The goals of microbiological sampling and study during Leg 204 were to determine the rate of microbial methane production and consumption, the composition of the microbial communities responsible for this methane production and consumption, and the effect of high methane concentrations and methane hydrates on sedimentary microbial biomass and community structure. Most of the onboard work was devoted to deciding where and how to sample and collecting subsamples to meet these goals. The bulk of the analyses necessary to achieve these goals are to be performed on shore.
Site 1244, located on the eastern slope of Hydrate Ridge, was the primary site chosen for comparison of microbiology with the Hydrate Ridge summit. The methane flux estimated from the sulfate gradient at this Site 1244 is 2.7 x 10-3 mmol/cm2/yr (see "Interstitial Water Geochemistry"), as compared to the summit, which has methane fluxes estimated at over 10,000 times these rates (see "Interstitial Water Geochemistry" in the "Site 1249" chapter and references therein). The methane flux at Site 1244 is comparable to the maximum fluxes found at the Blake Ridge (1.8 x 10-3 mmol/cm2/yr) (Borowski et al., 1996), where previous microbiological study on Leg 164 samples (Wellsbury et al., 2000) will also provide a framework for comparison.
Site 1244 was both the first and last site sampled for microbiology during Leg 204, and the samples taken and methods used reflect our learning process. Sampling in the upper sediment layers targeted the SMI, where microbial consumption of methane should peak. AMO, using sulfate as the electron acceptor, has been a topic of much recent study (Boetius et al., 2000; Orphan et al., 2002; Michaelis et al., 2002; Zhang et al., 2002) and is a major focus for the Leg 204 microbiological program. The initial holes drilled at Site 1244 were sampled for geochemistry, and once the sulfate and methane data had been examined, the SMI was targeted for intensive coupled microbiological and geochemical sampling. We found that this interface could also be estimated quickly by noting the presence of cracks (expanded gas) by eye, and more accurately, but still rapidly, by observing the sharp transition between high and low P-wave signal strength, causing dropouts in velocity values (see "Physical Properties" in the "Explanatory Notes" chapter). The SMI at Site 1244 was at 8.5 mbsf, based on measurements of sulfate (see "Interstitial Water Geochemistry") and methane (see "Hydrocarbon Gases" in "Organic Geochemistry"). The top three cores from Holes 1244C and 1244F were sampled intensively (one to two samples per section) in coordination with the geochemistry program (Table T13).
Methanogenesis can proceed in most anaerobic environments, but it becomes the major process when other electron donors such as nitrate and sulfate are depleted. We sampled regularly downhole to below the base of the GHSZ (Table T13) to quantify methanogenesis in these sediments. At the Blake Ridge, microbial biomass (as measured by direct counts) increased just below the GHSZ and then returned to background values (Wellsbury et al., 2000). Site 1244 will provide a comparison with these values.
Iron is another electron acceptor that can be used by microorganisms, but the iron data from the first hole at most sites were not available by the time microbiological sampling was initiated in the second hole. Because sampling at Site 1244 took place at both the beginning and the end of the leg, we had IW iron data available to guide microbiological sampling. Onboard IW analyses at Site 1244 indicated unusual iron geochemistry with a soluble iron maximum of 27.5 mM at 21 mbsf. Samples targeting this zone were taken from Holes 1244E and 1244F and placed in enrichment media for iron- or manganese-reducing organisms and for methanogens (see "Enrichment Cultures" in "Microbiology" in the "Explanatory Notes" chapter).
Sediments associated with hydrates were not deliberately sampled at this hole; however, IR anomalies indicated small amounts of disseminated or veined hydrates in Hole 1244E, which was sampled for microbiology.
The emphasis in sample processing was to work as quickly as possible without compromising microbiological technique, to maintain samples at or below ambient temperature of the formations from which they were taken, and to minimize exposure to oxygen. Cores were kept at 4°C and left undisturbed in their liners until they could be completely processed. When supply exceeded the rate of processing, cores were handled on a last-in, first-out basis. The processing of samples from Hole 1244C was conducted prior to completion of the refrigerated van laboratory, so the hold refrigerator was set up as a temporary sample processing laboratory and maintained at 4°C.
Samples for PFT analysis were not taken from Hole 1244C. Subsequent cores had 5-g subsamples taken from outer and inner layers for gas chromatography (GC) analysis, as described in "Perfluorocarbon Tracer" in "Shipboard Microbiological Procedures and Protocols" in the "Explanatory Notes" chapter. Samples have been analyzed as described, and raw data are presented in Table T14. Differences between outside and inside samples generally indicate minimal penetration. Notable exceptions may reflect penetration along fractures in sediments or discrepancies in handling and sampling, particularly when dealing with XCB cores.
A comparison of fluorescent microsphere penetration in core interiors and exteriors is summarized in Table T14. Microscopic analysis of outer core layers showed numbers of microspheres ranging upward from 104 spheres per gram of sediment, whereas microspheres were generally below the detection limit of 10 spheres per gram in samples taken from core interiors.
Samples were taken as described in the "Shipboard Microbiological Procedures and Protocols" in "Microbiology" in the "Explanatory Notes" chapter for inoculation of enrichment cultures targeting methane producing and iron- and manganese-reducing organisms. For methanogenic enrichments, one section from active upper sediments well below the SMI was sampled (Section 204-1244E-4H-5) as well as another section deeper in the hole (Section 204-1244E-18H-5). Samples were maintained at low temperature, and dilution series were inoculated to culture for microorganisms that produce methane from the disproportionation of acetate as well as from hydrogen and carbon dioxide. One series will be maintained at <10°C while a duplicate will be kept at 20°C. Deoxyribonucleic acid (DNA) sequence information from these enrichments will be used for comparison to nucleic acids extracted from frozen sediments, and isolates developed will be used for planned minimum metabolic energy, continuous culture experiments.
Enrichments in metal reducers were taken from regular intervals in Holes 1244F (0-15 mbsf) and 1244E to transect the dissolved iron peak near 30 mbsf. A total of 16 quadruplicate sets were inoculated. Media for growth of iron- and manganese-reducing organisms were inoculated with 1-2 g of freshly collected sediment. Four media formulations were used: acetate plus ferrihydrite, acetate plus manganese dioxide, formate plus ferrihydrite, and formate plus manganese dioxide. Formate-containing media were subsequently pressurized with 1-atm hydrogen gas.