Tuesday, June 24, 2008 : 2:20 p.m.

Identification of Native Aerobic Tba-Metabolizing Organisms in Long Island Ground Water Using 13C Stable Isotope Probing

Denise Aslett1, Joseph E. Haas II, M.S., PE2, Donald A. Trego3 and Michael Hyman1, (1)North Carolina State University, (2)New York State Department of Environmental Conservation, (3)Environmental Assessment and Remediations

Stable isotope probing describes a series of techniques that use incorporation of stable isotopes such as 15N and 13C into biological markers (lipids, proteins, nucleic acids) to identify microorganisms in complex communities that have specific metabolic capabilities. We have used 13C4-tertiary butyl alcohol (TBA) to identify aerobic TBA-utilizing organisms in samples from a self-inoculated Bio-GAC unit that was used as part of source area remediation activities at a site in Hampton Bays, Long Island.

One series of samples received three additions of unlabeled (12C) TBA, followed by a single addition of 13C4-TBA. A second series of samples received a total of four additions of 13C4 -TBA while a third series of samples received a single addition of 13C4-TBA. In all cases TBA consumption was followed by gas chromatography. Once the TBA had been consumed, community DNA was extracted and then subjected to high-speed density centrifugation to separate 13C and 12C-labeled DNA. The 16S rRNA genes in each pool were amplified by PCR and then analyzed by denaturing gradient gel electrophoresis. Individual bands were excised, sequenced and the resulting nucleotide sequences were analyzed for sequence similarities in national databases.

The clearest resolution of 12C and 13C-labeled DNA was obtained using a single addition of 13C4-TBA. Three bacterial species were subsequently identified.  A Xanthomonas strain was a dominant member of the microbial community that did not assimilate TBA (i.e. 12C-labeled DNA). Both a Ralstonia and a Polaromonas strain were identified as TBA-metabolizing bacteria in 13C-labeled DNA samples. Ralstonia strains are common soil microorganisms that often utilize H2 and synthesize polyhydroxybutyrate; both common characteristics seen in oxygenate-metabolizing organisms. Polaromonas strains have been previously identified in low temperature environments and GAC beds and degrade common environmental pollutants such as dichloroethylene (DCE).  Our results suggest there are diverse native TBA-utilizing bacteria in Long Island groundwater.

Denise Aslett, North Carolina State University Denise is a 3rd year PhD student in the Department of Microbiology at North Carolina State University. Denise's research focuses on the use of various stable isotope-based techniques to investigate the biochemistry and microbiology of fuel oxygenate biodegradation processes.

Joseph E. Haas II, M.S., PE, New York State Department of Environmental Conservation Joe has 19 years experience working as a state regulator managing a remedial section. He holds both Engineering Geologist and Hydrogeologist specialty licenses. Joe has been involved in research with representatives of the United States Environmental Protection Agency. These research efforts have been credited as having made significant contributions to the understanding of the characteristics of MTBE in the sub-surface environment and having influenced the national debate on the use of MTBE in gasoline. The Office of Research and Development acknowledged his contributions, in 2002, in the form of an award for Exceptional Support to ORD.

Donald A. Trego, Environmental Assessment and Remediations Don graduated in 1993 from the Pennsylvania State University with a bachelors of science in Environmental Resource Management. He has over 14 years of experience in the groundwater remediation industry. Don has been involved in numerous research initiatives with the New York State Department of Environmental Conservation (NYSDEC), the United States Environmental Protection Agency (USEPA), and the United States Geological Survey (USGS). As project manager, Don was a co-recipient of the 2004 National Ground Water Association’s (NGWA) – Outstanding Groundwater Remediation Project award. He currently is the Vice president of a respected environmental consulting firm located on Long Island, NY.

Michael Hyman, North Carolina State University Michael is an Associate Professor in the Department of Microbiology at North Carolina State University. Michael has over 20 years experience in the characterization of the microbial biodegradation processes. His main research interests are in the area of cometabolism and the biodegradation of fuel oxygenates.


2008 NGWA Conference on Eastern Regional Ground Water Issues