Microbiological sampling at Site 1231 covered both the iron- and manganese-rich zones in the upper half of the sediment column (0-68 mbsf) and the deeper sediment layers that are depleted of dissolved reduced metal ions (68-118 mbsf). In Hole 1231B, each core of the sediment column was sampled in at least two sections. One core per section was sampled for molecular microbiological analyses (deoxyribonucleic acid [DNA] and fluorescence in situ hybridization-secondary ion mass spectrometry [FISH-SIMS]), measurements of activity (sulfate reduction rates, hydrogen concentration and turnover, methanogenesis, acetate turnover, and thymidine incorporation), bacterial lipid biomarkers, adenosine triphosphate, and iron/manganese/sulfur solid phases. A second core section was sampled exclusively for iron/manganese/sulfur solid-phase analysis (Cores 201-1231B-1H through 13H) (see Fig. F4). In addition, selected depths were chosen for microbial enrichment and cultivation studies (Fig. F4)
The basalt/sediment interface at the bottom of Hole 1231D (Section 201-1231D-13H-CC; 114.35-114.7 mbsf) was sampled for DNA and biomarker analysis and microbial enrichments from sediment and basalt.
Sampling in Hole 1231E focused on intervals that had not been sufficiently sampled in Hole 1231B. These intervals were identified after the depth profiles of dissolved manganese and iron became available (Figs. F5, F3). Whole-round core (WRC) samples for enrichments of manganese(IV)- and iron(III)-reducing bacteria and some high-resolution samples for DNA analysis were taken. Several 1.5-m core sections showed conspicuous color gradients that corresponded to pronounced lithostratigraphic changes (see "Description of Lithostratigraphic Units" in "Lithostratigraphy"). Section 201-1231E-6H-2 (42.5-44.0 mbsf) changed from orange-yellow volcanic glass-rich clay at the top to brown clay at the bottom. Section 201-1231E-7H-4 (55.0-56.5 mbsf) changed from dark brown clay at the top to cream-colored nannofossil ooze at the bottom. Section 201-1231E-8H-4 (64.5-66 mbsf) was bisected by a sharp discontinuity at ~90 cm (65.4 mbsf), where the cream-colored nannofossil ooze at the top turned abruptly into chocolate-brown diatom-bearing clay at the bottom. In all three cases, DNA, biomarker, and enrichment samples were taken twice, from both ends of the core sections (Fig. F5).
Samples (1 cm3) for total prokaryotic cell enumeration were taken during core processing in the 4°C refrigerator from Holes 1231B (15 samples between the near surface and 113.1 mbsf), and 1231E (20 samples between 20 and 118.7 mbsf). Four depths of specific interest (~25, 53, 67, and 78 mbsf) identified by chemical profiles in Hole 1231B were sampled at high spatial resolution in Hole 1231E. All but the deepest sample (Sample 201-1231E-14H-3, 25-30 cm) from Hole 1231E were stored for shore-based processing.
Prokaryotes were present in all samples studied to the depth of 118.7 mbsf (Fig. F6). The highest number of prokaryotes, 1.72 x 108 cells/cm3, was found near the surface (Sample 201-1231B-1H-1, 0-1 cm). The lowest number was found at 81.64 mbsf, with 1.38 x 105 cells/cm3 (Sample 201-1231B-10H-2, 74-80 cm), was 1250-fold less than at the surface.
The overall depth profile of cell numbers per cubic centimeter (Fig. F7) follows a trend observed at other ODP sites (Parkes et al., 1994), although there are some substantial deviations. The sediment at Site 1231 is divided into an upper section of clay-rich sediment that overlies a nannofossil ooze (see "Description of Lithostratigraphic Units" in "Lithostratigraphy"). To a great extent, the prokaryotic cell profile mirrors the sedimentological change with considerably reduced numbers in the nannofossil ooze. In the upper 50 m, the data follow the lower prediction limit closely; however, small but significant (F-value = 7.21; degrees of freedom = 3 and 8; P < 0.05) increases in cell numbers occur at 9.45 and 15.23 mbsf (Samples 201-1231B-2H-5, 5-10 cm, and 201-1231B-3H-2, 83-89 cm). At 52.9 mbsf (Sample 201-1231B-7H-2, 50-56 cm), there is a substantial and significant (F-value = 12.66; degrees of freedom = 2 and 6; P < 0.02) increase in cell number by a factor of 5 to 4.8 x 106 cells/cm3. Below this depth, cell numbers decrease rapidly to a minimum of 1.38 x 105 cells/cm3 at 81.64 mbsf. This was the lowest count determined during Leg 201 and was below the limit where reliable counts are possible. The detection limit is based on calculations for a single membrane filter, and for each sample three replicate filters are used to provide a measure of variability. Where a zero count occurs, as it did in this case, the prokaryote population is estimated by combining all of the data from the three membranes and treating it as one subsample. This provides the only possible estimate of the population size in such samples but does not allow any measure of variability.
