Search

The detection and identification of the building blocks of life, from amino acids to more complex molecules such as certain lipids, is a crucial but highly challenging task for current and future space exploration missions in our Solar System. To date, Gas Chromatography Mass Spectrometry has been the main technology applied. Although it has shown excellent performance in laboratory research, it has not yet been able to provide a conclusive answer regarding the presence or absence of a signature of life, extinct or extant, in space exploration. In this contribution we present the current measurement capabilities of our space prototype laser-based mass spectrometer for organics detection. The developed mass spectrometer currently allows the detection and identification of small organic molecules, such as amino acids and nucleobases, at sample concentrations at the level of femtomole mm-2, using the same measurement protocol. The latter is highly relevant to space exploration, since with the instrumentation in use so far only one class of organics can be measured with one instrument configuration., (Copyright 2025 Andreas Riedo, Nikita J. Boeren, Peter Keresztes Schmidt, Marek Tulej, Peter Wurz. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

This study investigates the effects of three Martian-relevant salts-sodium chlorate, sodium perchlorate, and sodium chloride-on the viability and motility of Escherichia coli , a model organism for understanding microbial responses to environmental stress. These salts are abundant on Mars and play a crucial role in forming brines, one of the few sources of stable liquid water on the planet. We analyze the survivability under different salt concentrations using colony plating. Additionally, we perform a semi-automated motility analysis, analyzing microbial speeds and motility patterns. Our results show that sodium perchlorate is the most toxic, followed by sodium chlorate, with sodium chloride being the least harmful. Both survivability and motility are affected by salt concentration and exposure time. Notably, we observe a short-lived increase in motility at certain concentrations, particularly under sodium chlorate and sodium perchlorate stress, despite rapid declines in cell viability, suggesting a stress response mechanism. Given that motility might enhance an organism's ability to navigate harsh and variable environments, it holds promise as a key biosignature in the search for life on Mars.

The study of whether life exists, is extinct, or not depends on various sophisticated experimental studies, as many different signatures of life can be used. The experimental procedures that can be performed to identify life can be further restricted by time, resources, and mobility constraints. Therefore, any research analyzing the presence of extraterrestrial life must be precise and unambiguous. This research focuses on the objective of the extraterrestrial life detection domain and seeks to provide an efficient protocol that can produce life detection decisions based on empirical data obtained through chemical analysis under time and resource-constrained conditions. While the majority of existing frameworks in this field are designed to identify biomolecules, our goal is to accomplish the same with minimal operational expense and mission complexity. We argue that the thoughtful integration of multiple biomolecular detections with lesser complexity and a robust framework can improve overall mission performance by satisfying the necessary time and resource constraints. In this study, a rapid multiple biomolecules-based life detection protocol (MBLDP-R) from soil samples is developed from scratch and embedded in an operational scientific rover subsystem targeted for planetary analysis missions. The study uses artificial biomolecule samples and simulated extraterrestrial environments to illustrate the suggested protocol's end-to-end process. First, we list a few significant biomolecules, including lipids, proteins, carbohydrates, nucleic acids, ammonia, and pigments. Then, a weighted qualitative test scoring is applied to sort out the best test method for the finally selected biomolecules which are used as operational analogue to showcase the protocol's in-situ analysis and decision-making capabilities. Based on the suitable biomolecules, a scientific exploration subsystem is developed, and the implemented protocol is built to perform onboard sample analysis. Evaluation results show that: (1) the proposed MBLDP-R protocol could effectively predict the classes with an average f1-score of 98.65% (macro) and 90.00% (micro), (2) the area under the Receiver Operating Characteristics (AUC-ROC) curve shows the sample categories to be correctly predicted 92% of the time (97% for Extant, 88% for Extinct, and 92% in the case of NPL), and (3) the protocol is time-efficient with an average completion time of 17.60 min, demonstrating the protocol's rapid nature in detecting biosignatures in soil samples. The research outcome yields useful additional data for related future studies, particularly in the design of scientific frameworks for mission-specific requirements with limited resources while also serving as a reference point for constraint evaluation methods for similar systems., (© 2024. The Author(s).)

Low-molecular-mass organic chemicals are widely discussed as potential indicators of life in extraterrestrial habitats. However, demarcation lines between biotic chemicals and abiotic chemicals have been difficult to define. Here, we have analyzed the potential utility of the quantum chemical property, HOMO-LUMO gap (HLG), as a novel proxy variable of life, since a significant trend towards incrementally smaller HLGs has been described in the genetically encoded amino acids. The HLG is a zeroth-order predictor of chemical reactivity. Comparing a set of 134 abiotic organic molecules recovered from meteorites, with 570 microbial and plant secondary metabolites thought to be exclusively biotic, we found that the average HLG of biotic molecules was significantly narrower (-10.4 ± 0.9 eV versus -12.4 ± 1.6 eV), with an effect size of g = 1.87. Limitation to hydrophilic molecules (XlogP < 2) improved the separation of biotic from abiotic compounds (g = 2.52). The "hydrophilic reactivity" quadrant defined by |HLG| < 11.25 eV and XlogP < 2 was populated exclusively by 183 biotic compounds and 6 abiotic compounds, 5 of which were nucleobases. We conclude that hydrophilic molecules with small HLGs represent valuable indicators of biotic activity, and we discuss the evolutionary plausibility of this inference.

Recent advances in subsurface microbiology have demonstrated the habitability of multi-million-year-old igneous rocks, despite the scarce energy supply from rock-water interactions. Given the minimal evolution coupled with exceedingly slow metabolic rates in subsurface ecosystems, spatiotemporally stable igneous rocks can sustain microbes over geological time scales. This study investigated a 2-billion-year-old mafic rock in the Bushveld Igneous Complex, South Africa, where ultradeep drilling is being executed by the International Continental Scientific Drilling Program (ICDP). New procedures were successfully developed to simultaneously detect indigenous and contaminant microbial cells in a drill core sample. Precision rock sectioning coupled with infrared, fluorescence, and electron microscopy imaging of the rock section with submicron resolution revealed microbial colonization in veins filled with clay minerals. The entry and exit of microbial cells in the veins are severely limited by tight packing with clay minerals, the formation of which supplies energy sources for long-term habitability. Further microbiological characterization of drilled rock cores from the Bushveld Igneous Complex will expand the understanding of microbial evolution in deep igneous rocks over 2 billion years., (© 2024. The Author(s).)

Detecting life has driven research and exploration for centuries, but recent attempts to compile and generate a framework that summarizes life features, aimed to develop strategies for life detection missions beyond planet Earth, have disregarded a key life feature: behavior. Yet, some behaviors such as biomineralization or motility have occasionally been proposed as biosignatures to detect life. Here, we capitalize on a specific taxis' motility behavior, magnetotaxis, to experimentally provide insights in support of behavior as an unambiguous, sensitive biosignature, and magnetic forces as a prescreening option. Using a magnetotactic bacterial species, Magnetospirillum magneticum, we conducted a lab sensitivity experiment comparing PCR with the hanging drop behavioral assay, using a dilution series. The hanging drop behavioral assay visually shows the motility of MTB toward magnetic poles. Our findings reveal that the behavioral assay exhibits higher sensitivity in the detection of M. magneticum when compared to the established PCR protocol. While both methods present similar detection sensitivities at high concentrations, at ≥ 10 -7 fold dilutions, the behavioral method proved more sensitive. The behavioral method can detect bacteria even when samples are diluted at 10 -9 . Comparable results were obtained with environmental samples from the Hula Valley. We propose behavioral cues as valuable biosignatures in the ongoing efforts of life detection in unexplored aquatic habitats on Earth and to stimulate and support discussions about how to detect extant life beyond Earth. Generic and robust behavioral assays can represent a methodological revolution., (© 2024. The Author(s).)

