Supplementary Material for

Attached and suspended denitrifier communities in pristine limestone aquifers harbor high fractions of potential autotrophs oxidizing reduced iron and sulfur compounds

Herrmann M*, Opitz S, Harzer R, Totsche KU, Küsel K

*corresponding author:

Dr. Martina Herrmann

Friedrich Schiller University Jena

Institute of Ecology

Chair of Aquatic Geomicrobiology

Dornburger Strasse 159

D-07743 Jena

phone: +49 3641 949451

fax: +49 3641 949462

email:

Supplemental Figure S1. Gene ratios of nirK and nirS relative to bacterial + archaeal 16S rRNA gene numbers in the groundwater of (A) the upper (H42) and (B) the lower aquifer assemblage (H41) from April 2012 till May 2013 and in association with exposed rock material obtained from the two aquifer assemblages in May 2013 (PSH42, PSH41). nirS (grey bars), nirK (white bars). Ratios are calculated based on mean gene abundances determined by qPCR as shown in Figure 2

Supplemental Figure S2. Clustering of nirK-type (A) and nirS-type (B) denitrifier communities in association with groundwater (H41, H42) and exposed rock material (PSH41, PSH42) based on Yue & Clayton measures of dissimilarity

Supplemental Figure S3. Sequence read fractions of bacterial 16S rRNA genes affiliated with species or genera known to have the potential for denitrification, identified among the 200 most abundant OTUs across all four samples (0.03 distance cut-off for OTU assignment). Results are shown for bacterial communities in the groundwater (H41, H42; September 2013) and in association with exposed rock material (PSH41, PSH42; May 2013). Analysis based on amplicon sequencing of bacterial 16S rRNA genes using 454 pyrosequencing. Sequence identities with most closely related taxa are given in parentheses.

Supplemental Table S1 Overview of results obtained with 454 amplicon pyrosequencing targeting bacterial 16S rRNA genes in groundwater samples (H41, H42; September 2013) and in association with exposed rock material (PSH41, PSH42; May 2013) after subsampling (14415 sequence reads). OTUs were assigned based on a 0.03 distance cut-off.

sample / coverage / Number of observed OTUs / Number of estimated OTUs (Chao1) / Shannon index
H41 / 0.8644 / 2790 / 8369 / 5.57
PSH41 / 0.947 / 1582 / 2963 / 5.65
H42 / 0.9924 / 780 / 880 / 5.67
PSH42 / 0.9468 / 1568 / 2725 / 5.38