Introduction
The accumulation and preservation of organic molecules at or near planetary surfaces is an essential step in life's origins. Organic species accumulate by a combination of exogenous and endogenous processes (Chyba & Sagan Reference Chyba and Sagan1992; Hazen Reference Hazen2005; and references therein; Love & Brownlee Reference Love and Brownlee1993). At the same time, organic molecules may decompose through a variety of chemical and physical processes, including thermal degradation, mineral-mediated oxidation and photolysis (Bada et al. Reference Bada, Miller and Zhao1995; Bada & Lazcano Reference Bada and Lazcano2002; Ten Kate et al. Reference ten Kate, Gerry, Peters, Quinn, Foing and Ehrenfreund2005; Marshall-Bowman et al. Reference Marshall-Bowman, Ohara, Sverjensky, Hazen and Cleaves2010; Kim et al. Reference Kim, Yee, Nanda and Falkowski2013). As models of Martian meteoritic influx have suggested, considerable amounts of carbonaceous meteoritic materials should be present in the Martian regolith (Flynn & McKay Reference Flynn and McKay1990; Bland & Smith Reference Bland and Smith2000). Calculations by Kanavarioti & Mancinelli (Reference Kanavarioti and Mancinelli1990) based on the stability of amino acids revealed that remnants of these compounds, if they existed on Mars 3.5 billion years ago, might have been preserved buried beneath the surface-oxidizing layer. Therefore, assessing the stability of organic species in near-surface environments is one key to evaluating plausible scenarios for the origins of life.
The low level of organics on Mars has been puzzling because organic matter should be delivered continuously to the Martian surface, as it is to the Earth, from space via meteorites (Cronin & Chang Reference Cronin, Chang and Greenberg1993; Sephton et al. Reference Sephton, Wright, Gilmour, de Leeuw, Grady and Pillinger2002; Pizzarello et al. Reference Pizzarello, Cooper, Flynn, Lauretta and McSween2006), comets (Llorca Reference Llorca2005) and interplanetary dust particles (Schramm et al. Reference Schramm, Brownlee and Wheelock1989; Flynn Reference Flynn1996). However, exposure to ionizing radiation by charged, energetic particles arriving at the surface through the thin Martian atmosphere suggests that the fate of these organic molecules at or near the Martian surface must be very different than those on the Earth (e.g. ten Kate et al. Reference ten Kate, Gerry, Peters, Quinn, Foing and Ehrenfreund2005).
Accordingly, we are investigating the possible protective role of Martian analogue powdered minerals and soils for the survivability of biomolecules. Here, we report on the protective roles of Martian analogue minerals and soils against effects of ultraviolet (UV) and gamma radiation for purine, pyrimidine and uracil. Since RNA and DNA can be considered as derivatives of purine, pyrimidine, and uracil, these bases are crucial biomolecules in scenarios of prebiotic molecular organization, as well as in extant living systems.
The detection of organics on Mars by SAM at Gale Crater has been hampered by the contamination of the instrument by the derivatization reagents N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) and dimethylformamide and by the presence of perchlorate (ClO4 −) in the soil and sediments (Glavin et al. Reference Glavin2013; Leshin et al. Reference Leshin2013). The SAM results are consistent with the discovery by the Phoenix Wet Chemistry Laboratory of 0.6 wt.% ClO4 − in the soil (Hecht et al. Reference Hecht2009; Kounaves et al. Reference Kounaves2009). These discoveries suggest that ClO4 − is global in extent and that ClO4 − or intermediary oxychlorines such as ClO2 − or ClO− may be participants in the destructive oxidation of organics and/or their lack of detection by Viking and the Mars Science Laboratory (Navarro-González et al. Reference Navarro-González, Vargas, de la Rosa, Raga and McKay2010) and the reactivity detected by the Viking Biology Experiments (Quinn et al. Reference Quinn2013). A single organic, chlorobenzene, detected by SAM in a sample from the mudstone deposits at Yellowknife Bay in Gale Crater has been determined to derive from Martian organics (Freissinet et al. Reference Freissinet2015). The chlorobenzene detected is a reaction product of some unknown Martian organic species and, fortuitously, chlorobenzene does not appear as a strong reaction product of the MTBSTFA contaminant with perchlorate.
