- Email: [email protected]
Habitability: Where to look for life? Halophilic habitats: Earth analogs to study Mars habitability
Planetary and Space Science 68 (2012) 48–55
Contents lists available at SciVerse ScienceDirect
Planetary and Space Science journal homepage: www.elsevier.com/locate/pss
Habitability: Where to look for life? Halophilic habitats: Earth analogs to study Mars habitability F. Go´mez a,n, J.A. Rodrı´guez-Manfredi a, N. Rodrı´guez a, M. Ferna´ndez-Sampedro a, F.J. Caballero-Castrejo´n a, R. Amils a,b a b
´n a Ajalvir Km 4, Torrejo ´n de Ardoz, Madrid 28850, Spain Centro de Astrobiologı´a (INTA-CSIC), Crtra. Torrejo Centro de Biologı´a Molecular Universidad Autonoma de Madrid, Cantoblanco, Madrid 28049, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2010 Received in revised form 1 November 2011 Accepted 22 December 2011 Available online 10 January 2012
Oxidative stress, high radiation doses, low temperature and pressure are parameters which made Mars’s surface adverse for life. Those conditions found on Mars surface are harsh conditions for life to deal with. Life, as we know it on Earth, needs several requirements for its establishment but, the only ‘‘sine qua nom’’ element is water. Extremophilic microorganisms widened the window of possibilities for life to develop in the universe, and as a consequence on Mars. Recently reported results in extreme environments indicate the possibility of presence of ‘‘oasys’’ for life in microniches due to water deliquescence in salts deposits. The compilation of data produced by the ongoing missions (Mars Global Surveyor, Mars Odyssey, Mars Express and Mars Exploration Rover Opportunity) offers a completely different view from that reported by Viking missions: signs of an early wet Mars and rather recent volcanic activity. The discovery of important accumulations of sulfates, and the existence of iron minerals like jarosite, goethite and hematite in rocks of sedimentary origin has allowed specific terrestrial models related with this type of mineralogy to come into focus. Rı´o Tinto (Southwestern Spain, Iberian Pyritic Belt) is an extreme acidic environment, product of the chemolithotrophic activity of microorganisms that thrive in the massive pyrite-rich deposits of the Iberian Pyritic Belt. The high concentration of ferric iron and sulfates, products of the metabolism of pyrite, generate a collection of minerals, mainly gypsum, jarosite, goethite and hematites, all of which have been detected in different regions of Mars. Some particular protective environments or elements could house organic molecules or the first bacterial life forms on Mars surface. Terrestrial analogs could help us to afford its comprehension. We are reporting here some preliminary studies about endolithic niches inside salt deposits used by phototrophs for taking advantage of sheltering particular light wavelengths. These acidic salts deposits located in Rı´o Tinto shelter life forms which are difficult to localize by eye. Techniques for its localization and study during space missions are needed to develop. Extreme environments are good scenarios where to test and train those techniques and where hypothetical astrobiological space missions could be simulated for increasing possibilities of micro niches identification. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Endolithic micro environments Astrobiology
1. Introduction Current Mars exploration is producing a considerable amount of information which requires comparison with terrestrial analogs in order to interpret and evaluate compatibility with possible extinct and/or extant life on the planet. The first astrobiological mission specially designed to detect life on Mars, the Viking missions, thought life unlikely considering the amount of UV radiation bathing the surface of the planet, the resulting oxidative
n
Corresponding author. Tel.:þ 34 91 5206461. E-mail address: [email protected] (F. Go´mez).
0032-0633/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pss.2011.12.021
conditions, and the lack of adequate atmospheric protection. Recently reported results from Mars missions are changing our idea about Mars habitability. What it was a very dry scenario, with strong radiation and oxidative stress, now is becoming a less hostile environment. The existence of extinct or extant liquid water in Mars subsurface would increase its habitability potential. Evidence of water signals (ice water) on Mars was reported by the Mars Express mission (Poulet et al., 2005). OMEGA near-infrared spectrometer reported the presence of phyllosilicates on the Mars surface. These water bearing minerals located on early Mars surfaces are solid proof of the existence of water in early Mars. Other authors have recently reported a wider diversity of phyllosilicate mineralogy
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
using the Compact Reconnaissance Imaging Spectrometer (CRISM) on the Mars Reconnaissance Orbiter (MRO) (Mustard et al., 2008). Other evidence of a wet past of Mars come from the Opportunity rover at Meridiani Planum. The identification of hematite, goethite and sulfate rich deposits, such as jarosite (Squyres et al., 2004), gave a possible scenario for a past aqueous acidic environment on Mars. The study on Extreme Environments on Earth has contributed tremendously to the understanding of habitability conditions on other planetary bodies. Mars surface conditions are harsh conditions for life to exist. The possibilities for life on such place increase if we consider the possibility of micro niches where life could be located and protected against the adverse conditions (Rothschild, 1990). Water deliquescence in salts deposits on the surface of Mars increases water activity from which life could take advantage of (Davila et al., 2010). Some Atacama Desert locations which are tremendously adverse for life due to the low water activity reported the presence of microbial biodiversity when particular protected niches were studied (Go´mez-Silva et al., 2008). Some lessons could be taken from the study on extreme environments on Earth to learn about the possibilities of life on other planetary bodies (Go´mez et al., 2007, 2010). Rio Tinto, 100 km river located at South West of Spain, is being taken as a well reported Mars analog (Ferna´ndez-Remolar et al., 2004) due to the similarities in the mineralogy of the system which that reported by MER Opportunity Rover missions which landed in Meridiani Planum where sedimentary deposits have been identified in different craters (Squyres and Knoll, 2005). The Rio Tinto salts precipitation patters (Fig. 1) can contribute to biomineralization processes which could be of special interest for organics but also life preservation on environmental harsh conditions. These ‘‘oasys’’ for organics and/or life forms are of special astrobiological interest and should attract our attention in other planets and we should be looking for it during rover exploration missions. Endolithic micro niches in Rio Tinto salts precipitates determine controlled scenarios where phototrops develop under controlled conditions.
