Considerations for Detecting Organic Indicators of Metabolism on Enceladus.

Enceladus is of interest to astrobiology and the search for life since it is thought to host active hydrothermal activity and habitable conditions. It is also possible that the organics detected on Enceladus may indicate an active prebiotic or biotic system; in particular, the conditions on Enceladus may favor mineral-driven protometabolic reactions. When including metabolism-related biosignatures in Enceladus mission concepts, it is necessary to base these in a clearer understanding of how these signatures could also be produced prebiotically. In addition, postulating which biological metabolisms to look for on Enceladus requires a non-Earth-centric approach since the details of biological metabolic pathways are heavily shaped by adaptation to geochemical conditions over the planet's history. Creating metabolism-related organic detection objectives for Enceladus missions, therefore, requires consideration of how metabolic systems may operate differently on another world, while basing these speculations on observed Earth-specific microbial processes. In addition, advances in origin-of-life research can play a critical role in distinguishing between interpretations of any future organic detections on Enceladus, and the discovery of an extant prebiotic system would be a transformative astrobiological event in its own right.

Future of the Search for Life: Workshop Report.

The 2-week, virtual Future of the Search for Life science and engineering workshop brought together more than 100 scientists, engineers, and technologists in March and April 2022 to provide their expert opinion on the interconnections between life-detection science and technology. Participants identified the advances in measurement and sampling technologies they believed to be necessary to perform in situ searches for life elsewhere in our Solar System, 20 years or more in the future. Among suggested measurements for these searches, those pertaining to three potential indicators of life termed "dynamic disequilibrium," "catalysis," and "informational polymers" were identified as particularly promising avenues for further exploration. For these three indicators, small breakout groups of participants identified measurement needs and knowledge gaps, along with corresponding constraints on sample handling (acquisition and processing) approaches for a variety of environments on Enceladus, Europa, Mars, and Titan. Despite the diversity of these environments, sample processing approaches all tend to be more complex than those that have been implemented on missions or envisioned for mission concepts to date. The approaches considered by workshop breakout groups progress from nondestructive to destructive measurement techniques, and most involve the need for fluid (especially liquid) sample processing. Sample processing needs were identified as technology gaps. These gaps include technology and associated sampling strategies that allow the preservation of the thermal, mechanical, and chemical integrity of the samples upon acquisition; and to optimize the sample information obtained by operating suites of instruments on common samples. Crucially, the interplay between science-driven life-detection strategies and their technological implementation highlights the need for an unprecedented level of payload integration and extensive collaboration between scientists and engineers, starting from concept formulation through mission deployment of life-detection instruments and sample processing systems.

A microfluidic labyrinth self-assembled by a chemical garden.

Chemical gardens, self-assembling precipitates that spontaneously form when a metal salt is added to a solution of another precipitating anion, are of interest for various applications including producing reactive materials in controlled structures. Here, we report on two chemical garden reaction systems (CuCl2 and Cu(NO3)2 seed crystals submerged in sodium silicate) that produced self-assembled microfluidic labyrinths in a vertical 2D Hele-Shaw reactor. The formation of labyrinths as well as the specific growth modes of the precipitate were dependent on the silicate concentration: CuCl2 labyrinths formed only at 3 and 4 M silicate and Cu(NO3)2 labyrinths formed only at 4 and 5 M silicate. The labyrinth structures contained silicate on the exterior and crystalline material interpreted as hydrated minerals from the metal salt in their interiors. The bubble-guided tubes that form labyrinths can be controlled by changing the angle of the 2D reaction cell; this suggests that future experiments of this type could form self-organizing structures with controlled composition and orientation for use in microfluidics and various materials science applications.

Electron transport chains as a window into the earliest stages of evolution.

The origin and early evolution of life is generally studied under two different paradigms: bottom up and top down. Prebiotic chemistry and early Earth geochemistry allow researchers to explore possible origin of life scenarios. But for these "bottom-up" approaches, even successful experiments only amount to a proof of principle. On the other hand, "top-down" research on early evolutionary history is able to provide a historical account about ancient organisms, but is unable to investigate stages that occurred during and just after the origin of life. Here, we consider ancient electron transport chains (ETCs) as a potential bridge between early evolutionary history and a protocellular stage that preceded it. Current phylogenetic evidence suggests that ancestors of several extant ETC components were present at least as late as the last universal common ancestor of life. In addition, recent experiments have shown that some aspects of modern ETCs can be replicated by minerals, protocells, or organic cofactors in the absence of biological proteins. Here, we discuss the diversity of ETCs and other forms of chemiosmotic energy conservation, describe current work on the early evolution of membrane bioenergetics, and advocate for several lines of research to enhance this understanding by pairing top-down and bottom-up approaches.

