Chapter 9: Life as We Don't Know It.

While Earth contains the only known example of life in the universe, it is possible that life elsewhere is fundamentally different from what we are familiar with. There is an increased recognition in the astrobiology community that the search for life should steer away from terran-specific biosignatures to those that are more inclusive to all life-forms. To start exploring the space of possibilities that life could occupy, we can try to dissociate life from the chemistry that composes it on Earth by envisioning how different life elsewhere could be in composition, lifestyle, medium, and form, and by exploring how the general principles that govern living systems on Earth might be found in different forms and environments across the Solar System. Exotic life-forms could exist on Mars or Venus, or icy moons like Europa and Enceladus, or even as a shadow biosphere on Earth. New perspectives on agnostic biosignature detection have also begun to emerge, allowing for a broader and more inclusive approach to seeking exotic life with unknown chemistry that is distinct from life as we know it on Earth.

How to identify cell material in a single ice grain emitted from Enceladus or Europa.

Icy moons like Enceladus, and perhaps Europa, emit material sourced from their subsurface oceans into space via plumes of ice grains and gas. Both moons are prime targets for astrobiology investigations. Cassini measurements revealed a large compositional diversity of emitted ice grains with only 1 to 4% of Enceladus's plume ice grains containing organic material in high concentrations. Here, we report experiments simulating mass spectra of ice grains containing one bacterial cell, or fractions thereof, as encountered by advanced instruments on board future space missions to Enceladus or Europa, such as the SUrface Dust Analyzer onboard NASA's upcoming Europa Clipper mission at flyby speeds of 4 to 6 kilometers per second. Mass spectral signals characteristic of the bacteria are shown to be clearly identifiable by future missions, even if an ice grain contains much less than one cell. Our results demonstrate the advantage of analyses of individual ice grains compared to a diluted bulk sample in a heterogeneous plume.

Future of the Search for Life: Workshop Report.

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

Solid-State Single-Molecule Sensing with the Electronic Life-Detection Instrument for Enceladus/Europa (ELIE).

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.

Ice Transit and Performance Analysis for Cryorobotic Subglacial Access Missions on Earth and Europa.

Ice-covered ocean worlds, such as the Jovian moon Europa, are some of the prime targets for planetary exploration due to their high astrobiological potential. While upcoming space exploration missions, such as the Europa Clipper and JUICE missions, will give us further insight into the local cryoenvironment, any conclusive life detection investigation requires the capability to penetrate and transit the icy shell and access the subglacial ocean directly. Developing robust, autonomous cryorobotic technology for such a mission constitutes an extremely demanding multistakeholder challenge and requires a concentrated interdisciplinary effort between engineers, geoscientists, and astrobiologists. An important tool with which to foster cross-disciplinary work at an early stage of mission preparation is the virtual testbed. In this article, we report on recent progress in the development of an ice transit and performance model for later integration in such a virtual testbed. We introduce a trajectory model that, for the first time, allows for the evaluation of mission-critical parameters, such as transit time and average/overall power supply. Our workflow is applied to selected, existing cryobot designs while taking into consideration different terrestrial, as well as extraterrestrial, deployment scenarios. Specific analyses presented in this study show the tradeoff minimum transit time and maximum efficiency of a cryobot and allow for quantification of different sources of uncertainty to cryobot's trajectory models.

Recovery of Microbes from Subsurface Europa Analog Environments: An Efficient Mechanical-Thermal Probe for Collecting Biological Samples from the Subsurface of Icy Moons.

