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doi:10.2204/iodp.proc.318.102.2011

Geochemistry and microbiology

Organic geochemistry

Shipboard organic geochemistry for Expedition 318 was composed of routine analyses of hydrocarbon gas in sediment cores and lipid biomarker reconnaissance.

Description of routine procedures and instrumental analyses used during Expedition 318 are given by Pimmel and Claypool (2001). A detailed description of the nonroutine biomarker analyses is given below.

Hydrocarbon gases

Sediment gas compositions were typically determined once per core. Two sampling methods were employed, headspace sampling and gas void (VAC) sampling using a syringe. Headspace is the gas given off after heating a known quantity of sediment in a vial. VAC is direct extraction of gas from visible expansion voids within the core liner of the recovered core. Sediment plugs for headspace analysis were taken directly after the core was on the catwalk and prepared for analysis by two different methods.

A subsample consisted of 5 cm³ of sediment, sampled using a cork borer, put in a headspace vial, and crimp-sealed for standard IODP hydrocarbon safety monitoring. When the sediment was too lithified to extract using a cork borer, fragments of core were chiseled out and placed in the vial. The headspace samples for onboard analyses were heated at 70°C for 30 min before injection into the gas chromatograph.

Gases obtained by either headspace or VAC sampling were analyzed by one of two gas chromatography systems, the Agilent/HP 6890 Series II (GC3) or Agilent/HP 6890A natural gas analyzer (NGA). The gases were introduced by injection from a 5 mL syringe directly connected to the gas chromatograph by a 1 cm³ sample loop. Helium was used as the carrier gas.

The GC3 system determines concentrations of methane (C₁), ethane (C₂), ethene (C₂₌), propane (C₃), and propene (C₃₌) with a flame ionization detector (FID) using a 2.4 m × 3.2 mm stainless steel column packed with 100/120 mesh HayeSep R. Helium was used as the carrier gas, and the gas chromatograph oven temperature was programmed to hold for 0.5 min at 90°C, ramp at 30°C/min to 100°C, ramp at 15°C/min to 110°C, and remain at 110°C for 4.5 min before ramping at 50°C/min to 150°C, with a final holding time of 1.8 min. The FID temperature was 250°C.

The NGA system measures concentrations of C₁–C₇ hydrocarbons with a FID as well as measuring N₂, O₂, and CO₂ with a thermal conductivity detector (TCD). The TCD separation uses three columns, a 6 ft, 2.0 mm inner diameter stainless steel column (Poropak T, 50/80 mesh), a 3 ft, 2.0 mm inner diameter stainless steel molecular sieve column (13×, 60/80 mesh), and a 2.4 m × 3.2 mm stainless steel column packed with 80/100 mesh HayeSep R (Restek). The FID separation was on a DB-1 capillary column (60 m × 0.32 mm) with 1.5 µm phase thickness. The FID separation uses helium as the carrier gas, and the gas chromatograph oven temperature was programmed to hold for 2 min at 50°C, ramp at 8°C/min to 70°C, and then ramp at 25°C/min to 200°C, with a final holding time of 5 min. The FID temperature was 250°C.

Data were collected and evaluated with an Agilent ChemStation data-handling program. For both systems, chromatographic response was calibrated to nine different gas standards with variable quantities of low molecular–weight hydrocarbons, N₂, O₂, CO₂, Ar, and He and checked on a daily basis. The gas components are reported as parts per million by volume (ppmv) of the injected sample. Methane in uppermost headspace samples are also expressed as millimoles per liter of pore volume (mM), assuming a porosity of 0.45, a sample volume of 5 cm³, and a vial volume of 21.5 cm³,

Lipid biomarker analysis

Amenable lipids were extracted using a method adapted from Dickson et al. (2009) (Fig. F19). Sediments were freeze-dried using the onboard freeze-drier and homogenized using a grinding machine or pestle and mortar. From 2 to 20 g of the dried, powdered sediment was hydrolyzed and extracted using 10 mL of 0.3 M potassium hydroxide (KOH) in a solution of methanol and 5% water. This involved ultrasonicating for 5 min (3×) and heating in a heating block at 50°C for 2 h. The solvent is removed from the matrix by centrifugation of the sample for 5 min at 3300 rpm and decanted into a 50 mL pear-shaped flask. The sample was further extracted with 7 mL methylene chloride and methanol (3:1; 3×) under ultrasonication for 5 min. The extract solvent was centrifuged and decanted as above, and all extracts were combined in the 50 mL flask. This total lipid extract was concentrated to near dryness using a rotary evaporator under vacuum and was further separated into discrete chemical fractions using methods adapted from Dickson et al. (2009). First, the neutral fraction was recovered using liquid-liquid separation in a test tube. The neutral fraction was further separated into four subfractions using silica gel column chromatography: aliphatic hydrocarbons/n-alkanes (N₁), aromatic hydrocarbons (N₂), aldehydes and ketones (N₃), and alcohols (N₄) by elution with n-hexane, n-hexane/methylene chloride (2:1) mixture, methylene chloride, and methylene chloride/methanol (95:5) mixture, respectively. Alcohols in N₄ were derivatized to trimethylsilyl ethers using BSTFA reagent. Acidic components were extracted with methylene chloride from the remaining solution after acidifying (pH = 1) with HCl. The acidic fraction was concentrated and the carboxylic acids were derivatized to methyl esters with 14% BF₃/methanol at 100°C for 30 min. The methyl esters were recovered using liquid-liquid separation.

