Development of a gas chromatography compatible Sample Processing System (SPS) for the in-situ analysis of refractory organic matter in martian soil: preliminary results

In the frame of the 2009 Mars Science Laboratory (MSL) mission a new sample preparation system (SPS) compatible with gas chromatography–mass spectrometry (GC–MS) has been developed for the in situ analysis of complex organic molecules in the Martian soil. The goal is to detect, if they exist, some of the key compounds that play an important role in life on Earth including carboxylic acids, amino acids and nucleobases.     

Did life exist on Mars? Search for organic and inorganic signatures, one of the goals for “SAM” (sample analysis at Mars)

Observation of Mars shows signs of a past Earth-like climate, and, in that case, there is no objection to the possible development of life, in the underground or at the surface, as in the terrestrial primitive biosphere. Sample analysis at Mars (SAM) is an experiment which may be proposed for atmospheric, ground and underground in situ measurements. One of its goals is to bring direct or indirect information on the possibility for life to have developed on Mars, and to detect traces of past or present biological activity. With this aim, it focuses on the detection of organic molecules: volatile organics are extracted from the sample by simple heating, whereas refractory molecules are made analyzable (i.e. volatile), using derivatization technique or fragmentation by pyrolysis. Gaseous mixtures thus obtained are analyzed by gas chromatography associated to mass spectrometry. Beyond organics, carbonates and other salts are associated to the dense and moist atmosphere necessary to the development of life, and might have formed and accumulated in some places on Mars. They represent another target for SAM. Heating of the samples allows the analysis of structural gases of these minerals (CO₂ from carbonates, etc.), enabling to identify them. We also show, in this paper, that it may be possible to discriminate between abiotic minerals, and minerals (shells, etc.) created by living organisms.     

Possible complex organic compounds on Mars.

It is suggested that primitive Mars had somehow similar environments as primitive Earth. If life was born on the primitive earth using organic compounds which were produced from the early Earth environment, the same types of organic compounds were also formed on primitive Mars. Such organic compounds might have been preserved on Mars still now. We are studying possible organic formation on primitive and present Mars. A gaseous mixture of CO2, CO, N2 and H2O with various mixing ratios were irradiated with high energy protons (major components of cosmic rays). Hydrogen cyanide and formaldehyde were detected among volatile products, and yellow-brown-colored water-soluble non-volatile substances were produced, which gave amino acids after acid-hydrolysis. Major part of "amino acid precursors" were not simple molecules like aminonitriles, but complex compounds which eluted earlier than free amino acids in cation-exchange HPLC. These organic compounds should be major targets in the future Mars mission. Strategy for the detection of the complex organics on Mars will be discussed.     

Chemical evolution of primitive solar system bodies.

In this paper we summarize some of the most salient observations made recently on the organic molecules and other compounds of the biogenic elements present in the interstellar medium and in the primitive bodies of the solar system. They include the discovery of the first phosphorus molecular species in dense interstellar clouds, the presence of complex organic ions in the dust and gas phase of Halley's coma, the finding of unusual, probably presolar, deuterium-hydrogen ratios in the amino acids of carbonaceous chondrites, and new developments on the chemical evolution of Titan, the primitive Earth, and early Mars. Some of the outstanding problems concerning the synthesis of organic molecules on different cosmic bodies are also discussed from an exobiological perspective.     

Formation of bioorganic compounds in simulated planetary atmospheres by high energy particles or photons.