A Review on Hypothesized Metabolic Pathways on Europa and Enceladus: Space-Flight Detection Considerations.

Enceladus and Europa, icy moons of Saturn and Jupiter, respectively, are believed to be habitable with liquid water oceans and therefore are of interest for future life detection missions and mission concepts. With the limited data from missions to these moons, many studies have sought to better constrain these conditions. With these constraints, researchers have, based on modeling and experimental studies, hypothesized a number of possible metabolisms that could exist on Europa and Enceladus if these worlds host life. The most often hypothesized metabolisms are methanogenesis for Enceladus and methane oxidation/sulfate reduction on Europa. Here, we outline, review, and compare the best estimated conditions of each moon's ocean. We then discuss the hypothetical metabolisms that have been suggested to be present on these moons, based on laboratory studies and Earth analogs. We also detail different detection methods that could be used to detect these hypothetical metabolic reactions and make recommendations for future research and considerations for future missions.

Planetary Minerals Catalyze Conversion of a Polycyclic Aromatic Hydrocarbon to a Prebiotic Quinone: Implications for Origins of Life.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in astrochemical environments and are disbursed into planetary environments via meteorites and extraterrestrial infall where they may interact with mineral phases to produce quinones important for origins of life. In this study, we assessed the potential of the phyllosilicates montmorillonite (MONT) and kaolinite (KAO), and the enhanced Mojave Mars Simulant (MMS) to convert the PAH anthracene (ANTH) to the biologically important 9,10-anthraquinone (ANTHQ). All studied mineral substrates mediate conversion over the temperature range assessed (25-500°C). Apparent rate curves for conversion were sigmoidal for MONT and KAO, but quadratic for MMS. Conversion efficiency maxima for ANTHQ were 3.06% ± 0.42%, 1.15% ± 0.13%, and 0.56% ± 0.039% for MONT, KAO, and MMS, respectively. We hypothesized that differential substrate binding and compound loss account for the apparent conversion kinetics observed. Apparent loss rate curves for ANTH and ANTHQ were exponential for all substrates, suggesting a pathway for wide distribution of both compounds in warmer prebiotic environments. These findings improve upon our previously reported ANTHQ conversion efficiency on MONT and provide support for a plausible scenario in which PAH-mineral interactions could have produced prebiotically relevant quinones in early Earth environments.

Determining the "Biosignature Threshold" for Life Detection on Biotic, Abiotic, or Prebiotic Worlds.

The field of prebiotic chemistry has demonstrated that complex organic chemical systems that exhibit various life-like properties can be produced abiotically in the laboratory. Understanding these chemical systems is important for astrobiology and life detection since we do not know the extent to which prebiotic chemistry might exist or have existed on other worlds. Nor do we know what signatures are diagnostic of an extant or "failed" prebiotic system. On Earth, biology has suppressed most abiotic organic chemistry and overprints geologic records of prebiotic chemistry; therefore, it is difficult to validate whether chemical signatures from future planetary missions are remnant or extant prebiotic systems. The "biosignature threshold" between whether a chemical signature is more likely to be produced by abiotic versus biotic chemistry on a given world could vary significantly, depending on the particular environment, and could change over time, especially if life were to emerge and diversify on that world. To interpret organic signatures detected during a planetary mission, we advocate for (1) gaining a more complete understanding of prebiotic/abiotic chemical possibilities in diverse planetary environments and (2) involving experimental prebiotic samples as analogues when generating comparison libraries for "life-detection" mission instruments.

Testing Abiotic Reduction of NAD+ Directly Mediated by Iron/Sulfur Minerals.

