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Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29, pp. Tk_41-Tk_48, 2014 Topics

MELOS Life Search Plan: Search for Microbes on the Surface with Special Interest in -oxidizing Bacteria

1) 2) 3) 1) 4) By Akihiko YAMAGISHI , Yoshitaka YOSHIMURA , Hajime HONDA , Atsuo MIYAKAWA , Takehiko SATOH , 4) 4) 5) 6) 7) Genya ISHIGAMI , Junichi HARUYAMA , Kensei KOBAYASHI , Takeshi NAGANUMA , Sho SASAKI 8) and Hideaki MIYAMOTO

1) Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Tokyo, Japan 2) College of Agriculture, Tamagawa University, Tokyo, Japan 3) Nagaoka University of Technology, Niigata, Japan 4) Institute of Space and Astronautical Science, JAXA, Sagamihara, Japan 5) Yokohama National University, Yokohama, Japan 6) School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Japan 7) RISE Project, NAOJ, Oushu, Japan 8) The University of Tokyo, Tokyo, Japan (Received July 13th, 2013)

Our project aims to search for methane-oxidizing microbes on the Mars surface. The project is in preparation under the scheme of the MELOS working group. will be sampled from a depth of about 5 - 10 cm below the surface, where organisms are expected to be protected from the harsh hyper-oxidative environment of the Mars surface. The soils will be stained with a cocktail of fluorescent reagents, and examined by fluorescence microscopy. A combination of fluorescent dyes has been selected to identify life forms in samples. A combination of dyes will be used to detect membranes surrounding the “cell”. A substrate dye that emits fluorescence upon cleavage by a catalytic reaction will be used to detect the catalytic activity of the “cell”. This combination will also be useful for detecting pre-biotic organic material as well as remnants of ancient Martian life. Hydrolysis of the polymers in the “cell” followed by HPLC for amino acid analysis will be effective for examining whether Martian life is identical to or different from terrestrial life. The number and type of the amino acids as well as their chirality will be analyzed to distinguish whether the polymers are contaminants from .

Key Words: Fluorescence Microscopy, Cell Membrane, Microbes, Methane-oxidizer, Amino Acids

1. Introduction However, the presence of has become a focus of discussion again since the report of possible chemical Among the planets and giant satellites in our solar system, fossils in the ALH84001 meteorite that originated from Mars5). the characteristics of Mars, such as the size, mass, gravity, The results of the Viking experiments have been reexamined. surface temperature, and the existence of an atmosphere are The Viking GCMS instruments were not specifically designed quite similar to those of Earth. This may suggest that it is to search for living cells and the sensitivity was one part per possible for life similar to terrestrial life to arise and even billion for small organic compounds. such as survive on Mars. amino acids could not be detected if living cells were present The Viking missions in 1976, NASA’s early landing at levels less than 107 cells per gram6). Non-volatile organic missions to Mars, conducted several kinds of life science compounds, such as benzenecarboxylate, which have been experiments. The Gas Exchange (GEX) Experiments formed on the surface of Mars via oxidation of organic measured production of gases from surface samples after they material that arrived on Mars in meteorites, could not be easily were humidified and wetted with nutrient media. In these detected by the Viking GCMS instruments7). Thus some 1) experiments, the liberation of O2 gas was observed . The organic compounds may be present on Mars, although Labeled Release (LR) Experiment demonstrated immediate compounds near the surface may be destroyed by ionizing evolution of radioactive gas after adding 14C-labeled radiation8, 9). carbohydrates to the Martian regolith2). The Gas The evidence of past water activity and the existence of Chromatography / Mass Spectrometer (GCMS) Experiment methane in the Martian atmosphere also provided motivation showed absence of detectable organic compounds above a few for the re-examination of the organic compounds on Mars parts per billion in the upper 10 cm of surface soil3). The surface. The (MGS) Mars Orbiter combined results of these experiments were interpreted to Camera (MOC) acquired images of the with indicate the presence of oxidants that decomposed the organic fine spatial resolution, and showed large outflow channels as compounds added in GEX and LR, and no organisms were evidence for past, but quite recent, liquid water 10-12). The Mars present within the detection limits of the experiments4). Odyssey Neutron Spectrometer showed that poleward of ±60º

Copyright© 2014 by the Japan Society for Aeronautical and Space Sciences and ISTS. All rights reserved.

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was terrain rich in , probably H2O ice that the low surface gravity of Mars (0.376 g) would limit the existing beneath tens of centimeters of soil13). Hydrated existence and survival of terrestrial life. and phyllosilicates that provide evidence of the past presence The temperature range for terrestrial life to grow is, from of liquid water have been observed by the OMEGA imaging -20oC to the current record of 122oC 34, 35), partially overlaps spectrometer on board the spacecraft14, 15). The the temperature range of the current Mars surface, -87oC to also found sedimentary 20oC. Besides growth, storage and survival of seeds, spores, rocks formed by past aqueous processes on Mars16, 17). The vegetative cells and dried waterbears may not be significantly

