Articoli di riviste sul tema "Microbial populations – Antarctica"

Antarctic cryoconite holes, or small melt-holes in the surfaces of glaciers, create habitable oases for isolated microbial communities with tightly linked microbial population structures. Viruses may influence the dynamics of polar microbial communities, but the viromes of the Antarctic cryoconite holes have yet to be characterized. We characterize single-stranded DNA (ssDNA) viruses from three cryoconite holes in the Taylor Valley, Antarctica, using metagenomics. Half of the assembled metagenomes cluster with those in the viral family Microviridae (n = 7), and the rest with unclassified circular replication associated protein (Rep)-encoding single-stranded (CRESS) DNA viruses (n = 7). An additional 18 virus-like circular molecules encoding either a Rep, a capsid protein gene, or other unidentified but viral-like open reading frames were identified. The samples from which the genomes were identified show a strong gradient in microbial diversity and abundances, and the number of viral genomes detected in each sample mirror that gradient. Additionally, one of the CRESS genomes assembled here shares ~90% genome-wide pairwise identity with a virus identified from a freshwater pond on the McMurdo Ice Shelf (Antarctica). Otherwise, the similarity of these viruses to those previously identified is relatively low. Together, these patterns are consistent with the presence of a unique regional virome present in fresh water host populations of the McMurdo Dry Valley region.

Deception Island is a geothermal location in Antarctica that presents active fumaroles, which confers unique characteristics to this habitat. Several studies about microbial communities in Antarctica have been carried out, nevertheless, Antarctic microbiota is still partially unknown. Here we present a multidisciplinary study about sediments obtained by deposition during 4 years in which several approaches have been considered for their characterization. First, a physicochemical characterization, using ionic chromatography and mass spectrometry for the determination of most abundant ions (chloride and sulphate) and elements (mainly silicon), was conducted. In addition, the total microbial community was studied using a metataxonomical approach, revealing a bacterial community dominated by Proteobacteria and Thaumarchaeota as the main archaeal genera and a fungal community mainly composed by Aspergillaceae. Culture-dependent studies showed low microbial diversity, only achieving the isolation of Bacillus-related species, some of them thermophilic, and the isolation of common fungi of Aspergillus or Penicillium spp. Furthermore, diatoms were detected in the sediment and characterized attending to their morphological characteristics using scanning electron microscopy. The study reveals a high influence of the physicochemical conditions in the microbial populations and their distribution, offering valuable data on the interaction between the island and water microbiota.

AbstractThis study describes the ecology and distribution of the only two native Antarctic insects, the chironomid species Parochlus steinenii and Belgica antarctica, both found on Byers Peninsula (Livingston Island, South Shetland Islands). Parochlus steinenii inhabits lakes of the central plateau of Byers Peninsula associated with aquatic mosses on the bottom of lakes and in some streams of the South Beach area. Some streams have stable populations which are able to complete their life cycle while other streams have temporary, unstable populations. Belgica antarctica also inhabits streams running through mosses located in the South Beach area. Our data indicate that this species has a limited dispersal capability which is positively light activated for both adults and pupae. Both Antarctic midge species coexists on Byers Peninsula and share some stretches of streams. Isotopic studies show a non-selective feeding regime for both species with mixed carbon sources associated with both biofilm/microbial mats and mosses.

ABSTRACTOnce thought to be devoid of life, the Antarctic Ice Sheet is now known to be a dynamic reservoir of organic carbon and metabolically active microbial cells. At the ice-bed interface, subglacial lake and sedimentary environments support low diversity microbial populations, adapted to perennial cold, anoxia and lack of light. The dynamic exchange of water between these shallow environments conveys meltwaters and associated sediments into the coastal ocean. This, together with the release of iceberg-rafted debris to more distal coastal environments, could be important for sustaining primary productivity in the iron-limited Southern Ocean, via the release of associated nutrients and bioavailable iron. We estimate the magnitude and review the wider impacts of the potential export of nutrients (N, P, C, Si and bioavailable Fe) dissolved and associated with suspended sediments in Antarctic runoff and entombed in iceberg rafted debris. Located beneath subglacial lakes and the subglacial till complex are deep sedimentary basins up to 14 km thick, located largely around the Antarctic periphery. These sedimentary basins are largely hydrologically decoupled from shallower lake and till environments by the presence of highly consolidated sediments which limit the penetration of glacial meltwaters to depth. They provide extensive habitats for sustained microbial activity over Ma timescales, and are likely to be a focal point for the anaerobic cycling of organic carbon and other elements in the deep sub-surface. Organic carbon buried in these basins during ice sheet formation is thought to be microbially cycled to methane gas, and the methane largely stored as hydrate within sediments, stabilised by the high pressure/low temperature conditions. We conclude that the export of nutrients and biogenic gases from deep and shallow subglacial Antarctic environments designates Antarctica as a potentially important component of the Earth's carbon cycle, and highlight the importance of evaluating these potential impacts further via global and regional-scale biogeochemical modelling.

A field experiment investigating the effect of oil contamination on benthic microbial communities was conducted near Casey Station, East Antarctica. Defaunated sediment was treated with a mixture of Special Antarctic Blend diesel and lubricating oil and deployed in three different bays for eleven weeks. A molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE), was used to investigate the microbial community structure. The variation between replicate samples within treatment groups indicates that the benthic microbial populations are very diverse and evenly distributed. Comparisons to determine the significance of both deployment location and hydrocarbon treatment showed that the greatest effect was from a combination of location and treatment. Detailed analysis suggests that subtle differences may be obscured by variability introduced by PCR and gel stages in DGGE, undermining this experimental approach. It is concluded that both location and hydrocarbon contamination influenced the development of the microbial communities but that the effect of hydrocarbon treatment varied with location. This has important implications for the design of future experiments on the effect of hydrocarbons on benthic communities, especially if it is intended to generalize the conclusions drawn from site specific studies.

