Microbial diversity in the deep-subsurface hydrothermal aquifer feeding the giant gypsum crystal-bearing Naica Mine, Mexico

Abstract

The Naica Mine in northern Mexico is famous for its giant gypsum crystals, which may reach up to 11 m long and contain fluid inclusions that might have captured microorganisms during their formation. These crystals formed under particularly stable geochemical conditions in cavities filled by low salinity hydrothermal water at 54-58°C. We have explored the microbial diversity associated to these deep, saline hydrothermal waters collected in the deepest (ca. 700-760 m) mineshafts by amplifying, cloning and sequencing small-subunit ribosomal RNA genes using primers specific for archaea, bacteria, and eukaryotes. Eukaryotes were not detectable in the samples and the prokaryotic diversity identified was very low. Two archaeal operational taxonomic units (OTUs) were detected in one sample. They clustered with, respectively, basal Thaumarchaeota lineages and with a large clade of environmental sequences branching at the base of the Thermoplasmatales within the Euryarchaeota. Bacterial sequences belonged to the Candidate Division OP3, Firmicutes and the Alpha- and Beta-proteobacteria. Most of the lineages detected appear autochthonous to the Naica system, since they had as closest representatives environmental sequences retrieved from deep sediments or the deep subsurface. In addition, the high GC content of 16S rRNA gene sequences belonging to the archaea and to some OP3 OTUs suggests that at least these lineages are thermophilic. Attempts to amplify diagnostic functional genes for methanogenesis (mcrA) and sulfate reduction (dsrAB) were unsuccessful, suggesting that those activities, if present, are not important in the aquifer. By contrast, genes encoding archaeal ammonium monooxygenase (AamoA) were amplified, suggesting that Naica Thaumarchaeota are involved in nitrification. These organisms are likely thermophilic chemolithoautotrophs adapted to thrive in an extremely energy-limited environment.

Keywords: Candidate Division OP3; GC content; Thaumarchaeota; Thermoplasmatales; aerobic ammonium oxidation; hydrothermal; thermophile.

FIGURE 1

Hot spring sampling sites in the Naica Mine. Water sampling sites N1 and N2 are indicated in a not-to-scale hydrogeological sketch of the Naica deposit and associated caves. The horizontal groundwater level marks the position of the phreatic level immediately before the discovery of Cueva de la Espadas in 1910. The stratigraphic sequence on the right reports the lithology of a 1,150-m deep drillcore of the area (Garofalo et al., 2010). The figure was adapted from (Garcia-Ruiz et al., 2007) and (Forti and Sanna, 2010).

FIGURE 2

Relative proportions of archaeal and bacterial operational taxonomic units (OTUs) distributed in major high-rank taxa from Naica deep hot springs. Archaea were only detected in sample N1. Eukaryotes were not detected in any of the two samples.

FIGURE 3

Maximum likelihood phylogenetic tree of archaeal 16S rRNA genes retrieved from Naica deep hot springs. The tree was reconstructed using 1,079 positions. Sequences obtained in this work are shown in color. Accession numbers of sequences retrieved from GenBank are given between brackets. Only bootstrap values higher than 50% are given at nodes. The scale bar represents the number of substitutions per a unit branch length.

FIGURE 4

Maximum likelihood tree of bacterial 16S rRNA genes retrieved from Naica deep hot springs. The tree was reconstructed using 846 positions. Sequences obtained in this work are shown in color. Accession numbers of sequences retrieved from GenBank are given between brackets. Only bootstrap values higher than 50% are given at nodes. The scale bar represents the number of substitutions per a unit branch length.

FIGURE 5

Comparative histogram showing the GC content of the same 16S rRNA region in Naica clones and in a wide range of microbial species growing at different temperatures.

FIGURE 6

Maximum likelihood tree of archaeal amoA genes retrieved from Naica deep hot springs. The tree was reconstructed using 203 amino acid positions. Sequences obtained in this work are shown in color. Accession numbers of sequences retrieved from GenBank are given between brackets. Only bootstrap values higher than 50% are given at nodes. The scale bar represents the number of substitutions per a unit branch length.