Identification of benzene-degrading Proteobacteria in a constructed wetland by employing in situ microcosms and RNA-stable isotope probing

Constructed wetlands (CWs) are effective ecological remediation technologies for various contaminated water bodies. Here, we queried for benzene-degrading microbes in a horizontal subsurface flow CW with reducing conditions in the pore water and fed with benzene-contaminated groundwater. For identification of relevant microbes, we employed in situ microcosms (BACTRAPs, which are made from granulated activated carbon) coupled with C-13-stable isotope probing and Illumina sequencing of 16S rRNA amplicons. A significant incorporation of C-13 was detected in RNA isolated from BACTRAPs loaded with C-13-benzene and exposed in the CW for 28 days. A shorter incubation time did not result in detectable C-13 incorporation. After 28 days, members from four genera, namely Dechloromonas, Hydrogenophaga, and Zoogloea from the Betaproteobacteria and Arcobacter from the Epsilonproteobacteria were significantly labeled with C-13 and were abundant in the bacterial community on the BACTRAPs. Sequences affiliated to Geobacter were also numerous on the BACTRAPs but apparently those microbes did not metabolize benzene as no C-13 label incorporation was detected. Instead, they may have metabolized plant-derived organic compounds while using the BACTRAPs as electron sink. In representative wetland samples, sequences affiliated with Dechloromonas, Zoogloea, and Hydrogenophaga were present at relative proportions of up to a few percent. Sequences affiliated with Arcobacter were present at < 0.01% in wetland samples. In conclusion, we identified microbes of likely significance for benzene degradation in a CW used for remediation of contaminated water.