Delta plus

Soil samples were collected from all plots and microplots using a 5-cm i.d. auger and separated into 20-cm depth increments from 0 to 1m depth before planting and after the harvest of wheat and cotton (Fig 2). Soil samples were stored in an ice-box immediately after sampling and were then transported to the laboratory for analysis. Within 12 h all the fresh soil samples were extracted with 0.01 mol L^-1 CaCl₂ solution (ratio of soil to solution 1:10). The extracts were analyzed for NO₃^—N and NH₄⁺-N as mineral N (N_min) with an AA3 continuous flow analyzer (Seal Analytical, Southampton, UK). Soil samples from the microplots were air-dried and ground to pass a 0.15mm screen for total N and ¹⁵N isotope analysis. Irrigation water was also collected, filtered into sealed bottles and stored in an ice-box immediately until analysis.
Both crops were separated into grain and straw after the harvest and calculated the above biomass, grain and straw production. Grain and straw samples were subsequently dried at 105°C in a forced air oven and were ground to pass a 150-μm screen. Grain, straw and soil samples were analyzed for total N and ¹⁵N abundance by the micro-Kjeldahl procedure and by isotope ratio mass spectrometry (Delta Plus, Thermo Fisher, Waltham, MA).

Acid traps and homogenized dry soils were transferred into tin capsules and directly analyzed for N content and at%¹⁵N by EA-IRMS. The system consisted of an elemental analyzer (EA 1110, CE Instruments, Milan, Italy) connected via a ConFlo III interface (Thermo Fisher) to the IRMS (Delta^PLUS, Thermo Fisher). External calibration was performed using laboratory standards for isotope composition and N concentration [93] (link).

Dried and ground samples were simultaneously analyzed to determine the total C and N concentrations using an elemental analyzer (NA2500; CE Instruments, Milan, Italy). To determine the nitrate concentration in the tissues, samples of ~50 mg were extracted in 5 mL of distilled water in a hot bath at 100°C for 30 min and then centrifugated at 2600 g for 2 min. The nitrate concentration in the supernatant was colorimetrically determined according to Cataldo et al. (1975 (link)). The precipitate was collected and re-dried in an oven at 80°C for 48 h to measure the δ¹⁵N ratio. The dried precipitate from each plant part was thoroughly mixed based on the weight ratio of each part. The mixed sample for each plant was combusted in an elemental analyzer (NA2500; CE Instruments, Milan, Italy). A part of the combustion gases was introduced into an isotopic ratio mass spectrometer (Delta Plus, Thermo Fisher Scientific Inc. Worcester, MA, USA), and the δ¹⁵N value was determined.

CO₂ was trapped in 10 ml 3 M NaOH trap solution to measure soil respiration at 7 and 28 days of incubation as previously described (Zibilske, 1994 ). BaCO₃ pellets were dried at 50°C and stored for ¹³C analysis using an isotope ratio mass spectrometer (Delta Plus, IRMS; Thermo Fisher Scientific, Bremen, Germany). MBC at 7 and 28 days of incubation was determined using the chloroform fumigation-extraction method (Makarov et al., 2015 (link)). For ¹³C measurement of microbial biomass, an aliquot (ca. 10 mL) of the K₂SO₄ extract was freeze-dried, and the solid material was then analyzed.

For elemental analysis—isotope-ratio mass spectrometry (EA-IRMS), 5 mg intestinal biomass was washed to eliminate free L-threonine. After washing, the pellet was dried overnight in a speedvac (Eppendorf Concentrator 5301, Eppendorf, Hamburg, Germany) and for another 24 h at 60 °C. These samples were used for wet- and dry-weight analysis. Next, 0.05–0.3 mg (dry weight) was transferred into a tin capsule. Samples for elemental analysis and ¹³C and ¹⁵N quantification were analyzed with an elemental analyzer (EA 1110, CE Instruments, Wigen, United Kingdom) coupled via a ConFlo III device to the IRMS (DeltaPLUS, Thermo Fisher) [29 (link)].

Sediment samples were dried at 50 °C, weighed and washed through a 63 μm mesh, dried and weighed again. The size fraction >250 μm was separated by dry sieving. On average, 10–12 specimens of the planktonic foraminifera species G. ruber (white) were picked from the >250 μm size fraction. Only clean foraminifera specimens of approximately the same size were selected. Measurements of stable oxygen and carbon isotopes were performed on a Thermo DeltaPlus mass spectrometer equipped with a GasBench 2 carbonate preparation device. The isotope values were calibrated versus NBS 19 (National Bureau of Standards) and the in-house standard ‘Standard Bremen' (Solnhofen limestone). Isotope values are reported in per mil (‰) relative to the VPDB (Vienna Pee Dee Belemnite) scale. The analytical precision (1−sigma value) as obtained from 11 replicate Standard Bremen measurements was on average 0.058‰ for δ¹⁸O and 0.044‰ for δ¹⁸C. Only the oxygen-isotope data are reported in this paper.

After making all containers gas-tight and rinsing them with synthetic air (Westfalen AG, Münster, Germany), 12 ml of gas samples (IVA Analysentechnik, Meerbusch, Germany) were collected from the headspace of the Mason Jars on day 2, 3, 4, 8, 10, 15, 23, 31, 44, 65, 80, and 95. For each measurement of CO₂ respiration, two samplings of the container atmosphere were carried out, and the time in between the two samplings was adapted to the current respiration rates. During the incubation period of 95 d, the CO₂ concentration, as well as the ¹³C abundance in the respired CO₂, was measured via gas chromatography isotope ratio mass spectrometry (GC/IRMS; Delta Plus, Thermo Fisher, Dreieich, Germany). The CO₂ levels were calibrated against three calibration gases (890, 1500, and 3000 ppm CO₂; Linde AG, Pullach, Germany). Then, CO₂ with known isotopic composition, diluted in helium, was used as a lab standard. This standard was in turn calibrated against three international standards (RM 8562, RM 8563, and RM 8564; International Atomic Energy Agency, Vienna, Austria) with a dual inlet system. The temperature and water holding capacity were kept constant at 21 °C and 60%, respectively, along with the incubation period.