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Applied and Environmental Microbiology, June 2000, p. 2589-2598, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Functional Exoenzymes as Indicators of Metabolically Active
Bacteria in 124,000-Year-Old Sapropel Layers of the Eastern
Mediterranean Sea
Marco J. L.
Coolen and
Jörg
Overmann*
Paleomicrobiology Group, Institute for the
Chemistry and Biology of the Marine Environment, University of
Oldenburg, D-26111 Oldenburg, Germany
Received 18 January 2000/Accepted 28 March 2000
 |
ABSTRACT |
Hydrolytic exoenzymes as indicators of metabolically active
bacteria were investigated in four consecutive sapropel layers collected from bathyal sediments of the eastern Mediterranean Sea. For
comparison, the organic carbon-poor layers between the sapropels,
sediment from the anoxic Urania basin, and sediments of intertidal mud
flats of the German Wadden Sea were also analyzed. The sapropel layers
contained up to 1.5 · 108 bacterial cells
cm
3, whereas cell numbers in the intermediate layers were
lower by a factor of 10. In sapropels, the determination
of exoenzyme activity with fluorescently labeled substrate analogues
was impaired by the strong adsorption of up to 97% of the
enzymatically liberated fluorophores (4-methylumbelliferone [MUF] and
7-amino-4-methylcoumarin [MCA]) to the sediment particles. Because
all established methods for the extraction of adsorbed fluorophores
proved to be inadequate for sapropel sediments, we introduce a
correction method which is based on the measurement of equilibrium
adsorption isotherms for both compounds. Using this new approach, high
activities of aminopeptidase and alkaline phosphatase were detected
even in a 124,000-year-old sapropel layer, whereas the
activity of
-glucosidase was low in all layers. So far, it had been
assumed that the organic matter which constitutes the sapropels is
highly refractory. The high potential activities of bacterial
exoenzymes indicate that bacteria in Mediterranean sapropels are
metabolically active and utilize part of the subfossil kerogen. Since a
high adsorption capacity was determined not only for the
low-molecular-weight compounds MUF and MCA but also for DNA, the
extraordinarily strong adsorption of structurally different substrates
to the sapropel matrix appears to be the major reason for the long-term
preservation of biodegradable carbon in this environment.
 |
INTRODUCTION |
Deep-sea sediments of the eastern
Mediterranean Sea are characterized by the cyclic occurrence of dark,
more than 1-cm-thick sediment layers with high organic carbon contents
(>2% [wt/wt] total organic carbon [TOC]) (32). These
so-called sapropel layers are embedded in light-gray to brown
hemipelagic carbonate oozes which are poor in TOC (<0.5% [wt/wt]).
Sapropel formation has been related to increased influxes of
freshwater, which in turn was caused by changes in the climate of the
northern hemisphere (28, 38). The resulting isolation and
subsequent anoxia of deep Mediterranean bottom water (49, 54, 55,
57) and/or increased marine primary production (16,
17) may have caused the enhanced organic carbon preservation in
the sapropels.
The total numbers of bacterial cells in sapropel layers are
significantly enhanced compared to those in neighboring carbon-lean sediment layers. Dividing and divided bacterial cells have been detected even in 4.7-million-year-old sapropels and make up 10% of
total cell numbers (23). Furthermore, porewater sulfate is enriched in 34S with respect to modern Mediterranean
seawater (14). These observations indicate the presence of
metabolically active bacteria in the sapropel layers. Physiologically
active bacterial communities have been detected in diverse subsurface
environments, e.g., >750 m below the sea floor in marine sediments
(5, 48), in continental aquifer sediments (33,
36), in 230-million-year-old sandstone (62), and in
continental petroleum reservoirs (37). Deep crystalline rock
aquifers contain only traces of organic matter and harbor lithoautotrophic communities in which autotrophic methanogenesis predominates (60). Sulfate reduction has been demonstrated
in samples from deep marine sediments (48) and deep
subsurface sandstone (36). Sulfate-reducing bacteria
have been isolated from deep marine sediments (5); a
wider variety of physiological types was isolated from some continental
deep subsurface environments (4). However, little is known
about the physiological activity of the bacteria in situ and the
origins of their substrates (12, 36). This is especially
true for eastern-Mediterranean sapropels.
Sapropels differ from many other subsurface environments in that they
contain high concentrations of TOC (up to 30.5% [dry weight])
(23). The organic matter in the sapropels is up to 5.3 million years old (25) and consists mainly of dark-brown amorphous kerogen (1). The estimated burial efficiencies for sapropel organic matter are high, ranging between 20 and 80% (50, 51). Isotopic and geochemical tracers indicate that this organic matter is of marine origin and was predominantly formed by
prymnesiophytan (coccolithophoridan) and eustigmatophycean algae
and
in some cases
diatoms (15, 25). Apart from the
indirect evidence for postburial sulfate reduction, nothing is known of
the degradation potential and the actual physiological state of natural
bacterial communities in the subfossil sapropels.
A sensitive method for the detection of active microbial communities in
natural environments is the analysis of exoenzyme activities using
fluorescently labeled substrate analogues. From such measurements, the
affinity and potential rate of extracellular hydrolysis of biopolymers
can be inferred. Some exoenzymes, like alkaline phosphatase and
-glucosidase, are subject to substrate induction and catabolite
repression (20). Therefore short-term measurements of the
cell-specific hydrolysis rates of these exoenzymes also provide
information on the actual availability of bacterial carbon substrates
(20, 21, 22, 45). The present communication describes a
method for measurement of exoenzyme activities in sapropels. The
activities detected in the 124,000-year-old sapropels provide
conclusive evidence for the presence of active bacteria in the
subfossil sapropel layers.
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MATERIALS AND METHODS |
Sampling and sample preparation.
Gravity cores (average
length, 5 m) were obtained between January 22 and 30, 1998, at
three locations in the eastern Mediterranean during the R/V
Meteor cruise, leg 40/4 (Fig.
1A and Table
1). The core obtained at station 66 contained two sapropels (S1 and S3); this sapropel material was used
for method development. The second core, from station 69, contained
four different sapropels (S1, S3, S4, and S5 [according to the
numbering system in reference 57). From the latter
core, the top of the sediment (Z0; 4 to 6 cm below the surface), each
sapropel layer (S1, 25 to 27 cm; S3, 281 to 283 cm; S4, 337 to 339 cm;
and S5, 387 to 389 cm below the surface), and three intermediate
hemipelagic layers between the sapropels (denoted Z1, 89 to 91 cm; Z3,
304 to 306 cm; and Z4, 356 to 358 cm below the surface) were sampled. A
third core was obtained with a multicorer in the hypersaline Urania
basin, station 76, located between the crest of the Mediterranean Ridge and the Matapan Trench (40). The top 4 cm of surface
sediment was used for measurements.

