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Why are some microorganisms boring?
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Why are some microorganisms boring? Charles S. Cockell and Aude Herrera Centre for Earth, Planetary, Space and Astronomical Research (CEPSAR), Open University, Milton Keynes, MK7 6AA, UK
Microorganisms from diverse environments actively bore into rocks, contributing significantly to rock weathering. Carbonates are the most common substrate into which they bore, although there are also reports of microbial borings into volcanic glass. One of the most intriguing questions in microbial evolutionary biology is why some microorganisms bore. A variety of possible selection pressures, including nutrient acquisition, protection from UV radiation and predatory grazing could promote boring. None of these pressures is mutually exclusive and many of them could have acted in concert with varying strengths in different environments to favour the development of microorganisms that bore. We suggest that microbial boring might have begun in some environments as a mechanism against entombment by mineralization. Microbial boring by different microorganisms Some taxa of microorganisms are known to bore holes in rocks and this is an evolutionarily ancient characteristic (Table 1). Bore holes created by phototrophs in stromatolites from the Dahongyu Formation in China date back at least 1500 million years [1], but older borings made into 3.4 billion-year old Archean (see Glossary) volcanic glasses by microorganisms have been reported [2]. Thus the selection pressure for boring activity predates the evolution of animals. Organisms that actively bore (a process sometimes known as ‘biological corrosion’) are referred to as euendoliths, in contrast to chasmoendoliths, which grow in cracks or fissures, and cryptoendoliths, which grow in rock interstices or structural cavities inside rocks [3]. Some of the most recent discoveries of euendoliths have been new species from desert environments [4] and there have also been reports of euendolithic borings into volcanic glasses in deep ocean basalts [5]. Euendolithic activity is widely distributed among fungi, red and green algae and cyanobacteria [6,7]. Fungi need a supply of organic matter to survive and thrive, and they preferentially invade substrates where organic matter is available, such as the laminae of sea shells. In aquatic environments they tend to dominate at water depths where light levels preclude or limit the activity of boring phototrophs [8]. Fungal euendoliths also dominate in nonmarine and brackish aquatic environments and in soils [9,10]. The size of bore holes varies, depending on the organism that produced them [11] (Figure 1). Bore holes of 2–6 mm in diameter produced by the cyanobacterium Plectonema sp., and holes up to 20–40 mm in diameter produced by the chlorophyte (alga) Ostreobium sp. were found in corals in Corresponding author: Cockell, C.S. ([email protected]).
Moorea, French Polynesia [12]. Fungi often produce tunnels in rocks, which display swellings associated with sporangia in the hyphae [13]. For phototrophs the depth of boring is set by the maximum depth at which phototrophic growth can be sustained, typically on the order of 1–3 mm [14]. The distribution of different types of euendoliths, and thus their boring patterns at different depths, can be used as a basis for paleobathymetry, particularly if fossil evidence of euendoliths with known narrow depth ranges can be discriminated [15]. Boring rates in substrates can be high. For example, phototrophic euendoliths are significant contributors to biologically induced destruction of carbonate coastlines and corals, and the formation of ‘biokarst’ with bio-erosion estimates of up to 570 g CaCO3 m2 y.1 [12]. By contrast, the formation of carbonates within bore holes that cut across grains can also cause the cementation of the grains, potentially giving euendoliths a constructive role in stromatolite formation [16]. The mechanisms for microbial boring are not yet fully understood [17] and are probably different for different taxa. Possible mechanisms for cyanobacterial boring include the release of acid that would dissolve the rock or the sequestration of calcium ions using a calcium pump, which would lead to the dissolution of calcium carbonates. Fungal dissolution of minerals probably occurs by the production of acid [17]. In addition to the challenge of developing a mechanistic understanding of how boring occurs, an evolutionary question remains unanswered – why do microorganisms bore into rocks? In this article, we examine some of the selective advantages that organisms might obtain by boring into rocks. We
Glossary Archean: geological eon corresponding to the time period between 4 billion and 2.5 billion years ago. Biokarst: landscapes formed by biological activities. Biological corrosion: the destruction of rock by microbial borers. Cambrian: geological period corresponding to the time period between 543 and 490 million years ago. Chasmoendoliths: organisms that live within cracks or fissures in rocks. Cryptoendoliths: organisms that live within the interstices or structural cavities in rocks. Euendoliths: organisms that actively bore into rock. Feldspars: a class of aluminium-containing silicate rocks. Ichnofossils: trace fossils of life left in sediments. Paleobathymetry: the study of ancient water depth. PAR: Photosynthetically active radiation; radiation that can drive photosynthesis. Phototrophs: organisms that acquire their energy from photosynthesis. Proterozoic: geological eon corresponding to the time period between 2.5 billion and 543 million years ago. Stromatolite: A widely distributed structure consisting of laminated carbonate or silicate rocks, produced by the binding, precipitation or trapping of sediment by groups of microorganisms, primarily cyanobacteria.
