Metataxonomy and pigments analyses unravel microbial diversity and the relevance of retinal-based photoheterotrophy at different salinities in the Odiel Salterns (SW, Spain)

In this work, metabarcoding of 16S and 18S rRNA gene coding sequences were employed to characterize the prokaryotic and eukaryotic communities across the salinity gradient (3.5, 7.8, 14.4, and 31.8% NaCl) in Odiel salterns, located in the city of Huelva (SW Spain). Coastal salterns are excellent environments to study the dynamics of the microbiota across the salinity gradient since we find from seawater, the most abundant ecosystem on Earth, to crystallization pools, one of the most extreme. Diverse harsh conditions converge in solar salterns, including high salt concentration, temperature, and solar irradiance. Most environmental factors are similar in the different ponds that comprise the salterns, excepting salinity, which increases through the series of ponds until reaching saturation concentrations. Understanding the microbiology of these ponds is important for the salt production process, given that this microbiota can physically affect the evaporation process and chemically interact with dissolved ions. In addition, these microorganisms are crucial in these ecosystems, supporting bird populations, and potentially a source of enzymes and useful carotenoids for many biotechnological applications.

Methods

Water samples were collected from four ponds of the Odiel salt flats with 3.5, 7.5, 15, and 30% salinity. Fresh biomass was harvested from 2 L of each water sample by centrifugation at 19,800 ×g. The obtained pellet was used for the extraction of genomic DNA with the GeneJET Genomic Purification kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. The genomic DNA was used as a template for PCR amplification of specific regions of 16S rRNA and 18S rRNA coding genes. The hypervariable region V3-V4 of the 16S rRNA coding gene was amplified by using the primer set 341F/806R, while the primer pair 1380F/1510R was used for the amplification of the region V9 of the 18S rRNA coding gene. PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs, MA, USA). PCR products were mixed in equal ratios and purified following the Qiagen Gel Extraction Kit (Qiagen, Germany). Purified PCR products were employed for the generation of libraries with NEBNext® UltraTM DNA Library Prep Kit for Illumina and quantified via Qubit and Q-PCR. Finally, amplicons were sequenced on the Illumina platform to generate paired-end raw reads. To keep the reliability of the data, quality controls were performed at each step of the procedure, from the raw DNA samples to the final data (Q>36).