255 Synthesis and discussion in sulfide toxicity. Furthermore, gene-centric modelled biogeochemistry will benefit from coupling qPCR abundance data of archaea, bacteria, or specific microbial groups or functional markers (e.g., specific to ANME groups) with multi-omics approaches, which will help validate the modelled processes for each ecosystem of interest. Chapter 3 defined the Methylomonadaceae as a competitive methanotroph under nitrogen-reducing conditions, contributing under microaerophilic conditions to either denitrification or DNRA depending on nitrate excess or limitation, respectively. Moreover, we enriched for divergent Cu-MMO classified as Pseudomonadales IMCC2047 and Rugosibacter. Next steps should include the targeted enrichment of poorly characterized Methylomonadaceae with flexible electron acceptor potential and the putative short alkane degrading microorganisms. In this regard, we propose strategies such as Cu-MMO enzyme containing labelling via activity-based protein profiling (ABPP) combined with cell sorting. This approach has previously been successful to enrich for both for comammox bacteria, AOB, and MOB (Sakoula et al., 2022). In fact, this allows for the most unbiased approach to select for microorganisms in which their electron donor (diverse short alkanes e.g. methane, ethane, butane, propane, ammonium) or acceptor (oxygen, reduced nitrogen compounds, metals) are not defined and might not be exclusive to one specific microorganism. Highly enriched or pure cultures should then be used to test activity under different electron donors (for divergent Cu-MMO holders), acceptor affinity (oxygen, nitrate, or metals for Methylomonadaceae species) and/or low oxygen and high sulfide adaptation-mechanisms coupled to active expression via (meta)transcriptomics. In the case of Methylomonadaceae, considering the potential for EET via MHC, growth in bioelectrochemical systems or solid electron acceptors systems could be an exciting prospect. In Chapter 4, we monitored the activity and transcriptional changes of an anoxic stable co-culture of sulfide oxidizing denitrifiers, anammox, and N-DAMO to nitrogen deprivation and sulfide and nitric oxide stresses. We present the possibility to scale up a similar culture with or without sulfide oxidizing denitrifiers to a pilot saline or freshwater WWTP, respectively. This system could aim at highrate activate sludge combined with a controlled nitration step. To obtain highest 8
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