The National Aeronautics and Space Administration (NASA) and the European Space Agency (ESA) are studying how samples might be brought back to Earth from Mars safely. Backward planetary protection is key in this complex endeavour, as it is required to prevent potential adverse effects from returning materials to Earth's biosphere. As the question of whether or not life exists on Mars today or whether it ever did in the past is still unanswered, the effort to return samples from Mars is expected to be categorized as a ‘Restricted Earth Return’ mission, for which NASA policy requires the containment of any unsterilized material returned to Earth. NASA is investigating several solutions to contain Mars samples and sterilize any uncontained Martian particles. This effort has significant implications for both NASA's scientific mission, and the Earth's environment; and so special care and vigilance are needed in planning and execution in order to assure acceptance of safety to Earth's biosphere. To generate a technically acceptable sterilization process across a wide array of scientific and other stakeholders, on 30–31 January 2019, 10–11 June 2019 and 19–20 February 2020, NASA informally convened a Sterilization Working Group (SWG) composed of experts from industry, academia and government to assess methods for sterilization and inactivation, to identify future work needed to verify these methods against biological challenges, and to determine their feasibility for implementation on robotic spacecraft in deep space. The goals of the SWG were:

1. (1) Understand what it means to sterilize and/or inactivate Martian materials and how that understanding can be applied to the Mars Sample Return (MSR) mission.

2. (2) Assess methods for sterilization and inactivation, and identify future work needed to verify these methods.

3. (3) Provide an effective plan for communicating with other agencies and the public.

This paper provides a summary of the discussions and conclusions of the SWG over these three workshops. It reflects a consensus position based on qualitative discussion of how agencies might approach the problem of sterilization of Mars material. The SWG reached a consensus that sterilization options can be considered on the basis of biology as we know it, and that sterilization modalities that are effective on terrestrial materials and organisms should be part of the MSR planetary protection strategy. Conclusions pointed to several industry standards for sterilization to include heat, chemical, UV radiation and low-heat plasma. Technical trade-offs for each sterilization modality were discussed while simultaneously considering the engineering challenges and limitations for spaceflight. Future work includes more in-depth discussions on technical trade-offs of sterilization modalities, identifying and testing Earth analogue challenge organisms and proteinaceous molecules against chosen modalities, and executing collaborative agreements between NASA and external working group partners to help close data gaps, and to establish strong, scientifically grounded sterilization and inactivation standards for MSR.

The current advances in our exploration of Mars have made us think of the human species as a multiplanetary species. However, we have certain challenges before we can truly consider ourselves such a species, especially moral ones. Therefore, astrobioethics would be the right one to examine what it takes to consider ourselves a multiplanetary species. The purpose of this paper is to analyse the meaning and significance of being an inter- and multiplanetary species. To achieve this, a philosophical and critical analysis will be made, using as input aspects of biology, ethics and moral community. We conclude that to be a truly multiplanetary species, more than the technological aspects that allow us to reach other planets, a change at different levels will be needed.

In this work, we address the difficulty of reliably identifying traces of life on Mars. Several independent lines of evidence are required to build a compelling body of proof. In particular, we underline the importance of correctly interpreting the geological and mineralogical context of the sites to be explored for the presence of biosignatures. We use as examples to illustrate this, ALH84001 (where knowledge of the geological context was very limited) and other terrestrial deposits, for which this could be properly established. We also discuss promising locations and formations to be explored by ongoing and future rover missions, including Oxia Planum, which, dated at 4.0 Ga, is the most ancient Mars location targeted for investigation yet.

Plate tectonics drives variation in sea-level, over intervals of approximately107–108 years. These variations may have significant effects on the pace of (biological) evolution through the elimination of terrestrial niches and the expansion of shallow-water marine niches. However, within the solar system, only the Earth experiences this kind of tectonism. Venus displays regional tectonism, characterized by rising diapirs within the plastic mantle. Impinging on the lithosphere, these plumes produce a range of structures of varying dimensions; the uplift of which would raise sea-level, were Venus to have oceans. Using Magellan observations of Venus, we model the impact of regional tectonism on sea-level for given areas of Venusian ocean, then compare the effect with terrestrial tectonic processes for similar oceanic area. We show that despite variation in the geographical extent of Venusian-style tectonic processes, the styles of regional tectonism on Venus can produce the same order of magnitude changes in sea-level, for a given area of ocean, as plate tectonics. Consequently, we examine some of the impacts of marine transgression on habitability and the evolution of life.