Life emerged in a geochemical context, possibly in the midst of mineral substrates. However, it is not known to what extent minerals and dissolved inorganic ions could have facilitated the evolution of biochemical reactions. Herein, we have experimentally shown that iron sulfide minerals can act as electron transfer agents for the reduction of the ubiquitous biological protein cofactor nicotinamide adenine dinucleotide (NAD+) under anaerobic prebiotic conditions, observing the NAD+/NADH redox transition by using ultraviolet-visible spectroscopy and 1H nuclear magnetic resonance. This reaction was mediated with iron sulfide minerals, which were likely abundant on early Earth in seafloor and hydrothermal settings; and the NAD+/NADH redox reaction occurred in the absence of UV light, peptide ligand(s), or dissolved mediators. To better understand this reaction, thermodynamic modeling was also performed. The ability of an iron sulfide mineral to transfer electrons to a biochemical cofactor that is found in every living cell demonstrates how geologic materials could have played a direct role in the evolution of certain cofactor-driven metabolic pathways.

Phosphine Generation Pathways on Rocky Planets.

The possibility of life in the venusian clouds was proposed in the 1960s, and recently this hypothesis has been revived with the potential detection of phosphine (PH3) in Venus' atmosphere. These observations may have detected ∼5-20 ppb phosphine on Venus (Greaves et al., 2020), which raises questions about venusian atmospheric/geochemical processes and suggests that this phosphine could possibly be generated by biological processes. In such a claim, it is essential to understand the abiotic phosphorus chemistry that may occur under Venus-relevant conditions, particularly those processes that may result in phosphine generation. Here, we discuss two related abiotic routes for phosphine generation within the atmosphere of Venus. Based on our assessment, corrosion of large impactors as they ablate near Venus' cloud layer, and the presence of reduced phosphorus compounds in the subcloud layer could result in production of phosphine and may explain the phosphine detected in Venus' atmosphere or on other rocky planets. We end on a cautionary note: although there may be life in the clouds of Venus, the detection of a simple, single gas, phosphine, is likely not a decisive indicator.

A Proposed Geobiology-Driven Nomenclature for Astrobiological In Situ Observations and Sample Analyses.

As the exploration of Mars and other worlds for signs of life has increased, the need for a common nomenclature and consensus has become significantly important for proper identification of nonterrestrial/non-Earth biology, biogenic structures, and chemical processes generated from biological processes. The fact that Earth is our single data point for all life, diversity, and evolution means that there is an inherent bias toward life as we know it through our own planet's history. The search for life "as we don't know it" then brings this bias forward to decision-making regarding mission instruments and payloads. Understandably, this leads to several top-level scientific, theoretical, and philosophical questions regarding the definition of life and what it means for future life detection missions. How can we decide on how and where to detect known and unknown signs of life with a single biased data point? What features could act as universal biosignatures that support Darwinian evolution in the geological context of nonterrestrial time lines? The purpose of this article is to generate an improved nomenclature for terrestrial features that have mineral/microbial interactions within structures and to confirm which features can only exist from life (biotic), features that are modified by biological processes (biogenic), features that life does not affect (abiotic), and properties that can exist or not regardless of the presence of biology (abiogenic). These four categories are critical in understanding and deciphering future returned samples from Mars, signs of potential extinct/ancient and extant life on Mars, and in situ analyses from ocean worlds to distinguish and separate what physical structures and chemical patterns are due to life and which are not. Moreover, we discuss hypothetical detection and preservation environments for extant and extinct life, respectively. These proposed environments will take into account independent active and ancient in situ detection prospects by using previous planetary exploration studies and discuss the geobiological implications within an astrobiological context.

Plausible Emergence and Self Assembly of a Primitive Phospholipid from Reduced Phosphorus on the Primordial Earth.

