Chapter Three - Fungi in Deep Subsurface Environments

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Abstract

The igneous crust of the oceans and the continents represents the major part of Earth's lithosphere and has recently been recognized as a substantial, yet underexplored, microbial habitat. While prokaryotes have been the focus of most investigations, microeukaryotes have been surprisingly neglected. However, recent work acknowledges eukaryotes, and in particular fungi, as common inhabitants of the deep biosphere, including the deep igneous provinces. The fossil record of the subseafloor igneous crust, and to some extent the continental bedrock, establishes fungi or fungus-like organisms as inhabitants of deep rock since at least the Paleoproterozoic, which challenges the present notion of early fungal evolution. Additionally, deep fungi have been shown to play an important ecological role engaging in symbiosis-like relationships with prokaryotes, decomposing organic matter, and being responsible for mineral weathering and formation, thus mediating mobilization of biogeochemically important elements. In this review, we aim at covering the abundance and diversity of fungi in the various igneous rock provinces on Earth as well as describing the ecological impact of deep fungi. We further discuss what consequences recent findings might have for the understanding of the fungal distribution in extensive anoxic environments and for early fungal evolution.

Introduction

The igneous crust represents a major portion of Earth's habitable volume, yet its inhabitants are significantly underexplored compared to life on Earth's surface (Edwards, Fisher, & Wheat, 2012). The oceanic crust comprises about 30% of the Earth's total crust volume and is inhabited to at least 1 km depth below seafloor, probably more (Cogley, 1984; Orcutt, Sylvan, Knab, & Edwards, 2011). The continental crust represents the remaining 70% of Earth's total crust volume and is known to be inhabited to at least 5 km depth (Cogley, 1984; Lin et al., 2006; Pedersen et al., 2008). However, these known depths are restricted by limitations in sampling; the actual depth maximum for life is still unknown. Although the microbial processes are relatively slow because of the low energy supply (Wu et al., 2016), the deep ecosystems comprise a substantial biomass and are proposed to play an important role in the energy cycling of Earth (McMahon & Parnell, 2014). Estimates from the deep continental subsurface suggest that this environment accommodates up to 19% of Earth's biomass (McMahon & Parnell, 2014), while various estimates of the biomass in marine sediments have ranged between 30% and less than 1% of Earth's biomass (Colwell & D′Hondt, 2013), illustrating the difficulties in estimating biomass of such a remote and inaccessible environment. Estimations of the igneous oceanic crust have so far not been attempted.

Most investigations of the deep biosphere have been focused on prokaryotes (archaea and bacteria), while microeukaryotes have been more or less neglected. Lately, however, there has been a change in view, and especially fungi have been recognized as frequent inhabitants of deep subterranean settings. There is also a growing body of literature on fossilized fungi from deep environments, which shows fungi or fungus-like organisms as long-term residents of these habitats (Bengtson et al., 2017; Eickmann, Bach, Kiel, Reitner, & Peckmann, 2009; Ivarsson et al., 2012; Ivarsson, Bengtson, & Neubeck, 2016; Ivarsson, Holm, & Neubeck, 2015; Peckmann, Bach, Behrens, & Reitner, 2008; Schumann, Manz, Reitner, & Lustrino, 2004). It has become evident that fungi play an ecological key role in deep environments, as they are involved in abundant mineral weathering and formation, cycling of biogeochemically important elements like C, N, P, Fe, Mn, and S, as well as being engaged in close relationships with prokaryotes. The fungal diversity is surprisingly high and represent all major fungal phyla (Ivarsson, Bengtson, et al., 2016; Sohlberg et al., 2015), including several deep-branching novel lineages (Le Calvez, Burgaud, Mahe, Barbier, & Vandenkoornhuyse, 2009). Also, the presence of fungi in these environments, which have to be considered rather extreme from a fungal point of view, challenges the current view of fungal metabolism, tolerance for extreme conditions like temperature, dependence on oxygen, and their early evolution. Exploration of deep fungi will result in a more rich and diverse view of fungi, and hopefully enhance our knowledge of a group of organisms that is grossly underexplored. This chapter aims to cover the current knowledge about fungi in deep subsurface igneous settings and glance at the future challenges and goals.