49
dam or mining tunnel walls but always in places with specific environmental characteristics. It appears in not direct Sun light exposed places (shadow side of walls) with thermal and pH stability. Temperature above and below the precipitates was followed with PCE-ST 1 contact thermometer. 2.1. Absorbance records In acquiring the absorbance records along the diurnal hours, an Ocean Optics VIS-UV USB4000 fiber optic spectrometer was used. It covers a wavelength range from 200 to 850 nm with an
2. Material and methods Interesting multi layered salty deposits were identified in Rio Tinto source area (Fig. 2) where endolithic micro niches were settled. Green layers (Fig. 3) appear included in brown stratified salt precipitates. These salts deposits were stable along the year (not seasonal) no matter weather conditions on the zone. The crust deposit was between 5 mm and 1 cm width. The layered structure is deposited over rocks or over man made structures as
Fig. 2. Natrojarosite precipitates con Rio Tinto rocks. No signs of life underneath the precipitate are visible.
Fig. 1. Precipitation characteristics patterns on Rio Tinto. Salt saturation on the water allows spectacular precipitation on the river bed. Waves due to the seasonal precipitation and water movement can be recognized. Microorganisms are covered by mineral precipitation. Due to surface’s charges present on the bacteria and fungi, minerals and ions are attracted and precipitated on them. Microbes are the switch on element for biogeochemical structures formation.
50
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
Fig. 4. Absorbance spectra were acquired without natural conditions modifications.
The intact compact samples were placed directly on a flat aluminum sample holder. The powdered compact samples were packed into a standard aluminum sample holder and measured in the same way as the calibration samples. Two different samples of two locations of the salty crust were selected for XRD analysis. 2.3. Microscope observation
Fig. 3. Dark (green) layer below salt deposit where micro niches were generated by phototrophs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
optical resolution of 1.5 nm, providing a binary resolution of 16 bits. Additionally, an Ocean Optics QP400-2-UV/VIS 400 mm in diameter fiber optic was utilized to record the UV–vis spectra from behind the small vaulted salty crusts where the colonies were growing. By means of this procedure, the natural conditions were preserved in order to take the radiation measurements in the most non-altered state (Fig. 4). Radiation was measured along the day during four days in summer time (data not shown). As the radiation dose was similar at the same time during the four measured days, the plot at noon was chosen to be representative of the difference between the surface and subsurface measurements.
Microbial population of the salty crust was followed by optical microscopy (Axioskop 2, Zeiss). Bio-mineralization processes over the microbes were identified and followed through the observation of different samples from different stations which showed different level of dehydration. The samples were fixed using critical point (Critical Point Dryer Ball-Tec CPD 030) and mounted onto conductive graphite stubs and sputtered and gold-coated in a Bio-Rad SC 502 apparatus for electrical conductivity and to prevent charging under the electron beam. Samples were examined with a SEM (Electron Scanning Microscope JEOL JSM-5600 LV) (Figs. 5, 8 and 10) using an acceleration voltage of 20 kV and a working distance of 20 mm. The temperature of the sample stage during analysis was room temperature. The qualitative element composition of samples was determined by Energy-dispersive X-ray spectroscopy (EDX) microanalysis using an INCAx-sight with a Si–Li detector (Oxford, England) (Figs. 9 and 11), with detection limit of 10% of the main element. The INCAxsight with a Si–Li detector is able to detect the lighter elements (C, O and N), and the quantitative numerical data of the spectra obtained are referenced as default to the higher peaks obtained in each spectrum, which generally corresponded to C in our case.
2.2. X-ray diffraction (XRD) analysis 2.4. Microbial diversity XRPD was performed using a PANalytical X’Pert PRO MPD system (PW3040/60) (PANalytical B.V., The Netherlands) with ˚ and a divergence slit of 11. The X-ray Cu Ka radiation (l ¼1.542 A) generator was set to an acceleration voltage of 30 kV and a filament emission of 40 mA. Samples were scanned between 31 (2y) and 401 (2y) using a step size of 0.0081 (2y) and a count time of 2 s. Data were collected using X’Pert Data Collector and viewed using X’Pert Data Viewer (PANalytical B.V., The Netherlands).
Microbial diversity present in the samples was studied using molecular ecology techniques. Total DNA present in the sample was extracted using commercial kits from Promega (Maxwells) using company protocols, amplified by PCR using universal prokaryotes primers. The PCR products were directly sequenced with dye terminator cycle sequencing kit (Big-Dye 1.1 sequencing kit, Applied Biosystems) as described in the manufacturer’s instructions.
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
The sequences were aligned to 16S/18S rRNA sequences obtained from the National Center of Biotechnology Information Database by the BLAST search. The sequences were also checked for potential quimeras with the Bellerophon Chimera Check program and were subsequently aligned with 16S/18S rRNA reference sequences in the ARB package (http://www.arb-home.de). The rRNA aligment was corrected manually and aligment uncertainties were omitted. Only unambiguously aligned base position were used to construct phylogenetic trees with ARB. The phylogenetic relationship was recalculated and corroborate by Paup program Version 4.0b10
Fig. 5. Biological morphologies followed by SEM. Top: Dunaliella sp. as well as several spherical morphologies are present. Bottom: Mineralogical structures resembling tunnels appear on the biofilm.
51
(Sinauer Associates, Inc. Publishers). Percentage sequences similarities were calculated and corrected for substitution rates using Jukes–Cantor correction method (Jukes and Cantor, 1969). A phylogenetic analysis with maximum likelihood criterion was done in order to decide the number of different isolates.