How life arose on the primitive Earth is one of the biggest questions in science. Biomolecular emergence scenarios have proliferated in the literature but accounting for the ubiquity of oxidized (+ 5) phosphate (PO43-) in extant biochemistries has been challenging due to the dearth of phosphate and molecular oxygen on the primordial Earth. A compelling body of work suggests that exogenous schreibersite ((Fe,Ni)3P) was delivered to Earth via meteorite impacts during the Heavy Bombardment (ca. 4.1-3.8 Gya) and there converted to reduced P oxyanions (e.g., phosphite (HPO32-) and hypophosphite (H2PO2-)) and phosphonates. Inspired by this idea, we review the relevant literature to deduce a plausible reduced phospholipid analog of modern phosphatidylcholines that could have emerged in a primordial hydrothermal setting. A shallow alkaline lacustrine basin underlain by active hydrothermal fissures and meteoritic schreibersite-, clay-, and metal-enriched sediments is envisioned. The water column is laden with known and putative primordial hydrothermal reagents. Small system dimensions and thermal- and UV-driven evaporation further concentrate chemical precursors. We hypothesize that a reduced phospholipid arises from Fischer-Tropsch-type (FTT) production of a C8 alkanoic acid, which condenses with an organophosphinate (derived from schreibersite corrosion to hypophosphite with subsequent methylation/oxidation), to yield a reduced protophospholipid. This then condenses with an α-amino nitrile (derived from Strecker-type reactions) to form the polar head. Preliminary modeling results indicate that reduced phospholipids do not aggregate rapidly; however, single layer micelles are stable up to aggregates with approximately 100 molecules.

Investigation of Venus Cloud Aerosol and Gas Composition Including Potential Biogenic Materials via an Aerosol-Sampling Instrument Package.

A lightweight, low-power instrument package to measure, in situ, both (1) the local gaseous environment and (2) the composition and microphysical properties of attendant venusian aerosols is presented. This Aerosol-Sampling Instrument Package (ASIP) would be used to explore cloud chemical and possibly biotic processes on future aerial missions such as multiweek balloon missions and on short-duration (<1 h) probes on Venus and potentially on other cloudy worlds such as Titan, the Ice Giants, and Saturn. A quadrupole ion-trap mass spectrometer (QITMS; Madzunkov and Nikolic, J Am Soc Mass Spectrom 25:1841-1852, 2014) fed alternately by (1) an aerosol separator that injects only aerosols into a vaporizer and mass spectrometer and (2) the pure aerosol-filtered atmosphere, achieves the compositional measurements. Aerosols vaporized <600°C are measured over atomic mass ranges from 2 to 300 AMU at <0.02 AMU resolution, sufficient to measure trace materials, their isotopic ratios, and potential biogenic materials embedded within H2SO4 aerosols, to better than 20% in <300 s for H2SO4 -relative abundances of 2 × 10-9. An integrated lightweight, compact nephelometer/particle-counter determines the number density and particle sizes of the sampled aerosols.

Simulation of Early Earth Hydrothermal Chimneys in a Thermal Gradient Environment.

Deep sea hydrothermal vents are self-organizing precipitates generated from geochemical disequilibria and have been proposed as a possible setting for the emergence of life. The growth of hydrothermal chimneys in a thermal gradient environment within an early Earth vent system was successfully simulated by using different hydrothermal simulants, such as sodium sulfide, which were injected into an early Earth ocean simulant containing dissolved ferrous iron. Moreover, an apparatus was developed to sufficiently cool the ocean simulant to near 0 °C in a condenser vessel immersed in a cold water bath while injecting a sulfide solution at hot to room temperatures, effectively creating an artificial chimney structure in a temperature gradient environment over a period of a few hours. Such experiments with different chemistries and variable temperature gradients resulted in a variety of morphologies in the chimney structure. The use of ocean and hydrothermal fluid simulants at room temperature resulted in vertical chimneys, whereas the combination of a hot hydrothermal fluid and cold ocean simulant inhibited the formation of robust chimney structures. The customizable 3D printed condenser created for this study acts as a jacketed reaction vessel that can be easily modified and used by different researchers. It will allow the careful control of injection rate and chemical composition of vent and ocean simulants, which should help accurately simulate prebiotic reactions in chimney systems with thermal gradients similar to those of natural systems.

Chemical Gardens as Electrochemical Systems: In Situ Characterization of Simulated Prebiotic Hydrothermal Vents by Impedance Spectroscopy.

In an early earth or planetary chimney systems, hydrothermal fluid chemistry and flow durations play a large role in the chimney's ability to drive electrochemical reactions for the origin of life. We performed continuous electrochemical impedance spectroscopy (EIS) characterization on inorganic membranes representing prebiotic hydrothermal chimney vents in natural seafloor systems, by incorporating an electrode array into a chimney growth experiment. Localized potential and capacitances profiles in the chimney reveal a dynamic system where redox processes are driven by transport phenomena, increasing rapidly due to disequilibrium until achieving equilibrium at about 100 mV and 1000 µF/cm2 . The impedance in the chimney interior is three orders of magnitude lower (100 Ohms/cm2 vs 100 KOhms/cm2 ) than at the ocean or the ocean/chimney interface. The calculated peak dissipation factor (DF) values are more than ten times higher (40.0 vs 3.0) and also confirm the elevated chemical reactivity in the chimney interior.

