Enumeration of hydrocarbon-degrading bacteria in the environment

Introduction

Oil pollution is relatively new problem for the environment. It is only recently since the discovery of crude oil that nations have refined it into different types of fuel and use oil as an engine lubricant. Supertankers have been built that travel the worlds oceans delivering this "liquid gold" as part of an industry that is worth many billions of dollars.

Cleaning up after oil spills at home, at sea or at an industrial complex is a messy business. What if we could use bacteria that can breakdown these hydrocarbons to do the cleaning up for us? So, if you are a school student, your first mission is to tackle questions 1, 2, 3, and 4 below:

Detecting hydrocarbon-degrading bacteria:

Equipment and chemicals

Bushnell Haas Medium (made up in deionised water)

Procedure

1. Dilute 10g (wet weight) of soil in 90 mL 0.1% sterile sodium pyrophosphate solution containing 30 g of glass beads (1:10 dilution). Shake for 1 hour.

2. Serially dilute soil solution (10 mL in 90mL sterile sodium pyrophosphate solution ) to 1:10³ for pristine samples and 1:10⁹ for the oil-contaminated samples. For the first dilution vortex for one minute to separate bacteria from soil particles. If using sea water or river water as your source of bacteria simply carry out the dilution series but make sure you dilute the sample in sterile sea or river water and use also use to make your media.

3. Transfer 1 mL of appropriate dilutions into 10 mL BH medium four times (1 tube will serve as a sterile control, the other three are triplicate counts).

4. Add 50 µL of hydrocarbons as sole carbon and energy source. Sources of hydrocarbons can be either volatile fuel oil, parrafin oil, or pure compounds like alkanes or aromatic hydrocarbons. It is suggested for your first experiment that you use something like jet fuel.

5. Controls are sterilised by autoclaving twice on consecutive days.

6. All tubes are incubated at 25oC for 3-6 weeks.

Results

To determine if growth has occurred in the tubes they are compared with the controls and those that are both turbid and show disruption to the film of oil on the surface of the medium are scored as positive. Relate the dilution that still scored positive to the number of cells per gram of soil.

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Isolation of hydrocarbon-degrading bacteria:

Procedure

1. Environmental samples are diluted as above. Portions (0.1 mL) of each dilution are spread onto BH agar and supplied with hydrocarbons as sole carbon and energy source by placing it in a vapour tube (cut off micropipette tips, sealed at one end with heat) in the lid of the plate. As a hydrocarbon source it suggested you first try jet fuel or pure compounds like naphthalene (just place a few crystals in the lid of the plate) or dodecane. For jet fuel and dodecane place about 20 µL on filter paper in the lid of the dish or use vapour tubes.

2. Control plates without substrate are also inoculated.

3. Seal all plates with parafilm and then incubate in a tupperware box (to reduce loss of volatile hydrocarbons in to the lab). All plates are prepared in triplicate for each dilution and incubated at 25oC for at least 1 month.

Results

Colonies larger and different from those on the substrate-free control plates are selected and purified for further investigation. Hydrocarbon-degrading bacteria are removed from the isolation plates and purified on BH plates supplied with hydrocarbons as sole source of carbon.

(Original protocol courtesy of Dr Jackie Aislabie)