147 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea Polyhydroxyalkanoates (PHAs) quantification The PHA amount in the “Ca. Methanoperedens” sp. enriched biomass was measured at the same time points as the genomic samples were taken (Figure 1). The PHAs were hydrolyzed to polyhydroxy acids and these acids were then methylated. These methylated (poly)hydroxy acids were analyzed by GC-MS. Refer to Echeveste Medrano et al. (2024a) for detail extraction protocol. RESULTS AND DISCUSSION Methane oxidation by “Ca. Methanoperedens” is inhibited by sulfide exposure Batch incubations (“Batch”) were used to assess the sulfide inhibitory effect on the “Ca. Methanoperedens BLZ2” sp. enrichment using two sulfide pulses of 0.25 mM and 0.5 mM as stressors (Figure 2). At 0.25 mM sulfide exposure, we observed a drop of approximately 20% in activity within 24 h, but a recovery to 125% within 5 days. At 0.5 mM sulfide the activity was reduced to just 20% of the control, and after 5 days the activity was 70% compared to the control (Figure 2). Follow-up bioreactor experiment used 0.5 mM sulfide as the toxic threshold, whereas 0.25 mM for long term exposure. Briefly, in the long-term sulfide exposure experiment, the bioreactor (Figure 1) was spiked with 0.5 mM sulfide, followed by daily 0.25 mM for 6.5 weeks and followed by a second 0.5 mM spike. Exposure of the bioreactor biomass to 0.5 mM sulfide resulted in a drop to about 50% of the original activity at 24 h with a recovery to about 70% at 5 days. Surprisingly, the long-term exposure at approximately 0.25 mM/day did not help the culture adapt to sulfide. With the second 0.5 mM sulfide pulse (Figure 2), the activity dropped to only 25% with a slight recovery to 33% after 5 days compared to the control. Nitrate was almost always fully consumed during the monitoring period, with some short-term accumulation (200-400 µM) during the long-term sulfide exposure (Supplementary Figure 1A). Nitrite was mostly below the detection limit, except on days 18, 28 and 66 when 200 -800 µM nitrite was detected (Supplementary Figure 1A). In line with previous “Ca. Methanoperedens” experiments, DNRA occurred, 5
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