Metabolites Detected during Biodegradation of ¹³C₆-Benzene in Nitrate-Reducing and Methanogenic Enrichment Cultures

The mechanism for anaerobic metabolism of benzene remains unknown. To date, there have been only a few studies reporting metabolites of anaerobic benzene biodegradation, in part because anaerobic benzene-degrading enrichment cultures are not very common and only two isolates have been characterized to date. Phenol and benzoate, metabolites consistent with benzene hydroxylation or benzene carboxylation, have been identified previously in mixed cultures, and more recently benzene methylation to toluene has been proposed as another possible mechanism for anaerobic benzene degradation. In this study, 13C6-benzene was added to nitrate-reducing and methanogenic enrichment cultures and specific 13C-labeled metabolites were monitored over time. The putative metabolites were detected by gas chromatography/mass spectrometry in ether extracts of 100-mL samples of culture taken at each time point. This method of analysis provided the sensitivity required to accurately quantify low concentrations of these compounds. In addition, benzoate trapping was used in an attemptto increase concentrations of upstream metabolites. In both cultures, in the presence and absence of unlabeled benzoate (trap), [ring-13C]-toluene and [ring-13C]benzoate were detected transiently during degradation. The data strongly support initial methylation of benzene to toluene, followed bytransformation to benzoate. Although benzene methylation has been proposed previously, this is the first direct evidence to supportthis pathway. In the methanogenic culture only, 13C6-phenol was also detected. The transient appearance of phenol, which appeared to be further transformed to benzoate, suggests that a pathway involving hydroxylation to phenol, as proposed in other studies, was also operative.