AbstractCyanobacteria inhabit the Antarctic continent and have even been observed in the most southerly ice-free areas of Antarctica (86–87° S). The highest molecular diversity of cyanobacterial communities was found in the areas located between 70° S and 80° S. Further south and further north from this zone, the diversity abruptly decreased. Seventy-nine per cent (33 of 42 operational taxonomic units) of Antarctic terrestrial cyanobacteria have a cosmopolitan distribution. Analysis of the sampling efforts shows that only three regions (southern Victoria Land, the Sør Rondane Mountains and Alexander Island) have been particularly well studied, while other areas did not receive enough attention. Although cyanobacteria possess a capacity for long-range transport, regional populations in Antarctic ice-free areas seem to exist. The cyanobacterial communities of the three most intensively studied regions, separated from each other by a distance of 3000–3400 km, had a low degree of similarity with each other. Further development of microbial biogeography demands a standardized approach. For this purpose, as a minimal standard, we suggest using the sequence of cyanobacterial 16S rRNA gene between Escherichia coli positions 405 and 780.

The Ross Sea is one of the most productive areas of the Southern Ocean and includes several functionally different marine ecosystems. With the aim of identifying signs and patterns of microbial response to current climate change, seawater microbial populations were sampled at different depths, from surface to the bottom, at two Ross Sea mooring areas southeast of Victoria Land in Antarctica. This oceanographic experiment, the XX Italian Antarctic Expedition, 2004-05, was carried out in the framework of the ABIOCLEAR project as part of LTER-Italy. Here, microbial biogeochemical rates of respiration, carbon dioxide production, total community heterotrophic energy production, prokaryotic heterotrophic activity, production (by3H-leucine uptake) and prokaryotic biomass (by image analysis) were determined throughout the water column. As ancillary parameters, chlorophylla, adenosine-triphosphate concentrations, temperature and salinity were measured and reported. Microbial metabolism was highly variable amongst stations and depths. In epi- and mesopelagic zones, respiratory rates varied between 52.4–437.0 and 6.3–271.5 nanol O2l-1h-1; prokaryotic heterotrophic production varied between 0.46–29.5 and 0.3–6.11 nanog C l-1h-1; and prokaryotic biomass varied between 0.8–24.5 and 1.1–9.0 µg C l-1, respectively. The average heterotrophic energy production ranged between 570 and 103 mJ l-1h-1in upper and deeper layers, respectively. In the epipelagic layer, the Prokaryotic Carbon Demand and Prokaryotic Growth Efficiency averaged 9 times higher and 2 times lower, respectively, than in the mesopelagic one. The distribution of plankton metabolism and organic matter degradation was mainly related to the different hydrological and trophic conditions. In comparison with previous research, the Ross Sea results, here, evidenced a relatively impoverished oligotrophic microbial community, throughout the water column.

Diatom taxa present in the inland streams and lakes of the McMurdo Dry Valleys and James Ross Island, Antarctica, are presented in this paper. A total of nine taxa are illustrated, with descriptions of four new species ( Luticola austroatlantica sp. nov., Luticola dolia sp. nov., Luticola laeta sp. nov., Muelleria supra sp. nov.). In the perennially ice-covered lakes of the McMurdo Dry Valleys, diatoms are confined to benthic mats within the photic zone. In streams, diatoms are attached to benthic surfaces and within the microbial mat matrix. One species, L. austroatlantica, is found on James Ross Island, of the southern Atlantic archipelago, and the McMurdo Dry Valleys. The McMurdo Dry Valley populations are at the lower range of the size spectrum for the species. Streams flow for 6–10 weeks during the austral summer, when temperatures and solar radiation allow glacial ice to melt. The diatom flora of the region is characterized by species assemblages favored under harsh conditions, with naviculoid taxa as the dominant group and several major diatom groups conspicuously absent.

Quartz stone sublithic cyanobacterial communities are common throughout the Vestfold Hills, Eastern Antarctica (68°S 78°E) contributing biomass in areas otherwise devoid of any type of vegetation. In this study, the sublithic microbial community and underlying soil was investigated using a variety of traditional and molecular methods. Although direct epifluorescent counts of the sublithic growth (average 1.1 × 109 cells g−1 dry weight) and underlying soil (0.5 × 109 cells g−1 dry weight) were similar, sublith viable counts (2.1 × 107 cfu g−1 dry weight) were on average 3-orders of magnitude higher in the subliths. Enrichment and molecular analyses revealed the predominate cyanobacteria were non-halophilic, able to grow optimally at 15–20°C, and were related to the Phormidium subgroup with several distinct morphotypes and phylotypes present. Sublithic heterotrophic bacterial populations and those of underlying soils included mostly psychrotolerant taxa typical of Antarctic soil. However, psychrophilic and halophilic bacteria, mostly members of the alpha subdivision of the Proteobacteria and the order Cytophagales, were abundant in the sublithic growth film (20–40% of the viable count and about 50% of isolated individual taxa) but absent from underlying soils. It is suggested that quartz stone subliths might constitute a “refuge” for psychrophilic bacteria.

