HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 21 of 115 3.6. In vitro sulphate reduction with methane 3.6.1. In vitro rate determination from substrate concentration changes The sediment samples taken from the sulfate-methane transition zone are anoxically stored in butyl rubber sealed bottles(250 ml)without headspace (or methane in the headspace)at in situ temperature until further processing in the laboratory.All manipulations are done under an anoxic atmosphere of N2 using either the Hungate technique and its modifications(Widdel and Bak,1992;Breznak and Costilow,1994)or an anoxic glove box (Mecaplex).The original sediment is mixed with approximately the same volume of anoxic seawater.In this way,a slurry with 0.2 to 0.3 g sediment dry mass per ml is obtained which can be transferred to tubes by means of plastic tubes(length,150 mm;inner diameter,5 mm)connected to a syringe(preflushed with nitrogen).Sulphate reduction with methane at partial pressures of 0.1 MPa(1 atm)or less is measured in culture tubes(volume,approx.20 ml;length,135 mm; inner diameter,14 mm)with a tapered,Viton rubber-sealed orifice(inner diameter,9 mm). The culture tubes are provided with approx.3 ml of sediment slurry and 9 ml of anoxic sea water(pre-reduced with 0.5 mM sulphide)or artificial sea water medium with ammonium(4 mM),phosphate (1 mM),trace elements,vitamins,bicarbonate(30 mM)and sulphide(0.5-1 mM)as used for cultivation of SRB(Widdel and Bak,1992).The pH at the beginning of incubation is 7.5.Pure methane of atmospheric pressure(0.1 MPa)is applied in the headspace.In experiments with artificial seawater medium and methane of atmospheric pressure,an amount of CO2 corresponding to 1/10 of the headspace volume is in addition injected by means of a syringe.A commercial N2-CO2-mixture(90/10,[vol/vol])is applied for controls.Lower methane partial pressures are achieved in tubes with the indicated N2- CO2-mixture into which defined volumes of methane are injected.Tubes are incubated horizontally to facilitate diffusion of methane into the sediment;they are gently shaken for a few seconds once per day.Tubes are not continuously shaken to avoid possible disintegration of microbial associations.Samples for chemical analyses(100ul)are withdrawn during incubation via microliter syringes(preflushed with N2). The simultaneous determination of methane utilisation and sulphide production to investigate the stoichiometry of the process can be carried out in an incubation experiment without gas phase.A relatively large gas phase of methane compared to the aqueous phase would not allow reliable measurement of methane consumption,particularly at the beginning of the experiment when the decrease in the total amount of methane is still relatively small. Therefore,a special glass tube is used(modified from Alperin and Reeburgh,1985)that allows head space-free incubation of sediment samples with dissolved methane concentrations above the saturation limit given at ambient pressure.At one end,the glass tube (length,180 mm;inner diameter,17 mm)is tapered towards a regular orifice(inner diameter, 9 mm)with a stopper(Viton rubber,fixed by screw cap with hole)that allows withdrawal of aliquots with a syringe.At the other end,the tube is tapered towards a smaller elongated glass tube (length,65 mm;inner diameter,9 mm)that harbours a gas-tight piston(silicon- lubricated Viton rubber);the piston is held in position by a screwing device.If a sample is withdrawn via the opposite stopper,the piston is simultaneously pushed into the tube so as to avoid any underpressure and formation of gas bubbles.High dissolved methane concentrations are achieved via an initial small(5 ml)headspace in which 0.4 MPa(4 atm)of methane is kept for some hours under shaking.The gas phase is finally replaced by anoxic medium,and remaining bubbles are allowed to escape via an inserted hypodermic needle while the piston is slightly moved.This device,which is provided with 6 ml sediment slurry in a total volume of 40 ml,is incubated as the tubes described above