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Table 1. Examples of ancient euendolithic microborings or ichnofossilsa Location Strelley Pool Formation (Pilbara), Australia Barberton, South Africa Barberton, South Africa Hebei, China Eleonore Bay Group, central Greenland East Greenland Emei, China Scania, Sweden Henson Gletscher Formation, North Greenland Holm Dal Formation, North Greenland
Age 3.4 Ga 3.4–2.5 Ga 3.5–3.4 Ga 1.7 Ga Late Proterozoic 700–800 Ma Early Cambrian Early Cambrian Early Cambrian Early middle Cambrian Late middle Cambrian
Environment S V V C C C C C C C C
Refs [41,42] [2] [40] [1] [43,44] [45] [46] [47] [48] [49] [50]
a Symbols: Ga, billion years ago; Ma, million years ago; V, refers to volcanic environment of microborings; C, carbonate; S, other sedimentary rocks (although these other rocks might themselves be part of a volcanic setting, e.g. sandstones).
consider some of the challenges faced by microorganisms living on the surface or within the interstices of rocks. This can help to identify the pressures that might have favoured microorganisms that actively bore. As well as reviewing existing discourses, we also suggest that cell demineralization might have been one early cause of rock dissolution that ultimately could have led to rock tunnelling. Selection pressures that make boring advantageous Acquisition of nutrients Nutrient availability has been suggested as a possible factor controlling the distribution of microborers [18]. Although boring activity can potentially release nutrients from minerals, organisms within bore holes can be deprived of nutrients compared with those on the surface. For example, the availability of nutrients provided by flowing seawater to organisms growing on the surface could be greater than nutrient availability for organisms within a tube, where exchange is slowed by diffusion. Expending energy to bore into a rock will only be worthwhile if the substrate possesses a growth-limiting nutrient or trace element not found within the circulating water around the rock or if the energy derived from the rock equals or outweighs the energy put into boring. In evolving a mechanism for rock dissolution to acquire nutrients, microorganisms would be involved in an early form of euendolithic boring. For example, Garcia-Pichel
Figure 1. Euendolithic borings into a snail shell. A backscattered electron image from a thin section of a snail shell from the South Coast of England (Beer, Devon; C.Cockell, unpublished). The borings are evident as 10 micron-wide tunnels into the top 100 mm of the shells and are ubiquitous in snails living in the intertidal environment in this region. The borings are caused by cyanobacteria, including Plectonema sp. Scale bar, 100 mm.
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suggests that a calcium pump might be involved in cyanobacterial euendolithic boring [17]. One could speculate that originally the uptake of calcium as a nutrient from solution by organisms growing near carbonates would have the side-effect of changing the chemical equilibrium at the surface of the mineral to favour dissolution. The organism would then grow into the cavity that is created. In nutrientpoor environments, rock dissolution in this way would have been directly selected for and later it might have led to boring. This hypothesis would hold for any mechanism of nutrient acquisition that resulted in rock dissolution, including, for example, the production of organic acids by fungi leading to selection for euendolithic behaviour in nutrient-poor environments. This hypothesis would be consistent with the observations of fungal boring into soil feldspars. In a study of soil chronosequences [10], Smit and co-workers showed that fungal boring into feldspars began after more nutrient-rich minerals, such as biotite and hornblende, had been destroyed. However, based on the observation that the boring into feldspars was independent of feldspar abundance, they also suggested that tunnelling might be correlated with fungal density. A response to competition for other resources In many environments (such as deep water) boring into rock would reduce availability of photosynthetically active radiation (PAR) because the rock substrate would reduce exposure to PAR. Limitation of PAR in these circumstances would provide a selection pressure for upward movement, not downward boring. However, in high-light environments where surface PAR might cause photoinhibition, boring could provide an advantage. Euendolithic boring might be advantageous in competition for space, particularly within the interstices or cracks of rocks where biofilm development would be restricted. Although boring is energetically costly, if the cells survived in the newly exploited habitat where there was little or no competition, then the trait would persist in the absence of a selection pressure against it. Protection from physical extremes Microorganisms living inside a rock can be protected from desiccation and extreme temperatures on the surface of the rock [19]. The improved micro-environmental conditions experienced by cryptoendolithic lichens and their associated biota compared with conditions on rock surfaces have
Opinion been measured in extreme hot and cold deserts, such as the Negev desert of Israel and the Dry Valleys of the Antarctic [19]. In the Antarctic, cryptoendolithic lichens are protected from rapid freeze–thaw fluctuations that render the surface of the rock inhospitable. Where the porosity of the material is low, active boring would provide shielding similar to that experienced by chasmo- and cryptoendoliths. Euendoliths within carbonate materials in the intertidal zone are potentially exposed to desiccation cycles and moisture-retaining rock borings probably reduce or eliminate the time that organisms are exposed to desiccation. Euendoliths living in carbonate rocks near alpine glaciers are thought to have evolved to regulate their boring activity so as to prevent the destruction of the substrate, which is proposed to act as an environmental shield [20]. Protection from UV radiation Ultraviolet (UV) radiation is detrimental to various physiological processes in exposed cyanobacteria and fungi [21] and the interior of rocks can provide protection from UV radiation [22]. By tunnelling into rocks, an organism can gain the advantage of the UV-protecting properties of the rock surrounding it (Figure 2). The selection pressure to escape or mitigate UV radiation might have been stronger on the early Earth, specifi-
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cally prior to the oxygenation of the atmosphere approximately 2.4 billion years ago when the lack of oxygen might have also meant a lack of stratospheric ozone, meaning that wavelengths of UV radiation greater than 200 nm would have reached the surface of the Earth – in the absence of other atmospheric UV screens [23]. Protection from UV radiation as an initial selection pressure for boring is implausible for surface-dwelling organisms because it would require boring in the first place to obtain any beneficial screening. Boring to escape from UV radiation is also unlikely to be a plausible hypothesis for organisms inhabiting the deep oceans where there is no UV radiation. When the mechanisms of microbial boring are better understood it will be important to determine if the energetic cost of boring into rock is less than the energetic cost of repairing UV radiation damage or producing screening compounds to protect against UV damage on the surface. Prevention of detachment from rock substrates An organism that bores into a rock will be less likely to be detached by water currents. Many of the phototrophs that bore into rocks live in the intertidal zone, which experiences dynamic water regimens. Similarly, organisms living on volcanic glasses in hydrothermal environments are subjected to water flow over the surface of rocks, although
Figure 2. Protection from UV radiation in the euendolithic habitat. A rock sample exposed to UV radiation to demonstrate the efficacy of UV shielding. (a) Hyella sp. that occupy the interior of sandstone cliffs from Beer, Devon in England. (b) Surface of cliff material showing colonization by a diverse consortium of cyanobacteria (including Plectonema and Chroococcidiopsis spp.). (c) Rock sample exposed to 254 nm germicidal UV radiation (9.93 kJ cm2) after 48 hours in BG-11 medium. Note disappearance of surface biofilm. (d) Rock exposed to UV radiation incubated in BG-11 medium for five weeks. Cyanobacteria emerge from bore holes in the interior of the rock (arrow) in a localized manner and recolonize the surface. UV radiation of 254 nm is not found on the surface of the present-day Earth, but it could have penetrated to the surface of the early Earth. Scale bar, 10 mm in (a) and 0.5 cm in (b), (c) and (d) (C. Cockell, unpublished).
