The COSPAR planetary protection policy for missions to Icy Worlds: A review of history, current scientific knowledge, and future directions.

Recent discoveries related to the habitability and astrobiological relevance of the outer Solar System have expanded our understanding of where and how life may have originated. As a result, the Icy Worlds of the outer Solar System have become among the highest priority targets for future spacecraft missions dedicated to astrobiology-focused and/or direct life detection objectives. This, in turn, has led to a renewed interest in planetary protection concerns and policies for the exploration of these worlds and has been a topic of discussion within the COSPAR (Committee on Space Research) Panel on Planetary Protection. This paper summarizes the results of those discussions, reviewing the current knowledge and the history of planetary protection considerations for Icy Worlds as well as suggesting ways forward. Based on those discussions, we therefore suggest to (1) Establish a new definition for Icy Worlds for Planetary Protection that captures the outer Solar System moons and dwarf planets like Pluto, but excludes more primitive bodies such as comets, centaurs, and asteroids: Icy Worlds in our Solar System are defined as all bodies with an outermost layer that is believed to be greater than 50 % water ice by volume and have enough mass to assume a nearly round shape. (2) Establish indices for the lower limits of Earth life with regards to water activity (LLAw) and temperature (LLT) and apply them into all areas of the COSPAR Planetary Protection Policy. These values are currently set at 0.5 and -28 °C and were originally established for defining Mars Special Regions; (3) Establish LLT as a parameter to assign categorization for Icy Worlds missions. The suggested categorization will have a 1000-year period of biological exploration, to be applied to all Icy Worlds and not just Europa and Enceladus as is currently the case. (4) Have all missions consider the possibility of impact. Transient thermal anomalies caused by impact would be acceptable so long as there is less than 10-4 probability of a single microbe reaching deeper environments where temperature is >LLT in the period of biological exploration. (5) Restructure or remove Category II* from the policy as it becomes largely redundant with this new approach, (6) Establish that any sample return from an Icy World should be Category V restricted Earth return.

Bioweathering of nontronite colloids in hybrid silica gel: implications for iron mobilization.

AIMS: This study aimed to study biotic iron dissolution using a new hybrid material constituted of well-dispersed mineral colloids in a silica gel matrix. This permitted to prevent adsorption of colloidal mineral particles on bacteria. Hybrid silica gel (HSG) permitted to study bioweathering mechanisms by diffusing molecules. METHODS AND RESULTS: Hybrid silica gel was synthesized through a classical sol-gel procedure in which mineral colloidal particles (NAu-2) were embedded in a porous silica matrix. Rahnella aquatilis RA1, isolated from a wheat rhizosphere was chosen for its ability to dissolve minerals by producing various organic acids and siderophores. Pyruvic, acetic and lactic acids were the major organic acids produced by R. aquatilis RA1 followed by oxalic and citric acids at the end of incubation. Comparison of abiotic and biotic experiments revealed a high efficiency of R. aquatilis RA1 for iron dissolution suggesting an optimized action of different ligands that solubilized or mobilized iron. CONCLUSIONS: Hybrid silica gel allowed focusing on the colloidal mineral weathering by metabolites diffusion without mineral adsorption on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Hybrid silica gels are new and efficient tools to study colloidal mineral bioweathering. Adjusting HSG porosity and hydrophobicity should permit to precise the influence of limiting diffusion of siderophores or aliphatic organic acids on mineral weathering.

Bacterial and iron oxide aggregates mediate secondary iron mineral formation: green rust versus magnetite.

