Extremely Halophilic Bacteria in Crystallizer Ponds from Solar Salterns

Abstract

It is generally assumed that hypersaline environments with sodium chloride concentrations close to saturation are dominated by halophilic members of the domain Archaea, while Bacteria are not considered to be relevant in this kind of environment. Here, we report the high abundance and growth of a new group of hitherto-uncultured Bacteria in crystallizer ponds (salinity, from 30 to 37%) from multipond solar salterns. In the present study, these Bacteria constituted from 5 to 25% of the total prokaryotic community and were affiliated with the Cytophaga-Flavobacterium-Bacteroides phylum. Growth was demonstrated in saturated NaCl. A provisional classification of this new bacterial group as "Candidatus Salinibacter gen. nov." is proposed. The perception that Archaea are the only ecologically relevant prokaryotes in hypersaline aquatic environments should be revised.Hypersaline environments, such as the crystallizer ponds (i.e., ponds where sodium chloride precipitates) of multipond solar salterns, have been shown to harbor a very low prokaryotic diversity, to the point of having been described as "almost monospecific cultures of halophilic archaea" (9). The use of molecular techniques has also revealed a very low diversity, although the most abundant haloarchaeon (5) corresponding to the unique archaeal 16S ribosomal DNA (rDNA) sequences most frequently recovered from several crystallizer ponds by PCR-based methods did not correspond to any previously described microorganism (5,6,26). This apparent discrepancy between molecular and culture-based data has been found in other environments (4).When fluorescence in situ hybridization (FISH) was used to analyze the prokaryotic community inhabiting crystallizer ponds (around 37% salinity) of a marine solar saltern located in Alicante, Spain (5), Bacteria in high numbers (around 3 ϫ 10 6 /ml) were unexpectedly found. The cells, which accounted for 18% of total cell counts, contained large numbers of ribosomes, as indicated by the intense and uniform FISH signals obtained with the 16S rRNA-targeted oligonucleotide probe EUB338 (3), which is specific for members of the Bacteria domain. The finding of abundant Bacteria with high cellular rRNA content in such a hypersaline environment was unexpected in light of previous reports (20-23) suggesting that almost all the active biomass was of archaeal origin.Solar salterns consist of a series of shallow ponds connected in a sequence of increasingly saline brines. Crystallizers are the last ponds and have a salinity above 30% (6). Therefore, the presence in these ponds of Bacteria could be due either to their import from previous ponds with lower salinity or to their active growth in the crystallizers. The key point that would determine the relevance of finding Bacteria in the crystallizers was, then, whether they were in fact extreme halophiles forming part of the autochthonous microbiota of these ponds.In order to characterize this bacterial community and assess this point, we used the rRNA approach (4, 19), which allows direct, cu...

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists

“…Numbers of Bacteria and total cell counts showed a negative dependence with salinity, while Archaea was rather independent of this variable (Supplementary Tables S12 and S13). This may seem contradictory with previous results (Guixa-Boixereu et al, 1996;Antó n et al, 2000;Pedró s-Alió , 2005) that indicated that archaeal numbers increased with salinity. This discrepancy Figure 3 Temporal variation of total counts (grey symbols), Archaea (white symbols) and Bacteria (black symbols) in the different ponds.…”

“…Prokaryotic community dynamics and their relation to environmental parameters Total microbial counts were in the range previously observed in solar salterns (Guixa-Boixereu et al, 1996;Antó n et al 1999Antó n et al , 2000Benlloch et al 2002;Oren, 2002;Estrada et al, 2004;Burns et al, 2007;Mutlu et al 2008;Eulloumi et al, 2009) ranging from 6.3 Â 10 6 to 4.79 Â 10 7 cells ml À 1 in CR30 (OC) and CCAB (JL), respectively (Figure 3). Although throughout the concentration process from seawater to saturated brines there is an overall increase of cell density, at concentrations 420% this trend is not always observed (Guixa-Boixereu et al, 1996;Boujelben et al, 2012), as shown here.…”

Phylogenetic analysis of the bacterial community in a crystallizer pond, Pomorie salterns, Bulgaria

“…The domination of Firmicutes in PS is not in agreement with the opinion that Bacteroidetes is the most abundant bacterial phylum in clone libraries from crystallizer ponds [25,26]. According to Ant on et al [8], the presence of different bacterial taxa in them could be due either to their import from previous ponds with lower salinity or to their active growth in the crystallizers. The high number of the clones affiliated with this phylum suggested their active growth in PS.…”

“…The uncultured Bacteroidetes lineage has been reported to comprise 69% of all bacterial 16S rRNA clones from Pond 11 in Guerrero Negro salterns [22] and 50% in the Chula Vista salterns [32]. Although Salinibacter has been found ubiquitously in numerous hypersaline environments [8,34], it was not identified in either PS or in Maras salterns [21]. Abundance of new Bacteroidetes groups, different from Salinibacter, in the salterns has been established by single-cell genome analyses [35].…”

“…Investigations on the bacterial communities in several saline niches based on 16S rRNA sequences have revealed the presence of common phyla like Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes and Bacteroidetes [7]. Nonetheless, some distinctive microbial communities with different structures have been reported for different geographical location assemblages [8,9].…”