*2.4. PLFA Analysis*

Total lipids were extracted from 1.0 g freeze-dried litter soaked in a mixture of chloroform: methanol: phosphate buffer (1:2:0.8, *v*/*v*/*v*) according to the method of Zelles and Otaki [26,27]. Phospholipids were isolated on silica columns and hydrolyzed and methylated using a methanolic KOH solution. Fatty acid methyl esters (FAME) were separated into saturated, polyunsaturated and monounsaturated fatty acids using amino propyl-modified and silver-impregnated SPE columns. The PLFA module of the American MIDI Sherlock microbial identification system platform is used for sample analysis, with hydrogen as the carrier gas, Agilent 6890N meteorological chromatograph and FID detector as the hardware platform. The chromatographic column is Agilent 19091B-102 (25 m × 200 μm × 0.33 μm). The injection volume of each sample is 1 μL. According to the MIDI platform specification, all tests are calibrated with standards. PLFAs were identified and analyzed based on the MIDI map recognition software Sherlock system 6.2.
