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doi:10.2204/iodp.proc.329.112.2011 ProtocolsGeophysical protocols (Pockalny, Abrams, Ellett, Harris, Murphy)Geophysical mapping protocols (Pockalny, Abrams)Underway geophysical data collected during transits between core sites and during MCS acquisition at each site included the following:
SIMRAD EM120 swath mapping bathymetry was used for real-time assessment of potential coring targets during seismic surveys. The typical approach for a seismic survey was to orient the initial survey line oblique to the regional fabric so that the outer beams of the swath mapping system could be used to “forecast” promising sediment basins or avoid seamount chains. During this initial line, we determined the regional trend of abyssal hill fabric and used this trend as our heading for the crossing seismic line. The swath bathymetry data were also used in conjunction with the 3.5 kHz seismic system to confirm whether a reflector in the seismic record was from the subsurface or an artefact from a side-echo. Additional processing of the SIMRAD EM120 with MB system software was required to “edit” bad pings and create gridded bathymetry data for analysis. This process included using the following:
Knudsen 3.5 kHz seismic reflection data were also used in real time for coring target assessment. The on-screen display was continuously monitored during the survey to determine variation in sediment thickness and reflection characteristics. Sea SPY Overhauser total field magnetics data were used primarily during transits to identify the various magnetic anomalies traversed between coring stations. A C-Shell script (rr2mgd.csh) was written to calculate the anomalous magnetic field and merge the results with center-beam bathymetry and navigation data to create MGD77- and generic mapping tool–formatted files. The generic mapping tool–format file was then used as input into various generic mapping tool scripts for wiggle plots and forward modeling of the anomalous magnetic field data. The BGM-3 gravimeter data were recorded; however, the digital output was not consistent with either free-air anomaly or total gravity field data. Additional processing by Scripps University will be required to incorporate these data into the research program. Seismic protocols (Abrams, Pockalny, Ellett, Murphy)Knudsen 3.5 kHz seismic reflection data were recorded digitally and as an EPC paper record at 75 lines per inch. Recording depth range was generally set at 1000 m during transits and 500 m during site surveys. The 3.5 kHz was set for a 24 ms chirp, power of 3, and processing gain of 2. Data were digitally recorded in SEGY format and proprietary Knudson format (keb). Navigation data for each 3.5 kHz trace were also digitally recorded. The following file name conventions were used for 3.5 kHz data:
Approximately 8 km of 3.5 kHz seismic data from each line crossing a site were digitally processed and displayed with the following parameters:
MCS data were acquired at each site using one or two 150 inch3 GI guns (45 inch3 generator chamber, 105 inch3 injector) with a 48-channel digital streamer. Site survey data were acquired at 6 kt except at Site SPG-12 (IODP Site U1371), when weather conditions required surveying at 4.5 kt. The MCS acquisition geometry is shown in the figures for each individual site description (see KNOXRPTS in “Supplementary material”). Data were digitally recorded in SEGD format and converted to SEGY format with the following parameters:
The following file name convention was used for MCS data:
Approximately 13 km of MCS data from each line crossing a site were digitally processed and displayed with the following parameters:
Thermal gradient measurements (Harris)Thermal gradients were measured at most coring sites through the use of autonomous thermistor probes attached to the outside of the core barrel (Table T1). The autonomous data loggers are 175 mm in length and have a nominal temperature measurement range of –5°–60°C, with higher sensitivities at lower temperatures. Instrument precision is 1 mK, and the absolute accuracy is several milli-Kelvin based on laboratory calibrations. The time constant of the thermistor is ~2 s. Nonvolatile memory can hold up to 18 h of measurements collected at a sample rate of 1 s or data from a longer period recorded at a lower frequency. The data loggers were mounted to the outside of the core barrel using a finlike attachment. Two styles were used (Figs. F3, F4), a short fin and a longer fin that approximates an older style of outrigger thermistor probes. The fins were attached to the core barrel using banding in a spiral arrangement so that each logger would penetrate through relatively undisturbed sediment. The position of each fin was measured before and after deployment to determine the relative distance between thermistors and to ensure that the fins did not move during coring operations. A layer of rubber matting was placed between the attachments and the core barrel to increase the friction between the fin and core barrel. No movement during core operations on any of the fin attachments was observed. A logger for monitoring bottom water temperature and tilt was mounted inside the core weight. Measured tilts were small in all deployments in which the core did not fall over. When thermistors were attached to the core barrel, the core was left at bottom for ~7 min to record a time series of temperatures so that equilibrium temperatures could be estimated. A recurring concern during the cruise was the pullout tension in general and the added resistance that the attachments had on pullout. In general, thermistors were attached to 20 ft piston cores or the second 30 ft piston core when there appeared to be enough room so as not to exceed a maximum pullout tension of 20,000 lb. With the exception of the first site, where pullout was at a rate of 8 m/min, it appears that the thermistor attachments only added ~600 lb to pullout tension relative to cores in which the thermistors were not attached (Table T2). In most cases, four thermistors were mounted on the core barrel in two long-style and two short-style attachments. At coring Site SPG-1, the loggers were set to record for a 5 h duration at a 1 s interval. Shipboard attempts to read the data were unsuccessful. The manufacturer was contacted, but repeated attempts to download the data were unsuccessful. These loggers were set aside and were read after the cruise. Additionally, at this site, a data logger and long-style fin were lost during recovery. All subsequent loggers used a duration of 5 h at a 5 s interval, and data recovery was successful. Thermal conductivity measurements (Harris)Thermal conductivities were measured using the needle-probe technique (laboratory code THERM). The values were collected from equipment borrowed from University of California Santa Cruz. Needle probes were calibrated against a gelatin standard. Consistent offsets were found between probes and adjusted based on the standard to account for the offsets, greatly improving the consistency of the results. Heat flow determinations (Harris)If thermal conductivity is constant, heat flow can be calculated as the product of the thermal gradient and thermal conductivity. Core labeling protocols (Ferdelman, Anderson, Steinman)We employed the following scheme for labeling cores, lander samples, and samples from discrete intervals within the various types of cores. Our general labeling scheme included the following information, in the following order:
Stations are identified by site location (SPG1, SPG2, SPG12, etc.). No samples were taken for Station SPG8 because it was not included in the final ship track. The coring tools deployed are identified by the following designations:
A deployment number (1, 2, 3, etc.) is assigned for each trip down and back for each tool at each site, whether sediment was recovered or not. Because of the limited number of deployments, this number was usually 1 or 2 for each tool at each site. Section numbers (Sec1, Sec2, Sec3, Sec4, etc.) for the gravity trigger and piston corers represent core sections of 1.5 m or less and are numbered from the top. For the multicorer, the section numbers refer to tube numbers.
