1. Do They Eat Petroleum Hydrocarbons? Bioremediation of Toluene by Oil-Water Separator Bacteria LISA L. LOFTIS and JAMES E. CHAMPINE. Biology Department, Southeast Missouri State University.

2. Site History  Former Gasoline Station  Soil and Groundwater Contamination  Why Remediation System was chosen

3. Site Map

4. Soil Vapor Extraction Trailer

5. Hypothesis  Contaminated groundwater represents a natural enrichment for petroleum hydrocarbon degrading bacteria

6. Approach  Use toluene as a surrogate for petroleum related hydrocarbons that may be degraded in the aquifer  Test groundwater biofilm and pure cultures from the site for toluene degradation activity

7. Site Materials  Before and After groundwater samples  Oil/Water Separator Biofilm (Jar and Vial)  Collection of Bacterial Isolates

8. Gas Chromatograph

9. Chromatography Conditions  Carrier Gas  DB1 30 m capillary column  200ºC oven temperature  Flame Ionization Detector

10. Typical chromatogram

11. Serum Bottle Cultures  20 mL of sterile strength 0.1x TSB  Teflon Rubber Stoppers  Aluminum Crimps  Amended with 10  L of Toluene  Aerobic

12. Serum Bottle Cultures

13. Before Groundwater Experiment

14. After Groundwater Experiment

15. Isolations  Before  Beige (from After groundwater)  ECP 6  ECP 7

16. Toluene Degraders ECP 6 and ECP 7

17. Conclusions  Intrinsic Bioremediation did occur at the site  Site cleaned up in half of the estimated time  Bacteria with toluene degradation activity were isolated

18. Acknowledgments  Southeast Missouri State University Undergraduate Research Program  Heather DuShell  Dr. Bjorn Olesen and the Department of Chemistry  Vicki Howell, Biology Dept. Secretary