HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 16 of 115 3.4. Methanogenesis from CO2+H2 and from acetate Methanogenesis from H2 and CO2 or from acetate is measured experimentally in subcore samples using C-labeled bicarbonate or acetate as tracers.Methanogenic prokaryotes may reduce CO2 with hydrogen,or utilize organic compounds(e.g.acetate)to produce methane. This process generally occurs in marine sediments after sulphate-reduction in the upper few metres has depleted,or removed,pore-water sulphate concentrations thus allowing methanogens to successfully compete for hydrogen and organics.Methanogenesis is the dominant prokaryotic process in the deep sub seafloor.After injection and incubation evolved methane is oxidised to CO2 by flushing through a furnace containing copper oxide and collected with a CO2-scrubbing compound(phenethylamine),after the method of Whelan (1985). Field: At the core processing station sub-sample mini-cores(2.2 cm diameter)are taken with clean Perspex tubes forced into the cut core surface for 10 cm.During this process a vacuum is applied by sucking a tube attached to a stopper at the top of the tube to ensure that the sediment mini-core is not compressed during sampling.The tubes have been pre- drilled with 1 mm ports at 1 cm intervals along their length and these have been sealed with a silicone based aquarium sealant. The tubes are stoppered with a butyl rubber bung and stored temporarily,at the in situ temperature before being transported to the isotope station. Typically the mini-cores are left to equilibrate for 6-12 hours prior to injection.A 10 ul injection micro-syringe(Hamilton)is flushed thoroughly with the isotope(at least 5 times),ensuring that there are no air bubbles.The micro-syringe needle is inserted laterally through the ports in the side of the tube at 2 cm intervals and 2 ul of isotope are injected Amounts of radiotracer injected at each port are approximately; i)4C-bicarbonate 2 ul=50 Kbeq as sodium C-bicarbonate(Amersham,UK), previously diluted 1:4 with de-gassed,filter-sterilized(0.2 um)distilled water ii)14C-acetate 2 ul=15 Kbeq as sodium [1-(2)C]-acetate (Amersham,UK),used undiluted. At the conclusion of the injections the micro-syringe is thoroughly rinsed with distilled water(10 times),to remove any residual isotope. After injections all mini-cores are incubated at in situ temperature for 6 hours(acetate)or 18 hours (bicarbonate). Incubations are terminated by piston extrusion of 2 cm sections of mini-core that are sliced off and put immediately into glass jars containing 7 ml of 1.0 M NaOH.The jars are tightly sealed with a butyl rubber bung,shaken to reduce the core section to,a slurry, taped for security and stored upside down at room temperature to await processing
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 16 of 115 3.4. Methanogenesis from CO2 + H2 and from acetate Methanogenesis from H2 and CO2 or from acetate is measured experimentally in subcore samples using 14C-labeled bicarbonate or acetate as tracers. Methanogenic prokaryotes may reduce CO2 with hydrogen, or utilize organic compounds (e.g. acetate) to produce methane. This process generally occurs in marine sediments after sulphate-reduction in the upper few metres has depleted, or removed, pore-water sulphate concentrations thus allowing methanogens to successfully compete for hydrogen and organics. Methanogenesis is the dominant prokaryotic process in the deep sub seafloor. After injection and incubation evolved methane is oxidised to CO2 by flushing through a furnace containing copper oxide and collected with a CO2 – scrubbing compound (phenethylamine), after the method of Whelan (1985). Field: • At the core processing station sub-sample mini-cores (2.2 cm diameter) are taken with clean Perspex tubes forced into the cut core surface for 10 cm. During this process a vacuum is applied by sucking a tube attached to a stopper at the top of the tube to ensure that the sediment mini-core is not compressed during sampling. The tubes have been predrilled with 1 mm ports at 1 cm intervals along their length and these have been sealed with a silicone based aquarium sealant. • The tubes are stoppered with a butyl rubber bung and stored temporarily, at the in situ temperature before being transported