Soil PLFA analysis

Analysis of phospholipid fatty acids (PLFAs) was used to characterize the composition of the soil microbial community, according to the method of Bossio and Scow⁵³. Lipids were extracted from 8 g freeze-dried soil using a mixture of chloroform: methanol: citrate buffer (1:2:0.8). Polar lipids were separated from neutral lipids and glycolipids on solid phase extraction columns (Supelco Inc., Bellefonte, PA, USA). Phospholipids were treated with a mild-alkali methanolysis and the produced fatty acid methyl esters were subsequently extracted in hexane and dried under N₂. Samples were analysed using an Agilent 7890A series Gas Chromatograph equipped with MIDI peak identification software (Version 4.5; MIDI Inc., Newark, DE, USA). Before analysis, samples were dissolved in hexane that contained 19:0 as an internal standard. The following markers were chosen to represent bacterial biomass (i.e., 14:0, i14:0, 15:0, i15:0, a15:0, 16:0, i16:0, 16:1 ω7c, 16:1 ω9c, 17:0, i17:0, a17:0, 17:1 ω8c, cy 17:0, 18:0, 18:1 ω5c, 18:1 ω7c, 18:1 ω9c, and cy 19:0), and 18:2 ω6c represented fungal biomass⁴⁶.