The genus Serratia are opportunistic bacteria widely spread in natural environment. At the same time, this bacterial genus consists of the species associated with outbreaks of nosocomial infections. Serratia species are found in extreme habitats, but pathogenic potential of polyextremophilic strains in this genus remains unexplored. The aim of this study was to compare the genomes of two Serratia strains isolated in polar regions, primarily examining genetic factors of virulence and adaptation to cryogenic environment. During the 56th Russian Antarctic Expedition the Serratia liquefaciens 72 strain was isolated from a guano sample of the Adelie Penguin (Pygoscelis adeliae) colony on Tokarev Island (Haswell Archipelago, East Antarctica). The Serratia fonticola 5l strain was isolated from the frozen carcass of moose (Alces alces) fossils found on the Buor-Khaya Peninsula near the Laptev Sea coast (Yakutia Region, Russia). The whole-genome sequencing of such strains allowed to reveal genetic structures evidencing about their successful adaptation to low temperatures. Thus, it was found that both genomes contain genes encoding the main cold shock proteins, phylogenetically close to the corresponding genes in the hypobarotolerant Serratia liquefaciens strain ATCC 27592. Furthermore, both strains bear a cluster of tc-fABCD genes determining the bacterial adhesion to epithelial tissues, and the genes for RTX toxins — adhesins, crucial factors of biofilm formation in pathogenic Gram-negative bacteria. Experimental studies confirmed the ability of Serratia liquefaciens 72 and Serratia fonticola 5l to actively form biofilms in a wide temperature range (from 6°C to 37°C). The results obtained indicate that the examined genus Serratia strains isolated in Arctica and Antarctica exert overall similar adaptation strategies to polar climate, including the ability to produce pili, show active adhesion, and biofilm formation under low temperatures. Genetic adaptive factors may also act as pathogenicity factors allowing extremotolerant Serratia strains to exert traits of opportunistic and nosocomial pathogens and spread via chilled food-borne transmission. The wide use of food technologies, such as cooling and vacuum sealing, can potentially create a new ecological niche favourable for selection of psychrotolerant and hypobarotolerant pathogens. The data obtained allow to raise a question about necessity of further studies to monitor genetic diversity among psychrophilic hypobarotolerant microbial populations possessing pathogenic and epidemic potential.

ABSTRACT. Biodiversity and distribution of cyanobacteria at Dronning Maud Land, East Antarctica.The current study describes the biodiversity and distribution of cyanobacteria from the natural habitats of Schirmacher land, East Antarctica surveyed during 23rd Indian Antarctic Expedition (2003–2004). Cyanobacteria were mapped using the Global Positioning System (GPS). A total of 109 species (91 species were non-heterocystous and 18 species were heterocystous) from 30 genera and 9 families were recorded; 67, 86 and 14 species of cyanobacteria were identified at altitudes of sea level >100 m, 101–150 m and 398–461 m, respectively. The relative frequency and relative density of cyanobacterial populations in the microbial mats showed that 11 species from 8 genera were abundant and 6 species (Phormidium angustissimum, P. tenue, P. uncinatum Schizothrix vaginata, Nostoc kihlmanii and Plectonema terebrans) could be considered as dominant species in the study area.Key words. Antarctic, cyanobacteria, biodiversity, blue-green algae, Schirmacher oasis, Species distribution.RESUMEN. Biodiversidad y distribución de las cianobacterias de Dronning Maud Land, Antártida Oriental. En este estudio se describe la biodiversidad y distribución de las cianobacterias presentes en los hábitats naturales de Schirmacher, Antártida Oriental, muestreados durante la 23a Expedición India a la Antártida (2003-2004). Las muestras de cianobacterias fueron georreferenciadas mediante un GPS. Se identificaron 109 especies (91 no heterocistadas y 18 provistas de heterocistes) de 30 géneros y 9 familias; en los tramos de altitud sobre el nivel del mar >100 m, 101-150 m y 398-461 m se detectaron 67, 86 y 14 especies, respectivamente. La frecuencia y densidad relativas de las poblaciones de cianobacterias en los tapices microbianos mostraron que 11 especies de 8 géneros eran abundantes y que 6 especies (Phormidium angustissimum, P. tenue, P. uncinatum, Schizothrix vaginata, Nostoc kihlmanii y Plectonema terebrans) se pueden considerar como dominantes en el área de estudio.Palabras clave. Algas verde-azuladas, Antártida, cianobacterias, distribución de especies, oasis de Schirmacher.

Bacterial cultures obtained through selective enrichment of beach sand collected 60 days and one year after treatment of sites in a pilot oil spill trial conducted at Airport Beach, Vestfold Hills, East Antarctica, were examined for the ability to degrade n-alkanes and phenanthrene. The effects of different hydrocarbon mixtures (Special Antarctic Blend [SAB] and BP-Visco), fish oil [orange roughy]) and inoculation of replicate sites with water from Organic Lake (previously shown to contain hydrocarbon-degrading bacteria) on the indigenous microbial population were examined. Of the cultures obtained, those from sites treated with SAB and BP-Visco degraded n-alkanes most consistently and typically to the greatest extent. Two mixed cultures obtained from samples collected at 60 days and two isolates obtained from these cultures extensively degraded phenanthrene. 1-Hydroxy-naphthoic acid formed the major phenanthrene metabolite. Lower levels of salicylic acid, 1-naphthol, 1, 4-naphthaquinone and phenanthrene 9-10 dihydrodiol were detected in extracts of phenanthrene grown cultures. This study shows that under laboratory conditions indigenous Antarctic bacteria can degrade n-alkanes and the more recalcitrant polycyclic aromatic hydrocarbon, phenanthrene. The enrichment of hydrocarbon degrading microorganisms in Antarctic ecosystems exposed to hydrocarbons is relevant for the long term fate hydrocarbon spills in this environment.

This review seeks to highlight the potential importance of viruses in Antarctic ecosystems and describe the limited scope of Antarctic virus studies to date, including studies of marine, terrestrial and freshwater communities. Although much of the existing work focuses on the microbial community, there are also studies of virus infection in Antarctic animal and plant populations. We describe methodologies available for the study of viral ecology in the field and in calling for a more intensive research effort discuss how microbial ecology might benefit from the study of viruses in Antarctic ecosystems.

