oil is being degraded.

  9. Place your cumulative data into graphs and compare. What trends do you find, if any? Where did activity plateau? Why were the soil sample, inorganic nutrients, oxygen, etc. necessary for the oil concentration to be decreased?

Discussion

As previously stated, the soil sample provides bacteria to digest the oil. However, oil is composed only of hydrogen and carbon, and the bacteria need additional nutrients to grow. The inorganic nutrient mixture provides nitrogen and several essential minerals. The bacteria also require oxygen, provided by the air from the aquarium pumps.

Variations

Set up experiments to test:

  • whether air is required

  • the effect of temperature on the process (Be sure to use a fairly wide range of temperatures, such as from refrigerator temperature to above body temperature.)

  • the effect of using very different soil types


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