Picophytoplankton predominance in hypersaline lakes (Transylvanian Basin,Romania)

The occurrence and importance of photoautotrophic picoplankton (PPP, cells with a diameter <2 μm) was studied along a trophic and salinity gradient in hypersaline lakes of the Transylvanian Basin (Romania). The studied lakes were found to be rich in PPP, with abundances (maximum 7.6 × 10⁶ cells mL^?1) higher than in freshwater and marine environments of similar trophic conditions. The contribution of PPP to the total phytoplankton biovolume did not decrease with increasing trophic state as it was generally found in other aquatic environments. Regardless of the trophic conditions, the contribution of PPP could reach 90–100 % in these hypersaline lakes. We hypothesized that the PPP predominance might be the result of the low grazing pressure, since heterotrophic nanoflagellates (the main grazers of PPP) were absent in the studied samples. There were significant differences in community composition among the lakes along the salinity gradient. CyPPP predominated in less saline waters (mainly below 5 %), while EuPPP were present along the entire salinity range (up to 18.7 %), dominating the phytoplankton between 3 and 13 % salinity. Above 13 % salinity, the phytoplankton was composed mainly of Dunaliella species.     

Spatial and temporal distribution of archaeal diversity in meromictic,hypersaline Ocnei Lake (Transylvanian Basin,Romania)

Saline, meromictic lakes with significant depth are usually formed as a result of salt mining activity. Ocnei Lake is one of the largest Transylvanian (Central Romania) neutral, hypersaline lake of man-made origin. We aimed to survey the seasonal dynamics of archaeal diversity in the water column of Ocnei Lake by employing microbiological methods as well as molecular techniques based on the sequence analysis of the 16S rRNA gene. We found that archaeal diversity in the water column increased with depth and salinity, with 8 OTUs being detected in the epilimnion compared to 21 found in the chemocline, and 32 OTUs in the monimolimnion. Down to 3.5 m depth, the archaeal community was markedly dominated by the presence of an unclassified archaeon sharing 93 % sequence identity to Halogeometricum spp. At the chemocline, the shift in archaeal community composition was associated with an increase in salinity, the main factor affecting the vertical distribution of archaeal assemblages. It appears that the microoxic and hypersaline monimolimnion is populated by several major haloarchaeal taxa, with minor fluctuations in their relative abundances throughout all seasons. The culturable diversity was reasonably correlated to the dominant OTUs obtained by molecular methods. Our results indicate that Ocnei Lake represents a relatively stable extreme habitat, accommodating a diverse and putatively novel archaeal community, as 30 % of OTUs could not be classified at the genus level.     

Isolation of hydrocarbon-degrading extremely halophilic archaea from an uncontaminated hypersaline pond (Camargue, France)

Little information exists about the ability of halophilic archaea present in hypersaline environments to degrade hydrocarbons. In order to identify the potential actors of hydrocarbon degradation in these environments, enrichment cultures were prepared using samples collected from a shallow crystallizer pond with no known contamination history in Camargue, France, with n-alkanes provided as source of carbon and energy. Five alkane-degrading halophilic archaeal strains were isolated: one (strain MSNC 2) was closely related to Haloarcula and three (strains MSNC 4, MSNC 14, and MSNC 16) to Haloferax. Biodegradation assays showed that depending on the strain, 32 to 95% (0.5 g/l) of heptadecane was degraded after 30 days of incubation at 40°C in 225 g/l NaCl artificial medium. One of the strains (MSNC 14) was also able to degrade phenanthrene. This work clearly shows for the first time the potential role of halophilic archaea belonging to the genera Haloarcula and Haloferax in the degradation of hydrocarbons in both pristine and hydrocarbon-contaminated hypersaline environments.     

Diversity of halophilic archaea from six hypersaline environments in Turkey

The diversity of archaeal strains from six hypersaline environments in Turkey was analyzed by comparing their phenotypic characteristics and 16S rDNA sequences. Thirty-three isolates were characterized in terms of their phenotypic properties including morphological and biochemical characteristics, susceptibility to different antibiotics, and total lipid and plasmid contents, and finally compared by 16S rDNA gene sequences. The results showed that all isolates belong to the family Halobacteriaceae. Phylogenetic analyses using approximately 1,388 bp comparisions of 16S rDNA sequences demonstrated that all isolates clustered closely to species belonging to 9 genera, namely Halorubrum (8 isolates), Natrinema (5 isolates), Haloarcula (4 isolates), Natronococcus (4 isolates), Natrialba (4 isolates), Haloferax (3 isolates), Haloterrigena (3 isolates), Halalkalicoccus (1 isolate), and Halomicrobium (1 isolate). The results revealed a high diversity among the isolated halophilic strains and indicated that some of these strains constitute new taxa of extremely halophilic archaea.     

