239 Synthesis and discussion simpler, high resolution and cheaper technique than metatranscriptomics, as concluded for Chapters 3 and 6. Methane oxidation potential activity assays Activity assays with 13C-CH4 were employed for almost all Chapters (2, 5 to 7). This technique is the most frequently employed method to estimate methane oxidation potential in slow growing cultures. We utilized activity assays both in batch (Chapters 2, 5 to 7) and bioreactors (Chapters 5 and 6). Batch activity assays allowed for increased replicability (n=2-3) at the expenses of losing acclimation (direct exposure to salt in Chapter 6) and removal of toxic compound build up (sulfide in Chapter 7) that was circumvented using bioreactor-based activity assays (Chapters 5 and 6). Still, both batch and clumping bioreactor experiments required manual pressure and pH control together with close monitoring of electron acceptor limitation such as nitrate or toxic nitrite build up (Chapters 5 and 6). Visualization techniques For the microbial community characterization, FISH coupled to Confocal Laser Scanning Microcopy instead of Epifluorescence Microscopy or Transmission Electron Microscopy (TEM) remained the most adequate approach. In fact, we could combine both function (PHA) to identity (“Ca. Methanoperedens”) as in Chapter 6 and use the Z-stack depth to characterize the granular composition. In our case, for Chapter 6, Leica SP8-White Light Laser Confocal Microscope with Pulsed White Light Laser (WLL), offering simultaneously up to eight lines from 470 nm to 670 nm and excitation as low as 405 nm, was deemed very adequate to be able to adjust some low excitation fluorophore wave lengths such as the Alexa350-fluorophore, which did not overlap with the Nile Red PHA signal. Still, Confocal Microscopy coupled to Double Labeling of Oligonucleotide Probes (DOPE)-FISH remained insufficient to capture the free-living fraction of the “Ca. Methanoperedens” enrichments employed. This can be both due to low activity related to the stress that cells were subjected to or, the fact of employing a Sequencing Batch Reactor (SBR) that slowly removes suspended cells from the vessel. 8
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