HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 11 of 115 3.2. Leucine incorporation The method aims at measuring bacterial biomass(secondary)production by measuring the rate of incorporation of tritiated Leucine (H-Leu)into bacterial cells.Biomass production is calculated from rates of protein synthesis by using empirical factors.This method,indeed, has become the mostly common method for measuring bacterial production in both water column and sediments(Kirchman et al.,1986;van Duyl Kop,1994). Sediment samples are preferentially collected using multiple corers,which allow recovering undisturbed,intact sediment cores.Immediately after retrieval,sediment sub-samples are gently removed from the corer inserting cut-off 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 for 1 centimetre,in an overall procedure that is analogous to piston coring.The syringes are then removed from the sediment and the sediment is transferred into a sterile tube and resuspended in sterile seawater to form an homogeneous slurry (dilution 1:1). Sediment sub-samples(0.2 ml)are then transferred to 2-ml sterile Eppendorf tubes and incubated in the dark for 1 hour at in situ temperature with 30 ul of an aqueous solution of L- [4,5-H]leucine (Amersham).Saturation concentrations of leucine is generally at 0.5 uM (final concentration),but can vary in different areas/samples and should be thus assessed using different concentrations.The measurement of bacterial C production in deep sea samples generally is generally not carried out under in-situ pressure condition,so that estimates may be biased(Yayanos,1995). After incubation,bacterial incorporation is stopped with 1.7 ml of 80%ethanol.The sediment sub-sample is centrifuged(10000 x g,5 minutes),the supernatant gently removed and the sediment resuspended in 1.7 ml of 80%ethanol.This centrifugation-resuspension procedure is carried out twice.After the second procedure,the sediment-ethanol slurry is passed through a polycarbonate filter(0.2 um mesh size).The filters are rinsed four times with 2 ml of 5%TCA each(Trichloroacetic Acid),transferred to sterile pyrex tubes,added with 2ml of NaOH and heated for 2 hours in a water bath at 100C.After centrifugation of the tubes,1 ml of supernatant is transferred to scintillation vials containing 10 ml of scintillation liquid (Perkin Elmer or similar).Measurements of radioactivity are carried using a liquid scintillation counter. For each sediment sample,a total of three replicates and two blanks are analysed.Sediment blanks are run as described for sediment samples but adding 1.7 ml of 80%ethanol immediately before 'H-Leucine addition.Data are normalised to sediment dry weight after desiccation (60C,24h). Data of'H-leucine incorporation are converted into bacterial Carbon production by using the following formula: Bacterial C Production g"=[nmol incorporated leucine (100/7.3)M.0.86.R.2]/g Where: the ratio 100/7.3 is the percentage of leucine into the total bacterial aminoacid pool M is the molar weight of leucine -0.86 is the conversion factor of bacterial protein production to bacterial C production
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 11 of 115 3.2. Leucine incorporation The method aims at measuring bacterial biomass (secondary) production by measuring the rate of incorporation of tritiated Leucine (3 H-Leu) into bacterial cells. Biomass production is calculated from rates of protein synthesis by using empirical factors. This method, indeed, has become the mostly common method for measuring bacterial production in both water column and sediments (Kirchman et al., 1986; van Duyl & Kop, 1994). Sediment samples are preferentially collected using multiple corers, which allow recovering undisturbed, intact sediment cores. Immediately after retrieval, sediment sub-samples are gently removed from the corer inserting cut-off 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 for 1 centimetre, in an overall procedure that is analogous to piston coring. The syringes are then removed from the sediment and the sediment is transferred into a sterile tube and resuspended in sterile seawater to form an homogeneous slurry (dilution 1:1). Sediment sub-samples (0.2 ml) are then transferred to 2-ml sterile Eppendorf tubes and incubated in the dark for 1 hour at in situ temperature with 30 µl of an aqueous solution of L- [4,5-3 H] leucine (Amersham). Saturation concentrations of leucine is generally at 0.5 µM (final concentration), but can vary in different areas/samples and should be thus assessed using different concentrations. The measurement of bacterial C production in deep sea samples generally is generally not carried out under in-situ pressure condition, so that estimates