Various types of organic compounds have been detected in Jupiter, Titan, and cometary coma. It is probable that organic compounds were formed in primitive Earth and Mars atmospheres. Cosmic rays and solar UV are believed to be two major energy sources for organic formation in space. We examined energetics of organic formation in simulated planetary atmospheres. Gas mixtures including a C-source (carbon monoxide or methane) and a N-source (nitrogen or ammonia) was irradiated with the followings: High energy protons or electrons from accelerators, gamma-rays from 60Co, UV light from a deuterium lamp, and soft X-rays or UV light from an electron synchrotron. Amino acids were detected in the products of particles, gamma-rays and soft X-rays irradiation from each gas mixture examined. UV light gave, however, no amino acid precursors in the gas mixture of carbon monoxide, nitrogen and nitrogen. It gave only a trace of them in the gas mixture of carbon monoxide, ammonia and water or that of methane, nitrogen and water. Yield of amino acid precursors by photons greatly depended on their wavelength. These results suggest that nitrogen-containing organic compounds like amino acid precursors were formed chiefly with high energy particles, not UV photons, in Titan or primitive Earth/Mars atmospheres where ammonia is not available as a predominant N-source.     

Bio-markers and the search for extinct life on Mars.

Geologic and climatologic studies suggest that conditions on early Mars were similar to early Earth. Because life on Earth is believed to have originated during this early period (3.5 billion years ago), the Martian environment could have also been conducive to the origin of life. To investigate this possibility we must first define the attributes of an early Martian biota. Then, specific geographic locations on Mars must be chosen where life may have occurred (i.e. areas which had long standing water), and within these distinct locations search for key signatures or bio-markers of a possible extinct Martian biota. Some of the key signatures or bio-markers indicative of past biological activity on Earth may be applicable to Mars including: reduced carbon and nitrogen compounds, CO3(2-), SO4(2-), NO3-, NO2- [correction of NO2(2)], Mg, Mn, Fe, and certain other metals, and the isotopic ratios of C, N and S. However, we must also be able to distinguish abiotic from biologic origins for these bio-markers. For example, abiotically fixed N2 would form deposits of NO3- and NO2-, whereas biological processes would have reduced these to ammonium containing compounds, N2O, or N2, which would then be released to the atmosphere. A fully equipped Mars Rover might be able to perform analyses to measure most of these biomarkers while on the Martian surface.     

Estimation and assessment of Mars contamination.

Since the beginning of the exploration of Mars, more than fourty years ago, thirty-six missions have been launched, including fifty-nine different space systems such as fly-by spacecraft, orbiters, cruise modules, landing or penetrating systems. Taking into account failures at launch, about three missions out of four have been successfully sent toward the Red Planet. The fact today is that Mars orbital environment includes orbiters and perhaps debris, and that its atmosphere and its surface include terrestrial compounds and dormant microorganisms. Coming from the UN Outer Space Treaty [United Nations Treaty on Principles Governing the Activities of States in the Exploration and Use of Outer Space, including the Moon and Other Celestial Bodies (the "Outer Space Treaty") referenced 610 UNTS 205 - resolution 2222(XXI) of December 1966] and according to the COSPAR planetary protection policy recommendations [COSPAR Planetary Protection Policy (20 October 2002), accepted by the Council and Bureau, as moved for adoption by SC F and PPP, prepared by the COSPAR/IAU Workshop on Planetary Protection, 4/02 with updates 10/0, 2002], Mars environment has to be preserved so as not to jeopardize the scientific investigations, and the level of terrestrial material brought on and around Mars theoretically has to comply with this policy. It is useful to evaluate what and how many materials, compounds and microorganisms are on Mars, to list what is in orbit and to identify where all these items are. Considering assumptions about materials, spores and gas location and dispersion on Mars, average contamination levels can be estimated. It is clear now that as long as missions are sent to other extraterrestrial bodies, it is not possible to keep them perfectly clean. Mars is one of the most concerned body, and the large number of missions achieved, on-going and planned now raise the question about its possible contamination, not necessarily from a biological point of view, but with respect to all types of contamination. Answering this question, will help to assess the potential effects of such contamination on scientific results and will address concerns relative to any ethical considerations about the contamination of other planets.     

