Completion of the Viking labeled release experiment on Mars

Summary The final Labeled Release (LR) cycle on each Viking lander tested a surface sample that had been stored for several months at approximately 10°C prior to the onset of the active sequence. At each lander site, activity was strongly diminished. This thermal sensitivity of the active agent on the surface of Mars is consistent with a biological explanation of the LR experiment. At the end of one of these cycles, the incubation mixture was heated to 50°C to release any radioactive gas trapped in the sample matrix. The results suggest that more than one carbon substrate is involved in the LR reaction on Mars.The thermal data from the stored samples, coupled wth data from previous cycles, have formed the basis for evaluation of the thermal decomposition of the Mars active agent. The slope of the resulting Arrhenius plot has been used to test the fit of other flight data and to calculate the activation energy for thermal decomposition of the Mars agent. The results and their interpretation still leave unresolved the question of whether the Mars LR data were generated by biological or chemical activity.     

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.     

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.     

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.     

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.     

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.     

Laboratory simulations of the pyrolytic release experiments: An interim report

Summary During its operation on Mars the pyrolytic release experiment (PR) detected the fixation of small amounts of CO₂ and/or CO. Laboratory simulations of the experimental conditions were made in an attempt to substantiate the previous conclusion that these reactions were chemical rather than biological. The selection of model substrata for these tests was based on the known properties of the Martian surface material. After pretreatment and incubation under various conditions, pyrolytic analysis was used to indicate the extent of surface catalyzed conversion of¹⁴CO₂ or¹⁴CO to¹⁴C-organic compounds. This abiotic synthesis was detected in experiments with three iron oxides, viz. hematite, magnetite and maghemite. When the incubation atmosphere was supplemented with water vapor, the levels of synthesis were in a range comparable to that detected in the Viking PR tests. An abiotic synthesis was also detected in experiments with a mixture of clays and minerals (Mars analog soil) or with montmorillonite artifically enriched in iron. With either substratum the reaction appeared to be the result of a photocatalytic synthesis of¹⁴C-organics from¹⁴CO and surface hydroxyl groups. This process was not dependent on the presence of water vapor in the incubation atmosphere. Although a duplication of the Viking data has not been achieved, these findings support the abiotic interpretation of the PR results.     

A field enclosure apparatus for measuring crop photosynthesis

The field enclosure is a transparent box covering a soil area of 1.5 m². It is a semi-closed system in which concentrations of water vapour and carbon dioxide are maintained constant: the required rate of input of carbon dioxide being a measure of the photosynthesis rate, and the rate of condensation of water, on cooling coils, a measure of transpiration. The air within the enclosure is circulated rapidly by fans to decrease concentration gradients, and under steady radiation inputs the air temperature is controlled to ±0.5 °C. Both photosynthesis and transpiration rates are corrected for air exchange with the surroundings, as measured through the injection of the inert gas, nitrous oxide.     

Growth of Methanogens on a Mars Soil Simulant

Currently, the surface of Mars is probably too cold, too dry, and too oxidizing for life, as we know it, to exist. But the subsurface is another matter. Life forms that might exist below the surface could not obtain their energy from photosynthesis, but rather they would have to utilize chemical energy. Methanogens are one type of microorganism that might be able to survive below the surface of Mars. A potential habitat for existence of methanogens on Mars might be a geothermal source of hydrogen, possibly due to volcanic or hydrothermal activity, or the reaction of basalt and anaerobic water, carbon dioxide, which is abundant in the martian atmosphere, and of course, subsurface liquid water. We report here that certain methanogens can grow on a Mars soil simulant when supplied with carbon dioxide, molecular hydrogen, and varying amounts of water.     

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.     

Gas Exchange of Algae: III. Relation between the Concentration of Carbon Dioxide in the Nutrient Medium and the Oxygen Production of Chlorella pyrenoidosa

The oxygen production of a photosynthetic gas exchanger containing Chlorella pyrenoidosa (1% packed cell volume) was measured when various concentrations of carbon dioxide were present within the culture unit. The internal carbon dioxide concentrations were obtained by manipulating the entrance gas concentration and the flow rate. Carbon dioxide percentages were monitored by means of electrodes placed directly in the nutrient medium. The concentration of carbon dioxide in the nutrient medium which produced maximal photosynthesis was in the range of 1.5 to 2.5% by volume. Results were unaffected by either the level of carbon dioxide in the entrance gas or the rate of gas flow. Entrance gases containing 2% carbon dioxide flowing at 320 ml/min, 3% carbon dioxide at 135 ml/min, and 4% carbon dioxide at 55 ml/min yielded optimal carbon dioxide concentrations in the particular unit studied. By using carbon dioxide electrodes implanted directly in the gas exchanger to optimize the carbon dioxide concentration throughout the culture medium, it should be possible to design more efficient large-scale units.     

