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1.  Resistance of Bacterial Endospores to Outer Space for Planetary Protection Purposes—Experiment PROTECT of the EXPOSE-E Mission 
Astrobiology  2012;12(5):445-456.
Abstract
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. Key Words: Planetary protection—Bacterial spores—Space experiment—Simulated Mars mission. Astrobiology 12, 445–456.
doi:10.1089/ast.2011.0737
PMCID: PMC3371261  PMID: 22680691
2.  Bacterial Growth at the High Concentrations of Magnesium Sulfate Found in Martian Soils 
Astrobiology  2012;12(2):98-106.
Abstract
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 MgSO4 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 MgSO4 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 MgSO4. There was a complex physiological response to mixtures of MgSO4 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. Key Words: Analogue—Mars—Planetary protection—Salts—Life in extreme environments. Astrobiology 12, 98–106.
doi:10.1089/ast.2011.0720
PMCID: PMC3277918  PMID: 22248384
3.  Effects of Simulated Mars Conditions on the Survival and Growth of Escherichia coli and Serratia liquefaciens▿  
Escherichia coli and Serratia liquefaciens, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of E. coli and S. liquefaciens cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30°C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl2, MgSO4, NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for E. coli and S. liquefaciens at 30 or 20°C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO4 maintained at 20 or 30°C. Growth rates of E. coli and S. liquefaciens in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative E. coli cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and −50°C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m−2 for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of E. coli failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that E. coli may be able to survive, but not grow, in surficial soils on Mars.
doi:10.1128/AEM.02147-09
PMCID: PMC2849189  PMID: 20154104
4.  Searching for signatures of life on Mars: an Fe-isotope perspective 
Recent spacecraft and lander missions to Mars have reinforced previous interpretations that Mars was a wet and warm planet in the geological past. The role of liquid water in shaping many of the surface features on Mars has long been recognized. Since the presence of liquid water is essential for survival of life, conditions on early Mars might have been more favourable for the emergence and evolution of life. Until a sample return mission to Mars, one of the ways of studying the past environmental conditions on Mars is through chemical and isotopic studies of Martian meteorites. Over 35 individual meteorite samples, believed to have originated on Mars, are now available for lab-based studies. Fe is a key element that is present in both primary and secondary minerals in the Martian meteorites. Fe-isotope ratios can be fractionated by low-temperature processes which includes biological activity. Experimental investigations of Fe reduction and oxidation by bacteria have produced large fractionation in Fe-isotope ratios. Hence, it is considered likely that if there is/were any form of life present on Mars then it might be possible to detect its signature by Fe-isotope studies of Martian meteorites. In the present study, we have analysed a number of Martian meteorites for their bulk-Fe-isotope composition. In addition, a set of terrestrial analogue material has also been analysed to compare the results and draw inferences. So far, our studies have not found any measurable Fe-isotopic fractionation in bulk Martian meteorites that can be ascribed to any low-temperature process operative on Mars.
doi:10.1098/rstb.2006.1899
PMCID: PMC1664681  PMID: 17008212
Mars; Martian meteorites; SNC; terrestrial analogues; iron isotopes; life
5.  Astrobiological Aspects of Mars and Human Presence: Pros and Cons 
Hippokratia  2008;12(Suppl 1):49-52.
After the realization of the International Space Station, human exploratory missions to Moon or Mars, i.e. beyond low Earth orbit, are widely considered as the next logical step of peaceful cooperation in space on a global scale. Besides the human desire to extend the window of habitability, human exploratory missions are driven by several aspects of science, technology, culture and economy. Mars is currently considered as a major target in the search for life beyond the Earth. Understanding the history of water on Mars appears to be one of the clues to the puzzle on the probability of life on Mars. On Earth microorganisms have flourished for more than 3.5 Ga and have developed strategies to cope with so-called extreme conditions (e.g., hot vents, permafrost, subsurface regions, rocks or salt crystals). Therefore, in search for life on Mars, microorganisms are the most likely candidates for a putative biota on Mars and the search for morphological or chemical signatures of life or its relics is one of the primary and most exciting goals of Mars exploration. The presence of humans on the surface of Mars will substantially increase this research potential, e.g., by supporting deep subsurface drilling and by allowing intellectual collection and sophisticated in situ analysis of samples of astrobiological interest. On the other hand, such long-duration missions beyond LEO will add a new dimension to human space flight, concerning the distance of travel, the radiation environment, the gravity levels, the duration of the mission, and the level of confinement and isolation the crew will be exposed to. This will raise the significance of several health issues, above all radiation protection, gravity related effects as well as psychological issues. Furthermore, the import of internal and external microorganisms inevitably accompanying any human mission to Mars, or brought purposely to Mars as part of a bioregenerative life support system needs careful consideration with regard to planetary protection issues. Therefore, before planning any human exploratory mission, the critical issues concerning human health and wellbeing as well as protection of Mars in its pristine condition need to be investigated.
PMCID: PMC2577400  PMID: 19048093
Mars; Human exploratory missions; Astrobiology; Search for extraterrestrial life
6.  Perchlorate Radiolysis on Mars and the Origin of Martian Soil Reactivity 
Astrobiology  2013;13(6):515-520.
