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Figure 1 Map of the Frasassi cave system showing sample locations (open circles). The small inset shows the location of the Frasassi cave system in Italy. Major named caves in the Frasassi system are shown in different shades of gray. Topographic lines and elevations in meters refer to the surface topography. Base map courtesy of the Gruppo Speleologico CAI di Fabriano.

Figure 1 Map of the Frasassi cave system showing sample locations (open circles). The small inset shows the location of the Frasassi cave system in Italy. Major named caves in the Frasassi system are shown in different shades of gray. Topographic lines and elevations in meters refer to the surface topography. Base map courtesy of the Gruppo Speleologico CAI di Fabriano.

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“Combined in equimolar amounts to make EUBMIX. Table 1 Oligonucleotide probes used in this study
“Combined in equimolar amounts to make EUBMIX. Table 1 Oligonucleotide probes used in this study
Figure 2 Dissolved oxygen and total sulfide concentrations for waters hosting Frasassi biofilm samples. Concentration field for Lowe Kane Cave (LKC) (Engel et al., 2003) is shown in gray for comparison. Symbols are colored if more than 50% of the biofilm cell area i composed of a single population or group based on FISH. Colored squares with error bars show the mean + 1 s.d. for each major biofiln type. Samples analyzed by 16S rDNA cloning are circled. The open diamond symbol represents a filamentous epsilonproteobacteria biofilm from LKC reported in Engel et al. 2004. The range of conditions inside the RS05-21 (Thiothrix) biofilm based on voltammetry i indicated by the blue dashed box.
Figure 2 Dissolved oxygen and total sulfide concentrations for waters hosting Frasassi biofilm samples. Concentration field for Lowe Kane Cave (LKC) (Engel et al., 2003) is shown in gray for comparison. Symbols are colored if more than 50% of the biofilm cell area i composed of a single population or group based on FISH. Colored squares with error bars show the mean + 1 s.d. for each major biofiln type. Samples analyzed by 16S rDNA cloning are circled. The open diamond symbol represents a filamentous epsilonproteobacteria biofilm from LKC reported in Engel et al. 2004. The range of conditions inside the RS05-21 (Thiothrix) biofilm based on voltammetry i indicated by the blue dashed box.
Figure 3 Vertical sulfide concentration profiles measured using voltammetric microelectrodes. (a) Beggiatoa biofilm developed in thi sediment and at the sediment—water interface. Zero depth on the y axis corresponds to the upper surface of the biofilm. Dissolved oxyger concentrations were <15 uM (detection limit) for all points. Marine sediment curve is from a Beggiatoa mat described in Jorgensen anc Revsbech (1983). (b) Filamentous epsilonproteobacterial mat (‘streamers’) developed in turbulent water. The biofilm (gray box) i: attached at the upstream end to a rock several centimeters above the sediment—water interface. Zero depth on the y axis corresponds tc the upper surface of the biofilm. Stream water flows both above and below the biofilm.
Figure 3 Vertical sulfide concentration profiles measured using voltammetric microelectrodes. (a) Beggiatoa biofilm developed in thi sediment and at the sediment—water interface. Zero depth on the y axis corresponds to the upper surface of the biofilm. Dissolved oxyger concentrations were <15 uM (detection limit) for all points. Marine sediment curve is from a Beggiatoa mat described in Jorgensen anc Revsbech (1983). (b) Filamentous epsilonproteobacterial mat (‘streamers’) developed in turbulent water. The biofilm (gray box) i: attached at the upstream end to a rock several centimeters above the sediment—water interface. Zero depth on the y axis corresponds tc the upper surface of the biofilm. Stream water flows both above and below the biofilm.
Figure 4 Distribution of 16S rDNA clones in Frasassi stream biofilms. Shaded wedges represent known or putative sulfur-oxidizing clades. Deltaproteobacteria associated with sulfate-reducing clades are shown in black. White wedges include all other clones (see Supplementary Table 1). GS02-zEL and GS02-WM clone libraries are described in Macalady et al. (2006). Clones from both bacterial and universal libraries are combined for sample FS06-12.
Figure 4 Distribution of 16S rDNA clones in Frasassi stream biofilms. Shaded wedges represent known or putative sulfur-oxidizing clades. Deltaproteobacteria associated with sulfate-reducing clades are shown in black. White wedges include all other clones (see Supplementary Table 1). GS02-zEL and GS02-WM clone libraries are described in Macalady et al. (2006). Clones from both bacterial and universal libraries are combined for sample FS06-12.
Figure 5 Neighbor-joining phylogenetic tree showing gamma(beta)proteobacteria. Frasassi clones are shown in bold followed by the number of clones represented in each phylotype. Neighbor-joining bootstrap values >50% are shown. Filled circles indicate nodes present in the maximum likelihood phylogeny. Sequences identical to the probe BEG811 are indicated by the dashed line.
Figure 5 Neighbor-joining phylogenetic tree showing gamma(beta)proteobacteria. Frasassi clones are shown in bold followed by the number of clones represented in each phylotype. Neighbor-joining bootstrap values >50% are shown. Filled circles indicate nodes present in the maximum likelihood phylogeny. Sequences identical to the probe BEG811 are indicated by the dashed line.
Figure 6 Neighbor-joining phylogenetic tree showing epsilonproteobacteria. Frasassi clones are shown in bold followed by the number of clones represented in each phylotype. Neighbor-joining bootstrap values >50% are shown. Filled circles indicate nodes present in the maximum likelihood phylogeny. Clades which hybridize with probe EP404 are indicated by the dashed line.
Figure 6 Neighbor-joining phylogenetic tree showing epsilonproteobacteria. Frasassi clones are shown in bold followed by the number of clones represented in each phylotype. Neighbor-joining bootstrap values >50% are shown. Filled circles indicate nodes present in the maximum likelihood phylogeny. Clades which hybridize with probe EP404 are indicated by the dashed line.
Figure 7 Sulfide/oxygen ratios for Frasassi biofilms analyzed using fluorescence in situ hybridization (FISH). The upper panel shows a linear correlation (P<0.0001) between sulfide/oxygen ratio and the microbial composition of streamers. The open diamond (not included in correlation) represents a biofilm from Lower Kane Cave (Engel et al., 2004) and assumes 0.2uM O, (detection limit). The dashed arrow shows how the sulfide/ oxygen ratio for the sample would change assuming an O, concentration of 0.141M. The lower panel shows the distribution of biofilm types compared based on _ sulfide/oxygen ratios. Colored boxes and associated bars show the mean+1 s.d. for each major biofilm type. Mean sulfide/oxygen ratios associated with Thiothrix and filamentous epsilonproteobacteria habitats are significantly different (P= 0.0004).
Figure 7 Sulfide/oxygen ratios for Frasassi biofilms analyzed using fluorescence in situ hybridization (FISH). The upper panel shows a linear correlation (P<0.0001) between sulfide/oxygen ratio and the microbial composition of streamers. The open diamond (not included in correlation) represents a biofilm from Lower Kane Cave (Engel et al., 2004) and assumes 0.2uM O, (detection limit). The dashed arrow shows how the sulfide/ oxygen ratio for the sample would change assuming an O, concentration of 0.141M. The lower panel shows the distribution of biofilm types compared based on _ sulfide/oxygen ratios. Colored boxes and associated bars show the mean+1 s.d. for each major biofilm type. Mean sulfide/oxygen ratios associated with Thiothrix and filamentous epsilonproteobacteria habitats are significantly different (P= 0.0004).
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