3. Results The population was followed by optical and electron microscopes (Fig. 5 top and bottom) as well as by molecular ecology techniques. Table 1 shows the microbial similarities of the four different sequences obtained by DNA cloning and sequencing. The sequences were compared with those in Gene Data Bank. Microscope studies identified not complex biofilms on the sample (five different species were identified). Several morphologies belonging to Dunaliella sp. were observed in Fig. 5. Also some spherical morphologies belonging to phototrophs were identified. These data are congruent with the 16 rDNA sequences data (Table 1). High similarities were found with Gene Bank Sequences belonging to Cyanidium gen, as well as some uncultures cyanobacterium isolates. Bacterial Bacillus gen. morphologies represented the prokaryotes in the biofilm. Parameter as pH and temperature along the day in the micro niche intra salt deposit were followed. Salty crust was highly hydrated and free water was present over the crust. pH was measured in the free water over the crust. pH was stable in 1.0 which is very acidic value. The mineral precipitates were identified to be composed mainly by notrojarosite (Fig. 6) which is congruent with the very low pH value present in the precipitates. Also gypsum was present. Temperature was stable inside the salt deposit reaching 12 1C along the day. Fig. 7 shows the differences in the spectral conditions between the exterior and interior of the salty crust at noon. The dose at noon was choose for comparison between radiation at surface and beneath the crust because it was the maximum measured value along the day (Fig. 7). Higher curve in Fig. 7 is the spectral radiation over the salt deposit (micro niche outside) and lower curve below salt. Optimal micro niche was selected by the phototrophs for development. Radiation intensity inside salt deposit was attenuated. Phototrophs settled below the Natrojarosite and gypsum salt for getting better radiation conditions with photo active radiation (PAR) being optimal below salts. Microbial phosilization was followed by the microanalysis with EDX on wet and dry samples. Analytical composition wet sample (spot 1 of Fig. 8) reported clear carbon signal (Fig. 9) with left peak of the graph. When the same analysis was done on a driest sample (spot 3 in Fig. 10) it could be noticed that the left peak belonging to carbon reduced its level and started to grow the
Table 1 Blast comparison rendered high similarities among sequences from the sample with Gen Bank sequences. Sequence producing significant alignment
Microbial similarity
Sequence coverage (%)
Similarity (%)
AF022186.2 X55490.1 AY862772.2
Cyanidium caldarium strain RK1 chloroplast, complete genome C. caldarium chloroplast small subunit rRNA gene Uncultured Dunaliella sp. clone At18AugA12 16S ribosomal RNA gene, partial sequence; chloroplast Cyanidium sp. Monte Rotaro 16S ribosomal RNA gene, partial sequence; chloroplast Uncultured phototrophic eukaryote clone otu1 16S small subunit ribosomal RNA gene, partial sequence; chloroplast Uncultured Prochloron sp. clone AO-105 16S ribosomal RNA gene, partial sequence Uncultured cyanobacterium clone CSC14 small subunit ribosomal RNA gene, partial sequence Chlamydomonas applanata 16S ribosomal RNA gene, partial sequence; chloroplast gene for chloroplast product
100 100 100
97 97 95
99 99
92 92
99 100
92 92
100
91
AY391359.1 DQ471911.1 DQ357958.1 AY124354.1 AF394204.1
52
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
Fig. 6. XRD analysis of mineral covering the micro niche. Natrojarosite was the main component of the covering material.
Fig. 7. Radiation dose at noon at the surface (higher curve) and underneath (lower curve) the salty crust. Radiation was attenuated behind salt deposits. Micro niches seem to attenuate radiation conditions.
signals for elements as potassium, sodium, sulphur and metals as iron (Fig. 11).
4. Discussion Protected micro niches were identified in the source area of Rio Tinto, a very acidic environment drive by iron chemistry. Those micro niches are particular environments with different condition
to those previously reported for the whole Rio Tinto extreme ecosystem. Molecular ecology techniques and microscope observation reported the microbial population present in the system. Different microbial species to those present on the water column and sediments of the river were found inside the salty crust. Clear biofilm formations were identified. Not complex structures were observed as well as a low number in the species composition of the microbial population. Five different morphologies were identified in the SEM micro graphs. The number of the species was confirmed
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
Fig. 8. Micro structure of the sample. Several microbial morphologies are observable. Three spots were chosen for spectrum uptake.
Fig. 9. Spectrum 1 from sample in Fig. 6. Left hand peak is the carbon signal. It is strong due to the organic nature of the structure localized in spot 1 of Fig. 6.
Fig. 10. Sample that was taken in summer time. It is the driest and bio-mineralization process has increased in comparison with wet sample from Fig. 6.
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´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
Fig. 11. Spectrum from spot 1 in Fig. 8. Left hand peak of carbon is less than the same peak in Fig. 7. Otherwise peaks belonging to iron, potassium and lead are higher. Mineralization process is remarked in this driest sample.
