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

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. [...

Assessment of the adaptability of non-fastidious pathogenic bacteria to the Martian environment.

Understanding the extent to which non-fastidious pathogenic bacteria can survive in extraterrestrial conditions will help to improve astronaut safety. Despite stringent decontamination protocols, terrestrial microorganisms were previously found to travel on the bodies of astronauts, on spaceships and equipment. This might enable the microorganisms to adapt, grow and evolve in the new environment. In this study, we assessed the adaptability of clinically relevant bacteria species, which are able to grow on carbon-containing compounds identified in carbonaceous meteorites (Klebsiella pneumoniae, Burkholderia cepacia, Serratia marcescens and Pseudomonas aeruginosa), to the simulated Martian environment. Previous work has shown that bacterial survival and growth under these conditions led to the modification of their cell envelope, thereby altering their pathogenic potential. We continued with this line of research and explored the survival of these bacterial species to a range of simulated Martian conditions i.e., desiccation, UVC (254 nm) and polychromatic UV (200 - 400 nm) irradiation, growth in the presence of perchlorates, growth on Martian simulant and exposure to Martian atmospheric composition and pressure. Preliminary results showed that growth was enhanced by the addition of Mars Global simulant (mimicking Martian regolith) to the incubation media. Furthermore, these initial results showed that only two of the strains, K. pneumoniae and S. marcescens are tolerant to desiccation, up to 16 days. The UVC irradiation experiments have shown that the bacteria with the highest degree of survival are P. aeruginosa and S. marcescens. Likewise, the same two strains have shown higher survival rates compared to K. pneumoniae and B. cepacia when exposed to polychromatic UV irradiation. To investigate the consequences of survival and growth under simulated Martian conditions, on virulence and immune recognition, a follow-up study will analyze the response of immune cells placed in contact with bacteria exposed to the Martian environment. In addition, gene expression of the adapted bacteria will be further studied. This collaborative study between the DLR (German Aerospace Center) and the Radboud UMC, in the Netherlands has provided a starting point to the investigation into the adaptability of pathogenic bacteria to Martian conditions. Further studies are required in order to improve our insight on the effects of virulence and immune recognition of the exposed pathogens. This could enable us to potentially anticipate the risks of infection and inflammation during space-travel and exploration

Microbial Monitoring in the EDEN ISS Greenhouse, a Mobile Test Facility in Antarctica

The EDEN ISS greenhouse, integrated in two joined containers, is a confined mobile test facility in Antarctica for the development and optimization of new plant cultivation techniques for future space programs. The EDEN ISS greenhouse was used successfully from February to November 2018 for fresh food production for the overwintering crew at the Antarctic Neumayer III station. During the 9 months of operation, samples from the different plants, from the nutrition solution of the aeroponic planting system, and from diverse surfaces within the three different compartments of the container were taken [future exploration greenhouse (FEG), service section (SS), and cold porch (CP)]. Quantity as well as diversity of microorganisms was examined by cultivation. In case of the plant samples, microbial quantities were in a range from 102 to 104 colony forming units (CFU) per gram plant material. Compared to plants purchased from a German grocery, the produce hosted orders of magnitude more microorganisms than the EDEN ISS plants. The EDEN ISS plant samples contained mainly fungi and a few bacteria. No classical food associated pathogenic microorganism, like Escherichia and Salmonella, could be found. Probably due to the used cultivation approach, Archaea were not found in the samples. The bioburden in the nutrition solutions increased constantly over time but never reached critical values like 10² –10³ CFU per 100 mL in irrigation water as it is stated, e.g., for commercial European plant productions. The surface samples revealed high differences in the microbial burden between the greenhouse part of the container and the SS and CP part. However, the numbers of organisms (bacteria and fungi) found in the planted greenhouse were still not critical. The microbial loaded surfaces showed strong temporal as well as spatial fluctuations. In samples of the nutrition solution and the surface, the number of bacteria exceeded the amount of fungi by many times. For identification, 16S rRNA gene sequencing was performed for the isolated prokaryotic organisms. Phylogenetic analyses revealed that the most abundant bacterial phyla were Firmicutes and Actinobacteria. These phyla include plant- and human-associated bacterial species. In general, it could be shown that it is possible to produce edible fresh food in a remote environment and this food is safe for consumption from a microbiological point of view

