Abstract

Intraterrestrial life has been found at depths of several thousand metres in deep sub-sea floor sediments and in the basement crust beneath the sediments. It has also been found at up to 2800-m depth in continental sedimentary rocks, 5300-m depth in igneous rock aquifers and in fluid inclusions in ancient salt deposits from salt mines. The biomass of these intraterrestrial organisms may be equal to the total weight of all marine and terrestrial plants. The intraterrestrial microbes generally seem to be active at very low but significant rates and several investigations indicate chemolithoautotrophs to form a chemosynthetic base. Hydrogen, methane and carbon dioxide gases are continuously generated in the interior of our planet and probably constitute sustainable sources of carbon and energy for deep intraterrestrial biosphere ecosystems. Several prospective research areas are foreseen to focus on the importance of microbial communities for metabolic processes such as anaerobic utilisation of hydrocarbons and anaerobic methane oxidation.

Introduction

Jules Verne's famous book, Journey to the Centre of the Earth, reports how Professor Lidenbrock and his nephew, Axel, found advanced life in natural caves and tunnels deep below Europe. At the time it was written the story was genuine science fiction. Now, 135 years later, it is obvious that Jules Verne in a sense was correct because deep under the terrestrial ground as well as deep under the sea floor, life is very abundant, albeit in cavities smaller than imagined by Verne. Research directed towards exploring intraterrestrial microbial life is a rapidly growing field reflected by an increasing number of meetings and workshops in the research field and also by numerous intriguing articles in popular science journals [1–4]. The purpose of this mini-review is to give an overview of microbial life in various deep intraterrestrial habitats that are at present under exploration and to identify central questions regarding our understanding of microbial processes deep inside the earth, in the dark realm of deep sediments, rocks and minerals.

How deep is deep?

The superdeep well, SG-3, 12 262 m deep, in the Pechenga-Zapolyarny area of Kola Peninsula, Russia, is currently the deepest drilled hole in the world [5]. One of many important reasons for drilling this borehole was to see how deep a borehole could be drilled. The prospect of drilling a superdeep borehole becomes increasingly difficult with increasing depth and success depends on the geological formation drilled, the quality of equipment used and the skills of the drilling personnel. In addition, the drill string may get stuck in a layer with bad borehole stability and this is always a major uncertainty in deep drilling projects.

Other superdeep boreholes include several Cretaceous–Tertiary boundary (KTB) boreholes drilled by the German Continental Deep Drilling Programme into the crystalline rock of the Bavarian Black Forest (Schwarzwald) basement in Central Europe [5]. The deepest of the six wells drilled was 9100 m and it reached an in situ temperature of 265°C at that depth. One of these KTB wells was searched for hyperthermophiles at a depth of 4000 m. Culturable microorganisms could not be demonstrated, possibly because the temperature of the sampled fluids, 118°C, was too high for life [6]. So far, the highest culturing temperature for hyperthermophiles has not exceeded 113°C [7].

Another very deep borehole was drilled in Gravenberg, Sweden, in the search for deep earth gases [8]. It reached 6800 m and thermophilic bacteria were successfully enriched and isolated from a depth of 5278 m where the temperature was 65–75°C [9].

The borehole windows into superdeep environments are still very few and none have been drilled with microbiology as its major motive. Boreholes drilled with the purpose of exploring microbial life are rarely deeper than 1000 m [10,11]. However, depth is not the only limiting factor for survival of deep life. Rather, it is temperature that probably sets the ultimate limit for how deep life can penetrate into our planet. A temperature that is too high for life is reached at very different depths, from the sea floor surface at marine hot springs to 10.000 m down or deeper in massive sedimentary rock formations. The absolute majority of boreholes explored for microbial life does not reach such depths because the drilling cost for a borehole increases with depth which, together with increasing technical challenges, limits the number of superdeep boreholes drilled thus far to very few. The exploration of intraterrestrial life is consequently, in parallel to the exploration of possible life on other planets, strongly dependent on sophisticated technological equipment and skills. The exploration of the (super)deep intraterrestrial biosphere has merely begun.

