Phospholipid Fatty Acid Analysis
We used phospholipid fatty acids (PLFAs) to determine differences in the microbial biomass (MB) and the ratios of fungal to bacterial biomass. Briefly, total lipids were extracted using 10 ml of methanol, 5 ml chloroform, and 4 ml of a 50 mM phosphate buffer (pH = 7.4) from 5 g of lyophilized soil (White et al. 1979; DeForest et al.2004). To determine analytical recovery, phospholipid 19:0 (1,2-dinonadecanoyl-sn -glycero-3-phosphocholine) and 21:0 (1,2-diheneicosanoyl-sn -glycero-3-phosphocholine) standards (Avanti Polar Lipids, Inc., Alabaster, AL, USA) were added during the extraction phase (DeForest et al. 2012). Polar lipids were separated from other lipids using silicic acid solid-phase chromatography columns (500 mg 6 ml-1; Thermo Scientific, Waltham, MA, USA), and the separated polar lipids were converted to fatty acid methyl esters (FAME) through methanolysis (Guckert et al. 1985). The resulting FAMEs were separated using a HP GC-FID (HP6890 series, Agilent Technologies, Inc. Santa Clara, CA, USA) gas chromatograph, and peaks/biomarkers were identified using the Sherlock System (v. 6.1, MIDI, Inc., Newark, DE, USA). External FAME standards (K104 FAME mix, Grace, Deerfield, IL, USA) were used to determine concentrations. The sum of all detected 14–19 C-length PLFAs was used to calculate MB because longer PLFAs can be indicators of mosses and higher plants (Zelles 1999). Ratios of fungal to bacterial biomass (fungi:bacteria) were calculated by dividing the amount (mol) of the fungal biomarker 18:2ω6c by the sum of all other microbial biomarkers (i.e., mol 18:2ω6c /(mol MB – mol 18:2ω6c)).