The cortical anthraquinone yellow-orange pigment parietin is a secondary lichen substance providing UV-shielding properties that is produced by several lichen species. In our work, the secondary metabolite has been extracted from air-dried thalli of Xanthoria parietina. The aims of this study were to characterize parietin absorbance through UV-VIS spectrophotometry and with IR spectroscopy and to evaluate its photodegradability under UV radiation through in situ reflectance IR spectroscopy to understand to what extent the substance may have a photoprotective role. This allows us to relate parietin photo-degradability to the lichen UV tolerance in its natural terrestrial habitat and in extreme environments relevant for astrobiology such as Mars. Extracted crystals were UV irradiated for 5.59 h under N 2 flux. After the UV irradiation, we assessed relevant degradations in the 1614, 1227, 1202, 1160 and 755 cm -1 bands. However, in light of Xanthoria parietina survivability in extreme conditions such as space- and Mars-simulated ones, we highlight parietin UV photo-resistance and its relevance for astrobiology as photo-protective substance and possible bio-hint., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Committee on Space Research (COSPAR). Published by Elsevier B.V. All rights reserved.)

The possible existence of a microbial community in the venusian clouds is one of the most intriguing hypotheses in modern astrobiology. Such a community must be characterized by a high survivability potential under severe environmental conditions, the most extreme of which are very low pH levels and water activity. Considering different scenarios for the origin of life and geological history of our planet, a few of these scenarios are discussed in the context of the origin of hypothetical microbial life within the venusian cloud layer. The existence of liquid water on the surface of ancient Venus is one of the key outstanding questions influencing this possibility. We link the inherent attributes of microbial life as we know it that favor the persistence of life in such an environment and review the possible scenarios of life's origin and its evolution under a strong greenhouse effect and loss of water on Venus. We also propose a roadmap and describe a novel methodological approach for astrobiological research in the framework of future missions to Venus with the intent to reveal whether life exists today on the planet.

The search for life beyond Earth necessitates a rigorous and comprehensive examination of biosignatures, the types of observable imprints that life produces. These imprints and our ability to detect them with advanced instrumentation hold the key to our understanding of the presence and abundance of life in the universe. Biosignatures are the chemical or physical features associated with past or present life and may include the distribution of elements and molecules, alone or in combination, as well as changes in structural components or physical processes that would be distinct from an abiotic background. The scientific and technical strategies used to search for life on other planets include those that can be conducted in situ to planetary bodies and those that could be observed remotely. This chapter discusses numerous strategies that can be employed to look for biosignatures directly on other planetary bodies using robotic exploration including those that have been deployed to other planetary bodies, are currently being developed for flight, or will become a critical technology on future missions. Search strategies for remote observations using current and planned ground-based and space-based telescopes are also described. Evidence from spectral absorption, emission, or transmission features can be used to search for remote biosignatures and technosignatures. Improving our understanding of biosignatures, their production, transformation, and preservation on Earth can enhance our search efforts to detect life on other planets.

Organisms act stochastically. A not uncommon view in the ecological literature is that this is mainly due to the observer having insufficient information or a stochastic environment-and not partly because organisms themselves respond with inherent unpredictability. In this study, I compile the evidence that contradicts that view. Organisms generate uncertainty internally, which results in irreducible stochastic responses. I consider why: for instance, stochastic responses are associated with greater adaptability to changing environments and resource availability. Over longer timescales, biologically generated uncertainty influences behavior, evolution, and macroecological processes. Indeed, it could be stated that organisms are systems defined by the internal generation, magnification, and record-keeping of uncertainty as inputs to responses. Important practical implications arise if organisms can indeed be defined by an association with specific classes of inherent uncertainty: not least that isolating those signatures then provides a potential means for detecting life, for considering the forms that life could theoretically take, and for exploring the wider limits to how life might become distributed. These are all fundamental goals in astrobiology.

We report a simple, inexpensive design of a fluorescence microscope with light-emitting diode (LED) excitation for detection of labeled and unlabeled microorganisms in mineral substrates. The use of deep UV (DUV) excitation with visible emission requires no specialized optics or slides and can be implemented easily and inexpensively using an oblique illumination geometry. DUV excitation (<280 nm) is preferable to near UV (365 nm) for avoidance of mineral autofluorescence. When excited with DUV, unpigmented bacteria show two emission peaks: one in the near UV ∼320 nm, corresponding to proteins, and another peak in the blue to green range, corresponding to flavins and/or reduced nicotinamide adenine dinucleotide (NADH). Many commonly used dyes also show secondary excitation peaks in the DUV, with identical emission spectra and quantum yields as their primary peak. However, DUV fails to excite key biosignature molecules, especially chlorophyll in cyanobacteria. Visible excitation (violet to blue) also results in less mineral autofluorescence than near UV, and most autofluorescence in the minerals seen here is green, so that red dyes and red autofluorescence of chlorophyll and porphyrins are readily distinguished. The pairing of DUV and near UV or visible excitation, with emission across the visible, represents the most thorough approach to detection of labeled and unlabeled bacteria in soil and rock.

Astrobiology aims to determine the distribution and diversity of life in the universe. But as the word "biosignature" suggests, what will be detected is not life itself, but an observation implicating living systems. Our limited access to other worlds suggests this observation is more likely to reflect out-of-equilibrium gasses than a writhing octopus. Yet, anything short of a writhing octopus will raise skepticism about what has been detected. Resolving that skepticism requires a theory to delineate processes due to life and those due to abiotic mechanisms. This poses an existential question for life detection: How do astrobiologists plan to detect life on exoplanets via features shared between non-living and living systems? We argue that you cannot without an underlying theory of life. We illustrate this by analyzing the hypothetical detection of an "Earth 2.0" exoplanet. Without a theory of life, we argue the community should focus on identifying unambiguous features of life via four areas: examining life on Earth, building life in the lab, probing the solar system, and searching for technosignatures. Ultimately, we ask, what exactly do astrobiologists hope to learn by searching for life?, (© 2023 The Authors. BioEssays published by Wiley Periodicals LLC.)