Methods
Materials
Calcium carbonate (calcite; CaCO3), calcium sulphate (anhydrite; CaSO4), ferric oxide (Fe2O3), purine, pyrimidine, uracil and high-performance liquid chromatography (HPLC) grade acetonitrile were purchased from Sigma-Aldrich, St. Louis, MO. Atacama Desert soil was provided from Dr. Chris McKay's collection: ATC 01-6. It contains phyllosilicates, kaolinite and salts (Sutter et al. Reference Sutter, Amundson and Owen2006). The 7 Å clay mineral kaolinite [Al2Si2O5(OH)4, reference sample KGa-1b, 1127] was purchased from Source Clays Repository – The Clay Minerals Society. All of these minerals have been identified in Martian surface soil and are expected to have been significant mineralogical components of both Earth and Martian soils throughout most of their histories (Hazen Reference Hazen2005, Reference Hazen2013).
All glassware was wrapped in aluminium foil and heated at 500°C for 3 h before each use. Concentrations of purine, pyrimidine and uracil were measured by HPLC using an Alltech Alltima C-18 reverse phase column (Ertem et al. Reference Ertem, Hazen and Dworkin2007).
Preparation of minerals, removal of organics from minerals and soil samples
Minerals were washed three times with water (18 MΩ, Milli-Q; 150 mL/10 g of each mineral), once with methanol, and a final wash with water. Samples were then freeze-dried at −85°C and at 20–25 mbar for 24 h to remove water, where water and methanol, if any remained, undergo sublimation, to produce a fine powder. Further removal of organics from kaolinite and Atacama soil was carried out according to the procedure described in Wattel-Koekkoek et al. (Reference Wattel-Koekkoek, van Genuchten, Buurman and van Lagen2001): Following the water–methanol–water washings, minerals were shaken in aqueous 0.1 M Na4P2O7 solution, dialysed and freeze-dried. Organic-free minerals were passed through an 80 mesh sieve, which corresponds to a maximum particle size diameter of 177 µm, to obtain a product with uniform particle size distribution.
Preparation of organic compound – mineral mixtures for UV irradiation
The extent of binding of organic compounds to varied minerals varies with the structure of the organic molecules and the nature of the mineral (e.g. Ferris et al. Reference Ferris, Ertem and Agarwal1989; Hazen Reference Hazen2006). In order to have comparable quantities of each organic compound in each soil–organic mixture, physical mixtures of minerals with organic compounds were prepared and used throughout this research. We used 400 mg of mineral/soil powder for each experiment. We also established the volume of the organic compound solution required to completely wet the mineral without leaving excess solution above the surface of the powdered minerals. We then added the aqueous solutions prepared to contain 25 ppm of organic compound and 0.6% sodium perchlorate per 400 mg of each mineral.
Mineral–organic mixtures prepared for UV irradiation as described above were freeze-dried and placed onto aluminium plates with 3.0 cm diameter and 1.0 mm height, making sure that the surface of the organic compound–mineral mixture was optically flat and with a thickness of 1 mm. This experimental step is the most time-consuming and crucial, especially with hygroscopic minerals. Note that the penetration depth of UV light into soil, which depends on the wavelength and particle size, is about 1 mm or less (Sagan & Pollack Reference Sagan and Pollack1974; Keppler et al. Reference Keppler, Vigano, McLeod, Ott, Früchtl and Röckmann2012).