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Opinion within cracks this is probably less important. Many organisms successfully attach to the surface of rocks as epilithic biofilms in rivers, streams and the intertidal zone without boring into them [24] and euendolithic boring is probably more energetically costly than surface attachment (although the energy put into boring into rocks with high surface water-flow could be compensated by the associated greater nutrient flux). Perry [6] did not find a correlation between the extent of microboring and wave energy levels in Discovery Bay, North Jamaica, suggesting that waterflow was not a selection pressure in this environment. Escape from predation Growing into a rock would provide some protection from predation by macroscopic organisms, such as many fish and snails, because they are unable to penetrate the rock substrate (Box 1). However, predation is also possible by microscopic organisms. Euendolithic algae are extensively predated by euendolithic fungi [25]. The fungal hyphae grow around the algal filaments, eventually penetrating them and exuding dark tannin-like substances that turn the green algal bands in the rock black. Euendolithic fungal attack of coral algae leads to complex banding patterns within the corals. Thus, the euendolithic strategy does not provide an escape from predation for present-day algae [25,26]. On the Archean Earth, before the evolution of eukaryotes, fungal predation would not have constituted a selective pressure. However, fungal predation reflects the more general principle that bore holes do not provide escape from microbial attack per se, assuming that the attackers do not need to be able to bore themselves. For the same reasons it seems unlikely that filaments embedded in rocks would escape attack by phages, which could in principle attack along the length of the filament. Escape from mineralization We propose another hypothesis for the origin of boring behaviour. Microbial borings have been recorded most extensively in substrates associated with high mineralization rates [27]. This raises the possibility that euendolithic behaviour is a side-effect of an active demineralization process. In particular, cyanobacterial photosynthesis causes the precipitation of carbonates, and yet cyanobacteria are the best-characterized microbial borers. Organisms that are associated with hydrothermal regions [28] must contend with high rates of mineralization that threaten to engulf and immobilize cells. One side-effect of the innovation of a demineralization process would be the dissolution of the substrate to which the organism was attached. Mineralization on the top surface of euendolithic algae growing on rocks, but not around the cells within the rock carrying out boring, has been observed, demonstrating the presence of a mineralization challenge to the same organisms growing euendolithically [29]. Some iron-oxidizing bacteria do not mineralize on their surfaces [30,31], suggesting that they either have active demineralization processes, or passive processes that prevent mineralization. Iron-oxidizers have not been shown to bore, but the data provide evidence of a selection pressure to avoid mineralization in organisms typically found in hydrothermal environments. 104
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Box 1. Grazing and the pressure to bore On the present-day Earth, euendoliths are exposed to an intense grazing pressure by molluscs (e.g. Monodonta spp.) and sea urchins (Paracentrotus spp.). This pressure might have arisen early in the evolution of animals because phototrophs growing on and in rocks would have represented an early source of carbon and nutrients. Indeed, the evolution of grazing animals could have had a role in the decline of cyanobacterial stromatolites at the end of the Precambrian [32]. Grazers disaggregate and scrape away the carbonate matrix in which the euendoliths bore (Figure I). A study in French Polynesia [12] showed that grazing was intense. After two months, 22.4% of the surface area of rock had been colonized by euendoliths. After two years, grazers removed 2.3 kg m2 of fresh limestone blocks per year. Grazing was attributed to molluscs, sea urchins and herbivorous fish. After the two year period, just under 90% of the blocks had been removed by grazing. During grazing, the rocks break along the fracture planes established between the bore holes of the euendoliths, which represent the weakest regions of the rock [27]. As Figure I illustrates, the euendoliths that survive this process will be those that bore deepest. The ability to reach deep into the rock will be a function of both the ability to grow at low light intensities (deep in the rock) and the rate of boring, whereby euendoliths that bore rapidly are likely to reach below the grazing depth before the next grazing episode occurs. The intensity of grazing and the depth of scraping will determine the rate of boring required and the depth to which euendoliths must bore to escape grazing. The tendency of shallow water phototrophic borers to penetrate perpendicular to the rock, with deeper water organisms tending to bore parallel to the rock surface, is suggested to result from grazing pressure [8]. Deeper boring would be expected to result in deeper grazing. However, ultimately, the euendolith boring depth must be set by the depth at which light levels are below the minimum required for photosynthetic growth. Grazers that effectively scrape deeply and remove all euendoliths will run out of food, such that a balance must be established, potentially set by cyclical population growth.
