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.     

Germination and growth of wheat in simulated Martian atmospheres

One design for a manned Mars base incorporates a bioregenerative life support system based upon growing higher plants at a low atmospheric pressure in a greenhouse on the Martian surface. To determine the concept's feasibility, the germination and initial growth of wheat (Triticum aestivum) was evaluated at low atmospheric pressures in simulated Martian atmosphere (SMA) and in SMA supplemented with oxygen. Total atmospheric pressures ranged from 10 to 1013 mb. No seeds germinated in pure SMA, regardless of atmospheric pressure. In SMA plus oxygen at 60 mb total pressure, germination and growth occurred but were lower than in the Earth atmosphere controls.     

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.     

In situ microbial detection in Mojave Desert soil using native fluorescence

We report on the use of a portable instrument for microbial detection in the Mojave Desert soil and the potential for its use on Mars. The instrument is based on native fluorescence and employs four excitation wavelengths combined with four emission wavelengths. A soil dilution series in which known numbers of Bacillus subtilis spores were added to soil was used to determine the sensitivity of the instrument. We found that the fluorescence of the biological and organic components of the desert soil samples studied can be as strong as the fluorescence of the mineral component of these soils. Using the calibration derived from B. subtilis spores, we estimated that microbial content at our primary sampling site was 10(7) bacteria per gram of soil, a level confirmed by phospholipid fatty acid analysis. At a nearby site, but in a slightly different geological setting, we tested the instrument's ability to map out microbial concentrations in situ. Over a ～50 m diameter circle, soil microbial concentrations determined with the B. subtilis calibration indicate that the concentrations of microorganisms detected varies from 10(4) to 10(7) cells per gram of soil. We conclude that fluorescence is a promising method for detecting soil microbes in noncontact applications in extreme environments on Earth and may have applications on future missions to Mars.     

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.     

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.     

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.     

Terraforming Mars: conceptual solutions to the problem of plant growth in low concentrations of oxygen

The widespread growth of higher plants on Mars following ecopoiesis has often been invoked as a method of generating atmospheric oxygen. However, one issue that has been overlooked in this regard is the fact that terrestrial plants do not thrive under conditions of low oxygen tension. A review of the relevant botanical literature reveals that the high oxygen demands of root respiration could limit the introduction of most plants on Mars until after terraforming has raised the atmospheric pO2 to 20-100 mbar. A variety of physiological strategies are discussed which, if it is possible to implement them in a genetically engineered plant specifically designed for life on Mars, might allow this problem to be overcome.     

On laboratory simulation and the effect of small temperature oscillations about the freezing point and ice formation on the evaporation rate of water on Mars

We report measurements of the evaporation rate of water under Mars-like conditions (CO2 atmosphere at 7 mbar and approximately 0 degrees C) in which small temperature oscillations about the freezing point repeatedly formed and removed a thin layer of ice. We found that the average evaporation at 2.7 +/- 0.5 degrees C without an ice layer (corrected for the difference in gravity on Earth and on Mars) was 1.24 +/- 0.12 mm/h, while at -2.1 +/- 0.3 degrees C with an ice layer the average evaporation rate was 0.84 +/- 0.08 mm/h. These values are in good agreement with those calculated for the evaporation of liquid water and ice when it is assumed that evaporation only depends on diffusion and buoyancy. Our findings suggest that such differences in evaporation rates are entirely due to the temperature difference and that the ice layer has little effect on evaporation rate. We infer that the formation of thin layers of ice on pools of water on Mars does not significantly increase the stability of water on the surface of Mars.     

Olivine-respiring bacteria isolated from the rock-ice interface in a lava-tube cave, a Mars analog environment

The boundary between ice and basalt on Earth is an analogue for some near-surface environments of Mars. We investigated neutrophilic iron-oxidizing microorganisms from the basalt-ice interface in a lava tube from the Oregon Cascades with perennial ice. One of the isolates (Pseudomonas sp. HerB) can use ferrous iron Fe(II) from the igneous mineral olivine as an electron donor and O(2) as an electron acceptor. The optimum growth temperature is ～12-14°C, but growth also occurs at 5°C. Bicarbonate is a facultative source of carbon. Growth of Pseudomonas sp. HerB as a chemolithotrophic iron oxidizer with olivine as the source of energy is favored in low O(2) conditions (e.g., 1.6% O(2)). Most likely, microbial oxidation of olivine near pH 7 requires low O(2) to offset the abiotic oxidation of iron. The metabolic capabilities of this bacterium would allow it to live in near-surface, icy, volcanic environments of Mars in the present or recent geological past and make this type of physiology a prime candidate in the search for life on Mars.     

