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.     

The photolytic degradation and oxidation of organic compounds under simulated Martian conditions

Summary Cosmochemical considerations suggest various potential sources for the accumulation of organic matter on Mars. However the Viking Molecular Analysis did not indicate any indigenous organic compounds on the surface of Mars. Their disappearance from the top layer is most likely caused by the combined action of the high solar radiation flux and various oxidizing species in the Martian atmosphere and regolith. In this study the stability of several organic substances and a sample of the Murchison meteorite was tested under simulated Martian conditions. After adsorption on powdered quartz, samples of adenine, glycine and naphthalene were irradiated with UV light at various oxygen concentrations and exposure times. In the absence of oxygen, adenine and glycine appeared stable over the given irradiation period, whereas a definite loss was observed in the case of naphthalene, as well as in the volatilizable and pyrozable content of the Murchison meteorite. The presence of oxygen during UV exposure caused a significant increase in the degradation rate of all samples. It is likely that similar processes have led to the destruction of organic materials on the surface of Mars.     

Detection of organic compounds on Mars]

McKay et al. detected polycyclic aromatic hydrocarbons (PAHs) in Martian meteorite ALH 84001 by two-step laser mass spectrometry. From the presence of PAHs, together with other results, they concluded that there were past life of Mars. On the other hands, no organisms nor organic compounds were detected in Martian regolith in Viking experiments in 1976. In order to obtain solid evidence for organisms or bioorganic compounds compounds on Mars, further analyses of Martian samples are required. There may be four classes of organic compounds on Mars, which are (i) organic compounds abiotically formed from primitive Mars atmosphere, (ii) Organic compounds delivered out of Mars, (iii) Organic compounds biotically formed by Mars organisms, and (iv) Organic compounds abiotically formed from the present Mars atmosphere. Possible organic compounds on Mars and analytical methods for them are discussed.     

An automatically-returned Martian sample by 1985?

The success of the lunar sample analysis programs underscores the desirability of a returned Martian sample. A Mission which would bring back about 1 kg of soil is outlined. The vehicle would have a mass of about 15 tonnes on departure from Earth and would make extensive use of Viking and Mariner technology. Russian experience in the field of automatic soil sampling and automatic rendezvous would be invaluable and the Shuttle would make possible a tidier launch. Sterilisation or quarantine will be necessary to preclude back-contamination of Earth by hypothetical Martian micro-organisms. A prime quarantine facility designed to detect biogenic organic compounds and life processes could be set up at a Lunar base or in a Sky-lab. A single soil sample could be informative as to the general surface composition of Mars. Life detection would be a major task, followed closely by the chemistry of carbon and other life-related elements. However, knowledge of the detailed physics, chemistry and mineralogy of the Martian sample would be of inestimable value to planetary studies.     

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.     

The viking biology experiments: Epilogue and prologue

In looking ahead to possibe new attempts to search for extant life on Mars, the history of the Viking biological investigations is reviewed here. Scientific considerations that led to the selection of specific experimental approaches for life detection are discussed, as well as the overall results obtained from that mission. Despite extensive preflight testing of the concepts that were to be used, unanticipated artefacts arose in the actual mission. These almost certainly reflect the fact that, at that time, there were many gaps in our understanding of the physical and chemical characteristics of the Martian environment. After Viking, many of these issues still remain unresolved, and future attempts to search for extant biology should be restrained until adequate new information about potential habitable microenvironments is obtained.Presented at the International Symposium on the Biological Exploration of Mars, October 26–27, 1990, Tallahasee, Fla., U.S.A.     

Simulation of the Viking biology experiments: An overview

Summary Several ground-based investigations have been carried out since the Viking biology results were received from Mars. Many of these have resulted in reasonable simulations of the Martian data, using as analogues of Mars either strong oxidants, UV-treated materials, iron-containing clays, or iron salts. The ambiguity between the GCMS experiment, in which no organic compounds were found on Mars, and the Labeled Release experiment, in which added organics were decomposed, may well be accounted for by these simulations.     

