Metabolites formed during anaerobic transformation of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization

Abstract

Strain Tl is a facultative bacterium that is capable of anaerobic toluene degradation under denitrifying conditions. While 80% of the carbon from toluene is either oxidized to carbon dioxide or assimilated into cellular carbon, a significant portion of the remainder is transformed into two dead-end metabolites. These metabolites were produced simultaneous to the mineralization of toluene and were identified as benzylsuccinic acid and benzylfumaric acid. Identification was based on comparison of mass spectra of the methyl esters of the metabolites and authentic compounds that were chemically synthesized. Strain Tl is also capable of o-xylene transformation during growth on toluene. o-Xylene does not serve as a source of carbon and is not mineralized. Rather, it is transformed to analogous dead-end metabolites, (2-methylbenzyl)-succinic acid and (2-methylbenzyl)-fumaric acid. o-Xylene transformation also occurred during growth on succinic acid, which suggests that attack of the methyl group by succinyl-coenzyme A is a key reaction in this transformation. We reason that the main pathway for toluene oxidation to carbon dioxide involves a mechanism similar to that for the formation of the metabolites and involves an attack of the methyl group of toluene by acetyl-coenzyme A. Strain Ti is a denitrifying bacterium that has been shown to oxidize toluene under anaerobic conditions primarily to carbon dioxide and biomass (8). This organism is also capable of o-xylene transformation during growth on toluene; however, o-xylene cannot serve as a sole source of energy or cellular carbon. Metabolites were formed from both toluene and o-xylene during their transformation and were not further metabolized. These dead-end metabolites were determined not to be benzyl alcohol, benzaldehyde, benzoic acid, cresol, hydroxybenzyl alcohol, hydroxybenzaldehyde, hydroxybenzoic acid, methylbenzyl alcohol, tolualdehyde, or toluic acid (8). Since the metabolites were not further degraded, it was reasoned that they were not intermediates in the pathway of toluene oxidation to carbon dioxide. However, since a significant portion of toluene carbon was transformed to these metabolites, investigation of their formation was warranted. The toluene-dependent transformation of o-xylene is significant because o-xylene is relatively recalcitrant to biodegradation under both aerobic (2) and anaerobic (8) conditions. However, incomplete transformations have a potentially negative side since the metabolites formed may be more toxic than the original chemical that was the source of contamination. Therefore, identification of these metabolites can provide information on their toxicity, their formation, and how to initiate their mineralization.

Anaerobic bacterial metabolism of hydrocarbons

“…Krieger, M. Reinhard and A.M. Spormann, unpublished data). The o-and p-xylene isomers can also be co-metabolized with toluene and converted to dead-end products by some bacterial strains [33,44,47,68]. So far, only one pure culture growing on o-xylene has been obtained [34], and no pure cultures growing on p-xylene have yet been reported.…”

Functional analysis of an anaerobic m-xylene-degrading enrichment culture using protein-based stable isotope probing

“…A few isolates were reported to grow with m-xylene (Dolfing et al, 1990;Fries et al, 1994;Rabus & Widdel, 1995;Harms et al, 1999;Morasch et al, 2004) or o-xylene (Harms et al, 1999;Morasch et al, 2004); no pure culture was described yet degrading p-xylene anaerobically. A few studies showed that the anaerobic degradation of xylene isomers was initiated by addition of fumarate to a methyl moiety (Evans et al, 1992;Beller & Spormann, 1997;Krieger et al, 1999;Morasch et al, 2004;Morasch & Meckenstock, 2005), a reaction catalyzed by the enzyme benzylsuccinate synthase (BSS). The anaerobic degradation of several aliphatic hydrocarbons and alkylated aromatics is initiated by this reaction type (for an overview, see Heider, 2007), and the gene coding for the alpha subunit of BSS, bssA, was found in several sequence types (Winderl et al, 2007), indicating the existence of several isoenzymes of BSS.…”

Utilization of toluene and xylenes by a nitrate-reducing strain of Pseudomonas maltophilia under low oxygen and anoxic conditions

“…Pseudomonas maltophilia SU1 appears more versatile than other pure cultures of organisms that degrade aromatic hydrocarbons in the absence of oxygen. Pseudomonas T1 described by Evans et al [17,19] utilizes only toluene as a growth substrate, but converts o-xylene to a dead end metabolite when toluene is present in the medium [19]. Dolfing et al [5] and Schocher et al [6] studied a number of strains of Pseudomonas that could degrade toluene, and a number of toluene-related compound under denitrifying conditions.…”