Astrobiological Potential of Venus Atmosphere Chemical Anomalies and Other Unexplained Cloud Properties.

Long-standing unexplained Venus atmosphere observations and chemical anomalies point to unknown chemistry but also leave room for the possibility of life. The unexplained observations include several gases out of thermodynamic equilibrium (e.g., tens of ppm O2, the possible presence of PH3 and NH3, SO2 and H2O vertical abundance profiles), an unknown composition of large, lower cloud particles, and the "unknown absorber(s)." Here we first review relevant properties of the venusian atmosphere and then describe the atmospheric chemical anomalies and how they motivate future astrobiology missions to Venus.

Planetary Protection Knowledge Gap Closure Enabling Crewed Missions to Mars.

As focus for exploration of Mars transitions from current robotic explorers to development of crewed missions, it remains important to protect the integrity of scientific investigations at Mars, as well as protect the Earth's biosphere from any potential harmful effects from returned martian material. This is the discipline of planetary protection, and the Committee on Space Research (COSPAR) maintains the consensus international policy and guidelines on how this is implemented. Based on National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) studies that began in 2001, COSPAR adopted principles and guidelines for human missions to Mars in 2008. At that point, it was clear that to move from those qualitative provisions, a great deal of work and interaction with spacecraft designers would be necessary to generate meaningful quantitative recommendations that could embody the intent of the Outer Space Treaty (Article IX) in the design of such missions. Beginning in 2016, COSPAR then sponsored a multiyear interdisciplinary meeting series to address planetary protection "knowledge gaps" (KGs) with the intent of adapting and extending the current robotic mission-focused Planetary Protection Policy to support the design and implementation of crewed and hybrid exploration missions. This article describes the outcome of the interdisciplinary COSPAR meeting series, to describe and address these KGs, as well as identify potential paths to gap closure. It includes the background scientific basis for each topic area and knowledge updates since the meeting series ended. In particular, credible solutions for KG closure are described for the three topic areas of (1) microbial monitoring of spacecraft and crew health; (2) natural transport (and survival) of terrestrial microbial contamination at Mars, and (3) the technology and operation of spacecraft systems for contamination control. The article includes a KG data table on these topic areas, which is intended to be a point of departure for making future progress in developing an end-to-end planetary protection requirements implementation solution for a crewed mission to Mars. Overall, the workshop series has provided evidence of the feasibility of planetary protection implementation for a crewed Mars mission, given (1) the establishment of needed zoning, emission, transport, and survival parameters for terrestrial biological contamination and (2) the creation of an accepted risk-based compliance approach for adoption by spacefaring actors including national space agencies and commercial/nongovernment organizations.

Metabolomic and cultivation insights into the tolerance of the spacecraft-associated Acinetobacter toward Kleenol 30, a cleanroom floor detergent.

Introduction: Stringent cleaning procedures during spacecraft assembly are critical to maintaining the integrity of life-detection missions. To ensure cleanliness, NASA spacecraft are assembled in cleanroom facilities, where floors are routinely cleansed with Kleenol 30 (K30), an alkaline detergent. Methods: Through metabolomic and cultivation approaches, we show that cultures of spacecraft-associated Acinetobacter tolerate up to 1% v/v K30 and are fully inhibited at ≥2%; in comparison, NASA cleanrooms are cleansed with ~0.8-1.6% K30. Results: For A. johnsonii 2P08AA (isolated from a cleanroom floor), cultivations with 0.1% v/v K30 yield (1) no changes in cell density at late-log phase, (2) modest decreases in growth rate (~17%), (3) negligible lag phase times, (4) limited changes in the intracellular metabolome, and (5) increases in extracellular sugar acids, monosaccharides, organic acids, and fatty acids. For A. radioresistens 50v1 (isolated from a spacecraft surface), cultivations yield (1) ~50% survivals, (2) no changes in growth rate, (3) ~70% decreases in the lag phase time, (4) differential changes in intracellular amino acids, compatible solutes, nucleotide-related metabolites, dicarboxylic acids, and saturated fatty acids, and (5) substantial yet differential impacts to extracellular sugar acids, monosaccharides, and organic acids. Discussion: These combined results suggest that (1) K30 manifests strain-dependent impacts on the intracellular metabolomes, cultivation kinetics, and survivals, (2) K30 influences extracellular trace element acquisition in both strains, and (3) K30 is better tolerated by the floor-associated strain. Hence, this work lends support towards the hypothesis that repeated cleansing during spacecraft assembly serve as selective pressures that promote tolerances towards the cleaning conditions.

