5.4. Phospholipid Fatty Acid (PLFA) Analysis

The soil microbial community composition was analyzed under different P treatments by measuring soil PLFA concentrations. A chloroform–methanol–citrate buffer mixture (1:2:0.8, v/v/v; 0.15 M, pH 4.0) was used to extract the PLFAs from the lyophilized soil samples.³⁰ The obtained fatty acid methyl esters were isolated and distinguished on a gas chromatograph (Agilent 7890 N, Wilmington, DE) equipped with a MIDI Sherlock Microbial Identification System (version 4.5; MIDI, Newark, NJ). Chloroform, acetone, and methanol were added to the middle of the Supelclean Solid Phase Extraction Tubes that had been washed with acetone and chloroform to dissolve the glycolipid. This step was followed by saponification, methylation, and extraction steps, and finally 100 μL of chromatographically pure n-hexane was added to dissolve the sample to be tested. Based on 19:0 fatty acid methyl ester ratios, the abundance of each PLFA was calculated and presented as μg g^–1 soil.

Soil fungi, Gram-positive bacteria (G⁺), Gram-negative bacteria (G^–), and AMF were identified based on their specific PLFA biomarkers. In detail, the biomarkers included i14:0, i15:0, a15:0, i16:0, i17:0, and a17:0 for G⁺ bacteria; 16:1w7c, 18:1w7c, cy17:0, and cy19:0 for G^– bacteria; 10Me16:0, 18:2w6c, and 18:1w9c for fungi; and 16:1w5c for AMF.^30,31 To assess the changes in microbial community composition caused by different plant covers in the wetland soils, the ratios of fungi to bacteria (F/B, where bacteria is the sum of G⁺, G^–, 15:0, and 17:0) and of G⁺ to G^– (G⁺/G^–) were further calculated.