EXOA15

Two missions are on Mars looking for traces of life (MSL and Mars 2020) while the ExoMars mission, slated for launch later this year, is postponed. All missions are equipped with a powerful array of instrumentation for in situ analyses. In addition, the ExoMars rover, Rosalind Franklin, has the advantage of a 2 m drill to penetrate beneath the oxidised and UV-irradiated surface [1]. However, unless phototrophic life forms were able to develop on a planet that was never permanently habitable and that was characterised by punctuated and likely unconnected habitable niches [2, 3], martian life likely remained in a very primitive state of evolution, potentially similar to terrestrial chemotrophs, i.e. discrete, very small cells and colonies that leave even more subtle fossil remains. Indeed, only in the vicinity of nutrient-rich hydrothermal environments can chemotrophic biomass develop to create potentially visible morphological signatures in the form of a clotted texture in the associated sediments – a texture that is not immediately identifiable as a biosignature but that would warrant further analysis of such rocks by other instruments, such as SAM on Curiosity, SHERLOC on Perseverance, or MOMA on Rosalind Franklin.

Taking the early terrestrial environment and early life as analogues for habitable environments and primitive life forms on early Mars, it is becoming clearer that the most primitive of life forms known on Earth, chemolithotrophs that obtain energy from oxidation of inorganic substrates (e.g. ferrous iron, hydrogen sulfide, elemental sulfur, thiosulfate, or ammonia), were widely distributed but, because of their very small size (< 1 µm diameter) and very small, heterogeneously distributed colonies (5 µm to some tens of microns), are typically undetectable through bulk analyses (bulk carbon contents are generally 0.01-0.05 %.) (Fig. 1). To compound the challenge, morphologically-recognisable cells are rarely observed, while degraded cells mixed with generic extracellular polymeric substances (EPS) are more frequent. Chemolithotrophs can inhabit sediment particle surfaces and pore space close to the surface or at depth, as long as they have access to nutrients and water. But how to identify them in situ on Mars? Even on Earth with well-equipped, state of the art laboratories, detection and identification of fossil chemolithotrophs is difficult and, often, controversial. The solution requires seeking to simultaneously detect structural information of the organisms’ presence and analyse well-preserved organogeochemical signature with spatial precisions on the order of a micron and detection limits as low as possible. This would be a challenge for Mars Sample Return in the near future.

Figure 1. (A-C) distribution of tiny colonies and EPS clumps (red circles) in a silicified, volcanic sediment [3] at increasing magnifications. Individual dividing cells visible only at maximum resolution in (C).

References: [1] Vago, J.L., et al. (2017) Astrobiology, 17: 471–510. [2] Cockell et al., 2012. Icarus, 217:184–193. [3] Westall et al., (2015) Astrobiology, 17: 998-1029. [4] Author J. et al. (2002) LPS XXXIII, Abstract #2110.

How to cite: Westall, F., Clodore, L., Foucher, F., Milojevic, T., Kölbl, D., Guérillot, P., Hickman-Lewis, K., Cavalazzi, B., and Vago, J.: The search for extraterrestrial life and the problem of primitive life forms, now you see them, now you don’t, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-187, https://doi.org/10.5194/epsc2022-187, 2022.

INTRODUCTION

Over the past two decades, ground- and space-based observations have unveiled thousands exoplanets and planetary systems around other stars in our Galaxy. About 5000 exoplanets are currently confirmed, in large part detected as transits by the Kepler and TESS missions. Launched in 2019, PLATO mission represents the first-step characterisation towards the understanding of the structural properties of these planets. Nevertheless, a significant boost for the detection of transiting Earth-analogues around bright stars is expected from the PLATO mission. 
However, it's only through remote atmospheric spectroscopy of potentially habitable rocky planets, that one of the main goals of exoplanetary science, the quest for life outside the Solar System, can be tackled. This observational challenge should be partly within reach of the recently launched JWST and the next ground-based astronomical observatory, E-ELT.

To accomplish the demanding task of searching for and  deciphering spectral signatures, a thorough and holistic observational and theoretical characterization of carefully selected rocky exoplanets is required.
The selection, among the observationally reachable targets for high-resolution spectroscopy of thin atmospheres, requires habitability studies with climate models. 
These simulations will enable the identification of those exoplanets with the largest chance of potentially hosting a surface diffuse life, i.e. with the largest habitability, that must be evaluated over a wide range of mostly unknown conditions. 
A considerable effort of modelization that exploits all available observations will be needed in order to assess the global physical characterization of the selected exoplanets, and in particular precisely of their potential surface climate and habitability.

THE MODEL

Here we present EOS-ESTM, a flexible climate model aimed at simulating the surface and atmospheric conditions that characterize habitable planets. The model allows one to perform a fast exploration of the parameter space representative of planetary quantities, including those currently not measurable in rocky exoplanets. EOS-ESTM has been built up starting from ESTM (Vladilo et al. 2013, 2015), a seasonal-latitudinal EBM featuring an advanced treatment of surface and cloud components and a 2D (vertical and latitudinal) treatment of the energy transport.

The main features of the model that we have implemented can be summarised as follows.

Firstly, we have calculated the atmospheric radiative transfer using EOS (Simonetti et al. 2022), a procedure tailored for atmospheres of terrestrial-type planets, based on the opacity calculator HELIOS-K (Grimm & Heng 2015; Grimm et al. 2021) and the radiative transfer code HELIOS (Malik et al. 2017, 2019). Thanks to EOS, the ESTM radiative transfer can be now calculated for a variety of atmospheres with different bulk and greenhouse compositions, illuminated by stars with different SEDs.

