Combined Use of Cultivation-Dependent and Cultivation-Independent Methods Indicates that Members of Most Haloarchaeal Groups in an Australian Crystallizer Pond Are Cultivable

Abstract

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCRamplified 16S rRNA genes and by cultivation. High viable counts (10 6 CFU/ml) were obtained on solid media. Long incubation times (>8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

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

“…Three of them (Firmicutes, Proteobacteria and Bacteroidetes) are included in the list of six phyla with known halophiles [18]. The unexpected high-number genera differed from the common opinion for low bacterial diversity in most of the hypersaline environments and domination of only one cluster [19,20] and determined this niche as belonging to saltern ecosystems with highest bacterial diversity. It was significantly higher than that observed by other authors in hypersaline salterns with higher than 30% salt content of the sampling site (Table 2): three bacterial genera have been established in Maras salterns, Peru [21]; five bacteral genera in a 32% salt pond, Santa Pola salterns, Spain [19]; four bacterial genera in 30% salt ponds, Guerrero Negro saltern, Mexico [22]; and five bacterial genera in the 31% salt pond S5 from a solar saltern in Tunisia [23].…”

Halonotius pteroides gen. nov., sp. nov., an extremely halophilic archaeon recovered from a saltern crystallizer

“…One of the dominant groups, representing 16 % of the population, formed a phylogenetically novel clade, and appeared to be specifically related to sequences previously recovered from Deep Lake (Bowman et al, 2000), a hypersaline lake in Antarctica. We referred to this clade, which consisted of 16S rRNA gene sequences from isolates and cloned genes, as the 'Antarctic Deep Lake group' (Burns et al, 2004). The characteristics of four isolates from that group, 1.15.5 T , 2.27.5, 5.24.4 and 6.14.5, are presented here.…”

“…In a previous study, we described the cultivation and diversity of haloarchaea in an Australian saltern crystallizer (Burns et al, 2004). One of the dominant groups, representing 16 % of the population, formed a phylogenetically novel clade, and appeared to be specifically related to sequences previously recovered from Deep Lake (Bowman et al, 2000), a hypersaline lake in Antarctica.…”

Halophilic archaea isolated from man-made young (200 years) salt lakes in Slănic, Prahova, Romania

“…Although a few nonpigmented strains are reported, most strains have been shown to be pigmented pink or red [14]. Their diversity, mainly in saline crystallizer ponds, remains as one research focus point [15,16]. These artificial environments are characterized by a constant value of NaCl concentrations over the years [15] and may represent a selective environment for halophilic microorganisms.…”