Global microbial biomass distribution at a glance

The total number of intraterrestrial microorganisms varies notably, depending on the site studied. Values in the range of 103 to 108 per ml groundwater or g sediment are commonly reported [10,12]. Although the dry weight of 108 cells in 1 g of sediment is very small, viz. in the range of 1–10 μg, the weight of microorganisms over many square kilometres of ocean floor and continental shelf sediments, rock aquifers and so on may reach an impressive total. An attempt to estimate the total carbon in terrestrial and intraterrestrial environments was recently made [13]. Calculations indicate that the total amount of carbon in intraterrestrial organisms may equal that of all terrestrial and marine plants (Table 1). Although subject to a great deal of uncertainty, the estimates therefore suggest that the biomass of intraterrestrial life is very large. A wealth of microbial life may exist deep inside the earth, with many unknown species and microorganisms with unique physiological and biochemical features and is awaiting exploration.

1

Global carbon content in plant and prokaryotic biomass

Ecosystem Carbon content, 1012 kg of C 
 plant soil and aquatic prokaryotes intraterrestrial prokaryotes total 
Continental 560 26 22–215 608–801 
Oceanic 1.8 2.2 303 307 
Total 561.8 28.2 325–518 915–1108 
Source: [13
Ecosystem Carbon content, 1012 kg of C 
 plant soil and aquatic prokaryotes intraterrestrial prokaryotes total 
Continental 560 26 22–215 608–801 
Oceanic 1.8 2.2 303 307 
Total 561.8 28.2 325–518 915–1108 
Source: [13

Exploration in whose interests?

The interests in investigations of intraterrestrial life are represented by a very diverse array of general social, professional and industrial motives. Some of the current reasons for such investigations are listed below.

  1. Microbial activity in oil wells may have both negative (e.g. through corrosion and well souring) and positive (e.g. through surfactant production) effects on oil extraction. The oil industry, therefore, shows an interest in deep oil reservoir microbiology [14–16].

  2. The contamination of groundwater from surface and underground disposal sites, accidental spills, leakage and other human activities has triggered a widespread interest in the possibilities of restoring contaminated underground sites with the help of autochthonous and/or allochthonous microorganisms [17].

  3. Disposal of radioactive wastes and heavy metals in deep geological formations requires in-depth knowledge about the host rock environment, including possible effects of microbes on future repositories [18].

  4. There are enormous reservoirs of energy in the methane gas hydrates that are found globally in sub-ocean floor sediments, possibly twice the amount of energy contained in known oil and gas reservoirs [19]. Most of this methane is believed to have been produced by methanogens living deep below the deposits [20].

  5. An increasing number of scientists argue for an underground origin of life, possibly in the vicinity of hydrothermal systems [21]. If life did originate subterraneously, then it must have been present underground for as long as there has been life on our planet. A diverse and extended underground life on, or within, our planet suggests that life on other planets should be searched for underground rather than on the surface.

  6. Last but not least, the increasing knowledge about intraterrestrial life may significantly expand our knowledge of microbial diversity and, especially, of the metabolic capabilities of living organisms (see, for example, [22]).

Each of these interest areas focuses on more or less specific intraterrestrial environments and some of the currently studied ones are discussed below.

Environments for intraterrestrial life

General considerations

The realm of sediments, rocks and minerals offers environments for life that are very different from terrestrial and aquatic habitats. Water is common, but there is a very large solid surface-area-to-water-volume ratio and generally there is relatively little space for water and life per volume subsurface. The intraterrestrial microbes dwell in the pores of consolidated sediments and coarser, unconsolidated material, in fractures in hard rock and in fluid inclusions (see Section 2.4). A microscopic cell is ideally suited to these environments. Almost all very deep environments are anaerobic, with the exception of places where radioactivity may cause radiolysis of water, producing hydrogen and oxygen. With an increase in depth, the amount of dissolved solids in the groundwater tends to increase and so does the temperature, but the gradients of these increases vary, depending on the geological formation. The temperature trend implies that at all places on the planet, there will be a maximum depth below which life as we know it is impossible, but the maximum depth for life is highly variable. The upper known maximum temperature for microbial growth, 113°C, is reached at the ocean floor at hydrothermal vents, but not before 5000–10 000-m depth or more in shield rocks, mountains and deep sediments.

