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Organic geochemistry

The shipboard organic geochemistry program during Expedition 322 comprised analyses of dissolved gases including volatile hydrocarbons (C1–C4) and hydrogen (H2), as well as analysis of the sediment for total carbon, nitrogen, and sulfur content and inorganic and organic carbon content. In addition, the type and maturity of organic matter was characterized using Rock-Eval pyrolysis. Procedures used during Expedition 322 follow Pimmel and Claypool (2001) and Expedition 316 Scientists (2009). H2 analyses were an addition to the routine shipboard analytical program and employed a third-party laboratory instrument that was provided by the Organic Geochemistry Group of the MARUM Center for Marine Environmental Sciences (University of Bremen, Germany).

Hydrocarbon gases

Concentrations and distributions of light hydrocarbon gases, mainly methane (C1), ethane (C2), and propane (C3), were monitored for each core following standard headspace sampling protocols. A 5 cm3 sediment sample was collected with a cut-off plastic syringe from the freshly exposed end of the first section that was cut open in each core. In general, this was the section adjacent to the whole-round core cut for interstitial water sampling. When the sediments were too lithified, a cork borer was used to take the sample. The sample was extruded into a 24 mL glass vial and immediately sealed with a PTFE coated septum and metal crimp cap. The exact bulk mass of the wet sample was determined after the gas analysis was finished. For analysis of C1–C4 hydrocarbon gases, the vial was placed in a headspace sampler (Agilent Technologies G1888 network headspace sampler), where it was heated at 70°C for 30 min before an aliquot of the headspace gas was automatically injected into an Agilent 6890N gas chromatograph (GC) equipped with a packed column (GL HayeSep R) and flame ionization detector (FID). The carrier gas was helium. In the temperature program of the GC the initial temperature of 100°C was held for 5.5 min before the temperature was ramped up at a rate of 50°C/min to 140°C and held for 4 min. Chromatographic response of the GC was calibrated against five different authentic standards with variable quantities of low molecular weight hydrocarbons and checked on a daily basis.

Methane concentration in interstitial water was derived from the headspace concentration using the following mass balance approach:

CH4 = [χM × Patm × VH]/[R × T × VIW],



  • χM = molar fraction of methane in the headspace gas (obtained from GC analysis),

  • Patm = pressure in the vial headspace (assumed to be the measured atmospheric pressure when the vials were sealed),

  • VH = volume of headspace in the sample vial,

  • VIW = volume of interstitial water in the sediment sample,

  • R = universal gas constant, and

  • T = temperature of the vial headspace in Kelvin.

The interstitial water volume in the sediment sample was determined based on the bulk mass of the wet sample (Mb), the sediment's porosity (ϕ, which was extrapolated from shipboard MAD measurements in adjacent samples, grain density (ρg), and the density of interstitial water (ρIW) as:

VIW = MIWIW = [ϕ × ρIW]/[(1 – ϕ) × ρg] × MbIW,



  • MIW = interstitial water mass,

  • ρIW = 1.024 g/cm3, and

  • ρg = 2.8 g/cm3.


Hydrogen gas

Dissolved H2 concentration was monitored using two different headspace equilibration techniques. For the first method, hereafter called the incubation method, ~5 cm3 of sediment was collected from the freshly cut section ends of whole-round cores that were taken for deep biosphere research. Ideally, sampling was conducted immediately after core recovery, but in a few cases samples needed to be stored for a few hours in N2 flushed bags at 4°C prior to sampling. Samples were taken using sterile, cut-off plastic syringes and placed in 17 mL headspace vials that were closed with thick butyl rubber stoppers, crimp capped, and thoroughly flushed with N2 in order to establish an O2- and H2-free gas phase inside the vials. After analysis of the initial H2 concentration, samples were incubated at estimated in situ temperatures and H2 concentration in the gas phase was monitored as a time series. At each time point, 1 mL of gas was sampled with a gas-tight syringe. In order to maintain a constant pressure inside the vials, the withdrawn amount of gas was substituted through an injection of an equal volume of pure N2 after each analysis (using the bypass gas of the Peak Performer 1, see below). In principle, the time series is supposed to continue until the H2 concentration reaches a constant level that represents a steady state between H2 production and consumption. The incubation method allows the determination of dissolved H2 concentration based on two fundamental assumptions: (a) the analyzed gaseous H2 in the headspace is in equilibrium with dissolved H2 in the interstitial water, and (b) the incubation of samples in the laboratory allows the establishment of a steady state between production and consumption of H2 that is representative of in situ equilibrium.

The incubation method was initially developed for studies of freshwater sediments and microbial cultures (e.g., Lovley and Goodwin, 1988; Hoehler et al., 1998). In contrast to these metabolically active systems, deep marine subsurface sediments host microorganisms that metabolize at very low rates (D'Hondt et al., 2002; Parkes et al., 2005). Therefore, it is questionable whether the required steady state can be reached within an acceptable time frame in the laboratory and if such a steady state would be representative of in situ conditions.

