In the last two decades methyl tertiary-butyl ether (MTBE) gained widespread use as an octane enhancer and fuel oxygenate in gasoline, and has also become a frequently detected and problematic ground and surface water contaminant. One desirable fate process for oxygenated gasoline in contaminated environments is microbial biodegradation. The microbial oxidation of fuel constituents in contaminated environments is coupled to the reduction of a terminal electron acceptor (TEA) in a process that generates energy beneficial to microbial growth. In aerobic environments. oxygen is the TEA, while inorganic ions such as ferric iron, nitrate, sulfate, or bicarbonate, among others, typically serve as the TEA in anaerobic environments. Since oxygen is the most energetically favorable TEA, it frequently becomes depleted during the biodegradation of the more labile components of gasoline. Then, costly engineered systems may be used to increase the availability of oxygen, or the system may be left in an anaerobic state and the oxidation of contaminants can be coupled to the reduction of other available TEAs. This latter approach, known as "natural attenuation" or "intrinsic bioremediation", is preferred because it allows the more expensive engineered treatment approaches to be focused on sites that are under more acute health and environmental threat. However, natural attenuation is dependant upon the susceptibility of fuel components to anaerobic biodegradation. The biodegradation of gasoline hydrocarbons, including the benzene, toluene, - ethylbenzene, and xylene isomers (BTEX) of most regulatory concern, has been well documented to occur under both aerobic and anaerobic conditions. Furthermore, the aerobic biodegradation of the fuel additive MTBE has also been conclusively demonstrated. However, the greatest threat to the potential implementation of intrinsic bioremediation for MTBE-contaminated ground water is the relative recalcitrance of this oxygenate in anaerobic aquifers, and the paucity of thorough research conclusively demonstrating its susceptibility to anaerobic decay. This proposal seeks funding for a series of laboratory studies to conclusively demonstrate the susceptibility of MTBE, and its known metabolite tertiary-butyl alcohol (TBA), to anaerobic biodegradation by inocula from gasoline-contaminated aquifers where BP field monitoring suggests that this is occurring. These incubations will be conducted under the methanogenic conditions observed in the implicated field sites. However, the effect of addition of other TEAs, namely sulfate and nitrate, will also be tested. MTBE, TBA, and gasoline hydrocarbons will be measured by purge and trap gas chromatography (GC). GC with a radiometric detector will be used to monitor the production of 14C0, and 14CH4 from (?,?1-14C)-MTBE, methyl (?-14C-tertiary butyl) ether and ?-14C-TBA. This information will be coupled with information on the consumption of electron acceptors (sulfate, nitrate) and/or the production of metabolic end products (unlabeled methane, hydrogen sulfide) as appropriate. The experiments will be interpreted relative to appropriate sterile, substrate-unamended, and positive (benzoate) controls. Of particular interest to the prospect of methanogenic MTBE degradation is the question of whether MTBE is a substrate that is oxidized with the resulting electrons used to reduce bicarbonate to methane, or whether MTBE functions as the electron acceptor and is reductively demethylated to give TBA and methane. The reducing equivalents for the latter possibility might be provided by the anaerobic oxidation of gasoline hydrocarbons, including BTEX. Thus, the effect of addition of aromatic and simple substrates, such as benzoate and hydrogen, respectively, on anaerobic MTBE biodegradation will also be tested. Use of inhibitors of methanogenesis and sulfate reduction (bromoethane sulfonic acid and molybdate, respectively) will also lead to insight into the responsible microorganisms. Ultimately, demonstrating the anaerobic biodegradation of MTBE and understanding the process will help buttress claims for the intrinsic bioremediation of this gasoline-additive in contaminated aquifers.