Controls
-
(a) Non-irradiated samples: Three sets of each organic compound (purine, pyrimidine or uracil)–mineral mixture were prepared as described above. For each organic compound–mineral pair, one set was kept as control (i.e. was not irradiated), while two sets were UV-irradiated. Organic compounds were extracted from the mixtures of organics with calcium carbonate and calcium sulphate (both the controls and UV-irradiated samples) by shaking them with water and removing the extracts after centrifugation at 4000–6000 rpm for 30 min. This procedure was repeated three times. Three extracts were combined for each sample and solutions containing the organic compounds were freeze-dried. Freeze-dried samples were dissolved in 1.0 mL of water for HPLC analysis. Extraction of organics from Atacama soil– and kaolinite–organic mixtures was accomplished by two water extractions followed by one 0.1 M aqueous sodium pyrophospahate, Na4P2O7, extraction. Combined extracts were freeze-dried, dissolved in 1.0 mL of water, dialysed and analysed by HPLC (see Table 2).
-
(b) UV irradiation of organic compounds in the absence of minerals: The same volume of organic compound solution added to the minerals was placed onto aluminium plates, freeze-dried and UV irradiated in the absence of minerals under the same conditions employed for organic compound–mineral mixtures. After irradiation, aluminium plates were washed off with 1.0 mL of water three times. Combined extracts were freeze-dried, dissolved in 1.0 mL of water and analysed by HPLC.
UV irradiation experiments
UV irradiation of the organics in the absence of minerals and of mineral–organic compound mixtures were performed in a Martian Simulation Chamber at the University of Maryland under conditions mimicking the Martian surface, as listed in Hansen et al. (Reference Hansen, Merrison, Nørnberg, Aagaard and Finster2005) (Table 1). This chamber [Fig. 1(a) and (b)] consists of a stainless steel cylindrical assembly with an internal diameter of 25 cm and depth of 45 cm. The spectral irradiance of the 300 W ozone-free 6258 xenon-arc lamp, which provides collimated UV light, was measured using an actinometer at three wavelengths (Table 1).
Fig. 1. (a) Mars Simulation Chamber. (b) Mars Simulation Chamber-Gas tanks containing liquid nitrogen and Martian atmosphere gas mixture (gas mixture was custom prepared by Robert Oxygen Company, Rockville, Maryland).
Table 1. Parameters used in the simulation chamber and nominally present on Mars
a From Horneck et al. (Reference Horneck, Reitz, Rettberg, Schuber, Kochan, Möhlmann, Richter and Seidlitz2000).
b Annual average intensity at 11.6°N (personal communication between Horneck & Patel).
c Custom prepared by Roberts Oxygen Company.
The UV light was directed through a quartz window to mineral–organic mixtures containing 0.6% sodium perchlorate – a concentration similar to that found on Mars by Phoenix (Hecht et al. Reference Hecht2009; Kounaves et al. Reference Kounaves2009).
One set of duplicate mineral–organic compound samples was irradiated at 15–25 millibar, while the second set was irradiated at ambient pressure.
The dose of UV radiation received by the samples during 30 h was equivalent to 5 Martian Sol (Sol: Martian day = 24 h 37 min): 0.028 W m−2 nm−1.
Gamma irradiation experiments
For comparison, we also studied the effects of gamma radiation on mineral–biomolecule mixtures. Gamma rays have considerably higher energy [>2 × 10−14 J (Joule)], compared with UV radiation (5 × 10−19–2 × 10−17 J). Samples were prepared as described for UV irradiation experiments: organic–mineral mixtures were placed in 2 ml polyethylene tubes. Irradiation was performed at the Uniformed Services University, Bethesda, MD using Gamma Cell 40 from a 137Cs source at 25°C. Although Cs-137 itself is a beta emitter with a half-life of 30.1 years, its decay product metastable Ba-137 further decays by gamma emission to the stable Ba-137 with a half-life of only 2.6 min. In the Gamma Cell 40, electrons are trapped before reaching the target sample. In these experiments, samples received a total dose of 3 Gy (3 Gray) corresponding to approximately 15 000 days dosage on the Martian surface (Hassler et al. Reference Hassler2014).
