NEW MODELORGANISMS FOR ASTROBIOLOGY FROM MARS ANALOG ENVIRONMENTS

A selection of the core questions in astrobiology deal with the origin of life on Earth, life in extreme environments on Earth, and the search for past and present life on other celestial bodies. We are therefore searching for new model-organisms for astrobiology in extreme environments, the so-called Martian analog environments, which are similar to past and present-day Mars in some characteristics and properties (anoxic conditions, low nutrient availability, high salinity, low temperatures, etc.). At the moment we are working with three facultative anaerobic model-organisms, namely Yersinia intermedia MASE-LG-1, Buttiauxella sp. MASE-IM-9, and Salinisphaera shabanensis. These organisms are being evaluated for their tolerance to Mars relevant stress factors such as desiccation, Martian atmosphere, radiation (polychromatic / monochromatic UV; ionizing radiation), oxidizing compounds (perchlorates), and the presence of an analog Martian regolith. All these influencing factors were tested under anoxic conditions as single stresses and in combination [1, 2]. The results showed that the new model-organisms for the most part clearly survived the various stress factors, thus qualifying them as possible candidates for our future space experiment called MEXEM (Mars EXposed Extremophiles Mixture). MEXEM which will be an exposure experiment which is installed on the outside of the international space station

From Mars analogue environments to space: ground data evaluation of the survivability of Buttiauxella sp. MASE-IM-9 and Salinisphaera shabanensis

Mars analogues environments are some of the most extreme locations on Earth. Their unique combination of multiple extremes (e.g. high salinity, anoxia, and low nutrient availability) make them a valuable source of new polyextremophilic microbes in general, and for exploring the limits of life. These are seen as vital sources of information for Astrobiology, with implications for planetary protection and the search for life outside our planet. Despite this well-recognized relevance, current knowledge on the capability of (facultative) anaerobic microbes as single strains or in communities to withstand extraterrestrial conditions is still very sparse. Addressing this knowledge gap is one of the main goals of the project MEXEM (Mars EXposed Extremophiles Mixture), which is in preparation at the moment. As part of MEXEM, selected model organisms from all three domains of Life, will be exposed in a 3-month passive experiment with exposure to space conditions under anoxia followed by evaluation after their arrival back on Earth. The launch to the International Space Station is currently foreseen for 2024, and implies a series of preliminary tests and data collection on some of the selected strains. Here, we report on the survivability of Salinisphaera shabanensis, isolated from a deep-sea brine pool within the Red Sea, and of Buttiauxella sp. MASE-IM-9 isolated from a German sulphidic spring after exposure to Mars relevant stress factors (like desiccation and UV-radiation under anoxic conditions). Both organisms showed survival after anoxic desiccation for up to three months but this could be further extended by adding low amounts of artificial Mars regolith (MGS- 1S; 0.5 % wt/vol) and sucrose (0.1 M). The addition of these two components resulted in an elevation of the survival rate after desiccation of up to three orders of magnitude. Survival after desiccation could even be reproduced, if the cells were mixed, as an artificial community, before desiccation treatment. The presence of these two components also positively influenced survival after exposure to polychromatic UV (200 - 400 nm) up to 12 kJ/m2 in liquid and in a desiccated form

Ignicoccus hospitalis – understanding its extraordinary radiation tolerance and an unsolved archaeal repair system

Ignicoccus hospitalis is an obligate anaerobic, hyperthermophilic and chemolithoautotrophic archaeal microorganism that has exhibited an extraordinarily high tolerance against ionizing radiation (1). It was demonstrated by Koschnitzki, 2016 that I. hospitalis cells can remain viable after exposure to X-ray doses up to 12 kGy and it can completely repair DNA damages within one hour (2). I. hospitalis has a D10-value of ~5 kGy but it can remain metabolically active after being exposed up to 118 kGy (3). This exceptional radiotolerance is unexpected since ionizing radiation is not present in its natural environment - a submarine system of hydrothermal vents (4). Given that DNA damages induced by high temperature are similar to those induced by ionizing radiation (5), we hypothesize that the radiation tolerance of I. hospitalis is a consequence of the intrinsic biological properties it uses to cope the extreme conditions of its habitat. To unravel the mechanisms involved in the radiation tolerance of I. hospitalis, two approaches are currently being followed: exploring the intracellular-specific protection and monitoring the gene regulation of the DNA repair process. Having multiple genome copies (polyploidy) might allow microbes for genomic DNA protection, maintenance, and repair at extreme conditions (6). The possibility of polyploidy in I. hospitalis was addressed. The number of genome copies per cell under different growth stages was calculated based on the quantitation of the total DNA content and the cell density from a series of culture aliquots. It was found that during the beginning of Log phase, I. hospitalis cells have 0.85±0.35 genomes/cell, in the middle of Log phase this value doubles to 1.78±0.27 genomes/cell, and at the stationary phase it drops again to 0.59±0.37 genomes/cell. Compatible solutes have been extensively studied for their role in cellular protection against severe injuring influences like osmotic stress or heat shock, and for their function as radical scavenging molecules (7). A combination of different cultivation setups, like supra-optimal growth temperatures (92.5 – 95 ˚C) and high salinity (3 – 5 % w/v) were tested to influence the accumulation of compatible solutes. Then, desiccation survival was used as an indication of their presence within the cells. No cell survival after desiccation was detected, meaning there isn’t significant compatible solutes accumulation. An alternative intracellular protection mechanism in some microorganisms is based on the intracellular manganese/iron (Mn/Fe) ratio. It has been reported that Deinococcus radiodurans accumulates high amounts of intracellular manganese and low levels of iron (8). The determination of intracellular content of these two transition metals is currently ongoing, and it will be measured by ICP-MS. A set of transcriptomics experiments are currently in progress in order to investigate the up-or-downregulation of genes related with DNA repair mechanisms. We will use dRNA-seq analysis to contrast different irradiation conditions with pre-selected time points during the DNA repair process and optimal conditions. This project will help to gain knowledge on the DNA repair mechanisms in Archaea, and to better understand the limits of life

