Bacillus subtilis spore survival and expression of germination-induced bioluminescence after prolonged incubation under simulated Mars atmospheric pressure and composition: implications for planetary protection and lithopanspermia

Bacterial endospores in the genus Bacillus are considered good models for studying interplanetary transfer of microbes by natural or human processes. Although spore survival during transfer itself has been the subject of considerable study, the fate of spores in extraterrestrial environments has received less attention. In this report we subjected spores of a strain of Bacillus subtilis, containing luciferase resulting from expression of an sspB-luxAB gene fusion, to simulated martian atmospheric pressure (7-18 mbar) and composition (100% CO(2)) for up to 19 days in a Mars simulation chamber. We report here that survival was similar between spores exposed to Earth conditions and spores exposed up to 19 days to simulated martian conditions. However, germination-induced bioluminescence was lower in spores exposed to simulated martian atmosphere, which suggests sublethal impairment of some endogenous spore germination processes.     

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.     

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.     

Surface characteristics of spacecraft components affect the aggregation of microorganisms and may lead to different survival rates of bacteria on Mars landers

Layers of dormant endospores of Bacillus subtilis HA101 were applied to eight different spacecraft materials and exposed to martian conditions of low pressure (8.5 mbar), low temperature (-10 degrees C), and high CO(2) gas composition and irradiated with a Mars-normal ultraviolet (UV-visible- near-infrared spectrum. Bacterial layers were exposed to either 1 min or 1 h of Mars-normal UV irradiation, which simulated clear-sky conditions on equatorial Mars (0.1 tau). When exposed to 1 min of Mars UV irradiation, the numbers of viable endospores of B. subtilis were reduced three to four orders of magnitude for two brands of aluminum (Al), stainless steel, chemfilm-treated Al, clear-anodized Al, and black-anodized Al coupons. In contrast, bacterial survival was reduced only one to two orders of magnitude for endospores on the non-metal materials astroquartz and graphite composite when bacterial endospores were exposed to 1 min of Mars UV irradiation. When bacterial monolayers were exposed to 1 h of Mars UV irradiation, no viable bacteria were recovered from the six metal coupons listed above. In contrast, bacterial survival was reduced only two to three orders of magnitude for spore layers on astroquartz and graphite composite exposed to 1 h of Mars UV irradiation. Scanning electron microscopy images of the bacterial monolayers on all eight spacecraft materials revealed that endospores of B. subtilis formed large aggregates of multilayered spores on astroquartz and graphite composite, but not on the other six spacecraft materials. It is likely that the formation of multilayered aggregates of endospores on astroquartz and graphite composite is responsible for the enhanced survival of bacterial cells on these materials.     

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.     

The PROCESS experiment: amino and carboxylic acids under Mars-like surface UV radiation conditions in low-earth orbit

The search for organic molecules at the surface of Mars is a top priority of the next Mars exploration space missions: Mars Science Laboratory (NASA) and ExoMars (ESA). The detection of organic matter could provide information about the presence of a prebiotic chemistry or even biological activity on this planet. Therefore, a key step in interpretation of future data collected by these missions is to understand the preservation of organic matter in the martian environment. Several laboratory experiments have been devoted to quantifying and qualifying the evolution of organic molecules under simulated environmental conditions of Mars. However, these laboratory simulations are limited, and one major constraint is the reproduction of the UV spectrum that reaches the surface of Mars. As part of the PROCESS experiment of the European EXPOSE-E mission on board the International Space Station, a study was performed on the photodegradation of organics under filtered extraterrestrial solar electromagnetic radiation that mimics Mars-like surface UV radiation conditions. Glycine, serine, phthalic acid, phthalic acid in the presence of a mineral phase, and mellitic acid were exposed to these conditions for 1.5 years, and their evolution was determined by Fourier transform infrared spectroscopy after their retrieval. The results were compared with data from laboratory experiments. A 1.5-year exposure to Mars-like surface UV radiation conditions in space resulted in complete degradation of the organic compounds. Half-lives between 50 and 150?h for martian surface conditions were calculated from both laboratory and low-Earth orbit experiments. The results highlight that none of those organics are stable under low-Earth orbit solar UV radiation conditions.     