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FIG. 1.
Location of sampling sites in the eastern Mediterranean
(A) and in the German Wadden Sea (B). The asterisks denote sampling
points. Station numbers in the eastern Mediterranean are given in
italics.
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Each core was cut longitudinally, which left behind a potentially
contaminated surface. In order to collect samples aseptically,
the
surface was covered first with Saran Wrap and then by a layer
of
powdered dry ice. After 5 min of incubation, the underlying
0.5-cm-thick portion of the sediment was frozen and could easily
be
lifted off with a sterile scalpel, leaving behind an uncontaminated
area. Through this area, 5-cm
3 subsamples were retrieved
with sterile plastic syringes which
had their ends cut off. A total of
30 cm
3 of each layer was aseptically transferred into a
sterile 100-ml
serum flask, and 30 ml of filtered (0.2-µm pore size;
Sartorius,
Göttingen, Germany), autoclaved synthetic seawater was
added.
The synthetic seawater contained (per liter) NaCl, 24.3 g;
MgCl
2 · 6H
2O, 10 g;
CaCl
2 · 2H
2O, 1.5 g; KCl, 0.66 g; Na
2SO
4, 4 g; KBr,
0.1 g;
H
3BO
3, 25 mg; SrCl
2 · 6H
2O, 40 mg; NH
4Cl, 21 mg;
KH
2PO
4,
5.4 mg; NaF, 3 mg; trace element
solution SL12 (
47), 1 ml; and
selenite-tungstate solution
(
64), 1 ml. The pH was maintained
at 7.3 by buffering the
seawater with HEPES (10
mM).
The headspace of each slurry was immediately flushed with
N
2 for 3 min in order to create an anoxic atmosphere. The
samples
were stored at 4°C until the measurements of exoenzyme
activities,
which were performed on board within 48 h after
sampling.
Cores from intertidal mudflats of the German Wadden Sea in Jade Bay
near Dangast (53°27'N, 8°07'E) and Schillig (53°42'N,
8°01'E)
(Fig.
1B and Table
1) were obtained in November 1997.
The cores were
sampled with 25-cm-long, 4.5-cm-diameter plexiglass
tubes. The silty
Dangast sediment was homogenous and black. The
sediment from Schillig
consisted of a light-brown 5-cm-thick sandy
top layer overlying a gray
silty bottom sediment. After transport
to the laboratory, subsamples of
each type of sediment were taken
aseptically, and sediment slurries
were prepared using the methods
described
above.
Development of a method for the determination of exoenzyme
activities in sapropels.
Because we observed a strong adsorption
of free fluorophores, we initially determined the time required to
reach adsorption equilibrium for the two fluorophores
4-methylumbelliferone (MUF) and 7-amino-4-methylcoumarin (MCA). A
series of slurries was prepared for each sediment type (sapropels,
intermediate layers, and intertidal sediments), and MUF or MCA was
added to a final concentration of 37 or 7.5 µM, respectively. At
regular intervals between 5 and 480 min after the addition, a subset of
three samples was centrifuged (5 min at 10,000 × g),
and the supernatants were transferred to fresh Eppendorf tubes. The pHs
of samples containing MUF were increased to 11 by the addition of NaOH
(final concentration, 40 mM). Precipitation of carbonates was prevented
by the addition of Na4EDTA (1.7 M; final concentration, 0.1 M). Initial experiments demonstrated that EDTA did not affect the
fluorescence intensities of the samples. The concentrations of free
fluorophores in the supernatant were determined by fluorometry (RF1501
spectrofluorometer; Shimadzu, Duisburg, Germany) at an excitation
wavelength of 360 nm and an emission wavelength of 450 nm. For
calibration, MUF and MCA standards in artificial seawater at
concentrations of 10 to 2,500 nM were used. The amount of adsorbed
fluorophore was calculated as the difference between the total amount
added and the amount remaining in the supernatant.
At equilibrium, the amount of a substance adsorbed {
S; in
nanomoles · (gram [dry weight])
1} depends on
the concentration of the substance remaining in solution
(
Ce; in nanomoles milliliter
1)
according to the Freundlich equation (
44):
|
(1)
|
Here,
K is the affinity coefficient {in
milliliters · (gram [dry weight])
1} and
n is a dimensionless exponent.
S was calculated
from the
total concentration of fluorophore added,