ABSTRACT Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated.

The island species–area relationship (ISAR) is a positive association between the number of species and the area of an isolated, island-like habitat. ISARs are ubiquitous across domains of life, yet the processes generating ISARs remain poorly understood, particularly for microbes. Larger and more productive islands are hypothesized to have more species because they support larger populations of each species and thus reduce the probability of stochastic extinctions in small population sizes. Here, we disentangled the effects of “island” size and productivity on the ISAR of Antarctic cryoconite holes. We compared the species richness of bacteria and microbial eukaryotes on two glaciers that differ in their productivity across varying hole sizes. We found that cryoconite holes on the more productive Canada Glacier gained more species with increasing hole area than holes on the less productive Taylor Glacier. Within each glacier, neither productivity nor community evenness explained additional variation in the ISAR. Our results are, therefore, consistent with productivity shaping microbial ISARs at broad scales. More comparisons of microbial ISARs across environments with limited confounding factors, such as cryoconite holes, and experimental manipulations within these systems will further contribute to our understanding of the processes shaping microbial biogeography.

AbstractThe planktonic microbial communities of three meltwater ponds, located on the McMurdo Ice Shelf, were investigated from the end of January 2008 to early April, during which almost the entire pond volumes froze. The ponds were comprised of an upper mixed layer overlying a salt-stabilized density gradient in which planktonic communities were primarily embedded. Plankton comprised all components of the “microbial loop”, though carnivorous protists were rare. As the ponds froze and light became increasingly limited, it was expected conditions would induce physiological changes altering the functional role of autotrophic and heterotrophic microplankton within the ponds. The results showed that microbial groups responded to the onset of winter by declining in abundance, though an exception was the appearance of filamentous cyanobacteria in the water column in March. As freezing progressed, autotrophs declined more rapidly than heterotrophs and grazing rates and abundances of mixotrophic and heterotrophic organisms increased. Grazing pressure on bacteria and picophytoplankton also increased, in part explaining their decline over time. The results indicate that stressors imposed during freezing select for increasing heterotrophy within the remaining microbial communities, although all components of the food web eventually decline as the final freeze approaches.

It has been demonstrated that the englacial ecosystem in volcanic environments is inhabited by active bacteria. To know whether this result could be extrapolated to other Antarctic glaciers and to study the populations of microeukaryotes in addition to those of bacteria, a study was performed using ice samples from eight glaciers in the South Shetland archipelago. The identification of microbial communities of bacteria and microeukaryotes using 16S rRNA and 18S rRNA high throughput sequencing showed a great diversity when compared with microbiomes of other Antarctic glaciers or frozen deserts. Even the composition of the microbial communities identified in the glaciers from the same island was different, which may be due to the isolation of microbial clusters within the ice. A gradient in the abundance and diversity of the microbial communities from the volcano (west to the east) was observed. Additionally, a significant correlation was found between the chemical conditions of the ice samples and the composition of the prokaryotic populations inhabiting them along the volcanic gradient. The bacteria that participate in the sulfur cycle were those that best fit this trend. Furthermore, on the eastern island, a clear influence of human contamination was observed on the glacier microbiome.

Antibiotic resistance in aquatic bacteria has increased steadily as a consequence of the widespread use of antibiotics, but practice and international treaty should have limited antibiotic contamination in Antarctica. We estimated antibiotic resistance in microorganisms isolated from the Antarctic marine waters and a penguin rookery, for 2 reasons: (i) as a measure of human impact and (ii) as a potential “snapshot” of the preantibiotic world. Samples were taken at 4 established sampling sites near Palmer Station, which is situated at the southern end of the Palmer Archipelago (64°10′S, 61°50′W). Sites were chosen to provide different potentials for human contamination. Forty 50 mL samples of seawater were collected and colony-forming units (CFU)/mL were determined at 6 and 20 °C. For this study, presumed psychrophiles (growth at 6 °C) were assumed to be native to Antarctic waters, whereas presumed mesophiles (growth at 20 °C but not at 6 °C) were taken to represent introduced organisms. The 20–6 °C CFU/mL ratio was used as a measure of the relative impact to the ecosystem of presumably introduced organisms. This ratio was highest at the site nearest to Palmer Station and decreased with distance from it, suggesting that human presence has impacted the natural microbial flora of the site. The frequency of resistance to 5 common antibiotics was determined in each group of isolates. Overall drug resistance was higher among the presumed mesophiles than the presumed psychrophiles and increased with proximity to Palmer Station, with the presumed mesophiles showing higher frequencies of single and multiple drug resistance than the psychrophile population. The frequency of multidrug resistance followed the same pattern. It appears that multidrug resistance is low among native Antarctic bacteria but is increased by human habitation.

AbstractContrary to earlier beliefs, crustaceans are present in ice-covered lakes of Antarctica. Interpretation of the significance of this has been hampered by the absence of robust identification of taxa present. We examine cyclopoid copepods from three widely separated lakes. All belong to the michaelseni group of the genus Diacyclops, which is widespread across Continental Antarctica, but do not fit into any existing species. Two new species were identified from eastern Antarctica, D. walkeri from Pineapple Lake (Vestfold Hills) and D. kaupi from Transkriptsii Gulf (Bunger Hills). Most significant was a dense population of a new epibenthic species (D. joycei) associated with microbial mats in Lake Joyce, one of the smaller McMurdo Dry Valleys lakes. This represents the first record of adult cyclopoid copepods from the ice-covered lakes of the Transantarctic Mountains. Continental Antarctica is the centre of diversity for this group of crustaceans and we argue that this is better explained by persistence through past glacial advances rather than by recent post-glacial colonization. The existence of a species endemic to Lake Joyce but apparently absent from other Dry Valleys lakes is discussed in relation to our understanding of the history of the McMurdo Dry Valleys lakes and their faunas.