Potential for Phototrophy in Venus' Clouds.

We show that solar irradiances calculated across Venus' clouds support the potential for Earth-like phototrophy and that treatment of Venus' aerosols containing neutralized sulfuric acid favor a habitable zone. The phototrophic potential of Venus' atmosphere was assessed by calculating irradiances (200-2000 nm, 15° solar zenith angle, local noon) using a radiative transfer model that accounted for absorption and scattering by the major and minor atmospheric constituents. Comparisons to Earth's surface (46 W m-2, 280-400 nm) suggest that Venus' middle and lower clouds receive ∼87% less normalized UV flux (6-7 W m-2) across 200-400 nm, yet similar normalized photon flux densities (∼4400-6200 µmol m-2 s-1) across 350-1200 nm. Further, Venus' signature phototrophic windows and subwindows overlap with the absorption profiles of several photosynthetic pigments, especially bacteriochlorophyll b from intact cells and phycocyanin. Therefore, Venus' light, with limited UV flux in the middle and lower clouds, is likely quite favorable for phototrophy. We additionally present interpretations to refractive index and radio occultation measures for Venus' aerosols that suggest the presence of lower sulfuric abundances and/or neutralized forms of sulfuric acid, such as ammonium bisulfate. Under these considerations, the aerosols in Venus' middle clouds could harbor water activities (≥0.6) and buffered acidities (Hammett acidity factor, H0 -0.1 to -1.5) that lie within the limits of acidic cultivation (≥H0 -0.4) and are tantalizingly close to the limits of oxygenic photosynthesis (≥H0 0.1). Together, these photophysical and chemical considerations support a potential for phototrophy in Venus' clouds.

Venus' Mass Spectra Show Signs of Disequilibria in the Middle Clouds.

We present a re-examination of mass spectral data obtained from the Pioneer Venus Large Probe Neutral Mass Spectrometer. Our interpretations of differing trace chemical species are suggestive of redox disequilibria in Venus' middle clouds. Assignments to the data (at 51.3 km) include phosphine, hydrogen sulfide, nitrous acid, nitric acid, carbon monoxide, hydrochloric acid, hydrogen cyanide, ethane, and potentially ammonia, chlorous acid, and several tentative PxOy species. All parent ions were predicated upon assignment of corresponding fragmentation products, isotopologues, and atomic species. The data reveal parent ions at varying oxidation states, implying the presence of reducing power in the clouds, and illuminating the potential for chemistries yet to be discovered. When considering the hypothetical habitability of Venus' clouds, the assignments reveal a potential signature of anaerobic phosphorus metabolism (phosphine), an electron donor for anoxygenic photosynthesis (nitrite), and major constituents of the nitrogen cycle (nitrate, nitrite, ammonia, and N2).

Exobiology of the Venusian Clouds: New Insights into Habitability through Terrestrial Models and Methods of Detection.

The search for life beyond Earth has focused on Mars and the icy moons Europa and Enceladus, all of which are considered a safe haven for life due to evidence of current or past water. The surface of Venus, on the other hand, has extreme conditions that make it a nonhabitable environment to life as we know it. This is in contrast, however, to its cloud layer, which, while still an extreme environment, may prove to be a safe haven for some extreme forms of life similar to extremophiles on Earth. We consider the venusian clouds a habitable environment based on the presence of (1) a solvent for biochemical reactions, (2) appropriate physicochemical conditions, (3) available energy, and (4) biologically relevant elements. The diversity of extreme microbial ecosystems on Earth has allowed us to identify terrestrial chemolithoautotrophic microorganisms that may be analogs to putative venusian organisms. Here, we hypothesize and describe biological processes that may be performed by such organisms in the venusian clouds. To detect putative venusian organisms, we describe potential biosignature detection methods, which include metal-microbial interactions and optical methods. Finally, we describe currently available technology that can potentially be used for modeling and simulation experiments.

