161 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea 0 0.025 0.05 0.075 T0 T1 T2 T3 PHA derivatives (area/is/mg) * Figure 4. Polyhydroxyalkanoate (PHA) derivatives measured by Gas Chromatography-Mass Spectrometry (GC-MS) across the four different conditions (x-axis). PHA derivative quantities were normalized using an internal standard (IS) and dry weight (mg). Significant differences (t-test) are indicated with an asterisk (P<0.05). PHAs have been previously described as “Ca. Methanoperedens” storage polymers (Cai et al., 2019; Echeveste Medrano et al., 2024b, Chapter 6; Frank et al., 2023; Yu et al., 2018). Concomitant with a previous salt stress experiments on “Ca. Methanoperedens”, we observed PHA reduction upon 6.5 weeks of sulfide exposure (Figure 4) (Echeveste Medrano et al., 2024b, Chapter 6; Frank et al., 2023). Nitrate dependent PHA usage could therefore function as a stress mechanism saving the methane oxidation (reduced MCR gene expression from T0-T2 & T0-T3) for energy metabolism (Table 1 and Figure 4). DNA and RNA-based indications of morphotype shift upon long-term sulfide Our investigation included the monitoring of “Ca. Methanoperedens” morphotypes during the various exposure periods. Actually, previous salt-stress experiments transcriptional shifts and DNA-biomass fractionation of granulated versus suspended cells were described (McIlroy et al., 2023). In our study, we first filtered the “Ca. Methanoperedens” biomass into two fractions: retentate (granular) and filtrate (planktonic) (Figure 1). We were unable to obtain enough DNA for the planktonic fraction at time points T0 and T1 for metagenomic sequencing, whereas samples at T2 and T3 gave high enough values to proceed (Supplementary Figure 3). Read-based classification of the granular fraction of “Ca. Methanoperedens” 5
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