35 Introduction and thesis outline Figure 3 below depicts methods that can be utilized to explore the physiology of “Ca. Methanoperedens” in bioreactor systems. Common approaches to study “Ca. Methanoperedens” include activity assays (13C-CH₄) labeling to infer methane oxidation potential, as well as metagenomics and metatranscriptomics to analyze genetic potential and transcriptional activity. ‘Ca. Methanoperedens’ “Ca. Methanoperedens” marine ANME Metagenome Metatranscriptome Metaproteome Metabolomics rt-qPCR qPCR Sialic acids FISH NO3 - MnO2 Fe(OH)3 PHA Activity Assays CH4 13 C-CH4 13C-CO 2 Figure 3. Cartoon illustrating a wetland as an exemplary environment for enriching and characterizing “Ca. Methanoperedens” in a bioreactor. The most common habitats for “Ca. Methanoperedens” include freshwater (eutrophic) organic-rich anoxic environments, such as wetlands. In contrast, other anaerobic methanotrophic archaea (ANME) and those involved in sulfate-dependent anaerobic oxidation of methane are found in marine settings. Various methods employed in this PhD dissertation for characterizing an enrichment culture of “Ca. Methanoperedens” are categorized into activity, microbial community structure analysis, spatial distribution/morphology examination, and evaluation of granules or extracellular polymeric substance (EPS) layers. Methane oxidation potential assays are represented by the monitoring of 13C-CO₂ labeled production signal derived from 13C-CH₄. Reverse Transcriptase (RT)-quantitative PCR (RT-qPCR); Polyhydroxyalkanoates (PHAs). Images were obtained from Biorender and the Integration and Application Network Symbol Library from the Center for Environmental Science of the University of Maryland (USA) and edited using Adobe Illustrator 2024. 1
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