141 Physiological stress response to sulfide exposure of freshwater anaerobic methanotrophic archaea Here, we employed a “Ca. Methanoperedens” bioreactor enrichment culture as an ANME model organism to study the effects of and tolerance to sulfide stress. After short- and long-term exposure to sulfide, we measured methane oxidation potential via 13C-CH4 activity assays and physicochemical measurements. We also investigated the use of specific storage polymers, Polyhydroxyalkanoates (PHAs). Additionally, we employed metagenomics and metatranscriptomics to identify which genes and processes respond to sulfide stress. MATERIAL AND METHODS “Ca. Methanoperedens” enrichment bioreactor and medium The enrichment culture used in this study performed nitrate/nitrite-dependent anaerobic methane oxidation (N-DAMO), with a mixed microbial community dominated by “Ca. Methanoperedens BLZ2” sp. and nitrite-scavenging partner “Ca. Methylomirabilis oxyfera at 30 °C”. and nitrite-scavenging partner “Ca. Methylomirabilis oxyfera” (Arshad et al., 2015; Berger et al., 2017; Ettwig et al., 2009). The inoculum used for the original enrichments originated from Twentekanaal (52° 11′ 04″ N and 6° 24′ 40″ E, The Netherlands), as detailed by Raghoebarsing et al. (2006) and further enriched with “Ca. Methylomirabilis oxyfera” as described by Arshad et al. (2015). The mineral medium used for the microcosm and bioreactor experiment contained (per liter) 240 mg of CaCl2 ·2 H2O, 50 mg of KH2PO4, 160 mg of MgSO4 ·7 H2O together with 0.5mL of trace elements and 0.1 ml vitamins solution composition were employed as specified in (Kurth et al., 2019). To avoid iron-sulfur precipitates during the the microcosm and bioreactor sulfide pulse toxicity experiments (3 days before the start of “toxicity” and “exposure” activity assays), trace elements excluded iron. Nitrate and nitrite were monitored daily in the bioreactor using MQuantTM test strips (Merck, Darmstadt, Germany). Samples for sensitive nitrate, nitrite, and ammonium determination measurements were taken several times per week. Ammonium was determined using a high-sensitivity protocol (range from 0.5-5 mM) after reaction with 10% orthophthaldialdehyde as previously described (Taylor et al., 1974). Nitrate and nitrite concentrations were monitored using MQuantTM colorimetric test strips (Merck, Darmstadt, Germany) (Supplementary Figure 1A). 5
RkJQdWJsaXNoZXIy MTk4NDMw