Raman Spectroscopic Protocol for the Molecular Recognition of Key Biomarkers in Astrobiological Exploration

Raman spectroscopy is proposed as novel instrumentation for the remote, robotic exploration of planetary surfaces, especially Mars. In recent years, information about the chemicals produced by organisms at the terrestrial limits of life, such as those surviving in Antarctic habitats, has facilitated the assembly of a spectral database of key biomarkers. In addition biogeological modifications which are essential for the survival strategies of environmentally stressed organisms have been identified. In this paper, the requirements for Raman spectroscopic instrumental detection of key bio--and bio-geological markers are outlined and a preliminary protocol established for the molecular spectral recognition of biological signatures in remote astrobiological exploration.     

The universe: a cryogenic habitat for microbial life

Panspermia, an ancient idea, posits that microbial life is ubiquitous in the Universe. After several decades of almost irrational rejection, panspermia is at last coming to be regarded as a serious contender for the beginnings of life on our planet. Astronomical data is shown to be consistent with the widespread distribution of complex organic molecules and dust particles that may have a biological provenance. A minuscule (10(-21)) survival rate of freeze-dried bacteria in space is all that is needed to ensure the continual re-cycling of cosmic microbial life in the galaxy. Evidence that terrestrial life may have come from elsewhere in the solar system has accumulated over the past decade. Mars is seen by some as a possible source of terrestrial life, but some hundreds of billions of comets that enveloped the entire solar system, are a far more likely primordial reservoir of life. Comets would then have seeded Earth, Mars, and indeed all other habitable planetary bodies in the inner regions of the solar system. The implications of this point of view, which was developed in conjunction with the late Sir Fred Hoyle since the 1970s, are now becoming amenable to direct empirical test by studies of pristine organic material in the stratosphere. The ancient theory of panspermia may be on the verge of vindication, in which case the entire universe would be a grand crucible of cryomicrobiology.     

Martian paleolakes and waterways: Exobiological implications

The problems of how warm and wet Mars once was and when climate transitions may have occurred are not well understood. Mars may have had an early environment similar to Earth's that was conductive to the ermergence of life. In addition, increasing geologic evidence indicates that water, upon which terrestrial life depends, has been present on Mars throughout its history. This evidence suggests that life could have developed not only on early Mars but also over longer periods of time in longer lasting, more clement local environments. Indications of past or present life most likely would be found in areas where liquid water existed in sufficient quantities to provide for the needs of biological systems. We suggest that paleolakes may have provided such environments. Unlike the case on Earth, this record of the origin and evolution of life has probably not been erased by extensive deformation of the Martian surface. Our work has identified eleven prospective areas where large lacustrine basins may once have existed. These areas are important for future biological, geological, and climatological investigations.Presented at the International Symposium on The Biological Exploration of Mars, October 26–27, 1990, Tallahassee, FL, U.S.A.     

Labeled release — An experiment in radiorespirometry

The Labeled Release extraterrestrial life detection experiment onboard the Viking spacecraft is described as it will be implemented on the surface of Mars in 1976. This experiment is designed to detect heterotrophic life by supplying a dilute solution of radioactive organic substrates to a sample of Martian soil and monitoring for evolution of radioactive gas. A significantly attenuated response by a heat-sterilized control sample of the same soil would confirm a positive metabolic response. Experimental assumptions as well as criteria for the selection of organic substrates are presented. The Labeled Release nutrient has been widely tested, is versatile in eliciting terrestrial metabolic responses, and is stable to heat sterilization and to the long-term storage required before its use on Mars. A testing program has been conducted with flight-like instruments to acquire science data relevant to the interpretation of the Mars experiment. Factors involved in the delineation of a positive result are presented and the significance of the possible results discussed.     