CT (in nanomoles milliliter
1), and
the dry weight content of the slurry,
DSlurry
(in grams
[dry weight] · milliliter
1).
|
(2)
|
Equilibrium adsorption isotherms were determined by adding five
different concentrations of MUF or MCA to a set of slurry
aliquots of
the different sediment types. After an incubation
time of 8 h, the
concentrations of free fluorophores were measured
in the supernatant,
and the amount of fluorophores adsorbed per
gram (dry weight) of
sediment was calculated. Equation
1 was then
fitted to the data points
using the nonlinear-regression tool
of SigmaPlot 5.0 (Jandel
Scientific, Erkrath, Germany), which
yielded the two parameters
K and
n. For comparative purposes,
we also
measured the equilibrium adsorption isotherms of MUF and
MCA with
montmorillonite and cellulose as
adsorbers.
The total concentration of fluorophore,
CT,
liberated during the exoenzyme assays (see below) could be calculated
from the
equilibrium concentrations (
Ce) of MUF
or MCA in the supernatants
of the sediment slurries and from the values
of the parameters
K,
n, and
DSlurry determined for each type of sediment:
|
(3)
|
Comparison with established methods.
Our new method for the
quantification of exoenzyme activities was compared with established
methods, which rely on a quantitative desorption of the bound
fluorochromes from the sediment matrix (6, 13). Duplicate
samples of sediment slurries were first incubated for 8 h with MUF
(final concentration, 10 µM) in order to load the sediment with
fluorochromes. A third aliquot was incubated without MUF as a control
for autofluorescence. In one series of experiments, 0.5 ml of sterile
synthetic seawater and EDTA (final concentration, 0.1 M; in order to
prevent a subsequent precipitation of carbonates) was added to the
slurries. For desorption, NaOH was added to a final concentration of 90 mM, and the samples were stirred for 20 min. Subsamples (1.5 ml) were
centrifuged (5 min; 10,000 × g) in a microcentrifuge, and
the concentration of liberated MUF was measured fluorophotometrically
as described above. In a second series of desorption experiments, 0.5 ml of synthetic seawater and ethanol (final concentration, 80%
[vol/vol]) was added to the slurries after they were loaded with MUF.
Following centrifugation, EDTA (final concentration, 0.1 M) and NaOH
(final concentration, 0.04 N) were added to the supernatants, and the fluorescence was measured. A third series of sediment slurries was
incubated for 8 h with MCA at a final concentration of 8 µM, again leaving a parallel devoid of MCA as a control for
autofluorescence. After incubation, 0.5 ml of synthetic seawater and
acetone (final concentration, 10% [vol/vol]) was added, the samples
were centrifuged, and the fluorescence in the supernatant was determined.
Michaelis-Menten kinetics of exoenzymes in natural
sediments.
Our newly established correction method was used to
determine the Michaelis-Menten kinetics of the hydrolytic exoenzymes
alkaline phosphatase (EC 3.1.3.1),
-glucosidase (EC 3.1.21), and
leucine aminopeptidase (EC 3.4.1.1). Alkaline phosphatase and
-glucosidase were assayed with MUF-phosphate and
MUF-
-D-glucoside (Sigma, Deisenhofen, Germany).
Aminopeptidase activity was measured with MCA-labeled leucine (Sigma).
Since the hydrolytic activities of aminopeptidase and
-glucosidase
in up to 4,920-m-deep sediments have been demonstrated to be
independent of hydrostatic pressure (52), we did not use in
situ pressure during the incubation of exoenzyme assays.
Duplicate 1.4-ml aliquots of the sediment slurry were transferred to
autoclaved 10-ml serum vials containing stirring bars.
The enzymatic
reaction was started by the addition of 75 µl of
substrate analogue
solution to yield final concentrations of 5,
12, 24, 60, and 120 µM.
Each vial was sealed with a sterile butyl
rubber stopper and flushed
with N
2 for 1 min. During incubation,
all slurries were
stirred at 225 rpm and incubated at a temperature
of 15°C (the in
situ temperature). Sapropel sediments and hemipelagic
samples of cores
69 and 76 were incubated for 8 h. Samples from
intertidal mudflat
sediments had to be incubated for only 30 min
(alkaline phosphatase),
140 min (