may be biased (Yayanos, 1995). After incubation, bacterial incorporation is stopped with 1.7 ml of 80% ethanol. The sediment sub-sample is centrifuged (10000 x g, 5 minutes), the supernatant gently removed and the sediment resuspended in 1.7 ml of 80% ethanol. This centrifugation-resuspension procedure is carried out twice. After the second procedure, the sediment-ethanol slurry is passed through a polycarbonate filter (0.2 µm mesh size). The filters are rinsed four times with 2 ml of 5% TCA each (Trichloroacetic Acid), transferred to sterile pyrex tubes, added with 2ml of NaOH and heated for 2 hours in a water bath at 100°C. After centrifugation of the tubes, 1 ml of supernatant is transferred to scintillation vials containing 10 ml of scintillation liquid (Perkin Elmer or similar). Measurements of radioactivity are carried using a liquid scintillation counter. For each sediment sample, a total of three replicates and two blanks are analysed. Sediment blanks are run as described for sediment samples but adding 1.7 ml of 80% ethanol immediately before 3 H-Leucine addition. Data are normalised to sediment dry weight after desiccation (60°C, 24h). Data of 3 H-leucine incorporation are converted into bacterial Carbon production by using the following formula: Bacterial C Production g-1 = [nmol incorporated leucine (100/7.3) ·M · 0.86 · R · 2] / g Where: - the ratio 100/7.3 is the percentage of leucine into the total bacterial aminoacid pool - M is the molar weight of leucine - 0.86 is the conversion factor of bacterial protein production to bacterial C production
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 12 of 115 R is the correction for radioactive decay of the'H-leucine 2 is the intracellular isotope dilution g is sediment dry weight (grams). References: Kirchman DL,Newell SY,Hodson RE (1986)Incorporation versus biosynthesis of leucine:implications for measuring rates of protein synthesis and biomass production by bacteria in marine systems.Mar Ecol Prog Ser 32:47-59 van Duyl FC,&Kop AJ (1994)Bacterial production in North Sea sediments:clues to seasonal and spatial variations.Mar Biol 120:323-337 Yayanos AA (1995)Microbiology to 10,500 meters in the deep sea.Ann Rev Microbiol 49:777-805 Contact: Roberto Danovaro,Department of Marine Sciences,Polytechnic University of Marche, Ancona.(e-mail:danovaro@univpm.it )
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 12 of 115 - R is the correction for radioactive decay of the 3 H-leucine - 2 is the intracellular isotope dilution - g is sediment dry weight (grams). References: Kirchman DL, Newell SY, & Hodson RE (1986) Incorporation versus biosynthesis of leucine: implications for measuring rates of protein synthesis and biomass production by bacteria in marine systems. Mar Ecol Prog Ser 32:47-59 van Duyl FC, & Kop AJ (1994) Bacterial production in North Sea sediments: clues to seasonal and spatial variations. Mar Biol 120:323-337 Yayanos AA (1995) Microbiology to 10,500 meters in the deep sea. Ann Rev Microbiol 49:777-805 Contact: Roberto Danovaro, Department of Marine Sciences, Polytechnic University of Marche, Ancona. (e-mail: danovaro@univpm.it )
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 13 of 115 3.3. Sulphate reduction Sulphate reduction rates are measured experimentally in sediment cores using a whole-core injection method modified from Jorgensen(1978).The distillation procedures are performed either hot(Fossing and Jorgensen,1989)or cold(Kallmeyer et al.,2004)depending on the expected sulphate reduction rates. Field: Sediment subcores are sampled in 26 mm ID acrylic tubes with injection ports filled with silicone rubber. ● About 5 ul (400 kBq)radioactively labelled 34SO42solution is injected into the sediment in 1-cm depth intervals and incubated for 6-24 h at in situ temperature. The bacterial sulphate reduction is stopped in 20%(w/v)zinc acetate by mixing sediment with a known amount of 20%zinc acetate(ZnAc)solution(20 g zinc acetate dihydrate in 100 mL water).The ZnAc volume to sediment volume should be 2:1.ZnAc preserves the radiolabelled sulphide as ZnS and stops bacterial activity.It is important for optimal storage,samples should be frozen. Blank samples(i.e.sediment samples with no ZnS formed)are prepared when 5 cm3 sediment is preserved in 10 ml 20%-ZnAc (w/v)prior to 3S042 addition.About 10 ul (800 kBq)*SO4 is added to the preserved sample after at least 1 hour(i.e.when sulphate reduction has come to a complete stop).The blank samples are used during distillation to estimate the amount of non-reduced3S that are transferred from the labeled sediment to the trap.See Kallmeyer et al.