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Project Progress

Survivability on icy Worlds investigation examines the survivability of biological compounds under simulated icy world surface conditions, and compares the degradation products to abiotically synthesized compounds resulting from the radiation chemistry on icy worlds.

Summary of Objectives and Research
Investigation 2 focuses on survivability. As part of our Survivability investigation, we examine the similarities and differences between the abiotic chemistry of planetary ices irradiated with ultraviolet photons (UV), electrons, and ions, and the chemistry of biomolecules exposed to similar conditions. Can the chemical products resulting from these two scenarios be distinguished? Can viable microbes persist after exposure to such conditions? These are motivating questions for our investigation.

Project Progress

Summary:
The following co-Is and collaborators have been conducting research as summarized below.

Gudipati (JPL), PAH photochemistry in ice; Johnson & Hodyss (JPL), Amino acid and hydrocarbon photochemistry in ice, spectroscopy of spores in ice; Cooper (GMU), H₂O₂ production in ice; Strazzulla (INAF, Italy), Sulfur containing ices (ion induced radiation processing); Ponce & Hand (JPL), Monodispersed spores deposition chamber installed and first experiments conducted.

Co-investigator Cooper has continued research on the deposition of O and OH radicals to produce oxidants on icy satellite surfaces. This work is directed towards understanding the oxygen cycle that exists on icy satellites in which radiolytically produced oxidants are cycled within the ice, but also can be sputtered and redeposited, resulting in further chemistry. A manuscript by Do et al., 2012 describing reactions of O and OH in ices has been submitted to the Journal of Physical Chemistry A.

In related work, a paper by Cooper and team (Do and Cooper, 2012) was recently submitted for publication in Monthly Notices of the Royal Astronomical Society disputing the recent identification of OH radicals in water ice using infrared spectroscopy. In similar experiments performed to those in the paper claiming detection of OH, no evidence of unique IR spectral features of OH are observed. An alternative assignment has been suggested.

Additionally, another paper by the same group (Do and Cooper, 2012) has also been submitted to the Journal of Physical Chemistry A in which the intrinsic IR band strength of OH has been determined using matrix isolation spectroscopy. While the focus of the paper is related to Earth’s atmospheric chemistry, this value could be of importance in research related to ices if a definitive IR detection of OH is made in the future.

Two additional publications, one scientific journal article and an educational article, on icy worlds have been published. The first article describes research in which an alternative mechanism (O atom insertion into CH₄) is shown to likely be important in irradiated ices if O atoms are produces in significant quantities (Pearce et al., 2012). The second publication is an educational article by Cooper, 2012 aimed at describing the significance and detection of oxygen on icy satellite surfaces.

In a new study, conducted by Gudipati and Yang and published in the Astrophysical Journal Letters (Gudipati and Yang, 2012), provides the first direct look at the organic chemistry that takes place on icy particles in the frigid reaches of our solar system, and in the even chillier places between stars. Scientists think that the basic ingredients of life, including water and organics, began their journey to Earth on these lonesome ice particles. The ice and organics would have found their way into comets and asteroids, which then fell to Earth, delivering “prebiotic” ingredients that could have jump-started life. The organics looked at in the study are called polycyclic aromatic hydrocarbons, or PAHs for short. These carbon-rich molecules can be found on Earth as combustion products: for example, in barbecue pits, candle soot and even streaming out of the tail pipe of your car. They have also been spotted throughout space in comets, asteroids and more distant objects. NASA’s Spitzer Space Telescope has detected PAHs in the swirling planet-forming disks around stars, in the spaces between stars and in remote galaxies.

In this investigation, Murthy and his colleague Yang of JPL utilized their laboratory setup (Fig. 2-1) to mimic the environment of icy PAH molecules in the quiet cold of space, at temperatures as low as 5 K. First, they bombarded the particles with ultraviolet radiation similar to that from stars. Then, to determine the products of the chemical reaction, they used a type of laser system known as MALDI (for Matrix Assisted Laser Desorption and Ionization), which involves zapping the ice with both infrared and ultraviolet lasers.

The results revealed that the PAHs had transformed: they had incorporated hydrogen atoms into their structure and lost their circular, aromatic bonds, becoming more complex organics. According to the study, this is the type of change that would need to occur if the material were to eventually become amino acids and nucleotides — bits and pieces of protein and DNA, respectively.
A book chapter co-written by Cooper (with Murthy Gudipati) describing the fundamental chemical processes that occur in irradiated ices was published in the book The Science of Solar System Ices (Gudipati and Castillo, 2012) as a response to the growing need for cross-disciplinary dialog and communication in the Planetary Ices science community. The role of laboratory research and simulations in advancing our understanding of solar system ices (including satellites, KBOs, comets, and giant planets) is becoming increasingly important in recent years. Understanding ice surface radiation processing, particle and radiation penetration depths, surface and subsurface chemistry, morphology, phases, density, conductivity, etc., are only a few examples of the inventory of issues relevant to solar system ices. This book aims to achieve direct dialog and foster focused collaborations among the observational, modeling, and laboratory research communities.