Mars' surface is not universally biocidal

The Mars surface/near-surface is often considered to be biocidal. Here, diverse lines of evidence are presented indicating that some terrestrial microbes can survive the in-situ conditions albeit in an inactive state. For the purposes of planetary protection, it is important to consider what we mean by a planetary ‘surface’; this term has qualitatively distinct definitions fordifferent scientific disciplines, and can also have different meanings from a humanviewpoint versus that of a microbial cell. Most microbial cells spores or other cells deposited on Mars, even those that initially fall on the outward-facing part of the absolute surface, will fall within pores of the regolith or become covered by its dust. They are, therefore, protected from ultra-violet radiation. Desiccating conditions and low temperatures (−40 to −70°C) can act to preserve rather than kill all microbes, potentially maintaining cellular viability – especially for certain extremophiles – over geological timescales. Whereas salts are ubiquitous on Mars, many terrestrial microbes are highly tolerant to NaCl and other salts, and these substances (including potentially inhibitory chaotropes such as MgCl₂ and perchlorates) cannot access cells in the absence of a liquid milieu. Whereas the Mars regolith is nutrient-deplete and conditions may be acidic in places, oligotrophic conditions per se are not biocidal and many terrestrial microbes can thrive in acidic conditions (some acidophiles can proliferate at or below pH 0). The low temperatures of Mars' surface are not conducive to metabolic activity, but the biophysical sophistication and robust stress biology of many terrestrial microbes, and the protection afforded by Martian conditions, are likely to ensure the long-term viability of some extremophilic microbes if transported to Mars.     

Passive Detection of Biological Aerosols in the Atmosphere with a Fourier Transform Instrument (FTIR)—the Results of the Measurements in the Laboratory and in the Field

Fourier Transform Infrared Radiation (FTIR) spectroscopy is one of the most powerful methods for the detection of gaseous constituents, aerosols, and dust in planetary atmospheres. Infrared spectroscopy plays an important role in searching for biomarkers, organics and biological substances in the Universe. The possibility of detection and identifications with FTIR spectrometer of bio-aerosol spores (Bacillus atrophaeus var. globigii=BG) in the atmosphere is discussed in this paper. We describe the results of initial spectral measurements performed in the laboratory and in the field. The purpose of these experiments was to detect and to identify bio-aerosol spores in two conditions: 1) In a closed chamber where the thermal contrast between the background and aerosols was large, and 2) In open air where the thermal contrast between the background and aerosols was small. The extinction spectrum of BG spores was deduced by comparing our measurements with models, and other measurements known from the literature. Our theoretical and experimental studies indicate that, during passive remote sensing measurements, it is difficult-but possible to detect and to identify bio-aerosol clouds by their spectral signatures. The simple spectral analysis described in the paper can be useful for the detection of various kinds of trace aerosols-not only in the Earth's atmosphere, but also during planetary missions in the environments of other astronomical objects such as planets, comets etc. We expect that the interpretation of data from spectrometric sounding of Venus and Mars during the current missions Mars and Venus Express, and later during the Rosetta mission will benefit from our experimental work and numerical modelling.     

Soil and water and its relationship to the origin of life.

Soils of the terrestrial planets form at the boundaries between lithosphere, atmosphere and hydrosphere. Biogenesis occurred in these zones; thus, it is axiomatic that some, perhaps many, stages of biogensis occurred in intimate association with the mineral constituents of soils. Because of a high surface to mass ration and, consequently, a high surface reactivity, the layer lattice clay minerals are the most important of these. According to the geological record, clay minerals appeared very early on the primordial Earth. Recent investigations have confirmed their presence in carbonaceous meteorites and have indicated their occurrence on Mars. In this paper we collect pertinent physico-chemical data and summarize the organic reactions and interactions that are induced or catalyzed by clays. Many clay-organic reactions that do not occur readily at high water contents proceed rapidly at adsorbed water contents corresponding to surface coverages of one or two molecular layers. One or two monolayers of adsorbed water correspond to extremely dry or cold planetary environments. Some consequences of these facts vis á vis biogenesis on Mars are considered.     

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.     

Comparative Evaluation Of Raman Spectroscopy At Different Wavelengths For Extremophile Exemplars

Raman spectra have been obtained for extremophiles from several geological environments; selected examples have been taken from hot and cold deserts comprising psychrophiles, thermophiles and halophiles. The purpose of this study is the assessment of the effect of the wavelength of the laser excitation on the ability to determine unique information from the Raman spectra about the specificity of detection of biomolecules produced as a result of the survival strategies adopted by organisms in extreme terrestrial environments. It was concluded that whereas FT-Raman spectroscopy at 1064 nm gave good quality results the time required to record the data was relatively large compared with other wavelengths of excitation but that better access to the CH stretching region for organic molecules was given. Shorter wavelength excitation of biomolecules in the blue-green regions of the visible spectrum using a conventional dispersive spectrometer was more rapid but very dependent upon the type of chemical compound being studied; most relevant biomolecules fluoresced at these wavelengths but carotenoids exhibited a resonance effect which resulted in an improved detection capability. Minerals and geological materials, in contrast, were best studied at these visible wavelengths. In general, the best compromise system for the excitation of the Raman spectra of both geological and biological materials was provided using a 785 nm laser coupled with a dispersive spectrometer, especially for accessing the 1800–200 cm⁻¹ wavenumber shift region where much of the definitive analytical information resides. This work will have conclusions relevant to the use of miniaturised Raman spectrometers for the detection of biomolecules in extraterrestrial planetary exploration.     

