Life in the Ocean Crust

Life in the Ocean Crust

Chapter 2.5 Life in the Ocean Crust: Lessons from Subseafloor Laboratories Beth N. Orcutt,1,* Katrina J. Edwards2 1Bigelow Laboratory for Ocean Scien...

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Chapter 2.5

Life in the Ocean Crust: Lessons from Subseafloor Laboratories Beth N. Orcutt,1,* Katrina J. Edwards2 1Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA; 2Department of Earth Sciences, and Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA *Corresponding author: E-mail: [email protected]

2.5.1 INTRODUCTION One of the major discoveries of the scientific ocean drilling program over the past three decades is the confirmation of active and abundant life “buried alive” in the marine “deep biosphere,” including deeply buried sediment and igneous oceanic crust (IODP, 2001; IODP, 2011). Following pioneering studies using deep marine sediment that confirmed the biological production of methane (Hoehler, Borowski, Alperin, Rodrigiuez, & Paull, 2000; ­Oremland, ­Culbertson, & Simoneit, 1982) and visualized microbial cells (Cragg et al., 1990; Cragg, Harvey, Fry, Herbert, & Parkes, 1992; Cragg & Kemp, 1995; Parkes et al., 1994), subsequent dedicated ocean drilling research confirmed the widespread existence of a marine deep biosphere (D’Hondt et al., 2004; D’Hondt, ­Rutherford, & Spivack, 2002; Inagaki et al., 2006). These studies lead to a revolutionary idea that the sedimentary marine deep biosphere might contain the majority of Earth’s microorganisms and up to a third of all life on Earth (Parkes, Cragg, & Wellsbury, 2000; Whitman, Coleman, & Wiebe, 1998); however, these early estimates have since been revised downward by about an order of magnitude following increased sampling of a larger diversity of sedimentary systems, including the largely oligotrophic sedimentary deep biosphere (­Kallmeyer, Pockalny, Adhikari, Smith, & D’Hondt, 2012). Though diminished in scope, the novel function and genetic diversity of the sedimentary marine deep biosphere are still significant (Hinrichs & Inagaki, 2012; J­ ørgensen, 2012). By comparison, the existence and extent of a deep biosphere hosted in the igneous oceanic crust—the hard rock that resides beneath sediment or exposed near mid-ocean ridge spreading centers—are considerably less understood. Developments in Marine Geology, Volume 7. http://dx.doi.org/10.1016/B978-0-444-62617-2.00007-4 Copyright © 2014 Elsevier B.V. All rights reserved.

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Igneous oceanic crust is hydrologically active, with the entire volume of the oceans circulating through the rocky, porous, fractured crust on the order of hundreds of thousands of years (Wheat, McManus, Mottl, & Giambalvo, 2003). The crustal reservoir is also roughly 10 times larger than the sedimentary deep biosphere (Orcutt, Sylvan, Knab, & Edwards, 2011)—suggesting that this realm may harbor a larger abundance of microorganisms than sediment. Early molecular studies implicated microbial life in crustal samples (e.g., Giovannoni, Fisk, Mullins, & Furnes, 1996), but confirmation of an extensive biosphere in deep oceanic crust has been a challenging and long process. Unlike oceanic crust, deep sediment is readily sampled though the ocean drilling program, and previous studies have demonstrated that sediment can be recovered relatively uncontaminated and used for molecular biological surveys of life (Smith, Spivack, Fisk, H ­ aveman, &S ­ taudigel, 2000). There are significant challenges to sampling and study of igneous oceanic crust, in contrast. Coring in upper basement is notoriously difficult with the current drilling technologies used on the RV JOIDES Resolution, the drilling vessel most commonly used to sample igneous oceanic crust. ­Sample recovery during oceanic drilling is also low by comparison to sediment coring, with generally less than 25% of drilled material being recovered in cores (Edwards et al., 2012). Also, the sample material recovered generally skews to more massive rock that is easier to drill than highly fractured, altered, porous, and permeable basalts, although the latter is the more likely location for microbial colonization due to fractures allowing fluid and cell transport. Finally, drilling contamination can be a major concern in the rocks that are recovered (Lever et al., 2006), potentially obfuscating native rock-hosted microbial life. Persistence by the scientific community to overcome these challenges has resulted in a substantial body of evidence supporting the existence of a diverse and active deep biosphere hosted in igneous oceanic crust—the subject of this chapter. Here, we synthesize recent molecular biological and biogeochemical studies conducted on samples of igneous oceanic crust collected through the Integrated Ocean Drilling Program (IODP) from various locations around the globe (Figure 2.5.1), as well as from seafloor-exposed outcrops and hydrothermal deposits. We also highlight recent novel microbial experimental techniques using subseafloor observatory technologies that started during the recent phase of the ocean drilling program. Such microbial observatories present alternative methods for overcoming the challenges associated with drilling and coring in oceanic crust, opening a window into the hard-to-access deep igneous oceanic crust for microbiological analyses.

