The Ocean Drilling Program (ODP) is uniquely positioned to sample one of the least known and potentially strangest ecosystems on Earth—the microbial biosphere of deep marine sediments and the oceanic crust. The growing international interest in the study of this subsurface biosphere is driven by a variety of factors, including the role of subsurface microbial activity in Earth's biogeochemical cycles, the possibility of life on other planets, and sheer fascination with the nature of life at the apparent margin of existence.
Nearly 20 years ago, Deep Sea Drilling Project (DSDP) experiments with methane concentration and radiotracer uptake first demonstrated active microbial processes in cores recovered from deeply buried marine sediments (Oremland et al., 1982a; Whelan et al., 1986; Tarafa et al., 1987). Over the last 15 years, studies of ODP cores have extended our understanding of those processes (e.g., Cragg et al., 1992; Getliff et al., 1992) and consistently identified abundant prokaryotes in deeply buried oceanic sediments (e.g., Cragg et al., 1990, 1992; Thierstein and Störrlein, 1991; Parkes et al., 1994, 2000). Prokaryotes have been recovered from burial depths as great as 800 meters below the seafloor (mbsf) (Shipboard Scientific Party, 1999). Recent contamination tracer experiments suggest that the prokaryotes reported from ODP cores are indigenous to the drilled sediments (Smith et al., 2000b).
The number and biomass of prokaryotes in the subsurface biosphere of oceanic sediments have been estimated by extrapolation from direct counts of sedimentary prokaryotes at a small number of ODP sites. Based on that extrapolation, the marine subsurface biosphere has been estimated to compose as much as one-tenth (Parkes et al., 2000) or even one-third (Whitman et al., 1998) of the world's living biomass. In situ metabolic activity by at least a portion of this biosphere is spectacularly demonstrated by hydrates of methane produced by prokaryotes in deep-sea sediments. On a global scale, these hydrates contain 7,500 to 15,000 gigatons of carbon—four to eight times as much carbon as in living surface biosphere and soils combined (Kvenvolden, 1993). Interstitial water chemical studies (Borowski et al., 1996) and recent microbiological discoveries (Hinrichs et al., 1999; Boetius et al., 2000) suggest that, on an ongoing basis, the methane produced in deep-sea sediments is primarily destroyed by the sulfate-reducing activity of prokaryotes in overlying sediments.
Despite these recent advances, very little is known about the nature and activity of life in deep marine sediments. In particular, we know almost nothing about (1) the continuity of subsurface life from one oceanographic region to another, (2) metabolic interactions in deeply buried marine sediments, or (3) the conditions under which subsurface prokaryotes are active or inactive and living or dead.
There is abundant evidence of both microbial populations and microbial activity in subsurface marine sediments throughout the world ocean. Prokaryotic cells have been found in surprisingly high numbers in buried sediments at every site that has been assayed for their presence (Parkes et al., 2000). The abundance of those cells varies in a systematic and fairly predictable manner. For example, deeply buried shelf sediments from the Peru margin (high surface-ocean productivity and shallow water depth) contain 108-109 cells/cm3, and sediments from the eastern equatorial Pacific (low surface-ocean productivity and abyssal water depth) contain only 106 cells/cm3 (Parkes et al., 2000).
Interstitial water chemical data from hundreds of DSDP and ODP sites document the presence of subsurface metabolic activity in sediments throughout most of the deep ocean (D'Hondt et al., 2002). Bacterial sulfate reduction, methane production, and methanotrophy are common processes in deeply buried marine sediments. Other metabolic processes are known to occur in subsurface marine sediments but have been studied very little (such as manganese and iron reduction).