In 2019, the Atacama Rover Astrobiology Drilling Studies (ARADS) project field-tested an autonomous rover-mounted robotic drill prototype for a 6-Sol life detection mission to Mars (Icebreaker). ARADS drilled Mars-like materials in the Atacama Desert (Chile), one of the most life-diminished regions on Earth, where mitigating contamination transfer into life-detection instruments becomes critical. Our Contamination Control Strategy and Implementation (CCSI) for the Sample Handling and Transfer System (SHTS) hardware (drill, scoop and funnels) included out-of-simulation protocol testing (out-of-sim) for hardware decontamination and verification during the 6-Sol simulation (in-sim). The most effective five-step decontamination combined safer-to-use sterilants (3%&#95;hydrogen-peroxide-activated 5%&#95;sodium-hypochlorite), and in situ real-time verification by adenosine triphosphate (ATP) and Signs of Life Detector (SOLID) Fluorescence Immunoassay for characterization hardware bioburden and airborne contaminants. The 20- to 40-min protocol enabled a 4-log bioburden reduction down to <0.1 fmoles ATP detection limit (funnels and drill) to 0.2-0.7 fmoles (scoop) of total ATP. The (post-cleaning) hardware background was 0.3 to 1-2 attomoles ATP/cm 2 (cleanliness benchmark background values) equivalent to ca. 1-10 colony forming unit (CFU)/cm 2 . Further, 60-100% of the in-sim hardware background was ≤3-4 bacterial cells/cm 2 , the threshold limit for Class <7 aseptic operations. Across the six Sols, the flux of airborne contaminants to the drill sites was ∼5 and ∼22 amoles ATP/(cm 2 ·day), accounting for an unexpectedly high Fluorescence Intensity (FI) signal (FI: ∼6000) against aquatic cyanobacteria, but negligible anthropogenic contribution. The SOLID immunoassay also detected microorganisms from multiple habitats across the Atacama Desert (anoxic, alkaline/acidic microenvironments in halite fields, playas, and alluvial fans) in both airborne and post-cleaning hardware background. Finally, the hardware ATP background was 40-250 times lower than the ATP in cores. Similarly, the FI peaks (FI max ) against the microbial taxa and molecular biomarkers detected in the post-cleaned hardware (FI: ∼1500-1600) were 5-10 times lower than biomarkers in drilled sediments, excluding significant interference with putative biomarker found in cores. Similar protocols enable the acquisition of contamination-free materials for ultra-sensitive instruments analysis and the integrity of scientific results. Their application can augment our scientific knowledge of the distribution of cryptic life on Mars-like grounds and support life-detection robotic and human-operated missions to Mars.

The concept of a biosignature is widely used in astrobiology to suggest a link between some observation and a biological cause, given some context. The term itself has been defined and used in several ways in different parts of the scientific community involved in the search for past or present life on Earth and beyond. With the ongoing acceleration in the search for life in distant time and/or deep space, there is a need for clarity and accuracy in the formulation and reporting of claims. Here, we critically review the biosignature concept(s) and the associated nomenclature in light of several problems and ambiguities emphasized by recent works. One worry is that these terms and concepts may imply greater certainty than is usually justified by a rational interpretation of the data. A related worry is that terms such as "biosignature" may be inherently misleading, for example, because the divide between life and non-life-and their observable effects-is fuzzy. Another worry is that different parts of the multidisciplinary community may use non-equivalent or conflicting definitions and conceptions, leading to avoidable confusion. This review leads us to identify a number of pitfalls and to suggest how they can be circumvented. In general, we conclude that astrobiologists should exercise particular caution in deciding whether and how to use the concept of biosignature when thinking and communicating about habitability or life. Concepts and terms should be selected carefully and defined explicitly where appropriate. This would improve clarity and accuracy in the formulation of claims and subsequent technical and public communication about some of the most profound and important questions in science and society. With this objective in mind, we provide a checklist of questions that scientists and other interested parties should ask when assessing any reported detection of a "biosignature" to better understand exactly what is being claimed.

The origin of life and the detection of alien life have historically been treated as separate scientific research problems. However, they are not strictly independent. Here, we discuss the need for a better integration of the sciences of life detection and origins of life. Framing these dual problems within the formalism of Bayesian hypothesis testing, we demonstrate via simple examples how high confidence in life detection claims require either (1) a strong prior hypothesis about the existence of life in a particular alien environment, or conversely, (2) signatures of life that are not susceptible to false positives. As a case study, we discuss the role of priors and hypothesis testing in recent results reporting potential detection of life in the venusian atmosphere and in the icy plumes of Enceladus. While many current leading biosignature candidates are subject to false positives because they are not definitive of life, our analyses demonstrate why it is necessary to shift focus to candidate signatures that are definitive. This indicates a necessity to develop methods that lack substantial false positives, by using observables for life that rely on prior hypotheses with strong theoretical and empirical support in identifying defining features of life. Abstract theories developed in pursuit of understanding universal features of life are more likely to be definitive and to apply to life-as-we-don't-know-it. We discuss Molecular Assembly theory as an example of such an observable which is applicable to life detection within the solar system. In the absence of alien examples these are best validated in origin of life experiments, substantiating the need for better integration between origins of life and biosignature science research communities. This leads to a conclusion that extraordinary claims in astrobiology ( e.g., definitive detection of alien life) require extraordinary explanations, whereas the evidence itself could be quite ordinary.

We present a comparative study of the methods used in the search for extraterrestrial microorganism life, including a summary table where different life-detection techniques can be easily compared as an aid to mission and instrument design aimed at life detection. This is an extension of previous study, where detection techniques for a series of target characteristics and molecules that could constitute a positive life detection were evaluated. This comparison has been extended with a particular consideration to sources of false positives, the causes of negative detection, the results of detection techniques when presented regarding terrestrial life, and additional science objectives that could be achieved outside the primary aim of detecting life. These additions address both the scientific and programmatic side of exploration mission design, where a successful proposal must demonstrate probable outcomes and be able to return valuable results even if no life is found. The applicability of the life detection techniques is considered for Earth life, Earth-independent life (life emerging independently from that on Earth,) and Earth-kin life (sharing a common ancestor with life on Earth), and techniques effective in detecting Earth life should also be useful in the detection of Earth-kin life. However, their applicability is not guaranteed for Earth-independent life. As found in our previous study, there exists no realistic single detection method that can conclusively determine the discovery of extraterrestrial life, and no method is superior to all others. In this study, we further consider combinations of detection techniques and identify imaging as a valuable addition to molecule detection methods, even in cases where there is insufficient resolution to observe the detailed morphology of a microbial cell. The search for extraterrestrial life is further divided into a survey-and-detection and analysis-and-conclusion step. These steps benefit from different detection techniques, but imaging is necessary for both parts.

Flow cytometry is a potential technology for in situ life detection on icy moons (such as Enceladus and Europa) and on the polar ice caps of Mars. We developed a method for using flow cytometry to positively identify four classes of biomarkers using exogenous fluorescent stains: nucleic acids, proteins, carbohydrates, and lipids. We demonstrated the effectiveness of exogenous stains with six known organisms and known abiotic material and showed that the cytometer is easily able to distinguish between the known organisms and the known abiotic material using the exogenous stains. To simulate a life-detection experiment on an icy world lander, we used six natural samples with unknown biotic and abiotic content. We showed that flow cytometry can identify all four biomarkers using the exogenous stains and can separate the biotic material from the known abiotic material on scatter plots. Exogenous staining techniques would likely be used in conjunction with intrinsic fluorescence, clustering, and sorting for a more complete and capable life-detection instrument on an icy moon lander.

An ideal life detection instrument would have high sensitivity but be insensitive to abiotic processes and would be capable of detecting life with alternate molecular structures. In this study, we propose that catalytic activity can be the basis of a nearly ideal life detection instrument. There are several advantages to catalysis as an agnostic life detection method. Demonstrating catalysis does not necessarily require culturing/growing the alien life and in fact may persist even in dead biomass for some time, and the amplification by catalysis is large even by minute amounts of catalysts and, hence, can be readily detected against abiotic background rates. In specific, we propose a hydrolytic catalysis detection instrument that could detect activity in samples of extraterrestrial organic material from unknown life. The instrument uses chromogenic assay-based detection of various hydrolytic catalytic activities, which are matched to corresponding artificial substrates having the same, chromogenic (preferably fluorescent) upon release, group; D- and L-enantiomers of these substrates can be used to also answer the question whether unknown life is chiral. Since catalysis is a time-proportional product-concentration amplification process, hydrolytic catalytic activity can be measured on a sample of even a minute size, and with instruments based on, for example, optofluidic chip technology.

