Paull's Pingo is a seafloor mound feature (latitude 33.799° N, longitude 118.646° W; depth ~ 820 m) formed by the expansion of subsurface methane hydrate28 . We accessed active methane seeps at the pingo to collect sediment cores using Deep Submergence Vehicle (DSV) Alvin, during R/V Atlantis Leg AT15-53 (September 2009). Sediment core processing was conducted shipboard in an anaerobic chamber, flushed with a nitrogen headspace. One sediment core was sub-sectioned between 5 and 15 cm (relative to seafloor) and dedicated to methane-amended incubations. Two subsamples of 60 mL sediment were homogenized with 20 mL of sterile, anoxic artificial seawater medium29 . Incubations with the homogenized sediments were prepared in 120-mL serum vials, under a 40-mL headspace of ~3% CH4 and 97% N2. Incubations were amended with 13C-labeled methane (99 atom-% 13C) as an exogenous tracer to track methane oxidation (Supplementary Fig. 1 ). Stable isotope ratios (δ13C) for CO2 were measured by isotope ratio mass spectrometry (Thermo Finnigan Delta XP Plus in continuous flow mode). After one month of enrichment, the incubation was terminated and viruses were purified for DNA sequencing.
Finnigan delta plus xp
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Finnigan Delta Plus XP is a high-performance isotope ratio mass spectrometer (IRMS) designed for accurate and precise measurements of stable isotope ratios. It provides reliable and consistent data for a wide range of applications, including environmental, geological, and biological research.
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Lab products found in correlation
Thermo flash ea 1112, by Thermo Fisher Scientific (2 mentions)
Flash ea 1112, by Thermo Fisher Scientific (2 mentions)
Conflo 3, by Thermo Fisher Scientific (1 mentions)
2010 plus, by Shimadzu (1 mentions)
Gcms qp2010 ultra, by Shimadzu (1 mentions)
Hi9125, by Hanna Instruments (1 mentions)
K15no3, by Merck Group (1 mentions)
25 mm gf f filters, by Cytiva (1 mentions)
Tc ea, by Thermo Fisher Scientific (1 mentions)
12 protocols using finnigan delta plus xp
1
Methane Seep Sediment Incubation
2
Water Column SPM Isotopic Analysis
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Water column SPM data were obtained from water samples taken with Niskin bottles from 0 to 30 m depth at the inner and the outer cove during the austral summers 2007–2008 and 2008–2009. The water samples were filtered with GF-F glass fiber filters (47 mm diameter, 0.7 μm pore size), treated drop by drop with 1 M HCl and rinsed with distilled water to eliminate carbonates. Lipids were extracted in Chloroform-methanol (2:1 vol:vol). The isotopic analysis was performed in a mass spectrometer (IRMS) (Thermo Finnigan Delta XP Plus) connected by a Thermo Finnigan Conflo III to an elemental analyzer (Thermo Flash EA 1112).
3
Methane Stable Isotope Analysis Protocol
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Gas compositions were analysed by the same method as described above. δ3CCH4 was measured by coupling a Trace GC ultra gas chromatograph via a GC combustion interface (Thermo Surveyor) to a mass spectrometer (Thermo Finnigan Delta Plus XP).
Additional gas samples from gas emission spot 1 were analysed for δ13C and δ2H in CH4 at Imprint Analytics GmbH (Neutal, Austria) using a gas chromatograph (Shimadzu GC 2010Plus, Shimadzu Corp., Kyoto, Japan) coupled to a stable isotope ratio mass spectrometer (Nu Horizon; Nu Instruments Limited, Wrexham, UK). Gas samples were directly injected by an AOC5000 Autosampler (CTC Analytics, Zwingen, Switzerland) from the Labco Exetainers into the S/Sl Injector of the GC. The CH4 was separated from other gases in a Q-Plot GC column (Supelco, Bellefonte USA) at 35°C (isothermal). The separated analytes were identified in a quadrupole mass spectrometer (Shimadzu GCMS-QP2010Ultra). Methane was oxidized to CO2 for the δ13C analyses in an oxidation oven filled with oxidized Ni and Pt wires at a temperature of 1040°C. For the δ2H analysis, CH4 was pyrolysed at 1400°C to H2 in a ceramic tube (Hekatech, Wegberg, Germany). As reference material hexane vapour with a known δ13C and δ2H composition was used. δ13C is reported in ‰ VPDB, δ2H in ‰ SMOW.