An alternative approach is the complete extraction of dissolved H2 into a defined H2-free gas volume, hereafter called the extraction method, as previously used by Novelli et al. (1987) and D'Hondt et al. (2009). In principle, this method is based on the assumption that the initially present H2 exsolves from the liquid phase and can be captured in the defined headspace volume of a closed vial. Dissolved H2 concentration is then calculated based on mass balance considerations (see below).

For the extraction method, ~5 cm3 of sediment was sampled immediately after core recovery from the freshly exposed end of a core section. In general, this was the same section that was used for hydrocarbon gas analysis. The sample was extruded into a 17 mL headspace vial, which was immediately filled to the top with a NaCl solution (3.5%), sealed with a soft butyl stopper, and crimp capped. Excess water was allowed to escape through a hypodermic needle. Analysis was conducted as soon as possible after core recovery and sampling, but when analysis was delayed, samples were stored at 4°C. Prior to analysis, samples were allowed to equilibrate to room temperature. A headspace was created by displacing 5–10 mL of the aqueous phase with an equal volume of H2-free N2 (using the bypass gas of the Peak Performer 1, see below). The gas-in needle was removed first, and the liquid-out needle connected to a syringe was allowed to equilibrate the pressure in the vial headspace to atmospheric pressure. The volume offset in the liquid-out syringe was recorded. The vial was vortexed, and dissolved H2 was allowed to diffuse out of the interstitial water and equilibrate with the headspace for 20 min before H2 concentration was analyzed in the headspace gas.

Background control is essential for accurate H2 analysis by the extraction method, and blanks (i.e., vials filled with NaCl solution but without sediment) were analyzed frequently. On average, the reagent blank of the NaCl solution was 0.006 ± 0.007 µM H2. The reagent blank was subtracted from samples that were extracted with the NaCl solution.

In both methods, dissolved H2 concentration is determined based on the H2 concentration in the headspace gas, which was analyzed by gas chromatography with a reducing compound photometer (RCP) using a Peak Performer 1 (PP1) (Peak Laboratories LLC). Samples are injected into a flow of carrier gas and separated on a packed column before they react with a heated bed of mercuric oxide and form mercury vapor that is subsequently detected in a photometer cell. For H2, the reaction is: H2 + HgO(solid) → H2O + Hg(vapor). The instrument detection limit, evaluated statistically by a serial dilution of the primary standard with N2, is ~8 ppb. Here the instrument was operated using a column temperature of 105°C, an RCP bed temperature of 265°C and N2 as a carrier gas, and calibrated with a 10 ppm H2 primary standard on a daily basis. Typically, 1 mL of gas sample was injected to thoroughly flush the 0.1 mL sample loop and the tubing between the injection port and the loop.

The incubation and the extraction methods use different approaches to calculate the dissolved H2 concentration from the analyzed headspace concentration, but for both methods, the first step is to convert the H2 concentration in the headspace from molar fractions to molar concentration ([H2]g):

[H2]g = χH2 × P × R–1 × T–1,



  • [H2]g = expressed as nmol/L,

  • χH2 = molar fraction of H2 in the headspace gas (in ppb, obtained from GC analysis),

  • P = total gas pressure (in atm) in the headspace, which was atmospheric pressure,

  • R = universal gas constant, and

  • T = temperature of the gas phase in Kelvin.

For the incubation method, the concentration of H2 dissolved in the interstitial water ([H2]incub, expressed in nmol/L) is assumed to be in equilibrium with the gas phase and calculated as:

[H2]incub = β × [H2]g,


where β is an experimentally determined solubility constant corrected for temperature and salinity (Crozier and Yamamoto, 1974). The value of β is 0.01555 for seawater (salinity = 33.7‰) at 19.3°C.

For the extraction method, the concentration of H2 dissolved in the interstitial water ([H2]extract, expressed in nmol/L) is determined based on mass balance considerations:

[H2]extract = ([H2]g × VH + [H2]aq × Vaq)
× Vs–1 × ϕ–1,



  • [H2]g = calculated using Equation 46,

  • [H2]aq = H2 concentration in the aqueous phase: obtained from Equation 47, substituting [H2]aq with [H2]incub,

  • VH = volume of the headspace,

  • Vaq = volume of the aqueous phase, including the interstitial water and the solution added,

  • Vs = volume of the sediment sample, and,

  • ϕ = sediment porosity.

Lithification of sediments hampered the accurate measurement of sampled sediment volumes. Therefore, Vs was obtained based on the individual weights of the dried sediment samples and corresponding data on grain density and porosity from shipboard analysis.

