Flash ea 1112 series

The average hydrodynamic particle size and the size distribution were measured by dynamic light scattering (DLS) on a Nanosizer Nano Zetasizer instrument (Malvern Instruments, Malvern, UK), after the particles were dispersed in ethanol via ultrasonication for 30 min. The lateral diameter of the LDH nanoparticle was examined by scanning electron microscopy (SEM) on a JEOL 6300 SEM. LDH samples were dispersed in 75% ethanol solution via ultrasonication for 10 mins, and then, the SEM images were taken at 10–15 kV with magnifications of 20,000–80,000. Powder X-ray diffraction (XRD) patterns were recorded on a Rigaku Miniflex X-ray Diffractometer using Co Kα source (λ = 0.178897 nm) at a scanning rate of 0.02°/s (2θ) from 2θ = 2° to 2θ = 80°. Fourier transform infrared (FTIR) spectra were obtained on a Nicolet 6700 FTIR (Thermo Scientific, Waltham, MA, USA) in the range of 4000–400 cm⁻¹ by accumulating 32 scans at a resolution of 4 cm⁻¹. Inductively coupled plasma optical emission spectrometry (ICP-OES) was conducted on a Varian axial Vista CCD Simultaneous (Varian, Mulgrave, Australia) with the wavelength used for Mg and Al at 383.829 and 237.312 nm, respectively. The content of C, N and H was measured on a CHNS–O analyser (Flash EA 1112 Series, Thermo Scientific). The drug loading capacity was calculated as the drug mass divided by the LDH–drug complex mass.

The samples’ chemical composition was studied using a Thermo Fisher FlashEA 1112 Series elemental analyzer (Waltham, MA, USA).

All beaks were dried at 60 °C for 24–48 hours and ground into a fine powder. Powder sub-samples were weighed (to the nearest 0.3 mg) with a micro-balance and sterile-packed in tin containers. Stable isotope values were determined by a Flash EA 1112 Series elemental analyser coupled online via a Finnigan ConFlo II interface to a Delta VS mass spectrometer (Thermo Scientific) and expressed as: δ¹³C and δ¹⁵N = [(R_sample/R_standard) − 1] * 1000, where R = ¹³C/¹²C and ¹⁵N/¹⁴N, respectively. The carbon and nitrogen isotope ratios were expressed in delta (δ) notation relative to Vienna-PeeDee Belemnite limestone (V-PDB) for δ¹³C and atmospheric nitrogen (AIR) for δ¹⁵N. Replicate measurements of internal laboratory standards (acetanilide STD: Thermo Scientific PN 338 36700) in every batch (n = 14) indicated precision <0.2‰ for both δ¹³C and δ¹⁵N values. Mean mass C:N ratio of all samples was 3.19 ± 0.02.

The ¹³C isotopic discrimination in cyanobacterial cells was obtained from a 50 mL culture aliquot at OD₆₅₀ of 0.8–1, which was centrifuged at 10,000×g for 3 min. The pellet was freeze-dried overnight and homogenized. 0.2 g of the dried powder was transferred into metallic capsules (176980926, Lüdiswiss, Switzerland) and combusted in an elemental analyzer (Thermo Flash EA 1112 Series, Germany) where CO₂ was injected into a continuous-flow isotope mass spectrometer (Thermo-Finnigan Delta XP, Bremen, Germany). Peach leaf standards (NIST 1547) were measured every six samples. Results are presented as δ vs. PDB (Pee Dee Belemnite). The obtained ¹³C isotopic discrimination of the cyanobacterial biomass (δ¹³C) was corrected with the ¹³C isotopic composition of DIC found in the medium from either CO₂-enriched or control experiments, as described by Iñiguez et al. (2016 (link)).

Carbon isotope composition was estimated in leaves with the same leaf size and position, count as leaf three unless abnormal. Samples for stomatal characterization were taken first, and the remaining fresh leaf tissue was dried at 80°C for 2 days to be used for δ¹³C determination. δ¹³C was analyzed in 2 mg aliquots of leaf sample weighed in tin capsules. Samples were combusted in an elemental analyzer (Thermo Flash EA 1112 Series, Bremen, Germany), CO₂ was separated by chromatography and directly injected into a continuous-flow isotope ratio mass spectrometer (Thermo Finnigan Delta V, Bremen, Germany) through the interface ConFlo IV dilutor device (Thermo Finningan, Bremen, Germany). Samples were measured in duplicate. The isotope ratios were expressed in δ‰ against Vienna-Pee Dee Belemnite for δ¹³C according to the following equation: $\delta$ ‰ = ( $R_{SA}-R_{REF})/R_{REF}$ where R_SA is the isotope ratio measured for the sample and R_REF is the international standard isotope ratio. The isotopic values were calculated using a linear equation against working in-house standards, which were themselves calibrated against the international reference materials L-glutamic acid USGS 40 (US Geological Survey, Reston, VA, United States), fuel oil NBS-22 and IAEA-CH-6. The uncertainty of measurement (calculated as 2 standard deviations) was 0.1‰.

Five randomly selected pots of each treatment were harvested every 5 days until ear formation (when grain begins to form) was observed on the early Tammi cultivar (60 days). During this period both cultivars produced flag leaves, the stage prior to grain production, when most nitrogen has already been absorbed (Spink et al., 2015 ). This covered the period most likely to contain the peak nitrogen and biomass accumulation rate for both cultivars, the focus of this study. The plants were then removed from the pots, the roots washed, and individual shoot and root material separated. The root and shoot material of each plant were dried at 30°C until a stable weight was reached and weighed. Milled shoot samples were analyzed for carbon and nitrogen concentration (Flash EA 1112 Series, Thermo Fisher Scientific, Bremen, Germany).

Extraction of leaf starch was performed as described in previous studies (Wanek et al., 2001 (link); Goettlicher et al., 2006 (link); Richter et al., 2009 (link)). Leaf starch was isolated from 50mg leaf material with methanol/chloroform/water (MCW, 12:5:3, v/v/v) at 70°C for 30min. Samples were centrifuged (10 000 ×g, 2min) and supernatants removed, while the leaf-starch-containing pellets were washed with MCW and deionized water and dried at room temperature (RT). Pellets were then re-suspended in water and boiled at 99°C for 15min to facilitate starch gelatinization. Subsequently, leaf starch was enzymatically digested with α-amylase (EC 3.2.1.1, Sigma-Aldrich, Buchs, Switzerland) at 85°C for 2h, and cleaned with centrifugation filters to remove enzymes (Vivaspin, Sartorius, Göttingen, Germany). To determine δ¹³C of bulk leaves (δ¹³C_leaf) and starch, an elemental analyser (Flash EA 1112 Series) coupled to a Delta^plusXP-IRMS was used (both Thermo Fisher, Bremen, Germany; Werner et al., 1999 (link)). Measurements of samples, blanks, and reference material followed the identical treatment principle described by Werner and Brand (2001) (link). The long-term precision of a quality control standard for all sequences was SD≤0.12‰.