In order to examine the potential survivability of life in the Martian deep subsurface, we have investigated the effects of temperature (45°C, 55°C and 65°C) and pressure (1, 400, 800 and 1200 atm) on the growth, carbon isotopic data and morphology of chemolithoautotrophic anaerobic methanogenic archaea, Methanothermobacter wolfeii. The growth and survivability of this methanogen were determined by measuring the methane concentration in headspace gas samples after the cells were returned to their conventional growth conditions. Interestingly, this methanogen survived at all the temperatures and pressures tested. M. wolfeii demonstrated the highest methane concentration following exposure to pressure of 800 atm and a temperature of 65°C. We found that the stable carbon isotopic fractionation of methane, δ13C(CH4), was slightly more enriched in 12C at 1 atm and 55°C than the carbon isotopic data obtained in other temperature and pressure conditions. A comparison of the images of the cells before and after the exposure to different temperatures and pressures did not show any obvious alteration in the morphology of M. wolfeii. The research reported here suggests that at least one methanogen, M. wolfeii, may be able to survive under hypothetical Martian subsurface conditions with respect to temperature and pressure.

Astrobioethics is an emerging discipline that studies, evaluates and analyses the moral, legal and social issues of the search for life in the Universe. As a new field of study, it requires an epistemological foundation to be able to be recognized as an academic discipline. In order to achieve this, it is important to understand the disciplinary nature of astrobiology as a transdisciplinary form of research, above the multi and interdisciplinary levels, although this does not necessarily mean discarding them. In addition, the questions and topics of each subdivision of astrobioethics studies are specified and explained in this paper.

The appellation ‘habitable zone’ in astrobiology in sooth evinces an overlooked and winding history that can be traced back to the 19th century. This paper sketches how this term from geography was generalized to encompass planetary habitability. The people involved in this narrative are numerous, but the bulk of their musings were rather nebulous. Yet, during this period appear the first true insights, although sadly this saga is not altogether sans blights.

Previous exploration missions have revealed Mars as a potential candidate for the existence of extraterrestrial life. If life could have existed beneath the Martian subsurface, biosignatures would have been preserved in iron-rich minerals. Prior investigations of terrestrial biosignatures and metabolic processes of geological analogues would be beneficial for identifying past metabolic processes on Mars, particularly morphological and chemical signatures indicative of past life, where biological components could potentially be denatured following continued exposure to extreme conditions. The objective of the research was to find potential implications for Martian subsurface life by characterizing morphological, mineralogical and microbial signatures of hematite deposits, both hematite rock and related soil samples, collected from Highland Complex of Sri Lanka. Rock samples examined through scanning electron microscopy-energy dispersive X-ray (SEM-EDX) spectroscopy. Analysis showed globular and spherical growth layers nucleated by bacteria. EDX results showed a higher iron to oxygen ratio in nuclei colonies compared to growth layers, which indicated a compositional variation due to microbial interaction. X-ray diffraction analysis of the hematite samples revealed variations in chemical composition along the vertical soil profile, with the top surface soil layer being particularly enriched with Fe2O3, suggesting internal dissolution of hematite through weathering. Furthermore, inductively coupled plasma-mass spectrometry analyses carried out on both rock and soil samples showed a possible indication of microbially induced mineral-weathering, particularly release of trapped trace metals in the parent rock. Microbial diversity analysis using 16S rRNA gene sequencing revealed that the rock sample was dominated by Actinobacteria and Proteobacteria, specifically, members of iron-metabolizing bacterial genera, including Mycobacterium, Arthrobacter, Amycolatopsis, Nocardia and Pedomicrobium. These results suggest that morphological and biogeochemical clues derived from studying the role of bacterial activity in hematite weathering and precipitation processes can be implemented as potential comparative tools to interpret similar processes that could have occurred on early Mars.

