Sites 1225 and 1226 were selected as drilling targets because their microbial activities and cell counts were expected to be far below those in ocean-margin settings but above those in the lowest activity open-ocean environments.
The principal objectives at Site 1225 were
Site 1225 is located in the eastern equatorial Pacific near the present-day boundary between the South Equatorial Current and the North Equatorial Countercurrent at 3760 m water depth. It lies in the sedimentary bulge created by the rain of biogenic debris from the relatively high productivity equatorial ocean. Geochemical studies of DSDP and ODP sites throughout this region have shown that seawater flows through the basaltic basement that underlies the sediments throughout this region (Baker et al., 1991; Oyun et al., 1995). Anomalously low conductive heat flow occurs throughout the region (Von Herzen and Uyeda, 1963; Sclater et al., 1976), possibly because the large-scale advection of relatively cool seawater through the basalts depresses conductive heat flow (Oyun et al., 1995).
The lithologies, sediment age, and many geophysical characteristics of the target site were well characterized by earlier studies of nearby Site 851 (Mayer, Pisias, Janecek, et al., 1992; Pisias, Mayer, Janecek, Palmer-Julson, and van Andel, 1995). Those studies indicated that the site is representative of a large portion of the eastern equatorial Pacific region. The gross lithologic and physical properties of the carbonate and siliceous oozes and chalk at Site 851 are characteristic of sediments throughout the region (Mayer, Pisias, Janecek, et al., 1992). The pore water chemical profiles at Site 851 exhibit clear evidence of seawater flow through the underlying basalts (and perhaps the lower part of the sediment column) (Oyun et al., 1995; Spivack and You, 1997).
Cragg and Kemp (1995) documented the presence of microbial cells and activity throughout the sediment column at Site 851. For the first few tens of meters below seafloor, counts of both total cells and dividing cells were low relative to counts from similar depths at sites from the Peru shelf and the Japan Sea (Cragg and Kemp, 1995). At greater depths, Site 851 cell counts approached the averaged values from all previously counted sites.
Leg 138 shipboard chemistry showed that concentrations of several dissolved chemical species (ammonium, strontium, and silica) and alkalinity peaked midway down the sediment column. In contrast, dissolved sulfate exhibited maximum values near the sediment/water interface and the basement/sediment interface (Mayer, Pisias, Janecek, et al., 1992). These patterns of sedimentary pore water concentration are inferred to result from low levels of biological activity throughout the sediment column, coupled with diffusive exchange with the overlying ocean and seawater flowing through the underlying basalts (and perhaps the lower part of the sediment column) (Spivack and You, 1997). Geochemical modeling suggests that net microbial sulfate reduction in the upper half of the Site 851 sediment column is only 2.8 (x 10–9 mol/cm2/yr (D'Hondt et al., 2002). This rate of sulfate reduction corresponds to a respiration rate of 5.6 (x 10–9 mol CO2/cm2/yr. This rate of respiration is only the barest fraction of the rate of CO2 reduction by photosynthesis in the overlying equatorial ocean (9.3 x 10–4 mol/cm2/yr) (D'Hondt et al., 2002). The subsurface extent of electron acceptors with higher standard free-energy yields (oxygen, nitrate, manganese oxide, and iron oxides) in this region was not determined for Site 851.
At Site 1225, concentrations of CH4, NH4+, DIC, and alkalinity peak in the middle of the sediment column and decline toward both the sediment/ocean interface and the sediment/basement interface. In contrast, SO42– concentration is lowest in the middle part of the sediment column and NO3– and O2 are present only at the ocean and basement interfaces. These profiles result from the balance between net subsurface microbial activities and small net fluxes of biologically utilized chemicals across the ocean/sediment and sediment/basement interfaces into and out of the subsurface sediments. Although slight upward advection of ~0.1 mm/yr is inferred from chlorinity profiles, microbial activity occurs at high enough rates in the lowermost sediment column to maintain downward diffusion gradients of NH4+, DIC, and alkalinity.
Pore water data also document O2 penetration into the top 2 m of the sediment column, a zone of NO3– in the top 1.5 m of the sediment, a peak concentration of dissolved Mn at 3.6 mbsf, a broad zone of relatively high dissolved Fe concentration centered at ~25 mbsf, and sinks for reduced Mn and dissolved Fe at 100 mbsf. SO42– concentration decreases downhole by only ~10% from seawater values; most of this decrease occurs in the upper 30 m. This vertically extended sequence of successive pore water chemical zones closely resembles the sequence seen in nearshore sediments on centimeter to decimeter depth scales (with depth-dependent transitions from a zone of oxygen reduction to successive zones of nitrate, manganese oxide, iron oxide, and sulfate reduction). These data are consistent with the hypothesis that subseafloor microbial communities preferentially utilize the available electron acceptor that yields the highest free energy of reaction.
In the lower portion of the sediment column, this sequence of successive reduction zones is reversed by seawater flow through the underlying basaltic basement. Diffusion of solutes from this seawater to the overlying sediment delivers NO3– to the lowermost 20 m of the sediment column (300 mbsf to basement) and possibly also O2 to the lowermost meter of the column (319.3 mbsf to basement). This short interval of dissolved O2 and NO3– is overlain by a broad zone of dissolved Mn centered near 250 mbsf and a broad peak of dissolved Fe centered at ~230 mbsf. These profiles show that electron acceptors yielding high free energies of reaction are introduced to at least some portions of the deep subseafloor biosphere by hydrologic processes. They also indicate that microbial activity in the underlying basement is insufficient to strip even the scarcest preferentially utilized electron acceptors from the seawater that flows through the basement.
