BACKGROUND

Pore water chemical data from hundreds of DSDP and ODP sites documents the occurrence of subsurface catabolic activity in sediments throughout most of the deep ocean (D'Hondt et al., unpubl. data). Microbial sulfate reduction, methane production, and methanotrophy are common processes in deeply buried marine sediments. Other catabolic 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 microbial cells in deeply buried marine sediments and the clear pore water evidence of in situ microbial catabolism, the identity and structure of these communities and the metabolic adaptations of the microbes that constitute them remain largely unknown. Most-probable number (MPN) experiments have demonstrated that viable cells occur 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., unpubl. data). If all of these enumerated cells are alive, their rates of SO₄^-2 reduction are two to five orders of magnitude lower in the ocean margin anaerobic methanotrophy zone and five to nine or more orders of magnitude lower in open ocean sediments than per-cell rates observed in pure cultures and inferred in surface marine sediments (Jørgensen, 1978; Knoblauch et al., 1999; Ravenschlag et al., 2000; D'Hondt et al., in prep.). In contrast, if subsurface cells actually utilize SO₄^-2 at the lowest rates measured for cells in laboratory cultures or inferred for cells in surface marine sediments, fewer than 1 in 100 is actively respiring in the sulfate-reducing methanotrophy zone of the most microbially active sites and fewer than 1 in 1,000,000 is actively respiring at the least microbially active open ocean sites. In short, most of the subsurface microbes enumerated by direct microscopy in marine sediments must be either adapted for extraordinarily low levels of metabolic activity—or dead. This conclusion is supported by available estimates of mean generation times of up to 1 m.y. for deep subsurface microbes (Parkes et al., 2000).

Metabolic Diversity
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 pore 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 appear to occur deep in 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 build up of bicarbonate and ammonium are indicators of the diagenesis of organic material in all marine sediments. Sulfate reduction dominates 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 pore 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 is 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). Based on pore water modeling of sulfate and methane profiles, the same process appears to drive a significant part of sulfate reduction in the sea bed (e.g., Borowski et al., 1999; D'Hondt et al., unpubl. data). 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 proposed for Leg 201, manganese oxide may provide an important oxidant of organic material and its reduction can be traced tens of meters into the seafloor (Yeats, Hart, et al., 1976). The reduction of iron oxides expectedly plays a greater quantitative role and iron(III) bound in sheet silicates may provide a slow but continuous source of oxidation potential over 105-106 years (Raiswell and Canfield, 1996).

H₂ is an important intermediate in the microbial degradation pathways of ocean margin sediments, and its pore water concentration is strictly regulated by the uptake potential of the microbial community and the energy yield of their H₂ metabolism. Thus, the H₂ 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 H₂, 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.

Microbial Diversity
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 fully 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 Fe(III). Whether deeply buried sulfate-reducing communities throughout the world ocean are dominated by close relatives of D. profundus or composed of a host of other organisms remains to 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, to our knowledge, not lead 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 near nshore surface sediments have been biomarker fingerprinted and phylogenetically identified, but not yet cultured (Hinrichs et al., 1999; Boetius et al., 2000; Teske et al., 2000; Lanoil et al., 2000). Whether or not the same microbial community (composed of a sulfate-reducing bacterium and a previously unknown member of the archeal Methanosarcinales) is 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 bacterial 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 Fe(III) reduction (Lonergan et al. 1996). A Thermus sp. isolated from a deep South African gold mine used O₂, NO₃^-, Fe(III), S, Mn(IV), Co(III), Cr(VI), and U(VI) as electron acceptors (Kieft et al. 1998). Respiration of metal oxides could allow bacteria and archaea a respiratory mode of life even after other electron acceptors, including oxidized sulfur species, become extinguished with increasing distance from the oxidized biosphere. The metabolic flexibility of D. profundus and the South African Thermus sp. allow multiple scenarios of subsurface phylogenetic diversity. One possible scenario is that a certain microbial 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 NO₃^- reduction, manganese reduction, iron reduction, and SO₄^-2 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° to 30°C or 30° to 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.

Global Biogeochemical Effects
The subsurface biosphere of marine sediments may affect the surface Earth in a variety of ways. It is now widely recognized that release of CH₄ 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 SO₄^-2 reduction by the buried biosphere may also change Earth's surface chemistry and climate. Such reduction is a major sink of SO₄^-2 from the world ocean (Holland, 1984). Because SO₄^-2 is the second most abundant anion in seawater (Pilson, 1998) and SO₄^-2 reduction followed by sulfide precipitation results in the production of two equivalents of alkalinity per mole, subsurface
SO₄^-2 reduction may affect total oceanic alkalinity and, consequently, the partitioning of CO₂ between atmosphere and ocean over geologic time. 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. Furthermore, NO₃^- reduction by the subsurface biosphere may be a net sink of biologically accessible nitrogen from the world ocean (Moore et al., 2001).

Why the Equatorial Pacific and Peru Margin?
In short, we know almost nothing about the population structure, metabolic strategies, community composition, and global biogeochemical influence of the marine deep biosphere. If we are to develop a coherent understanding of the microbial communities that are deeply buried in marine sediments, a focused and interdisciplinary program of deep biosphere study is required. Leg 201 presents such a program.

Sampling of the Leg 201 sedimentary environments will allow 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 (¾~25°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; Equatorial Pacific ODP Leg 138) (Yeats, Hart, et al., 1976; Suess, von Huene, et al., 1990; Pisias et al., 1995). In short, several widely different marine sedimentary regimes will be 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 to be 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-rich deep-water sequence off the continental shelf of Peru (see Fig. 1).

The first two environments are characteristic of open-ocean sedimentary regimes. ODP Leg 138 studies identified abundant subsurface microbes in this equatorial Pacific region (Cragg and Kemp, 1995). Shipboard chemistry from Leg 138 and DSDP Leg 34 (Pisias et al., 1995; Yeats, Hart, et al., 1976) suggest that the deeply buried microbial communities of these two regions, respectively, rely on sulfate and manganese reduction. The subsurface extent of electron acceptors with similar or intermediate standard free energy yields (nitrate, oxygen, and iron oxides) in these regions remains unknown.

The second two environments are characteristic of ocean margin regimes. Studies of ODP Leg 112 samples identified abundant subsurface microbes in Peru Shelf sediments (Cragg et al., 1990). At all sites but one, these shallow-water sediments and the deep-water hydrate-rich 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, thus indicating relatively low microbial activity.

Subsurface flow affects the subsurface environment at both the shallow-water Peru Shelf sediments and the open-ocean equatorial Pacific sites. In the former region, it is brine flow through the sediments. In the latter region, it is seawater flow through the underlying crust and perhaps the deepest sediments.