Phoenix mission found a shallow H2O ice table in the center limited by the low Martian temperatures. and edge of a nearby polygon at depths of 5 to 18 centimeters Although the terrestrial bacterium Shewanella oneidensis by excavating with the and associated Icy Soil tolerates pressures as high as 1.6 GPa36), the effect of low Acquisition Device18). These findings suggested that there was pressures found on the current Mars surface against microbes water activity on (or beneath) the surface and suitable has not been examined. As microorganisms are often conditions for life in the past, although there has been no lyophilized in a vacuum for long-term preservation (e.g., ref. direct observation of liquid water at present. 37), the low Martian atmospheric pressure may reduce On the other hand, The Planetary Fourier Spectrometer biological activity but is unlikely to limit the survivability of onboard the Mars Express detected methane at a terrestrial-type life. concentration of approximately 10 ppbv in global Terrestrial life forms are found over the entire range abundance19,20). Similar abundance was observed by the from freshwater to salt-saturated waters. Some halophilic Fourier Transform Spectrometer at the Canada-France-Hawaii (salt-loving) microorganisms such as Halomonas species Telescope21). Methane was released in large plumes and peak maintain activity over the entire range of salinity (e.g., ref. 38, amounts were observed during Martian summer with 39). It is likely that massive evaporation of liquid water high-dispersion infrared spectrometers22). Methane is occurred on Mars in the past, leaving massive evaporites that produced by both biotic and abiotic processes on Earth, so the are salt-saturated. If NaCl is the major salt, many terrestrial origin of Martian methane is the subject of considerable halophilic microorganisms would be capable of survival. If 23-26) + + 2+ 2- discussion . Isotopic measurement of methane is planned Martian sea salts consist mainly of Li , K , Ca , and SO4 , on the mission (MSL)24) and the their possible influence on terrestrial-type life is not well source may be revealed by the Mars Trace Gas Mission, defined. Associated with high salinity, the extreme dryness of which will spatially map important trace gases including the Mars surface is likely to limit biological activities, but not methane using an orbiter27). The MSL will analyze not only survivability, as evidenced by long-term storage of lyophilized the atmospheric composition, but also solids near the surface (freeze-dried) microorganisms. with a GCMS (gas chromatograph and quadrupole mass Radiation is likely the most seriously limiting factor for spectrometer) and a tunable spectrometer to measure activity and survivability of terrestrial-type life; however, organic compounds and isotope compositions, respectively28). removal of water would increase tolerance and survivability. The ’s ExoMars mission is planning This hypothesis is based on the dried states of water bears, i.e., life detection experiments on Mars with a Life Marker Chip, tardigrades 40, 33) and chiromid larvae 41, 42) as well as which detects biomolecules based on antibody microarray microorganisms 43). The extremely high radiation-tolerance of technology using subsurface samples taken by drilling or Deinococcus radiodurans has been well studied and ascribed excavating29). to a by-product of adaptation to extreme desiccation44). We propose a new life detection project on Mars to search UV radiation on the Mars surface can be estimated to be 20 for methane-oxidizing microbes by fluorescence microscopy W m-2 based on the assumption that (1) UV radiation combined with amino acid analysis and . contributes about 10% of the Martian “solar constant” of ca. We also propose to search for “cells” from a depth of about 5 - 600 W m-2, calculated from the Earth’s solar constant of 1366 10 cm below the surface, which is feasible with current W m-2 and the -Mars distance of 1.5 AU; and, (2) the thin technology30). Microscopic observation can be done using low Martian atmosphere cuts solar radiation by ca. 7/8 (ca. 1/8 mass equipment with low electric power consumption, and has reaching the Mars surface). The Earth’s atmosphere cuts the potential to detect single “cells”. The subsequent analysis radiation to ca. 1/500, from a comparison of surface and space of amino acids will provide the information needed to define station altitudes45, 46); and the Martian atmospheric pressure the origin of the “cell”. and surface gravity, 0.006 and 0.38 of the Earth’s, respectively, would cut radiation to ca. 1/8 (from 1/500 ÷ 0.006 x 0.38), 2. Survivability of Life in the Mars Environment which is a rough estimate of the Martian atmospheric shield performance. If the same shield performance holds for other Physical and chemical limits for terrestrial life have been ionizing radiations, ca. 1.2 mGy -1 as determined during major foci in astrobiology31, 32), and are summarized in Table 1 the mission on its orbit, a dose of 0.4 in the previous publication30). Microgravity may affect mGy day-1 is estimated for the Mars surface. structural organization and functional harmonization of The Mars performed47, 48) wet chemical biological systems of higher organisms such as animals and analyses of the surface soil, and determined the soil pH to be plants (e.g., ref. 31, 33). In contrast, microorganisms are less 7.7±0.5. For reference, the lowest (acidic) pH limit for affected by near-zero gravity. There is no evidence indicating terrestrial life is currently -0.06, i.e., [H+] = 1.15 mol L-1, as

Tk_42 A. YAMAGISHI et al.: MELOS Life Search Plan: Search for Microbes on the Mars Surface shown by the archaea Picrophilus oshimae and P. permeable and impermeable pigments. The first type of the torridus 49, 50), while the highest (alkaline) pH12.5 is known pigment will be used to detect membrane surrounding the for the bacterium Alkaliphilus transvaalensis 51). “cell”.

The redox potential (Eh) of the Martian soil has not been The second characteristic is metabolism. All life forms reported so far, but occurrences of sulfate and depend on free energy, obtained from metabolism. strongly suggest that the Martian soil is highly oxidative and Metabolism, in turn, consists of a complex series of biological coincides with the presence of ferric oxides on the surface. reactions called metabolic pathways, which are catalyzed by Perchlorate, an efficient oxidizer, is present in the Martian soil, enzymes. We plan to detect esterase, one of the most but may not restrict the survivability of life on Mars, since commonly found enzymes in cells on Earth. certain terrestrial microorganisms exploit perchlorate in The third characteristic is division or proliferation of a anaerobic oxidation metabolism52, 53). “cell”. However, even for the professional microbiologist Results of the Viking biological experiments were with adequate facilities, less than 1% of the microbe can be interpreted to indicate the presence of highly oxidizing H2O2, cultivated, because it is not easy to find appropriate conditions which could be generated by photochemical reactions and/or for proliferation. Accordingly, the direct observation of the dust storms54). Many microorganisms show high tolerance to proliferation process is less feasible. Instead, we will target the

