Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View.

Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.

Rapid recovery of life at ground zero of the end-Cretaceous mass extinction.

The Cretaceous/Palaeogene mass extinction eradicated 76% of species on Earth1,2. It was caused by the impact of an asteroid3,4 on the Yucatán carbonate platform in the southern Gulf of Mexico 66 million years ago 5 , forming the Chicxulub impact crater6,7. After the mass extinction, the recovery of the global marine ecosystem-measured as primary productivity-was geographically heterogeneous 8 ; export production in the Gulf of Mexico and North Atlantic-western Tethys was slower than in most other regions8-11, taking 300 thousand years (kyr) to return to levels similar to those of the Late Cretaceous period. Delayed recovery of marine productivity closer to the crater implies an impact-related environmental control, such as toxic metal poisoning 12 , on recovery times. If no such geographic pattern exists, the best explanation for the observed heterogeneity is a combination of ecological factors-trophic interactions 13 , species incumbency and competitive exclusion by opportunists 14 -and 'chance'8,15,16. The question of whether the post-impact recovery of marine productivity was delayed closer to the crater has a bearing on the predictability of future patterns of recovery in anthropogenically perturbed ecosystems. If there is a relationship between the distance from the impact and the recovery of marine productivity, we would expect recovery rates to be slowest in the crater itself. Here we present a record of foraminifera, calcareous nannoplankton, trace fossils and elemental abundance data from within the Chicxulub crater, dated to approximately the first 200 kyr of the Palaeocene. We show that life reappeared in the basin just years after the impact and a high-productivity ecosystem was established within 30 kyr, which indicates that proximity to the impact did not delay recovery and that there was therefore no impact-related environmental control on recovery. Ecological processes probably controlled the recovery of productivity after the Cretaceous/Palaeogene mass extinction and are therefore likely to be important for the response of the ocean ecosystem to other rapid extinction events.

The smallest space miners: principles of space biomining.

As we aim to expand human presence in space, we need to find viable approaches to achieve independence from terrestrial resources. Space biomining of the Moon, Mars and asteroids has been indicated as one of the promising approaches to achieve in-situ resource utilization by the main space agencies. Structural and expensive metals, essential mineral nutrients, water, oxygen and volatiles could be potentially extracted from extraterrestrial regolith and rocks using microbial-based biotechnologies. The use of bioleaching microorganisms could also be applied to space bioremediation, recycling of waste and to reinforce regenerative life support systems. However, the science around space biomining is still young. Relevant differences between terrestrial and extraterrestrial conditions exist, including the rock types and ores available for mining, and a direct application of established terrestrial biomining techniques may not be a possibility. It is, therefore, necessary to invest in terrestrial and space-based research of specific methods for space applications to learn the effects of space conditions on biomining and bioremediation, expand our knowledge on organotrophic and community-based bioleaching mechanisms, as well as on anaerobic biomining, and investigate the use of synthetic biology to overcome limitations posed by the space environments.

Are microorganisms everywhere they can be?

Baas-Becking is famously attributed with the conjecture that 'everything is everywhere, but the environment selects'. Although this aphorism is largely challenged by microbial biogeographical data, even weak versions of the claim leave unanswered the question about whether all environments that could theoretically support life contain life. In the last decade, the discovery of thermally sterilized habitable environments disconnected from inhabited regions, and habitats within organisms such as the sterile, but habitable human fetal gut, suggest the existence of a diversity of macroscopic habitable environments apparently devoid of actively metabolizing or reproducing life. Less clear is the status of such environments at the micron scale, for example, between colonies of organisms within rock interstices or on and within other substrates. I discuss recent evidence for these types of environments. These environments have practical uses in: (i) being negative controls for understanding the role of microbial processes in geochemical cycles and geological processes, (ii) yielding insights into the extent to which the biosphere extends into all spaces it theoretically can, (iii) suggesting caution in interpreting the results of life detection instrumentation, and (iv) being useful for establishing the conditions for the origin of life and its prevalence on other planetary bodies.

Structural responses of model biomembranes to Mars-relevant salts.

