Table 1. Overview of blue samples and analyses performed.
3.2.1 X-Ray Fluorescence XRF analysis of sample H1 (Table 1) of the mat substrate versus the background (collected from a region close to the sampling spot not covered by mat) revealed several differences between the elements present. Iron, calcium, silicon, magnesium, titanium, and manganese were decreased in mat substrate samples as compared to the background, while copper, phosphorus, and aluminum were lower in the background and higher in the mat-covered samples (Figure 5a). The mat substrate samples showed a striking enrichment in copper, which given the amount of aluminum and silicon present, could be in the form of a hydrated copper silicate. The Al:Cu:Si ratio of the mat substrate versus the background is shown in Figure 5b.Figure 5. XRF measurements of blue precipitates in sample H1 vs. background. (a) Major elements were measured by pointing the XRF onto the cave wall where the mat was collected, vs. the background, i.e., a spot on the wall nearby not covered in mat.(b) Ratios of select elements aluminum, copper, and silicon in blue precipitate vs. background. Copper is markedly absent in the background. (c) Al:Cu:Si ratios measured in all blue precipitates. Figure 5c shows the Al:Cu:Si ratio of samples C2 (0.33:3.3:1), H1a (0.81:0.35:1), H1b (0.73:0.29:1), H7a (0:12.8:1) and H7b (0.07:1.6:1). A typical Al:Cu:Si ratio of chrysocolla, an amorphous, hydrated copper silicate, is 0.12:1.98:1 (Anthony, 1990), and is in the same range as samples C2 and H7b. 3.2.2 DNA sequencing Blue samples C2 (from cave C in southern Iceland) and H7 (from cave H in northern Iceland) were analyzed for bacterial community composition based on DNA extracted using phenol/chloroform with ethanol precipitation and amplified using V4-specific 16S rRNA gene primers (Figure 6). At the phylum level (Figure 6a, b), Proteobacteria strongly dominated the bacterial community composition. The most common family was Burkholderiaceae in both samples (Figure 6a, b). C2 and H7 contained a number of similar genera (Figure 6c), notably Ralstonia(Proteobacteria) (74% in C2 and 74% in H7) andCaulobacter (Proteobacteria) (20% in C2 and 5% in H7). The genera Corynebacterium (Actinobacteria), Cutibacterium(Actinobacteria), and H1 (Bacteroidetes) were only found in sample C2, while Cupriavidus (Proteobacteria),Methylobacterium-Methylorubrum (Proteobacteria), andThermovirga (Synergistaceae) were found exclusively in sample H7. No archaeal 16S rRNA sequences were detected.
3.2.3 Confocal Raman Spectroscopy
Sample C2 in Cave C was analyzed using confocal Raman spectroscopy. A drop of water hanging from the ceiling containing a significant amount of blue precipitate was observed under the microscope and identified with brown speckles incorporated in the crystalline deposit (Figure 6d). The Raman spectrum of the brown speckle shows prominent peaks at 1008, 1155, and 1513 cm-1, typical of a carotenoid signature (Figure 6e). The 1008 cm-1 peak corresponds to the in-plane rocking modes of the CH3 groups attached to the polyene chain, while the peak at 1155 cm-1 is associated with C-C stretching and C-H deformation, and the one at 1516 cm-1 with C=C stretching (Vítek et al., 2009; Baqué et al., 2018).