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.