Appendix A. Methods for the hybrid procedure of phospholipid fatty acid (PLFA) and fatty acid methyl ester (FAME) analysis used to characterize microbial community composition.
Using a modified Bligh and Dyer (1959) technique, membrane lipids were extracted from 4 g of lyophilized whole soil using a chloroform-methanol extraction with a phosphate buffer (potassium phosphate (3.6 mL), methanol (8 mL), and CHCl3 (4 mL)) in 25-mL glass tubes, shaken for 1 hour and centrifuged. Supernatant was then decanted to 30-mL tubes and potassium phosphate buffer and CHCl3 were re-added, and the tubes were vortexed for 30 s. The phases were allowed to separate overnight at room temperature. The top layer was aspirated off (saving the chloroform phase), and volume was reduced in a RapidVap (Labconco Corporation, Kansas City, Missouri, USA). Samples were then purified following the procedure for FAME as given by Microbial ID Inc. (Hayward, California, USA). Sodium hydroxide was added for saponification and the solution heated in a water bath for 30 min, followed by mild alkaline methanolysis.
Microbial profiles were identified by analyzing the methyl-ester derivatives from the phospholipid extractions on a gas chromatograph (Hewlett-Packard 6890) equipped with a flame ionization detector and split/splitless inlet and a 25 m × 0.2 mm inside diameter × 0.33 µm film thickness Ultra 2 (5%-phenyl, 95% methyl) capillary column (Agilent) using hydrogen as the carrier gas, N2 as the make up gas, and air to support the flame. Gas chromatograph conditions were set by the MIDI Sherlock program (MIDI, Inc., Newark, Delaware, USA). Peaks were identified with bacterial fatty acid standards and Sherlock peak identification software. Fatty acids were quantified by comparisons of peak areas from the sample compared to peak areas of two internal standards, 9:0 (nonanoic methyl ester) and 19:0 (nonadeconoic methyl ester), of known concentration.