Seasonal and diurnal variations in Martian surface ultraviolet irradiation: biological and chemical implications for the Martian regolith

The issue of the variation of the surface ultraviolet (UV) environment on Mars was investigated with particular emphasis being placed on the interpretation of data in a biological context. A UV model has been developed to yield the surface UV irradiance at any time and place over the Martian year. Seasonal and diurnal variations were calculated and dose rates evaluated. Biological interpretation of UV doses is performed through the calculation of DNA damage effects upon phage T7 and Uracil, used as examples for biological dosimeters. A solar UV 'hotspot' was revealed towards perihelion in the southern hemisphere, with a significant damaging effect upon these species. Diurnal profiles of UV irradiance are also seen to vary markedly between aphelion and perihelion. The effect of UV dose is also discussed in terms of the chemical environment of the Martian regolith, since UV irradiance can reach high enough levels so as to have a significant effect upon the soil chemistry. We show, by assuming that H2O is the main source of hydrogen in the Martian atmosphere, that the stoichiometrically desirable ratio of 2:1 for atmospheric H and O loss rates to space are not maintained and at present the ratio is about 20:1. A large planetary oxygen surface sink is therefore necessary, in contrast with escape to space. This surface oxygen sink has important implications for the oxidation potential and the toxicology of the Martian soil. UV-induced adsorption of {\rm O}_{2}^{-} super-radicals plays an important role in the oxidative environment of the Martian surface, and the biologically damaging areas found in this study are also shown to be regions of high subsurface oxidation. Furthermore, we briefly cover the astrobiological implications for landing sites that are planned for future Mars missions

The microbial diversity of the Su Bentu cave, Italy and the influence of human exploration.

Introduction: The microbial diversity in the Su Bentu Cave (Sardinia, Italy) was investigated by means of Illumina MiSeq analysis. The hypogean environment is of great interest for astrobiological research as cave conditions may resemble those in extra-terrestrial regions. Furthermore, they hold high potential to identify novel, extremely adapted organisms to severely oligo-trophic habitats. However, the influence of human is not neglectable and in-depth investigations are needed to determine the impact of exploration on an otherwise mostly pristine ecosystem. The cave investigated in this study develops for several kilometres into the mountain, two hundred metres below the topographic surface and is characterized by a strong air circulation. Its structure is composed of huge passages carved in limestone where an ephemeral underground stream creates some lakes, close to which seven samples of visible calcite rafts, manganese deposits and moonmilk (a hydrated calcium carbonate speleothem), were sampled during an expedition in 2014. Other samples were re-trieved from a frequently used campsite and from some dry cave passages leading deeper into the cave

Surviving Mars: new insights into the persistence of facultative anaerobic microbes from analogue sites

Mars analogue environments are some of the most extreme locations on Earth. Their unique combination of multiples extremes (e.g. high salinity, anoxia and low nutrient availability) make them valuable sources for finding new polyextremophilic microbes, and for exploring the limits of life. Mars, especially at its surface, is still considered to be very hostile to life but it probably possesses geological subsurface niches where the occurrence of (polyextremophilic) life is conceivable. Despite their well-recognized relevance, current knowledge on the capability of (facultative) anaerobic microbes to withstand extraterrestrial/Martian conditions, either as single strains or in communities, is still very sparse. Therefore, space experiments simulating the Martian environmental conditions by using space as a tool for astrobiological research are needed to substantiate the hypotheses of habitability of Mars. Addressing this knowledge gap is one of the main goals of the project MEXEM (Mars EXposed Extremophiles Mixture), where selected model organisms will be subjected to space for a period of 3 months. These experiments will take place on the Exobiology facility (currently under development and implementation), located outside the International Space Station. Such space experiments require a series of preliminary tests and ground data collection for the selected microbial strains. Here, we report on the survivability of Salinisphaera shabanensis and Buttiauxella sp. MASE-IM-9 after exposure to Mars-relevant stress factors (such as desiccation and ultraviolet (UV) radiation under anoxia). Both organisms showed survival after anoxic desiccation for up to 3 months but this could be further extended (nearly doubled) by adding artificial Mars regolith (MGS-1S; 0.5% wt/v) and sucrose (0.1 M). Survival after desiccation was also observed when both organisms were mixed before treatment. Mixing also positively influenced survival after exposure to polychromatic Mars-like UV radiation (200–400 nm) up to 12 kJ m−2, both in suspension and in a desiccated for

