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Analysis of phospholipid fatty acids

JS José A. Siles
BÖ Birgit Öhlinger
TC Tomas Cajthaml
EK Erich Kistler
RM Rosa Margesin
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Phospholipid fatty acid (PLFA) analysis was used as a proxy to quantify living microbial biomass in each soil sample. Extraction and analysis of PLFAs were carried out as previously described34. Briefly, microbial lipids were firstly extracted from 0.5 g of soil sample using a mixture of chloroform:methanol:phosphate buffer (1:2:0.8; v/v/v) according to Bligh and Dyer35. Phospholipids were then separated by solid-phase extraction cartridges (LiChrolut Si 60, Merck), and the samples were subjected to mild alkaline methanolysis. The free methyl esters of phospholipid fatty acids were analyzed by gas chromatography-mass spectrometry (GC-MS; 450-GC, 240-MS ion trap detector, Varian, Walnut Creek, CA).

The PLFA i14:0, i15:0, a15:0, 16:1ω9, 16:1ω7, 16:1ω5, 10Me-16:0, i17:0, a17:0, cy17:0, 17:0, 10Me-17:0, 10Me-18:0 and cy19:0 were addressed as bacterial signature markers. The PLFA 10Me-16:0, 10Me-17:0 and 10Me-18:0 were used as actinobacterial markers, while the fatty acid 18:2ω6,9 was selected as fungal marker. The fatty acids found both in bacteria and fungi, such as 15:0 and 18:1ω7, were excluded from the analysis characterizing the different microbial groups36. The sum of all the identified phospholipids was used to estimate the total microbial biomass. Ratios between PLFA markers derived from fungi and bacteria (F/B) were calculated.

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