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Geochemistry and microbiology

Inorganic geochemistry

Whole-round pore water sample collection

Shipboard analyses were performed on interstitial waters extracted from 5 cm long whole-round sediment samples. Samples were collected every 1.5 m to the estimated base of the sulfate reduction zone at 50 mbsf. Below 50 mbsf, whole rounds were collected every 10 m to terminal depth (TD). The higher sampling frequency above 50 mbsf provided a better definition of the sulfate/​methane interface (SMI). The whole-round sections were cut from the core immediately after recovery on deck. The surfaces of the samples were scraped with a polytetrafluoroethylene (PTFE)-coated spatula to remove sediment potentially contaminated with drilling fluids. Interstitial water was extracted by applying up to 40,000 lb of pressure in a titanium squeezer. The pore water samples were collected directly into a 60 mL plastic syringe, and sample splits were filtered through a 0.45 µm filter. The pore water volume extracted was ~40 mL. Whole-round intervals were increased to 10 cm long when pore water recovery was not sufficient to provide the volume required for analysis. Sample splits were sealed in plastic vials for shipboard analysis, IODP archives, and for shore-based elemental and isotopic analysis.

Rhyzon sample collection

High-resolution pore water sampling was conducted on the upper 15 m of selected holes using soil moisture probes called Rhyzon samplers. Rhyzon samplers enable discrete sampling with minimal disturbance to the sediment surface. The method does not require filtering or removal of wet sediment from the piston core. Rhyzon samplers used during Expedition 308 were 2.5 mm wide × 30 mm long filters with a pore size of 1 µM. The use of Rhyzon samplers to extract pore waters from marine sediments is potentially a powerful and novel alternative to the technique of squeezing whole-round sediments.

Samplers were inserted into the sediment at ~10 cm intervals. Pore water was withdrawn under vacuum into acid-washed 60 mL syringes or He-purged headspace vials. Whole-round cores were sampled prior to splitting for sedimentological description. Shipboard samples were stored in plastic vials in a refrigerator prior to analysis. Sediment samples were also collected for physical property testing including permeability and porosity. MST scans (see “Physical properties”) were run before and after sampling to assess potential alteration of sediment properties (see “Rhyzon” in “Supplementary material”).

Shipboard analyses

Interstitial water samples were analyzed for routine shipboard measurements according to standard procedures and calibrated against International Association of the Physical Sciences of the Oceans (IAPSO) standards (Gieskes et al., 1991). Alkalinity and pH were determined immediately following sample collection using the Metrohm autotitrator with a Brinkman combination pH electrode. The electrode was calibrated before the analyses and was checked periodically for drift. Salinity was estimated using an Index Instruments digital refractometer to measure the total dissolved solids. Chlorinity was determined by titration with silver nitrate. Concentrations of anions (SO42– and Cl) and cations (Ca2+, Mg2+, K+, and Na+) were determined in duplicate using a Dionex ion chromatograph. All samples were run at 1:200 dilution. Standards were measured at the start and end of each run to test for drift in the response of the conductivity detector. Precision on separate dilutions was better than 2%. Concentrations of NH4+ and PO43– were determined using a Milton Roy spectrophotometer. Minor element concentrations (Fe2+, Mn2+, Li+, B3+, Si2+, Sr2+, and Ba2+) were analyzed using a JY2000 inductively coupled plasma–atomic emission spectrometer (ICP-AES) run at 1:10 dilution. Dissolved inorganic carbon (DIC) content was determined by coulometry.

Total inorganic carbon (TIC) content of the sediment was determined by coulometry. Total carbon (TC), nitrogen, and hydrogen sediment concentrations were determined with a Carlo Erba NCS analyzer. Total organic carbon (TOC) content was calculated as the difference between TC and TIC, where


Total sulfur analyses were inaccurate and of poor precision. These results were considered qualitative and are not reported.

Sample collection for shore-based analyses

Pore water splits were collected from whole-round interstitial water samples for shore-based elemental (minor and trace metal constituents) and isotopic (δ13C, δ15N, δ18O, δD, δ34S, δ11B, and 87Sr/86Sr) analyses. Samples for elemental analysis were acidified with ultrapure HNO3, whereas samples for isotopic analysis were preserved with mercuric chloride or zinc acetate, with exception of water samples collected for hydrogen and oxygen isotopic analysis. Pore waters were stored in cryogenic vials. Nontreated interstitial water aliquots were prepared for isotopic analysis of oxygen, hydrogen, DIC, carbon, boron, and strontium immediately after arrival in a shore-based laboratory.

Sedimentary pyrite was sampled for δ34S analysis when collected in sufficient quantities. Sediments were collected from squeezed whole rounds for ICP-mass spectrometry (MS) analysis of major (Na, Mg, K, Ca, Ti, Mn, Fe, Al, Si, and P) and trace (Sr, Ba, Y, Zr, Co, Ni, Sc, and Rb) elements.