There is a set of questions that are commonly addressed relating to the microbiology of most intraterrestrial environments. These are largely equivalent to the questions posed in studies of terrestrial and aquatic ecosystems. Numbers of individual cells and the members of various physiological groups, as well as the diversity of microbial populations are frequently analysed. Once the presence, abundance and guild structure of microorganisms have been demonstrated, species diversity may be addressed, but a key question is the metabolic state of the community, that is, are the intraterrestrial microbes metabolically active at any level or are they resting in dormant states?

Microbes are experts on utilising any energy that becomes thermodynamically available for biochemical reactions in the environment. Such energy can be extracted from various sources, depending on the type of geological formation. Recharging groundwater may carry a slow flow of organic carbon from the ground surface down into hard rock aquifers. In sedimentary rock layers, utilisable organic carbon may have remained since their formation in an ancient sea. Finally, a flow of reduced gases such as hydrogen and methane from the mantle of our planet could ultimately be a driving force for active life of the microbes dwelling in both deep rock aquifers and sediments. The intraterrestrial microbes can, however, not be more active than the support of energy over time allows and this may be a very slow, diffusion-limited process.

The sub-sea floor sediment and basement rock

The Ocean Drilling Program (ODP) is an international partnership of scientists and research institutions organised to explore the evolution and structure of the earth (http://www.oceandrilling.org/). With its drill ship, JOIDES Resolution, the ODP can drill cores (long cylinders of sediment and rock) in water depths of up to 8.2 km. Since January 1985, the ODP has recovered more than 160 000 m of cores. Results obtained thus far show microbial life to be abundant both in the deep sub-sea floor sediments and in the basement crust under the sediments [23,24]. As a result, exploration of the deep sub-sea floor biosphere is now a high-priority research objective of the ODP.

The basement of the sea floor is born at spreading ocean ridges that commonly stretch over long distances on the bottom of the sea. Lava and mantle rock move up at the site of these ridges and thus reach the ocean floor. At the ridges, hydrothermal vents are the obvious indicators of an ongoing convective circulation of saltwater through fissures and cracks in the newborn hot basement. Some calculations indicate that all oceanic water passes through the basement rocks every 30 million years. Large parts of the new ocean floor consist of pillow lavas with amorphic volcanic glass on the pillow surface. Microscopic and culturing investigations strongly suggest that microorganisms are involved in the weathering of this volcanic glass [23,25]. Channels, cavities and holes in the micrometer range penetrate the glass and the tips of the tunnels commonly stain with nucleic acid specific stains, suggesting the presence of microorganisms. The circulation of saltwater through the basement and the weathering activities of microorganisms in the flow paths therefore may have a significant impact on the composition of saltwater [26].

Due to plate tectonic forces the new ocean basin that forms at spreading ocean ridges migrates towards subduction zones where it is engulfed under continental shelves and returned to the interior of our planet. This tour takes at most 170 million years. During that time, more and more sediments build up on top of the hard basement rock and eventually layers are formed that can be thousands of metres thick. During the sedimentation process, microorganisms are mixed with the sediment and they appear to be important factors in the diagenesis of the sediments [24]. The continued presence of active bacterial populations in deep sediments that are over 10 million years old may relate to acetate generation from organic matter during burial. This was indicated by the fact that at two sites in the Atlantic Ocean, pore-water acetate concentration has been found to increase at depths below about 150 m [24]. The increase appears to be associated with a significant increase in bacterial acetogenic activity.

The acetate produced in the sediments is available for acetoclastic methanogenesis and this may be a possible mechanism for formation of the enormous reservoirs of methane in gas hydrates found globally in sub-sea floor sediments [19]. Methane formation by bacterial methanogenesis based on the burial of and acetogenic decomposition of organic carbon in marine sediments has been demonstrated to roughly explain the content of gas hydrates in the marine sediments investigated [20]. However, for the local formation of massive concentrations of gas hydrates, some accumulation processes are required. Accumulation of gas hydrates near the base of the gas hydrate stability zone (which depends on pressure and temperature) is possible if methane migrates from depths below the hydrate deposits. The enormous reservoirs of energy in the form of methane gas hydrates in sub-sea floor sediments seem to have been produced by methanogens living deep underneath the deposits.