by molecular ecology techniques which rendered high similarities with eukaryotes as Dunaliella sp., Cyanidium sp., and others phototrophs. Some prokaryotes were also identified. The stability in pH and temperature seems to indicate a microbial adapted protected ecosystems what was confirmed when spectral analysis outside and inside the salts deposits were took. Optimal attenuated irradiative conditions for phototrophs were identified inside the salt deposits. Thermal and pH stability, humidity and radiation conditions seem to indicate a protected ecosystem controlled by the biology which settled on the endolithic deposit. Those ambient conditions are optimal for phototrophs taking advantage of protected micro niches with optimal light conditions for their development. The green think layer (phototrophs developing inside the salt deposits) disappears when exposed to air without the protection of the salts over them. There was a clear relationship between the salt deposits and those microorganisms. Both of them are clearly associated and microorganisms are always localized in the same layer of the deposits. The number of cells in the localized niches was higher than in the surroundings, and what is more important, the number of different species low, and not widely distributed. There was a direct relationship between the species and the niche and the ambient physico-chemical conditions on it. This is the first time that these niches are reported in Rio Tinto. Some microorganisms developing in the biofilms inside the salt deposits were covered by mineral precipitation with time. This particular fossilization process can be followed by electron microscopy (Fig. 5). Mineralogical structures resembling tunnels appear on the biological biofilm (Fig. 5, bottom) which seem to be the results of the disappearance of the microorganism after dryness process for electron microscopy observation. It seems a biological driven process which could be the origin of some of the mineral deposits on the river. The mineralogical similarities between some Mars surface areas and this extreme ecosystem (Ferna´ndez-Remolar et al., 2005) transformed the extreme ecosystem of Rio Tinto into an Earth analog of special interest. The observed bio-mineralization process in those described micro-niches could be of relevance for habitability on Mars due to the importance of Rio Tinto as Earth analog. The localization of those protected micro niches inside salts deposits in such Earth analog suppose an important step forward in the habitability potential of Mars surface. Similar protected micro niches could be established by microbes on the surface of Mars on areas were salty deposits have been identified (Fig. 12). Life could take advantage of water activity increases by
Fig. 12. Distributary fan with salty deposits Image credit: (Mars Global Surveyor NASA/JPL/Malin Space Science System).
water deliquescence in salt deposits, thermal stability due to controlled solar exposition and irradiative protection due to the salty crust, for its colonization of protected micro niches under harsh conditions and in adverse extreme locations, as it was reported here for salt deposits micro niches in Rio Tinto. Important conclusions can be extracted from these results from the habitability point of view. Future missions to study habitability potential on Mars should be focused on mineral deposits with biological potential.
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
Acknowledgments This study was funded by the project AYA2010-20213 from Spanish Ministry of Education and Science and FEDER funds from European Community. References ¨ Davila, A.F., Duport, L.G., Melchiorri, R., Janchen, J., Valea, S., de Los Rios, A., Faire´n, ¨ A.G., Mohlmann, D., McKay, C.P., Ascaso, C., Wierzchos, J., 2010. Hygroscopic salts and the potential for life on Mars. Astrobiology 10 (6), 617–628. Ferna´ndez-Remolar, D., Go´mez-Elvira, J., Go´mez, F., Sebastia´n, E., Martı´n, J., Manfredi, J.A., Torres, J., Gonza´lez Kesler, C., Amils, R., 2004. The Tinto River, an extreme acidic environment under control of iron, as an analog of the Terra Meridiani hematite site of Mars. Planetary and Space Science 52, 239–248. Ferna´ndez-Remolar, D.C., Morris, R.V., Gruener, J.E., Amils, R., Knoll, A.H., 2005. The Rı´o Tinto Basin, Spain: mineralogy, sedimentary geobiology, and implications for interpretation of outcrop rocks at Meridiani Planum, Mars. Earth and Planetary Science Letters 240 (1), 149–167. Go´mez, F., Aguilera, A., Amils, R., 2007. Soluble ferric iron as an effective protective agent against UV radiation: implications for early life. Icarus 191, 352–359. doi:10.1016/j.icarus.2007.04.008. Go´mez, F., Mateo-Marti, E., Prieto-Ballesteros, O., Gago, J., Amils, R., 2010. Protection of chemolithoautotrophic bacteria exposed to Mars environmental conditions. Icarus 209 (2), 482–487.
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Go´mez-Silva, B., Rainey, F.A., Warren-Rhodes, K.A., McKay, C.P., Navarro-Gonza´lez, R., 2008. Atacama desert soil microbiology. In: Dion, P., Nautiyal, C.S. (Eds.), Microbiology of Extreme Soils. Soil Biology, 13. , & Springer-Verlag, Berlin Heidelberg, pp. 117. Jukes, T.H., Cantor, C.R., 1969. Evolution of protein molecules. In: Munro, H.N. (Ed.), Mammalian protein metabolism. , Academic Press, New York, pp. 21–132. Mustard, J.F., Murchie, S.L., Pelkey, S.M., Ehlmann, B.L., Milliken, R.E., Grant, J.A., Bibring, J.-P., Poulet, F., Bishop, J., Dobrea, E.N., Roach, L., Seelos, F., Arvidson, R.E., Wiseman, S., Green, R., Hash, C., Humm, D., Malaret, E., McGovern, J.A., Seelos, K., Clancy, T., Clark, R., Des Marais, D., Izenberg, N., Knudson, A., Langevin, Y., Martin, T., McGuire, P., Morris, R., Robinson, M., Roush, T., Smith, M., Swayze, G., Taylor, H., Titus, T., Wolff, M., 2008. Hydrated silicate minerals on Mars observed by the Mars reconnaissance orbiter CRISM instrument. Nature 454, 305. doi:10.1038/nature07097. Poulet, F., Bibring, J.P., Mustard, J.F., Gendrin, A., Mangold, N., Langevin, Y., Arvidson, R.E., Gondet, B., Go´mez, C., The Omega Team, 2005. Phyllosilacates on Mars and implications for early martian climate. Nature 438, 623–627. Rothschild, L.J., 1990. Earth analogs for martian life. microbes in evaporites, a new model system for life on Mars. Icarus 88, 246–260. Squyres, S.W., Grotzinger, J.P., Arvidson, R.E., Bell III, J.F., Calvin, W., Christensen, P.R., Clark, B.C., Crisp, J.A., Farrand, W.H., Herkenhoff, K.E., Johnson, J.R., Klingelhofer, G., Knoll, A.H., McLennan, S.M., McSween Jr., H.Y., Morris, R.V., Rice Jr., J.W., Rieder, R., Soderblom, L.A., 2004. In situ evidence for an ancient aqueous environment at Meridiani Planum, Mars. Science 306 (5702), 1709–1714. Squyres, S.W., Knoll, A.H., 2005. Sedimentary rocks at Meridiani Planum: origin, diagenesis, and implications for life on Mars. Earth and Planetary Science Letters 240 (1), 1–10.