Results
Results of UV irradiation for 30 h (equivalent to 5 Martian Sol irradiation) shown in Table 2 indicate only a minor loss of organic compounds (<2%) when they are mixed with Mars analogue minerals/soils. Furthermore, the extent of loss does not vary with pressure in the chamber during irradiation. UV-irradiated purine, pyrimidine and uracil in the absence of minerals completely decompose to compounds without any chromophore group(s) in their structure.
Table 2. Results showing the percentages of organic destroyed after 30 h of UV irradiation under conditions listed in Table 1. As explained in the text, experiments were run in duplicates
Predetermined volumes of solutions to wet each mineral–organic mixture containing 25 ppm organic compound and 0.6% NaClO4 were added to each 400 mg of mineral. The mixtures were freeze-dried and irradiated in the Martian simulation chamber. One set of samples was irradiated at ambient pressure, while the second set was irradiated at 15–25 millibar. As controls, the same amounts of organic compounds added to the minerals were irradiated under the same conditions but in the absence of minerals. Organics were extracted from the minerals and analysed by HPLC along with the organics irradiated in the absence of minerals.
Organic compounds were completely decomposed by oxidation when they were mixed with Fe2O3 before being subjected to UV light. These results are not included in Table 2.
Irradiation performed in the absence of perchlorate ions, where chlorine is at its highest oxidation state of 7+, produced comparable results (i.e. 1–2% loss of organics). In comparison, gamma irradiation of mineral–organic mixtures with a dose of 3 Gray corresponding to ~15 000 days on the Martian surface (Hassler et al. Reference Hassler2014) results in about 10% loss, as shown in Table 3.
Table 3. Percentages of organic lost after 13 Gray gamma irradiation
Biomolecule–mineral mixtures were irradiated with gamma radiation from a 137Cs source, extracted and organics were analysed by HPLC. Numbers indicate in mAU (milli Absorption Units) obtained by HPLC.
Discussion
These results are in sharp contrast to the behaviour of mixtures of purine, pyrimidine and uracil with Fe2O3, which decomposes these compounds in ambient light upon mixing, before UV irradiation. Similarly, rapid photo-oxidation has been observed for other transition metal oxides, for example in the rapid decomposition of the pentose sugars arabinose, lyxose, ribose and xylose in the presence of rutile (TiO2) under ambient light conditions, but not in darkness (Klochko et al. Reference Klochko, Hazen, Sverjensky and Cody2012).
These results do not necessarily distinguish whether the loss of organics occurred only at the very outer surface of mineral–organic mixtures or throughout the whole mixture. It is conceivable, for example, that some of the 1–2% loss of organics during irradiation of samples was due to the location of organics; that is, it is possible that organic species situated on the exposed outer surface of mineral–organic mixtures, namely above the penetration depth of UV radiation, were not protected by the minerals/soils.
Kaolinite, which has a layer structure made up of one tetrahedral sheet and one octahedral sheet, does not strongly bind biomolecules studied here. We are currently investigating the protective role of clay minerals composed of three-sheet layers with varying charge densities, namely phyllosilicates (charge density arises from the isomorphic substitution of Al3+ ions in the octahedral sheet by Mg2+ ions and Si4+ ions in the tetrahedral sheets by Al3+ ions. It is counterbalanced by the equivalent number of interlayer cations, mostly Na+ or Ca2+, held in the interlayer region of the clays). Phyllosilicates, along with sulphates, have been identified on Mars in the southern hemisphere (Bibring et al. Reference Bibring2005). Since The Wet Chemistry Laboratory on the Phoenix Lander Mission can analyse the chemistry and mineralogy of the soil (Kounaves et al. Reference Kounaves2009), the charge density of the smectites can be calculated from chemical analysis data (Köster Reference Köster1977).