Growing evidence of the potential habitability of Ocean Worlds across our solar system is motivating the advancement of technologies capable of detecting life as we know it-sharing a common ancestry or physicochemical origin with life on Earth-or don't know it, representing a distinct emergence of life different than our one known example. Here, we propose the Electronic Life-detection Instrument for Enceladus/Europa (ELIE), a solid-state single-molecule instrument payload that aims to search for life based on the detection of amino acids and informational polymers (IPs) at the parts per billion to trillion level. As a first proof-of-principle in a laboratory environment, we demonstrate the single-molecule detection of the amino acid L-proline at a 10 μM concentration in a compact system. Based on ELIE's solid-state quantum electronic tunneling sensing mechanism, we further propose the quantum property of the HOMO-LUMO gap (energy difference between a molecule's highest energy-occupied molecular orbital and lowest energy-unoccupied molecular orbital) as a novel metric to assess amino acid complexity. Finally, we assess the potential of ELIE to discriminate between abiotically and biotically derived α-amino acid abundance distributions to reduce the false positive risk for life detection. Nanogap technology can also be applied to the detection of nucleobases and short sequences of IPs such as, but not limited to, RNA and DNA. Future missions may utilize ELIE to target preserved biosignatures on the surface of Mars, extant life in its deep subsurface, or life or its biosignatures in a plume, surface, or subsurface of ice moons such as Enceladus or Europa. One-Sentence Summary: A solid-state nanogap can determine the abundance distribution of amino acids, detect nucleic acids, and shows potential for detecting life as we know it and life as we don't know it.

In recent years, strong evidence has emerged indicating the potential habitability of the subsurface of Mars. Occasional discharge events that bring subsurface fluids to the surface may carry with them the biological traces of subsurface organisms. Similar events are known to take place on Earth and are frequently associated with long-term mineralogical preservation of organic material, including DNA. Taking advantage of this process may allow for the development of life-detection strategies targeting biosignatures from the more habitable subsurface environment without the need for direct subsurface exploration. To test the potential for this approach to life-detection, we adapted a protocol to extract microbial DNA preserved in carbonate rocks and tested its efficacy in detecting subsurface organisms at a Mars analog site in southeastern Utah, USA, using samples from ancient and modern carbonate deposits associated with natural and artificial springs. Our results indicated that DNA from deep-subsurface organisms preserved in carbonate deposits can remain recoverable for up to 100,000 years, supporting life-detection strategies based on the detection of deep-subsurface biosignatures in surface-exposed rocks on Mars.

The French-Italian Concordia Research Station, situated on the Antarctic Polar Plateau at an elevation of 3233 m above sea level, offers a unique opportunity to study the presence and variation of microbes introduced by abiotic or biotic vectors and, consequently, appraise the amplitude of human impact in such a pristine environment. This research built upon a previous work, which explored microbial diversity in the surface snow surrounding the Concordia Research Station. While that study successfully characterized the bacterial assemblage, detecting fungal diversity was hampered by the low DNA content. To address this knowledge gap, in the present study, we optimized the sampling by increasing ice/snow collected to leverage the final DNA yield. The V4 variable region of the 16S rDNA and Internal Transcribed Spacer (ITS1) rDNA was used to evaluate bacterial and fungal diversity. From the sequencing, we obtained 3,352,661 and 4,433,595 reads clustered in 930 and 3182 amplicon sequence variants (ASVs) for fungi and bacteria, respectively. Amplicon sequencing revealed a predominance of Basidiomycota (49%) and Ascomycota (42%) in the fungal component; Bacteroidota (65.8%) is the main representative among the bacterial phyla. Basidiomycetes are almost exclusively represented by yeast-like fungi. Our findings provide the first comprehensive overview of both fungal and bacterial diversity in the Antarctic Polar Plateau's surface snow/ice near Concordia Station and to identify seasonality as the main driver of microbial diversity; we also detected the most sensitive microorganisms to these factors, which could serve as indicators of human impact in this pristine environment and aid in planetary protection for future exploration missions.

Good feature engineering is a prerequisite for accurate classification, especially in challenging scenarios such as detecting the breathing of living persons trapped under building rubble using bioradar. Unlike monitoring patients' breathing through the air, the measuring conditions of a rescue bioradar are very complex. The ultimate goal of search and rescue is to determine the presence of a living person, which requires extracting representative features that can distinguish measurements with the presence of a person and without. To address this challenge, we conducted a bioradar test scenario under laboratory conditions and decomposed the radar signal into different range intervals to derive multiple virtual scenes from the real one. We then extracted physical and statistical quantitative features that represent a measurement, aiming to find those features that are robust to the complexity of rescue-radar measuring conditions, including different rubble sites, breathing rates, signal strengths, and short-duration disturbances. To this end, we utilized two methods, Analysis of Variance (ANOVA), and Minimum Redundancy Maximum Relevance (MRMR), to analyze the significance of the extracted features. We then trained the classification model using a linear kernel support vector machine (SVM). As the main result of this work, we identified an optimal feature set of four features based on the feature ranking and the improvement in the classification accuracy of the SVM model. These four features are related to four different physical quantities and independent from different rubble sites.

The search for extant microbial life will be a major focus of future astrobiology missions; however, no direct extant life detection instrumentation is included in current missions to Mars. In this study, we developed the semiautomated MicroLife detection platform that collects and processes environmental samples, detects biosignatures, and characterizes microbial activity. This platform is composed of a drill for sample collection, a redox dye colorimetric system for microbial metabolic activity detection and assessment (μMAMA [microfluidics Microbial Activity MicroAssay]), and a MinION sequencer for biosignature detection and characterization of microbial communities. The MicroLife platform was field-tested on White Glacier on Axel Heiberg Island in the Canadian high Arctic, with two extracted ice cores. The μMAMA successfully detected microbial metabolism from the ice cores within 1 day of incubation. The MinION sequencing of the ice cores and the positive μMAMA card identified a microbial community consistent with cold and oligotrophic environments. Furthermore, isolation and identification of microbial isolates from the μMAMA card corroborated the MinION sequencing. Together, these analyses support the MicroLife platform's efficacy in identifying microbes natively present in cryoenvironments and detecting their metabolic activity. Given our MicroLife platform's size and low energy requirements, it could be incorporated into a future landed platform or rovers for life detection.