Then, we have upgraded the parameterizations that describe the clouds properties. New equations have been introduced for the albedo of the clouds and its dependence on the albedo of the underlying surface. The clouds coverage over ice is now a function of the global planetary ice coverage. A specific treatment for the transmittance and OLR forcing of clouds at very low temperature has been introduced.

Lastly, we have introduced a generalized logistic function to estimate the ice coverage as a function of mean zonal surface temperature. Based on a detailed study of the ice distribution on Earth, the adopted algorithm discriminates between ice over lands and oceans. The albedo and thermal capacity of transitional ice is now estimated using the fractional ice coverage. 

RESULTS

With the aim of providing a reference model for studies of habitable planets, we calibrated EOS-ESTM using a large set of Earth satellite and reanalysis data.  
The reference Earth model satisfies a variety of diagnostic tests, including mean latitudinal profiles of surface temperature (Figure 1), TOA albedo, OLR and ice coverage.

Fig. 1. Mean annual latitude profile of surface temperature predicted by the reference Earth model The temperature profile is compared with ERA5 temperatures averaged in the period 2005-2015 (blue dots).

To test the consistency of EOS-ESTM with previous studies of non-terrestrial climate conditions we performed a series of comparisons with a hierarchy of climate models, varying the levels of insolation (Figure 2), the stellar spectrum and planetary parameters (radius and rotation rate).

Fig. 2. Comparison of global and annual  mean surface temperature obtained from different climate Earth's models by increasing the solar constant. Red, solid line: EOS-ESTM (this work). Black, solid line: 3D model CAM4 (Wolf & Toon, 2015). Cyan, solid line: 3D model CAM3 (Wolf & Toon, 2014). Green, solid line: 3D model by Leconte et al. (2013).

The application of EOS-ESTM to the case of a CO₂-dominated atmosphere in maximum greenhouse conditions (Kasting et al. 1993) yields a detailed description of the transition to a snowball state that takes place when the insolation decreases in the proximity of the outer edge of the HZ. Thanks to the flexibility of our model we can explore how this transition develops in different planetary conditions (e.g. rotation rate, Figure 3).

Fig. 3. Dependence on planetary rotation period rotation period, P_rot, of the fractional ice coverage calculated at the outer edge of the HZ. The results were obtained for an Earth-like planet with a CO₂-dominated, maximum greenhouse atmosphere, the remaining parameters being fixed to Earth values.

REFERENCES

Grimm S. L., Heng K., 2015, HELIOS-K: Opacity Calculator for Radiative Transfer (ascl:1503.004)

Grimm S. L., et al., 2021, ApJS, 253, 30

Kasting J. F., Whitmire D. P., Reynolds R. T., 1993, Icarus, 101, 108

Leconte J., Forget F., Charnay B., Wordsworth R., Pottier A., 2013, Nature, 504, 268

Malik M., et al., 2017, AJ, 153, 56

Malik M., Kitzmann D., Mendonça J. M., Grimm S. L., Marleau G.-D., Linder E. F., Tsai S.-M., Heng K., 2019, AJ, 157, 170

Simonetti P., Vladilo G., Silva L., Maris M., Ivanovski S. L., Biasiotti L., Malik M., von Hardenberg J., 2022, ApJ, 925, 105

Vladilo G., Murante G., Silva L., Provenzale A., Ferri G., Ragazzini G., 2013, ApJ, 767, 65

Vladilo G., Silva L., Murante G., Filippi L., Provenzale A., 2015, ApJ, 804,50

Wolf E. T., Toon O. B., 2014, Geophys. Res. Lett., 41, 167

Wolf E. T., Toon O. B., 2015, Journal of Geophysical Research (Atmospheres), 120, 5775

How to cite: Biasiotti, L., Simonetti, P., Vladilo, G., Silva, L., Murante, G., Ivanovski, S., Maris, M., Monai, S., Bisesi, E., and von Hardenberg, J.: EOS-ESTM: a flexible climate model for habitable exoplanets, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-1195, https://doi.org/10.5194/epsc2022-1195, 2022.

Some organisms have shown to be able to naturally survive environments which we consider extreme, including the Low Earth Orbit, or even Outer Space. These microorganisms have natural mechanisms to repair severe DNA damage, such as the caused by ionizing and non-ionizing radiation or extreme temperatures and pressures. Some examples are Deinococcus radiodurans, which proved to be capable of surviving in the Exposure Facility of the International Space Station (ISS) for up to three years, and tardigrade species, such as Ramazzottius varieornatus, which are some of the most resilient known organisms. In this study, performed at the Barcelona Biomedical Research Park in collaboration with Hospital del Mar, survival under simulated Low Earth Orbit environmental conditions was tested in engineered and wild-type Escherichia coli strains. Ionizing radiation resistance was enhanced by transforming the Dsup gene from R. varieornatus and two genes from D. radiodurans involved in DNA damage repair, RecA and uvrD. This enhancement, together with a directed evolution process, resulted in a significant increase in the surviving fraction of the E. coli strain protected with the Dsup gene after a high dose, up to 3000 Gy, of ionizing radiation exposure in the form of a continuous spectrum of X-ray photons. Additionally, the survival to wide ranges of temperatures and low pressures was tested for the same strains, revealing a lack of relevance of cell aggregation for survival under the mentioned conditions in contrast with the case of D. radiodurans. However, survival rates showed no enhancement for any of the new E. coli strains. In a new collaboration with the Subterranean Laboratory of Canfranc, both the absence of radiation and extreme levels of radiation will be further studied. Additionally, an extreme environments analogue for several environmental conditions will be built, allowing for more specific testing on a controlled environment. This research represents a first step in the creation of new bacterial strains engineered to survive severe conditions and adapting existing species for their survival in remote environments, like extra-terrestrial habitats. These species could pave the road for future human expeditions, helping develop environments hospitable to life. In addition, studying the efficacy and the functioning of the genetic mechanisms used in this study could be beneficial for fields such as ecological restoration and medical and life sciences engineering, addressing treatments and/or diseases caused or related to radiation and DNA damage. Space is believed to be the last frontier, but the truth is, we are still a frontier to ourselves.