The extent of smectites’ ability to serve as catalyst for the formation of RNA-like oligomers widely varies with their charge density (Ertem et al. Reference Ertem, Steudel, Emmerich and Lagaly2010). Our research designed to test the protective effect of smectite with varying charge densities against radiation will demonstrate the correlation, if any, between the charge density and the protective effect (that is, whether the protective effect of three-sheet smectites varies with the charge density of the mineral). These studies in turn will provide useful information to find the best target sites to look for organics on the Martian surface. Although the presence of perchlorate ions in Martian soil has been established by several groups, we did run a set of experiments in the absence of perchlorate ions and obtained comparable results.
Results of the UV irradiation carried out at 15–25 millibar and at ambient pressure were comparable and demonstrated that pressure has no significant effect on the irradiation products, as was also shown by previous research (Horneck et al. Reference Horneck, Reitz, Rettberg, Schuber, Kochan, Möhlmann, Richter and Seidlitz2000; Schuerger et al. Reference Schuerger, Mancinelli, Kern, Rothschild and McKay2003, Reference Schuerger, Richards, Newcombe and Venkateswaran2006).
Conclusions
We have chosen to study the survivability of purine, pyrimidine and uracil against the effects of UV and gamma radiation because RNA and DNA are derivatives of these biomolecules. Our results demonstrate that in the absence of minerals, or in the presence of ferric oxide, purine, pyrimidine and uracil decomposed into products without any chromophore group. If they turned into gaseous products upon being exposed to UV irradiation, it would have been very difficult to determine their identity under the 98% CO2 atmospheric conditions of the experiments.
In the presence of powdered minerals and soil analogues, by contrast, these organic species are largely preserved in surface layers only about 1 mm thick, and the extent of preservation does not vary with the nature of the mineral/soil.
A number of missions to Mars and other planets are being planned to search for organic molecules and possible biosignatures. Therefore, it is of the utmost importance to investigate the survivability of organics under plausible surface conditions on the surface of each planet. Our results demonstrated that 25 ppm (which corresponds to 25 mg of organics per kilogram of soil) of purine, pyrimidine, and uracil mixed with soils present on the Martian surface would enjoy the possible protective effect of minerals, as was first proposed by Reference BernalBernal (Reference Bernal1949) as early as in 1947 (published in Reference Bernal1949).
These experiments also point to the need for additional research. For example, studies in preparation of organic survivability in layers both significantly thinner and thicker than 1 mm with a wider range of minerals and soils may reveal the extent to which mineral–molecule interactions, potentially independent of UV or visible light flux, lead to organic survival or degradation.
Currently, we are investigating the effects of gamma radiation, asteroid impacts and cosmic radiation and particles on the survivability of a suite of biomolecules and alkyl derivatives of sulfonic acid and phosphonic acids in the presence and absence of Martian analogue minerals. Results of gamma irradiation indicated that purine and uracil undergo a 10–13% lost upon irradiating the CaCO3–purine and CaCO3–uracil mixtures with gamma rays of 3 Gray, which corresponds to a dosage equivalent to ~15 000 days on the Martian surface. Experiments designed to study the effects of gamma radiation at a dose corresponding to 500 000 years and 1 000 000 years on the Martian surface are in progress.
Acknowledgements
This research was supported by a grant from NASA NNX10AT27G-EXOB 2009. The Mars Simulation Chamber was constructed by funds awarded by NAI-DDF. We are grateful to NASA and NAI for their generous support of this research. R.M.H. thanks NSF for support of mineral surface studies and the Deep Carbon Observatory and NAI for support of mineral evolution studies. G.E. is greatly thankful to Dr. Sanford P. Markey of National Institutes of Health for the opportunity to work in his laboratory as a Special Volunteer and for the permission to use the instruments, without which this research would not be completed. She also thanks Professor Russell R. Dickerson of the University of Maryland for the opportunity to join his team as a Visiting Senior Research Scientist.