Life detection technology using ultra-wideband (UWB) radar is a non-contact, active detection technology, which can be used to search for survivors in disaster rescues. The existing multi-target detection method based on UWB radar echo signals has low accuracy and has difficulty extracting breathing and heartbeat information at the same time. Therefore, this paper proposes a new multi-target localization and vital sign detection method using ultra-wide band radar. A target recognition and localization method based on permutation entropy (PE) and K means++ clustering is proposed to determine the number and position of targets in the environment. An adaptive denoising method for vital sign extraction based on ensemble empirical mode decomposition (EEMD) and wavelet analysis (WA) is proposed to reconstruct the breathing and heartbeat signals of human targets. A heartbeat frequency extraction method based on particle swarm optimization (PSO) and stochastic resonance (SR) is proposed to detect the heartbeat frequency of human targets. Experimental results show that the PE-K means++ method can successfully recognize and locate multiple human targets in the environment, and its average relative error is 1.83%. Using the EEMD-WA method can effectively filter the clutter signal, and the average relative error of the reconstructed respiratory signal frequency is 4.27%. The average relative error of heartbeat frequency detected by the PSO-SR method was 6.23%. The multi-target localization and vital sign detection method proposed in this paper can effectively recognize all human targets in the multi-target scene and provide their accurate location and vital signs information. This provides a theoretical basis for the technical system of emergency rescue and technical support for post-disaster rescue.

The plume of Enceladus is thought to originate from the dispersion of a liquid source beneath the icy crust. Cryovolcanic activity on Enceladus may present a direct way of accessing material originating from the potentially habitable subsurface ocean. One way to test the hypothesis of whether life is present within the ocean of Enceladus would be to investigate the plume material for the presence of microbial life. In this study, we investigated the entrainment of Bacillus subtilis within Enceladus-like fluids under boiling conditions caused by exposure of the fluids to low pressure. We show that boiling, associated with exposure of a fluid to low pressure, works as a mechanism for dispersing bacteria in Enceladus plume-like environments. Exposure of Enceladus-type fluids (0.01-0.1 molal Na 2 CO 3 and 0.05-0.2 molal NaCl) to low pressure (5 mbar) results in the dispersion of bacteria in droplets that evaporate to produce particles of salt. We find that, for particles with radius ( r ) ≤ 10 μm, the number of dispersed particles containing cells was between 7.7% and 10.9%. However, for larger particles 10 <  r  ≤ 50 μm, 64.4% and 56.4% contained cells for lower and upper end-member solutions, respectively. Our results suggest that the gravity-induced size sorting of plume particles will result in plume deposits closer to the vent source containing a larger volume of biological material than within the plume. If life is present in the ocean of Enceladus, we would expect that it would be effectively entrained and deposited on the surface; therefore, it would be accessible with a surface-lander-based instrument.

The Concordia Research Station provides a unique location for preparatory activities for future human journey to Mars, to explore microbial diversity at subzero temperatures, and monitor the dissemination of human-associated microorganisms within the pristine surrounding environment. Amplicon sequencing was leveraged to investigate the microbial diversity of surface snow samples collected monthly over a two-year period, at three distances from the Station (10, 500, and 1000 m). Even when the extracted total DNA was below the detection limit, 16S rRNA gene sequencing was successfully performed on all samples, while 18S rRNA was amplified on 19 samples out of 51. No significant relationships were observed between microbial diversity and seasonality (summer or winter) or distance from the Concordia base. This suggested that if present, the anthropogenic impact should have been below the detectable limit. While harboring low microbial diversity, the surface snow samples were characterized by heterogeneous microbiomes. Ultimately, our study corroborated the use of DNA sequencing-based techniques for revealing microbial presence in remote and hostile environments, with implications for Planetary Protection during space missions and for life-detection in astrobiology relevant targets.

Raman spectroscopy, an emerging technology for in situ space exploration, has been suggested for life detection for the Europa Lander Mission. However, obtaining spectra of samples from the europan icy shell requires measurements at temperatures down to -233°C, which will affect the Raman spectra of any potential biosignatures. In this study, we obtained Raman spectra of amino acids using a 785 nm Raman system at temperatures ranging down to -196°C, analogous to Europa's surface and near subsurface. Significant Raman band width narrowing and decreasing variance were observed at lower temperatures leading to higher-precision Raman measurements, which required higher spectral resolution that could be as high as 2 cm -1 for full identification of amino acids. Such spectral resolution is much higher than the resolution of contemporary Raman instruments for planetary exploration and may be particularly problematic for miniaturized instruments. Shifting of Raman bands to both higher and lower frequencies by as much as ∼25 cm -1 together with changes in the Raman band intensity were recorded. The emergence of new bands and diminishing of the original bands also occurred for some amino acids. A significantly increased fluorescence background was observed in spectra of fluorescent molecules ( i.e., tryptophan). A link between the type of vibrational modes associated with Raman bands and the change in their Raman shift at extreme low temperatures was identified and described. This link offers an exciting new method of molecule identification solely based on the comparison of spectra collected at two different temperatures and could greatly improve the identification capabilities in Raman spectroscopy for a wide array of applications.

Before the first humans depart for Mars in the next decade, hundreds of tons of martian water-ice must be harvested to produce propellant for the return vehicle, a process known as in situ resource utilization (ISRU). We describe here an instrument, the Agnostic Life Finder (ALF), that is an inexpensive life-detection add-on to ISRU. ALF exploits a well-supported view that informational genetic biopolymers in life in water must have two structural features: (1) Informational biopolymers must carry a repeating charge; they must be polyelectrolytes. (2) Their building blocks must fit into an aperiodic crystal structure; the building blocks must be size-shape regular. ALF exploits the first structural feature to extract polyelectrolytes from ∼10 cubic meters of mined martian water by applying a voltage gradient perpendicularly to the water's flow. This gradient diverts polyelectrolytes from the flow toward their respective electrodes (polyanions to the anode, polycations to the cathode), where they are captured in cartridges before they encounter the electrodes. There, they can later be released to analyze their building blocks, for example, by mass spectrometry or nanopore. Upstream, martian cells holding martian informational polyelectrolytes are disrupted by ultrasound. To manage the (unknown) conductivity of the water due to the presence of salts, the mined water is preconditioned by electrodialysis using porous membranes. ALF uses only resources and technology that must already be available for ISRU. Thus, life detection is easily and inexpensively integrated into SpaceX or NASA ISRU missions.

Homochirality is a generic and unique property of all biochemical life and it is considered a universal and agnostic biosignature. Upon interaction with unpolarized light, homochirality induces fractional circular polarization in the light that is scattered from it, which can be sensed remotely. As such, it can be a prime candidate biosignature in the context of future life detection missions and observatories. The linear polarizance of vegetation is also sometimes envisaged as a biosignature, although it does not share the same molecular origin as circular polarization. It is known that linear polarization of surfaces is strongly dependent on the phase angle. The relationship between the phase angle and circular polarization stemming from macromolecular assemblies such as in vegetation, however, remained unclear. In this study, using the average of 27 different species, we demonstrate that the circular polarization-phase angle dependency of vegetation induces relatively small changes in spectral shape and mostly affects the signal magnitude. With these results, we underline the use of circular spectropolarimetry as a promising agnostic biosignature complementary to the use of linear spectropolarimetry and scalar reflectance.

Life detection on Mars is an important topic that includes a direct search for biomarkers. This requires instruments for in situ biomarker detection that are compact, lightweight, and able to withstand operations in space. Solid-state nanopores are excellent candidates that allow fast single-molecule detection. They can withstand high temperatures and be sterilized to minimize planetary contamination. The instruments are portable with low-power requirements. We demonstrate a few key results in advancing the use of nanopores for in-space applications. First, we developed modified deoxyribonucleic acid (DNA) extraction protocols to extract DNA from Mars analog soils. Second, we used silicon nitride nanopores to demonstrate the detection of extracted DNA and corresponding current characteristics. The yields and properties of extracted DNA ( e.g. , estimated diameters) varied somewhat by soil types, extraction methods, and nanopores used. The yields varied from a minimum of 0.9 ng DNA/g soil for a magnesium carbonate sample from Lake Salda to a maximum of 210 ng DNA/g soil for a calcium carbonate sample from Trona Pinnacles. For a given soil type, yields from different methods varied by a factor of up to 50. These observations motivate future studies with a broader range of Mars-like soils and improved instruments to increase signal-to-noise-ratio at higher measurement bandwidths.

