| Groundwater contamination with the synthetic fuel oxygenate, methyl tert-butyl ether (MTBE), is an extensive problem. Microbial mediated biodegradation holds promise as a tool for remediation of contaminated water supplies. However, MTBE biotransformation processes are slow and MTBE degrading organisms are difficult to isolate, creating challenges relating to site assessment, enhancement of natural attenuation and monitoring bioremediation in situ. In this study we analyzed MTBE degrading cultures using a variety of isolation independent techniques. A majority of the experiments used previously established anaerobic enrichment cultures that had been maintained on MTBE for several years. We demonstrated that low concentrations of some aryl O-methyl ether compounds enhanced the rate of MTBE degradation. Propyl iodide caused a light-reversible inhibition of MTBE depletion, suggesting that the anaerobic MTBE O-demethylation reaction was corrinoid dependent. Terminal-restriction fragment length polymorphism (T-RFLP) and sequence analysis of 16S rRNA genes from one anaerobic MTBE degrading enrichment culture showed a phylogenetically diverse population with no exact matches to previously isolated or described species. Stable isotope probing experiments verified that microorganisms from anaerobic MTBE degrading enrichment culture used 13C from 13C-MTBE for growth and cell division and that a particular subpopulation assimilated this carbon prior to the rest of the population. We also analyzed carbon and hydrogen stable isotope fractionation occurring during MTBE degradation. In anaerobic cultures, substantial fractionation of hydrogen was found only in cultures supplied with syringic acid during MTBE degradation, providing the first experimental suggestion of multiple anaerobic MTBE O-demethylation mechanisms. During aerobic MTBE degradation by the psychrophilic bacterium, Variovorax paradoxus, carbon and hydrogen fractionation were not influenced by incubation temperature during degradation. This work represents a significant contribution to the current body of knowledge about MTBE degradation and the data presented will be useful in many aspects of studying, enhancing and monitoring MTBE degradation under a variety of conditions. |
