No abstract

The biosphere of planet Earth is delineated by physico-chemical conditions that are too harsh for, or inconsistent with, life processes and maintenance of the structure and function of biomolecules. To define the window of life on Earth (and perhaps gain insights into the limits that life could tolerate elsewhere), and hence understand some of the most unusual biological activities that operate at such extremes, it is necessary to understand the causes and cellular basis of systems failure beyond these windows. Because water plays such a central role in biomolecules and bioprocesses, its availability, properties and behaviour are among the key life-limiting parameters. Saline waters dominate the Earth, with the oceans holding 96.5% of the planet's water. Saline groundwater, inland seas or saltwater lakes hold another 1%, a quantity that exceeds the world's available freshwater. About one quarter of Earth's land mass is underlain by salt, often more than 100 m thick. Evaporite deposits contain hypersaline waters within and between their salt crystals, and even contain large subterranean salt lakes, and therefore represent significant microbial habitats. Salts have a major impact on the nature and extent of the biosphere, because solutes radically influence water's availability (water activity) and exert other activities that also affect biological systems (e.g. ionic, kosmotropic, chaotropic and those that affect cell turgor), and as a consequence can be major stressors of cellular systems. Despite the stressor effects of salts, hypersaline environments can be heavily populated with salt-tolerant or -dependent microbes, the halophiles. The most common salt in hypersaline environments is NaCl, but many evaporite deposits and brines are also rich in other salts, including MgCl 2 (several hundred million tonnes of bischofite, MgCl2·6H2O, occur in one formation alone). Magnesium (Mg) is the third most abundant element dissolved in seawater and is ubiquitous in the Earth's crust, and throughout the Solar System, where it exists in association with a variety of anions. Magnesium chloride is exceptionally soluble in water, so can achieve high concentrations (> 5 M) in brines. However, while NaCldominated hypersaline environments are habitats for a rich variety of salt-adapted microbes, there are contradictory indications of life in MgCl 2-rich environments. In this work, we have sought to obtain new insights into how MgCl2 affects cellular systems, to assess whether MgCl2 can determine the window of life, and, if so, to derive a value for this window. We have dissected two relevant cellular stress-related activities of MgCl2 solutions, namely water activity reduction and chaotropicity, and analysed signatures of life at different concentrations of MgCl2 in a natural environment, namely the 0.05-5.05 M MgCl2 gradient of the seawater : hypersaline brine interface of Discovery Basin -a large, stable brine lake almost saturated with MgCl2, located on the Mediterranean Sea floor. We document here the exceptional chaotropicity...

During screening for biosurfactant-producing, n-alkane-degrading marine bacteria, six heterotrophic bacterial strains were isolated from enriched mixed cultures, obtained from sea waterkediment samples collected near the Isle of Borkum (North Sea), using Mihagol-S (C,,,,-n-al kanes)as principal carbon source. These Gram-negative, aerobic, rod-shaped bacteria use a limited number of organic compounds, including aliphatic hydrocarbons, volatile fatty acids, and pyruvate and its methyl ether. During cultivation on n-alkanes as sole source of carbon and energy, all strains produced both extracellular and cell-bound surface-active glucose lipids which reduced the surface tension of water from 72 to 29 mN m-' (16). This novel class of glycolipids was found to be produced only by these strains. The 165 rRNA gene sequence analysis showed that these strains are all members of the y-subclass of the Proteobacteria. Their phospholipid ester-linked fatty acid composition was shown to be similar to that of members of the genus Halomonas, although they did not demonstrate a close phylogenetic relationship to any previously described species. On the basis of the information summarized above, a new genus and species, Akaniworax borkumensis, is described to include these bacteria. Strain SKZT is the type strain of A. borkumensis.