Abstract
Results from the Viking biology experiments indicate the presence of reactive oxidants in martian soils that have previously been attributed to peroxide and superoxide. Instruments on the Mars Phoenix Lander and the Mars Science Laboratory detected perchlorate in martian soil, which is nonreactive under the conditions of the Viking biology experiments. We show that calcium perchlorate exposed to gamma rays decomposes in a CO2 atmosphere to form hypochlorite (ClO−), trapped oxygen (O2), and chlorine dioxide (ClO2). Our results show that the release of trapped O2 (g) from radiation-damaged perchlorate salts and the reaction of ClO− with amino acids that were added to the martian soils can explain the results of the Viking biology experiments. We conclude that neither hydrogen peroxide nor superoxide is required to explain the results of the Viking biology experiments. Key Words: Mars—Radiolysis—Organic degradation—in situ measurement—Planetary habitability and biosignatures. Astrobiology 13, 515–520.
doi:10.1089/ast.2013.0999
PMCID: PMC3691774  PMID: 23746165
7.  Models of Formation and Activity of Spring Mounds in the Mechertate-Chrita-Sidi El Hani System, Eastern Tunisia: Implications for the Habitability of Mars 
Life : Open Access Journal  2014;4(3):386-432.
Spring mounds on Earth and on Mars could represent optimal niches of life development. If life ever occurred on Mars, ancient spring deposits would be excellent localities to search for morphological or chemical remnants of an ancient biosphere. In this work, we investigate models of formation and activity of well-exposed spring mounds in the Mechertate-Chrita-Sidi El Hani (MCSH) system, eastern Tunisia. We then use these models to explore possible spring mound formation on Mars. In the MCSH system, the genesis of the spring mounds is a direct consequence of groundwater upwelling, triggered by tectonics and/or hydraulics. As they are oriented preferentially along faults, they can be considered as fault spring mounds, implying a tectonic influence in their formation process. However, the hydraulic pressure generated by the convergence of aquifers towards the surface of the system also allows consideration of an origin as artesian spring mounds. In the case of the MCSH system, our geologic data presented here show that both models are valid, and we propose a combined hydro-tectonic model as the likely formation mechanism of artesian-fault spring mounds. During their evolution from the embryonic (early) to the islet (“island”) stages, spring mounds are also shaped by eolian accumulations and induration processes. Similarly, spring mounds have been suggested to be relatively common in certain provinces on the Martian surface, but their mode of formation is still a matter of debate. We propose that the tectonic, hydraulic, and combined hydro-tectonic models describing the spring mounds at MCSH could be relevant as Martian analogs because: (i) the Martian subsurface may be over pressured, potentially expelling mineral-enriched waters as spring mounds on the surface; (ii) the Martian subsurface may be fractured, causing alignment of the spring mounds in preferential orientations; and (iii) indurated eolian sedimentation and erosional remnants are common features on Mars. The spring mounds further bear diagnostic mineralogic and magnetic properties, in comparison with their immediate surroundings. Consequently, remote sensing techniques can be very useful to identify similar spring mounds on Mars. The mechanisms (tectonic and/or hydraulic) of formation and evolution of spring mounds at the MCSH system are suitable for the proliferation and protection of life respectively. Similarly, life or its resulting biomarkers on Mars may have been protected or preserved under the spring mounds.
doi:10.3390/life4030386
PMCID: PMC4206853  PMID: 25370379
Mechertate-Chrita-Sidi El Hani system; Mars habitability; terrestrial analogs; groundwater upwelling; seepage; tectonic model; hydraulic model; fault spring mounds; artesian spring mounds
8.  Investigating the Effects of Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and Other Extremely Halophilic Archaebacteria 
Astrobiology  2009;9(1):104-112.
The isolation of viable extremely halophilic archaea from 250-million-year-old rock salt suggests the possibility of their long-term survival under desiccation. Since halite has been found on Mars and in meteorites, haloarchaeal survival of martian surface conditions is being explored. Halococcus dombrowskii H4 DSM 14522T was exposed to UV doses over a wavelength range of 200–400 nm to simulate martian UV flux. Cells embedded in a thin layer of laboratory-grown halite were found to accumulate preferentially within fluid inclusions. Survival was assessed by staining with the LIVE/DEAD kit dyes, determining colony-forming units, and using growth tests. Halite-embedded cells showed no loss of viability after exposure to about 21 kJ/m2, and they resumed growth in liquid medium with lag phases of 12 days or more after exposure up to 148 kJ/m2. The estimated D37 (dose of 37 % survival) for Hcc. dombrowskii was ≥ 400 kJ/m2. However, exposure of cells to UV flux while in liquid culture reduced D37 by 2 orders of magnitude (to about 1 kJ/m2); similar results were obtained with Halobacterium salinarum NRC-1 and Haloarcula japonica. The absorption of incoming light of shorter wavelength by color centers resulting from defects in the halite crystal structure likely contributed to these results. Under natural conditions, haloarchaeal cells become embedded in salt upon evaporation; therefore, dispersal of potential microscopic life within small crystals, perhaps in dust, on the surface of Mars could resist damage by UV radiation.
doi:10.1089/ast.2007.0234
PMCID: PMC3182532  PMID: 19215203
Halococcus dombrowskii; Simulated martian UV radiation; LIVE/DEAD staining; Halite fluid inclusions; UV transmittance and reflectance; Desiccation
9.  Persistence of Biomarker ATP and ATP-Generating Capability in Bacterial Cells and Spores Contaminating Spacecraft Materials under Earth Conditions and in a Simulated Martian Environment▿  
Applied and Environmental Microbiology  2008;74(16):5159-5167.
Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5′-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m−2 of UV-C (200 to 280 nm), −10°C, and a Mars gas mix of CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.
doi:10.1128/AEM.00891-08
PMCID: PMC2519281  PMID: 18567687
10.  Radiation-associated cardiovascular risks for future deep-space missions 
Journal of Radiation Research  2014;55(Suppl 1):i37-i39.
Background: During the future Moon and Mars missions, astronauts will be exposed to space radiation (IR) for extended time. The majority of space flight-associated risks identified for the cardiovascular (CV) system to date were determined shortly after low Earth orbit (LEO) short- and long-duration space flights that include: serious cardiac dysrhythmias, compromised orthostatic CV response and manifestation of previously asymptomatic CV disease. Further ground-based experiments using a surrogate model of microgravity supported the space flight data for significant cardiac remodeling due to prolonged exposure to microgravity. These symptoms were determined to be a consequence of adaptation to microgravity that could be ameliorated by a post-mission exercise program, and were not identified as risk factors that were causatively related to space IR. Long-term degenerative effects of cosmic IR during and after space flights on CV system are unknown.
It was suggested that due to GCR, each cell in an astronaut's body will be traversed by 1H every 3 days, helium (2He) nuclei every few weeks and high charge and energy (HZE) nuclei (e.g. 28Si, 56Fe) every few months. Despite the fact that only 1% of GCR is composed of ions heavier than helium, ∼41% of the IR dose-equivalent is predicted to be HZE particles with 13% being from 56Fe particles, only. During an exploration-class space mission to Mars, astronauts will not have access to comprehensive healthcare services for a period of at least 2–3 years. Since the majority of experienced astronauts are middle-aged (average age is 46, and the range is 33–58 years), they are at risk for developing serious CV events which could be life-threatening for the astronaut and mission-threatening for NASA. Therefore, it is important to evaluate the effects and potential CV risks caused by space IR. We hypothesized that: (i) low-dose space IR-induced biological responses may be long-lasting and are IR type-dependent; (ii) IR may increase CV risks in the aging heart (IR + AGING model) and affect the heart recovery after an adverse CV event, such as acute myocardial infarct (IR + AGING + AMI model).
Methods: Eight- to 9-month-old C57BL/6N male mice were IR once with proton (1H) 50 cGy, 1 GeV/n or iron (56Fe) 15 cGy, 1 GeV/n. We evaluated IR-induced biological tissue responses—underlying molecular mechanisms, calcium handling, signal transduction, gene expression and cardiac fibrosis. Cardiac function was assessed by echocardiography (ECHO) and hemodynamic measurements (HEMO) as detailed in Fig. 1. AMI was induced by ligation of left anterior descending coronary artery 1 and 3 months post-IR as detailed in Fig. 2.Fig. 1.Radiation + aging model. Fig. 2.Radiation + aging model + adverse CV event model.
Results: In the IR + AGING model, cardiac function was not different among the control and 1H-IR group, whereas left ventricular end-diastolic pressure (LVEDP) was significantly increased in 56Fe mice 1 and 3 months post-IR. There was a small but statistically significant (P < 0.04) improvement of ejection fraction % (EF%) in 1H-IR vs control mice. One month post-IR, compared with control, 1H- and 56Fe-IR hearts had a significant up-regulation of sarcolemmal Na+–Ca2+ exchanger (NCX) (∼200% P<0.007), sarco(endo)plasmic reticulum calcium-ATPase (SERCA2a, >200% increases, P < 0.02) and 400% decreases in p-p38 MAPK (P < 0.05), suggesting activation of compensatory mechanisms in [Ca2+]i handling in these hearts. By 3 months, compared with control, 1H- and 56Fe-IR hearts had 200–500% (P < 0.02) decreases in SERCA2a and more than 200% decreases in p-Creb-1 (P < 0.02), suggesting reduced capacity in intracellular [Ca2+]i handling. These data suggest that dysfunction in [Ca2+]i handling combined with LVEDP increase after 56Fe-IR may arise from the excessive demand on the heart due to prolonged activation of compensatory mechanisms that lead to changes in SERCA2a and p-Creb1 levels. This may represent a possible intracellular mechanism of heart failure in development in 56Fe-IR hearts.