The individual lipid fractions were analyzed and structural identification confirmed using an HP 5973 gas chromatograph–mass spectrometer equipped with a 7683 autosampler and fused silica capillary column (DB-1 60 m × 0.317 mm internal diameter × 1.50 µm) and detector. The gas chromatograph oven temperature was programmed to ramp at 30°C/min from 50° to 120°C and then ramp at 5°C/min to 300°C, with a final hold time of 22 min.

Inorganic geochemistry

Interstitial water analyses

Interstitial water analyses for the uppermost 20 mbsf were carried out on cores from Sites U1357 and U1359 in conjunction with sampling for microbiology studies. The sampling strategy is detailed in “Microbiology.”

Traditional interstitial water squeezing pushes the water out of the sediment by placing it into a titanium squeezer, modified after the stainless steel squeezer of Manheim and Sayles (1974). Gauge pressures up to 20 MPa are applied using a laboratory hydraulic press to extract interstitial water. The interstitial water squeezed out of the sediment was extruded into a prewashed (10% HCl) 50 mL plastic syringe attached to the bottom of the squeezer assembly. The solution was subsequently aliquoted into four parts:

1. One part (10 mL) was filtered through a 0.45 µm polysulfone disposable filter (Whatman) into a centrifuge tube for shipboard routine analyses of salinity, alkalinity and pH, chlorinity, dissolved inorganic carbon (DIC), anions and cations (Cl^–, SO₄^2–, Na⁺, K⁺, Ca²⁺, and Mg²⁺), nutrients (PO₄^3–, NH₄⁺, and NO₂^– + NO₃^–), and minor elements (Li, B, Mn, Fe, Sr, Ba, Si, and P).

2. One part (5 mL) was filtered through a 0.45 µm polysulfone disposable filter (Whatman) into a baked small ampoule for shore-based analyses of total organic carbon. HgCl₂ (5 µL) was added to each vial.

3. One part (10 mL) was filtered through a 0.45 µm polysulfone disposable filter (Whatman) into a baked large ampoule for shore-based DIC isotope analyses (δ¹³C). HgCl₂ (5 µL) was added to each vial.

4. The remaining interstitial water (at least 7 mL) was filtered though a 0.22 µm polysulfone disposable filter (Millipore) into a baked glass bottle for shore-based analyses of carbohydrate and amino acids.

Salinity, pH, and alkalinity analyses

Interstitial water analyses followed the procedures outlined by Gieskes et al. (1991), Murray et al. (2000), and user manuals for new shipboard instrumentation with modifications as indicated. Interstitial water was analyzed for salinity with a Reichert special scale, previously calibrated using the International Association of Physical Sciences of the Ocean (IAPSO) seawater standard. Alkalinity and pH were measured after squeezing by Gran titration with a Metrohm autotitrator. The IAPSO seawater standard was used for standardization of alkalinity.

Ion chromatography

Sulfate, chloride, magnesium, calcium, sodium, and potassium concentrations in interstitial water were determined with a Dionex ICS-3000 ion chromatograph on 1:200 diluted aliquots in 18 MΩ water. The IAPSO seawater standard was used for standardization of measurements made on the ion chromatograph.

