Vario el 3

The rice husk, harvested from Huaian city, China, was selected as the feedstock in the present experiments. The shattered rice husk was sieved and the particles sizes ranging from 20 mesh to 40 mesh were used for the present study. The chosen materials were then dried for 12 h at 105 °C prior to the tests. Proximate analysis was conducted on the basis of the American Society of Testing Materials (ASTM) procedures. The ultimate analysis was conducted using a Vario EL-III elementar (ELEMENTAR Analysensysteme GmbH). The obtained results are indicated in Table 1.

Seeds were ground in liquid nitrogen. Subsequently, the homogenized powder was lyophilized and total N-content was measured by a CN-element analyzer (Elementar Vario EL III, Elementar Analysensysteme GmbH, Langenselbold, Germany) via heat combustion at 1150 °C and thermal conductivity detection. The stable isotope ratio ¹⁵N/¹⁴N was measured as described in Franzaring, Gensheimer, Weller, Schmid and Fangmeier [20 (link)] using an isotope ratio mass spectrometer (Deltaplus XL, Thermo Finnigan, Bremen, Germany) connected by an open split device (ConFlow II, Thermo Finnigan, Bremen, Germany). The ¹⁴N/¹⁵N ratio was expressed relative to the isotopic signature of N₂ in the air as δ values (in ‰).

Since the amount of rhizosphere soil was limited to 2 to 3 g per sample, basic soil parameters were determined using respective bulk soil. For pH analysis, 12 g of air-dried soil was suspended in 30 ml of 0.01 M CaCl₂ solution (1:2.5 [wt/vol]). The soil suspension was equilibrated at room temperature and thoroughly mixed every 20 min. After 1 h, the pH was measured with a pH electrode. Total carbon and nitrogen contents were determined from air-dried soil using an elemental analyzer (Elementar Vario EL III; Elementar, Hanau, Germany). For analysis of mineral nitrogen, 5 g of fresh soil was suspended in 20 ml of 1 M KCl solution and measured via flow injection analysis (FlAstar 5000; Foss GmbH, Rellingen, Germany). Available phosphorus was extracted from fresh soil with double lactate (1:50 [wt/vol], pH 3.6) and quantified using the colorimetrical molybdenum blue method (77 (link)).

Soil properties were measured as follows: pH was measured with a glass electrode at a 1:2.5 (w/v) soil‐to‐water ratio; total carbon (C) and total nitrogen (N) with an Elementar Vario EL III (Elementar Analysensysteme GmbH, Germany); total phosphorus (P), total calcium (Ca), and total magnesium (Mg) with an Inductively Coupled Plasma‐Atomic Emission Spectrometer (ICP‐AES) (iCAP 6300, Thermo Jarrell Ash Co.); particle size distribution (PSD) with an X‐ray diffractometer (Rigaku D/Max 2550pc, Rigaku Corporation) at a voltage of 40 kV and current of 300 mA.

Since the amount of rhizosphere soil was limited to 2 to 3 g per sample, basic soil parameters were determined using respective bulk soil. For pH analysis, 12 g of air-dried soil was suspended in 30 ml of 0.01 M CaCl₂ solution (1:2.5 [wt/vol]). The soil suspension was equilibrated at room temperature and thoroughly mixed every 20 min. After 1 h, the pH was measured with a pH electrode. Total carbon and nitrogen contents were determined from air-dried soil using an elemental analyzer (Elementar Vario EL III; Elementar, Hanau, Germany). For analysis of mineral nitrogen, 5 g of fresh soil was suspended in 20 ml of 1 M KCl solution and measured via flow injection analysis (FlAstar 5000; Foss GmbH, Rellingen, Germany). Available phosphorus was extracted from fresh soil with double lactate (1:50 [wt/vol], pH 3.6) and quantified using the colorimetrical molybdenum blue method (77 (link)).

Soil samples were analyzed for the following soil properties: soil pH, total carbon (TC), total nitrogen (TN), total organic carbon (TOC), mineral nitrogen (NO₃⁻-N and NH₄⁺-N), total phosphorus (TP), and available phosphorus (AP). Leaf total nitrogen and leaf total phosphorus were tested as leaf properties. Briefly, soil pH was determined with a soil-to-water ratio of 2:5 (wt/vol) using a glass electrode (FE20, Mettler Toledo). Soil total carbon (TC) and total N (TN) were determined with an Elementar Vario EL III (Elementar Analysensysteme GmbH, Germany). Soil total organic carbon (TOC) was determined using the K₂Cr₂O₇ oxidation-reduction titration method. Soil mineral N (NH₄⁺-N and NO₃⁻-N, extracted with 2 M KCl) and soil total P (TP, extracted with H₂SO₄:HClO₄ [4:1, vol/vol] solution) were determined using a continuous flow analyzer (SAN++, Skalar, Netherlands) (63 (link)). Soil available P (AP) was determined using the Mehlich-3 method (64 (link)). Leaf N and P concentrations were determined using the standard micro-Kjeldahl and vanadomolybdo phosphoric acid yellow color methods, respectively (65 (link)).

For water content determination, fresh soil samples were weighed and subsequently
dried at 105 °C until weight stability. pH was measured
in a suspension of 10 g air dried soil in 25 mL of a
0.01 M CaCl₂-solution. For determination of leachable
chloride and leachable organic carbon, 10 g of soil were mixed with
100 mL deionized water and shaken at 150 rpm for
24 h on a rotary shaker. Samples were centrifuged for
5 minutes at 4000 × g and
filtered through a 0.45 μm pore size cellulose ester
filter (Millex HA filter, EMD Millipore Corporation, USA). Dissolved organic
carbon was measured with a High TOC Elementar system (Elementar Analysensysteme
GmbH, Hanau, Germany) and chloride was determined by ion chromatography (Dionex
DX 120, Thermo Scientific, Sunnyvale, CA, USA). For total organic carbon
analysis soil samples were dried at 40 °C and sieved
(2 mm mesh) to exclude large roots and stones. The organic carbon
content was determined by heat combustion (1150 °C) and
thermal conductivity analysis on a CNS element analyzer (Elementar Vario EL III,
Elementar Analysensysteme GmbH, Hanau, Germany). Adsorbable organic halogen
(AOX) content in the soil samples was determined according to the standard
protocol (DIN EN ISO 9562) for soil leachates (DIN EN 12457-4) at the Laboratory
for Environmental and Product Analytics (DEKRA GmbH, Halle, Germany).