Automated life-detection experiments for the Viking mission to Mars

As part of the Viking mission to Mars in 1975, an automated set of instruments is being built to test for the presence of metabolizing organisms on that planet. Three separate modules are combined in this instrument so that samples of the Martian surface can be subjected to a broad array of experimental conditions so as to measure biological activity. The first, the Pyrolytic Release Module, will expose surface samples to a mixture of C¹⁴O and C¹⁴O₂ in the presence of Martian atmosphere and a light source that simulates the Martian visible spectrum. The assay system is designed to determine the extent of assimilation of CO or CO₂ into organic compounds. A small amount of water can be injected into the gas phase during incubation upon command. The Gas Exchange Module will incubate surface samples in a humidified CO₂ atmosphere. At specified times, portions of the incubation atmosphere will be analyzed by gas chromatography to detect the release or uptake of CO₂ and several additional gases. A rich and diversified source of organic nutrients and trace compounds will be available as further additions to the incubating samples. The Label Release Module will incubate surface samples with a dilute aqueous solution of simple radioactive organic substrates in Martian atmosphere, and the gas phase will be monitored continuously for the release of labeled CO₂. Each module, in addition to its gas and nutrient sources, incubation chambers, and detector systems, contains heaters capable of sterilizing surface samples to serve as controls. Since the instrument is designed to operate under Martian conditions and to detect Martian, not terrestrial, organisms, and because the final flight instruments can perform only four assays for each module, formidable problems exist in testing the hardware. The implications of this situation are discussed.     

The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight

The environment in space and on planets such as Mars can be lethal to microorganisms because of the high vacuum and high solar radiation flux, in particular UV radiation, in such environments. Spores of various Bacillus species are among the organisms most resistant to the lethal effects of high vacuum and UV radiation, and as a consequence are of major concern for planetary contamination via unmanned spacecraft or even natural processes. This review focuses on the spores of various Bacillus species: (i) their mechanisms of UV resistance; (ii) their survival in unmanned spacecraft, space flight and simulated space flight and Martian conditions; (iii) the UV flux in space and on Mars; (iv) factors affecting spore survival in such high UV flux environments.     

Laboratory investigations of mars: Chemical and spectroscopic characteristics of a suite of clays as Mars Soil Analogs

Two major questions have been raised by prior explorations of Mars. Has there ever been abundant water on Mars? Why is the iron found in the Martian soil not readily seen in the reflectance spectra of the surface? The work reported here describes a model soil system of Mars Soil Analog Materials, MarSAM, with attributes which could help resolve both of these dilemmas. The first set of MarSAM consisted of a suite of variably iron/calcium-exchanged montmorillonite clays. Several properties, including chemical composition, surface-ion composition, water adsorption isotherms, and reflectance spectra, of these clays have been examined. Also, simulations of the Viking Labeled Release Experiment using the MarSAM were performed. The results of these studies show that surface iron and adsorbed water are important determinants of clay behavior as evidenced by changes in reflectance, water absorption, and clay surface reactions. Thus, these materials provide a model soil system which reasonably satisfies the constraints imposed by the Viking analyses and remote spectral observations of the Martian surface, and which offers a sink for significant amounts of water. Finally, our initial results may provide insights into the mechanisms of reactions that occur on clay surfaces as well as a more specific approach to determining the mineralogy of Martian soils.     

The analysis of water in the Martian regolith

Summary One of the scientific objectives of the Viking Mission to Mars was to accomplish an analysis of water in the Martian regolith. The analytical scheme originally envisioned was severly compromised in the latter stages of the Lander instrument package design. Nevertheless, a crude soil water analysis was accomplished. Samples from each of the two widely separated sites yielded roughly 1 to 3% water by weight when heated successively to several temperatures up to 500°C. A significant portion of this water was released in the 200° to 350°C interval indicating the presence of mineral hydrates of relatively low thermal stability, a finding in keeping with the low temperatures generally prevailing on Mars. The presence of a duricrust at one of the Lander sites is taken as possible evidence for the presence of hygroscopic minerals on Mars. The demonstrated presence of atmospheric water vapor and thermodynamic calculations lead to the belief that adsorbed water could provide a relatively favorable environment for endolithic organisms on Mars similar to types recently discovered in the dry antarctic deserts.     

Mars ultraviolet simulation facility

Summary A facility was established for long-duration ultraviolet (UV) radiation exposure of natural and synthetic materials in order to test hypotheses concerning Martian soil chemistry observed by the Viking Mars landers. The system utilized a 2500 watt xenon lamp as the radiation source, with the beam passing through a heat-dissipating water filter before impinging upon an exposure chamber containing the samples to be irradiated. The chamber was designed to allow for continuous tumbling of the samples, maintenance of temperatures below 0° during exposure, and monitoring of beam intensity. The facility also provided for sample preparation under a variety of atmospheric conditions, in addition to the Mars nominal. As many as 33 sealed sample ampules have been irradiated in a single exposure. Over 100 samples have been irradiated for approximately 100 to 700 h. The facility has performed well in providing continuous UV irradiation of multiple samples for long periods of time under simulated Mars atmospheric and thermal conditions.     