Lipid membranes are a key component of contemporary living systems and are thought to have been essential to the origin of life. Most research on membranes has focused on situations restricted to ambient physiological or benchtop conditions. However, the influence of more extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments are less well understood. The deep subsurface environments of Mars, for instance, may harbor high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments for cellular life. Here, we investigated the combined effects of high concentrations of salts, including sodium and magnesium perchlorate and sulfate, and high hydrostatic pressure on the stability and structure of model biomembranes of varying complexity. To this end, a variety of biophysical techniques have been applied, which include calorimetry, fluorescence spectroscopies, small-angle X-ray scattering, dynamic light scattering, and microscopy techniques. We show that the structure and phase behavior of lipid membranes is sensitively dictated by the nature of the salt, in particular its anion and its concentration. We demonstrate that, with the exception of magnesium perchlorate, which can also induce cubic lipid arrangements, long-chain saturated lipid bilayer structures can still persist at high salt concentrations across a range of pressures. The lateral organization of complex heterogeneous raft-like membranes is affected by all salts. For simple, in particular bacterial membrane-type bilayer systems with unsaturated chains, vesicular structures are still stable at Martian brine conditions, also up to the kbar pressure range, demonstrating the potential compatibility of environments containing such ionic and pressure extremes to lipid-encapsulated life.

Ions in the Deep Subsurface of Earth, Mars, and Icy Moons: Their Effects in Combination with Temperature and Pressure on tRNA-Ligand Binding.

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions' role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl2, MgSO4, and Mg(ClO4)2) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg2+ salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid-ligand interactions of the type studied here.

Microbial Life in Impact Craters.

Asteroid and comet impacts are known to have caused profound disruption to multicellular life, yet their influence on habitats for microorganisms, which comprise the majority of Earth's biomass, is less well understood. Of particular interest are geological changes in the target lithology at and near the point of impact that can persist for billions of years. Deep subsurface and surface-dwelling microorganisms are shown to gain advantages from impact-induced fracturing of rocks. Deleterious changes are associated with impact-induced closure of pore spaces in rocks. Superimposed on these long-term geological changes are post-impact alterations such as changes in the hydrological system in and around a crater. The close coupling between geological changes and the conditions for microorganisms yields a synthesis of the fields of microbiology and impact cratering. We use these data to discuss how craters can be used in the search for life beyond Earth.

Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

An Estimate of the Total DNA in the Biosphere.

Modern whole-organism genome analysis, in combination with biomass estimates, allows us to estimate a lower bound on the total information content in the biosphere: 5.3 × 1031 (±3.6 × 1031) megabases (Mb) of DNA. Given conservative estimates regarding DNA transcription rates, this information content suggests biosphere processing speeds exceeding yottaNOPS values (1024 Nucleotide Operations Per Second). Although prokaryotes evolved at least 3 billion years before plants and animals, we find that the information content of prokaryotes is similar to plants and animals at the present day. This information-based approach offers a new way to quantify anthropogenic and natural processes in the biosphere and its information diversity over time.

Rock geochemistry induces stress and starvation responses in the bacterial proteome.

Interactions between microorganisms and rocks play an important role in Earth system processes. However, little is known about the molecular capabilities microorganisms require to live in rocky environments. Using a quantitative label-free proteomics approach, we show that a model bacterium (Cupriavidus metallidurans CH34) can use volcanic rock to satisfy some elemental requirements, resulting in increased rates of cell division in both magnesium- and iron-limited media. However, the rocks also introduced multiple new stresses via chemical changes associated with pH, elemental leaching and surface adsorption of nutrients that were reflected in the proteome. For example, the loss of bioavailable phosphorus was observed and resulted in the upregulation of diverse phosphate limitation proteins, which facilitate increase phosphate uptake and scavenging within the cell. Our results revealed that despite the provision of essential elements, rock chemistry drives complex metabolic reorganization within rock-dwelling organisms, requiring tight regulation of cellular processes at the protein level. This study advances our ability to identify key microbial responses that enable life to persist in rock environments.

Multiplication of microbes below 0.690 water activity: implications for terrestrial and extraterrestrial life.

Since a key requirement of known life forms is available water (water activity; aw ), recent searches for signatures of past life in terrestrial and extraterrestrial environments have targeted places known to have contained significant quantities of biologically available water. However, early life on Earth inhabited high-salt environments, suggesting an ability to withstand low water-activity. The lower limit of water activity that enables cell division appears to be ∼ 0.605 which, until now, was only known to be exhibited by a single eukaryote, the sugar-tolerant, fungal xerophile Xeromyces bisporus. The first forms of life on Earth were, though, prokaryotic. Recent evidence now indicates that some halophilic Archaea and Bacteria have water-activity limits more or less equal to those of X. bisporus. We discuss water activity in relation to the limits of Earth's present-day biosphere; the possibility of microbial multiplication by utilizing water from thin, aqueous films or non-liquid sources; whether prokaryotes were the first organisms able to multiply close to the 0.605-aw limit; and whether extraterrestrial aqueous milieux of ≥ 0.605 aw can resemble fertile microbial habitats found on Earth.

