Soil Microbial Community Analysis via PLFA

Soil microbial composition was determined by analyzing the ester-linked phospholipid fatty acid (PLFA) composition of the soil. About 8.0 g of frozen soil was weighed, and lipids were extracted overnight by the modified Bossio and Scow (1998) (link) method, using 23 mL of chloroform: methanol: phosphate buffer (1:2:0.8 v/v/v) solution. The extracts were separated on silica acid columns by sequential elution using organic solvents with increasing polarity, followed by evaporation under N₂. Phospholipids were sequentially saponified and methylated to form fatty acid methyl esters. Individual fatty acid methyl esters were identified and quantified using a gas chromatograph (Agilent 6890N) equipped with the MIDI software package Sherlock MIS version 4.5 (MIDI Inc., Newark, Delaware, USA), and PLFA was analyzed. The MIDI package automatically controlled all gas chromatography operations, including calibration, subsequent sample sequencing, peak integration, and nomenclature. The calibration standards contained a mixture of linear saturated methyl hydroxy fatty acid esters with a length of 10–20 carbons (MIDI Part No. 1208) (He et al., 2019 (link)).