Total nitrogen content in grain was determined as described by Lu (1999 ). Briefly, the dried samples of ground grains (about 0.3 g) were digested using the H2SO4-HClO4 method. The digestion was then used to determine the total nitrogen content by the Kjeldahl method with a 2300 Kjeltec Analyzer Unit (Foss Tecator AB, Sweden).
Kjeltec 2300 analyzer unit
Manufactured by Foss
Sourced in Sweden, Denmark
The Kjeltec® 2300 Analyzer Unit is a laboratory instrument designed for the determination of nitrogen content in a variety of samples. It utilizes the Kjeldahl method of nitrogen analysis, providing accurate and reliable results.
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48 protocols using kjeltec 2300 analyzer unit
1
Grain Nitrogen Content Analysis
2
Grassland and Forest Soil Characterization
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Soil samples were collected at the end of July 2011. Four 2 × 2 m sampling quadrats were established in the grassland and forest plots (each about 100 × 100 m), respectively. Quadrats were placed randomly within the plots, and were separated from each other by at least 15 m. Soil samples (0–20 cm depth) were collected from 15 points within each quadrat using a 4‐cm‐diameter auger. Totally, four composite soil samples (>10 kg) were obtained from each plot and hand‐cleared off roots and visible organic debris, and then, approximately 100 g of each soil sample was air‐dried in a ventilation room for analyses of soil properties (e.g., C, N, and pH). The remaining soils were stored at 4 °C. Soil organic C (SOC, %) was measured using the modified Mebius method (Nelson and Sommers 1982 ). Total soil N (%) was measured using the modified Kjeldahl wet digestion procedure (Gallaher et al. 1976 ) and a 2300 Kjeltec Analyzer Unit (FOSS, Sweden). Soil pH was determined by a pH meter placed in soil mixed with distilled water at a ratio of 1:2.5. Selected soil properties are shown in the Table S1.
3
Soil Properties Measurement Protocol
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The content of organic C in all samples was measured by using the modified Mebius method [18 ]. Total soil nitrogen (TN) was measured with a modified Kjeldahl wet digestion procedure [19 ], using a 2300 Kjeltec Analyzer Unit (FOSS Tecator, Hoganas, Sweden). Total phosphorus (TP) was determined by the ammonium molybdate method after persulfate oxidation [20 ]. Soil pH was determined using a pH meter and a slurry of soil mixed with distilled water (1:2.5). In this study, the measurements for soil properties were conducted in four replicates.
4
Soil and Litter Nutrient Analysis
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The C concentration in soil and litter (%) was measured using a modified Mebius method54 . The N concentration of soil and litter (%) was measured with a modified Kjeldahl wet-digestion procedure55 , using a 2300 Kjeltec Analyzer Unit (FOSS, Sweden). Soil pH was measured in a soil-water slurry (1:2.5, w/w) with an Ultrameter-2 pH meter (Myron L. Company, California, USA). Soil water-holding capacity (WHC, %) and gravimetric moisture content (%) were measured at the Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing45 (link).
5
Soil and Leaf Nutrient Analysis
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Total N content of leaf and soil was measured by a 2300 Kjeltec Analyzer Unit (FOSS, Höganäs, Sweden). Total P and total K, total Mg and total Fe of leaf, soil TP, TK, AP and AK were extracted according to literature31 (link) and were determined by ICP (Kleve, Germany).
6
Soil Chemical Characterization Protocol
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Soil pH was measured using a soil water suspension (in a ratio of 1:2.5 w/v). Soil OC was determined by the K 2 Cr 2 O 7 -H 2 SO 4 oxidation method [39] (link). TN was measured using a 2300 Kjeltec Analyzer Unit (FOSS, Höganäs, Sweden) [40] (link). Following previously described methods, TP and TK were extracted [8] (link), AN content was determined [41] , and AP and AK were extracted and assayed [42] (link). TP, TK, AP, and AK contents were measured by inductively coupled plasma emission spectrometry (Spectro Analytical Instruments, Spectro Arcos ICP, Kleve, Germany) [43, (link)44] (link).