-glucosidase), and 85 min (aminopeptidase)
due to the
significantly higher enzyme
activities.
For each concentration, three different blanks (B
1,
B
2, and B
3) were incubated in parallel. One
blank (B
1), used to assess
nonenzymatic hydrolytic cleavage
of the substrate analogues, was
boiled for 30 min prior to incubation
in order to inhibit the
enzyme. This method of inactivation was chosen
because of the
adsorptive capacities of the sapropels. Mercuric
chloride fails
to block exoenzyme activity in sediments or in aquatic
samples
containing particulate material (
6,
19). Similarly,
various
detergents, denaturing agents, and organic solvents do not
completely
inhibit aminopeptidase activity (
6). However,
boiling sediment
slurries prior to the addition of substrate represents
a reliable
control for abiotic cleavage of fluorogenic substrates in
various
samples (
6,
30,
34). In order to correct for the
fluorescent
compounds released from the sediments during boiling, a
second
blank (B
2) was incubated without substrate
analogues. The fluorescence
caused by the compounds extracted from the
sediments without boiling
was determined in a third blank
(B
3) which was also incubated
without substrate analogues.
After incubation, the slurries were
transferred to microcentrifuge
tubes and centrifuged for 5 min
at 10,000 ×
g, and the
concentrations of free dissolved fluorophores
were determined
fluorometrically as detailed above. Using this
new approach, the
detection limit for the concentration of free
fluorophores was 0.024 nmol · ml
1 for the intermediate layers and 0.12 nmol · ml
1 for the sapropel layers as a result of
the higher autofluorescence
in these
samples.
The resulting Michaelis-Menten plot was linearized by the formula of
Wright and Hobbie (
65), and the maximum enzyme activity,
Vmax (in nanomoles centimeter
3
sediment hour
1), and
Km + Sn (the sum of the half saturation constant plus the
concentration of natural substrate [in micromolar units]) were
estimated:
|
(4)
|
Here,
tinc is the incubation time (in
hours) and
Ahydrol is the concentration of
substrate analogue enzymatically hydrolyzed
during the incubation;
A denotes the total concentration of substrate
analogue
added.
Vertical distribution of exoenzyme activities.
Because of
the limited amount of sapropel material which was available from core
69, only exoenzyme activities at saturating substrate concentrations
were determined for the vertical series of sapropel layers. Based on
the Michaelis-Menten kinetics determined in sapropel S3 of core 66 and
in the Dangast and Schillig sediments (see Table 4), a final
concentration of 250 µM was chosen for the substrate analogues. For
each sample, measurements were performed in duplicate and included the
three blanks (B1, B2, and B3; see above). The rates of enzymatic hydrolysis were calculated from the
equilibrium concentrations of the free fluorophores and from the
adsorption isotherms for MUF and MCA in the respective sediment material (equation 3).
Acridine orange direct cell count.
For staining of sediment
samples, screw-cap glass tubes (22 ml) containing one glass bead each
were heat sterilized at 160°C for 4 h. Sediment slurries (1:10
dilution) were prepared with sterile filtered (0.1-µm pore size;
Sartorius) artificial sea water, and 5 µl of the slurries was added
to 9.9 ml of a sterile filtered (0.1-µm pore size) formaldehyde
solution (4% [vol/vol] in artificial seawater). After the mixture
was vortexed for 1 min, 0.1 ml of sterile filtered (0.1-µm pore size)
acridine orange staining solution (200 µg · ml
1)
was added. Staining proceeded for 10 min in the dark, after which the
entire sample was filtered with a black polycarbonate filter (0.2-µm
pore size; Millipore, Eschborn, Germany). Bacterial cells were counted
by epifluorescence microscopy (Zeiss Axiolab, Jena, Germany) at a
magnification of ×1,000. As a contamination control, sterile filtered
and autoclaved artificial seawater was used instead of sediment slurries.
Most probable numbers of chemoorganotrophic bacteria.
Plate
counts of aerobic chemoorganoheterotrophic bacteria were performed on
CPSm agar. This medium contains (per liter of artificial seawater [see
above]) casein peptone, 0.5 g; Bacto Peptone, 0.5 g; soluble
starch, 0.5 g; glycerol, 1 ml; SL12, 1 ml; selenite-tungstate
solution, 1 ml; and washed agar, 15 g. The pH was set to 7.4.
The most probable numbers of anaerobic chemotrophic bacteria were
determined in liquid dilution series which were set up in
polystyrene
microtiter plates. All manipulations were carried
out within an
anaerobic chamber on board the research vessel.
The medium MM consisted
of artificial seawater containing a suite
of carbon substrates
(glucose, cellobiose,
N-acetyl glucosamine,
glycerol,
pyruvate, 2-oxoglutarate, succinate, fumarate, formate,
acetate,
propionate, butyrate, ethanol, mannitol, salicylate,
dimethyl sulfide,
alanine, arginine, asparagine, isoleucine, cysteine,
tyrosine, and
glutamine; final concentrations, 200 µM each), as
well as soluble
starch, 0.1% (wt/vol); Tween 80, 0.01% (wt/vol);
SL10
(
64), 1 ml · liter
1; selenite-tungstate
solution, 1 ml · liter
1; and 10-vitamin solution
(
3), 10 ml · liter
1. The liquid medium
was reduced by the addition of sulfide to
a final concentration of 400 µM, and the pH was set to 7.4. After
inoculation, the microtiter
plates were transferred to incubation
bags containing an oxygen removal
and CO
2 generation system (Anaerocult
C mini; Merck,
Darmstadt, Germany). The microtiter plates were
incubated at a
temperature of 20°C for 3
months.
After incubation, each well was monitored for bacterial growth.
Subsamples (20 µl) were transferred to a 96-well dot blot
apparatus
containing one sheet of black polycarbonate filter membrane
(0.2-µm
pore size; Millipore). To each well, 180 µl of sterile
filtered
(0.1-µm pore size) autoclaved water and 10 µl of sterile
filtered
acridine orange staining solution were added. After 10
min of
incubation in the dark, the acridine orange-stained microtiter
plate
samples were blotted onto the polycarbonate membrane, and
each dot was
screened for the presence of bacterial cells by epifluorescence
microscopy at a magnification of ×1,000.
TOC in sediments.
For carbon analyses, all sediment samples
were ground in a bead mill. Total carbon was measured with an
elementary analyzer (SC444; Leco, Kirchheim, Germany), inorganic carbon
was measured with a CO2-coulometer (CM 5012; UIC
Coulometrics, Joliet, Ill.), and organic carbon in the sediments was
calculated as the difference. The water content was determined by
weighing each type of sediment before and after drying it for 24 h
at a temperature of 70°C, after which the sediments had reached a
constant weight.
 |
RESULTS |
Adsorption kinetics.
Initially, we determined the time course
for the adsorption of MUF and MCA to the different types of sediments.
The adsorption of both fluorophores reached saturation as early as
after 1 h of incubation (Fig. 2).
After this time interval, 95.4% of the MUF and 97.1% of the MCA which
had been added to the sapropel sediment slurries had disappeared from
the dissolved phase. In contrast, only 52% of the MUF and 59% of the
MCA adsorbed to Dangast sediments (Fig. 2). Since the measurement of
exoenzyme activities in sapropels requires an incubation time of 8 h (see Materials and Methods), the major fraction of the liberated
fluorophores will rapidly adsorb to the sapropel matrix concomitant to
their enzymatic liberation from the substrate analogues and thus will not be detectable at the end of the experiments.

