Soil Microbial Community Analysis via PLFA

Soil microbial community composition was assessed using PLFA analysis performed by the Laboratory of Environmental Biotechnology, Institute of Microbiology of the Czech Academy of Sciences, following the methodology described in Garcia-Sanchez et al. (2019 (link)). The PLFA were extracted from 1 g of freeze-dried soil samples with a mixture of chloroform–methanol-phosphate buffer (1:2:0.8, v/v/v), as previously described by Bligh and Dyer (1959 (link)). The lipids were fractionated into neutral lipids (NLFA), glycolipids and polar lipids (PLFAs) using an extraction cartridge (LiChrolut Si-60, Merck, White-house Station, USA), and NLFA and PLFA were subjected to mild alkaline methanolysis as described in Snajdr et al. (2008 (link)). The free methyl esters of NLFA and PLFAs were analyzed by gas chromatography-mass spectrometry (450-GC, 240-MS ion trap detector, Varian, Walnut Creek, CA) following the same procedure described by Sampedro et al. (2009 (link)).
The soil microbial community composition was characterized using the following PLFAs: fungal biomass was estimated on the basis of 18:2w6,9 content (Snajdr et al. 2008 (link)), bacterial biomass was quantified as the sum of i14:0, i15:0, a15:0, 16:1w5, 16:1w7; 16:1w9, 10Me-16:0, i16:0, i17:0, a17:0, cy17:0, 17:0, 10Me-17:0, 18:1w7, 10Me-18:0, and cy19:0. Actinobacterial biomass was determined as the sum of 10Me-16:0, 10Me-17:0, and 10-Me18:0, Gram-positive bacteria (G +) as sum of i14:0, i15:0, a15:0, i16:0, i17:0, and a17:0, and Gram-negative bacteria (G-) as the sum of 16:1w7, 16:1w9, 18:1w7, cy17:0, and cy19:0. The NLFA 16:1w5 was assigned as a marker for the quantification of AMF and total PLFA concentration was used to estimate the total viable microbial biomass (Olsson et al. 2003 (link)). Last, we calculated microbial ratios F:B (fungi: bacteria), G + :G- (Gram-positive bacteria: Gram-negative bacteria), and F:AMF (fungi: AMF).