7
Measuring Soil Carbon and Nitrogen Mineralization
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We measured soil C and N mineralization rates via the aerobic incubation method (Chen et al., 2019) . For each sample, 20 g of fresh soil was incubated in a 250-ml brown bottle at 25°C. During incubation, we added water to maintain the soil at 60% of field capacity based on weight. On incubation days 1, 2, 3, 7, 10, 15, and 21, we measured the CO2 released from the soil via gas chromatography (Agilent HP 5890 SERIES II, USA), and the average value was used as the soil C mineralization rate. NH + 4 -N and NO - 3 -N were extracted from a 20-g sample of fresh soil that was not incubated and a 20-g sample of incubated soil with 50 ml of 2 mol L -1 KCl; the contents of NH + 4 -N and NO - 3 -N were determined with a 2300 Kjeltec Analyzer Unit (FOSS, Höganäs, Sweden).
The difference in total inorganic N before and after incubation was the soil N mineralization rate.
The difference in total inorganic N before and after incubation was the soil N mineralization rate.
8
Soil Organic Carbon and Nitrogen Analysis
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The content of organic carbon and nitrogen in soil and organic materials (chicken manure, cow manure, and straw) were determined by dichromate oxidation and Kjeldahl digestion, respectively (Nelson et al., 1996) . Brie y, for soil organic carbon analysis, 0.5 g of air-dried soil or soil organic materials was sieved through 0.15-mm sieves and placed in conical asks. Next, 10 mL of K 2 Cr 2 O 7 -H 2 SO 4 solution was added, and the wetted soil samples were then baked for 30 min in an oven at 170-180℃. o-Phenanthroline was used as an indicator. For organic nitrogen analysis, 1-g (< 0.15 mm) soil samples were placed in a rigid test tube, and 10 mL H 2 SO 4 was added. The N concentration of soil was measured using a 2300 Kjeltec Analyzer Unit (FOSS, Sweden).
9
Nitrogen Content Quantification Protocol
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Kjeldahl nitrogen was measured by the method of Wada et al. (2015) . Total kjeldahl nitrogen was determined by using a micro Kjeldahl procedure with sulphuric acid, digestion catalyst and conversion of organic nitrogen into ammonium form according to the Total Kjeldahl nitrogen method (2300 Kjeltec Analyzer Unit, Foss Tecator AB, Sweden). Nitrate nitrogen content was measured according to Patterson et al. (2010) . For tissue analysis, 100 mg of fresh root tissue was frozen in liquid nitrogen, pulverized, and added to 1 ml of deionized water.
The suspension was incubated at 45 o C for 1 h and then centrifuged at 5000 × g for 15 min. The supernatant was utilized for nitrate quantization.
The suspension was incubated at 45 o C for 1 h and then centrifuged at 5000 × g for 15 min. The supernatant was utilized for nitrate quantization.
10
Proximate Composition and Biomarker Analysis
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Proximate composition analysis of all samples was conducted using the methods described by AOAC (2003) . Moisture content was determined by oven drying at 105 °C to constant weight. Crude protein content was determined by Kjeldahl method after acid digestion using 2300 Kjeltec Analyzer Unit (FOSS Tecator, Haganas, Sweden). Crude lipid content was determined by chloroform-methanol extraction. Ash was determined by combustion in muffle furnace at 550°C.
Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride, total cholesterol (TC), glucose (Glu), HDL-Cholesterol (HDL-C), LDL-Cholesterol (LDL-C) were tested by automatic biochemistry analyzer BS-460 produced by Mindray, using standard kits according to the instructions (Mindary Bio Medical Electronic Limited by Share Ltd, Shenzhen, China). Tissue glycogen (liver and muscle) was determined using the amyloglucosidase method, and the reagents were prepared by Nanjing Jiancheng Bioengineering Institute, Nanjing, China.
Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride, total cholesterol (TC), glucose (Glu), HDL-Cholesterol (HDL-C), LDL-Cholesterol (LDL-C) were tested by automatic biochemistry analyzer BS-460 produced by Mindray, using standard kits according to the instructions (Mindary Bio Medical Electronic Limited by Share Ltd, Shenzhen, China). Tissue glycogen (liver and muscle) was determined using the amyloglucosidase method, and the reagents were prepared by Nanjing Jiancheng Bioengineering Institute, Nanjing, China.
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