207 Methanotrophic flexibility of “Ca. Methanoperedens” and SRB in meromictic Lake Cadagno of Figures 1, 4 and Supplementary Figure 4 from Su et al. (2020). Based on these results we selected specific sediment layers for metagenomic sequencing (23 cm & 25 cm) and for further long-term slurry incubation experiments (depths 19-24 cm) (Figure 1B). Sediment core (cm) 19 25 Unamended control Nitrate FeOx MnO2 MnO2 + MoO4 2SO4 - + MoO4 2- SO4 20 35 0 35 0 35 0 8 0 35 8 Relative abundance (%) AOM-R Sulfate Sulfide Depth (cm) All incubations included: - 13C-CH4 activity assays -Chemical characterization - 16S rRNA gene amp bacteria and archaea 5 conditions selected for metagenomics 23 “Ca. Methanoperedens” (ZOTU: 202, 928,1102,1150) SRB Desulfobacteraceae Uncultured Desulfobacterota (ZOTU 307) Methane 19 25 3-year incubated slurry samples (mix 19-24 cm) A B Figure 1. A Cartoon summarizing previous anaerobic oxidation of methane (AOM) data from Lake Cadagno sediment (Su et al., 2020). Top: Depth‐specific in situ anaerobic oxidation of methane oxidation rates (AOM-R) determined using 13C-CH4 tracer whole‐core incubations, and profiles of porewater concentrations of dissolved methane, sulfate, and sulfide. Bottom: Depth profiles of 16S rRNA gene amplicons (relative abundances) of “Ca. Methanoperedens spp.” and SRBs in the sediment. B Overview of long-term slurry incubation assays presented in the current study, including the seven conditions employed and the parameters analyzed. The sediment samples and incubations selected for metagenomics are colored in pink. Long-term slurry incubations We established static slurry incubations with fresh sediment material (depths 19 - 24 cm), collected from the center of Lake Cadagno (46°33”01.89”“N, 8°42”42.04E) in October 2017. In an anoxic chamber with an N2 atmosphere, we mixed the sediment material with anoxic freshwater medium in a volume ratio of ~ 1:4, and dispensed 100 mL of this slurry into 250 mL serum bottles. We supplemented the slurries with either NO3 - (0.5 mM), synthesized δ-MnO 2 (10 mM), ferrihydrite (Fe5O8H·H2O, 10 mM), or sulfate (4.8 mM). Molybdate (MoO4 2-) (20 mM) was added as 7
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