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.     

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.     

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.     

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.     

Oxidant enhancement in martian dust devils and storms: implications for life and habitability

We investigate a new mechanism for producing oxidants, especially hydrogen peroxide (H2O2), on Mars. Large-scale electrostatic fields generated by charged sand and dust in the martian dust devils and storms, as well as during normal saltation, can induce chemical changes near and above the surface of Mars. The most dramatic effect is found in the production of H2O2 whose atmospheric abundance in the "vapor" phase can exceed 200 times that produced by photochemistry alone. With large electric fields, H2O2 abundance gets large enough for condensation to occur, followed by precipitation out of the atmosphere. Large quantities of H2O2 would then be adsorbed into the regolith, either as solid H2O2 "dust" or as re-evaporated vapor if the solid does not survive as it diffuses from its production region close to the surface. We suggest that this H2O2, or another superoxide processed from it in the surface, may be responsible for scavenging organic material from Mars. The presence of H2O2 in the surface could also accelerate the loss of methane from the atmosphere, thus requiring a larger source for maintaining a steady-state abundance of methane on Mars. The surface oxidants, together with storm electric fields and the harmful ultraviolet radiation that readily passes through the thin martian atmosphere, are likely to render the surface of Mars inhospitable to life as we know it.     

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 and germinability of Bacillus subtilis spores exposed to simulated Mars solar radiation: implications for life detection and planetary protection

Bacterial spores have been considered as microbial life that could survive interplanetary transport by natural impact processes or human spaceflight activity. Deposition of terrestrial microbes or their biosignature molecules onto the surface of Mars could negatively impact life detection experiments and planetary protection measures. Simulated Mars solar radiation, particularly the ultraviolet component, has been shown to reduce spore viability, but its effect on spore germination and resulting production of biosignature molecules has not been explored. We examined the survival and germinability of Bacillus subtilis spores exposed to simulated martian conditions that include solar radiation. Spores of B. subtilis that contain luciferase resulting from expression of an sspB-luxAB gene fusion were deposited on aluminum coupons to simulate deposition on spacecraft surfaces and exposed to simulated Mars atmosphere and solar radiation. The equivalent of 42 min of simulated Mars solar radiation exposure reduced spore viability by nearly 3 logs, while germination-induced bioluminescence, a measure of germination metabolism, was reduced by less than 1 log. The data indicate that spores can retain the potential to initiate germination-associated metabolic processes and produce biological signature molecules after being rendered nonviable by exposure to Mars solar radiation.     

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.     

Bacterial growth at the high concentrations of magnesium sulfate found in martian soils

The martian surface environment exhibits extremes of salinity, temperature, desiccation, and radiation that would make it difficult for terrestrial microbes to survive. Recent evidence suggests that martian soils contain high concentrations of MgSO? minerals. Through warming of the soils, meltwater derived from subterranean ice-rich regolith may exist for an extended period of time and thus allow the propagation of terrestrial microbes and create significant bioburden at the near surface of Mars. The current report demonstrates that halotolerant bacteria from the Great Salt Plains (GSP) of Oklahoma are capable of growing at high concentrations of MgSO? in the form of 2 M solutions of epsomite. The epsotolerance of isolates in the GSP bacterial collection was determined, with 35% growing at 2 M MgSO?. There was a complex physiological response to mixtures of MgSO? and NaCl coupled with other environmental stressors. Growth also was measured at 1 M concentrations of other magnesium and sulfate salts. The complex responses may be partially explained by the pattern of chaotropicity observed for high-salt solutions as measured by agar gelation temperature. Select isolates could grow at the high salt concentrations and low temperatures found on Mars. Survival during repetitive freeze-thaw or drying-rewetting cycles was used as other measures of potential success on the martian surface. Our results indicate that terrestrial microbes might survive under the high-salt, low-temperature, anaerobic conditions on Mars and present significant potential for forward contamination. Stringent planetary protection requirements are needed for future life-detection missions to Mars.     

