1. Cleaning Oil Spills Through Bio Remediation Becky Otter March 3, 2006

2. Where and How Often Do Oil Spills Occur?  Last year alone there were 21 spills between 7-700 tonnes of oil and 3 spills over 700 tonnes of oil. (www.itopf.com/stats.html )www.itopf.com/stats.html

3. (www.itopf.com/stats.html )www.itopf.com/stats.html ~ 2 million gallons of bilge water is dumped per year into the oceans with an average of 100ppm of oil http://www.acnatsci.org/educatio n/kye/hi/kye52002.html#sec3)

4. Making Refined Oil Structure of “Heavy” Crude oil. (http://www.l loydminster heavyoil.co m/whatislau nch.htm) (www.eia.doe.gov/.../ Refining_text.htm)www.eia.doe.gov/.../ Refining_text.htm Oil Distillation Column in Fawley UK

5. Clean Up Methods Clean Up Methods  Mechanical containment or recovery  Chemical and biological  Physical

6. What Affects How Diesel Fuel is Degraded by Microbes?  Hydrophobic nature of oil limits transfer to cell surfaces of micro-organisms  Concentration of oil  Composition of oil  Temperature  pH if the oil is on soil  Viscosity Leahy and Colwell, Microbiol Rev. 1990 September; 54(3): 305–315 (Leahy and Colwell, Microbiol Rev. 1990 September; 54(3): 305–315 ) Lee, et al.Journal of Applied Microbiology. 2006; 100: 325-333.) (Lee, et al., Journal of Applied Microbiology. 2006; 100: 325-333.) R. erythropolis, a close relative to EN3 strain

7. How The Surfactant Interacts with the Hydrocarbon (Makkar and Rockne, Enviro. Tox and Chem 2003) The surfactant surrounds the hydrocarbon allowing the bacterial cell to take up the hydrocarbon The hydrophobic tails of the surfactants interact which each other forming a micelle in an aqueous solution

8. “Enhanced biodegration of diesel oil by a newly identified Rhodococcus baikonurensis EN3 in presence of mycolic acid”  The EN3 strain can undergo the following process  Mycolic acid (a surfactant) was synthesized to increase the bioavailability of diesel to microbes  Scientists compared mycolic acid enhancement to enhancement by other surfactants at 20,000mg/l of oil.  Used gas chromatography to measure the amounts of diesel degraded Lee, et al.Journal of Applied Microbiology. 2006; 100: 325-333.) (Lee, et al., Journal of Applied Microbiology. 2006; 100: 325-333.) Mycolic Acid ANY HYDROCARBON

9. Results Figure 4 Degradation of diesel oil by Rhodococcus baikonurensis strain EN3. Strain EN3 was inoculated into MS solutions with diesel oil at different concentrations: (a) 1000, (b) 5000, (c) 10 000, and (d) 20 000 mg l 1. Initial inoculated cell concentration was 6 ×104 CFU ml 1., Uninoculated cultures as controls; ○, inoculated cultures for the experiments.

10. Figure 5 Chromatograms of diesel oil degradation [(a) 0 day, (b) first day, (c) second day, (d) third day, (e) fourth day, (f) fifth day, (g) sixth day and (h) seventh day]. The added diesel oil concentration was 10 000 mg l 1. Two of the residual peaks at the final day (h) were identified as the recalcitrant hydrocarbons pristane (Pr) and phytane (Ph).

11. Figure 6 Effect of surfactants (100 mg l 1) on the degradation of diesel oil by Rhodococcus baikonurensis strain EN3. Initial concentration of the diesel oil was 20 000 mg l 1, and the initial cell concentration 6 × 104 CFU ml 1. Figure 7 Effect of mycolic acid on the degradation of diesel oil by Rhodococcus baikonurensis strain EN3. Initial concentration of the diesel oil was 20 000 mg l 1, and the initial cell concentration 6 × 104 CFU ml 1. Initial concentration of mycolic acid: ○, 10 mg l 1;, 50 mg l 1;, 100 mg l 1., Uninoculated cultures without mycolic acid as control 1;, inoculated cultures without mycolic acid as control 2.

12. Results  After 7 days EN3 alone degraded almost Most of the degradation occurring in the first 3 days. Most of the degradation occurring in the first 3 days.  Other surfactants can enhance degradation of diesel, however mycolic acid is the most effectiveat 20,000mg/l.  There was no real difference between maximum level of degradation with samples containing mycolic acid at 10, 50, & 100 mg/l. Amount of Diesel FuelPercent degraded after 7 days 1000mg/l~100% 5000mg/l64.90% 10000mg/l60.70% 20000mg/l30.50%

13. Conclusion  The bacterial strain EN3 along with synthetic mycolic acid has the potential to degrade fairly large amounts of fuel over a short period of time in both aqueous and soil environments  This is new alternative or additive to current methods of cleaning oil spills without the other worries such as how to dispose of the diesel soaked sorbents or environmental effects caused from burning off the oil

14. Sources  Academy of Natural Sciences website, accessed Feb 2006. http://www.acnatsci.org/education/kye/hi/kye52002.html#sec3  Biswas, et al. “Microbial uptake of diesel oil sorbed on soil and oil spill clean-up sorbents.” Journal of Chemical Technology and Biotechnology. 2005; 80: 587- 593.  Burford and Hien. “Effect of Foam Density, Oil Viscosity, and Temperature on Oil Sorption Behavior of Polyurethane.” Journal of Applied Polymer Science. 2006; 99: 360-367.  ITOPF website, accessed Feb 2006.(http://www.itopf.com/stats.html)  Leahy and Colwell.“Microbial degradation of hydrocarbons in the environment.” Microbiol Rev. 1990 September; 54(3): 305–315  Lee, et al. “Enhanced biodegration of diesel oil by newly identified Rhodococcus baiknurensis EN3 in the presence of mycolic acid.” Journal of Applied Microbiology. 2006; 100: 325-333.  Makkar R. and Rockne K. “Comparison of synthetic surfactants and biosuractants in enhancing biodegration of polycyclic aromatic hydrocarbons” Environmental Toxicology and Chemistry. 2003; 22 (10): 2280-2292.  Oil Spills-EPA website, accessed Feb 2006 (http://www.epa.gov/oilspill/oiltech.htm). http://www.epa.gov/oilspill/oiltech.htm