Machine Learning Analysis of the Thermodynamic Responses of In Situ Dielectric Spectroscopy Data in Amino Acids and Inorganic Electrolytes.

Dielectric spectroscopy (DS) can be a robust in situ technique for geochemical applications. In this study, we applied deep-learning techniques to DS measurement data to enable rapid science interrogation and identification of electrolyte solutions containing salts and amino acids over a wide temperature range (20 to -60 °C). For the purpose of searching for signs of life, detecting amino acids is a fundamental high priority for field and planetary instruments as amino acids are one of the building blocks for life as we know it. A convolutional neural network (CNN) with channel-wise one-dimensional filters is proposed to fulfill the task, using the DS data of amino acid and inorganic salt solutions. Experimental results show that the CNN with two convolutional layers and one fully connected layer can effectively differentiate solutions containing amino acids from those containing salts in both the liquid and solid (water ice) states. To complement the experimental measurements and CNN analysis, the diffusive behaviors of ions (K+, Cl-, and OH-) were further discussed with atomistic molecular dynamics simulations performed in this work as well as the quantum simulation published in the literature. Combining DS with machine-learning techniques and simulations will greatly facilitate more real-time decision-making of mobility systems for future exploratory endeavors in other worlds beyond Earth.

3D Printed Minerals as Astrobiology Analogs of Hydrothermal Vent Chimneys.

Hydrothermal vents, which are highly plausible habitable environments for life and of interest for some origin-of-life scenarios, may exist on icy moons such as Europa or Enceladus in addition to Earth. Some hydrothermal vent chimney structures are extremely porous and friable, making their reconstruction in the lab challenging (e.g., brucite or saponite in alkaline hydrothermal settings). Here, we present the results from our efforts to reconstruct a simplified chimney structure directly out of mineral powder using binder jet additive manufacturing. Olivine sand was chosen for this initial method development effort since it represents a naturally occurring seafloor material and is inexpensively available in large quantities in powder form. The crystal structure of olivine used for the print was not modified during the process, as confirmed by powder X-ray diffraction (XRD). To characterize the microstructure of our 3D printed precipitates, we used computed tomography (CT) X-ray scan techniques. We also evaluated a chimney precipitate from a sample collected from the Prony Hydrothermal Field (PHF), southern New Caledonia, an alkaline system driven by serpentinization with mineralogy composed of brucite and carbonates. While not directly comparable from a mineralogical point of view, the microstructure and porosity of both precipitates was similar, suggesting that our 3D printing technique may be a valuable tool for future astrobiology research on hydrothermal vent precipitates.

CO2 reduction driven by a pH gradient.

All life on Earth is built of organic molecules, so the primordial sources of reduced carbon remain a major open question in studies of the origin of life. A variant of the alkaline-hydrothermal-vent theory for life's emergence suggests that organics could have been produced by the reduction of CO2 via H2 oxidation, facilitated by geologically sustained pH gradients. The process would be an abiotic analog-and proposed evolutionary predecessor-of the Wood-Ljungdahl acetyl-CoA pathway of modern archaea and bacteria. The first energetic bottleneck of the pathway involves the endergonic reduction of CO2 with H2 to formate (HCOO-), which has proven elusive in mild abiotic settings. Here we show the reduction of CO2 with H2 at room temperature under moderate pressures (1.5 bar), driven by microfluidic pH gradients across inorganic Fe(Ni)S precipitates. Isotopic labeling with 13C confirmed formate production. Separately, deuterium (2H) labeling indicated that electron transfer to CO2 does not occur via direct hydrogenation with H2 but instead, freshly deposited Fe(Ni)S precipitates appear to facilitate electron transfer in an electrochemical-cell mechanism with two distinct half-reactions. Decreasing the pH gradient significantly, removing H2, or eliminating the precipitate yielded no detectable product. Our work demonstrates the feasibility of spatially separated yet electrically coupled geochemical reactions as drivers of otherwise endergonic processes. Beyond corroborating the ability of early-Earth alkaline hydrothermal systems to couple carbon reduction to hydrogen oxidation through biologically relevant mechanisms, these results may also be of significance for industrial and environmental applications, where other redox reactions could be facilitated using similarly mild approaches.

