EXPOSE, an astrobiological exposure facility on the international space station - from proposal to flight.

Following an European Space Agency announcement of opportunity in 1996 for "Externally mounted payloads for 1st utilization phase" on the International Space Station (ISS), scientists working in the fields of astrobiology proposed experiments aiming at longterm exposure of a variety of chemical compounds and extremely resistant microorganisms to the hostile space environment. The ESA exposure facility EXPOSE was built and an operations' concept was prepared. The EXPOSE experiments were developed through an intensive pre-flight experiment verification test program. 12 years later, two sets of astrobiological experiments in two EXPOSE facilities have been successfully launched to the ISS for external exposure for up to 1.5 years. EXPOSE-E, now installed at the balcony of the European Columbus module, was launched in February 2008, while EXPOSE-R took off to the ISS in November 2008 and was installed on the external URM-D platform of the Russian Zvezda module in March 2009.

Tolerances of Deinococcus geothermalis Biofilms and Planktonic Cells Exposed to Space and Simulated Martian Conditions in Low Earth Orbit for Almost Two Years.

Fossilized biofilms represent one of the oldest known confirmations of life on the Earth. The success of microbes in biofilms results from properties that are inherent in the biofilm, including enhanced interaction, protection, and biodiversity. Given the diversity of microbes that live in biofilms in harsh environments on the Earth, it is logical to hypothesize that, if microbes inhabit other bodies in the Universe, there are also biofilms on those bodies. The Biofilm Organisms Surfing Space experiment was conducted as part of the EXPOSE-R2 mission on the International Space Station. The experiment was an international collaboration designed to perform a comparative study regarding the survival of biofilms versus planktonic cells of various microorganisms, exposed to space and Mars-like conditions. The objective was to determine whether there are lifestyle-dependent differences to cope with the unique mixture of stress factors, including desiccation, temperature oscillations, vacuum, or a Mars-like gas atmosphere and pressure in combination with extraterrestrial or Mars-like ultraviolet (UV) radiation residing during the long-term space mission. In this study, the outcome of the flight and mission ground reference analysis of Deinococcus geothermalis is presented. Cultural tests demonstrated that D. geothermalis remained viable in the desiccated state, being able to survive space and Mars-like conditions and tolerating high extraterrestrial UV radiation for more than 2 years. Culturability decreased, but was better preserved, in the biofilm consortium than in planktonic cells. These results are correlated to differences in genomic integrity after exposure, as visualized by random amplified polymorphic DNA-polymerase chain reaction. Interestingly, cultivation-independent viability markers such as membrane integrity, ATP content, and intracellular esterase activity remained nearly unaffected, indicating that subpopulations of the cells had survived in a viable but nonculturable state. These findings support the hypothesis of long-term survival of microorganisms under the harsh environmental conditions in space and on Mars to a higher degree if exposed as biofilm.

Survival of Deinococcus geothermalis in Biofilms under Desiccation and Simulated Space and Martian Conditions.

Biofilm formation represents a successful survival strategy for bacteria. In biofilms, cells are embedded in a matrix of extracellular polymeric substances (EPS). As they are often more stress-tolerant than single cells, biofilm cells might survive the conditions present in space and on Mars. To investigate this topic, the bacterium Deinococcus geothermalis was chosen as a model organism due to its tolerance toward desiccation and radiation. Biofilms cultivated on membranes and, for comparison, planktonically grown cells deposited on membranes were air-dried and exposed to individual stressors that included prolonged desiccation, extreme temperatures, vacuum, simulated martian atmosphere, and UV irradiation, and they were exposed to combinations of stressors that simulate space (desiccation + vacuum + UV) or martian (desiccation + Mars atmosphere + UV) conditions. The effect of sulfatic Mars regolith simulant on cell viability during stress was investigated separately. The EPS produced by the biofilm cells contained mainly polysaccharides and proteins. To detect viable but nonculturable (VBNC) cells, cultivation-independent viability indicators (membrane integrity, ATP, 16S rRNA) were determined in addition to colony counts. Desiccation for 2 months resulted in a decrease of culturability with minor changes of membrane integrity in biofilm cells and major loss of membrane integrity in planktonic bacteria. Temperatures between -25°C and +60°C, vacuum, and Mars atmosphere affected neither culturability nor membrane integrity in both phenotypes. Monochromatic (254 nm; ≥1 kJ m-2) and polychromatic (200-400 nm; >5.5 MJ m-2 for planktonic cells and >270 MJ m-2 for biofilms) UV irradiation significantly reduced the culturability of D. geothermalis but did not affect cultivation-independent viability markers, indicating the induction of a VBNC state in UV-irradiated cells. In conclusion, a substantial proportion of the D. geothermalis population remained viable under all stress conditions tested, and in most cases the biofilm form proved advantageous for surviving space and Mars-like conditions. Key Words: Biofilms-Desiccation-UV radiation-Mars-Lithopanspermia. Astrobiology 17, 431-447.

