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doi:10.2204/iodp.proc.329.112.2011

Objectives

Geophysical objectives

Our principal geophysical objectives were to locate potential coring locations and to characterize the tectonic setting, the sediment characteristics, and the potential for hydrologic processes at each coring site. These objectives were achieved with a suite of geophysical measurements, including multibeam bathymetry, seismic reflection profiling, magnetics, gravity, and heat flow.

Seafloor mapping objectives

Multibeam bathymetry surveys of each coring site were conducted with SIMRAD EM120 swath mapping system prior to coring and on-station operations. The primary goals of seafloor mapping were to

• Identify and locate coring sites and

• Characterize the tectonic, volcanic, and kinematic environment of the survey area.

Swath bathymetry data were used in real time during seismic surveys to identify regions of the seafloor where sediments were most likely to be imaged with seismic methods. These bathymetry data were later processed and gridded to generate regional views of the survey areas to determine the orientation (i.e., spreading direction) and roughness (i.e., approximate spreading rate) of original abyssal hill fabric. These bathymetric charts were also used to identify the presence and magnitude of off-axis volcanism.

Total magnetic field measurements were made with the SeaSPY Overhauser magnetometer/gradiometer during the approach and departure of each coring site. These data were used in real time to identify individual magnetic anomalies on approach to a new site. Additional forward modeling of the calculated anomalous magnetic field was used to calculate spreading rates and determine the kinematic environment of crustal accretion.

Postcruise activities included additional processing of the multibeam data at each station and creation of a complete catalog of multibeam and backscatter plots for each 3° of transit.

Seismic objectives

Seismic surveys of each coring site were conducted with the Knudsen digitally recorded 3.5 kHz seismic reflection system and multichannel seismic reflection (MCS) system using one or two 150 inch³ generator injector (GI) guns (45 inch³ generator chamber, 105 inch³ injector) with a 48-channel digital streamer. The primary goals of the seismic surveys were to

• Locate suitable/representative coring sites,

• Estimate sediment thickness, and

• Identify potential lithologic variations within the seismic section.

Seismic surveys of 6 to 12 h duration were conducted and included crossing survey lines at/near the intended coring locations. Sediment thicknesses and potential sediment layers were determined from onscreen and hard copies of seismic sections. Additional postcruise seismic processing was conducted to generate stacked seismic sections from the MCS data.

Heat flow objectives

Thermal measurements were made to understand thermal processes at each coring site and to supplement the geophysical characterization of coring sites. This work is intended to address several questions:

• What is the thermal environment from which the cores are taken?

• What are the rates of advective heat flow and fluid flow through the sediments?

• What are the fluxes of heat through each coring site as a function of plate age, sediment thickness, and basement relief?

The coring sites (Fig. F2) span a broad range of tectonic settings, crustal conditions, and crustal ages. Tectonic settings include the East Pacific Rise ridge flank, the South Pacific abyssal plain, and the bathymetrically anomalous South Pacific Superswell (McNutt, 1998). Crustal ages range from ~15 to >100 Ma along the coring transects. These transects cross an area that is characterized by thin and discontinuous sediments and a relatively high population of seamounts (Wessel, 2001). All of these characteristics increase buoyancy-driving forces, promoting the likelihood of fluid flow through the crust and sediments (Harris et al., 2004).

Heat flow data within and near the South Pacific Gyre prior to the cruise are sparse (Fig. F2). Most of the existing heat flow data lie north of 20°S, with little data near the northern and southern transects. The existing heat flow data are predominantly single, widely spaced probe measurements. Interpretations of heat flow data in the South Pacific Superswell suggest advective fluid flow (Stein and Abbott, 1991; McNutt, 1998).

Sedimentology objectives

Our principal sedimentological objective was to document the composition, distribution, approximate age-depth relationships, and biologically relevant physical properties (e.g., formation factor, porosity) of the subseafloor sedimentary habitats in the South Pacific Gyre region.

Additional (postcruise) objectives are to determine the histories of aeolian sedimentation and nutrient dynamics in the South Pacific Gyre. These studies entail

1. Developing detailed strontium isotope stratigraphies from ichthyoliths and carbonate microfossils (to refine estimates of sediment age, sedimentation rates, and mass accumulation rates),

2. Determining grain size of terrigenous extracts,

3. Using Sr-Nd-Pb isotopes for determining provenance of the terrigenous input (e.g., the sites are located in the trades and therefore may show a positive ε_Nd signature, indicative of the younger volcanics from the Andes), and

4. Determining carbon isotopic and elemental signatures of planktonic and benthic carbonate microfossils to explore the history of nutrient dynamics.

Biogeochemistry objectives

Our biogeochemical objectives included documenting the biogeochemical environment of the subseafloor sediments and shallow basaltic basement beneath the low-productivity South Pacific Gyre to identify the principal redox processes that occur in those sediments (e.g., D’Hondt et al., 2002, 2004), to quantify the rates at which those processes occur (D’Hondt et al., 2002, 2004), to calculate mineral stabilities and, where possible, test thermodynamic models of microbial competition (Hoehler et al., 1998; Wang et al., 2004) and quantify the potential of water radiolysis for supporting life in these clay-rich sediments.