H2O2, and thus any possible Martian life may not be damaged genetic molecules needed for inheritance of the genetic 55) 2- too seriously by H2O2 . Oxidation by superoxide ions (O ) information. In life on Earth, DNA is the molecule that stores in the Mars soil has also been proposed56), and many terrestrial genetic information; DNA is replicated into two molecules organisms possess the enzyme superoxide dismutase to that are transferred to the two daughter cells upon cell division. 2- catalyze transformation of O to O2 and H2O2. Therefore, DNA may not be used in “cells” on Mars. However, the survivability of Martian life may not be heavily damaged by elegant structure that retains and duplicates genetic O2-. information in DNA is also retained in RNA. RNA can be the molecule to store and transfer genetic information to 3. How to Find Microbes on Mars daughter cells. The double helices made of RNA are expected in such cells. Double-stranded helices made of To determine the location where to find life, several factors RNA is often stained with nucleotide specific pigments57). have to be considered including the presence of liquid water The essential element for retaining information is pairs of and free energy. Free energy is required for life forms to organic bases in the DNA double helix, and the target site of sustain life for geological time span. The terrestrial life can DNA-staining pigments is the pair-structure of the organic obtain free energy from certain types of disequilibria. bases at the center of the double helix. Similar but not Chemical disequilibria of the system consisting of reducing identical structural characteristics may be retained in genetic and oxidizing compounds are the typical source of free energy. molecules of Martian “cells”. The range of detectable Because of the generally oxidative conditions on surface of molecules will be tested in the future. The DNA staining Mars, location with any reducing compounds can be the place reagents will be the third type of the fluorescent dye used to for search for life. The report of the methane on the Mars identify Martian “cells”. surface discussed above can be the clue to find life in Mars. Upon identification of candidate “cells” by fluorescence The initial effort to find microbes on Mars should be microscopy, they will be analyzed by second stage analytical concentrate on the location where reducing compound is process. In the second stage, the “cells” will be hydrolysed. available especially where methane is emitted from Living cells on Earth consist of 70% water and 15% protein. underground. The exact location within the reach of a rover Cells contain many types of proteins, from as many as several should be defined prior to the landing approach of the rover thousand proteins in prokaryotes, to several tens of thousands onto the Martian surface. Options for defining locations of in eukaryotes, each having a molecular weight from a few methane emission are discussed in a later section. thousand to several hundred thousand. The molecular weight Methane may be emitted either through holes or through spectra are too complex to be resolved by any type of mass sand or soil. In either case, soil around the methane emitting spectral detectors. However, once proteins are hydrolyzed, area will be collected by robotic arms on the rover. The soil they produce a mixture of 19 chiral-specific amino acids and will be inspected with a fluorescence microscope. Using a glycine, which has no optical isomer. A specific set of 20 combination of fluorescent dyes, specific components of amino acids is commonly found in all cells on Earth. Based on biological cells can be distinguished from non-biological research on chemical evolution, which must have occurred inorganic and organic matters. prior to the emergence of life, amino acids are known to be Adequate combination of pigments used will be determined abiotically produced in a wide range of possible pre-biological to detect biological characteristics that define life. The first environments. Accordingly, there is a fair chance that Martian characteristic is the membrane surrounding the “cell”. The “cells” contain polymers of amino acids. However, the “cell” should be surrounded by an impermeable membrane to number, types and chirality of the amino acids may not be the define “self” and “non-self” and to separate inside from same as those in living cells on Earth. The number and outside. The presence of this defining characteristic will be characteristics of amino acids will be analyzed in the second tested either by detecting the surrounding membrane or by stage of “cell” analysis. detecting the boundaries using a combination of membrane