The presence of perchlorate in the Martian soil may limit in-situ resource utilization (ISRU) technologies to support human outposts. In order to exploit the desiccation, radiation-tolerant cyanobacterium Chroococcidopsis in Biological Life Support Systems based on ISRU, we investigated the perchlorate tolerance of Chroococcidopsis sp. CCMEE 029 and its derivative CCMEE 029 P-MRS. This strain was obtained from dried cells mixed with Martian regolith simulant and exposed to Mars-like conditions during the BIOMEX space experiment. After a 55-day exposure of up to 200 mM perchlorate ions, a tolerance threshold value of 100 mM perchlorate ions was identified for both Chroococcidopsis strains. After 40-day incubation, a Mars-relevant perchlorate concentration of 2.4 mM perchlorate ions, provided as a 60 and 40% mixture of Mg- and Ca-perchlorate, had no negative effect on the growth rate of the two strains. A proof-of-concept experiment was conducted using Chroococcidopsis lysate in ISRU technologies to feed a heterotrophic bacterium, i.e. an Escherichia coli strain capable of metabolizing sucrose. The sucrose content was fivefold increased in Chroococcidopsis cells through air-drying and the yielded lysate successfully supported the bacterial growth. This suggested that Chroococcidopsis is a suitable candidate for ISRU technologies to support heterotrophic BLSS components in a Mars-relevant perchlorate environment that would prove challenging to many other cyanobacteria, allowing a ‘live off the land’ approach on Mars.

Mars exploration motivates the search for extraterrestrial life, the development of space technologies, and the design of human missions and habitations. Here, we seek new insights and pose unresolved questions relating to the natural history of Mars, habitability, robotic and human exploration, planetary protection, and the impacts on human society. Key observations and findings include:

1. – high escape rates of early Mars' atmosphere, including loss of water, impact present-day habitability;

2. – putative fossils on Mars will likely be ambiguous biomarkers for life;

3. – microbial contamination resulting from human habitation is unavoidable; and

4. – based on Mars' current planetary protection category, robotic payload(s) should characterize the local martian environment for any life-forms prior to human habitation.

Some of the outstanding questions are:

1. – which interpretation of the hemispheric dichotomy of the planet is correct;

2. – to what degree did deep-penetrating faults transport subsurface liquids to Mars' surface;

3. – in what abundance are carbonates formed by atmospheric processes;

4. – what properties of martian meteorites could be used to constrain their source locations;

5. – the origin(s) of organic macromolecules;

6. – was/is Mars inhabited;

7. – how can missions designed to uncover microbial activity in the subsurface eliminate potential false positives caused by microbial contaminants from Earth;

8. – how can we ensure that humans and microbes form a stable and benign biosphere; and

9. – should humans relate to putative extraterrestrial life from a biocentric viewpoint (preservation of all biology), or anthropocentric viewpoint of expanding habitation of space?

Studies of Mars' evolution can shed light on the habitability of extrasolar planets. In addition, Mars exploration can drive future policy developments and confirm (or put into question) the feasibility and/or extent of human habitability of space.

Laboratory generation of water nanoclusters from amorphous ice and strong terahertz (THz) radiation from water nanoclusters ejected from water vapour into a vacuum suggest the possibility of water nanoclusters ejected into interstellar space from abundant amorphous ice-coated cosmic dust produced by supernovae explosions. Water nanoclusters (section ‘Water nanoclusters’) offer a hypothetical scenario connecting major mysteries of our Universe: dark matter (section ‘Baryonic dark matter’), dark energy (section ‘Dark energy’), cosmology (section ‘Cosmology’), astrobiology (section ‘Astrobiology’) and the RNA world (section ‘The RNA world’) as the origin of life on Earth and habitable exoplanets. Despite their expected low density in space compared to hydrogen, their quantum-entangled diffuse Rydberg electronic states make cosmic water nanoclusters a candidate for baryonic dark matter that can also absorb, via the microscopic dynamical Casimir effect, the virtual photons of zero-point-energy vacuum fluctuations above the nanocluster cut-off vibrational frequencies, leaving only vacuum fluctuations below these frequencies to be gravitationally active, thus leading to a possible common origin of dark matter and dark energy. This picture includes novel explanations of the small cosmological constant, the coincidence of energy and matter densities, possible contributions of the red-shifted THz radiation from cosmic water nanoclusters at redshift z ≅ 10 to the cosmic microwave background (CMB) spectrum, the Hubble constant crisis, the role of water as a known coolant for rapid early star formation and ultimately, how life may have originated from RNA protocells on Earth and exoplanets and moons in the habitable zones of developed solar systems. Together, they lead to a cyclic universe cosmology – based on the proposed equivalence of cosmic water nanoclusters to a quintessence scalar field – instead of a multiverse based on cosmic inflation theory. Recent CMB birefringence measurements may support quintessence. Finally, from the quantum chemistry of water nanoclusters interacting with prebiotic organic molecules, amino acids and RNA protocells on early Earth and habitable exoplanets, this scenario is consistent with the anthropic principle that our Universe must have those properties which allow life, as we know it – based on water, to develop at the present stage of its history.