Figure I. Schematic of the results of grazing on phototrophic euendolithic bacteria. Grazing by animals on coastal limestones and corals removes the surface layer of rock and its phototrophic euendolithic inhabitants. Only euendoliths that have bored below the grazing depth survive.
Directionality of boring Whatever evolutionary pressures are postulated they must explain the directionality of boring [i.e. the formation of welldefined tunnels (Box 2)]. Physical extremes on the surface of a rock might favour the end of filaments actively boring away from the surface into the rock, thus generating a selection pressure for tunnelling. Boring resulting from a demineralization process would be consistent with tunnelling behaviour. At least in filamentous organisms, those that evolved a way to demineralize the filament end would be able to escape the region of mineralization. Demineralizing the complete cell might be energetically unfavourable and unnecessary. One consequence of demineralization around the end of a cell, particularly a filament, would be that when it met mineral faces the microorganisms would dissolve them, forming a tunnel.
Opinion Box 2. Euendoliths in volcanic glass Microorganisms have an active role in the weathering of volcanic rocks, forming pitted and corroded granular textures, particularly on reactive volcanic glasses where zones of elemental depletion develop and silicate enrichment occurs [33–35]. Less commonly, euendolithic borings into volcanic glasses have been reported, including deep ocean pillow lavas [35–39] (Figure I). Furnes et al. suggest a dominant role for bioalteration of volcanic glasses in the upper 300 m of oceanic crust [38]. The origin of some of these borings has been controversial in that the organisms responsible for the borings have not been cultured. Abiotic ambient inclusion trails, generated by the high-pressure forcing of mineral grains such as sulphides through other grains, have been suggested as a mechanism for the abiotic production of tunnels. Research that suggests the involvement of biological activity has used numerous methods, including isotopic and elemental analysis, which taken together suggest a biological role for alteration [38]. Glass recovered by drilling from more than 250 m below the sea floor shows tunnels into altered zones that are 1–10 mm wide and penetrate to a depth of 150 mm [5]. In other settings, larger tubes up to 20 mm in diameter and with a depth of 0.5 mm have been observed [38]. Binding of the nucleic acid stain DAPI (4 0 ,6diamidino-2-phenylindole) to DNA and fluorescence signals obtained using FISH (fluorescent in situ hybridization) probes against archaea and bacteria in the tunnels suggest that organisms might be responsible for the tunnelling, although this only proves occupancy and does not prove that microorganisms created the tunnels in the first place. The binding of these dyes and probes to particles in which no minerals were detected and the binding of nucleic acid probes to the same particles as DAPI fluorescent particles suggest a biological explanation. Microprobe analysis shows the presence of carbon not associated with carbonates. Low carbon isotope values were also suggested as evidence supporting a biological role in glass alteration. The age dating of materials with biological isotopic signatures has been used to argue that the age of fossils of putative ancient microbial borers is similar to the age of the rocks [40].
Figure I. Fine boring channels in volcanic glass. Glass rim of a basalt collected by the Deep Sea Drilling Program from the Mid-Atlantic Ridge. The sample was cored from 480 m below the sea floor where the ambient temperature was approximately 20 8C. The sample is 15 million years old. The pale gray-green area is unaltered glass. The reddish-orange area is altered glass and iron oxyhydroxides. The dark zone between the altered and unaltered glass is a zone of iron accumulation. Fine branching channels extend from the dark zone into the fresh glass (M. Fisk, unpublished). Scale bar, 20 mm.
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The nutrient acquisition hypothesis does not readily account for directionality. If the nutrient gain makes it energetically worthwhile to dissolve the rock then it would be beneficial for all cells in a filament (for example) to dissolve the rock, releasing nutrients and generating a pit or cavity in the rock rather than a tunnel. On the other hand, the alteration of minerals around a cell might limit further nutrient uptake such that an actively advancing cell has a greater chance of moving into fresh mineral regions where nutrients are more readily acquired. Concluding remarks and future perspectives A range of environmental stressors and microbial physiological requirements that might act as selection pressures to encourage boring have been described here. The acquisition of nutrients, finding a niche with limited competition, selection owing to adverse conditions on the surface of rocks or the prevention of mineralization are the most promising explanations for the evolution and persistence of boring behaviour. There is no reason why these hypotheses should be mutually exclusive. A combination of these pressures might have occurred in different environments, with some pressures being more evolutionarily recent, such as the grazing pressure, which has been placed on borers in the intertidal environment since the Cambrian. To resolve the relative importance of the different factors there are several promising areas of enquiry. The elucidation of the mechanisms of boring will provide information on energetic requirements, which can be used to understand the costs, and therefore the advantages and disadvantages, of the boring habit in different environments. Further investigations on the physiological attributes and tolerances of borers will unravel what implications boring behaviour has with respect to resource acquisition and escape from environmental stressors. Unravelling the molecular biology of boring, the minimum number of genes required for boring, the origin of these genes and their possible regulatory links to processes such as nutrient acquisition will greatly assist in understanding the genetic steps that led to directional boring in rocks. Ecological approaches could better define the distribution of borers and thus the environmental characteristics that encourage boring. The matching of these data with evolutionary biological considerations of how competition, environmental stressors and resource availability have changed over time will create a better understanding of how and why (and even where) boring activity might have evolved, and how it became directional. In conclusion, boring is one of the most remarkable behaviours exhibited among microorganisms. Explaining its origin and persistence is a fascinating and important conundrum in microbial evolutionary biology. Acknowledgments We would like to thank Ferran Garcia-Pichel, Arizona State University, Martin Fisk, Oregon State University and three anonymous reviewers for critical reviews of the manuscript and valuable improvements. This paper was written under support from the Leverhulme Trust (Project No. F/00 269/N). 105