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4. Fluorescent Microscopy probe, because microorganisms must possess catalytic activities to maintain their metabolism. Fluorescent microscopy is a method to detect localized Cell membrane detecting probes bind to electrically neutral in situ. Biosignatures are labeled with hydrophobic molecules, including proteins, lipids, and cell fluorescent dyes. Stained objects are observed with an membranes72). CFW (fluorescent brightener calcofluor white epifluorescence microscope with a resolution of 0.6 Pm M2R), Mg-ANS ( salt of 1-anilino-8-naphthalene using NA 0.6 objective lens. Because the fluorescent cell can sulfonic acid), and FM1-43 have been used65, 73-76). The cell be detected in the dark back ground in fluorescent microscopy, membranes or cell walls surrounding the “cells” will be 1 Pm is sufficient to detect any fluorescent object as small as detected, which indicates the boundary between the inside and 0.2 Pm, the size of virus. This method is as highly sensitive as outside of the “cell”. Alternatively, cell membranes about 104 cells per g-soil can be detected and will provide surrounding “cells” may be identified by using a combination clear evidence for as images. Many of cell-membrane permeable and impermeable nucleotide fluorescent dyes are commercially available and used specific dyes such as LIVE/DEAD BacLight77). However, routinely to study terrestrial microorganisms57, 58). Thus, their nucleotides may or may not be the genetic components of validities have been well established. Analytical procedures possible Martian “cells”, thus a combination of pigments with are simple and can be done in a short time. Weinstein et al. broad staining specificities for not only nucleotide but also (2008)59) tested a detection instrument, Fluorescence Imager, protein or protenous molecules will be used to search for in the in Chile. They sprayed several mL of candidate “cells” surrounded by membranes. dye solutions on soil and rocks, and detected a variety of In addition, fluorescent dyes that detect protein or amino visible life forms such as lichens with CCD camera although acids could be useful to search for pre-biotic organic materials. their system was limited to detect life larger than 1 mm. SYPRO-RED78), fluorescamine79) and other amine reactive To apply this method to detect Martian microorganisms, we dyes that react specifically with amino groups57) are have designed a strategy to detect “cells”, although the actual candidates for this type of probe. species of fluorescent dyes remain to be selected. Our method Many fluorescent probes were extensively studied by is based on multilabeling with different types of fluorescent Nadeau et al. (2008)65). They showed that the usefulness of a dyes. The principle is to detect biosignatures that are particular fluorophore strongly depended on the particular responsible for the basic characteristics of life: genetic sample and means of detection, and that all fluorescent information, metabolism, and discrimination of self from pigments showed some nonspecific binding. We will select non-self60). Each characteristic will be detected using a fluorescent dyes and staining methods that minimize different type of fluorescent dye that is specific for nucleic nonspecific binding using simulated Martian samples. acids, enzymes, or cytoplasmic membranes. This combination Multilabeling with different types of dyes will be tested to of dyes has a potential to distinguish living “cells” from dead increase the detection accuracies and to decrease the “cells” and other non-biological organic compounds. identification of false positive (abiotic) structures. Nucleic acid probes are most commonly used to study High-resolution images with a micron scale will also microbial ecology. AO (acridine orange), DAPI provide information about the morphology of the candidate (4’,6-diamidino-2-phenylindole), SYBR Green I and II, and “cells”. Terrestrial microorganisms often have specific SYBR Gold are examples of this type of probe61-64). Although morphological characteristics, such as spherical and rod many probes cannot be used on soil samples because of strong shapes, and a characteristic size distribution. Size is usually binding to mineral particles, some probes such as AO were defined within a range of a single species or group of similar reported to be useful for endolith samples65). species. Together with the fluorescence intensity from the Metabolic activities are detected by using enzyme substrate combination of different fluorescence probes and the fluorescent dyes. FDA (fluorescein diacetate), CFDA morphological information, we will be able to define the (5-carboxyfluorescein diacetate), SFDA (5-(and 6-) characteristics of the candidate “cells”. sulfofluorescein diacetate), and CFDA-AM The microscopic instrument which is to be used on Mars (5-carboxyfluorescein diacetate, acetoxymethyl ester) have should be lightweight and energy efficient. We have been been used for this purpose66-71). These dyes are often used as designing a several-watt consumption microscope that is 1-2 viability indicators. They remain non-fluorescent unless kg in weight. To save electricity, a laser diode (LD) will be hydrolyzed by the intracellular enzyme, esterase, which is a used as the illuminant to excite dye-labeled samples. The laser ubiquitous enzyme in organisms on earth. In the case of soil, diodes will be used to excite multiple wavelengths such as 405 this type of dye often shows intense background fluorescence nm or 488 nm for different types of fluorescent dyes. To due to abiotic catalytic activities and leakage of fluorescent detect the low fluorescence emitted by the 10 mW optical product from the cells, which can mask stained output power LD, back-illuminated charge-coupled device microorganisms. SFDA used with ethanol can solve this (BI-CCD) detectors are also considered. The combination of problem70). Some dyes were also reported to detect artificial LD and BI-CCD will provide high sensitivity that can detect a protenoid microspheres, which could have enzymatic single microbe cell. This microscope automatically scans in x activity60). Accordingly, this type of dye has the potential to and y direction to explore a wide range, and automatically detect enzyme-like catalysts. If living microorganisms are observe the complex sample with the irregular shapes such as present on Mars, they could be detected with this type of sand and soil particles.

