238 Chapter 8 The two metaproteomes of the “Ca. Methanoperedens Vercelli” sp. enrichment culture presented in Chapter 6, with more than 10 weeks of difference between sampling points (0% salinity and 1.5%) did not yield significant changes in the identified proteins. This suggests that for such a slow-growing microbial community, either longer time differences or, shorter Hydraulic Retention Times (HRT) are needed to observe measurable changes. Insights into Metabolomics In Chapter 6, we utilized untargeted metabolomics with Liquid ChromatographyMass Spectrometry using a Quadrupole Time-of-Flight detector (LC-MS-qTOF), contrasting this approach with more conventional targeted methods like HighPerformance Liquid Chromatography (HPLC). The key findings in Chapter 6 were primarily driven by data from the untargeted search for compatible solutes accumulating inside the cell as salinity increased, which allowed us to later reconstruct the production pathway using metagenomics data and to target active expression through reverse transcription quantitative PCR (rt-qPCR). One limitation of LC-MS-qTOF is the size limit of the metabolites of interest, especially meant for small sugars or amino acids. In addition, when an interesting candidate metabolite is identified, further MS/MS spectra must be combined with reference metabolites which can be very expensive to purchase or, even need to be chemically synthesized. In the case of N(ε)-acetyl-β-L-lysine, prior knowledge of its function as an osmolyte allowed us to use a methanogenic culture to extract the metabolite from a biological source, thereby reducing the production costs associated with synthesizing it chemically (Chapter 6). Reverse Transcriptase quantitative PCR (RT-qPCR) and qPCR Another key complementary molecular technique was the use of rt-qPCR and qPCR in Chapters 4 and 6, respectively. qPCR is a relatively simple technique compared to metagenomics or 16S rRNA gene amplicon sequencing for obtaining cell abundance (archaea to bacteria) (Chapter 6). In the case of rt-qPCR, additional biases of differential RT efficiencies, primer specificity and normalizer gene (mixed culture) and DNA amplification control are key to allow for a robust measurement. Having said that, if targeted active expression is desired rt-qPCR serves as a much
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