(2004)for a complete discussion of blanks and detection limits. Laboratory: The weight of each vial sediment ZnAc is determined.The empty vial weight(mean of several weighings of empty vials)and the weight of ZnAc solution may be subtracted to give the sediment wet weight. The samples are centrifuged(4500 rpm,5 min;sandy sediments 6000 rpm)and the supernatant carefully removed and kept for further analysis. ● 35SOradioactivity of the supernatant is determined on a0.1 ml sample in 1 ml H2O and 7 ml Lumasafe plus(Lumac BV.)in the scintillation counter. Distillation techniques: Hot distillations can be performed on samples with an expected sulphate reduction rate 1 nmol SO4cm day (see also Fossing and Jorgensen,1989): Approx.1-2 g sediment (exact weight must be known)is transferred to a flask and mixed with 10 ml 50%ethanol.The flask is attached to the distillation apparatus and connected to a distillation trap through a condenser. The flask is flushed with N2 for 10 min,after which 8 ml HCI(6 M)and 16 ml CrlI- solution(1M)are added to liberate all reduced inorganic sulfur species(mono-and disulphides and elemental sulfur)when boiled for 40 min. The released H2S is trapped in 10 ml 5%ZnAc as ZnS.To reduce foaming in the trap a drop of antifoam is added to the pipet tip above the trap solution
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 13 of 115 3.3. Sulphate reduction Sulphate reduction rates are measured experimentally in sediment cores using a whole-core injection method modified from Jørgensen (1978). The distillation procedures are performed either hot (Fossing and Jørgensen, 1989) or cold (Kallmeyer et al., 2004) depending on the expected sulphate reduction rates. Field: • Sediment subcores are sampled in 26 mm ID acrylic tubes with injection ports filled with silicone rubber. • About 5 µl (400 kBq) radioactively labelled 34SO4 2- solution is injected into the sediment in 1-cm depth intervals and incubated for 6-24 h at in situ temperature. • The bacterial sulphate reduction is stopped in 20% (w/v) zinc acetate by mixing sediment with a known amount of 20% zinc acetate (ZnAc) solution (20 g zinc acetate dihydrate in 100 mL water) . The ZnAc volume to sediment volume should be 2:1. ZnAc preserves the radiolabelled sulphide as Zn35S and stops bacterial activity. It is important for optimal storage, samples should be frozen. • Blank samples (i.e. sediment samples with no Zn35S formed) are prepared when 5 cm3 sediment is preserved in 10 ml 20 %-ZnAc (w/v) prior to 35SO4 2- addition. About 10 µl (800 kBq) 35SO4 2- is added to the preserved sample after at least 1 hour (i.e. when sulphate reduction has come to a complete stop). The blank samples are used during distillation to estimate the amount of non-reduced 35S that are transferred from the labeled sediment to the trap. See Kallmeyer et al.(2004) for a complete discussion of blanks and detection limits. Laboratory: • The weight of each vial + sediment + ZnAc is determined. The empty vial weight (mean of several weighings of empty vials) and the weight of ZnAc solution may be subtracted to give the sediment wet weight. • The samples are centrifuged (4500 rpm, 5 min; sandy sediments 6000 rpm) and the supernatant carefully removed and kept for further analysis. • 35SO4 2- radioactivity of the supernatant is determined on a 0.1 ml sample in 1 ml H2O and 7 ml Lumasafe plus (Lumac BV.) in the scintillation counter. Distillation techniques: Hot distillations can be performed on samples with an expected sulphate reduction rate > 1 nmol SO4 2- cm-3 day-1 (see also Fossing and Jørgensen, 1989): • Approx. 1-2 g sediment (exact weight must be known) is transferred to a flask and mixed with 10 ml 50% ethanol. The flask is attached to the distillation apparatus and connected to a distillation trap through a condenser. • The flask is flushed with N2 for 10 min, after which 8 ml HCl (6 M) and 16 ml CrIIsolution (1M) are added to liberate all reduced inorganic sulfur species (mono- and disulphides and elemental sulfur) when boiled for 40 min. • The released H2S is trapped in 10 ml 5% ZnAc as ZnS. To reduce foaming in the trap a drop of antifoam is added to the pipet tip above the trap solution
