Delta 5 advantage

Body water and muscle protein enrichment was measured as previously described [18] (link). Saliva was heated in a vial at 100 °C, then cooled rapidly on ice and the condensate transferred to a clean vial ready for analysis. Deuterium enrichment was measured on a high-temperature conversion elemental analyser connected to an isotope ratio mass spectrometer (TC/EA-IRMS Thermo Finnigan, Hemel Hempstead, UK). Myofibrillar proteins were extracted as previously described; briefly, the myofibrillar fraction was isolated by centrifugation from the sarcoplasmic protein solubilized in 0.3M NaOH and separated from the insoluble collagen by centrifugation. The protein was then precipitated, acid hydrolysed, and the free amino acids were purified and derivatised as their n-methoxycarbonyl methyl esters (MCME) (Wilkinson 2014). Incorporation of deuterium into protein bound alanine was determined by gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-pyrolysis-IRMS, Delta V Advantage; Thermo Finnigan, Hemel Hempstead, UK) alongside a standard curve of known dl-Alanine-2,3,3,3-d4 enrichment to validate measurement accuracy.

Two micrograms of DNA was placed into a tin capsule (Thermo Scientific), and dried for approximately 2.5 h at 50°C. Freeze-dried rhizosphere soil samples from the unlabeled and ¹³CO₂-labeled chambers were milled to pass through a 200-mesh sieve. Between 2 and 4 mg of soil from each sample was weighed, and both the DNA and soil samples were examined with an elemental analyzer (Thermo Scientific FLASH 2000, Germany) coupled online to an isotope ratio mass spectrometer (Delta V advantage, Thermo Finnigan, Germany). The δ¹³C (‰) was calculated as δ¹³C (‰) = [(Rsample – Rstandard)/Rstandard] × 1000, where R is the molar ratio of ¹³C to ¹²C. DNA concentration in the ¹³C-labeled sample was calculated as ¹³C-DNA (ng g^–1 soil) = ¹³C atom% excess × DNA concentration (ng g^–1 soil), where ¹³C atom% excess = ¹³C atom% of the samples from ¹³CO₂-labeled chamber –unlabeled chamber.

The ¹³C isotopic discrimination in the algal samples (δ¹³C_alga) was determined by mass spectrometry using a DELTA V Advantage (Thermo Electron Corporation, USA) Isotope Ratio Mass Spectrometer (IRMS) connected to a Flash EA 1112 CNH analyser, as described by Iñiguez et al. (2016) . The ¹³C isotopic discrimination of the dissolved inorganic carbon found in the medium (δ¹³C_DIC) was measured with the same IRMS connected to a GasBench II (Thermo Electron Corporation) system, using 20 ml FSW collected from each cylinder, previously filtered (Whatman GF/F). The δ¹³C_alga was corrected with the δ¹³C_DIC values from the medium, since the CO₂ source used in the experiment for the CO₂-enriched treatment came from previously fixed CO₂ that had been already discriminated.

The value of δ¹³C is used as a proxy of HCO₃⁻versus CO₂ only used by an aquatic primary producer, the former requiring a carbon concentrating mechanism (CCM). Typically, a value below (more negative than) − 30‰ indicates an inactive or absent CCM. However, this reference value should be taken cautiously, since it can be influenced by the specific δ¹³C value of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) for CO₂ fixation in a given species. The abundance of ¹³C relative to ¹²C in E. huxleyi samples was determined by mass spectrometry using a DELTA V Advantage (Thermo Electron Corporation, USA) Isotope Ratio Mass Spectrometer (IRMS) connected to a Flash EA 1112 CNH analyser. δ¹³C isotopic discrimination in the microalgae samples (δ¹³C_sample) was expressed in the unit notation as deviations from the ¹³C/¹²C ratio of the Pee-Dee Belemnite CaCO₃ (PDB, which is the same as VPDB) calculated according to (Eq. 3): $${\mathrm{\delta }}^{13}\mathrm{C}\left(‰\right)=[({\mathrm{\delta }}^{13}\mathrm{C}/{\mathrm{\delta }}^{12}\mathrm{C}{)}_{\mathrm{sample}}/({\mathrm{\delta }}^{13}\mathrm{C}/{\mathrm{\delta }}^{12}\mathrm{C}{)}_{\mathrm{PDB}}-1]\bullet {10}^3$$
To determine the isotopic composition of dissolved inorganic carbon (δ¹³C_DIC), 25 mL from each cylinder were filtered (Whatman GF/F). Measurements of δ¹³C_DIC were performed with the same IRMS mentioned above connected to a GasBench II (Thermo Electron Corporation) system. The δ¹³C_sample was corrected by δ¹³C_DIC values from the medium, previously tested in a TOC-L analyser.

For analysis, ¹³C-labeled NaHCO₃ (99% ¹³C, Cambridge Isotope Laboratories, MA, USA) was added into the culture medium as described above. Each sample was collected at different culture times using 0.3-μm glass fiber filters (GF-75, Advantec, Japan) that had been weighed and burned at 450 °C for 4 h. Sample filters were then freeze dried for about 2 days, after which they were weighed and packed into tin cans. The ¹³C content in cells was determined using a Flash EA 1112 Series elemental analyzer coupled with a ConfloIII interface to a Delta V Advantage isotope ratio mass spectrometer (Thermo Electron, San Jose, CA, USA). All defined samples were collected and analyzed in triplicate.

The gas components (H₂, CH₄, and CO₂) were measured by gas chromatography (GC112A; Shanghai Precision and Scientific Instrument Co., Ltd, Shanghai, China) equipped with a flame ionization detector and thermal conductivity detector. The volume of the gas was determined using a gas meter (TG05/6; Ritter, Bochum, Germany). The stable carbon isotopic compositions of the produced CH₄ (δ¹³CH₄) and CO₂ (δ¹³CO₂) were periodically monitored using isotope ratio mass spectrometry (Delta V Advantage; Thermo Electron Corporation, United States) linked to gas chromatography (6980N; Agilent Technologies, Santa Clara, CA, United States). The CO₂ gas standard (δ¹³C_VPDB = –27.5‰) was injected before and after each gas analysis. The pH of the liquid samples was determined using a pH meter (PXSJ-216F; Shanghai Precision and Scientific Instrument Co., Ltd., Shanghai, China). Subsequently, the samples were centrifuged at 4,460 g and 4°C for 10 min using a high-speed refrigerated centrifuge (TL-18 M; Shanghai Centrifugal Machinery Research Institute, Shanghai, China). The supernatant was collected for further analyses. Dissolved organic carbon (DOC) and inorganic carbon were measured using a Total Carbon/Total Nitrogen analyzer (TOC-VCPN, TNM-1, Shimadzu, Kyoto, Japan).

Determination of carbon and oxygen isotope abundance on ground bulk samples of leaves and seed was completed using a Delta V Advantage isotope ratio mass spectrometer (IRMS) (Thermo Electron) with a Conflo IV interface (ThermoFisher Scientific, Bremen, Germany).