Origins of life: A comparison of theories and application to Mars

The field of study that deals with the origins of life does not have a consensus for a theory of life's origin. An analysis of the range of theories offered shows that they share some common features that may be reliable predictors when considering the possible origins of life on another planet. The fundamental datum dealing with the origins of life is that life appeared early in the history of the Earth, probably before 3.5 Ga and possibly before 3.8 Ga. What might be called the standard theory (the Oparin-Haldane theory) posits the production of organic molecules on the early Earth followed by chemical reactions that produced increased organic complexity leading eventually to organic life capable of reproduction, mutation, and selection using organic material as nutrients. A distinct class of other theories (panspermia theories) suggests that life was carried to Earth from elsewhere — these theories receive some support from recent work on planetary impact processes. Other alternatives to the standard model suggest that life arose as an inorganic (clay) form and/or that the initial energy source was not organic material but chemical energy or sunlight. We find that the entire range of current theories suggests that liquid water is the quintessential environmental criterion for both the origin and sustenance of life. It is therefore of interest that during the time that life appeared on Earth we have evidence for liquid water present on the surface of Mars.     

Why biologists should support the exploration of Mars

Physicists, chemists and geologists in the USA and Europe propose that the search for extraterrestrial life is an important justification for the exploration of Mars. Biologists, however, much more excited by the advent of the postgenome sequencing era, in general display little enthusiasm for planetary exploration. We argue that the search for traces of life on Mars represents a major thought-provoking challenge for the life sciences that should be taken up by the biological community.     

From exobiology to cosmobiology at LISA and elsewhere.

Since the emergence of Exobiology, back to the l960ties, this field drastically increased and, although differently named, is today a largely recognized scientific domain of wild interdisciplinarity. It includes not only the search for extraterrestrial living Systems, in particular by direct exploration of planetary bodies and studies of extraterrestrial materials, but also the study on the origins of life on Earth and, in connection to this field, the study of extraterrestrial organic chemistry. The exobiology programmes currently developed at LISA are related to this last aspect. They include the study of prebiotic-like chemistry in the gas and solid phases, based on laboratory simulation experiments, theoretical modeling and future in situ measurements in Titan's atmosphere and in cometary nuclei. A national program of exobiology, coordinated by LISA is under development in France, it covers many of the various aspects of Exobiology, including the study of life in extreme environments, as a reference tool for extraterrestrial life, the study of the primitive environment of the Earth, of the organic chemistry in comets and on Titan, of Mars and Europa and even of extrasolar planets as potential niches for extraterrestrial living systems, associated to the determination of the electromagnetic signatures of life. In parallel to this general program, a proposal for a large simulation chamber to be used as a national facility in particular to simulate the organic chemistry in various planetary environments, and in the interstellar medium, is under preparation. International cooperations linked to these programmes, in particular in the frame of the development of an exobiology facility on the International Space Station, would be of crucial interest.     

Soil and water and its relationship to the origin of life

Soils of the terrestrial planets form at the boundaries between lithosphere, atmosphere and hydrosphere. Biogenesis occurred in these zones; thus, it is axiomatic that some, perhaps many, stages of biogenesis occurred in intimate association with the mineral constituents of soils. Because of a high surface to mass ratio and, consequently, a high surface reactivity, the layer lattice clay minerals are the most important of these. according to the geological record, clay minerals appeared very early on the primordial Earth. Recent investigations have confirmed their presence in carbonaceous meteorites and have indicated their occurrence on Mars. In this paper we collect pertinent physico-chemical data and summarize the organic reactions and interactions that are induced or catalyzed by clays. Many clay-organic reactions that do not occur readily at high water contents proceed rapidly at adsorbed water contents corresponding to surface coverages of one or two molecular layers. One or two monolayers of adsorbed water correspond to extremely dry on cold planetary environments. Some consequences of these factsvis à vis biogenesis on Mars are considered.     