Reduction of the temperature sensitivity of Halomonas hydrothermalis by iron starvation combined with microaerobic conditions.

The limits to biological processes on Earth are determined by physicochemical parameters, such as extremes of temperature and low water availability. Research into microbial extremophiles has enhanced our understanding of the biophysical boundaries which define the biosphere. However, there remains a paucity of information on the degree to which rates of microbial multiplication within extreme environments are determined by the availability of specific chemical elements. Here, we show that iron availability and the composition of the gaseous phase (aerobic versus microaerobic) determine the susceptibility of a marine bacterium, Halomonas hydrothermalis, to suboptimal and elevated temperature and salinity by impacting rates of cell division (but not viability). In particular, iron starvation combined with microaerobic conditions (5% [vol/vol] O2, 10% [vol/vol] CO2, reduced pH) reduced sensitivity to temperature across the 13°C range tested. These data demonstrate that nutrient limitation interacts with physicochemical parameters to determine biological permissiveness for extreme environments. The interplay between resource availability and stress tolerance, therefore, may shape the distribution and ecology of microorganisms within Earth's biosphere.

Pioneer microbial communities of the Fimmvörðuháls lava flow, Eyjafjallajökull, Iceland.

Little is understood regarding the phylogeny and metabolic capabilities of the earliest colonists of volcanic rocks, yet these data are essential for understanding how life becomes established in and interacts with the planetary crust, ultimately contributing to critical zone processes and soil formation. Here, we report the use of molecular and culture-dependent methods to determine the composition of pioneer microbial communities colonising the basaltic Fimmvörðuháls lava flow at Eyjafjallajökull, Iceland, formed in 2010. Our data show that 3 to 5 months post eruption, the lava was colonised by a low-diversity microbial community dominated by Betaproteobacteria, primarily taxa related to non-phototrophic diazotrophs such as Herbaspirillum spp. and chemolithotrophs such as Thiobacillus. Although successfully cultured following enrichment, phototrophs were not abundant members of the Fimmvörðuháls communities, as revealed by molecular analysis, and phototrophy is therefore not likely to be a dominant biogeochemical process in these early successional basalt communities. These results contrast with older Icelandic lava of comparable mineralogy, in which phototrophs comprised a significant fraction of microbial communities, and the non-phototrophic community fractions were dominated by Acidobacteria and Actinobacteria.

The minimum energy required to build a cell.

Understanding the energy requirements for cell synthesis accurately and comprehensively has been a longstanding challenge. We introduce a computational model that estimates the minimum energy necessary to build any cell from its constituent parts. This method combines omics and internal cell compositions from various sources to calculate the Gibbs Free Energy of biosynthesis independently of specific metabolic pathways. Our public tool, Synercell, can be used with other models for minumum species-specific energy estimations in any well-sequenced species. The energy for synthesising the genome, transcriptome, proteome, and lipid bilayer of four cell types: Escherichia coli, Saccharomyces cerevisiae, an average mammalian cell and JCVI-syn3A were estimated. Their modelled minimum synthesis energies at 298 K were 9.54 × 10 - 11 J/cell, 4.99 × 10 - 9 J/cell, 3.71 × 10 - 7 J/cell and 3.69 × 10 - 12 respectively. Gram-for-gram synthesis of lipid bilayers requires the most energy, followed by the proteome, genome, and transcriptome. The average per gram cost of biomass synthesis is in the 300s of J/g for all four cells. Implications for the generalisability of cell construction and applications to biogeosciences, cellular biology, biotechnology, and astrobiology are discussed.

Preservation of Bacillus subtilis' cellular liquid state at deep sub-zero temperatures in perchlorate brines.

