t g.. i •

Figure 4. FISH analysis of biofilm from a deep subsurface thermal spring. Probes labeled with the fluorescent dye Cy3 were used, which were specific for eubacteria (left panel) or archaea (right panel); fluorescence was observed with a Leica DM4000 B microscope.

Figure 4. FISH analysis of biofilm from a deep subsurface thermal spring. Probes labeled with the fluorescent dye Cy3 were used, which were specific for eubacteria (left panel) or archaea (right panel); fluorescence was observed with a Leica DM4000 B microscope.

5. How Old Are Microorganisms in the Subsurface?

The results presented above provided evidence for an extensive biodiversity of extremely halophilic archaea in salt sediments, which are thought to have been deposited about 280 to 192 million years ago, and in brines, which are associated with the deposits. The salt sediments can be viewed as remnants from ancient hypersaline oceans. The fluid inclusions in Permian rock salt were found to contain cations and anions in a similar composition as today's sea water (Horita et al., 1991). Dating of the salt deposits by sulfur-isotope analysis (ratios of 32S/34S as measured by mass spectrometry), in connection with information from stratigraphy, indicated a Permo-Triassic age for Alpine and Zechstein deposits (Holser and Kaplan, 1966). This estimate was independently confirmed by the identification of pollen grains, which possessed distinct morphological features and could be assigned to extinct plants, in the sediments (Klaus, 1974). While there is no direct proof that viable haloarchaea have been entrapped in rock salt since its sedimentation, it would also be difficult to prove the opposite, namely that masses of diverse microorganisms entered the evaporites in recent times (see also McGenity et al., 2000 for further discussion). Especially for the Alpine deposits, an influx of meteoric waters containing microorganisms is rather improbable, because these sediments have been folded up and are located at altitudes of 1,000 m or higher for at least the last 100 million years (Einsele, 1992). Besides, the Alpine salt deposits are covered by layers of dolomite, limestone, marl, clay and other rocks; most of them are water-impermeable, and thus have contributed to the preservation of the salt deposits during the ice ages with their heavy precipitation.

If a Permo-Triassic age is postulated for the haloarchaeal isolates, then it becomes necessary to explain the biological mechanisms for such extreme longevity. Grant (2004) and Grant et al. (1998) suggested several possibilities, such as the formation of resting stages other than spores - since archaea are not known to form spores -, or the maintenance of cellular functions with traces of carbon and energy sources within the salt sediments, which would imply an almost infinitely slow metabolism. At this time, there are no methods available to prove directly a great prokaryotic age, whether it be a bacterium or a haloarchaeon. The mass of an average prokaryotic cell is only about 10-12 g (picograms); it is composed of about 3,000 different biomolecules, some of which are present at femtogram levels at best; therefore, no current dating procedures can be applied.

The deep surface thermal springs in the Central Alps are located in igneous rocks (basalt, granite etc.). These are the predominant solid constituents of the Earth, formed through cooling of molten material, as described by Pedersen ( Igneous rocks are too hot when formed to contain life of any kind. Therefore, if prokaryotic life is found in igneous granitic rocks, it must have entered after cooling and fracturing of the rock mass. From geological studies it has become clear that groundwater at depths of about 500 m in such rocks can be very old and ages of 10,000 years are not unusual (Pedersen;

The water of the thermal springs in the Central Alps is known to circulate with an interval of at least 3,000 years (see Weidler et al., 2007).

6. Considerations for Astrobiology

The interest in subterranean microbial life has increased considerably since the discovery of bacteria-like microfossils in the Martian meteorite ALH84001, together with polycyclic aromatic hydrocarbons and low temperature carbonate globules (McKay et al., 1996). It was suggested that if these features constitute a proof for past or extant life on Mars, such life must have existed within the surface of Mars. The apparent longevity of haloarchaeal strains in dry salty environments is of interest for astrobiological studies and in particular, for the search for life on Mars. On Earth, microorganisms were the first life forms to emerge and were present perhaps as early as 3.8 billion years ago (Schidlowski, 1988, 2001). If Mars and Earth had a similar geological past, as has been suggested (Schidlowski, 2001; Nisbet and Sleep, 2001), then microbial life, or the remnants of it, could still be present on Mars.