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 21 of 115 3.6. In vitro sulphate reduction with methane 3.6.1. In vitro rate determination from substrate concentration changes The sediment samples taken from the sulfate-methane transition zone are anoxically stored in butyl rubber sealed bottles (250 ml) without headspace (or methane in the headspace) at in situ temperature until further processing in the laboratory. All manipulations are done under an anoxic atmosphere of N2 using either the Hungate technique and its modifications (Widdel and Bak, 1992; Breznak and Costilow, 1994) or an anoxic glove box (Mecaplex). The original sediment is mixed with approximately the same volume of anoxic seawater. In this way, a slurry with 0.2 to 0.3 g sediment dry mass per ml is obtained which can be transferred to tubes by means of plastic tubes (length, 150 mm; inner diameter, 5 mm) connected to a syringe (preflushed with nitrogen). Sulphate reduction with methane at partial pressures of 0.1 MPa (1 atm) or less is measured in culture tubes (volume, approx. 20 ml; length, 135 mm; inner diameter, 14 mm) with a tapered, Viton rubber-sealed orifice (inner diameter, 9 mm). The culture tubes are provided with approx. 3 ml of sediment slurry and 9 ml of anoxic sea water (pre-reduced with 0.5 mM sulphide) or artificial sea water medium with ammonium (4 mM), phosphate (1 mM), trace elements, vitamins, bicarbonate (30 mM) and sulphide (0.5−1 mM) as used for cultivation of SRB (Widdel and Bak, 1992). The pH at the beginning of incubation is 7.5. Pure methane of atmospheric pressure (0.1 MPa) is applied in the headspace. In experiments with artificial seawater medium and methane of atmospheric pressure, an amount of CO2 corresponding to 1/10 of the headspace volume is in addition injected by means of a syringe. A commercial N2-CO2-mixture (90/10, [vol/vol]) is applied for controls. Lower methane partial pressures are achieved in tubes with the indicated N2- CO2-mixture into which defined volumes of methane are injected. Tubes are incubated horizontally to facilitate diffusion of methane into the sediment; they are gently shaken for a few seconds once per day. Tubes are not continuously shaken to avoid possible disintegration of microbial associations. Samples for chemical analyses (100µl) are withdrawn during incubation via microliter syringes (preflushed with N2). The simultaneous determination of methane utilisation and sulphide production to investigate the stoichiometry of the process can be carried out in an incubation experiment without gas phase. A relatively large gas phase of methane compared to the aqueous phase would not allow reliable measurement of methane consumption, particularly at the beginning of the experiment when the decrease in the total amount of methane is still relatively small. Therefore, a special glass tube is used (modified from Alperin and Reeburgh, 1985) that allows head space-free incubation of sediment samples with dissolved methane concentrations above the saturation limit given at ambient pressure. At one end, the glass tube (length, 180 mm; inner diameter, 17 mm) is tapered towards a regular orifice (inner diameter, 9 mm) with a stopper (Viton rubber, fixed by screw cap with hole) that allows withdrawal of aliquots with a syringe. At the other end, the tube is tapered towards a smaller elongated glass tube (length, 65 mm; inner diameter, 9 mm) that harbours a gas-tight piston (siliconlubricated Viton rubber); the piston is held in position by a screwing device. If a sample is withdrawn via the opposite stopper, the piston is simultaneously pushed into the tube so as to avoid any underpressure and formation of gas bubbles. High dissolved methane concentrations are achieved via an initial small (5 ml) headspace in which 0.4 MPa (4 atm) of methane is kept for some hours under shaking. The gas phase is finally replaced by anoxic medium, and remaining bubbles are allowed to escape via an inserted hypodermic needle while the piston is slightly moved. This device, which is provided with 6 ml sediment slurry in a total volume of 40 ml, is incubated as the tubes described above
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 22 of 115 Analyses: Sediment dry mass is determined after drying at 80C for 2 days. Sulphide is determined colorimetrically using the methylene blue formation reaction in a miniaturised assay (Aeckersberg et al.,1991)or the formation of colloidal copper sulphide (Cord-Ruwisch,1985). For the quantification of sulphate,1.5 ml of a particle-free water sample is mixed with 0.1 ml of 2 M HCI.After heating in a boiling water bath,0.4 ml of 0.5 M BaCl2 solution is added. Precipitated BaSO4 is quantitatively collected on a nitrocellulose filter(25 mm diameter,0.2 um pore size),washed with 10 ml distilled water,dried at 60C and quantified by weighing Methane is determined using a GC 14B gas chromatograph(Shimadzu)equipped with a Supel-Q Plot column (30 m x 0.53 mm;Supelco)and a flame ionisation detector.The carrier gas was N2 at a flow rate of 3 ml min.The column temperature was 110C. Calculations: The geometry inside the inoculated tubes and their handling(occasional shaking)do not allow application of diffusion models to calculate the