This work aims to summarize the results of research carried out by Brazilian researchers on the plant communities of Antarctic ice free areas during the last twenty five years. Since 1988 field work has been carried out in Elephant Island, King George Island, Nelson Island and Deception Island. During this period six papers were published on the chemistry of lichens, seven papers on plant taxonomy, five papers on plant biology, two studies on UVB photoprotection, three studies about the relationships between plant communities and bird colonies and eleven papers on plant communities from ice free areas. At the present, Brazilian botanists are researching the plant communities of Antarctic ice free areas in order to understand their relationships to soil microbial communities, the biodiversity, the distribution of the plants populations and their relationship with birds colonies. In addition to these activities, a group of Brazilian researchers are undertaking studies related to Antarctic plant genetic diversity, plant chemistry and their biotechnological applications.

During the attempt to directly access, measure and sample Subglacial Lake Ellsworth in 2012–2013, we conducted microbiological analyses of the drilling equipment, scientific instrumentation, field camp and natural surroundings. From these studies, a number of lessons can be learned about the cleanliness of deep Antarctic subglacial lake access leading to, in particular, knowledge of the limitations of some of the most basic relevant microbiological principles. Here, we focus on five of the core challenges faced and describe how cleanliness and sterilization were implemented in the field. In the light of our field experiences, we consider how effective these actions were, and what can be learnt for future subglacial exploration missions. The five areas covered are: (i) field camp environment and activities, (ii) the engineering processes surrounding the hot water drilling, (iii) sample handling, including recovery, stability and preservation, (iv) clean access methodologies and removal of sample material, and (v) the biodiversity and distribution of bacteria around the Antarctic. Comparisons are made between the microbiology of the Lake Ellsworth field site and other Antarctic systems, including the lakes on Signy Island, and on the Antarctic Peninsula at Lake Hodgson. Ongoing research to better define and characterize the behaviour of natural and introduced microbial populations in response to deep-ice drilling is also discussed. We recommend that future access programmes: (i) assess each specific local environment in enhanced detail due to the potential for local contamination, (ii) consider the sterility of the access in more detail, specifically focusing on single cell colonization and the introduction of new species through contamination of pre-existing microbial communities, (iii) consider experimental bias in methodological approaches, (iv) undertake in situ biodiversity detection to mitigate risk of non-sample return and post-sample contamination, and (v) address the critical question of how important these microbes are in the functioning of Antarctic ecosystems.

In Antarctic lakes, organisms are confronted by continuous low temperatures as well as a poor light climate and nutrient limitation. Such extreme environments support truncated food webs with no fish, few metazoans and a dominance of microbial plankton. The key to success lies in entering the short Antarctic summer with actively growing populations. In many cases, the most successful organisms continue to function throughout the year. The few crustacean zooplankton remain active in the winter months, surviving on endogenous energy reserves and, in some cases, continuing development. Among the Protozoa, mixotrophy is an important nutritional strategy. In the extreme lakes of the McMurdo Dry Valleys, planktonic cryptophytes are forced to sustain a mixotrophic strategy and cannot survive by photosynthesis alone. The dependence on ingesting bacteria varies seasonally and with depth in the water column. In the Vestfold Hills, Pyramimonas , which dominates the plankton of some of the saline lakes, also resorts to mixotrophy, but does become entirely photosynthetic at mid–summer. Mixotrophic ciliates are also common and the entirely photosynthetic ciliate Mesodinium rubrum has a widespread distribution in the saline lakes of the Vestfold Hills, where it attains high concentrations. Bacteria continue to grow all year, showing cycles that appear to be related to the availability of dissolved organic carbon. In saline lakes, bacteria experience sub–zero temperatures for long periods of the year and have developed biochemical adaptations that include anti–freeze proteins, changes in the concentrations of polyunsaturated fatty acids in their membranes and suites of low–temperature enzymes.

ABSTRACTThe anoxic and freezing brine that permeates Lake Vida's perennial ice below 16 m contains an abundance of very small (≤0.2-μm) particles mixed with a less abundant population of microbial cells ranging from >0.2 to 1.5 μm in length. Fluorescent DNA staining, electron microscopy (EM) observations, elemental analysis, and extraction of high-molecular-weight genomic DNA indicated that a significant portion of these ultrasmall particles are cells. A continuous electron-dense layer surrounding a less electron-dense region was observed by EM, indicating the presence of a biological membrane surrounding a cytoplasm. The ultrasmall cells are 0.192 ± 0.065 μm, with morphology characteristic of coccoid and diplococcic bacterial cells, often surrounded by iron-rich capsular structures. EM observations also detected the presence of smaller unidentified nanoparticles of 0.020 to 0.140 μm among the brine cells. A 16S rRNA gene clone library from the brine 0.1- to 0.2-μm-size fraction revealed a relatively low-diversity assemblage ofBacteriasequences distinct from the previously reported >0.2-μm-cell-size Lake Vida brine assemblage. The brine 0.1- to 0.2-μm-size fraction was dominated by theProteobacteria-affiliated generaHerbaspirillum,Pseudoalteromonas, andMarinobacter. Cultivation efforts of the 0.1- to 0.2-μm-size fraction led to the isolation ofActinobacteria-affiliated generaMicrobacteriumandKocuria. Based on phylogenetic relatedness and microscopic observations, we hypothesize that the ultrasmall cells in Lake Vida brine are ultramicrocells that are likely in a reduced size state as a result of environmental stress or life cycle-related conditions.