The eclectic range of artefacts and ‘messages’ we dispatch into the vast expanse of space may become one of the most enduring remnants of our present civilization, but how does his protracted legacy adequately document the plurality of societal values and common, cultural heritage on our heterogeneous world? For decades now, this rendition of the egalitarian principle has been explored by the Search for Extra-Terrestrial Intelligence community in order to draft theoretical responses to ‘who speaks for Earth?’ for hypothetical extra-terrestrial communication strategies. However, besides the moral, ethical and democratic advancements made by this particular enterprise, there remains little practical exemplars of implementing this garnered knowledge into other experimental elements that could function as mutual emissaries of Earth; physical artefacts that could provide accessible details about our present world for future archaeological observations by our space-faring progeny, potential visiting extrasolar denizens or even for posterity. While some initiatives have been founded to investigate this enduring dilemma of humanity over the last half-century, there are very few comparative studies in regards to how these objects, time capsules and transmission events collectively disseminate content about the aggregate of our species and the Earth system it inhabits. This catalogue, assembled for extended study as part of the Beyond the Earth foundation, is intended as an initial, dialogic step towards evaluating such a ‘profile of humanity’. This investigation will endeavour to collate all cultural resources that can presently be garnered from spacecraft (non-mission orientated, cultural material that conveys an impression of Earth) and non-terrestrial transmissions (electromagnetic signals that are intentionally aimed off-world to embody humanities’ evolving, philosophical identity) in the expanse beyond our planetary borders in order to cross-analyse how we presently illustrate the diversity of our planet before, subsequently, deducing how we could appropriately depict our collective human civilization [and biosphere] within deep space and cosmic time.

Nucleobases are nitrogenous bases composed of monomers that are a major constituent of RNA and DNA, which are an essential part of any cellular life on the Earth. The search for nucleobases in the interstellar medium remains a major challenge, however, the recent detection of nucleobases in meteorite samples and laboratory synthesis in simulated analogue experiments have confirmed their abiotic origin and a possible route for their delivery to the Earth. Nevertheless, cellular life is based on the interacting network of complex structures, and there is substantial lack of information on the possible routes by which such ordered structures may be formed in the prebiotic environment. In the current study, we present the evidence for the synthesis of complex structures due to shock processing of nucleobases. The nucleobases were subjected to the reflected shock temperature of 3500–7000 K (estimated) and pressure of about 15–34 bar for over ~2 ms timescale. Under such extreme thermodynamic conditions, the nucleobases sample experiences superheating and subsequent cooling. Electron microscopic studies of shock processed residue show that nucleobases result in spontaneous formation of complex structures when subjected to extreme conditions of shock. These results suggest that impact shock processes might have contributed to the self-assembly of biologically relevant structures and the origin of life.

Since their discovery, submarine hydrothermal vent systems have been pointed out as important places where chemical evolution on Earth could have occurred; and their role in the process has been highlighted. Similarly, some hypotheses have considered these systems in origin of life scenarios. In this way, many experiments have been developed, and the knowledge about these systems has increased. Due to their complexity, many experimental simulations have only included a few of the geochemical variables present in these environments, pressure and temperature. Other main variables have hardly been included, such as mineralogy, thermal and pH gradients, dissolved ions and/or redox reactions. As it has been understood, the dynamism and heterogeneity of these environments are huge, and it comprises different scales, from single vents to full hydrothermal fields. However, the vast majority of experiments focus on a specific part of these systems and do not include salinity, mineralogy and pH gradients. For this reason, in this paper, we pointed out some considerations about how this dynamism can be interpreted, and included in some models, as well their importance in prebiotic chemistry experiments and their extrapolations regarding the hypothesis about the origins of life.