Spectrophotometric analyses

Phosphate, ammonium, and total nitrate and nitrite concentrations in the interstitial water were determined by an OI Analytical DA3500 discrete analyzer spectrophotometer unit, an automated system that controls sample analysis and reagent aspiration, dispensing, heating, and mixing. In the phosphate method, orthophosphate reacts with Mo(VI) and Sb(III) in an acidic solution to form an antimony phosphomolybdate complex. Ascorbic acid reduces this complex to form a blue color, measured at 880 nm. Potassium phosphate monobasic (KH₂PO₄) was used to produce a calibration curve and as an internal standard. In the ammonium method, phenol undergoes diazotization and the subsequent diazo compound is oxidized by sodium hypochlorite to yield a blue color, measured spectrophotometrically at 640 nm. Ammonium chloride (NH₄Cl) was used to produce a calibration curve and as an internal standard. In the nitrate/nitrite method, the sample passes through an open-tubular copperized cadmium coil, which reduces nitrate to nitrite. Both the reduced nitrate and any pre-existing nitrite are diazotized with sulfanilamide and coupled with N-(1-naphthyl)ethylenediamine dihydrochloride to form a colored azo dye, measured spectrophotometrically at 540 nm.

Major and trace element analyses by ICP-AES

Concentrations of selected elements (Li, B, Mn, Fe, Sr, Ba, and Si) in the interstitial waters were determined by inductively coupled plasma–atomic emission spectroscopy (ICP-AES) with a Teledyne Prodigy high-dispersion ICP-AES. The method for shipboard ICP-AES analysis of samples is described in detail in ODP Technical Note 29 (Murray et al., 2000).

Each batch of ~20 samples run on the ICP-AES contained 6 artificial standards of known increasing concentrations for all elements of interest, as well as 2 additional standards to monitor instrumental drift. Samples were analyzed in batches to take advantage of achieved calibration and each sample was analyzed three times from the same dilute solution (i.e., in triplicate) within a given sample run. Samples and standards were diluted 1:10 prior to analyses using 2% HNO₃.

Following each run of the instrument, the measured raw-intensity values are transferred to a data file. Instrumental drift was negligible during swift analyses of pore waters, and therefore no correction was applied. A calibration line for each element was calculated using the results for the known standard solutions. Element concentrations in the samples were subsequently calculated from the relevant calibration line. Replicate analyses of one of the artificial standards was used to estimate precision and accuracy for minor elements, which typically was between 2% and 5%, except for Si runs from Site U1357 (5%–10% errors).

Dissolved inorganic carbon

A portion of undiluted interstitial water (2 mL) was used to determine DIC using a UIC 5011 CO₂ coulometer. Accuracy during individual batches of analyses was determined by running 500 µL of a 100 mM Na₂CO₃ standard every 10 samples. Reproducibility for this standard throughout the course of sample analyses was within 1%.

Sediment samples

We routinely took one 5 cm³ sample of sediment for bulk geochemistry from the working half of each core. The samples were selected in collaboration with the sedimentologists to represent the major lithologic units recovered, or any layer of special interest. Samples for geochemistry were freeze-dried for ~12 h and crushed to a fine powder using a pestle and agate mortar. Geochemical analyses carried out included percent carbonate and elemental analyses of carbon, nitrogen, and sulfur, as well as major and minor element geochemistry (ICP-AES).

For intervals sampled for microbiology, squeeze cakes from the interstitial water samples were freeze-dried for onshore elemental analyses (C/N) and analyses of amino acids and carbohydrates.

Inorganic carbon and carbonate

Freeze-dried and ground sediment (10–20 mg) was reacted with 1 N HCl. The liberated CO₂ was backtitrated to a colorimetric end point. Sediment carbonate content (in weight percent) is calculated from inorganic carbon (IC) content by assuming all carbonate occurs as calcium carbonate:

Inorganic carbon content of sediment samples was determined using a UIC 5011 CO₂ coulometer. Accuracy during individual batches of analyses was determined by running a carbonate standard (100 wt% CaCO₃) every 10 samples. Reproducibility of the standard was found to be within 1%.

Elemental analyses (C, N, and S)

Freeze-dried and ground sediment (10–15 mg) was weighed into tin cups. After addition of a vanadium pentoxide catalyst, the sample was combusted in a stream of oxygen at 1800°C. The reaction gases were passed through a reduction chamber to reduce nitrogen oxides to nitrogen and sulfur trioxide to sulfur dioxide. Gases were subsequently separated by a chromatographic column before detection by thermal conductivity.

Total carbon, nitrogen, and sulfur contents of sediment samples were determined with a ThermoElectron Corporation FlashEA 1112 carbon-hydrogen-nitrogen-sulfur (CHNS) elemental analyzer equipped with a ThermoElectron packed column CHNS/NCS and a thermal conductivity detector. All measurements were calibrated to a sulfanilamide standard, which was run every 10 samples and yielded reproducibilities of 1–2 wt% for carbon and nitrogen and 3–5 wt% for sulfur.

