Goal: The objectives of this study are to provide knowledge on the metabolic reactions and intermediates involved in anaerobic ethylbenzene oxidation, and to get a better understanding of the factors that control expression of the degradative enzyme activities.
Rationale: BTEX (benzene, toluene, ethylbenzene, and xylenes) are the most water soluble components of fuel. Leaking underground storage tanks and surface spills often contaminate soils and aquifers. Anaerobic degradation of BTEX has been observed to occur under natural conditions opening the potential to use the degradative activity of microorganisms to remediate contaminated sites. Only recently, pure cultures of toluene or xylene degrading bacteria have been described. At the time this project was started, EB1 which was isolated at Stanford's Environmental Engineering and Science program, was the only anaerobic organism capable of complete ethylbenzene oxidation to CO2 under anoxic conditions. To predict when and to what extent intrinsic bioremediation of fuel components in general, and ethylbenzene in particular, occur and how these processes can be enhanced, a better understanding of the biochemical pathways and reactions involved is necessary.
Approach: Studies with strain EB1 metabolizing ethylbenzene under denitrifying conditions were conducted. Cells grown anaerobically with ethylbenzene and nitrate as substrates are harvested anaerobically, concentrated, and subjected to cell suspension experiments. Suspensions are ammended with ethylbenzene and nitrate, and the concentrations of hydrocarbons and nitrate are followed by GC and IC respectively. Potential intermediates during anaerobic degradation of ethylbenzene are detected using HPLC and the chemical nature of the compounds is identified by GC-MS. Based on the finding of intermediates isotope exchange studies will be imployed to substantiate the proposed pathway. Results from the cell suspension studies will then be used to design enzyme assays with cell-free extracts.
Status: During anaerobic ethylbenzene mineralization by EB1 in cell suspensions 1-phenyl-ethanol and acetophenone were formed. The formation was transient and was paralleled by nitrate and nitrite consumption. In additon, it was found that EB1 is able to grow anaerobically with 1-phenyl-ethanol and acetophenone as substrates under denitrifying conditions. These results suggest that anerobic degradation of ethylbenzene in strain EB1 may proceed with 1-phenyl-ethanol and acetophenone as intermediates.