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

Microbial Metabolism of Amino Acids—Biologically Induced Removal of Glycine and the Resulting Fingerprint as a Potential Biosignature

The identification of reliable biomarkers, such as amino acids, is key for the search of extraterrestrial life. A large number of microorganisms metabolize, synthesize, take up and excrete amino acids as part of the amino acid metabolism during aerobic and/or anaerobic respiration or in fermentation. In this work, we investigated whether the anaerobic microbial metabolism of amino acids could leave a secondary biosignature indicating biological activity in the environment around the cells. The observed fingerprints would reflect the physiological capabilities of the specific microbial community under investigation. The metabolic processing of an amino acid mixture by two distinct anaerobic microbial communities collected from Islinger Mühlbach (ISM) and Sippenauer Moor (SM), Germany was examined. The amino acid mixture contained L-alanine, β-alanine, L-aspartic acid, DL-proline, L-leucine, L-valine, glycine, L-phenylalanine and L-isoleucine. In parallel, an amino acid spiked medium without microorganisms was used as a control to determine abiotic changes over time. Liquid chromatography mass spectrometry (LC-MS) was used to track amino acid changes over time. When comparing to the control samples that did not show significant changes of amino acids concentrations over time, we found that glycine was almost completely depleted from both microbial samples to less than 3% after the first two weeks- This results indicates a preferential use of this simple amino acid by these microbial communities. Although glycine degradation can be caused by abiotic processes, these results show that its preferential depletion in an environment would be consistent with the presence of life. We found changes in most other amino acids that varied between amino acids and communities, suggesting complex dynamics with no clear universal pattern that might be used as a signature of life. However, marked increases in amino acids, caused by cellular synthesis and release into the extracellular environment (e.g., alanine), were observed and could be considered a signature of metabolic activity. We conclude, that substantial anomalous enhancements of some amino acids against the expected abiotic background concentration may be an agnostic signature of the presence of biological processes

BIOMARKERS DETECTION IN MARS ANALOGUE SITES WITHIN MASE PROJECT

Life is a physico-chemical process by which tell-tale signals or traces are left on the environment. These signals are indicators of life and are known as biomarkers. Besides, the traces of some kinds of microorganisms can be well preserved, provided that they are rapidly mineralized and that the sediments in which they occur are rapidly cemented [1]. The search for these traces of life is one of the main objectives of Mars exploration [1] and to improve and optimize the search and detection of them forms part of MASE project targets. In MASE project (Mars Analogues for Space Exploration) we work to improve approaches and methods for biomarker detection in samples with low biomass from Mars analogue sites. A developed antibody multiarray competitive immunoassay (MACIA) for the simultaneous detection of compounds of a wide range of molecular sizes or whole spores and cells [2] [3] has revealed as suitable option to achieve this purpose

The responses of an anaerobic microorganism, Yersinia intermedia MASE-LG-1 to individual and combined simulated Martian stresses

The limits of life of aerobic microorganisms are well understood, but the responses of anaerobic microorganisms to individual and combined extreme stressors are less well known. Motivated by an interest in understanding the survivability of anaerobic microorganisms under Martian conditions, we investigated the responses of a new isolate, Yersinia intermedia MASE-LG-1 to individual and combined stresses associated with the Martian surface. This organism belongs to an adaptable and persistent genus of anaerobic microorganisms found in many environments worldwide. The effects of desiccation, low pressure, ionizing radiation, varying temperature, osmotic pressure, and oxidizing chemical compounds were investigated. The strain showed a high tolerance to desiccation, with a decline of survivability by four orders of magnitude during a storage time of 85 days. Exposure to X-rays resulted in dose-dependent inactivation for exposure up to 600 Gy while applied doses above 750 Gy led to complete inactivation. The effects of the combination of desiccation and irradiation were additive and the survivability was influenced by the order in which they were imposed. Ionizing irradiation and subsequent desiccation was more deleterious than vice versa. By contrast, the presence of perchlorates was not found to significantly affect the survival of the Yersinia strain after ionizing radiation. These data show that the organism has the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environment. Eventually it lost its viability showing that many of the most adaptable anaerobic organisms on Earth would be killed on Mars today