2.5.2 GENERAL OVERVIEW OF THE DIVERSITY, ACTIVITY, AND ABUNDANCE OF MICROBIAL LIFE IN IGNEOUS OCEANIC CRUST At the start of IODP back in 2003, little was then known about the existence of microbial life in oceanic crust. The Initial Science Plan for IODP (IODP, 2001) hinted at the existence of microbial life in oceanic crust, based on pioneering

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FIGURE 2.5.1  Global map of locations where seafloor and/or subsurface crustal rocks have been collected for microbiological investigation (see text for more details). East Pacific Rise (EPR). Inset in upper left indicates water depth in meters, and scale bar in bottom left indicates horizontal distance in kilometers. Map was created using the default Global Multi-Resolution Topography Synthesis (Ryan et al., 2009) basemap in GeoMapApp version 3.2.1 (www.­geomapapp.org).

studies that identified structures seen in oceanic crust rock thin sections that were attributed to microbial activity (Furnes et al., 2001; Fisk, Giovannoni, & ­Thoreth, 1998; Furnes & Staudigel, 1999; Giovannoni et al., 1996; Torsvik, Furnes, Muehlenbacks, Thorseth, & Tumyr, 1998). Other work also documented the existence of conspicuous stalked filaments on seafloor and subsurface basalts (­Thorseth et al., 2001; Thorseth, Pedersen, & Christie, 2003), which are only known to be produced by iron-oxidizing bacteria (Chan, Fakra, Emerson, F ­ leming, & Edwards, 2010; Emerson et al., 2007; Emerson & Moyer, 2002). Studies to identify the identity and function of the microorganisms that colonized the rock surfaces indicated that various groups of Proteobacteria—a diverse phylum of the Domain Bacteria—were involved in iron oxidation on basalt surfaces through unknown biochemical mechanisms (Edwards, McCollom, Konishi, & Buseck, 2003; ­Rogers, Santelli, & Edwards, 2003; Thorseth et al., 2001). These early studies prompted a wave of research into the composition of microbial communities inhabiting seafloor-exposed and subsurface basalts (Figure 2.5.2), buoyed by the concurrent findings mentioned above of a vast deep biosphere hosted in deep marine sediment. A series of papers documented the relatively high diversity of bacterial communities supported on seafloor-exposed basalts as compared to deep seawater and other deep-sea habitats (Mason et al.,

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FIGURE 2.5.2  Examples of seafloor-exposed and subsurface crustal rocks collected for microbiological investigation. (A) Collection of a seafloor-exposed basalt covered in iron-oxidizing bacteria from the Loihi Seamount by the ROV Jason II. (B) Example subsurface drill cores of fractured massive basalt from the Juan de Fuca Ridge flank. (C) Close-up of serpentinized breccia collected from the subsurface of the western Mid-Atlantic Ridge flank at North Pond. (D) Close-up of secondary mineral formation in veins of basalt collected from North Pond. (E) Close-up of vein-filling secondary minerals in serpentinized breccia collected from North Pond. (F) Oxidized glassy basalt recovered from North Pond. Photograph in A courtesy of Woods Hole Oceanographic Institution; photographs in B–F copyright Beth Orcutt.

2008; Santelli et al., 2008; Santelli, Edgcomb, Bach, & Edwards, 2009; Toner et al., 2013). These works also indicated that bacterial community diversity increased as the rock surfaces became progressively more altered and oxidized (Santelli et al., 2008, 2009), although another study did not observe an increase in bacterial abundance with age of basalts (Einen, Thorseth, & Ovreas, 2008).

