Analytical research plan

Core flow for onboard measurements and storage

Despite the multiple coring strategies and disconnected intervals, we will follow IODP standard measurements in the shipboard laboratory. All sections (1.5 m in length) go through X-ray CT scan and further analysis/sampling strategies will be determined based on the CT observation. Intervals for whole-round core sampling will be determined at this point for microbiological analysis/sampling and interstitial water extraction. The fluid samples will be subjected to standard analyses of interstitial water chemistry.

Cores will be split into working and archive halves after physical property measurements by multisensor core logger. Archive halves will be used for visual core descriptions and image/color scan. Working halves will be used for physical property measurements, such as thermal conductivity and shear strength. Discrete samples will be used for measurements of moisture and density, P-wave velocity, and bulk chemistry. Bulk carbon and sulfur analyses and organic matter maturity measurements by Rock-Eval will provide constraints on chemical processes in and around the coalbeds.

The cryogenic magnetometer will be unavailable through the expedition because of current upgrading of the equipment. Considering the sparse coring, we will have to omit shipboard measurements of remnant magnetism.

After shipboard processing, all cores will be packed anaerobically in oxygen-impermeable bags filled with N2 and stored at 4°C. The anaerobic storage at 4°C will maintain subseafloor microbial activities; hence, these sediments can still be used for a variety of additional postcruise microbiological and biogeochemical studies.

Cuttings analysis

In addition to core samples, cuttings material recovered from circulating drilling mud will also be available for scientific analysis. Because of the sparse coring, we will rely on micropaleontological observation of cuttings for age determination. Lithology observation will also be important for decisions regarding the drilling/coring strategy on board the ship.

Gas monitoring

During the riser drilling operation, we will continuously monitor the chemical composition (C1/C2+) of mud gas with gas chromatography deployed in a newly constructed mud-gas container lab on the Chikyu. Circulating drilling mud will be sampled as soon as possible, and the resulting gas sample will be transferred to the mud-gas container lab via the shipboard flow-through pipeline. Carbon isotopic composition of methane (δ13CCH4) will be continuously monitored using an automated wavelength-scanned cavity ring-down spectrometer. These mud-gas measurements will fill the gap of coring intervals, and be useful in monitoring how biological and nonbiological diagenetic processes affect the vertical transition of the coalbed hydrocarbon system as well as the relationship between gaseous components and lithostratigraphy. Mud-gas samples will be also available for more detailed shore-based analysis, such as hydrogen isotopic composition of methane.

High-pressure core

During riserless operations using Hybrid-PCS and PCATS, a 3.5 m length high-pressure core will be transferred to an aluminum high-pressure chamber with PCATS in the cooled container. During the transfer operation, P-wave velocity and gamma-density data as well as X-ray CT scan images will be simultaneously obtained by nondestructive measurement in PCATS. Using the high-pressure chamber system, we will measure in situ chemical composition and concentration of free hydrocarbon gas by conducting a gas-production test using PCATS.

Sampling strategy

Shipboard and shore-based researchers should refer to the IODP Sample, Data, and Obligations policy posted on the Web at This document outlines the policy for distributing IODP samples and data to research scientists, curators, and educators. The document also defines the obligations that sample and data recipients incur. The Sample Allocation Committee (SAC) (composed of Co-Chief Scientists, Staff Scientist, the IODP curator on shore, and the curatorial representative on board the ship) will work with the entire scientific party to formulate a formal expedition-specific sampling plan for shipboard and postcruise sampling.

Shipboard scientists are expected to submit sample requests (at 2 months before the beginning of the expedition. Based on sample requests (shore-based and shipboard) submitted by this deadline, the SAC will prepare a tentative sampling plan that will be revised on the ship as dictated by recovery and expedition objectives. The sampling plan will be subject to modification depending on the actual material recovered and collaborations that may evolve between scientists during the expedition. Modification of the strategy during the expedition must be approved by the Co-Chief Scientists, Staff Scientist, and curatorial representative on board the ship.

