Taxonomic and functional analyses of intact microbial communities thriving in extreme, astrobiology-relevant, anoxic sites.

BACKGROUND: Extreme terrestrial, analogue environments are widely used models to study the limits of life and to infer habitability of extraterrestrial settings. In contrast to Earth's ecosystems, potential extraterrestrial biotopes are usually characterized by a lack of oxygen. METHODS: In the MASE project (Mars Analogues for Space Exploration), we selected representative anoxic analogue environments (permafrost, salt-mine, acidic lake and river, sulfur springs) for the comprehensive analysis of their microbial communities. We assessed the microbiome profile of intact cells by propidium monoazide-based amplicon and shotgun metagenome sequencing, supplemented with an extensive cultivation effort. RESULTS: The information retrieved from microbiome analyses on the intact microbial community thriving in the MASE sites, together with the isolation of 31 model microorganisms and successful binning of 15 high-quality genomes allowed us to observe principle pathways, which pinpoint specific microbial functions in the MASE sites compared to moderate environments. The microorganisms were characterized by an impressive machinery to withstand physical and chemical pressures. All levels of our analyses revealed the strong and omnipresent dependency of the microbial communities on complex organic matter. Moreover, we identified an extremotolerant cosmopolitan group of 34 poly-extremophiles thriving in all sites. CONCLUSIONS: Our results reveal the presence of a core microbiome and microbial taxonomic similarities between saline and acidic anoxic environments. Our work further emphasizes the importance of the environmental, terrestrial parameters for the functionality of a microbial community, but also reveals a high proportion of living microorganisms in extreme environments with a high adaptation potential within habitability borders. Video abstract.

Impact of Simulated Martian Conditions on (Facultatively) Anaerobic Bacterial Strains from Different Mars Analogue Sites.

Five bacterial (facultatively) anaerobic strains, namely Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1 isolated from different extreme natural environments were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions. These stress factors encompassed low water activity, oxidizing compounds, and ionizing radiation. Stress tests were performed under permanently anoxic conditions. The survival rate after addition of sodium perchlorate (Na-perchlorate) was found to be species-specific. The inter-comparison of the five microorganisms revealed that Clostridium sp. MASE-IM-4 was the most sensitive strain (D10-value (15 min, NaClO4) = 0.6 M). The most tolerant microorganism was Trichococcus sp. MASE-IM-5 with a calculated D10-value (15 min, NaClO4) of 1.9 M. Cultivation in the presence of Na-perchlorate in Martian relevant concentrations up to 1 wt% led to the observation of chains of cells in all strains. Exposure to Na-perchlorate led to a lowering of the survival rate after desiccation. Consecutive exposure to desiccating conditions and ionizing radiation led to additive effects. Moreover, in a desiccated state, an enhanced radiation tolerance could be observed for the strains Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5. These data show that anaerobic microorganisms from Mars analogue environments can resist a variety of Martian-simulated stresses either individually or in combination. However, responses were species-specific and some Mars-simulated extremes killed certain organisms. Thus, although Martian stresses would be expected to act differentially on microorganisms, none of the expected extremes tested here and found on Mars prevent the growth of anaerobic microorganisms.

Microbial Markers Profile in Anaerobic Mars Analogue Environments Using the LDChip (Life Detector Chip) Antibody Microarray Core of the SOLID (Signs of Life Detector) Platform.

One of the main objectives for astrobiology is to unravel and explore the habitability of environments beyond Earth, paying special attention to Mars. If the combined environmental stress factors on Mars are compatible with life or if they were less harsh in the past, to investigate the traces of past or present life is critical to understand its potential habitability. Essential for this research is the characterization of Mars analogue environments on Earth through the development of techniques for biomarker detection in them. Biosensing techniques based on fluorescence sandwich microarray immunoassays (FSMI) have shown to be a powerful tool to detect biosignatures and depict the microbial profiles of different environments. In this study, we described the microbial biomarker profile of five anoxic Mars analogues sites using the Life Detector Chip (LDChip), an antibody microarray for multiple microbial marker detection. Furthermore, we contributed to new targets by developing a new 26-polyclonal antibodies microarray using crude extracts from anaerobic sampling sites, halophilic microorganisms, and anaerobic isolates obtained in the framework of the European Mars Analogues for Space Exploration (MASE) project. The new subset of antibodies was characterized and implemented into a microarray platform (MASE-Chip) for microbial marker searching in salty and anaerobic environments.

Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS.

BIOMEX (BIOlogy and Mars EXperiment) is an ESA/Roscosmos space exposure experiment housed within the exposure facility EXPOSE-R2 outside the Zvezda module on the International Space Station (ISS). The design of the multiuser facility supports-among others-the BIOMEX investigations into the stability and level of degradation of space-exposed biosignatures such as pigments, secondary metabolites, and cell surfaces in contact with a terrestrial and Mars analog mineral environment. In parallel, analysis on the viability of the investigated organisms has provided relevant data for evaluation of the habitability of Mars, for the limits of life, and for the likelihood of an interplanetary transfer of life (theory of lithopanspermia). In this project, lichens, archaea, bacteria, cyanobacteria, snow/permafrost algae, meristematic black fungi, and bryophytes from alpine and polar habitats were embedded, grown, and cultured on a mixture of martian and lunar regolith analogs or other terrestrial minerals. The organisms and regolith analogs and terrestrial mineral mixtures were then exposed to space and to simulated Mars-like conditions by way of the EXPOSE-R2 facility. In this special issue, we present the first set of data obtained in reference to our investigation into the habitability of Mars and limits of life. This project was initiated and implemented by the BIOMEX group, an international and interdisciplinary consortium of 30 institutes in 12 countries on 3 continents. Preflight tests for sample selection, results from ground-based simulation experiments, and the space experiments themselves are presented and include a complete overview of the scientific processes required for this space experiment and postflight analysis. The presented BIOMEX concept could be scaled up to future exposure experiments on the Moon and will serve as a pretest in low Earth orbit.

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.

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

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.

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.

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.

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-, ß-, 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.

Lessons learned from the microbial analysis of the Herschel spacecraft during assembly, integration, and test operations.

Understanding microbial diversity in spacecraft assembly clean rooms is of major interest with respect to planetary protection considerations. A coordinated screening of different clean rooms in Europe and South America by three German institutes [Deutsches Zentrum für Luft- und Raumfahrt (DLR), Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), and the Institute of Microbiology and Archaea Center, University of Regensburg] took place during the assembly, test, and launch operations of the Herschel spacecraft in 2006-2009. Through this campaign, we retrieved critical information regarding the microbiome within these clean rooms and on the Herschel spacecraft, which served as a model for upcoming ESA mission preparations. This "lessons learned" document summarizes and discusses the data we obtained during this sampling campaign. Additionally, we have taken the opportunity to create a database that includes all 16S rRNA gene sequences ever retrieved from molecular and cultivable diversity studies of spacecraft assembly clean rooms to compare the microbiomes of US, European, and South American facilities.

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 NASA's and ESA's 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.

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.