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The dominant members of the basalt hosted microbial communities grouped with uncultured members of the Proteobacteria phylum, and in particular in the Gamma- and Alpha-Proteobacteria classes (Mason et al., 2008; Santelli et al., 2009). Archaea were also detected on seafloor-exposed basalts, although in relatively low abundance and diversity (Mason et al., 2008; Santelli et al., 2008, 2009). The function of the Archaeal members in the rock-hosted communities is unclear, however, as the phylogenetic affiliation of the observed members was predominantly to uncultured representatives with unknown function. These seafloor-exposed basalts have been estimated to harbor 105–109 cells g−1 of rock (Santelli et al., 2008), and there was some indication that genes for carbon fixation and methane and nitrogen cycling were expressed in these microbial communities (Mason et al., 2008). Concurrently, efforts were underway to document microbial life in deeper oceanic crust samples (Figure 2.5.2). Geochemical methods including stable isotope analysis of mineral deposits in crustal samples—not as susceptible to contamination issues as microbiological analysis—had already indicated microbial sulfate reduction in upper basement and deep mantle-type rocks (Alt et al., 2007; Alt et al., 2003), although the timing of the microbial activity was unclear as stable isotopic signatures can be a record of past activity. New methods to collect deep basalt samples suitable for molecular biological investigation were developed in 2004 during IODP Expedition 301 (Lever et al., 2006). Earlier attempts to document microbial diversity in crustal samples were stymied by contamination (Santelli, Bach, Banerjee, & Edwards, 2010), although another study with gabbroic rocks from the Atlantis Massif was not as impacted by contamination issues (Mason et al., 2010). These stringent methods have paid off with the recent documentation of functional genes for methane and sulfur cycling—indicating recent microbial activity—coregistered with stable isotopic evidence of carbon and sulfur cycling in mineral deposits in subseafloor basalts collected from the warm and chemically reducing upper basement of the eastern flank of the Juan de Fuca Ridge (Lever et al., 2013). These recent results document a relatively low diversity of microbial members involved in methane and sulfur cycling in this system, with only two groups of methane cyclers and one group of sulfate reducers present (Lever et al., 2013). Interestingly, although sulfate reduction and methane cycling were confirmed metabolic strategies in these samples, the regional fluid chemistry of the Juan de Fuca Ridge flank suggests that microbial sulfate reduction rates are very low in this chemically reducing environment (Hulme & Wheat, submitted for publication; Wheat et al., 2003b; Wheat & Mottl, 2000). More recently, basement samples have been collected for microbiological analysis during several recent IODP expeditions to a variety of settings (­Figure 2.5.1). Building off previous work (Lever et al., 2013), a return expedition to the eastern flank of the Juan de Fuca Ridge allowed collection of more upper basement basalts (Expedition 327 Scientists, 2011). Upper basement samples were

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collected from aged oceanic crust during IODP Expedition 329 to the South Pacific Gyre (Expedition 329 Scientists, 2011), and seamount basalts were also recently collected from the Louisville Seamounts during IODP Expedition 330 (Expedition 330 Scientists, 2011). Hard rock samples from hydrothermal vent stock work sampling during IODP Expedition 331 are also being analyzed for hydrothermal end-members in the deep biosphere (Expedition 331 Scientists, 2010). In 2011, a major IODP expedition to document life in oceanic crust focused on upper basement of the western flank of the Mid-Atlantic Ridge for microbiological investigation (Expedition 336 Scientists, 2012). This site has been the focus of several expeditions over the history of the ocean drilling program, as it represents a relatively young (∼8 Ma) and cool (<25 °C) region of upper basement (Bartetzko, Pezard, Goldberg, Sun, & Becker, 2001; Becker, 1990; Becker, Bartetzko, & Davis, 2001; Becker, Langseth, & Hyndman, 1984; Lawrence, Drever, & Kastner, 1979; McDuff, 1984; Ziebis et al., 2012). Some of the first results from this recent expedition indicate oxygen consumption in upper basement, likely a product of microbial activity as abiotic reactions would be sluggish at these cooler temperatures (Orcutt et al., 2013b), confirming projections made from shallow sediment gravity cores in the area that indicated upward flow of oxygen from upper basement into basal sediments (Ziebis et al., 2012). Studies are currently underway to identify and quantify microbial groups in the upper basement rocks from this site (Expedition 336 Scientists, 2012). At the time of this review, analyses of these recently collected samples are in full swing but results are not available yet. Two important improvements have been critical in enabling these studies within IODP—careful sample collection to minimize contamination (with routine contamination control), and modified DNA extraction and amplification protocols to maximize yield from low-biomass samples. A more extensive review of these improvements and their importance to the next phase of IODP has recently been published (Orcutt et al., 2013a). The recent demonstration of the ability to extract and amplify actively expressed RNA molecules from deep marine sediment (Orsi, Edgcomb, Christman, & Biddle, 2013) offers a tantalizing prospect for soon being able to do the same with deep igneous oceanic crust.

2.5.3 SUBSEAFLOOR OBSERVATORIES: ANOTHER TOOL FOR STUDYING LIFE IN OCEANIC CRUST To overcome the limitations in hard rock sample collection from deep oceanic crust, microbiologists began collaborating around the turn of the century with geophysical, geochemical, and hydrogeological scientists using subseafloor observatories set in upper basaltic basement (Figures 2.5.3 and 2.5.4). The use of subseafloor observatories began in the 1990s, with the first Circulation Obviation Retrofit Kit (CORK) installed on the eastern flank of the Juan de Fuca Ridge during Ocean Drilling Program Leg 139 (Davis, Becker, Pettigrew, ­Carson, & Macdonald, 1992). In brief, a CORK is similar to a well used on land