actual methane concentration in the sediment during incubation.Only rough estimation of a lower limit appears possible below which the methane concentration in the sediment is unlikely to drop.In the horizontally incubated culture tubes,the settled sediment forms a loose layer nearly over the whole length. The height of the liquid,which can be regarded as the approximate diffusion distance,is Ax= 0.8 cm(maximum in the middle).The loose thin sediment layer is not expected to impede diffusion significantly.In the incubation experiment with various methane pressures, application of 0.1 MPa(1 atm)methane results in an increase of the sulphide concentration in the medium of approx.0.07 umol cmd,which is 0.84 umol d for the culture volume (12 ml).With a surface area of the settled sediment of roughly 8 cm2,the flux of sulphide into the medium and hence the flux of methane into the sediment would beJ=0.105 umol cmd The diffusion coefficient for methane in seawater at 12C is D=0.86 cm2d(1.10>cm2 s;Iversen and Jorgensen,1985).Hence,the calculated flux would be associated with an approximate concentration difference between the sediment and the medium surface of-AC =0.1 umol cm(0.1 mM),according to Fick's first law of diffusion (for linear gradient,J= -D AC/Ax).The occasional shaking favours the methane supply to the sediment.Hence,if the concentration of methane in the upper medium remains at 1.4 mM(dissolved methane upon addition of 0.1 MPa,12C),the concentration in the sediment under the given conditions should not be lower than 1.3 mM.If the rate v of sulphide production(and hence of methane oxidation)depends on the methane concentration C according to Michaelis- Menten kinetics,an increase of the concentration from Ci to C2 would result in an increase of the rate by a factor of v2/v [C2(KM+C1]/[Ci(KM C2)]. Free energy changes(AG values)under in situ or incubation conditions can be calculated from Gvalues via AG values.For SO4,HCO3 and HS in seawater,activity coefficients of 0.1,0.5 and 0.5,respectively,were estimated(Stumm and Morgan,1996).The influence of temperature on AG can be calculated via the integrated Gibbs-Helmholtz equation including the enthalpy (AH).Redox potentials of half-reactions(viewed as being in equilibrium)are calculated from AG values via E values using concentrations and activity coefficients as for the calculation of AG,and assuming a pH of 7.5
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 22 of 115 Analyses: Sediment dry mass is determined after drying at 80°C for 2 days. Sulphide is determined colorimetrically using the methylene blue formation reaction in a miniaturised assay (Aeckersberg et al., 1991) or the formation of colloidal copper sulphide (Cord-Ruwisch, 1985). For the quantification of sulphate, 1.5 ml of a particle-free water sample is mixed with 0.1 ml of 2 M HCl. After heating in a boiling water bath, 0.4 ml of 0.5 M BaCl2 solution is added. Precipitated BaSO4 is quantitatively collected on a nitrocellulose filter (25 mm diameter, 0.2 µm pore size), washed with 10 ml distilled water, dried at 60°C and quantified by weighing. Methane is determined using a GC 14B gas chromatograph (Shimadzu) equipped with a Supel-Q Plot column (30 m x 0.53 mm; Supelco) and a flame ionisation detector. The carrier gas was N2 at a flow rate of 3 ml min-1. The column temperature was 110°C. Calculations: The geometry inside the inoculated tubes and their handling (occasional shaking) do not allow application of diffusion models to calculate the actual methane concentration in the sediment during incubation. Only rough estimation of a lower limit appears possible below which the methane concentration in the sediment is unlikely to drop. In the horizontally incubated culture tubes, the settled sediment forms a loose layer nearly over the whole length. The height of the liquid, which can be regarded as the approximate diffusion distance, is ∆x = 0.8 cm (maximum in the middle). The loose thin sediment layer is not expected to impede diffusion significantly. In the incubation experiment with various methane pressures, application of 0.1 MPa (1 atm) methane results in an increase of the sulphide concentration in the medium of approx. 