INTRODUCTION

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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 andin some casesdiatoms (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.

	MATERIALS AND METHODS

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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.

View larger version (22K): [in this window] [in a new window]   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.

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.

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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.

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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 · ml¹) 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.

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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

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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.

View larger version (16K): [in this window] [in a new window]   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.

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 · g¹ and thus was comparable to that of pure montmorillonite (Table 2). However, the affinity of sapropel material for MCA (37.4 ± 3.3 ml · g¹) was lower than that for MUF.

View larger version (14K): [in this window] [in a new window]   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.

View larger version (17K): [in this window] [in a new window]   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.

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.

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 K_m + S_n of 27.5 µM, which is in the same range as the values determined for the other sediments (Table 4).

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Michaelis-Menten kinetics of alkaline phosphatase activity measured in sapropel S3 of core 66. The error bars indicate standard deviations.

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 sediment³ h¹) (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.

View larger version (21K): [in this window] [in a new window]   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.

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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.

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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).

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View larger version (25K): [in this window] [in a new window]   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

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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.

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.

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.

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