To meet these objectives, our shipboard analyses focused on determining dissolved concentrations of transitory compounds (e.g., O₂ and H₂) and compounds of special interest for documenting the biogeochemical environment and defining microbiological sampling strategies (e.g., O₂, NO₃^–, and, for the southern transect, SO₄^2–). Also to meet these objectives, samples were taken for determining solid-phase chemistry and concentrations of a diverse range of other dissolved chemicals postcruise.

The results of these studies will allow us to test our hypotheses that (1) net metabolic activities are low and oxygen (O₂) is the principal net terminal electron acceptor in this subseafloor ecosystem (and that consequently the diversity of anaerobic activities will be far less than in previously examined subseafloor sediments) and (2) radiolysis of water is a relatively significant source of microbially harvestable energy in this subseafloor environment.

Objectives of activity experiments

Distributions of metabolic products and isotopic signatures in pore waters and solid phases provide the basic information for estimating net microbial activities in subseafloor environments. However, not all processes are evident in the distributions of geochemical constituents. Metabolic reactants or products may be too low in concentration, indistinguishable from nonreacting components, or subject to secondary reactions that obscure the original signal. In certain cases, we can employ tracer experiments to reveal key underlying processes or activities.

In a classic tracer experiment, a compound that bears a radioactive or stable isotope of one of the elements in the compound of interest is added in trace (typically <5% total concentration) amounts. In subseafloor sediments, we are time-limited and working at the edge of detection limits (i.e., we cannot perform experiments on geological timescales). Consequently, we are often compelled to add compounds at higher than typical concentrations. Furthermore, we manipulate the sediments in ways that may critically depart from in situ conditions. It needs to be emphasized that tracer experiments demonstrate potential activities. How close rates determined in an experiment reflect in situ rates and activities must be judged for every experiment. Nonetheless, in the search for microbial activity in the ultra-oligotrophic subseafloor ocean, tracer experiments may provide key insights or constraints on processes.

Hydrogenase activity

Hydrogenase enzymes are key components of all known biochemical pathways that involve hydrogen as a product or as a substrate. In typical sediments, virtually all of the dissolved hydrogen present in the interstitial water is produced biologically from anaerobic fermentation of organic matter. In the extremely organic poor clays of the South Pacific Gyre, hydrogen production from radiolysis of water may be a significant source of reducing power for microorganisms. Thus, measurement of hydrogenase activity is significant for understanding the activity of microorganisms in relation to the possible sources of energy in these sediments.

Nitrogen cycling

After respiration using dissolved oxygen, nitrate reduction to dinitrogen gas (denitrification) yields the most energy per mole of typical organic carbon compound (e.g., sugar) or hydrogen. Furthermore, reduced nitrogen compounds, principally ammonia, are released during decomposition of organic matter. Nitrification of dissolved ammonia with oxygen (or even with nitrate) may form a secondary and significant pathway of oxygen consumption in these ultra-oligotrophic sediments. Nitrification and denitrification pathways can be identified by amending sediments with ¹⁵N-labeled NH₄ and NO₃^–, respectively, and then examining isotopic ratios in the NO₃^– and N₂ produced during time-course experiments.

Thymidine incorporation

The incorporation of thymidine into DNA is a commonly used method to estimate microbial growth. In deep subsurface sedimentary samples, the method has been adapted for use in identifying zones of microbial activity. We inject ³H-labeled thymidine into subsamples from multicores and piston cores to determine the incorporation of tritium into DNA over a 3-day time course experiment.

Cysteine degradation experiments

Bacterial communities in abyssal sediments that can oxidize reduced sulfur compounds have been reported (Teske et al., 2000). These reports raise the question of the provenance of the reduced sulfur for such a metabolism. Abyssal sediments from the north-central Pacific also contain low but measurable concentrations of reduced sulfur minerals (Berelson et al., 1990). Reduced sulfur may originate during degradation of sulfur-containing amino acids such as cysteine or methionine.

Microbiology objectives

The principal objective of our microbiological studies was to characterize the composition and biomass of the living microbial communities in the relatively shallow subseafloor sediments of this oxic and organic-poor environment. To meet this broad objective, science party members are undertook many specific projects with narrower objectives. For example, individual postcruise studies are:

1. Examining trends in community composition within and between sites related to changing geochemical and lithological regimes and sediment age or depth,

2. Searching for evidence of selective patterns of microbial colonization on surfaces of specific sedimentary components (e.g., to determine if minerals, micrometeorites, and so on, provide key nutrients for microbial metabolism under oligotrophic conditions),

3. Testing the role of microbes in formation of manganese nodules,

4. Documenting the potential isolation and connectedness of microbial communities in the sediment relative to that in the underlying basalt, and

5. Examining the biosynthetic potential of the microbial communities, among others.

To meet these objectives, we undertook select microbiological studies during the cruise and we are undertaking a diverse range of studies postcruise.