Co –Invesigators Paul Johnson and Robert Hodyss, along with JPL summer intern, Tucker Ely, began an investigation into the spectral properties of bacterial spores in cryogenic ices as well as their viability under solar radiation. Bacterial spores are one of the toughest and most durable forms of life on earth, and are consequently fascinating subjects of investigation for astrobiology. Can bacterial spores embedded in near surface Europan ice survive the Jovian radiation environment? Can either the spores or their organic radiolysis products be detected spectroscopically through remote sensing?

This work was in collaboration with Co-Investigator Adrian Ponce, whose group (Dr. Aaron Noell and Arin Greenwood) prepared and provided the Bacillus subtilis spore samples investigated (Fig 2-2).

B. subtillus was chosen for not only its ability to readily generate spores, but its pervasiveness in the available literature. Noell and Greenwood deposited purified spores onto silicon-oxide coated aluminum mirrors in both a monolayer, and multilayer form. The multilayer form, which generated a substantially increased FT-IR signal, was initially used to more clearly identify the known absorptive features of spores at standard temperature. The monolayer was then substituted in, not only to make the signal strength more quantifiable, but also to allow for more accurate post-UV viability testing (carried out by Noell and Greenwood as part of a separate and ongoing investigation), ensuring that all spores received an equal UV dosage and were not protecting one another. The monolayer spores were stamped onto half of the mirror (the remaining half used as a background blank), whereas the multilayer took the form of a noticeable spore spot on the mirror ~2mm in diameter. Once prepared, the mirrors were mounted on a cryostat and transferred to a vacuum apparatus for UV irradiation and FT-IR analysis.

Briefly, we found that Bacillus subtilis spores generate unique low molecular weight photoproducts when irradiated with UV at low temperature and pressures (100K and 10^-9 Torr). After irradiation, absorption bands were observed at 2249.4 and 2168.7 cm^-1, which is within the general region of nitrile containing groups, though no positive identification has yet been achieved (Fig. 2-3). Looking at the evolved gases with a mass spectrometer while heating the samples showed that the associated irradiation-induced photoproducts generate secondary products upon the ionization with masses of 14.2 and 40.2 amu (no order association). The addition of a thin (0.25μm) water-ice layer on top of the spores did not appear to alter or interfere with the UV induced photochemistry and accompanying products.

Future research will greatly expand the scope of the project to include (i) completion of viability assessment of post-irradiated spores (ii) spore survivability as a function of ice thickness, (iii) spore spectral signature alteration as a function of ice thickness, and (iv) the identification of the aforementioned photoproducts with accompanying chemistry.

Co-Investigators Johnson and Hodyss also published a paper describing work related to amino acids (See Fig. 2-4). Specifically, the paper describes a wavelength resolved study of the ultraviolet photolysis of glycine and phenylalanine. Studying these reactions at multiple discreet wavelengths distinguishes the present work by resolving the important contribution of photons with energies much lower than Lyman-alpha (121.6 nm). We report that although the half-lives of glycine and phenylalanine are essentially identical at 147 nm, they diverge at 206 nm and diverge significantly at 254 nm with glycine having longer half-lives at these longer wavelengths. Scaling the results to account for the wavelength dependent variation in solar irradiance shows that despite the reduction of photon energies in the 200–250 nm range, versus 147 nm, it is the longer wavelengths that will dominate the destruction of amino acids in icy surfaces. It seems unlikely that organics can survive long enough on the surface of an icy planetary body to be detected without being frequently replenished from a shielded source such as a subsurface ocean.

Co-Investigator Adrian Ponce carried out the following four major tasks:

Task 1: Viability Assessment of Microbes in Simulated Europan Radiation Environment

Motivation: This task focuses on the viability of spores and radiation resistant microbes in ices under radiation environment. Planetary ice sheets are likely depositories of putative microbial extant/extinct life from the potential biospheres on, for example, Mars, Europa, and Enceladus. Hardy microorganisms embedded within these ices would have the potential to survive over millions of years. Experiments require the preparation of uniform monolayer distributions of spores and resistant microbes on aluminum targets for electron radiation inactivation experiments performed by Co-I Kevin Hand, and UV inaction experiments performed by Co-I Paul Johnson at temperature regimes of the Europa surface. Viability assessment of spores and microbes after irradiation are performed by culture, germinability, and ATP assays.