For example:,
Labeling work and archive halvesThe piston cores, trigger cores, and gravity cores that were cut for visual description, sampling, and archiving were labeled in the following manner. The cores are ultimately destined to be split perpendicular to the P-Mag line. Therefore, each section half of the core liner was labeled (i.e., two times per section) prior to its placement in the coring device to indicate the working and archive halves of the cores. Prior to deployment, the sections were lettered with small Roman numerals starting from the bottom and going up to the top of the core (Fig. F5). This kept the order of sections clear until we established where the top of the sediment is, and therefore, the top of Section 1. Archive halves received the designation “A” and working halves received the designation “W.” After corer deployment and recovery, the core liners were removed from the core pipe and permanent section numbers were assigned in normal order from top (1) to bottom (2, 3, etc.) as appropriate. The core caps are color coded, with blue indicating the top of the core or section and red indicating the bottom. Station number, tool and deployment number, and section were labeled on each of the archive and working halves of the top and bottom core caps. Core cutting protocols (Ferdelman, Anderson, Steinman)Piston and trigger gravity coresAt each site, two piston or relatively long gravity cores were usually obtained. These cores were designated either for microbiological/interstitial water analysis (MBIO/IW) or for oxygen measurements and sedimentological studies. Cores meant for MBIO/IW sectioning were moved as quickly as possible to the scientific cold room until they could be sectioned and subsampled. Individual sections were cut into whole-round intervals at predefined depths for interstitial water sampling and microbiological subsampling. Remaining subsections of the core were labeled “MST” (for multisensor track) and set aside for sedimentological and physical properties measurements. From the working halves of the MST intervals, additional samples were taken for headspace and high-resolution cell counting purposes. The MST intervals were then forwarded to the MST van for physical properties measurements. The remainders of the MBIO intervals were placed in the refrigerated container at 5°C. Sample codes for subsamples are given in Table T3. The sections of cores that were designated for oxygen measurements (O2), thermal conductivity (THERM), and sedimentological analysis were placed in the main laboratory to achieve temperature equilibration with the laboratory. After temperature equilibration, dissolved oxygen concentration was measured at 20–30 cm intervals in each core section (see below for optode and electrode oxygen determinations). After the oxygen measurements, thermal conductivity measurements were performed. On a small number of these cores, pore waters were also subsampled using Rhizon samplers (RHIZ). MulticoresMulticores were removed immediately to the scientific cold room. Subsampling generally followed the scheme listed in Table T4. This table provides a general scheme for sample distribution. Sampling combinations were often changed due to recovery of fewer than eight intact multicores or other reasons. Water overlying the sediments in the multicores (OW) was often subsampled. Starting with Site SPG-9, the multicore designated for interstitial water sampling was processed immediately on the deck. This accelerated delivery of the samples to the Chemistry Laboratory for squeezing and processing. The lithology and physical properties (LPP) cores were also sampled outside. Core-logging protocols (Rogers)Every core retrieved during Cruise KNOX-02RR was run through Oregon State University’s GEOTEK multisensor core logger. The core track was set up for up to 1.5 m sections of 4 inch (~0 cm) polyvinyl chloride (PVC) core liner and had instruments to measure gamma ray attenuation, core thickness deviation, P-wave traveltime, temperature, and magnetic susceptibility. Gamma ray attenuation was measured using a directed 137C source and detector. This pair of instruments was mounted horizontally such that any gaps caused by slumping should end up at the top. The raw data are in counts per second (cps). Typical maximum counts were around 20,000 cps, whereas a typical core section was near 8,000 cps. Variations in these attenuated gamma counts are related to changes in core density or thickness. The velocity of sound, or P-wave velocity, was measured by two rollers kept in contact with the core liner. This measurement is dependent on a good connection by both rollers and accurate measurement of core thickness. Calipers attached to these rollers measure deviation in millimeters from a calibration piece. Magnetic susceptibility was measured with a loop sensor just large enough to fit the core through. Sensitive circuits measure the strength of the magnetic field supported by minerals in the core. Any proximity of metal interferes with this instrument, and the edge effects of this measurement are significant. The temperature of the core and the temperature of the instrument also affect the magnetic measurement. A thermal probe was inserted at the bottom of the last section of each core. The HVAC system in the van attempted to keep a constant temperature of 21°C (69°–71°F) to minimize variations in temperature. A number of calibrations were made to the instrument and software prior to collecting data. The temperature probe was calibrated against water of different temperatures measured with a thermometer from another laboratory. Oregon State University provided a 4 inch core section with solid aluminum cylinders of different diameters and freshwater. With the gamma attenuation of this calibration piece and the known densities of water and aluminum, the density of other cores of the same size should be accurately measured. This standard was rerun every few stations, as the gamma detector changes with time. In addition to these calibrations, a deionized water standard constructed using a section