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

Site HistorySite History Former Gasoline StationFormer Gasoline Station Soil and Groundwater ContaminationSoil and Groundwater Contamination Why Remediation System was chosenWhy Remediation System was chosen

Site MapSite Map

Soil Vapor Extraction TrailerSoil Vapor Extraction Trailer

HypothesisHypothesis Contaminated groundwater represents a Contaminated groundwater represents a

natural enrichment for petroleum natural enrichment for petroleum hydrocarbon degrading bacteriahydrocarbon degrading bacteria

ApproachApproach Use toluene as a surrogate for petroleum Use toluene as a surrogate for petroleum

related hydrocarbons that may be related hydrocarbons that may be degraded in the aquiferdegraded in the aquifer

Test groundwater biofilm and pure Test groundwater biofilm and pure cultures from the site for toluene cultures from the site for toluene degradation activitydegradation activity

Site MaterialsSite Materials Before and After Before and After

groundwater samples groundwater samples Oil/Water Separator Oil/Water Separator

Biofilm (Jar and Vial)Biofilm (Jar and Vial) Collection of Collection of

Bacterial IsolatesBacterial Isolates

Gas ChromatographGas Chromatograph

Chromatography Chromatography ConditionsConditions

Carrier GasCarrier Gas DB1 30 m capillary columnDB1 30 m capillary column 200200ºC oven temperatureºC oven temperature Flame Ionization DetectorFlame Ionization Detector

Typical chromatogramTypical chromatogram

Serum Bottle CulturesSerum Bottle Cultures 20 mL of sterile 20 mL of sterile

strength 0.1x TSBstrength 0.1x TSB Teflon Rubber Teflon Rubber

StoppersStoppers Aluminum CrimpsAluminum Crimps Amended with 10 Amended with 10

L of TolueneL of Toluene AerobicAerobic

Serum Bottle CulturesSerum Bottle Cultures

Before Groundwater Before Groundwater ExperimentExperimentBefore Groundwater

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Date

Area

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After Groundwater After Groundwater ExperimentExperiment

After Groundwater (red bacteria)

0200000400000600000800000

1000000

Date

Area

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IsolationsIsolations Before Before Beige (from After Beige (from After

groundwater)groundwater) ECP 6ECP 6 ECP 7ECP 7

Toluene Degraders Toluene Degraders ECP 6 and ECP 7ECP 6 and ECP 7

ECP 6 and ECP 7

020000000400000006000000080000000

100000000

Area

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Series2Series1

ConclusionsConclusions Intrinsic Bioremediation did occur at the Intrinsic Bioremediation did occur at the

sitesite Site cleaned up in half of the estimated Site cleaned up in half of the estimated

timetime Bacteria with toluene degradation activity Bacteria with toluene degradation activity

were isolatedwere isolated

AcknowledgmentsAcknowledgments Southeast Missouri State University Southeast Missouri State University

Undergraduate Research ProgramUndergraduate Research Program Heather DuShellHeather DuShell Dr. Bjorn Olesen and the Department of Dr. Bjorn Olesen and the Department of

ChemistryChemistry Vicki Howell, Biology Dept. Secretary Vicki Howell, Biology Dept. Secretary