A recently discovered global warming catastrophe occurred 55 million years ago as a result of rapid melting of methane gas hydrates [27]. That event demonstrates the delicate balance of our global climate and should be taken as a serious warning. We cannot ignore the possibility that a continuation of the newly observed rapid melting of the arctic ice [28] may trigger a similar, but modern such catastrophe. A future Antarctic sea without its ice cover would in summer time take up 80% solar energy instead of reflecting 80%, as done presently with warming of the oceans as an obvious result. The past event of global warming is a good example of how microbes may have an enormous influence on global processes, albeit indirectly, via methane formation in deep sub-ocean floor environments.

Successful enrichments of thermophiles have been performed from samples from sub-sea floor and continental oil reservoirs [14–16,29], indicating that microorganisms survive in deep hot oil reservoirs. However, the need for quality assurance for good microbiology sampling, with protocols for contamination control, has not been emphasised in the oil industry. It has still not been conclusively shown that these isolates were indigenous to the deep oil wells. This is surprising, since it is well known to oil drillers that microorganisms, especially sulfate-reducing bacteria, may cause expensive problems related to corrosion and souring of wells. Solving the question of the origin of microbes in deep oil wells would definitely reduce uncertainties about how these problems can best be attacked. The costs for proper contamination control would be extremely small in comparison with the costs that can be saved by reducing microbial corrosion and oil well souring.

Continental sedimentary rocks

A significant effort was devoted to a good contamination control during the US Department of Energy's investigative programme on the microbiology of subsurface sediments [10]. For instance, the need for rapid initiation of analyses after sediment sample acquisition was demonstrated for measurements of in situ microbial properties [30]. Subsequently, the experience gained was used in studies of the environmental conditions under which microorganisms exist in deep continental hydrocarbon reservoirs. The samples included sidewall cores collected from a natural gas-bearing formation 2800 m below the surface in Taylorsville Basin, Virginia, in the USA [31]. Microbiological data, and data from chemical and microbial tracers and controls indicated that the interiors of some sidewall cores contained microorganisms indigenous to the deep hot rock formation. The cultured microorganisms were composed primarily of saline-tolerant, thermophilic, fermenting, Fe(III)-reducing and sulfate-reducing bacteria with physiological capabilities compatible with the in situ temperature (76°C) and pressure (32 MPa).

Large phylogenetically and culturally diverse communities of Archaea and Bacteria in North American deep subsurface continental sediments have been demonstrated [32], which is in line with reported biodiversity from other investigated underground sites in Africa and Scandinavia [33,34]. The possible in situ activity of microorganisms in the subsurface sedimentary rocks studied was also addressed. As in other oligotrophic habitats, activity occurred, but at very slow rates. Also, there was usually a significant correlation between the type of geological strata, for instance in the pore size and organic content, and activity [35].

The research of continental sedimentary rocks shows that living microbes are present and active down to the deepest levels studied (about 3000 m). They will probably be present even deeper, as deep as the temperature allows, as discussed in Section 1.1. The results have important implications for long-term maintenance of anoxic conditions and the impact of anaerobic biochemical processes on groundwater chemistry. They also imply that natural attenuation of contaminated groundwater by indigenous microbes can occur at depth. Further work is needed to unravel details about these significant processes.

Ancient salt deposits

Salt crystals are formed when saline water evaporates. During this process, small parts of the brine become trapped in the salt and end up as fluid inclusions. Any halophilic microorganisms present in the fluid will be trapped as well. Large, ancient deep deposits of salt exist all over our planet. For instance, there are extensive salt deposits deep under Germany, Denmark and the North Sea, formed 250 million years ago from shallow seas which evaporated when the climate in Europe was much warmer because of an equatorial position of the European continent caused by plate tectonics. Similar formations are found elsewhere on the planet. When ancient fluid inclusions from such deposits are opened and the fluid is transferred to culture media for halophiles, growth occurs and the growing organisms exhibit a large species diversity [36]. It seems very probable that these growing halophiles were captured 250 million years ago during the formation of the salt deposits. This finding invokes many intriguing questions, regarding how long microorganisms can survive and specifically, how halophiles could stay alive for 250 million years in their small, saline prisons. These observations do indeed imply that some microorganisms may have eternal life, at least in relation to the brief lifetime of most plants and animals.