Below this depth, prokaryotic cell concentrations seem to increase; however, this may not be true, as with the exception of the deepest sample prokaryote populations in the three samples between 96.2 and 113.1 mbsf are around the detection limit. They all have large variances, and there is no statistically significant difference between them. This situation may be clarified with additional shore-based enumeration when additional subsamples can be counted, thus reducing sample variability.
The deepest sample at 118.7 mbsf had similar prokaryotic cell numbers to those at 62.64 mbsf. The reasons for this unexpected increase in cell numbers are not clear. However, this sample was very different from others higher in the sediment column in being adjacent to the basement and containing basaltic fragments.
Two increases in cell numbers occur at ~10-15 mbsf and again at ~53 mbsf. These two depths are near the upper and lower boundaries for methane concentrations found only in the upper sediments at this site. Additionally, the lower increase in prokaryotic cell counts is located near a small but significant decrease in interstitial water sulfate concentrations (see "Biogeochemistry"). This suggests that enhanced bacterial activity, albeit at a low level, is occurring at this site.
Numbers of dividing cells (suggested as an index of growth 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. F6). They then decrease rapidly to ~1.8% at 9.5 mbsf. Below 15 mbsf, only two depths (52 and 118.7 mbsf) gave reliable enumeration of dividing cells of 9%-13%. At all other depths, the percentages of dividing cells were either zero or the total cell population was so low that determining the percentage of dividing cells became highly variable and unreliable and so are not reported here.
The nature of the sediments at this site made direct counting more problematic than usual because the presence of manganese oxide in the sediments produced flocculent material that blocked filter membranes. Hence, where normally subsamples of 15-40 µL can be processed, subsample size was occasionally restricted to 5 µL at Site 1231. Consequently, the detection limit increased to 4 x 105 cells/cm3; thus, counting sensitivity decreased.
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 1231B and 1231D (see "Perfluorocarbon Tracer Contamination Tests" in "Microbiological 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 PFT concentrations in the center of a core to PFT concentrations at the periphery of the same core, a 5-cm3 subcore sample was taken adjacent to the core liner and two 5-cm3 subcores were taken from the center. Whenever possible, the samples were taken directly on the catwalk and capped away from nearby cores because the PFT content of catwalk air under these conditions was usually negligible.
Low levels of potential seawater contamination (<0.05 µL seawater/g sediment; average = 0.023 µL seawater/g sediment) (Table T8) were found for the center portions of all tested cores of Holes 1231B and 1231D, including the core catcher from Core 201-1231D-13X. The outer portions of all tested cores had substantially higher levels of PFT, and therefore, higher potential seawater contamination.
Of the four slurry samples taken from Site 1231 (Table T9), none showed high concentrations of PFT that suggest potential prokaryotic cell contamination from seawater drilling fluid.
Assuming 5 x 108 prokaryotic cells/L surface seawater, each 0.1 µL of seawater contamination may represent as many as 50 contaminating cells if the sediment is permeable enough to allow cells to travel with the PFT.
Fluorescent microspheres (beads) were deployed on all four cores from which slurries were intended to be made 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 beads.
Bead deployment was confirmed from the outer core scrapings in all four cores that were sampled for slurry preparation. No beads were detected in samples from the prepared slurries, indicating that they were probably free of contamination (data not shown).
Cultivations were normally conducted with slurries that were prepared by subcoring with 60-mL syringes from freshly broken whole-round core surfaces. Quantitative (most probable number [MPN]) and qualitative (enrichment) cultivations were started at temperatures between 4° and 80°C (Table T10). The media used were selective for fermenters, sulfate reducers, methanogens, and various anaerobic chemolithotrophic and heterotrophic prokaryotes that use iron(III) or manganese(IV) as an electron acceptor. Additionally, sulfite was added to some media. Sulfite can be used as an electron acceptor by sulfate-reducing bacteria or can be disproportionated to sulfide plus sulfate (suffix -SO3 in Table T10). In order to determine the spore-forming portion of the community, some media were inoculated with pasteurized (10 min at 80°C) sediment (prefix past- in Table T10). Furthermore, some MPN assays were incubated under air (suffix -O2 in Table T10)in order to enrich facultative or strict aerobic prokaryotes (see Table T10 in the "Explanatory Notes" chapter).
Many recent and relict worm burrows were filled with what appeared to be iron sulfide and pyrite, indicating that sulfate reduction was occurring or had occurred (see "Unit I" in "Description of Lithostratigraphic Units" in "Lithostratigraphy"). Therefore, samples from the burrows were used for enrichments of sulfate-reducing bacteria at 4° and 19°-21°C.
13C substrate incubations were initiated for postcruise analysis by FISH-SIMS using master slurry material from Cores 201-1231B-1H, 2H, 6H, and 12H. The 13C substrates used were methane, acetate, and glucose. Glucose was used in Cores 201-1231B-1H and 2H. Acetate was used at all four depths, with multiple bottles at each depth. Additionally, incubations with methane were initiated with material from Cores 201-1231E-3H, 4H, and 5H.