The ultra-low temperatures (<173K) and ultra-low pressures (<0.1 Pa) that exist on the surface of icy moons present a formidable challenge for collecting biological samples. Standard drilling technology is not efficient in these conditions, where conduction of thermal energy leads to the possibility of freezing in place and shear forces impart a strenuous test on microbial viability. If microbes exist within the first few meters of the surface, an extraction process must be gentle enough to recover them intact. This report describes a substantial improvement from the study by Davis in 2017, who presented a concave conical thermal probe capable of penetrating -65°C ice in 1000 Pa pressure. The current report describes a mechanical-thermal device for penetrating ≤ -150°C ice in 10 Pa pressure, which is analogous to the physical conditions on the surface of icy moons. The mechanism has an efficiency of >68% with -65°C ice and >61% with -150°C ice, which is well above the expected 10-15% for a Philberth-type probe. In addition, the probe can harvest a sensitive bacterium (Escherichia coli) from under a layer of acidified peroxide ice (pH 1.1), which is analogous to the expected surface chemical composition of the icy moon Europa. In field tests at -20°C air and -6°C ice temperatures, multiple organisms were extracted in a viable state, and chemical analysis indicated high-resolution separation of stratified layers. Finally, attaching the thermal tip to a telescopic mechanism allowed the probe to penetrate through 1.0 m of -65°C ice, which is well below the depth of harmful radiation expected at the subsurface of Europa. The current work opens the door for a lander vehicle to penetrate the upper subsurface of Europa and analyze biologically active samples.

The Impact of Extreme Low Temperatures on Raman Spectra of Amino Acids Relevant for the Search for Life on Europa.

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.

Contamination analysis of Arctic ice samples as planetary field analogs and implications for future life-detection missions to Europa and Enceladus.

Missions to detect extraterrestrial life are being designed to visit Europa and Enceladus in the next decades. The contact between the mission payload and the habitable subsurface of these satellites involves significant risk of forward contamination. The standardization of protocols to decontaminate ice cores from planetary field analogs of icy moons, and monitor the contamination in downstream analysis, has a direct application for developing clean approaches crucial to life detection missions in these satellites. Here we developed a comprehensive protocol that can be used to monitor and minimize the contamination of Arctic ice cores in processing and downstream analysis. We physically removed the exterior layers of ice cores to minimize bioburden from sampling. To monitor contamination, we constructed artificial controls and applied culture-dependent and culture-independent techniques such as 16S rRNA amplicon sequencing. We identified 13 bacterial contaminants, including a radioresistant species. This protocol decreases the contamination risk, provides quantitative and qualitative information about contamination agents, and allows validation of the results obtained. This study highlights the importance of decreasing and evaluating prokaryotic contamination in the processing of polar ice cores, including in their use as analogs of Europa and Enceladus.

The Bioburden and Ionic Composition of Hypersaline Lake Ices: Novel Habitats on Earth and Their Astrobiological Implications.

We present thermophysical, biological, and chemical observations of ice and brine samples from five compositionally diverse hypersaline lakes in British Columbia's interior plateau. Possessing a spectrum of magnesium, sodium, sulfate, carbonate, and chloride salts, these low-temperature high-salinity lakes are analogs for planetary ice-brine environments, including the ice shells of Europa and Enceladus and ice-brine systems on Mars. As such, understanding the thermodynamics and biogeochemistry of these systems can provide insights into the evolution, habitability, and detectability of high-priority astrobiology targets. We show that biomass is typically concentrated in a layer near the base of the ice cover, but that chemical and biological impurities are present throughout the ice. Coupling bioburden, ionic concentration, and seasonal temperature measurements, we demonstrate that impurity entrainment in the ice is directly correlated to ice formation rate and parent fluid composition. We highlight unique phenomena, including brine supercooling, salt hydrate precipitation, and internal brine layers in the ice cover, important processes to be considered for planetary ice-brine environments. These systems can be leveraged to constrain the distribution, longevity, and habitability of low-temperature solar system brines-relevant to interpreting spacecraft data and planning future missions in the lens of both planetary exploration and planetary protection.

Double ridge formation over shallow water sills on Jupiter's moon Europa.

