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Oil Spill in a Test Tube

By Judy Brown



Abstract

The purpose of this activity is to develop a test tube model of an oil spill in order to experiment with conditions needed for bioremediation.

During each phase of this project, students have the opportunity to experience the work of science research. Lab teams of three to four students develop flow charts of their procedures before moving to the laboratory to work.

Teams are required to keep accurate and detailed lab logs of their work. Each team is given minimal media, RAG-1 streak plates and new and used motor oil. They first follow an established protocol to establish a test tube model and then are challenged to design an experiment to enhance the oil degrading properties of marine bacteria.

Students learn to apply the principals of experimental design by analyzing a research article on the EXXON Valdez bioremediation efforts and developing and using positive and negative controls. Specrophotometric measurement techniques are used to quantify data. The Student's t-test is used to determine if the data are statistically significant. At the conclusion of this activity, students develop seminar presentations and defend their research design and findings.


Background

A large oil spill is one of the most dramatic and terrible environmental disasters. Heart wrenching photographs of oil soaked birds and marine mammals capture our attention and concern. Oil spills have a predictable progression. Usually the oil spill is crude oil which contains volatile substances that have low boiling points. These substances evaporate immediately, reducing the spill by 25% but releasing toxic substances into the atmosphere. The remaining oil is very thick and sticky and adheres to anything it touches including rocks, sand and marine life. Some of this oil is degraded by marine bacteria which degrades the hydrocarbon in oil for use as a carbon source.

Scientists have been experimenting with the use of oil degrading marine bacteria in oil spill clean ups. Marine bacteria which naturally degrade oil grow much more slowly than other bacteria strains. E. coli bacteria used in biotechnology labs divides every twenty minutes and is a vigorous and hardy species. If the gene which enables marine species to degrade oil can be isolated and inserted into E. coli bacteria, the ability to use bioremediation to clean up oil spills might be enhanced.

Acinetobacter calcoaceticus RAG-1 is a marine bacteria which can utilize the hydrocarbons in oil as a source of carbon. When these bacteria are grown in a carbon minimal medium, they will breakdown hydrocarbons. RAG-1 releases an emulsan - a polysaccharide that will emulsify oil. Emulsan accumulates on RAG 1 cell surfaces as minicapsules and is released into the media as an active emulsifier as the cell growth approaches the stationary phase. The absence of a carbon nutrient source accelerates the release of emulsans.


Purpose

To develop a test tube model of an oil spill in order to experiment with conditions needed for bioremediation.

Materials

  • two sterile 10 ml test tubes containing 3 ml carbon deficient media
  • 2 snap covers for tubes - size 13
  • 1 agar plate of RAG-1
  • 60 ul used motor oil
  • 60 ul fresh motor oil
  • overnight growth of Acinetobacter calcoaceticus RAG-1
  • test tube rack
  • flame source
  • goggles
  • 10-100 ul micropipet

  • Procedure for Establishing Test Tube Model

    1. Using the spectrophotometer make a baseline reading of the carbon deficient media and record in your lab log.

    2. Use the inoculating loop to scrape up a colony of RAG-1 and inoculate each cuvette with bacteria. Be sure to use proper aseptic technique.

    3. Use the micropipet to layer 60 ul of used oil on one of the cuvette tubes and label the tube "used oil".

    4. Layer 60 ul of fresh oil on the other cuvette tube.

    5. Be sure both tubes are securely covered and labeled with your team name. Place tubes in the shaker water bath.

    6. Check tubes every 24 hours using the spectrophotometer. There is a correlation between the optical density of the media, the growth rate of the bacteria and the amount of oil degraded. Graph this correlation.

    7. After establishing this model, design an experiment to determine if this process can be improved and used to bioremediate future oil spills. You might want to consider:

      a. enhancing the growth media with nutrients to increase bacterial activity keeping in mind the challenge of the scale up needed for an actual oil spill.

      b. isolating the RAG-1 plasmid and transforming hardier strains of bacteria.

      c. developing a method which allows the quick delivery of ready-to-grow bacterial cultures to an oil spill site.

      d. using another organism such as algae as a model for bioremediation.

      e. any creative ideas you can generate!


    Prep for Oil Degrading Bacteria Lab

    Acinetobacter calcoaceticus RAG-1 - available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20807 # ATCC 31012

    Carbon Minimal Salts Culture Media

  • 2.2 g K2 HPO4 . 3 H2 0
  • 0.73 g KH2 PO4
  • 1 g (NH4 )2 SO4
  • 30 g NaCl
  • d H2 0 to total volume of 1 liter. pH solution to 7.0 with NaO.
  • 0.2 g MgSO4*
  • * Add after medium is autoclaved and cooled to room temperature.

    Using sterile technique aliquot 3 ml media into sterile10 ml disposable culture tubes with #13 caps. These can be inserted directly into the sample case of most Spectrophotometer 20 instruments. Innoculate media with 1 ml of an overnight growth of Acinetobacter calcoaceticus RAG-1 grown in LB broth. Students add 60 ul oil to each tube.

    Grow cultures at 30 degrees C for 72 hours with a 250 rpm shake. ( If you don't have a shaker, rig up one by using an old phonograph player. Drill an off-center hole in a large record you don't want anymore and use liquid nails to stick a couple of test tube racks to the record. Set the phonograph at 33.)


    Student's t-Test

    In 1908, William S. Gosset, a statistician for Guiness' Brewery in Dublin, developed a formula for measuring the probability that two, small random samples of continuous variables might have been drawn from the same normally distributed population. He published his work, known as the t-test under the pseudonym "A Student", perhaps to conceal his work from competing breweries.

    This is a useful instrument for students to use to analyze results of their oil spill in a test tube experiments in order to determine if the difference in their samples is statistically significant.

    Symbols used for t-test

    X1 = mean of sample 1

    X2 = mean of sample 2

    n1 = number of subjects in sample 1

    n2 = number of subjects in sample 2

    S12 = variance of sample 1
    (remember S12 doesn't mean to square this number.)

    S22 = variance of sample 2

    ____ = the sum of

    Strategy             Formula in Math        Formula in English                  
    mean                    X1 = _ X1           mean = sum of sample 
                                X1     n        divided by the number 
                                                of subjects in each sample
    
    Find variance        S12= _ (X1 - X2)2      subtract X from each number. 
                              S12 n-1           Square each answer. Add up the       
                                                squared numbers.  Divide by the
                                                number of numbers -1
    
    


    References

    Exocellular Esterase and Emulsan Release from the Cell Surface of Acinetobacter calcoaceticus, Yossef Shabtai and David L. Gutnick, Journal of Bacteriology, March 1985, p. 1176-1181.

    Enchanced Removal of Exxon Valdez Spilled Oil From Alaskan Gravel by a Mircrobial Surfactant, S. Harvey, I. Elashvili, J.J. Valdes, D. Kamely and A. M. Chakrabarty, Biotechnology, Volume 8, March 1990, p. 228-230.


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