PRINCIPAL RESULTS

The principal objective of Leg 201 was to document the nature, extent, and biogeochemical consequences of microbial activity and prokaryotic communities in several different deeply buried marine sedimentary environments. To maximize the scientific utility of Leg 201 results, biogeochemical, microbiological, physical property, and sedimentological sampling and analyses and downhole tool deployment and downhole logging were all closely integrated. Leg 201 physical property, sedimentology, and downhole studies provided detailed evidence of the environmental factors that influence subseafloor microbial life and are in turn influenced by it. To build comprehensive records of net microbial activities and their consequences, the Leg 201 shipboard party conducted a broad range of biogeochemical studies. To develop comprehensive records of subseafloor prokaryotic communities, the shipboard party also undertook and initiated a multifaceted array of microbiological studies. The extent of biogeochemical and microbiological analyses on Leg 201 samples is unprecedented in ODP history.

During Leg 201, a variety of sediments (Fig. F2) were cored in both open-ocean and ocean-margin provinces in the eastern tropical Pacific Ocean. Neogene deep-sea clays and Paleogene nannofossil ooze were cored at Peru Basin Site 1231. Miocene to Holocene carbonate and siliceous oozes and chalk were cored at Sites 1225 and 1226. Miocene to Holocene biogenic oozes and terrigenous sediments of the shallow Peru shelf were cored at Sites 1227, 1228, and 1229. Organic-rich Miocene to Holocene sediments were cored at Site 1230 on the Peru slope, in the accretionary wedge just landward of the Peru Trench.

In situ data from Leg 201 demonstrate that comparable temperature ranges are found at all the sites, allowing direct comparisons of microbial activities and communities in very different environments under similar thermal conditions (Fig. F3). In situ temperatures of the organic-poor sediments of Sites 1225 and 1231 and the organic-rich Peru shelf Site 1230 are in the range preferred by psychrophilic prokaryotes (0°-10°C). In situ temperatures of the organic-rich Peru shelf sediments and throughout most of the sediment column at eastern equatorial Pacific Site 1226 are in the low end of the range preferred by mesophilic prokaryotes (which inhabit 10° to 35°C environments).

AODCs showed that prokaryotic cell concentrations of the Leg 201 sediments generally followed the well-established trend of exponential declines in cell concentration with subsurface depth. Cell counts are generally higher at the ocean-margin sites than at the open-ocean sites (Fig. F4). Cell concentrations progressively increase from Peru Basin Site 1231 to equatorial Pacific Site 1225 to eastern equatorial Site 1226 to Peru shelf Site 1227 to Peru shelf Site 1229 and Peru slope hydrate Site 1230. A similar difference between open-ocean and ocean-margin sites was noted in a recent survey of previously studied ODP sites (Parkes et al., 2000).

The geometric mean prokaryotic cell concentrations for all of the Leg 201 sites are close to, or even slightly below, the mean concentrations for all previously studied ODP sites (Fig. F5). This finding is somewhat surprising because so many of the Leg 201 sites contained organic-rich sediments, including the Peru shelf sites, Peru slope hydrate Site 1230, and eastern equatorial Site 1226. Because previously enumerated sites targeted a representative range of ODP sampled environments, the similarity of our results to the average for previously studied sites may reflect a collective ODP bias toward drilling at the ocean margins and away from drilling in such regions as the oceans' central gyres.

Interstitial water studies of the Leg 201 sites showed that net subseafloor microbial activity is much higher at the ocean-margin sites than at the open-ocean sites. For example, subseafloor concentrations of dissolved inorganic carbon (DIC = HCO₃^- + CO₂ + CO₃^2-) and ammonium are much higher at ocean-margin Sites 1227, 1228, 1229, and 1230 than at open-ocean Sites 1231, 1225, and 1226 (Fig. F6). DIC and ammonium are generic products of microbial activity, regardless of the principal electron-accepting pathway. Consequently, the very high concentrations of those chemical species at the ocean-margin sites and the generally low concentrations at the open-ocean sites indicate that net respiration of organic carbon to carbon dioxide and net mineralization of organic nitrogen to ammonium is much higher in subseafloor sediments of the ocean margin than subseafloor sediments of the open ocean.