UV photochemistry of DNA in vitro and in Bacillus subtilis spores at earth-ambient and low atmospheric pressure: implications for spore survival on other planets or moons in the solar system

Two major parameters influencing the survival of Bacillus subtilis spores in space and on bodies within the Solar System are UV radiation and vacuum, both of which induce inactivating damage to DNA. To date, however, spore survival and DNA photochemistry have been explored only at the extremes of Earth-normal atmospheric pressure (101.3 kPa) and at simulated space vacuum (10(-3)-10(-6) Pa). In this study, wild-type spores, mutant spores lacking alpha/beta-type small, acid-soluble spore proteins (SASP), naked DNA, and complexes between SASP SspC and DNA were exposed simultaneously to UV (254 nm) at intermediate pressure (1-2 Pa), and the UV photoproducts cis,syn-thymine-thymine cyclobutane dimer (c,sTT), trans,syn-thymine-thymine cyclobutane dimer (t,sTT), and "spore photoproduct" (SP) were quantified. At 101.3 kPa, UV-treated wild-type spores accumulated only SP, but spores treated with UV radiation at 1-2 Pa exhibited a spectrum of DNA damage similar to that of spores treated at 10(-6) Pa, with accumulation of SP, c,sTT, and t,sTT. The presence or absence of alpha/beta-type SASP in spores was partly responsible for the shift observed between levels of SP and c,sTT, but not t,sTT. The changes observed in spore DNA photochemistry at 1-2 Pa in vivo were not reproduced by irradiation of naked DNA or SspC:DNA complexes in vitro, suggesting that factors other than SASP are involved in spore DNA photochemistry at low pressure.     

Preservation of martian organic and environmental records: final report of the Mars biosignature working group

The Mars Science Laboratory (MSL) has an instrument package capable of making measurements of past and present environmental conditions. The data generated may tell us if Mars is, or ever was, able to support life. However, the knowledge of Mars' past history and the geological processes most likely to preserve a record of that history remain sparse and, in some instances, ambiguous. Physical, chemical, and geological processes relevant to biosignature preservation on Earth, especially under conditions early in its history when microbial life predominated, are also imperfectly known. Here, we present the report of a working group chartered by the Co-Chairs of NASA's MSL Project Science Group, John P. Grotzinger and Michael A. Meyer, to review and evaluate potential for biosignature formation and preservation on Mars. Orbital images confirm that layered rocks achieved kilometer-scale thicknesses in some regions of ancient Mars. Clearly, interplays of sedimentation and erosional processes govern present-day exposures, and our understanding of these processes is incomplete. MSL can document and evaluate patterns of stratigraphic development as well as the sources of layered materials and their subsequent diagenesis. It can also document other potential biosignature repositories such as hydrothermal environments. These capabilities offer an unprecedented opportunity to decipher key aspects of the environmental evolution of Mars' early surface and aspects of the diagenetic processes that have operated since that time. Considering the MSL instrument payload package, we identified the following classes of biosignatures as within the MSL detection window: organism morphologies (cells, body fossils, casts), biofabrics (including microbial mats), diagnostic organic molecules, isotopic signatures, evidence of biomineralization and bioalteration, spatial patterns in chemistry, and biogenic gases. Of these, biogenic organic molecules and biogenic atmospheric gases are considered the most definitive and most readily detectable by MSL.     

Mutagenesis in bacterial spores exposed to space and simulated martian conditions: data from the EXPOSE-E spaceflight experiment PROTECT

As part of the PROTECT experiment of the EXPOSE-E mission on board the International Space Station (ISS), the mutagenic efficiency of space was studied in spores of Bacillus subtilis 168. After 1.5 years' exposure to selected parameters of outer space or simulated martian conditions, the rates of induced mutations to rifampicin resistance (Rif(R)) and sporulation deficiency (Spo(-)) were quantified. In all flight samples, both mutations, Rif(R) and Spo(-), were induced and their rates increased by several orders of magnitude. Extraterrestrial solar UV radiation (>110?nm) as well as simulated martian UV radiation (>200?nm) led to the most pronounced increase (up to nearly 4 orders of magnitude); however, mutations were also induced in flight samples shielded from insolation, which were exposed to the same conditions except solar irradiation. Nucleotide sequencing located the Rif(R) mutations in the rpoB gene encoding the β-subunit of RNA polymerase. Mutations isolated from flight and parallel mission ground reference (MGR) samples were exclusively localized to Cluster I. The 21 Rif(R) mutations isolated from the flight experiment showed all a C to T transition and were all localized to one hotspot: H482Y. In mutants isolated from the MGR, the spectrum was wider with predicted amino acid changes at residues Q469K/L/R, H482D/P/R/Y, and S487L. The data show the unique mutagenic power of space and martian surface conditions as a consequence of DNA injuries induced by solar UV radiation and space vacuum or the low pressure of Mars.     