Despite the ubiquity of prokaryotic cells in deeply buried marine sediments and the clear interstitial water evidence of in situ microbial metabolism, the identity and structure of these communities and the metabolic adaptations of the prokaryotes that constitute them remain largely unknown. Most probable number (MPN) experiments have demonstrated that viable cells are present in deeply buried marine sediments (Parkes et al., 2000). However, these viable cells represent only the barest fraction (0.00001% to 0.6%) of the total cells enumerated in the sediments sampled (Parkes et al., 2000). The extent to which this discrepancy between enumerated and viable cells reflects a culturing bias, known also from surface sediments, or the extent to which it reflects a real difference between a small active population and a very large inactive (dormant or dead) population remains to be determined.
The importance of this issue for our understanding of subsurface population structure and metabolic adaptation is underscored by estimates of the mean sulfate reduction per enumerated subsurface cell (D'Hondt et al., 2002). If all of these enumerated cells are alive, their rates of sulfate reduction are zero to two orders of magnitude lower in the ocean-margin anaerobic methanotrophy zone and four or more orders of magnitude lower in open-ocean sediments than per-cell rates inferred in surface marine sediments (Jřrgensen, 1978; Knoblauch et al., 1999; Ravenschlag et al., 2000; D'Hondt et al., 2002). In contrast, if subsurface cells actually utilize sulfate at the lowest rates inferred for cells in surface marine sediments, as few as 1 in 100 may be actively respiring in the sulfate-reducing methanotrophy zone of the most microbially active sites and fewer than 1 in 10,000 is actively respiring at the most microbially active open-ocean sites. In short, most of the subsurface prokaryotes enumerated by direct microscopy in marine sediments must be either adapted for extraordinarily low levels of metabolic activity or dormant—or even dead. This conclusion is supported by available estimates of mean generation times of up to 1 m.y. for deep subsurface prokaryotes (Parkes et al., 2000).
The metabolic diversity and rates of microbial processes in deep subsurface sediments can be inferred from a broad range of geochemical information, including modeling of interstitial water profiles of ions, gases, and low molecular weight organic molecules, mass balance calculations of changes in solid-phase constituents, and stable isotope fractionation. Basically, the same types and sequences of microbial processes may occur deep beneath the seafloor as are known from the much more active surface sediments of ocean margins. The mechanisms and regulation of the exceedingly slow hydrolytic degradation of macromolecular organic compounds are, however, only poorly understood. So, too, are the fermentative pathways that produce substrates for the terminal mineralization processes such as sulfate reduction or methanogenesis.
The buildup of bicarbonate and ammonium are indicators of the diagenesis of organic material in all marine sediments. Sulfate reduction appears to dominate biogeochemical processes down to the depth of sulfate depletion, many tens or hundreds of meters below the seafloor, where it is followed by methanogenesis. Although methane formation is very slow, the continuous production of a diffusible gas over millions of years results in vast methane accumulations, either dissolved in the interstitial water or in the condensed form of gas hydrates. In both cases, an upward flux of methane reaches the sulfate zone and supports an interface of enhanced microbial activity based on methane oxidation. In shallow marine sediments, this anaerobic process appears to be catalyzed by a syntrophic community consisting of archaea, which convert methane back to an intermediate such as hydrogen or acetate, and sulfate-reducing bacteria, which oxidize the intermediate (e.g., Hoehler et al., 1994; Valentine and Reeburgh, 2000; Boetius et al., 2000; Orphan et al., 2001). Based on interstitial water modeling of sulfate and methane profiles, the same process appears to drive a significant part of sulfate reduction in the seabed (e.g., Borowski et al., 1999; D'Hondt et al., 2002). This interface is of particular importance, since it constitutes a barrier against the escape of methane up into the ocean water and eventually into the atmosphere.
In deep-sea sediments, such as the Peru Basin sites drilled during Leg 201, manganese oxide may provide an important oxidant of organic material and its reduction can be traced tens of meters below the seafloor (Yeats, Hart, et al., 1976). The reduction of iron oxides is also expected to play a greater quantitative role in deep-sea sediments than in ocean-margin sediments. Iron(III) bound in mineral phases may provide a slow but continuous source of oxidation potential.