In the IR + AGING + AMI model, no mortality was observed among three different groups 1 or 3 months post-IR and up to 28 days post-AMI. However, 1 month post-IR and 28 days post-AMI, the infarct size was significantly smaller in 56Fe-IR (p < 0.003) and 1H-IR (p = n.s.) vs control-IR mice, suggesting that at 1 month, 56Fe-IR primes the heart to recover better after AMI. In contrast, 3 months post AMI, 1H-AMI mice had a better cardiac functional recovery compared with control-AMI and 56Fe-AMI mice. The ejection fraction (EF%) was most decreased in 56Fe-AMI mice (56Fe-AMI vs 1H-AMI: 18 vs 48%, P < 0.007, ∼65–70% pre-AMI EF% for all groups). There was a 2- to 4-fold increase in LVEDP in 56Fe-AMI vs 1H-AMI (P < 0.04), suggesting that 56Fe-AMI hearts developed cardiac de-compensation. Western blots showed that 3 days post-AMI, compared with control- and 1H-IR-AMI mice, 56Fe-IR-AMI hearts had a 4- to 7-fold (P < 0.04) decreases in p-Akt (Thr308), p-Erk1/2 (P < 0.007) and ∼2-fold (P < 0.01) increase in phosphorylated ribosomal protein S6 kinase (p-S6k, a readout for mTORC1 pathway activation), suggesting decreased survival and angiogenesis signaling and decreased autophagy in these hearts. Seven days post-AMI, the levels of p-pErk1/2 were comparable between all three treatment conditions. However, in 56Fe-IR-AMI hearts, the p-Akt (Thr308) levels remained 4-fold decreased. Additionally, here was a 3-fold (P<0.05) decrease in p-S6k levels and >10-fold increase in p-p38 MAPK level in 56Fe vs control and 1H-IR-AMI hearts, suggesting continuous decreases in the survival, proliferation and angiogenesis signaling (p-Akt and p-S6k) and increase in the apoptotic signaling (p-p38 MAPK) up to Day 7 post-AMI in 56Fe-IR-AMI mice.
In summary, our results revealed that by 1 and 3 months post-IR in IR + AGING, 56Fe-IR but not 1H-IR mice had worse cardiac function. Further, a single 1H-IR 3 months prior to AMI improved, whereas 56Fe-IR worsened, recovery from AMI recovery. Our data in the IR + AGING and IR + AGING + AMI groups strongly suggest that low-dose HZE particle IR (56Fe) have long-lasting negative effect on heart homeostasis during normal aging, and present a significant CV risk for recovery after adverse CV event, such as AMI.
doi:10.1093/jrr/rrt202
PMCID: PMC3941505
HZE; iron; proton; low-dose; cardiovascular risks; Ca2+
11.  Effect of Shadowing on Survival of Bacteria under Conditions Simulating the Martian Atmosphere and UV Radiation▿ †  
Spacecraft-associated spores and four non-spore-forming bacterial isolates were prepared in Atacama Desert soil suspensions and tested both in solution and in a desiccated state to elucidate the shadowing effect of soil particulates on bacterial survival under simulated Martian atmospheric and UV irradiation conditions. All non-spore-forming cells that were prepared in nutrient-depleted, 0.2-μm-filtered desert soil (DSE) microcosms and desiccated for 75 days on aluminum died, whereas cells prepared similarly in 60-μm-filtered desert soil (DS) microcosms survived such conditions. Among the bacterial cells tested, Microbacterium schleiferi and Arthrobacter sp. exhibited elevated resistance to 254-nm UV irradiation (low-pressure Hg lamp), and their survival indices were comparable to those of DS- and DSE-associated Bacillus pumilus spores. Desiccated DSE-associated spores survived exposure to full Martian UV irradiation (200 to 400 nm) for 5 min and were only slightly affected by Martian atmospheric conditions in the absence of UV irradiation. Although prolonged UV irradiation (5 min to 12 h) killed substantial portions of the spores in DSE microcosms (∼5- to 6-log reduction with Martian UV irradiation), dramatic survival of spores was apparent in DS-spore microcosms. The survival of soil-associated wild-type spores under Martian conditions could have repercussions for forward contamination of extraterrestrial environments, especially Mars.
doi:10.1128/AEM.01973-07
PMCID: PMC2258572  PMID: 18083857
12.  The Apparent Involvement of ANMEs in Mineral Dependent Methane Oxidation, as an Analog for Possible Martian Methanotrophy 
Life : Open Access Journal  2011;1(1):19-33.
On Earth, marine anaerobic methane oxidation (AOM) can be driven by the microbial reduction of sulfate, iron, and manganese. Here, we have further characterized marine sediment incubations to determine if the mineral dependent methane oxidation involves similar microorganisms to those found for sulfate-dependent methane oxidation. Through FISH and FISH-SIMS analyses using 13C and 15N labeled substrates, we find that the most active cells during manganese dependent AOM are primarily mixed and mixed-cluster aggregates of archaea and bacteria. Overall, our control experiment using sulfate showed two active bacterial clusters, two active shell aggregates, one active mixed aggregate, and an active archaeal sarcina, the last of which appeared to take up methane in the absence of a closely-associated bacterial partner. A single example of a shell aggregate appeared to be active in the manganese incubation, along with three mixed aggregates and an archaeal sarcina. These results suggest that the microorganisms (e.g., ANME-2) found active in the manganese-dependent incubations are likely capable of sulfate-dependent AOM. Similar metabolic flexibility for Martian methanotrophs would mean that the same microbial groups could inhabit a diverse set of Martian mineralogical crustal environments. The recently discovered seasonal Martian plumes of methane outgassing could be coupled to the reduction of abundant surface sulfates and extensive metal oxides, providing a feasible metabolism for present and past Mars. In an optimistic scenario Martian methanotrophy consumes much of the periodic methane released supporting on the order of 10,000 microbial cells per cm2 of Martian surface. Alternatively, most of the methane released each year could be oxidized through an abiotic process requiring biological methane oxidation to be more limited. If under this scenario, 1% of this methane flux were oxidized by biology in surface soils or in subsurface aquifers (prior to release), a total of about 1020 microbial cells could be supported through methanotrophy with the cells concentrated in regions of methane release.
doi:10.3390/life1010019
PMCID: PMC4187123  PMID: 25382054
Archaea; methane; methanotrophy; Mars; subsurface biosphere
13.  Boron Enrichment in Martian Clay 
PLoS ONE  2013;8(6):e64624.