Jupiter's moon Europa is a prime candidate for extraterrestrial habitability in our solar system. The surface landforms of its ice shell express the subsurface structure, dynamics, and exchange governing this potential. Double ridges are the most common surface feature on Europa and occur across every sector of the moon, but their formation is poorly understood, with current hypotheses providing competing and incomplete mechanisms for the development of their distinct morphology. Here we present the discovery and analysis of a double ridge in Northwest Greenland with the same gravity-scaled geometry as those found on Europa. Using surface elevation and radar sounding data, we show that this double ridge was formed by successive refreezing, pressurization, and fracture of a shallow water sill within the ice sheet. If the same process is responsible for Europa's double ridges, our results suggest that shallow liquid water is spatially and temporally ubiquitous across Europa's ice shell.

Trawl impacts on the relative status of biotic communities of seabed sedimentary habitats in 24 regions worldwide.

Bottom trawling is widespread globally and impacts seabed habitats. However, risks from trawling remain unquantified at large scales in most regions. We address these issues by synthesizing evidence on the impacts of different trawl-gear types, seabed recovery rates, and spatial distributions of trawling intensity in a quantitative indicator of biotic status (relative amount of pretrawling biota) for sedimentary habitats, where most bottom-trawling occurs, in 24 regions worldwide. Regional average status relative to an untrawled state (=1) was high (>0.9) in 15 regions, but <0.7 in three (European) regions and only 0.25 in the Adriatic Sea. Across all regions, 66% of seabed area was not trawled (status = 1), 1.5% was depleted (status = 0), and 93% had status > 0.8. These assessments are first order, based on parameters estimated with uncertainty from meta-analyses; we recommend regional analyses to refine parameters for local specificity. Nevertheless, our results are sufficiently robust to highlight regions needing more effective management to reduce exploitation and improve stock sustainability and seabed environmental status-while also showing seabed status was high (>0.95) in regions where catches of trawled fish stocks meet accepted benchmarks for sustainable exploitation, demonstrating that environmental benefits accrue from effective fisheries management. Furthermore, regional seabed status was related to the proportional area swept by trawling, enabling preliminary predictions of regional status when only the total amount of trawling is known. This research advances seascape-scale understanding of trawl impacts in regions around the world, enables quantitative assessment of sustainability risks, and facilitates implementation of an ecosystem approach to trawl fisheries management globally.

Comparative activity of newer ß-lactam/ß-lactamase inhibitor combinations against Pseudomonas aeruginosa from patients hospitalized with pneumonia in European medical centers in 2020.

Pseudomonas aeruginosa isolates were consecutively collected from patients with pneumonia in 29 medical centers in 2020 and susceptibility tested by broth microdilution method. Ceftazidime-avibactam (95.5% susceptible), imipenem-relebactam (94.3% susceptible), and ceftolozane-tazobactam (93.3% susceptible) were the most active compounds after colistin (99.5% susceptible). Susceptibility rates for the ß-lactam/ß-lactamase inhibitor combinations (BL/BLIs) varied against isolates resistant to piperacillin-tazobactam, meropenem, imipenem, and/or ceftazidime. Ceftazidime-avibactam was the most active BL/BLI against resistant subsets from Western Europe, whereas imipenem-relebactam was slightly more active than other BL/BLIs against resistant subsets from Eastern Europe. Susceptibility rates were markedly lower in Eastern Europe than Western Europe.

Technology's Palette: Voyager's Eyes and the Hyperchromatic Enhancement of Jupiter and Saturn.

In the late 1970s and early 1980s, NASA's Voyager mission offered the first clear pictures of Jupiter and Saturn. These images show the planets in strikingly brilliant, recognizably engineered, psychedelic colors: technology's palette. The use of color was justified on epistemological grounds; it made visible scientifically compelling features. But color palette also has a history, one that has not been previously considered. This article takes up this history and adds to the literature on the visual culture of science. It establishes that the Jet Propulsion Laboratory's pioneering role in digital image processing, the color conventions adopted for representing Earth, and American counterculture of the 1960s and its attitudes toward technology together created the conditions that allowed for hyperchromatic views of the planets. Technology's palette enhanced the scientific understanding of Jupiter and Saturn, while simultaneously celebrating technologically enhanced vision and the promise of seeing by means of humanmachine collaborations.