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Why are some microorganisms boring? Charles S. Cockell and Aude Herrera Centre for Earth, Planetary, Space and Astronomical Research (CEPSAR), Open University, Milton Keynes, MK7 6AA, UK
Microorganisms from diverse environments actively bore into rocks, contributing significantly to rock weathering. Carbonates are the most common substrate into which they bore, although there are also reports of microbial borings into volcanic glass. One of the most intriguing questions in microbial evolutionary biology is why some microorganisms bore. A variety of possible selection pressures, including nutrient acquisition, protection from UV radiation and predatory grazing could promote boring. None of these pressures is mutually exclusive and many of them could have acted in concert with varying strengths in different environments to favour the development of microorganisms that bore. We suggest that microbial boring might have begun in some environments as a mechanism against entombment by mineralization. Microbial boring by different microorganisms Some taxa of microorganisms are known to bore holes in rocks and this is an evolutionarily ancient characteristic (Table 1). Bore holes created by phototrophs in stromatolites from the Dahongyu Formation in China date back at least 1500 million years [1], but older borings made into 3.4 billion-year old Archean (see Glossary) volcanic glasses by microorganisms have been reported [2]. Thus the selection pressure for boring activity predates the evolution of animals. Organisms that actively bore (a process sometimes known as ‘biological corrosion’) are referred to as euendoliths, in contrast to chasmoendoliths, which grow in cracks or fissures, and cryptoendoliths, which grow in rock interstices or structural cavities inside rocks [3]. Some of the most recent discoveries of euendoliths have been new species from desert environments [4] and there have also been reports of euendolithic borings into volcanic glasses in deep ocean basalts [5]. Euendolithic activity is widely distributed among fungi, red and green algae and cyanobacteria [6,7]. Fungi need a supply of organic matter to survive and thrive, and they preferentially invade substrates where organic matter is available, such as the laminae of sea shells. In aquatic environments they tend to dominate at water depths where light levels preclude or limit the activity of boring phototrophs [8]. Fungal euendoliths also dominate in nonmarine and brackish aquatic environments and in soils [9,10]. The size of bore holes varies, depending on the organism that produced them [11] (Figure 1). Bore holes of 2–6 mm in diameter produced by the cyanobacterium Plectonema sp., and holes up to 20–40 mm in diameter produced by the chlorophyte (alga) Ostreobium sp. were found in corals in Corresponding author: Cockell, C.S. ([email protected]).
Moorea, French Polynesia [12]. Fungi often produce tunnels in rocks, which display swellings associated with sporangia in the hyphae [13]. For phototrophs the depth of boring is set by the maximum depth at which phototrophic growth can be sustained, typically on the order of 1–3 mm [14]. The distribution of different types of euendoliths, and thus their boring patterns at different depths, can be used as a basis for paleobathymetry, particularly if fossil evidence of euendoliths with known narrow depth ranges can be discriminated [15]. Boring rates in substrates can be high. For example, phototrophic euendoliths are significant contributors to biologically induced destruction of carbonate coastlines and corals, and the formation of ‘biokarst’ with bio-erosion estimates of up to 570 g CaCO3 m2 y.1 [12]. By contrast, the formation of carbonates within bore holes that cut across grains can also cause the cementation of the grains, potentially giving euendoliths a constructive role in stromatolite formation [16]. The mechanisms for microbial boring are not yet fully understood [17] and are probably different for different taxa. Possible mechanisms for cyanobacterial boring include the release of acid that would dissolve the rock or the sequestration of calcium ions using a calcium pump, which would lead to the dissolution of calcium carbonates. Fungal dissolution of minerals probably occurs by the production of acid [17]. In addition to the challenge of developing a mechanistic understanding of how boring occurs, an evolutionary question remains unanswered – why do microorganisms bore into rocks? In this article, we examine some of the selective advantages that organisms might obtain by boring into rocks. We
Glossary Archean: geological eon corresponding to the time period between 4 billion and 2.5 billion years ago. Biokarst: landscapes formed by biological activities. Biological corrosion: the destruction of rock by microbial borers. Cambrian: geological period corresponding to the time period between 543 and 490 million years ago. Chasmoendoliths: organisms that live within cracks or fissures in rocks. Cryptoendoliths: organisms that live within the interstices or structural cavities in rocks. Euendoliths: organisms that actively bore into rock. Feldspars: a class of aluminium-containing silicate rocks. Ichnofossils: trace fossils of life left in sediments. Paleobathymetry: the study of ancient water depth. PAR: Photosynthetically active radiation; radiation that can drive photosynthesis. Phototrophs: organisms that acquire their energy from photosynthesis. Proterozoic: geological eon corresponding to the time period between 2.5 billion and 543 million years ago. Stromatolite: A widely distributed structure consisting of laminated carbonate or silicate rocks, produced by the binding, precipitation or trapping of sediment by groups of microorganisms, primarily cyanobacteria.