0.07 µmol cm−3 d−1 , which is 0.84 µmol d−1 for the culture volume (12 ml). With a surface area of the settled sediment of roughly 8 cm2 , the flux of sulphide into the medium and hence the flux of methane into the sediment would be J = 0.105 µmol cm−2 d−1 . The diffusion coefficient for methane in seawater at 12°C is D = 0.86 cm2 d−1 (1 · 10−5 cm2 s −1 ; Iversen and Jørgensen, 1985). Hence, the calculated flux would be associated with an approximate concentration difference between the sediment and the medium surface of −∆C = 0.1 µmol cm−3 (0.1 mM), according to Fick's first law of diffusion (for linear gradient, J = −D ∆C/∆x). The occasional shaking favours the methane supply to the sediment. Hence, if the concentration of methane in the upper medium remains at 1.4 mM (dissolved methane upon addition of 0.1 MPa, 12°C), the concentration in the sediment under the given conditions should not be lower than 1.3 mM. If the rate v of sulphide production (and hence of methane oxidation) depends on the methane concentration C according to MichaelisMenten kinetics, an increase of the concentration from C1 to C2 would result in an increase of the rate by a factor of v2/v1 = [C2(KM + C1)]/[C1(KM + C2)]. Free energy changes (∆G values) under in situ or incubation conditions can be calculated from G°f values via ∆G° values. For SO4 2− , HCO3 − and HS− in seawater, activity coefficients of 0.1, 0.5 and 0.5, respectively, were estimated (Stumm and Morgan, 1996). The influence of temperature on ∆G° can be calculated via the integrated Gibbs-Helmholtz equation including the enthalpy (∆H°). Redox potentials of half-reactions (viewed as being in equilibrium) are calculated from ∆G° values via E° values using concentrations and activity coefficients as for the calculation of ∆G, and assuming a pH of 7.5
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 23 of 115 Reference: Nauhaus K,Boetius A,Kruger M,Widdel F (2002).In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area.Environmental Microbiology 4,296-305. Widdel,F.and Bak F.(1992).The gram negative mesophilic sulfate reducing bacteria,In: The Prokaryotes(ed.Dworkin,M.),pp.3352-3378.Springer Verlag. Contact: Helge Niemann,Max Planck Institute for Marine Microbiology,Bremen,Germany (e-mail: hniemann@mpi-bremen.de)
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 23 of 115 Reference: Nauhaus K, Boetius A, Krüger M, Widdel F (2002). In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area. Environmental Microbiology 4, 296-305. Widdel, F. and Bak F. (1992). The gram negative mesophilic sulfate reducing bacteria, In: The Prokaryotes (ed. Dworkin, M.), pp. 3352-3378. Springer Verlag. Contact: Helge Niemann, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: hniemann@mpi-bremen.de )
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 24 of 115 3.6.2. In vitro rate determination from radio-labelled tracer turnover In vitro methane and sulphate turn over in sediment slurries can be measured in long term incubations with radio-labelled methane and sulphate according to a modified method of Nauhaus et al.(2002;see section 2.6.1).For this purpose,Hungate tubes(20 ml;n=5 for AOM and SR,respectively)are provided with 3 ml of sediment slurry (containing ca.1.5 ml of sediment)and 15 ml of anoxic,artificial seawater medium(Widdel and Bak,1992)in a glove box under strictly anoxic conditions.The remaining headspace is flushed with a CH4/N2 mixture to adjust methane concentration in the media.The slurry is then pre- incubated in a horizontal position to facilitate diffusion of methane into the sediment for 1 day to 1 week(depending on the expected activity).During this time period,the headspace is flushed several times with the CH4/N2 mixture to maintain constant methane concentrations in the medium.After the pre-incubation period,the methane headspace is replaced with artificial seawater medium containing the same methane concentration as the sediment slurry 50 ul 4C-labelled methane and 5 ul 35S-labelled sulphate(tracer dissolved in water,10 kBq and 50 kBq,respectively)are injected in the tubes in equilibrium with artificial seawater medium.The Hungate tubes are then incubated in a horizontal position to facilitate diffusion of methane into the sediment for 1 day to 1 week (depending on the expected activity).The incubations are stopped by fixing the sediment slurries in glass jars containing 25 ml NaOH (2.5 %w/v)and in falcon tubes containing 20 ml of Zn-Acetate solution (20%,w/v)for AOM and SR rate measurements,respectively.Further processing and rate calculations are according to the ex situ AOM and SR rate measurements(sections 2.5 and 2.3,respectively). Reference: Nauhaus K,Boetius A,Kruger M,Widdel F(2002).In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area.Environmental Microbiology 4,296-305. Widdel,F.and Bak F.(1992).The gram negative mesophilic sulfate reducing bacteria,In: The Prokaryotes(ed.Dworkin,M.),pp.3352-3378.Springer Verlag Contact: Helge Niemann,Max Planck Institute for Marine Microbiology,Bremen,Germany (e-mail: hniemann@mpi-bremen.de