Aquifers in igneous terrestrial rocks

Igneous rocks are the predominant solid constituents of the earth, formed through cooling of molten or partly molten material at or beneath the earth's surface. These rocks are penetrated by humans for a variety of purposes such as mining for metals, the extraction of groundwater and the building of tunnels and vaults for communication, transport, defence, storage, deposition and waste disposal. The concept of underground storage of hazardous wastes is commonly applied to materials that are impossible to transform to a non-hazardous form. One group of such materials is the toxic metals, especially heavy metals and radionuclides. The disposal concepts vary from country to country, but igneous rocks are commonly in target for countries with access to these types of geological structures. Deposition of long-lived hazardous wastes requires extremely good knowledge about the igneous rock environment to be used as a host. Particularly the nuclear waste industry invests large sums in research on the safe underground disposal of all types of radioactive wastes [37].

Early studies in the Swedish long-term nuclear waste disposal research programme on subterranean microbiology revealed previously unknown microbial ecosystems in igneous rock aquifers at depths exceeding 1000 m [38]. This discovery triggered a thorough exploration of the subterranean biosphere in the aquifers of the Fennoscandian (Baltic) Shield [39]. Similarly, the Canadian radioactive waste disposal programme has stimulated investigations of microorganisms in deep igneous rock aquifers of the Canadian Shield [40].

Other investigations examined the potential risk of radionuclide migration caused by microorganisms able to survive in the deep groundwater systems [41]. It soon became apparent that microbial communities exist in most, if not all, deep aquifers [38]. Attention was then shifted to assay the activity potential of these microorganisms using radiotracer methods [42–45]. The results suggested remarkable metabolic and species diversity, which led to DNA extraction and 16/18S rDNA cloning and sequencing for assessment of subterranean microbial diversity [34,46,47]. This work has also revealed several hitherto unknown microbial species adapted to life in igneous rock aquifers [48–50].

The repeated observations of autotrophic, hydrogen-dependent microorganisms in the deep aquifers imply that hydrogen may be an important electron and energy source and carbon dioxide an important carbon source in deep subsurface ecosystems. Hydrogen, methane and carbon dioxide have been found in μM concentrations at all sites that have been tested for these gases [51]. Methane is a major product of autotrophic methanogens, which have been shown to be present at the Åspö Hard Rock Laboratory in Sweden, situated 400 km south of Stockholm [51]. Therefore, a model has been proposed of a hydrogen-driven biosphere in deep igneous rock aquifers in the Fennoscandian Shield [39]. A similar model has been suggested for deep basalt aquifers [52]. The organisms at the base of these ecosystems are assumed to be autotrophic acetogens capable of reacting hydrogen with carbon dioxide to produce acetate, autotrophic methanogens that produce methane from hydrogen and carbon dioxide, and acetoclastic methanogens that produce methane from the acetate product of the autotrophic acetogens (Fig. 1). All components needed for the life cycle in Fig. 1 have been found in deep igneous rock aquifers and the microbial activities expected have been demonstrated [51]. Consequently, the model is supported by the qualitative data obtained so far. Ideally, the next step will be to obtain quantitative data, which would require very sensitive experimental conditions, because of the very slow metabolic rates that are expected under non-disturbed conditions. The central question in such an experimental endeavour is whether or not in situ hydrogen-driven microbial chemolithoautotrophic activities at depth are in balance with estimated renewal rates of hydrogen. An indisputable positive answer to this question is crucial for the unequivocal confirmation of a deep hydrogen-driven biosphere.

1

The deep hydrogen-driven biosphere hypothesis, illustrated by its carbon cycle. At relevant temperature and water availability conditions, intraterrestrial microorganisms are capable of performing a life cycle that is independent of sun-driven ecosystems. Hydrogen and carbon dioxide from the deep crust of the earth are used as energy and carbon sources.

1

The deep hydrogen-driven biosphere hypothesis, illustrated by its carbon cycle. At relevant temperature and water availability conditions, intraterrestrial microorganisms are capable of performing a life cycle that is independent of sun-driven ecosystems. Hydrogen and carbon dioxide from the deep crust of the earth are used as energy and carbon sources.