Capillary electrophoresis (CE) is a promising liquid-based technique for in situ chemical analysis on ocean worlds that allows the detection of a wide range of organic molecules relevant to the search for life. CE coupled with mass spectrometry (MS) is particularly valuable as it also enables the discovery of unknown compounds. Here we demonstrate that CE coupled to MS via electrospray ionization (ESI) can readily analyze samples containing up to half the saturation levels of salts relevant to ocean worlds when using 5 M acetic acid as the separation media. A mixture containing amino acids, peptides, nucleobases, and nucleosides was analyzed in the presence of two salts, NaCl and MgSO 4 , based on their relevance to Europa and Enceladus. We demonstrate here CE-MS limits of detection for these organics ranging from 0.05 to 1 μM (8 to 89 ppb) in the absence of salts. More importantly, we demonstrate here for the first time that organics in the low micromolar range (1-50 μM) are detected by CE-MS in the presence of 3 M NaCl without desalting, preconcentration, or derivatization. This demonstration highlights how CE-MS is uniquely suited for organic analysis on future missions to ocean worlds.

Cassini revealed that Saturn's Moon Enceladus hosts a subsurface ocean that meets the accepted criteria for habitability with bio-essential elements and compounds, liquid water, and energy sources available in the environment. Whether these conditions are sufficiently abundant and collocated to support life remains unknown and cannot be determined from Cassini data. However, thanks to the plume of oceanic material emanating from Enceladus' south pole, a new mission to Enceladus could search for evidence of life without having to descend through kilometers of ice. In this article, we outline the science motivations for such a successor to Cassini , choosing the primary science goal to be determining whether Enceladus is inhabited and assuming a resource level equivalent to NASA's Flagship-class missions. We selected a set of potential biosignature measurements that are complementary and orthogonal to build a robust case for any life detection result. This result would be further informed by quantifications of the habitability of the environment through geochemical and geophysical investigations into the ocean and ice shell crust. This study demonstrates that Enceladus' plume offers an unparalleled opportunity for in situ exploration of an Ocean World and that the planetary science and astrobiology community is well equipped to take full advantage of it in the coming decades.

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.

With no direct extant-life detection instrumentation included in a space mission since the 1970s, the advancement of new technologies to be included in future space missions is imperative. We developed, optimized, and tested a semi-automated prototype, the microfluidics Microbial Activity MicroAssay (μMAMA). This system metabolically characterizes and detects extant microbial life by way of metabolism-indicator redox dyes. We first evaluated the robustness and sensitivity of six redox dye/buffer combinations, and we then tested their responses to metabolic activity in astrobiological analog high-Arctic samples. We determined that the Biolog Inoculating Fluid (IF)-C and AlamarBlue buffered in IF-0a (aB-IF0a) dye/buffer combinations were optimal, as they detected metabolic activity from the fewest microbial cells (10 2 cells/mL) while maintaining efficacy over a broad physiochemical range of pH (0-13), temperature (-10°C to 37°C), salinity and perchlorate (tested up to 30%), and in the presence of a Mars regolith simulant (MMS-2). The μMAMA, which incorporated these redox dyes, detected extant active cold-adapted microbial life from high Arctic analog sites, including samples amended with substrates targeting chemolithoautotrophic metabolisms. Given μMAMA's small size (we estimate a complete planetary instrument could occupy as little as 3 L) and potential for automation, it could easily be incorporated into almost any landed platform for life detection missions.

The utilization of nanopore technologies for the detection of organic biogenic compounds has garnered significant focus in recent years. Oxford Nanopore Technologies' (ONT) MinION instrument, which can detect and sequence nucleic acids (NAs), is one such example. These technologies have much promise for unambiguous life detection but require significant development in terms of methods for extraction and preparation of NAs for biosignature detection and their feasibility for use in astrobiology-focused field missions. In this study, we tested pre-existing, automated, or semiautomated NA extraction technologies, coupled with automated ONT VolTRAX NA sample preparation, and verification with Nanopore MinION sequencing. All of the extraction systems tested (SuperFastPrep2, ClaremontX1, and SOLID-Sample Preparation Unit) showed potential for extracting DNA from Canadian High Arctic environments analogous to Mars, Europa, and Enceladus, which could subsequently be detected and sequenced with the MinION. However, they differed with regard to efficacy, yield, purity, and sequencing and annotation quality. Overall, bead beating-based systems performed the best for these parameters. In addition, we showed that the MinION could sequence unpurified DNA contained in crude cell lysates. This is valuable from an astrobiology perspective because purification steps are time-consuming and complicate the requirements for an automated extraction and life detection system. Our results indicate that semiautomated NA extraction and preparation technologies hold much promise, and with increased optimization and automation could be coupled to a larger platform incorporating nanopore detection and sequencing of NAs for life detection applications.

Answering the question of whether life ever existed on Mars is a key goal of both NASA's and ESA's imminent Mars rover missions. The obfuscatory effects of oxidizing salts, such as perchlorates and sulfates, on organic matter during thermal decomposition analysis techniques are well established. Less well studied are the transformative effects of iron oxides and (oxy)hydroxides, which are present in great abundances in the martian regolith. We examined the products of flash pyrolysis-gas chromatography-mass spectrometry (a technique analogous to the thermal techniques employed by past, current, and future landed Mars missions) which form when the cyanobacteria Arthrospira platensis are heated in the presence of a variety of Mars-relevant iron-bearing minerals. We found that iron oxides/(oxy)hydroxides have transformative effects on the pyrolytic products of cyanobacterial biomolecules. Both the abundance and variety of molecular species detected were decreased as iron substrates transformed biomolecules, by both oxidative and reductive processes, into lower fidelity alkanes, aromatic and aryl-bonded hydrocarbons. Despite the loss of fidelity, a suite that contains mid-length alkanes and polyaromatic hydrocarbons and/or aryl-bonded molecules in iron-rich samples subjected to pyrolysis may allude to the transformation of cyanobacterially derived mid-long chain length fatty acids (particularly unsaturated fatty acids) originally present in the sample. Hematite was found to be the iron oxide with the lowest transformation potential, and because this iron oxide has a high affinity for codeposition of organic matter and preservation over geological timescales, sampling at Mars should target sediments/strata that have undergone a diagenetic history encouraging the dehydration, dihydroxylation, and oxidation of more reactive iron-bearing phases to hematite by looking for (mineralogical) evidence of the activity of oxidizing, acidic/neutral, and either hot or long-lived fluids.

