List of experiments

 

  

 

 

Bioremediation - Sheen Screen
Environmental Microbiology
Experiments for middle and high school

Christine L. Case
Skyline College

 

Objectives  
  1. Define bioremediation.
  2. Demonstrate enrichment of oil-degrading bacteria.

Background  

Although many bacteria have dietary requirements similar to ours-that's why they cause food spoilage-others metabolize substances which are toxic to many plants and animals-heavy metals, sulfur, petroleum, and even PCBs and mercury. The use of bacteria to eliminate pollutants is called bioremediation. Unlike some forms of environmental clean-up, in which dangerous substances are removed from one place only to be dumped in another, bacterial clean-up eliminates the toxic substance, and often returns a harmless or useful substance to the environment.

In this exercise we will investigate bacterial degradation of hydrocarbons. A hydrocarbon is a compound that contains only carbon and hydrogen atoms. The carbon chains can be straight, branched, or rings. One of the most promising successes occurred on an Alaskan beach following the 1989 Valdez oil spill. Several naturally-occurring bacteria in the genus Pseudomonas are able to degrade oil for their carbon and energy requirements. In the presence of air, they remove two carbons at a time, in a process called beta-oxidation, from a large petroleum molecule. These bacteria degrade the oil too slowly to be helpful cleaning up an oil spill. However, scientists hit upon a very simple way to speed them up. They dumped nitrogen and phosphorous plant fertilizers onto the beach (a process called bioaugmentation). The number of oil-degrading bacteria increased compared to unfertilized control beaches, and the test beach is now free of oil.

In this exercise, we will enrich for hydrocarbon-degrading bacteria in various samples using the sheen screen method developed by Ed Brown in Valdez, Alaska. Degradation of the hydrocarbon should result in the opaque emulsified oil becoming small, soluble molecules such as fatty acids and acetyl groups.

 

 

 

Materials  

Small, wide-mouth jars (4)

Refined oil

1-mL pipette (1)

Plant fertilizer containing nitrogen and phosphorous

Soil. You might compare different soils including one from an area contaminated with oil.

Procedure
Part A: An oil spill

 

 

 

  

1. Label two jars:

#1: Soil

#2: Uninoculated

2. Add approximately 5 mL distilled water to each jar. Add 4-5 drops of oil to each jar. Carefully record the general appearance, color, and texture of the oil.

3. Inoculate jar #1 with 0.5 g of soil. Do not inoculate jar #2. What is the purpose of jar #2?

4. Screw the lid on each jar and invert several time to mix.

5. Incubate the jars with the lids loosened a half-turn at 30°C.

6. Invert the jars once or twice daily to increase the dissolved oxygen content in the water and to mix the microbes with the oil.

7. Observe jars every 24 hr for 3-4 days. Make careful observations of the general appearance, color, texture, and turbidity of the water.

Procedure
Part B: Bioaugmentation

 

 

 

  

1. Label two jars:

#1: Soil

#2: Uninoculated

2. Add approximately 5 mL distilled water to each jar. Add 15-20 drops of oil to each jar. Carefully record the general appearance, color, and texture of the oil.

3. Add a pinch of fertilizer over the entire oil layer in each jar.

4. Inoculate jar #1 with 0.5 g of soil. Do not inoculate jar #2. What is the purpose of jar #2?

5. Screw the lid on each jar and invert several time to mix.

6. Incubate the jars with the lids loosened a half-turn at 30°C.

7. Invert the jars once or twice daily to increase the dissolved oxygen content in the water and to mix the microbes with the oil.

8. Observe jars every 24 hr for 3-4 days. Make careful observations of the general appearance, color, texture, and turbidity of the water.

Questions  
  1. How can you tell whether the oil was degraded?
  2. What are the limiting factors in Part A?
  3. Show beta-oxidation of this hydrocarbon.
  4. Here are the formulas of two detergents that have been manufactured: Which of these would be readily degraded by bacteria? Why?
  5. How could the oil-degrading bacteria you isolated be used to clean-up an oil spill?

Additional Activities

 

 

  
  1. Use sand and gravel in a Petri plate or shallow pan instead of a liquid culture to simulate an oil spill. Test the rates of degradation with (a) no inoculum; (b) a soil inoculum; (c) soil and fertilizer.
  2. Change the hydrocarbon to compare rates of degradation.
  3. The jars labeled #2 are enrichments for hydrocarbon-degrading bacteria. Use samples from these jars as inocula for the experiments on Bioremediation of Gasoline and Bioremediation of Oils.


 

 

Qu. 3. Show beta-oxidation of this hydrocarbon.

 

 

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Qu. 4. Which of these would be readily degraded by bacteria?

 

 

 

 

 

 

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