143 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea Long term sulfide exposure in bioreactor A 2 L bioreactor (Applikon, Delft, The Netherlands) was anoxically inoculated with 2 L of granular biomass from the anaerobic methanotrophs described above. The bioreactor experiment ran for 73 days and was operated as a sequencing fed-batch reactor (SBR). The sequences consisted of a 24-hour cycle: 22 h 50 min of medium feed (~ 600 ml/day) or sulfide (2.5 mM/day with ~ 100 ml/day, during long-term exposure), 15 min of settling, 45 min of supernatant removal and 5 min of buffer time in between cycle changes (total of 10 min), resulting in approximately three days of hydraulic retention time. The bioreactor contained two standard sixblade turbines operating at 180 rpm and was maintained at room temperature (18 -22 ºC). The pH was buffered with a 100 g/l KHCO3 solution and controlled by a BL 931700 pH controller Black Stone (Hanna Instruments, Rhode Island, USA) (Supplementary Fig. 1B). The bioreactor was continuously fed with CH4 at a flow rate of 10 ml/min and sparged with additional Ar:CO2 (95/5) (~ 100 ml/min) during the settling time and supernatant removal. After an acclimatization period, the bioreactor experiment was divided into three parts: (i) sulfide toxic pulse response (0.5 mM) (from T0 to T1), (ii) sulfide long-term exposure period (0.25 mM/day) for approximately 6.5 weeks and (iii) second toxic pulse response (0.5 mM) (from T2 to T3) (Figure 1). The methane oxidation rate was determined during the three periods: control, toxicity and exposure. The 13C-CH4 activity assays were performed with the entire bioreactor and lasted for 5 days. During the test, the bioreactor was operated in batch mode with 20% 13C-CH4 and additional N2 in the headspace to achieve an overpressure of 1.2-1.4 bar. The 0.5 L headspace was first flushed for 2-3 h with Ar:CO2 (95:5) to remove residual methane traces and stirring was increased to 250 rpm to allow for increased methane diffusion. The initial nitrate concentration was approximately 1-1.5 mM. Over the 5 days, labelled 13C-CO 2 and 12C-CO 2 were measured by GC-MS as aforementioned. The nitrate concentration (MQuantTM strips), overpressure and pH were measured and controlled manually. The methylene blue assay was employed for sulfide analysis using the HACH 8131 method (1.5-50 µM) (HACH, Loveland, CO, USA) and measured immediately at specified time points before and after DNA sampling (T0-T1 and T2-T3) (Figure 1). The dry weight (n=3, 10 ml) of the biomass used was determined after drying for 3 days at 100ºC. 5
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