Hydrogen is an important intermediate in the microbial degradation pathways of shallowly buried ocean-margin sediments, where its interstitial water concentration appears to be strictly regulated by the uptake potential of the prokaryotic community and the energy yield of their hydrogen metabolism. Thus, the hydrogen partial pressure in the sulfate reduction zone is maintained below the threshold level required by archaea to drive methanogenesis (Hoehler et al., 1998). The key role of hydrogen, known from the metabolic competition among microbial populations in surface sediments, may also be critical for the deep subsurface biosphere. The potential sources for microbial energy metabolism need to be surveyed with an open mind toward new and unexpected types of redox processes and mineral surface reactions.
The phylogenetic and physiological diversity of deep sediment communities remains virtually unknown. Only two isolates of sulfate-reducing bacteria from subsurface sediments (80 and 500 mbsf) have been characterized (Bale et al., 1997). These isolates (from a single site in the Japan Sea) are of a new barophilic species, Desulfovibrio profundus. Its unusually wide growth temperature range (15°-65°C) has no counterpart in any other known sulfate-reducing bacterium. It is metabolically flexible; it possesses the capability to reduce oxidized sulfur species, nitrate, and iron(III). Whether deeply buried sulfate-reducing communities throughout the world ocean are dominated by close relatives of D. profundus or are composed of a host of other organisms remains to be tested.
The record of methanogenic isolates from the subsurface is surprisingly spare. MPN enumerations of methanogens in deep marine sediments have yielded cultured methanogens in much smaller numbers than sulfate-reducing bacteria (in the Peru margin; Cragg et al., 1990) or not at all (in the Japan Sea; Cragg et al., 1992). These surveys have not, to our knowledge, led to the description of new methanogen species from the marine subsurface. Hence, the phylogenetic composition of marine subsurface methanogenic communities remains essentially unknown.
Organisms responsible for methanotrophy in nearshore surface sediments have been biomarker fingerprinted and phylogenetically identified but not yet cultured (Hinrichs et al., 1999; Boetius et al., 2000; Lanoil et al., 2001; Teske et al., 2002). Whether or not similar microbial communities (composed of sulfate-reducing bacteria and previously unknown members of the archaea Methanosarcinales) are responsible for methanotrophy in the more deeply buried biosphere throughout the world ocean (in both ocean-margin and open-ocean environments) remains to be seen.
Novel forms of prokaryotic metabolism with subsurface potential are constantly being discovered. For example, systematic studies of sulfate- and sulfur-reducing bacteria and archaea have shown that many representatives of these organisms, among them an astonishing set of phylogenetically very deep lineages (Vargas et al., 1998), share an unexpected capacity for iron(III) reduction (Lonergan et al., 1996). A Thermus sp. isolated from a deep South African gold mine used dissolved oxygen, nitrate, iron(III), S0, manganese(IV), cobalt(III), chromium(VI), and uranium(VI) as electron acceptors (Kieft et al., 1999). Respiration of metal oxides could allow bacteria and archaea a respiratory mode of life even after other electron acceptors, including oxidized sulfur species, become depleted with increasing distance from the oxidized biosphere. The metabolic flexibility of D. profundus and the South African Thermus sp. allows multiple scenarios of subsurface phylogenetic diversity. One possible scenario is that a certain prokaryotic community becomes buried below the sediment surface and basically persists in its phylogenetic diversity and physiological potential over millions of years. This community may be responsible for nitrate reduction, manganese reduction, iron reduction, and sulfate reduction throughout the vertical expanse of a single sediment column—and even dominate the subsurface respiratory realm throughout the sediments of the world ocean (at least within a broad temperature range, such as 0°-30°C or 30°-60°C). A second (and perhaps more likely) scenario is that the phylogenetic composition of subsurface communities may be shaped by variables other than the type of electron acceptors available. For example, it may be controlled by electron donor availability, micronutrient availability, or the ability of well-tuned species or communities to out-compete each other under slightly different local environmental conditions, such as different concentrations of metabolic products and reactants. Also, physical factors such as available pore space, ability to migrate in the sediment, interactions with mineral surfaces, or distance to solid substrates may be important.