Metabolie Fate of Glycyl-14C-prolylhydroxyproline in Young Rats

To examine the origin of urinary hydroxyproline peptides, the metabolism of the radioactive tripeptide, glycyl-¹⁴C-prolylhydroxyproline, was investigated in normal young rats in vivo. The radioactive tripeptide was synthesized from glycine, l-(U-¹⁴C)proline and hydroxy-l-proline in our laboratory. The distributions of the radioactivity in body protein, lipid and soluble fractions were 23.7, 1.8 and 0.12% of the injected dose, respectively, 56 hr after the intraperitoneal injection of the ¹⁴C-tripeptide. The excretions of the radioactivity into expired carbon dioxide and urine were 29.6 and 34.2% of the injected dose, respectively, and large proportions of both the ¹⁴C excretions occurred during the first 12hr.

The results suggest that not a small amount of the glycylprolylhydroxyproline peptide injected is hydrolyzed in tissues of animals and the free proline derived is used for protein synthesis and/or further degraded to expired carbon dioxide.>     

Plant- and soil related controls of the flow of carbon from roots through the soil microbial biomass

The flow of carbon from plant roots through the microbial biomass is one of the key processes in terrestrial ecosystems. Roots release considerable amounts of organic materials which are utilized by microbes as substrate for biosynthesis and energy supply. The fate of photosynthates and other organic material in the soil-root environment under different conditions was studied using¹⁴C-tracers. Soil structure and texture had a large effect on the turnover of the¹⁴C-labelled materials through the microbial biomas. Finer, clayey soils tended to be more preservative than coarser, sandy soils,i.e., larger amounts of¹⁴C were incorporated in microbial biomass and soil organic matter fractions in clayey soils than in sandy soils.The soil nutrient status also appeared to affect organic matter turnover. At limiting plant-nutrient concentrations the utilization of¹⁴C-labelled photosynthates seem to be hampered. Plant roots influenced the transformation of glucose and crop residues and the effect was attributed to plant-induced changes in mineral nutrient status. The mechanisms of this process and the consequences are discussed.A number of areas for future research are identified, including the potentials for manipulating rhizodeposition.     

Sagebrush carbon allocation patterns and grasshopper nutrition: the influence of CO2 enrichment and soil mineral limitation

Summary Artemisia tridentata seedlings were grown under carbon dioxide concentrations of 350 and 650 l l^–1 and two levels of soil nutrition. In the high nutrient treatment, increasing CO₂ led to a doubling of shoot mass, whereas nutrient limitation completely constrained the response to elevated CO₂. Root biomass was unaffected by any treatment. Plant root/shoot ratios declined under carbon dioxide enrichment but increased under low nutrient availability, thus the ratio was apparently controlled by changes in carbon allocation to shoot mass alone. Growth under CO₂ enrichment increased the starch concentrations of leaves grown under both nutrient regimes, while increased CO₂ and low nutrient availability acted in concert to reduce leaf nitrogen concentration and water content. Carbon dioxide enrichment and soil nutrient limitation both acted to increase the balance of leaf storage carbohydrate versus nitrogen (C/N). The two treatment effects were significantly interactive in that nutrient limitation slightly reduced the C/N balance among the high-CO₂ plants. Leaf volatile terpene concentration increased only in the nutrient limited plants and did not follow the overall increase in leaf C/N ratio. Grasshopper consumption was significantly greater on host leaves grown under CO₂ enrichment but was reduced on leaves grown under low nutrient availability. An overall negative relationship of consumption versus leaf volatile concentration suggests that terpenes may have been one of several important leaf characteristics limiting consumption of the low nutrient hosts. Digestibility of host leaves grown under the high CO₂ treatment was significantly increased and was related to high leaf starch content. Grasshopper growth efficiency (ECI) was significantly reduced by the nutrient limitation treatment but co-varied with leaf water content.     

Increased root exudation of14C-compounds by sorghum seedlings inoculated with nitrogen-fixing bacteria

Summary Organic components leaked fromSorghum bicolor seedlings (‘root exudates’) were examined by recovering¹⁴C labelled compounds from root solutions of seedlings inoculated withAzospirillum brasilense, Azotobacter vinelandii orKlebsiella pneumoniae nif-. Up to 3.5% of the total¹⁴C recovered from shoots, roots, and nutrient solutions was found in the root solutions. Inoculation with Azospirillum and Azotobacter increased the amounts of¹⁴C and decreased the amounts of carbohydrates in the root solutions. When sucrose was added as a carbon source for the bacteria, the increase of¹⁴C in the solutions did not occur. Quantities of¹⁴C found in the root solutions were proportional to amounts of mineral nitrogen supplied to the plants. Bacterial growth also was proportional to nitrogen levels. When sorghum plants were grown in soil and labelled with¹⁴CO₂, about 15% of the total¹⁴C recovered within 48 hours exposure was found in soil leachates.     