We have detected a concentration of boron in martian clay far in excess of that in any previously reported extra-terrestrial object. This enrichment indicates that the chemistry necessary for the formation of ribose, a key component of RNA, could have existed on Mars since the formation of early clay deposits, contemporary to the emergence of life on Earth. Given the greater similarity of Earth and Mars early in their geological history, and the extensive disruption of Earth's earliest mineralogy by plate tectonics, we suggest that the conditions for prebiotic ribose synthesis may be better understood by further Mars exploration.
doi:10.1371/journal.pone.0064624
PMCID: PMC3675118  PMID: 23762242
14.  How Safe Is Safe Enough? Radiation Risk for a Human Mission to Mars 
PLoS ONE  2013;8(10):e74988.
Astronauts on a mission to Mars would be exposed for up to 3 years to galactic cosmic rays (GCR) — made up of high-energy protons and high charge (Z) and energy (E) (HZE) nuclei. GCR exposure rate increases about three times as spacecraft venture out of Earth orbit into deep space where protection of the Earth's magnetosphere and solid body are lost. NASA's radiation standard limits astronaut exposures to a 3% risk of exposure induced death (REID) at the upper 95% confidence interval (CI) of the risk estimate. Fatal cancer risk has been considered the dominant risk for GCR, however recent epidemiological analysis of radiation risks for circulatory diseases allow for predictions of REID for circulatory diseases to be included with cancer risk predictions for space missions. Using NASA's models of risks and uncertainties, we predicted that central estimates for radiation induced mortality and morbidity could exceed 5% and 10% with upper 95% CI near 10% and 20%, respectively for a Mars mission. Additional risks to the central nervous system (CNS) and qualitative differences in the biological effects of GCR compared to terrestrial radiation may significantly increase these estimates, and will require new knowledge to evaluate.
doi:10.1371/journal.pone.0074988
PMCID: PMC3797711  PMID: 24146746
15.  Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars 
Radiation research  2003;159(6):722-729.
Kminek, G., Bada, J. L., Pogliano, K. and Ward, J. F. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars. Radiat. Res. 159, 722–729 (2003).
When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897–900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627–2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars.
PMCID: PMC3919141  PMID: 12751954
16.  Growth Performance and Root Transcriptome Remodeling of Arabidopsis in Response to Mars-Like Levels of Magnesium Sulfate 
PLoS ONE  2010;5(8):e12348.
Background
Martian regolith (unconsolidated surface material) is a potential medium for plant growth in bioregenerative life support systems during manned missions on Mars. However, hydrated magnesium sulfate mineral levels in the regolith of Mars can reach as high as 10 wt%, and would be expected to be highly inhibitory to plant growth.
Methodology and Principal Findings
Disabling ion transporters AtMRS2-10 and AtSULTR1;2, which are plasma membrane localized in peripheral root cells, is not an effective way to confer tolerance to magnesium sulfate soils. Arabidopsis mrs2-10 and sel1-10 knockout lines do not mitigate the growth inhibiting impacts of high MgSO4·7H2O concentrations observed with wildtype plants. A global approach was used to identify novel genes with potential to enhance tolerance to high MgSO4·7H2O (magnesium sulfate) stress. The early Arabidopsis root transcriptome response to elevated concentrations of magnesium sulfate was characterized in Col-0, and also between Col-0 and the mutant line cax1-1, which was confirmed to be relatively tolerant of high levels of MgSO4·7H2O in soil solution. Differentially expressed genes in Col-0 treated for 45 min. encode enzymes primarily involved in hormone metabolism, transcription factors, calcium-binding proteins, kinases, cell wall related proteins and membrane-based transporters. Over 200 genes encoding transporters were differentially expressed in Col-0 up to 180 min. of exposure, and one of the first down-regulated genes was CAX1. The importance of this early response in wildtype Arabidopsis is exemplified in the fact that only four transcripts were differentially expressed between Col-0 and cax1-1 at 180 min. after initiation of treatment.
Conclusions/Significance
The results provide a solid basis for the understanding of the metabolic response of plants to elevated magnesium sulfate soils; it is the first transcriptome analysis of plants in this environment. The results foster the development of Mars soil-compatible plants by showing that cax1 mutants exhibit partial tolerance to magnesium sulfate, and by elucidating a small subset (500 vs. >10,000) of candidate genes for mutation or metabolic engineering that will enhance tolerance to magnesium sulfate soils.
doi:10.1371/journal.pone.0012348
PMCID: PMC2925951  PMID: 20808807
17.  Biota and Biomolecules in Extreme Environments on Earth: Implications for Life Detection on Mars 
Life  2014;4(4):535-565.