Tk_44 A. YAMAGISHI et al.: MELOS Life Search Plan: Search for Microbes on the Mars Surface

Microspectrofluorometry has been reported to be effective methane is mostly biological in origin, while that on Mars is a for discriminating biotic particles from abiotic matter of debate. could be formed autofluorescence, and multilabeled samples can be detected geologically, for example, as a by-product of the process with a long pass emission filter under single excitation light65). called serpentinization. This process results from reactions Otherwise, color separation to target the fluorescent between water and olivine, whose existence on Mars is widely wavelength by three or more CCD devices could be useful. recognized79). We would like to note that, in either case, The plural CCDs is useful for distinguishing fluorescence methane production occurs in the subsurface, which requires signals from background fluorescence, and identification of an eruption/release process through ephemeral vents. respective fluorescence signal under the conditions where the The determination of the exact location of methane sample is stained with multiple pigments. Appropriate emissions may be difficult, because the erupted methane in the software is also important to automatically select particles atmosphere may diffuse rapidly. On the other hand, possessing multiple fluorescent peaks. Staining procedures subsurface sources of methane may have a number of vent would be done in an air lock chamber to avoid freezing and systems to release methane, and their fluxes can vary over evaporation of the dye solutions. time. Thus, we propose the following procedure to determine the exact location of the vent; (1) an orbiter (or carrier of a 5. Where to Search: Location of Methane Source rover) identifies a region with a local concentration of methane, (2) a long-range, long-lifetime, and autonomous Measurements by PFS (Planetary Fourier Spectrometer) rover lands within the area (hopefully within a few hundred onboard Mars Express indicated that the Martian atmosphere kilometers) identified by the orbiter immediately before the does contain methane, although at an extremely low level (10 decent of the rover, and (3) the rover autonomously searches parts per billion)19). However, the existence of methane is for higher concentrations of methane with continuous in situ surprising because it requires a recent and potentially measurements of methane concentration and wind, which sustained source of methane, otherwise light would should be occasionally supported by theoretical estimates of break down the methane in less than several hundred years the local climate based on the observations of an orbiter. under current Martian conditions. Observations of In searching for methane vents, detailed morphological and ground-based telescopes showed that Martian methane has spectral studies of the target area from orbiters will be also regional variations in concentration22), with some areas having useful. Possible ephemeral vents could be small volcanic high concentrations such as Nili Fossae, Terra Sabae and vents, surface fractures, collapsed structures, and mud Syrtis Major. If this observation is correct, the spatial volcanoes. Spectral detection of serpentine and possible variations must result from either heterogeneous production clathrate hydrates would be also helpful. These and release of methane from source areas, or rapid regional morphological and spectral observations will help to prioritize removal of methane from the Martian atmosphere. The latter the route of the surface rover for detecting methane. is unlikely because the current understanding of An orbiter (or a carrier of rover) may narrow the potential photochemistry and rock-atmospheric interactions on Mars are source region to, say, several hundred kilometers through difficult to reconcile with the short timescale of atmospheric orbital observations. However, to reach the vent, the rover circulation (~10 days, horizontally). On the other hand, the may have to travel a long distance that exceeds the maximum former process has many examples, such as volcanic activity, range of the rover. Considering the possibility of variations in magmatic intrusions, hydrothermal vents, outgassing of methane production, perhaps the best way to identify the exact clathrate hydrates, and microbial processes. All of these location of the methane vent is the following procedure: (1) processes require a subsurface source of methane at an first roughly determine the location using an orbiter before the appropriate depth, which allows slow leakage through vent rover is released, (2) in-situ measurements with higher systems. resolution to detect methane distribution within the We have to note that technical difficulties in the detection atmosphere, and (3) surface measurements by a rover. For (2), of methane require careful interpretation of the current a balloon might be a good idea to provide atmospheric observations. Different views exist for the source of measurements at various heights (the daily temperature cycle atmospheric methane on Mars, such as (1) the source of makes it rise and sink), or an airplane flying over the surface Martian methane is concentrated in both space and time22), and of Mars studying methane concentrations and detailed (2) trace amounts of methane are released from seasonal topography. Alternatively, a widely-distributed sensor melting of the surface of the north polar cap as suggested by network as proposed by Fink et al., 200680) might be suitable PFS onboard the Mars Express19, 20). At this point, we do not for this purpose. know if the sources of observed methane are persistent or transient. Thus, an orbiter mission to measure methane in the 6. Conclusions atmosphere to determine how it varies over time and space, such as the NASA and ESA' , will be In conclusion, we propose to search for microbes on Mars, important to understand the nature of the source of methane. 5 to 10 cm below the surface. The first effort should be to We favor localized release of methane since several identify locations where methane is emitted from underground. independent observations suggest local concentrations of The rover will approach the methane-emitting site, where soil methane (up to 30 ppb or much higher)22). On the Earth, will be collected and analyzed. A combination of fluorescent

