Project Abstract

Pathways of Anaerobic Toluene Metabolism by a Sulfate-Reducing Bacterium, Strain PRTOL1

Principal Investigator

Alfred M. Spormann
Stanford University
E-mail: spormann@Ce.stanford.edu

Goal

The objectives of this study are to determine the metabolic pathways involved in anaerobic toluene degradation in sufate-reducing strain PRTOL1, and to identify metabolic intermediates that can be used as indicators for anaerobic bioremediation of BTEX compounds.

Rationale

Although some information is available about anaerobic mineralization of toluene in one denitrifying strain, it is not clear if all anaerobes that degrade toluene utilize the same biochemical pathway. Concomitant to toluene metabolism in all toluene metabolizing denitrifying cultures that have been investigated as well as in PRTOL1, is the formation of the metabolic by-products, benzylsuccinate and benzylfumarate. The by-products are not further metabolized by pure cultures. Recently, we found that these compounds can be demonstrated to be present in field sites where BTEX compounds were released. We focus on experiments investigating the connection between by-product formation and toluene metabolism.

Approach

Studies using PRTOL1 cells that metabolize toluene with sulfate as the electron acceptor involve cell suspensions and cell extracts. Both sets of experiments are designed to identify the initial biochemical steps involved in toluene oxidation and by-product formation. By manipulating the metabolic performance of PRTOL1 cells in suspensions, intermediates are expected to be transiently formed and consumed. Results of these cell suspension experiments will then be used to design assays to demonstrate specific enzyme activities in cell extracts. In cell extract studies, we expect to specifically investigate the biochemical reactions involved in the formation of benzylsuccinate and benzylfumarate.

Status

Cell growth experiments with PRTOL1 revealed that toluene-metabolizing cells co-metabolically transform o- and p-xylene to the corresponding methyl derivatives of benzylsuccinate and benzylfumarate, although PRTOL1 is unable to grow on o- and p-xylene. In cell extract studies, we found that PRTOL1 contains activities of the key enzymes of the carbon monoxide dehydrogenase pathway for complete acetyl-CoA oxidation rather than activities of the citric acid cycle. This finding is important becasue it has been proposed that succinyl-CoA, originating from a catabolic citric acid cycle, may be involved in by-product formation.