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doi:10.2204/iodp.proc.347.104.2015

Microbiology

Hole M0060B was drilled specifically for microbiology, interstitial water chemistry, and unstable geochemical parameters at Site M0060. Counts of microbial cells were made on board the ship by fluorescence microscopy using the acridine orange direct count (AODC) method and flow cytometry (FCM) using SYBR green DNA stain. Additional AODC counts were made during the OSP. Further counts by fluorescence microscopy will be done after the OSP using both acridine orange and SYBR green staining.

Microbial cells were enumerated at 28 sediment depths from independently taken samples for FCM (27 samples) and AODC (13 samples), both on the ship and during the OSP (Table T11). Microbial cell abundance is relatively low in the upper few meters (Fig. F18) with ~107 cells/cm3 at 2.58 mbsf. The upper 6 mbsf at this site is well-sorted sand with high porosities and a very low TOC content (see “Geochemistry”), which is usually associated with smaller microbial cell densities. At 7.63 mbsf, microbial cell abundance increases to 3.61 × 108 cells/cm3, and this community size is maintained for all the remaining samples in this hole to 84.43 mbsf with no significant difference between the counts (t = 0.198; degree of freedom [df] = 35 [not significant]).

Apart from the upper 6 m of sand, there are no major lithologic changes in the upper 80 m of this hole, which comprises silt-grade material (Fig. F18). It is interesting to note that there appears to be a higher TOC content in the upper parts of Unit II (see “Geochemistry”) where cell numbers concomitantly increase. The alkalinity profile shows a broad maximum between 7 and 30 mbsf reaching 24 meq/L compared to a background level of 10–12 meq/L for the rest of the hole. This suggests that microbial mineralization of buried organic matter is enhanced in the upper 6–30 m. However, there is no indication of a corresponding enhancement of the microbial community size over this depth range (Fig. F18).

Apart from the upper 6 m of this hole, all cell concentrations measured in Hole M0060B were extremely high, with values far above the upper prediction limit of the global regression of prokaryotic cells with depth (Fig. F18). The maximum deviation from the global regression occurred at 53.83 mbsf, where cell numbers were ~110 times greater than the global regression.

Cell counts were made by both AODC and FCM at the same sediment depths in 12 samples from Hole M0060B. A paired sample t-test on these data showed no significant difference between the two techniques (t = 0.044; df = 11 [not significant]). In Figure F19, FCM counts are plotted against AODC from the same depths. A calculated regression of this data set (data not shown), however, shows poor agreement between the two techniques, and a slope test showed a highly significant deviation from a slope of 1 (t = 5.735; df = 10; P < 0.001). This is not a surprise with this data set. The unvarying nature of the counts with depth means the data are clustered around one area on this graph and the result of the paired sample t-test should be regarded as indicative for the successful methods comparison.

Perfluorocarbon (PFC) contamination tracer was well above detection in the liner fluid and exteriors of all cores, indicating continuous PFC delivery into the borehole (Table T12). Liner fluid PFC concentrations fluctuated over 2–3 orders of magnitude (Fig. F20A), indicating variations in the rates of PFC delivery or mixing into the drilling fluid stream. Generally, the measured PFC concentrations were much below the target concentration of 1 mg PFC/L. Despite the fluctuations, PFC was above detection in the vast majority of core halfway and interior sections (Fig. F20B). Based on the fraction of liner fluid in cores, contamination was highest in the surface core, which consisted of sand, and it remained relatively high in Core 347-M0060B-3H, which consisted of organic-poor clay (Fig. F20C). Below that depth, drilling fluid intrusion (as estimated from liner fluid PFC values) fluctuated greatly with no clear relation to depth or lithology, except that PFC was below detection in the halfway and interior parts of the two lowermost cores (347-M0060B-25H and 27H). In addition to the surface cores, several deeper cores were highly contaminated (347-M0060B-6H, 10H, 17H, and 19H) and were estimated to potentially have 103–105 contaminant cells/cm3 in the interiors (Fig. F20D).

Based on PFC data in core interiors, cores that are suitable for microbiological analyses include, in addition to Cores 347-M0060B-25H and 27H, Cores 5H, 9H, and 11H, in all of which PFC was below detection in the core interiors. Moreover, Cores 21H and 23H show a only moderate level of contamination, estimated to be <100 cells/cm3 of sediment in the interiors (Table T12; Fig. F20D).