List of experiments

Bioremediation of Fats and Oils
Environmental Microbiology
Experiments for middle and high school

Christine L. Case
Skyline College

• Define the following terms: bioremediation, lipid, fat.

• Isolate lipid-degrading bacteria.

• Describe the digestion of fats.
  

Many bacteria use exoenzymes such as lipase to hydrolytically decompose lipids. The lipid molecule is broken down into glycerol and three fatty acids (figure 1). Some bacteria can ferment the glycerol; others oxidize the fatty acids. When lipid hydrolysis occurs in decomposition of foods such as butter, it results in rancid flavor and aroma due to the fatty acids (such as butyic acid). Sewage systems have a great deal of difficulty decomposing lipids because the process is slow and requires large amount of oxygen. Bacterial cultures are being used to degrade grease from restaurants and meat-processors before disposal.

In this exercise, we will select for oil-degrading bacteria. Degradation of the hydrocarbon should result in clearing around the colonies. Lipids are insoluble in water causing the culture medium to be opaque. Degradation will result in soluble molecules causing the clearing; the fatty acids will also decrease the pH of the medium.

Figure 1.

Tributyrin digestion by hydrolysis. Lipids are composed of a glycerol and three fatty acid molecules. Back

Oil-agar. Prepare 500 mL minimal salts agar (w/o glucose). If the agar solidifes in a glass bottle, liquefy it in a microwave oven. Add 1% oil to the agar. Blend at high speed for 5 min. Transfer oil-agar to a flask and sterilize in an autoclave or pressure cooker. The oil will remain emulsified.

Aseptically dispense 15 mL of the oil-agar in each Petri plate. It isn't necessary to pipette the agar, it can be poured to a depth of approximately 5 mm.

Soil slurry (a pinch of soil mixed in a few milliliters of water).

1. Spread a drop of the soil slurry onto the surface of each place. Incubate the plates at 30°C.
   

2. Examine the plates for the number of oil-degrading bacteria.

1. How could the bacteria you have grown be used commercially in a detergent or drain cleaner?
   

2. Beside clearing, how else could you determine if lipids had been hydrolysed?
   

3. Which oil was the most readily digested?
   

4. Look at the chemical equation for digestion of tributyrin. Why do you get thirsty after eating?