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FIG. 2.
Time course of fluorophore adsorption in slurries of
sapropel S3 of core 66 ( and ) and of Dangast sediment ( and
). For both sediments, the adsorption kinetics of MUF ( and ;
final concentration, 36 µM) and of MCA ( and ; final
concentration, 7.5 µM) are shown. The error bars indicate standard
deviations.
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Equilibrium adsorption isotherms of fluorophores.
For each
type of sediment, MUF or MCA was added at five different concentrations
and the equilibrium concentrations in solution were determined (Fig.
3). The adsorption of fluorochromes to
sediments from Dangast and Schillig and to the carbonaceous
intermediate sediment layers from the eastern Mediterranean was
significantly lower than the absorption to the sapropels (compare Fig.
3 and 4). The equilibrium adsorption
isotherms of fluorophores for sapropel S3 of core 66 followed the
Freundlich adsorption model (equation 1) rather than Langmuir
adsorption kinetics (44). The adsorptive affinity,
K, of MUF to sapropel S3 was (69.5 ± 7.9) ml · g
1 and thus was comparable to that of pure
montmorillonite (Table 2). However, the
affinity of sapropel material for MCA (37.4 ± 3.3 ml · g
1) was lower than that for MUF.

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FIG. 3.
Concentrations of dissolved fluorophores in sediment
slurries of sapropel S3 ( and ), Dangast sediment ( and ),
and Schillig surface sediment ( and ) after 8 h of
incubation. The solid symbols represent adsorption of MUF, and the open
symbols represent that of MCA. The error bars indicate standard
deviations. The concentrations of free fluorophores expected in the
absence of adsorption are indicated by the dotted line.
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FIG. 4.
Equilibrium adsorption isotherms of MUF (A) and MCA (B)
for each type of sediment fitted to the Freundlich equation (lines).
Sapropel S3, core 66 ( and ); Dangast sediment ( and ),
Schillig silty bottom sediment ( and ), Schillig sandy top
sediment ( and ), and the top layer, Z0, of core 66 ( and )
are shown. The error bars indicate standard deviations.
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Desorption experiments.
MUF which has been adsorbed to
estuarine sediments has been shown to be completely extracted with 90 mM NaOH. As an alternative extraction method, 80% ethanol has been
used successfully to recover MUF adsorbed to a freshwater sediment rich
in organic carbon (13). Adsorbed MCA can be desorbed with
10% acetone (6). For comparison with our newly established
method, we assessed the efficiencies of the three extraction methods
described above with slurries of sapropel S3 and with intertidal
sediments from Dangast.
Treatment with 90 mM NaOH resulted in a complete extraction of MUF from
the particulates in Dangast sediment, but only 34%
of the MUF was
desorbed from the sapropel sample (Table
3). Treatment
with 80% ethanol increased
the recovery of MUF, but the desorption
was unsatisfactory (54%).
Similarly, the efficiency of extraction
of MCA with diluted
acetone was high (83%) for Dangast sediment.
However, this method was
not suitable for sapropel slurries, from
which only 16% of the MCA
could be liberated. A second drawback
of the extraction method
was the large amounts of autofluorescent
compounds which
were concomitantly extracted from the sapropel
matrix. This
autofluorescence interferes with the fluorometric
quantification of MUF
or MCA.
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TABLE 3.
Efficiency of established extraction methods in
liberating adsorbed fluorophores from two types
of sedimentsa
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Obviously, the efficiency of extraction by NaOH, ethanol, or acetone
depends critically on the composition of the sediment,
and an
alternative method is required for the quantification of
exoenzyme
activities in sediments containing large amounts of
kerogen, like the
sapropels from the eastern Mediterranean. For
this reason we
established and applied a correction procedure
based on equilibrium
adsorption isotherms for the two
fluorochromes.
Michaelis-Menten kinetics of the three exoenzymes.
Because of
the limited amount of core material available, the saturation
characteristics of only one enzyme, alkaline phosphatase, could be
determined for sapropel material (Fig.
5). For the other two exoenzymes, the
maximum rates were measured. Alkaline phosphatase in sapropel S3
exhibited a Km + Sn of 27.5 µM, which is in the same range as the values determined for the other
sediments (Table 4).

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FIG. 5.
Michaelis-Menten kinetics of alkaline phosphatase
activity measured in sapropel S3 of core 66. The error bars indicate
standard deviations.
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TABLE 4.
Estimate of the half saturation constant,
Km + Sn, and of the
maximum velocity, Vmax, of the three
hydrolytic enzymes determined for the different sediment types
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Vertical distribution of exoenzyme activities.
In core 69, the
highest values for alkaline phosphatase were detected in the youngest
sapropel, S1 (6.55 ± 0.19 nmol cm of sediment
3
h
1) (Fig. 6), but
activities were only threefold lower in the oldest sapropels. The
activity of alkaline phosphatase was significantly higher within the
sapropels than in the intermediate hemipelagic layers. In the anoxic
surface sediment of the Urania basin, alkaline phosphatase activity was
lower by a factor of 2 to 5 than that in the sapropels, while the
activities in Dangast and Schillig sediments were 60 and 30 times
higher.