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 14 of 115 The activity of the total reduced inorganic sulfur species(TRIS)is determined by liquid scintillation counting.Half the volume of the ZnAc trap is quantitatively transferred into a counting vial and mixed with 10 ml of Lumasafe plus (Lumac BV.) PEEK Tubing Citrate trap Zinc Acetate trap icals Magnetic stirre Cold distillation is recommended for samples having expected sulphate reduction rates of<1 nmol SO42cm day(see also Kallmeyer et al.,2004): The sediment is resuspended in 20 ml 1,2 N-N dimethylformamide(technical grade), transferred to a flask with a magnetic stirrer and a drop of antifoam,and the flask isattached to the distillation apparatus. In cases where the samples contain only small amount of reduced sulfur carrier is added, usually 0.5 mL of 50 mM ZnS suspension The flask is flushed with N2 for 10 min,after which 8 ml HCI(6 M)and 16 ml CrII- solution(1M)are added to reduce all reduced inorganic sulfur species(mono-and disulphides and elemental sulfur).The released H2S is trapped in 7 ml ZnAc(5%)during 2 h of destillation.To reduce foaming in the trap a drop of antifoam is added to the pipet tip above the trap solution. The activity of the total reduced inorganic sulfur species(TRIS)is determined by liquid scintillation counting.The entire volume of the ZnAc trap is quantitatively transferred into a counting vial and mixed with 14 ml of Lumasafe plus (Lumac BV.) Calculation: The sulphate reduction rate is calculated as SRR=a. s106 nmol cm day A+a 1
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 14 of 115 • The activity of the total reduced inorganic sulfur species (TRIS) is determined by liquid scintillation counting. Half the volume of the ZnAc trap is quantitatively transferred into a counting vial and mixed with 10 ml of Lumasafe plus (Lumac BV.) Cold distillation is recommended for samples having expected sulphate reduction rates of < 1 nmol SO4 2- cm-3 day-1 (see also Kallmeyer et al., 2004): • The sediment is resuspended in 20 ml 1,2 N-N dimethylformamide (technical grade), transferred to a flask with a magnetic stirrer and a drop of antifoam, and the flask isattached to the distillation apparatus. • In cases where the samples contain only small amount of reduced sulfur carrier is added, usually 0.5 mL of 50 mM ZnS suspension • The flask is flushed with N2 for 10 min, after which 8 ml HCl (6 M) and 16 ml CrIIsolution (1M) are added to reduce all reduced inorganic sulfur species (mono- and disulphides and elemental sulfur). The released H2S is trapped in 7 ml ZnAc (5 %) during 2 h of destillation. To reduce foaming in the trap a drop of antifoam is added to the pipet tip above the trap solution. • The activity of the total reduced inorganic sulfur species (TRIS) is determined by liquid scintillation counting. The entire volume of the ZnAc trap is quantitatively transferred into a counting vial and mixed with 14 ml of Lumasafe plus (Lumac BV.) Calculation: The sulphate reduction rate is calculated as [ ] 1.06 24 2 4 ⋅ ⋅ ⋅ + = − SO A a t a SRR nmol SO4 2- cm-3 day-1
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 15 of 115 where a is the total radioactivity of ZnS in one volume of fresh sediment,4 is the total radioactivity of 3SOin one volume of fresh sediment after incubation,is the incubation time in hours,/SO/is the sulphate concentration in nmol per cm fresh sediment,and 1.06 is the correction factor for the expected isotope fractionation. References: Jorgensen,B.B.(1978)A comparison of methods for the quantification of bacterial sulfate reduction in coastal marine sediments.I.Measurement with radiotracer techniques. Geomicrobiol.J.1:11-27. Fossing,H.,and Jorgensen B.B.(1989)Measurement of bacterial sulfate reduction in sediments.Evaluation of a single-step chromium reduction method.Biogeochemistry 8:205- 222 Kallmeyer,J.,Ferdelman T.,Weber,A.,Fossing,H.,and Jorgensen B.B.(2004).A cold chromium distillation procedure for radiolabeled sulfide applied to sulfate reduction measurements.Limnol.Oceanogr.Methods.2:171-180. Contact: Tim Ferdelman,Max Planck Institute for Marine Microbiology,Bremen,Germany(e-mail: tferdelm@mpi-bremen.de )
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 15 of 115 where a is the total radioactivity of ZnS in one volume of fresh sediment, A is the total radioactivity of 35SO4 2- in one volume of fresh sediment after incubation, t is the incubation time in hours, [SO4 2-] is the sulphate concentration in nmol per cm-3 fresh sediment, and 1.06 is the correction factor for the expected isotope fractionation. References: Jørgensen, B.B. (1978) A comparison of methods for the quantification of bacterial sulfate reduction in coastal marine sediments. I. Measurement with radiotracer techniques. Geomicrobiol. J. 1:11-27. Fossing, H., and Jørgensen B.B.(1989) Measurement of bacterial sulfate reduction in sediments. Evaluation of a single-step chromium reduction method. Biogeochemistry 8: 205- 222. Kallmeyer, J., Ferdelman T., Weber, A., Fossing, H., and Jørgensen B.B. (2004). A cold chromium distillation procedure for radiolabeled sulfide applied to sulfate reduction measurements. Limnol. Oceanogr. Methods. 2:171-180. Contact: Tim Ferdelman, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: tferdelm@mpi-bremen.de )