The subsurface biosphere of marine sediments may affect the surface Earth in a variety of ways. It is now widely recognized that release of methane from marine sediments may affect atmospheric carbon stocks and climate (Dickens et al., 1995; Dickens, 2000; Kennett et al., 2000; Hesselbo et al., 2000; Hinrichs, 2001). It is less widely recognized that sulfate reduction by the buried biosphere may also change Earth's surface chemistry and climate. Such reduction is a major sink of sulfate from the world ocean (Holland, 1984). Because sulfate is the second most abundant anion in seawater (Pilson, 1998) and sulfate reduction followed by sulfide precipitation results in the production of two equivalents of alkalinity per mole, subsurface sulfate reduction may affect total oceanic alkalinity and, consequently, the partitioning of carbon dioxide between atmosphere and ocean over geologic time (D'Hondt et al., 2002). The ultimate effect of this process on the surface Earth will depend on the extent to which reduced sulfur is fixed in the sediment rather than diffusing back into the overlying ocean to be oxidized.
In short, we know almost nothing about the population structure, metabolic strategies, community composition, and global biogeochemical influence of the marine deep biosphere. We also know almost nothing about how the chemical and physical characteristics of subseafloor sediments control the prokaryotic communities and activities that occur within those sediments. Consequently, we also know little about how modern prokaryotic communities are constrained by past oceanographic history. If we are to develop a coherent understanding of the prokaryotic communities that are deeply buried in marine sediments, a focused and interdisciplinary program of deep biosphere study is required. Leg 201 presents a crucial early step in the development of such a program.
Sampling of the Leg 201 sedimentary environments allows us to document the activity, composition, and biogeochemical effects of the subsurface biosphere in environments representative of essentially the entire range of subsurface conditions that can be found in relatively cool (2°-25°C) marine sediments. These include equatorial Pacific sediments typical of the open ocean, Peru margin sediments typical of a nearshore upwelling regime, and Peru Basin sediments. Much of the geochemical and sedimentological character of these sediments has been documented during previous ODP and DSDP legs (DSDP Leg 34, Peru margin ODP Leg 112, and equatorial Pacific ODP Leg 138) (Yeats, Hart, et al., 1976; Mayer, Pisias, Janecek, et al., 1992; Suess, von Huene, et al., 1990; Pisias, Mayer, Janecek, Palmer-Julson, and van Andel, 1995). In short, several widely different marine sedimentary regimes were explored during this single drilling leg. Few regions in the world contain within a relatively short distance so many marine sedimentary regimes that have been so well characterized.
The environments that were examined include (1) carbonate and siliceous oozes of the equatorial Pacific, (2) clays and nannofossil-rich oozes of the Peru Basin, (3) organic-rich silty sediments of the shallow Peru shelf, and (4) a hydrate-bearing deepwater sequence off the continental shelf of Peru (see Fig. F1).
The first two environments are characteristic of open-ocean sedimentary regimes. Leg 138 studies identified the presence of subsurface prokaryotes throughout the sediment column in this equatorial Pacific region (Cragg and Kemp, 1995). Shipboard chemical analyses from Legs 138 and 34 (Pisias et al., 1995; Yeats, Hart, et al., 1976) suggest that the deeply buried prokaryotic communities of these two regions rely primarily on sulfate and manganese reduction, respectively. Despite these studies, the subsurface extent of electron acceptors with similar or intermediate standard free-energy yields (nitrate, oxygen, and iron oxides) in these regions was unknown prior to Leg 201.
The second two environments are characteristic of ocean-margin regimes. Studies of Leg 112 samples identified abundant subsurface prokaryotes in Peru shelf sediments (Cragg et al., 1990). At all sites but one, these shallow-water sediments and the deepwater hydrate-bearing sediments are rich in dissolved sulfate at shallow burial depths (down to a few meters below seafloor) and rich in methane at greater burial depths (starting a few meters below seafloor or tens of meters below seafloor) (Suess, von Huene, et al., 1990). The remaining site is sulfate rich and methane poor throughout the targeted drilling interval.