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|
FIG. 6.
Comparison of TOC levels (A), total cell
numbers (B), and exoenzyme activities of alkaline phosphatase (C),
-glucosidase (D), and aminopeptidase (E) for each type of sediment.
The error bars indicate standard deviations.
|
|
Of the three exoenzyme activities,

-glucosidase exhibited by far the
lowest activities in the sapropels and did not exceed
0.034 nmol cm of
sediment
3 h
1 (Fig.
6 and Table
5). The

-glucosidase activity in the
Urania
sediment was three times higher than those in the sapropels and
intermediate layers of the core from station M40/4-69-2SL. The
two
intertidal sediments exhibited

-glucosidase activities exceeding
those in the upper sapropel sediments by a factor of 490 to 670
(Fig.
6
and Table
5). With a detection limit for free fluorophores
of 0.01 µM
MUF, the minimum detectable

-glucosidase activity
after correction
for the adsorption and for the backround fluorescence
of the sapropel
sediments was 0.015 nmol h
1 cm
3. At this
detection limit, no significant

-glucosidase activity
could be
determined in the older sapropel layers, S4 and S5.
The highest aminopeptidase activity was measured in the uppermost
sediment layer (Z0) of core 69. In the intermediate layers
below, the
activity of this exoenzyme declined rapidly from 2.52
to 0.022 nmol cm
3 h
1. In contrast, the
aminopeptidase activity remained almost constant
in sapropels
S1 through S5. Values in the Urania basin sediment
were significantly
lower than those in the sapropel layers, whereas
the activities in the
intertidal sediments of Schillig exceeded
those in the sapropels by 25 and 66 times (Fig.
6 and Table
5).
TOC and total and culturable bacterial cell numbers.
The
sapropels investigated in the present study had TOC contents between
2.3 and 8.5% (Fig. 6A). The TOC content of surface sediment from
Dangast was comparable to that of sapropel S1. In contrast, the
intermediate hemipelagic layers of the Mediterranean sediment core 69, as well as the surface sediments obtained from the Urania basin and
from Schillig, all contained TOC at concentrations of less than 0.5%
(dry weight) of the sediment.
Numbers of bacteria were higher in the surface sediment of core 69 (8.5 · 10
7 cells · cm
3) than in
deeper hemipelagic layers (1.7 · 10
7 to 3.5 · 10
7 cells · cm
3). The bacterial
numbers in all four sapropels investigated (0.87
· 10
8 to 1.48 · 10
8 cells · cm
3) exceeded those in the carbon-poor layers (Fig.
6B).
The frequency
of dividing and divided cells in Mediterranean sediments
ranged
between 2.1 and 5.2%; no significant differences were found
between
the sapropels and adjacent carbon-poor layers. In the present
study, the highest concentration of bacterial cells was observed
in
Dangast
sediment.
Plate counts of aerobic chemoorganoheterotrophic bacteria on CPSm agar
yielded positive results only in the case of two out
of eight layers of
core 69. The numbers of culturable cells were
very low, reaching a
total of 1.5 · 10
2 CFU per ml in the uppermost
carbon-poor layer, Z0, which corresponds
to a culturability of
0.00017%. The second positive sample was
sapropel S3, which contained
3.6 · 10
1 CFU ml
1 (culturability,
0.000024%). In the liquid dilution series, no
growth of bacteria from
the sediment layers of core 69 could be
detected even after 3 months of
incubation under anoxic conditions.
For comparison, plate counts of
aerobic chemoorganoheterotrophic
bacteria from the intertidal sediment
layers of Dangast and Schillig
were much higher and ranged from
2.3 · 10
6 to 2.3 · 10
8 CFU
ml
1 (culturability, 0.23 to 2.3%). Similarly, the
culturability of
anaerobic bacteria in the liquid medium MM was much
higher when
samples from Dangast (0.006%) and other surface sediments
from
the Wadden Sea (0.8% [S. Droege, H. Cypionka, J. Overmann, and
H. Sass, unpublished data]) were used. This indicates that specific
physiological features of the bacteria in Mediterranean sapropels
are
the reason for their low
culturability.
Correlation analysis and cell-specific exoenzyme activity.
For
Mediterranean sapropels, no correlation was found between bacterial
cell numbers and TOC (r2 = 0.002) or
between TOC and the three exoenzyme activities
(r2
0.072).
The cell-specific alkaline phosphatase activities were comparable in
the sapropels (1.28 · 10
17 to 5.3 · 10
17 mol · cell
1 · h
1) and intermediate layers (0.21 · 10
17 to 5.85 · 10
17 mol · cell
1 · h
1) (Fig.
7). The specific activities of

-glucosidase were much
lower and reached only 25.1 · 10
20 mol · cell
1 · h
1 in the sapropels and 9.9 · 10
20
to 228 · 10
20 mol · cell
1 · h
1 in the intermediate
layers. Interestingly, a consistent trend
with depth was observed for
the specific aminopeptidase activity.
We determined a maximum value of
the specific activity at the
surface of the Mediterranean sediments
(2.97 · 10
17 mol · cell
1
· h
1). In the hemipelagic layers below, the activity
continuously
declined from 0.38 · 10
17 mol
· cell
1 · h
1 (Z1) to 0.08 · 10
17 mol · cell
1 · h
1 (Z4). In contrast to the organic carbon-poor layers,
however,
the specific aminopeptidase activities remained at a higher
level
even in the oldest subfossil sapropel layers (0.53 · 10
17 to 1.45 · 10
17 mol · cell
1 · h
1) (Fig.
7).

