Nicotinamide br Conclusion br Introduction Biological wastew
Introduction Biological wastewater treatment is targeted towards the removal of phosphorous, nitrogen and organic substances by the metabolic activity of a diverse activated sludge microbial community. More precisely, it is based on the versatile catalytic activity of the microbial enzymes. It is generally recognized that the metabolic potential of microorganisms and especially of bacteria is immense. Despite the manifold experimental evidence that microbial isolates are able to transform a large variety of xenobiotics (Abu Laban et al., 2009, Kolvenbach et al., 2014), analytical surveys have shown that many polar organic micropollutants such as pharmaceuticals, biocides and personal care products are not or incompletely removed in conventional wastewater treatment (Daughton and Ternes, 1999). As a consequence, micropollutants and their transformation products (TPs) are released into rivers and streams, from where some even pass into ground water and drinking water, potentially causing adverse effects on aquatic ecosystems and human health (Garcia-Rodriguez et al., 2014). Efforts to characterize the microbial Nicotinamide and its function on a molecular level have been hindered by the huge complexity of activated sludge and the dynamics of the wastewater treatment process (wastewater composition, seasonal changes, geographical differences, etc.). In the past decade, at least some of these methodological limitations were overcome through the advent of high-throughput sequencing techniques. As in many other research areas, the rapid increase in sequence information pushed forward techniques which facilitated the elucidation and systemic understanding of biodegradation pathways. These techniques were also applied to wastewater treatment to varying extents, ranging from qPCR (Helbling et al., 2012b) to FISH (Ettwig et al., 2009, Lolas et al., 2012), metagenomics (Martin et al., 2006) or metatranscriptomics (Helbling et al., 2012a), 16S-amplicon based taxonomic community profiling (Vanwonterghem et al., 2014) and metaproteomics (Collado et al., 2013, Hansen et al., 2014, Wilmes et al., 2008). The abovementioned molecular techniques are state-of-the-art for the identification of candidate organisms, genes or gene products (transcripts, enzymes) likely to be involved in micropollutant biotransformation. The advantage of these molecular techniques is that they avoid culturing biases as they are directly applicable to complex environmental samples. Meta-omics methodologies furthermore aspire to characterize the entire pool of genes or gene products present in a community at a given time, to establish statistical associations with biotransformations. These meta-omics techniques, however, still depend on the results of biochemical experiments with extracted, native enzymes to establish causality to micropollutant biotransformation (Johnson et al., 2015). Studies with native enzymes originating from specialized or pure cultures help to reliably assign function to unknown proteins, thereby improving database annotations (Mills et al., 2015) and mechanistic understanding of degradation processes (Prior et al., 2009, Xu et al., 2015). However, pure or enriched laboratory cultures cannot provide reliable information about the diversity and activity of the enzymes actually involved in micropollutant degradation under environmental conditions. Therefore, the exact identities, organismal origins and subcellular localizations of the involved candidate enzymes in situ are still largely unknown. Furthermore, the variety of enzymes (and organisms) and the metabolic or co-metabolic nature of the biotransformation of a certain micropollutant remain unclear (Fischer and Majewsky, 2014). To unravel the complex biodegradation processes in environmental systems, it is therefore essential to complement meta-omic studies with experiments using native, i.e. active enzymes from environmental samples (Arnosti et al., 2014). Studies of native activated sludge enzymes so far mainly focused on enzymes which are thought to convert conventional wastewater substrates like organic and inorganic nitrogen and phosphorous compounds. These are predominantly hydrolytic enzymes such as peptidases (formerly known as proteases (Barrett et al., 2013)), phosphatases, glucosidases, and lipases, which are being monitored via robust colorimetric or fluorometric assays based on the turnover of commercially available substrate derivatives (Burgess and Pletschke, 2008a, Cortés-Lorenzo et al., 2012, Gómez-Silván et al., 2013). Also some non-hydrolytic enzymes, e.g. catechol dioxygenases have been assayed, relying on the use of proxy substrates (Grekova-Vasileva and Topalova, 2009, Khunjar et al., 2011).