The three main requirements for life as we know it are the presence of organic compounds, liquid water, and free energy. Several groups of organic compounds (e.g., amino acids, nucleobases, lipids) occur in all life forms on Earth and are used as diagnostic molecules, i.e., biomarkers, for the characterization of extant or extinct life. Due to their indispensability for life on Earth, these biomarkers are also prime targets in the search for life on Mars. Biomarkers degrade over time; in situ environmental conditions influence the preservation of those molecules. Nonetheless, upon shielding (e.g., by mineral surfaces), particular biomarkers can persist for billions of years, making them of vital importance in answering questions about the origins and limits of life on early Earth and Mars. The search for organic material and biosignatures on Mars is particularly challenging due to the hostile environment and its effect on organic compounds near the surface. In support of life detection on Mars, it is crucial to investigate analogue environments on Earth that resemble best past and present Mars conditions. Terrestrial extreme environments offer a rich source of information allowing us to determine how extreme conditions affect life and molecules associated with it. Extremophilic organisms have adapted to the most stunning conditions on Earth in environments with often unique geological and chemical features. One challenge in detecting biomarkers is to optimize extraction, since organic molecules can be low in abundance and can strongly adsorb to mineral surfaces. Methods and analytical tools in the field of life science are continuously improving. Amplification methods are very useful for the detection of low concentrations of genomic material but most other organic molecules are not prone to amplification methods. Therefore, a great deal depends on the extraction efficiency. The questions “what to look for”, “where to look”, and “how to look for it” require more of our attention to ensure the success of future life detection missions on Mars.
doi:10.3390/life4040535
PMCID: PMC4284457  PMID: 25370528
biomarkers; Mars; minerals; adsorption; extreme environments; life detection; extraction techniques; origin of life
18.  Removal of Pharmaceutical Residues by Ferrate(VI) 
PLoS ONE  2013;8(2):e55729.
Background
Pharmaceuticals and their metabolites are inevitably emitted into the waters. The adverse environmental and human health effects of pharmaceutical residues in water could take place under a very low concentration range; from several µg/L to ng/L. These are challenges to the global water industries as there is no unit process specifically designed to remove these pollutants. An efficient technology is thus sought to treat these pollutants in water and waste water.
Methodology/Major Results
A novel chemical, ferrate, was assessed using a standard jar test procedure for the removal of pharmaceuticals. The analytical protocols of pharmaceuticals were standard solid phase extraction together with various instrumentation methods including LC-MS, HPLC-UV and UV/Vis spectroscopy. Ferrate can remove more than 80% of ciprofloxacin (CIP) at ferrate dose of 1 mg Fe/L and 30% of ibuprofen (IBU) at ferrate dose of 2 mg Fe/L. Removal of pharmaceuticals by ferrate was pH dependant and this was in coordinate to the chemical/physical properties of pharmaceuticals. Ferrate has shown higher capability in the degradation of CIP than IBU; this is because CIP has electron-rich organic moieties (EOM) which can be readily degraded by ferrate oxidation and IBU has electron-withdrawing groups which has slow reaction rate with ferrate. Promising performance of ferrate in the treatment of real waste water effluent at both pH 6 and 8 and dose range of 1–5 mg Fe/L was observed. Removal efficiency of ciprofloxacin was the highest among the target compounds (63%), followed by naproxen (43%). On the other hand, n-acetyl sulphamethoxazole was the hardest to be removed by ferrate (8% only).
Conclusions
Ferrate is a promising chemical to be used to treat pharmaceuticals in waste water. Adjusting operating conditions in terms of the properties of target pharmaceuticals can maximise the pharmaceutical removal efficiency.
doi:10.1371/journal.pone.0055729
PMCID: PMC3567129  PMID: 23409029
19.  Alliance of Proteomics and Genomics to Unravel the Specificities of Sahara Bacterium Deinococcus deserti 
PLoS Genetics  2009;5(3):e1000434.
To better understand adaptation to harsh conditions encountered in hot arid deserts, we report the first complete genome sequence and proteome analysis of a bacterium, Deinococcus deserti VCD115, isolated from Sahara surface sand. Its genome consists of a 2.8-Mb chromosome and three large plasmids of 324 kb, 314 kb, and 396 kb. Accurate primary genome annotation of its 3,455 genes was guided by extensive proteome shotgun analysis. From the large corpus of MS/MS spectra recorded, 1,348 proteins were uncovered and semiquantified by spectral counting. Among the highly detected proteins are several orphans and Deinococcus-specific proteins of unknown function. The alliance of proteomics and genomics high-throughput techniques allowed identification of 15 unpredicted genes and, surprisingly, reversal of incorrectly predicted orientation of 11 genes. Reversal of orientation of two Deinococcus-specific radiation-induced genes, ddrC and ddrH, and identification in D. deserti of supplementary genes involved in manganese import extend our knowledge of the radiotolerance toolbox of Deinococcaceae. Additional genes involved in nutrient import and in DNA repair (i.e., two extra recA, three translesion DNA polymerases, a photolyase) were also identified and found to be expressed under standard growth conditions, and, for these DNA repair genes, after exposure of the cells to UV. The supplementary nutrient import and DNA repair genes are likely important for survival and adaptation of D. deserti to its nutrient-poor, dry, and UV-exposed extreme environment.