Survival of Bacillus pumilus spores for a prolonged period of time in real space conditions

To prevent forward contamination and maintain the scientific integrity of future life-detection missions, it is important to characterize and attempt to eliminate terrestrial microorganisms associated with exploratory spacecraft and landing vehicles. Among the organisms isolated from spacecraft-associated surfaces, spores of Bacillus pumilus SAFR-032 exhibited unusually high resistance to decontamination techniques such as UV radiation and peroxide treatment. Subsequently, B. pumilus SAFR-032 was flown to the International Space Station (ISS) and exposed to a variety of space conditions via the European Technology Exposure Facility (EuTEF). After 18 months of exposure in the EXPOSE facility of the European Space Agency (ESA) on EuTEF under dark space conditions, SAFR-032 spores showed 10-40% survivability, whereas a survival rate of 85-100% was observed when these spores were kept aboard the ISS under dark simulated martian atmospheric conditions. In contrast, when UV (>110?nm) was applied on SAFR-032 spores for the same time period and under the same conditions used in EXPOSE, a ～7-log reduction in viability was observed. A parallel experiment was conducted on Earth with identical samples under simulated space conditions. Spores exposed to ground simulations showed less of a reduction in viability when compared with the "real space" exposed spores (～3-log reduction in viability for "UV-Mars," and ～4-log reduction in viability for "UV-Space"). A comparative proteomics analysis indicated that proteins conferring resistant traits (superoxide dismutase) were present in higher concentration in space-exposed spores when compared to controls. Also, the first-generation cells and spores derived from space-exposed samples exhibited elevated UVC resistance when compared with their ground control counterparts. The data generated are important for calculating the probability and mechanisms of microbial survival in space conditions and assessing microbial contaminants as risks for forward contamination and in situ life detection.     

Planetary quarantine in the solar system. Survival rates of some terrestrial organisms under simulated space conditions by proton irradiation

We have been studying the survival rates of some species of terrestrial unicellular and multicellular organism (viruses, bacteria, yeasts, fungi, algae, etc.) under simulated interstellar conditions, in connection with planetary quarantine. The interstellar environment in the solar system has been simulated by low temperature, high vacuum (77 K, 4 x 10(-8) torr), and proton irradiation from a Van de Graaff generator. After exposure to a barrage of protons corresponding to about 250 years of irradiation in solar space, tobacco mosaic virus, Bacillus subtilis spores, Staphylococcus aureus, Micrococcus flavus, Aspergillus niger spores, and Clostridium mangenoti spores showed survival rates of 82, 45, 74, 13, 28, and 25%, respectively.     

Radiation transport modeling and assessment to better predict radiation exposure, dose, and toxicological effects to human organs on long duration space flights.

NASA is very interested in improving its ability to monitor and forecast the radiation levels that pose a health risk to space-walking astronauts as they construct the International Space Station and astronauts that will participate in long-term and deep-space missions. Human exploratory missions to the moon and Mars within the next quarter century, will expose crews to transient radiation from solar particle events which include high-energy galactic cosmic rays and high-energy protons. Because the radiation levels in space are high and solar activity is presently unpredictable, adequate shielding is needed to minimize the deleterious health effects of exposure to radiation. Today, numerous models have been developed and used to predict radiation exposure. Such a model is the Space Environment Information Systems (SPENVIS) modeling program, developed by the Belgian Institute for Space Aeronautics. SPENVIS, which has been assessed to be an excellent tool in characterizing the radiation environment for microelectronics and investigating orbital debris, is being evaluated for its usefulness with determining the dose and dose-equivalent for human exposure. Thus far. the calculations for dose-depth relations under varying shielding conditions have been in agreement with calculations done using HZETRN and PDOSE, which are well-known and widely used models for characterizing the environments for human exploratory missions. There is disagreement when assessing the impact of secondary radiation particles since SPENVIS does a crude estimation of the secondary radiation particles when calculating LET versus Flux. SPENVIS was used to model dose-depth relations for the blood-forming organs. Radiation sickness and cancer are life-threatening consequences resulting from radiation exposure. In space. exposure to radiation generally includes all of the critical organs. Biological and toxicological impacts have been included for discussion along with alternative risk mitigation methods--shielding and anti-carcinogens.     