On a more precise level of comparison, the profiles of DIC show that the Leg 201 sites span a wide range of subsurface activity levels (Fig. F7). The lowest DIC concentration is present at clay-rich open-ocean Site 1231. DIC concentrations are visibly higher at equatorial Site 1225 and still higher at eastern equatorial Site 1226. Still higher DIC concentrations are present at the Peru shelf sites (1227, 1228, and 1229). The highest concentrations are present at Peru slope hydrate Site 1230, downslope of the shelf sites and directly beneath the Peru upwelling zone. Ammonium shows the same site-to-site progression as DIC. This site-to-site progression of increasing DIC and ammonium concentrations matches the progression of increasing cell concentrations seen in Figure F4. The similarity of these progressions suggests that subsurface cell concentrations are related to net subsurface microbial activity.

Leg 201 profiles of dissolved chemicals in interstitial waters also document the subseafloor occurrence of specific microbial processes. Despite the large apparent differences between net microbial activities of the ocean-margin sites and those of the open-ocean sites, all of the microbial processes that we inferred to occur in subsurface ocean-margin sediments also appear to occur in the subseafloor open-ocean sediments.

Downhole depletion of dissolved sulfate occurs at all of the Leg 201 sites (Fig. F8). The lowest magnitude of subseafloor depletion occurs at open-ocean Site 1231, where dissolved sulfate declines by <2 mM. The highest magnitude of subseafloor sulfate depletion occurs at the Peru slope hydrate Site 1230, where all of the sulfate diffusing downward from the seafloor disappears by 9 mbsf.

Concentration profiles of dissolved manganese (Fig. F8) and iron suggest that manganese and iron reduction also occur at all of the sites sampled during this cruise. Reduced manganese and iron are, respectively, products of manganese(IV) and iron(III) reduction. Consequently, concentration profiles of dissolved manganese and iron can be used to infer net manganese and iron reduction in subseafloor environments. Concentrations of dissolved manganese (Fig. F8) and iron are generally higher in the deeply buried open-ocean sediments than in the deeply buried ocean-margin sediments. By inference, manganese and iron reduction are overall more important for diagenesis in subseafloor open-ocean sediments. This finding is consistent with the general perception that manganese oxides and iron oxides are largely depleted at shallow sediment depths in shelf environments (Canfield et al., 1993).

Methane is a stable product of some microbial activities. It is a source of carbon and energy for other microbial activities. The presence of distinct minima and maxima in the subsurface profiles of dissolved methane at all Leg 201 sites indicates that methane is biologically created and destroyed in the subsurface realm of both the ocean-margin and open-ocean provinces (Fig. F9). Documentation of methane profiles at these open-ocean sites required Leg 201 scientists to develop new sample handling protocols and to accurately measure methane at extremely low concentrations. The discovery of methane at all Leg 201 sites strongly suggests that methanogenesis occurs in deeply buried sediments throughout the world ocean. This discovery builds on the recent observation that methane is commonly present in subseafloor sediments of the open ocean, despite the presence of high sulfate concentrations (D'Hondt et al., 2002). Unexpectedly, the Leg 201 studies also indicate that methanogenesis occurs in subseafloor realms of active manganese and iron reduction. This discovery echoes recent evidence that methanogenesis and iron reduction co-occur in the continental subsurface environment of a freshwater aquifer (Bekins et al., 2001). Even more unexpectedly, Leg 201 studies also showed that both ethane and propane are biologically created and destroyed in parallel with methane at both open-ocean and ocean-margin sites (see example from Site 1227 in Fig. F10).

