Microbiome and ecology of a hot spring-microbialite system on the Trans-Himalayan Plateau

Abstract

Little is known about life in the boron-rich hot springs of Trans-Himalayas. Here, we explore the geomicrobiology of a 4438-m-high spring which emanates ~70 °C-water from a boratic microbialite called Shivlinga. Due to low atmospheric pressure, the vent-water is close to boiling point so can entropically destabilize biomacromolecular systems. Starting from the vent, Shivlinga's geomicrobiology was revealed along the thermal gradients of an outflow-channel and a progressively-drying mineral matrix that has no running water; ecosystem constraints were then considered in relation to those of entropically comparable environments. The spring-water chemistry and sinter mineralogy were dominated by borates, sodium, thiosulfate, sulfate, sulfite, sulfide, bicarbonate, and other macromolecule-stabilizing (kosmotropic) substances. Microbial diversity was high along both of the hydrothermal gradients. Bacteria, Eukarya and Archaea constituted >98%, ~1% and <1% of Shivlinga's microbiome, respectively. Temperature constrained the biodiversity at ~50 °C and ~60 °C, but not below 46 °C. Along each thermal gradient, in the vent-to-apron trajectory, communities were dominated by Aquificae/Deinococcus-Thermus, then Chlorobi/Chloroflexi/Cyanobacteria, and finally Bacteroidetes/Proteobacteria/Firmicutes. Interestingly, sites of >45 °C were inhabited by phylogenetic relatives of taxa for which laboratory growth is not known at >45 °C. Shivlinga's geomicrobiology highlights the possibility that the system's kosmotrope-dominated chemistry mitigates against the biomacromolecule-disordering effects of its thermal water.

Figure 1

The environmental context of the Shivlinga microbialite hot spring system at the time of sampling on 23 July 2013: (A) the three geomicrobiological zones, scale bar represents 1 m; (B) view of Shivlinga’s microbial communities showing the spring-water transit that represented the wet thermal gradient, scale bar represents 1 m; (C) microbialite body showing the vent and position of the two thermal gradients that were sampled, scale bar represents 10 cm; (D) bedrock slope around Shivlinga’s base, showing the microflora, scale bar represents 10 cm; (E and F) WG6 and WG7, respectively, scale bars represent 5 cm for E and 10 cm for F. For (G), the light blue circle indicates the microbialite body, the yellow oval demarcation indicates the bedrock slope around the base, the purple oval demarcation shows Shivlinga’s apron, and the cyan arrow shows the direction of water flow across the apron, away from the microbialite hot spring. For (H), the cyan curved-line with an arrow-head shows the meandering path of the spring-water across the apron, away from the microbialite body (this also represents part of the wet thermal gradient), the pink triangles indicate the sample sites for WG6 and WG7, the orange triangle indicates the location of the Sinter-Sample 4 (SS4). For (I), the green curved-line with an arrow-head indicates the trajectory of the drying thermal gradient, while the cyan curved-line with an arrow-head indicates part of the wet thermal gradient which starts at the vent and leads to the first mat of the bedrock slope (the latter also represents the direction of water flow); the red triangles indicate the position of the sampled communities VW and VWM; the green triangles indicate the sampling-positions for communities DG3 and DG4; the pink triangles indicate the sample sites for WG3 and WG4; and the orange triangles indicate the locations of the Sinter-Samples 1, 2 and 5 (SS1, SS2 and SS5, respectively). For (J), the cyan curved-line with an arrow-head indicates part of the wet thermal gradient that runs along the bedrock slope surrounding Shivlinga’s base; the pink triangles indicate the sample sites for WG4 and WG5; and the orange triangle indicates the location of the Sinter-Sample 3 (SS3). For (K) and (L), the pink triangles indicate the sampling positions for communities WG6 and WG7, while the cyan curved-lines with an arrow-head each show the direction of water flow.

Figure 2

Macro-/micro-scale microbe-mineral structures at the Shivlinga site: (A) green microbial mat (pink arrows), and white spherules (red arrow) that collectively form shrub-like mineral bodies (orange circle), on the upper surfaces of the vent’s rim (blue scale bar = 10 mm); (B) sinter particles from 5-cm-deep inside the wall of the microbialite, associated with bacterial filaments (red arrow) (image was taken using SEM, blue scale bar = 150 µm); and (C) a diatom cell (golden arrow) associated with the particles shown in (B) (using SEM, blue scale bar = 20 µm).

Figure 3

Microbial structures from the Shivlinga site: (A and B) microbial cell filaments from streamers of VWM and WG3, respectively; (C) microbial biomass of DG3; (D) diatoms from the surface of the microbial mass which makes up WG4 and DG4 (D1 and D2 respectively); (E and F) cells from the core of the microbial mass which makes up DG4 and WG5, respectively; (G) Chroococcales-like cyanobacteria in the core of the microbial mass which makes up DG3; (H) fungal hyphae and diatoms near the edge of the microbial mass which makes up DG4. All images were produced using phase contrast microscopy except for (C) that used laser scanning confocal microscopy. For (C1), excitation was carried out at 488 nm and detection at 630–650 nm; (C2) is a differential interference contrast image with blue arrows indicating diatom cells; and for (C3), excitation was carried out at 543 nm and detection at 650 nm.

Figure 4

Microbial cells present in the different layers of the WG7 sample: (row A) top-most layer of the microbial mat; (row B) sub-surface layer 1 (from a depth of 1–2 mm); (row C) sub-surface layer 2 (from a depth of 3–4 mm); and (row D) diatom cells and borax crystals in sub-surface layer 3 (from a depth of 5–8 mm); (Column I) images were taken using phase contrast microscopy; (column II, except for row D) images were taken using laser scanning confocal microscopy, at 488 nm excitation and 630–650 nm detection; (column III, except for row D) used differential interference contrast to modify the image from column II; and (column IV, except for row D) used laser scanning confocal microscopy (543 nm excitation and long pass 650 nm detection) to further modify the image from column II. Photographs in row D were taken using phase contrast microscopy.

Figure 5

Mean percentage of reads ascribed to bacterial phyla, and classes within the Proteobacteria, in the duplicate metagenomes obtained from each mat community of (A) the drying thermal gradient and (B) the wet thermal gradient. The 21 phyla and five proteobacterial classes represented account for >0.1% reads in at least one of the 16 metagenomes analyzed. The category ‘others’ encompasses the phyla that accounted for <0.1% reads in every metagenome analyzed. Statistical significance of the fluctuations in the relative abundance of the taxa along the hydrothermal gradients can be seen in Supplementary Figs. 4 and 5 where their mean relative abundance within each mat community has been plotted alongside the two original relative abundance values obtained from the duplicate metagenomes (shown as vertical range bar in Supplementary Figs. 4 and 5).

Figure 6

Simpson Dominance (A,E), Shannon Diversity (B,F) and Shannon Equitability (C,G) Indices, and total genus count (D,H), along the drying (A–D), and the wet (E–H), thermal gradients.

Figure 7

Functional analysis of the metagenomes isolated from the eight microbial mat communities of Shivlinga: (A) heat map comparing the richness of the metabolic/functional categories across the communities, determined in terms of the number of Clusters of Orthologous Groups (COGs) of Proteins that are ascribed to the categories in individual communities; a two-dimensional clustering is also shown, involving the eight mat communities on one hand and the 18 functional categories of COGs on the other; color gradient of the heat map varied from high (red) to low (green), through moderate (yellow), richness of the categories across the communities; (B) Statistically significant high (green circles) or low (red circles) richness of the functional categories across the communities, as determined by Chi Square test with p < 0.001.