EXPOSE-R2: The Astrobiological ESA Mission on Board of the International Space Station.

On July 23, 2014, the Progress cargo spacecraft 56P was launched from Baikonur to the International Space Station (ISS), carrying EXPOSE-R2, the third ESA (European Space Agency) EXPOSE facility, the second EXPOSE on the outside platform of the Russian Zvezda module, with four international astrobiological experiments into space. More than 600 biological samples of archaea, bacteria (as biofilms and in planktonic form), lichens, fungi, plant seeds, triops eggs, mosses and 150 samples of organic compounds were exposed to the harsh space environment and to parameters similar to those on the Mars surface. Radiation dosimeters distributed over the whole facility complemented the scientific payload. Three extravehicular activities later the chemical samples were returned to Earth on March 2, 2016, with Soyuz 44S, having spent 588 days in space. The biological samples arrived back later, on June 18, 2016, with 45S, after a total duration in space of 531 days. The exposure of the samples to Low Earth Orbit vacuum lasted for 531 days and was divided in two parts: protected against solar irradiation during the first 62 days, followed by exposure to solar radiation during the subsequent 469 days. In parallel to the space mission, a Mission Ground Reference (MGR) experiment with a flight identical Hardware and a complete flight identical set of samples was performed at the premises of DLR (German Aerospace Center) in Cologne by MUSC (Microgravity User Support Center), according to the mission data either downloaded from the ISS (temperature data, facility status, inner pressure status) or provided by RedShift Design and Engineering BVBA, Belgium (calculated ultra violet radiation fluence data). In this paper, the EXPOSE-R2 facility, the experimental samples, mission parameters, environmental parameters, and the overall mission and MGR sequences are described, building the background for the research papers of the individual experiments, their analysis and results.

Exposure of phage T7 to simulated space environment: the effect of vacuum and UV-C radiation.

The experiment "Phage and Uracil Response" (PUR) will be accommodated in the EXPOSE facility of the International Space Station (ISS). Its objective is to examine and quantify the effect of specific space conditions on bacteriophage T7 and isolated T7 DNA thin films. In order to define the environmental and technical requirements of the EXPOSE, the samples were subjected to the Experiment Verification Test (EVT). During EVT the samples were exposed to selected space conditions: high vacuum (10(-4) to 10(-6) Pa) and UV-C radiation (254 nm) alone and in combination. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA/phage and the decrease of the amount of PCR products have been detected indicating the damage of isolated and intraphage T7 DNA. Intraphage DNA is more sensitive to simulated space parameters than isolated T7 DNA in thin layers as well. We obtained substantial evidence that DNA lesions accumulate throughout exposure, and the amount of damage depends on the thickness of the layers. According to our preliminary results, the damages by exposure to conditions of dehydration and UV irradiation are larger than the sum of vacuum alone, or radiation alone case, suggesting a synergistic action of space vacuum and UV radiation with DNA being the critical target.

Effects of a simulated martian UV flux on the cyanobacterium, Chroococcidiopsis sp. 029.

Dried monolayers of Chroococcidiopsis sp. 029, a desiccation-tolerant, endolithic cyanobacterium, were exposed to a simulated martian-surface UV and visible light flux, which may also approximate to the worst-case scenario for the Archean Earth. After 5 min, there was a 99% loss of cell viability, and there were no survivors after 30 min. However, this survival was approximately 10 times higher than that previously reported for Bacillus subtilis. We show that under 1 mm of rock, Chroococcidiopsis sp. could survive (and potentially grow) under the high martian UV flux if water and nutrient requirements for growth were met. In isolated cells, phycobilisomes and esterases remained intact hours after viability was lost. Esterase activity was reduced by 99% after a 1-h exposure, while 99% loss of autofluorescence required a 4-h exposure. However, cell morphology was not changed, and DNA was still detectable by 4',6-diamidino-2-phenylindole staining after an 8-h exposure (equivalent to approximately 1 day on Mars at the equator). Under 1 mm of simulant martian soil or gneiss, the effect of UV radiation could not be detected on esterase activity or autofluorescence after 4 h. These results show that under the intense martian UV flux the morphological signatures of life can persist even after viability, enzymatic activity, and pigmentation have been destroyed. Finally, the global dispersal of viable, isolated cells of even this desiccation-tolerant, ionizing-radiation-resistant microorganism on Mars is unlikely as they are killed quickly by unattenuated UV radiation when in a desiccated state. These findings have implications for the survival of diverse microbial contaminants dispersed during the course of human exploratory class missions on the surface of Mars.

Space experiment "Cellular Responses to Radiation in Space (CellRad)": Hardware and biological system tests.