Contents lists available at SciVerse ScienceDirect
Planetary and Space Science journal homepage: www.elsevier.com/locate/pss
Habitability: Where to look for life? Halophilic habitats: Earth analogs to study Mars habitability F. Go´mez a,n, J.A. Rodrı´guez-Manfredi a, N. Rodrı´guez a, M. Ferna´ndez-Sampedro a, F.J. Caballero-Castrejo´n a, R. Amils a,b a b
´n a Ajalvir Km 4, Torrejo ´n de Ardoz, Madrid 28850, Spain Centro de Astrobiologı´a (INTA-CSIC), Crtra. Torrejo Centro de Biologı´a Molecular Universidad Autonoma de Madrid, Cantoblanco, Madrid 28049, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2010 Received in revised form 1 November 2011 Accepted 22 December 2011 Available online 10 January 2012
Oxidative stress, high radiation doses, low temperature and pressure are parameters which made Mars’s surface adverse for life. Those conditions found on Mars surface are harsh conditions for life to deal with. Life, as we know it on Earth, needs several requirements for its establishment but, the only ‘‘sine qua nom’’ element is water. Extremophilic microorganisms widened the window of possibilities for life to develop in the universe, and as a consequence on Mars. Recently reported results in extreme environments indicate the possibility of presence of ‘‘oasys’’ for life in microniches due to water deliquescence in salts deposits. The compilation of data produced by the ongoing missions (Mars Global Surveyor, Mars Odyssey, Mars Express and Mars Exploration Rover Opportunity) offers a completely different view from that reported by Viking missions: signs of an early wet Mars and rather recent volcanic activity. The discovery of important accumulations of sulfates, and the existence of iron minerals like jarosite, goethite and hematite in rocks of sedimentary origin has allowed specific terrestrial models related with this type of mineralogy to come into focus. Rı´o Tinto (Southwestern Spain, Iberian Pyritic Belt) is an extreme acidic environment, product of the chemolithotrophic activity of microorganisms that thrive in the massive pyrite-rich deposits of the Iberian Pyritic Belt. The high concentration of ferric iron and sulfates, products of the metabolism of pyrite, generate a collection of minerals, mainly gypsum, jarosite, goethite and hematites, all of which have been detected in different regions of Mars. Some particular protective environments or elements could house organic molecules or the first bacterial life forms on Mars surface. Terrestrial analogs could help us to afford its comprehension. We are reporting here some preliminary studies about endolithic niches inside salt deposits used by phototrophs for taking advantage of sheltering particular light wavelengths. These acidic salts deposits located in Rı´o Tinto shelter life forms which are difficult to localize by eye. Techniques for its localization and study during space missions are needed to develop. Extreme environments are good scenarios where to test and train those techniques and where hypothetical astrobiological space missions could be simulated for increasing possibilities of micro niches identification. & 2011 Elsevier Ltd. All rights reserved.
Keywords: Endolithic micro environments Astrobiology
1. Introduction Current Mars exploration is producing a considerable amount of information which requires comparison with terrestrial analogs in order to interpret and evaluate compatibility with possible extinct and/or extant life on the planet. The first astrobiological mission specially designed to detect life on Mars, the Viking missions, thought life unlikely considering the amount of UV radiation bathing the surface of the planet, the resulting oxidative
n
Corresponding author. Tel.:þ 34 91 5206461. E-mail address: [email protected] (F. Go´mez).
0032-0633/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pss.2011.12.021
conditions, and the lack of adequate atmospheric protection. Recently reported results from Mars missions are changing our idea about Mars habitability. What it was a very dry scenario, with strong radiation and oxidative stress, now is becoming a less hostile environment. The existence of extinct or extant liquid water in Mars subsurface would increase its habitability potential. Evidence of water signals (ice water) on Mars was reported by the Mars Express mission (Poulet et al., 2005). OMEGA near-infrared spectrometer reported the presence of phyllosilicates on the Mars surface. These water bearing minerals located on early Mars surfaces are solid proof of the existence of water in early Mars. Other authors have recently reported a wider diversity of phyllosilicate mineralogy
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
using the Compact Reconnaissance Imaging Spectrometer (CRISM) on the Mars Reconnaissance Orbiter (MRO) (Mustard et al., 2008). Other evidence of a wet past of Mars come from the Opportunity rover at Meridiani Planum. The identification of hematite, goethite and sulfate rich deposits, such as jarosite (Squyres et al., 2004), gave a possible scenario for a past aqueous acidic environment on Mars. The study on Extreme Environments on Earth has contributed tremendously to the understanding of habitability conditions on other planetary bodies. Mars surface conditions are harsh conditions for life to exist. The possibilities for life on such place increase if we consider the possibility of micro niches where life could be located and protected against the adverse conditions (Rothschild, 1990). Water deliquescence in salts deposits on the surface of Mars increases water activity from which life could take advantage of (Davila et al., 2010). Some Atacama Desert locations which are tremendously adverse for life due to the low water activity reported the presence of microbial biodiversity when particular protected niches were studied (Go´mez-Silva et al., 2008). Some lessons could be taken from the study on extreme environments on Earth to learn about the possibilities of life on other planetary bodies (Go´mez et al., 2007, 2010). Rio Tinto, 100 km river located at South West of Spain, is being taken as a well reported Mars analog (Ferna´ndez-Remolar et al., 2004) due to the similarities in the mineralogy of the system which that reported by MER Opportunity Rover missions which landed in Meridiani Planum where sedimentary deposits have been identified in different craters (Squyres and Knoll, 2005). The Rio Tinto salts precipitation patters (Fig. 1) can contribute to biomineralization processes which could be of special interest for organics but also life preservation on environmental harsh conditions. These ‘‘oasys’’ for organics and/or life forms are of special astrobiological interest and should attract our attention in other planets and we should be looking for it during rover exploration missions. Endolithic micro niches in Rio Tinto salts precipitates determine controlled scenarios where phototrops develop under controlled conditions.
