In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited.

We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and ¹³C-toluene in situ protein-based stable isotope probing (Protein-SIP).

The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales which contributed each about 20% to total taxon abundances. Protein-SIP data revealed that members of Burkholderiaceae, which proteins showed about 73% of ¹³C-incorporation, were main degraders of toluene in the planted system, while members of the Comamonadaceae were involved to a lesser extent in degradation (about 64% ¹³C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii.

We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes which are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.