Assessing biodegradation potential using in situ microcosms and 13C-labeled hydrocarbons

EPA Grant Number: X83-2428-01
Title: Assessing biodegradation potential using in situ microcosms and 13C-labeled hydrocarbons

Kerry L. Sublette, Sarkeys Professor of Environmental Engineering, University of Tulsa, 600 S. College Ave., Tulsa, OK 74104, (918)631-3085 Phone, (918)631-3268 Fax, 

David White, UTK/ORNL Distinguished Scientist and Professor of Microbiology and Director for the Center for Biomarker Analysis, University of Tennessee, 10515 Research Dr., Suite 300, Knoxville, TN 37932, (865)974-8001 Phone, (865)974-8027 Fax,  

Aaron Peacock,  Center for Biomarker Analysis, University of Tennessee, 10515 Research Dr., Suite 300, Knoxville, TN 37932, (865)974-8014 Phone, (865)974-8027 Fax,

William E. Holmes, School of Natural Resources, University of Michigan, 430 E. University Ave, Ann Arbor, MI 48109, (743)647-5925 Phone,;

Institutions: University of Tulsa; University of Tennessee; University of Michigan
EPA Project Officer: Bala Krishnan
Project Period: 10/5/05-10/4/06
Project Amount: $86,986
Research Category: Environmental forensics


Description:  This project will develop and field test groundwater monitoring tools which can be used to demonstrate in situ biodegradation potential for target hydrocarbons.

Objective:  This project seeks to further prove the efficacy of employing Bio-Sep bio-traps pre-loaded with 13C-hydrocarbons in BTEX-contaminated aquifers to provide absolute proof of in situ biodegradation potential and obtain regulatory approval for the use of the technique in Oklahoma, Colorado, Texas, and California.  We will work with industrial partners and state regulatory agencies to identify appropriate aquifers for testing the method.  We will seek to demonstrate the technology in a variety of different aquifers based on hydrocarbon concentration, redox conditions, and prevalent electron acceptors.  Prior to deployment we will demonstrate the low leachability of the hydrocarbon in laboratory simulations using an artificial groundwater with similar geochemistry to test site aquifers.  Based on our experience in California, this is likely to be a prerequisite to obtaining permission for field deployment.

Approach:  As noted above we anticipate the need to do leaching tests to demonstrate to regulators that significant amounts of hydrocarbon are not lost from the hydrocarbon-baited bio-traps during incubation in the aquifer.  We anticipate that these will be conducted in two-inch glass columns with suspended bio-traps.  The columns will receive a continuous feed of a simulated groundwater matching as closely as possible the geochemistry of the intended site of deployment.  The simulated groundwater will be supplemented with sodium azide to prevent microbial growth.  The aqueous feedrate to the columns will be determined from the seepage velocity in the target aquifer.  The column effluent will be monitored for the hydrocarbon bait for a 15-day period.  Effluent samples will be immediately extracted with methylene chloride and analyzed by GC/MS.

Prior to deployment of bio-traps Bio-Sep beads will be amended with isotopically labeled hydrocarbons as follows.  The beads will be heated at 300 °C for 4 hours to render the material free of organic residues and fossil biomarkers.   After cooling beads will be transferred to a vacuum desiccator containing a mixture of 13C-hydrocarbon (20%) and 12C-hydrocarbon (80%).   Pressure in the desiccator will be reduced until the hydrocarbon begins to boil.  The desiccator will then be sealed and maintained in this condition for at least 48 hrs to allow the hydrocarbons to become fixed to the beads.  A mixture of 13C- and 12C-labeled hydrocarbon will be used to reduce costs.  This mixture provides enough hydrocarbon in the beads to promote significant biofilm formation while providing enough 13C to be easily detected in biomass lipids.  The desiccator will be pre-fired at 300 oC prior to use and washed with methanol.  Loaded beads will be rehydrated by incremental addition of nanopure water at 60 oC in a clean and pre-fired vessel until rehydrated and then transferred to the bio-traps.

Bio-traps will be composed of about 100 Bio-Sep beads in 11.4-cm PFA tubing (12.7 mm OD, 9.5 mm ID).  The tubing will be perforated with six rows of 2.3-mm holes spaced 0.6 cm apart to provide contact between the beads and the groundwater.  Beads will held in place in the tubing with glass wool and nylon plugs.   Prior to use all bio-trap materials which cannot be fired will be washed with methanol.  Assembly of the bio-traps will take place on a surface of aluminum foil which has been fired at 300 oC.  All parts will be handled with sterile gloves and assembled bio-traps will be stored in sterile Whirl-pak bags at 4 oC until deployed.  Bio-traps will be transported to the field site in a cooler on Blue Ice as a further precaution against contamination.