Venus, an Astrobiology Target.

We present a case for the exploration of Venus as an astrobiology target-(1) investigations focused on the likelihood that liquid water existed on the surface in the past, leading to the potential for the origin and evolution of life, (2) investigations into the potential for habitable zones within Venus' present-day clouds and Venus-like exo atmospheres, (3) theoretical investigations into how active aerobiology may impact the radiative energy balance of Venus' clouds and Venus-like atmospheres, and (4) application of these investigative approaches toward better understanding the atmospheric dynamics and habitability of exoplanets. The proximity of Venus to Earth, guidance for exoplanet habitability investigations, and access to the potential cloud habitable layer and surface for prolonged in situ extended measurements together make the planet a very attractive target for near term astrobiological exploration.

Simple kinetics, assay, and trends for soil microbial catalases.

In this report, we expand upon the enzymology and ecology of soil catalases through development and application of a simple kinetic model and field-amenable assay based upon volume displacement. Through this approach, we (A) directly relate apparent Michaelis-Menten terms to the catalase reaction mechanism, (B) obtain upper estimates of the intrinsic rate constants for the catalase community (k3'), along with moles of catalase per 16S rRNA gene copy number, (C) utilize catalase specific activities (SAs) to obtain biomass estimates of soil and permafrost communities (LOD, ~104 copy number gdw-1), and (D) relate kinetic trends to changes in bacterial community structure. In addition, this novel kinetic approach simultaneously incorporates barometric adjustments to afford comparisons across field measurements. As per our model, and when compared to garden soils, biological soil crusts exhibited ~2-fold lower values for k3', ≥105-fold higher catalase moles per biomass (250-1200 zmol copy number-1), and ~104-fold higher SAs per biomass (74-230 fkat copy number-1); whereas the highest SAs were obtained from permafrost and high-elevation soil communities (5900-6700 fkat copy number-1). In sum, the total trends suggest that microbial communities which experience higher degrees of native oxidative stress possess higher basal intracellular catalase concentrations and SAs per biomass.

Metabolism and Biodegradation of Spacecraft Cleaning Reagents by Strains of Spacecraft-Associated Acinetobacter.

Spacecraft assembly facilities are oligotrophic and low-humidity environments, which are routinely cleaned using alcohol wipes for benchtops and spacecraft materials, and alkaline detergents for floors. Despite these cleaning protocols, spacecraft assembly facilities possess a persistent, diverse, dynamic, and low abundant core microbiome, where the Acinetobacter are among the dominant members of the community. In this report, we show that several spacecraft-associated Acinetobacter metabolize or biodegrade the spacecraft cleaning reagents of ethanol (ethyl alcohol), 2-propanol (isopropyl alcohol), and Kleenol 30 (floor detergent) under ultraminimal conditions. Using cultivation and stable isotope labeling studies, we show that ethanol is a sole carbon source when cultivating in 0.2 × M9 minimal medium containing 26 µM Fe(NH4)2(SO4)2. Although cultures expectedly did not grow solely on 2-propanol, cultivations on mixtures of ethanol and 2-propanol exhibited enhanced plate counts at mole ratios of ≤0.50. In support, enzymology experiments on cellular extracts were consistent with oxidation of ethanol and 2-propanol by a membrane-bound alcohol dehydrogenase. In the presence of Kleenol 30, untargeted metabolite profiling on ultraminimal cultures of Acinetobacter radioresistens 50v1 indicated (1) biodegradation of Kleenol 30 into products including ethylene glycols, (2) the potential metabolism of decanoate (formed during incubation of Kleenol 30 in 0.2 × M9), and (3) decreases in the abundances of several hydroxy- and ketoacids in the extracellular metabolome. In ultraminimal medium (when using ethanol as a sole carbon source), A. radioresistens 50v1 also exhibits a remarkable survival against hydrogen peroxide (∼1.5-log loss, ∼108 colony forming units (cfu)/mL, 10 mM H2O2), indicating a considerable tolerance toward oxidative stress under nutrient-restricted conditions. Together, these results suggest that the spacecraft cleaning reagents may (1) serve as nutrient sources under oligotrophic conditions and (2) sustain extremotolerances against the oxidative stresses associated with low-humidity environments. In perspective, this study provides a plausible biochemical rationale to the observed microbial ecology dynamics of spacecraft-associated environments.

