233 Synthesis and discussion BRIDGING ENVIRONMENTAL MICROBIOLOGY WITH PHYSIOLOGY IN BIOREACTORS This dissertation combines bioreactor culturing with meta-omics analyses, activity measurements, and physiological response or adaptation. Here, we reflect on the observed physiological potential biases, costs, and technical difficulties as well as - in the case of bioreactors - the environmental relevance and biotechnological implications inferred per method (Table 1). Exploring Microbial Communities: Amplicon Sequencing vs. Metagenomics To track microbial community structure, we relied on 16S rRNA gene amplicon sequences combined with metagenomics, where sequencing was outsourced to Macrogen (The Netherlands, Europe). Both these sequencing techniques have pros and cons which are discussed below. 16S rRNA amplicon proved to be a very powerful technique to obtain a relatively low-cost high resolution taxonomic profiling of the microbial community across multiple sediment depths (Chapter 2 and 7), incubations (Chapter 7) and diverse experimental time points (Chapter 3 and 6). Still, the functional metabolic data deduced from the taxonomical affiliation obtained through 16S rRNA gene amplicon are limited. Moreover, the SILVA classification obtained is specific to the time of analysis and can vary over time, particularly for rarer microorganisms. For example, in Chapter 7, our targeted putative syntrophic SRB was initially classified as Desulfobulbaceae and later as “uncultured Desulfobacterota”, which more closely matched the GTDB classification obtained from the metagenome. In Chapters 2 to 4 and 6, distinct primer sets were employed for either bacterial or archaeal amplification, in contrast to the more general prokaryotic targeting primers employed in Chapter 7. By using a designated archaeal primer pair, we were able to obtain a higher resolution on the usually less abundant archaeal phyla at the expense of needing to run a complementary qPCR to obtain the bacteria to archaeal ratios (Chapter 5). 8
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