Characterization of the Microbial Population Inhabiting a Solar Saltern Pond of the Odiel Marshlands (SW Spain)

Abstract

The solar salterns located in the Odiel marshlands, in southwest Spain, are an excellent example of a hypersaline environment inhabited by microbial populations specialized in thriving under conditions of high salinity, which remains poorly explored. Traditional culture-dependent taxonomic studies have usually under-estimated the biodiversity in saline environments due to the difficulties that many of these species have to grow at laboratory conditions. Here we compare two molecular methods to profile the microbial population present in the Odiel saltern hypersaline water ponds (33% salinity). On the one hand, the construction and characterization of two clone PCR amplified-16S rRNA libraries, and on the other, a high throughput 16S rRNA sequencing approach based on the Illumina MiSeq platform. The results reveal that both methods are comparable for the estimation of major genera, although massive sequencing provides more information about the less abundant ones. The obtained data indicate that Salinibacter ruber is the most abundant genus, followed by the archaea genera, Halorubrum and Haloquadratum. However, more than 100 additional species can be detected by Next Generation Sequencing (NGS). In addition, a preliminary study to test the biotechnological applications of this microbial population, based on its ability to produce and excrete haloenzymes, is shown.

Keywords: 16S rRNA metagenomics; Odiel marshlands; archaea; halo-extremophyles; haloenzymes.

Figure 1

Distribution of the clones from the archaeal 16S rRNA clone library among different genera. 16S rRNA fragments obtained by amplification with archaeal specific primers were cloned into pGEMT vector for the construction of a clone library. The inserted sequence of 25 of the obtained clones were analysed and compared with the NCBI to identify the original genera. Data are expressed as percentages of the total archaeal population. Only sequences that shared over 95% 16S rRNA sequence identity with a known one was assigned to a specific genus.

Figure 2

Molecular Phylogenetic Analysis by Maximum Likelihood method. The tree represents the relationship among the 16S rRNA sequences from strains isolated from the saltern ponds of the Odiel Marshlands and reference archaeal sequences. Multiple alignments were generated by MUSCLE and the tree was constructed with MEGA 7, using 1000 bootstrap replicates. The name and the NCBI access number are indicated for all the reference sequences. Black diamonds represent 16S rDNA Sequences from the isolates and “Col. N” denotes the colony number. When an identical sequence was obtained from different colonies it was denoted as “Col. N1/N2.” The tree is drawn to scale, branch lengths represent the number of substitutions per site. Scale bar indicate 5% sequence divergence. The sequence of Methanococcus vulcanus was used as the outgroup.

Figure 3

Relative abundance of the genera found by the metagenomic approaches. Operational Taxonomic Units (OTUs) distribution in Odiel saline ponds (33% salinity) obtained from Illumina MiSeq sequencing of the 16S rRNA V3, V4 hypervariable regions. Four data sets were obtained from two different sequencing services. The graphic shows the percentage of the genera with more than 0.2% abundance. Minor genera include all the genera below 0.2%. The sequences that clustered together but could not been affiliated to a genus are named as “clusters” and have been divided in “Major clusters” (>0.2%) and “Minor clusters” (<0.2%). “No hit” represents the sequences which did not cluster with any other obtained sequence.

Figure 4

Comparison of the main archaeal genera present in the Odiel saline ponds. Percentage of the different archaea genera over the total archaea population found in the Odiel saline ponds obtained by the two different culture-independent approaches previously described: construction of a clone library (Library) and massive 16S rRNA sequencing, including the two replicates from each Metagenomic Service (LFS and SBV). Only the most abundant genera are shown.

Figure 5

Detection of extracellular halocin and enzymatic activities in the enriched archaeal biomass by plate assay. The biomass obtained from the Odiel salterns water was enriched and concentrated as detailed in Material and Methods and used to test extracellular: halocin (A), amylase (B), protease (C), lipase (D), cellulase (E) and laccase (F) activities by the plate assays described in Materials and Methods.