The chemical composition of the Bannock basin has been studied in some detail 1,2 . We recently showed that unusual microbial populations, including a new division of Archaea (MSBL1) 3 , inhabit the NaCl-rich hypersaline brine. High salinities tend to reduce biodiversity 4 , but when brines come into contact with fresher water the natural haloclines formed frequently contain gradients of other chemicals, including permutations of electron donors and acceptors, that may enhance microbial diversity, activity and biogeochemical cycling 5,6 . Here we report a 2.5-mthick chemocline with a steep NaCl gradient at 3.3 km within the water column betweeen Bannock anoxic hypersaline brine 7 and overlying sea water. The chemocline supports some of the most biomass-rich and active microbial communities in the deep sea, dominated by Bacteria rather than Archaea, and including four major new divisions of Bacteria. Significantly higher metabolic activities were measured in the chemocline than in the overlying sea water and underlying brine; functional analyses indicate that a range of biological processes is likely to occur in the chemocline. Many prokaryotic taxa, including the phylogenetically new groups, were confined to defined salinities, and collectively formed a diverse, sharply stratified, deep-sea ecosystem with sufficient biomass to potentially contribute to organic geological deposits.High-precision sampling was conducted during cruises of the research vessel Urania equipped with the Modus-Scipack system (http://www.geo.unimib.it/BioDeep/Project.html; Fig. 1a). The vehicle Modus, connected by cable to the research vessel, held a second instrument, the Scipack, with a 10-m data transmission cable. The Scipack, consisting of a Rosette sampler equipped with a CTD (conductivity-temperature-depth probe) and a series of Niskin bottles, was connected to the Modus through the Sciskid, a module equipped with a pressure sensor for recording the pressure at which the Niskin bottles were closed (Fig. 1c). A camera on the Modus could provide an image of the Scipack entering the brine lake (Fig. 1b, and Supplementary Fig. S1). Immediately after sampling, the Modus-Scipack was raised, the Niskin bottles were retrieved and their contents were carefully fractionated on board ship by slowly recovering 0.5-litre, 1-litre or 2-litre fractions from the bottom tap. These were then immediately analysed for salinity (Fig. 1d). The reconstructed interface salinity profile was strongly positively correlated (r ¼ 0.98, P , 0.001) with the CTD conductivity profile recorded in independent non-sampling casts (Fig. 2d), indicating that little or no mixing had occurred.The interface halocline was about 2.5 m deep, in agreement with previous estimates that employed alternative sampling strategies 1 . Although biomass values fluctuated along the halocline, there were significantly greater numbers of microbial cells in the interface (about 10 6 cells ml 21 ) than in either the deep sea water or the underlying hypersaline brine, both of which had about...

Methanogenic archaea are major players in the global carbon cycle and in the biotechnology of anaerobic digestion. The phylum Euryarchaeota includes diverse groups of methanogens that are interspersed with non-methanogenic lineages. So far methanogens inhabiting hypersaline environments have been identified only within the order Methanosarcinales. We report the discovery of a deep phylogenetic lineage of extremophilic methanogens in hypersaline lakes, and present analysis of two nearly complete genomes from this group. Within the phylum Euryarchaeota, these isolates form a separate, class-level lineage “Methanonatronarchaeia” that is most closely related to the class Halobacteria. Similar to the Halobacteria, “Methanonatronarchaeia” are extremely halophilic and do not accumulate organic osmoprotectants. The high intracellular concentration of potassium implies that “Methanonatronarchaeia” employ the “salt-in” osmoprotection strategy. These methanogens are heterotrophic methyl-reducers that utilize C1-methylated compounds as electron acceptors and formate or hydrogen as electron donors. The genomes contain an incomplete and apparently inactivated set of genes encoding the upper branch of methyl group oxidation to CO2 as well as membrane-bound heterosulfide reductase and cytochromes. These features differentiates “Methanonatronarchaeia” from all known methyl-reducing methanogens. The discovery of extremely halophilic, methyl-reducing methanogens related to haloarchaea provides insights into the origin of methanogenesis and shows that the strategies employed by methanogens to thrive in salt-saturating conditions are not limited to the classical methylotrophic pathway.

Mesophilic Crenarchaeota have recently been thought to be significant contributors to nitrogen (N) and carbon (C) cycling. In this study, we examined the vertical distribution of ammonia-oxidizing Crenarchaeota at offshore site in Southern Tyrrhenian Sea. The median value of the crenachaeal cell to amoA gene ratio was close to one suggesting that virtually all deep-sea Crenarchaeota possess the capacity to oxidize ammonia. Crenarchaea-specific genes, nirK and ureC, for nitrite reductase and urease were identified and their affiliation demonstrated the presence of 'deep-sea' clades distinct from 'shallow' representatives. Measured deep-sea dark CO 2 fixation estimates were comparable to the median value of photosynthetic biomass production calculated for this area of Tyrrhenian Sea, pointing to the significance of this process in the C cycle of aphotic marine ecosystems. To elucidate the pivotal organisms in this process, we targeted known marine crenarchaeal autotrophy-related genes, coding for acetyl-CoA carboxylase (accA) and 4-hydroxybutyryl-CoA dehydratase (4-hbd). As in case of nirK and ureC, these genes are grouped with deepsea sequences being distantly related to those retrieved from the epipelagic zone. To pair the molecular data with specific functional attributes we performed [ 14 C]HCO 3 incorporation experiments followed by analyses of radiolabeled proteins using shotgun proteomics approach. More than 100 oligopeptides were attributed to 40 marine crenarchaeal-specific proteins that are involved in 10 different metabolic processes, including autotrophy. Obtained results provided a clear proof of chemolithoautotrophic physiology of bathypelagic crenarchaeota and indicated that this numerically predominant group of microorganisms facilitate a hitherto unrecognized sink for inorganic C of a global importance.

