Microbiological sampling of Site 1228 covered the sulfate-reducing zone near the surface, the sulfate minimum between 30 and 80 mbsf, and the deeper brine interval layer at depths between 80 and 193 mbsf. One or two sections of each core were routinely sampled for deoxyribonucleic acid (DNA) analysis, measurements of sulfate reduction rates, hydrogen concentrations and turnover, methanogenesis rates, acetate turnover, thymidine incorporation, bacterial lipid biomarkers, adenosine triphosphate, fluorescent in situ hybridation (FISH), and iron/manganese/sulfur solid phases (Fig. F6). An additional abridged sampling program for iron/manganese/sulfur solid phases and sulfate reduction rates was performed at higher resolution throughout the upper part of the core (Cores 201-1228A-2H through 7H). Core 201-1228A-6H (43-52 mbsf) was not sampled because the core barrel shattered, resulting in a severely disturbed core. It was substituted with a core section from the same depth interval of Hole 1228B (Section 201-1228B-6H-3, 49.3-50.8 mbsf). A full suite of microbiological samples was taken on this section (Fig. F7). Every core that could be obtained from the deeper layers of the sediment column was sampled to analyze the prokaryote communities and activities in the deep sulfate-rich brine layer below the sulfate minimum (Sections 201-1228A-14H-3 through 22H-2) (Fig. F6). Sampling of four sections of the mudline core from Hole 1228E (Sections 201-1228E-1H-1, 1H-2, 1H-3, and 1H-4) aimed at fine resolution of the highly compressed chemical and prokaryote gradients at the top of the sediment column in order to provide good end-member data for the microbiological and geochemical analyses in deep sediment layers (Fig. F8).
Samples of 1-cm3 plugs for prokaryotic cell enumeration were taken on the catwalk from a total of 20 depths between the sediment surface and 187.4 mbsf at this site. These were between 7.9 and 187.4 mbsf in Hole 1228A (14 samples), at 48.9 mbsf in Hole 1228B, and between the sediment surface and 5.7 mbsf in Hole 1228E (5 samples). Additionally, 2-mL samples of 25% slurry were taken from the four slurries prepared in the laboratory. These samples will be processed as part of shore-based activities, and no data are presented here.
While drilling cores for microbiology, the potential for contamination with bacteria from the surface is highly critical. Contamination tests were continuously conducted using solutes (PFT) or bacterial-sized particles (fluorescent microspheres) to check for the potential intrusion of drill water from the periphery toward the center of cores and thus to confirm the suitability of the core material for microbiological research. We used the chemical and particle tracer techniques described in ODP Technical Note 28 (Smith et al., 2000). Furthermore, the freshly collected cores were visually examined for possible cracks and other signs of disturbance by observation through the transparent core liner. Core sections observed to be disturbed before or after subsampling were not analyzed further. Such disturbance phenomena are critical to the integrity of the core material and therefore also to its usefulness for microbiological studies.
The PFT was injected continuously into the drilling fluid during drilling of Holes 1228A and 1228E (see "Perfluorocarbon Tracer Contamination Tests" in "Procedures and Protocols" in "Microbiology" in the "Explanatory Notes" chapter). PFT sampling focused on microbiology cores and especially on sections that were used for slurry preparation and cultivations.
To compare the PFT concentration in the center of a core to the PFT concentration at the periphery of the same core, two 5-cm3 samples were taken from the center of a core end and another 5-cm3 sample was taken at the core periphery, adjacent to the core liner. Whenever possible, the samples were taken directly on the catwalk because the PFT content of catwalk air was usually near zero.
Low levels of potential seawater contamination (Table T5) were found for the center portions of all tested cores of Holes 1228A and 1228E, not higher than 0.11 無 seawater/g sediment (average = 0.03 無 seawater/g sediment), with two of nine below detection (<0.01 無 seawater/g sediment). The outer portions of all tested cores had a significantly higher level of PFT tracer and potential seawater contamination (average = 0.17 無 seawater/g sediment). In all cases, the PFT content and potential seawater contamination levels were higher at the periphery of the core than in the center. Cores 201-1228A-15H and 22H were particularly wet cores and were, therefore, subsampled to investigate whether or not the visible water was contaminating seawater drilling fluid. In the case of Core 201-1228A-15H, the PFT concentration was measured in two different subcores. Both of these subcores indicated concentrations of PFT lower than would expected for samples contaminated with drilling fluid. In the case of Core 201-1228A-22H, two samples were taken, one in a dry and one in a wet region of the microbiology section. Both showed similar low concentrations of PFT, representing 0.1 無 potential seawater contamination/g sediment.
Of the four master slurry samples taken from Site 1228 (Table T6), three showed small but detectable concentrations of PFT. These small concentrations represent 0.03 無 potential seawater contamination/mL slurry. Assuming 5 x 108 bacterial cells/L surface seawater, each 0.1 無 seawater contamination may represent as many as 50 contaminating cells if the sediment is porous enough to allow cells to travel with the PFT.
Fluorescent microspheres (beads) were deployed on all four cores from which slurries were made at this site. The mode of deployment for bead packs was altered from that of the previous site, as a consequence of some previous deployment failures. During previous deployments, Whirl-Pak bags (4 oz) containing 20 mL of microsphere suspension were attached to one side of a spacer within the core catcher. However, at the previous site, some sediment cores were soft enough to slide past the attached bag without bursting it, so the microspheres were not released. As a modification, the bag was filled with twice the volume (40 mL), while keeping the number of microspheres constant, and heat-sealed at the ends. The bag was then placed across the spacer unit and tied in on both sides, with the heat seals outside of the spacer lumen. Sediment cores were consequently forced to burst through the bead bag when a core was taken. No failures were reported at this site.
For each slurry two subsamples were processed: (1) a sample of the slurry to check contamination and (2) a scraping from the outer surface of the core to confirm deployment of microspheres. Microsphere deployment was confirmed from the outer core scrapings in all four cores that were sampled for slurry preparation (Table T7). In two slurry samples, microspheres were detected (1/sample in each case). Further microscopic searching of the membrane failed to detect any additional microspheres. These single microspheres represent potential contaminations of 42 to 56 prokaryotic cells/mL sediment. It is believed that these data represent filter handling and processing effects rather than true contamination (see "Fluorescent Microparticle Tracer" in "Procedures and Protocols" in "Microbiology" in the "Explanatory Notes" chapter).
Slurries for cultivation were usually prepared by subcoring with two 60-mL syringes from the center of two freshly broken surfaces (after precutting the core liner with the ODP cutter). Bending the ends of the whole-round core upward allowed released particles to drop down into a bin. This technique provided untouched (although not always smooth) surfaces that were immediately sampled. All MPN dilutions and enrichments inoculated using samples from Site 1228 are listed in Table T8.
13C substrate incubations were initiated for postcruise analysis by FISH-secondary ion mass spectrometry (SIMS) using material from Cores 201-1228A-2H, 5H, and 14H. In each case, 10 mL of the master slurry was injected into a bottle. The 13C substrates used were methane, acetate, and glucose. For Cores 201-1228A-2H and 5H, one of each bottle was inoculated. For Core 201-1228A-14H, two bottles with each substrate were inoculated.