Dissolved H2 concentration is generally in the range of 1–2 nM. Lovley and Goodwin (1988) and Hoehler et al. (2001) observed similar concentration in experiments with near-surface aquatic sediments where sulfate reduction is the primary electron-accepting reaction. On the basis of their observations, Lovley and Goodwin (1988) hypothesized that H2 concentration in aquatic environments is controlled by competition between different metabolic pathways. According to this hypothesis, microbes using electron acceptors that yield higher free energies of reaction are able to operate at lower electron donor concentration and thereby out-compete microbes limited to electron acceptors that yield lower free energies of reaction. Documentation of this concentration at Site 1225 suggests that even in low-activity subseafloor sediments, H2 concentration may be controlled by the same thermodynamic competition between electron-accepting pathways as in high-activity sediments and can be predicted from the dominant pathway.
Methane is present at a trace concentration of <250 nM throughout the sediment column. This finding demonstrates the presence of CH4 in subseafloor sediments with SO42– concentration that is very close to seawater values. The generation of CH4 in these sediments challenges models of microbial activity that are based on standard free energies. There are a number of possible reasons for the occurrence of methanogenesis in sulfate-rich sediments. For example, the methanogens and sulfate reducers may rely on different electron donors (e.g., the methanogens may utilize methylated amines and the sulfate-reducers may rely on H2 and/or acetate) (Oremland and Polcin, 1982; Oremland et al., 1982b; King, 1984).
The steady-state maintenance of CH4 in the subseafloor sediments of Site 1225 indicates that if anaerobic methanotrophy occurs here, it does not drive the CH4 concentration below a threshold concentration of a few tens to hundreds of nanomolar. Concentration is lowest near the sediment/ocean and sediment/basement interfaces, where CH4 may be oxidized by microbes using electron acceptors that yield relatively high energies of reaction (such as NO3– or O2). The highest CH4 concentration is present in the middle of the sediment column, where SO42– appears to be the only electron acceptor available. We hypothesize that the peak CH4 concentration is held at the observed level (~150–250 nM) because sulfate-reducing methanotrophs cannot oxidize CH4 at lower concentration under in situ conditions.
Concentrations of acetate and formate were <1 and <0.5 µM, respectively, throughout the sediment column. These concentrations are an order of magnitude lower than those measured in continental shelf sediments (Sørensen et al., 1981; Wellsbury and Parkes, 1995) and are also lower than in other deep sediment sites (Wellsbury et al., in press). These very low concentrations appear unlikely to be regulated by limiting energy yields but may be limited by the kinetics of active uptake by the anaerobic respiring bacteria. Since these results are among the first to demonstrate very low concentrations of short-chain fatty acids in cold, low-activity subsurface sediments, there is no database for comparison.
Comparison of Site 1225 physical property, sedimentology, and chemical records suggests that broad-scale patterns of past oceanographic change exert strong influence on present subseafloor metabolic activity. The concentration of dissolved iron closely follows downhole variation in magnetic susceptibility and split-core reflectance, with peak concentrations of dissolved iron and solid-phase iron compounds (inferred from magnetic susceptibility) in the intervals from ~0 to 70 mbsf and 200 to 270 mbsf. The intact magnetic reversal record suggests that the magnetic compounds were created during or shortly after sediment deposition. Dissolved SO42– is the terminal electron acceptor in the intervening sediments, which are depleted in dissolved iron and have low magnetic susceptibility. These intervening sediments are characterized by the most intensely bioturbated intervals and were deposited during a late Miocene–early Pliocene biogenic bloom that occurred throughout much of the global ocean (van Andel et al., 1975; Farrell et al., 1995; Dickens and Owen, 1999).
Four Adara tool deployments plus two deployments of the Davis-Villinger Temperature Probe (DVTP) defined a sediment/water interface temperature of 1.4°C and an estimated sediment/basement interface temperature of 7.0°C. The downhole temperature gradient curved slightly downward. The slight curvature appears to be best explained by a geologically recent decrease in basement temperature, perhaps a result of an increased rate of seawater flow through the basement. Throughout the sediment column, in situ temperatures were well within the range inhabited by psychrophilic bacteria.
Experiments on major bacterial processes and experiments for enumeration of viable bacteria were initiated at selected depths ranging from near the mudline to near the basement, where samples were obtained within centimeters of the basalt. Subsamples for postcruise biomolecular assays and microbiological experiments were routinely taken from all of the distinct geochemical zones and lithologic subunits. Total bacterial numbers were enumerated on board. These bacterial counts are very close to data obtained from nearby Site 851 and consequently demonstrate the high reproducibility of AODC tests in subseafloor microbial studies.
Contamination of microbial samples was closely monitored at Site 1225 by injection of PFT and microbe-sized fluorescent microspheres during the drilling process. Based on the PFT results, microbial contamination is very low throughout most of the sampled sedimentary column. Potential bacterial contamination from the drilling fluid is statistically less than the detection limit of 50 cells/g sediment in microbiology samples from nine different sedimentary horizons and <200–2200 cells/g sediment in samples from seven additional horizons. The potential for microbial contamination is much greater in the deepest core taken at this site (Core 201-1225A-35X), especially in the Core 35X core catcher, where PFT concentration indicates possible contamination as high as 11,400–167,900 cells/g sediment. The only samples of basaltic basement recovered at this site were in this core catcher. Fluorescent bead experiments demonstrated potential contamination in the first two APC samples taken for cultivation experiments. After small modifications to microbiological sample handling routines, the remaining APC cultivation samples were free of the contaminant tracer beads.
At this site, novel experiments with core temperatures and contamination tracers were undertaken to determine how handling of cores and samples for microbiological studies might be improved. Catwalk experiments with an IR camera were used to assess the effects of different core handling procedures on transient warming of the core and, consequently, on the recovery of temperature-sensitive microbes. Detailed PFT experiments were used to assess within-core variation in drilling-induced contamination.