The definition of the deep biosphere tends to vary a lot, except in the sense that it covers life at great depths. Commonly, the deep biosphere is taken to comprise ecosystems in both oceanic and continental settings beneath the rhizosphere or bioturbated zone. The habitable depth is limited by space (rock porosity), the availability of water, and the maximum viable temperature depending on the local geothermal gradient (Heim, 2011). In this chapter, we will focus on the igneous portions of the subsurface.

All rock dwelling microorganisms are termed endoliths. Depending on their spatial relationship to, and interaction with, the host rock and minerals they are divided into subcategories. Microorganisms that actively penetrate into rock interiors and create habitable cavities are called euendoliths. Microorganisms that invade preexistent fissures and cracks are called chasmoendoliths, and microorganisms that invade preexistent structural cavities are called cryptoendoliths (Golubic, Friedmann, & Schneider, 1981; Ivarsson, Holm, et al., 2015).

A number of reviews have been produced that cover both the continental and the oceanic deep realm, the latter with emphasis on sediments (Colwell & D′Hondt, 2013; Heim, 2011; Orcutt et al., 2011). Our understanding of the deep biosphere has come a long way since the first investigations of cells in water and oil pumped from oil wells in the 1920s (Bastin, Greer, Merritt, & Moulton, 1926), or from the first short cores from the seafloor in the 1930s showing an increase of anaerobes and decrease of aerobes with depths (Zobell & Anderson, 1936). The last 30 years have involved an upswing in literature much due to the establishment of large-scale underground facilities for nuclear waste repository research, or the establishment of international deep drilling programs such as ICDP and DCDP, later ODP and IODP.

The subsurface realm is everything but a homogenous environment. It is highly variable and characterized by physical and chemical gradients. Large-scale geology like plate tectonics as well as local geochemical conditions like the composition of hydrothermal fluids gives rise to specialized macro- or microniches with ecologically different prerequisites. The microbial populations differ depending on whether they live in a serpentinized rock, a geothermally active area, or in a granite aquifer. Common for all metabolically active life forms is that they require a gradient (Kappler et al., 2005). Living cells may survive environments lacking gradient through becoming thermodynamically static by entering dormant states, or by transiting through harsh environments encased in substrates such as amber or halites (Colwell & D′Hondt, 2013). However, if cells are active in any sense they must take advantage of a thermodynamical disequilibrium (Colwell & D′Hondt, 2013). Thermodynamic disequilibria are established in any environment by one or several chemical or physical regimes that determine the supply of electron donors and acceptors. Under such conditions microbes can make a living. If conditions are ample in a large volume of Earth then there may be enough active cells in such pockets to make a global biogeochemical difference through their collective activities.

In the dark subsurface environment, in the absence of sunlight, microorganisms gain energy from coupling of reducing and oxidizing (redox) reactions that are thermodynamically favorable. The base of the deep biosphere is thought to consist of chemoautotrophs, organisms that obtain energy by the oxidation of electron-donating molecules in their environment in contrast to photoautotrophs that utilize solar energy. Organisms that use inorganic substrates like CO2 and minerals to obtain energy are called lithoautotrophs. The opposite to autotrophs are heterotrophs, organisms that are unable to synthesize their own carbon-based compounds from inorganic sources and, hence, feed on organic carbon produced by, or available in, other organisms.

The most accessible electron donors in subsurface settings are H2, CH4, H2S, S0, S2O32, Fe2 +, Mn2 +, NH4+, NO2, and organic matter. Possible electron acceptors are CO2, SO42, or O2. Possible metabolic pathways differ depending on in what type of host rock the microorganisms exist. Basalt, which makes up the majority of the oceanic crust, is comprised of roughly 9% FeO and 0.1% each of MnO and S. Whereas alteration of ultramafic rocks (serpentinization) produces high pH fluids poor in Fe and S but highly enriched in H2, CH4, and simple hydrocarbons, alteration of basalt results in acidic fluids enriched in reduced Fe and S compounds and to a smaller extent in H2 and CH4 (Bach & Edwards, 2004). Reduced Fe, Mn, and S components are, thus, the most likely energy sources for microorganisms in basalt systems, while H2 and CH4 are the most bioavailable compounds in ultramafic settings. This is supported by molecular analyses and cultivation approaches on seafloor-exposed basalt (Ivarsson, Holm, et al., 2015).