Tk_45 Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29 (2014) dyes will be used to detect candidate “cells” using a Planum: Origin, diagenesis, and implications for life on Mars, fluorescence microscope. Putative “cells” will be hydrolyzed Earth Planet. Sci. Lett., 240 (2005), pp. 1-10. 18) Smith, P. H., Tamppari, L. K., Arvidson, R. E., Bass, D., Blaney, and analyzed by mass spectral analysis to define the et al.: H2O at the Phoenix landing site, Science, 325 (2009), pp. characteristics of the candidate “cells”, which will indicate the 58-61. origin of the candidate. 19) Formisano, V., Atreya, S., Encrenaz, T., Ignatiev, N. and Giuranna, M.: Detection of methane in the , Acknowledgments Science, 306 (2004), pp. 1758-1761. 20) Geminale, A., Formisano, V. and Giuranna, M.: Methane in Martian atmosphere: Average spatial, diurnal, and seasonal The research was supported in part by ISAS/JAXA fund for behaviour, Planet. Space Sci., 56 (2008), pp. 1194-1203. MELOS working group. 21) Krasnopolsky, V. A., Maillard, J. P. and Owen, T. C.: Detection of methane in the martian atmosphere: evidence for life? Icarus, 172 (2004), pp. 537-547. References 22) Mumma, M. J., Villanueva, G. L., Novak, R. E., Hewagama, T., Bonev, B. P., Disanti, M. A., Mandell, A. M. and Smith, M. D.: 1) Oyama, V. I. and Berdahl, B. J.: The Viking Gas Exchange Strong release of methane on Mars in northern summer 2003, Experiment Results From Chryse and Utopia Surface Samples, J. Science, 323 (2009), pp. 1041-1045. Geophys. Res., 82 (1977), pp. 4669-4676. 23) Krasnopolsky, V. A.: Some problems related to the origin of 2) Levin, G. V. and Straat, P. A.: Recent results from the Viking methane on Mars, Icarus, 180 (2006), pp. 359-367. labeled release experiment on Mars, J. Geophys. Res., 82, (1977), 24) Atreya, S. K., Mahaffy, P. R. and Wong, A.-S.: Methane and pp. 4663-4667. related trace species on Mars: Origin, loss, implications for life, 3) Biemann, K., Oro, J., Toulmin, P. III, Orgel, L. E., Nier, A. O., and habitability, Planet. Space Sci., 55 (2007), pp. 358-369. Anderson, D. M., Simmonds, P. G., Flory, D., Diaz, A. V., 25) Lefevre, F. and Forget, F.: Observed variations of methane on Rushneck, D. R., Biller, J. E. and Lafleur, A. L.: The search for Mars unexplained by known atmospheric chemistry and physics, organic substances and inorganic volatile compounds in the Nature, 460 (2009), pp. 720-723. surface of Mars, J. Geophys. Res., 82, (1977), pp. 4641-4658. 26) Shkrob, I. A., Chemerisov, S. D. and Marin, T. W.: Photocatalytic 4) Klein, H. P.: The Viking biological investigation: general aspects. Decomposition of Carboxylated Molecules on Light-Exposed J. Geophys. Res., 82, (1977), pp. 4677-4680. Martian and Its Relation to Methane Production on 5) McKay, D. S., Gibson, E. K. Jr., Thomas-Keprta, K. L., Vali, H., Mars, , 10 (2010), pp. 425-436. Romanek, C. S., Clemett, S. J., Chillier, X. D. F., Maechling, C. 27) Smith, M. D., Allen, M., Banfield, D., Barnes, J. R., Clancy, R. R. and Zare R. N.: Search for past life on Mars: possible relic T., James, P., Kasting, J., Wennberg, P., Winterhalter, D., Wolff, biogenic activity in Martian meteorite ALH84001, Science, 273 M. and Zurek. R.: MARS TRACE GAS MISSION: Scientific (1996), pp. 924-930. Goals and Measurement Objectives, A White Paper submitted to 6) Glavin, D. P., Schubert, M., Botta, O., Kminek, G. and Bada, J. the NRC Planet. Sci. Decadal Survey Panel September 15, 2009. L.: Detecting pyrolysis products from bacteria on Mars, Earth 28) Mahaffy, P.: Exploration of the Habitability of Mars: Planet. Scie. Lett., 185 (2001), pp. 1-5. Development of Analytical Protocols for Measurement of Organic 7) Benner, S. A., Devine, K. G., Matveeva, L. N. and Powell, D. H.: Carbon on the 2009 Mars Science Laboratory, Space Sci. Rev., (2000) The missing organic molecules on Mars, Proc. Natl. Acad. 135 (2008), pp. 255-268. Sci. USA, 97 (2001), pp. 2425-2430. 29) Parnell, J., Cullen, D., Sims, M. R., Bowden, S., Cockell, S., 8) Dartnell, L. R., Desorgher, L., Ward, J. M. and Coates, A. J.: Court, R., Ehrenfreund, P., Gaubert, F., Grant, W., Parro, V., Martian sub-surface ionising radiation: biosignatures and , Rohmer, M., Stephton, M., Stan-Lotter, H., Steele, A., Toporski, Biogeosciences, 4 (2007), pp. 545-558. J. and Vago, J.: Searching for life on Mars: selection of molecular 9) Kminek, G. and Bada, J. L.: The effect of ionizing radiation on targets for ESA’s Aurora ExoMars Mission, Astrobiol., 7 (2007), the preservation of amino acids on Mars, Earth and Planetary pp. 478-604. Science Letters, 245 (2006), pp. 1-5. 30) Yamagishi, A., Yokobori, S., Yoshimura, Y., Yamashita, M., 10) Malin, M. C. and Carr, M. H.: Groundwater formation of martian Hashimoto, H., Kubota, T., Yano, H., Haruyama, J., Tabata, M., valleys, Nature, 397(6720) (1999), pp. 589-591. Kobayashi, K., Honda, H., Utsumi, Y., Saiki, T., Itoh, T., 11) Malin, M. C. and Edgett, K. S.: Evidence for persistent flow and Miyakawa, A., Hamase, K., Naganuma, T., Mita, H., Tonokura, aqueous sedimentation on early Mars, Science, 302 (2003), pp. K., Sasaki, S. and Miyamoto, H.: Japan Astrobiology Mars 1931-1934. Project (JAMP): Search for microbes on the Mars surface with 12) Masson, P., Carr, M. H., Costard, F., Greeley, R., Hauber, E. and special interest in methane-oxidizing bacteria, Biol. Sci. Space 24 Jaumann, R.: Geomorphologic evidence for liquid water, Space (2010), pp. 67-82 Sci. Rev., 96 (2001), pp. 333-364. 31) Rothschild, L. J. and Mancinelli R. L.: Life in extreme 13) Feldman, W. C., Boynton, W. V., Tokar, R. L., Prettyman, T. H., environments, Nature, 409 (2001), pp. 1092-1101. et al.: Global distribution of neutrons from Mars: results from 32) Marion, G. M., Fritsen, C. H., Eicken, H. and Payne, M. C.: The Mars odyssey, Science, 297 (2002), pp. 75-78. search for life on : Limiting environmental factors, 14) Gendrin, A., Mangold, N., Bibring, J. P., Langevin, Y., Gondet, potential habitats, an Earth analogues, Astrobiology, 3 (2003), pp. B., Poulet, F., Bonello, G., Quantin, C., Mustard, J., Arvidson, R. 785-811. and LeMouelic, S.: in Martian layered terrains: the 33) Jönsson, K. I., Rabbow, E., Schill, R. O., Harms-Ringdahl, M. OMEGA/Mars Express view, Science, 307 (2005), pp. and Rettberg P.: Tardigrades survive exposure to space in low 1587-1591. Earth orbit, Curr. Biol., 18 (2008), pp. R729-R731. 15) Poulet, F., Bibring, J. P., Mustard, J. F., Gendrin, A., Mangold, et 34) Junge, K., Eicken, H. and Deming, J. W.: Bacterial activity at -2 al.: Phyllosilicates on Mars and implications for early martian to -20°C in Arctic wintertime sea ice, Appl. Environ. Microbiol., climate, Nature, 438 (2005), pp. 623-627. 70 (2004), pp. 550-557. 16) Squyres, S. W., Arvidson, R. E., Bell, J. F.: 3rd, Bruckner, J., et 35) Takai, K., Nakamura, K., Toki, T., Tsunogai, U., Miyazaki, M., al. The Opportunity Rover's Athena science investigation at Miyazaki, J., Hirayama, H., Nakagawa, S., Nunoura, T. and Meridiani Planum, Mars, Science, 306 (2004), pp. 1698-1703. Horikoshi, K.: Cell proliferation at 122°C and isotopically heavy 17) Squyres, S. W. and Knoll, A. H.: Sedimentary rocks at Meridiani CH4 production by a hyperthermophilic methanogen under high-pressure cultivation, Proc. Natl. Acad. Sci. USA, 105 (2008),