Physiological diversity and adaptations of aerobic heterotrophic bacteria from different depths of hypersaline, heliothermal and meromictic Ekho Lake (East Antarctica)

The hypersaline and meromictic Ekho Lake provides, with increasing depth, a large variety of different habitats to microorganisms. Out of 250 isolates, 51 bacterial strains were studied for physiological adaptations to their environment (i.e. to the depth layer from which they were obtained). Their salinity and temperature tolerance ranges for growth reflected the very conditions that were measured in the samples of their origin collected in the summer of 1989/1990. These observations indicated that the isolates are capable of growth at the depth of their origin and that they conceivably were endemics. Additionally, a separation of physiologically different populations of heterotrophic bacteria was indicated with respect to the utilization of C-compounds: the uppermost 4 m of the oxylimnion (with frequent water turnover and more extreme temperature conditions) supported primarily sugar-utilizing and halotolerant !-Proteobacteria and Gram-positives. The deeper, hypersaline and heliothermally heated layers (>4-24 m) had more moderately halophilic and amino acid-utilizing %-Proteobacteria possibly of marine origin.     

Diversity of cultivable halophilic archaea and bacteria from superficial hypersaline sediments of Tunisian solar salterns

Prokaryotes in the superficial sediments are ecologically important microorganisms that are responsible for the decomposition, mineralization and subsequent recycling of organic matter. The aim of this study was to explore the phylogenetic and functional diversity of halophilic archaea and bacteria isolated from the superficial sediments of solar salterns at Sfax, Tunisia. Sixty four strains were isolated from crystallizer (TS18) and non-crystallizer (M1) ponds and submitted to genotypic characterization and evaluation by amplified ribosomal RNA restriction analysis (ARDRA) techniques. Our findings revealed that the archaeal diversity observed for 29 isolates generated five distinct patterns from the non-crystallizer M1 pond, with Halorubrum chaoviator as the most prevalent cultivable species. However, in the TS18 crystallizer pond, ten restriction patterns were observed, with the prevalence of haloarchaea EB27K, a not yet identified genotype. The construction of a neighbour-joining tree of 16S rRNA gene sequences resulted in the division of the potential new species into two major groups, with four strains closely related to the sequence of the unculturable haloarchaeon EB27K and one strain to the recently described Halovenus aranensis strain. The 35 bacterial strains observed in this work were present only in the non-crystallizer pond (M1) and presented two distinct ARDRA patterns. These strains belonged to the γ-proteobacteria subdivision, with members of Salicola marasensis (83 %) being the most predominant species among the isolates. 16S rRNA gene sequencing revealed that Salicola strains displayed different degrees of homogeneity. The results from pulsed field gel electrophoresis assays showed that the Salicola isolates could be clustered in two distinct groups with different genome sizes.     

Heterogeneity of small plasmids from halophilic archaea.

Small multicopy plasmids in three strains of halophilic archaea, SB3, GRB, and GN101, were found to be present in a cell as a population of related but not identical sequences. Two types of heterogeneity were observed: macroheterogeneity, represented by two major plasmid sequence versions homologous to each other by 80%, and microheterogeneity, in which individual plasmids differ by one or a few nucleotide substitutions.     

新疆两盐湖可培养嗜盐古菌多样性研究

从新疆地区艾比盐湖和艾丁盐湖卤水及泥土样品中分离到86株嗜盐古菌。16S rRNA基因序列分析结果表明,分离自艾比湖的嗜盐古菌分别属于Haloarcula、Halobacterium、Halorubrum、Haloterrigena、Natrinema和Natronorubrum6个属的11个分类单元,而分离自艾丁湖的嗜盐古菌分别属于Haloarcula、Halobiforma、Halorubrum、Haloterrigena、Natrialba、Natrinema6个属的8个分类单元,这一结果表明艾比湖可培养嗜盐古菌生物多样性稍高于艾丁湖。基于16S rRNA基因序列的系统发育分析表明代表菌株ABH15应为Natronorubrum属的中性嗜盐古菌新种,代表菌株ABH07、ABH12、ABH17、ABH19、ABH51和AD30可能是Halobacterium、Halorubrum、Haloterrigena、Haloarcula的新成员。     

Diversity of culturable halophilic archaea isolated from Rambla Salada, Murcia (Spain)