From extreme environments on Earth to space: Buttiauxella sp. MASE-IM-9 and Salinisphaera shabanensis as new model organisms in Astrobiology

Mars analogue environments are some of the most extreme locations on Earth. Their unique combination of multiples extremes (e.g. high salinity, anoxia, and low nutrient availability) make them a valuable source of new polyextremophilic microbes in general and for exploring the limits of life. These are seen as vital sources of information for Astrobiology, with implications for planetary protection and the search for life outside our planet. [...

The application of Cold Atmospheric Plasma (CAP) for the sterilisation of spacecraft components

The search for past or present extraterrestrial life is one main driver for space missions to habitable planets and moons in our solar system. Especially our neighbour planet Mars, but also Europa and Enceladus, icy moons in the outer solar system, are of high astrobiological interest. In order to find traces of life on another celestial body by in-situ measurements the instruments for life detection have to be very sensitive and the unintended contamination with organic compounds and microorganisms from Earth has to be avoided. The COSPAR’s Planetary Protection Policy and Guidelines specify the maximally allowed bioburden of spacecraft depending on the type of mission, the target and, in the case of Mars, of the landing site (Kminek et al., 2017)

An ultraviolet simulator for the incident Martian surface radiation and its applications

Ultraviolet (UV) radiation can act on putative organic/biological matter at the Martian surface in several ways. Only absorbed, but not transmitted or reflected, radiation energy can be photo-chemically effective. The most important biological UV effects are due to photochemical reactions in nucleic acids, DNA or RNA, which constitute the genetic material of all cellular organisms and viruses. Protein or lipid effects generally play a minor role, but they are also relevant in some cases. UV radiation can induce wavelengths-specific types of DNA damage. At the same time it can also induce the photo-reversion reaction of a UV induced DNA photoproduct of nucleic acid bases, the pyrimidine dimers. Intense UVB and UVC radiation, experienced on early Earth and present-day Mars, has been revealed to be harmful to all organisms, including extremophile bacteria and spores. Moreover, the formation of oxidants, catalytically produced in the Martian environment through UV irradiation, may be responsible for the destruction of organic matter on Mars. Following this, more laboratory simulations are vital in order to investigate and understand UV effects on organic matter in the case of Mars. We have designed a radiation apparatus that simulates the anticipated Martian UV surface spectrum between 200 and 400 nm (UVC-UVA). The system comprises a UV enhanced xenon arc lamp, special filter-sets and mirrors to simulate the effects of the Martian atmospheric column and dust loading. We describe the technical setup and performance of the system and discuss its uses for different applications. The design is focused on portability, therefore, the Mars-UV simulator represents a device for several different Mars simulation facilities with specific emphasis on Mars research topics

MICROORGANISMS FROM MARS ANALOGUE ENVIRONMENTS IN EARTH - COULD THEY SURVIVE ON MARS?

Assessing the habitability of Mars and detecting life, if it was ever there, depends on knowledge of whether the combined environmental stresses experienced on Mars are compatible with life and whether a record of that life could ever be detected. Many combinations of Mars relevant stress factors, such as high radiation dose rates and high UV uences combined with high salt concentrations, and low water activity, have not been investigated. In particular, the response of anaerobic organisms to Mars-like stress factors and combinations thereof are not known. In the EC project MASE (Mars Analogues for Space Exploration) we address these limitations by characterising different Mars analogue environments on Earth, isolating microorganisms from these sites and exposing them to Mars relevant stress factors alone and in combination. We want to find out, if these bacteria respond in an additive or synergistic way and if they would be able to survive on Mars. So far, eight only distantly related microorganisms are under detailed investigation, e.g Yersinia sp., Halanaerobium sp., Acidiphilum sp. Desulfovibrio sp.. Unexpectedly, a Yersinia strain turned out to be quite resistant, especially against desicca- tion and oxidising compounds, whereas a Desulfovibrio sp. strain exhibit a relatively high radiation resistance. The future experiments aim at the identification of the underlying cellu- lar and molecular mechanisms and the comparison to other new isolates from Mars analogue environments on Earth in the MASE project