Organic geochemistry

Sediment hydrocarbon accumulations were quantified by headspace gas analysis (Pimmel and Claypool, 2001). A 5 cm3 sediment headspace sample was collected every 10 m, heated, and the evolved gases were analyzed on gas chromatographs (GCs) dedicated to gas monitoring. Compositions of gases in sediment were determined at least once per core. Headspace gases were analyzed on either a Hewlett Packard 5890 Series II GC or a Hewlett-Packard 5890A natural gas analyzer (NGA). The first system determines concentrations of C1 through C3 hydrocarbons with a flame ionization detector (FID). A broader suite of hydrocarbon concentrations, ranging from C1 to C7, was detected with an FID and, concentrations of N2, O2, CO2, Ar, and He were measured with a thermal conductivity detector (TCD) in the NGA. Chromatographic response on both GC instruments was calibrated against six different gas standards with variable quantities of low molecular weight hydrocarbons, N2, O2, CO2, Ar, and He. Representative expansion void gases (EVGs) were collected directly through the core liner with an EVG sampler to prevent contact with ambient air. Gas concentrations were quantified, without the heating step, using the headspace GC method described above. Sediment samples were collected for land-based analysis of amino acid chirality, adenosine triphosphate (ATP), and acid and alkaline phosphatases (enzymatic activity).


Most subseafloor environments (deeper than 10 cm) are now recognized as very silent and low-activity biospheres based on recent advancements of subsurface microbiology (Whitman et al., 1998; D’Hondt et al., 2004; DeLong, 2004). However, the environments where energy sources for microbes are provided by geohydrological flow or sedimentation of organic matter are candidates for active or activated microbial processes. In the Gulf of Mexico, high sedimentation rates have been observed and strong geohydrological activity was expected. This situation suggests the possible presence of an active shallow subseafloor biosphere dependent on organic carbon as well as microbial communities in the deep subseafloor, activated by geohydrological flow.

Contamination testing

It is important to assess potential seawater contamination of sediments sampled for microbial analysis. Sediment microbial biomass decreases with depth and is in low abundance relative to the microbial biomass found in seawater. The fluorescent microparticle tracer method, as described in ODP Technical Note 28 (Smith et al., 2000), was applied during the expedition to quantify the extent of contamination produced during the coring process.

Latex fluorescent microspheres (Fluoresbrite carboxylate microspheres, 0.5 µm YG) were used as a particulate tracer. A 2 mL stock solution of microspheres was diluted with 40 mL of distilled water and sealed into a plastic bag. The bag was attached to the inside of the core catcher. The bag burst on impact into the sediments, and microspheres mixed with seawater and coated the core surface. During processing of whole-round cores, sediments from outer and inner layers were sampled by syringe for microscopic examination. The weighed samples were suspended in saturated NaCl solution to extract microspheres. The mixture was centrifuged at 1000 rpm for 5 min (Minispin, Eppendorf), and the supernatant was filtered onto black polycarbonate filters (Millipore; 0.2 µm). Fluorescent microsphere analysis was conducted with a Zeiss Axioplan fluorescence microscope equipped with the Zeiss number 9 filter set (BP 450–490; LP 520). The number of spheres observed was used to quantify contamination in spheres per gram of sample.

Core collection and sampling

In order to avoid contact with oxygen and monitor contamination, samples for microbiology were sampled immediately on the catwalk and brought into a glove box following subsampling for contamination testing. In the glove box, 2–3 cm3 and 20–30 cm3 of sediment were collected with a syringe for cultivation and tracer incubation analyses, respectively. Samples for cultivation analysis were suspended in 20 mL of sterilized seawater, with and without Na2S in glass vials, and sediment designated for tracer analysis was packed in anaerobic bags and stored at –80°C. The cultivation slurries were pressurized with nitrogen gas at 1.5 atm and stored at 5°C. An additional 1 cm3 of sediment was extracted for microscopic observation (acridine orange direct counts [AODC] and fluorescence in situ hybridization [FISH], etc.) using a syringe. After these subsampling routines, the sediments for culture-independent molecular analyses based on deoxyribonucleic acid (DNA) (16S ribosomal ribonucleic acid [rRNA] gene clone analysis, quantitative polymerase chain reaction [PCR], etc.) were taken by spatula and stored in 50 mL plastic tubes at –80°C.

Microscopic observation (biomass count)

The sediments (1 cm3) were sampled by syringe from whole-round core samples and suspended in 9 mL of sterilized seawater containing 3.7% neutralized formaldehyde. A 1 mL aliquot of the sediment suspension was stained with acridine orange. After staining for 1 h at 4°C, the sediment suspension was centrifuged for 5 s at 2000 × g and the supernatant was filtered through a 0.2 µm Isopore membrane filter (Millipore). The cells on the filters were counted under fluorescent light using a microscope. At least 50 microscopic fields for each sample were examined to determine the direct cell counts.