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Benzene is an aromatic compound and harmful for the environment. Biodegradation of benzene can reduce the toxicological risk after accidental or controlled release of this chemical in the environment. In this study, we further characterized an anaerobic continuous biofilm culture grown for more than 14 years on benzene with nitrate as electron acceptor. We determined steady state degradation rates, microbial community composition dynamics in the biofilm, and the initial anaerobic benzene degradation reactions. Benzene was degraded at a rate of 0.15 μmol/mg protein/day and a first-order rate constant of 3.04/day which was fourfold higher than rates reported previously. Bacteria belonging to the Peptococcaceae were found to play an important role in this anaerobic benzene-degrading biofilm culture, but also members of the Anaerolineaceae were predicted to be involved in benzene degradation or benzene metabolite degradation based on Illumina MiSeq analysis of 16S ribosomal RNA genes. Biomass retention in the reactor using a filtration finger resulted in reduction of benzene degradation capacity. Detection of the benzene carboxylase encoding gene, abcA, and benzoic acid in the culture vessel indicated that benzene degradation proceeds through an initial carboxylation step.

Anaerobic benzene degradation was studied with a highly enriched iron-reducing culture (BF) composed of mainly Peptococcaceae-related Gram-positive microorganisms. The proteomes of benzene-, phenol- and benzoate-grown cells of culture BF were compared by SDS-PAGE. A specific benzene-expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI-MS/MS-based shotgun proteomic analysis revealed other specifically benzene-expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY) of Geobacter metallireducens and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (∼ 17 kb) composed of carboxylase-related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX)., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

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.

Here, we present a detailed functional and phylogenetic characterization of an iron-reducing enrichment culture maintained in our lab with benzene as sole carbon and energy source. We used DNA-stable isotope probing to identify microbes within the enrichment most active in the assimilation of (13)C-label. When (12)C(6)- and (13)C(6)-benzene were added as comparative substrates, marked differences in the quantitative buoyant density distribution became apparent especially for uncultured microbes within the Gram-positive Peptococcaceae, closely related to environmental clones retrieved from contaminated aquifers world wide and only distantly related to cultured representatives of the genus Thermincola. Prominent among the other constituents of the enrichment were uncultured Deltaproteobacteria, as well as members of the Actinobacteria. Although their presence within the enrichment seems to be stable they did not assimilate (13)C-label as significantly as the Clostridia within the time course of our experiment. We hypothesize that benzene degradation in our enrichment involves an unusual syntrophy, where members of the Clostridia primarily oxidize benzene. Electrons from the contaminant are both directly transferred to ferric iron by the primary oxidizers, but also partially shared with the Desulfobulbaceae as syntrophic partners. Alternatively, electrons may also be quantitatively transferred to the partners, which then reduce the ferric iron. Thus our results provide evidence for the importance of a novel clade of Gram-positive iron-reducers in anaerobic benzene degradation, and a role of syntrophic interactions in this process. These findings shed a totally new light on the factors controlling benzene degradation in anaerobic contaminated environments.