The physics,biology, and environmental ethics of making mars habitable

The considerable evidence that Mars once had a wetter, more clement, environment motivates the search for past or present life on that planet. This evidence also suggests the possibility of restoring habitable conditions on Mars. While the total amounts of the key molecules--carbon dioxide, water, and nitrogen--needed for creating a biosphere on Mars are unknown, estimates suggest that there may be enough in the subsurface. Super greenhouse gases, in particular, perfluorocarbons, are currently the most effective and practical way to warm Mars and thicken its atmosphere so that liquid water is stable on the surface. This process could take approximately 100 years. If enough carbon dioxide is frozen in the South Polar Cap and absorbed in the regolith, the resulting thick and warm carbon dioxide atmosphere could support many types of microorganisms, plants, and invertebrates. If a planet-wide martian biosphere converted carbon dioxide into oxygen with an average efficiency equal to that for Earth's biosphere, it would take > 100,000 years to create Earth-like oxygen levels. Ethical issues associated with bringing life to Mars center on the possibility of indigenous martian life and the relative value of a planet with or without a global biosphere.     

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.     

Oxidant enhancement in martian dust devils and storms: storm electric fields and electron dissociative attachment

Laboratory studies, numerical simulations, and desert field tests indicate that aeolian dust transport can generate atmospheric electricity via contact electrification or "triboelectricity." In convective structures such as dust devils and dust storms, grain stratification leads to macroscopic charge separations and gives rise to an overall electric dipole moment in the aeolian feature, similar in nature to the dipolar electric field generated in terrestrial thunderstorms. Previous numerical simulations indicate that these storm electric fields on Mars can approach the ambient breakdown field strength of approximately 25 kV/m. In terrestrial dust phenomena, potentials ranging from approximately 20 to 160 kV/m have been directly measured. The large electrostatic fields predicted in martian dust devils and storms can energize electrons in the low pressure martian atmosphere to values exceeding the electron dissociative attachment energy of both CO2 and H2O, which results in the formation of the new chemical products CO/O- and OH/H-, respectively. Using a collisional plasma physics model, we present calculations of the CO/O- and OH/H- reaction and production rates. We demonstrate that these rates vary geometrically with the ambient electric field, with substantial production of dissociative products when fields approach the breakdown value of approximately 25 kV/m. The dissociation of H2O into OH/H- provides a key ingredient for the generation of oxidants; thus electrically charged dust may significantly impact the habitability of Mars.     

An extensive phase space for the potential martian biosphere

We present a comprehensive model of martian pressure-temperature (P-T) phase space and compare it with that of Earth. Martian P-T conditions compatible with liquid water extend to a depth of ～310?km. We use our phase space model of Mars and of terrestrial life to estimate the depths and extent of the water on Mars that is habitable for terrestrial life. We find an extensive overlap between inhabited terrestrial phase space and martian phase space. The lower martian surface temperatures and shallower martian geotherm suggest that, if there is a hot deep biosphere on Mars, it could extend 7 times deeper than the ～5?km depth of the hot deep terrestrial biosphere in the crust inhabited by hyperthermophilic chemolithotrophs. This corresponds to ～3.2% of the volume of present-day Mars being potentially habitable for terrestrial-like life.     

On the existence and stability of liquid water on the surface of mars today

The recent discovery of high concentrations of hydrogen just below the surface of Mars' polar regions by Mars Odyssey has enlivened the debate about past or present life on Mars. The prevailing assumption prior to the discovery was that the liquid water essential for its existence is absent. That assumption was based largely on the calculation of heat and mass transfer coefficients or theoretical climate models. This research uses an experimental approach to determine the feasibility of liquid water under martian conditions, setting the stage for a more empirical approach to the question of life on Mars. Experiments were conducted in three parts: Liquid water's existence was confirmed by droplets observed under martian conditions in part 1; the evolution of frost melting on the surface of various rocks under martian conditions was observed in part 2; and the evaporation rate of water in Petri dishes under Mars-like conditions was determined and compared with the theoretical predictions of various investigators in part 3. The results led to the conclusion that liquid water can be stable for extended periods of time on the martian surface under present-day conditions.     