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 24 of 115 3.6.2. In vitro rate determination from radio-labelled tracer turnover In vitro methane and sulphate turn over in sediment slurries can be measured in long term incubations with radio-labelled methane and sulphate according to a modified method of Nauhaus et al. (2002; see section 2.6.1). For this purpose, Hungate tubes (20 ml; n = 5 for AOM and SR, respectively) are provided with 3 ml of sediment slurry (containing ca. 1.5 ml of sediment) and 15 ml of anoxic, artificial seawater medium (Widdel and Bak, 1992) in a glove box under strictly anoxic conditions. The remaining headspace is flushed with a CH4/N2 mixture to adjust methane concentration in the media. The slurry is then preincubated in a horizontal position to facilitate diffusion of methane into the sediment for 1 day to 1 week (depending on the expected activity). During this time period, the headspace is flushed several times with the CH4/N2 mixture to maintain constant methane concentrations in the medium. After the pre-incubation period, the methane headspace is replaced with artificial seawater medium containing the same methane concentration as the sediment slurry. 50 µl 14C-labelled methane and 5 µl 35S-labelled sulphate (tracer dissolved in water, 10 kBq and 50 kBq, respectively) are injected in the tubes in equilibrium with artificial seawater medium. The Hungate tubes are then incubated in a horizontal position to facilitate diffusion of methane into the sediment for 1 day to 1 week (depending on the expected activity). The incubations are stopped by fixing the sediment slurries in glass jars containing 25 ml NaOH (2.5 %, w/v) and in falcon tubes containing 20 ml of Zn-Acetate solution (20%, w/v) for AOM and SR rate measurements, respectively. Further processing and rate calculations are according to the ex situ AOM and SR rate measurements (sections 2.5 and 2.3, respectively). Reference: Nauhaus K, Boetius A, Krüger M, Widdel F (2002). In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area. Environmental Microbiology 4, 296-305. Widdel, F. and Bak F. (1992). The gram negative mesophilic sulfate reducing bacteria, In: The Prokaryotes (ed. Dworkin, M.), pp. 3352-3378. Springer Verlag. Contact: Helge Niemann, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: hniemann@mpi-bremen.de )
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 25 of 115 3.7. Assessment of extracellular enzymatic activities of benthic assemblages This method allows measuring the degradation rates of high-molecular-weight(HMW) organic compounds by extracellular microbial enzymes.These enzymatic activities,which are recognised as the key step in the degradation and utilisation of organic polymers by bacteria(Hoppe 1991;Meyer-Reil 1991),are measured by using fluorogenic model substrates(Hendel and Marxen 1997).The in vitro degradation of these fluorogenic analogues provides a reliable estimation of the rates of enzymatic activity and,in the deep sea, has been primarily focused on leucine aminopeptidase,B-D-glucosidase and alkaline phosphatase Extracellular enzymatic activity is measured immediately after sediment retrieval(Meyer- Reil,1987;Meyer-Reil and Koster,1992).Activities of L-aminopeptidase,B-D-glucosidase and alkaline-phosphatase are quantified fluorometrically by the cleavage of artificial fluorogenic substrates(Hoppe,1993),using L-Leucine-4-methylcoumarinyl-7-amide(Leu- MCA),4-methylumbelliferone B-D-glucopyranoside(MUF-Glu),4-methylumbelliferone phosphate(MUF-P)as substrates,respectively Marine sediment collection for the assessment of extracellular enzymatic activities must be carried out avoiding any contamination of the sample that could affect estimates.Sediment samples from deep localities for enzymatic assays are preferentially collected using multiple corers.Immediately after retrieval,sediment sub-samples are gently removed from the corer inserting 10-ml plastic syringes along the axis of the core.The syringe plunger is held fixed at the sediment surface while the barrel is pushed into the sediment,in an overall procedure that is analogous to piston coring.The syringes are then removed from the sediment and the sediment is transferred in a sterile tube and resuspended with sterile seawater to produce a sediment slurry (1:1 ratio). Substrate incubations are performed in the dark at in situ temperature for 1 hour(enzymatic activities generally increase linearly with time up to 3 hours),in a final volume of 5 ml containing sterile seawater,an aliquot of the sediment slurry (500 ul)and the fluorogenic substrate.The substrate is added at saturating concentrations(which