Additional gas samples from gas emission spot 1 were analysed for δ13C and δ2H in CH4 at Imprint Analytics GmbH (Neutal, Austria) using a gas chromatograph (Shimadzu GC 2010Plus, Shimadzu Corp., Kyoto, Japan) coupled to a stable isotope ratio mass spectrometer (Nu Horizon; Nu Instruments Limited, Wrexham, UK). Gas samples were directly injected by an AOC5000 Autosampler (CTC Analytics, Zwingen, Switzerland) from the Labco Exetainers into the S/Sl Injector of the GC. The CH4 was separated from other gases in a Q-Plot GC column (Supelco, Bellefonte USA) at 35°C (isothermal). The separated analytes were identified in a quadrupole mass spectrometer (Shimadzu GCMS-QP2010Ultra). Methane was oxidized to CO2 for the δ13C analyses in an oxidation oven filled with oxidized Ni and Pt wires at a temperature of 1040°C. For the δ2H analysis, CH4 was pyrolysed at 1400°C to H2 in a ceramic tube (Hekatech, Wegberg, Germany). As reference material hexane vapour with a known δ13C and δ2H composition was used. δ13C is reported in ‰ VPDB, δ2H in ‰ SMOW.
4
Carbon Isotope Measurements via irm-GC/MS
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Carbon concentration and isotopic measurements were conducted on 0.2 μm-filtered effluent water samples <24 h after collection. Measurements were performed on an isotope-monitoring gas chromatography/mass spectrometry (irm-GC/MS) Thermo Finnigan Delta Plus XP isotope-ratio mass spectrometer connected to TRACE GC as previously described (Ijiri et al., 2012 (link)).
5
Deadwood Carbon Isotope Analysis
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The 49 freeze‐dried and milled deadwood samples were weighed into tin capsules for C% and δ13C measurements in an elemental analyser (EA; Costech 4010; Costech Analytical Technologies Inc., Valencia, CA, USA) connected in continuous‐flow mode to an isotope ratio mass spectrometer (IRMS; Finnigan Delta Plus XP; Thermo Fisher Scientific, Waltham, MA, USA). The standard deviations for repeated analyses of reference materials were better than 0.2‰. The isotope signature is expressed in the delta notation δ13C = (Rsample/Rstandard − 1) × 1,000 (‰) relative to the international standard VPDB, where R = 13C/12C of the sample or standard.
6
Soil Nitrogen Isotope Analysis Protocol
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The δ15N value of soil sample was measured by a Finnigan DeltaPlusXP (Thermo scientific, Waltham, Massachusetts, USA) coupled with an automatic elemental analyzer (Flash EA1112, Thermo Finnigan). The isotope result of soil N was calculated with the following equation: δ15N (‰) = (Rsample/Rstandard -1)×1000, where Rsample and Rstandard represent the ratios of 15N/14N in the sample standard. The standard of δ15N is atmospheric N2. The standard deviation of above isotopic repeated measurements was ±0.15‰.
Soil pH value was determined using a pH meter (HI-9125, Hanna Instruments Inc, Woonsocket, RI) with a dry soil-water ratio of 1:2.5. Total organic carbon (TOC) and nitrogen (TN) concentrations of soil samples were measured by a TOC/TN analyzer (Multi N/C 3100CLD, Jena, Germany). Soil C/N ratio was expressed by the quotient of TOC and TN content. Another subsample was separated into clay (<2μm) by ultrasonic energy method. The result of particle size analysis was expressed as a percentage of the weight of oven-dried soil. The values of all the above observed variables, which includes the MAP and MAT of sampling locations, soil N, soil pH, soil C: N, soil clay and soil δ15N, were listed inS1 Appendix .
