Fractionation of fatty acids derived from soil lipids by solid phase extraction and their quantitative analysis by GC-MS.

To provide a procedure to estimate microbial biomass and community structure, an analytical method for the quantitative measurement of the profile of fatty acids derived from phospholipid (PL) and lipopolysacchride (LPS) in agricultural soil was developed and evaluated. The lipids were extracted by the one-phase procedure, and the PL was separated from other fractions by a solid phase column. Then, the PL was subjected to a mild alkaline hydrolysis to form fatty acid methyl esters (FAMEs). The hydroxy fatty acids methyl esters (OHFAMEs) derived from LPS in the soil studied were obtained by acidic hydrolysis of the extracted soil residue, followed by methylation. The FAMEs were further cleaned up and separated into several subgroups by utilization of consecutive solid phase columns. To measure the OHFAMEs and monoenoic FAMEs, these compounds needed further trimethylsilylization and dimethyl disulphide derivatization, respectively, before injection. Qualitative and quantitative measurements were made by an internal standard method using a GC-MS system. More than 160 fatty acids derived from PL and several dozens of hydroxy fatty acids derived from LPS in the soil have been found. The total amount of PL-FAs in the soil were found to be 23μg g-1. In contrast, the OHFAs derived from LPS amounted to only 519 ng g-1. As little as a few ng g-1 of these fatty acids present in the PL-fraction could be detected by this method. The recovery of bacterial standard FAMEs was between 73 and 104% after consecutive separation of FAMEs into various subgroups using solid phase columns. The diversity of the fatty acids may reflect the complexity of microorganisms present in the soil studied