Previous studies have shown that a significant part of the bacterial communities of Antarctic soils is represented by cells passing through filters with pore sizes of 0.2 µm. These results raised new research questions about the composition and diversity of the filterable forms of bacteria (FFB) in Antarctic soils and their role in the adaptation of bacteria to the extreme living conditions. To answer such questions, we analyzed the succession of bacterial communities during incubation of Antarctic soil samples from the Bunger Hills at increased humidity and positive temperatures (5 °C and 20 °C). We determined the total number of viable cells by fluorescence microscopy in all samples and assessed the taxonomic diversity of bacteria by next-generation sequencing of the 16S rRNA gene region. Our results have shown that at those checkpoints where the total number of cells reached the maximum, the FFB fraction reached its minimum, and vice versa. We did not observe significant changes in taxonomic diversity in the soil bacterial communities during succession. During our study, we found that the soil bacterial communities as a whole and the FFB fraction consist of almost the same phylogenetic groups. We suppose rapid transition of the cells of the active part of the bacterial population to small dormant forms is one of the survival strategies in extreme conditions and contributes to the stable functioning of microbial communities in Antarctic soils.

Microbes are the foundation of marine ecosystems [Falkowski PG, Fenchel T, Delong EF (2008) Science 320(5879):1034–1039]. Until now, the analytical framework for understanding the implications of ocean warming on microbes has not considered thermal exposure during transport in dynamic seascapes, implying that our current view of change for these critical organisms may be inaccurate. Here we show that upper-ocean microbes experience along-trajectory temperature variability up to 10 °C greater than seasonal fluctuations estimated in a static frame, and that this variability depends strongly on location. These findings demonstrate that drift in ocean currents can increase the thermal exposure of microbes and suggests that microbial populations with broad thermal tolerance will survive transport to distant regions of the ocean and invade new habitats. Our findings also suggest that advection has the capacity to influence microbial community assemblies, such that regions with strong currents and large thermal fluctuations select for communities with greatest plasticity and evolvability, and communities with narrow thermal performance are found where ocean currents are weak or along-trajectory temperature variation is low. Given that fluctuating environments select for individual plasticity in microbial lineages, and that physiological plasticity of ancestors can predict the magnitude of evolutionary responses of subsequent generations to environmental change [Schaum CE, Collins S (2014) Proc Biol Soc 281(1793):20141486], our findings suggest that microbial populations in the sub-Antarctic (∼40°S), North Pacific, and North Atlantic will have the most capacity to adapt to contemporary ocean warming.

AbstractSubglacial ecosystems have recently become of interest within the astrobiological community, as they represent a potentially habitable location in otherwise uninhabitable environments. We used data from Blood Falls, particularly the periodic discharge from the subglacial reservoir beneath Taylor Glacier, Antarctica, to construct an ecosystem model of the putative subglacial microbial community residing there using system dynamics modelling. The model results were, for the most part, within an order of magnitude of the geochemical field data. Productivity was quite low, at 6.4×10−5g carbon l−1yr−1. Based on the results, we draw the following conjectures for the search for life on Mars: A similar ecosystem would require a continual supply of oxidized iron for energy and generate significant amounts of reduced iron as a waste product, be relatively resilient to temporary disturbances, and, thermodynamically, would require at least 0.003 kJ mol l−1of energy to survive at that level of productivity. These results may help to better identify the constraints and boundaries of ecosystems in extreme environments, on Earth and other planetary bodies.

ABSTRACT The permanently frozen freshwater Lake Fryxell, located in the Dry Valleys of Antarctica, exhibits an ideal geochemistry for microbial sulfate reduction. To investigate the population of sulfate-reducing bacteria in Lake Fryxell, both 16S rRNA gene and metabolic primer sets targeting the dsrA gene for the dissimilatory sulfite reductase alpha subunit were employed to analyze environmental DNA obtained from the water column and sediments of Lake Fryxell. In addition, enrichment cultures of sulfate-reducing bacteria established at 4°C from Lake Fryxell water were also screened using the dsrA primer set. The sequence information obtained showed that a diverse group of sulfate-reducing prokaryotes of the domain Bacteria inhabit Lake Fryxell. With one exception, the enrichment culture sequences were not represented within the environmental sequences. Sequence data were compared with the geochemical profile of Lake Fryxell to identify possible connections between the diversity of sulfate-reducing bacteria and limnological conditions. Several clone groups were highly localized with respect to lake depth and, therefore, experienced specific physiochemical conditions. However, all sulfate-reducing bacteria inhabiting Lake Fryxell must function under the constantly cold conditions characteristic of this extreme environment.

ABSTRACT Depth profiles of metals in Lake Vanda, a permanently ice-covered, stratified Antarctic lake, suggest the importance of particulate manganese oxides in the scavenging, transport, and release of metals. Since manganese oxides can be solubilized by manganese-reducing bacteria, microbially mediated manganese reduction was investigated in Lake Vanda. Microbes concentrated from oxic regions of the water column, encompassing a peak of soluble manganese [Mn(II)], reduced synthetic manganese oxides (MnO2) when incubated aerobically. Pure cultures of manganese-reducing bacteria were readily isolated from waters collected near the oxic Mn(II) peak. Based on phylogenetic analysis of the 16S rRNA gene sequence, most of the isolated manganese reducers belong to the genusCarnobacterium. Cultures of a phylogenetically representative strain of Carnobacterium reduced synthetic MnO2 in the presence of sodium azide, as was seen in field assays. Unlike anaerobes that utilize manganese oxides as terminal electron acceptors in respiration, isolates of the genusCarnobacterium reduced Mn(IV) via a diffusible compound under oxic conditions. The release of adsorbed trace metals accompanying the solubilization of manganese oxides may provide populations of Carnobacterium with a source of nutrients in this extremely oligotrophic environment.

