Summary: Within the complex of deep, hypersaline anoxic lakes ( DHALs ) of the Mediterranean Ridge, we identified a new, unexplored DHAL and named it ‘ L ake K ryos ’ after a nearby depression. This lake is filled with magnesium chloride ( MgCl ₂ )‐rich, athalassohaline brine (salinity > 470 practical salinity units), presumably formed by the dissolution of Messinian bischofite. Compared with the DHAL Discovery , it contains elevated concentrations of kosmotropic sodium and sulfate ions, which are capable of reducing the net chaotropicily of MgCl ₂ ‐rich solutions. The brine of L ake K ryos may therefore be biologically permissive at MgCl ₂ concentrations previously considered incompatible with life. We characterized the microbiology of the seawater– K ryos brine interface and managed to recover mRNA from the 2.27–3.03 M MgCl ₂ layer (equivalent to 0.747–0.631 water activity), thereby expanding the established chaotropicity window‐for‐life. The primary bacterial taxa present there were Kebrit Deep Bacteria 1 candidate division and DHAL ‐specific group of organisms, distantly related to D esulfohalobium . Two euryarchaeal candidate divisions, Mediterranean Sea Brine Lakes group 1 and halophilic cluster 1, accounted for > 85% of the rRNA ‐containing archaeal clones derived from the 2.27–3.03 M MgCl ₂ layer, but were minority community‐members in the overlying interface‐layers. These findings shed light on the plausibility of life in highly chaotropic environments, geochemical windows for microbial extremophiles, and have implications for habitability elsewhere in the Solar System.

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 CO₂ 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 deep-sea sequences being distantly related to those retrieved from the epipelagic zone. To pair the molecular data with specific functional attributes we performed [¹⁴C]HCO₃ 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.

Meso- and bathypelagic ecosystems represent the most common marine ecological niche on Earth and contain complex communities of microorganisms that are for the most part ecophysiologically poorly characterized. Gradients of physico-chemical factors (for example, depth-related gradients of light, temperature, salinity, nutrients and pressure) constitute major forces shaping ecosystems at activity 'hot spots' on the ocean floor, such as hydrothermal vents, cold seepages and mud volcanoes and hypersaline lakes, though the relationships between community composition, activities and environmental parameters remain largely elusive. We report here results of a detailed study of primary producing microbial communities in the deep Eastern Mediterranean Sea. The brine column of the deep anoxic hypersaline brine lake, L'Atalante, the overlying water column and the brine-seawater interface, were characterized physico- and geochemically, and microbiologically, in terms of their microbial community compositions, functional gene distributions and [¹⁴C]bicarbonate assimilation activities. The depth distribution of genes encoding the crenarchaeal ammonia monooxygenase subunit (amoA), and the bacterial ribulose-1,5-biphosphate carboxylase/oxygenase large subunit (RuBisCO), was found to coincide with two different types of chemoautotrophy. Meso- and bathypelagic microbial communities were enriched in ammonia-oxidizing Crenarchaeota, whereas the autotrophic community at the oxic/anoxic interface of L'Atalante lake was dominated by Epsilonproteobacteria and sulfur-oxidizing Gammaproteobacteria. These autotrophic microbes are thus the basis of the food webs populating these deep-sea ecosystems.

Deep-sea hypersaline anoxic lakes (DHALs) of the Eastern Mediterranean represent some of the most hostile environments on our planet. We investigated microbial life in the recently discovered Lake Medee, the largest DHAL found to-date. Medee has two unique features: a complex geobiochemical stratification and an absence of chemolithoautotrophic Epsilonproteobacteria, which usually play the primary role in dark bicarbonate assimilation in DHALs interfaces. Presumably because of these features, Medee is less productive and exhibits reduced diversity of autochthonous prokaryotes in its interior. Indeed, the brine community almost exclusively consists of the members of euryarchaeal MSBL1 and bacterial KB1 candidate divisions. Our experiments utilizing cultivation and [¹⁴C]-assimilation, showed that these organisms at least partially rely on reductive cleavage of osmoprotectant glycine betaine and are engaged in trophic cooperation. These findings provide novel insights into how prokaryotic communities can adapt to salt-saturated conditions and sustain active metabolism at the thermodynamic edge of life.

Summary: In September 2008, an expedition of the RV Urania was devoted to exploration of the genomic richness of deep hypersaline anoxic lakes (DHALs) located in the Western part of the Mediterranean Ridge. Approximately 40 nautical miles SE from Urania Lake, the presence of anoxic hypersaline lake, which we named Thetis , was confirmed by swath bathymetry profiling and through immediate sampling casts. The brine surface of the Thetis Lake is located at a depth of 3258 m with a thickness of ∼157 m. Brine composition was found to be thalassohaline, saturated by NaCl with a total salinity of 348‰, which is one of highest value reported for DHALs. Similarly to other Mediterranean DHALs, seawater‐brine interface of Thetis represents a steep pycno‐ and chemocline with gradients of salinity, electron donors and acceptors and posseses a remarkable stratification of prokaryotic communities, observed to be more metabolically active in the upper interface where redox gradient was sharper. [ ¹⁴ C]‐bicarbonate fixation analysis revealed that microbial communities are sustained by sulfur‐oxidizing chemolithoautotrophic primary producers that thrive within upper interface. Besides microaerophilic autotrophy, heterotrophic sulfate reduction, methanogenesis and anaerobic methane oxidation are likely the predominant processes driving the ecosystem of Thetis Lake.