Biotransformation of halogenated aromatics and proteomic profiling of microbial communities in cultures from urban hyporheic zones

The increased presence of trace organic contaminants in wastewater, including halogenated aromatics, poses a challenge for urban water management. Halogenated aromatics are often only partially removed by conventional wastewater treatment and may accumulate in the environment, particularly in partially closed urban water cycles, threatening environmental and human health. Hyporheic zones, regions within riverbeds where groundwater and surface water mix, feature diverse redox conditions and high microbial activity that can facilitate trace organic compound attenuation. Yet, the specific mechanisms driving the attenuation of halogenated aromatics remain poorly understood. Understanding microbial transformation processes in hyporheic zones requires methods that link microbial community composition to metabolic activity. Protein mass spectrometry, including metaproteomics and protein-stable isotope probing (protein-SIP), can link microbial taxa to activity but depend for environmental samples on metagenome-derived databases, which are costly and time-intensive to obtain, limiting their broader use. The aim of this thesis was to develop and apply different protein mass spectrometric approaches to analyze microbial community compositions in environmental samples without relying on metagenome-derived databases. Three distinct strategies were explored: (1) GroEL-proteotyping, which uses the conserved chaperonin GroEL as a universal, separable taxonomic marker, enabling profiling with a sample-independent database but limited taxonomic resolution; (2) GroELproteotyping-based SIP, integrating GroEL-proteotyping with stable isotope probing to link identified taxa to metabolic activity via isotope incorporation into GroEL; and (3) de novo peptide sequencing, which generates sample-specific databases for metaproteomics and protein-SIP directly from mass spectra, enabling species-level resolution while preserving functional insights. Batch experiments with defined bacterial mixtures demonstrated that GroEL SIP can distinguish taxa based on substrate usage and transformation pathways. Microcosm experiments with inocula from the hyporheic zone showed that sitagliptin was biotransformed under sequential anoxic/oxic conditions, while other halogenated aromatic pharmaceuticals were not. Applying the developed methods to microcosms enabled benchmarking against 16S rRNA gene amplicon sequencing and metaproteomics with metagenome-derived databases, revealing microbial taxa and enzymes potentially involved in sitagliptin transformation. Several sitagliptin transformation products were detected in both microcosms and river sediment pore water. The abundance of transformation products observed in anoxic batch experiments correlated with redox indicators in the pore water, underscoring the environmental relevance of the observed processes. The transformation products were less toxic than sitagliptin, highlighting the detoxification potential of hyporheic zones. Overall, this work introduces broadly applicable protein mass spectrometry methods for microbial ecology, elucidates the fate of sitagliptin, its transformation products and potentially involved microbial populations in the transformation process, highlights the importance of difference redox conditions in the transformation process and provides evidence for the detoxification of trace organic contaminants in hyporheic zones. By this, this work paves the way for harnessing microbial processes in hyporheic zones to mitigate trace organic contaminants in urban water systems.