We have studied the diversity of culturable halophilic Archaea at Rambla Salada, Murcia (south-eastern Spain). We made 8 samplings at different places in this habitat during the years 2006 and 2007 and isolated a total of 49 strains, which were identified by means of phenotypic tests and the hypervariable V1–V3 region of the 16S rRNA gene sequences (around 500 bp). The ribosomal data showed that the isolates belonged to 12 genera within the Halobacteriaceae family, with Haloferax and Natrinema being the most abundant. Five strains showed less than 97% sequence identity with validly described species and may well represent new taxa. All the strains grew best with around 25% w/v salts, required high concentrations of NaCl and magnesium and produced red to pink colonies. They were facultative anaerobes with both respiratory and fermentative metabolisms. The diversity of the archaeal community was analysed with the MOTHUR package. We identified 14 OTUs at the 3% genetic distance level and found quite high diversity. Rarefaction curves of richness estimators and diversity indices demonstrated that our collection of isolates represented the archaeal community at Rambla Salada that can be isolated under the conditions used in this work. This is the first report to be published on the culturable archaea at Rambla Salada, an area of considerable ecological interest.     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

Phylogenetic diversity of archaea and bacteria in the anoxic zone of a meromictic lake (Lake Pavin, France)

The compositions of archaeal and bacterial populations at different depths (60 m [mixolimnion-chemocline interface], 70 m [chemocline-subchemocline interface], 90 m, and 92 m [the water-sediment interface]) in the anoxic zone of the water column in Lake Pavin, a freshwater permanently stratified mountain lake in France, were determined. Phylogenetic trees were constructed from sequences to assess archaeal and bacterial diversity at the four sites.     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

Biodegradation of crude oil and pure hydrocarbons by extreme halophilic archaea from hypersaline coasts of the Arabian Gulf

Two extreme halophilic Haloferax strains and one strain each of Halobacterium and Halococcus were isolated from a hypersaline coastal area of the Arabian Gulf on a mineral salt medium with crude oil vapor as a sole source of carbon and energy. These archaea needed at least 1 M NaCl for growth in culture, and grew best in the presence of 4 M NaCl or more. Optimum growth temperatures lied between 40 and 45oC. The four archaea were resistant to the antibiotics chloramphenicol, cycloheximide, nalidixic acid, penicillin, streptomycin and tetracycline. The strains could grow on a wide scope of aliphatic and aromatic (both mono-and polynuclear) hydrocarbons, as sole sources of carbon and energy. Quantitative measurements revealed that these extreme halophilic prokaryotes could biodegrade crude oil (13–47%, depending on the strain and medium salinity), n-octadecane (28–67%) and phenanthrene (13–30%) in culture after 3 weeks of incubation. The rates of biodegradation by all strains were enhanced with increasing NaCl concentration in the medium. Optimal concentration was 3 M NaCl, but even with 4 M NaCl the hydrocarbon-biodegradation rates were higher than with 1 and 2 M NaCl. It was concluded that these archaea could contribute to self-cleaning and bioremediation of oil-polluted hypersaline environments.     

Distribution and diversity of archaea corresponding to the limnological cycle of a hypersaline stratified lake (Solar lake, Sinai, Egypt)

The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to the Halobacteriaceae family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the genera Methanobacterium and Methanococcus. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genus Thermoplasma. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.     

Phylogenetic diversity and activity of aerobic heterotrophic bacteria from a hypersaline oil-polluted microbial mat

The diversity and function of aerobic heterotrophic bacteria (AHB) in cyanobacterial mats have been largely overlooked. We used culture-dependent and molecular techniques to explore the species diversity, degradative capacities and functional guilds of AHB in the photic layer (2mm) of an oil-polluted microbial mat from Saudi Arabia. Enrichment isolation was carried out at different salinities (5% and 12%) and temperatures (28 and 45 degrees C) and on various substrates (acetate, glycolate, Spirulina extract and crude oils). Counts of most probable number showed a numerical abundance of AHB in the range of 1.15-8.13x10(6) cellsg(-1) and suggested the presence of halotolerant and thermotolerant populations. Most of the 16S rRNA sequences of the obtained clones and isolates were phylogenetically affiliated to the groups Gammaproteobacteria, Bacteriodetes and Alphaproteobacteria. Groups like Deltaproteobacteria, Verrucomicrobia, Planctomycetes, Spirochaetes, Acidobacteria and Deinococcus-Thermus were only detected by cloning. The strains isolated on acetate and glycolate belonged to the genera Marinobacter, Halomonas, Roseobacter and Rhodobacter whereas the strains enriched on crude oil belonged to Marinobacter and Alcanivorax. Members of the Bacteriodetes group were only enriched on Spirulina extract indicating their specialization in the degradation of cyanobacterial dead cells. The substrate spectra of representative strains showed the ability of all AHB to metabolize cyanobacterial photosynthetic and fermentation products. However, the unique in situ conditions of the mat apparently favored the enrichment of versatile strains that grew on both the cyanobacterial exudates and the hydrocarbons. We conclude that AHB in cyanobacterial mats represent a diverse community that plays an important role in carbon-cycling within microbial mats.     

