In situ protein‐SIP highlights Burkholderiaceae as key players degrading toluene by para ring hydroxylation in a constructed wetland model

Abstract

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 (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-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 that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.

Aerobic Toluene Degraders in the Rhizosphere of a Constructed Wetland Model Show Diurnal Polyhydroxyalkanoate Metabolism

“…Such PFR systems were further used to investigate microbial degradation of the aromatic hydrocarbon toluene by isotope fractionation and DNA microarray analysis, 16S rRNA amplicon sequencing (24), and protein-based stable isotope probing (protein-SIP) (25). These studies revealed that toluene was predominantly or even exclusively degraded aerobically by members of the Burkholderiales via a sequential monooxygenation of the aromatic ring followed by ring cleavage through an extradiol dioxygenase.…”

Microbial Community Assessment in Wetlands for Water Pollution Control: Past, Present, and Future Outlook

“…Proteins corresponding to quantified mRNA were not found, however the proteomic survey was a general success. A follow up study by Lünsmann et al [41] utilized proteomics, stable isotope analysis and metagenomics to further elucidate a specific bacterial family (Burkholderiaceae) and functional pathways mainly responsible for toluene degradation in lab-scale TWs.…”

“…Stable isotope analysis was also sometimes classified as a type of activity measure. For example, Braeckevelt et al [45] used stable isotope analysis to study the degradation of monochlorobenzene, and Lünsmann et al [41] for the study of toluene degradation. It was however admittedly difficult to properly categorize these studies (and other isotope studies) within the four-category construct (activity, enumeration, function, and structure) of this meta-analysis.…”

“…It was however admittedly difficult to properly categorize these studies (and other isotope studies) within the four-category construct (activity, enumeration, function, and structure) of this meta-analysis. For example, Lünsmann et al [41] used isotope analysis for identifying specific bacteria involved in toluene degradation which could perhaps be said to be aimed at a more structural analysis objective. Another example was that of Martinez-Lavanchy et al [40] who used isotope analysis as a second line of evidence to verify functional pathways, which could have been classified as a functional assessment measure rather than an activity based measure.…”

“…For example, the study of Lünsmann et al [41] required collaboration among departments of Biotechnology, Proteomics, Environmental Biotechnology, Isotope Biogeochemistry, Metabolomics, and a Microbial Interactions and Processes Research Group. It is expected that additional collaborations will be developed in the coming years and that TW microbial community research will be multiphasic reaching beyond the current horizons and starting new paradigms.…”