49
dam or mining tunnel walls but always in places with specific environmental characteristics. It appears in not direct Sun light exposed places (shadow side of walls) with thermal and pH stability. Temperature above and below the precipitates was followed with PCE-ST 1 contact thermometer. 2.1. Absorbance records In acquiring the absorbance records along the diurnal hours, an Ocean Optics VIS-UV USB4000 fiber optic spectrometer was used. It covers a wavelength range from 200 to 850 nm with an
2. Material and methods Interesting multi layered salty deposits were identified in Rio Tinto source area (Fig. 2) where endolithic micro niches were settled. Green layers (Fig. 3) appear included in brown stratified salt precipitates. These salts deposits were stable along the year (not seasonal) no matter weather conditions on the zone. The crust deposit was between 5 mm and 1 cm width. The layered structure is deposited over rocks or over man made structures as
Fig. 2. Natrojarosite precipitates con Rio Tinto rocks. No signs of life underneath the precipitate are visible.
Fig. 1. Precipitation characteristics patterns on Rio Tinto. Salt saturation on the water allows spectacular precipitation on the river bed. Waves due to the seasonal precipitation and water movement can be recognized. Microorganisms are covered by mineral precipitation. Due to surface’s charges present on the bacteria and fungi, minerals and ions are attracted and precipitated on them. Microbes are the switch on element for biogeochemical structures formation.
50
´mez et al. / Planetary and Space Science 68 (2012) 48–55 F. Go
Fig. 4. Absorbance spectra were acquired without natural conditions modifications.
The intact compact samples were placed directly on a flat aluminum sample holder. The powdered compact samples were packed into a standard aluminum sample holder and measured in the same way as the calibration samples. Two different samples of two locations of the salty crust were selected for XRD analysis. 2.3. Microscope observation
Fig. 3. Dark (green) layer below salt deposit where micro niches were generated by phototrophs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
optical resolution of 1.5 nm, providing a binary resolution of 16 bits. Additionally, an Ocean Optics QP400-2-UV/VIS 400 mm in diameter fiber optic was utilized to record the UV–vis spectra from behind the small vaulted salty crusts where the colonies were growing. By means of this procedure, the natural conditions were preserved in order to take the radiation measurements in the most non-altered state (Fig. 4). Radiation was measured along the day during four days in summer time (data not shown). As the radiation dose was similar at the same time during the four measured days, the plot at noon was chosen to be representative of the difference between the surface and subsurface measurements.
Microbial population of the salty crust was followed by optical microscopy (Axioskop 2, Zeiss). Bio-mineralization processes over the microbes were identified and followed through the observation of different samples from different stations which showed different level of dehydration. The samples were fixed using critical point (Critical Point Dryer Ball-Tec CPD 030) and mounted onto conductive graphite stubs and sputtered and gold-coated in a Bio-Rad SC 502 apparatus for electrical conductivity and to prevent charging under the electron beam. Samples were examined with a SEM (Electron Scanning Microscope JEOL JSM-5600 LV) (Figs. 5, 8 and 10) using an acceleration voltage of 20 kV and a working distance of 20 mm. The temperature of the sample stage during analysis was room temperature. The qualitative element composition of samples was determined by Energy-dispersive X-ray spectroscopy (EDX) microanalysis using an INCAx-sight with a Si–Li detector (Oxford, England) (Figs. 9 and 11), with detection limit of 10% of the main element. The INCAxsight with a Si–Li detector is able to detect the lighter elements (C, O and N), and the quantitative numerical data of the spectra obtained are referenced as default to the higher peaks obtained in each spectrum, which generally corresponded to C in our case.
2.2. X-ray diffraction (XRD) analysis 2.4. Microbial diversity XRPD was performed using a PANalytical X’Pert PRO MPD system (PW3040/60) (PANalytical B.V., The Netherlands) with ˚ and a divergence slit of 11. The X-ray Cu Ka radiation (l ¼1.542 A) generator was set to an acceleration voltage of 30 kV and a filament emission of 40 mA. Samples were scanned between 31 (2y) and 401 (2y) using a step size of 0.0081 (2y) and a count time of 2 s. Data were collected using X’Pert Data Collector and viewed using X’Pert Data Viewer (PANalytical B.V., The Netherlands).
Microbial diversity present in the samples was studied using molecular ecology techniques. Total DNA present in the sample was extracted using commercial kits from Promega (Maxwells) using company protocols, amplified by PCR using universal prokaryotes primers. The PCR products were directly sequenced with dye terminator cycle sequencing kit (Big-Dye 1.1 sequencing kit, Applied Biosystems) as described in the manufacturer’s instructions.
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The sequences were aligned to 16S/18S rRNA sequences obtained from the National Center of Biotechnology Information Database by the BLAST search. The sequences were also checked for potential quimeras with the Bellerophon Chimera Check program and were subsequently aligned with 16S/18S rRNA reference sequences in the ARB package (http://www.arb-home.de). The rRNA aligment was corrected manually and aligment uncertainties were omitted. Only unambiguously aligned base position were used to construct phylogenetic trees with ARB. The phylogenetic relationship was recalculated and corroborate by Paup program Version 4.0b10
Fig. 5. Biological morphologies followed by SEM. Top: Dunaliella sp. as well as several spherical morphologies are present. Bottom: Mineralogical structures resembling tunnels appear on the biofilm.
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(Sinauer Associates, Inc. Publishers). Percentage sequences similarities were calculated and corrected for substitution rates using Jukes–Cantor correction method (Jukes and Cantor, 1969). A phylogenetic analysis with maximum likelihood criterion was done in order to decide the number of different isolates.