Total organic carbon (TOC_DIFF) content was calculated as the difference between total carbon (TC) and inorganic carbon (IC) from coulometry:

Major and trace element analyses by ICP-AES

The method for shipboard ICP-AES analysis of bulk rock samples was described in detail in ODP Technical Note 29 (Murray et al., 2000). The following protocol is an abbreviated form of this procedure with minor modifications. For flux fusion, 100 mg of ground and homogenized sediment was weighed and added to a vial containing 400 mg of preweighed lithium metaborate (LiBO₂) powder (sample flux = 1:4). All samples and standards were weighed on the Cahn C-31 microbalance, with typical weighing errors around ±0.05 mg under relatively smooth sea-surface conditions and ±0.1 mg in rougher seas. When weighing errors were significantly larger, sample processing was halted until calmer seas were reached again. After homogenization of both powders, the mixture was poured into a Pt-Au crucible and 10 µL of 0.172 µM LiBr was added to prevent the cooled bead from sticking to the crucible. Samples were fused individually at 1050°C for ~12 min in an internal-rotating induction furnace (Bead Sampler NT-2100) and subsequently cooled to form a bead. After cooling, the bead was dissolved in 50 mL of 10% HNO₃ (trace metal grade) in an acid-washed high-density polypropylene (HDPE) Nalgene wide-mouth bottle, which was then agitated on a Burrell wrist-action bottle shaker for 1.5 h. After complete sample dissolution, samples were filtered through a 0.45 µm Acrodisc into an acid-washed 60 mL HDPE Nalgene bottle. Next, 1.25 mL aliquots of this filtered solution were transferred to scintillation vials and diluted with 8.75 mL of 10% HNO₃ for triplicate analysis by ICP-AES (final dilution factor for each sample = ~4000).

Each batch of 20–30 samples run on the ICP-AES contained two procedural blanks, six referenced standards (BHVO-2, BCR-2, JG-2, SO-1, LKSD-1, and RGM-1), and 5–10 drift standards (BHVO-2) to monitor instrumental drift. Samples were analyzed in batches to take advantage of achieved calibration, and each sample was analyzed three times from the same dilute solution (i.e., in triplicate) within a given sample run.

We determined major (Si, Ti, Al, Fe, Mn, Ca, Na, K, and P) and trace (Ba, Sr, V, Sc, and Co) element concentrations. Following each run of the instrument, the measured raw-intensity values were transferred to a data file and corrected for instrument drift and procedural blank. Drift correction was applied to each element by linear interpolation between the drift-monitoring solutions. After drift correction and blank subtraction, a calibration line for each element was calculated using the results for certified rock standards. Element concentrations in the samples were then calculated from the relevant calibration lines. Replicate analyses of rock standards was used to estimate precision and accuracy for major and trace elements, which were between 5% and 20%, dependent on the element of interest.

Microbiology

Core handling and sampling

Microbiology sampling was carried out for Holes U1357C and U1359B. Core sections dedicated to microbiology were handled with aseptic techniques to minimize contamination, as previously described for ODP Leg 201 (Shipboard Scientific Party, 2003; adjustments as described below).

Whole-round samples 10 cm in length were taken at high resolution for microbiology in the uppermost 20 mbsf. Sampling intervals were approximately every 20 cm within the top 4 mbsf, every 30 cm from 4 to 6 mbsf, every 50 cm from 5 to 10 mbsf, and every 1 m from 10 to 20 mbsf. Whole-round samples for interstitial waters were taken adjacent to all microbiology samples. Microbiology whole-round samples were cut using sterilized spatulas, capped with sterilized end caps, and immediately brought to the cold room (<8°C) for subsampling.

Beyond the uppermost 20 m, 5 mL samples were taken with a sterile 5 mL syringe barrel from the bottom of approximately every fourth section at Sites U1357 and U1359. Before sampling, the bottom of each section was scraped with a sterile spatula. The samples were immediately frozen at –80°C for onshore deoxyribonucleic acid analyses.

Further handling of the microbiology whole-round samples was done under a laminar flow hood in the cold room utilizing sterile techniques. Within the hood, each whole-round was removed from the core liner, and the surface was carefully scraped with a sterile scalpel to remove potential contamination from seawater and sediment smearing. A subsample from each whole-round sample was taken with a sterile 5 mL syringe barrel, stored frozen at –80°C in a 2 oz whirlpak bag, and saved for onshore molecular analysis. After subsampling, the remaining whole-round sample was placed into large whirlpak bags, which were gently folded over and placed into vacuum seal bags. Oxygen scrubbers were added to each vacuum-sealed bag to scavenge any remaining oxygen. Sealed samples were stored at –80°C for onshore analysis.

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