of our liner was run before every core as a basis for future comparison and postprocessing. A number of different types of cores were logged on this track. Trigger cores, large gravity cores, and piston cores were in 4 inch PVC liners with a 0.61 cm wall thickness, and small gravity cores were in 2.5 inch clear plastic liners. Each core was split into sections of 1.5 m or less, with most sections being 1.5 m and the topmost section being of various shorter lengths, depending on recovery. Typically, many whole-round samples were removed from the first piston core for biogeochemical and microbiological sampling purposes. For logging, the remaining pieces of the core were spaced with empty core liner to reconstruct the length and spacing of the original core in the logger. Because of rounding errors and end cap thickness, these reconstructions are accurate only to a couple of centimeters. The multisensor track was not set up for the smaller 2.5 inch gravity cores. To log these cores, a half-section of 4 inch liner was placed on the track and lined with bubble wrap to reduce the density error. The 2.5 inch core was placed on this plastic spacer such that its thickest point was in line with the gamma pathway. The only three measurements valid for this method are raw gamma data, temperature, and magnetic susceptibility. Proper logging of these cores would require changing to a different track setup every station and, consequently, would be impractical for underway work. Sedimentology protocols (Hasiuk, Stancin)Material for sedimentological analysis came as either as whole-core material (in PVC liners) from the MST van or as miscellaneous samples from various points in the core stream. Whole-core sections were split for description using a jig-mounted circular saw and delivered to the shipboard sedimentology station for description and analysis. To aid with core description, plastic buttons were inserted into the core every 10 cm from top of section and were labeled every 50 cm, with a minimum of one labeled button per section. The basic record of core descriptions is the paper log sheet, which contains identification, location, date of description, and all descriptive and sampling information. A smear slide was made at least once per section (more often if interest arose). It was analyzed under both reflected light and polarized light microscope at magnifications between 1× and 100×. Bulk sediment and relevant sedimentary features were color-matched to Munsell soil color charts. Samples for sediment porosity determination were taken in close proximity to interstitial water samples and where oxygen measurements were made. Porosity samples were adjacent to interstitial water samples when possible or at regular intervals in whole cores (generally 10, 40, 70, 100, and 130 cm). Samples were collected for elemental and isotopic carbonate geochemistry at Sites SPG-5 and SPG-6 (IODP Site U1368) (25 cm interval) and at Site SPG-7 (10 cm interval). At Sites SPG-9 and SPG-10 (IODP Site U1369), samples were collected every 25 cm for determination of mineralogy postcruise using X-ray diffraction (XRD). For postcruise studies of the sediment’s radiolytic potential, ~50 g of wet sediment was taken from the leftovers of the MBIO sections from Site SPG-3 for high-resolution gamma spectroscopy (GAMMA) at MPI. Subsequent to sampling, cores were plastic wrapped and sealed at both ends (and in the middle of full 1.5 m sections) with masking tape and placed in D-tubes, which were labeled with site, coring device, section, interval, up arrow, and archive/working designation. D-tubes were then boxed and stored in a refrigerated cargo container for shipment to the URI core repository. Conductivity protocols (Hasiuk, Stancin)Conductivity measurements were made to supplement interstitial water chemistry. They were performed every 5 cm on cores where interstitial water samples were taken, as well as on some other whole cores to produce longer uninterrupted data sets. A Brinkmann/Metrohm conductometer was used for the procedure. For calibration, standards were made from 100% seawater; 75% seawater and 25% 18 MΩ deionized water; 50% seawater and 50% deionized water; 25% seawater and 75% deionized water; and 100% deionized water. These standard solutions were analyzed at the beginning of each section of core. Generally this calibration yielded a linear relationship with a correlation coefficient >0.95. If this metric fell below 0.95, the standard solutions were remade. If the correlation coefficient was <<0.95, the probe was replatinized. The probe consisted of two metal prongs 1 cm apart set in a plastic block. The probes were inserted into the sediment until the block rested on the sediment surface. The prongs were aligned parallel to the depth axis of the core to measure vertical conductivity. The probe was cleaned with deionized water between uses. Every five measurements, the 100% seawater standard solution was analyzed to provide a measurement of instrument drift. However, sediments analyzed varied little downcore, with the exception of Site SPG-5, where a change in lithology from clay to carbonate gave a large change. Poor data were generated when analyzing excessively liquid sediment (such as at the top of the first section of cores), air pockets (often created by sampling), or edge material. Biogeochemistry protocols (Spivack, Fischer, Fuldauer, Graham, Griffith, Nordhausen, Schrum, Smith)Interstitial water analyses (Spivack, Fuldauer, Graham, Griffith, Schrum)Interstitial water samples were recovered from 5 cm long whole rounds sliced from cores and with Rhizon samplers. Water was extracted from the whole rounds using a Manheim-type squeezer. Depending on the length of the recovered core, sampling intervals were varied to maximize the number of interstitial water samples and still leave sufficient core for microbiological sampling between interstitial water samples (12–27 cm, depending on core length). This sampling strategy was designed to produce high-resolution chemical profiles suitable for inferring rates of metabolic reactions. The collected interstitial water was split into aliquots for the various shipboard and postcruise chemical analyses. Shipboard analyses included alkalinity titrations; methane quantification by gas chromatography; and sulfate, chloride, bromide, and nitrate determinations by ion chromatography. Alkalinity titrations were run on a Metrohm 809 Titrando autotitrator with a Metrohm pH microelectrode. An aliquot of 3 mL of interstitial water was titrated with 0.1 M HCl at 25°C. Alkalinity was calculated with the Gran determination: where