How to cite: Puig, J., Knödlseder, N., Quera, J., Algara, M., and Güell, M.: DNA Damage Protection for Enhanced Bacterial Survival Under Simulated Low Earth Orbit Environmental Conditions in Escherichia coli, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-378, https://doi.org/10.5194/epsc2022-378, 2022.

The search for extraterrestrial life and finding habitable environments on other planets and satellites like Mars, Europa, Enceladus and Titan are a priority of NASA and ESA, since the last decade. To contribute to these highly significant challenges, research has been done with established exposure platforms like those on the Foton satellite and EXPOSE on the ISS expanding now to ESA’s platform Bartolomeo. These were used to expose samples to space vacuum and space radiation, but also to provide gas supply and selected planetary radiation environments. Results obtained by these experiments have allowed to get supplemental knowledge necessary for supporting future investigations to search for life in the universe.

Several extremophile lichen species, have been exposed to extraterrestrial environments, i.e. space- and Mars like parameters, during short and long periods on board of ESA’s space missions (Foton M2 and M3, EXPOSE E and R2) to investigate the limits of terrestrial life. To maximize the scientific outcome of these experiments, LICHENS [1], LITHOPANSPERMIA [2], LIFE [3], and BIOMEX [4], a common elaboration and analysis of the results obtained on analogue field studies with results obtained in planetary simulation facilities was necessary to check the survival potential and vitality of the samples before flight. Tests and experiments at different simulation facilities at DLR, and at INTA, included the exposure to space vacuum, space UV radiation and space cosmic radiation, and to Mars-like environment, i.e. Mars atmospheric composition and pressure, as well as Mars UV radiation. Not microgravity or reduced gravity, which is present in space and on Mars, was tried.

Here we show the results of the resistance of two extremophile vagrant lichen species, Xanthoparmelia hueana and Circinaria gyrosa, to simulated microgravity (rotation speed of clinostate: 1 rpm) using the UNZIP clinostate at CIB-CSIC (Centro Investigaciones Biológicas Margarita Salas). This is the first time that lichens will be exposed to weightlessness environment in an attempt to isolate the potential contribution of microgravity from other extraterrestrial factors (radiation, vacuum). Combinations of simulated spaceflight conditions, including microgravity, will be necessary to check how this parameter affects the biomolecular level of lichens and their microbiome.

References

[1] Sancho, L.G., de la Torre, R., Horneck, G., Ascaso, C., de los Rios, A., Pintado, A., and Schuster, M. (2007) Lichens survive in space: results from the 2005 LICHENS experiment. Astrobiology 7:443–454. https://doi.org/10.1089/ast.2006.0046

[2] de la Torre, R., Sancho, L.G., Horneck, G., de los Ríos, A., Wierzchos, J., Olsson-Francis, K., and Ott, S. (2010) Survival of lichens and bacteria exposed to outer space conditions—results of the Lithopanspermia experiments. Icarus 208:735–748. https://doi.org/10.1016/j.icarus.2010.03.010

[3] Onofri, S., de la Torre, R., de Vera, J.P., Ott, S., Zucconi, L., Selbmann, L., Scalzi, G., Venkateswaran, K.J., Rabbow, E., Sánchez, F.J., and Horneck, G. (2012) Survival of rock-colonizing organisms after 1.5 year in outer space. Astrobiology 12: 508-516. https://doi.org/10.1089/ast.2011.0736

[4] de Vera, J.P., , and the BIOMEX-Team and associatesTeam (Ute Boettger, Rosa de la Torre Noetzel et al.) (2019) Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS. Astrobiology, 19-2. https://doi.org/10.1089/ast.2018.1897.

How to cite: de la Torre Noetzel, R., Alvarez, O. B., Ortega-García, M. V., García Sancho, L., Villasante, A., del Olmo, J., de Vera, J. P., and Herranz, R.: Limits of life at spaceflight conditions: survival of lichens to simulated microgravity, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-1055, https://doi.org/10.5194/epsc2022-1055, 2022.

Introduction

Icy moons of the giant planets contain liquid water oceans where habitability is feasible. A prime example is Jupiter’s moon, Europa, which could contain multiple regions where life might be sustained under its thick ice layer ^[1]. However, whether the physical and chemical conditions in Europa’s ocean are conducive to life is currently unknown. Terrestrial analogue environments provide one opportunity to study microbial dynamics under chemical conditions relevant to Europa. Knowledge of Europa’s ocean chemistry comes from models based on Earth-based observations and spacecraft flybys ^[2,3], which suggest that the ocean might be favourable to support life, specifically, chemolithotrophs that obtain energy by oxidising inorganic compounds ^[1,4]. Developments in machine learning (ML) algorithms offer the possibility of selecting terrestrial analogues with a high degree of similarity to the physicochemical environment proposed for Europa ^[5].

However, there are limitations to terrestrial analogues; conditions in these environments do not perfectly match the physicochemical conditions in Europa, especially when the moon’s ocean pressure ranges from a few hundred bar to over 1200 bar ^[1,2]. Thus, controlled lab experiments must also be used to fully simulate the desired extraterrestrial environment. The aim of this study was to integrate analogue and experimental approaches to cultivate a microbial community capable of growth under the specific physical and chemical conditions of Europa’s sub-surface ocean that can serve as a proxy to study the habitability of Europa.