Deeply buried brine affects the subsurface environment at the shallow-water Peru shelf sediments. At equatorial Pacific open-ocean sites, the subsurface environment is affected by water flow through the underlying basaltic basement.
The overarching objective of Leg 201 is to investigate the nature, extent, and biogeochemical consequences of prokaryotic activity in several different deeply buried marine sedimentary environments.
During Leg 201, we addressed several fundamental questions about the deeply buried biosphere:
Several aspects of these questions require extensive postcruise research to fully address. This reliance on postcruise research is necessary for at least two reasons. First, some experiments initiated during the cruise will take months (radiotracer experiments) or years (cultivation experiments) to complete. Second, some key studies, such as genetic assays of the prokaryotic communities and isotopic studies of biogeochemical fluxes, will be undertaken postcruise because they require technical facilities and expenditures of time beyond those available to a shipboard party.
Despite these limitations, other aspects of the above questions were successfully addressed during Leg 201. In particular, shipboard biogeochemical, geophysical, and sedimentological studies provide new understanding of the effects of interstitial water chemistry, sediment composition and structure, fluid flow, and past oceanographic conditions on metabolic diversity, prokaryotic activities, and the nature of metabolic competition in these subsurface environments. These shipboard studies improve our understanding of how deep subsurface biogeochemical processes affect both their local environments and the surface world.
The study of deep subsurface prokaryotes and their activity is a methodological and experimental challenge at the frontiers of modern life and earth sciences. Leg 201 is the first deep-sea drilling expedition to be primarily focused on subsurface prokaryotic communities and their geochemical activities. Many of the studies carried out during this cruise were undertaken by ODP shipboard scientists for the first time. Many of these approaches had not been previously used to study the deep biosphere. A number of methods and concepts had to be further developed, refined, or even completely changed during the expedition. The scientific approaches were consequently chosen on the basis of extensive discussions and experiences of many colleagues and are still very much in the development phase. Some of these approaches may need further refinement before they are recommended for future routine application.
The research objectives of Leg 201 required shipboard scientists to address the following specific questions regarding the subseafloor sedimentary biosphere:
To address these questions effectively, a very wide range of sedimentological, geophysical, geochemical, and microbiological analyses were undertaken during Leg 201. To maximize understanding of the interplay between subsurface prokaryotes and their environment, whenever possible, these diverse analyses were conducted on the same sediment samples or samples immediately adjacent to each other.
A full suite of standard sedimentary analyses was used to document the physical and compositional nature of subsurface environments explored during Leg 201. These included visual core descriptions, digital color scanning and optical reflectance scanning of the split cores, and microscopic observation and/or X-ray diffraction analyses of individual sediment samples.
Core logging of magnetic susceptibility and intensity was used to identify environments of particular interest for Leg 201 objectives, such as pronounced biogeochemical fronts or lithostratigraphic boundaries. Core logs (magnetic susceptibility, gamma ray attenuation [GRA] bulk density, and natural gamma radiation [NGR]) were also used to correlate intervals of particular interest from hole to hole at the same site. Where possible, magnetic reversal logs were used to determine sediment age and correlate from hole to hole and site to site. NGR was measured both on cores in the laboratory and using wireline logs for in situ formation properties. Wireline logs (resistivity, NGR, and geophysical proxies for density and porosity) were also used to characterize the physical and compositional nature of subseafloor environments.
Analysis of the physical environment also included study of environmental properties such as temperature and pressure, which are important for the selection of cell properties and regulation of metabolic activity. Physical properties critical for quantifying transport processes, such as porosity and diffusivity, were analyzed in order to interpret the chemical gradients with respect to subsurface flow, chemical diffusion, and the availability of substrates for prokaryotes.
