Maria Bohmeier

EXPOSE-E: an ESA astrobiology mission 1.5 years in space.

The multi-user facility EXPOSE-E was designed by the European Space Agency to enable astrobiology research in space (low-Earth orbit). On 7 February 2008, EXPOSE-E was carried to the International Space Station (ISS) on the European Technology Exposure Facility (EuTEF) platform in the cargo bay of Space Shuttle STS-122 Atlantis. The facility was installed at the starboard cone of the Columbus module by extravehicular activity, where it remained in space for 1.5 years. EXPOSE-E was returned to Earth with STS-128 Discovery on 12 September 2009 for subsequent sample analysis. EXPOSE-E provided accommodation in three exposure trays for a variety of astrobiological test samples that were exposed to selected space conditions: either to space vacuum, solar electromagnetic radiation at >110 nm and cosmic radiation (trays 1 and 3) or to simulated martian surface conditions (tray 2). Data on UV radiation, cosmic radiation, and temperature were measured every 10 s and downlinked by telemetry. A parallel mission ground reference (MGR) experiment was performed on ground with a parallel set of hardware and samples under simulated space conditions. EXPOSE-E performed a successful 1.5-year mission in space.

Abundance and Diversity of Microbial Inhabitants in European Spacecraft-Associated Clean Rooms

The determination of the microbial load of a spacecraft en route to interesting extraterrestrial environments is mandatory and currently based on the culturable, heat-shock-surviving portion of microbial contaminants. Our study compared these classical bioburden measurements as required by NASAs and ESAs guidelines for the microbial examination of flight hardware, with molecular analysis methods (16S rRNA gene cloning and quantitative PCR) to further develop our understanding of the diversity and abundance of the microbial communities of spacecraft-associated clean rooms. Three samplings of the Herschel Space Observatory and its surrounding clean rooms were performed in two different European facilities. Molecular analyses detected a broad diversity of microbes typically found in the human microbiome with three bacterial genera (Staphylococcus, Propionibacterium, and Brevundimonas) common to all three locations. Bioburden measurements revealed a low, but heterogeneous, abundance of spore-forming and other heat-resistant microorganisms. Total cell numbers estimated by quantitative real-time PCR were typically 3 orders of magnitude greater than those determined by viable counts, which indicates a tendency for traditional methods to underestimate the extent of clean room bioburden. Furthermore, the molecular methods allowed the detection of a much broader diversity than traditional culture-based methods.

Natural microbial populations in a water-based biowaste management system for space life support

The reutilization of wastewater is a key issue with regard to long-term space missions and planetary habitation. This study reports the design, test runs and microbiological analyses of a fixed bed biofiltration system which applies pumice grain (16-25 mm grain size, 90 m(2)/m(3) active surface) as matrix and calcium carbonate as buffer. For activation, the pumice was inoculated with garden soil known to contain a diverse community of microorganisms, thus enabling the filtration system to potentially degrade all kinds of organic matter. Current experiments over 194 days with diluted synthetic urine (7% and 20%) showed that the 7% filter units produced nitrate slowly but steadily (max. 2191 mg NO3-N/day). In the 20% units nitrate production was slower and less stable (max. 1411 mg NO3-N/day). 84% and 76% of the contained nitrogen was converted into nitrate. The low conversion rate is assumed to be due to the high flow rate, which keeps the biofilm on the pumice thin. At the same time the thin biofilm seems to prevent the activity of denitrifiers implicating the existence of a trade off between rate and the amount of nitrogen loss. Microbiological analyses identified a comparatively low number of species (26 in the filter material, 12 in the filtrate) indicating that urine serves as a strongly selective medium and filter units for the degradation of mixed feedstock have to be pre-conditioned on the intended substrates from the beginning.

