Archaeal overdominance close to life-limiting conditions in geothermally influenced hypersaline lakes at the Danakil Depression, Ethiopia.

The Dallol protovolcanic area on the Danakil Depression (Afar region, Ethiopia) exhibits unique hydrothermal manifestations in hypersaline context, yielding varied polyextreme physicochemical conditions. Previous studies identified a wide archaeal diversity in less extreme brines but failed to identify microorganisms thriving in either high-chaotropicity, low-water-activity brines or hyperacidic-hypersaline Na-Fe-rich brines. Recently, we accessed several small lakes under intense degassing activity adjacent to the Round Mountain, west to the Dallol dome [Western Canyon Lakes (WCL); WCL1-5]. They exhibited intermediate parameter combinations (pH ~ 5, 34%-41% (weight/volume) NaCl-dominated salts with relatively high levels of chaotropic Mg-Ca salts) that should allow to better constrain life limits. These lakes were overwhelmingly dominated by Archaea, encompassing up to 99% of prokaryotic 16S rRNA gene amplicon sequences in metabarcoding studies. The majority belonged to Halobacteriota and Nanohaloarchaeota, the latter representing up to half of prokaryotic sequences. Optical and epifluorescence microscopy showed active cells in natural samples and diverse morphotypes in enrichment cultures. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy revealed tiny cells (200-300 nm diameter) epibiotically associated with somewhat larger cells (0.6-1 µm) but also the presence of silica-dominated precipitates of similar size and shape, highlighting the difficulty of distinguishing microbes from mineral biomorphs in this kind of low-biomass systems.

Active Microbial Airborne Dispersal and Biomorphs as Confounding Factors for Life Detection in the Cell-Degrading Brines of the Polyextreme Dallol Geothermal Field.

Determining the precise limits of life in polyextreme environments is challenging. Studies along gradients of polyextreme conditions in the Dallol proto-volcano area (Danakil salt desert, Ethiopia) showed the occurrence of archaea-dominated communities (up to 99%) in several hypersaline systems but strongly suggested that life did not thrive in the hyperacidic (pH ∼0), hypersaline (∼35% [wt/vol],) and sometimes hot (up to 108°C) ponds of the Dallol dome. However, it was recently claimed that archaea flourish in these brines based on the detection of one Nanohaloarchaeotas 16S rRNA gene and fluorescent in situ hybridization (FISH) experiments with archaea-specific probes. Here, we characterized the diversity of microorganisms in aerosols over Dallol, and we show that, in addition to typical bacteria from soil/dust, they transport halophilic archaea likely originating from neighboring hypersaline ecosystems. We also show that cells and DNA from cultures and natural local halophilic communities are rapidly destroyed upon contact with Dallol brine. Furthermore, we confirm the widespread occurrence of mineral particles, including silica-based biomorphs, in Dallol brines. FISH experiments using appropriate controls show that DNA fluorescent probes and dyes unspecifically bind to mineral precipitates in Dallol brines; cellular morphologies were unambiguously observed only in nearby hypersaline ecosystems. Our results show that airborne cell dispersal and unspecific binding of fluorescent probes are confounding factors likely affecting previous inferences of archaea thriving in Dallol. They highlight the need for controls and the consideration of alternative abiotic explanations before safely drawing conclusions about the presence of life in polyextreme terrestrial or extraterrestrial systems. IMPORTANCE 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. Here, we explored how these factors can affect contrasting reports about microbial life thriving in the hypersaline and hyperacidic brines of the Dallol geothermal field (Danakil Depression, Ethiopia). We show not only that aerosols actively transport a wide diversity of prokaryotic and eukaryotic cells over Dallol but also that, upon contact with the chaotropic hyperacidic brine, cells and DNA are rapidly degraded. We also show the extant occurrence of mineral (mostly silica-based) biomorphs that unspecifically bind fluorescent probes and dyes. Our study highlights the need for controls and the consideration of alternative abiotic explanations before safely drawing conclusions on the presence of life in polyextreme terrestrial or extraterrestrial systems.

Hyperdiverse archaea near life limits at the polyextreme geothermal Dallol area.

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 ribosomal RNA gene 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 in Ethiopia. We identified two physicochemical barriers to life in the presence of surface liquid water defined by (1) high chaotropicity-low water activity in Mg2+/Ca2+-dominated brines and (2) hyperacidity-salt combinations (pH ~0/NaCl-dominated salt saturation). When detected, life was dominated by highly diverse ultrasmall archaea that were widely distributed across phyla with and without previously known halophilic members. We hypothesize that a high cytoplasmic K+-level was an original archaeal adaptation to hyperthermophily, subsequently exapted during several transitions to extreme halophily. 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.