Alkalinity (in mM) is equal to the y-intercept divided by the slope of the F versus Va plot times 33.33 (Gieskes et al., 1991). The electrode was calibrated with three pH standards. The A value was determined from the slope of an E versus (Va × Na)/(Va + Vo) plot, where Na is the normality of the titrant. An International Association for the Physical Sciences of the Oceans (IAPSO) seawater standard was analyzed at the beginning and end of a set of samples for each station. Surface seawater was analyzed after every fourth sample to measure precision. For methane analyses, two 5 cm3 sediment plugs were collected in 20 mL headspace vials and sealed with Teflon/silicon septa and crimp caps. One set of samples was treated with 2 mL of 1 M sodium hydroxide, flushed with He for 30 s, inverted, and stored for future analysis. The other set of samples was flushed with He and run on a Shimadzu GC-17A gas chromatograph. The sample was injected as soon after He flushing as possible and injected again after being heated to 70°C for a minimum of 30 min. The gas chromatograph was equipped with a 1 mL sample loop coupled to a 2.5 m 80/100 mesh Hayesep R packed column and flame ionization detector. The gas chromatograph was calibrated using He as the zero point, ambient laboratory air as 2 ppm methane, and a certified 5 ppm methane gas standard. Sulfate, chloride, and bromide were quantified with a Metrohm 861 Advanced Compact ion chromatograph. The ion chromatograph was comprised of an 853 CO2 suppressor, a thermal conductivity detector, a 150 mm × 4.0 mm Metrosep A SUPP 5 150 column, and a 20 µL sample loop. A Metrohm 837 ion chromatography eluent/sample degasser was coupled to the system. The column oven was set at 25°C. The eluent solution was 3.2 mM Na2CO3, 1.0 mM NaHCO3. A 1:50 dilution of interstitial water and 18 MΩ deionized water was analyzed. Interstitial water, blanks (18 MΩ deionized water), a 1:50 dilution of filtered surface seawater, and standards (a 1:50 dilution of IAPSO seawater standard) were loaded on a Metrohm 813 compact autosampler. Duplicates of interstitial water were analyzed consecutively, and each sequence was run twice. A standard was run after every fourth interstitial water sample. The surface seawater was run in duplicate at the beginning and end of the sequence to measure precision within and between the sequences. Nitrate concentrations were analyzed with a Metrohm 844 UV/Vis compact ion chromatograph. A 150 mm × 4.0 mm Metrosep A SUPP 8 150 column was used. The column oven was set at 30°C. The eluent was a 10% NaCl solution. An aliquot of 0.75 mL of interstitial water was injected manually into a 250 µL sample loop. The sample and eluent were measured spectroscopically with an ultraviolet lamp as the light source. Wavelengths of 210 and 215 nm were used. Duplicates were run on each sample. A standard made up of 35.72 µM NO3 in surface seawater was run after every fourth sample. Seawater spiked with 35 µM NO3 was analyzed at the beginning and end of each sequence to measure precision. Samples were collected for postcruise determination of concentrations of diverse dissolved chemicals (Ca2+, Mg2+, K+, Na+, NH4+, dissolved organic carbon [DOC], dissolved organic nitrogen, carbohydrate, dissolved amino acids, fatty acids, dissolved inorganic carbon [DIC], He, Fe2+, Mn2+, and PO4), as well as for determination of δ15N of dissolved NO3– and/or NH4+ and δ13C of DOC/DIC. Squeezed sediment was retained for postcruise determination of uranium, thorium, and potassium concentrations, total organic carbon, carbonate, δ13C of bulk organic matter, and mineralogy. Chemical treatments for postcruise analyses are tabulated in Table T5. Oxygen analysis (Fischer, Nordhausen)Ex situ dissolved oxygen measurements were performed on sections of cores (Fischer et al., 2009). Except at Site SPG-3, these measurements were performed on complete core sections, which were typically 150 cm long, with the top and bottom sections usually shorter. Because only one piston core was successfully recovered at Site SPG-3, the oxygen measurements were done on the sections sampled for interstitial water analysis and microbiology; the subsections that remained for oxygen measurements were ~30 cm long. At Sites SPG-11 (IODP Site U1370) and SPG-12, large gravity cores were used. Prior to the measurements, cores were placed for at least 12 h at 20°C in the air-conditioned main laboratory with the closest air exchange vents blocked to ensure thermal equilibration. Holes were then drilled every 30 cm, and the concentration of dissolved oxygen was determined by inserting a probe radially into the center of the core. Model calculations and radial profiles showed that the oxygen concentration in the center of the core was not affected by ambient air on these timescales. At Sites SPG-1 and SPG-2 (IODP Site U1366), both custom-made Clark-type microelectrodes (Revsbech and Jørgensen, 1986) and optodes (Klimant et al., 1995) were used to evaluate which method is better suited for application to whole-round cores. The oxygen optodes, connected to a Microsensor Oxygen Meter Microx TX3 (Presens GmbH, Regensburg, Germany), turned out to be more stable and were used for all other measurements. For the in situ measurements, a free-falling benthic lander was used. Once released, the lander sinks to the sediment surface and starts the measurements autonomously. After finishing the measurements, the lander releases weights and rises back to the surface. This can be triggered by an acoustic signal or by a burn wire timer. The