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     

Smectite clays in Mars soil: Evidence for their presence and role in Viking biology experimental results

Summary Various chemical, physical and geological observations indicate that smectite clays are probably the major components of the Martian soil. Satisfactory ground-based chemical simulation of the Viking biology experimental results was obtained with the smectite clays nontronite and montmorillonite when they contained iron and hydrogen as adsorbed ions. Radioactive gas was released from the medium solution used in the Viking Labeled Release (LR) experiment when interacted with the clays, at rates and quantities similar to those measured by Viking on Mars. Heating of the active clay (mixed with soluble salts) to 160°C in CO₂ atmosphere reduced the decomposition activity considerably, again, as was observed on Mars. The decomposition reaction in LR experiment is postulated to be iron-catalyzed formate decomposition on the clay surface. The main features of the Viking Pyrolytic Release (PR) experiment were also simulated recently (Hubbard, 1979) which the iron clays, including a relatively low 1st peak and significant 2nd peak.The accumulated observations on various Martian soil properties and the results of simulation experiments, thus indicate that smectite clays are major and active components of the Martian soil. It now appears that many of the results of the Viking biology experiments can be explained on the basis of their surface activity in catalysis and adsorption.     

Metabolism of Di- and Mono-n-Butyl Phthalate by Soil Bacteria

Samples for mycological analysis were collected from surfaces in the Skylab spacecraft before launch and during flight for each manned mission. Fungal contamination levels were low during the first two flights; however, the species recovered were different for each mission. On the third mission, widespread contamination of the Skylab spacecraft with Aspergillus and Pencillium spp. was detected. This contamination was traced to several contaminated space suit undergarments.     

Did Viking Discover Life on Mars?

A major argument in the claim that life had been discovered during the Viking mission to Mars is that the results obtained in the Labeled Release (LR) experiment are analogous to those observed with terrestrial microorganisms. This assertion is critically examined and found to be implausible.     

Life on Mars? The Viking labeled release experiment.

Viking radiorespirometry ("Labeled Release" [LR]) experiments conducted on surface material obtained at two sites on Mars have produced results which on Earth would clearly establish the presence of microbial activity in the soil. However, two factors on Mars keep the question open. First, the intense UV flux striking Mars has given rise to several theories postulating the production of highly oxidative compounds. Such compounds might be responsible for the observed results. Second, the molecular analysis experiment has not found organic matter in the Mars surface material, and therefore, does not support the presence of roganisms. However, sensitivity limitations of the organic analysis instrument could permit as many as one million terrestrial type bacteria to go undetected. Terrestrial experiments with UV irradiation of Mars Analog Soil did not produce Mars type LR results. Gamma irradiation of silica gel did produce positive results, but not mimicking those on Mars. The life question remains open.     

Terrestrial contamination in Apollo lunar samples

The Apollo lunar samples were seen to offer a unique opportunity in the search for extraterrestrial organic matter without the ambiguity surrounding meteorite analysis due to their unknown contamination histories. The recognition that only a small amount of indigenous organic material was likely to be present in lunar samples combined with the extreme sensitivity of organic analysis methods made it clear that this opportunity could be realized only by carefully controlling the collection, processing, and analysis of the samples in order that they might remain free of significant levels of contamination. The contamination control procedures adopted are described and the analytical evidence obtained throughout the program on potential contamination sources is presented. The organic contaminants actually found in the lunar samples by the various investigators are summarized. It is shown that the program succeeded in providing investigators with samples containing less than 0.1 ppm total contamination.     

Clean Chemistry for Elemental Impurities Analysis of Pharmaceuticals in Compliance with USP 232

United States Pharmacopeia updated its 100 years old metal analysis method with inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). These sensitive instruments require that sample preparation be at least as sophisticated as the instrumentation used in the analysis. Sample contamination during sample preparation has to be controlled to an acceptable level given the low detection limit of these instruments and the ubiquitous presence of elements. This article focused on sample contamination during sample preparation. Contaminations from environment, reagents, and lab apparatus were investigated for their impact on trace element analysis. Advice on clean lab practice was offered to the pharmaceutical industry in regard to contamination control in elemental analysis labs at a time when the industry is preparing for compliance with elemental impurities in drug products.