The hot springs of Yellowstone National Park (YNP) provide a natural laboratory for investigating the influence of chemical speciation and energy availability on the distribution of microbes. Among 133 inorganic chemotrophic energy supplies, total dissolved ammonia oxidation was consistently one of the highest across 63 YNP hot spring samples. Despite this, the distribution of ammonia-oxidizing archaea (AOA) does not reflect the energy supply calculated using total chemical abundances, and AOA were detected in only similar to 25% of the hot springs sampled and constituted <5% of any microbial community. To investigate the role of substrate speciation preference, total dissolved ammonia (NH3(aq) + NH4+) measurements were made in coordination with the collection of biological samples. DNA was extracted from these samples for 16S rRNA gene sequencing. By speciating the total dissolved ammonia concentrations into NH3(aq) and NH4+, a minimum concentration for substrate availability was identified at 0.07 mu molal NH3(aq). A maximum NH4+ concentration of 20.5 mu molal, indicates that NH4+ may be inhibitory at higher concentrations. Energy supply calculations performed using speciated chemical abundances reveal a minimum of 0.017 J (kg of water)(-1) required for NH3(aq) oxidation. Both the NH3(aq) concentration and NH3(aq) oxidation energy supply minimums, as well as the NH4+ maximum concentration, lead to significant differences in AOA distribution (p < 0.0001; Mann-Whitney U test). Thus, microbial distribution is better defined by speciated substrate concentrations, such as NH3(aq) and NH4+, and the energy supply from NH3(aq) oxidation, than by total chemical abundances or energy supplies calculated from total chemical abundances.

ABSTRACT Evidence from the Cassini mission confirmed that Saturn’s moon Enceladus hosts a subsurface alkaline ocean where rock-water reactions may generate redox disequilibria capable of supporting microbial metabolisms. To investigate potential microbial survival under simulated Enceladus ocean conditions, we used thermodynamic modeling to develop a salt formulation consistent with one possible Enceladus ocean composition and supplemented it with putative microbial energy sources to create a growth medium. The medium was inoculated with samples from diverse ocean world analog environments on Earth to determine which microorganisms could persist under Enceladus-like conditions. The microorganisms persisting in this geochemically bounded medium were heterotrophic, metabolically versatile bacteria with low carbon requirements. Genomic and physiological analyses further showed the presence of multiple stress-response pathways, sodium-based bioenergetic systems, osmoregulation strategies, and other adaptations consistent with survival in alkaline, low-nutrient settings. These results suggest that some stress-tolerant heterotrophic bacteria may serve as useful model organisms for life in Enceladus’ subsurface ocean. These findings demonstrate the value of geochemically modeled media as a framework for constraining habitability, identifying relevant biosignatures, and probing potential microbial survival strategies beyond Earth.

The search for life in our solar system often involves efforts to detect organic molecules, which have been found on many extraterrestrial bodies, including planets, moons, meteorites, comets, and asteroids. These chemical signatures are not typically thought of as biosignatures because we know that organic synthesis can occur through abiotic processes. Therefore, development of methods for distinguishing biotic and abiotic biosignatures would enable interpretation of data collected from habitability and life-detection missions. Life on Earth harnesses energy-releasing reactions to power biosynthesis reactions, which often require energy. Using thermodynamic data, we can quantify the energy required for organic synthesis. If an organic molecule is detected in an abundance that is thermodynamically unstable, then it is possible that life coupled its synthesis to other energy-releasing reactions. On the other hand, if an organic molecule is detected in an abundance that is thermodynamically stable, then abiotic synthesis was plausible. This sorting framework can be applied to the search for life wherever we have geochemical data. One such example is Saturn’s moon Enceladus. Small compounds involving the elements that comprise the majority of biomass were detected by the Cassini spacecraft in the plume gas erupting from the subsurface ocean. Using Enceladus as an example, we demonstrate the utility of thermodynamic calculations for distinguishing biosignatures and show that organic synthesis is often favorable using the carbon sources available on Enceladus. While these results may lead us to conclude that hypothetical organic signatures on Enceladus are abiotic, this framework can be applied to other environments in the search for genuine biosignatures.

