2.3 PLFA extraction
For the PLFA extraction, 2 g of freeze-dried (2-mm sieved) soil was extracted and derivatized to fatty acid methyl esters following the previously reported method of Bligh and Dyer (1959) as adapted by White et al.(1979). Briefly, the soil was first extracted twice using a 22.8-mL single-phase solution of chloroform: methanol: citrate buffer (1:2:0.8 v/v/v,0.15 M, pH 4.0). The supernatant was transferred to test tubes and split into two phases by adding equal amounts of CHCl3 and citrate buffer. Phospholipids were then separated from neutral lipids and glycolipids on a silica acid column (Supelco, Bellefonte, PA, USA). Methyl nonadecanoate fatty acid (19:0) was added prior to derivatization as an internal standard to quantify the concentration of phospholipids. Following methylation of the phospholipids, the PLFA methyl esters were separated and identified using gas chromatography (GC; N6890; Agilent Technologies, Inc., Santa Clara, CA, USA) fitted with a MIDI Sherlock microbial identification system (version 4.5; MIDI, Inc., Newark, DE, USA). Individual PLFAs were quantified from the combined area of the peaks with mass-to-charge values (m/z 44, 45, and 46) relative to the internal standard added to each sample (Thornton et al., 2011). Iso- and anteiso-branched fatty acids (except for 10Me-branched PLFAs) were used as indicators for G+ bacteria, while monounsaturated and cyclopropyl fatty acids were used as indicators for G− bacteria (Wang et al., 2016; Ma et al., 2018). The 10Me-branched PLFAs were used as actinomycete biomarkers, while 18:2 ω6c and arbuscular mycorrhizal fungi 16:1 ω5c were used as fungal biomarkers. The sum of the G+ bacteria, G− bacteria, and actinomycetes was used for ratio calculation as bacteria PLFAs.