Bio-Traps Coupled with Molecular Biological Methods and Stable Isotope Probing Demonstrate the In Situ Biodegradation Potential of MTBE and TBA in Gasoline-Contaminated Aquifers

Biofilms characteristic of aquifer conditions can be rapidly and efficiently collected using in situ microcosms or bio-traps containing Bio-Sep((R)) beads (25% Nomex and 75% powdered activated carbon [PAC]) (University of Tulsa, Tulsa, Oklahoma). Bio-Sep beads can be baited with a variety of organic compounds by vapor-phase adsorption onto the PAC component of the beads under reduced pressure. In the aquifer, the bait or the amendment does not substantially leach into the aquifer but is available to the bacteria as a carbon source within the bead. When the organic compound is labeled with C-13, phospholipids may be extracted from bead biofilms postincubation and derived fatty acid methyl esters analyzed for C-13. Incorporation of C-13 into biomass provides proof of current in situ degradation potential. We have now Bio-Sep beads with C-13-labeled methyl-tert-butyl ether (MTBE) and tert-butyl alcohol (TBA). Deployment of these C-13-amended bio-traps has been coupled with molecular biological methods and stable isotope probing to demonstrate the biodegradation of MTBE and TBA in an anaerobic gasoline plume in southern California. Nonamended bio-traps were deployed at the same site as a preexperiment to determine if spatial variations in microbial community structure could be linked to concentrations of oxygenates or other electron donors. Analysis of biofilm phospholipids and DNA from nonamended bio-traps demonstrated a clear relationship between various aspects of the subsurface microbial community structure and the concentration of TBA. There was no correlation of any phospholipid fatty acid or DNA attributes with the concentrations of MTBE; benzene, toluene, ethylbenzene, and xylenes (BTEX); or gasoline range total petroleum hydrocarbons (TPHg). Deployment of C-13-MTBE- or C-13-TBA-amended bio-traps in the plume clearly demonstrated the degradation of both MTBE and TBA under aquifer conditions through incorporation of C-13 into membrane phospholipids.