The optical constants of gypsum particles as analog of Martian sulfates

Various indications seem to suggest that in the past the Martian climate allowed the presence of liquid water on the surface of the planet. It is therefore reasonable to think that bodies of standing water were once present in basins such as craters or depressions and that evaporite deposits of sulfates could have formed in these basins. The identification of such deposits, which might have survived in some regions of the planet, could confirm the present hypothesis about the ancient climate on Mars. Recent analyses of Mars spectra, obtained by ground-based observations, show bands consistent with sulfate features. If such data have to be interpreted with the aid of synthetic spectra, the knowledge of the complex refractive index of some sulfate is necessary. In this work we present the optical constants of submicron particles of gypsum (a typical hydrate–sulfate very common on Earth), derived by means of the dispersion theory from transmission spectra. We have performed the calculation of the optical constants in the wavelength range 2–70 μm, also in view of the wide spectral range covered by the Planetary Fourier Spectrometer (PFS) on board of the ESA space mission Mars Express. The data have been compared with optical constants obtained by other authors from reflectance measurements of particulate samples.     

Search for life on Mars: evaluation of techniques.

An important question for exobiology is, did life evolve on Mars? To answer this question, experiments must be conducted on the martian surface. Given current mission constraints on mass, power, and volume, these experiments can only be performed using proposed analytical techniques such as: electron microscopy, X-ray fluorescence, X-ray diffraction, alpha-proton backscatter, gamma-ray spectrometry, differential thermal analysis, differential scanning calorimetry, pyrolysis gas chromatography, mass spectrometry, and specific element detectors. Using prepared test samples consisting of 1% organic matter (bovine serum albumin) in palagonite and a mixture of palagonite, clays, iron oxides, and evaporites, it was determined that a combination of X-ray diffraction and differential thermal analysis coupled with gas chromatography provides the best insight into the chemistry, mineralogy, and geological history of the samples.     

Planetary protection and the search for life beneath the surface of Mars.

The search for traces of extinct and extant life on Mars will be extended to beneath the surface of the planet. Current data from Mars missions suggesting the presence of liquid water early in Mars' history and mathematical modeling of the fate of water on Mars imply that liquid water may exist deep beneath the surface of Mars. This leads to the hypothesis that life may exist deep beneath the Martian surface. One possible scenario to look for life on Mars involves a series of unmanned missions culminating with a manned mission drilling deep into the Martian subsurface (approximately 3Km), collecting samples, and conducting preliminary analyses to select samples for return to earth. This mission must address both forward and back contamination issues, and falls under planetary protection category V. Planetary protection issues to be addressed include provisions stating that the inevitable deposition of earth microbes by humans should be minimized and localized, and that earth microbes and organic material must not contaminate the Martian subsurface. This requires that the drilling equipment be sterilized prior to use. Further, the collection, containment and retrieval of the sample must be conducted such that the crew is protected and that any materials returning to earth are contained (i.e., physically and biologically isolated) and the chain of connection with Mars is broken.     

Biological nitrogen fixation under primordial Martian partial pressures of dinitrogen.

Early Earth and early Mars were similar enough such that past geochemical and climatic conditions on Mars may have also been favorable for the origin of life. However, one of the most striking differences between the two planets was the low partial pressure of dinitrogen (pN2) on early Mars (18 mb). On Earth, nitrogen is a key biological element and in many ecosystems the low availability of fixed nitrogen compounds is the main factor limiting growth. Biological fixation of dinitrogen on Earth is a crucial source of fixed nitrogen. Could the low availability of dinitrogen in the primordial Martian atmosphere have prevented the existence, or evolution of Martian microbiota? Azotobacter vinelandii and Azomonas agilis were grown in nitrogen free synthetic medium under various partial pressures of dinitrogen ranging from 780-0 mb (total atmosphere=1 bar). Below 400 mb the biomass, cell number, and growth rate decreased with decreasing pN2. Both microorganisms were capable of growth at a pN2 as low as 5 mb, but no growth was observed at a pN2 < or = 1 mb. The data appear to indicate that biological nitrogen fixation could have occurred on primordial Mars (pN2=18 mb) making it possible for a biotic system to have played a role in the Martian nitrogen cycle. It is possible that nitrogen may have played a key role in the early evolution of life on Mars, and that later a lack of available nitrogen on that planet (currently, pN2=0.2 mb) may have been involved in its subsequent extinction.     

Distribution and variability of accelerated electrons at Mars

We investigate accelerated electrons observed by Mars Global Surveyor (MGS), using data from the Electron Reflectometer (ER) instrument. We find three different types of accelerated electron events. Current sheet events occur over regions with weak or no crustal fields, have the highest electron energy fluxes, and are likely located on draped magnetotail fields. Extended events occur over regions with moderate crustal magnetic fields, and are most often observed on closed magnetic field lines. Localized events have the lowest energy fluxes, occur in strong magnetic cusp regions, and are the most likely kind of event to be found on open magnetic field lines. Some localized events have clear signatures of field-aligned currents; these events have much higher electron fluxes, and are preferentially observed on radially oriented open magnetic field lines. Electron acceleration events, especially localized events, are similar in many ways to events observed in the terrestrial auroral zone. However, physical processes related to those found in the terrestrial cusp and/or plasmasheet could also be responsible for accelerating electrons at Mars.     

Planetary protection issues in advance of human exploration of Mars.

Current planetary quarantine considerations focus on robotic missions and attempt a policy of no biological contamination. The presence of humans on Mars, however, will inevitably result in biological contamination and physical alteration of the local environment. The focus of planetary quarantine must therefore shift toward defining and minimizing the inevitable contamination associated with humans. This will involve first determining those areas that will be affected by the presence of a human base, then verifying that these environments do not harbor indigenous life nor provide sites for Earth bacteria to grow. Precursor missions can provide salient information that can make more efficient the planning and design of human exploration missions. In particular, a robotic sample return mission can help to eliminate the concern about returning samples with humans or the return of humans themselves from a planetary quarantine perspective. Without a robotic return the cost of quarantine that would have to be added to a human mission may well exceed the cost of a robotic return mission. Even if the preponderance of scientific evidence argues against the presence of indigenous life, it must be considered as part of any serious planetary quarantine analysis for missions to Mars. If there is life on Mars, the question of human exploration assumes an ethical dimension.     

Life support approaches for Mars missions.

Life support approaches for Mars missions are evaluated using an equivalent system mass (ESM) approach, in which all significant costs are converted into mass units. The best approach, as defined by the lowest mission ESM, depends on several mission parameters, notably duration, environment and consequent infrastructure costs, and crew size, as well as the characteristics of the technologies which are available. Generally, for the missions under consideration, physicochemical regeneration is most cost effective. However, bioregeneration is likely to be of use for producing salad crops for any mission, for producing staple crops for medium duration missions, and for most food, air and water regeneration for long missions (durations of a decade). Potential applications of in situ resource utilization need to be considered further.     

The organic aerosols of Titan

A dark reddish organic solid, called tholin, is synthesized from simulated Titanian atmospheres by irradiation with high energy electrons in a plasma discharge. The visible reflection spectrum of this tholin is found to be similar to that of high altitude aerosols responsible for the albedo and reddish color of Titan. The real (n) and imaginary (k) parts of the complex refractive index of thin films of Titan tholin prepared by continuous D.C. discharge through a 0.9 N₂/0.1 CH₄ gas mixture at 0.2 mb is determined from x-ray to microwave frequencies. Values of n (1.65) and k (0.004 to 0.08) in the visible are consistent with deductions made by ground-based and spaceborne observations of Titan. Many infrared absorption features are present in k(λ), including the 4.6 μm nitrile band. Molecular analysis of the volatile component of this tholin was performed by sequential and non-sequential pyrolytic gas chromatography/mass spectrometry. More than one hundred organic compounds are released; tentative identifications include saturated and unsaturated aliphatic hydrocarbons, substituted polycyclic aromatics, nitriles, amines, pyrroles, pyrazines, pyridines, pyrimidines, and the purine, adenine. In addition, acid hydrolysis produces a racemic mixture of biological and non-biological amino acids. Many of these molecules are implicated in the origin of life on Earth, suggesting Titan as a contemporary laboratory environment for prebiological organic chemistry on a planetary scale.     

Life support systems for Mars transit.

The long-held human dream of travel to the stars and planets will probably be realized within the next quarter century. Preliminary analyses by U.S. scientists and engineers suggests that a first trip to Mars could begin as early as 2016. A proposal by U.S.S.R. space planners has suggested that an effort involving the cooperation and collaboration of many nations could begin by 2011. Among the major considerations that must be made in preparation for such an excursion are solidification of the scientific, economic and philosophical rationales for such a trip made by humans, and realistic evaluations of current and projected technical capabilities. Issues in the latter category include launch and propulsion systems, long term system stability and reliability, the psychological and physiological consequences of long term exposure to the space environment, the development and use of countermeasures to deleterious human physiological responses to the space environment, and life support systems that are both capable of the immense journey and reliable enough to assure their continued operation for the duration of the voyage. Many of the issues important in the design of a life support system for a Mars trip are based on reasonably well understood data: the human requirements for food, oxygen and water. However, other issues are less well-defined, such as the demands that will be made on the system for personal cleanliness and hygiene, environmental cleanliness, prevention or reduction of environmental toxins, and psychological responses to the environment and to the diet. It is much too early to make final decisions about the characteristics of the long-duration life support system needed for travel to Mars, or for use on its surface. However, it is clear that life support systems will evolve during the next few decades form the relatively straightforward systems that are used on Shuttle and Soyuz, to increasingly more complex and regenerative systems. The Soviet Union has an operating life support system on Mir that can apparently evolve, and the United States is currently planning the one for Space Station Freedom that will use partial regeneration. It is essential to develop concepts now for life support systems on an advanced Space Station, the lunar outpost (to be launched in about 2004) and the lunar base. Such concepts will build on current technology and capabilities. But because of the variety of different technologies that can be developed, and the potential for coordinating the functions of very diverse sub-systems within the same life support system, the possibility of developing an efficient, reliable mixed process system is high. It is likely that a life support system for Mars transit and base will use a composite of physical, chemical, and biological processes. The purpose of this paper is to explore the potentially useful structural elements of a life support system for use on a Mars trip, and to identify the features that, at this time, appear to be most appropriate for inclusion in the system.     

Simulation of the environmental climate conditions on martian surface and its effect on Deinococcus radiodurans

The resistance of terrestrial microorganisms under the thermo-physical conditions of Mars (diurnal temperature variations, UV climate, atmospheric pressure and gas composition) at mid-latitudes was studied for the understanding and assessment of potential life processes on Mars. In order to accomplish a targeted search for life on other planets, e.g. Mars, it is necessary to know the limiting physical and chemical parameters of terrestrial life. Therefore the polyextremophile bacterium Deinococcus radiodurans was chosen as test organism for these investigations. For the simulation studies at the Planetary and Space Simulation Facilities (PSI) at DLR, Cologne, Germany, conditions that are present during the southern summer at latitude of 60° on Mars were applied.We could simulate several environmental parameters of Mars in one single experiment: vacuum/low pressure, anoxic atmosphere and diurnal cycles in temperature and relative humidity, energy-rich ultraviolet (UV) radiation as well as shielding by different martian soil analogue materials. These parameters have been applied both single and in different combinations in laboratory experiments. Astonishingly the diurnal Mars-like cycles in temperature and relative humidity affected the viability of D. radiodurans cells quite severely. But the martian UV climate turned out to be the most deleterious factor, though D. radiodurans is red-pigmented due to carotenoids incorporated in its cell wall, which have been assigned not only a possible role as free radical scavenger but also as a UV-protectant. An additional UV-protection was accomplished by mixing the bacteria with nano-sized hematite.     