Tk_46 A. YAMAGISHI et al.: MELOS Life Search Plan: Search for Microbes on the Mars Surface

pp. 10949-10954. Syst. Evol. Microbiol., 51 (2001), pp. 1245-1256. 36) Sharma, A., Scott, J. H., Cody, G. D., Fogel, M. L., Hazen, R. M., 52) Coates, J. D., Michaelidou, U., Bruce, R. A., O’Connor, S. M., Hemley, R. J. and Huntress, W. T.: Microbial activity at Crespi, J. A. and Achenbach, L. A.: Ubiquity and diversity of gigapascal pressures, Science, 295 (2002), pp. 1514-1516. dissimilatory (per)chlorate-reducing bacteria, Appl. Environ. 37) Dewald, R. R.: Preservation of Serratia marcescens by Mircobiol., 65 (1999), pp. 5234-41. high-vacuum lyophilization, Appl. Microbiol., 14 (1966), pp. 53) Coates, J.D. and Achenbach, L. A.: Microbial perchlorate 561-567. reduction: rocket-fuelled metabolism, Nat. Rev. Microbiol., 38) Naganuma, T. and Horikoshi, K.: Extremely halotolerant 2(2004), pp. 569-580. Halomonas sp. isolated from a tar ball and its cellular fatty acid 54) Atreya, S. K., Wong, A., Renno, N. O., Farrell, W. M., Delory, G. composition, J. Mar. Biotechnol., 1 (1993), pp. 151-157. T., Sentman, D. D., Cummer, S. A., Marshall, J. R., Rafkin, S. C. 39) Okamoto, T., Maruyama, A., Imura, S., Takeyama, H. and R. and Catling, D. C.: Oxidant enhancement in Martian dust Naganuma, T.: Comparative phylogenetic analyses of Halomonas devils and storms: implications for life and habitability, variabilis and related organisms based on 16S rRNA, gyrB and Astrobiology, 6 (2006), pp. 439-450. ectBC gene sequences, Syst. Appl. Microbiol., 27 (2004), pp. 55) Mancinelli, R. L.: Peroxides and the survivability of 323-333. microorganisms on the surface of Mars, Adv. Space Res., 9 40) Horikawa, D. D., Sakashita, T., Katagiri, C., Watanabe, M., (1989), pp. 191-195. Kikawada, T., Nakahara, Y., Hamada, N., Wada, S., Funayama, 56) Yen, A. S., Kim, S. S., Hecht, M. H., Frant, M. S. and Murray, B.: T., Higashi, S., Kobayashi, Y., Okuda, T. and Kuwabara, M.: Evidence that the reactivity of the Martian soil is due to Radiation tolerance in the tardigrade Milnesium tardigradum, Int. superoxide ions, Science, 289 (2000), pp. 1909-1912. J. Rad. Biol., 82 (2006), pp. 843-848. 57) Haugland, R. P.: Handbook of fluorescent probes and research 41) Watanabe, M., Sakashita, T., Fujita, A., Kikawada, T., Horikawa, chemicals, 6th ed., Molecular Probes Inc. Eugend OR USA D. D., Nakahara, Y., Wada, S., Funayama, T., Hamada, N., (1996). Kobayashi, Y. and Okuda, T.: Biological effects of anhydrobiosis 58) Herman, B.: Fluorescence microscopy, 2nd ed., Bios Scientific in an African chironomid, Polypedilum vanderplanki on radiation Publishers, Oxford UK (1998). tolerance, Int. J. Radiat. Biol., 82 (2006), pp. 587-592. 59) Weinstein, S., Pane, D., Ernst, L.A., Warren-Rhodes, K., Dohm, 42) Watanabe, M., Sakashita, T., Fujita, A., Kikawada, T., Nakahara, J.M., et al.: Application of pulsed-excitation fluorescence imager Y., Hamada, N., Horikawa, D. D., Wada, S., Funayama, T., for daylight detection of sparse life in tests in the Atacama Desert, Kobayashi, Y. and Okuda, T.: Estimation of radiation tolerance to J. Geophys. Res. 113(2008), G01S90, doi:10.1029/2006JG000319 high LET heavy ions in an anhydrobiotic insect, Polypedilum 60) Kawasaki, Y.: Direct detection of Martian microorganisms based vanderplanki, Int. J. Radiat. Biol., 82 (2007), pp. 835-842. on fluorescence microscopy, Adv. Space Res., 23 (1999), pp. 43) Mashino, Y., Nakai, R., Nakamura, T. K., Yukimura, K., Shibuya, 309-317. E., Migiyama, E., Kuroda, A., Takahashi, H., Naganuma, T., 61) Kepner, R. L. Jr. and Pratt, J. R.: Use of fluorochromes for direct Imura, S., Iwatsuki, T., Kaneko, T., Kobayashi, K., Kobayashi, enumeration of total bacteria in environmental samples: past and K., Sato, H., Saigusa, M., Shimada, K., Shirakabe, Y., Takano, present, Microbiol. Rev., 58 (1994), pp. 603-15. Y., Takayama, K., Tawara, H., Hashimoto, H., Morita, Y., 62) Klauth, P., Wilhelm, R., Klumpp, E., Poschen, L. and Groeneweg, Yasuda, H. and Yamashita, M.: Microbial survivability against J.: Enumeration of soil bacteria with the green fluorescent nucleic high dose X-ray and high energy Fe ion, Space Utiliz. Res., 26 acid dye Sytox green in the presence of soil particles, J. (2010), pp. 143-146. Microbiol. Methods, 59 (2004), pp. 189-198. 44) Battista, J. R.: Against all odds: the survival strategies of 63) Weinbauer, M. G., Beckmann, C. and Hofle, M. G.: Utility of Deinococcus radiodurans, Annu. Rev. Microbiol., 51 (1997), pp. green fluorescent nucleic acid dyes and aluminum oxide 203-24. membrane filters for rapid epifluorescence enumeration of soil 45) Yasuda, H., Badhwar, G. D., Komiyama, T. and Fujitaka, K.: and sediment bacteria, Appl. Environ. Microbiol., 64 (1998), pp. Effective dose equivalent on the ninth Shuttle-Mir mission 5000-5003. (STS-91), Radiat. Res., 154 (2000), pp. 705-713. 64) Shibata, A., Goto, Y., Saito, H., Kikuchi, T., Toda, T. and 46) Takahashi, A. and Ohnishi, T.: The significance of the study Taguchi, S.: Comparison of SYBR Green I and SYBR Gold stains about the biological effects of solar ultraviolet radiation using the for enumerating bacteria and viruses by epifluorescence Exposed Facility on the International Space Station, Biol. Sci. microscopy, Aquatic Microbial Ecology, 43 (2006), pp. 223-231. Space, 18 (2004), pp. 255-260. 65) Nadeau, J. L., Perreault, N. N., Niederberger, T. D., Whyte, L. G., 47) Boynton, W. V., Ming, D. W., Kounaves, S. P., Young, S. M. M., Sun, H. J. and Leon, R.: Fluorescence microscopy as a tool for in Arvidson, R. E., Hecht, M. H., Hoffman, J., Niles, P. B., Hamara, situ life detection. Astrobiology, 8 (2008), pp. 859-874. D. K., Quinn, R. C., Smith, P. H., Sutter, B., Catling, D. C. and 66) Lundgren, B.: Fluorescein diacetate as a stain of metabolically Morris R. V.: Evidence for carbonate at the Mars active bacteria in soil, Oikos, 36 (1981), pp. 17-22. Phoenix Landing site, Science, 325 (2009), pp. 61-64. 67) Porter, J., Diaper, J., Edwards, C. and Pickup, R.: Direct 48) Hecht, M. H., Kounaves, S. P., Quinn, R. C., West, S. J., Young, measurements of natural planktonic bacterial community viability S. M. M., Ming, D. W., Catling, D. C., Clark, B. C., Boynton, by flow cytometry, Appl. Environ. Microbiol., 61 (1995), pp. W. V., Hoffman, J., DeFlores, L. P., Gospodinova, K., Kapit, J., 2783-2786. Smith, P. H.: Detection of perchlorate and the soluble chemistry 68) Soderstrom, B. E.: Vital staining of fungi in pure cultures and in of Martian soil at the Phoenix Lander site, Science, 325 (2009), soil with fluorescein diacetate, Soil Biol. Biochem., 9 (1977), pp. pp. 64-67. 59-63. 49) Schleper, C., Piihler, G., Kuhlmorgen, B. and Zillig, W.: Life at 69) Soderstrom, B. E.: Some problems in assessing the fluorescein extremely low pH, Nature, 375 (1995), pp. 741-742. diacetate-active fungal biomass in the soil, Soil Biol. Biochem., 11 50) Schleper, C., Puehler G., Holz, I., Gambacorta, A., Janekovic, D., (1979), pp. 147-148. Santarius, U., Klenk, H. P. and Zillig, W.: Picrophilus gen. nov., 70) Tsuji, T., Kawasaki, Y., Takeshima, S., Sekiya, T. and Tanaka, S.: fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic A new fluorescence staining assay for visualizing living genus and family comprising archaea capable of growth around microorganisms in soil, Appl. Environ. Microbiol., 61 (1995), pp. pH 0, J. Bacteriol., 177 (1995), pp. 7050–7059. 3415-3421. 51) Takai, K., Moser, D. P., Onstott, T. C., Spoelstra, N., Pfiffner, S. 71) Ashida, N., Ishii, S., Hayano, S., Tago, K., Tsuji, T.: Yoshimura, M., Dohnalkova, A. and Fredrickson, J. K.: Alkaliphilus Y., Otsuka S. and Senoo, K. Isolation of functional single cells transvaalensis gen. nov., sp. nov., an extremely alkaliphilic from environments using a micromanipulator: application to study bacterium isolated from a deep South African gold mine, Int. J. denitrifying bacteria, Appl. Microbiol. Biotech., 85 (2009), pp.

