Chemical Analyses

Liquid samples and gas samples were taken with sterile syringes. Liquid samples were centrifuged at 13,000 × g (Himac CT15E, Hitachi Koki Co., Ltd., Tokyo, Japan) for 15 min, and the supernatant was filtered (HPLC nylon filter, pore volume 0.2 μm, Infochroma, Zug, Switzerland). Organic acids and sugars were identified using high-performance liquid chromatography with ion exclusion chromatography (1,090 series II with UV detector, Hewlett Packard, Palo Alto, CA) (Wieczorek et al., 2014). Carbon dioxide, molecular hydrogen, and methane were measured with a gas chromatograph; the pH was measured with a pH electrode; and the ammonium, nitrate, and sulfate concentrations were assessed with ion chromatography, as described previously (Wieczorek et al., 2014).

For the analysis of ¹²CO₂ and ¹³CO₂ in the samples, gas chromatography-mass spectrometry (GC-MS) analysis was performed using a Perkin-Elmer GC Clarus 600 system with a Rtx®-1 capillary column (60 m × 320 μM). For GC-MS detection, an electron ionization system was operated with an ionization energy of 70 eV. Mass spectra were taken from 14 to 70 Da. Helium was used as carrier gas at a constant flow with 300 kPa, and an injection volume of 10 μl (split ratio 10:1) was employed manually by use of gastight syringes. Each sample was measured five times. The total amount of ¹²CO₂ and ¹³CO₂ was analyzed by extraction of the masses 44 and 45 followed by peak integration. The peak areas were corrected with ¹²CO₂ and ¹³CO₂ indoor air values and finally the ratio of the masses 45/44 was calculated. The soil moisture content was determined by weighing sieved soil before and after drying at 105°C for 48 h.