The minimum permanent archive will be the standard archive half of each core. All sample frequencies and sizes must be justified on a scientific basis and will depend on core recovery, the full spectrum of other requests, and the cruise objectives. Some redundancy of measurement is unavoidable, but minimizing the duplication of measurements among the shipboard party and identified shore-based collaborators will be a factor in evaluating sample requests.

If some critical intervals are recovered, there may be considerable demand for samples from a limited amount of cored material. These intervals may require special handling, a higher sampling density, reduced sample size, or continuous core sampling by a single investigator. A sampling plan coordinated by the SAC may be required before critical intervals are sampled.

Routine microbiology samples will be reserved for archiving purposes. A 10 cm whole-round core sample is taken at every 10 m of core and is kept at –80°C. These samples will be available for future microbiological studies on shore.

Contamination assessment for riser coring

For geochemistry and microbiology, determining a sample's degree of contamination with alkaline (~pH 10) mud circulation fluids will be crucial because some facultative anaerobic halophilic or halo-tolerant microbes such as Halomonas may grow in the circulation mud tank (Masui et al., 2008). Also, the pore waters squeezed from consolidated sedimentary rocks will be highly sensitive to contaminating chemicals. During Expedition 337, we will test the use of perfluorocarbon tracers (PFTs) for all riser drilling cores. In the circulation mud tank, PFT concentrations will be kept at 1 part per million (approximately half the concentration of saturation in seawater). During the mud circulation, 1 kg of PFT will be supplemented with 100 m3 of mud in the tank. The tracer permeation in core sections will be evaluated with a gas chromatograph in the microbiology lab on the Chikyu according to previously established protocols (Smith et al., 2000; House et al., 2003; Lever et al., 2006).

Subseafloor biomass profiling with multiple methods to 2200 mbsf

Expedition 337 will provide an unprecedented opportunity to study deep subseafloor microbial communities inhabiting organic-rich, gassy sediments to 2200 mbsf. The targeted depth of maximum penetration is similar to or even extends the previous depth record of scientific ocean drilling, which is currently 2111 mbsf and held by the R/V JOIDES Resolution during ODP Leg 148 in Hole 504B off Costa Rica (Alt, Kinoshita, Stokking, et al., 1993). Because the current depth record of the existence of subseafloor life is at 1626 mbsf at the Newfoundland margin (Roussel et al., 2008), our study of vertical distribution of microbial biomass will significantly extend our understanding of the extent of subseafloor life and the biosphere on Earth. In addition, the analysis of the distribution and quantity of subseafloor biomass will provide primary information on how microbial populations are sustained by flux of nutrients and electron donors and acceptors, sediment porosity and fluid flow regimes along the unconformity layers, and other lithostratigraphic interfaces (e.g., coal/sand interface).

To detect and quantify the biomass of deep biosphere microbial populations, we will use a newly developed computer-based image analysis (Morono et al., 2009). To detach the cells effectively, the sediments will be washed with hydrofluoric acid and the microbial cells will be separated from solid sediment particles using the Nicodenz gradient method (Kallmeyer et al., 2008). Using SYBR-stained cells, the cell detection and enumeration will be performed with an automated slide-leader system equipped with autofocused fluorescent microscopy (Morono and Inagaki, 2010). We will also compare results using the new high-throughput cell counting technique for geological habitats, based on high-specification flowcytometry (Y. Morono, J. Kallmeyer, and F. Inagaki, pers. comm., 2010) (Fig. F11). In addition, we will use established protocols involving the analysis of intact polar membrane lipids (e.g., Lipp et al., 2008).

Microbial community composition across the marine/terrestrial interface

We will study microbial diversities and community structures using molecular ecological approaches. Most microorganisms are expected to be uncultured heterotrophs, as previously observed at other organic-rich subseafloor sedimentary environments like ODP Sites 1229 and 1230 off Peru (e.g., Parkes et al., 2005; Biddle et al., 2006; Inagaki et al., 2006b). For deep biosphere communities, the relevance of paleoenvironmental conditions during sediment deposition in structuring the microbial community composition, diversity richness, and evenness remains unknown. What are the chemical or geophysical constraints on the microbial community structures? How do lithostratigraphic variations play a role for the migration or stratification of microbial communities? To address these fundamental questions related to the diversity and community structure, we will study samples from all recovered lithostratigraphic units corresponding to marine and terrestrial deposits and coal layers.

