METHODS
Sediment
Sampling and Preservation
Whole-round cores (10 cm)
were taken from hydraulic piston cores at depths picked on the basis of
estimated temperatures. No acetone was used to seal the end caps of the cut
cores. Whole-round cores were immediately processed inside an ethanol-sterilized
glove bag filled with argon. As the surface of the cut core was potentially
contaminated, a fresh face was exposed by removing the top 1 cm with a sterile
spatula. The 1 cm of core closest to the core liner was similarly avoided when
sampling. Subsamples were preserved for Acridine orange direct counts and lipid
analyses. Samples for Acridine orange direct counts were taken with sterile 5-
or 10-cm3 truncated syringes inserted 3-4 cm into the sediment.
Samples of 1-2 cm3 were sectioned with an ethanol-sterilized spatula
or razor blade into a clean scintillation vial containing 5 mL of sterile,
filtered 4% formalin in artificial seawater. The vials were shaken or vortexed
to disperse the sediment and stored at 4°C. Samples for lipid analysis were
taken similarly, but with a 60-cm3 truncated syringe; 30-40 cm3
were sectioned into a sterile 50-cm3 Falcon centrifuge tube and
immediately stored at -20°C. Aliquots of sediment were also removed for culture
of high-temperature, anaerobic microorganisms and DNA extraction.
Acridine
Orange Direct Counts
A 1-cm3 sample
of sediment was diluted in 10 mL of filter-sterilized (0.2 µm) 4% formalin in
artificial seawater (FFSW). This slurry was vortexed vigorously, 100 µL was
removed with a wide-bore pipet tip, diluted in 10 mL of FFSW with 0.02% Triton
X-100 (Sigma), and sonicated for 10 min at 1 mA. Samples were mixed thoroughly
before removing an aliquot for filtration; the volume was adjusted so that
50%-75% of the slide was covered by sediment (generally 0.2-0.4 mL). This
aliquot was diluted in 5-8 mL of FFSW to aid in dispersion and filtered onto
0.2-µm black-stained polycarbonate filters (Poretics).
A filter-sterile solution
of 0.1% Acridine orange (Sigma) in artificial seawater was overlain on the
filter for 10 min, and the filter was rinsed with 10 mL of FFSW. Some filters
were counterstained with the fluorochrome DAPI (4',6-diamidino-2-phenylindole)
(Sigma); a filter-sterile solution of 20 µg/mL DAPI in deionized water was
overlain on the Acridine orange-stained, rinsed filters for 1 min, and the
filters were given a final rinse with 1 mL of FFSW. Filters were removed from
the filtration unit under vacuum and allowed to air dry before mounting in
paraffin oil (Fluka).
Samples were examined
under blue excitation on a Zeiss epifluorescence microscope using a 100×
Neofluor lens with the optivar set at 1.25. Particles that fluoresced bright
green (or red orange, in some cases), were 0.2-1 µm in size, round or rod
shaped, and had well-defined edges were classed as bacteria for this study.
Random grids were counted until 200 bacteria were found or 500 grids had been
examined. Two slides were counted from each sample and, following the
recommendation of Cragg and Parkes (1994), a third was prepared and counted if
the calculated number of bacteria per cubic centimeter for the first two filters
varied by more than half an order of magnitude. Blanks were made and examined
with every batch of slides.
Lipid
Analyses
Frozen sediment samples
were extracted with the single-phase chloroform-methanol-buffer system of Bligh
and Dyer (1954), as modified by White et al. (1979a). Next, 37 mL of chloroform,
75 mL of methanol, and 30 mL of phosphate buffer (50 mM, pH 7.4) were added to
the sediment, mixed, and allowed to extract for 6 hr. The single-phase
extractant was separated from the solid material by centrifugation at 2000 rpm
for 20 min and decanting into a separatory funnel. A further 37 mL of chloroform
was used to wash the pelleted solids, which were then recentrifuged, and the
chloroform added to the extract. An additional 37.5 mL of water was added to the
extract to force the separation of the aqueous from the organic phase. After
separation for ~12 hr, the organic phase was drained into a round-bottomed flask
and the solvent removed with a rotary evaporator.