Effects of Amino Acids on Iron-Silicate Chemical Garden Precipitation.

Understanding the structure and behavior of chemical gardens is of interest for materials science, for understanding organic-mineral interactions, and for simulating geological mineral structures in hydrothermal systems on Earth and other worlds. Herein, we explored the effects of amino acids on inorganic chemical garden precipitate systems of iron chloride and sodium silicate to determine if/how the addition of organics can affect self-assembling morphologies or crystal growth. Amino acids affect chemical garden growth and morphology at the macro-scale and at the nanoscale. In this reaction system, the concentration of amino acid had a greater impact than the amino acid side chain, and increasing concentrations of organics caused structures to have smoother exteriors as amino acids accumulated on the outside surface. These results provide an example of how organic compounds can become incorporated into and influence the growth of inorganic self-organizing precipitates in far-from-equilibrium systems. Additionally, sample handing methods were developed to successfully image these delicate structures.

Reactivity of Metabolic Intermediates and Cofactor Stability under Model Early Earth Conditions.

Understanding the emergence of metabolic pathways is key to unraveling the factors that promoted the origin of life. One popular view is that protein cofactors acted as catalysts prior to the evolution of the protein enzymes with which they are now associated. We investigated the stability of acetyl coenzyme A (Acetyl Co-A, the group transfer cofactor in citric acid synthesis in the TCA cycle) under early Earth conditions, as well as whether Acetyl Co-A or its small molecule analogs thioacetate or acetate can catalyze the transfer of an acetyl group onto oxaloacetate in the absence of the citrate synthase enzyme. Several different temperatures, pH ranges, and compositions of aqueous environments were tested to simulate the Earth's early ocean and its possible components; the effect of these variables on oxaloacetate and cofactor chemistry were assessed under ambient and anoxic conditions. The cofactors tested are chemically stable under early Earth conditions, but none of the three compounds (Acetyl Co-A, thioacetate, or acetate) promoted synthesis of citric acid from oxaloacetate under the conditions tested. Oxaloacetate reacted with itself and/or decomposed to form a sequence of other products under ambient conditions, and under anoxic conditions was more stable; under ambient conditions the specific chemical pathways observed depended on the environmental conditions such as pH and presence/absence of bicarbonate or salt ions in early Earth ocean simulants. This work demonstrates the stability of these metabolic intermediates under anoxic conditions. However, even though free cofactors may be stable in a geological environmental setting, an enzyme or other mechanism to promote reaction specificity would likely be necessary for at least this particular reaction to proceed.

Detecting Endogenous Microbial Metabolism and Differentiating Between Abiotic and Biotic Signals Observed by Bioelectrochemical Systems in Soils.

Unambiguous detection of chemical and physical signatures of microbial life on Mars or other solar system bodies requires differentiation between signals produced by biotic and abiotic processes; instruments aimed at generalized in situ extant life detection would therefore increase the science return of a life-detection mission. Here, we investigate Bioelectrochemical Systems (BES) as a technique to measure microbial metabolism (which produces electrical current and redox changes) and distinguish between potential abiotic and biotic responses in environmental samples. Samples from inhabited niches should contain everything necessary to produce current, that is, catalysts (microorganisms) and fuel (nutrients). BES can also probe for inactive organisms in less energetically rich areas by adding a fuel to drive metabolism. A commercial potting soil and a Mars simulant soil were inoculated in the anodic chamber of microbial fuel cells, and current was monitored over time. Addition of a fuel (electron donor) source was tested for metabolic stimulation of endogenous microbes. Redox reactions between Mars simulant soil and the introduced electron donor (lactate) produced false-positive results, emphasizing the importance of careful interpretation of signals obtained. The addition of lactate to both soils resulted in enhanced biologically produced current, allowing stimulation and detection of dormant microbes. Our results demonstrate that BES provide an approach to detect metabolism in samples without prior knowledge of the organisms present, and that thorough electrochemical analyses and experimental design are necessary to determine if signals are biotic.