Author Summary
D. deserti belongs to the Deinococcaceae, a family of bacteria characterized by an exceptional ability to withstand the lethal effects of DNA-damaging agents, including ionizing radiation, UV light, and desiccation. It was isolated from Sahara surface sands, an extreme and nutrient-poor environment, regularly exposed to intense UV radiation, cycles of extreme temperatures, and desiccation. The evolution of organisms that are able to survive acute irradiation doses of 15,000 Gy is difficult to explain given the apparent absence of highly radioactive habitats on Earth over geologic time. Thus, it seems more likely that the natural selection pressure for the evolution of radiation-resistant bacteria was chronic exposure to nonradioactive forms of DNA damage, in particular those promoted by desiccation. Here, we report the first complete genome sequence of a bacterium, D. deserti VCD115, isolated from hot, arid desert surface sand. Accurate genome annotation of its 3,455 genes was guided by extensive proteome analysis in which 1,348 proteins were uncovered after growth in standard conditions. Supplementary genes involved in manganese import, in nutrient import, and in DNA repair were identified and are likely important for survival and adaptation of D. deserti to its hostile environment.
doi:10.1371/journal.pgen.1000434
PMCID: PMC2669436  PMID: 19370165
20.  Olivine-Respiring Bacteria Isolated from the Rock-Ice Interface in a Lava-Tube Cave, a Mars Analog Environment 
Astrobiology  2012;12(1):9-18.
Abstract
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 O2 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 O2 conditions (e.g., 1.6% O2). Most likely, microbial oxidation of olivine near pH 7 requires low O2 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. Key Words: Extremophiles—Mars—Olivine—Iron-oxidizing bacteria—Redox. Astrobiology 12, 9–18.
doi:10.1089/ast.2011.0639
PMCID: PMC3264960  PMID: 22165996
21.  Venus-Earth-Mars: Comparative Climatology and the Search for Life in the Solar System 
Life : Open Access Journal  2012;2(3):255-273.
Both Venus and Mars have captured the human imagination during the twentieth century as possible abodes of life. Venus had long enchanted humans—all the more so after astronomers realized it was shrouded in a mysterious cloak of clouds permanently hiding the surface from view. It was also the closest planet to Earth, with nearly the same size and surface gravity. These attributes brought myriad speculations about the nature of Venus, its climate, and the possibility of life existing there in some form. Mars also harbored interest as a place where life had or might still exist. Seasonal changes on Mars were interpreted as due to the possible spread and retreat of ice caps and lichen-like vegetation. A core element of this belief rested with the climatology of these two planets, as observed by astronomers, but these ideas were significantly altered, if not dashed during the space age. Missions to Venus and Mars revealed strikingly different worlds. The high temperatures and pressures found on Venus supported a “runaway greenhouse theory,” and Mars harbored an apparently lifeless landscape similar to the surface of the Moon. While hopes for Venus as an abode of life ended, the search for evidence of past life on Mars, possibly microbial, remains a central theme in space exploration. This survey explores the evolution of thinking about the climates of Venus and Mars as life-support systems, in comparison to Earth.
doi:10.3390/life2030255
PMCID: PMC4187128  PMID: 25371106
Venus; Mars; Earth; space exploration; astrobiology; Percival Lowell; NASA; Carl Sagan; Percival Lowell; James C. Fletcher
22.  Towards synthetic biological approaches to resource utilization on space missions 
Journal of the Royal Society Interface  2015;12(102):20140715.
This paper demonstrates the significant utility of deploying non-traditional biological techniques to harness available volatiles and waste resources on manned missions to explore the Moon and Mars. Compared with anticipated non-biological approaches, it is determined that for 916 day Martian missions: 205 days of high-quality methane and oxygen Mars bioproduction with Methanobacterium thermoautotrophicum can reduce the mass of a Martian fuel-manufacture plant by 56%; 496 days of biomass generation with Arthrospira platensis and Arthrospira maxima on Mars can decrease the shipped wet-food mixed-menu mass for a Mars stay and a one-way voyage by 38%; 202 days of Mars polyhydroxybutyrate synthesis with Cupriavidus necator can lower the shipped mass to three-dimensional print a 120 m3 six-person habitat by 85% and a few days of acetaminophen production with engineered Synechocystis sp. PCC 6803 can completely replenish expired or irradiated stocks of the pharmaceutical, thereby providing independence from unmanned resupply spacecraft that take up to 210 days to arrive. Analogous outcomes are included for lunar missions. Because of the benign assumptions involved, the results provide a glimpse of the intriguing potential of ‘space synthetic biology’, and help focus related efforts for immediate, near-term impact.
doi:10.1098/rsif.2014.0715
PMCID: PMC4277073  PMID: 25376875
space synthetic biology; methanogens; cyanobacteria; Spirulina; polyhydroxybutyrate; acetaminophen
23.  Correlation Between the Extent of Catalytic Activity and Charge Density of Montmorillonites 
Astrobiology  2010;10(7):743-749.