lander was equipped with an array of four custom-made optodes, mounted on stainless steel tubes (6 mm diameter), which could profile 45 cm into the sediment. A microprofiler that held four oxygen microelectrodes was used to determine the oxygen profile in the uppermost 20–50 mm of the sediment, as well as the diffusive oxygen uptake. To measure the total oxygen uptake directly, an incubation chamber with two optodes and a syringe sampler was used. In addition, a Niskin bottle for bottom water samples was attached to the lander. Hydrogen analysis (Smith, Spivack)The concentration of hydrogen gas (H2) dissolved in the interstitial water was measured at each site. This was done by placing ~3 cm3 of sediment into a vial and filling the vial completely with deionized water, taking care to not include any headspace. Hydrogen-free gas (500 µL) was then injected through the septum while allowing an equal volume of water to escape to create a headspace. The H2 was then given time to diffuse out of the interstitial water (>24 h). After allowing the H2 to accumulate in the headspace, 300 µL was removed and injected into a reduced gas analyzer (Trace Analytic ta3000). The instrument was calibrated with a 100.6 ppm H2 standard (Scott Specialty Gases). Blanks were prepared by using vials with deionized water and the H2-free headspace. In this configuration, the system had an average detection limit of 67 nM H2 with a range of 2–229 nM. At Site SPG-1, 3 cm3 plugs of sediment were placed in glass vials, filled with deionized water and a ~1 cm3 headspace consisting of laboratory air. The blanks using laboratory air contained too much H2 relative to the samples and severely increased the detection limit. Beginning with Site SPG-2, the procedure was modified to use bypass gas (carrier gas [N2] that had passed over the mercury bed to remove traces of H2) for the headspace. For these analyses, samples were collected at each site either directly above or below each whole-round sample taken for geochemical analysis. The samples were taken from the core in 3 cm3 cut-off syringes and extruded directly into the vial and immediately filled with water and capped. Microbial activity protocols (Ferdelman, Soffientino)Protocol for tritiated H2 assay of hydrogenase activity (Soffientino)Measurement of hydrogenase activity is carried out using the tritium-based method of Soffientino et al. (2006). Briefly, 3 cm3 of sediment was slurried with 10 mL sterile filtered seawater in a 30 cm3 glass syringe. A headspace of tritiated hydrogen and nitrogen was added, and the samples were incubated at ambient temperature (20°C) with gentle shaking for 12 h. Subsamples of the slurry were withdrawn every hour, degassed, and scintillation counted. Four samples per core were assayed in triplicate, with paired killed controls made by slurrying sediment with saturated mercuric chloride solution instead of seawater. The hydrogenase activity is calculated as the linear slope obtained by regression of the scintillation counts against time. The detection limit of the assay is between 1 × 10–12 and 1 × 10–13 mol hydrogen/cm3 sediment/min. Protocols for other microbial activities (Ferdelman)Slurry preparationA 60 cm3 subcore is taken from the piston or multicorer core and extruded into aluminium-polyethylene “wine-bag.” A total of 60 mL of filtered overlying water is added to the sediment, and the sediment water mixture is slurried. At the surface, these volumes are doubled. From this slurry we took the following:
The remaining slurry was frozen at –20°C. Nitrate and ammonium experiments
Thymidine experiment
Cysteine degradation experiment
Microbiology protocols (Smith, Durbin, Forschner, Harrison, Horn, Kallmeyer, Lever, Puschell, Soffientino)Sample handling protocols (Smith, Durbin, Forschner, Harrison, Horn, Kallmeyer, Lever, Puschell, Soffientino)All microbiological sampling of the sediments was done with sterile cut-off syringes. These syringes were prepared in two ways. For the first two sites, syringes were cut by hand with either a knife blade or a small saw, wrapped in foil, and autoclaved. For subsequent sites, syringes were cut using a webcutter heated knife. Syringes of 3 mL volume were then modified by hand using a knife blade to angle the leading edge of the syringe inward. After cutting, the outside leading edge of larger syringes was run across the hot knife to give a clean edge that angled outward. Then the syringes were wrapped in foil and autoclaved with the plunger pulled back. The sampling surface of the sediment cores was scraped using a sterilized spatula and sampled at a distance of several millimeters from the edge of the core to avoid contamination. The filled syringes were recapped with foil and sealed either in foil bags or sterile bags and distributed for the various projects. Cell enumeration protocols (Kallmeyer, Puschell, Harrison)At all sites, sediment samples were taken for four different categories of cell enumeration:
Because cell concentrations were expected to be below the detection limit of standard acridine orange direct count techniques (~105 cells/cm3), shipboard efforts focused on cell counts using the first approach and on appropriate preparation of samples for the second, third, and fourth approaches. For comparison to sedimentary cell counts, surface water samples were taken by bucket from the ship at each site and bottom water samples were taken from the water in the multicorer tube sampled for cell counts. The surface water samples were from approximately the upper 10 cm of the water column. Shipboard separation of cells from the sedimentary matrix was achieved with a technique developed by Kallmeyer et al. (2008). The sediment is treated with a mixture of different detergents, solvents, and complexing agents, followed by separation of the cells from the sediment particles by density centrifugation. The cell extracts are then filtered and stained with SYBR Green I, a stain with high specificity for double-stranded DNA. At each site, samples were taken for this purpose from a multicore and from the longest piston