There are different criteria that are usually analysed independently before identifying a new trace fossil, such as morphological regularity, completeness, dispersion, recurrence and complexity, surface morphology, and context. The synthesis of these criteria, as utilized in paleosol ichnology, composes a protocol that is presented herein for the first time and can be used for testing the ichnogenicity of trace-like structures in any paleoenvironment of Earth or Mars. As a study case, the Martian ‘stick-like structures’ do not fulfil any of the requirements posed by this protocol to be postulated as trace fossils. The ichnogenicity test, focussed exclusively on morphology and context, is simpler but equally useful as the biogenicity ones. It may be applied in the future with other potential cases before carrying on more complex analyses or to evaluate the astrobiological interest of trace-like structures.

The term habitable is used to describe planets that can harbour life. Debate exists as to specific conditions that allow for habitability but the use of the term as a planetary variable has become ubiquitous. This paper poses a meta-level question: What type of variable is habitability? Is it akin to temperature, in that it is something that characterizes a planet, or is something that flows through a planet, akin to heat? That is, is habitability a state or a process variable? Forth coming observations can be used to discriminate between these end-member hypotheses. Each has different implications for the factors that lead to differences between planets (e.g. the differences between Earth and Venus). Observational tests can proceed independent of any new modelling of planetary habitability. However, the viability of habitability as a process can influence future modelling. We discuss a specific modelling framework based on anticipating observations that can discriminate between different views of habitability.

Previously conducted space missions revealed the presence of perchlorates, which are known to have a high oxidizing potential in Martian regolith, at the level of 0.5%. Due to hygroscopic properties and crystallization features of perchlorate-containing solutions, assumptions leading to the possibility of the existence of liquid water in the form of brines, which can contribute to the vital activity of microorganisms, have been made. At the same time, high concentrations of perchlorates can inhibit the growth of microorganisms and cause their death. Previously performed studies have discovered the presence of highly diverse microbial communities in terrestrial perchlorate-containing soils and have also demonstrated the stability and activity of some prokaryotes cultured on highly concentrated perchlorates media (over 10%). Nevertheless, the limits of microbial tolerance to perchlorates and whether microbial communities are able to withstand the effects of high concentrations of perchlorates remain uncertain. The aim of this research was to study the reaction of microbial communities of hot-arid and cryo-arid soils and sedimentary rocks to the adding of a highly concentrated solution of sodium perchlorate (5%) in situ. An increase in the total number of prokaryotes, the number of metabolically active Bacteria and Archaea, and the variety of the consumed substrates were revealed. It was observed that in samples incubated with sodium perchlorate, a high taxonomic diversity of the microbial community is preserved at a level comparable to control sample. The study shows that the presence of high concentrations of sodium perchlorate (5%) in the soil does not lead to the death or significant inhibition of microbial communities.

We are witnessing the enormous breakthroughs of space technology, which will eventually allow us to reach Mars. However, it seems that the technological evolution is expanding at a faster rate than the moral development. Are we ethically ready to take human beings to Mars? Will it be a private company the first one that manages to take us there? Should we colonize Mars or leave it like it is right now? Are astrobiological interests being contemplated when discussing human presence in Mars? These are some of the questions that we must answer since the moment of stepping on Mars does not seem to be far away. Therefore, the objective of this article is to evaluate the idea of Mars being a free planet from any of Earth's governments, and to analyse the idea of colonizing Mars considering that by doing that we could seriously endanger native life. What it proposed is that its unavoidable that we will reach Mars, however, we may not be prepared as humanity and this is something that we must face.

Mars is a primary target of astrobiological interest: its past environmental conditions may have been favourable to the emergence of a prebiotic chemistry and, potentially, biological activity. In situ exploration is currently underway at the Mars surface, and the subsurface (2 m depth) will be explored in the future ESA ExoMars mission. In this context, BIOlogy and Mars EXperiment was performed to evaluate the stability and detectability of organic biomarkers under space and Mars-like conditions. Our data suggested that some target molecules, namely melanin, azelaic acid and nucleic acids, can be detected even after 16 months exposure to Low Earth Orbit conditions by multidisciplinary approaches. We used the same techniques as onboard the ExoMars rover, as Raman and infrared spectroscopies and gas chromatograph-mass spectrometer, and polymerase chain reaction even if this is not planned for the imminent mission to Mars. These results should be taken into account for future Mars exploration.