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doi:10.2204/iodp.proc.347.103.2015

Microbiology

Two holes, M0059C and M0059E, were drilled specifically for microbiology, interstitial water chemistry, and unstable geochemical parameters at Site M0059. Counts of microbial cells were made on board the ship by fluorescence microscopy using acridine orange direct counts (AODC) and by flow cytometry (FCM) using SYBR green I DNA stain. Additional AODC were also made during the OSP. Further counts by fluorescence microscopy will be done after the OSP using both acridine orange and SYBR green I staining.

Hole M0059C

Microbial cells were enumerated at 29 depths from independently taken samples for FCM (28 samples) and AODC (16 samples), both on the ship and during the OSP (Table T11). Microbial cell abundance in the clay of possible Holocene age (0 to ~50 mbsf) was very high (~10⁸ to 10⁹ cells/cm³), with a maximum value of 1.15 × 10⁹ cells/cm³ at 4.53 mbsf (Fig. F24). There was only a small decrease in numbers until ~35 mbsf, below which cell numbers decreased more steeply. The change at 35 mbsf does not appear to be related to any lithologic change, as this depth is well within the clay interval (Fig. F24). Interestingly, there was also no abrupt change in cell abundance between the organic-rich Holocene sequence and the organic-poor glacial clay at ~51 mbsf. Cell numbers reached a minimum of 4.16 × 10⁷ cells/cm³ at 68.15 mbsf. A small increase in cell numbers in the lower two samples of this hole was detected by both methods.

The cell count profiles have some resemblance to the salinity profile, which also shows an overall decrease with depth (Fig. F24). The small increase in salinity at the base of Hole M0059C replicates the increase in total cell numbers. The alkalinity profile, indicative of bacterial mineralization of organic matter, appears less related to the cell count profile.

Cell concentrations measured in Hole M0059C were extremely high, with all data values above the upper prediction limit of the global regression of prokaryotes cells with depth (Fig. F24). The maximum deviation from the global regression occurred at 20–30 mbsf, where cell numbers were ~40 times greater than the global regression.

Hole M0059E

Microbial cell numbers were enumerated at 24 sediment depths from samples taken for FCM (9 samples) and AODC (19 samples), both offshore and during the OSP (Table T12). Microbial cell abundance in the upper Holocene clay was very high (~10⁸ to >10⁹ cells/cm³), with a maximum value of 4.35 × 10⁹ cells/cm³ at 1.52 mbsf (Fig. F25). There was a hiatus in count data between 18 and 49 mbsf. Although samples were taken for this interval, they have proven difficult to count because of the clumped nature of the prokaryotic cells around organic particles. A different approach to sample processing will need to be developed for these samples before counting can be achieved. In order to check whether there was a distinct change in cell abundance from the Holocene sequence to the late glacial clay, detailed sampling was made across this lithologic boundary (~51 mbsf). A drop in cell numbers (Fig. F25) was observed below the transition from the Holocene organic-rich clay to the late glacial clay.

Similar to Hole M0059C, the cell count profile is similar to the salinity profile in this hole (Fig. F25). More importantly, the higher cell numbers in the Holocene sequence coincide with the zone of enhanced alkalinity production and, thus, of organic matter mineralization. The minimum cell count was 2.29 × 10⁷ cells/cm³ at 84.02 mbsf.

Similar to Hole M0059C, all cell abundances measured in this core were extremely high with almost all data values above the upper prediction limit of the global regression of prokaryotic cells with depth (Fig. F25). The maximum deviation occurred around 8 mbsf, where cell numbers based on cytometer counts were ~88 times greater than the global regression.

Cell counts were made by both AODC and FCM at the same sediment depths in 15 samples from Hole M0059C and 6 samples from Hole M0059E. Separate paired sample t-tests on these two data sets showed no significant difference between the two techniques (t = 1.633; degree of freedom [df] = 14 [not significant]; t = 2.031; df = 5 [not significant]) for Holes M0059C and M0059E, respectively. In Figure F26, FCM counts are plotted against AODC counts from the same depths for both holes combined. The calculated regression of this data set shows strong agreement between the two techniques, and a slope test showed no significant deviation from a slope of 1 (t = 0.0024; df = 19 [not significant]).

High throughput cell counting by FCM has previously been attempted using samples from IODP Expeditions 329 and 337. This application was not successful, however, because of the extremely low cell abundance in sediments from both these expeditions.

Figure F27 shows fluorescence microscopic images of cells from Hole M0059C (1.53 mbsf) stained by SYBR green I. Figure F27A shows the microscopic image without any treatment to detach cells or remove particles. Compared to no treatment, sonication detached cells from large particles of diatoms and sediments enabling improved cell visualization (Fig. F27B). Further treatment with hydrofluoric acid cleared the background by removing clay minerals and other fluorescing particles and facilitated cell counting (Fig. F27C).

In order to assess potential contamination during the coring operation, measurements of the microbial loading of drilling fluid were made in Hole M0059E (Table T13). Cell numbers in the drilling fluid varied little over time: a maximum of 1.11 × 10⁶ cells/mL when coring at 3 mbsf and a minimum of 5.14 × 10⁵ cells/mL when coring at 26 mbsf. Average cell numbers in the drilling fluid were 7 × 10⁵ cells/mL.

Perfluorocarbon tracer (PFC) for contamination testing was detected in the liner fluid and exteriors of all cores, indicating continuous PFC delivery into the borehole. An example is shown from Hole M0059E (Table T14). Liner fluid PFC concentrations at this site fluctuated over more than two orders of magnitude (Fig. F28A), indicating variations in the rates of PFC delivery and mixing into the drilling fluid stream. Generally, the measured PFC concentrations were considerably 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. F28B). No clear depth-related trends in contamination are apparent, except that the two uppermost cores (347-M0059C-1H and 2H) show the highest level of contamination of interior parts of all cores sampled at this site (Fig. F28C). Similarly, there was no clear trend in relation to lithology, which consisted of organic-rich black clays and varved green clays throughout the hole (Fig. F25). In addition to Cores 347-M0059C-1H and 2H, Core 13H shows a high level of contamination in the interior. Cores 1H, 2H, and 13H may each potentially have 10³–10⁴ contaminant cells/cm³ of sediment within their interiors (Fig. F28D). This, however, is still only ~0.001% of the indigenous community cell numbers (cf. Fig. F25).

Apart from these highly contaminated cores, numerous cores were potentially contaminated to a much smaller degree (~100 cells/cm³ or less) in the interior. These include Cores 347-M0059C-6H, 7H, 11H, 15H, 17H, and 21H. No contamination was detected in the interiors of Cores 4H, 5H, 8H, 10H, 19H, and 25H (Fig. F28D). Taken together, these results indicate that 12 of the 18 cores sampled for PFC show very moderate to no evidence of contamination with foreign microbes from drilling fluid.

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