Fatty Acid Methyl Ester Analysis and Microbial Biomass

Phospholipid-derived fatty acids (PLFAs) were extracted from 1 g of sieved, root-free, freeze-dried soil. We used a modified Bligh and Dyer (1959) extraction procedure (White et al., 1979; Guckert et al., 1985) where a single-phase solvent system (chloroform) was modified to include a phosphate buffer. This initially extracts lipids from only viable microorganisms captured at the time of sampling. Lipid extracts were then fractionated on silicic acid columns into neutral, glyco- and polar lipids. Polar lipids were collected and then methylated with 0.2 M methanolic KOH to form fatty acid methyl esters (FAMEs). Purified FAMEs were brought to volume with hexane before injection onto a Varian 3800 FID GC. FAME identification and quantification of each peak was based on retention time data with known standards from Matreya, LLC^®. The polyenoic unsaturated fatty acids, 18:2ω6 and 18:1ω9c, were considered as fungal biomarkers (Bardgett et al., 1996; Bååth, 2003). Bacterial markers included saturated Gram-positive fatty acids (i15:0, a15:0, i16:0, i17:0, and a17:0), monoenoic and cyclopropane unsaturated Gram-negative fatty acids (18:1ω7c and cy19:0), and general bacterial markers (15:0, 16:1ω7c, and 16:1ω7t) and fungal markers included non arbuscular mycorrhizal fungi (18:2ω6,9 and 18:1ω9) and arbuscular mycorrhizal fungi (AMF)(16:1ω5) (Ekelund et al., 2003; Leckie et al., 2004).