On the stability of Laplace resonance for Galilean moons (Io, Europa, Ganymede).

This paper presents the application of recent ansatz for estimation of stability of the Laplace resonance for Galilean moons (Io, Europa, Ganymede). We estimate over time the eccentricity + semi-major axis in a binary system experiencing the net tidal friction, including the additional tidal heating which comes from the transformation of net transfer of angular momentum between the Galilean moons of Jupiter (due to dynamical features of the Laplace resonance). Presumably, there should be a net transfer of angular momentum between Io and Europa (for the reason that tidal heating on Ganymede seems to be negligible with respect to Io and Europa). We established the fact that Laplace resonance should be valid and stable on a timescale of centuries in the future, but there might be chaotic perturbations less than 0.1\% for the accuracy of such phenomenon. Moreover, the presented ansatz can be used to predict a scheme for optimizing the maneuvers of spacecrafts in the vicinity of Ganymede (due to absence of net transfer of angular momentum between Ganymede and other Galilean moons). The main conclusion stems from previously suggested approach (\cite{ershkov2017tidal.

Exobiology of the Venusian Clouds: New Insights into Habitability through Terrestrial Models and Methods of Detection.

The search for life beyond Earth has focused on Mars and the icy moons Europa and Enceladus, all of which are considered a safe haven for life due to evidence of current or past water. The surface of Venus, on the other hand, has extreme conditions that make it a nonhabitable environment to life as we know it. This is in contrast, however, to its cloud layer, which, while still an extreme environment, may prove to be a safe haven for some extreme forms of life similar to extremophiles on Earth. We consider the venusian clouds a habitable environment based on the presence of (1) a solvent for biochemical reactions, (2) appropriate physicochemical conditions, (3) available energy, and (4) biologically relevant elements. The diversity of extreme microbial ecosystems on Earth has allowed us to identify terrestrial chemolithoautotrophic microorganisms that may be analogs to putative venusian organisms. Here, we hypothesize and describe biological processes that may be performed by such organisms in the venusian clouds. To detect putative venusian organisms, we describe potential biosignature detection methods, which include metal-microbial interactions and optical methods. Finally, we describe currently available technology that can potentially be used for modeling and simulation experiments.

Genetic diversity of the Mycobacterium tuberculosis complex strains from newly diagnosed tuberculosis patients in Northwest Ethiopia reveals a predominance of East-African-Indian and Euro-American lineages.

OBJECTIVES: This study described the population structure of M. tuberculosis complex (MTBc) strains among patients with pulmonary or lymph node tuberculosis (TB) in Northwest Ethiopia and tested the performance of culture isolation and MPT64-based speciation for Lineage 7 (L7). METHODS: Patients were recruited between April 2017 and June 2019 in North Gondar, Ethiopia. The MPT64 assay was used to confirm MTBc, and spoligotyping was used to characterize mycobacterial lineages. Line probe assay (LPA) was used to detect resistance to rifampicin and isoniazid. RESULTS: Among 274 MTBc genotyped isolates, there were five MTBc lineages: L1-L4 and L7 were identified, with predominant East-African-Indian (L3) (53.6%) and Euro-American (L4) (40.1%) strains, and low prevalence (2.6%) of Ethiopia L7. The genotypes were similarly distributed between pulmonary and lymph node TB, and all lineages were equally isolated by culture and recognized as MTBc by the MPT64 assay. Additionally, LPA showed that 259 (94.5%) MTBc were susceptible to both rifampicin and isoniazid, and one (0.4%) was multi-drug resistant (resistant to both rifampicin and isoniazid). CONCLUSION: These findings show that TB in North Gondar, Ethiopia, is mainly caused by L3 and L4 strains, with low rates of L7, confirmed as MTBc by MPT64 assay and with limited resistance to rifampicin and isoniazid.