Environmental effects on CO2 efflux from riparian tundra in the northern foothills of the Brooks Range,Alaska, USA

Summary Carbon dioxide efflux and soil microenvironmental factors were measured diurnally in Carex aquatilus-and Eriophorum angustifolium-dominated riparian tundra communities to determine the relative importance of soil environmental factors controlling ecosystem carbon dioxide exchange with the atmosphere. Measurements were made weekly between 18 June and 24 July 1990. Diurnal patterns in carbon dioxide efflux were best explained by changes in soil temperature, while seasonal changes in efflux were correlated with changes in depth to water table, depth to frozen soil and soil moisture. Carbon dioxide efflux rates were lowest early in the growing season when high water tables and low soil temperatures limited microbial and root activity. Individual rainfall events that raised the water table were found to strongly reduce carbon dioxide efflux. As the growing season progressed, rainfall was low and depth to water table and soil temperatures increased. In response, carbon dioxide efflux increased strongly, attaining rates late in the season of approximately 10 g CO₂ m^–2 day^–1. These rates are as high as maxima recorded for other arctic sites. A mathematical model is developed which demonstrates that soil temperature and depth to water table may be used as efficient predictors of ecosystem CO₂ efflux in this habitat. In parallel with the field measurements of CO₂ efflux, microbial respiration was studied in the laboratory as a function of temperature and water content. Estimates of microbial respiration per square meter under field conditions were made by adjusting for potential respiring soil volume as water table changed and using measured soil temperatures. The results indicate that the effect of these factors on microbial respiration may explain a large part of the diurnal and seasonal variation observed in CO₂ efflux. As in coastal tundra sites, environmental changes that alter water table depth in riparian tundra communities will have large effects on ecosystem CO₂ efflux and carbon balance.     

Errors in differential infrared carbon dioxide analysis resulting from water vapor

Water vapor was added differentially to the gas streams entering the cells of three makes of differential infrared carbon dioxide analysers. Analyser deflections were compared with those expected from dilution of the carbon dioxide by the additional gas. Tests were made at 0, 365, and 730 cm³ m^–3 concentrations of carbon dioxide, and with the dewpoint in one cell of the analysers held constant at 15, 20, or 25°C. None of the analysers always responded in the ways predicted from dilution. The results showed that errors of a few cm³ m^–3 could occur in estimates of carbon dioxide differentials using the theoretical correction for dilution. Furthermore the amount of error varied with the carbon dioxide range, the difference in water content, and in some cases the dewpoint range.     

Carbon dioxide and biearbonate in cell division

Summary The effects of carbon dioxide and of bicarbonate on cell division were studied on synchronized cells of the high-temperature green alga, Chlorella 7-11-05. After 7 hours of growth in nutrient medium in light, cells were centrifuged and resuspended in distilled water or in bicarbonate and placed in darkness. Atmospheric air, or a mixture of carbon dioxide and air, was bubbled through algal suspensions during the dark period. In distilled water cells readily divided in atmospheric air but not in 1% (2.6·10^-4 M) or in higher concentrations of carbon dioxide. The suspension of cells in bicarbonate counteracted the inhibitory action of carbon dioxide. A minimum molar concentration of bicarbonate necessary to counteract the inhibitory effect of carbon dioxide was found to be equal to the molar concentration of carbon dioxide in the suspending fluid. The highest concentration of carbon dioxide, the adverse effect of which could not be balanced by any concentration of bicarbonate, was found to be in the vicinity of 1.3·10^-2 M (50% CO₂ in air). Possible effects on cell division of the change in Ph and the implicated role of carbon dioxide in normal and neoplastic growth were discussed.     

Evaluation of the use of a model rhizodeposition technique to separate root and microbial respiration in soil

A model rhizodeposition technique to estimate the root and microbial components of ¹⁴C soil/root respiration in pulse-labelling experiments is described. The method involves the injection of model rhizodeposits, consisting of ¹⁴C-labelled glucose, root extract or root cell wall material, into the rooted soil of an unlabelled plant, simultaneously with the pulse-labelling of a separate but similar plant with ¹⁴CO₂. In a growth chamber experiment with 30 day old wheat and barley the contribution of direct root respiration to ¹⁴C soil/root respiration over a 26 day period after labelling was estimated 89–95%. Estimates of direct root respiration in field-grown wheat and barley at different development stages in most cases accounted for at least 75% of ¹⁴C soil/root respiration over a 21 day period after labelling. The mineralization rate of injected ¹⁴C-glucose was positively correlated with the concentration of glucose-C established in soil. The use of the method in rhizosphere carbon budget estimations is evaluated. Communication No. 73 of the Dutch Programme on Soil Ecology of Arable Farming Systems. Communication No. 73 of the Dutch Programme on Soil Ecology of Arable Farming Systems.