Small step or giant leap? Human locomotion on Mars

Human locomotion on Mars will be considerably different from on Earth. Optimum walking speeds will be approximately 30% lower and transitioning from a walk to a run will occur at a speed 25% slower. Peak vertical forces will be reduced by as much as 50%, and although ground contact time will remain constant with locomotion in 1 g, stride length and stride time will increase. During running on Mars airborne time will increase by approximately 80% in comparison to running on the Earth. On Mars, half as much energy will be required to travel the equivalent distance on Earth and it will be 65% more economical to run rather than to walk. Crews will, therefore, find themselves using a loping gait--a running-like action, with a slight upper body lean and an extended aerial phase, an unfamiliar gait in terrestrial locomotion.     

Exposure of Arabidopsis thaliana to hypobaric environments: implications for low-pressure bioregenerative life support systems for human exploration missions and terraforming on Mars

Understanding how hypobaria can affect net photosynthetic (P (net)) and net evapotranspiration rates of plants is important for the Mars Exploration Program because low-pressured environments may be used to reduce the equivalent system mass of near-term plant biology experiments on landers or future bioregenerative advanced life support systems. Furthermore, introductions of plants to the surface of a partially terraformed Mars will be constrained by the limits of sustainable growth and reproduction of plants to hypobaric conditions. To explore the effects of hypobaria on plant physiology, a low-pressure growth chamber (LPGC) was constructed that maintained hypobaric environments capable of supporting short-term plant physiological studies. Experiments were conducted on Arabidopsis thaliana maintained in the LPGC with total atmospheric pressures set at 101 (Earth sea-level control), 75, 50, 25 or 10 kPa. Plants were grown in a separate incubator at 101 kPa for 6 weeks, transferred to the LPGC, and acclimated to low-pressure atmospheres for either 1 or 16 h. After 1 or 16 h of acclimation, CO(2) levels were allowed to drawdown from 0.1 kPa to CO(2) compensation points to assess P (net) rates under different hypobaric conditions. Results showed that P (net) increased as the pressures decreased from 101 to 10 kPa when CO(2) partial pressure (pp) values were below 0.04 kPa (i.e., when ppCO2 was considered limiting). In contrast, when ppCO(2) was in the nonlimiting range from 0.10 to 0.07 kPa, the P (net) rates were insensitive to decreasing pressures. Thus, if CO(2 )concentrations can be kept elevated in hypobaric plant growth modules or on the surface of a partially terraformed Mars, P (net) rates may be relatively unaffected by hypobaria. Results support the conclusions that (i) hypobaric plant growth modules might be operated around 10 kPa without undue inhibition of photosynthesis and (ii) terraforming efforts on Mars might require a surface pressure of at least 10 kPa (100 mb) for normal growth of deployed plant species.     

一种三元结构的火星采样返回任务方案分析

介绍了目前国外提出的一种三元结构的火星采样返回任务方案,整个方案分3次发射,分别发射漫游车、着陆器和轨道器,每次发射间隔为4年,最终目的是将火星样品带回地球。该方案的优势在于,通过3次发射分别完成漫游车巡视勘察、着陆器现场探测、轨道器数据中继和在轨探测,最终综合完成火星采样返回,能够极大地缓解项目进度和资金压力,充分利用每次发射窗口分步骤完成探测任务。文章重点对方案涉及的关键技术进行了分析,包括样品获取与封装、行星保护、精准着陆、漫游车的危险规避能力和移动性、火星上升器、交会与样品捕获、地球再入器技术等;对方案的前景和优势进行了探讨,并给出几点启示,如精准着陆或成为今后行星探测着陆方式的新趋势,火星采样返回任务将是人类火星探测的里程碑,今后的深空探测任务趋向国际合作模式等。     

The Mars Sample Return Project.

The Mars Sample Return (MSR) Project is underway. A 2003 mission to be launched on a Delta III Class vehicle and a 2005 mission launched on an Ariane 5 will culminate in carefully selected Mars samples arriving on Earth in 2008. NASA is the lead agency and will provide the Mars landed elements, namely, landers, rovers, and Mars ascent vehicles (MAVs). The French Space Agency CNES is the largest international partner and will provide for the joint NASA/CNES 2005 Mission the Ariane 5 launch and the Earth Return Mars Orbiter that will capture the sample canisters from the Mars parking orbits the MAVs place them in. The sample canisters will be returned to Earth aboard the CNES Orbiter in the Earth Entry Vehicles provided by NASA. Other national space agencies are also expected to participate in substantial roles. Italy is planning to provide a drill that will operate from the Landers to provide subsurface samples. Other experiments in addition to the MSR payload will also be carried on the Landers. This paper will present the current status of the design of the MSR missions and flight articles.     