Kinetic and Thermodynamic Analysis During Dissimilatory γ-FeOOH Reduction: Formation of Green Rust 1 and Magnetite

The oldest sedimentary rocks on Earth, the 3.8‐Ga Isua Iron‐Formation in southwestern Greenland, are metamorphosed past the point where organic‐walled fossils would remain. Acid residues and thin sections of these rocks reveal ferric microstructures that have filamentous, hollow rod, and spherical shapes not characteristic of crystalline minerals. Instead, they resemble ferric‐coated remains of bacteria. Modern so‐called iron bacteria were therefore studied to enhance a search image for oxide minerals precipitated by early bacteria. Iron bacteria become coated with ferrihydrite, a metastable mineral that converts to hematite, which is stable under high temperatures. If these unusual morphotypes are mineral remains of microfossils, then life must have evolved somewhat earlier than 3.8 Ga, and may have involved the interaction of sediments and molecular oxygen in water, with iron as a catalyst. Timing is constrained by the early in fall of planetary materials that would have heated the planet's surface.

Because there are no earlier sedimentary rocks to study on Earth, it may be necessary to expand the search elsewhere in the solar system for clues to any biotic precursors or other types of early life. Evidence from Mars shows geophysical and geochemical differentiation at a very early stage, which makes it an important candidate for such a search if sedimentation is an important process in life's origins. Not only does Mars have iron oxide‐rich soils, but its oldest regions have river channels where surface water and sediment may have been carried, and seepage areas where groundwater may have discharged. Mars may have had an atmosphere and liquid water in the crucial time frame of 3.9–4.0 Ga. A study of morphologies of iron oxide minerals collected in the southern highlands during a Mars sample return mission may therefore help to fill in important gaps in the history of Earth's earliest biosphere.     

Survival of Methanogenic Archaea from Siberian Permafrost under Simulated Martian Thermal Conditions

Methanogenic archaea from Siberian permafrost complementary to the already well-studied methanogens from non-permafrost habitats were exposed to simulated Martian conditions. After 22 days of exposure to thermo-physical conditions at Martian low- and mid-latitudes up to 90% of methanogenic archaea from Siberian permafrost survived in pure cultures as well as in environmental samples. In contrast, only 0.3%–5.8% of reference organisms from non-permafrost habitats survived at these conditions. This suggests that methanogens from terrestrial permafrost seem to be remarkably resistant to Martian conditions. Our data also suggest that in scenario of subsurface lithoautotrophic life on Mars, methanogenic archaea from Siberian permafrost could be used as appropriate candidates for the microbial life on Mars.     

Optimisation of Wavelength Modulated Raman Spectroscopy: Towards High Throughput Cell Screening

In the field of biomedicine, Raman spectroscopy is a powerful technique to discriminate between normal and cancerous cells. However the strong background signal from the sample and the instrumentation affects the efficiency of this discrimination technique. Wavelength Modulated Raman spectroscopy (WMRS) may suppress the background from the Raman spectra. In this study we demonstrate a systematic approach for optimizing the various parameters of WMRS to achieve a reduction in the acquisition time for potential applications such as higher throughput cell screening. The Signal to Noise Ratio (SNR) of the Raman bands depends on the modulation amplitude, time constant and total acquisition time. It was observed that the sampling rate does not influence the signal to noise ratio of the Raman bands if three or more wavelengths are sampled. With these optimised WMRS parameters, we increased the throughput in the binary classification of normal human urothelial cells and bladder cancer cells by reducing the total acquisition time to 6 s which is significantly lower in comparison to previous acquisition times required for the discrimination between similar cell types.     