to the isotope station. • Typically the mini-cores are left to equilibrate for 6 – 12 hours prior to injection. A 10 µl injection micro-syringe (Hamilton) is flushed thoroughly with the isotope (at least 5 times), ensuring that there are no air bubbles. The micro-syringe needle is inserted laterally through the ports in the side of the tube at 2 cm intervals and 2 µl of isotope are injected. • Amounts of radiotracer injected at each port are approximately; i) 14C-bicarbonate 2 µl = 50 Kbeq as sodium 14C-bicarbonate (Amersham, UK), previously diluted 1:4 with de-gassed, filter-sterilized (0.2 µm) distilled water ii) 14C-acetate 2 µl = 15 Kbeq as sodium [1-(2)14C] -acetate (Amersham, UK), used undiluted. At the conclusion of the injections the micro-syringe is thoroughly rinsed with distilled water (10 times), to remove any residual isotope. • After injections all mini-cores are incubated at in situ temperature for 6 hours (acetate) or 18 hours (bicarbonate). • Incubations are terminated by piston extrusion of 2 cm sections of mini-core that are sliced off and put immediately into glass jars containing 7 ml of 1.0 M NaOH. The jars are tightly sealed with a butyl rubber bung, shaken to reduce the core section to, a slurry, taped for security and stored upside down at room temperature to await processing
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 17 of 115 Laboratory: Samples are processed through a"methane-furnace rig"using a method adapted from that described by Whelan et al(1985).The principle of this apparatus is that the sample vials are connected,using large diameter needles(19G),to a stream of carrier gas(99%N2,1% O2)flowing at 70 ml/min.The headspace is blown along copper tubing through a Vost tube of indicating silica-gel desiccant,followed by a CO2 trap(Supelco,UK)and a second desiccant trap.After this the gas sample passes through a wider(6 mm)steel tube packed with copper oxide in a cylindrical furnace(Carbolite,UK)at 800C where the 4CH4 is oxidized to 4CO2.The CO2 is collected by bubbling the gas flow through a series of two rubber-stoppered scintillation vials containing 10 ml of scintillant(Opti- Phase 3,Perkin Elmer,UK)mixed (93:7)with B-phenethylamine to capture the 4CO2. The scintillations are then counted on a scintillation counter and rates of methanogenesis are calculated. Reference: Whelan,J.K.,Oremland,R.,Tarafa,M.,Smith,R.,Howarth,R.,and Lee,C.,(1985). Evidence for sulfate-reducing and methane producing organisms in sediments from Sites 618, 619 and 622.In;Bouma,A.H.,Coleman,J.,Meyer,A.W.,et al.,Init.Repts.DSDP,96: Washington(U.S.Govt.Printing Office),767-775 Contact: Barry Cragg,School of Earth,Ocean and Planetary Sciences,Cardiff University,UK (e-mail: b.cragg@earth.cf.ac.uk
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 17 of 115 Laboratory: • Samples are processed through a “methane-furnace rig” using a method adapted from that described by Whelan et al (1985). The principle of this apparatus is that the sample vials are connected, using large diameter needles (19G), to a stream of carrier gas (99% N2, 1% O2) flowing at 70 ml/min. The headspace is blown along copper tubing through a Vost tube of indicating silica-gel desiccant, followed by a CO2 trap (Supelco, UK) and a second desiccant trap. After this the gas sample passes through a wider (6 mm) steel tube packed with copper oxide in a cylindrical furnace (Carbolite, UK) at 800°C where the 14CH4 is oxidized to 14CO2. The CO2 is collected by bubbling the gas flow through a series of two rubber-stoppered scintillation vials containing 10 ml of scintillant (OptiPhase 3, Perkin Elmer, UK) mixed (93:7) with β-phenethylamine to capture the 14CO2. The scintillations are then counted on a scintillation counter and rates of methanogenesis are calculated. Reference: Whelan, J.K., Oremland, R., Tarafa, M., Smith, R., Howarth, R., and Lee, C., (1985). Evidence for sulfate-reducing and methane producing organisms in sediments from Sites 618, 619 and 622. In; Bouma, A.H., Coleman, J., Meyer, A.W., et al., Init.Repts. DSDP, 96: Washington (U.S. Govt. Printing Office), 767-775 Contact: Barry Cragg, School of Earth, Ocean and Planetary Sciences, Cardiff University, UK (e-mail: b.cragg@earth.cf.ac.uk )
