Environmental Engineering Reference
In-Depth Information
370
◦
C) with in situ pumps. The GFF were extracted for 24 h in a Soxhlet
apparatus to obtain the total lipid extracts. Aliquots of the total extracts were
saponified after addition of an internal standard and separated into fatty acid
and neutral lipid fractions. The fatty acid fractions were methylated (BF
3
-
MeOH, Sigma) and the neutral fractions were derivatized (BSTFA, Sigma) and
analyzed by gas chromatography and gas chromatography-mass spectrometry
for the quantification and identification of lipids, respectively.
2.5 Fluorescence in Situ Hybridization (FISH) and Cell
Counts
Bacterial abundance was determined by epifluorescence microscopy (Zeiss
Axioscope 2, 1,000 magnification) of DAPI (4',6-diamidino-2-phenylindole)-
stained cells. Bacterial cells were fixed by the addition of concentrated formald-
ehyde solution (5 % final concentration) for 15 min at room temperature and
thereafter recovered by gentle vacuum filtration (20 and 50 ml for each sam-
ple) on to polycarbonate filters with a pore size of 0.2 µm (GTPB, Millipore).
After washing with PBS and water, the filters were transferred into sterile PP
petri dishes, sealed and stored frozen at -20
◦
C for FISH. The protocol of
Pernthaler et al. [33] was used for the hybridization procedure. The following
oligonucleotide probes (MWG, Germany) were used to describe the micro-
bial communities: Arch915 for members of the domain
Archaea
; Eel MS 932
(ANME-2 group); ANME-1, distantly related to
Methanosarcinales
[4]; and
MG84/705 and MA450, describing methanotroph groups I and II [9], respec-
tively. Probes were labeled with the indocarbocyanine fluorescent dye CY3 and
fluorescein (MWG, Germany).
3. RESULTS
3.1 Oxygen Profiles
The water column of the sampled stations clearly showed a chemocline
separating the water column in an oxic and anoxic zone. Figure 4 shows the
oxygen profiles of the investigated stations. At the NW station 7617 and at
the central station 7605 oxygen concentrations reached up to 220 µM, the SW
station 7623 had surface water oxygen concentrations below 67 µM. These low
concentrations may be caused by the Sakarya river inflow and its particulate
organic matter load at the sampling site. The central station 7605 showed
relatively stable values of 220 µM at the surface down to 55 m and then a very
fast decrease to values around zero below 100 m. Oxygen depletion was also
found below approximately 100 m at the SW station and below 140 m at the
NW station. Here, the transition between the oxic and the anoxic layer was not
as abrupt as observed at the other two stations. Stations 064 and 072 on the