0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.12.007 Available online 14 February 2008
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Table 1. Examples of ancient euendolithic microborings or ichnofossilsa Location Strelley Pool Formation (Pilbara), Australia Barberton, South Africa Barberton, South Africa Hebei, China Eleonore Bay Group, central Greenland East Greenland Emei, China Scania, Sweden Henson Gletscher Formation, North Greenland Holm Dal Formation, North Greenland
Age 3.4 Ga 3.4–2.5 Ga 3.5–3.4 Ga 1.7 Ga Late Proterozoic 700–800 Ma Early Cambrian Early Cambrian Early Cambrian Early middle Cambrian Late middle Cambrian
Environment S V V C C C C C C C C
Refs [41,42] [2] [40] [1] [43,44] [45] [46] [47] [48] [49] [50]
a Symbols: Ga, billion years ago; Ma, million years ago; V, refers to volcanic environment of microborings; C, carbonate; S, other sedimentary rocks (although these other rocks might themselves be part of a volcanic setting, e.g. sandstones).
consider some of the challenges faced by microorganisms living on the surface or within the interstices of rocks. This can help to identify the pressures that might have favoured microorganisms that actively bore. As well as reviewing existing discourses, we also suggest that cell demineralization might have been one early cause of rock dissolution that ultimately could have led to rock tunnelling. Selection pressures that make boring advantageous Acquisition of nutrients Nutrient availability has been suggested as a possible factor controlling the distribution of microborers [18]. Although boring activity can potentially release nutrients from minerals, organisms within bore holes can be deprived of nutrients compared with those on the surface. For example, the availability of nutrients provided by flowing seawater to organisms growing on the surface could be greater than nutrient availability for organisms within a tube, where exchange is slowed by diffusion. Expending energy to bore into a rock will only be worthwhile if the substrate possesses a growth-limiting nutrient or trace element not found within the circulating water around the rock or if the energy derived from the rock equals or outweighs the energy put into boring. In evolving a mechanism for rock dissolution to acquire nutrients, microorganisms would be involved in an early form of euendolithic boring. For example, Garcia-Pichel
Figure 1. Euendolithic borings into a snail shell. A backscattered electron image from a thin section of a snail shell from the South Coast of England (Beer, Devon; C.Cockell, unpublished). The borings are evident as 10 micron-wide tunnels into the top 100 mm of the shells and are ubiquitous in snails living in the intertidal environment in this region. The borings are caused by cyanobacteria, including Plectonema sp. Scale bar, 100 mm.
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suggests that a calcium pump might be involved in cyanobacterial euendolithic boring [17]. One could speculate that originally the uptake of calcium as a nutrient from solution by organisms growing near carbonates would have the side-effect of changing the chemical equilibrium at the surface of the mineral to favour dissolution. The organism would then grow into the cavity that is created. In nutrientpoor environments, rock dissolution in this way would have been directly selected for and later it might have led to boring. This hypothesis would hold for any mechanism of nutrient acquisition that resulted in rock dissolution, including, for example, the production of organic acids by fungi leading to selection for euendolithic behaviour in nutrient-poor environments. This hypothesis would be consistent with the observations of fungal boring into soil feldspars. In a study of soil chronosequences [10], Smit and co-workers showed that fungal boring into feldspars began after more nutrient-rich minerals, such as biotite and hornblende, had been destroyed. However, based on the observation that the boring into feldspars was independent of feldspar abundance, they also suggested that tunnelling might be correlated with fungal density. A response to competition for other resources In many environments (such as deep water) boring into rock would reduce availability of photosynthetically active radiation (PAR) because the rock substrate would reduce exposure to PAR. Limitation of PAR in these circumstances would provide a selection pressure for upward movement, not downward boring. However, in high-light environments where surface PAR might cause photoinhibition, boring could provide an advantage. Euendolithic boring might be advantageous in competition for space, particularly within the interstices or cracks of rocks where biofilm development would be restricted. Although boring is energetically costly, if the cells survived in the newly exploited habitat where there was little or no competition, then the trait would persist in the absence of a selection pressure against it. Protection from physical extremes Microorganisms living inside a rock can be protected from desiccation and extreme temperatures on the surface of the rock [19]. The improved micro-environmental conditions experienced by cryptoendolithic lichens and their associated biota compared with conditions on rock surfaces have
Opinion been measured in extreme hot and cold deserts, such as the Negev desert of Israel and the Dry Valleys of the Antarctic [19]. In the Antarctic, cryptoendolithic lichens are protected from rapid freeze–thaw fluctuations that render the surface of the rock inhospitable. Where the porosity of the material is low, active boring would provide shielding similar to that experienced by chasmo- and cryptoendoliths. Euendoliths within carbonate materials in the intertidal zone are potentially exposed to desiccation cycles and moisture-retaining rock borings probably reduce or eliminate the time that organisms are exposed to desiccation. Euendoliths living in carbonate rocks near alpine glaciers are thought to have evolved to regulate their boring activity so as to prevent the destruction of the substrate, which is proposed to act as an environmental shield [20]. Protection from UV radiation Ultraviolet (UV) radiation is detrimental to various physiological processes in exposed cyanobacteria and fungi [21] and the interior of rocks can provide protection from UV radiation [22]. By tunnelling into rocks, an organism can gain the advantage of the UV-protecting properties of the rock surrounding it (Figure 2). The selection pressure to escape or mitigate UV radiation might have been stronger on the early Earth, specifi-
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cally prior to the oxygenation of the atmosphere approximately 2.4 billion years ago when the lack of oxygen might have also meant a lack of stratospheric ozone, meaning that wavelengths of UV radiation greater than 200 nm would have reached the surface of the Earth – in the absence of other atmospheric UV screens [23]. Protection from UV radiation as an initial selection pressure for boring is implausible for surface-dwelling organisms because it would require boring in the first place to obtain any beneficial screening. Boring to escape from UV radiation is also unlikely to be a plausible hypothesis for organisms inhabiting the deep oceans where there is no UV radiation. When the mechanisms of microbial boring are better understood it will be important to determine if the energetic cost of boring into rock is less than the energetic cost of repairing UV radiation damage or producing screening compounds to protect against UV damage on the surface. Prevention of detachment from rock substrates An organism that bores into a rock will be less likely to be detached by water currents. Many of the phototrophs that bore into rocks live in the intertidal zone, which experiences dynamic water regimens. Similarly, organisms living on volcanic glasses in hydrothermal environments are subjected to water flow over the surface of rocks, although
Figure 2. Protection from UV radiation in the euendolithic habitat. A rock sample exposed to UV radiation to demonstrate the efficacy of UV shielding. (a) Hyella sp. that occupy the interior of sandstone cliffs from Beer, Devon in England. (b) Surface of cliff material showing colonization by a diverse consortium of cyanobacteria (including Plectonema and Chroococcidiopsis spp.). (c) Rock sample exposed to 254 nm germicidal UV radiation (9.93 kJ cm2) after 48 hours in BG-11 medium. Note disappearance of surface biofilm. (d) Rock exposed to UV radiation incubated in BG-11 medium for five weeks. Cyanobacteria emerge from bore holes in the interior of the rock (arrow) in a localized manner and recolonize the surface. UV radiation of 254 nm is not found on the surface of the present-day Earth, but it could have penetrated to the surface of the early Earth. Scale bar, 10 mm in (a) and 0.5 cm in (b), (c) and (d) (C. Cockell, unpublished).