In felsic rock systems, Mn- and Fe-oxidizing communities live in near-surface aquifers in the presence of O2, and below the redox front the most significant communities are (with increasing depth) manganese reducers, iron reducers, sulfate reducers, and methanogens (Hallbeck & Pedersen, 2008; Pedersen et al., 2008). There is a shift from heterotrophic processes (fueled by descending dissolved organic carbon) to autotrophic, probably hydrogen-based regime (Wu et al., 2016). Nitrate-reducing bacteria and acetogens are also important (Hallbeck & Pedersen, 2008).

Microorganisms engaged in an endolithic lifestyle are dependent on open pore space to dwell in. Igneous rocks contain various amounts of pore space in the form of fissures, cracks, or vesicles depending on their composition, origin, age, and geological history. Such pores often form interconnected networks of pore space for microorganisms to migrate through and colonize. The oceanic crust, for instance, consists of an upper layer characterized by extensive fracturing, about 10% porosity, and with permeabilities of about 10 12 to 10 15 m2 (Bach & Edwards, 2004; Orcutt et al., 2011). Fractures created by tension release or quick cooling occur with varying size and frequency, as do vesicles as a result of pressure release during magma extrusion. Thus, subseafloor basalts contain coherent systems of interconnected microfractures and vesicles in which seawater and hydrothermal fluids circulate. Roughly 60% of the oceanic crust is hydrologically active, and the total fluid volume that is held within the oceanic crust corresponds to 2% of the total ocean (Orcutt et al., 2011). The entire water volume of the ocean circulates through the oceanic igneous crust every 105–107 years, which means that the oceanic igneous crust is the largest aquifer system on Earth (Fisher & Becker, 2000; Orcutt et al., 2011). Microorganisms are passively transported or actively migrating through this system wherever pore space and fluid flow permit. The host rock and secondary mineralizations of the fracture walls are used for colonization and anchoring of microbial communities (Fig. 1, Fig. 2), and the minerals of the host rock can be used as energy sources for certain lithoautotrophic microorganisms. The continental crystalline bedrock is generally of low porosity (at most a couple of percent) and groundwater flow and microbial activity are mainly restricted to interconnected networks of fractures, formed and reactivated episodically by tectonic forces (e.g., extension, compression), pressure release close to the ground surface, and cooling (Drake, Tullborg, & Page, 2009). With age, pore space becomes filled by secondary mineralizations, which will thwart or eventually even stop fluid flow and, thus, microbial migration.

Studies of fossilized communities in both oceanic and continental igneous rock have shown that the initial stages of microbial colonization are characterized by the formation of a biofilm that is laid down on the walls of the open pore space and usually cover the entire surface. This initial biofilm can be formed either by fungi or by prokaryotes (Ivarsson, Holm, et al., 2015). A fungal biofilm is usually relatively smooth, between 10 and 30 μm thick, from which fungal sporophores or hyphae protrude. Further growth of hyphae into the open pore space will form complex mycelia, which can eventually fill the entire pore space. Prokaryotic initial biofilms are usually different in nature, as they are built up in cycles overgrowing each other, resulting in a layered microstromatolitic structure. Such microstromatolites usually consist of oxides of iron or manganese and are interpreted as the result of iron- or manganese-oxidizing bacteria (Bengtson et al., 2014; Ivarsson, Bengtson, et al., 2015; Ivarsson, Holm, et al., 2015). Bacterial biofilms can, later on, be overgrown by fungal biofilms and mycelia that graze the prokaryotic biomass (Ivarsson, Bengtson, et al., 2015). There are also examples of a type of microstromatolites called Frutexites, formed by iron-oxidizing bacteria, which use the fungal mycelium as a framework for growth (Bengtson et al., 2014).

