Microorganisms are the kinetic controls on both methane production and consumption. Their distribution and activity must, therefore, be estimated in order to model the dynamics of the gas hydrate system. Target zones of interest include (1) the top of the sediment column, where microbial sulfate reduction and microbial methane oxidation are coupled; (2) the base of the gas hydrate stability zone (GHSZ), which was a region of increased microbial numbers at Blake Ridge (Wellsbury et al., 2000); and (3) sediments hosting massive and disseminated hydrates, which may also host distinct microbial communities.
Because good microbiology samples can only be taken when the cores are fresh, the shipboard microbiologists focused on getting the best samples possible for future investigations. In addition to processing cores to accommodate sample requests, a whole-round sample from every section sampled for microbiology was frozen as an ODP archive sample. Cores were sampled for microbiology at Sites 1244, 1245, 1249, 1250, and 1251 and may be identified in the ODP database query "Hole, Core" summaries by the notation "Tracer/Whirl-Pac."
Because of the need to monitor contamination and control core temperature, cores for microbiological sampling were specified prior to core collection. Two types of tracers were used to monitor seawater infiltration into these cores: soluble perfluorocarbons and fluorescent microparticles. The perfluoro(methylcyclohexane) tracer was pumped into the drilling fluid (surface seawater) by a variable-speed, high-pressure liquid chromatography pump connected to the mud-pump system. The tracer pump operated at a fixed rate relative to the mud-pump speed so that the concentration of tracer was always 1 ppm (v/v). The pump was started for each designated microbiology core immediately after recovery of the previous core so that tracer would be "washed down" with the core barrel and arrive at the drill bit before coring began. The tracer pump was then run continuously until the core had been completely cut. The fluorescent microspheres were delivered in a plastic bag (designed to rupture on contact with the sediment) attached to the core catcher (see "Contamination Tests" in "Shipboard Microbiological Procedures and Protocols").
Core temperature is an important consideration when taking microbiological samples. Ideally, cores should be maintained as close to in situ temperatures as possible; in practice, cooling cores below in situ temperatures is acceptable, whereas warming cores above in situ temperatures can yield disastrous results. Our goal was to maintain core temperature at or below in situ temperatures at all times. Shallow cores at Hydrate Ridge have in situ temperatures below the temperature of the surface seawater (12°C), which is used as drilling fluid and through which the cores must travel to reach the rig floor. Cores with in situ temperatures <10°C that warmed to >10°C were rejected for microbiological sampling. To keep warming to a minimum, microbiology cores were retrieved and sent to the catwalk using the expedited core-retrieval protocol implemented during Leg 201 (see the "Introduction" in "Microbiology" in the "Explanatory Notes" chapter of D'Hondt, Jørgensen, Miller, et al., 2003). When a core barrel was retrieved, the core was immediately removed from the barrel and sent to the catwalk. Once the core was completely in the hands of the core technicians, the drilling crew sent the next core barrel down the wireline. This core-handling protocol increased the coring time but was necessary to keep core temperatures <10°C. Temperatures were monitored using the IR camera on freshly cut core ends.
Core sections were cut on the catwalk as usual, but no acetone was used to seal the end caps. Most sections that were sampled for microbiology had whole rounds for IW removed from the bottom end. A refrigerated cargo van (provided by Dr. Scott Dallimore of the Geologic Survey of Canada [GSC] Pacific for use during this cruise) mounted on the port side of the ship, aft of the rig floor, was maintained at 4°C and used as a microbiological laboratory space. All cores for microbiological study were transported directly from the catwalk to the van, where they were subsampled and packaged.
APC core sections sampled for microbiology were generally intact and undisturbed. Whole-round samples were removed from these sections in the refrigerated van by first cutting the liner with the rotary knife cutter (as normally used on the catwalk) and then fracturing, rather than cutting, the sediment. The whole rounds were dropped into Whirl-Pak bags (Nasco) or the Fisher equivalent and frozen, refrigerated, or further subsampled. Although this method leaves most samples in contact with contaminated material near the core liner, a decision was made that, in view of the very slow nature of the diffusion process and of the low storage temperatures involved, such treatment was preferable to additional processing under adverse shipboard conditions.
XCB core sections were processed by splitting the liner, but not the core itself, on the core cutting table. In the refrigerated van, the top of the liner was removed to expose the core and individual biscuits were removed and repeatedly pared on fresh sterile sheets of aluminum foil. Pared biscuits were placed in Whirl-Pak bags and frozen, refrigerated, or subsampled. Although we removed the contaminated material from XCB core samples to the best of our ability, they should be pared again in a sterile environment.
Whole-round samples or pared biscuits were subsampled with sterile, truncated 5-mL syringes or alcohol-flamed spatulas. Subsamples were placed in preservative solutions for direct microscopic counts, fluorescent in situ hybridization (FISH) with nucleic acid probes, and microsphere enumeration. Whole-round samples, biscuits, or subsamples that were stored unpreserved at 4°C were sealed in nitrogen-flushed triple-ply Mylar heat-seal bags (Kapak Corp., Minneapolis, Minnesota) to maintain an anoxic environment. Samples to be frozen were stored at -80°C.
A glove bag (Coy, Grass Lake, Michigan) containing a nitrogen atmosphere with 5% CO2 and 5% H2 was used for anaerobic handling of core sections to inoculate growth media selective for methane-producing organisms and iron- and manganese-reducing microorganisms. Subcores were brought into the glove bag in nitrogen-filled jars, maintained cold on blue ice, and work was performed quickly to minimize warming. All media and sterile tools were also precooled and kept on blue ice during processing. Hydrogen is present to combine with residual oxygen in a reaction catalyzed by palladium pellets maintained within the bag. The bag was maintained regularly, and several hours before each use was flushed with a gas mixture and provided with freshly baked (140°C) catalyst. As an additional precaution to minimize oxygen contamination, tools and glassware to be used for manipulation and storage of samples for strict anaerobic work were stored within the glove bag.