Methods

The selection of a suitable analogue sample through ML^[5] and the design of a growth media was based on a model for Europa’s ocean composition^[6]. The environmental sample came from Basque Lake, Canada and the media was dominated by a carbonate-chloride rich chemistry at a salinity of 12.6 g/L and under 2 bar of 80%-20% H₂/CO₂ gas headspace ^[4,6]. Sub-culturing was performed to eliminate contaminants from the initial sample, with final cultures reaching cell densities of 10⁵-10⁷ cells per millilitre. This community was then transferred to a bespoke environmental simulation chamber (Figure 1) where it could be gradually conditioned to the physical conditions within Europa’s ocean. Specifically, the pressure was initially set at 20 bar (10-fold increase from the benchtop control) and then increased to 100, 200 and 300 bar to selectively cultivate communities adapted for growth under Europa ocean conditions. At the same time, samples were taken to monitor pressure-induced changes in the microbial community composition and their physiology. The chemical composition of the media was monitored using Ion Chromatography (IC) and Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). The growth of the microbial community was monitored using fluorescence cell microscopy and its composition using 16s rRNA sequencing. Changes in microbial morphology due to pressure were investigated using Transmission Electron Microscopy (TEM).

Figure 1 Pressure vessel used as an environmental chamber where microbial communities can be exposed to different pressure and temperature regimes.

Results

Preliminary results demonstrate that microbial communities from Basque Lake can adapt to live under the conditions of Europa’s sub-surface ocean. There was an increased number of cells at 100 bar (6.35 x 10⁶ cells ml^-1 compared to 1.88 x 10⁶ cells ml^-1 at 2 bar) and growth was still measured at 300 bar (1.98 x 10⁷ cells ml^-1). However, pressure influenced the size of the cells, making them smaller and the generation of extracellular material (Figure 2) which will be further investigated using TEM. Future work will focus on isolating microorganisms from Europa’s simulated environment.

Figure 2 Fluorescence microscopy images of microbial communities growing in Europan chemical conditions under 2 bars of pressure (A) and 300 bars of pressure (B).

Conclusion

These high-pressure experiments have shown that microbes can adapt and grow under the conditions in the sub-surface ocean of Europa even after several sub-cultures. These microbial communities can now serve as a proxy to study life in this moon. This project could change how we approach the search for life in these moons and where we expect to find these organisms. The increased cell numbers under a pressure equivalent to the sub-surface environment of Europa point at a possible habitat where life might concentrate. Future work will investigate the effect on the morphology of the cells, as well as the formation of extracellular components, on the nucleation of ice at the sub-surface interface and thus how potential biosignatures might be captured into the ice shell and expressed to the surface.

References

1. Russell, M. J. et al. The possible emergence of life and differentiation of a shallow biosphere on irradiated icy worlds: The example of Europa. Astrobiology 17, 1265–1273 (2017).

2. Kivelson, M. G. et al. Galileo magnetometer measurements: A stronger case for a subsurface ocean at Europa. Science (1979) 289, 1340–1343 (2000).

3. Paganini, L. et al. A measurement of water vapour amid a largely quiescent environment on Europa. Nature Astronomy 4, 266–272 (2020).

4. Russell, M. J. et al. Serpentinization as a source of energy at the origin of life. Geobiology 8, 355–371 (2010).

5. del Moral, A. et al. Using supervised machine learning methods to improve the selection of analogue sites for studying habitability of the sub- surface ocean of Europa. 14, 10–12 (2022).

6. Melwani Daswani, M. et al. A Metamorphic Origin for Europa’s Ocean. Geophysical Research Letters 48, (2021).

How to cite: del Moral, A., Siggs, D., Fox-Powell, M. G., Pearson, V. K., and Olsson-Francis, K.: Exploring Europa’s biological potential using machine learning and laboratory simulations, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-1227, https://doi.org/10.5194/epsc2022-1227, 2022.

Detecting unquestionable signatures of biological activity in extra-terrestrial Solar System bodies is one of the most challenging objectives in Astrobiology. Discerning if these signatures have been generated by extinct or actual extant organisms would also be of principal interest. While most of the latest exploration missions to Mars launched on 2020 (i.e. NASA’s Mars 2020) or being launched in the near future (ESA’s ExoMars) carry instruments - Raman spectrometers and MOMA (Mars Organic Molecule Analyser) - able to detect organic compounds, they lack the capabilities to undoubtedly confirm those signatures have been generated either by past or extant life.

Here we present the MICRO-life detection platform, a comprehensive, multi-technique instrumentation for molecular microbial ecology studies, intended to take part in future exploratory space missions. The MICRO-life detection platform already includes three complementary tools that, in the next future, would be operated at once without any human intervention. Thus, findings coming out from all the included techniques would be combined for a better assignment of putative positive results.

The first instrument included in the platform is MagLysis, a small, lightweight, solid-state, automatable instrument designed to help in the extraterrestrial detection of the most unambiguous and informative biosignatures: nucleic acids (NA). MagLysis aims to bead-beat the biomass in a sample to disrupt the cell walls and membranes, thus releasing the contents of the cell cytoplasm, including DNA and RNA molecules. The unique feature of MagLysis is that only ferromagnetic beads (made of stainless steel) are agitated inside the vessel by two opposing, low-inductance electromagnets, resulting in mechanical lysis (Fig. 1A). Reducing the mass actuated by the device to only the beads means that the power required is potentially low and that there are fewer moving parts, both extremely important features for space exploratory missions. Moreover, extracted DNA was successfully amplified by PCR and subsequently sequenced by the second instrument included in the platform, Oxford Nanopore Technologies’ (ONT) MinION.