Clearly, all these microbial communities are dependent on and spatially controlled by the physical nature of their habitats. The host rock and secondary mineralizations shape the habitable space. However, the microorganisms also interact with their close environment by active biomediated weathering. Fungal hyphae have been reported to abundantly dissolve and penetrate secondary minerals like zeolites and carbonates, forming cavities or tunnel-like structures (Bengtson et al., 2014; Drake, Heim, et al., 2017; Drake, Ivarsson, et al., 2017; Ivarsson, Bengtson, et al., 2015). Growth of microstromatolites has also been shown to dissolve carbonates, as formation of iron oxides lower pH and carbonates will passively dissolve in contact with the stromatolites (Bengtson et al., 2014).

The microbial activity also results in mineral formation like carbonates, oxides, sulfides, or sulfates. Fossilization of microbial consortia goes through mineralization of iron oxides and clays that will eventually fill voids, to various degrees. Fungi can also trigger zeolite formation directly on their hyphae, which function as nuclei for crystal growth. It is reasonable to assume that microbially mediated weathering of the rock would mobilize elements like Si, Al, Fe, Mn, and Mg, and stimulate the formation of zeolites, clays, or even oxides. Fungi are known from soils to form oxalates on their hyphae and, even though oxalates have not yet been observed in subsurface environments, it appears to be a viable process.

Section snippets

Fungi in Deep Continental Environments

The presence of microorganisms in continental subsurface environments has been known since the 1930s, but from the 1990s and onward there has been a tremendous increase in deep-biosphere investigations in crystalline basement (Heim, 2011; Pedersen, 1993, Pedersen, 1997). One major reason for this is the planning and construction of final repositories for spent nuclear fuel in European countries like Sweden, Finland, United Kingdom, and Germany as well as in the United States (Boivin-Jahns,

Fungi in the Oceanic Crust

Coordinated scientific drilling and exploration have during the last three decades recognized a deep biosphere in the deep-sea sediments and a seafloor microbial biota that was previously unknown (Schrenk, Huber, & Edwards, 2009). So far, the emphasis has been on prokaryotes, but, although neglected at the beginning, the presence of eukaryotes including fungi in these environments is now being investigated (Orsi, Biddle, & Edgcomb, 2013; Orsi, Edgcomb, et al., 2013). It is evident that fungi

Metabolic Pathways and Cycling of Elements in the Subsurface Realm

The presence of fungi in subsurface environments raises questions regarding metabolic pathways, access to bioavailable elements, metals, and carbon sources (Ivarsson et al., 2012; Schumann et al., 2004). Being heterotrophs, fungi need a constant supply of carbohydrates for their metabolism. Marine sediments are relative rich in bioavailable carbon and nutrients, but in a nutrient-poor environment such as the oceanic or continental igneous crust, occurrence of accessible organic compounds is

The Igneous Crust: A Long-Standing Habitat

The ocean crust has served as a habitat for fungi or fungus-like mycelium-forming microorganisms since, at least, the Paleoproterozoic (Bengtson et al., 2017). During this long period of time the planet went through major geological, biological, and chemical transitions like the oxygenation of the oceans and the atmosphere and the associated rise of phototrophs and eukaryotes; the global ice ages, the Cambrian explosion of animal life, and the colonization of land by fungi, plants, and animals.

Future Prospects

The recurrent preservation of fossil deep-biosphere biotas in igneous rocks was until recently unsuspected, and little attention had therefore been given to paleontological investigation of these environments. Particularly surprising was the almost ubiquitous presence in such rocks of fungi and fungus-like organisms, often in association with symbiotic prokaryotes. We are now in the beginning of an exploratory journey into this realm. The early results are very encouraging, showing that voids

Acknowledgments

We thank Marianne Ahlbom at the Department of Geological Sciences, Stockholm University, for assistance with ESEM analyses, Veneta Belivanova at the Department of Palaeobiology, Swedish Museum of Natural History, and Federica Marone at the Paul Scherrer Institute, Switzerland, for assistance with SRXTM analyses. This work was supported by the Swedish Research Council (Contracts No. 2010-3929, 2012-4364, and 2013-4290), Danish National Research Foundation (DNRF53), Paul Scherrer Institute

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