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.
Highlights
The study of the microbial population inhabiting extreme saline environments has gained increasing interest in the last years due to its usually uncompleted characterization, which is essential to understand the ecology of these ecosystems [1] and because archaea have revealed themselves as the key to understand the origin of eukaryotic cells [2]
Two 16S rRNA libraries clone libraries, one for archaea and another one for bacteria, were constructed from an environmental water sample collected at the end of the summer, in the crystallizer ponds located in the Marshlands of the Odiel river in the southwest coast of Spain
The chosen primers have been shown to be very specific for each prokaryotic group studied, since archaea sequences have not been obtained in the bacterial library, nor have bacterial sequences been detected in the archaeal library
Summary
The study of the microbial population inhabiting extreme saline environments has gained increasing interest in the last years due to its usually uncompleted characterization, which is essential to understand the ecology of these ecosystems [1] and because archaea have revealed themselves as the key to understand the origin of eukaryotic cells [2]. The application of molecular techniques, based on the comparison of highly conserved DNA reference sequences, such as the genes encoding for ribosomal RNA (16S rRNA, 5S rRNA), has allowed to overcome this limitation, making possible the comparison of different microbial communities and the discovery of a good number of uncultured new species Examples of these culture-independent methods include: random fragment length polymorphisms (RFLP) [14], fluorescence in situ hybridization with rRNA-targeted probes [15], denaturing gradient gel electrophoresis (DGGE) [16]. 16S rRNA massive sequencing approach based on the Illumina MiSeq platform to profile the same genomic sample This double approach has allowed us, to explore the microbial diversity of this water environment and to validate the results of the PCR gene library by comparison with the NGS approach. The ability of this microbial population to produce and excrete haloenzymes with applied interest has been studied
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