One factor contributing to the high uncertainty in radiation risk assessment for long-term space missions is the insufficient knowledge about possible interactions of radiation with other spaceflight environmental factors. Such factors, e.g. microgravity, have to be considered as possibly additive or even synergistic factors in cancerogenesis. Regarding the effects of microgravity on signal transduction, it cannot be excluded that microgravity alters the cellular response to cosmic radiation, which comprises a complex network of signaling pathways. The purpose of the experiment "Cellular Responses to Radiation in Space" (CellRad, formerly CERASP) is to study the effects of combined exposure to microgravity, radiation and general space flight conditions on mammalian cells, in particular Human Embryonic Kidney (HEK) cells that are stably transfected with different plasmids allowing monitoring of proliferation and the Nuclear Factor κB (NF-κB) pathway by means of fluorescent proteins. The cells will be seeded on ground in multiwell plate units (MPUs), transported to the ISS, and irradiated by an artificial radiation source after an adaptation period at 0 × g and 1 × g. After different incubation periods, the cells will be fixed by pumping a formaldehyde solution into the MPUs. Ground control samples will be treated in the same way. For implementation of CellRad in the Biolab on the International Space Station (ISS), tests of the hardware and the biological systems were performed. The sequence of different steps in MPU fabrication (cutting, drilling, cleaning, growth surface coating, and sterilization) was optimized in order to reach full biocompatibility. Different coatings of the foil used as growth surface revealed that coating with 0.1 mg/ml poly-D-lysine supports cell attachment better than collagen type I. The tests of prototype hardware (Science Model) proved its full functionality for automated medium change, irradiation and fixation of cells. Exposure of HEK cells to the ß-rays emitted by the radiation source dose-dependently decreased cell growth and increased NF-κB activation. The signal of the fluorescent proteins after formaldehyde fixation was stable for at least six months after fixation, allowing storage of the MPUs after fixation for several months before the transport back to Earth and evaluation of the fluorescence intensity. In conclusion, these tests show the feasibility of CellRad on the ISS with the currently available transport mechanisms.

Resistance of bacterial endospores to outer space for planetary protection purposes--experiment PROTECT of the EXPOSE-E mission.

Spore-forming bacteria are of particular concern in the context of planetary protection because their tough endospores may withstand certain sterilization procedures as well as the harsh environments of outer space or planetary surfaces. To test their hardiness on a hypothetical mission to Mars, spores of Bacillus subtilis 168 and Bacillus pumilus SAFR-032 were exposed for 1.5 years to selected parameters of space in the experiment PROTECT during the EXPOSE-E mission on board the International Space Station. Mounted as dry layers on spacecraft-qualified aluminum coupons, the "trip to Mars" spores experienced space vacuum, cosmic and extraterrestrial solar radiation, and temperature fluctuations, whereas the "stay on Mars" spores were subjected to a simulated martian environment that included atmospheric pressure and composition, and UV and cosmic radiation. The survival of spores from both assays was determined after retrieval. It was clearly shown that solar extraterrestrial UV radiation (λ≥110 nm) as well as the martian UV spectrum (λ≥200 nm) was the most deleterious factor applied; in some samples only a few survivors were recovered from spores exposed in monolayers. Spores in multilayers survived better by several orders of magnitude. All other environmental parameters encountered by the "trip to Mars" or "stay on Mars" spores did little harm to the spores, which showed about 50% survival or more. The data demonstrate the high chance of survival of spores on a Mars mission, if protected against solar irradiation. These results will have implications for planetary protection considerations.

Survival of spores of the UV-resistant Bacillus subtilis strain MW01 after exposure to low-earth orbit and simulated martian conditions: data from the space experiment ADAPT on EXPOSE-E.

In the space experiment "Molecular adaptation strategies of microorganisms to different space and planetary UV climate conditions" (ADAPT), bacterial endospores of the highly UV-resistant Bacillus subtilis strain MW01 were exposed to low-Earth orbit (LEO) and simulated martian surface conditions for 559 days on board the European Space Agency's exposure facility EXPOSE-E, mounted outside the International Space Station. The survival of B. subtilis MW01 spores from both assays (LEO and simulated martian conditions) was determined by a colony-formation assay after retrieval. It was clearly shown that solar extraterrestrial UV radiation (λ≥110 nm) as well as the martian UV spectrum (λ≥200 nm) was the most deleterious factor applied; in some samples only a few spore survivors were recovered from B. subtilis MW01 spores exposed in monolayers. However, if shielded from solar irradiation, about 8% of MW01 spores survived in LEO conditions, and 100% survived in simulated martian conditions, compared to the laboratory controls. The results demonstrate the effect of shielding against the high inactivation potential of extraterrestrial solar UV radiation, which limits the chances of survival of even the highly UV-resistant strain of B. subtilis MW01 in the harsh environments of outer space and the martian surface.

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.