Additional file 6: of A new family of uncultivated bacteria involved in methanogenesis from the ubiquitous osmolyte glycine betaine in coastal saltmarsh sediments

Figure S1. Microcosm incubation of salt marsh sediments with trimethylamine (TMA). Samples were taken at 234 hr (T1), 307 hr (T2) and 427 hr (T3) for amplicon sequencing of microbial 16S rRNA genes. Methane was quantified using a gas chromatograph and TMA was quantified using an ion exchange chromatograph. Error bars indicate standard deviation from 3-6 biological replicates. Figure S2. Phylogenetic analyses of 16S rRNA genes of the sulfate-reducing proteobacterial OTUs that were enriched by GBT. Relative abundance of the Desulfobacteraceae, Desulforomonadaceae and Desulfobulbaceae was plotted during GBT enrichment overtime. GBT addition significantly enriched two OTUs (OTU822440212; OTU167393497) that are affiliated to Desulfobacterium of the Desulfobacteraceae family. Figure S3. Genome characteristics and comparative genomes of two Desulfobacterium MAGs against cultivated representatives. Figure S4. Phylogenetic analyses of the archaeal 16S rRNA genes of the Methanococcoides OTUs that were enriched by GBT (OTU810315212) and TMA (OTU823373423). Relative abundance of Methanococcoides in GBT- and TMA- amended microcosms was plotted during the course of enrichment. Figure S5. Genome characteristics and comparative genomes of two Methanococcoides MAGs against cultivated representatives. Figure S6. Amplicon sequencing analyse of 16S rRNA genes from ‘heavy’ (H) and ‘light’ (L) fractions of DNA-stable isotope probing incubations using a) 13C2-GBT and b) 13C3-TMA respectively. All incubations were set up in three biological replicates. Figure S7. Phylogenetic analyses of the RpoB showing the evolutionary relationship of Candidatus ‘Betaina sedimentti’ in the Clostridiales order. Scale bar represents substitution per amino acid position. Figure S8. An overview of the metabolic reconstruction of Desulfobacterium (Bin 22, Heavy_R3, Table 1). This sulfate-reducing bacterium is enriched by GBT in microcosm incubations (Figure S2). (PDF 2133 kb)