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Beyond pollutant removal - understanding the biochemical mechanism of sulfonamide degradation in wastewater and the role of ipso-substitution

Beyond pollutant removal - understanding the biochemical mechanism of sulfonamide degradation in wastewater and the role of ipso-substitution

Harald Horn
Marius Majewsky








01.09.2015 - 31.08.2017


Sulfonamide antibiotics are widely used both for the treatment of humans and livestock. Due to their recalcitrance, they are only insufficiently removed in wastewater treatment plants and can therefore be readily detected in effluents, in surface waters and groundwater. Constant exposure of microorganisms even to non-inhibitory concentrations has been shown to induce the propagation of antibiotic resistances, which is a cause of concern with regard to the rising number of reports on multi-resistant pathogens. The biodegradation processes of this class of antibiotics have to be more intensively studied to allow for successful optimization of removal during wastewater treatment.

Microbacterium sp. strain BR1 has been isolated from an enrichment culture from activated sludge and is able to degrade sulfonamide antibiotics as a source of carbon and energy. In this strain, enzymes and their encoding genes have been identified which are responsible sulfonamide degradation. An FMN-dependent monooxygenase, encoded by the sadA gene, was shown to catalyze ipso-substitution of sulfonamides, initiating their fragmentation to 4-benzoquinone-imine, sulfite and the moiety previously attached to the sulfo group. While 4-benzoquinone-imine serves as the source of carbon and energy, the mostly heterocyclic moiety was shown to remain a dead-end metabolite in culture supernatants.

In the proposed project, we will evaluate whether the identified dead-end metabolites can also be linked to biological sulfonamide degradation. At the same time it will be investigated if the presence of homolog bacterial genes detected in selected wastewater treatment plants can be attributed to sulfonamide degrading activity. We will obtain genome sequences from further isolated sulfonamide degrading strains to investigate the presence of sadA homologs. From homolog sequences, a consensus sequence will be calculated to serve as a base for PCR primers to specifically amplify and quantify sadA homologs in activated sludge. Sequences flanking these homologs will be analyzed for the presence of gene clusters resembling that in Microbacterium sp. strain BR1 Sulfonamide removal rate of the sludges will be determined by quantification of sulfonamides and dead-end metabolites in influent and effluent of the wastewater treatment plants. Additionally, the ability of the sludge to degrade artificially added sulfamethoxazole will be assayed. For pure strains, sulfonamide degradation at various nutrient conditions will be assayed to establish thresholds for up- or down-regulation of genes associated with ipso-substitution of sulfonamides. Our overall aim is to transfer findings from the ongoing project funded by the SNF and others to provide a better understanding of sulfonamide degradation in wastewater treatment plants, compared to the current practice, which is a mere balance of influent and effluent concentrations of the parent compound, or at best acetylated derivatives of that. Bringing all ends together, it will be possible to assess how much sulfonamides are really degraded, and which contribution to that stems from homologs of the FMN-dependent monooxygenase isolated in the currently ongoing project.