core or gravity core available. Depending on the length of the core, between 12 and 25 samples were taken at each site, evenly spaced over the entire depth range. Selected samples were immediately analyzed; the remainder were analyzed postexpedition. For each depth, one 2 cm3 sample was fixed in 8 mL of 2.5% NaCl solution with 2% formalin as a fixative. From this slurry, three 500 µL aliquots were extracted and counted as described above. At every site, one blank sample was processed by treating 500 µL of 0.2 µm filtered NaCl solution like a sediment sample throughout the entire extraction and counting procedure. Sediment samples were also collected for postcruise microscopy at different depths throughout a multicore and the longest piston core or gravity core at each site. The sampling resolution for these samples was one sample every 5 cm in the multicore and roughly one sample every 0.5 m in the longer core. These microscopy samples were then processed with five different fixation methods. For each sample collected, 1 cm3 was used for acridine orange counts (fixed in 9 mL of 2% formaldehyde in artificial seawater and stored at 4°C), 0.5 cm3 was used for CARD-FISH (fixed in 1.5 mL of 5.3% formaldehyde in artificial seawater overnight, then washed two times with artificial seawater and stored in 50% phosphate buffered saline [PBS]/ethanol buffer at –20°C), 0.5 cm3 was used for CARD-FISH with an alternative fixation technique (fixed in 1.5 mL of 70% ethanol for 4 h and then washed two times and stored in 50% PBS/ethanol buffer), and 1.0 cm3 was used for incubations of 2 and 48 h with BrDU followed by fixation with 5.3% formaldehyde and washing steps and then storage in 50% PBS/ethanol. The purpose of using several methods was to look for evidence of cell viability in the sediment. Because acridine orange stains nucleic acid, that method was used to count cells independently of the SYBR Green counts. Samples were stained with CARD-FISH to look for the activity of cells by detection of rRNA. The BrDU incubations will allow the detection of cells that are synthesizing DNA, an indicator for cell division. This is achieved by staining the cells with a BrDU-specific antibody and then doing a secondary stain with a fluorescent tagged antibody. Additional 5 cm3 samples for postcruise microscopy were collected and fixed for 4–12 h with 1% formaldehyde, rinsed twice with 1:1 PBS/ethanol, and resuspended in 5 mL ethanol. These samples were divided into discrete mineral partitions on shore for additional CARD-FISH studies. Sediment plugs of 3 cm3 were collected at 1 m intervals and fixed as above for postcruise cell separation and filtration for FISH. Molecular protocols for sediments (Durbin, Forschner, Harrison,Lever, Puschell)Sediment samples, used to characterize microbial community composition and changes within sediment columns and between sites, were obtained from both piston and multicores (Durbin and Teske, 2010). Piston cores were sampled at regular depth intervals. Samples designated for molecular diversity characterization and genomics were sampled using two 60 cm3 syringes of sediments every 30–100 cm core interval. Both 60 cm3 syringes were aliquotted into 5 cm3 subsamples, some of which were frozen at –80°C without further processing, whereas others were slurried in saline solution (30 g/L NaCl, 60–240 mM NaPh) or in 3.5% saline/10% glycerol prior to freezing at –80°C. Multicores for high-resolution microbial diversity and genomics studies in surface sediments were sampled continuously, from top to bottom. Sampling occurred with three 60 cm3 syringes, each of which had a depth scale on the side, which allowed high-resolution subsampling of sediment in 1 cm depth intervals. The top 20 cm was sectioned into 1 cm depth intervals, and each interval was pooled. Depending on multicore recovery, we subsampled sediments below similarly, but using 2 cm depth intervals from 20–30 centimeters below seafloor (cmbsf) and 5 cm intervals in sediments below 30 cmbsf. All subsamples obtained from multicores were immediately frozen at –80°C. To generate a distinct and reproducible pattern that can be used for comparing the biodiversity of surface sediments from different sites, sediment samples (60 cm3) for terminal restriction fragment length polymorphism (tRFLP) analysis were taken at selected sites from the top layer of the multicore and stored at –80°C. This analysis will be used to determine the microbial diversity by amplifying 16S rRNA genes from the sediment and cutting them with restriction enzymes and sorting the fragments by size. To assess the total bacterial diversity of the sedimentary communities in select horizons of all the sites, 60 cm3 samples were taken at regular intervals and frozen for postcruise 16S tag sequencing. To determine biosynthetic potential, a 60 cm3 sample was collected at ~1 m intervals from the piston corer and a 10 cm3 sample was taken in 10 cm increments from the multicorer. Samples were preserved at –80°C for shore-based molecular analysis of bacterial genes associated with polyketide and nonribosomal peptide synthesis. Because of the low biomass, DNA extractions will be subjected to whole genome amplification prior to PCR amplification for detection of the genes. Presence or absence of biosynthetic gene clusters will be put in context of depth and geochemical properties to help direct future culturing and sampling efforts. To document microbe-mineral associations, plugs of sediment from piston and multicores were frozen at –80°C for shore-based DNA extraction, after splitting the sediment into component minerals using magnetic separation followed by density separation in sodium metatungstate (a nontoxic heavy liquid adjustable up to 3.1 g/cm3). For these studies, DNA will be extracted from bulk samples and mineral fractions for construction of clone libraries, 16s rRNA gene sequencing, and tRFLP. XRD and energy dispersive spectroscopy using a scanning electron microscope (SEM-EDS) will be performed on extracted samples to confirm compositional heterogeneity. Mn nodules have also been