Although cores were processed as quickly and as carefully as possible, shipboard handling should not be simply accepted as aseptic. We recommend that investigators receiving samples treat them as potentially contaminated and subsample accordingly whenever possible. Microsphere enumerations, summarized in the relevant site chapters, and additional microsphere evaluations performed postcruise at individual laboratories should be used to evaluate the quality of individual samples.
The greatest challenge for subsurface microbiological investigations is verification that observed populations and activities are authigenic and not the result of introduced contaminants. Chemical (perfluorocarbon) and particulate (latex microsphere) tracers were used during microbiological coring to check for the potential intrusion of drilling fluid (surface seawater) and confirm the suitability of the core material for microbiological research. The presence or absence of these two tracers also acts as a quality assurance check on core-handling methods. These tracer techniques were used during Leg 201 and are described in ODP Technical Note, 28 (Smith et al., 2000b). Freshly collected cores were also examined through the transparent core liner for signs of drilling disturbance (shearing or slurrying). Core sections observed to be disturbed before or during subsampling were considered unsuitable for further study and were discarded.
Perfluorocarbon tracer (PFT) was continuously fed into the drilling fluid at a concentration close to the limit of solubility (1 µg/g) and well above the detection limit for gas chromatographic analysis (1 pg/g) of that material. Samples for PFT analysis were taken from all cores intended for microbiological studies. Syringe subcores were taken from the interior (to monitor intrusion) and exterior (to verify delivery) of a freshly broken core or biscuit surface, extruded into headspace vials, and sealed with Teflon septa. Air samples were occasionally taken to monitor the background (blank) level of PFT in the refrigerated van. Samples were kept at -80°C until ready for analysis by gas chromatography (GC). Methods described in Smith et al. (2000b) were used for PFT analysis, with minor modifications. The same GC setup was used, but the instrument was modified by installation of a 1-cm3 sample loop and injector valve to standardize injection volumes.
Latex fluorescent microspheres (Polysciences, Inc., Warrington, Pennsylvania) (YG; 0.5 µm diameter) were used as a particulate tracer complementary to the volatile PFT. A 2-mL aliquot of microsphere stock (2.69% solids) was diluted with 40-mL distilled water, sonicated for 2 min, and heat sealed into a 4-oz Whirl-Pak bag. The bag was then attached as described in Smith et al. (2000a, 2000b) to the inside of the core catcher and positioned to rupture upon impact of the core tube with bottom sediments, where the microspheres will mix with seawater and coat the outside of the core as it is pushed into the liner. During core processing, subsamples of sediments were collected from outer and inner layers for microscopic examination. Weighed samples were mixed thoroughly with saturated sodium chloride solution to extract microspheres. The slurry was then centrifuged to separate the liquid phase (Marathon 21K; 5 min; 1000 relative centrifugal force), the supernatant was filtered onto a black polycarbonate filter (Millipore; 0.2-µm pore size), and the filter was mounted on a clean slide for microscopic examination. Microspheres in slide preparations were counted using a Zeiss Axioplan fluorescence microscope equipped with the Zeiss No. 9 filter set (BP 450-490; LP 520), and the number of spheres observed was used to quantify contamination in spheres per gram of sample. Comparison of microsphere numbers between paired samples from inner and outer core layers provides a relative measure of fluid intrusion. A sample with many spheres in outer layers and few or none within may be considered "higher quality" than one with very few spheres in the outer layers and few or none within.
Liquid-culture enrichments were begun for methanogenic organisms that use either hydrogen or acetate as an energy source and for organisms or consortia capable of dissimilatory iron or manganese reduction coupled with either acetate or formate oxidation. Media, as described in Boone et al. (1989) and Lovley and Phillips (1988), were prepared at Idaho National Energy and Environmental Laboratories (INEEL) prior to the voyage.
Samples for enrichments were taken from cores that had been maintained at low temperature and placed in oxygen-free conditions as quickly as possible. For sediment transfers, subcores were brought into the glove bag in nitrogen-filled jars and insulated on blue ice; work was performed quickly to minimize warming. All media and sterile tools were also precooled and kept on blue ice during processing. One to two grams of sediment were aseptically transferred to culture tubes containing 10 mL of selective medium. Tubes were opened only briefly for the transfer and immediately resealed. Methanogen enrichments were serially diluted as a first step in isolating novel organisms. The tubes containing 2 g of sediment were vigorously mixed with a vortexer and serially diluted 10-, 100-, and 1000-fold by transferring with a syringe into sealed culture tubes containing 10 mL of medium. Inoculated tubes were then placed in a 10°C incubator until shipment, at the end of the voyage, to INEEL for long-term enrichment and isolation study.
The bulk of the samples taken were for shore-based investigations. Whole-round samples were taken for lipid analysis, adenosine triphosphate (ATP) analysis, and deoxyribonucleic acid (DNA) extraction and stored double-bagged in Whirl-Pak bags at -80°C. Subsamples were preserved for direct counts (stored in 0.2-µm-filtered artificial seawater containing 2% formaldehyde) and FISH with nucleic acid probes as well as FISH-secondary ion mass spectroscopy (both stored in a 1:1 solution of ethanol and phosphate-buffered saline); all of these samples were stored at 4°C. Cores to be used for culture-based analyses and for methods that require living cells as starting material (e.g., rate measurements) were stored under nitrogen either in triple-ply Mylar heat-seal bags or in Whirl-Pak bags inside canning jars that were flushed with nitrogen before sealing.