Insights into the Microbial Diversity and Bioburden in a South American Spacecraft Assembly Clean Room

In this study, samples from the spacecraft assembly clean room BAF (final assembly building), located at Centre Spatial Guyanais in Kourou, French Guiana, were characterized by qualitative and quantitative methods to determine the bioburden and biodiversity. The cultivation assays mainly focused on extremotolerant microorganisms that have special metabolic skills, such as the ability to grow without oxygen, fix nitrogen, grow autotrophically, or reduce sulfate. A broad range of media and growth conditions were used to simulate possible extraterrestrial environments and clean room buildings. In addition to these alternative cultivation assays, the ESA standard protocol for bioburden estimation was also applied. The phylogenetic analysis of the isolates (mainly facultative anaerobes) showed an extraordinarily broad cultivable biodiversity. Overall, 49 species were isolated and identified as members of the bacterial phyla Actinobacteria, Firmicutes, α-, β-, γ-Proteobacteria, and Bacteroidetes/Chlorobi. In addition to cultivation-based analyses, molecular techniques were also applied, including construction of a 16S rRNA gene clone library. The results indicate a wide-ranging microbial diversity (12 bacterial phyla, 34 families) that not only confirms the results of the cultivation efforts but also deepens our understanding of the noncultivable variety. Our investigations hint at a very broad, mainly uncultivated microbial diversity.

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.

Anaerobic microorganisms in astrobiological analogue environments: from field site to culture collection

Astrobiology seeks to understand the limits of life and to determine the physiology of organisms in order to better assess the habitability of other worlds. To successfully achieve these goals we require microorganisms from environments on Earth that approximate to extraterrestrial environments in terms of physical and/or chemical conditions. The most challenging of these environments with respect to sample collection, isolation and cultivation of microorganisms are anoxic environments. In this paper, an approach to this challenge was implemented within the European Unions MASE (Mars Analogues for Space Exploration) project. In this review paper, we aim to provide a set of methods for future field work and sampling campaigns. A number of anoxic environment based on characteristics that make them analogous to past and present locations on Mars were selected. They included anoxic sulphur-rich springs (Germany), the salt-rich Boulby Mine (UK), a lake in a basaltic context (Iceland), acidic sediments in the Rio Tinto (Spain), glacier samples (Austria) and permafrost samples (Russia and Canada). Samples were collected under strict anoxic conditions to be used for cultivation and genomic community analysis. Using the samples, a culturing approach was implemented to enrich anaerobic organisms using a defined medium that would allow for organisms to be grown under identical conditions in future physiological comparisons. Anaerobic microorganisms were isolated and deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) culture collection to make them available to other scientists. In MASE, the selected organisms are studied with respect to survival and growth under Mars relevant stresses. They are artificially fossilized and the resulting biosignatures studied and used to investigate the efficacy of life detection instrumentation for planetary missions. Some of the organisms belong to genera with medical and environmental importance such as Yersinia spp., illustrating how astrobiology field research can be used to increase the availability of microbial isolates for applied terrestrial purposes.

Beyond Chloride Brines: Variable Metabolomic Responses in the Anaerobic Organism Yersinia intermedia MASE-LG-1 to NaCl and MgSO4 at Identical Water Activity

Growth in sodium chloride (NaCl) is known to induce stress in non-halophilic microorganisms leading to effects on the microbial metabolism and cell structure. Microorganisms have evolved a number of adaptations, both structural and metabolic, to counteract osmotic stress. These strategies are well-understood for organisms in NaCl-rich brines such as the accumulation of certain organic solutes (known as either compatible solutes or osmolytes). Less well studied are responses to ionic environments such as sulfate-rich brines which are prevalent on Earth but can also be found on Mars. In this paper, we investigated the global metabolic response of the anaerobic bacterium Yersinia intermedia MASE-LG-1 to osmotic salt stress induced by either magnesium sulfate (MgSO4) or NaCl at the same water activity (0.975). Using a non-targeted mass spectrometry approach, the intensity of hundreds of metabolites was measured. The compatible solutes L-asparagine and sucrose were found to be increased in both MgSO4 and NaCl compared to the control sample, suggesting a similar osmotic response to different ionic environments. We were able to demonstrate that Yersinia intermedia MASE-LG-1 accumulated a range of other compatible solutes. However, we also found the global metabolic responses, especially with regard to amino acid metabolism and carbohydrate metabolism, to be salt-specific, thus, suggesting ion-specific regulation of specific metabolic pathways.