3. Results The population was followed by optical and electron microscopes (Fig. 5 top and bottom) as well as by molecular ecology techniques. Table 1 shows the microbial similarities of the four different sequences obtained by DNA cloning and sequencing. The sequences were compared with those in Gene Data Bank. Microscope studies identified not complex biofilms on the sample (five different species were identified). Several morphologies belonging to Dunaliella sp. were observed in Fig. 5. Also some spherical morphologies belonging to phototrophs were identified. These data are congruent with the 16 rDNA sequences data (Table 1). High similarities were found with Gene Bank Sequences belonging to Cyanidium gen, as well as some uncultures cyanobacterium isolates. Bacterial Bacillus gen. morphologies represented the prokaryotes in the biofilm. Parameter as pH and temperature along the day in the micro niche intra salt deposit were followed. Salty crust was highly hydrated and free water was present over the crust. pH was measured in the free water over the crust. pH was stable in 1.0 which is very acidic value. The mineral precipitates were identified to be composed mainly by notrojarosite (Fig. 6) which is congruent with the very low pH value present in the precipitates. Also gypsum was present. Temperature was stable inside the salt deposit reaching 12 1C along the day. Fig. 7 shows the differences in the spectral conditions between the exterior and interior of the salty crust at noon. The dose at noon was choose for comparison between radiation at surface and beneath the crust because it was the maximum measured value along the day (Fig. 7). Higher curve in Fig. 7 is the spectral radiation over the salt deposit (micro niche outside) and lower curve below salt. Optimal micro niche was selected by the phototrophs for development. Radiation intensity inside salt deposit was attenuated. Phototrophs settled below the Natrojarosite and gypsum salt for getting better radiation conditions with photo active radiation (PAR) being optimal below salts. Microbial phosilization was followed by the microanalysis with EDX on wet and dry samples. Analytical composition wet sample (spot 1 of Fig. 8) reported clear carbon signal (Fig. 9) with left peak of the graph. When the same analysis was done on a driest sample (spot 3 in Fig. 10) it could be noticed that the left peak belonging to carbon reduced its level and started to grow the
Table 1 Blast comparison rendered high similarities among sequences from the sample with Gen Bank sequences. Sequence producing significant alignment
Microbial similarity
Sequence coverage (%)
Similarity (%)
AF022186.2 X55490.1 AY862772.2
Cyanidium caldarium strain RK1 chloroplast, complete genome C. caldarium chloroplast small subunit rRNA gene Uncultured Dunaliella sp. clone At18AugA12 16S ribosomal RNA gene, partial sequence; chloroplast Cyanidium sp. Monte Rotaro 16S ribosomal RNA gene, partial sequence; chloroplast Uncultured phototrophic eukaryote clone otu1 16S small subunit ribosomal RNA gene, partial sequence; chloroplast Uncultured Prochloron sp. clone AO-105 16S ribosomal RNA gene, partial sequence Uncultured cyanobacterium clone CSC14 small subunit ribosomal RNA gene, partial sequence Chlamydomonas applanata 16S ribosomal RNA gene, partial sequence; chloroplast gene for chloroplast product
100 100 100
97 97 95
99 99
92 92
99 100
92 92
100
91
AY391359.1 DQ471911.1 DQ357958.1 AY124354.1 AF394204.1
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Fig. 6. XRD analysis of mineral covering the micro niche. Natrojarosite was the main component of the covering material.
Fig. 7. Radiation dose at noon at the surface (higher curve) and underneath (lower curve) the salty crust. Radiation was attenuated behind salt deposits. Micro niches seem to attenuate radiation conditions.
signals for elements as potassium, sodium, sulphur and metals as iron (Fig. 11).
4. Discussion Protected micro niches were identified in the source area of Rio Tinto, a very acidic environment drive by iron chemistry. Those micro niches are particular environments with different condition
to those previously reported for the whole Rio Tinto extreme ecosystem. Molecular ecology techniques and microscope observation reported the microbial population present in the system. Different microbial species to those present on the water column and sediments of the river were found inside the salty crust. Clear biofilm formations were identified. Not complex structures were observed as well as a low number in the species composition of the microbial population. Five different morphologies were identified in the SEM micro graphs. The number of the species was confirmed
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Fig. 8. Micro structure of the sample. Several microbial morphologies are observable. Three spots were chosen for spectrum uptake.
Fig. 9. Spectrum 1 from sample in Fig. 6. Left hand peak is the carbon signal. It is strong due to the organic nature of the structure localized in spot 1 of Fig. 6.
Fig. 10. Sample that was taken in summer time. It is the driest and bio-mineralization process has increased in comparison with wet sample from Fig. 6.
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Fig. 11. Spectrum from spot 1 in Fig. 8. Left hand peak of carbon is less than the same peak in Fig. 7. Otherwise peaks belonging to iron, potassium and lead are higher. Mineralization process is remarked in this driest sample.