Caves

Caves may provide the link between surface and subsurface environments. The cave environment varies tremendously. One very large cave which may serve as an example is the Lechuguilla Cave in Carlsbad Caverns National Park in New Mexico, USA. It is a deep, extensive, gypsum- and sulfur-bearing hypogenic cave, at least 90% of which lies more than 300 m beneath the entrance. This cave is therefore a good example of an intraterrestrial cave environment. Allochthonous organic input to the cave is limited due to the depth, but bacterial and fungal colonisation is relatively extensive [53]. Various points of evidence suggest that autotrophic bacteria are present in the ceiling-bound residues and could act as primary producers in a unique subterranean microbial food chain. Because other major sources of organic matter have not been detected, it is suggested that these bacteria provide requisite organic matter to the known heterotrophic bacteria and fungi in the residues. The cave-wide bacterial and fungal distribution, the large volumes of corrosion residues and the presence of ancient bacterial filaments in unusual calcite speleothems (biothems) attest to the apparent longevity of microbial occupation in this cave. More research should be invested in this and other caves to reveal the ecology and the biogeochemistry of cave ecosystems.

What's next?

The search for the maximum depths for life in various intraterrestrial environments will probably always be one major desire in the exploration of deep life. Knowing more about these limits to life within our planet will enable more precise calculations of the total amount of intraterrestrial organisms. It is possible that the maximum in situ temperature for life overrides the 113°C shown at laboratory conditions. Deep, high-pressure environments are required to keep water liquid above the boiling point (100°C) at atmospheric pressure. Therefore, these deep crustal environments are relevant targets for the search of the maximum temperature for life.

A paper on anaerobic degradation and methane production from long chain alkanes by a consortium of microorganisms was recently published [54]. A team of seven different microorganisms were observed to crack the hydrocarbon, to produce methane and carbon dioxide and also to expel hydrogen sulfide. This is an encouraging result for those who believe that any catabolic reaction that can be linked to oxygen as the electron acceptor, may also occur in the absence of oxygen, albeit slower and as a result of collaboration between species. The experiment showing anaerobic degradation of alkanes continued for almost two years before conclusive results were obtained. One implication deduced from these results is that intraterrestrial microbes play in a very different league from those in well-fed, aerobic, pure cultures. Patience, a great deal of time and advanced culturing methods will be required for successful exploration of the metabolic activities of intraterrestrial microbial communities.

Most deep environments contain methane in varying concentrations. Methane oxidation in aerobic environments is well documented and very widespread on the planet. As yet, there exists no microorganism, or community of microorganisms, in culture that oxidises methane under anaerobic conditions, although much indirect evidence for the occurrence of this process in nature has been published (see, e.g. [55]). If a metabolic process for anaerobic methane oxidation can be demonstrated, then a tremendous source of energy and carbon will become available for models of how intraterrestrial life is fuelled.

The theory of a deep biosphere driven by hydrogen generated in deep geological strata (Fig. 1) requires more research. There are at least six possible processes whereby crustal hydrogen is generated [56]:

  1. The reaction between dissolved gases in the carbon–hydrogen–oxygen–sulfur system in magmas, especially in those with basaltic affinities.

  2. The decomposition of methane to carbon (graphite) and hydrogen at temperatures above 600°C.

  3. The reaction between CO2, H2O and CH4 at elevated temperatures in vapours.

  4. Radiolysis of water by radioactive isotopes of uranium and thorium and their daughter isotopes, and potassium.

  5. Cataclasis of silicates under stress in the presence of water.

  6. Hydrolysis by ferrous minerals in mafic and ultramafic rocks.

It is important to explore the scale of these processes and the rates at which the produced hydrogen is becoming available for deep microbial ecosystems.

The idea, in conclusion, that life originated on the surface of our planet, where it was strongly dependent on a hypothetical primordial soup, has recently come up against strong competition. Today there are several suggestions that life originated in the form of a thermophilic lithotroph [57] and that the birthplace was intraterrestrial, perhaps a hydrothermal vent area [58]. Consequently it should be obvious that the search for extraterrestrial life should concentrate on samples from under the surface of other planets [59]. However, that is another topic and far too speculative for this mini-review.

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