Acetate and hydrogen are generally understood to be the most common products of microbial fermentation. They are also generally understood to be the most common energy sources in microbial respiration reactions. Consequently, they are expected to be key intermediates in subsurface microbial activities. Leg 201 studies showed that acetate, formate, and hydrogen are present throughout the subsurface environment at all sites. The properties that control subsurface concentrations of acetate, formate, and hydrogen, whether kinetic or thermodynamic, remain to be determined.

Classic models of microbial activity in marine sediments assume that the oxidants (electron acceptors) that are used to support respiration by sedimentary prokaryotes are introduced to the sediment through the sediment/water interface. Sulfate is the most common electron acceptor in marine sediments. Oxygen and nitrate are the electron acceptors that yield the highest free energies. Sulfate, dissolved oxygen, and nitrate are all introduced to the subseafloor realm by diffusion from the overlying ocean. Leg 201 studies of open-ocean Sites 1225 and 1231 showed that nitrate and traces of oxygen also enter at least some deep-sea sediments by diffusing or advecting upward from waters flowing through the underlying basaltic crust (Fig. F11). In this manner, electron acceptors with high free-energy yields enter deep in open-ocean sediment columns. Conversely, downward diffusion from the sediment into the water flowing through the basement strips away products of microbial activity, such as methane, ammonium, and DIC. In both of these ways, chemical exchange between the sediment column and the underlying basement mirrors diffusive exchange across the sediment/ocean interface.

The discovery that nitrate and dissolved oxygen enter the sediment column from the formation waters of the underlying basalt has an additional interesting implication. It indicates that at least in some areas microbial activity within the basalt is insufficient to strip even the scarcest preferentially utilized electron acceptors from the water that flows through it.

At the Peru shelf sites, chemical species also enter the sediment sequences from below; however, at these sites, the chemicals diffuse upward from underlying Miocene brine. At Site 1229, dissolved sulfate diffuses upward into methane-rich Pleistocene sediment. Downhole profiles of dissolved sulfate, methane, acetate, and barium show that chemical distributions at the lower (brine controlled) sulfate/methane interface mirror those of the overlying "normal" sulfate/methane interface (Fig. F12). Cell counts and the pronounced inflections in chemical profiles demonstrate that the subseafloor prokaryotic cell populations and activity are focused at the lower sulfate/methane interface. Dissolved barium profiles indicate that microbial activity at this lower interface directly influences sediment chemistry by causing the precipitation of barite immediately below the lower interface and the dissolution of barite immediately above it. Perhaps the most striking feature of this interface is its thousandfold increase in cell concentrations relative to the sediments that lie immediately above and below it (Fig. F12). The observed cell concentrations at this interface are an order of magnitude higher than the concentrations observed at the seafloor.

In all of the sedimentary environments sampled during Leg 201, sedimentary properties and, by inference, past oceanographic conditions affect the current rates and sediment depths of several principal microbial activities. This point is most broadly demonstrated by the general correspondence between each site's geographic location (open ocean, equatorial upwelling, or coastal upwelling) and its current cell concentrations and concentrations of metabolic products (e.g., DIC and methane) (Figs. F5, F6, F7, F9).

At individual sites, the depths at which specific microbial activities are most pronounced are commonly directly related to sediment properties. These properties in turn were largely determined by oceanographic conditions at the time of the sediment's original deposition. For example, correspondences between dissolved iron concentration and magnetic susceptibility suggest that current subseafloor rates and foci of microbial iron reduction may be ultimately controlled by the availability of oxidized iron in mineral form. This relationship is illustrated at Site 1225, where the concentration of dissolved iron closely follows magnetic susceptibility (Fig. F13). At that site, the peak iron concentration is present in lithologic Subunit IC, where magnetic susceptibility is highest. These sediments were deposited during the middle-late Miocene "carbonate crash" of the equatorial Pacific (Farrell et al., 1995). Leg 201 magnetic reversal records suggest that the magnetic susceptibility of these sediments dates to the time of their original deposition. The overlying interval of near-zero magnetic susceptibility and very low dissolved iron concentration was deposited during the late Miocene "biogenic bloom" of the Indo-Pacific tropical ocean (Farrell et al., 1995).

