Bacteria play a dominant role in the degradation of organic matter within sediments and, as a consequence, drive chemical changes and early diagenesis. The existence of a deep bacterial biosphere in marine sediments has only recently been established (Parkes et al., 1994), but already the activity of bacteria in depths to 750 mbsf and their direct involvement in geochemical changes have been demonstrated.

Recent research (Wellsbury et al., 1997) has shown that temperature increases during burial can result in organic matter becoming easier to degrade by bacteria, and that bacterial populations and their activity can increase in deeper layers below 100 m. Increasing organic matter bioavailability was reflected in increases up to a thousandfold in substrates for bacterial activity (volatile fatty acids, particularly acetate) in deep sediments (Leg 164; Paull, Matsumoto, Wallace, et al., 1996b). Thus, bacterial populations should exist at much greater depths and may even increase with depth rather than decrease as the energy sources "improve" with increasing depth and temperature.

This work aims to determine the bacterial mechanisms involved in, and the impact these have on, the modification of deeply buried organic matter. Leg 180 also provided the deepest samples yet collected for bacterial analysis.

Two types of samples were taken for microbiological analysis:

1. Sediment in 1-cm³ samples was taken for direct microscopic determination of bacterial numbers. The samples were taken from the end of selected 1.5-m core sections immediately after the sections were cut on the catwalk and before the sections were sealed with an end cap. Potentially contaminated sediment was first removed using a sterile scalpel. Then, using a sterile 5-cm³ plastic syringe with the luer end removed, a 1-cm³ minicore sample was taken and sealed with a sterile suba-seal stopper. In a clean area of the laboratory, the 1-cm³ sample was extruded into a sterile serum vial containing 9 mL of filter-sterilized (0.2 µm) 4% formaldehyde in artificial seawater. The vial was crimped and shaken vigorously to disperse the sediment particles and then stored at 4ºC.
2. Whole-round core samples were taken and stored intact for shore-based laboratory analysis. These were cut from 1.5-m core sections on which the end caps had not been sealed with acetone. The core sections were removed from the catwalk and brought into the core reception area, where they were cleaned, wiped with ethanol, and placed into a sterile cutting rig (Cragg et al., 1992) flushed constantly with sterile oxygen-free nitrogen (OFN) to maintain anoxia. Sterile whole-round core samples were taken adjacent to the whole-round cores for IW sampling and organic geochemical analysis. One end of the whole-round core was cut with a sterile hacksaw blade, removed from the rig, and immediately capped with a sterile core end cap while being flushed with sterile OFN from a gassing jet. The core end cap was then taped to the liner. The cutting rig was cleaned, alcohol washed, and heat sterilized, and the process was repeated with the other end of the whole-round core. Once fitted with end caps, cores were sealed in a gas-tight laminated plastic/aluminum bag containing a chemical oxygen scrubber (Anaerocult A, Merck-BDH) to produce anaerobic conditions. Sealed, anaerobic core sections were stored at 4ºC before analysis onshore.

Total bacterial numbers and numbers of dividing and divided cells were determined using acridine orange as a fluorochrome dye with epifluorescence microscopy (Fry, 1988). Fixed samples were mixed thoroughly, and a 5- to 10-µL subsample was added to 10 mL of 2% (v/v) filter-sterilized (0.2 µm) formaldehyde in artificial seawater containing 2% (v/v) acetic acid to dissolve excess carbonate. Acridine orange (50 µL of a 5-g·L^-1 filter-sterilized (0.1 µm) stock solution) was added and the sample was incubated for 3 min. Stained cells and sediment were trapped on a 0.2-µm black polycarbonate membrane (Costar, High Wycombe, United Kingdom). Excess dye was removed from the membrane by rinsing with an additional 10 mL of 2% (v/v) filter-sterilized formaldehyde in artificial seawater containing 2% (v/v) acetic acid, and the membrane was mounted for microscopic analysis in a minimum of paraffin oil under a coverslip. At least three membranes were prepared for each sample: where 95% confidence limits of the mean count exceeded 0.5 log10 units, further replicate filters were prepared. A minimum of 200 fields of view were counted.

The mounted membrane filters were viewed under incident illumination with a Zeiss Axioskop microscope fitted with a 50-W mercury vapor lamp, a wide-band interference filter set for blue excitation, a 100× (numerical aperture = 1.3) Plan Neofluar objective lens, and 10× eyepieces. Bacterially shaped fluorescing objects were enumerated, with the numbers of cells on particles doubled in the final calculations to account for masking. Dividing cells (those with a clear invagination) and divided cells (pairs of cells of identical morphology) were also counted. The detection limit for bacterial cells is ~1 × 10⁵ cells/cm³ (Cragg, 1994).