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FIGURE 2.5.3  Examples of seafloor platforms of Circulation Obviation Retrofit Kit (CORK) subseafloor observatories used for microbiological research. (A) View of the Hole U1383C North Pond CORK observatory instrumented with a GeoMICROBE sled (unit on left-hand side of image) connected to several fluid sampling lines, and with a OsmoSampler package also connected to fluid sampling lines (unit on lower right-hand side of image). The ball-valve sampling unit is located in the center of the image, and pressure monitoring equipment is mounted on the back of the CORK platform in this configuration. (B) View of the Hole U1362A Juan de Fuca CORK observatory instrumented with OsmoSampler packages (in the green milk crates) being connected to the fluid sampling valves with the ROV Jason-II. (C) View of OsmoSampler packages being mounted on the Hole U1301A CORK observatory by the ROV Jason-II. (D) Close-up view of various OsmoSampler units for microbiological investigation housed inside a sampling milk crate, including several spools of Teflon tubing, osmotic pumps, and flow-through colonization experiments packaged with various mineral substrates. Photographs in A–C courtesy of the Woods Hole Oceanographic Institution; photograph in D copyright of Beth Orcutt.

to access freshwater in an underground aquifer, except that CORKs are designed to access hydrological units in upper oceanic crust, where modified seawater circulates beneath the seafloor. CORKs are installed from a drilling ship like the RV JOIDES Resolution following drilling and/or coring of the hole as well as placement of a seafloor Re-Entry Cone and Casing and a Borehole Instrument

182  Earth and Life Processes Discovered from Subseafloor Environments FIGURE 2.5.4  Schematic of Circulation Obviation Retrofit Kit subseafloor borehole observatories. Modified from Orcutt et al. (2011) ISME Journal and reprinted here with permission.

Hanger for latching the CORK hardware into place (Graber, ­Pollard, Jonasson, & Shulte, 2002). CORK hardware can reach several hundreds of meters below the seafloor using a series of nested pipes, often referred to as casings, and the pipes can be slotted or perforated to allow fluid movement into and out of the pipe. Once installed, a CORK can then serve as a long-term monitoring and access point into the subsurface. CORK designs (Figures 2.5.3 and 2.5.4) have evolved over the decades, ranging from relatively simple open-pipe designs in the early stages for thermal and pressure characterization of subseafloor hydrology (Davis et al., 1992) to designs with subseafloor seals to isolate different hydrological units (Becker & Davis, 2005; Edwards et al., 2012; Fisher et al., 2005, 2011). Similarly, the construction materials used to fabricate CORKs have evolved from generic steel, which corrodes easily and can confound microbiological experiments, to corrosion-resistant fiberglass and epoxy-coated steel (Edwards et al., 2012; Fisher et al., 2011; Orcutt, Barco, Joye, & Edwards, 2012). Most CORKs feature a seafloor installation, often referred to as a “Christmas tree,” where various valves and sensors can be accessed with a remotely operated vehicle or submersible (Figures 2.5.3 and 2.5.4). The valves and sensors at the seafloor are connected to small-bore tubing (roughly 3–12 mm inner diameter) that reaches to different depths into the oceanic crust on the outside of the CORK casing, allowing sampling of fluids. Traditionally, stainless steel tubing has been used for these “umbilicals”; however, problems with corrosion and biofouling have lead to the usage of Teflon-coated umbilicals as well (Edwards et al., 2012; Fisher et al., 2005, 2011). For sampling, valves can be opened to allow free flow of overpressured fluids, or pumps can be used to pull fluids up from depth (Cowen et al., 2011; Wheat et al., 2010, 2011). More recently, modified “lateral CORKs” or “L-CORKs” are designed with a ball valve on the seafloor installation to allow easier access to fluids sourced from depth (Edwards et al., 2012; Fisher et al., 2011). Another sampling and monitoring mechanism associated with CORKs is the deployment of instrument strings on the inside of the CORK (Edwards et al.,