Relationships between dissolved manganese concentrations, lithology, and physical properties similarly suggest that current rates and stratigraphic foci of microbial manganese reduction are contingent on oceanographic history. Such a relationship is seen at Site 1226, where the peak dissolved manganese concentration is present in the oldest sediments (Fig. F14). This sedimentary interval exhibits the lowest natural gamma radiation and, as shown by Leg 138 shipboard studies, the lowest average total organic carbon (TOC) concentration of this 16-m.y. sedimentary sequence (Shipboard Scientific Party, 1992a). This interval of low gamma radiation and low TOC was deposited at very slow rates over 8 m.y. (Shipboard Scientific Party, 1992a). The overlying interval of higher gamma radiation, higher TOC, and much lower dissolved manganese concentration was deposited during the late Miocene biogenic bloom (Shipboard Scientific Party, 1992a). These relationships suggest that manganese reducers now active in these sediments are utilizing oxidized manganese that accumulated during an interval of unusually low carbon accumulation from 8 to 16 m.y. ago.

Profiles of magnetic susceptibility and concentrations of dissolved iron, manganese, and sulfide suggest that current iron- and manganese-reducing activity may be similarly contingent on depositional history at the Peru shelf sites. For example, at Site 1229 the peak dissolved iron concentration and lowest dissolved sulfide concentration coincide with intervals of relatively high magnetic susceptibility at 87 mbsf and below 123 mbsf (Fig. F15). High magnetic susceptibility is present in intervals of terrigenous-dominated sediments deposited during intervals of low relative sea level. The peak iron concentration at 87 mbsf is within the well-developed methanogenic zone at this site (Fig. F16). The peak concentration below 123 mbsf lies within the underlying sulfate-reducing zone. In short, higher manganese and iron concentrations are consistently associated with relatively high susceptibility intervals at both open-ocean and ocean-margin sites. This association illustrates one way in which current rates and stratigraphic depths of an individual microbial process may depend on past ocean history.

At the Peru shelf sites, the lithologic context of anaerobic oxidation of methane (AOM) zones suggests another way that past ocean history may affect current microbial activity. At these sites, AOM and related processes predominantly occur in narrow subsurface zones that are associated with thin sedimentary intervals characterized by high grain density (Fig. F17), accompanied by high NGR, high resistivity, and low porosity. These thin low-porosity intervals are unusually rich in terrigenous sediment and are interpreted to have been deposited during times of low relative sea level. This lithologic association of AOM zones with high-density, low-porosity offlap sediments provides intriguing evidence that on the Peru shelf the position of AOM zones may be presently pinned within the sediment column by lithologic properties and, by extension, depositional history. More detailed determination of the extent to which physical and compositional properties control subseafloor biogeochemical zonations in this region will require further investigation.

A different aspect of the Peru shelf records provides evidence that geologically recent oceanographic changes may also affect the activity of subseafloor organisms at shallow burial depths. At Sites 1228 and 1229, a brief positive excursion in alkalinity, DIC, ammonium, and sulfide coincides with a brief negative excursion in dissolved sulfate at 2-3 mbsf (Fig. F18). This near-surface interstitial water anomaly indicates that the steady-state diffusion of biologically active chemicals past the upper sediment column was disrupted by late Pleistocene environmental change and has not yet fully recovered. There are at least three possible explanations for this anomaly. It may result from ongoing activity in a microbial "hotspot" at this shallow sediment depth, it may be a chemical relic of past microbial activity and is now relaxing back to a diffusional steady state, or it may be due to the recent establishment of an oxygen minimum at this water depth, causing the extinction of a bioirrigating benthos and a stimulation of sulfate reduction. The first and third of these explanations imply that this anomaly is a fingerprint of current microbial activity.

