Stable-isotope probing combined with amplicon sequencing and metagenomics identifies key bacterial benzene degraders under microaerobic conditions.

The primary aim of the present study was to reveal the major differences between benzene-degrading bacterial communities evolve under aerobic versus microaerobic conditions and to reveal the diversity of those bacteria, which can relatively quickly degrade benzene even under microaerobic conditions. For this, parallel aerobic and microaerobic microcosms were set up by using groundwater sediment of a BTEX-contaminated site and ¹³C labelled benzene. The evolved total bacterial communities were first investigated by 16S rRNA gene Illumina amplicon sequencing, followed by a density gradient fractionation of DNA and a separate investigation of "heavy" and "light" DNA fractions. Results shed light on the fact that the availability of oxygen strongly determined the structure of the degrading bacterial communities. While members of the genus Pseudomonas were overwhelmingly dominant under clear aerobic conditions, they were almost completely replaced by members of genera Malikia and Azovibrio in the microaerobic microcosms. Investigation of the density resolved DNA fractions further confirmed the key role of these two latter genera in the microaerobic degradation of benzene. Moreover, analysis of a previously acquired metagenome-assembled Azovibrio genome suggested that benzene was degraded through the meta-cleavage pathway by this bacterium, with the help of a subfamily I.2.I-type catechol 2,3-dioxygenase. Overall, results of the present study implicate that under limited oxygen availability, some potentially microaerophilic bacteria play crucial role in the aerobic degradation of aromatic hydrocarbons.