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J. Seckbach andM. Walsh (eds.), From Fossils to Astrobiology, 233-248. © Springer Science + Business Media B. V. 2009

MICROORGANISMS IN THE ANCIENT TERRESTRIAL SUBSURFACE - AND IN OUTER SPACE?

HELGA STAN-LOTTER, SERGIU FENDRIHAN, MARION DORNMAYR-PFAFFENHUEMER, FRIEDRICH GERBL, ANDREA LEGAT, CLAUDIA GRUBER AND GERHARD WEIDLER

University of Salzburg, Division of Molecular Biology, Department of Microbiology, Billrothstr 11, A-5020 Salzburg, Austria

1. Introduction

Microorganisms have been detected in great depth in subterranean environments, such as granite, sediments, permafrost areas, caves, rocks in gold - and uranium mines. Research directed towards exploring intraterrestrial microbial communities is a rapidly growing field and since the appearance of the first book, covering this subject, by Amy and Haldeman (1997), several aspects have been reviewed by Pedersen (2000), Gilichinsky (2002), Stan-Lotter et al. (2004) and Teske and Sorensen (2008). The surfaces on rocky planets and moons, which may be envisaged for the search for extraterrestrial life, can be considered as sterilizing environments, due to the high incidence of ultraviolet radiation, if there is no shielding atmosphere. This applies to Mars, where data about the conditions on the surface have been published (Ronto et al., 2003), and it is likely valid also for other bodies such as Jupiter's moon Europa. However, the subsurface of Mars and the presumed underground ocean of Europa hold great promise, since life may have survived there, protected from the harsh conditions on the surface. Thus, extraterrestrial subsurfaces will likely be probed for signs of life, following the development of suitable methods and instruments. Therefore, results from the often practical and applied microbial research in the terrestrial underground, which deal with storage problems for radioactive waste, aquifers and their cleanup from pollution, or restoration of contaminated sites in mining operations, could have consequences for the planning of missions to outer space in search for organics and life.

New issues have emerged as a result of the subterranean research, and many as yet unanswered questions have been raised: Are microorganisms from geological sites as old as the deposits from which they were isolated or identified? If so, how can this be proven beyond all doubt? And if proven, how could these organisms survive? Which mechanisms would allow them to endure prolonged states of starvation and/or desiccation? Are there specific dormant survival forms other than bacterial endospores? If so, could those be recognized in geological materials, such as rocks or sediments? Can dormant forms be resuscitated? Are subterranean prokaryotes able to divide almost infinitely slowly (as has been suggested by Fredrickson and Onstott, 1996), which would imply the maintenance of minimal metabolic activity? Answers to these questions will impact the search for life in extraterrestrial subsurfaces.

In this chapter, current evidence for several examples of subterranean micro-bial life is reviewed, two environments (evaporites and subsurface springs) and their inhabitants are described in more detail, and potential lessons for astrobio-logical issues are considered.

2. Extent of Intraterrestrial Life and the Availability of Living Prokaryotic Fossils

The number of intraterrestrial microbes varies notably depending on the environments and sites being studied. Whitman et al. (1998) provided estimates for aquatic and soil environments and for the terrestrial subsurface, which probably contributes the major part of all biomass. Values in the range of several thousand up to hundreds of million microbes per millilitre of groundwater or gram of sediment are commonly reported. Although the wet weight of 100 million microbes, which may be present in 1 g of sediment, is only in the range of 100-1,000 |LLg, the total weight of microorganisms in many square kilometres of seafloor and continental shelve sediments, rock aquifers etc. may reach an impressive number. The carbon content of prokaryotes in intraterrestrial environments was estimated by Pedersen (2000) between 325 and 518 x 1012 kg, who pointed out that the total amount of carbon in intraterrestrial micro-organisms may equal that of all terrestrial and marine plant life together, which is in the order of 562 x 1012 kg (Pedersen, 2000).

The activities of intraterrestrial microbes, let alone their roles in the maintenance and evolution of the geosphere, are only insufficiently known. These issues provide important challenges for future research. The microorganisms in the deep subsurface of the Earth can be considered living fossils, since many of them must have been in their underground environments for very long periods of time and have probably survived in a dormant or nearly dormant state. The age of the microbial populations is currently not known, since accurate methods for dating individual cells are lacking (see below).

The depth of terrestrial subsurface layers, in which microorganisms can be found, is apparently limited just by the temperature, which increases with depth. The current records are in the range of about 3,000-3,500 m, e.g. a thermophilic Geobacillus was isolated in a deep South African gold mine from 3,200 m below surface; its temperature optimum was 65°C (Deflaun et al., 2007). Hyperthermophilic archaea can grow at very high temperatures; current record holders are Pyrolobus fumarii, which grows optimally at 105°C and survives up to 113°C (Blochl et al., 1997), and an unclassified archaeon, which was reported to grow up to 121°C (Kashefi and Lovley, 2003). Therefore, in greater depths, archaeal representatives could be expected. Since many of the thermophilic archaea are lithoautotrophs, they should be well adapted to live in subterranean environments. However, the questions of energy and carbon sources of the subterranean microbial communities are not clarified yet, and suggestions for and against evidence for a hydrogen-driven subterranean biosphere are being discussed (Stevens and McKinley, 1995; Nealson et al., 2005). Alternatively, reduced metals in the surroundings could serve as energy sources; typical microbially mediated redox pairs are manganese (II) oxidizing to manganese (IV), ferrous iron to ferric iron, sulfide to sulfate and methane to carbon dioxide (Madigan and Martinko, 2006).

3. Methods for Detection and Identification

The prokaryotic subterranean population has been analysed by conventional enrichment procedures and plate count methods. However, these approaches generally suffer from the phenomenon of the "great plate count anomaly", as known from environmental sites, since only a small fraction of the existing microbial community can be cultured in the usual types of nutrient media (Amann et al., 1995). In addition, microorganisms from the subsurface were often found to grow extremely slowly, even when appropriate media were used, making enumeration and analysis difficult. Therefore, the amplification of diagnostic molecules, such as the 16S rRNA genes, by the polymerase chain reaction (PCR), which obviates culturing of microorganisms, is being applied widely, and has permitted the detection of novel and unexpected phylogenetic groups in numerous environmental samples (Ward et al., 1990). Other molecular methods, which are increasingly used, are denaturing gradient gel electrophoresis (DGGE; see Muyzer and Smalla, 1998) and fluorescence in situ hybridisation (FISH; Bottari et al., 2006).

Direct light microscopic observations, combined with specific staining methods, are another technique for estimation of environmental microbial content. Staining procedures often use the fluorescent dyes DAPI (4', 6-diamidino-2-phenylindole), as originally suggested by Porter and Feig (1980) and, more recently, the LIVE/ DEAD BacLight™ Bacterial Viability kit (Leuko et al., 2004). These procedures facilitate the enumeration of cells and the latter can also provide judgment about their viability. In some subterranean environments, such as Permafrost, direct electron microscopy of samples has been applied and has revealed dwarf forms and cyst-like cells of non-spore forming bacteria (Soina et al., 2004).

4. Specific Subterranean Environments

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