Survival of Bacillus subtilis endospores on ultraviolet-irradiated rover wheels and Mars regolith under simulated Martian conditions

Endospores of Bacillus subtilis HA101 were applied to a simulated Mars Exploration Rover (MER) wheel and exposed to Mars-normal UV irradiation for 1, 3, or 6 h. The experiment was designed to simulate a contaminated rover wheel sitting on its landing platform before rolling off onto the martian terrain, as was encountered during the Spirit and Opportunity missions. When exposed to 1 h of Mars UV, a reduction of 81% of viable endospores was observed compared to the non-UV irradiated controls. When exposed for 3 or 6 h, reductions of 94.6% and 96.6%, respectively, were observed compared to controls. In a second experiment, the contaminated rover wheel was rolled over a bed of heat-sterilized Mars analog soil; then the analog soil was exposed to full martian conditions of UV irradiation, low pressure (6.9 mbar), low temperature (-10°C), and an anaerobic CO(2) martian atmosphere for 24 h to determine whether endospores of B. subtilis on the contaminated rover wheel could be transferred to the surface of the analog soil and survive martian conditions. The experiment simulated conditions in which a rover wheel might come into contact with martian regolith immediately after landing, such as is designed for the upcoming Mars Science Laboratory (MSL) rover. The contaminated rover wheel transferred viable endospores of B. subtilis to the Mars analog soil, as demonstrated by 31.7% of samples showing positive growth. However, when contaminated soil samples were exposed to full martian conditions for 24 h, only 16.7% of samples exhibited positive growth-a 50% reduction in the number of soil samples positive for the transferred viable endospores.     

Cave biosignature suites: microbes,minerals, and Mars

Earth's subsurface offers one of the best possible sites to search for microbial life and the characteristic lithologies that life leaves behind. The subterrain may be equally valuable for astrobiology. Where surface conditions are particularly hostile, like on Mars, the subsurface may offer the only habitat for extant lifeforms and access to recognizable biosignatures. We have identified numerous unequivocally biogenic macroscopic, microscopic, and chemical/geochemical cave biosignatures. However, to be especially useful for astrobiology, we are looking for suites of characteristics. Ideally, "biosignature suites" should be both macroscopically and microscopically detectable, independently verifiable by nonmorphological means, and as independent as possible of specific details of life chemistries--demanding (and sometimes conflicting) criteria. Working in fragile, legally protected environments, we developed noninvasive and minimal impact techniques for life and biosignature detection/characterization analogous to Planetary Protection Protocols. Our difficult field conditions have shared limitations common to extraterrestrial robotic and human missions. Thus, the cave/subsurface astrobiology model addresses the most important goals from both scientific and operational points of view. We present details of cave biosignature suites involving manganese and iron oxides, calcite, and sulfur minerals. Suites include morphological fossils, mineral-coated filaments, living microbial mats and preserved biofabrics, 13C and 34S values consistent with microbial metabolism, genetic data, unusual elemental abundances and ratios, and crystallographic mineral forms.     

Spore UV and acceleration resistance of endolithic Bacillus pumilus and Bacillus subtilis isolates obtained from Sonoran desert basalt: implications for lithopanspermia

Bacterial spores have been used as model systems for studying the theory of interplanetary transport of life by natural processes such as asteroidal or cometary impacts (i.e., lithopanspermia). Because current spallation theory predicts that near-surface rocks are ideal candidates for planetary ejection and surface basalts are widely distributed throughout the rocky planets, we isolated spore-forming bacteria from the interior of near-subsurface basalt rocks collected in the Sonoran desert near Tucson, Arizona. Spores were found to inhabit basalt at very low concentrations (相似文献   

The BOSS and BIOMEX space experiments on the EXPOSE-R2 mission: Endurance of the desert cyanobacterium Chroococcidiopsis under simulated space vacuum,Martian atmosphere,UVC radiation and temperature extremes.