Serpentinization, the reaction of water with ultramafic rock, produces reduced, hyperalkaline, and H2-rich fluids that support a variety of hydrogenotrophic microbial metabolisms. Previous work indicates the occurrence of methanogenesis in fluids from the actively serpentinizing Samail Ophiolite in the Sultanate of Oman. While those fluids contain abundant H2 to fuel hydrogenotrophic methanogenesis (CO2 + 4H2 ➔ CH4 + 2H2O), the concentration of CO2 is very low due to the hyperalkalinity (> pH 11) and geochemistry of the fluids. As a result, species such as formate and acetate may be important as alternative methanogenic substrates. In this study we quantified the impact of inorganic carbon, formate and acetate availability for methanogenic metabolisms, across a range of fluid chemistries, in terms of (1) the potential diffusive flux of substrates to the cell, (2) the Affinity (Gibbs energy change) associated with methanogenic metabolism, and (3) the energy “inventory” per kg fluid. In parallel, we assessed the genomic potential for the conduct of those three methanogenic modes across the same set of fluids and consider the results within the quantitative framework of energy availability. We find that formatotrophic methanogenesis affords a higher Affinity (greater energetic yield) than acetoclastic and hydrogenotrophic methanogenesis in pristine serpentinized fluids and, in agreement with previous studies, find genomic evidence for a methanogen of the genus Methanobacterium to carry out formatotrophic and hydrogenotrophic methanogenesis, with the possibility of even using bicarbonate as a supply of CO2. Acetoclastic methanogenesis is also shown to be energetically favorable in these fluids, and we report the first detection of a potential acetoclastic methanogen of the family Methanosarcinaceae, which forms a distinct clade with a genome from the serpentinizing seafloor hydrothermal vent field, Lost City. These results demonstrate the applicability of an energy availability framework for interpreting methanogen ecology in serpentinizing systems.

Linear free energy relations were obtained from existing experimental data and used to estimate stability constants for over 16,000 metal complexes with monovalent oxygen-bearing ligands comprising 75 metal species and 220 ligands. Similar relationships for metal-ligand entropy of association were also obtained which facilitate computation of stability constants from 0 to 125°C for over 6000 metal-ligand complexes. Our inventory for monovalent oxygen-bearing ligands includes carboxylic acids, phenols, alkyl alcohols, substituted alcohols and inorganic ligands with a special emphasis on geobiologically prevalent ligands like lactate, pyruvate, ascorbate and borate. Methods were also devised to estimate metal-ligand stability constants and complexation entropy if only the pKa of the ligand were known. The slopes and intercepts of these linear free energy relations can be explained by ligand denticity and metal ionic radius, as well as inductive and steric effects, thus providing chemical foundations for these estimation strategies. The stability constant estimates derived from this work were applied to model the speciation of metals and hexanoic acid in two hypothetical hydrothermal mixing fluids representative of the Rainbow and Lau vent fields. This enabled prediction of the distribution of biologically relevant chemical species across key state variables of the chemical system, including temperature, pH, and chemical composition when no stability constant information is available. We further explore the implications of these findings in the areas of metal transport in terrestrial and extra-terrestrial aqueous systems, deep-sea biology, origin and evolution of life, and environmental chemistry and biology.