Although a low temperature limit for life has not been established, it is thought that there exists a physical limit imposed by the onset of intracellular vitrification, typically occurring at ~-20 °C for unicellular organisms. Here, we show, through differential scanning calorimetry, that molar concentrations of magnesium perchlorate can depress the intracellular vitrification point of Bacillus subtilis cells to temperatures much lower than those previously reported. At 2.5 M Mg(ClO4)2, the peak vitrification temperature was lowered to -83 °C. Our results show that inorganic eutectic salts can in principle maintain liquid water in cells at much lower temperatures than those previously claimed as a lower limit to life, raising the prospects of active biochemical processes in low temperature natural settings. Our results may have implications for the habitability of Mars, where perchlorate salts are pervasive and potentially other terrestrial and extraterrestrial, cryosphere environments.

Life on Earth can grow on extraterrestrial organic carbon.

The universe is a vast store of organic abiotic carbon that could potentially drive heterotrophy on habitable planets. Meteorites are one of the transporters of this carbon to planetary surfaces. Meteoritic material was accumulating on early Earth when life emerged and proliferated. Yet it is not known if this organic carbon from space was accessible to life. In this research, an anaerobic microbial community was grown with the CM2 carbonaceous chondrite Aguas Zarcas as the sole carbon, energy and nutrient source. Using a reversed 13C-stable isotope labelling experiment in combination with optical photothermal infrared (O-PTIR) spectroscopy of single cells, this paper demonstrates the direct transfer of carbon from meteorite into microbial biomass. This implies that meteoritic organics could have been used as a carbon source on early Earth and other habitable planets, and supports the potential for a heterotrophic metabolism in early living systems.

The Concept of Life on Venus Informs the Concept of Habitability.

An enduring question in astrobiology is how we assess extraterrestrial environments as being suitable for life. We suggest that the most reliable assessments of the habitability of extraterrestrial environments are made with respect to the empirically determined limits to known life. We discuss qualitatively distinct categories of habitability: empirical habitability that is constrained by the observed limits to biological activity; habitability sensu stricto, which is defined with reference to the known or unknown limits to the activity of all known organisms; and habitability sensu lato (habitability in the broadest sense), which is circumscribed by the limit of all possible life in the universe, which is the most difficult (and perhaps impossible) to determine. We use the cloud deck of Venus, which is temperate but incompatible with known life, as an example to elaborate and hypothesize on these limits.

Detecting Microbiology in the Upper Atmosphere: Relative-Velocity Filtered Sampling.

The purpose of this article is to reopen from a practical perspective the question of the extent in altitude of Earth's biosphere. We make a number of different suggestions for how searches for biological material could be conducted in the mesosphere and lower thermosphere, colloquially referred to as the "ignore-osphere" because it has been generally ignored in the meteorological community compared to other regions. Relatively recent technological advances such as CubeSats in very low Earth orbit or more standard approaches such as the rocket-borne MAGIC meteoric smoke particle sampler are shown as potentially viable for sampling biological material in the ignore-osphere. The issue of contamination is discussed, and a potential solution to the problem is proposed by means of a new detector design that filters for particles based on their size and relative velocity to the detector.

Linking Methanogenesis in Low-Temperature Hydrothermal Vent Systems to Planetary Spectra: Methane Biosignatures on an Archean-Earth-like Exoplanet.

In this work, the viability of the detection of methane produced by microbial activity in low-temperature hydrothermal vents on an Archean-Earth-like exoplanet in the habitable zone is explored via a simplified bottom-up approach using a toy model. By simulating methanogens at hydrothermal vent sites in the deep ocean, biological methane production for a range of substrate inflow rates was determined and compared to literature values. These production rates were then used, along with a range of ocean floor vent coverage fractions, to determine likely methane concentrations in the simplified atmosphere. At maximum production rates, a vent coverage of 4-15 × 10-4 % (roughly 2000-6500 times that of modern Earth) is required to achieve 0.25% atmospheric methane. At minimum production rates, 100% vent coverage is not enough to produce 0.25% atmospheric methane. NASA's Planetary Spectrum Generator was then used to assess the detectability of methane features at various atmospheric concentrations. Even with future space-based observatory concepts (such as LUVOIR and HabEx), our results show the importance of both mirror size and distance to the observed planet. Planets with a substantial biomass of methanogens in hydrothermal vents can still lack a detectable, convincingly biological methane signature if they are beyond the scope of the chosen instrument. This work shows the value of coupling microbial ecological modeling with exoplanet science to better understand the constraints on biosignature gas production and its detectability.

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.