Anaerobic toluene activation by benzylsuccinate synthase in a highly enriched methanogenic culture

Abstract

Permeabilized cells of a highly enriched, toluene-mineralizing, methanogenic culture catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formation was >85% of the specific in vivo rate of toluene consumption. This is the first report of benzylsuccinate synthase activity in a methanogenic culture; the activity has previously been reported to occur in denitrifying, sulfate-reducing, and anoxygenic phototrophic bacteria.

FIG. 1

Kinetics of in vitro benzylsuccinate-d₈ formation from toluene-d₈ and fumarate by permeabilized cells of a methanogenic enrichment culture. The data points represent the averages of results of duplicate assays. The linear regression used to calculate the rate of benzylsuccinate formation is shown.

FIG. 2

Cumulative toluene consumption by whole cells of a methanogenic enrichment culture. The linear regression used to calculate the rate of toluene consumption is shown.

FIG. 3

Normalized plot of toluene-d₈ and benzylsuccinate-d₈ concentration in a methanogenic enrichment culture. Toluene concentration is normalized to the total consumed (∼500 μM), and labeled benzylsuccinate is normalized to its maximum observed concentration (∼0.01 μM). Benzylsuccinate-d₈ was quantified by liquid chromatography-MS-MS, and toluene-d₈ was quantified by GC-flame ionization detection.