Life detection in a universe of false positives: Can the Fatal Flaws of Exoplanet Biosignatures be Overcome Absent a Theory of Life?

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?

Subsurface Microbial Colonization at Mineral-Filled Veins in 2-Billion-Year-Old Mafic Rock from the Bushveld Igneous Complex, South Africa.

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.

A Multi-Target Localization and Vital Sign Detection Method Using Ultra-Wide Band Radar.

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.

Automatic Life Detection Based on Efficient Features of Ground-Penetrating Rescue Radar Signals.

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.

Long-term thermal stability of fluorescent dye used for chiral amino acid analysis on future spaceflight missions.

Future spaceflight missions focused on life detection will carry with them new, state-of-the-art instrumentation capable of highly selective and sensitive organic analysis. CE-LIF is an ideal candidate for such a mission due to its high separation efficiency and low LODs. One perceived risk of utilizing this technique on a future mission is the stability of the chemical reagents in the spaceflight environment. Here, we present an investigation of the thermal stability of the fluorescent dye (5-carboxyfluorescein succinimidyl ester) used for amino acid analysis. The dye was stored at 4, 25, and 60°C for 1 month, 6 months, 1 year, and 2 years. When stored at 4°C for 2 years, 25°C for 6 months, or 60°C for 1 month there was no effect on CE-LIF assay performance due to dye degradation. Beyond these time points, while the dye degradation begins to interfere with the analysis, it is still possible to perform the analysis and achieve the majority of amino acid biosignature science goals described in the science definition team report for the potential Europa Lander mission. This work indicates that thermal control of the dye at ≤4°C will be needed during transit on future spaceflight missions to maintain dye stability.

Stability of reagents used for chiral amino acid analysis during spaceflight missions in high-radiation environments.

The search for biosignatures on spaceflight missions requires in situ instrumentation capable of highly selective and sensitive organic analyses. To this end, CE-LIF is a uniquely promising technique, capable of determining the type, abundance, and chirality of amino acids present in environmental samples at nanomolar concentrations. However, this type of assay requires several reagents that have not yet been used on spaceflight missions. A key concern, particularly for future missions to Europa, is the survivability of these critical components for CE separation and LIF detection under high levels of radiation. Here we present an investigation of the chemical stability of the reagents and associated fused silica capillary after a total ionizing dose of 300 krad, exceeding the predicted total ionizing dose for the potential Europa Lander Mission payload by two-fold. Neither the fused silica capillary nor the fluorescent dye (5-carboxyfluorescein succinimidyl ester) showed significant change in performance following irradiation. Following the irradiation of the pre-mixed background electrolyte, both migration time and resolution were affected. However, when the reagents (sodium tetraborate, sodium taurocholate, and γ-cyclodextrin) and the acetonitrile solution were irradiated separately and mixed afterwards, there was no change in the separation performance.

Evaluation of a Sensor System for Detecting Humans Trapped under Rubble: A Pilot Study.

Rapid localization of injured survivors by rescue teams to prevent death is a major issue. In this paper, a sensor system for human rescue including three different types of sensors, a CO2 sensor, a thermal camera, and a microphone, is proposed. The performance of this system in detecting living victims under the rubble has been tested in a high-fidelity simulated disaster area. Results show that the CO2 sensor is useful to effectively reduce the possible concerned area, while the thermal camera can confirm the correct position of the victim. Moreover, it is believed that the use of microphones in connection with other sensors would be of great benefit for the detection of casualties. In this work, an algorithm to recognize voices or suspected human noise under rubble has also been developed and tested.

Simultaneous Life Detection and Localization Using a Wideband Chaotic Signal with an Embedded Tone.

A hybrid life detection radar system which transmits a wideband chaotic signal containing an embedded single-tone is proposed. The chaotic signal is used for target localization by the time-domain correlation method and synthetic aperture technique, and the single-tone signal is used to measure the frequencies of breathing and heartbeat based on an on-chip split-ring integrated sensor and Michelson interference principle. Experimental results in free space and in through-wall scenarios demonstrate that the system can realize human detection and localization simultaneously with high range resolution, high sensitivity, and large dynamic range without complex signal processing. The range resolution is about 10 cm, and the dynamic range is 35 dB for the respiration signal detection and 25 dB for the heartbeat signal detection. Due to its good immunity to interference/jamming and high spectrum efficiency, the proposed system is suitable for post-disaster rescue, elder/infant/patient vitality monitoring, and anti-terrorism enforcement applications.

Life Is Uncertain: Inherent Variability Exhibited by Organisms, and at Higher Levels of Biological Organization.

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.

A multiple biomolecules-based rapid life detection protocol embedded in a rover scientific subsystem for soil sample analysis.

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.

UV photo-degradation of the secondary lichen substance parietin: A multi-spectroscopic analysis in astrobiology perspective.

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 N2 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.

Chapter 8: Searching for Life Beyond Earth.

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.

Fluorescence Microscopy with Deep UV, Near UV, and Visible Excitation for In Situ Detection of Microorganisms.

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.

Different Scenarios for the Origin and the Subsequent Succession of a Hypothetical Microbial Community in the Cloud Layer of Venus.

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.

Detecting life by behavior, the overlooked sensitivity of behavioral assays.

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.

The HOMO-LUMO Gap as Discriminator of Biotic from Abiotic Chemistries.

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.

A Comparative Study of Methods for Detecting Extraterrestrial Life in Exploration Missions to Mars and the Solar System II: Targeted Characteristics, Detection Techniques, and Their Combination for Survey, Detection, and Analysis.

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.

Organic Catalytic Activity as a Method for Agnostic Life Detection.

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.

Genetic Biosignatures of Deep-Subsurface Organisms Preserved in Carbonates Over a 100,000 Year Timescale at a Surface-Accessible Mars Analog Site in Southeastern Utah.

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.

False Positives and the Challenge of Testing the Alien Hypothesis.

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.