16S rRNA Gene Diversity in the Salt Crust of Salar de Uyuni, Bolivia, the World’s Largest Salt Flat

Salar de Uyuni is the world’s largest salt flat, located in the southwestern Altiplano of Bolivia at an altitude of approximately 3,650 m above sea level (1, 2). The geochemical composition of brines and salt crusts (high in lithium, boron, magnesium, potassium, sodium, and chloride) and other physical conditions (high daily temperature fluctuations, UV radiation, and albedo) create a polyextreme environment close to multiple limits of life (3–5). In addition to an understanding of extremophilic life on Earth, characterization of the microbial communities of Salar de Uyuni will contribute to determining the potential habitability of other planets, as well as new biotechnology applications (6–9). To date, however, few microbial genomic studies have been conducted (10–13). This study provides an initial snapshot of microbial diversity in the crust.

Samples were collected in March 2015 from a remote site (20°33′28.58ʺS, 67°12′29.56ʺW) in Salar de Uyuni from the surface salt crust (SSC) (BOL4) and at a depth of 5 to 15 cm (near-surface salt crust [NSSC] [BOL3]). DNA was extracted with the PowerLyzer PowerSoil DNA extraction kit (MO BIO Laboratories, Inc., Carlsbad, CA). Library construction and 16S amplicon sequencing of the V3 to V4 region were performed on a MiSeq platform according to the manufacturer’s recommendations (Illumina, Inc., San Diego, CA) using the primers Bakt_341F and Bakt_805R (14–16).

Raw reads were processed with mothur (v1.39.5), and sequence data were aggregated with R (v3.4.1) (16–19) (https://www.mothur.org/wiki/MiSeq_SOP). Paired-end sequencing generated 213,999 raw reads (median length, 459 bp [range, 35 to 600 bp]). Reads were assembled with a quality score threshold of 20. Sequences longer than 475 bp and those with ambiguities and homopolymers (>8 bp), as well as chimeras, were removed, and sequences were aligned against the SILVA small subunit (SSU) Ref NR 99 database (v132) (20). Based on analysis using mothur, sequences with at least 97% similarity were binned into operational taxonomic units and classified (using a pseudobootstrap value of 80) against the reference database trimmed to positions 201 to 1000 of the 16S sequence of Escherichia coli (GenBank accession number J01859.1). Singletons were removed and only prokaryotic sequences were retained, resulting in 21,636 (SSC) and 3,625 (NSSC) sequences with a median length of 457 bp (range, 419 to 466 bp).

Archaea constituted 13.47% of the SSC sequences and 11.50% of the NSSC sequences, and bacteria constituted 86.53% and 88.50%, respectively. For archaea, all sequences were classified at the phylum level and 96.57% (SSC) and 91.85% (NSSC) at the genus level; 98.73% (SSC) and 93.29% (NSSC) were Euryarchaeota, and 1.27% (SSC) and 6.24% (NSSC) were Nanoarchaeota. Hadesarchaeota were present only in NSSC (0.48%). The most prevalent genera were Halonotius in SSC (53.09%) and Halodesulfurarchaeum in NSSC (47.96%) (Table 1). For bacteria, 99.71% (SSC) and 93.95% (NSSC) of sequences were classified at the phylum level and 91.36% (SSC) and 73.72% (NSSC) at the genus level. Bacteroidetes (58.25% [SSC] and 11.69% [NSSC]) and Proteobacteria (40.63% [SSC] and 73.72% [NSSC]) were most prevalent at the phylum level, and Salinibacter in SSC (53.21%) and Halorhodospira in NSSC (30.17%) were most prevalent at the genus level (Table 1). These findings represent a snapshot of considerable prokaryotic diversity in the largest salt flat on Earth.

The DasSarma laboratory was supported by NASA Exobiology grant 80NSSC19K0463, and F.L.M. was supported by the Fulbright Fellowship Program. D.G. thanks the Swedish International Development Cooperation Agency for supporting his work. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.