Using Stable Isotope Probing and Bio-Traps to Identify TBA Degraders In Situ Under Sulfate Reducing Conditions

Kerry Sublette (The University of Tulsa), Jennifer Busch-Harris (ConocoPhillips), Kathleen Duncan (University of Oklahoma), Greg Davis and Dora Ogles (Microbial Insights) and Doug Mackay (University of California-Davis)

We have demonstrated that biofilms characteristic of aquifer conditions can be rapidly and efficiently collected using in situ microcosms or “bio-traps” containing Bio-Sep® beads. Bio-Sep® beads are 3-4 mm in diameter and composed of 25% aramid polymer and 75% powdered activated carbon (PAC). They have a porous, sponge-like structure with 74% porosity and 600 m²/g of surface area surrounded by an ultrafiltration-like membrane that contains holes and tears of 1-10 microns in size. Bio-Sep® beads are heated to 300° C for sterilization and to render the beads free of fossil biomarkers. When bio-traps are deployed in groundwater, indigenous bacteria enter through the outside membrane and migrate into the porous internal matrix. Microbes then attach to this internal structure and reproduce to form biofilms. In this manner, microbes can be concentrated for analysis despite their relatively low density of microbes in the sampled groundwater. Microorganisms must grow and reproduce within the bead to be detected. Thus the beads will collect only those organisms which are active under the specific subsurface conditions. Those microorganisms that are not capable of growth under in situ conditions due to a nutrient limitation, unfavorable redox potential, or adverse environmental conditions will not be collected in the beads at detectable levels.  Bio-Sep® beads can also be “baited” with a variety of organic compounds by vapor phase adsorption onto the PAC component of the beads. The adsorbed organic has been shown to be bioavailable to bacteria that form biofilms in the beads during incubation in a contaminated aquifer. If the compound is labeled with 13C, polar lipids may be extracted from bead biofilms and the derived fatty acid methyl esters analyzed for 13C incorporation using GC-IRMS. Since the beads are clean of biomarkers and sterile when deployed, incorporation of 13C in phospholipids provides proof of in situ biodegradation of a target compound by indigenous microorganisms under actual aquifer conditions.  We have successfully used bio-traps amended with 13C6-benzene, 13C5-MTBE, and 13C4-TBA to demonstrate the in situ biodegradation potential of these compounds at several sites. One such site has been the Vandenberg AFB site where bio-traps amended with 13C4-TBA were deployed in an anaerobic TBA plume. Several phospholipid fatty acids extracted from bead biofilms were found to be labeled with 13C indicating biodegradation of TBA. Fatty acid and DNA labeling patterns suggest the participation of sulfate-reducing bacteria in the degradation process.