The proposed space experiments BOSS (Biofilm Organisms Surfing Space) and BIOMEX (BIOlogy and Mars experiment) will take place on the space exposure facility EXPOSE-R2 on the International Space Station (ISS), which is set to be launched in 2014. In BOSS the hypothesis to be tested is that microorganisms grown as biofilms, hence embedded in self-produced extracellular polymeric substances, are more tolerant to space and Martian conditions compared to their planktonic counterparts. Various microbial biofilms have been developed including those obtained from the cyanobacterium Chroococcidiopsis isolated from hot and cold deserts. The prime objective of BIOMEX is to evaluate to what extent biomolecules are resistant to, and can maintain their stability under, space and Mars-like conditions; therefore a variety of pigments and cell components are under investigation to establish a biosignature data base; e.g. a Raman spectral library to be used for extraterrestrial life biosignatures. The secondary objective of BIOMEX is to investigate the endurance of extremophiles, focusing on their interactions with Lunar and Martian mineral analogues. Ground-based studies are currently being carried out in the framework of EVTs (Experiment Verification Tests) by exposing selected organisms to space and Martian simulations. Results on a desert strain of Chroococcidiopsis obtained from the first set of EVT, e.g. space vacuum, Mars atmosphere, UVC radiation, temperature cycles and extremes, suggested that dried biofilms exhibited an enhanced survival compared to planktonic lifestyle. Moreover the protection provided by a Martian mineral analogue (S-MRS) to the sub-cellular integrities of Chroococcidiopsis against UVC radiation supports the endurance of this cyanobacterium under extraterrestrial conditions and its relevance in the development of life detection strategies.     

Issues in planetary protection: policy, protocol and implementation

Planetary protection is NASA's term for the practice of protecting solar system bodies from Earth life while protecting Earth from life that may be brought back from other solar system bodies. Spacefaring nations will soon begin retrieving samples from Mars and other solar system bodies. For these samples, planetary protection is in order, and measures are already in place to prevent the forward contamination of Mars and other bodies by Earth microbes and the backward contamination of Earth by possible extraterrestrial life. A major goal of planetary protection controls on forward contamination is to preserve the planetary record of natural processes by preventing human-caused microbial introductions.     

Estimates of Carrington-class solar particle event radiation exposures as a function of altitude in the atmosphere of Mars

Radiation exposure estimates for crew members on the surface of Mars may vary widely because of the large variations in terrain altitude. The maximum altitude difference between the highest (top of Olympus Mons) and the lowest (bottom of the Hellas impact basin) points on Mars is about 32 km. In this work estimates of radiation exposures as a function of altitude, from the Hellas impact basin to Olympus Mons, are made for a solar particle event proton radiation environment comparable to the Carrington event of 1859. We assume that the proton energy distribution for this Carrington-type event is similar to that of the Band Function fit of the February 1956 event. In this work we use the HZETRN 2010 radiation transport code, originally developed at NASA Langley Research Center, and the Computerized Anatomical Male and Female human geometry models to estimate exposures for aluminum shield areal densities similar to those provided by a spacesuit, surface lander, and permanent habitat as a function of altitude in the Mars atmosphere. Comparisons of the predicted organ exposures with current NASA Permissible Exposure Limits (PELs) are made.     

The O/OREOS mission: first science data from the Space Environment Survivability of Living Organisms (SESLO) payload

We report the first telemetered spaceflight science results from the orbiting Space Environment Survivability of Living Organisms (SESLO) experiment, executed by one of the two 10?cm cube-format payloads aboard the 5.5?kg Organism/Organic Exposure to Orbital Stresses (O/OREOS) free-flying nanosatellite. The O/OREOS spacecraft was launched successfully to a 72° inclination, 650?km Earth orbit on 19 November 2010. This satellite provides access to the radiation environment of space in relatively weak regions of Earth's protective magnetosphere as it passes close to the north and south magnetic poles; the total dose rate is about 15 times that in the orbit of the International Space Station. The SESLO experiment measures the long-term survival, germination, and growth responses, including metabolic activity, of Bacillus subtilis spores exposed to the microgravity, ionizing radiation, and heavy-ion bombardment of its high-inclination orbit. Six microwells containing wild-type (168) and six more containing radiation-sensitive mutant (WN1087) strains of dried B. subtilis spores were rehydrated with nutrient medium after 14 days in space to allow the spores to germinate and grow. Similarly, the same distribution of organisms in a different set of microwells was rehydrated with nutrient medium after 97 days in space. The nutrient medium included the redox dye Alamar blue, which changes color in response to cellular metabolic activity. Three-color transmitted intensity measurements of all microwells were telemetered to Earth within days of each of the 48?h growth experiments. We report here on the evaluation and interpretation of these spaceflight data in comparison to delayed-synchronous laboratory ground control experiments.