Petroleum Bioremediation in Seawater Using Guano as the Fertilizer

ABSTRACT

The biodegradation of hydrocarbon pollutants in open systems, such as oceans, is generally limited by the availability of utilizable nitrogen and phosphorus sources. Here the authors demonstrate the potential of overcoming this problem with guano as the fertilizer. In the first set of experiments, the principle and conditions for growing bacteria on a water insoluble fertilizer was established, using uric acid as the nitrogen source and a pure culture of an isolated hydrocarbon-degrading bacterium, Alcanivorax sp. OK2. Using a simulated open system, it was demonstrated that uric acid (the major nitrogen component of guano) binds to crude oil and is available for the growth of strain OK2 and petroleum degradation. In the second set of experiments, using a simulated open system, it was demonstrated that commercial guano was an effective source of nitrogen and phosphorus for the growth of marine bacteria on crude oil. Bacterial cultures reached over 10⁸ cells per ml and 70% of the crude oil was degraded. Controls using ammonium sulfate and phosphate in place of guano in the simulated open system reached only 10⁶ cells per ml and showed no detectable hydrocarbon degradation. Isolation and characterization of the bacteria in the crude oil/guano cultures indicated that they were primarily strains of Alcanivorax and Alteromonas.

KEYWORDS:

• Alcanivorax
• Alteromonas
• guano
• petroleum degradation
• uric acid

ACKNOWLEDGEMENTS

This investigation was supported by the Pasha Gol Chair for Applied Microbiology, the Manja and Morris Leigh Chair in Biophysics and Biotechnology, and EU Project COMMODE.

Notes

^a After mixing 5 mg/ml crude oil and the nitrogen supplement in sterile artificial seawater (SAS), the clear aqueous phase was removed and replaced with fresh SAS. The procedure was repeated three times to remove water soluble components. The medium was then supplemented with 1 mM potassium phosphate buffer and inoculated with a 0.01 ml of an OK2 culture grown in PU medium.

^b Maximum cell yield was obtained at 7 days.

^c Petroleum degradation was measured as described in Materials and Methods after 20 days.

^a Samples from “guano” and “no guano” flasks after 3 days of growth were extracted and then subjected to column fractionation and gas chromatography as described in Materials and Methods. The analysis was repeated twice and in no case was the deviation greater than 4%. The percent degradation relates to the no guano control.