火星探测的主要科学问题

在太阳系的行星中,火星与地球之间存在最多的相似之处,因此,火星是一颗承载人类最多梦想的星球。火星有水和生命存在的问题,激发了人类火星探索的好奇心,成为人类持续探测火星的推动力。火星的起源和演化与太阳系形成过程的关系,火星与类地行星的共性和特性,是当代行星科学研究的重要内容。为了人类社会的可持续发展,火星可否改造成为适宜人类居住的绿色星球——这些是人类在火星探测中必须面对的重大科学问题。只有这些重大科学问题被一一解答,我们才能清晰地去思考地球和人类自身的未来！     

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.     

Bacillus subtilis spores on artificial meteorites survive hypervelocity atmospheric entry: implications for Lithopanspermia

An important but untested aspect of the lithopanspermia hypothesis is that microbes situated on or within meteorites could survive hypervelocity entry from space through Earth's atmosphere. The use of high-altitude sounding rockets to test this notion was explored. Granite samples permeated with spores of Bacillus subtilis strain WN511 were attached to the exterior telemetry module of a sounding rocket and launched from White Sands Missile Range, New Mexico into space, reaching maximum atmospheric entry velocity of 1.2 km/s. Maximum recorded temperature during the flight was measured at 145 degrees C. The surfaces of the post-flight granite samples were swabbed and tested for recovery and survival of WN511 spores, using genetic markers and the unique DNA fingerprint of WN511 as recovery criteria. Spore survivors were isolated at high frequency, ranging from 1.2% to 4.4% compared with ground controls, from all surfaces except the forward-facing surface. Sporulation-defective mutants were noted among the spaceflight survivors at high frequency (4%). These experiments constitute the first report of spore survival to hypervelocity atmospheric transit, and indicate that sounding rocket flights can be used to model the high-speed atmospheric entry of bacteria-laden artificial meteorites.     

Physiological and biomechanical considerations for a human Mars mission

Evolving on Earth has made humans perfectly adapted, both physiologically and biomechanically, to its gravity and atmospheric conditions. Leaving the Earth and its protective environment, therefore, results in the degradation of a number of human systems. Long-duration stays on the International Space Station (ISS) are accompanied by significant effects on crew's cardiovascular, vestibular and musculoskeletal systems. Bone loss and muscle atrophy are experienced at a rate of 1-3% and 5% per month respectively, while VO2 (oxygen consumption) measurements are reduced by approximately 25% after a few weeks in space. If these figures are simply extrapolated, a future human mission to Mars will be seriously jeopardised and crews may find they cross the threshold of bone and muscle loss and aerobic fitness--ultimately with them being unable to return to Earth. When arriving on Mars, considerable biomechanical alterations will also occur. Optimum walking speeds will be approximately 30% lower and transitioning from a walk to a run will occur at a speed 25% slower. Peak vertical forces will be reduced by as much as 50%, while stride length, stride time and airborne time will all increase. On Mars, half as much energy will be required to travel the equivalent distance on Earth and it will be 65% more economical to run rather than to walk.     

Results of measurements with the Planetary Fourier Spectrometer onboard Mars Express: Clouds and dust at the end of southern summer. A comparison with OMEGA images

We discuss the results of measurements made with the Planetary Fourier Spectrometer (PFS) onboard the Mars Express spacecraft. The data were obtained in the beginning of the mission and correspond to the end of summer in the southern hemisphere of Mars (L _s ～ 340°). Three orbits are considered, two of which passed through volcanoes Olympus and Ascraeus Mons (the height above the surface is about +20 km), while the third orbit intersects lowland Hellas (?7 km). The influence of the relief on the properties of the aerosol observed is demonstrated: clouds of water ice with a visual optical thickness of 0.1–0.5 were observed above volcanoes, while only dust was found during the observations (close in time) along the orbit passing through Hellas in low and middle latitudes. This dust is homogeneously mixed with gas and has a reduced optical thickness of 0.25±0.05 (at v = 1100 cm^?1). In addition to orographic clouds, ice clouds were observed in this season in the northern polar region. The clouds seen in the images obtained simultaneously by the mapping spectrometer OMEGA confirm the PFS results. Temperature inversion is discovered in the north polar hood below the level 1 mbar with a temperature maximum at about 0.6 mbar. This inversion is associated with descending movements in the Hadley cell.