Microbiological sampling for Site 1225 was conducted at regular depth intervals in order to enable a general microbiological inventory throughout the entire sediment column. Microbiology sample intervals of ~19 m resulted from routinely sampling the third section of every second core from Hole 1225A (Fig. F8). Routine subsamples for sulfate reduction rate determinations, deoxyribonucleic acid (DNA), fluorescence in situ hybridization (FISH), adenosine triphosphate (ATP), hydrogen, and lipid biomarker analyses were accordingly taken from each of these samples and processed, fixed, or frozen, as appropriate. Samples for AODC were fixed from the same intervals, and the cells were counted on the ship. A 15-cm segment from every microbiology core section was immediately transferred to the geochemistry laboratory for chemical analysis of interstitial water. Activity measurements, such as bicarbonate turnover, acetate turnover, thymidine incorporation, and hydrogen turnover, were conducted on a subset of the routine samples, in particular, at the upper and lower ends of the sediment column. Samples for cultivations were taken with the aim of comparing distinct depths and chemical zones in the sediment column. Cultivation samples were taken from the surface or the near-surface sediment column (Core 201-1225A-2H), from intermediate depths near 100 and 200 mbsf (Cores 12H and 22H), and from the deepest core of the sediment column (Core 34H) (Fig. F8).
Microbiological sampling in Hole 1225C focused on two additional sample sets (Fig. F9). The mudline core of Hole 1225C was sampled at three depths (Sections 201-1225A-1H-1, 1H-3, and 1H-6) to obtain finer resolution near the sediment surface, where the steepest biogeochemical gradients had been encountered during ODP Leg 138 and were confirmed by interstitial water analyses from Hole 1225A. Sampling depths for cultivation of manganese-reducing bacteria in Hole 1225C were chosen according to the analyzed manganese profile of Hole 1225A (Fig. F5).
To explore the composition of microbial communities associated with oceanic crust and their contribution to basalt weathering, XCB drilling into basement rock was performed (Core 201-1225A-35X; 319.6 mbsf). The core catcher yielded a few fragments of basaltic rock, which were sampled separately using a specially designed sample processing scheme (see "Rock Sampling and Distribution").
Samples of 1-cm3 plugs for prokaryotic cell enumeration were taken on the catwalk from a total of 25 depths between 0.8 and 8.37 mbsf in Hole 1225C (3 samples) and between 8.8 and 319.6 mbsf in Hole 1225A (22 samples). Prokaryotes were present at all 25 depths (Fig. F10). The greatest abundance of cells was found in the near-surface sample (Sample 201-1225A-1H-1, 80-85 cm), which contained 7.5 x 106 cells/cm3, and the smallest number of cells was found near the sediment/basement interface at 320.45 mbsf (Sample 201-1225A-35X-CC, 0-1 cm), which contained 1.8 x 105 cells/cm3, a factor of 42 decrease in cell density. The overall depth profile follows a trend observed at other ODP sites (Parkes et al., 1994); however, absolute numbers of prokaryotes are considerably lower than the average numbers for all previously examined sites, particularly in the upper 65 m (Fig. F11), apart from a small increase at ~35 mbsf. Between ~70 and 280 mbsf, data generally agree with the average of previously studied ODP sites, albeit at the lower end of the range, but from ~300 mbsf, prokaryote numbers are again significantly lower than the average.
Site 1225 is located within 100 m of Site 851 (Leg 138), from which prokaryotic counts were previously made (Cragg and Kemp, 1995). There is broad agreement between the two data sets, with an upper zone containing low prokaryotic cell numbers, a central zone of relatively elevated numbers, and low numbers near the basement. Overall, prokaryotic cell counts made at Site 851 are higher than those presented here. At Site 851, prokaryotic cell numbers decreased significantly near basement, reaching 3.7 x 105 cells/cm3 at 317.4 mbsf. This value is not significantly different than that determined here.
Counting techniques have improved since Leg 138, allowing lower detection limits—estimated here at 4.7 x 104 cells/cm3 (compared to 1.8 x 105 cells/cm3; Cragg and Kemp, 1995)—and therefore increased sensitivity. Numbers of dividing cells (suggested as an index of population activity) are typically <10% of the total count. As expected, dividing cells as a percentage of the total count are high near the surface (Fig. F10) and are also elevated between ~55 and 200 mbsf. This agrees with data from Site 851. However, two more increases are identified in the data presented here: one at ~30-50 mbsf and another at 230-270 mbsf. The reasons for these increases are not clear. However, the lower increase occurs at the same depth as increases in dissolved manganese, dissolved iron, TOC, and a probable increase in methane, all indicative of bacterial activity (see Fig. F5).
Samples for prokaryotic cell counts were taken from five slurries prepared in the laboratory, as 2 mL of 25% slurry. Slurries were made from Cores 201-1225A-2H, 12H, 22H, and 34H and from Core 201-1225C-1H. Total counts on five slurry samples did not differ significantly from total counts on adjacent plug samples.
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 experiments use a dissolved organic tracer, perfluoromethylcyclohexane, to estimate the level of seawater contamination that has penetrated into sediment cores during drilling. The PFT was injected continuously into the drilling fluid during drilling of Holes 1225A and 1225C. Because the PFT is dissolved in the drilling fluid rather than suspended as a particle, these results are estimates of the potential for microbial contamination and are expected to provide an upper limit of actual microbial contamination. Subcores of 5 cm3 were taken from the bottom cut of each microbiology (MBIO) section or from the adjacent top of the immediately underlying section, and 5-mL aliquots were taken from each master slurry. Samples from the core catcher were collected to measure the concentration of PFT in samples recovered from the near-basement sample. The delivery of PFT to the drill bit was positively confirmed by measuring the concentration of PFT in drilling fluid removed from the top of the core or by detection of PFT in sediment smeared along the periphery of the core.