Life on Mars: chemical arguments and clues from Martian meteorites

Primitive terrestrial life – defined as a chemical system able to transfer its molecular information via self-replication and to evolve – probably originated from the evolution of reduced organic molecules in liquid water. Several sources have been proposed for the prebiotic organic molecules: terrestrial primitive atmosphere (methane or carbon dioxide), deep-sea hydrothermal systems, and extraterrestrial meteoritic and cometary dust grains. The study of carbonaceous chondrites, which contain up to 5% by weight of organic matter, has allowed close examination of the delivery of extraterrestrial organic material. Eight proteinaceous amino acids have been identified in the Murchison meteorite among more than 70 amino acids. Engel reported that l-alanine was surprisingly more abundant than d-alanine in the Murchison meteorite. Cronin also found excesses of l-enantiomers for nonprotein amino acids. A large collection of micrometeorites has been recently extracted from Antarctic old blue ice. In the 50- to 100-μm size range, carbonaceous micrometeorites represent 80% of the samples and contain 2% of carbon, on average. They might have brought more carbon than that involved in the present surficial biomass. The early histories of Mars and Earth clearly show similarities. Liquid water was once stable on the surface of Mars, attesting the presence of an atmosphere capable of deccelerating C-rich micrometeorites. Therefore, primitive life may have developed on Mars as well and fossilized microorganisms may still be present in the near subsurface. The Viking missions to Mars in 1976 did not find evidence of either contemporary or past life, but the mass spectrometer on the lander aeroshell determined the atmospheric composition, which has allowed a family of meteorites to be identified as Martian. Although these samples are essentially volcanic in origin, it has been recognized that some of them contain carbonate inclusions and even veins that have a carbon isotopic composition indicative of an origin from Martian atmospheric carbon dioxide. The oxygen isotopic composition of these carbonate deposits allows calculation of the temperature regime existing during formation from a fluid that dissolved the carbon dioxide. As the composition of the fluid is unknown, only a temperature range can be estimated, but this falls between 0° and 90°C, which would seem entirely appropriate for life processes. It was such carbonate veins that were found to host putative microfossils. Irrespective of the existence of features that could be considered to be fossils, carbonate-rich portions of Martian meteorites tend to have material, at more than 1000 ppm, that combusts at a low temperature; i.e., it is an organic form of carbon. Unfortunately, this organic matter does not have a diagnostic isotopic signature so it cannot be unambiguously said to be indigenous to the samples. However, many circumstantial arguments can be made to the effect that it is cogenetic with the carbonate and hence Martian. If it could be proved that the organic matter was preterrestrial, then the isotopic fractionation between it and the carbon is in the right sense for a biological origin. Received: January 22, 1998 / Accepted: February 16, 1998     

Cataclysm No More: New Views on the Timing and Delivery of Lunar Impactors

If properly interpreted, the impact record of the Moon, Earth’s nearest neighbour, can be used to gain insights into how the Earth has been influenced by impacting events since its formation ~4.5 billion years (Ga) ago. However, the nature and timing of the lunar impactors – and indeed the lunar impact record itself – are not well understood. Of particular interest are the ages of lunar impact basins and what they tell us about the proposed “lunar cataclysm” and/or the late heavy bombardment (LHB), and how this impact episode may have affected early life on Earth or other planets. Investigations of the lunar impactor population over time have been undertaken and include analyses of orbital data and images; lunar, terrestrial, and other planetary sample data; and dynamical modelling. Here, the existing information regarding the nature of the lunar impact record is reviewed and new interpretations are presented. Importantly, it is demonstrated that most evidence supports a prolonged lunar (and thus, terrestrial) bombardment from ~4.2 to 3.4 Ga and not a cataclysmic spike at ~3.9 Ga. Implications for the conditions required for the origin of life are addressed.     

Efficient inhibition of class A and class D beta-lactamases by Michaelis complexes

A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 beta-lactamases with K(I) = 42 +/- 4 nm and 1.0 +/- 0.1 microm, respectively. To gain insight into the reaction chemistry of SA-1-204, the reactions between this inhibitor and SHV-1 and OXA-1 were studied by Raman spectroscopy in single crystals and in solution. Raman signatures characteristic of the unreacted beta-lactam ring show that in both phases the inhibitor binds as a noncovalent Michaelis-like complex. This complex is present as the major population for periods of up to an hour. On longer time scales, the Raman data show that beta-lactam ring opening eventually leads to a complex mixture of reaction products. However, the data clearly demonstrate that the key species for inhibition on the time scale of bacterial half-lives is the noncovalent complex preceding acylation.     

Validation of the drop coating deposition Raman method for protein analysis

Drop coating deposition Raman (DCDR) spectroscopy is critically evaluated to establish the limits to which it may be used to detect changes in protein conformation, binding, and purity. Difference spectroscopy is used to evaluate the reproducibility of the DCDR spectra under various experimental conditions. The results indicate (i) the absence of thermal/photochemical laser damage induced by the Raman excitation laser under typical DCDR data collection conditions, (ii) the reproducibility of DCDR spectra from samples with different volumes or concentrations, (iii) the water content of DCDR protein deposits and associated spectral signatures, and (iv) the degree of similarity between solution Raman spectra and DCDR spectra.