ONT MinION is a fully portable device (Fig. 1B) able to carry out real-time analysis of DNA, RNA, proteins and other smaller molecules. This instrument has been tested in the International Space Station (ISS) and in field campaigns at our lab, with results that have been already published. With it, feasible sequence data can be obtained within 48 h from natural samples with only ~ 0.001 ng of DNA, as it has been already tested by us on terrestrial analogues of Mars and the Icy Moons, such as the High Arctic, Antarctica and Utah and Atacama Desert samples, as well as on samples that have been subjected to long periods of Mars-like conditions. Additionally, MinION results have been proved to reflect very similar microbial community compositions to those obtained by other techniques, such as Illumina MiSeq sequencing or LDChip.

Complementary to MinION, MICRO-life detection platform also includes a method for the detection and characterization of viable microorganisms based on their metabolic capacities: Microfluidic microbial activity microassay (μMAMA). μMAMA is a multi-well plate that focus on the capability of extant microorganisms - including yet unculturable species - to become metabolically active when they are supplied with different organic and inorganic substrates. The technique itself is based on redox-indicator dyes chemically associated to the substrates than can subsequently be measured by a spectrophotometer (Fig. 1C); additionally, it has been proved to work in a wide range of pH values or temperatures. This has been the case of samples from the High Arctic, where this technique has already shown that metabolically active communities were differentially found at 5 °C or 20 °C. Subsequently, those communities yielded positive results in a MinION sequencing process, showing that most of the members in the active communities belonged to Pseudomonas genus and demonstrating the complementarity of both techniques.

Attending to the results already obtained from cold extreme environments in polar regions and deserts (Mars and Icy Moons analogs), MICRO-life detection platform and self-developed techniques within are very promising tools for Astrobiological research. Their ease-of-use and the proof of being resistant to the most extreme conditions including space travel ones, make the platform a strong candidate to be employed in future extreme environment research and, in the mid-term, included in space exploratory missions as an important element to the search for extant extra-terrestrial life.

How to cite: Fernández-Martínez, M. Á., O'Connor, B., Bourdages, L.-J., Maggiori, C., and Whyte, L.: The comprehensive ‘MICRO-life detection platform’ applied to in situ research at Mars and Icy Moons terrestrial analogs, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-918, https://doi.org/10.5194/epsc2022-918, 2022.

How to cite: Clodoré, L., Foucher, F., Hickman-Lewis, K., Sorieul, S., Réfrégiers, M., Collet, G., and Westall, F.: Identifying biosignatures in a Mars-analogue volcanic rock: The ~3.5 Ga Kitty’s Gap chert, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-15, https://doi.org/10.5194/epsc2022-15, 2022.

Introduction

One of the main topics of astrobiology research is the study of life’s limits in stressful environments. The study of organisms in extreme environments might give an indication about their potential adaptive plasticity, in the view of a climate change perspective, the terrestrial geological past and future scenarios, as well as extra-terrestrial habitats such as Mars’ surface. Lichens - with their excellent adaptive abilities - represents an extremely interesting case study. Several astrobiological studies involving lichens - that are symbiotic association between a fungus and an alga and/or acyanobacterium - proved the ability of these organisms to resist and thrive in extreme environments such as space and Mars’ surface simulated conditions [1, 2]. We have already tested the lichen species Xanthoria parietina (L.) Th. Fr. in simulated space conditions, that was able to survive and to reactivate after exposure [3]. X. parietina is a cosmopolitan foliose lichen that grows on barks and rocks [4]. This species shows high tolerance to air pollutants, heavy metals, and resistance to UV-radiation thanks to the shielding properties of the secondary metabolite parietin [5, 6]. Here we present a new study on the survival of X. parietina under simulated Mars conditions performed at the Mars Simulation Facility of the DLR Institute of Planetary Research in Berlin (Fig.1).

Figure 1 - Mars Simulation Facility at DLR with the opened experiment chamber.

Methods

The aim of the study was to assess the survivability of Xanthoria parietina under simulated Mars conditions for 30 days [7, 8]. Inside the Mars simulation chamber, eight samples (Fig.2) were exposed to the simulated atmospheric conditions of Mars of which four were fully UV-irradiated with day-night cycles (FM, Full-Mars) and the other four kept in darkness (DM, Dark-Mars). A three-gas mixture of 95% CO₂, 4% N₂ and 1% O₂ was used as best approximation of Mars-like atmospheric conditions, with a constant pressure of 600Pa. Temperature and humidity were subjected to day-night cycles, reaching during daytime 15°C and 0% RH, and during night -55°C and 100% RH (Fig.3) according to Martian thermophysical conditions at mid-latitudes. UV-radiation for FM samples was simulated using a Xenon UV-lamp (spectral range 200 nm – 2200 nm) that was automatically turned on for 16 h (day) and turned off for 8 h (night) daily. The total average radiation dose for FM was 2452.32 J/cm² and the average instantaneous irradiance on the sample spots was 14,2 W/m² [9]. Four other samples (Fig.2) were kept in control conditions during the experiment, at the constant temperature of 25°C, daily wetted and 12h dark and 12h light (ca. 50 μmol m^-2 s^-1 PAR photons). Several analyses were carried out to study all the samples before, during and after the exposure to the extreme Mars conditions. In detail, this experiment was performed aiming:

• to monitor the lichen vitality through chlorophyll a fluorescence (F_V/F_M) as photosynthetic efficiency parameter, carrying out in situ and after treatment analyses,
• to evaluate the oxidative stress due to the extreme conditions, highlighting eventual changes in the lichen carotenoids’ Raman signatures,
• to verify eventual modifications in the infrared features (peak shifting) in the lichen FTIR reflectance spectrum possibly related to UV-photodegradation effects,
• to highlight possible variations in the lichen ultrastructure through TEM analysis.

Figure 2 - Xanthoria parietina samples ready for the experiment. First row (from above): full Mars samples, second row: dark Mars samples, third row: control samples.