The total lipid extract
was fractionated into neutral lipids, glycolipids, and polar lipids by silicic
acid column chromatography (Guckert et al., 1985). Large Pasteur pipets (1 cm
diameter) partially blocked with a plug of glass wool were prepared, and 0.5 g
of silicic acid was added as a slurry in chloroform. The columns were pre-eluted
with 2 mL of chloroform, and the sample was transferred to the column with three
100-µL washes of chloroform. Neutral lipids were eluted with 5 mL of
chloroform, glycolipids with 5 mL of acetone, and polar lipids with 5 mL of
methanol. The solvent was removed from the polar lipids under a stream of dry
nitrogen at 37°C.
The polar lipids were
transesterified to the fatty acid methyl esters by a mild alkaline methanolysis
(Guckert et al., 1985). The polar lipid extract was dissolved in 1 mL of
chloroform/methanol (1:1), 1 mL of methanolic KOH was added, and the mixture was
heated at 37°C for 1 hr. Fatty acid methyl esters were recovered from the
organic fraction of the sample after adding 2 mL of hexane and 2 mL of water to
break phase.
A strong acid methanolysis
was performed on the glycolipid and polar lipid residue (Hedrick et al., 1992).
The lipid was dissolved in 1 mL of methanol:chloroform:concentrated hydrochloric
acid (10:1:1) and heated for 1 hr at 100°C. Next, 2 mL each of lipid-free water
and hexane:chloroform (4:1) were added to the cooled methanolyzate. The mixture
was thoroughly mixed and centrifuged, and the upper organic was layer
transferred.
The methanolyzate was
extracted twice more with 2 mL hexane:chloroform (4:1), and the solvent was
removed from the pooled organic extracts in a stream of dry N2 at
37°C. The samples were then derivatized to the tetramethyl silyl derivative
using O-bis(trimethylsilyl)trifluoroacetamide (Pierce) before analyzing on the
gas chromatograph-mass spectrometer.
The fatty acid methyl
esters were analyzed by capillary gas chromatography with flame ionization
detection on a Hewlett-Packard 5890 Series 2 chromatograph with a 50-m nonpolar
column (0.2-mm ID; 0.11-µm film thickness). The injector and detector were
maintained at 270°and 290°C, respectively. The column temperature was
programmed from 60°C for 2 min, ramped at 10°C/min to 150°C, and then ramped
to 312°C at 3°C/min. Preliminary peak identification was by comparison of
retention times with known standards. Detailed identification of peaks was by
gas chromatography-mass spectroscopy (GC-MS) of selected samples using a
Hewlett-Packard 5890 series 2 gas chromatograph interfaced to a Hewlett-Packard
5971 mass selective detector using the same column and temperature program
previously described. Mass spectra were determined by electron impact at 70 eV.
Methyl nonodecanoate was used as the internal standard, and the phospholipid
fatty acid (PLFA) was expressed as equivalent peak response to the internal
standard.
GC-MS analysis of the
ether-lipid cores was performed with high-temperature gas chromatography as
described by Nichols et al. (1993). Samples were analyzed by a Hewlett Packard
6890 gas chromatograph (GC) interfaced with a 5973 mass selective detector. The
GC was equipped with a 3 m × 0.25 mm ID, 5% phenylsiloxane fused silica column
and an on-column injector. The samples were injected at 90°C, and after 2 min
the GC oven temperature was programmed to 190°C at 30°C/min and then to 380°C
at 10°C/min. The final temperature was held for 15 min. Helium was used as the
carrier gas, and column head pressure was 3 psi.
Lipid
Nomenclature
Fatty acids were named
according to the convention X:Y
Z,
where "X" is the number of carbon atoms in the chain, "Y" is
the number of unsaturations, and "Z" is the number of carbon atoms
from the methyl end of the molecule to the first unsaturation encountered.
Prefixes are as follows: "i" = iso-branched (methyl branch on the
second carbon from the methyl end), "a" = anteiso-branched (methyl
branch on the third carbon from the methyl end), "10Me" = methyl
branch on the tenth carbon from the carboxylate end, and "cy" =
cyclopropyl. The suffixes "c" and "t" stand for the cis
and trans geometric isomers of the unsaturation, respectively. A subtle
ambiguity in this naming convention is that the number of carbons given for iso,
anteiso, and cyclopropyl fatty acids includes all of the carbons in the fatty
acid, but 10Me16:0 indicates a methyl group attached (17 carbons in the fatty
acid).