Populations of the acidophilic purple nonsulfur bacterium Rhodopila globiformis were identified in two geographically distinct thermal areas in Yellowstone National Park (Wyoming, USA), as confirmed by 16S rRNA gene sequencing and detection of characteristic methoxylated ketocarotenoids. Microcosm-based carbon uptake assays where oxygenic photosynthesis was excluded via addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea yielded a light-driven dissolved inorganic carbon (DIC) assimilation rate (7 ± 2 mg C g C-1 h-1) comparable to those of highly productive algal mats in acidic hot springs, suggesting that R. globiformis may be performing photoautotrophy at the time of the assay. Rates of acetate assimilation were more than two orders of magnitude lower than DIC assimilation and did not differ between light and dark treatments, indicating photoheterotrophic use of acetate was not occurring, though photoheterotrophic assimilation of other organic compounds cannot be excluded. The tepid (35°C) spring waters are acidic (pH = 3.7) with moderate dissolved hydrogen sulfide (0.2 mM) and abundant DIC (11 mM), an apparently rare set of conditions thought to arise from extremely shallow mixing of oxygenated meteoric water and volcanic gases. Though originally isolated and cultured photoheterotrophically, in nature, R. globiformis may grow photoautotrophically under the normal conditions of its habitat, utilizing a stable supply of DIC afforded by the injection of CO2-rich volcanic gases. To our knowledge, these are the most acidic conditions under which light-driven DIC assimilation has been observed in the domain Bacteria.IMPORTANCEPurple nonsulfur bacteria are ecologically diverse and metabolically versatile anoxygenic phototrophs; however, only a few acid-tolerant species are known. We identified populations of the purple nonsulfur bacterium Rhodopila globiformis in warm, acidic springs with moderate (~0.2 mM) concentrations of dissolved hydrogen sulfide in two thermal areas of Yellowstone National Park (Wyoming, USA). Comprehensive geochemical analyses of the spring waters illustrate that they are formed by mixing of groundwater and CO2-rich volcanic gases extremely close to the surface, relatively rare conditions that lead to characterization of R. globiformis as an endangered species. A high rate of light-driven assimilation of dissolved CO2 that rivals rates for acidophilic algae was observed, indicating that R. globiformis is responsible for a significant amount of primary production and suggesting it may primarily grow photoautotrophically in nature. These observations constitute the first insights into the physiological ecology of the most acidophilic anaerobic anoxygenic phototroph presently known.

The geochemistry of trace elements in volcanic gas emissions at Vulcano (Sicily, Italy) was investigated. Trace element concentrations in 94-412 degrees C fumarole gases span over 10 orders of magnitude, from 0.01 pmol/mol to 300 mu mol/mol, with some metalloids (B, Si) being the most abundant, followed by alkali, alkaline earth, and certain transition metals, and rare earth elements typically displaying the lowest concentrations. Thermodynamic modeling predicts most trace elements to be transported as chloride, hydroxide, and mixed hydroxychloro gas species (LiCl, KCl, NaCl, RbCl and CsCl, Be(OH)2, Mg(OH)2, MgCl2, CaCl2, SrCl2, CaCl(OH), TiOCl2, VOCl, VOCl2, VOCl3, NbOCl3, Cr(OH)3, CrCl3, Fe(OH)2, FeCl2 Co(OH)2, CoCl2, Ni(OH)2 to NiCl2, Cd(OH)2, CdCl2, Re(OH)3, ReCl3, ZnCl2, AgCl, WO2(OH)2, Al(OH)3, Si(OH)4, B(OH)4, TlO, GaCl3, SbCl, MnCl2, CuCl). Sulfide, hydrate, and elemental gas species are also important for some elements (Cd, AuS, Hg, PbS2, BiS, Bi, AsS, As2S3, TeS, SeH, SeS). However, for many trace elements, speciation remains uncertain or unknown due to a lack of thermodynamic data. Upon cooling and decompression of the volcanic gas, most trace elements are predicted to reach gas-solid equilibrium, resulting in the formation of secondary minerals. At high temperatures ( 700-1000 degrees C), the mineral assemblage forming is dominated by quartz, Ca-Na-K feldspars, and Mg-pyroxene, containing minor concentrations of other alkali and alkaline earth metals. Further cooling and decompression leads to the formation of minerals including magnetite, pyrite, chalcocite, and chalcopyrite together with other less abundant oxides (V, Cr, Ga, W, and Sn) and sulfides (Zn, Pb, Ni, Co, Cd, Mo, Ag, As, and Bi), and eventually a range of sulfates and sulfosalts (Li, K, Na, Rb, Cs, Be, Mg, Ca, Sr, Bi, Mn, Fe, Zn, Pb, and Sn) at the lowest temperatures ( 100-300 degrees C). For most trace elements, fumarole emission concentrations reflect higher gas-solid equilibrium temperatures than those observed during sampling, suggesting gas-solid equilibria at high temperatures followed by incomplete re-equilibration upon further cooling near the surface. Trace element fluxes span over eight orders of magnitude, ranging from >100 kg/day to 1 & sdot;10- 6 kg/day. Silica, Al, and B consistently exhibit the highest fluxes, followed by alkali and alkaline earth metals, various transition metals and metalloids, with rare earth elements and actinides displaying the lowest fluxes. Generally, the trace element fluxes are lower compared to neighboring Stromboli and Etna, except for Pb, Bi, B, As, Sb, and Te.