Figure 3 - Detail of the day-night cycles of the simulated Mars conditions (temperature, red thick line; humidity, blue thin line) and fluorescence variation values for both the treatments (FM and DM).

Results

The results showed significant differences between FM and DM photosynthetic efficiency parameter during exposure to Mars environment, exhibiting F_V/F_M values correlated with temperature and humidity day-night cycles (Fig.3). The F_V/F_M recovery values showed significant differences between the treatments too, highlighting that FM conditions caused stronger effects on fluorescence values. Additional analyses show possible changes in the Raman and FTIR spectra of the irradiated samples with several features involved. Overall, Xanthoria parietina was able to survive to FM conditions, and for this reason it may be considered a candidate for long exposure in space and evaluations on the photodegradability of parietin in extreme conditions.

Reference

[1] Onofri, S., de la Torre, R., de Vera, J. P., Ott, S., Zucconi, L., Selbmann, L., Scalzi, G., Venkateswaran, K. J., Rabbow, E., Sánchez Iñigo, F. J., and Horneck, G. (2012). Survival of rock-colonizing organisms after 1.5 years in outer space. Astrobiology, 12(5), 508-516.

[2] De Vera, J. P., Möhlmann, D., Butina, F., Lorek, A., Wernecke, R., and Ott, S. (2010). Survival potential and photosynthetic activity of lichens under Mars-like conditions: a laboratory study. Astrobiology, 10(2), 215-227.

[3] Lorenz, C., Bianchi, E., Benesperi, R., Loppi, S., Papini, A., Poggiali, G., & Brucato, J. R. (2022). Survival of Xanthoria parietina in simulated space conditions: vitality assessment and spectroscopic analysis. International Journal of Astrobiology, 1-17.

[4] Nimis P.L., 2016. ITALIC - The Information System on Italian Lichens. Version 5.0. University of Trieste, Dept. of Biology, (http://dryades.units.it/italic), accessed on 2022, 05, 09. for all data contained in the taxon pages, including notes, descriptions, and ecological indicator values. 

[5] Silberstein, L., Siegel, B., Siegel, S., Mukhtar, A., and Galun, M. (1996). Comparative Studies on Xanthoria parietina, a Pollution Resistant Lichen, and Ramalina duriaei, a Sensitive Species. I. Effects of Air Pollution on Physiological Processes. The Lichenologist, 28:355-365.

[6] Solhaug, K. A., and Gauslaa, Y. (1996). Parietin, a photoprotective secondary product of the lichen Xanthoria parietina. Oecologia, 108:412-418.

[7] Lorek, A., and Koncz, A. (2013). Simulation and measurement of extraterrestrial conditions for experiments on habitability with respect to Mars. In Habitability of Other Planets and Satellites (pp. 145-162). Springer, Dordrecht.

[8] De Vera, J. P., Schulze-Makuch, D., Khan, A., Lorek, A., Koncz, A., Möhlmann, D., and Spohn, T. (2014). Adaptation of an Antarctic lichen to Martian niche conditions can occur within 34 days. Planetary and Space Science, 98, 182-190.

[9] Cockell, C. S., Catling, D. C., Davis, W. L., Snook, K., Kepner, R. L., Lee, P., and McKay, C. P. (2000). The ultraviolet environment of Mars: biological implications past, present, and future. Icarus, 146(2), 343-359.

How to cite: Lorenz, C., Bianchi, E., Poggiali, G., Alemanno, G., Benesperi, R., Brucato, J. R., Garland, S., Helbert, J., Lorek, A., Maturilli, A., Papini, A., de Vera, J.-P., and Baqué, M.: Survivability of Xanthoria parietina in simulated Mars conditions for 30 days, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-189, https://doi.org/10.5194/epsc2022-189, 2022.

Introduction: While perchlorate (ClO₄^-) salts occur only occasionally in natural environments on Earth, they seem to be widely distributed on Mars [1]. The hygroscopicity of these salts and their potential to reduce the freezing point of pure water might enable the provision of liquid water by the formation of temporarily stable liquid brines close to the surface of Mars [2]. However, low water activities and the enhanced chaotropicity (i.e. the potential of biomacromolecule destabilization) of perchlorate pose a huge challenge for putative microorganisms in these brines [3]. Here we present the results of the first study investigating perchlorate-specific stress responses (i.e., with a significant distinction compared to general salt stress) of a halotolerant model organism, Debaryomyces hansenii, with an untargeted proteomic approach to provide fundamental understanding of the required cellular adaptation mechanisms for life in perchlorate-rich habitats on Mars.

Methodology: Growth experiments with D. hansenii were conducted in liquid growth media DMSZ #90 containing either NaCl, NaClO₄ or no additional salt. Water activities (a_w) of the growth media were measured with the Rotronics® ‘HC2-AW-USB’ a_w meter. Proteomics analyses of the grown cultures were conducted using the SPEED (Sample Preparation by Easy Extraction and Digestion) protocol described previously [4].

Results: While D. hansenii grew at NaCl concentrations of up to 4.0 mol/kg (a_w = 0.854), growth in perchlorate-containing media was obtained only at NaClO₄ concentrations up to 2.5 mol/kg (a_w = 0.926) [5]. The proteomics data [6] indicated that the chaotropicity of perchlorate is likely to be the major factor of the reduced tolerance of D. hansenii towards NaClO₄ compared to NaCl, while the perchlorate´s oxidative properties seem to play only a subordinated role in growth limitation. This is consistent with previous studies finding a high kinetic barrier for the reduction reaction of the perchlorate anion in solution [7].