In the presence of methanoate as electron donor, Shewanella putrefaciens, a Gram-negative, facultative anaerobe, is able to transform lepidocrocite (gamma-FeOOH) to secondary Fe (II-III) minerals such as carbonated green rust (GR1) and magnetite. When bacterial cells were added to a gamma-FeOOH suspension, aggregates were produced consisting of both bacteria and gamma-FeOOH particles. Recently, we showed that the production of secondary minerals (GR1 vs. magnetite) was dependent on bacterial cell density and not only on iron reduction rates. Thus, gamma-FeOOH and S. putrefaciens aggregation pattern was suggested as the main mechanism driving mineralization. In this study, lepidocrocite bioreduction experiments, in the presence of anthraquinone disulfonate, were conducted by varying the [cell]/[lepidocrocite] ratio in order to determine whether different types of aggregate are formed, which may facilitate precipitation of GR1 as opposed to magnetite. Confocal laser scanning microscopy was used to analyze the relative cell surface area and lepidocrocite concentration within the aggregates and captured images were characterized by statistical methods for spatial data (i.e. variograms). These results suggest that the [cell]/[lepidocrocite] ratio influenced both the aggregate structure and the nature of the secondary iron mineral formed. Subsequently, a [cell]/[lepidocrocite] ratio above 1 x 10(7) cells mmol(-1) leads to densely packed aggregates and to the formation of GR1. Below this ratio, looser aggregates are formed and magnetite was systematically produced. The data presented in this study bring us closer to a more comprehensive understanding of the parameters governing the formation of minerals in dense bacterial suspensions and suggest that screening mineral-bacteria aggregate structure is critical to understanding (bio)mineralization pathways.

Isotopic tracing of clear water sources in an urban sewer: A combined water and dissolved sulfate stable isotope approach.

This paper investigates the potential of stable isotopes of both water (deltaD and deltaOH(2)O18) and dissolved sulfate (delta(34)S and deltaOSO(4)18) for determining the origin and the amount of clear waters entering an urban sewer. The dynamics of various hydrological processes that commonly occur within the sewer system such as groundwater infiltration, rainwater percolation, or stormwater release from retention basins, can be readily described using water isotope ratios. In particular, stable water isotopes indicate that the relative volumes of infiltrated groundwater and sewage remain approximately constant and independent of wastewater flow rate during the day, thus demonstrating that the usual quantification of parasitic discharge from minimal nocturnal flow measurements can lead to completely erroneous results. The isotopic signature of dissolved sulfate can also provide valuable information about the nature of water inputs to the sewage flow, but could not be used in our case to quantify the infiltrating water. Indeed, even though the microbial activity had a limited effect on the isotopic composition of dissolved sulfate at the sampling sites investigated, the dissolved sulfate concentration in sewage was regulated by the formation of barite and calcium-phosphate mineral species. Sulfate originating from urine was also detected as a source using the oxygen isotopic composition of sulfate, which suggests that deltaOSO(4)18 might find use as a urine tracer.

Selenium bioavailability and uptake as affected by four different plants in a loamy clay soil with particular attention to mycorrhizae inoculated ryegrass.

The aim of this study was to investigate the influence of plant species, especially of their rhizosphere soil, and of inoculation with an arbuscular mycorrhizal (AM) fungus on the bioavailability of selenium and its transfer in soil-plant systems. A pot experiment was performed with a loamy clay soil and four plant species: maize, lettuce, radish and ryegrass, the last one being inoculated or not with an arbuscular mycorrhizal fungus (Glomus mosseae). Plant biomass and Se concentration in shoots and roots were estimated at harvest. Se bioavailability in rhizosphere and unplanted soil was evaluated using sequential extractions. Plant biomass and selenium uptake varied with plant species. The quantity of rhizosphere soil also differed between plants and was not proportional to plant biomass. The highest plant biomass, Se concentration in plants, and soil to plant transfer factor were obtained with radish. The lowest Se transfer factors were obtained with ryegrass. For the latter, mycorrhizal inoculation did not significantly affect plant growth, but reduced selenium transfer from soil to plant by 30%. In unplanted soil after 65 days aging, more than 90% of added Se was water-extractable. On the contrary, Se concentration in water extracts of rhizosphere soil represented less than 1% and 20% of added Se for ryegrass and maize, respectively. No correlation was found between the water-extractable fraction and Se concentration in plants. The speciation of selenium in the water extracts indicated that selenate was reduced, may be under organic forms, in the rhizosphere soil.

Effect of the mycorrhizosphere on the genotypic and metabolic diversity of the bacterial communities involved in mineral weathering in a forest soil.

