Association of Acidotolerant Cyanobacteria to Microbial Mats below pH 1 in Acidic Mineral Precipitates in Río Tinto River in Spain

Abstract

This report describes acidic microbial mats containing cyanobacteria that are strongly associated to precipitated minerals in the source area of Río Tinto. Río Tinto (Huelva, Southwestern Spain) is an extreme acidic environment where iron and sulfur cycles play a fundamental role in sustaining the extremely low pH and the high concentration of heavy metals, while maintaining a high level of microbial diversity. These multi-layered mineral deposits are stable all year round and are characterized by a succession of thick greenish-blue and brownish layers mainly composed of natrojarosite. The temperature and absorbance above and below the mineral precipitates were followed and stable conditions were detected inside the mineral precipitates. Different methodologies, scanning and transmission electron microscopy, immunological detection, fluorescence in situ hybridization, and metagenomic analysis were used to describe the biodiversity existing in these microbial mats, demonstrating, for the first time, the existence of acid-tolerant cyanobacteria in a hyperacidic environment of below pH 1. Up to 0.46% of the classified sequences belong to cyanobacterial microorganisms, and 1.47% of the aligned DNA reads belong to the Cyanobacteria clade.

Keywords: acidophiles; astrobiology; cyanobacteria; earth analogues; endolithic ecosystems; extremophiles; natrojarosite.

Figure 1

Microbial mat sample consisting of a mineral layer on the surface, with a blue-green layer in the middle and finally, a brown ocher layer at the bottom.

Figure 2

Radiation spectrum at the surface (red) and inside the microbial mat (green).

Figure 3

XRD analysis of the mineral layers. On the left is the outer layer. On the right is the brownish inner layer.

Figure 4

(a) Scanning electron microscopy images of the green-blue layer in which spherical morphologies with some surface features are observed; (b) close-up of some individual cells attached to the mineral surface.

Figure 5

EDX analysis in two spots (arrowheads) of the green-blue layer. Location of the spot (a) for the EDX analysis (b). With more zoom another analysis locations was selected (c) for the EDX analysis in (d).

Figure 6

Cells from the green-blue layer observed by TEM. Left image: individual spherical cell morphology. Right image: consortium of multicellular morphologies. (a) Fibrous protein and mucilaginous layer outside the cell envelope; (b,c) polysaccharide capsule; (d) cell membranes; (e) thylakoid membrane system; (f) putative carboxy-type corpuscles; (h) granules of variable density that may contain glycogen; (g) polyphosphates; and (i) lipid drops.

Figure 7

Detailed close-up section of the membrane: (a) fibrous protein and mucilaginous layer outside of the cell envelope; (b,c) polysaccharide capsule; (d) cell membrane; and (e) thylakoid membrane system.

Figure 8

α-major carboxysome shell protein immunolocalization in the cyanobacteria cells indicated by the arrows.

Figure 9

Cyanobacteria and Bacteria detected by CARD-FISH. (a): DAPI general stain; (b): bacteria detected by EUB338 I–III probe (red); (c): cyanobacteria detected by CYA 361 (green); (d): merge of (b,c) images.

Figure 10

Taxonomic distribution of shotgun metagenomic reads assigned to Cyanobacteria phylum.

Figure 11

Microbial mats spectral analysis. (a) View of the microbial mats, the colored circles indicate the points where the spectra were collected. The color of the circles corresponds to the color of the spectra in the panels (b,c). (b) Visible spectra collected in situ. (c) Micro-Raman spectrum collected in the laboratory on a sample of the green area.