The temperature in the Arctic region has been increasing in the recent past accompanied by melting of its glaciers. We took a snapshot of the current microbial inhabitation of an Alaskan glacier (which can be considered as one of the simplest possible ecosystems) by using metagenomic sequencing of 16S rRNA recovered from ice/snow samples. Somewhat contrary to our expectations and earlier estimates, a rich and diverse microbial population of more than 2,500 species was revealed including several species of Archaea that has been identified for the first time in the glaciers of the Northern hemisphere. The most prominent bacterial groups found were Proteobacteria, Bacteroidetes, and Firmicutes. Firmicutes were not reported in large numbers in a previously studied Alpine glacier but were dominant in an Antarctic subglacial lake. Representatives of Cyanobacteria, Actinobacteria and Planctomycetes were among the most numerous, likely reflecting the dependence of the ecosystem on the energy obtained through photosynthesis and close links with the microbial community of the soil. Principal component analysis (PCA) of nucleotide word frequency revealed distinct sequence clusters for different taxonomic groups in the Alaskan glacier community and separate clusters for the glacial communities from other regions of the world. Comparative analysis of the community composition and bacterial diversity present in the Byron glacier in Alaska with other environments showed larger overlap with an Arctic soil than with a high Arctic lake, indicating patterns of community exchange and suggesting that these bacteria may play an important role in soil development during glacial retreat.

ABSTRACTAppropriate remediation targets or universal guidelines for polar regions do not currently exist, and a comprehensive understanding of the effects of diesel fuel on the natural microbial populations in polar and subpolar soils is lacking. Our aim was to investigate the response of the bacterial community to diesel fuel and to evaluate if these responses have the potential to be used as indicators of soil toxicity thresholds. We set up short- and long-exposure tests across a soil organic carbon gradient. Utilizing broad and targeted community indices, as well as functional genes involved in the nitrogen cycle, we investigated the bacterial community structure and its potential functioning in response to special Antarctic blend (SAB) diesel fuel. We found the primary effect of diesel fuel toxicity was a reduction in species richness, evenness, and phylogenetic diversity, with the resulting community heavily dominated by a few species, principallyPseudomonas. The decline in richness and phylogenetic diversity was linked to disruption of the nitrogen cycle, with species and functional genes involved in nitrification significantly reduced. Of the 11 targets we evaluated, we found the bacterialamoAgene indicative of potential ammonium oxidation, the most suitable indicator of toxicity. Dose-response modeling for this target generated an average effective concentration responsible for 20% change (EC20) of 155 mg kg−1, which is consistent with previous Macquarie Island ecotoxicology assays. Unlike traditional single-species tolerance testing, bacterial targets allowed us to simultaneously evaluate more than 1,700 species from 39 phyla, inclusive of rare, sensitive, and functionally relevant portions of the community.

AbstractWe base our search for the right instrumentation for detecting biosignatures on Europa on the analogy suggested by the recent work on polar ecosystems in the Canadian Arctic at Ellesmere Island. In that location sulphur patches (analogous to the Europan patches) are accumulating on glacial ice lying over saline springs rich in sulphate and sulphide. Their work reinforces earlier analogies in Antarctic ecosystems that are appropriate models for possible habitats that will be explored by the European Space Agency JUpiter ICy Moons Explorer (JUICE) mission to the Jovian System. Its Jupiter Ganymede Orbiter (JGO) will include orbits around Europa and Ganymede. The Galileo orbital mission discovered surficial patches of non-ice elements on Europa that were widespread and, in some cases possibly endogenous. This suggests the possibility that the observed chemical elements in the exoatmosphere may be from the subsurface ocean. Spatial resolution calculations of Cassidy and co-workers are available, suggesting that the atmospheric S content can be mapped by a neutral mass spectrometer, now included among the selected JUICE instruments. In some cases, large S-fractionations are due to microbial reduction and disproportionation (although sometimes providing a test for ecosystem fingerprints, even though with Sim – Bosak – Ono we maintain that microbial sulphate reduction large sulphur isotope fractionation does not require disproportionation. We address the question of the possible role of oxygen in the Europan ocean. Instrument issues are discussed for measuring stable S-isotope fractionations up to the known limits in natural populations of δ34 ≈ −70‰. We state the hypothesis of a Europa anaerobic oceanic population of sulphate reducers and disproportionators that would have the effect of fractionating the sulphate that reaches the low-albedo surficial regions. This hypothesis is compatible with the time-honoured expectation of Kaplan and co-workers (going back to the 1960s) that the distribution range of 32S/34S in analysed extra-terrestrial material appears to be narrower than the isotopic ratio of H, C or N and may be the most reliable for estimating biological effects. In addition, we discuss the necessary instruments that can test our biogenic hypothesis. First of all we hasten to clarify that the last-generation miniaturized mass spectrometer we discuss in the present paper are capable of reaching the required accuracy of ‰ for the all-important measurements with JGO of the thin atmospheres of the icy satellites. To implement the measurements, we single out miniature laser ablation time-of-flight mass spectrometers that are ideal for the forthcoming JUICE probing of the exoatmospheres, ionospheres and, indirectly, surficial low-albedo regions. Ganymede's surface, besides having ancient dark terrains covering about one-third of the total surface, has bright terrains of more recent origin, possibly due to some internal processes, not excluding biological ones. The geochemical test could identify bioindicators on Europa and exclude them on its large neighbour by probing relatively recent bright terrains on Ganymede's Polar Regions.