selectively sampled to be processed as stated above. Homogenized mineral samples were taken from stations 2, 7, and 9 to be processed for mineral separation, XRD, and SEM-EDS in order to better characterize sediment mineralogy and to extract micrometeorites. Cultivation protocols for sediments (Puschell, Forschner)For our basic cultivation studies, 20 cm3 sediment samples were taken at different depths throughout a multicore and a piston core at each site. The sample resolution obtained for the multicore was one sample every 5 cm. For the piston core three samples were taken from different depths depending on the length of the core. Also, at sites where the piston core retrieved the basalt/sediment interface, a cultivation sample of 10 cm3 was taken from the lowermost sediment. The total number of cultivation samples per site was between 7 and 10. The number of samples taken was much greater than could be processed during the cruise. Inoculations of aerobic cultures were done at all sites with the top layer of sediment sampled from the multicore. At selected sites, aerobic cultures were also started at multiple depths within the multicore. Seven types of media were used for aerobic enrichments. These were designed to target ammonium oxidizers, manganese oxidizers, thiosulfate oxidizers, nitrite oxidizers, nitrogen fixers, heterotrophs adapted for oligotrophy, and methyltrophs. Inoculations of anaerobic cultures were done using the top layer of sediment from the multicore and the deepest sediment sample from the piston core for select sites. Eight types of media were used for anaerobic enrichments. These were designed to target denitrifiers, manganese reducers, iron reducers, sulfate reducers with acetate, sulfate reducers with malate, methyltrophs, nitrogen fixers, and fermenters. Further inoculations were carried out promptly when the samples return to MPI. For every site, a most probable number analysis was done for aerobic heterotrophs on samples taken from the top of the multicore. This is another way for determining the viability of the cells in the sediment and to give an estimate of cultivable cell counts. For studies of the biosynthetic potential of microbes from subseafloor sedimentary communities in the South Pacific Gyre, a 3 cm3 sample was collected from the piston core at intervals of ~1 m and from the multicorer at 10 cm increments. When possible, the sediment from around the subsampled site of the multicorer was collected, extracted for nutrients, and sterilized for use as a natural growth media. Cultivation efforts focused on the isolation of marine actinobacteria and Bacillus sp. since these have historically been rich resources for the discovery of biomedically promising metabolites. Aliquots of sediment were subjected to heat shock, alcohol treatment, or dry stamping to help select for the desired bacteria. Treated samples, in addition to an untreated aliquot, were plated onto various oligotrophic media and incubated at 4°C. The Yayanos pour-tube method was used to culture selected sediment samples under environmentally relevant pressures. This method involves suspending cells in agar just prior to solidifying, immobilizing the cells. Individual colonies grown under high pressure can then be easily isolated to determine if pressure helps select for novel strains. Due to space restraints, the samples incubated under 5800 psi at 4°C were composed mostly of the untreated aliquots from the uppermost sediments. On the ship, there was little visible growth. However, upon growth, reisolations of the cultured bacteria will be continued until pure cultures are obtained for further studies to be conducted at URI. In these postexpedition studies, DNA will be extracted from all isolated bacteria for phylogenetic analysis. Additionally, isolates will be cultivated in seawater broth media, and culture extracts will be evaluated for the production of antibiotics using bioassays. Chemical and spectroscopic analysis (natural magnetic remanence and high-pressure liquid chromatography–mass spectroscopy) will be used to probe for the production of novel metabolites. Protocols for microbiological studies of manganese nodules (Harrison, Horn)Manganese nodules were recovered from the cored sediment at most sites. Two separate suites of nodules were carefully sampled for postcruise study of their microbial communities. Sampling of the first set of manganese nodules will be used to characterize the microbial communities by DNA analysis, FISH, and electron microscopy. The separate sample sets were taken for various regions of the nodule (up to two distinct regions within the nodule and the surface). Nodule samples were saved for further geochemical, isotopic, and mineralogical analyses to determine specific microbe-mineral interactions on and within nodules, the extent to which these influence nodule formation and growth, and to use isotopic analyses to attempt a measure of nodule growth rates. The goal of nodule sampling is to characterize the microbial community on nodules with the hope of providing clues as to nodule formation and growth. The first suite of nodules (from Sites SPG-2, SPG-9, and SPG-10) was processed by chipping off 0.1 to 1.5 mm diameter pieces of whole nodules with a sterilized hammer and chisel. The chips were then transferred to subsamples using sterilized tweezers. Each subsampling set consists of up to fourteen 1 to 1.5 mL samples processed as follows: up to 10 subsamples frozen at –80°C for DNA extraction, up to 2 samples fixed in 4% paraformaldehyde, then centrifuged and rinsed twice with a 1:1 mixture of PBS/ethanol and stored at 4°C for FISH, and up to 2 samples fixed in 2.5% glutaraldehyde and stored at 4°C for electron microscopy (EM). Separate subsample sets are taken for various regions of the nodule (up to three distinct regions within the nodule and the exterior surface). For example, the nodule sampled for Core SPG10-M1 was split into four sections: the outer and inner surfaces of the external layers, sediment trapped within the nodule, and the inner core. Remaining nodule samples