by molecular ecology techniques which rendered high similarities with eukaryotes as Dunaliella sp., Cyanidium sp., and others phototrophs. Some prokaryotes were also identified. The stability in pH and temperature seems to indicate a microbial adapted protected ecosystems what was confirmed when spectral analysis outside and inside the salts deposits were took. Optimal attenuated irradiative conditions for phototrophs were identified inside the salt deposits. Thermal and pH stability, humidity and radiation conditions seem to indicate a protected ecosystem controlled by the biology which settled on the endolithic deposit. Those ambient conditions are optimal for phototrophs taking advantage of protected micro niches with optimal light conditions for their development. The green think layer (phototrophs developing inside the salt deposits) disappears when exposed to air without the protection of the salts over them. There was a clear relationship between the salt deposits and those microorganisms. Both of them are clearly associated and microorganisms are always localized in the same layer of the deposits. The number of cells in the localized niches was higher than in the surroundings, and what is more important, the number of different species low, and not widely distributed. There was a direct relationship between the species and the niche and the ambient physico-chemical conditions on it. This is the first time that these niches are reported in Rio Tinto. Some microorganisms developing in the biofilms inside the salt deposits were covered by mineral precipitation with time. This particular fossilization process can be followed by electron microscopy (Fig. 5). Mineralogical structures resembling tunnels appear on the biological biofilm (Fig. 5, bottom) which seem to be the results of the disappearance of the microorganism after dryness process for electron microscopy observation. It seems a biological driven process which could be the origin of some of the mineral deposits on the river. The mineralogical similarities between some Mars surface areas and this extreme ecosystem (Ferna´ndez-Remolar et al., 2005) transformed the extreme ecosystem of Rio Tinto into an Earth analog of special interest. The observed bio-mineralization process in those described micro-niches could be of relevance for habitability on Mars due to the importance of Rio Tinto as Earth analog. The localization of those protected micro niches inside salts deposits in such Earth analog suppose an important step forward in the habitability potential of Mars surface. Similar protected micro niches could be established by microbes on the surface of Mars on areas were salty deposits have been identified (Fig. 12). Life could take advantage of water activity increases by
Fig. 12. Distributary fan with salty deposits Image credit: (Mars Global Surveyor NASA/JPL/Malin Space Science System).
water deliquescence in salt deposits, thermal stability due to controlled solar exposition and irradiative protection due to the salty crust, for its colonization of protected micro niches under harsh conditions and in adverse extreme locations, as it was reported here for salt deposits micro niches in Rio Tinto. Important conclusions can be extracted from these results from the habitability point of view. Future missions to study habitability potential on Mars should be focused on mineral deposits with biological potential.
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Acknowledgments This study was funded by the project AYA2010-20213 from Spanish Ministry of Education and Science and FEDER funds from European Community. References ¨ Davila, A.F., Duport, L.G., Melchiorri, R., Janchen, J., Valea, S., de Los Rios, A., Faire´n, ¨ A.G., Mohlmann, D., McKay, C.P., Ascaso, C., Wierzchos, J., 2010. Hygroscopic salts and the potential for life on Mars. Astrobiology 10 (6), 617–628. Ferna´ndez-Remolar, D., Go´mez-Elvira, J., Go´mez, F., Sebastia´n, E., Martı´n, J., Manfredi, J.A., Torres, J., Gonza´lez Kesler, C., Amils, R., 2004. The Tinto River, an extreme acidic environment under control of iron, as an analog of the Terra Meridiani hematite site of Mars. Planetary and Space Science 52, 239–248. Ferna´ndez-Remolar, D.C., Morris, R.V., Gruener, J.E., Amils, R., Knoll, A.H., 2005. The Rı´o Tinto Basin, Spain: mineralogy, sedimentary geobiology, and implications for interpretation of outcrop rocks at Meridiani Planum, Mars. Earth and Planetary Science Letters 240 (1), 149–167. Go´mez, F., Aguilera, A., Amils, R., 2007. Soluble ferric iron as an effective protective agent against UV radiation: implications for early life. Icarus 191, 352–359. doi:10.1016/j.icarus.2007.04.008. Go´mez, F., Mateo-Marti, E., Prieto-Ballesteros, O., Gago, J., Amils, R., 2010. Protection of chemolithoautotrophic bacteria exposed to Mars environmental conditions. Icarus 209 (2), 482–487.
55
Go´mez-Silva, B., Rainey, F.A., Warren-Rhodes, K.A., McKay, C.P., Navarro-Gonza´lez, R., 2008. Atacama desert soil microbiology. In: Dion, P., Nautiyal, C.S. (Eds.), Microbiology of Extreme Soils. Soil Biology, 13. , & Springer-Verlag, Berlin Heidelberg, pp. 117. Jukes, T.H., Cantor, C.R., 1969. Evolution of protein molecules. In: Munro, H.N. (Ed.), Mammalian protein metabolism. , Academic Press, New York, pp. 21–132. Mustard, J.F., Murchie, S.L., Pelkey, S.M., Ehlmann, B.L., Milliken, R.E., Grant, J.A., Bibring, J.-P., Poulet, F., Bishop, J., Dobrea, E.N., Roach, L., Seelos, F., Arvidson, R.E., Wiseman, S., Green, R., Hash, C., Humm, D., Malaret, E., McGovern, J.A., Seelos, K., Clancy, T., Clark, R., Des Marais, D., Izenberg, N., Knudson, A., Langevin, Y., Martin, T., McGuire, P., Morris, R., Robinson, M., Roush, T., Smith, M., Swayze, G., Taylor, H., Titus, T., Wolff, M., 2008. Hydrated silicate minerals on Mars observed by the Mars reconnaissance orbiter CRISM instrument. Nature 454, 305. doi:10.1038/nature07097. Poulet, F., Bibring, J.P., Mustard, J.F., Gendrin, A., Mangold, N., Langevin, Y., Arvidson, R.E., Gondet, B., Go´mez, C., The Omega Team, 2005. Phyllosilacates on Mars and implications for early martian climate. Nature 438, 623–627. Rothschild, L.J., 1990. Earth analogs for martian life. microbes in evaporites, a new model system for life on Mars. Icarus 88, 246–260. Squyres, S.W., Grotzinger, J.P., Arvidson, R.E., Bell III, J.F., Calvin, W., Christensen, P.R., Clark, B.C., Crisp, J.A., Farrand, W.H., Herkenhoff, K.E., Johnson, J.R., Klingelhofer, G., Knoll, A.H., McLennan, S.M., McSween Jr., H.Y., Morris, R.V., Rice Jr., J.W., Rieder, R., Soderblom, L.A., 2004. In situ evidence for an ancient aqueous environment at Meridiani Planum, Mars. Science 306 (5702), 1709–1714. Squyres, S.W., Knoll, A.H., 2005. Sedimentary rocks at Meridiani Planum: origin, diagenesis, and implications for life on Mars. Earth and Planetary Science Letters 240 (1), 1–10.