With regard to molecular techniques, DNA (and/or ribonucleic acid [RNA]) will be extracted from sediment core samples by newly developed extraction techniques that minimize bias (Y. Morono et al., unpubl. data). Cell lysis efficiency will be determined for all samples by checking the number of SYBR-stainable cell particles using an automated fluorescent image analysis (Morono et al., 2009; Morono and Inagaki, 2010). 16S rRNA gene and other functional gene fragments will be amplified with tagged primer sets, sequenced with 454 pyrosequencing and/or other high-throughput sequencing technologies, and then statistically and phylogentically analyzed. In addition, some specific phylotypes of biogeochemically relevant key players (e.g., methanogens and acetogens) will be visualized by FISH-based techniques using specifically designed probes.

Geobiological studies of autotrophic microbes

To understand the potential carbon flow patterns in the coalbed subseafloor microbial ecosystem, understanding of distribution, diversity and functioning of autotrophic microbial communities (i.e., CO2-assimilating microbes) is important because of their capability to convert inorganic substrates into organic matter. Autotrophs such as homoacetogens may play an important role by converting CO2 to acetate and biomass in an artificial CO2 deposit. Both reactions, CO2-reduction to methane and to acetate, are likely exergonic in pore water with elevated dissolved H2 concentrations as a result of lignite diagenesis. Therefore, we will pay special attention to the population and activity of methanogens and other autotrophic communities using multiple cultivation and culture-independent approaches, including an analysis of their sensitivity toward high CO2 and low pH.

To understand the methanogenic archaeal populations, a key gene for methanogenesis pathway, methyl coenzyme-M reductase (MCR) genes will be phylogenetically characterized and quantified by PCR-based molecular ecological techniques (e.g., Colwell et al., 2008). We will also study the coenzyme F430, the specific nickel-containing prosthetic group by multiple structural analyses, such as spectroscopic analysis, high-resolution X-ray structure analysis, and time-of-flight mass spectrometry (TOF-MS) analysis. In addition to these culture-independent analyses on shore, we will cultivate methanogens and other deep subseafloor microbes using a flow-through bioreactor system under the in situ high pressure and temperature. A variety of methanogens have already been successfully retrieved from the shallow subsurface sediments at the same site (Imachi et al., submitted). However, we still do not know what kinds of methanogens and associated communities are mainly fueling the significant accumulation of biogenic methane above the coal layer. Using the high-pressure flow-through reactor system, we will also study carbon- and hydrogen-isotopic fractionation of methanogens in the subseafloor sedimentary microbial ecosystem. The expected cultivation-based evidence will contribute to the understanding of the hydrocarbon system and the microbial potential for CO2 conversion.

Inorganic and organic geochemistry: understanding the deep carbon cycle

During the Chikyu shakedown cruise (Expedition CK06-06) in 2006, the shallow sedimentary unit above the BSR at 365 mbsf contained methane hydrate in porous ash and sandy layers. Given the moderately low concentrations of organic carbon (0.8%–1.8%) in the strata, it is conceivable that the methane originates from greater depths, presumably the deeply buried coalbed. Diffusion of biologically produced methane from organic-rich sediments into overlying organic-lean sediments has been demonstrated at other deep drilling sites (e.g., from deeply buried Cretaceous black shales drilled during ODP Leg 207 at Demerara Rise [Arndt et al., 2006]). Using cored materials, we will conduct detailed analysis of numerous geochemical parameters that will enable us to quantify the fluxes of various compounds from and into the Eocene coalbed. Concentrations of major anions and cations (e.g., sulfate, chloride, alkalinity, sulfide, phosphate, ammonium, magnesium, potassium, and calcium) in pore water samples and formation fluids will be determined. We will determine concentrations and stable carbon isotopic compositions of various carbon-bearing compounds (i.e., CH4 and C2+ hydrocarbons, volatile fatty acids, dissolved organic carbon, and dissolved inorganic carbon [DIC] [e.g., Heuer et al., 2009: Lever et al., 2010]). We will determine δD values of CH4 in order to distinguish between different pathways of methanogenesis (cf. Whiticar, 1999). The dissolved organic matter in the pore and formation water and its structural link to lignite-derived organic compounds will be studied by Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS) (e.g., Schmidt et al., 2009). We will seek to apply new assays based on solid-phase microextraction for the quantification and isotopic analysis of methylotrophic substrates such as methylamines and methanol in pore fluids. This set of analyses will provide information pertinent to the geobiological carbon cycling and will enable us to model fluxes of carbon-bearing compounds in and out of the coalbed (e.g., Sivan et al., 2007; Wang et al., 2008). We will determine concentrations and stable isotopic compositions of organic carbon, carbonate, nitrogen, and sulfur and hydrogen indexes by Rock-Eval pyrolysis to further characterize the diagenetic setting and broadly distinguish sources of organic material. Some of these techniques will be applied to selected, cleaned core cuttings in order to supplement geochemical data with continuous information. In addition, we will measure 129I/127I ratios of pore waters to examine the age distribution of pore water enriched in iodine and methane in gas-rich strata (e.g., Tomaru et al., 2009).