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 18 of 115 3.5. Anaerobic oxidation of methane The oxidation of methane is measured experimentally in sediments,either using a whole core injection technique or small glass tubes sealed with butyl rubber stoppers.Sediments are incubated with CH4 as a tracer.The method is modified from Iversen and Jorgensen (1985). The samples are incubated anaerobically withCHa-tracer,which is oxidized to CO2. Depending on the expected rate,the incubation time varies between 8 and 24 hours. The ongoing AOM process with the tracer is stopped by transferring the sample to 50 ml glass jars containing 25 ml NaOH(2,5 %w/v)->separation of CO2(dissolved in NaOH) and CH4(gaseous in headspace and is measured first) a)12+14CH4 concentration is measured by gas-chromatography: 200 ul of the headspace are subsampled for GC analyses.This amount is negligible in comparison to the total volume of the headspace.Artefacts in theCH4 measurements are therefore not introduced. the methane concentration is calculated from the methane peak area of the GC measurement: CH,[nmol ml-sedGC-Area*C*Vo 22.4*Volsediment (ml) Cr is the calibration factor(0.0007 at the MPI GC system),which is determined with standard gas mixtures for 200 ul injection volume;22.4 is the gas constant(mol 1)at room temperature. b)Headspace(containing 214CH)is burned and 4CH is measured indirectly as 14CO after burning: The sample headspace is connected to the oven by rubber tubing.It is then purged with air and the air/methane mixture is subsequently burned toCO2.TheCO2 is trapped in two succeeding 20 ml scintillation vials containing 1 ml phenylethylamine +7 ml ethylenglycolmonomethylether. Both vials are measured in the wet scintillation counter after the addition of 10 ml Ultima-Gold.The resulting counts (i.e.,CPMB)of both vials are summed and the amount ofCO2 calculated from the activity and the blank counts determined from a vial containing only the scintillation chemicals: 4 CH [KBq]= CPMBsamplea+b-CPMBlak 60000
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 18 of 115 3.5. Anaerobic oxidation of methane The oxidation of methane is measured experimentally in sediments, either using a whole core injection technique or small glass tubes sealed with butyl rubber stoppers. Sediments are incubated with 14CH4 as a tracer. The method is modified from Iversen and Jørgensen (1985). • The samples are incubated anaerobically with 14CH4-tracer, which is oxidized to 14CO2. Depending on the expected rate, the incubation time varies between 8 and 24 hours. • The ongoing AOM process with the tracer is stopped by transferring the sample to 50 ml glass jars containing 25 ml NaOH (2,5 %, w/v) → separation of CO2 (dissolved in NaOH) and CH4 (gaseous in headspace and is measured first) a) 12+14CH4 concentration is measured by gas-chromatography: • 200 µl of the headspace are subsampled for GC analyses. This amount is negligible in comparison to the total volume of the headspace. Artefacts in the 14CH4 measurements are therefore not introduced. • the methane concentration is calculated from the methane peak area of the GC measurement: Sediment (ml) -1 F Headspace (ml) 4 22.4*Vol GC - Area *C *Vol CH [nmol ml-sed ] = CF is the calibration factor (0.0007 at the MPI GC system), which is determined with standard gas mixtures for 200 µl injection volume; 22.4 is the gas constant (mol l-1) at room temperature. b) Headspace (containing 12+14CH4) is burned and 14CH4 is measured indirectly as 14CO2 after burning: • The sample headspace is connected to the oven by rubber tubing. It is then purged with air and the air/methane mixture is subsequently burned to 14CO2. The 14CO2 is trapped in two succeeding 20 ml scintillation vials containing 1 ml phenylethylamine + 7 ml ethylenglycolmonomethylether. • Both vials are measured in the wet scintillation counter after the addition of 10 ml Ultima-Gold. The resulting counts (i.e., CPMB) of both vials are summed and the amount of 14CO2 calculated from the activity and the blank counts determined from a vial containing only the scintillation chemicals: 60000 CPMB - CPMB CH [KBq] Sample a b Blank 4 14 + =
