2.3. Quantification of the microbial biomass
The samples used in the phospholipid fatty acid (PLFA) analysis
were extracted using a chloroform–methanol–phosphate buffer
(1:2:0.8). Phospholipids were separated using solid-phase extraction
cartridges (LiChrolut Si 60, Merck), after which the samples
were subjected to mild alkaline methanolysis (Oravecz et al., 2004).
The free methyl esters of the phospholipid fatty acids were analyzed
using gas chromatography–mass spectrometry (Varian 3400; ITS-
40, Finnigan). The GC instrument was equipped with split/splitless
injector and a DB-5MS column was used for separation (60 m,
0.25mm i.d., 0.25_m film thickness). The temperature program
started at 60 ◦C and was held for 1min in splitless mode. Then
the splitter was opened and the oven was heated to 160 ◦C at
a rate of 25 ◦Cmin−1. The second temperature ramp was up to
280 ◦C at a rate of 2.5 ◦Cmin−1, this temperature being maintained
for 10 min. The solvent delay time was set to 8 min. The transfer
line temperature was set to 280 ◦C. Mass spectra were recorded at
1 scan s−1 under electron impact at 70 eV, mass range 50–350 amu.
Methylated fatty acids were identified according to their mass