PLFA analyses

The PLFA patterns were analyzed⁴⁶ and adjusted according to the ISO/TS 29843-2:2011F standard. In summary, the soil lipids from 3 g of soil (freeze-dried aliquots) were extracted with a Bligh and Dyer solution [methanol, chloroform, and citrate buffer (pH = 4 ± 0.1), 2:1:0.8, v/v/v]. A biphasic system was achieved by adding chloroform and citrate buffer from which the lipid phase was evaporated at 30 °C under a nitrogen stream. The phospholipids were separated from neutral lipids and glycolipids by solid-phase extraction on silica tubes (SPE DSC-Si, 500 mg, Discovery^®) and evaporated. The PLFA were turned into fatty acid methyl esters (FAMEs) via alkaline methanolysis⁴⁷ and later quantified via gas chromatic retention time comparison with a gas chromatograph (GC Agilent HP6890, G1530A, Chemstation, Santa Clara, USA) connected to a flame ionization detector equipped with a capillary column (SGE, BPX5, 60 m × 0,25 mm × 0,25 mm). The FAME concentrations were quantified relative to methyl nonadecanoate (19:0), enabling methylated lipids to be identified. A standard soil was used and extracted in parallel to detect potential deviations between the extraction rounds, expressed in nmol C-FA per g of soil. Mono-unsaturated and cyclopropylated PLFA (C16:1w7c, C18:1w9c, and C18:1w9t) were assigned to gram-negative bacteria, iso-branched and anteiso-branched PLFA (iC15:0, aC15:0, iC16:0, i-C17:0, C:17, and C18:0) were assigned to gram-positive bacteria and C18:2w6c, C18:3w3c, respectively C20:5w3c were assigned to fungi⁴⁸. The total content of bacteria was expressed by adding gram-positive, gram-negative together with the markers C14:0, C16:0, C20:0, and C15:1. Lastly, the ¹³C-labeling of FAME was concluded by correcting for the added methyl moieties during methanolysis and relating it to the chain length of fatty acids (Eq. ⁶).

where δ¹³CF_A represents the δ¹³C of the fatty acid, C_n the number of C atoms in the fatty acid, δ¹³C_FAME is the δ¹³C of the fatty acid methyl ester, and δ¹³C_MeOH is the δ¹³C of the methanol used for the methylation (−63%) to calculate the isotope ratios of the fatty acids. The relative incorporation of ¹³C into four microbial groups was calculated by relating the proportions of each fatty acid to the total ¹³C incorporation, and the absolute incorporation of ¹³C in each microbial group was calculated by dividing the amount of ¹³C enriched fatty acid by the total amount of extracted fatty acid for that particular group.