Q-195. A Comparative Analysis of In Situ BTEX Degraders at Different LUST Sites Using Stable Isotope Probing

W. Sun1, S. Xie2, A. M. Cupples1;
1Michigan State Univ., East Lansing, MI, 2Peking Univ., Beijing, CHINA.

Sites containing leaking underground storage tanks (LUST sites) are a national problem, with over 443 568 releases confirmed as of 2003, resulting in BTEX (benzene, toluene, ethylbenzene, xylenes) or oxygenate (eg MTBE or TBA) contamination and risk to human health through polluted drinking water supplies. Although many aerobic BTEX degraders have been identified through enrichment and isolation procedures, data on the microorganisms actually responsible for in situ biodegradation is still lacking. Here, recent advances in molecular methods, specifically stable isotope probing (SIP), were utilized to identify such bacteria. Toluene and m-xylene were chosen as initial model contaminants for these experiments. Sediments obtained from different LUST sites were used to construct aerobic microcosms and SIP was conducted on samples illustrating significant contaminant removal. SIP experiments consisted of triplicate control microcosms (no contaminant added), or triplicate sample microcosms with either labeled (13C) or unlabelled toluene or m-xylene. DNA was extracted at several time points during the experimental period to control for label cross feeding. Following extraction, DNA was subject to ultracentrifugation, fractionation, and terminal restriction fragment length polymorphism (T-RFLP). T-RFLP profiles in each fraction were then compared to determine which T-RFLP fragments were more dominant in the heavy fractions of the samples exposed to label compared to those exposed to the unlabelled contaminant. The comparison of these profiles provided an effective control for false positive results. Gene sequences (16S rRNA) obtained from labeled, heavy DNA fractions were then compared to in silico enzyme cut sites to identify the microorganisms responsible for label uptake at each site. A comparative analysis of the microorganisms responsible for 13C-toluene and 13C-m-xylene uptake from different sites is presented.