Single-cell analysis in hypersaline brines predicts a water-activity limit of microbial anabolic activity.

Hypersaline brines provide excellent opportunities to study extreme microbial life. Here, we investigated anabolic activity in nearly 6000 individual cells from solar saltern sites with water activities (aw) ranging from 0.982 to 0.409 (seawater to extreme brine). Average anabolic activity decreased exponentially with aw, with nuanced trends evident at the single-cell level: The proportion of active cells remained high (>50%) even after NaCl saturation, and subsets of cells spiked in activity as aw decreased. Intracommunity heterogeneity in activity increased as seawater transitioned to brine, suggesting increased phenotypic heterogeneity with increased physiological stress. No microbial activity was detected in the 0.409-aw brine (an MgCl2-dominated site) despite the presence of cell-like structures. Extrapolating our data, we predict an aw limit for detectable anabolic activity of 0.540, which is beyond the currently accepted limit of life based on cell division. This work demonstrates the utility of single-cell, metabolism-based techniques for detecting active life and expands the potential habitable space on Earth and beyond.

Phosphate availability and implications for life on ocean worlds.

Several moons in the outer solar system host liquid water oceans. A key next step in assessing the habitability of these ocean worlds is to determine whether life's elemental and energy requirements are also met. Phosphorus is required by all known life and is often limited to biological productivity in Earth's oceans. This raises the possibility that its availability may limit the abundance or productivity of Earth-like life on ocean worlds. To address this potential problem, here we calculate the equilibrium dissolved phosphate concentrations associated with the reaction of water and rocks-a key driver of ocean chemical evolution-across a broad range of compositional inputs and reaction conditions. Equilibrium dissolved phosphate concentrations range from 10-11 to 10-1 mol/kg across the full range of carbonaceous chondrite compositions and reaction conditions considered, but are generally > 10-5 mol/kg for most plausible scenarios. Relative to the phosphate requirements and uptake kinetics of microorganisms in Earth's oceans, such concentrations would be sufficient to support initially rapid cell growth and construction of global ocean cell populations larger than those observed in Earth's deep oceans.

Microbial diversity and activity in Southern California salterns and bitterns: analogues for remnant ocean worlds.

Concurrent osmotic and chaotropic stress make MgCl2 -rich brines extremely inhospitable environments. Understanding the limits of life in these brines is essential to the search for extraterrestrial life on contemporary and relict ocean worlds, like Mars, which could host similar environments. We sequenced environmental 16S rRNA genes and quantified microbial activity across a broad range of salinity and chaotropicity at a Mars-analogue salt harvesting facility in Southern California, where seawater is evaporated in a series of ponds ranging from kosmotropic NaCl brines to highly chaotropic MgCl2 brines. Within NaCl brines, we observed a proliferation of specialized halophilic Euryarchaeota, which corresponded closely with the dominant taxa found in salterns around the world. These communities were characterized by very slow growth rates and high biomass accumulation. As salinity and chaotropicity increased, we found that the MgCl2 -rich brines eventually exceeded the limits of microbial activity. We found evidence that exogenous genetic material is preserved in these chaotropic brines, producing an unexpected increase in diversity in the presumably sterile MgCl2 -saturated brines. Because of their high potential for biomarker preservation, chaotropic brines could therefore serve as repositories of genetic biomarkers from nearby environments (both on Earth and beyond) making them prime targets for future life-detection missions.

Morphological and isotopic changes of heterocystous cyanobacteria in response to N2 partial pressure.

Earth's atmospheric composition has changed significantly over geologic time. Many redox active atmospheric constituents have left evidence of their presence, while inert constituents such as dinitrogen gas (N2 ) are more elusive. In this study, we examine two potential biological indicators of atmospheric N2 : the morphological and isotopic signatures of heterocystous cyanobacteria. Biological nitrogen fixation constitutes the primary source of fixed nitrogen to the global biosphere and is catalyzed by the oxygen-sensitive enzyme nitrogenase. To protect this enzyme, some filamentous cyanobacteria restrict nitrogen fixation to microoxic cells (heterocysts) while carrying out oxygenic photosynthesis in vegetative cells. Heterocysts terminally differentiate in a pattern that is maintained as the filaments grow, and nitrogen fixation imparts a measurable isotope effect, creating two biosignatures that have previously been interrogated under modern N2 partial pressure (pN2 ) conditions. Here, we examine the effect of variable pN2 on these biosignatures for two species of the filamentous cyanobacterium Anabaena. We provide the first in vivo estimate of the intrinsic isotope fractionation factor of Mo-nitrogenase (εfix  = -2.71 ± 0.09‰) and show that, with decreasing pN2 , the net nitrogen isotope fractionation decreases for both species, while the heterocyst spacing decreases for Anabaena cylindrica and remains unchanged for Anabaena variabilis. These results are consistent with the nitrogen fixation mechanisms available in the two species. Application of these quantifiable effects to the geologic record may lead to new paleobarometric measurements for pN2 , ultimately contributing to a better understanding of Earth's atmospheric evolution.

Air density 2.7 billion years ago limited to less than twice modern levels by fossil raindrop imprints.

According to the 'Faint Young Sun' paradox, during the late Archaean eon a Sun approximately 20% dimmer warmed the early Earth such that it had liquid water and a clement climate. Explanations for this phenomenon have invoked a denser atmosphere that provided warmth by nitrogen pressure broadening or enhanced greenhouse gas concentrations. Such solutions are allowed by geochemical studies and numerical investigations that place approximate concentration limits on Archaean atmospheric gases, including methane, carbon dioxide and oxygen. But no field data constraining ground-level air density and barometric pressure have been reported, leaving the plausibility of these various hypotheses in doubt. Here we show that raindrop imprints in tuffs of the Ventersdorp Supergroup, South Africa, constrain surface air density 2.7 billion years ago to less than twice modern levels. We interpret the raindrop fossils using experiments in which water droplets of known size fall at terminal velocity into fresh and weathered volcanic ash, thus defining a relationship between imprint size and raindrop impact momentum. Fragmentation following raindrop flattening limits raindrop size to a maximum value independent of air density, whereas raindrop terminal velocity varies as the inverse of the square root of air density. If the Archaean raindrops reached the modern maximum measured size, air density must have been less than 2.3 kg m(-3), compared to today's 1.2 kg m(-3), but because such drops rarely occur, air density was more probably below 1.3 kg m(-3). The upper estimate for air density renders the pressure broadening explanation possible, but it is improbable under the likely lower estimates. Our results also disallow the extreme CO(2) levels required for hot Archaean climates.