To date, several bacterial species have been described as mineral-weathering agents which improve plant nutrition and growth. However, the possible relationships between mineral-weathering potential, taxonomic identity, and metabolic ability have not been investigated thus far. In this study, we characterized a collection of 61 bacterial strains isolated from Scleroderma citrinum mycorrhizae, the mycorrhizosphere, and the adjacent bulk soil in an oak forest. The ability of bacteria to weather biotite was assessed with a new microplate bioassay that measures the pH and the quantity of iron released from this mineral. We showed that weathering bacteria occurred more frequently in the vicinity of S. citrinum than in the bulk soil. Moreover, the weathering efficacy of the mycorrhizosphere bacterial isolates was significantly greater than that of the bulk soil isolates. All the bacterial isolates were identified by partial 16S rRNA gene sequence analysis as members of the genera Burkholderia, Collimonas, Pseudomonas, and Sphingomonas, and their carbon metabolism was characterized by the BIOLOG method. The most efficient isolates belonged to the genera Burkholderia and Collimonas. Multivariate analysis resulted in identification of three metabolic groups, one of which contained mainly bacterial isolates associated with S. citrinum and exhibiting high mineral-weathering potential. Therefore, our results support the hypothesis that by its carbon metabolism this fungus selects in the bulk soil reservoir a bacterial community with high weathering potential, and they also address the question of functional complementation between mycorrhizal fungi and bacteria in the ectomycorrhizal complex for the promotion of tree nutrition.

Association constants of Pb2+ with binding sites of fungal biomass using metal-based titrations.

Biosorption is perceived as an alternative method for toxic heavy metal removal/recovery from aqueous effluents. This work focused on derivation of some key quantitative physico-chemical characteristics of a representative biosorbent material required for its further effective exploitation. The newly developed acid-base and metal-based titrator allowed the characterization of the chemisorption active sites of Rhizopus arrhizus biomass and the study of their metal affinity. This experimental approach, combined with an analytical method consisting of transforming the initial data enabled the calculation of the number and capacity of the reactive sites (Qads) and the metal affinity constants (Km) for lead sorption by R. arrhizus biomass. The pKm values for Rhizopus biomass varied between -3 and -6 for sites releasing no protons, -1 and 1 for sites releasing one proton, and > 8 for sites releasing two protons - combined with the Pb precipitation phenomenon. At low temperatures, the active binding site number was lower at lower lead concentrations whereas the precipitation was promoted at higher lead concentration values. Lead adsorption contributed modestly (11%) to its overall uptake and occurred at low lead concentrations onto strong and medium affinity binding sites. Micro-precipitation quickly commenced around active binding sites distinguished by their weak affinity whenever the solution lead concentrations reached 10(-6) or 10(-5) M and represented more than 85% of the total sorbed metal quantity. The work also demonstrated the usefulnes of the methodology reported here for characterizing complex biosorbent materials.

Evolution of size distribution and transfer of mineral particles between flocs in activated sludges: an insight into floc exchange dynamics.

The aggregation behavior of activated sludge flocs was investigated by monitoring the size distribution of flocs and transfer of mineral particles between flocs, under various conditions of agitation and dilution. The results showed that (i) the shape of the floc size distribution can be fitted with a gamma function, (ii) a steady-state mean floc size is reached for a given stirring rate, (iii) this stable floc size is shifted towards floc growth as sludge concentration is increased, (iv) under cycled-shear conditions, microbial aggregates break up and re-form in an almost reversible manner, (v) blending of raw sludge and sludge spiked with Aquatal mineral particles results in particle exchange between flocs and (vi) the detailed study of exchange kinetics indicates that some flocs do not participate to the aggregation dynamics. These experimental results suggest that the activated sludge floc size is governed by a flocculation/deflocculation balance, implying an exchange of floc constituents between microbial aggregates.

Composition, structure and size distribution of suspended particulates from the Rhine River.

Fluvial suspended particulates collected from the Rhine River were investigated in terms of composition, structure and size distribution. Elemental analysis and Diffuse Reflectance Infrared Spectroscopy reveal that most particulate organic matter is formed from material derived from microorganisms. Transmission Electron Microscopy observations on resin-embedded samples and structural characterization from break-up experiments, show that fluvial particulate matter should be viewed as fractal aggregates organized by bacterial exopolymeric substances. The shape of particulate size distribution suggests that the formation and dynamics of suspended particulate matter are controlled mainly by physical processes. Finally, particulate growth and structure are consistent with a cluster-cluster aggregation scheme.

Occurrences at mineral-bacteria interface during oxidation of arsenopyrite by Thiobacillus ferrooxidans.

