Ultra 2 column

The δ¹⁵N values were measured using an online GC/C/IRMS system manufactured by Thermo Fisher Scientific, which included the GC apparatus (Trace 1310), a GC Isolink II, a ConFlo IV interface, and a Delta V Advantage isotope-ratio mass spectrometer. An Ultra 2 column (50 m × 0.32 mm i.d., 0.54 μm film; Agilent Technologies, Santa Clara, CA, US) was used as the GC column, and the temperature of the column oven was set to 40 °C initially (2.5 min hold) and then increased at 20 °C min⁻¹ to 110 °C (0 min hold), 3.2 °C min⁻¹ to 150 °C (0 min hold), 9 °C min⁻¹ to 220 °C (10 min hold), and 30 °C min⁻¹ to 250 °C (5 min hold). The temperature at the inlet was 270 °C, the injection dose was 1 μL (split-less), the flow rate was 1.4 mL min⁻¹, and the temperature in the reactor was 1,000 °C. The amino acid standard was prepared by weighing and mixing each standard reagent (1 mg, ʟ-aspartate, glycine (B), ʟ-phenylalanine, ʟ-hydroxyproline, and ʟ-norleucine), followed by derivatization as described in Section 2.4.1.

PLFA analysis was performed using the methods of White et al. [31 (link)]. Lipids were extracted from 8 g dry-weight-equivalent of fresh soil from each sample in a monophasic solution of chloroform, methanol, and citrate buffer (1.0:2.0:0.8 v/v/v) [32 (link)]. Phospholipids were separated on solid-phase columns. Subsequently, the phospholipids were collected and methylated, and the resulting fatty acid methyl esters were analyzed using an Agilent 6850N gas chromatographer (Agilent Technologies, Palo Alto, USA) equipped with an ULTRA-2 column (25 m length × 0.20 mm internal diameter, 0.33 μm film thickness). Peaks were identified based on retention time and mass spectral information. Quantification of each PLFAs was achieved by comparing the peak areas with the internal standard methyl nonadecanoate (19:0) [33 ]. PLFA-values were reported as nmol g⁻¹ soil. Biomarkers for the soil microbial community [34 –37 (link)] are shown in Table 1. Other PLFAs detected in the current experiment, such as 10 me17:0, 18:1w7c, and 20:1w9c, were used to assess microbial compositions. The ratios of the sum of saturated (SAT) and mono-unsaturated (MONO) PLFAs were used as physiological or nutritional stress indicators [17 ]. For differences in PLFA composition and individual PLFAs between forest age and season, we used log-transformed mole percentages of total PLFAs within a sample.