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|
FIG. 7.
Specific activities of alkaline (alk.) phosphatase
( ), peptidase ( ), and -glucosidase ( ) in sapropels S1 and
S3 to S5 (vertical positions are indicated by black rectangles) and
organic carbon-poor layers of core 69. The values were calculated from
maximum enzyme activities and total bacterial cell counts and represent
potential rates (see Discussion).
|
|
 |
DISCUSSION |
Methodology.
In the present investigation, exoenzyme
activities were determined in sediment slurries. The injection of
substrate analogues into intact sediment cores has been proposed to
yield more realistic values of enzyme activities (41).
However, because of the extraordinarily high adsorption capacity, it
appears to be very difficult to create a homogenous distribution of
substrate analogues in sapropels by the core injection technique. In
addition, the small amount of sediment material which was available
from the Mediterranean sapropels made subsampling and the preparation
of sediment slurries inevitable. The disruption of in situ
microstructures during the preparation of sediment slurries can cause
an increase in exoenzyme activity (27, 41, 43); this effect
may be especially pronounced in ancient sapropels. Consequently, our
values should be taken as potential rather than actual rates of
exoenzyme activity. An adsorption of substrate analogues could slow
down their degradation in the sediment slurries. Since uncleaved
fluorescently labeled substrate analogues cannot be detected by the
present methods, we did not attempt to quantify their adsorption to the
sapropel matrix. However, adsorption of substrate analogues would lead to an underestimation of exoenzyme activities, and the potential rates
determined in the present study should thus be taken as minimum values.
In contrast to other sediments with even higher organic-matter content
(i.e., 16% of the dry weight [
13]), a desorption
of
bound fluorophores by chemical treatment was not feasible in
the case
of the sapropels. Obviously, the extremely high adsorption
of the
fluorophores is caused by the chemical properties of the
kerogen in
sapropels. Furthermore, adsorption of the fluorophores
MUF and MCA
depends on their concentrations in a nonlinear manner.
Consequently, a
linear correction for adsorption (
9,
34,
61) was not
adequate in the present study. Rather, equilibrium
adsorption isotherms
had to be recorded prior to measurements
of exoenzyme activities
in subfossil Mediterranean
sapropels.
Exoenzymes as indicators of physiologically active bacteria.
Since the culturability of bacteria in the sapropels was
extraordinarily low, the majority of bacterial cells counted in
this habitat may be physiologically inactive. At present, no method is
available to distinguish between exoenzymes associated with live
bacterial cells (so-called ectoenzymes [20]) and
liberated extracellular enzymes which have been immobilized by
adsorption to the nonliving particulate fraction of sapropel sediments.
In the adsorbed state, peroxidase, catalase, urease, and phosphatase can withstand proteolysis at least for several days (42,
58). Nothing is known about the long-term stability of
extracellular enzymes in the natural environment; however, a
persistence for 124,000 years appears highly unlikely.
Indeed, several lines of evidence indicate that the functional
exoenzymes detected in the present study are associated with
live
bacterial cells. Firstly, alkaline phosphatase immobilized
on soil
particles is extremely heat stable and retains its full
activity even
during 2 h of incubation at 80°C (
42). In contrast,
the alkaline phosphatase activity measured in Mediterranean sapropels
and other sediments was significantly reduced (e.g., to 5.9% of
the
initial activity in sapropel S1) by heating. Secondly, even
the
124,000-year-old sapropel harbors bacteria which are still
capable of
uptake and degradation of glucose (
46). Since glucose
is
liberated by

-glucosidase, this finding matches the presence
of the
active exoenzyme. Thirdly, a parallel investigation of
the bacterial
diversity in the sapropel layers revealed the presence
of 16S rRNA
sequences of exclusively gram-negative bacteria, most
of them (92%)
being members of the
Chloroflexus subdivision (M.
J. L. Coolen, A. Smock, H. Sass, H. Cypionka, and J. Overmann,
unpublished data). Gram-negative cells, unlike their gram-positive
counterparts, generally release only a little of their periplasmic
enzymes (
18). Based on this cumulative evidence, we conclude
that the exoenzyme activities determined in the present study
are
associated with extant bacterial
populations.
Implications for the microbial ecology of Mediterranean
sapropels.
Previously, the presence of active bacterial
populations in Mediterranean sapropels has been inferred from elevated
numbers of bacterial cells and the high frequency of dividing bacteria (reference 23 and this study). The exoenzymes
alkaline phosphatase and
-glucosidase are inducible by their
respective substrates (organic phosphoesters and cellobiose) and are
subject to catabolite repression by their products glucose and
phosphate, respectively (20, 59). Since the cell-specific
activities of both enzymes may thus be used as indicators for the
presence of degradable biopolymers (20, 21, 22, 45), our
data provide new insights into the physiological state of the bacteria
present in the sapropel layers.
Compared to the cell-specific phosphatase activity determined for
bacteria in the sapropels, that of phosphate-deficient natural
bacterial communities reaches much higher values (3 · 10
15 to 7.7 · 10
15 mol · cell
1 · h
1 [
45]).
Although phosphorus is the main limiting inorganic nutrient
in the
ultraoligotrophic eastern Mediterranean (
66), neither
the bacteria in sapropels nor those in the intermediate layers
appear
to be limited by inorganic phosphate. The specific activities
of

