The primary microbiology objective for Leg 200 was to explore the composition of the microbial communities associated with deep marine sediments and the pore space as well as pore water fluids in the oceanic crust. To address these topics, a multiphasic approach comprising cultivation-based and culture-independent methods was conducted. For that purpose, sediment and rock samples were collected, and the presence of microorganisms was determined by fluorescent total cell count staining techniques (Live/Dead staining, deoxyribonucleic acid [DNA]-binding fluorochromes like 4´,6-diamidino-2´-phenylindole-dihydrochloride [DAPI], SYBR Green, and SYTO 62) and by fluorescent in situ hybridization (FISH) using domain- and group-specific oligonucleotide probes. The most probable number (MPN) was estimated for sulfate-reducing bacteria and fermentative bacteria. In addition, enrichment cultures of microorganisms putatively inhabiting these environments were established under aerobic and anaerobic conditions. Further samples were taken in order to extract the total DNA of the microbial communities for postcruise analysis of microbial diversity and to assess proxies of the microbial activity by the determination of different biomarkers including eubacterial phospholipid fatty acids and other lipids.
Whole-round samples were cut from the cores for microbiological investigations. The cores were cut and capped on the catwalk and immediately transported to the microbiology laboratory. The surface of the samples was removed with sterile spatulas and spoons, and subsamples ranging from 1 to 5 g were taken under sterile conditions.
Sediment samples (3-5 g) were taken from the inner part of the whole-round sample, suspended in 1x PBS (130-mM sodium chloride, 10-mM sodium phosphate buffer, pH 7.2 [Dulbecco's phosphate-buffered saline]) and fixed with 0.2-µm filtered formaldehyde solution (2% final concentration) for at least 4 hr at 4°C. Sediment samples were centrifuged at 5000 rpm for 20 min (Marathon 21K; Fisher Scientific), thoroughly washed twice with 1x PBS and stored in a 1:1 mixture of 1x PBS and 99.9% ethanol at 4°C. To determine the microbial abundance, fixed samples were stained with DAPI, SYBR Green, or SYTO 62. To determine the amount of living bacteria, aliquots of the samples (1 mL) were filtered onto black, 13-mm-diameter polycarbonate filters (0.2-µm pore size) in a filtration tower, mounted on microscope slides with a drop of nonfluorescent mounting oil, and examined using the Live/Dead staining kit and epifluorescence microscopy.
For shore-based scanning and transmission electron microscopy analyses (SEM and TEM, respectively), sediment samples were fixed with freshly prepared buffered glutaraldehyde solution (2% final concentration), washed twice with 1x PBS and stored in 1x PBS at 4°C.
Subsamples from the suspended sediments were plated onto solid media plates of different trophic levels and incubated under aerobic and anaerobic conditions, respectively. Enrichment cultures selective for sulfate-reducing, fermentative, and facultative anaerobic bacteria were established. The cultures were incubated at 4°C and room temperature. For further cultivation, 800-µL aliquots were preserved and frozen immediately after sampling at -76°C using the commercial Mikrobank system.
To study the distribution and phylogenetic affiliations from the sediment associated bacteria in more detail, FISH was performed by applying probes of different phylogenetic levels on the bacteria within the suspended formaldehyde-fixed sediments. Fixed samples were also transported to the University of Göttingen for embedding and analyzing thin sections of the sediments with the FISH technique.
Samples obtained from the native sediment as well as of the sediment suspensions were taken and frozen immediately at -76°C. These frozen samples were transported to the University of Göttingen for further postcruise DNA extraction and DNA-based analysis of the microbial diversity and in-depth phylogenetical characterization.
Subsamples of the sediments were frozen immediately at -76°C in sterile aluminum foil and were transported to the University of Göttingen for extraction and determination of different biomarkers including eubacterial phospholipid fatty acids and other lipids.
Whole-round core samples were collected on the catwalk from the end of an unsplit core liner or in the core splitting room immediately after the core liner was split, or a minicore was taken. Cores were handled with ethanol-treated latex gloves, and the surfaces of the cores were disinfected with isopropanol. The cores were subsequently crushed, and inner parts were ground under aseptic conditions and suspended in 1x PBS. The suspensions were used for the inoculation of the agar plates and enrichment cultures. Subsamples for DNA extraction were obtained as described below.
Small pieces of crushed rock (3-6 mm in diameter) as well as ground rock material were fixed with formaldehyde and glutaraldehyde solutions as described above. Samples were stained with different DNA staining dyes. To assess the inherent autofluorescence of the samples, untreated control rock samples were investigated.
For the establishment of enrichment cultures as described above, small pieces obtained from the inner parts of crushed rocks as well as suspended ground material were used as inocula. Suspended ground material was additionally plated onto the agar plates.
Whole-rock pieces split from the interior part of the cores were fixed as described above and stored for further embedding and thin sectioning at the University of Göttingen. FISH will also be performed on thin sections of the basaltic rock samples.
For shore-based SEM and TEM analyses, samples were fixed with glutaraldehyde as described above. Thin sections will be made at the University of Göttingen.
Whole-rock pieces split from the interior part of the cores were frozen at -76°C in sterile aluminum foil immediately after sampling for shore-based DNA extraction and analysis.
Large pieces of crushed rocks were frozen at -76°C in sterile aluminum foil immediately after sampling and were transported to the University of Göttingen for the investigation of different biomarkers.