In this work, metabarcoding of 16S and 18S rRNA gene coding sequences were employed to characterize the prokaryotic and eukaryotic communities in the phosphogypsum stacks located in the city of Huelva (SW Spain). In this environment around 100 Mt of phosphogypsum with extreme acidity and high concentrations of heavy metals and radionuclides have been accumulated for more than forty years on the marshlands of the Tinto River estuary. The microbial community inhabiting these adverse conditions remains unknown, although it can have an effect on the biogeochemical cycle of the phosphogypsum components and contain new species with biotechnological interest.

Water samples were collected from seawater, the Tinto River lower course, and two different points in phosphogypsum stacks, the perimeter channel and the piezometer. Fresh biomass was harvested from each water sample by filtering through a 0.7 μm glass fiber filter (Whatman, GF/G), and DNA was extracted by using 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 gen 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).

Description of samples in Readme file.