Mass transfer processes between fluids and ultramafic rocks produce subsurface environments encompassing a wide range of redox conditions. A notable locality where an extensive range of redox conditions is observed in one location is Hole BA1B, a similar to 400 m borehole drilled by the Oman Drilling Project. A sulfur-enriched serpentinite zone, containing up to 0.6 wt% S, occurs between shallow oxidized serpentinites (<30 m) and deep partially serpentinized harzburgite (>150 m). All three alteration zones are predominantly composed of serpentine. However, microanalysis of samples from the sulfur-enriched zone shows that mesh textures after olivine are composed of serpentine, brucite, and tochilinite mixtures, yielding optically black thin-section samples that characterize this sulfidic zone. It is proposed that sulfur accumulates in this zone via a process similar to those found in supergene ore deposits. Reaction-path models show that at shallow conditions open to atmospheric input, sulfur is mobilized via oxidative weathering of serpentinized dunite and harzburgite. Sulfatebearing fluids percolate deeper and react with host rocks in a system closed to atmospheric input. As fluids become more reduced, dissolved sulfate is precipitated as sulfide minerals yielding rocks with similar to 0.4 wt% S, like those observed in Hole BA1B. Despite enrichment of S in the sulfidic zone in Hole BA1B, Ni and Co contents are uniform throughout all three layers in the borehole. This is consistent with model results which show that Ni (and, by analogy, Co) is less mobile than S, and can be hosted in serpentine and NiFe alloys in addition to sulfides. The sulfur enrichment process may occur abiotically. However, sulfide enrichment via microbial reduction of sulfate and other sulfur species can also facilitate the formation of the sulfidic zone. Bioenergetic calculations show that abundant energy is available for sulfur reducing microbes, consistent with previous work demonstrating the presence of active, sulfate-reducing microorganisms in Hole BA1B and other nearby boreholes. This suggests that the observed sulfur enrichment is an ongoing process. Overall, this work shows that variable redox conditions are attained as fluids percolate and react with serpentinized ultramafic rocks at variable extents of interaction between aquifer fluids, host ultramafic rocks, and the atmosphere.

The MAss Spectrometer for Planetary EXploration (MASPEX) is a high-mass-resolution, high-sensitivity, multi-bounce time-of-flight mass spectrometer (MBTOF) capable of measuring minor species with abundances of sub-parts-per-million in Europa’s sputter-produced and radiolytically modified exosphere and in its oceanic plumes. The goal of the MASPEX-Europa investigation is to determine, through in-situ measurement of the exosphere and plume composition, whether the conditions for habitability exist or have existed on Europa. As conventionally defined, based on our knowledge of Earth life, the three fundamental conditions for habitability are: (1) the presence of liquid water; (2) the presence of organic compounds and the biogenic elements CHNOPS; and (3) a source of energy available for metabolic processes, which for Europa will most probably be chemosynthetic rather than photosynthetic. Condition (1) is already established by previous indirect (magnetic field) measurements, while MASPEX will contribute directly to the evaluation of condition (2) through highly specific compositional measurements in the Europan exosphere and plumes. The composition measurements will also contribute to the test of condition (3) through disequilibrium states of chemical reactions. Thus, the primary goal of MASPEX for Europa Clipper is to assess the habitability of Europa and specifically of its interior ocean. MASPEX has been developed successfully, and its calibration has demonstrated that it meets its specified requirements for sensitivity, dynamic range, and mass resolution. This paper reports the development of the MASPEX scientific investigation, the instrument, its performance, and calibration.