Magnetism, iron minerals, and life on Mars

A short critical review is provided on two questions linking magnetism and possible early life on Mars: (1) Did Mars have an Earth-like internal magnetic field, and, if so, during which period and was it a requisite for life? (2) Is there a connection between iron minerals in the martian regolith and life? We also discuss the possible astrobiological implications of magnetic measurements at the surface of Mars using two proposed instruments. A magnetic remanence device based on magnetic field measurements can be used to identify Noachian age rocks and lightning impacts. A contact magnetic susceptibility probe can be used to investigate weathering rinds on martian rocks and identify meteorites among the small regolith rocks. Both materials are considered possible specific niches for microorganisms and, thus, potential astrobiological targets. Experimental results on analogues are presented to support the suitability of such in situ measurements.     

火星探测用金属/CO2燃烧技术研究进展与展望

火星原位资源利用指利用火星当地资源生产火星探测所需原料和能源,减少任务载荷,降低发射成本,是火星探测不可或缺的关键技术。金属和二氧化碳是火星重要的原位资源,部分金属可以在二氧化碳气氛中燃烧,使得金属/CO_2燃烧体系在火星上扮演地球上化石燃料/空气燃烧体系的角色成为可能。从拓展金属/CO_2燃烧技术在火星探测中应用的角度出发,梳理了火星二氧化碳收集方式、火星矿物分布和冶炼、金属/CO_2燃烧技术的主要应用方式(Mg/CO_2火箭发动机和Mg/CO_2金属燃烧器)的研究进展,并对今后的研究进行了展望。     

Low-temperature ionizing radiation resistance of Deinococcus radiodurans and Antarctic Dry Valley bacteria

The high flux of cosmic rays onto the unshielded surface of Mars poses a significant hazard to the survival of martian microbial life. Here, we determined the survival responses of several bacterial strains to ionizing radiation exposure while frozen at a low temperature characteristic of the martian near-subsurface. Novel psychrotolerant bacterial strains were isolated from the Antarctic Dry Valleys, an environmental analogue of the martian surface, and identified by 16S rRNA gene phylogeny as representatives of Brevundimonas, Rhodococcus, and Pseudomonas genera. These isolates, in addition to the known radioresistant extremophile Deinococcus radiodurans, were exposed to gamma rays while frozen on dry ice (-79°C). We found D. radiodurans to exhibit far greater radiation resistance when irradiated at -79°C than was observed in similar studies performed at higher temperatures. This greater radiation resistance has important implications for the estimation of potential survival times of microorganisms near the martian surface. Furthermore, the most radiation resistant of these Dry Valley isolates, Brevundimonas sp. MV.7, was found to show 99% 16S rRNA gene similarity to contaminant bacteria discovered in clean rooms at both Kennedy and Johnson Space Centers and so is of prime concern to efforts in the planetary protection of Mars from our lander probes. Results from this experimental irradiation, combined with previous radiation modeling, indicate that Brevundimonas sp. MV.7 emplaced only 30?cm deep in martian dust could survive the cosmic radiation for up to 100,000 years before suffering 10? population reduction.     

Special regions in Mars exploration: Problems and potential

“Special regions” on Mars are areas designated in the COSPAR planetary protection policy as areas that may support Earth microbes inadvertently introduced to Mars, or that may have a high probability of supporting indigenous martian life. Since absolutely nothing is known about martian life, the operational definition of a special region is a place that may allow the formation and maintenance of liquid water, on or under the surface of Mars. This paper will review the special-regions concept, the implications of recent recommendations on avoiding them, and the work of the Mars science community in providing an operational definition of those areas on Mars that are “non-special.”