The application of molecular methods including stable isotope probing to identify the microorganisms involved in toluene and MTBE degradation in mixed microbial systems

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 (e.g. MTBE or TBA) contamination. These chemicals are a threat to drinking water supplies because of their human health effects and relatively high aqueous solubilities. Bioremediation can be a cost-effective method to remove such groundwater contaminants either through natural attenuation or by advanced engineering methods. Understanding the microbial processes involved in the biodegradation of BTEX and MTBE has the potential to improve the efficiency of LUST site remediation. The overall aims of this project were to: (1) characterize the microorganisms able to degrade toluene under a range of redox conditions from a variety of soil and sediment sources using stable isotope probing (SIP); (2) investigate the diversity of the bssA and bamA genes in a wide range of anaerobic toluene-degrading consortia; and (3) identify the microorganisms able to transform MTBE anaerobically using SIP.;The first study in this research involved using SIP to identify the active members in an aerobic toluene degrading consortium. Specifically, SIP was used with terminal restriction fragment length polymorphism (TRFLP) and the results indicated that a 313 bp terminal restriction fragment (T-RF) incorporated the majority of the 13C from 13C labeled toluene. Sequencing of 16S rRNA genes from these communities indicated the organism represented by this T-RF was a Polaromonas spp. Real-time PCR was also utilized to provide quantitative patterns in gradient fractions and document increases in Polaromonas populations as toluene was degraded. In the second study, SIP was applied to five toluene-degrading consortia under sulfate and nitrate amended conditions. In all, five different phylotypes were found to be responsible for toluene degradation and these included previously identified toluene degraders as well as novel toluene degrading microorganisms. In nitrate amended microcosms, inoculated from granular sludge, microorganisms classifying within the genus Thauera were the primary toluene degraders. Whereas in nitrate amended microcosms, inoculated from a different source (agricultural soil), microorganisms in the family Comamonadaceae (genus unclassified) were the key degraders. In one set of sulfate amended microcosms (agricultural soil), the primary degrader affiliated within the class Clostridia (genus Desulfosporosinus), while in other sulfate amended microcosms, the primary degraders affiliated with the class Deltaproteobacteria, classifying within the families Syntrophobacteraceae (digester sludge) or Desulfobulbaceae (contaminated soil) (genus unclassified for both). The third study involved an investigation into the diversity of anaerobic toluene-degrading functional genes (bssA and bamA genes) in a number of inocula sources. The results suggest that targeting the bamA and bssA genes in a quantitative or non-quantitative manner could be a productive approach for investigating toluene biodegradation potential over a range of samples and redox conditions. The final study involved using SIP to investigate the dominant degraders in an anaerobic MTBE degrading microcosms. These experiments indicated bacteria in the phyla Firmicutes (family Ruminococcaceae) and Alphaproteobacteria (genus Sphingopyxis) were the dominant MTBE degraders in a methanogenic MTBE-degrading consortium seeded from activated sludge.