Chemical profiles of the Leg 201 sites provide consistent evidence of microbial influence on their sedimentary environment. This evidence is most clearly expressed at the ocean-margin sites, where microbial activity is highest. At these sites, the precipitation and dissolution of a number of minerals, including pyrite, barite, dolomite, and apatite, are catalyzed by microbial activities.

Dissolved barium, sulfate, and methane profiles provide a particularly clear illustration of the interplay between microbial activity and authigenic mineral formation and dissolution at these sites (1227, 1228, 1229, and 1230). The dissolved barium profiles at these sites are broadly similar to their methane profiles and are inversely related to their dissolved sulfate profiles (Fig. F19). The inverse relationship between sulfate and barium is inferred to be controlled by the solubility product of barite and the in situ activity of sulfate-reducing bacteria. Within the AOM zone, dissolved sulfate concentration declines toward 0 mM, barite dissolves, and the dissolved barium concentration rises. Diffusion of the dissolved barium past the AOM zone is suspected to sustain modern barite formation in the zone of dissolved sulfate. At sites with particularly high dissolved barium concentrations (such as Site 1230), a significant fraction of the sulfate diffusing toward the AOM zone may participate in this cycle of barite precipitation and dissolution before finally being reduced by microbial activity.

A broad range of microbiological studies was initiated during Leg 201 in order to study the diversity and population size of subsurface prokaryotic communities and to analyze the pathways and rates of their metabolic activities. These studies will not yield immediate results, partly because of the expected slow growth of deep subsurface prokaryotes and partly because the research requires special analytical equipment available only in shore-based laboratories.

A very large number of cultivation experiments and viable counts based on MPN methods were among the studies initiated on board. These cultivation experiments target a broad physiological spectrum of heterotrophic and autotrophic prokaryotes that utilize diverse electron acceptors and donors in their energy metabolism. Some experiments target chemoautotrophs using different electron donor/acceptor combinations. Others focus on heterotrophs using different electron acceptors for respiration. Others focus on methanogens and acetogens or fermenting or spore-forming prokaryotes. Still others target prokaryotes adapted to different monomeric or polymeric carbon sources, different temperature adaptations (psychrophilic, mesophilic, or thermophilic), and/or different pH, salinity, and pressure requirements. Some cultivation experiments focus on consortia depending on syntrophic degradation of organic substrates. Gradient cultures were initiated to screen for different substrate concentration requirements. Radiotracer MPN cultivations were inoculated for detection of minimal growth.

A wide range of other microbiological studies will be conducted postcruise. An extensive sampling scheme was developed for postcruise studies of prokaryotic populations using culture-independent molecular analyses. Molecular techniques based on 16S rRNA (ribosomal ribonucleic acid) gene sequence information will be used to analyze prokaryotic diversity and function. Sequence libraries for populations of different sediment depths and geochemical interfaces are expected to demonstrate differences related to the special conditions for life in the deep subsurface. Quantitative analyses of the prokaryotic communities will be done using, for example, FISH and real-time polymerase chain reaction. Secondary ion mass spectrometry (SIMS) will be used to analyze the stable carbon isotope composition of individual cells or cell clusters that are identified with nonspecific fluorescence stains or FISH probes. The potential metabolic activity of dominant members of these communities will be analyzed by sequencing functional genes for key enzymes, such as dissimilatory sulfite reductase and adenosine-5´-phosphosulfate reductase for bacterial sulfate reduction, coenzyme-M methyl reductase for methanogenesis, and formyl tetrahydrofolate synthase for acetogenesis. Finally, the carbon isotopic signature of biomarkers will be analyzed in order to identify the carbon substrate of the dominant prokaryotic populations.