Biogeochemical and geobiological experiments: activities and fluxes

We will use large-volume samples of live sediments that are taken in regular intervals of 1 per recovered core (i.e., whole-round core) to conduct laboratory incubation to monitor the production potential for methane and methanogenic substrates such as methyl-compounds, acetate, and H2. To better assess fluxes of compounds in organic-rich strata and in the adjacent sediment horizons, we will quantify microbial respiration processes by high-sensitivity methods such as radiotracer turnover based on 14C-, 35S-, and deuterium-labeled compounds. These ex situ experiments will inform us on the potential activity of the coalbed microbial communities and provide information on the reactivity of the lignite. These experiments will be anaerobically conducted at near in situ temperature in the newly developed radioisotope container lab on the Chikyu. Rate measurements directly related to methanogenesis are the priority, such as acetate turnover, methanol turnover, methane turnover, and CO2 turnover. In addition, bulk community growth can be studied by thymidine incorporation. As microbial communities in these experiments, we will use both the natural indigenous microbial population and microbial inoculates that have been tested in coal-to-methane degradation (e.g., Jones et al., 2008a, 2008b; Orem et al., 2010). The inocula also include mesophilic to thermophilic methanogens isolated from subseafloor sediments in shallow zones of the same drilling site (C9001) and from high-CO2 hydrothermal fluids in the Okinawa Trough hydrothermal fields. In selected experiments, we will use stable isotope–labeled substrates in order to establish reactant-product relationships (e.g., 13C-methanol, 13C-DIC, etc.) and quantify the relative importance of the various pathways metabolizing C1 compounds (e.g., Wegener et al., 2008). The rate of 13C-labeled substrate incorporations will be determined at single-cell levels using a combined FISH-NanoSIMS approach (Musat et al., 2008) as well as on the basis of microbial biomarker analysis (e.g., Wegener et al., 2008). Overall, these experiments will provide information crucial to the assessment of the lignite's potential to generate methane and other dissolved organic species and will inform the design of experiments under in situ pressure and temperature.

Shore-based study of offshore CO2 sequestration potentials: does a deeply buried coalbed act as a "Subsea Forest?"

One of the great concerns regarding geological CO2 storage is the behavior of CO2 and its impact on ecological balance of carbon cycling. To store substantial quantities of CO2 in the deep underground or marine subsurface, the captured CO2 is condensed as liquid. At the high pressure and high temperature in the deep subsurface, the CO2 will be present in supercritical state. Where liquid or supercritical CO2 is in contact with a water phase, the CO2 levels will be in equilibrium with the liquid CO2, thus be saturated. The pH of pore water or solvent water for CO2 sequestration will be significantly decreased; hence, the mineral trap through carbonation is extremely low. A diffusion of liquid CO2 through the surrounding pore spaces is expected. However, the dense liquid or supercritical CO2 is hydrophobic and thus would barely mix with pore water. These physical and chemical characteristics of CO2 suggest that once liquid and/or supercritical CO2 is injected in deep subsurface repositories, CO2 may be retained and these unusual artificial environments may remain stable over geologic timescales. Biological conversion of the injected CO2 in the subsurface to organic compounds such as methane will depend on how microorganisms will respond to the chemical perturbation and on the intrinsic reducing power of the subsurface environment. In fact, C1-metabolizing microbial life has been observed in the natural deep-sea CO2-seep and hydrothermal environments (Inagaki et al., 2006a).