If halophilic prokaryotes on Earth can remain viable for very long periods of time, then it is reasonable to consider the possibility that viable microorganisms may exist - or may have existed in the past - in similar subsurface salt deposits on other planets or moons. This notion becomes all the more plausible in view of the detection of halite in extraterrestrial materials: the SCN meteorites (Gooding, 1992), which stem from Mars (Treiman et al., 2000), were found to contain halite. Quite recently, the data from the US rovers Spirit ( id/5166705/) and Opportunity ( saltwater.html) suggested the formation of at least some Martian deposits from concentrated salt water. Macroscopic crystals of extraterrestrial halite, together with sylvite (KCl) and water inclusions, were found in the Monahans meteorite, which fell in Texas in 1998; the pieces were inspected days after being collected (Zolensky et al., 1999). Another line of evidence for the existence of extraterrestrial salt was provided by the Galileo spacecraft; its onboard magnetometer detected fluctuations that are consistent with the magnetic effects of currents flowing in a salty ocean on the Jovian moon Europa (McCord et al., 1998).

The examples presented here imply that much of prokaryotic life on Earth is indeed adapted to a subterranean lifestyle. This suggests that a planet with a lifeless surface may hide life deep beneath its surface, provided there is liquid water and energy available; alternatively, microbes may be present in a dormant state.

The search for life in the solar system and beyond is a goal of several space agencies in the twenty-first century (Foing, 2002). Current planning of the European Space Agency includes the ExoMars concept, which consists of a mobile rover capable of drilling into the surface of Mars at least 2 m deep and of probing for traces of organics and biomolecules (Vago and Kminek, 2007). In addition, return samples from Mars or other celestial bodies might become available.

Other well-characterized samples may stem from meteorites, which might contain halite, as described above. In any case, the development of sensitive and specific methods for life detection in extraterrestrial samples will be crucial, since the requirement for authenticity - i.e. proof, that any detected substances are not stemming from Earth - is severe, and this will likely spawn applications to terrestrial samples as well.

7. Summary

The deep terrestrial subsurface harbors large microbial communities which have not yet been fully explored. Evidence from geological findings suggests that certain prokaryotes may have remained viable in sediments and subterranean water for thousand and perhaps millions of years. Considering the harsh environments on other planets and moons as well as in interplanetary space, the chances for finding extraterrestrial life should be greater when drilling into the subsurface and searching with highly sensitive instrumentation for organic molecules and perhaps living fossils.

8. Acknowledgements

This work was supported by the Austrian Science Foundation (FWF), projects P16260-B07 and P19250-B17. We thank M. Mayr, Salinen Austria and J. Knoll, City of Bad Gastein, for help in obtaining rock salt samples and spring water samples, respectively.

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Biodata of Tomas Hode, Sherry L. Cady, Ilka von Dalwigk, and Per Kristiansson, authors of "Evidence of Ancient Microbial Life in an Impact Structure and Its Implications for Astrobiology - A Case Study"

Dr. Tomas Hode is currently a research associate at the Department of Geology at Portland State University, Oregon, USA. Tomas Hode obtained his Ph.D. in Astrobiology from the Swedish Museum of Natural History, and Stockholm University in 2005, and continued his research as a post-doc and research associate at Portland State University. Dr. Hode's scientific interests include a variety of subjects relating to early Earth studies, impact geology, fluid inclusions, biosignatures, and development of new microanalytical methods.

E-mail: [email protected]

Professor Sherry L. Cady is currently an Associate Professor at the Department of Geology, Portland State University, in Portland, Oregon. She obtained her Ph.D. in Geology from the University of California in Berkeley in 1994, and continued her research at the SETI Institute and NASA Ames Research Center before moving to Oregon. Her current scientific interests focus on microbial biosignatures, habitable environments beyond Earth, and early and extreme environments on Earth. Dr. Cady is the founding and current Editor-in-Chief of Astrobiology, the leading peer-reviewed journal that explores the secrets of life's origin, evolution, distribution, and destiny in the universe.

E-mail: [email protected]

Ilka von Dalwigk is currently employed at the research department at Vattenfall, a Swedish Energy company, working with issues related to CO2 capture and storage. She started to study geosciences at the Phillipps University, Marburg before she moved to Stockholm, Sweden where she wrote her Master thesis on marine impact deposits. She continued her academic career with Ph.D. studies on the Siljan Impact Structure with special focus on deformation structures related to large impacts.

E-mail: [email protected]

Professor Per Kristiansson is currently the head of the division of Nuclear Physics at Lund University, Sweden. He obtained his Ph.D. from the Lund University, division of Cosmic and Subatomic Physics in 1985 and continued his research as Post Doc at the Lawrence Berkeley Lab working with relativistic heavy ion collisions. He returned to Lund University in 1987 and became a professor in Applied Nuclear Physics in 2002. Professor Per Kristianssons scientific interests are in the areas of: Ion Beam Analysis, specially with focused MeV ion beams. Technical

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