Functional characterization of an anaerobic benzene‐degrading enrichment culture by DNA stable isotope probing

The flow of carbon under sulfate‐reducing conditions within a benzene‐mineralizing enrichment culture was analysed using fully labelled [¹³C₆]‐benzene. Over 180 days of incubation, 95% of added ¹³C‐benzene was released as ¹³C‐carbon dioxide. DNA extracted from cultures that had degraded different amounts of unlabelled or ¹³C‐labelled benzene was centrifuged in CsCl density gradients to identify ¹³C‐benzene‐assimilating organisms by density‐resolved terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA gene fragments. Two phylotypes showed significantly increased relative abundance of their terminal restriction fragments in ‘heavy’ fractions of ¹³C‐benzene‐incubated microcosms compared with a ¹²C‐benzene‐incubated control: a member of the Cryptanaerobacter/Pelotomaculum group within the Peptococcaceae, and a phylotype belonging to the Epsilonproteobacteria. The Cryptanaerobacter/Pelotomaculum phylotype was the most frequent sequence type. A small amount of ¹³C‐methane was aceticlastically produced, as concluded from the linear relationship between methane production and benzene degradation and the detection of Methanosaetaceae as the only methanogens present. Other phylotypes detected but not ¹³C‐labelled belong to several genera of sulfate‐reducing bacteria, that may act as hydrogen scavengers for benzene oxidation. Our results strongly support the hypothesis that benzene is mineralized by a consortium consisting of syntrophs, hydrogenotrophic sulfate reducers and to a minor extent of aceticlastic methanogens.