Abstract
The clay mineral montmorillonite is a member of the phyllosilicate group of minerals, which has been detected on martian soil. Montmorillonite catalyzes the condensation of activated monomers to form RNA-like oligomers. Extent of catalysis, that is, the yield of oligomers, and the length of the longest oligomer formed in these reactions widely varies with the source of montmorillonite (i.e., the locality where the mineral is mined). This study was undertaken to establish whether there exists a correlation between the extent of catalytic property and the charge density of montmorillonites. Charge density was determined by saturating the montmorillonites with alkyl ammonium cations that contained increasing lengths of alkyl chains, [CH3-(CH2)n-NH3]+, where n = 3–16 and 18, and then measuring d(001), interlayer spacing of the resulting montmorillonite-alkyl ammonium-montmorillonite complex by X-ray diffractometry (XRD).
Results demonstrate that catalytic activity of montmorillonites with lower charge density is superior to that of higher charge density montmorillonite. They produce longer oligomers that contain 9 to 10 monomer units, while montmorillonite with high charge density catalyzes the formation of oligomers that contain only 4 monomer units.
The charge density of montmorillonites can also be calculated from the chemical composition if elemental analysis data of the pure mineral are available. In the next mission to Mars, CheMin (Chemistry and Mineralogy), a combined X-ray diffraction/X-ray fluorescence instrument, will provide information on the mineralogical and elemental analysis of the samples. Possible significance of these results for planning the future missions to Mars for the search of organic compounds and extinct or extant life is discussed. Key Words: Mars—Origin of life—Montmorillonite—Mineral catalysis—Layer charge density—X–ray diffractometry. Astrobiology 10, 743–749.
doi:10.1089/ast.2009.0436
PMCID: PMC2992397  PMID: 20854214
24.  Natural Fumarolic Alteration of Fluorapatite, Olivine, and Basaltic Glass, and Implications for Habitable Environments on Mars 
Astrobiology  2013;13(11):1049-1064.
Abstract
Fumaroles represent a very important potential habitat on Mars because they contain water and nutrients. Global deposition of volcanic sulfate aerosols may also have been an important soil-forming process affecting large areas of Mars. Here we identify alteration from the Senator fumarole, northwest Nevada, USA, and in low-temperature environments near the fumarole to help interpret fumarolic and acid vapor alteration of rocks and soils on Mars. We analyzed soil samples and fluorapatite, olivine, and basaltic glass placed at and near the fumarole in in situ mineral alteration experiments designed to measure weathering under natural field conditions.
Using synchrotron X-ray diffraction, we clearly observe hydroxyl-carbonate-bearing fluorapatite as a fumarolic alteration product of the original material, fluorapatite. The composition of apatites as well as secondary phosphates has been previously used to infer magmatic conditions as well as fumarolic conditions on Mars. To our knowledge, the observations reported here represent the first documented instance of formation of hydroxyl-carbonate-bearing apatite from fluorapatite in a field experiment. Retreat of olivine surfaces, as well as abundant NH4-containing minerals, was also characteristic of fumarolic alteration. In contrast, alteration in the nearby low-temperature environment resulted in formation of large pits on olivine surfaces, which were clearly distinguishable from the fumarolic alteration. Raman signatures of some fumarolically impacted surfaces are consistent with detection of the biological molecules chlorophyll and scytenomin, potentially useful biosignatures. Observations of altered minerals on Mars may therefore help identify the environment of formation and understand the aqueous history and potential habitability of that planet. Key Words: Fumaroles—Mars—Olivine—Acidophile—Geothermal—Search for life (biosignatures)—Synchrotron X-ray diffraction. Astrobiology 13, 1049–1064.
doi:10.1089/ast.2013.0985
PMCID: PMC3865726  PMID: 24283927
25.  Raman Spectroscopic Analysis of Geological and Biogeological Specimens of Relevance to the ExoMars Mission 
Astrobiology  2013;13(6):543-549.
Abstract
A novel miniaturized Raman spectrometer is scheduled to fly as part of the analytical instrumentation package on an ESA remote robotic lander in the ESA/Roscosmos ExoMars mission to search for evidence for extant or extinct life on Mars in 2018. The Raman spectrometer will be part of the first-pass analytical stage of the sampling procedure, following detailed surface examination by the PanCam scanning camera unit on the ExoMars rover vehicle. The requirements of the analytical protocol are stringent and critical; this study represents a laboratory blind interrogation of specimens that form a list of materials that are of relevance to martian exploration and at this stage simulates a test of current laboratory instrumentation to highlight the Raman technique strengths and possible weaknesses that may be encountered in practice on the martian surface and from which future studies could be formulated. In this preliminary exercise, some 10 samples that are considered terrestrial representatives of the mineralogy and possible biogeologically modified structures that may be identified on Mars have been examined with Raman spectroscopy, and conclusions have been drawn about the viability of the unambiguous spectral identification of biomolecular life signatures. It is concluded that the Raman spectroscopic technique does indeed demonstrate the capability to identify biomolecular signatures and the mineralogy in real-world terrestrial samples with a very high degree of success without any preconception being made about their origin and classification. Key Words: Biosignatures—Mars Exploration Rovers—Raman spectroscopy—Search for life (biosignatures)—Planetary instrumentation. Astrobiology 13, 543–549.
doi:10.1089/ast.2012.0872
PMCID: PMC3689185  PMID: 23758166

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