Mineralization and Preservation of an extremotolerant Bacterium Isolated from an Early Mars Analog Environment

The artificial mineralization of a polyresistant bacterial strain isolated from an acidic, oligotrophic lake was carried out to better understand microbial (i) early mineralization and (ii) potential for further fossilisation. Mineralization was conducted in mineral matrixes commonly found on Mars and Early-Earth, silica and gypsum, for 6 months. Samples were analyzed using microbiological (survival rates), morphological (electron microscopy), biochemical (GC-MS, Microarray immunoassay, Rock-Eval) and spectroscopic (EDX, FTIR, RAMAN spectroscopy) methods. We also investigated the impact of physiological status on mineralization and long-term fossilisation by exposing cells or not to Mars-related stresses (desiccation and radiation). Bacterial populations remained viable after 6 months although the kinetics of mineralization and cell-mineral interactions depended on the nature of minerals. Detection of biosignatures strongly depended on analytical methods, successful with FTIR and EDX but not with RAMAN and immunoassays. Neither influence of stress exposure, nor qualitative and quantitative changes of detected molecules were observed as a function of mineralization time and matrix. Rock-Eval analysis suggests that potential for preservation on geological times may be possible only with moderate diagenetic and metamorphic conditions. The implications of our results for microfossil preservation in the geological record of Earth as well as on Mars are discussed.

On the Stability of Deinoxanthin Exposed to Mars Conditions during a Long-Term Space Mission and Implications for Biomarker Detection on Other Planets

Outer space, the final frontier, is a hostile and unforgiving place for any form of life as we know it. The unique environment of space allows for a close simulation of Mars surface conditions that cannot be simulated as accurately on the Earth. For this experiment, we tested the resistance of Deinococcus radiodurans to survive exposure to simulated Mars-like conditions in low-Earth orbit for a prolonged period of time as part of the Biology and Mars experiment (BIOMEX) project. Special focus was placed on the integrity of the carotenoid deinoxanthin, which may serve as a potential biomarker to search for remnants of life on other planets. Survival was investigated by evaluating colony forming units, damage inflicted to the 16S rRNA gene by quantitative PCR, and the integrity and detectability of deinoxanthin by Raman spectroscopy. Exposure to space conditions had a strong detrimental effect on the survival of the strains and the 16S rRNA integrity, yet results show that deinoxanthin survives exposure to conditions as they prevail on Mars. Solar radiation is not only strongly detrimental to the survival and 16S rRNA integrity but also to the Raman signal of deinoxanthin. Samples not exposed to solar radiation showed only minuscule signs of deterioration. To test whether deinoxanthin is able to withstand the tested parameters without the protection of the cell, it was extracted from cell homogenate and exposed to high/low temperatures, vacuum, germicidal UV-C radiation, and simulated solar radiation. Results obtained by Raman investigations showed a strong resistance of deinoxanthin against outer space and Mars conditions, with the only exception of the exposure to simulated solar radiation. Therefore, deinoxanthin proved to be a suitable easily detectable biomarker for the search of Earth-like organic pigment-containing life on other planets.

Lack of correlation of desiccation and radiation tolerance in microorganisms from diverse extreme environments tested under anoxic conditions

Abstract Four facultative anaerobic and two obligate anaerobic bacteria were isolated from extreme environments (deep subsurface halite mine, sulfidic anoxic spring, mineral-rich river) in the frame MASE (Mars Analogues for Space Exploration) project. The isolates were investigated under anoxic conditions for their survivability after desiccation up to 6 months and their tolerance to ionizing radiation up to 3000 Gy. The results indicated that tolerances to both stresses are strain-specific features. Yersinia intermedia MASE-LG-1 showed a high desiccation tolerance but its radiation tolerance was very low. The most radiation-tolerant strains were Buttiauxella sp. MASE-IM-9 and Halanaerobium sp. MASE-BB-1. In both cases, cultivable cells were detectable after an exposure to 3 kGy of ionizing radiation, but cells only survived desiccation for 90 and 30 days, respectively. Although a correlation between desiccation and ionizing radiation resistance has been hypothesized for some aerobic microorganisms, our data showed that there was no correlation between tolerance to desiccation and ionizing radiation, suggesting that the physiological basis of both forms of tolerances is not necessarily linked. In addition, these results indicated that facultative and obligate anaerobic organisms living in extreme environments possess varied species-specific tolerances to extremes.