We hope to identify two test sites in each of the target states.  At each site we will deploy bio-traps in a minimum of two wells, one in the plume and one background or unimpacted well.  Existing groundwater monitoring data will be used to choose the best impacted well for deployment of bio-traps; that is, a well where intrinsic bioremediation is suggested by geochemistry.  At sites where the geochemistry is inconclusive or the hydrogeology is complex, bio-traps may be installed in multiple impacted wells.

Bio-traps with 13C-enriched benzene, 13C-enriched toluene, and non-amended bio-traps will be deployed at each test site in triplicate.  Bio-traps will be suspended in groundwater monitoring wells using a braided nylon line at a depth to place the bio-traps where the highest concentrations of dissolved BTEX exist.  If the vertical distribution of BTEX is not known we will assume that the highest concentrations are near the water table and the bio-traps will be suspended at a depth of 1-2 ft below the water table.  If the site has a history of significant variation in the depth of the water table the bio-traps will be suspended from a float to maintain a fixed distance from the water table.

Bio-traps will typically be deployed for 30 – 45 days.  Once retrieved bio-traps will be placed in sterile Whirl-pak bags and shipped cold by overnight delivery service to Microbial Insights (MI) for analysis.  At MI ten beads will be taken at random from each bio-trap, placed in methylene chloride (2 mL/bead), and shipped to TU for benzene and toluene analysis.  Beads from all bio-traps (amended and non-amended) will be analyzed for 13C-benzene, 12C-benzene, 13C-toluene, and 12C-toluene to check for cross talk between bio-traps and to document hydrocarbon loss.  GC/MS will be used for these analyses. The isotopic composition of the 13C-enriched hydrocarbons will be determined by comparing the abundance of the respective molecular ions of the labelled and the non-labelled compound.  The remaining beads from each bio-trap will be extracted with a single-phase chloroform-methanol-buffer system (Bligh and Dyer, 1954; White et al., 1998).  The dried chloroform phases (after phase separation by addition of water and chloroform) will be subjected to derivatization with trimethylchlorosilane (TMCS) in methanol yielding fatty acid methyl esters (FAMEs).  Gas chromatography coupled to isotopic ratio monitoring mass spectrometry (GC-IRMS) will be used to measure 13C incorporation in total lipid fatty acids (TLFA).  Absolute concentrations of TLFAs will be determined by parallel injections on a GC/MS.  13C enrichment of TLFAs will prove biodegradation of the hydrocarbons by indigenous microorganisms under in situ aquifer conditions. 

Site specific milestones include:

Expected Results: The ultimate goal of this line of work is to develop an accurate, cost-effective tool to document the biodegradation potential of specific hydrocarbons in contaminated groundwater under in situ conditions.  This tool will be extremely useful to the industry and the regulatory community in risk-based decision making especially in those circumstances where the geochemistry is unclear.  It has not escaped our attention that this methodology could also ultimately yield information about exactly which microorganisms in a contaminated aquifer are responsible for the biodegradation process.  If the total lipid extract were fractioned to recover polar phospholipids from the cellular membranes, the identification of specific labelled fatty acids could provide insight into the organisms responsible for biodegradation.  Further, if labeled DNA could be recovered sequencing of the 16S rDNA gene could provide an even more precise identification.   Many other chemicals, such as nutrients, electron acceptors, etc., can be also incorporated into Bio-Sep beads during fabrication to produce slow-release repositories of these growth-supporting substances within the bio-traps.  Biofilms will be formed which are reflective of the available carbon sources as well as prevailing environmental conditions (e.g., temperature, pH, redox potential) in the aquifer and may thus be suitable to delineate operative mechanisms of natural attenuation and evaluate remediation amendments.  The diffusional resistances that slow the release of amendments also slow the release of products of microbial metabolism.  Chemical analysis of the aqueous and adsorbed phases can provide further evidence of operative microbial activities.  Although the current project focuses exclusively on the use of bio-traps baited with only isotopically labeled hydrocarbons, clearly this technology can have far reaching applications in both industry use and in research.

Key Words: Benzene, microbial ecology, groundwater, intrinsic bioremediation