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2012; Fisher et al., 2005, 2011). Here, narrow-bore (less than 10 cm diameter) sensors, samplers, and experiments are connected together on a long Spectra® cable that hangs from the top of the CORK installation, with instruments then suspended at various depths within the CORK casings. Monitoring, sampling, and experimentation for deep biosphere ­investigations are available both at the seafloor and at depth within the CORK (Figures 2.5.3 and 2.5.4). In addition to the battery-powered temperature and pressure sensors located at the wellheads and at depth on instrument strings (Edwards et al., 2012; Fisher et al., 2005), which provide important contextual information about the nature of fluid flow in the subsurface, other sensors can also be deployed to measure fluid chemistry. For example, in situ electrochemistry analyzers (Luther et al., 2008) have been deployed on CORK wellheads connected to fluid sampling valves to record the redox chemistry (i.e., iron, sulfur, and oxygen concentrations) of CORK fluids (Cowen et al., 2011; Wheat et al., 2011). More recently, in situ oxygen sensors have been deployed on CORK instrument strings to monitor oxygen concentrations over time (Edwards et al., 2012). Fluid chemistry within upper basement can also be documented using nonbattery-powered fluid sampling systems called “OsmoSamplers” (­Jannasch, Wheat, Plant, Kastner, & Stakes, 2004) that can be deployed both at the wellheads and at depth for extended periods of time (i.e., years; Kastner et al., 2006; Wheat, Elderfield, Mottl, & Monnin, 2000; Wheat et al., 2011). With OsmoSamplers, fluid samples are continuously collected at rates of roughly 0.5 ml d−1 into small-bore (∼1 mm inner diameter) tubing, with the pumping rate dependent on the surface area of membranes contained with the osmotic pumps that “power” the samplers (Jannasch et al., 2004). Major ion concentrations can be determined from fluids collected into acid-washed Teflon coils, while minor and trace element concentrations can be determined from fluids fixed in situ with acid in Teflon tubing coils using an acid-addition OsmoSampler (Wheat et al., 2011). Gas concentrations can be determined on fluids contained within copper tubing, which prevents outgassing and loss of pressure (Jannasch et al., 2004; Lapham et al., 2008; Wheat et al., 2011). These fluid sampling instruments provide important chemical information about the fluids circulating within basement, allowing constraint of the redox reactions occurring that might support a deep biosphere within oceanic crust. Most excitingly for deep biosphere research, sampling and in situ experimentation for microbiological investigations are also possible with CORKs— recent microbiological research using such experimentation is described in more detail in the next section. In situ fluid sampling, filtering and concentration have occurred using BioColumns (Cowen et al., 2003) and in situ pumping systems called GeoMICROBE sleds and mobile pumping units (Cowen et al., 2011; Jungbluth, Grote, Lin, Cowen, & Rappe, 2012; Jungbluth, Lin, Cowen, Glazer, & Rappe, 2014; Lin, Cowen, Olson, Amend, & Lilley, 2012). These sample collection systems allow the collection at snapshots in time of discrete volumes of fluids, up to several liters at a time, for shipboard and shore-based investigations of fluid chemistry and cell abundance, diversity, and activity.

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More recently, fluid sampling has also occurred with customized syringe samplers via wellhead ball valves and fluid sampling lines. Continuous and longterm fluid collection for cell abundance and diversity studies is also possible through a Biological OsmoSampler (BOSS) that introduces a preservative into the sampling stream, fixing the sample for DNA- and RNA-based laboratory investigation (Robidart et al., 2013). BOSS systems can be deployed both at CORK wellheads and at depth within a CORK on instrument strings (Edwards et al., 2012; Wheat et al., 2011). Increasingly, OsmoSampler systems are also being used to pump in situ fluids through defined colonization substrates to study microbe-mineral interactions in deep oceanic crust (Edwards et al., 2012; Fisher et al., 2005, 2011; Orcutt et al., 2011; Smith et al., 2011; Wheat et al., 2011) (Figure 2.5.5). Here, sterilized substrates such as basalts, pyrites, and other solid materials are deployed in flow-through columns connected to osmotic pumps (Figures 2.5.3 and 2.5.4), allowing in situ microorganisms to colonize the materials (Orcutt et al., 2011; Smith et al., 2011). Recently, OsmoSamplers have also been designed to “feed” enrichment substrates into the flowthrough columns to encourage microbial growth (Edwards et al., 2012; Fisher et al., 2011; Orcutt et al., 2011; Wheat et al., 2011). As with other OsmoSampler systems, these microbiology OsmoSamplers can be deployed both at CORK wellheads and at depth on the instrument string for years at a time. Combined, these technologies represent avenues for sampling the deep biosphere hosted in deep oceanic crust, although care must be taken to interpret colonization results in the context of the experimental conditions, since flow-through observatory experiments have different water–rock ratios than the native crustal environment.

2.5.4 RECENT DEEP BIOSPHERE DISCOVERIES FROM SUBSEAFLOOR OBSERVATORIES The earliest deep biosphere studies using CORK observatories were conducted in the late 1990s at an early-generation CORK installed at ODP Hole 1026B on the eastern flank of the Juan de Fuca Ridge (Cowen et al., 2003). A BioColumn filtering unit was attached to the wellhead fluid outflow valve, designed to scavenge particles and organics from the escaping warm (54–64 °C) basement fluids sourced from the upper 50 m of basaltic basement underlying roughly 250 m of sediment. In these early studies, the cell density in the basement fluids was determined to be 8.5 ± 4.1 × 104 cells ml−1, and the dominant microorganisms based on a sequencing survey of small-subunit rRNA genes from environmental DNA extracts grouped near the Desulfotomaculum genus of the Firmicutes phylum and the Archaeoglobus genus of the Euryarchaeota (Cowen et al., 2003). This pioneering study documented the existence of a moderately diverse microbial community sourced from upper basaltic basement, although the degree of influence

