Soil gas was sampled at 1 cm depth in control plots at intervals before and after glycine label addition. A 20 ml sample was extracted directly from the soil by hand from four points with a 25 mL plastic syringe and immediately flushed through the septum of a crimp sealed 1.8 ml vial. The vials were left over-pressurized with c. 1 ml sample until analysis of δ13CO2 at gas chromatograph (GC) (Hewlett–Packard 6890) in continuous flow mode with a Preparation Concentration unit (PreCon, Thermo Scientific, Germany) and stable isotope ratio mass spectrometer (IRMS) (Finnigan Delta PLUS, Thermo Scientific, Germany). These analyses were made at Technical University of Denmark.
Finnigan delta plus
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Finnigan Delta Plus is a high-performance isotope ratio mass spectrometer designed for precise and accurate measurement of stable isotope ratios. It is capable of analyzing a wide range of sample types, including gases, liquids, and solids.
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8 protocols using finnigan delta plus
1
Soil CO2 Isotope Sampling Protocol
2
Carbon Isotope Ratio Analysis
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One and a half mg of the dried tissue samples used for DM determinations, were placed in tin capsules to determine the amount of 13C and 12C in an elemental analyzer (Flash® EA, 1112 Series) coupled to an isotope ratio mass spectrometer (Thermo Finnigan Delta Plus®). The R-value (13C/12C molar abundance ratio of the sample) is expressed in relation to the Pee Dee Belemnite standard (PDB). The values of ∂13C ‰ were obtained through comparison of 13C/12C molar abundances of leaf samples from the different treatments with respect to the PDB standard according to O’Leary (1993) (link):
where ∂13C is the ratio of carbon isotopes, Rsample is the ratio of the 13C/12C molar abundance of the plant sample and RPDB is the ratio of the Standard 13C/12C molar abundance (PDB), which is equal to 0.0112372 (Squeo and Ehleringer, 2004 ).
where ∂13C is the ratio of carbon isotopes, Rsample is the ratio of the 13C/12C molar abundance of the plant sample and RPDB is the ratio of the Standard 13C/12C molar abundance (PDB), which is equal to 0.0112372 (Squeo and Ehleringer, 2004 ).
3
Sediment Nitrogen Isotope Analysis
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We took one 50-ml surface sediment sample per transect for δ15N analysis. The samples were stored at −20°C and transported frozen to the ZMT. They were then dried at 50°C in a forced air oven until constant DW, ground to a fine powder with mortar and pestle, and weighed into tin capsules prior to analysis for nitrogen stable isotope composition (δ15N) with a gas isotope ratio mass spectrometer (Thermo Finnigan Delta Plus, Waltham, MA, USA). Results are expressed in δ notation (‰) where the standard for δ15N is atmospheric N2.
4
Pore Water Nutrient Analysis
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Pore water was extracted from 2.5 mm sediment slices from 0 to 2 cm depth, and from 1 cm sediment slices from 2 to 12 cm depth by centrifugation (∼4000 × g, 5 min, through a 0.45 μm 25 mm Acrodisc HT Tuffryn Membrane syringe filter), and the obtained concentrations of the first 2 cm were averaged for the first and second centimeter of the sediment to estimate pore water nutrient concentrations in a 1 cm resolution. Pore water samples were stored in pre-rinsed pony vials and NH4+, NO3-, NO2-, and PO43- concentrations were analyzed as described by Bale et al. (2013) (link). The total organic carbon (TOC) and total organic nitrogen (TON) content per gram of freeze dried sediment were determined after acidification of freeze dried sediment (0.5-1 g) with 2 N HCl. Residues were analyzed by using a Thermo Finnigan Delta plus isotope ratio monitoring mass spectrometer (irmMS) connected to a Flash 2000 elemental analyzer (Thermo Fisher Scientific, Milan, Italy).
5
Urine Sampling for Deuterium and Oxygen-18 Analysis
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The urine samples were collected five times (at baseline and on days 1, 2, 13, and 14 after initiating DLW testing). Subjects were requested not to eat or drink for 30 minutes before each urine collection. Subjects were instructed to collect and record details of urine sampling to ensure result accuracy. During mornings when urine samples were collected, subjects discarded the first urine voided after waking up and sampled urine an hour later. Urine samples were stored in a freezer below −20°C before analysis. 2H and 18O in urine samples were analyzed using an isotopic mass analyzer (Finnigan Delta Plus; Thermo Fisher Scientific, Waltham, MA, USA).
6
Bulk Elemental and Isotopic Analysis
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Bulk elemental and stable isotope analyses were conducted following the methods described by Kim et al.43 (link). In brief, the TOC content of samples decalcified with 10% HCl for 24 h, the nitrogen contents in both bulk (total nitrogen, Ntot) and KOBr/KOH-treated (inorganic nitrogen, Ninorg) samples, and carbon isotopic compositions were determined at KOPRI using an elemental analyzer (Thermo Electron Corporation Flash EA 2000, Thermo Fisher Scientific, Germany) coupled with an isotope ratio mass spectrometer (Finnigan Delta Plus, Thermo Fisher Scientific, Germany). The carbon isotope ratios of TOC (δ13Corg) were reported using δ notation (per mil) with respect to the Vienna Pee Dee Belemnite (VPDB). The analytical precision was better than 0.5 wt.% and 0.5‰ for carbon and 0.5 wt.% for nitrogen. Accelerator mass spectrometry (AMS) radiocarbon (14C) analyses of TOC were performed at the MICADAS radiocarbon laboratory at the Alfred-Wegener Institute (AWI, Bremerhaven, Germany) and the Center for Applied Isotope Studies at the University of Georgia (CAIS, Georgia, USA), following standard procedures. Radiocarbon results were presented in Delta notation (Δ14Corg, ‰), as defined by Stuiver and Polach68 (link).
7
Stable Carbon Isotope Analysis
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Between three and six samples from young leaves were taken for each cultivar and treatment at sampling time. Samples were dried for 48 h in an oven at 60 • C and ground into powder. Subsamples of 2 mg were analyzed for isotope ratio (δ 13 C). Samples were combusted in an elemental analyzer (Thermo Flash EA 1112 Series, Bremen, Germany); CO 2 was separated by chromatography and directly injected into a continuous-flow isotope ratio mass spectrometer (Thermo Finnigan Delta Plus, Bremen, Germany). Peach leaf standards (NIST 1547) were run every eight samples. δ 13 C was calculated as: δ 13 C sample (% ) = ((R sample/R standard) -1) × 1000 [49] (link).
8
Carbonate Isotopic Analysis Protocol
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For carbonate-bound carbon and oxygen isotopes, untreated powders were weighed into glass vials and reacted with concentrated phosphoric acid at 80°C in a Kiel III Carbonate-Device. The resulting CO2 gas was purified cryogenically and analyzed with a dual-inlet IRMS (Thermo Finnigan Delta Plus). As for carbon and nitrogen isotopes, we used a two-point calibration to correct the raw data. The average precision was 0.04‰ for both δ 13 Ccarb and δ 18 Ocarb. We assumed calcite mineralogy for the oxygen isotope measurements, as confirmed by high molar Ca/Mg ratios measured in Na-acetate extracts (range 4 to 283, median 40, excluding samples with less than 2% carbonate; see below). The CO2 gas pressure was calibrated for carbonate quantity (relative precision 6.4%). Carbon and oxygen isotope data are reported in standard delta notation, referenced to VPDB for C and O.
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