The chaotropic activity of perchlorate leads to a destabilization of biomacromolecules such as proteins. Consequently, cells of D. hansenii showed upregulation of metabolic pathways involved in glycan biosynthesis, protein glycosylation and the requisite protein transport mechanisms when exposed to perchlorate stress, presumably in order to increase protein stability. Furthermore, the biosynthesis and reorganization of cell wall components, e.g. chitin, was significantly upregulated compared to NaCl-induced stress, indicating the necessity of stabilizing the cell envelope. In addition to these perchlorate-specific stress responses, many other metabolic pathways were expressed similarly in NaCl and NaClO₄-stressed cells, such as signaling pathways, elevated energy metabolism, or osmolyte biosynthesis.

Conclusions: To counteract perchlorate-induced chaotropic stresses, cells initiate metabolic pathways that stabilize biomacromolecules and cell envelopes. We hypothesize that these stress responses would also be relevant for putative Martian microorganisms, which likely developed chaotropic defense strategies in order to counteract the relatively high perchlorate concentrations in the Martian regolith. Chaotropic-specific adaptations might be the evolutionary development of stabilized confirmations of biomacromolecules in which structures with covalent bounds and cross-linking are favored over looser electrostatic interactions, hydrogen bonding or hydrophobic effects. Additionally, cell components susceptible to chaotropic stress might be stabilized by the attachment of polymers similar to stabilization effects via protein glycosylation as observed in our experiments. Characteristic biomarkers might result from these adaptions which will likely prompt further investigations.

References:

[1] Clark, B.C. and Kounaves, S.P. (2016) IJA, 15, 311–318; doi: 10.1017/S1473550415000385.

[2] Martín-Torres, F.J., et al. (2015) Nat. Geosci. 8, 357–361; doi: 10.1038/ngeo2412.

[3] Hallsworth, et al. (2007) Environ. Microbiol., 9, 801–813; doi: 10.1111/j.1462-2920.2006.01212.x.

[4] Doellinger, J., et al. (2020) MCP, 19, 209–222; doi: 10.1074/mcp.TIR119.001616.

[5] Heinz, J., et al. (2021) Life, 11; doi: 10.3390/life11111194.

[6] Heinz, J., et al. (2022) preprint at biorxiv.org; doi: 10.1101/2022.05.02.490276.

[7] Urbansky, E.T. (1998) Bioremediat. J., 2, 81–95; doi: 10.1080/10889869891214231.

How to cite: Heinz, J., Doellinger, J., Maus, D., Schneider, A., Lasch, P., Grossart, H.-P., and Schulze-Makuch, D.: Perchlorate-induced proteomic stress responses of Debaryomyces hansenii and their consequences for the habitability of Martian brines, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-275, https://doi.org/10.5194/epsc2022-275, 2022.

Laboratory simulations of the Martian conditions are essential in order to interpret results collected on Mars exploration missions. Among them, the study of the degradation and/or the evolution of possible organic biomarkers adsorbed on minerals is very important. The thin atmosphere of Mars allows ionizing radiations to reach the surface of the planet and drive the photochemical production of strong oxidants in the soil which possibly degrade the organics on the Mars´ surface¹. However, traces of ancient life forms may be found in old mineral deposits preserved within protected environments recently accessible due to erosion or impacts.It is known that mid-UV radiation is among the main degradation agents on Mars². Thus, inspection the stability and reactivity of possible biomarkers in the eolian-mobile layer and in the different fresh subsurface minerals exposed at the surface of Mars is important. Laboratory simulations of the harsh Martian conditions can evaluate the likelihood of preservation of potential biomarkers on different minerals on Mars. This provides a critical support to rover missions on Mars, helping to select the most appropriate minerals to find organic molecules and therefore, possible biomarkers.

In this work, the interaction and stability of L-histidine in saponite are studied. The saponite mineral was chosen because is a common product of low-temperature reaction between water and the mafic minerals on Mars. In addition, saponite in old cold basaltic crust is favourable for microbial life.³ L-histidine, an α- amino acid used in the biosynthesis of proteins, was chosen as biomarker because it is very diagnostic of life. Moreover, it presents different protonation states depending on the pH, which might result in different interaction with the mineral at different pHs.

The samples were prepared using the equilibrium adsorption method. In particular, pure synthetic saponite was put in contact with different solutions of L-histidine at pH 2.7 and 9.6, in order to investigate the effect of acidic or alkaline conditions on the adsorption process. The suspensions were kept for 24 hours on rotation to reach the equilibrium state during molecular adsorption, and then they were dried in an oven at 40^◦C.

The samples were analyzed by X-Ray Diffraction to understand if any intercalation of L-histidine in the negatively-charged interlayer sites of saponite occurs at acidic pH. Further characterization of molecule-mineral interactions was carried out by Transmittance and Diffuse Reflectance InfraRed Fourier Transform Spectroscopy. Finally, the samples were processed under Martian-like UV irradiation conditions using an experimental setup which allows to monitor the degradation kinetics in situ by infrared spectroscopy analysis during UV irradiation. These experiments allowed us to investigate the catalytic/protective behaviour of saponite towards L-hystidine in different pH conditions, and obtain the degradation cross section of L-histidine adsorbed on saponite compared to the pure molecule, along with the half-lifetime of degradation under Martian UV flux.

References

 ¹Quinn, R. C.; Martucci, H. F. H.; Miller, S. R.; Bryson, C. E. Perchlorate Radiolysis on Mars and the Origin of Martian Soil Reactivity. Astrobiology 2013, 13 (6), 515–520.

²Fornaro, T.; Steele, A.; Brucato, J. R. Catalytic/Protective Properties of Martian Minerals and Implications for Possible Origin of Life on Mars. Life 2018, 8.

³Y. Sueoka, S. Yamashita, M. Kouduka, Y. Suzuki, Deep Microbial Colonization in Saponite-Bearing Fractures in Aged Basaltic Crust: Implications for Subsurface Life on Mars, Frontiers in Microbiology. 10 (2019).