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 19 of 115 Quarz tube with copper(ID)oxide and quarz wool Direct connection flow-meter 1.stop-cock Oven Headspace Wahing bottle Sample vial with artificial air NaOH and sediment Scintillation 2.stop-cock vials 西 Washing bottle c)CO2-diffusion method:24CO2 is removed from the NaOH by acidification: The lid is removed and the 50 ml jars containing the samples are weighed(if sediment volume is unknown). The samples are transferred from the jars into 250 ml Erlenmeyer flasks.The empty glass jar is weighed again to determine sample mass,which can be calculated to volume with porosity values. 6 ml scintillation vial containing 1ml phenylethylamine and Iml 0,5M NaOH is connected with the rubber stopper(rubber-stopper construction)in a"free floating" position. 6ml 6M HCl are injected between rubber stopper and flask neck to acidify the sample in order draw out the 4CH4. .The closed Erlenmeyer flask are shacked for 4 hrs to promote trapping of the CO2 in the phenylethylamine. the radioactivity is measured in the wet scintillation counter after the addition 3ml of Ultima Gold.The amount ofCO2 calculated from the activity of the sample and a blank containing only the scintillation chemicals: 4C02[KBq]= CPMBsample -CPMBk 60000 d)Rate calculation: Two formulae may be applied to calculate AOM: 14CO conc.CH AOM=CHincubat.Time (1)
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 19 of 115 c) CO2-diffusion method: 12+14CO2 is removed from the NaOH by acidification: • The lid is removed and the 50 ml jars containing the samples are weighed (if sediment volume is unknown). • The samples are transferred from the jars into 250 ml Erlenmeyer flasks. The empty glass jar is weighed again to determine sample mass, which can be calculated to volume with porosity values. • 6 ml scintillation vial containing 1ml phenylethylamine and 1ml 0,5M NaOH is connected with the rubber stopper (rubber-stopper construction) in a “free floating” position. • 6ml 6M HCl are injected between rubber stopper and flask neck to acidify the sample in order draw out the 14CH4. • The closed Erlenmeyer flask are shacked for 4 hrs to promote trapping of the 14CO2 in the phenylethylamine. • the radioactivity is measured in the wet scintillation counter after the addition 3ml of Ultima Gold. The amount of 14CO2 calculated from the activity of the sample and a blank containing only the scintillation chemicals: 60000 CPMB - CPMB CO [KBq] Sample Blank 2 14 = d) Rate calculation: Two formulae may be applied to calculate AOM: incubat.Time conc.CH CH CO CO AOM 4 2 14 4 14 2 14 × + = (1) Wahing bottle Sample vial with NaOH and sediment Washing bottle 2. stop-cock Scintillation vials Quarz tube with copper(II)oxide and quarz wool 1. stop-cock flow-meter artificial air Direct connection Headspace Oven
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 20 of 115 Here,CO2 and CH are the activities(Bq)of carbon dioxide and methane,whereas conc. CH4 is the concentrations of methane at the beginning of the incubation. If significant amounts of methane have escaped during the incubation,the following formula can be used as it accounts for this loss. “C02 conc..CH, AOM=CHincubat.Time (2) Here,CO2 and 4CH are the activities(Bq)of carbon dioxide and methane,whereas conc. CH4 is the concentrations of methane at the end of the incubation. Reference: Iversen,N.and Jorgensen B.B.(1985)Anaerobic methane oxidation rates at the sulfate- methane transition in marine sediments from Kattegat and Skagerrak (Denmark).Limnol. Oceanogr.30:944-955. 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 20 of 115 Here, 14CO2 and 14CH4 are the activities (Bq) of carbon dioxide and methane, whereas conc. CH4 is the concentrations of methane at the beginning of the incubation. If significant amounts of methane have escaped during the incubation, the following formula can be used as it accounts for this loss. incubat.Time conc.CH CH CO AOM 4 4 14 2 14 = × (2) Here, 14CO2 and 14CH4 are the activities (Bq) of carbon dioxide and methane, whereas conc. CH4 is the concentrations of methane at the end of the incubation. Reference: Iversen, N. and Jørgensen B.B. (1985) Anaerobic methane oxidation rates at the sulfatemethane transition in marine sediments from Kattegat and Skagerrak (Denmark). Limnol. Oceanogr. 30: 944-955. Contact: Helge Niemann, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: hniemann@mpi-bremen.de )