IODP

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doi:10.2204/iodp.sp.329.2010

Sampling strategy

All standard IODP shipboard analyses will be performed during the proposed expedition. Shipboard procedures that are not standard for IODP are discussed here. Many of the shipboard microbiological and geochemical analyses will follow those of Leg 201 (D'Hondt, Jørgensen, Miller, et al., 2003). Many analyses that will be performed postcruise are also described here.

Chemistry and microbial activities

Documentation of microbial processes in South Pacific Gyre sediment requires a wide range of biogeochemical analyses. Transport-reaction modeling of dissolved metabolite concentrations will allow us to quantify rates of principal net activities in the sediment (e.g., reductions of O2, NO3–, SO42–, Mn[IV], and Fe[II]) (Jørgensen, 2000). Chemical data (e.g., the position of the chloride maximum from the LGM) and thermal data will be used to account for advective transport in this modeling (D'Hondt et al., 2004). Concentration data will also allow us to calculate mineral stabilities and, in anoxic sediment, to test thermodynamic models of metabolic competition (Hoehler et al., 1998; Wang et al., 2004). Mineralogical, textural, and isotopic studies will be used to evaluate the extent of microbial mineral alteration (Boyd and Scott, 2001; Rouxel et al., 2003).

We will undertake key biogeochemical analyses and radiotracer incubations on the ship and collect appropriate samples for critical categories of postcruise biogeochemical studies. Particular shipboard effort will be placed on obtaining rapid, high-resolution concentrations of key dissolved chemicals that we expect to be present in the sediment at each site. In oxic sediment, these species include O2, NO3–, H2, SO42–, and many other ions. In anoxic sediment, they include NO3–, H2, SO42–, and many other dissolved metabolites, including, but not limited to, Fe(II), Mn(II), sulfide, acetate, formate, lactate, methane, ethane, and propane. Dissolved O2 concentrations will be measured by using micro-electrodes or micro-optodes on thermally equilibrated core sections (using methodology fine-tuned on recent coring cruises to the South Pacific Gyre, North Pond, and North Pacific Gyre). Concentrations of other dissolved species will be determined by ion chromatography (NO3–, SO42–, acetate, formate, and lactate), gas chromatography (dissolved gases), and ICP (dissolved Fe and Mn).

Gross rates of activities in sediment and basalt will be quantified by radiotracer incubations (Soffientino et al., 2006). The potential for autotrophic activity in basalt and sediment will be assessed by addition of Na14CO2/NaH14CO3 followed by short incubations to quantify 14C incorporation into cellular biomass (method adapted from Wirsen et al. [1993], who used it to demonstrate autotrophy associated with hydrothermal sulfides). This shipboard assay has been successfully used on young basalt from Loihi Seamount (Clement, Tebo, and Edwards, unpubl. data, 2006). The 14C-incorporated cells can be phylogenetically identified and visualized by micro-autoradiography–fluorescence in situ hybridization (MAR-FISH) technique (Lee et al., 1999; Ouverney and Fuhrman, 1999). Alternatively, assimilation ratio of 13C- and/or 15N-labeled substrates will be determined by nano-scale secondary mass spectrometry (NanoSIMS) at single-cell level (Dekas et al., 2009; Morono and Kallmeyer, pers. comm., 2010). Stimulation of in situ communities by lithotrophic substrates (sulfide, Fe[II], and Mn[II]) has also been successful with young basalt.

Uncontaminated samples of sediment and basalt will be collected and frozen for (1) shore-based solid-phase and isotopic analyses, (2) microbial biomarker studies, and (3) analyses of mineralogy and solid-phase geochemistry. These analyses will include studies of Fe and S isotopes and elemental composition.

Community composition

To document community composition in uncontaminated sediment and basalt, a large number of samples will be taken for postcruise molecular studies, including, but not limited to, FISH and 16S rRNA gene-tagged sequencing with 454 technology (e.g., Sogin et al., 2006). The molecular studies will allow us to determine (1) the microbial community structure (i.e., diversity richness and community evenness) and (2) their phylogenetic compositions. The RNA sequencing studies, targeting both 16S rRNA and messenger RNA (mRNA), will document the genetic and functional taxonomy of the actively growing and metabolizing fraction of the microbial community (Fukui et al., 1996; Poulsen et al., 1993). If the extracted DNA or RNA concentration is not enough for subsequent molecular and metagenomic analyses, whole genomes will be amplified by multiple displacement amplification with phi29 polymerase under strict experimental condition. These high-throughput molecular studies will quantify bacterial and archaeal diversity in these subseafloor environments and will enable detection of rare members that are not able to be sampled using conventional capillary sequencing approaches.

To determine the physiology, potential biogeochemical function, diversity, and habitat range of low-activity subseafloor microorganisms we will initiate a very broad range of cultivations during the expedition. As during Leg 201 (Shipboard Scientific Party, 2003b), many of these cultivations will use complex substrates, diversified electron acceptors, gradient media, and helper cultures. Cultivations will also allow ground-truthing of molecular analyses and provide estimates of minimum cultivable populations.

Biomass estimates

To document microbial biomass in the sediment and basement, we will use general nucleic acid stains [SybrGreen] for shipboard counts of total cell concentrations (Morono et al., 2009). The cell concentrations observed on expedition KNOX-02RR are well below the detection limit for the standard technique of counting cells in a slurried sediment (Kallmeyer et al., 2009). To undertake counts at this concentration requires separation and concentration of the cells from their sedimentary matrix (Kallmeyer et al., 2008). The accuracy of this approach has been quantified by parallel standard counts and cell-separation counts on samples from previous IODP sites (Kallmeyer et al., 2008) and Cruise KNOX-02RR (A. Puschell, unpubl. data, 2007). By adding autofluorescent microspheres to cell count slurries as a dilution standard, we will quantify the dilution/concentration factor of the cell-separation step and improve estimates of total cell density.