At Site 1225, the results of PFT experiments were for the most part ambiguous because of analytical problems and because of problems with sample handling on the catwalk. In particular, gas chromatograph (GC) traces had small peaks (other than the PFT) of similar retention time to that of the PFT. Further, catwalk samples were taken by syringe at the end of sections without using a fresh, sterile scalpel to remove the surface layer contaminated by the slicing of the sections. These problems mostly resulted in ambiguous and unusable results for Site 1225.
However, in a few cases, the results clearly indicated no PFT contamination. These were subcores taken from Cores 201-1225A-16H, 30H, 32H, and the "master slurry" taken from Core 22H. All of these samples had PFT concentrations below detection (corresponding to <0.02 µL seawater/g sediment). Additionally, there were several cases with PFT concentrations so high that there is no ambiguity as to whether or not these were contaminated by seawater during the drilling process. These were all the samples taken from the core catcher from Core 201-1225A-35X, with PFT concentrations suggesting contamination of ~3 µL seawater/g sediment.
Assuming 5 x 108 bacterial cells/L surface seawater, each 0.1 µL of seawater contamination may represent as many as 50 contaminating cells if the sediment is porous enough to allow the cell to travel with the PFT.
Fluorescent microspheres were used as a particulate tracer on all five cores from which slurries were subsequently made in the laboratory. A 10-mL sample of slurry was processed from each slurry for microsphere detection. Microspheres were detected in two of the five slurry samples (Table T6), indicating potential contamination from drilling fluid. The method of delivery prevents a usefully quantitative estimate of the amount of contamination and can only give a minimum for the amount of contamination. In all cases, the sample taken for direct prokaryotic cell counts was also used as a confirmation sample for the presence or absence of microspheres. Where microspheres were not detected in either sample, further samples from the outside of the relevant cores were taken to confirm microsphere delivery.
Overall, both the PFT analyses and the bead counts suggested that the slurry from Core 201-1225A-22H was not contaminated. Only trace amounts of PFT were detected in the subcore, and neither PFT nor beads were detected in the slurry itself. The most substantial contamination, with respect to PFT levels, was found in the XCB core catcher (Core 1225A-35X-CC), from which the basaltic rock samples were obtained. The high bead counts occurring simultaneously with low PFT values in two of the slurries from Sections 201-1225A-2H-3 and 12H-3 suggests that contamination did not occur during drilling but rather occurred during sample handling and processing.
All most probable number (MPN) dilutions and enrichments inoculated using samples from Site 1225 are listed in Table T7. A strong indication for manganese(IV) reduction was given by the manganese(II) interstitial water profile, showing a pronounced peak of manganese(II) close to the sediment surface (Fig. F5). Consequently, MPN enrichments of manganese(IV)-reducing bacteria were made at high resolution in Hole 1225A.
Growth was observed after the first few days of incubation in some of the following enrichment experiments:
These first positive results need to be confirmed by further subculturing and verifying by molecular data. Furthermore, the potential for previous contamination of the samples used for enrichment was higher at this first site of Leg 201 than at the following sites. This is particularly true of the first two examples shown above, where high levels of contamination were observed, and illustrates the potential problems of contamination in XCB cores.
Samples were routinely taken for postcruise FISH-secondary ion mass spectrometry (SIMS) analyses from each sampled core section (Fig. F8). In most cases, a 5-cm3 subcore was injected into 10 mL of filtered ethanol:phosphate buffered saline (PBS) solution and stored at -20°C. However, the samples taken from Cores 201-1225A-2H and 12H and Section 201-1225C-1H-6 were fixed in 3.7% formaldehyde, washed, and stored at -20°C. In addition, a single subsample of the basalt from Core 201-1225A-35X was also collected for postcruise SIMS analysis. This sample was stored in 10 mL of filtered ethanol:PBS solution and stored at -20°C.
13C substrate incubations were initiated for postcruise analysis by FISH-SIMS using material from Cores 201-1225A-2H, 12H, and 22H. In this case, 10 mL of the master slurry was injected into each bottle. For Cores 201-1225A-2H and 12H, the 13C substrates used were methane, acetate, or glucose. For Core 201-1225A-22H, the 13C substrates used were acetate or glucose, with two of each initiated on board.
Two rock pieces (dark gray to black basalt) were obtained from the core catcher of the lowermost core (Core 201-1225A-35X). These pieces included an oval rock (Fig. F12) (3 cm x 2 cm x 2 cm) and an elongated, thinner rock (2 cm x 1 cm x 1 cm). The rocks were embedded in disturbed sediment, indicative of seawater contamination. Adhering sediment particles were removed by washing the rocks individually in a 250-mL Pyrex bottle containing 50 mL of nitrogen-flushed marine salts solution (see "Rock Sampling" in "Microbiology" and Table T8, both in the "Explanatory Notes" chapter). After the first washing step, the rocks were transferred into a second wash bottle with fresh nitrogen-flushed marine salts solution and washed again by shaking and rinsing. After this step, the rocks were handled with a sterile forceps and cleaned individually by scraping small remaining pockets of metal oxide and whitish sediment from the surface using sterile syringe needles. The loosened particles were rinsed off with nitrogen-flushed marine salts solution from a syringe.
One rock piece was too large for the shipboard rock crusher. It was wrapped in sterile aluminum foil and broken into pieces by repeated energetic strokes with a hammer. The pieces were divided as follows:
The smaller rock piece was crushed in a sterile rock crusher and the pieces were divided up as follows:
The combined first washes of the two rocks had the consistency of a sediment slurry (100 mL volume; ~10% vol/vol sediment at the bottom), very similar to the slurry from Section 201-1225A-34H-3. This rock-associated sediment was split for the following: