Flame ionization detector

The fatty acids present in the cold-extracted methanol:chloroform [21 ] layer were acid methylated [23 –25 (link)], and analyzed in a gas chromatograph coupled with a flame ionization detector (Perkin Elmer, Waltham, MA, USA). The fatty acid methyl esters were separated in an Omegawax-320 column (30 m long, 0.32 mm internal diameter and 0.25 mm film thickness; Sulpeco, USA). The injected sample volume was 2 μL and the temperature of the injector (split of 1:20) and the detector temperatures were 260°C and 280°C, respectively. The column temperature started at 110°C with a ramp of 40°C/min until 233°C which was held for 2 min. After that, the oven temperature reached 240°C at 1°C/min and held for 21 min. The identification of the different fatty acid methyl esters was based on the specific retention times obtained from a mixture containing 37 different standards (Supelco 18919-1AMP, Sigma-Aldrich, St. Louis, MO, USA). Helium was utilized as the carrier gas with a flow rate of 1.8 mL/min at 10 psi.

The soil pH was determined (AB15 pH meter, Accumet, Fisher Scientific) at a soil-to-deionized water ratio of 1:2.5 (w/v). The contents of soil organic carbon (SOC) and total nitrogen (TN) in dried soil ground with a ball mill (Retsch PM 200 Planetary Ball Mill, Haan, Germany) were determined by combustion (CNS-2000, LECO, St. Joseph, MI, United States). Available phosphorus (AP) was extracted with 0.5 M NaHCO₃ (pH 8.5) at a soil-to-solution ratio of 1:20 (w/v) for 30 min and measured by a colorimetric procedure. Available potassium (AK) was extracted with 1 M CH₃COONH₄ (pH 7.0) at a soil-to-solution ratio of 1:10 (w/v) for 30 min and determined by flame atomic absorption spectrometry. Available nitrogen (AN) was determined by the alkali hydrolysis diffusion method (Supplementary Table 2). The water content (WC) and electrical conductivity (EC) were measured by standard methods. Soil temperature (ST) at 10-cm depth was measured using a mercury-in-glass geothermometer with bent stems (Hongxing Thermal Instruments, Wuqiang, Hebei, China) at 13:00 on the soil sampling day. Soil nitrogenase activity was determined using the acetylene reduction assay method; ethylene concentrations were measured using a gas chromatograph with a flame-ionization detector (Perkin Elmer, Waltham, MA, United States) (Christophe et al., 2010 (link)).

GC analyses for detection and quantification of MCFA were carried out on a Perkin Elmer Clarus 400 instrument (Perkin Elmer, Germany) equipped with an Elite 5MS capillary column (30 m × 0.25 mm, film thickness 1.00 µm, Perkin Elmer, Germany) and a flame ionization detector (Perkin Elmer, Germany). 1 μL of the sample was analyzed after split injection (1:10) and helium was used as carrier gas (90 kPa). For quantification of FAMEs, the temperatures of the injector and detector were 250 and 300 °C, respectively. The following temperature program was used: 50 °C for 5 min; increase of 10 °C/min to 120 °C and hold for 5 min; increase of 15 °C/min to 220 °C and hold for 10 min; increase of 20 °C/min to 300 °C and hold for 5 min. Medium-chain FAMEs were identified and quantified by comparison to FAMEs in standard samples.