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.
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).