are saved for further geochemical, isotopic, and mineralogical studies. The second suite of samples consists of three small nodules fixed with 1% paraformaldehyde for shore-based microscopy and additional nodules frozen at –80°C for later DNA extraction along with associated sediment. This sample set will further be mineralogically and chemically characterized by XRD and SEM-EDS. Protocols for microbiological studies of altered basalts (Horn)Altered basalts were recovered from the base of the cored sediment at two sites for a total of three samples (SPG3-P1, SPG3-P2, and SPG7-P1). These were carefully sampled for postcruise study of their microbial communities. For comparison to microbial communities in the underlying basaltic glass, the deepest sediments recovered at these sites were sampled for characterization of the microbial communities by DNA analysis, FISH, and EM. All samples will be processed at USC. Sediment volume (10 mL) of sediment was sampled from the sediment/basalt interface using a cut-off autoclaved syringe. The sediment was suspended in a 1:1 ratio of bottom water that had been filtered at 0.2 µm and autoclaved (FABW) to create a sediment slurry. Fourteen 1–1.5 mL subsamples were taken with 10 subsamples for DNA extraction. Two samples were fixed in 4% paraformaldehyde, centrifuged and rinsed twice with 1:1 PBS/ethanol, and stored at 4°C for FISH. Two samples were fixed in 2.5% glutaraldehyde and stored at 4°C for EM. The remaining sample was then used for a series of four enrichment cultures in FABW stored at 4°C, unamended, 10 mM nitrate, 10 mM ammonia, and 5 mM nitrite and 5 mM ammonia enriched. The goal of the DNA analysis and microscopy is to compare the bottommost recovered sediment microbial communities to the basalt glass communities recovered. The goal of the enrichment cultures is to target microbes involved in the nitrogen cycle in the subsurface for further characterization and potential isolation. Sampling of the basaltic glass followed the same sampling plan as the overlying sediments, with the addition of FeS gradient tubes, culturing, and geology samples taken. Chips of basalt 0.5 to 1.5 mm diameter were used instead of a slurry. The gradient tubes are as described by Emerson and Floyd (2005) with the substitution of FABW for modified Wolfe’s mineral medium. Additional gradient tubes were amended with 100 µL of 200 mM nitrate or nitrite added to the top of the overlying plug and allowed to diffuse into the medium before inoculation with chips of glass 1–4 mm in diameter. The inoculated tubes were stored at 4°C. The culturing samples consist of basalt glass immersed in FABW and stored at 4°C for later use. The geology samples are for geochemical, isotopic, and mineralogical characterization. Water column sampling and sample handling protocols (Halm, Dorrance)At each site, water from two to three depths was taken by a CTD Rosette. Surface water was also taken. The principal shipboard focus of water column analyses was on nitrogen fixation rates and assimilation rates for organic and inorganic nitrogen. For this work, water samples were incubated with 15N-labeled nitrogen gas, ammonium, and leucine. With the isotopic ratios, rates can be calculated. N2 fixation was expected to be the main source of nutrient nitrogen. Subsamples were also taken for CARD-FISH study and other molecular analyses, to identify diazotroph bacteria groups and to quantify the activity of the nifH gene (which is responsible for nitrogen fixation) (Halm et al., submitted). Nutrient concentrations define the chemical environment of the samples. Concentration/temperature/depth recorder samplingAt all 11 sites, samples were taken from three or four depths with a CTD rosette. The depths sampled depended on the occurrence of chlorophyll in the water. These water samples (15–25 L) were taken from the chlorophyll maximum and two other depths. Seawater from the seawater supply line in the Roger Revelle Hydro Lab was taken as a surface water sample. Nitrogen fixationTo 1 L of seawater, 4 mL 15N2 gas and 50 µL 13C-bicarbonate (0.1 M end concentration) were added and incubated for 3–6 h under in situ conditions. The samples were filtered on GF/F filters and frozen at –20°C for transport to MPI. For each depth, 1 L was incubated in light and one in dark. As a control, 1 L of surface water was handled like the other samples, but labels were added immediately before filtering and freezing. Nitrogen assimilationFor organic and inorganic N-uptake at nine stations, 15NH4 (2 mM end concentration) or 15N-leucine (0.2 mM end concentration) was added together with 13C-bicarbonate to 250 mL of surface water, and at four stations to 250 mL of water from 40 m depth, incubated for 3–6 h in light and dark, filtered, and frozen at –20°C. At every station, 4–8 L of water was filtered as background. The isotopic ratios were measured by mass spectrometry in Bremen postcruise. SortingAt some stations, 1 L of water from the chlorophyll maximum was incubated with 15N2 and 13C-bicarbonate, filtered on two GTTP filters, fixed with paraformaldehyde (PFA), and frozen at –20°C. In Bremen, the filtered cells were resuspended and sorted by flow cytometry. After sorting, the isotopic ratios of sorted bacteria groups were measured. NutrientsFor nutrient measurement, 50 mL of every depth was frozen. Ammonium was measured on board. CARD-FISHAliquots of 15–30 mL of every depth were fixed with PFA (1 M end concentration), filtered on GTTP filters, and stored at –20°C. DNA/RNA filtersFor molecular work, 2–10 L from every depth were filtered and frozen at –80°C. DiversityFor postcruise tag sequencing studies of bacterial diversity, 5 L of seawater was taken from the depth of the chlorophyll maximum of each site, filtered, and stored at –80°C for postcruise analysis at the Marine Biological Laboratory (Woods Hole, USA) as part of the International Census of Marine Microbes. Cell enumerationFor postcruise cell counts, 18–20 mL of seawater was stored with formaldehyde in a Falcon tube and refrigerated. |
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