Tk_47 Trans. JSASS Aerospace Tech. Japan Vol. 12, No. ists29 (2014)

1211-1217. Microbiol., 67 (2001), pp. 420-425. 72) Azzi, A.: The application of fluorescent probes in membrane 78) Zubkov, M. V., Fuchs, B. M., Eilers, H., Burkill, P. H., Amann, studies, Quart. Rev. Biophys., 8 (1975), pp. 237-316. R.: Determination of total protein content of bacterial cells using 73) Mayfield, C. I.: A simple fluorescence staining technique for in SYPRO staining and flow cytometry, Appl. Environ. Microbiol.. situ soil microorganisms, Canad. J. Microbiol., 21 (1975), pp. 65 (1999), pp. 3251–3257. 727-729. 79) Oze, C. and Sharma, M.: Have olivine, will gas: Serpentinization 74) Mayfield, C. I.: A fluorescence-staining method for and the abiogenic production of methane on Mars, Geophys. Res. microscopically counting viable microorganisms in soil, Canad. Lett., 32 (2005), pp. L10203. J. Microbiol., 23 (1977), pp. 75-83. 80) Fink, W., Dohm, J. M., Tarbell, M. A., Hare, T. M. and Baker, V. 75) Postma, J. and Altemüller, H. J.: Bacteria in thin soil sections R.: Next-generation robotic planetary reconnaissance missions: A stained with the fluorescent brightener calcofluor white M2R, Soil paradigm shift, Planet. Space Sci., 53 (14-15) (2005), pp. Biol. Biochem., 22 (1990), pp. 89-96. 1419-1426. 76) Weaver, R. W. and Zibilske, L.: Affinity of Cellular Constituents of Two Bacteria for Fluorescent Brighteners, Appl. Environ. Microbiol., 29 (1975), pp. 287-292. 77) Auty, M. A., Gardiner, G. E., McBrearty, S. J., O'Sullivan, E. O., Mulvihill, D. M., Collins, J. K., Fitzgerald, G. F., Stanton, C. and Ross, R. P.: Direct In situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy, Appl. Environ.

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