Planetary protection issues related to human missions to Mars

In accordance with the United Nations Outer Space Treaties [United Nations, Agreement Governing the Activities of States on the Moon and Other Celestial Bodies, UN doc A/RES/34/68, resolution 38/68 of December 1979], currently maintained and promulgated by the Committee on Space Research [COSPAR Planetary Protection Panel, Planetary Protection Policy accepted by the COSPAR Council and Bureau, 20 October 2002, amended 24 March 2005, http://www.cosparhq.org/scistr/PPPolicy.htm], missions exploring the Solar system must meet planetary protection requirements. Planetary protection aims to protect celestial bodies from terrestrial contamination and to protect the Earth environment from potential biological contamination carried by returned samples or space systems that have been in contact with an extraterrestrial environment. From an exobiology perspective, Mars is one of the major targets, and several missions are currently in operation, in transit, or scheduled for its exploration. Some of them include payloads dedicated to the detection of life or traces of life. The next step, over the coming years, will be to return samples from Mars to Earth, with a view to increasing our knowledge in preparation for the first manned mission that is likely to take place within the next few decades. Robotic missions to Mars shall meet planetary protection specifications, currently well documented, and planetary protection programs are implemented in a very reliable manner given that experience in the field spans some 40 years. With regards to sample return missions, a set of stringent requirements has been approved by COSPAR [COSPAR Planetary Protection Panel, Planetary Protection Policy accepted by the COSPAR Council and Bureau, 20 October 2002, amended 24 March 2005, http://www.cosparhq.org/scistr/PPPolicy.htm], and technical challenges must now be overcome in order to preserve the Earth’s biosphere from any eventual contamination risk. In addition to the human dimension of the mission, sending astronauts to Mars will entail meeting all these constraints. Astronauts present huge sources of contamination for Mars and are also potential carriers of biohazardous material on their return to Earth. If they were to have the misfortune of being contaminated, they themselves would become a biohazard, and, as a consequence, in addition to the technical constraints, human and ethical considerations must also be taken into account.     

Stable isotope analysis at the molecular level: a new approach for determining the origins of amino acids in the Murchison meteorite.

A combined gas chromatography/isotope ratio mass spectrometry (GC/IRMS) method has been developed that permits the direct stable carbon isotope analysis of N(O)-trifluoroacetyl-isopropyl esters of individual amino acids and their respective enantiomers at nanomole abundances. Calculation of the original delta 13C values of the amino acids is accomplished via a correction for the carbon introduced during the derivatization process. Previous GC/IRMS analyses of individual amino acids in the non-hydrolyzed water extract of an interior sample of a Murchison meteorite stone revealed an enrichment in 13C relative to terrestrial organic matter, in agreement with previous findings for bulk extracts. The range of amino acid delta 13C values (+5 to +30%, PDB) suggests possible kinetic effects during synthesis. In this study, an apparent kinetic isotope effect was also observed for the amino acid products of a spark discharge experiment. These preliminary results are supportive of a similar mechanism for the abiotic synthesis of amino acids in the Murchison meteorite.     

Life on Mars? I. The chemical environment.

The origin of life at its abiotic evolutionary stage, requires a combination of constituents and environmental conditions that enable the synthesis of complex replicating macromolecules from simpler monomeric molecules. It is very likely that the early stages of this evolutionary process have been spontaneous, rapid and widespread on the surface of the primitive Earth, resulting in the formation of quite sophisticated living organisms within less than a billion years. To what extent did such conditions prevail on Mars? Two companion-papers (Life on Mars? I and II) will review and discuss the available information related to the chemical, physical and environmental conditions on Mars and assess it from the perspective of potential exobiological evolution.