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Opinion within cracks this is probably less important. Many organisms successfully attach to the surface of rocks as epilithic biofilms in rivers, streams and the intertidal zone without boring into them [24] and euendolithic boring is probably more energetically costly than surface attachment (although the energy put into boring into rocks with high surface water-flow could be compensated by the associated greater nutrient flux). Perry [6] did not find a correlation between the extent of microboring and wave energy levels in Discovery Bay, North Jamaica, suggesting that waterflow was not a selection pressure in this environment. Escape from predation Growing into a rock would provide some protection from predation by macroscopic organisms, such as many fish and snails, because they are unable to penetrate the rock substrate (Box 1). However, predation is also possible by microscopic organisms. Euendolithic algae are extensively predated by euendolithic fungi [25]. The fungal hyphae grow around the algal filaments, eventually penetrating them and exuding dark tannin-like substances that turn the green algal bands in the rock black. Euendolithic fungal attack of coral algae leads to complex banding patterns within the corals. Thus, the euendolithic strategy does not provide an escape from predation for present-day algae [25,26]. On the Archean Earth, before the evolution of eukaryotes, fungal predation would not have constituted a selective pressure. However, fungal predation reflects the more general principle that bore holes do not provide escape from microbial attack per se, assuming that the attackers do not need to be able to bore themselves. For the same reasons it seems unlikely that filaments embedded in rocks would escape attack by phages, which could in principle attack along the length of the filament. Escape from mineralization We propose another hypothesis for the origin of boring behaviour. Microbial borings have been recorded most extensively in substrates associated with high mineralization rates [27]. This raises the possibility that euendolithic behaviour is a side-effect of an active demineralization process. In particular, cyanobacterial photosynthesis causes the precipitation of carbonates, and yet cyanobacteria are the best-characterized microbial borers. Organisms that are associated with hydrothermal regions [28] must contend with high rates of mineralization that threaten to engulf and immobilize cells. One side-effect of the innovation of a demineralization process would be the dissolution of the substrate to which the organism was attached. Mineralization on the top surface of euendolithic algae growing on rocks, but not around the cells within the rock carrying out boring, has been observed, demonstrating the presence of a mineralization challenge to the same organisms growing euendolithically [29]. Some iron-oxidizing bacteria do not mineralize on their surfaces [30,31], suggesting that they either have active demineralization processes, or passive processes that prevent mineralization. Iron-oxidizers have not been shown to bore, but the data provide evidence of a selection pressure to avoid mineralization in organisms typically found in hydrothermal environments. 104
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Box 1. Grazing and the pressure to bore On the present-day Earth, euendoliths are exposed to an intense grazing pressure by molluscs (e.g. Monodonta spp.) and sea urchins (Paracentrotus spp.). This pressure might have arisen early in the evolution of animals because phototrophs growing on and in rocks would have represented an early source of carbon and nutrients. Indeed, the evolution of grazing animals could have had a role in the decline of cyanobacterial stromatolites at the end of the Precambrian [32]. Grazers disaggregate and scrape away the carbonate matrix in which the euendoliths bore (Figure I). A study in French Polynesia [12] showed that grazing was intense. After two months, 22.4% of the surface area of rock had been colonized by euendoliths. After two years, grazers removed 2.3 kg m2 of fresh limestone blocks per year. Grazing was attributed to molluscs, sea urchins and herbivorous fish. After the two year period, just under 90% of the blocks had been removed by grazing. During grazing, the rocks break along the fracture planes established between the bore holes of the euendoliths, which represent the weakest regions of the rock [27]. As Figure I illustrates, the euendoliths that survive this process will be those that bore deepest. The ability to reach deep into the rock will be a function of both the ability to grow at low light intensities (deep in the rock) and the rate of boring, whereby euendoliths that bore rapidly are likely to reach below the grazing depth before the next grazing episode occurs. The intensity of grazing and the depth of scraping will determine the rate of boring required and the depth to which euendoliths must bore to escape grazing. The tendency of shallow water phototrophic borers to penetrate perpendicular to the rock, with deeper water organisms tending to bore parallel to the rock surface, is suggested to result from grazing pressure [8]. Deeper boring would be expected to result in deeper grazing. However, ultimately, the euendolith boring depth must be set by the depth at which light levels are below the minimum required for photosynthetic growth. Grazers that effectively scrape deeply and remove all euendoliths will run out of food, such that a balance must be established, potentially set by cyclical population growth.