Methane production and archaeal community composition were studied in samples from an acidic peat bog incubated at different temperatures and pH values. H 2 -dependent methanogenesis increased strongly at the lowest pH, 3.8, and Methanobacteriaceae became important except for Methanomicrobiaceae and Methanosarcinaceae. An acidophilic and psychrotolerant Methanobacterium sp. was isolated using H 2 -plus-CO 2 -supplemented medium at pH 4.5.Wetlands are considered to be the largest natural sources of atmospheric CH 4 . Acidic peatlands are the most typical type of northern wetlands and are responsible for about 60% of total wetland emission (26). Peat bogs are characterized by low concentrations of mineral salts, low pH, and low temperature. Various factors have been identified as important controls of methanogenesis, with temperature, water table level, and content of organic matter being the most notable ones (4,9,12,27,32,35,38). However, there is little information on how pH influences the composition and functioning of the methanogenic community.In peatlands, hydrogentrophic methanogenesis is the predominant pathway of CH 4 formation, especially in deeper layers, accounting for 50 to 100% of total CH 4 production (12,18,28,40). However, acetoclastic methanogenesis has also been found to play an important role in acidic bogs (1, 2, 21). Relatively little is known about the archaeal communities inhabiting peatlands. Recent studies of different wetlands revealed the presence of methanogens belonging to the Methanomicrobiaceae, Methanobacteriaceae, Methanococcaceae, Methanosarcinaceae, and Methanosaetaceae as well as new archaeal lineages within the Euryarchaeota (3,7,8,13,17,34,36,37). However, the role of the methanogenic populations in CH 4 production under different in situ conditions is unknown.Attempts to isolate acidophilic or acidotolerant methanogens in pure culture have failed until very recently, although acid-tolerant enrichment cultures have been reported (6,11,15,34,41). It was only after we finished our study that Bräuer and coworkers reported the successful isolation of a moderately acidophilic methanogen belonging to the Methanomicrobiales order (5).The aim of the present study was to investigate how high acidity and low temperature can affect the functioning of the methanogenic community, its structure, and, hence, methane production in a peat bog, as well as to obtain a pure culture of an acidophilic methanogen. We used the same bog samples as in our previous study (21).We obtained peat samples from Bakchar Bog, which is located in West Siberia (57°N, 83°E). The main unforested part of the bog is covered with continuous Sphagnum moss and patches of vascular plants (Carex, Menyanthes, and Equisetum spp.). The detailed location of the bog and structure of the plant community have been described earlier (21, 29). The samples were taken in July 1999 at a depth of 30 to 50 cm below the water table from the site covered with Equisetum. The peat pH values were in a range of 3.5 to 5.5, with pH 4.8 at t...

Microbial community of the deep-sea brine Lake Kryos seawater-brine interface is active below the chaotropicity limit of life as revealed by recovery of mRNA Yakimov, M. M., La Cono, V., Spada, G. L., Bortoluzzi, G., Messina, E., Smedile, F., ... Giuliano, L. (2015). Microbial community of the deep-sea brine Lake Kryos seawater-brine interface is active below the chaotropicity limit of life as revealed by recovery of mRNA. Environmental Microbiology, 17 (2) This is the accepted version of the following article: Microbial community of the deep-sea brine Lake Kryos seawater-brine interface is active below the chaotropicity limit of life as revealed by recovery of mRNA, Michail M. Yakimov, Violetta La Cono, Gina La Spada, Giovanni Bortoluzzi, Enzo Messina, Francesco Smedile, Erika Arcadi, Mireno Borghini, Manuel Ferrer, Phillippe Schmitt-Kopplin, Norbert Hertkorn, Jonathan A. Cray, John E. Hallsworth, Peter N. Golyshin andLaura Giuliano, which has been published in final form at ttp://onlinelibrary.wiley.com/doi/10.1111/1462-2920.12587/abstract. General rightsCopyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights.Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. 25This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12587 Accepted ArticleThis article is protected by copyright. All rights reserved. the microbiology of the seawater-Kryos brine interface and managed to recover mRNA from the 10 2.27-3.03 M MgCl 2 layer (equivalent to 0.747-0.631 water-activity) thereby expanding the established 11 chaotropicity window-for-life. The primary bacterial taxa present there were KB1 candidate division 12 and DHAL-specific group of organisms, distantly related to Desulfohalobium. Two euryarchaeal 13 candidate divisions MSBL1 and HC1, detected in minority in the overlaying layers, accounted for 14 more than 85% of the rRNA-containing archaeal clones analyzed in 2.27-3.03 M MgCl 2 layer. These 15 findings shed light on the plausibility of life in highly chaotropic environments, geochemical 16 windows for microbial extremophiles, and have implications for habitability elsewhere in the Solar 17 System.