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FIGURE 2.5.5  Overview of flow-through microbial colonization experiments (FLOCS) used in subsurface observatories. (A) Close-up of mineral coupons mounted on a plastic grid, including (from L to R) two types of basalt, chalcopyrite, pyrite, pyrrhotite, and magnetite. (B) View of flow-through “FLOCS” columns packed with (from bottom to top) basalt chips, pyrite chips, and various mineral coupons suspended in a glass wool matrix, in preparation for deployment on a Circulation Obviation Retrofit Kit (CORK) wellhead. (C) Mineral coupons mounted on the outside of FLOCS columns in preparation for deployment on a CORK instrument string. (D) A different downhole FLOCS design with internal cassettes packed with mineral substrates and glass beads. (E) Assembled downhole OsmoSampler packages in preparation for deployment in North Pond CORKs, with the osmotic pumps (top right) connected to sampling coils of Teflon (left) and copper (right). (F) Close-up view of FLOCS colonization experiment recovered from the Hole 1026B wellhead after 1 year of deployment. All photographs copyright Beth Orcutt.

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that the corroding CORK hardware had on the microbial ecology was not previously possible to distinguish. These microbial groups were thought to be involved in sulfur and/or nitrogen cycling in this system. In 2004, the early-generation CORK at Hole 1026B was upgraded to a second-generation CORK system, and new CORKs were also installed in the same area at IODP Holes U1301A and U1301B during IODP Expedition 301 (Fisher et al., 2005). For the first time, instrument strings were deployed in these CORKs containing mineral colonization experiments along with a variety of OsmoSamplers for measuring fluid chemistry over time (Fisher et al., 2005) (Figure 2.5.5). In 2008, these instrument strings were recovered after a dynamic recovery of the basement aquifer to warm and reducing “predrilling” conditions following up to 3 years of cool conditions due to seawater inflow into the borehole through a bad seal (Wheat et al., 2010). Mineral coupons of basalt and pyrite deployed at depth in the CORKs in Holes U1301A and 1026B were colonized during the cool (and presumably oxic) period of seawater inflow by iron oxide stalk-forming microorganisms (Figure 2.5.6)—likely iron-oxidizing bacteria such as Mariprofundus (Emerson et al., 2007; Emerson & Moyer, 2002) as evidenced by “fossil” stalks observed in biofilms on the mineral surfaces (Orcutt et al., 2011). Sequencing of small-subunit rRNA genes in DNA extracts from these chips revealed a community similar in composition to that observed in the Cowen et al. (2003) study—Firmicutes-group bacteria and Archaeoglobus Archaea being the dominant clones (Orcutt et al., 2011)—documenting roughly similar microbial community compositions under consistent thermal and chemical conditions in the upper basaltic basement at this location. In contrast, multiple years of fluid sampling at Hole U1301A following the recovery of the instrument strings revealed a fluctuating composition of the basement fluid microbial communities, transitioning from a Firmicutes-dominated system in 2008 to a Gammaproteobacteria-dominated system in 2010 (Jungbluth, Grote, Lin, Cowen, & Rappe, 2012), and more recent sampling at the Hole 1026B observatory also reveals a potential shift in microbial community structure (Jungbluth et al., 2014). It is likely that the repeated recovery and deployment of CORK instrument strings during those years caused shifts in the fluid sources to the basement aquifer, overprinting on the microbial community composition observed. These fluids had cell densities of roughly 1 × 104 cells ml−1 (Jungbluth et al., 2012), which is lower than that observed in the earlier BioColumn studies (Cowen et al., 2003). Flow-through columns packed with various mineral substrates and connected to osmotic pumps in these same CORK intervals were also colonized by cells, with cell densities ranging from 2.3 to 39 × 107 cells g−1, with the highest densities observed on iron-bearing olivines (Smith et al., 2011). Heterotrophic enrichment cultures inoculated with the colonized minerals yielded colonies phylogenetically related to known marine halophiles such as Alcanivorax, Halomonas, and Marinobacter (Smith et al., 2011), although these phylotypes were rare or absent in other studies (Cowen et al., 2003; Jungbluth et al., 2012; Orcutt et al., 2011).

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FIGURE 2.5.6  Microbial cells and iron-oxide stalks observed on FLOCS colonization substrates deployed at the Juan de Fuca CORKs. (A and B) Microbial cells colonizing two different basalt chips deployed at the Hole 1026B CORK wellhead for 1 year. Cells were stained with propidium iodide and SYBR Green DNA stains as described in Orcutt, Wheat, and Edwards (2010). (C–F) Stalks formed from iron-oxidizing bacteria on chips of basalt (C and E) and pyrite (D and F) after 4 years of deployment in Hole U1301A CORK observed by epifluorescence microscopy (C and D) and scanning electron microscopy (E and F) as described in Orcutt et al. (2011). Images copyright of Beth Orcutt (A–D) and Katrina Edwards (E and F).