In addition to routine cell counts, we will take uncontaminated samples of both sediment and basalt for FISH counting to determine the extent to which counted cells are truly living, dormant, or dead. Quantifications of phylogenetic groups of active cells using FISH probes will be checked against DNA-based quantification using quantitative polymerase chain reaction (Schippers et al., 2005).

Potential for radiolysis

We will directly assess the potential of water radiolysis for supporting life in the organic-poor sediment and underlying basalt of the gyre. To estimate water radiolysis rates, we will determine uranium, thorium, and potassium concentrations by (1) natural gamma ray logging of representative cores and (2) analysis of selected samples by ICP-MS.

To verify rates of water radiation, we will determine the flux of He-4 to interstitial water. In subseafloor environments, radiolytic H2 production will principally result from production of He-4 (alpha particles) (Blair et al., 2007). Consequently, measurement and transport modeling of He-4 concentration profiles will constrain estimates of radiolysis rates independently of estimates based on abundances of radioactive elements.

To verify rates of H2 production by water radiolysis, we will undertake postcruise experiments with killed samples (of the cored sediment and basalt) plus tritium-labeled water and quantify the appearance of labeled H2 over the course of the experiments. Similar (unlabeled) experiments with artificial radiation sources were done by Lin et al. (2005b).

To quantify rates of microbial uptake of radiolytic H2, we will measure dissolved H2 concentrations in the cored sediment and at the sediment/basalt interface and compare them to the concentrations expected from the water radiolysis rates with no uptake (Fig. F10). The expected rate of radiolytic H2 production is so high (Blair et al., 2007) that in situ H2 concentrations will be measurable shipboard if the H2 is not biologically utilized. If H2 is biologically utilized and held to a thermodynamic minimum, its in situ concentrations will be below detection.

Finally, we will compare radiolysis rates to measured hydrogen turnover in sediment incubations with known numbers of microbial cells, using an array of relevant electron acceptors (O2, NO3–, and oxidized metals). In this way, the microbial rates of hydrogen oxidation that are thermodynamically possible in the subsurface ecosystem can be constrained. Bacterially catalyzed redox reactions are many orders of magnitude faster than abiotic reactions. Because of its high activation enthalpy, the recombination of O2 and H2 will not occur at all with measurable rates at temperatures below 400°C; bacterial catalysis allows this reaction on a timescale of minutes.

Contamination tracing

To evaluate the extent to which contaminating cells may have penetrated a sample, we will use (1) perfluorocarbon tracer in the drilling fluid (in sediment and basalt), (2) fluorescent microspheres injected at the drill bit (in basalt), and (3) genomic comparison of the basalt or sediment sample with drilling fluid from the time of coring. The first two techniques have been successfully used on multiple ODP legs and Expedition 301 (Smith et al., 2000a, 2000b; House et al., 2003; Lever et al., 2006). The third technique is common for studies of seafloor samples, where contamination is ubiquitous and genomic signatures of the contaminating material are subtracted from those of the seafloor sample. Previous results have consistently shown that core centers are much less contaminated than core peripheries (by factors of 3–300) and that APC cores are generally not significantly contaminated (House et al., 2003). The uppermost 1.5 m section of APC cores tends to be more contaminated than deeper sections (Lever et al., 2006). Contamination tends be much greater in RCB cores (e.g., on surfaces of cored basalt) and extended core barrel (XCB) cores than in APC cores. In all categories of core, potential contamination varies considerably from sample to sample. Consequently, to avoid contamination of microbiological results, contamination tests must be conducted on each core or hard rock sample that is used for a microbiological experiment (D'Hondt, Jørgensen, Miller, et al., 2003).

Geophysical properties

Physical properties will be used to quantify chemical fluxes through the sediment and to quantify the extent of basement alteration as a function of basement age and fluid flow. Formation factor will be measured for calculating fluxes of chemical species through the sediment. At all sites, sediment temperatures will be logged with the advanced piston corer temperature tool (APCT-3) and analyzed for active fluid flow. Basement holes will be logged with a suite of geophysical tools, including velocity (DSI), density, porosity, resistivity, magnetometer, natural gamma radiation, and FMS logs. For both sediment and basalt, core-based physical properties (bulk density, matrix [or grain] density, porosity, thermal conductivity, core temperature, P-wave velocity, and light absorption spectroscopy [LAS]) will be measured.

Alteration of the basaltic oceanic crust

Assessment of the extent and relative importance of secondary alteration to the basaltic crust will require an integrated program of petrographic, geochemical, and borehole analyses. At hand-sample and thin section scales, we will carefully describe general alteration textures and characteristics (e.g., veins, halos, vesicle filling, mineral/matrix replacement, and glass palagonitization), principal secondary mineralogy (e.g., saponite, celadonite, calcite, Fe oxyhydroxide, etc.), and the size, distribution, and orientation of veins and other structural features in the crust. Discrete samples of the core, representing "fresh" (e.g., pristine glass, if recovered), average, and end-member altered domains, will be powdered and analyzed for bulk major, trace, and volatile element chemistry, as a means of characterizing the bulk crustal composition and geochemical effects of alteration. Borehole logging and core-log integration are invaluable for reconstructing recovery gaps and estimating bulk geochemical and structural characteristics of deep basement drill sites (Barr et al., 2002; Révillon et al., 2002; Kelley et al., 2003; Pockalny and Larson, 2003). Radiogenic isotope measurements will place important constraints on the timing of alteration at each site. For example, calcite formed during crust alteration often contains high concentrations of uranium but little to no lead, making the lead isotopic system a potentially useful calcite precipitation geochronometer, especially in old oceanic crust (Hauff et al., 2003).

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