Figure I. Schematic of the results of grazing on phototrophic euendolithic bacteria. Grazing by animals on coastal limestones and corals removes the surface layer of rock and its phototrophic euendolithic inhabitants. Only euendoliths that have bored below the grazing depth survive.
Directionality of boring Whatever evolutionary pressures are postulated they must explain the directionality of boring [i.e. the formation of welldefined tunnels (Box 2)]. Physical extremes on the surface of a rock might favour the end of filaments actively boring away from the surface into the rock, thus generating a selection pressure for tunnelling. Boring resulting from a demineralization process would be consistent with tunnelling behaviour. At least in filamentous organisms, those that evolved a way to demineralize the filament end would be able to escape the region of mineralization. Demineralizing the complete cell might be energetically unfavourable and unnecessary. One consequence of demineralization around the end of a cell, particularly a filament, would be that when it met mineral faces the microorganisms would dissolve them, forming a tunnel.
Opinion Box 2. Euendoliths in volcanic glass Microorganisms have an active role in the weathering of volcanic rocks, forming pitted and corroded granular textures, particularly on reactive volcanic glasses where zones of elemental depletion develop and silicate enrichment occurs [33–35]. Less commonly, euendolithic borings into volcanic glasses have been reported, including deep ocean pillow lavas [35–39] (Figure I). Furnes et al. suggest a dominant role for bioalteration of volcanic glasses in the upper 300 m of oceanic crust [38]. The origin of some of these borings has been controversial in that the organisms responsible for the borings have not been cultured. Abiotic ambient inclusion trails, generated by the high-pressure forcing of mineral grains such as sulphides through other grains, have been suggested as a mechanism for the abiotic production of tunnels. Research that suggests the involvement of biological activity has used numerous methods, including isotopic and elemental analysis, which taken together suggest a biological role for alteration [38]. Glass recovered by drilling from more than 250 m below the sea floor shows tunnels into altered zones that are 1–10 mm wide and penetrate to a depth of 150 mm [5]. In other settings, larger tubes up to 20 mm in diameter and with a depth of 0.5 mm have been observed [38]. Binding of the nucleic acid stain DAPI (4 0 ,6diamidino-2-phenylindole) to DNA and fluorescence signals obtained using FISH (fluorescent in situ hybridization) probes against archaea and bacteria in the tunnels suggest that organisms might be responsible for the tunnelling, although this only proves occupancy and does not prove that microorganisms created the tunnels in the first place. The binding of these dyes and probes to particles in which no minerals were detected and the binding of nucleic acid probes to the same particles as DAPI fluorescent particles suggest a biological explanation. Microprobe analysis shows the presence of carbon not associated with carbonates. Low carbon isotope values were also suggested as evidence supporting a biological role in glass alteration. The age dating of materials with biological isotopic signatures has been used to argue that the age of fossils of putative ancient microbial borers is similar to the age of the rocks [40].
Figure I. Fine boring channels in volcanic glass. Glass rim of a basalt collected by the Deep Sea Drilling Program from the Mid-Atlantic Ridge. The sample was cored from 480 m below the sea floor where the ambient temperature was approximately 20 8C. The sample is 15 million years old. The pale gray-green area is unaltered glass. The reddish-orange area is altered glass and iron oxyhydroxides. The dark zone between the altered and unaltered glass is a zone of iron accumulation. Fine branching channels extend from the dark zone into the fresh glass (M. Fisk, unpublished). Scale bar, 20 mm.
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The nutrient acquisition hypothesis does not readily account for directionality. If the nutrient gain makes it energetically worthwhile to dissolve the rock then it would be beneficial for all cells in a filament (for example) to dissolve the rock, releasing nutrients and generating a pit or cavity in the rock rather than a tunnel. On the other hand, the alteration of minerals around a cell might limit further nutrient uptake such that an actively advancing cell has a greater chance of moving into fresh mineral regions where nutrients are more readily acquired. Concluding remarks and future perspectives A range of environmental stressors and microbial physiological requirements that might act as selection pressures to encourage boring have been described here. The acquisition of nutrients, finding a niche with limited competition, selection owing to adverse conditions on the surface of rocks or the prevention of mineralization are the most promising explanations for the evolution and persistence of boring behaviour. There is no reason why these hypotheses should be mutually exclusive. A combination of these pressures might have occurred in different environments, with some pressures being more evolutionarily recent, such as the grazing pressure, which has been placed on borers in the intertidal environment since the Cambrian. To resolve the relative importance of the different factors there are several promising areas of enquiry. The elucidation of the mechanisms of boring will provide information on energetic requirements, which can be used to understand the costs, and therefore the advantages and disadvantages, of the boring habit in different environments. Further investigations on the physiological attributes and tolerances of borers will unravel what implications boring behaviour has with respect to resource acquisition and escape from environmental stressors. Unravelling the molecular biology of boring, the minimum number of genes required for boring, the origin of these genes and their possible regulatory links to processes such as nutrient acquisition will greatly assist in understanding the genetic steps that led to directional boring in rocks. Ecological approaches could better define the distribution of borers and thus the environmental characteristics that encourage boring. The matching of these data with evolutionary biological considerations of how competition, environmental stressors and resource availability have changed over time will create a better understanding of how and why (and even where) boring activity might have evolved, and how it became directional. In conclusion, boring is one of the most remarkable behaviours exhibited among microorganisms. Explaining its origin and persistence is a fascinating and important conundrum in microbial evolutionary biology. Acknowledgments We would like to thank Ferran Garcia-Pichel, Arizona State University, Martin Fisk, Oregon State University and three anonymous reviewers for critical reviews of the manuscript and valuable improvements. This paper was written under support from the Leverhulme Trust (Project No. F/00 269/N). 105
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