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This same area of the Juan de Fuca Ridge flank was again targeted with newer-generation CORKs during IODP Expedition 327 in 2010 (Fisher et al., 2011). Unlike earlier CORKs in this region, the new CORKs installed at IODP Holes U1362A and U1362B were constructed with epoxy-coated steel casing to minimize impacts from steel corrosion on microbial community composition and activity (Orcutt, Barco, Joye, & Edwards, 2012). Flow-through, osmotic pump-driven, colonization experiments called “FLOCS” (Orcutt, Wheat, & Edwards, 2010) (Figure 2.5.5) were deployed on the instrument strings in these Holes (Fisher et al., 2011; J.P. Baquiran, G. Ramirez, B. N. Orcutt, K. J. Edwards, unpublished data) as well as on the CORK wellheads. Similar flow-through colonization experiments recently deployed for 1 year on the Hole U1301A CORK wellhead revealed microbial communities that were more similar in composition to seafloor-exposed basalts and sulfides than to the earlier studies of the Hole U1301A subsurface, suggesting an important role for temperature in controlling microbial community structure in the basaltic deep biosphere, as the wellhead experiments were conducted at bottom seawater temperatures (J.P. Baquiran, G. Ramirez, B. N. Orcutt, K. J. Edwards, unpublished data). Although the vast majority of subsurface observatory research has been conducted on the eastern flank of the Juan de Fuca Ridge, other deep crustal systems also yield important clues to the nature of a crustal deep biosphere. A borehole observatory system deployed in Hole 896A on the Costa Rica Rift flank produced fluids that were recently sampled for microbial community composition analysis (Nigro et al., 2012). In many ways, the Hole 896A environment is similar to that of the Juan de Fuca Ridge flank, including similar rock types in upper basement of roughly the same age and similar thermal structure and fluid composition, although possible seawater leakage into the observatory cannot be excluded (Nigro et al., 2012). Based on a sequencing survey of small-subunit rRNA genes in environmental DNA extracts, the dominant microbial groups in the Hole 896A samples were related to the Thiomicrospira genus, with sulfur-oxidizing cultivated members, although other forms of metabolism may also be possible. The community structure was more similar to seafloor-exposed basalts and hydrothermal sulfides than to the microbial communities observed in the Juan de Fuca Ridge flank subsurface, though, suggesting an important role for seawater mixing with deeply sourced hydrothermal fluids in controlling microbial community composition in the crustal subsurface (Nigro et al., 2012). More recently, a series of next-generation CORKs constructed of fiberglass and epoxy-coated steel was deployed into young (<8 Ma) and cool (<25 °C) ridge flank crust on the western flank of the Mid-Atlantic Ridge at “North Pond” during IODP Expedition 336 in 2011 (Edwards, Bach, & Klaus, 2010; Edwards et al., 2012). These new CORKs are replete with downhole and wellhead fluid samplers and colonization experiments to examine the nature of microbial communities resident in this type of crust (Edwards et al., 2010).

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2.5.5 THE FUTURE OF SUBSEAFLOOR LABORATORIES FOR DEEP BIOSPHERE RESEARCH The utility of subseafloor laboratories for deep biosphere research is expected to continue in the new drilling program, as they offer unprecedented access to the most difficult to reach habitat on Earth. The CORK network on the Juan de Fuca Ridge flank represents a model study for collaboration between geophysicists, geochemists, hydrologists, and microbiologists in understanding the understudied upper basement environment, allowing key questions about crustal permeability and fluid flow (Fisher & Becker, 2000) and global chemical cycling (Wheat, Jannasch, Kastner, Plant, & DeCarlo, 2003a; Wheat et al., 2004) to be answered in addition to opening a window into the crustal subsurface biosphere. This network will undoubtedly be targeted for continued investigation, although it represents only one end-member (i.e., warm and reducing chemical environment) of the global crustal system. On the another end of the spectrum, the recently installed CORK network on the Mid-Atlantic Ridge flank (Edwards et al., 2012) is poised to yield new insights into the nature of microbial communities harbored in young, cool, and oxic upper basaltic crust—an end-­member of crustal conditions that has not received much attention until recently. Other CORKs and subseafloor observatories are also being tapped for microbiological research. As was the case for the Juan de Fuca CORKs, these globally distributed observatories were established for nonmicrobiological research purposes, but microbiological experimentation techniques are being adapted to integrate into established research programs. As mentioned above, fluid samples from the observatory currently in Hole 896A allowed comparison of the microbial communities hosted in another warm and chemically reducing basement environment with the Juan de Fuca Ridge flank system (Nigro et al., 2012). Microbial colonization experiments were also recently (2009) deployed in CORK observatories positioned across the Costa Rica convergent margin at ODP Leg 205 Holes 1253 and 1255; some of the samples were only recently recovered in December 2013. Ongoing research in the Nankai Trough has seen the incorporation of downhole fluid sampling and microbial colonization experiments associated with the GeniusPlug retrievable observatory systems (Kopf et al., 2011). Elsewhere in the Nankai system, a CORK system installed in ODP Hole 808I was recently revisited in late 2011 for fluid collection for microbiological analysis, allowing investigation of the microbial communities present in deep sediment above the Nankai subduction zone décollement (J.P. Baquiran, B. N. Orcutt, K. J. Edwards, S. Hulme, G. Wheat, unpublished data). During IODP Expedition 331 to the Okinawa Trough, a new wireline in situ borehole fluid sampler was also used to access hydrothermal fluids made accessible through drilling (IODP, 2011). Finally, recent results from other, older-generation observatories elsewhere on the Juan de Fuca Ridge flank offer a unique look at the possible evolution of microbial communities in deep oceanic crust under different thermal and age conditions (Jungbluth et al., 2014).