Venus' Spectral Signatures and the Potential for Life in the Clouds.

The lower cloud layer of Venus (47.5-50.5 km) is an exceptional target for exploration due to the favorable conditions for microbial life, including moderate temperatures and pressures (∼60°C and 1 atm), and the presence of micron-sized sulfuric acid aerosols. Nearly a century after the ultraviolet (UV) contrasts of Venus' cloud layer were discovered with Earth-based photographs, the substances and mechanisms responsible for the changes in Venus' contrasts and albedo are still unknown. While current models include sulfur dioxide and iron chloride as the UV absorbers, the temporal and spatial changes in contrasts, and albedo, between 330 and 500 nm, remain to be fully explained. Within this context, we present a discussion regarding the potential for microorganisms to survive in Venus' lower clouds and contribute to the observed bulk spectra. In this article, we provide an overview of relevant Venus observations, compare the spectral and physical properties of Venus' clouds to terrestrial biological materials, review the potential for an iron- and sulfur-centered metabolism in the clouds, discuss conceivable mechanisms of transport from the surface toward a more habitable zone in the clouds, and identify spectral and biological experiments that could measure the habitability of Venus' clouds and terrestrial analogues. Together, our lines of reasoning suggest that particles in Venus' lower clouds contain sufficient mass balance to harbor microorganisms, water, and solutes, and potentially sufficient biomass to be detected by optical methods. As such, the comparisons presented in this article warrant further investigations into the prospect of biosignatures in Venus' clouds.

Microbial Community and Biochemical Dynamics of Biological Soil Crusts across a Gradient of Surface Coverage in the Central Mojave Desert.

In this study, we expand upon the biogeography of biological soil crusts (BSCs) and provide molecular insights into the microbial community and biochemical dynamics along the vertical BSC column structure, and across a transect of increasing BSC surface coverage in the central Mojave Desert, CA, United States. Next generation sequencing reveals a bacterial community profile that is distinct among BSCs in the southwestern United States. Distribution of major phyla in the BSC topsoils included Cyanobacteria (33 ± 8%), Proteobacteria (26 ± 6%), and Chloroflexi (12 ± 4%), with Phormidium being the numerically dominant genus. Furthermore, BSC subsurfaces contained Proteobacteria (23 ± 5%), Actinobacteria (20 ± 5%), and Chloroflexi (18 ± 3%), with an unidentified genus from Chloroflexi (AKIW781, order) being numerically dominant. Across the transect, changes in distribution at the phylum (p < 0.0439) and genus (p < 0.006) levels, including multiple biochemical and geochemical trends (p < 0.05), positively correlated with increasing BSC surface coverage. This included increases in (a) Chloroflexi abundance, (b) abundance and diversity of Cyanobacteria, (b) OTU-level diversity in the topsoil, (c) OTU-level differentiation between the topsoil and subsurface, (d) intracellular ATP abundances and catalase activities, and (e) enrichments in clay, silt, and varying elements, including S, Mn, Co, As, and Pb, in the BSC topsoils. In sum, these studies suggest that BSCs from regions of differing surface coverage represent early successional stages, which exhibit increasing bacterial diversity, metabolic activities, and capacity to restructure the soil. Further, these trends suggest that BSC successional maturation and colonization across the transect are inhibited by metals/metalloids such as B, Ca, Ti, Mn, Co, Ni, Mo, and Pb.

A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays.

Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here, we introduce a general technique for patterning nanoparticle arrays using two-dimensional crystals of genetically modified hollow protein structures called chaperonins. Constrained chemical synthesis of transition metal nanoparticles is initiated using templates functionalized with polyhistidine sequences. These nanoparticles are ordered into arrays because the template-driven synthesis is constrained by the nanoscale structure of the crystallized protein. We anticipate that this system may be used to pattern different classes of nanoparticles based on the growing library of sequences shown to specifically bind or direct the growth of materials.

Tryptophan 500 and arginine 707 define product and substrate active site binding in soybean lipoxygenase-1.

There is much debate whether the fatty acid substrate of lipoxygenase binds "carboxylate-end first" or "methyl-end first" in the active site of soybean lipoxygenase-1 (sLO-1). To address this issue, we investigated the sLO-1 mutants Trp500Leu, Trp500Phe, Lys260Leu, and Arg707Leu with steady-state and stopped-flow kinetics. Our data indicate that the substrates (linoleic acid (LA), arachidonic acid (AA)), and the products (13-(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (HPOD) and 15-(S)-hydroperoxyeicosatetraeonic acid (15-(S)-HPETE)) interact with the aromatic residue Trp500 (possibly pi-pi interaction) and with the positively charged amino acid residue Arg707 (charge-charge interaction). Residue Lys260 of soybean lipoxygenase-1 had little effect on either the activation or steady-state kinetics, indicating that both the substrates and products bind "carboxylate-end first" with sLO-1 and not "methyl-end first" as has been proposed for human 15-lipoxygenase.

Near-infrared detection of potential evidence for microscopic organisms on Europa.

The possibility of an ocean within the icy shell of Jupiter's moon Europa has established that world as a primary candidate in the search for extraterrestrial life within our Solar System. This paper evaluates the potential to detect evidence for microbial life by comparing laboratory studies of terrestrial microorganisms with measurements from the Galileo Near Infrared Imaging Spectrometer (NIMS). If the interior of Europa at one time harbored life, some evidence may remain in the surface materials. Examination of laboratory spectra of terrestrial extremophiles measured at cryogenic temperatures reveals distorted, asymmetric nearinfrared absorption features due to water of hydration. The band centers, widths, and shapes of these features closely match those observed in the Europa spectra. These features are strongest in reddish-brown, disrupted terrains such as linea and chaos regions. Narrow spectral features due to amide bonds in the microbe proteins provide a means of constraining the abundances of such materials using the NIMS data. The NIMS data of disrupted terrains exhibit distorted, asymmetric near-infrared absorption features consistent with the presence of water ice, sulfuric acid octahydrate, hydrated salts, and possibly as much as 0.2 mg cm(-3) of carbonaceous material that could be of biological origin. However, inherent noise in the observations and limitations of spectral sampling must be taken into account when discussing these findings.

Impact of low-temperature plasmas on Deinococcus radiodurans and biomolecules.

The effects of cold plasma on Deinococcus radiodurans, plasmid DNA, and model proteins were assessed using microbiological, spectrometric, and biochemical techniques. In low power O(2) plasma (approximately 25 W, approximately 45 mTorr, 90 min), D. radiodurans, a radiation-resistant bacterium, showed a 99.999% reduction in bioburden. In higher power O(2) plasma (100 W and 500 mTorr), the reduction rate increased about 10-fold and observation by atomic force microscopy showed significant damage to the cell. Damage to cellular lipids, proteins, and chromosome was indicated by losses of infrared spectroscopic peaks at 2930, 1651, 1538, and 1245 cm(-1), respectively. In vitro experiments show that O(2) plasmas induce DNA strand scissions and cross-linking as well as reduction of enzyme activity. The observed degradation and removal of biomolecules was power-dependent. Exposures to 200 W at 500 mTorr removed biomolecules to below detection limits in 60 s. Emission spectroscopy indicated that D. radiodurans cells were volatilized into CO(2), CO, N(2), and H(2)O, confirming that these plasmas were removing complex biological matter from surfaces. A CO(2) plasma was not as effective as the O(2) plasma, indicating the importance of plasma composition and the dominant role of chemical degradation. Together, these findings have implications for NASA planetary protection schemes and for the contamination of Mars.