How to cite: Brucato, J. R., Garcia Florentino, C., Alberini, A., Fornaro, T., Madariaga, J. M., and Poggiali, G.: L-histidine in Saponite: Detection, Characterization and UV Degradation Studies for Biosignature Identification on Mars, Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-584, https://doi.org/10.5194/epsc2022-584, 2022.

Introduction. The harsh oxidative and radiative conditions of the Martian environment influence the fate of organic molecules present on its surface. Formation of radical species was suggested to transform organic macromolecules into carboxylic acid through Fenton chemistry (1, 2) or irradiation semiconductor surfaces (3). Aromatic carboxylic acids such as phthalic acid or benzoic acid are thought to be abundant on the Martian surface as they are in stable intermediate oxidation states and can be formed from the oxidation of Polycyclic Aromatic Hydrocarbons (PAHs) (1, 4) or alkylbenzene compounds (4) coming from endogenous or exogenous sources. Because benzene carboxylates are metastable, they should not be entirely oxidized into volatile molecules such as CO₂ or O₂, but instead, ionized by solar radiation to form organic salts (2, 4, 5). Benner et al. (2000) suggested that the low volatility of these salts could compromise their in situ detection through thermal extraction analyses as performed by analytic chemistry laboratories onboard Martian surface probes, such as the Sample Analyzer at Mars (SAM) experiment onboard Curiosity rover or the Mars Organic Molecular Analyzer (MOMA) instrument of the Rosalind Franklin Exomars rover (4-6). To determine the possibility to identify these molecules through direct or indirect detection on Mars, we examined laboratory results from SAM and MOMA-like Gas Chromatography-Mass Spectrometry (GC-MS) analyses of two acid/salt couples (phthalic acid/calcium phthalate and benzoic acid/calcium benzoate). We analyzed the difference in behavior and signatures of both molecular forms when using pyrolysis and wet chemistry experiments used in SAM and MOMA, and the relevance of these results in the search of organic molecules on Mars.

Method. Synthetic samples were made by mixing the carboxylic acid molecules or their organic salts standards at 1wt.% in fused silica, to simulate a relatively inert mineral matrix. The samples were pyrolyzed in SAM-like conditions with a ramp of 35°C.min^-1 and in MOMA-like conditions in flash pyrolysis at 500°C and 800°C.  The volatiles released from each sample were analyzed by Evolved Gas Analysis (EGA) and Gas Chromatography-Mass Spectrometry (GC-MS). We also performed derivatization experiments to help detect refractory organic compounds, with N,N-methyl-tert-butyl-dimethylsilyltrifluoroacetamide (MTBSTFA), used for wet chemistry experiments in SAM, and N,N-Dimethylformamide dimethyl acetal (DMF-DMA), to be used in the MOMA experiment of the ExoMars mission.

Organic acid/salt behavior under pyrolysis conditions. As predicted by Benner et al. (4), when analyzed through pyrolysis-GC-MS, the organic salts species did not produce the organic parent molecule (phthalic acid or benzoic acid (Fig. 1 (a)). However, we have identified two by-products characteristic of the degradation of the organic salts, diphenylmethane (Fig. 1 (b)) and triphenylmethane (Fig.1 (b)) which were absent of the chromatograms of the acid species. These results show that for both carboxylic acid couples studied, the acid and the salt don’t follow the same degradation pathway resulting in differences in the species detected as well as major differences in the abundance of products observed in the chromatograms. This means that if carboxylic acids are present on Mars in their saline form linked to calcium cations, we would not be able to identify it through the detection of its acid form with the SAM nor MOMA pyrolysis set-ups, but rather through the detection of characteristic by-products that would serve as indirect clues for identification.

Figure 1. Chromatograms obtained under the same analytical conditions as the pyrolysis in SAM. (a) benzoic acid mixed at 1 wt. % in fused silica and (b) calcium benzoate mixed at 1 wt. % in fused silica.

Organic acid/salt derivatized with MTBSTFA and DMFDMA.

Figure 2. Bar chart representing the abundance of derivatized benzoic acid obtained with calcium benzoate and benzoic acid. The derivatization reagent used was DMFDMA (a) and MTBSTFA (b).

When derivatized with DMFDMA or MTBSTFA, both the acid and the organic salt produced the derivatized product of the carboxylic acid. Both the phthalic acid and benzoic acids have a higher derivatization yield than their salt counterpart with both derivatization reagents. This is likely due to a better availability of the hydrogen on the carboxylic acid function. Moreover, we obtained a higher yield of both the acid and the salt with MTBSTFA than with DMF-DMA, with the loss of detection of calcium phthalate derivative with the latter. In conclusion, if present in the Martian soil, aromatic organic salts could be directly detected through wet chemistry experiments, showing the complementarity of this technique with pyrolysis.

References. (1) Oró et al. (1979) Life Sciences and Space Research, 77-86. (2) Donald et al. (2013) Science. (3) Fox et al. (2019) Journal of Geophysical Research: Planets 124, 3257-3266. (4) Benner et al. (2000) Proceedings of the National Academy of Sciences 97, 2425-2430. (5) Lasne et al. (2016) Astrobiology 16, 977-996. (6) Hakkinen et al. (2014) Environ Sci Technol 48, 13718-13726.

How to cite: Mcintosh, O., Szopa, C., Freissinet, C., Buch, A., and Boulesteix, D.: Analysis of aromatic organic salts with gas chromatography-mass spectrometry and implications for their detection at Mars surface with in situ experiments , Europlanet Science Congress 2022, Granada, Spain, 18–23 Sep 2022, EPSC2022-590, https://doi.org/10.5194/epsc2022-590, 2022.