M Microbial life has adapted to various individual extreme conditions; yet, organisms simultaneously adapted to very low pH, high salt and high temperature are unknown. We combined environmental 16S/18S rRNAgene metabarcoding, cultural approaches, fluorescence-activated cell sorting, scanning electron microscopy and chemical analyses to study samples along such unique polyextreme gradients in the Dallol-Danakil area (Ethiopia). We identify two physicochemical barriers to life in the presence of surface liquid water defined by: i) high chaotropicity-low water activity in Mg 2+ /Ca 2+-dominated brines and ii) hyperacidity-salt combinations (pH~0/ NaCl-dominated salt-saturation). When detected, life was dominated by highly diverse ultrasmall archaea widely distributed across phyla with and without previously known halophilic members. We hypothesize that high cytoplasmic K +-level was an original archaeal adaptation to hyperthermophily, subsequently exapted during multiple transitions to extreme halophily. W We detect active silica encrustment/fossilization of cells but also abiotic biomorphs of varied chemistry. Our work helps circumscribing habitability and calls for cautionary interpretations of morphological biosignatures on Earth and beyond. Belilla et al. 3 Microbial life has adapted to so-called extreme values of temperature, pH or salinity, but also to several polyextreme, e.g. hot acidic or salty alkaline, ecosystems 1,2. Various microbial lineages have been identified in acidic brines in the pH range 1.5-4.5, e.g. in Western Australia 3,4 and Chile 3. However, although some acidophilic archaea thrive at pH~0 (Picrophilus oshimae grows at an optimal pH of 0.7) 5 and many halophilic archaea live in hypersaline systems (>30%; NaCl-saturation conditions), organisms adapted simultaneously to very low pH (<1) and high salt, and eventually also high temperature, are not known among cultured prokaryotic species 1. Are molecular adaptations to these combinations incompatible or (hot) hyperacidic hypersaline environments simply rare and unexplored? The Dallol geothermal dome and its surroundings (Danakil Depression, Afar, Ethiopia) allow to address this question by offering unique polyextreme gradients combining high salt content (33 to >50%; either Mg 2+ /Ca 2+ or Na + (/Fe 2+/3+)-rich), high temperature (25-110°C) and low pH (≤-1.5 to 6). Dallol is an uplifted (~40 m) dome structure located in the North of the Danakil depression (~120 m below-sea-level), a 200 km-long basin within the Afar rift, at the triple junction between the Nubian, Somalian and Arabian Plates 6. Lying only 30 km north of the hypersaline, hydrothermally-influenced, Lake Assale (Karum) and the Erta Ale volcanic range, Dallol does not display volcanic outcrops but intense degassing and hydrothermalism. These activities are observed on the salt dome and the adjacent Black Mountain and Yellow Lake (Gaet'Ale) areas 6,7 (Fig. 1a-b). Gas and fluid isotopic measurements indicate that meteoritic waters, notably infiltrating from the high Ethiopian plat...

T he Dallol protovolcanic area on the Danakil Depres s ion (Afar region, E thiopia) exhibits unique hydrothermal manifes tations in hypers aline context, yielding varied polyextreme phys icochemical conditions . P revious s tudies identified a wide archaeal divers ity in les s extreme brines but failed to identify microorganis ms thriving in either high-chaotropicity, low-wateractivity brines or hyperacidic-hypers aline Na-F e-rich brines . R ecently, we acces s ed s everal s mall lakes under intens e degas s ing activity adjacent to the R ound Mountain, wes t to the Dallol dome (Wes tern C anyon Lakes ; WC L1-5). T hey exhibited intermediate parameter combinations (pH~5, 34-41% NaC l-dominated s alts with relatively high levels of chaotropic Mg-C a s alts ) that s hould allow to better cons train life limits . T hes e lakes were overwhelmingly dominated by Archaea, encompas s ing up to 99% of prokaryotic 16S rR NA gene amplicon s equences in metabarcoding s tudies . T he majority belonged to Halobacteriota and Nanohaloarchaeota, the latter repres enting up to half of prokaryotic s equences . Optical and epifluores cence micros copy s howed active cells in natural s amples and divers e morphotypes in enrichment cultures . S canning electron micros copy coupled with energy-dis pers ive x-ray s pectros copy revealed tiny cells (200-300 nm diameter) epibiotically as s ociated with s omewhat larger cells (0.6-1 µm) but als o the pres ence of s ilica-dominated precipitates of s imilar s ize and s hape, highlighting the difficulty of dis tinguis hing microbes from mineral biomorphs in this kind of low-biomas s s ys tems .

Microbial life has adapted to various individual extreme conditions; yet, organisms simultaneously adapted to low pH, high salt and high temperature are unknown. We combined environmental 16S/18S rRNA-gene metabarcoding, cultural approaches, fluorescence-activated cell sorting, scanning electron microscopy and chemical analyses to study samples along such 25 unique polyextreme gradients in the Dallol-Danakil area (Ethiopia). We identify two physicochemical barriers to life in the presence of surface liquid water defined by high chaotropicity-low water activity in Mg 2+ /Ca 2+ -dominated brines and hyperacidity-salt combinations. When detected, life was dominated by highly diverse ultrasmall archaea widely distributed across phyla with and without previously known hyperhalophilic members. We detect 30 active silica encrustment/fossilization of cells but also abiotic biomorphs of varied chemistry, raising warnings for the interpretation of morphological biosignatures on Earth and beyond. One Sentence Summary:The absence of life from some polyextreme sites in the presence of liquid surface water on Earth 35 helps to circumscribe habitability Belilla et al. 2 Main Text:Microbial life has adapted to so-called extreme values of temperature, pH or salinity, but also to 5 several polyextreme, e.g. hot acidic or salty alkaline, ecosystems (1, 2). However, organisms adapted simultaneously to low pH and high salt, and eventually also high temperature, are not known (1). Are molecular adaptations to those combinations incompatible or (hot) acidic hypersaline environments simply rare and unexplored? The Dallol geothermal dome and its surroundings (Danakil Depression, Afar, Ethiopia) allow to address this question by offering 10 unique polyextreme gradients combining high salt content (20 to >50%; either Mg 2+ /Ca 2+ or Na + (/Fe 2+/3+ )-rich), high temperature (25-110°C) and low pH (≤-1.5 to 6). Dallol is an up-lifted (~40 m) dome structure located in the North of the Danakil depression (~120 m below-sea-level), a 200 km-long basin within the Afar rift, at the triple junction between the Nubian, Somalian and Arabian Plates (3). Lying only 30 km north of the hypersaline, hydrothermally-influenced, Lake 15 Assale (Karum) and the Erta Ale volcanic range, Dallol does not display volcanic outcrops but intense degassing and hydrothermal activity. These activities are observed on the salt dome and the adjacent Black Mountain and Yellow Lake (Gaet'Ale) areas (3, 4) ( Fig. 1a-b). Gas and fluid isotopic measurements indicate that meteoritic waters, notably infiltrating from the high Ethiopian plateau (>2,500 m), interact with an underlying geothermal reservoir (280-370°C) (4, 20 5). Further interaction of those fluids with the km-thick marine evaporites filling the Danakil depression results in unique combinations of polyextreme conditions and salt chemistries (3,4,6, 7), which have led some authors consider Dallol as a Mars analog (8).Here, we use environmental 16S/18S rRNA-gene metabarcoding, cultural approaches, fluorescence-activa...

Determining the precise limits of life in polyextreme environments is challenging. Confounding factors such as exogenous contamination and the abiotic formation of structures resembling cells need to be considered before concluding on the unambiguous development of microbial life in low-biomass environments.

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