A Differential Metabarcoding Approach to Describe Taxonomy Profiles of Bacteria and Archaea in the Saltern of Margherita di Savoia (Italy)

Abstract

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9–36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9–14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1–36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial “Candidatus Aquiluna” (~19% at 14.5% salinity). Interestingly, “Candidatus Aquiluna” had not been identified before in thalassohaline waters.

Spatio-Temporal Variation of the Bacterial Communities along a Salinity Gradient within a Thalassohaline Environment (Saline di Tarquinia Salterns, Italy)

“…In any case, in all sites both Proteobacteria and Actinobacteria were higher at low-intermediate salinities (26%–180%). Similar results were reported by other authors [ 24 , 26 , 28 , 31 , 34 , 36 , 53 ]. However, a decrease in the relative abundance of these phyla along the salinity gradient was often reported and, at salinities higher than 200‰, Bacteroidetes abundance highly increased [ 4 , 19 , 22 , 28 , 30 , 31 , 34 , 36 , 37 , 54 ].…”

“…However, a decrease in the relative abundance of these phyla along the salinity gradient was often reported and, at salinities higher than 200‰, Bacteroidetes abundance highly increased [ 4 , 19 , 22 , 28 , 30 , 31 , 34 , 36 , 37 , 54 ]. Moreover, various studies evidenced that at the highest salinities (≥ 300‰), where diversity is low, Euryarchaeota (Archaea) represented the greatest proportion of the community, but Bacteroidetes was the most abundant phylum among Bacteria (often representing almost the entire bacterial community) [ 4 , 17 , 28 , 29 , 31 , 34 , 37 , 53 , 54 , 55 , 56 , 57 ]. This was not confirmed by our work, since in ST Bacteroidetes generally showed low abundances, excluding some peaks in months characterised by high salinities.…”

“…However, the majority were based on low-resolution methods (i.e., 16S rDNA clone libraries or Denaturing Gradient Gel Electrophoresis, DGGE) [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ], which returned a limited vision of the diversity, affecting also community structure analysis. In addition, the majority of the investigations are based on one-time sampling [ 17 , 18 , 19 , 21 , 23 , 25 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ], generating scant information on the prokaryotic taxa fluctuations over time.…”

“…Moreover, general prokaryotic community pattern and distribution along the salinity gradient within the MS environments have often been described [ 4 , 17 , 22 , 25 , 28 , 31 , 36 , 37 ]. Besides, it has been shown that MS of different geographic areas revealed only partially similar patterns and distribution of prokaryotes [ 18 , 20 , 21 , 23 , 26 , 27 , 32 , 33 , 34 , 35 , 38 , 39 ]. Thus, tracing and identifying common and different features in the prokaryotic assemblages of the worldwide MS is of valuable biological and ecological interest.…”

Transient Dynamics of Archaea and Bacteria in Sediments and Brine Across a Salinity Gradient in a Solar Saltern of Goa, India

“…In the intermediate salinity samples (13–18%), bacterial phylotypes affiliated to phyla Proteobacteria , Bacteroidetes , Actinobacteria , and Verrumicrobia were usually encountered while the archaeal members belonged to genera Halorubrum , Haloarcula , and Haloquadratum (phylum Euryarchaeota ). High salinity samples (>25%) were dominated by halophilic archaea belonging to genera such as Haloquadratum and Halorubrum along with a minor contribution from bacterial phylotypes belonging to Bacteroidetes (genus Salinibacter ) ( Mouné et al, 2003 ; Pašić et al, 2005 ; Sørensen et al, 2005 ; Park et al, 2006 ; Wang et al, 2007 ; Baati et al, 2008 , 2010 ; Tsiamis et al, 2008 ; Manikandan et al, 2009 ; Oren et al, 2009 ; Oh et al, 2010 ; López-López et al, 2010 ; Zafrilla et al, 2010 ; Tkavc et al, 2011 ; Trigui et al, 2011 ; Boujelben et al, 2012 ; Dillon et al, 2013 ; Fernandez et al, 2014a , b ; Mani et al, 2015 ; Çinar and Mutlu, 2016 ; Di Meglio et al, 2016 ; Zhang et al, 2016 ; Kalwasińska et al, 2018 ; Leoni et al, 2020 ). In general, archaeal composition from the total prokaryotic community was observed to be between 27–46% at intermediate salinities (13–19%) and 80–90% at high salinities (>25%) ( Ghai et al, 2011 ; Fernandez et al, 2014a , b ).…”