Total carbon, nitrogen, and sulfur contents of the solid phase

In general, solid phase samples for organic geochemistry were taken every 2–3 m downcore. In each core, one sample was associated with a cluster sample for a suite of solid phase analyses. This sample was taken from the whole-round core for interstitial water sampling and adjacent to the headspace samples for gas analysis. After freeze-drying and homogenizing the sample, total carbon, nitrogen, and sulfur concentrations were determined by elemental analysis using a Thermo Finnigan Flash EA 1112 carbon-hydrogen-nitrogen-sulfur (CHNS) analyzer. Calibration was based on the synthetic standard sulfanilamide, which contains 41.81 wt% C, 16.27 wt% N, and 18.62 wt% S. About 20–50 mg of freeze-dried ground sediment was weighed and placed in a tin container for carbon and nitrogen analyses. For sulfur analysis, the same amount of freeze-dried sediment was weighed and put in a tin container with the same amount of a V2O5 catalyst. Sediment samples were combusted at 1000°C in a stream of oxygen. Nitrogen oxides were reduced to N2, and the mixture of CO2, N2, and SO2 was separated by gas chromatography and detected by a thermal conductivity detector (TCD). The standard deviation of carbon, nitrogen, and sulfur concentrations is less than ±0.1%. Accuracy for carbon and sulfur analysis was confirmed using two GSJ reference samples.

Inorganic carbon, organic carbon, and carbonate content of the solid phase

In the same set of samples that was used for the analysis of total carbon, nitrogen, and sulfur contents of the solid phase, inorganic carbon concentration was determined using a Coulometrics 5012 CO2 coulometer. About 10–20 mg of freeze-dried ground sediment was weighed and reacted with 2M HCl. The liberated CO2 was titrated, and the change in light transmittance was monitored with a photodetection cell. The weight percentage of calcium carbonate was calculated from the inorganic carbon content, assuming that all the evolved CO2 was derived from dissolution of calcium carbonate, by the following equation:

CaCO3 (wt%) = inorganic carbon (wt%) × 100/12.


No correction was made for the presence of other carbonate minerals. National Institute of Standards and Technology–Standard Reference Material 88b was used to confirm accuracy, which was ±0.2 wt% from the certified value (12–64 wt%) of inorganic carbon content. Total organic carbon (TOC) contents were calculated by subtraction of inorganic carbon from total carbon contents as determined by elemental analysis.

Characterization of the type and maturity of organic matter by Rock-Eval pyrolysis

Rock-Eval pyrolysis was used to characterize the type and maturity of the organic matter in the sediments and to identify its hydrocarbon potential. In principle, Rock-Eval pyrolysis consists of sequentially heating a sample in an inert atmosphere (helium) within a pyrolysis oven. It allows us to quantitatively and selectively determine (1) the free hydrocarbons contained in the sample and (2) the hydrocarbon- and oxygen-containing compounds (CO2) that are volatilized during the cracking of unextractable organic matter (kerogen) in the sample. In addition, the shipboard instrument, a Rock-Eval6 Standard, can also be used to oxidize and quantify the residual organic carbon (i.e., organic matter remaining after pyrolysis).

Rock-Eval pyrolysis yields the following basic parameters (Pimmel and Claypool, 2001). S1 is the amount of free hydrocarbons (gas and oil) in the sample (in milligrams of hydrocarbon per gram of sediment). S2 is the amount of hydrocarbons generated through volatilization of very heavy hydrocarbons compounds (>C40) and thermal cracking of nonvolatile organic matter (in milligrams of hydrocarbon per gram of sediment); it is an indication of the quantity of hydrocarbons that the sediment can potentially produce should burial and maturation continue. S3 is the amount of CO2 (in milligrams CO2 per gram of sediment) produced during pyrolysis of kerogen. S3 is an indication of the amount of oxygen in the kerogen. Tmax is the temperature at which the maximum release of hydrocarbons from cracking of kerogen occurs during pyrolysis. Tmax is an indication of the stage of maturation of the organic matter. HI is the hydrogen index (HI = [100 × S2]/TOC; in milligrams of hydrocarbon per gram of TOC). HI correlates with the ratio of H to C, which is high for lipid- and protein-rich organic matter of marine algae. OI is the oxygen index (OI = [100 × S3]/TOC; in milligrams CO2 per gram of TOC). OI is a parameter that correlates with the ratio of O to C, which is high for polysacharride-rich remains of land plants and inert organic material (residual organic matter). PI is the production index (PI = S1/[S1 + S2]). PI is used to characterize the evolution level of the organic matter. PC is the pyrolyzable carbon (PC = 0.083 × [S1 + S2]). PC corresponds to carbon content of hydrocarbons volatilized and pyrolyzed during the analysis. RC is the residual organic carbon.

During Expedition 322, Rock-Eval analysis was conducted on samples from depth intervals that had been sampled for hydrocarbon gases and dissolved H2. For analysis, subsamples of ~60 mg dry sediment were obtained from the same freeze-dried and homogenized bulk sample that had been used to determine total carbon, nitrogen, and sulfur contents of the sediment's solid phase. The pyrolysis oven temperature program used the following procedures. For 3 min, the oven was kept at 300°C and the volatilized free hydrocarbons were measured as the S1 peak (detected by FID). The temperature was then increased from 300° to 550°C (at 25°C/min). The hydrocarbons released from this thermal cracking were measured as the S2 peak (by FID), and the temperature at which S2 reached its maximum was recorded as Tmax. The CO2 released from kerogen cracking was trapped in the 300°–390°C range. The trap was heated and the released CO2 was detected as S3 peak (by TCD). The residual organic matter was oxidized in an oxidation oven kept at 600°C.