The combination of an improved bacterial desorption method, scanning electron microscopy (SEM), diffuse reflectance and transmission infrared Fourier transform spectroscopy, and a desorption-leaching device like high-pressure liquid chromatography (HPLC) was used to analyze bacterial populations (adhering and free bacteria) and surface-oxidized phases (ferric arsenates and elemental sulfur) during the arsenopyrite biooxidation by Thiobacillus ferrooxidans. The bacterial distribution, the physicochemical composition of the leachate, the evolution of corrosion patterns, and the nature and amount of the surface-oxidized chemical species characterized different behavior for each step of arsenopyrite bioleaching. The first step is characterized by a slow but strong adhesion of bacteria to mineral surfaces, the appearance of a surface phase of elemental sulfur, the weak solubilization of Fe(II), As(III), and As(V), and the presence of the first corrosion patterns, which follow the fragility zones and the crystallographic orientation of mineral grains. After this short step, growth of the unattached bacteria begins, while ferrous ions in solution are oxidized by them. Ferric ions produced by the bacteria can oxidize the sulfide directly and are regenerated by Fe(II) bacterial oxidation. At this time, a bioleaching cycle takes place and a coarse surface phase of ferric arsenate (FeAsO(4) . xH(2)O where x approximately 2) and deep ovoid pores appear. At the end of the bioleaching cycle, the high concentration of Fe(III) and As(V) in solution promotes the precipitation of a second phase of amorphous ferric arsenate (FeAsO(4) . xH(2)O where x approximately 4) in the leachate. Then the biooxidation process ceases: The bacteria adhering to the mineral sufaces are coated by the ferric arsenates and the concentration of Fe(III) on the leachate is found to have decreased greatly. Both oxidation mechanisms (direct and indirect oxidation) have been stopped. (c) 1995 John Wiley & Sons, Inc.

Quantification of the intragranular porosity formed in bioleaching of pyrite by Thiobacillus ferroxidans.

During the bacterial oxidation of a pure pyrite by Thiobacillus ferrooxidans, a great number of corrosion tunnels appear that are easily revealed by scanning electron microscopy observations. This involves an increase in the surface area without significant granulometric reduction of mineral grains. Thus, the evaluation of intragranular porosity, determined by elution front analysis, allows one to estimate accurately the fraction of oxidized sulphide, because of the development of deep holes (propagating pore mechanism). After 60 days of bioleaching, the intragranular porosity represents about 34% of the initial sulphide volume, which corresponds to 25 km of tunnels (2 microm i.d.) per gram of pyrite. On other hand, the granulometric reduction ( approximately 7%) is responsible for a 23% decrease of the initial sulphide volume. The elution front analysis appears as a nondestructive method for measuring the intragranular porosity of the bioleached pyrite.

Corrosion and Electrochemical Oxidation of a Pyrite by Thiobacillus ferrooxidans.

The oxidation of a pure pyrite by Thiobacillus ferrooxidans is not really a constant phenomenon; it must be considered to be more like a succession of different steps which need characterization. Electrochemical studies using a combination of a platinum electrode and a specific pyrite electrode (packed-ground-pyrite electrode) revealed four steps in the bioleaching process. Each step can be identified by the electrochemical behavior (redox potentials) of pyrite, which in turn can be related to chemical (leachate content), bacterial (growth), and physical (corrosion patterns) parameters of the leaching process. A comparison of the oxidation rates of iron and sulfur indicated the nonstoichiometric bacterial oxidation of a pure pyrite in which superficial phenomena, aqueous oxidation, and deep crystal dissolution are successively involved.

[Radioimmunoassay of trypsin in dried blood. Importance for the neonatal detection of cystic fibrosis (author's transl)]. / Dosage radio-immunologique de la trypsine dans le sang séché. Intérêt pour le dépistage néonatal de la mucoviscidose.

The demonstration of very high levels of immunoreactive trypsin in the blood of newborn infants with cystic fibrosis has provided a new way of detecting the disease soon after birth. A radioimmunoassay of trypsin in the eluate of blood dried on filter paper has now been developed. The sensitivity and accuracy of the method, as well as the good correlation observed between the values obtained and those of the conventional plasma assay, indicate that it is reliable and well adapted to the newborn. The new assay can easily be inserted into the present system of neonatal disease detection. A preliminary assessment of more than 5,000 tests enables the authors to report an early diagnosis of proven cystic fibrosis and to discuss an essential aspect of mass-detection methods: the indicence of false-positive results.