212 Chapter 7 RESULTS Long-term incubations with different electron acceptors preserve and enrich “Ca. Methanoperedens” as the key driver of AOM The original carbon isotopic composition of the organic matter in the sediment used for the incubation experiment was -29.11±1.99 ‰ δ13C-TOC. As we used 13C-labeled methane to trace AOM in the slurry incubations with different electron acceptors (Supplementary Table 1), any change towards more positive δ13C-TOC values is caused by the production of new biomass from 13C-methane. Similarly, production of enriched δ13C-DIC indicates active AOM. Accordingly, all incubations showed more or less AOM activity, as indicated by the different enrichment in 13C. Most strikingly, δ13C-DIC in the incubation with added MnO 2 was significantly enriched in 13C relative to the control or bottles with sulfate addition, whereas the δ¹³C value was less positive in the sample when both MnO2 and MoO4 2- were present (one order of magnitude lower than the control). Likewise, both TOC and TC had much more positive δ¹³C values in the solid phase of the MnO2-amended slurry compared to the incubation with sulfate addition or the unamended control. Notably, substantial amounts of sulfate were produced during the longterm incubation with MnO2, while sulfide remained close to the detection limit. By comparison, high concentrations of sulfide (~ 0.5 mM) were detected in the slurry with sulfate addition at the end of incubation (Supplementary Table 1). By 16S rRNA gene sequencing at the end of the incubation period, we find that all incubations were dominated by bacteria, predominantly composed of Chloroflexi and Proteobacteria, including the phylum Aminecenantes (Supplementary Figure 1A and C). Within the archaeal fraction, Euryarchaeota dominated all incubations, particularly the manganese oxide incubation, followed by Woesearchaeota (Supplementary Figure 1A and B). “Ca. Methanoperedens” was retained under all conditions and was most enriched (19.2% relative abundance) in the manganese oxide incubation (Supplementary Figure 1D). In contrast, incubation with both sulfate and molybdate had the lowest relative abundance (0.04%). In slurries amended with other electron acceptors, including the incubation with both manganese oxides and molybdate, the relative abundances of “Ca. Methanoperedens” spp. were between 0.52%
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