-glucosidase determined in the present study fall within the
range
observed for marine sediments (17.8 · 10
20 to
232 · 10
20 mol · cell
1
· h
1 [
8,
34]) and pelagic water
samples (50 · 10
20 mol · cell
1 · h
1 [
29]).
Thus, a complete anaerobic food chain still proceeds
in most if not all
of the sapropels investigated, despite the
great age of the bulk
sapropel organic matter. Based on the observation
that almost all of
the bacterial 16S rRNA sequences recovered
from sapropel layers are
affiliated with the
Chloroflexus subdivision
(Coolen et al.,
unpublished), the primary steps of the anaerobic
microbial food chain
in Mediterranean sapropel sediments may be
mediated by unknown
mesophilic chemoorganoheterotrophic members
of this bacterial
division.
The activity of leucine aminopeptidase is exclusively associated with
heterotrophic bacteria (
20). This exoenzyme, in contrast
to
alkaline phosphatase and

-glucosidase, is not induced by its
natural
substrate (polypeptides or proteins) in marine sediments;
it is
inhibited by glycine and other amino acids (
10). The
specific
aminopeptidase activity at the surfaces of Mediterranean
sediments
(2.97 · 10
17 mol · cell
1 · h
1) was comparable to that
in other sediments (0.5 · 10
17 to 2.94 · 10
17 mol · cell
1 · h
1 [
8,
29]) but continuously declined
with depth in the hemipelagic
layers, whereas specific activities were
elevated in the subfossil
sapropel layers. The specific aminopeptidase
activity increases
during initial stages of energy and nutrient
starvation and declines
thereafter (
2); hence, starvation
may be less pronounced for
bacteria living in the sapropels than for
those in intermediate
layers.
Extracellular enzymatic hydrolysis of biopolymers is the rate-limiting
step for the utilization of organic matter in surface
aquatic
environments (
7,
20,
56). In most marine sediments,
exoenzyme activities decrease rapidly with depth (
8,
9,
11,
35,
41,
53), thus following the gradients of easily
degradable
substances. This general pattern was also observed
for aminopeptidase
and alkaline phosphatase in the successive
hemipelagic layers. However,
localized maxima of specific exoenzyme
activities were confined to the
sapropels and thus cannot be explained
by a percolation of easily
degradable organic carbon into subfossil
sapropel layers. Compared to
other deep-sea sediments, those from
the eastern Mediterranean contain
much lower concentrations of
labile organic compounds (
24).
We conclude that organic carbon
provided by the sapropel layers
themselves must support bacterial
metabolism in
situ.
In continental aquifers and deep marine sediments, microbial biomass
correlates with total organic carbon content (
23,
33).
This
correlation has not been observed for sapropels (
23),
suggesting
that either the bioavailability of organic carbon substrates
or
the supply of electron-accepting substrates limits bacterial
population
size within the sapropels. Bacteria in Miocene subsurface
sediments
appear to be limited by the supply of electron acceptors
rather
than organic carbon substrates (
26,
33). This does
not apply
to Mediterranean sediments, including sapropels, in which the
concentrations of sulfate range between 20 and 50 mM
(
14; H.-J.
Brumsack, personal communication).
Consequently, a low bioavailability
of organic carbon most likely
limits bacterial metabolism in the
sapropel layers. Adsorption of
organic matter to mineral surfaces
slows down the degradation rates by
5 orders of magnitude (
31).
Even in geologically young
sediment layers, a major fraction (20
to 100%) of the detectable
acetate is not bioavailable (
63).
As shown here for the
first time, the sapropel matrix exhibits
an extraordinarily high
adsorption capacity for low-molecular-weight
organic carbon compounds,
as exemplified by MUF and MCA and also
demonstrated for DNA (Coolen et
al., unpublished). Hence, a strong
adsorption of organic carbon
substrates to the sapropel kerogen
may be the major limiting factor for
bacterial growth in Mediterranean
sapropels.
Despite the significant exoenzyme activities and cell numbers, we were
able to cultivate only a minute fraction of the bacteria
from the
various Mediterranean sapropels by conventional isolation
techniques or
by employing improved liquid media. Taken together,
our results
indicate that (i) survival and metabolism of nonsporulating
bacteria
can be supported even by 124,000-year-old sediment organic
matter and
(ii) new cultivation approaches will have to be developed
for the
isolation of bacteria from such extreme
environments.
 |
ACKNOWLEDGMENTS |
We are indebted to Andrea Smock for help with determination of
cell numbers and the TOC contents of the various sediments and Henrike
Oertel for help with the initial experiments. Heribert Cypionka and
Henrik Sass are gratefully acknowledged for their support during the
cruise, and the master and crew of the R/V Meteor for their
help during collection of the sediment cores.
The present work was supported by grants from the Deutsche
Forschungsgemeinschaft to H. Cypionka and J.O. (Cy 1/8-1 and Cy 1/10-1).
 |
FOOTNOTES |
*
Corresponding author. Mailing address:
Paleomicrobiology Group, Institute for the Chemistry and Biology
of the Marine Environment, University of Oldenburg, D-26111 Oldenburg,
Germany. Phone: 49-441-798-5376. Fax: 49-441-798-3583. E-mail:
j.overmann{at}icbm.de.
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Applied and Environmental Microbiology, June 2000, p. 2589-2598, Vol. 66, No. 6
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