Status: After one round of sample preparation, irradiation, and viability assessment a number of challenges in sample preparation were identified that need to be addressed before inactivation parameters can be obtained. Specifically, deposition of uniform monolayers in the target area of the electron beam has been difficult to obtain. At this point, we have evaluated numerous strategies, including various detergents and drying conditions, and evaluated their suitability with electron microscopy. We found that Bacillus subtilis endospores deposited in a high-density, homogeneously distributed single layer on metal, glass, and silicone substrates using vacuum filtration followed by a wetted filter transfer step. Quantitative transfer of spores onto these substrates was achieved as evidenced by ESEM and culturing, and will enable endospore inactivation studies. This sample preparation method is being submitted for peer review, titled “High-Density, Homogeneous Endospore Monolayer Deposition on Test Surfaces”(Noell, Greenwood et al., 2012 in preparation). We estimate that irradiation experiments will be possible in the first quarter of 2013, and that subsequent data collection will proceed on target for completing this task on time and on budget.

Task 2: Viability of Microbes Embedded in Kilimanjaro’s Glacial Ice

Motivation: The summit of Kilimanjaro in equatorial East Africa hosts melting/subliming glaciers with stratigraphic layering, including embedded atmospheric dust, that are believed to be nearly 12,000 years old. The periglacial soils and supraglacial meltwater ponds are among the most extreme microbial habitats on Earth, as microbes eking out a living in these near-sterile environments face extreme diurnal freeze-thaw cycles, high UV flux, half an atmosphere of pressure, and extreme low organic carbon content. The Kilimanjaro summit also contains fissure vents and fumaroles that afford the possibility of investigating microbial habitability in terms of viability, diversity and abundance across temperature and water gradients that represent a continuum of microenvironments. These factors make Kilimanjaro an excellent Europa analog that will further our understanding of microbial growth boundary conditions, habitability, and preservation of organics.

Status: The equatorial glaciers atop Mount Kilimanjaro, Tanzania, contain stratified layers of ice with embedded particulate matter and associated microorganisms. While the environmental extremes of supraglacial habitats on Kilimanjaro are potential analog site for investigations of microbial survival and growth on Mars, Europa and other icy worlds, there are currently no investigations published on the microbiology of Kilimanjaro glaciers. We aseptically collected particulate matter from ice layers, including two layers with visible dust inclusions, near the base of a ~30 m vertical glacial cliff at the southwest corner of the Northern Ice Field, and found that the two dust-rich layers contained sufficient material for microbial community composition analysis. Four hundred seventy-four clones containing amplified ¹⁶S rRNA genes were prepared, and the gene sequences were analyzed phylogenetically. Actinobacteria dominate both dust layers, and while the upper layer also contains Gammaproteobacteria, the lower layer is more diverse, including Betaproteobacteria, Alphaproteobacteria, and Bacteroidetes. Of the fourteen distinct species-groups in these phyla, twelve (85%) are most closely related to species found in cold-water environments. We postulate that the microbes embedded in the dust layers were once part of a supraglacial cold-water ecosystem, where post-depositional growth of cold-water tolerant species produced the observed microbial communities. We anticipate that future investigations of this tropical-alpine, supraglacial, cold-water ecosystem will yield insights into microbial activity at the temperature, water and organic carbon limits for life on Earth. This work is being submitted for peer review in a manuscript titled “Microbial Diversity of Kilimanjaro’s Glacial Ice”(Beaty, Connon et al., 2012, in preparation).

Current age estimates from ice core data and glacial dynamics modeling disagree by an order of magnitude (11,700 vs. ~1,000 years old). We measured radiocarbon dates of glacial dust and proximal soil samples collected from, in, and around the NIF that better constrain glacier age estimates. At the oldest glacier sampling site we accessed, a dust-rich ice layer, 1.5 m above the base of the ~40 m high glacier, we measured an age range of 1600 – 790 years old from four separate samples. This variation likely arises from radiocarbon heterogeneity in the volcanic soils at the peak, which are the main source of the dust recovered from the glacier. Sediment samples from supraglacial ponds contained modern carbon, in contrast to the 1520 – 3840 year old age range measured for the surrounding soils, indicating that sufficient microbial activity has occurred in the pond to reset the carbon clock. While the dust layer sampling strategy did not enable mapping of the entire NIF, as visibly dusty sediment layers are of limited extent, the reported ice layer dates are in better agreement with glacial modeling that suggests a younger and more dynamic glacial system on Kilimanjaro. Our findings have been submitted for peer review in a manuscript titled “Age of Glacial Dust Layers and Soils at Kilimanjaro’s Northern Ice Field”(Noell, Abbey et al. 2012 submitted)

Task 3: High Pressure Microbiology

Motivation: The emerging field of high pressure microbiology is relevant to the largest segment of Earth’s biosphere, as 95% of water containing 55% of all waterborne microbes is located more than 200 m (i.e., >2 MPa) below sea level (i.e., 0.1 MPa). On Europa the fraction of water volume at high hydrostatic pressure is expected to be even greater. A growing body of literature documents investigations of high-pressure physiology of piezophiles and non-piezophiles, and their adaptations to extreme high-pressure environments. However, much less is known about microbial adaption to high pressure versus effects of temperature, pH and osmotic pressure.