The goal of NASA’s Europa Clipper mission is to assess the habitability of Jupiter’s moon Europa. After entering Jupiter orbit in 2030, the flight system will collect science data while flying past Europa 49 times at typical closest approach distances of 25–100 km. The mission’s objectives are to investigate Europa’s interior (ice shell and ocean), composition, and geology; the mission will also search for and characterize any current activity including possible plumes. The science objectives will be accomplished with a payload consisting of remote sensing and in-situ instruments. Remote sensing investigations cover the ultraviolet, visible, near infrared, and thermal infrared wavelength ranges of the electromagnetic spectrum, as well as an ice-penetrating radar. In-situ investigations measure the magnetic field, dust grains, neutral gas, and plasma surrounding Europa. Gravity science will be achieved using the telecommunication system, and a radiation monitoring engineering subsystem will provide complementary science data. The flight system is designed to enable all science instruments to operate and gather data simultaneously. Mission planning and operations are guided by scientific requirements and observation strategies, while appropriate updates to the plan will be made tactically as the instruments and Europa are characterized and discoveries emerge. Following collection and validation, all science data will be archived in NASA’s Planetary Data System. Communication, data sharing, and publication policies promote visibility, collaboration, and mutual interdependence across the full Europa Clipper science team, to best achieve the interdisciplinary science necessary to understand Europa.

On Earth and beyond, organic chemistry often occurs in the presence of water within environments that deviate drastically from ambient conditions (25 degrees C and 1 bar). Accurately predicting aqueous organic reaction pathways is crucial toward understanding planetary scale processes such as the cycling of elements crucial for life (e.g., carbon and nitrogen). Advanced thermodynamic modeling can be utilized to determine the favorability of various organic reactions based on geologically relevant ranges of temperature and pressure, as well as compositional variables (e.g., pH). However, data that would otherwise allow for a diversity of organic compounds and environmental conditions to be modeled are sparse and rarely tested with experiments, particularly with regard to organic-nitrogen compounds. In this work, we develop a framework to estimate thermodynamic properties at ambient conditions that can then be extrapolated across ranges of temperature and pressure for aqueous primary, secondary, and tertiary amines and aminiums (protonated amines), specifically those structures containing linear alkyl chains and benzyl functional groups. We also performed hydrothermal experiments (250 degrees C, similar to 40 bar) involving reactions of methylamines to test our resulting thermodynamic models, and we compare our models for other alkylamines and benzylamines to previous empirical measurements from the literature. Specifically, we use existing thermodynamic data along with our estimates at ambient conditions in combination with a variety of existing extrapolation methods related to the revised Helgeson-Kirkham-Flowers (HKF) equations of state to generate temperature- and pressure-dependent predictions of acid dissociation constants (i.e., pK(a) values) that strongly agree with previous empirical measurements. We use similar methods to predict product distributions for reactions involving primary, secondary, and tertiary amines/aminiums, as well as ammonia/ammonium and corresponding alcohols whose collective distributions depend on reversible substitution reactions. Our predictions are in good agreement with our experimental results involving the methylamine reaction system as well as previous experiments involving the benzylamine reaction system, for which we also produced thermodynamic estimates involving benzyl alcohol. The agreement between independent theoretical predictions and experimental measurements suggests that our estimated properties can be applied to modeling amine chemistry in other experimental and natural aqueous systems that range in temperature and pressure, providing new tools for planetary exploration.

Jupiter's icy moon, Europa, harbors a subsurface liquid water ocean; the prospect of this ocean being habitable motivates further exploration of the moon with the upcoming NASA Europa Clipper mission. Key among the mission goals is a comprehensive assessment of the moon's composition, which is essential for assessing Europa's habitability. Through powerful remote sensing and in situ investigations, the Europa Clipper mission will explore the composition of Europa's surface and subsurface, its tenuous atmosphere, and the local space environment surrounding the moon. Clues on the interior composition of Europa will be gathered through these assessments, especially in regions that may expose subsurface materials, including compelling geologic landforms or locations indicative of recent or current activity such as potential plumes. The planned reconnaissance of the icy world will constrain models that simulate the ongoing external and internal processes that act to alter its composition. This paper presents the composition-themed goals for the Europa Clipper mission, the synergistic, composition-focused investigations that will be conducted, and how the anticipated scientific return will advance our understanding of the origin, evolution, and current state of Europa.

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.