Detecting evidence of life on other planetary bodies requires a certain understanding of known biomarkers and their chemical nature, preservation potential, or biological specificity. In a planetary search for life, carbonates are of special interest due to their known association with life as we know it. On Earth, carbonates serve as an invaluable paleogeochemical archive of fossils of up to billions of years old. Here, we investigated biomarker profiles on three Chilean Triassic-Jurassic sedimentary records regarding our search for signs of past and present life over ∼200 Ma. A multianalytical platform that combines lipid-derived biomarkers, metaproteomics, and a life detector chip (LDChip) is considered in the detection of biomolecules with different perdurability and source-diagnosis potential. The combined identification of proteins with positive LDChip inmunodetections provides metabolic information and taxonomic affiliation of modern/subrecent biosignatures. Molecular and isotopic analysis of more perdurable hydrocarbon cores allows for the identification of general biosources and dominant autotrophic pathways over time, as well as recreation of prevailing redox conditions over ∼200 Ma. We demonstrate how extraterrestrial life detection can benefit from the use of different biomarkers to overcome diagnosis limitations due to a lack of specificity and/or alteration over time. Our findings have implications for future astrobiological missions to Mars.

Digital holographic microscopy provides the ability to observe throughout a volume that is large compared to its resolution without the need to actively refocus to capture the entire volume. This enables simultaneous observations of large numbers of small objects within such a volume. We have constructed a microscope that can observe a volume of 0.4 µm × 0.4 µm × 1.0 µm with submicrometer resolution (in xy) and 2 µm resolution (in z) for observation of microorganisms and minerals in liquid environments on Earth and on potential planetary missions. Because environmental samples are likely to contain mixtures of inorganics and microorganisms of comparable sizes near the resolution limit of the instrument, discrimination between living and non-living objects may be difficult. The active motion of motile organisms can be used to readily distinguish them from non-motile objects (live or inorganic), but additional methods are required to distinguish non-motile organisms and inorganic objects that are of comparable size but different composition and structure. We demonstrate the use of passive motion to make this discrimination by evaluating diffusion and buoyancy characteristics of cells, styrene beads, alumina particles, and gas-filled vesicles of micron scale in the field of view.

The search for, and characterization of, organic matter on Mars is central to efforts in identifying habitable environments and detecting evidence of life in the martian surface and near surface. Iron oxides are ubiquitous in the martian regolith and are known to be associated with the deposition and preservation of organic matter in certain terrestrial environments, thus iron oxide-rich sediments are potential targets for life-detection missions. The most frequently used protocol for martian organic matter characterization (also planned for use on ExoMars) has been thermal extraction for the transfer of organic matter to gas chromatography-mass spectrometry (GC-MS) detectors. For the effective use of thermal extraction for martian samples, it is necessary to explore how potential biomarker organic molecules evolve during this process in the presence of iron oxides. We have thermally decomposed iron oxides simultaneously with (z)-octadec-9-enoic and n -octadecanoic acids and analyzed the products through pyrolysis-GC-MS. We found that the thermally driven dehydration, reduction, and recrystallization of iron oxides transformed fatty acids. Overall detectability of products greatly reduced, molecular diversity decreased, unsaturated products decreased, and aromatization increased. The severity of this effect increased as reduction potential of the iron oxide and inferred free radical formation increased. Of the iron oxides tested hematite showed the least transformative effects, followed by magnetite, goethite, then ferrihydrite. It was possible to identify the saturation state of the parent carboxylic acid at high (0.5 wt %) concentrations by the distribution of n -alkylbenzenes in the pyrolysis products. When selecting life-detection targets on Mars, localities where hematite is the dominant iron oxide could be targeted preferentially, otherwise thermal analysis of carboxylic acids, or similar biomarker molecules, will lead to enhanced polymerization, aromatization, and breakdown, which will in turn reduce the fidelity of the original biomarker, similar to changes normally observed during thermal maturation.

In situ instrumentation that can detect amino acids at parts-per-billion concentration levels and distinguish an enantiomeric excess of either d- or l-amino acids is vital for future robotic life-detection missions to promising targets in our solar system. In this article, a novel chiral amino acid analysis method is described, which reduces the risk of organic contamination and spurious signals from by-products by avoiding organic solvents and organic additives. Online solid-phase extraction, chiral liquid chromatography, and mass spectrometry were used for automated analysis of amino acids from solid and aqueous environmental samples. Carbonated water (pH ∼3, ∼5 wt % CO 2 achieved at 6 MPa) was used as the extraction solvent for solid samples at 150°C and as the mobile phase at ambient temperature for chiral chromatographic separation. Of 18 enantiomeric amino acids, 5 enantiomeric pairs were separated with a chromatographic resolution >1.5 and 12 pairs with a resolution >0.7. The median lower limit of detection of amino acids was 2.5 μg/L, with the lowest experimentally verified as low as 0.25 μg/L. Samples from a geyser site (Great Fountain Geyser) and a geothermal spring site (Lemon Spring) in Yellowstone National Park were analyzed to demonstrate the viability of the method for future in situ missions to Ocean Worlds.

Preferential uptake of either levorotatory (L) or dextrorotatory (D) enantiomer of a chiral molecule is a potential planetary life-detection method. On Earth, bacteria, as a rule, metabolize D-sugars and L-amino acids. Here, we use growth experiments to identify exceptions to the rule and their potential impact on the method's reliability. Our experiments involve six strains of Bacillus and collective uptake of the sugars glucose and arabinose, and the amino acids alanine, glutamic acid, leucine, cysteine, and serine-all of which are highly soluble. We find that selective uptake is not evident unless (1) each sugar is tested individually and (2) multiple amino acids are tested together in a mixture. Combining sugars should be avoided because, as we show in Bacillus bacteria, the same organisms may catabolize one sugar, glucose, in D-form and another sugar, arabinose, in L-form. Single amino acids should be avoided because bacteria can access certain proteinogenically incompatible enantiomers using specific racemases. Specifically, bacteria contain an alanine acid racemase and can catabolize D-alanine if no other D-amino acids are present. The proposed improvements would reliably separate nonselective chemical reactions from biological reactions and, if life is indicated, inform whether the selective patterns for amino acids and sugars are the same as on Earth.

Searching for life in the Universe depends on unambiguously distinguishing biological features from background signals, which could take the form of chemical, morphological, or spectral signatures. The discovery and direct measurement of organic compounds unambiguously indicative of extraterrestrial (ET) life is a major goal of Solar System exploration. Biology processes matter and energy differently from abiological systems, and materials produced by biological systems may become enriched in planetary environments where biology is operative. However, ET biology might be composed of different components than terrestrial life. As ET sample return is difficult, in situ methods for identifying biology will be useful. Mass spectrometry (MS) is a potentially versatile life detection technique, which will be used to analyze numerous Solar System environments in the near future. We show here that simple algorithmic analysis of MS data from abiotic synthesis (natural and synthetic), microbial cells, and thermally processed biological materials (lab-grown organisms and petroleum) easily identifies relational organic compound distributions that distinguish pristine and aged biological and abiological materials, which likely can be attributed to the types of compounds these processes produce, as well as how they are formed and decompose. To our knowledge this is the first comprehensive demonstration of the utility of this analytical technique for the detection of biology. This method is independent of the detection of particular masses or molecular species samples may contain. This suggests a general method to agnostically detect evidence of biology using MS given a sufficiently strong signal in which the majority of the material in a sample has either a biological or abiological origin. Such metrics are also likely to be useful for studies of possible emergent living phenomena, and paleobiological samples.

