Supplementary Information Formate-H 2 -lyase, a Driver of Hydrogenotrophic Processes in the Root-zone of a Methane-emitting Fen Sindy Hunger, Oliver Schmidt, Anita S. Gößner, and Harold L. Drake Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany Additional references cited in Supplement: Hurlbert, S.H. (1971) Nonconcept of species diversity-critique and alternative parameters. Ecology 52:

Supplemental Figure S1. Rarefaction analysis of in silico-translated amino acid sequences of amplified hydrogenase gene fragments and mcrA/mrtA fragments. 95% confidence intervals are shown. Phylotypes were calculated based on a 80% similarity cut-off for hydrogenase gene sequences (Schmidt et al., 2011) and 85.7% similarity cut- off for mcrA/mrtA-encoded amino acid sequences (Hunger et al., 2011a). Curves were calculated according to the Hurlbert rarefaction (Hurlbert, 1971) using the “aRarefact” software ( Color Code: blue, roots before incubation; brown, unsupplemented root slurries after 28 days of incubation; green, formate-supplemented root slurries after 28 days of incubation (Fig. 1).

Supplemental Figure S2. Phylogenetic tree of in silico-translated amino acid sequences derived from [FeFe]- hydrogenase genes (boldface) and closely related sequences. GenBank accession numbers are indicated. Relative abundances of phylotypes (PLT) in the different clone libraries are given in parentheses: Roots before incubation/ unsupplemented root slurries after 28 days of incubation/ formate-supplemented root slurries after 28 days of incubation. Sequences correspond to residues 183 to 375 of the Desulfovibrio vulgaris hydrogenase (AAS96246). The phylogenetic tree was calculated using the maximum parsimony method. Empty circles at nodes indicate congruent nodes in either the neighbor joining or the maximum likelihood tree. Filled circles indicate congruent nodes in all three trees. Bootstrap values are from the maximum parsimony tree (100 resamplings) and are only displayed at nodes congruent in all three trees. The bar indicates a 0.1 change per amino acid. The hydrogenase of Thermotoga maritima (AAD36496) was used as outgroup. Color code: blue, Firmicutes; yellow, Proteobacteria; green, Bacteroidetes; orange, Spirochaetes; pink, Verrucomicrobia; purple, Chlorobi; uncolored, unaffiliated.

Supplemental Figure S3. Phylogenetic tree of in silico-translated amino acid sequences derived from group 4 [NiFe]-hydrogenase genes (boldface) and closely related sequences. GenBank accession numbers are indicated. Relative abundances of phylotypes (PLT) in the different clone libraries are given in parentheses: Roots before incubation/ unsupplemented root slurries after 28 days of incubation/ formate- supplemented root slurries after 28 days of incubation. Sequences correspond to residues 246 to 528 of the Escherichia coli hydrogenase 3 HycE protein (AAC75763). The phylogenetic tree was calculated using the neighbor joining method. Empty circles at nodes indicate congruent nodes in either the maximum parsimony or the maximum likelihood tree. Filled circles indicate congruent nodes in all three trees. Bootstrap values are averages from the maximum parsimony tree (1000 resamplings), the neighbor joining tree (1000), and the maximum likelihood tree (100), and are only displayed at nodes congruent in all three trees. The bar indicates a 0.1 change per amino acid. The hydrogenase of Methanocaldococcus jannaschii (AAB99031) was used as outgroup. Color code: blue, Firmicutes; orange, Acidobacteria; green, Actinobacteria; uncolored, unaffiliated.

Supplemental Figure S4. Phylogenetic tree of in silico-translated amino acid sequences derived from mcrA/mrtA genes sequences (boldface) and closely related sequences. GenBank accession numbers are indicated. Relative abundances of phylotypes in the different clone libraries are given in parentheses: Roots before incubation/ unsupplemented root slurries after 28 days of incubation/ formate- supplemented root slurries after 28 days of incubation. Sequences correspond to residues 98 to 227 of the mcrA amino acid sequence of Methanocella paludicola (AB300467). The phylogenetic tree was calculated using the maximum parsimony method. Empty circles at nodes indicate congruent nodes in either the neighbor joining or the maximum likelihood tree. Filled circles indicate congruent nodes in all three trees. Bootstrap values are averages from the maximum parsimony tree (1000 resamplings), the neighbor joining tree (1000), and the maximum likelihood tree (100), and are only displayed at nodes congruent in all three trees. The bar indicates a 0.1 change per amino acid. The amino acid sequence of Methanopyrus kandleri (AE009439) was used as outgroup. Methanosaetaceae are in quotes due to it’s current status as a illegitimate name (

Supplemental Figure S5. Effect of formate on the product profiles of isolates SB1 and SB2. Substrate provided: open circle, none (unsupplemented control); closed circle, glucose; open square, formate; closed square, glucose plus formate. Additional product profiles are shown in Fig. 6.

Supplemental Figure S6. Phylogenetic tree of 16S rRNA gene sequences of the acetogenic enrichment and closely related sequences. GenBank accession numbers are indicated. Sequences correspond to nucleic acids 107 to 816 of the 16S rRNA gene of E. coli (AB035923). The phylogenetic tree was calculated using the maximum parsimony method. Filled circles indicate congruent nodes in the neighbor joining and the maximum likelihood tree. Bootstrap values are from the maximum parsimony tree (1000 resamplings) and are only displayed at nodes congruent in all three trees. The bar indicates a 0.1 change per amino acid. The 16S rRNA gene sequences of M. kandleri (M59932) was used as outgroup.