Sulfidogenesis in hypersaline chloride-sulfate lakes of Kulunda Steppe (Altai, Russia)

The activity and culturable diversity of sulfidogens were investigated in anoxic sediments of four hypersaline lakes with pH 7.6-8.2 in the Kulunda Steppe (Altai, Russia). Sulfate reduction rates were low, varying from 0.1 to 6.0 nmol HS(-) /(cm(3) h) with a maximum in the top 10 cm layer. Potential sulfidogenic rates with thiosulfate and sulfur as the e-acceptors were higher than with sulfate and were stimulated by formate, lactate, and acetate. Sulfidogenesis was optimal at salt concentrations below 2 M NaCl. Cultivation at 2 M NaCl resulted in the isolation of several strains of moderately halophilic SRB, but no growth of SRB was observed at 4 M NaCl. At lithotrophic conditions (i.e., with formate or H(2) as e-donors), several closely related alkalitolerant strains belonging to the genus Desulfonatronovibrio were isolated. Enrichments at heterotrophic conditions with lactate, propionate, acetate, or butyrate using sulfate or thiosulfate as e-acceptors yielded isolates related to Desulfosalsimonas propionicica, Desulfohalobium utahense, and Desulfocella halophila. Sulfur-reducing enrichments at 2 M NaCl with ethanol produced a member of the genus Halanaerobium, while enrichments at 4 M NaCl with acetate were dominated by archaea, demonstrating for the first time such type of catabolism in haloarchaea.     

Halapricum desulfuricans sp. nov., carbohydrate-utilizing,sulfur-respiring haloarchaea from hypersaline lakes

Nine pure cultures of neutrophilic haloaloarchaea capable of anaerobic growth by carbohydrate-dependent sulfur respiration were isolated from hypersaline lakes in southwestern Siberia and southern Russia. According to phylogenomic analysis the isolates were closely related to each other and formed a new species within the genus Halapricum (family Haloarculaceae). They have three types of catabolism: fermentative, resulting in H₂ formation; anaerobic respiration using sulfur compounds as e-acceptors and aerobic respiration. Apart from elemental sulfur, all isolates can also use three different sulfoxides as acceptors and the type strain also grows with thiosulfate, reducing it partially to sulfide and sulfite. All strains utilized sugars and glycerol as the e-donors and C source for anaerobic growth and some can also grow with alpha-glucans, such as starch and dextrins. The major respiratory menaquinones are MK-8:8 and MK-8:7, but 5–19% consists of “thermoplasmata” quinones (MMK-8:8 and MMK-8:7), whose occurrence in haloarchaea is unprecedented. On the basis of their unique physiological properties and results of phylogenomic analysis, the isolates are suggested to be classified into a novel species Halapricum desulfuricans sp. nov. (type strain HSR12-2^T = JCM 34032^T = UNIQEM U1001^T).     

Sulfate reduction and methanogenesis in the Shira and Shunet meromictic lakes (Khakasia, Russia)

The biogeochemical and molecular biological study of the chemocline and sediments of saline meromictic lakes Shira and Shunet (Khakasia, Russia) was performed. A marked increase in the rates of sulfate reduction and methanogenesis was revealed at the medium depths of the chemocline. The rates of these processes in the bottom sediments decreased with depth. The numbers of the members of domains Bacteria, Archaea, and of sulfate-reducing bacteria (SRB) were determined by fluorescence in situ hybridization with rRNA specific oligonucleotide probes labeled with horseradish peroxidase and subsequent tyramide signal amplification. In the chemocline, both the total microbial numbers and those of Bacteria were shown to increase with depth. The archaea and SRB were present in almost equal numbers. In the lake sediments, a drastic decrease in microbial numbers with depth was revealed. SRB were found to prevail in the upper sediment layer and archaea in the lower one. This finding correlated with the measured rates of sulfate reduction and methanogenesis.