It was an important goal of Leg 201 to identify and quantify the dominant microbial processes in the deep subsurface. To identify gross rates of microbial processes and rates that involve intermediate metabolites, experiments using radioactive tracers were conducted on the ship. Such use of radiotracers increases the sensitivity of process rate measurements by several orders of magnitude. The following microbial processes were analyzed at all relevant sites with samples from many depths and geochemical zones: sulfate reduction (³⁵S), methanogenesis (¹⁴C) and acetogenesis (¹⁴C), anaerobic methane oxidation (¹⁴C), acetate (¹⁴C) and hydrogen (³H) turnover, and prokaryotic growth using thymidine (¹⁴C) and leucine (³H) incorporation. Most of these experiments were terminated by the end of the cruise. Their activities will be measured in shore-based laboratories after the cruise. The results of these experiments will be compared to net rates obtained by modeling of interstitial water chemical gradients.

The study of deep subsurface prokaryotic communities is highly dependent on rigorous contamination control. Contamination tests are necessary to assess the extent to which prokaryotes from the surface environment may have reached the microbiological sediment samples at any point. Contaminating prokaryotes may be introduced from drilling fluid during the coring operation, they may penetrate into cores from their contaminated periphery during the wireline trip, or they may be introduced during the subsequent sectioning and subsampling of sediment. Consequently, refinement of routine methods for testing and avoiding potential contamination was an important part of the microbiological work initiated during Leg 201 (see House et al., this volume).

During the drilling operation at all sites, perfluorocarbon tracer (PFT) was fed in trace quantities into the seawater pumped through the drill string (Smith et al., 2000a, 2000b). This practice ensured that the core liner was bathed in a dissolved tracer that could later be analyzed at high sensitivity in retrieved cores and microbiological samples. The quantity of PFT detected in a sample provided an estimate of the maximal amount of seawater introduced into that sample. The PFT provides an upper-bound estimate of prokaryotic contamination, as it may penetrate by molecular diffusion through pore space inaccessible to prokaryotes. To further evaluate the extent to which contaminating prokaryotes may have penetrated into a sample, fluorescent beads of approximate prokaryotic cell size were applied as an additional contamination tracer. A suspension of >10¹¹ 0.5-µm-sized beads was released in the core cutting shoe at the critical moment of impact against the sediment. Microscopic counts of beads subsequently indicated whether samples used for microbiology had become "infected." This test may be more realistic with respect to the possibility of prokaryote-sized particles penetrating a sediment core. However, it is rather qualitative because the beads are not uniformly delivered to all surfaces of the core. Taken together, the two approaches provide a critical test on which to base confidence as to the noncontaminated nature of microbiological samples.

A summary of the extensive contamination tests for all Leg 201 sites is given in Figure F20 (this figure is based on the uncorrected data set and may include data points that were discarded as unreliable later [Site 1225]), which shows PFT and bead concentrations observed in selected samples. The two contamination indicators are positively correlated, thus improving confidence in the prediction of potential contamination by their combined use. The subset of samples presented in Figure F20 was chosen to reflect the wide range of tracer results observed during Leg 201. Out of the 154 PFT tests conducted on sediment samples, 62% had <0.1 µL seawater/g sediment potential contamination and 16% had no detectable contamination (<0.01 µL seawater/g sediment potential contamination). Out of 22 bead tests performed at all sites, >50% had no detectable beads. The centers of the cores, which were used for microbiological sampling, were compared to the periphery in contact with the core liner. The results show that core centers are much less contaminated than core peripheries (see House et al., this volume). Advanced hydraulic piston corer (APC) cores generally show far less potential contamination than the "biscuits" of sediment retrieved in extended core barrel (XCB) cores. The large scatter in all data shows that potential contamination is highly irregular from sample to sample. Consequently, contamination tests must be conducted specifically on the same sample that is used for a microbiological experiment in order to judge its contamination potential. It is not sufficient to rely on general experience with a specific coring/sampling technique or sediment type or to examine a sample from a different core portion.

Samples specifically used for isolations and viable bacterial counts during Leg 201 were generally proven by both PFT and bead tests to have very low or undetectable contamination. Through the extensive experience gained during the cruise, our confidence has strengthened that, with rigorous contamination controls and aseptic sampling techniques, deep subsurface samples can routinely be obtained without the introduction of microorganisms from the surface environment.