182 Chapter 6 plus Orbitrap, ThermoFisher Scientific, Bleiswijk, Netherlands). For the sialidase activity measurements, lyophilized biomass was added into enough volume of PBS for 60 min to make the biomass swell. Afterwards, the samples were centrifuged at 2,000 g for 10 min at 4°C. 10 mg of the pellet was collected and added into 1 ml of PBS. After vortexing for 1 min, the sialidase activity of the samples was measured. 10 μl of the homogenized sample was mixed with 40 μl of PBS and 50 μl of 100 μM 4MU-Neu5Ac (2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic Acid) and incubated at 37°C for 60 min. After the addition of 100 μl of 0.5 M sodium carbonate buffer, the fluorescent intensity was measured (ex/em, 355 nm/460 nm) with using 96 well black plates. 10 µl of PBS was applied as negative control whereas 10 µl of 0.63 μg/ml AUSA was the positive control. The autofluorescence intensity of each sample was also determined by adding 10 μl of the sample into 90 μl of PBS. The sialidase activity was calculated by subtracting the autofluorescence intensity of the sample from the fluorescence intensity of the sample. RESULTS “Ca. Methanoperedens” partially adapted to increasing salt concentrations We investigated the biogeography of our “Ca. Methanoperedens” species through a database study of publicly available metagenomes. We found that our Methanoperedenaceae archaea, “Ca. Methanoperedens Vercelli Strain 1”, predominated in permafrost metagenomes (~ 50% of the total metagenomesource recovered), followed by freshwater sediments (~ 25%), bioreactors, and peat ecosystems (~ 25%) (Supplementary Fig. 2 and Supplementary Table 2). To get a first impression on the salt stress response of “Ca. Methanoperedens”, we performed microcosm experiments with biomass of a “Ca. Methanoperedens” enrichment culture containing “Ca. Methanoperedens” originating from a eutrophic freshwater rice paddy field in Italy. With respect to freshwater conditions, direct exposure to 0.5 % salinity resulted in a loss of almost half of the activity at the final time point, and a total loss of activity at 2% salinity (Supplementary Fig. 3). We still observed methane oxidation at 1%, although with a slower acclimation (Supplementary Fig. 3). To test if gradual exposure to salt stress would result in
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