We sought to understand the spatiotemporal characteristics of biological motion perception. We presented observers with biological motion walkers that differed in terms of form coherence or kinematics (i.e., the presence or absence of natural acceleration). Participants were asked to discriminate the facing direction of the stimuli while their magnetoencephalographic responses were concurrently imaged. We found that two univariate response components can be observed around ~200 msec and ~650 msec post-stimulus onset, each engaging lateral-occipital and parietal cortex prior to temporal and frontal cortex. Moreover, while univariate responses show biological motion form-specificity only after 300 msec, multivariate patterns specific to form can be well discriminated from those for local cues as early as 100 msec after stimulus onset. By finally examining the representational similarity of fMRI and MEG patterned responses, we show that early responses to biological motion are most likely sourced to occipital cortex while later responses likely originate from extrastriate body areas., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

(1) Background: Future missions to potentially habitable places in the Solar System require biochemistry-independent methods for detecting potential alien life forms. The technology was not advanced enough for onboard machine analysis of microscopic observations to be performed in past missions, but recent increases in computational power make the use of automated in-situ analyses feasible. (2) Methods: Here, we present a semi-automated experimental setup, capable of distinguishing the movement of abiotic particles due to Brownian motion from the motility behavior of the bacteria Pseudoalteromonas haloplanktis, Planococcus halocryophilus, Bacillus subtilis, and Escherichia coli . Supervised machine learning algorithms were also used to specifically identify these species based on their characteristic motility behavior. (3) Results: While we were able to distinguish microbial motility from the abiotic movements due to Brownian motion with an accuracy exceeding 99%, the accuracy of the automated identification rates for the selected species does not exceed 82%. (4) Conclusions: Motility is an excellent biosignature, which can be used as a tool for upcoming life-detection missions. This study serves as the basis for the further development of a microscopic life recognition system for upcoming missions to Mars or the ocean worlds of the outer Solar System.

Perchlorate anions are produced by chemical industries and are important contaminants in certain natural ecosystems. Perchlorate also occurs in some natural and uncontaminated environments such as the Atacama Desert, the high Arctic or the Antarctic Dry Valleys, and is especially abundant on the surface of Mars. As some bacterial strains are capable of using perchlorate as an electron acceptor under anaerobic conditions, their detection is relevant for environmental monitoring on Earth as well as for the search for life on Mars. We have developed an antibody microarray with 20 polyclonal antibodies to detect perchlorate-reducing bacteria (PRB) strains and two crucial and highly conserved enzymes involved in perchlorate respiration: perchlorate reductase and chlorite dismutase. We determined the cross-reactivity, the working concentration, and the limit of detection of each antibody individually and in a multiplex format by Fluorescent Sandwich Microarray Immunoassay. Although most of them exhibited relatively high sensitivity and specificity, we applied a deconvolution method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<10 3 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration., Competing Interests: LA was employed by company Inmunología y Genética Aplicada, S.A. (INGENASA). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Gallardo-Carreño, Moreno-Paz, Aguirre, Blanco, Alonso-Pintado, Raymond-Bouchard, Maggiori, Rivas, Engelbrektson, Whyte and Parro.)

It has been experimentally demonstrated that, under alkaline conditions, silica is able to induce the formation of mineral self-assembled inorganic-inorganic composite materials similar in morphology, texture and nanostructure to the hybrid biomineral structures that, millions of years later, life was able to self-organize. These mineral self-organized structures (MISOS) have been also shown to work as effective catalysts for prebiotic chemical reactions and to easily create compartmentalization within the solutions where they form. We reason that, during the very earliest history of this planet, there was a geochemical scenario that inevitably led to the existence of a large-scale factory of simple and complex organic compounds, many of which were relevant to prebiotic chemistry. The factory was built on a silica-rich high-pH ocean and powered by two main factors: a) a quasi-infinite source of simple carbon molecules synthesized abiotically from reactions associated with serpentinization, or transported from meteorites and produced from their impact on that alkaline ocean, and b) the formation of self-organized silica-metal mineral composites that catalyze the condensation of simple molecules in a methane-rich reduced atmosphere. We discuss the plausibility of this geochemical scenario, review the details of the formation of MISOS and its catalytic properties and the transition towards a slightly alkaline to neutral ocean., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Conceptual frameworks are developed for evaluating the ability of different biosignatures to provide evidence for the presence of life in planned missions or observational studies. The focus is on intrinsic characteristics of biosignatures in space environments rather than on their detection, which depends on technology. Evaluation procedures are drawn from extensive studies in decision theory on related problems in business, engineering, medical fields, and the social arena. Three approaches are particularly useful. Two of them, Signal Detection Theory and Bayesian hypothesis testing, are based on probabilities. The third approach is based on utility theory. In all the frameworks, knowledge about a subject matter has to be translated into probabilities and/or utilities in a multistep process called elicitation. We present the first attempt to cover all steps, from acquiring knowledge about biosignatures to assigning probabilities or utilities to global quantities, such as false positives and false negatives. Since elicitation involves human judgment that is always prone to perceptual and cognitive biases, the relevant biases are discussed and illustrated in examples. We further discuss at which stage of elicitation human judgment should be involved to ensure the most reliable outcomes. An example, how evaluating biosignatures might be implemented, is given in the Supplementary Information.

Sulfate and iron oxide deposits in Río Tinto (Southwestern Spain) are a terrestrial analog of early martian hematite-rich regions. Understanding the distribution and drivers of microbial life in iron-rich environments can give critical clues on how to search for biosignatures on Mars. We simulated a robotic drilling mission searching for signs of life in the martian subsurface, by using a 1m-class planetary prototype drill mounted on a full-scale mockup of NASA's Phoenix and InSight lander platforms. We demonstrated fully automated and aseptic drilling on iron and sulfur rich sediments at the Río Tinto riverbanks, and sample transfer and delivery to sterile containers and analytical instruments. As a ground-truth study, samples were analyzed in the field with the life detector chip immunoassay for searching microbial markers, and then in the laboratory with X-ray diffraction to determine mineralogy, gas chromatography/mass spectrometry for lipid composition, isotope-ratio mass spectrometry for isotopic ratios, and 16S/18S rRNA genes sequencing for biodiversity. A ubiquitous presence of microbial biomarkers distributed along the 1m-depth subsurface was influenced by the local mineralogy and geochemistry. The spatial heterogeneity of abiotic variables at local scale highlights the importance of considering drill replicates in future martian drilling missions. The multi-analytical approach provided proof of concept that molecular biomarkers varying in compositional nature, preservation potential, and taxonomic specificity can be recovered from shallow drilling on iron-rich Mars analogues by using an automated life-detection lander prototype, such as the one proposed for NASA's IceBreaker mission proposal.

With the discovery of the persistent jets of water being ejected to space from Enceladus, an understanding of the effect of the space environment on potential organisms and biosignatures in them is necessary for planning life detection missions. We experimentally determine the survivability of microbial cells in liquid medium when ejected into vacuum. Epifluorescence microscopy, using a lipid stain, and SEM imaging were used to interrogate the cellular integrity of E. coli after ejected through a pressurized nozzle into a vacuum chamber. The experimental samples showed a 94% decrease in visible intact E. coli cells but showed a fluorescence residue in the shape of the sublimated droplets that indicated the presence of lipids. The differences in the experimental conditions versus those expected on Enceladus should not change the analog value because the process a sample would undergo when ejected into space was representative. E. coli was selected for testing although other cell types could vary physiologically which would affect their response to a vacuum environment. More testing is needed to determine the dynamic range in concentration of cells expected to survive the plume environment. However, these results suggest that lipids may be directly detectable evidence of life in icy world plumes.