are generally at 100-200 um,final concentrations),but saturating conditions must be estimated with caution,after kinetic runs,using the Michaelis-Menten equation.The measurement of enzymatic activities in deep sea samples is generally not carried out under in-situ pressure condition.However, previous studies reported that aminopeptidase activities did not change significantly between decompressed and recompressed abyssal sediment samples (Poremba 1995),but Deming and Baross(2000)reported a 5-fold increase of aminopeptidase activity after abyssal sediment recompression.These contrasting results indicate that the extracellular enzymatic activities measured in the deep sea must be considered with caution. After incubation,the slurries are centrifuged (3000 x g,5 minutes)and supernatants analysed fluorometrically (at 380 nm excitation,440 nm emission for Leu-MCA and 365 nm excitation, 455 nm emission for Glu-MUF and MUF-P).Immediately after substrate inoculation(at t=0),the fluorescence of each sample is measured (blank)and then subtracted from fluorescence after 1 hour of incubation.Data are normalised to sediment dry weight(60C
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 25 of 115 3.7. Assessment of extracellular enzymatic activities of benthic assemblages This method allows measuring the degradation rates of high-molecular-weight (HMW) organic compounds by extracellular microbial enzymes. These enzymatic activities, which are recognised as the key step in the degradation and utilisation of organic polymers by bacteria (Hoppe 1991; Meyer-Reil 1991), are measured by using fluorogenic model substrates (Hendel and Marxen 1997). The in vitro degradation of these fluorogenic analogues provides a reliable estimation of the rates of enzymatic activity and, in the deep sea, has been primarily focused on leucine aminopeptidase, ß-D-glucosidase and alkaline phosphatase. Extracellular enzymatic activity is measured immediately after sediment retrieval (MeyerReil, 1987; Meyer-Reil and Koster, 1992). Activities of L-aminopeptidase, β-D-glucosidase and alkaline-phosphatase are quantified fluorometrically by the cleavage of artificial fluorogenic substrates (Hoppe, 1993), using L-Leucine-4-methylcoumarinyl-7-amide (LeuMCA), 4-methylumbelliferone β-D-glucopyranoside (MUF-Glu), 4-methylumbelliferone phosphate (MUF-P) as substrates, respectively. Marine sediment collection for the assessment of extracellular enzymatic activities must be carried out avoiding any contamination of the sample that could affect estimates. Sediment samples from deep localities for enzymatic assays are preferentially collected using multiple corers. Immediately after retrieval, sediment sub-samples are gently removed from the corer inserting 10-ml plastic syringes along the axis of the core. The syringe plunger is held fixed at the sediment surface while the barrel is pushed into the sediment, in an overall procedure that is analogous to piston coring. The syringes are then removed from the sediment and the sediment is transferred in a sterile tube and resuspended with sterile seawater to produce a sediment slurry (1:1 ratio). Substrate incubations are performed in the dark at in situ temperature for 1 hour (enzymatic activities generally increase linearly with time up to 3 hours), in a final volume of 5 ml containing sterile seawater, an aliquot of the sediment slurry (500 µl) and the fluorogenic substrate. The substrate is added at saturating concentrations (which are generally at 100-200 µm, final concentrations), but saturating conditions must be estimated with caution, after kinetic runs, using the Michaelis-Menten equation. The measurement of enzymatic activities in deep sea samples is generally not carried out under in-situ pressure condition. However, previous studies reported that aminopeptidase activities did not change significantly between decompressed and recompressed abyssal sediment samples (Poremba 1995), but Deming and Baross (2000) reported a 5-fold increase of aminopeptidase activity after abyssal sediment recompression. These contrasting results indicate that the extracellular enzymatic activities measured in the deep sea must be considered with caution. After incubation, the slurries are centrifuged (3000 x g, 5 minutes) and supernatants analysed fluorometrically (at 380 nm excitation, 440 nm emission for Leu-MCA and 365 nm excitation, 455 nm emission for Glu-MUF and MUF-P). Immediately after substrate inoculation (at t = 0), the fluorescence of each sample is measured (blank) and then subtracted from fluorescence after 1 hour of incubation. Data are normalised to sediment dry weight (60°C