Bacterial spores are the most resilient and long lived microbial forms, with report of longevity up to several hundred million years, making bacterial spores the most likely organisms to survive an interplanetary journey. Once embedded into Europan ice and subdued into its ocean by convection, the possibility of germination and outgrowth remains plausible. Recently, we and others have shown that aerobic Bacillus spores germinate as a result of being exposed to high hydrostatic pressures (50-800 MPa) (Figure 2-4). However, this organism is aerobic, and it is expected that the Europan ocean will be anoxic. Here we propose to investigate the effects of high hydrostatic pressure on the germination of Clostridium sporogenes and C. frigoris spores, which are anaerobic species and the latter one also being psychrophilic (i.e., cold loving). Germination can be induced at high pressures (100 mPa).

Status: This project has been proposed as an NAI DDF, but a funding decision has not yet been announced at the time of this report.

Task 4: Endospores in Lake Vida, Antarctica
Ponce and his team also supported Co-I Alison Murray’s investigation of the microbial ecology of Lake Vida, which serves as a Europa analog environment, by measuring germinable endospore populations and their size distribution. This work has been accepted by Proceeding of the National Academy of Sciences in a manuscript titled “Microbial Life at -13 ºC in the Brine of an Ice-Sealed Antarctic Lake” (see abstract below; Kenig et al. 2012 accepted).

Abstract of PNAS paper: The permanent ice cover of Lake Vida (Antarctica) encapsulates an extreme cryogenic brine ecosystem (-13 oC; salinity, 200). This aphotic ecosystem is anoxic and consists of a slightly acidic (pH 6.2) sodium chloride dominated brine. Expeditions in 2005 and 2010 were conducted to investigate the biogeochemistry of Lake Vida’s brine system. A phylogenetically diverse and metabolically active Bacteria-dominated microbial assemblage was observed in the brine. These bacteria live under very high levels of reduced metals, ammonia, molecular hydrogen (H₂), and dissolved organic carbon, as well as high concentrations of oxidized species of nitrogen (i.e. supersaturated nitrous oxide and ~ 1 mmol L-1 nitrate) and sulfur (as sulfate). The existence of this system, with active biota, and a suite of reduced as well as oxidized compounds, is unusual given the millennial scale of its isolation from external sources of energy. The geochemistry of the brine suggests that abiotic brine-rock reactions may occur in this system and that the rich sources of dissolved electron acceptors prevent sulfate reduction and methanogenesis from being energetically favorable. The discovery of this ecosystem and the in situ biotic and abiotic processes occurring at low temperature provides a tractable system to study habitability of isolated terrestrial cryo-environments (e.g. permafrost croypegs and subglacial ecosystems) and is a potential analog for habitats on other icy worlds where water-rock reactions may co-occur with saline deposits and subsurface oceans.

Collaborator G. Strazzulla of the INAF-Catania Astrophysical Observatory group in collaboration with the group at CIMAP-GANIL in Caen (France) performed experiments on effects induced by fast ions colliding with solids of astrophysical interest. The project is aimed at measuring of formation yields of specific molecules after implantation of reactive (H, C, N, O, S etc), multiply charged ions at different energies in water ice with relevant application to the chemistry of the icy moons in the outer solar system. Ion beams have been obtained at GANIL after a selective call for proposals to which we successfully applied.

The results obtained so far are relative to implantation of 30 keV ¹³Cⁿ⁺ (n =2, 3) into thick films (i.e. thicker than the penetration depth of the ions) of water ice, and indicate that:

o ¹³CO₂ was produced after implantation with different charge states at different temperatures.
o No effects related to the temperature of the ices have been observed.
o No effects related to the charge states of the incident ions have been found.

They conclude that although a relevant quantity of CO₂ can be formed by implantation of Jovian magnetospheric carbon ions on the Galilean icy moons, this is not the dominant formation mechanism. As alternative mechanism, they propose that the synthesis of carbon dioxide occurs by ion bombardment at the interface ice-solid carbon.