A Cruise-Phase Microbial Survival Model for Calculating Bioburden Reductions on Past or Future Spacecraft Throughout Their Missions with Application to Europa Clipper.

During transit between the Earth and planetary destinations, spacecraft encounter conditions that are deleterious to the survival of terrestrial microorganisms. To model the resulting bioburden reduction, a Cruise-Phase Microbial Survival (CPMS) model was prepared based upon the Lunar Microbial Survival model, which considers the effects of temperature, vacuum, ultraviolet (UV), and ionizing radiation found in the space environment. As an example, the CPMS was used to determine the expected bioburden reductions on the Europa Clipper spacecraft upon arrival at Jupiter under two different transit scenarios. Under a direct trajectory scenario, exterior surfaces are rapidly sterilized with tens of thousands of lethal doses (LDs) absorbed to the spacecraft exterior and at least one LD to all interior spaces of the spacecraft heated to at least 240 K. Under a Venus-Earth-Earth gravity assist (VEEGA) trajectory, we find substantially higher bioburden reductions resulting from the spacecraft spending much more time near the Sun and more time in transit overall. With VEEGA, the exterior absorbs hundreds of thousands of LDs and interior surfaces heated above 230 K would absorb at least one LD. From these simulations, we are able to generalize about bioburden reduction in transit on spacecraft in general, finding that all spacecraft surfaces would sustain at least one LD in ≤38.5 years even if completely unheated. Temperature and vacuum synergy dominates surface reductions out to at most 3.3 AU (for gold multilayer insulation), UV irradiation and temperature between 3.3 and 600 AU, and past 600 AU the effect of vacuum acting alone is the primary factor for all exterior and interior surfaces. Even under the most conservative estimates, if the average interior temperature of the Cassini spacecraft exceeded 218 K, or the Galileo spacecraft interior exceeded 222 K, neither spacecraft would have likely had any viable bioburdens onboard at disposal.

Viability of Bacillus subtilis Spores Exposed to Ultraviolet Light at Ocean World Surface Temperatures.

This work investigated microorganism survival under temperature and ultraviolet (UV) radiation conditions found at the surface of ice-covered ocean worlds. These studies were motivated by a desire to understand the ability of resilient forms of life to survive under such conditions as a proxy for potential endogenic life and to inform planetary protection protocols for future missions. To accomplish this, we irradiated Bacillus subtilis spores with solar-like UV photons at temperatures ranging from room temperature down to 11 K and reported survival fractions with respect to fluence. We observed an increase in survival at low temperatures and found that the inactivation rate follows an Arrhenius-type behavior above 60 K. For solar-photon fluxes and surface temperatures at Europa and Enceladus, we found that Bacillus subtilis spores would be inactivated in less than an hour when in direct sunlight.

Exploring Deep-Sea Brines as Potential Terrestrial Analogues of Oceans in the Icy Moons of the Outer Solar System.

Several icy moons of the outer solar system have been receiving considerable attention and are currently seen as major targets for astrobiological research and the search for life beyond our planet. Despite the limited amount of data on the oceans of these moon, we expect them to be composed of brines with variable chemistry, some degree of hydrothermal input, and be under high pressure conditions. The combination of these different conditions significantly limits the number of extreme locations, which can be used as terrestrial analogues. Here we propose the use of deep-sea brines as potential terrestrial analogues to the oceans in the outer solar system. We provide an overview of what is currently known about the conditions on the icy moons of the outer solar system and their oceans as well as on deep-sea brines of the Red Sea and the Mediterranean and their microbiology. We also identify several threads of future research, which would be particularly useful in the context of future exploration of these extra-terrestrial oceans.