What are the drivers in microbial succession in the deep subseafloor biosphere? Previous deep biosphere studies using 14C tracers demonstrated that potential rates of CO2-reducing methanogenesis in typical coastal organic-rich marine sediments are a few tens of picomols per cubic centimeter per day (as reviewed by Parkes et al., 2000). In contrast, the activity of batch-cultured thermophilic methanogens is ~0.1 to 1 mmol/cm3/day, that is, roughly eight to nine orders of magnitude higher than subseafloor methanogenesis activity. As a consequence of hypothetical carbon storage, CO2 concentration could approach 1 mol/L, the conversion of CO2 to current ambient levels by the indigenous subseafloor microbial ecosystem would take over hundreds of millions of years. However, if there are substantial sources of energy and reducing power, such as H2 or acetate generated by the diagenesis of organic matter at elevated temperatures (Parkes et al., 2007), the CO2 turnover time may be significantly reduced based on the potential activity of methanogens. We suggest that the potentially active and abundant microbial communities associated with the deeply buried coalbeds—the so called "Subsea Forest"—constitute a highly interesting target for testing CO2 and pH effects on subsurface life, including effects on C1-metabolisms, heterotrophic consumption, lipid and DNA formation, and carbon assimilation.

To produce such energy and nutrient sources for microbes, the coal needs to be relatively immature, because then it still contains not only hydrogen but also N- and P-bearing compounds and the porosity of lignite is generally higher than that of more mature graphite coal. Indeed, microbiological studies of terrestrial coal environments revealed the presence of hydrogenotrophic methanogens (Shimizu et al., 2007; Krüger et al., 2008: Strapoc et al., 2008). The coals in the Shimokita gas field are mostly composed of lignite and are intercalated with porous sandy layers, like lens structure (Osawa et al., 2002). Along with the migrating CO2, it is possible that dissolved organics or reduced chemical compounds may be advected by the CO2 conveyor and fuel heterotrophic respiration (Onstott, 2005). Especially, energy sources co-migrating with liquid CO2 such as sulfide, methane, or H2 may be oxidized by the subsurface microbiota driving autotrophic growth, or—if physiological functions are repressed by low pH—will not be utilized and transported further. Using high-pressure reactors, sediments samples from Expedition 337 will be incubated under high CO2 and compared to in situ conditions to assess whether the communities can adapt to high CO2 and, if so, over which timescale.

Geophysical implications for CO2 sequestration potentials

During the proposed hydrocarbon expedition, a variety of logging and experimental data will be obtained from the borehole and drilled cores. In particular, the high-pressure immersion experiments using representative core materials and CO2-rich fluids will provide critical information for simulating the behavior of CO2 in the deep subseafloor environment. As the significant geophysical and sedimentologial parameters, permeability, porosity and capacity of CO2 storage will be experimentally determined using fluid flow reaction chambers ex situ and under varied temperature and pressure conditions. During and after the incubation, we will evaluate how the CO2 fluid-rock reaction changes mineral compositions and physical properties of sediments. Using a supercomputer device (e.g., Earth Simulator at JAMSTEC), the analysis of these in situ and ex situ data, including the seismic data set, will constitute regional 3-D models of CO2 dispersal with time, migration behaviors, and environmental changes (e.g., porosity, pH, and pCO2). The modeling will include rates of biological CO2 turnover to CH4 or other hydrocarbons, representing the feedback velocity of CO2 disposal and the enhanced gas production rates. Also, these computational simulations will provide significant information to plan a scientifically sound set of experiments for the large-scale active experimentation in the future and will contribute to the preparation of large-scale carbon storage in similar environmental settings around the western coast of Pacific Ocean.