Cell enumeration objectives

The principal objective of our cell enumeration studies was to estimate the concentration of living biomass in these very organic poor subseafloor sediments and to develop a conservative estimate of the extent to which the observed cells from this environment are truly living and active. The first of these objectives was met by census of cells using DNA-specific stains (SYBR Green and acridine orange). To determine the extent to which counted cells are truly active, we took uncontaminated shipboard samples for postcruise catalyzed reporter deposition—fluorescence in situ hybridization (CARD-FISH) counts on both bulk sediments and separated mineral components. This approach uses an oligonucleotide probe that hybridizes to a target rRNA molecule. Consequently, it differentiates metabolically active, rRNA-rich cells from rRNA-depleted, inactive, or dead cells. To determine if the cells are growing, bulk sediment samples were taken for incubations with a DNA nucleotide analog, bromodeoxyuridine (BrDU). Cells that take up BrDU will incorporate it into their DNA only during DNA synthesis, a key step in cell division.

The results of these studies have allowed us to test our hypothesis that current estimates of active subseafloor biomass (Whitman et al., 1998; Parkes et al., 2000) are far too high because the abundance of active cells in this habitat is at least two orders of magnitude lower than current estimates of average cell concentrations in deep-sea sediments (Kallmeyer et al., 2009).

Molecular biological objectives

Our principal molecular biological objectives were to determine the community composition, active members, potential activities, diversity, and distributions of microbial communities in the shallow subseafloor sediments of the South Pacific Gyre. Secondary molecular objectives included the characterization of microbial communities associated with manganese nodules and basaltic glass.

Postexpedition investigations into microbial diversity, as well as depth-related and geographic distribution patterns thereof, focused on 16S/18S rRNA and 16S/18S rRNA genes using polymerase chain reaction (PCR) (e.g., Durbin and Teske, 2010). Samples were also taken to

1. Target functional genes, including genes involved in carbon fixation and nitrogen metabolism, using PCR;

2. Detect biosynthetic gene clusters (polyketide synthase and nonribosomal peptide sythetase) through PCR amplification of whole sediment DNA extractions; and

3. Pursue further investigations into microbial activities and evolution using phi-29 polymerase-mediated whole genome amplification and community metagenomics.

Finally, samples were taken to target select community members for population or single-cell genomics using cells extracted from bulk sediments and separated mineral components and sorted using flow cytometry. Extraction of DNA from separated mineral components is intended to assess microscalar variations in microbial diversity in the sediment column as a function of mineralogy and substrate composition.

The results of these and other studies will ultimately allow us to test our hypothesis that the composition of the subseafloor sedimentary community in this province is distinctly different from the communities observed in the higher activity anaerobic subseafloor ecosystems that have been examined to date.

Cultivation objectives

The unique nature of the sediments collected during the South Pacific Gyre coring expedition provides an opportunity to study potentially novel microbial diversity and adaptations to subsurface life. A principal objective of our cultivation experiments was to test the potential diversity of metabolic activities in these oxic and organic-poor subseafloor sediments. An additional objective was to determine the viability of aerobic heterotrophs in the sediment and to estimate the number of cultivable cells.

Recent studies have suggested that the deep ocean may harbor new genera of obligate marine microbes with extraordinary biosynthetic potential. Consequently, another objective of our studies was to extend knowledge of the cultivable marine actinobacteria from deep-ocean sediments. In particular, we cultivated Gram-positive bacteria from sediments collected during the South Pacific Gyre coring cruise to determine the phylogenetic affiliations of bacterial isolates and to assess the biosynthetic potential of selected Gram-positive isolates using genetic, chemical, and bioassay analyses.

Objectives of manganese nodule studies

The goal of our manganese nodule studies was to characterize the microbial community of the nodules with the hope of providing clues about the processes of nodule formation and growth. Specific objectives were to determine microbe-mineral interactions on and within nodules and the extent to which these influence nodule formation and growth and to use isotopic analyses to measure nodule growth rates. We further hope to distinguish microbial diversity specifically associated with Mn nodules, whether they provide a growth substrate or are themselves indicative of microbial activity (i.e., Mn oxidation). If the microbial community has a role in mediating the formation of Mn nodules, we hope to identify which organisms are responsible for the process using FISH and DNA analysis.

Objectives of basalt studies

At sites where we recovered the basaltic basement that underlies the sediment, we took samples for DNA analysis and microscopy of the basaltic glass to characterize its microbial communities for comparison to seafloor basaltic communities at other locations and look for differences based upon age and nutrient availability. These studies place a special focus on microbes involved in the iron and nitrogen cycles. To understand the potential isolation and connectedness of microbial communities in the basaltic basement to that in the overlying sediment, samples were taken to compare the bottommost recovered sediment microbial communities to communities in the basaltic glass recovered.

Objectives of water column studies

The South Pacific Gyre is the most oligotrophic region of Earth’s oceans. Our concentration/temperature/depth recorder (CTD) water column analyses and sampling focused on

1. Documenting key properties of the near-surface waters,

2. Sampling to determine bacterial diversity, and

3. Documenting the nature of the biological nitrogen cycle (nitrogen fixation rates, assimilation rates of organic and inorganic nitrogen, diazotrophic bacterial groups present, and the activity of nifH genes, which are responsible for nitrogen fixation) in this little-studied oceanic region (Halm et al., submitted).

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