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. 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Most of the microbial biogeographic patterns in the oceans have been depicted at the whole community level, leaving out finer taxonomic resolution (i.e., microdiversity) that is crucial to conduct intra-population phylogeographic study, as commonly done for macroorganisms. Here, we present a new approach to unravel the bacterial phylogeographic patterns combining community-wide survey by 16S rRNA gene metabarcoding and intra-species resolution through the oligotyping method, allowing robust estimations of genetic and phylogeographic indices, and migration parameters. As a proof-of-concept, we focused on the bacterial genus Spirochaeta across three distant biogeographic provinces of the Southern Ocean; maritime Antarctica, sub-Antarctic Islands, and Patagonia. Each targeted Spirochaeta operational taxonomic units were characterized by a substantial intrapopulation microdiversity, and significant genetic differentiation and phylogeographic structure among the three provinces. Gene flow estimations among Spirochaeta populations support the role of the Antarctic Polar Front as a biogeographic barrier to bacterial dispersal between Antarctic and sub-Antarctic provinces. Conversely, the Antarctic Circumpolar Current appears as the main driver of gene flow, connecting sub-Antarctic Islands with Patagonia and maritime Antarctica. Additionally, historical processes (drift and dispersal limitation) govern up to 86% of the spatial turnover among Spirochaeta populations. Overall, our approach bridges the gap between microbial and macrobial ecology by revealing strong congruency with macroorganisms distribution patterns at the populational level, shaped by the same oceanographic structures and ecological processes.

Background: Sabalan (Savalan) Lake is a stable crater lake locating at the summit of Sabalan, an inactive stratovolcano and the third highest mountain of Iran. Because of cold weather conditions, the lake is frozen in most months of the year. The biodiversity of microbial flora in this area needs to be explored to find its similarity with the arctic regions’ biodiversity. Objective: The psychrophilic bacterial population of Sabalan (Savalan) Crater Lake was identified. The current research is the first report of aquatic bacterial strains isolation and characterization from Sabalan Lake. Methods: Water sample collections were cultured on four different media, then colonies were isolated by the plating method. The phylogenetic features of the isolates were scrutinized and finally, the phenotypic characteristics were investigated using specific culture methods. Results: The results of morphological tests indicated that most isolates were Gram-negative and rod shape, which were able to grow between ˗4 and +37 ºC.‎ According to the phylogenetic analysis the isolated strains belong to Pseudomonas, Yersinia, Kocuria, and Micrococcus genera and about 60% of the isolates belong to the various species of Pseudomonas as a ‎dominant genus with abounded frequency. ‎In addition, several isolates showed 99% similarity with bacteria, which were previously isolated from Antarctic regions such as Pseudomonas antarctica and Micrococcus antarctica. Conclusion: It can be concluded that the microbial populations of cold areas is the same across the geographical distances. In addition, these bacterial strains could be a primitive source of new enzymes for technological applications such as biosurfactant production.

ABSTRACT Ecological communities are regulated by the flow of energy through environments. Energy flow is typically limited by access to photosynthetically active radiation (PAR) and oxygen concentration (O2). The microbial mats growing on the bottom of Lake Fryxell, Antarctica, have well-defined environmental gradients in PAR and (O2). We analyzed the metagenomes of layers from these microbial mats to test the extent to which access to oxygen and light controls community structure. We found variation in the diversity and relative abundances of Archaea, Bacteria and Eukaryotes across three (O2) and PAR conditions: high (O2) and maximum PAR, variable (O2) with lower maximum PAR, and low (O2) and maximum PAR. We found distinct communities structured by the optimization of energy use on a millimeter-scale across these conditions. In mat layers where (O2) was saturated, PAR structured the community. In contrast, (O2) positively correlated with diversity and affected the distribution of dominant populations across the three habitats, suggesting that meter-scale diversity is structured by energy availability. Microbial communities changed across covarying gradients of PAR and (O2). The comprehensive metagenomic analysis suggests that the benthic microbial communities in Lake Fryxell are structured by energy flow across both meter- and millimeter-scales.

ABSTRACTThe skin is the first line of defense between an animal and its environment, and disruptions in skin-associated microorganisms can be linked to an animal's health and nutritional state. To better understand the skin microbiome of large whales, high-throughput sequencing of partial small subunit rRNA genes was used to study the skin-associated bacteria of 89 seemingly healthy humpback whales (Megaptera novaeangliae) sampled along the Western Antarctic Peninsula (WAP) during early (2010) and late (2013) austral summers. Six core groups of bacteria were present in 93% or more of all humpback skin samples. A shift was observed in the average relative abundances of these core bacteria over time, with the emergence of four additional core groups of bacteria that corresponded to a decrease in water temperature, possibly caused by season- or foraging-related changes in skin biochemistry that influenced microbial growth, or other temporal factors. The skin microbiome differed between whales sampled at several regional locations along the WAP, suggesting that environmental factors or population may also influence the whale skin microbiome. Overall, the skin microbiome of humpback whales appears to provide insight into animal- and environment-related factors and may serve as a useful indicator for animal health or ecosystem alterations.IMPORTANCEThe microbiomes of wild animals are currently understudied but may provide information about animal health and/or animal-environment interactions. In the largest sampling of any marine mammal microbiome, this study demonstrates conservation in the skin microbiome of 89 seemingly healthy humpback whales sampled in the Western Antarctic Peninsula, with shifts in the bacterial community composition related to temporal and regional variability. This study is important because it suggests that the skin microbiome of humpback whales could provide insight into animal nutritional or seasonal/environment-related factors, which are becoming increasingly important to recognize due to unprecedented rates of climate change and anthropogenic impact on ocean ecosystems.