The detailed analysis of interstitial water chemistry was a major emphasis during Leg 201 and was probably more comprehensive than during any previous ODP leg. A broad spectrum of dissolved inorganic ions, gases, and organic solutes was measured with close vertical resolution throughout the sediment column at each site in order to identify potential substrates and products of microbial metabolism and provide the chemical data necessary to quantitatively model steady-state net rates of microbial activities.
Additional shore-based analyses of solid-phase geochemistry and interstitial water chemistry will enable mass balance calculations of burial rates and diagenetic transformations of organic compounds and mineral phases. Stable isotope analyses of carbon, hydrogen, oxygen, sulfur, nitrogen, and iron in both dissolved and solid chemical phases will serve as a complementary approach to interpret the biogeochemical alterations. The sensitivity of isotopic analyses allows inferences about processes that are too slow to detect with other experimental process studies. Furthermore, analyses of specific biomarkers and their isotopic signals will help to identify which prokaryotes were involved in these slow alterations in the past.
A variety of experiments were undertaken during Leg 201 to estimate in situ activities of specific samples. Most of these experiments relied on minute quantities of radioactive chemicals to trace rates of specific activities. Although specific activities are generally limited to a subset of the prokaryotic community, they typically serve a crucial role in the flow of energy and material through the entire community. Well-established 35S, 14C, and 3H techniques were used to quantify within-sample rates of sulfate reduction, methanogenesis, and thymidine uptake. Innovative experiments with 3H2 were used to trace hydrogen uptake and turnover. A few additional experiments included incubating samples with stable isotopes as tracers; some used 18O to study oxygen exchange between water and phosphate, and others used 13C to trace assimilation of carbon from acetate into biomass.
A wide variety of microbial cultivation experiments were initiated during Leg 201 to identify and quantify subseafloor prokaryotic populations. These included a large number of incubations that utilize selective media for enrichments in order to isolate and identify a broad spectrum of physiological types with respect to energy metabolism and temperature adaptation (general heterotrophs; fermenters; autotrophic and heterotrophic sulfate reducers, methanogens, and acetogens; iron and manganese reducers; anaerobic ammonium and methane oxidizers; and psychrophiles, mesophiles, and thermophiles). Serial dilution (MPN) cultivation experiments were initiated to enumerate those prokaryotes able to show growth and metabolic activity under nutrient-rich laboratory conditions. Homogenized sediment slurries were diluted in tenfold steps into liquid media, which should support the growth of specific physiological types of prokaryotes. In such experiments, the highest dilutions that still have growth are classically interpreted to indicate the MPN of these organisms (American Public Health Association, 1989). MPN counts typically provide only a minimum estimate of the true numbers of organisms that were viable in situ because many prokaryotes (perhaps the vast majority) are not cultivable with currently available methods. MPN cultivations also serve as starting material for enrichments and isolations of the prokaryotes. The isolation of prokaryotes from the highest dilutions with positive growth maximizes the chance of finding organisms that are quantitatively dominant and, therefore, geochemically most important.
As for many ODP legs over the past decade, total cell numbers of prokaryotes were determined during Leg 201 by direct microscopy of fluorescently stained cells (acridine orange direct count [AODC]). Similar counts of defined groups of prokaryotes will be done postcruise by using fluorescence in situ hybridization (FISH) to specifically stain cells that share genetic sequence information.
The genetic diversity and geographic continuity of subseafloor prokaryotic populations will be addressed by postcruise research. This research will largely rely on the recently developed approach of analyzing deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from entire communities in natural samples. This approach allows analysis of natural communities of prokaryotes that are unavailable in culture, either because cultivation attempts have not yet succeeded in providing suitable growth conditions or because cultivation-based approaches have limited capacity to deal with great prokaryotic diversity. DNA and RNA in Leg 201 sediment samples will be extracted by several participating groups, who will use them to analyze subseafloor prokaryotic diversity based on genetic sequence information. This will, for the first time, establish a database on the diversity of prokaryotes from the deep subsurface and the key genes of their energy metabolism.