Soil pH value was determined using a pH meter (HI-9125, Hanna Instruments Inc, Woonsocket, RI) with a dry soil-water ratio of 1:2.5. Total organic carbon (TOC) and nitrogen (TN) concentrations of soil samples were measured by a TOC/TN analyzer (Multi N/C 3100CLD, Jena, Germany). Soil C/N ratio was expressed by the quotient of TOC and TN content. Another subsample was separated into clay (<2μm) by ultrasonic energy method. The result of particle size analysis was expressed as a percentage of the weight of oven-dried soil. The values of all the above observed variables, which includes the MAP and MAT of sampling locations, soil N, soil pH, soil C: N, soil clay and soil δ15N, were listed in
7
Moisture, COD, and δ13C Analysis
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Moisture content was determined by drying samples for 24 h at 105°C. Chemical oxygen demand (CODCr) was measured using the closed reflux colorimetric method in reaction tubes with prepared reagents provided by Hach Company (Loveland, CO, USA), according to the manufacturer’s instructions. Analysis of δ13C was carried out at the International Research Center for Agricultural Sciences (Tsukuba, Japan) using an infrared mass spectrometer (Finnigan Delta PLUS XP; Thermo Scientific, Hamburg, Germany) connected to an element analyzer (EA Flash 1112; Carlo Erba, Milan, Italy). To detect total carbon, freeze-dried slurry samples were incinerated in the element analyzer furnace and separated as pure CO2 gas, a small quantity of which was used to measure the ratio of 13CO2/12CO2 as different mass weights of 45/44 to obtain δ13C (‰).
8
Measuring 15N Uptake in Wheat Roots
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For 15N uptake analysis, wheat seedlings were cultured in the hydroponic culture (supplemented with 2.5 mM KNO3, pH 5.8) for two weeks. After N starvation by culturing the seedlings in a hydroponic solution without N for 2 days, wheat roots were treated with K15NO3 (98 atom% 15N; SigmaAldrich, number 335134) for 30 min. After washing with 0.1 mM CaSO4 solution and deionized water as described previously38 (link), roots of seedlings were collected and dried at 70 °C for 3 days. After grinding the sample into powder, the 15N content in the root was measured using an isotope ratio mass spectrometer (Thermo Finnigan Delta Plus XP; Flash EA 1112) with three biological replicates.
9
Determining Bulk 13C Uptake and POC/PON
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Bulk DI13C uptake rates and POC and PON concentrations were determined from triplicate incubation bottles from each time point as described in Ref.37 (link). Briefly, 2.75 L of seawater were filtered onto pre-combusted (450 °C, 6 h) 25 mm GF/F filters (Whatman, St Louis, MO, USA) and stored at − 20 °C until further processing. Filters were acidified with fuming HCl in a desiccator overnight, oven dried for 1 h at 55 °C and folded in tin cups. Particulate organic C and N as well as the relative abundances of each isotope (i.e. 12C/13C) was determined by a Thermo Flash EA 1,112 elemental analyzer coupled to an isotopic ratio mass spectrometer (Finnigan Delta Plus XP, Thermo Fisher Scientific). CO2 fixation rates were subsequently calculated from the incorporation of 13C into biomass according to Ref.37 (link).
10
Stable Isotope Analysis of Wood and Soil
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All pooled wood and soil samples were analysed for δ13C and δ15N by combustion under excess oxygen in an elemental analyser (EA, Costech 4010, Costech Analytical Technologies Inc., Valencia, CA, USA) connected in continuous-flow mode to an isotope ratio mass spectrometer (IRMS, Finnigan Delta Plus XP, Thermo Fisher Scientific, Waltham, MA, USA). We used 6–18 mg of a sample for the analysis, depending on the sample’s N percentage. The standard deviation derived from repeated analyses of reference materials was smaller than 0.2‰ for δ13C and smaller than 0.3‰ for δ15N. We expressed the isotope signature in the delta notation δ13C = (Rsample/Rstandard−1) × 1000 (‰) relative to the international standard, which is VPDB ‘Vienna Pee Dee Belemnite’ for carbon and atmospheric N2 for nitrogen.
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