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New subseafloor observatory networks and designs are in the pipeline, as well. A CORK network is planned for the Cascadia Margin gas hydrate system, which incorporates the new SCIMPI (Simple Cabled Instrument for Measuring Properties In Situ) observatory design for simplified geophysical monitoring (Moran et al., 2006). The first SCIMPI was recently deployed in 2013 during IODP Expedition 341S. SCIMPI is not designed for microbiological research; however, other CORKs in the network are expected to incorporate some microbiological experimentation including BOSS OsmoSampler systems (Robidart et al., 2013; Wheat et al., 2011). Another new observatory design recently on the scene is CORKLite, an observatory system designed to be placed into previously drilled and cased “legacy” boreholes to enable geophysical, geochemical, and microbiological monitoring (Wheat et al., 2012). The first CORK-Lite deployment occurred in 2012 in the legacy borehole at IODP Hole U1382B, making it part of the observatory network at North Pond. The successful deployment of CORK-Lite in this system opens up the opportunity for turning other legacy boreholes around the globe into observatory systems, too (Edwards et al., 2012).

2.5.6 THE SIZE OF THE DEEP BIOSPHERE HOSTED IN IGNEOUS OCEANIC CRUST Given the appreciable size of the igneous oceanic crust as a reservoir for life on Earth coupled with the Deep Biosphere initiative of IODP, it is tempting to speculate on the quantity of biomass in the crustal deep biosphere. Is igneous oceanic crust a significant reservoir of life, and if so, what are the impacts on global models of elemental cycling? A model-driven estimate of the size of the crustal deep biosphere—based on assumptions on the volume or igneous oceanic crust, the percentage of pore space occupied by cells, and assumed cell sizes—suggests that roughly 200 Pg of biomass carbon is stored in the crust (Heberling, Lowell, Liu, & Fisk, 2010). At the time of this model, this amount was comparable to the amount of biomass predicted to be harbored in deep marine sediments (Lipp, Morono, Inagaki, & ­Hinrichs, 2008; Whitman et al., 1998); however, more recent estimates of the sedimentary deep biosphere at about 4 Pg carbon (Kallmeyer, Pockalny, Adhikari, Smith, & D’Hondt, 2012) would indicate a larger biosphere in the crustal realm, if these estimates are confirmed. Furthermore, if such estimates are accurate even within an order of magnitude, then the size of the crustal deep biosphere would make it a significant contributor to global biomass on Earth (Whitman et al., 1998). It should be emphasized, though, that these estimates were made in a vacuum of data due to a lack of suitable samples for ground-truthing estimates. It is hoped that the recent underway efforts to analyze upper basement materials from a variety of settings (Figure 2.5.1) will enable more robust estimates to be made.

2.5.7 CONCLUSIONS The last decade of deep biosphere research within IODP and associated programs has confirmed the existence of an active deep biosphere hosted in

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igneous oceanic crust, making this the largest habitat for life on Earth if it is inhabited throughout. News studies are now needed in a range of settings to explore the extent of life in the crustal ecosystem, as there is a basic lack of understanding about the limits of life in the deep biosphere. Is the extent of life limited by availability of carbon or other energy sources or even nutrients? By the age of the crust? By depth or pressure or temperature? These are fundamental questions that need to be addressed in the next phase of ocean drilling (IODP, 2011). The upcoming ship track for the RV JOIDES Resolution in the new drilling program (Humpris & Koppers, 2013) passes by a number of sites ripe with possibilities for exploring deep life in different oceanic crust settings (Edwards, Fisher, & Wheat, 2012). Recent advances in DNA sequencing techniques, sample collection, and handling have made possible recent analyses of igneous oceanic crust, revealing the diversity of microbial groups resident in oceanic crust under different conditions. Pioneering studies are now needed to push the analytical boundary forward to understand the biochemical mechanisms used by deep crustal microorganisms to sustain life “on the rocks” and to understand the genetic connection of deep crustal microbial ecosystems to the rest of the Earth environment.

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