Characterization of Lipid Fatty Acids in Whole-Cell Microorganisms Using in Situ Supercritical Fluid Derivatization/Extraction and Gas Chromatography/Mass Spectrometry

Abstract

In situ supercritical fluid derivatization and extraction was used as a sample preparation technique for the classification of bacteria using fatty acid profiling. Addition of a quaternary ammonium salt such as phenyltrimethylammonium hydroxide under static supercritical conditions directly to lyophilized, whole-cell bacteria in an extraction vessel resulted in the saponification of the bacterial lipids and derivatization of their fatty acids. The derivatized fatty acid methyl esters (FAMEs) were then extracted with supercritical CO2 and analyzed without additional treatment using GC/MS. Iso and anteiso C15:0 and C17:0 along with C18:0 were predominant in Gram-positive bacteria, while C16:1, C16:0, C18:1, and cyclopropyl cyC17:0 and cyC19:0 were significant in Gram-negative bacteria. Application of principal components analysis to the FAME GC/MS data resulted in the differentiation between Gram-positive and Gram-negative type bacteria. Differentiation between species among the same genera was also observed.

Supercritical Fluid Application in Food and Bioprocess Technology

“…The CO 2 extraction process is selective in the presence of chlorophyll a. Moreover, there are some reports which describe the SFE of bacterial (Gharaibeh and Voorhees, 1996) and fungal lipids (Cygnarowicz et al, 1992) for use in the classification of them by fatty acid profiles. A simple two-step process was developed to extract and purify medium chain length polyhydroxyalkanoates (MCL-PHA) from bacterial cells (Pseudomonas resinovorans) grown on lard and tallow (Hampson and Ashby, 1999).…”

MALDI‐FTMS of Whole‐Cell Bacteria

“…1-3 Identification of glycerides, phospholipids, and glycolipids is the basis of numerous bacterial pyrolysis [38][39][40] and the FAME [41][42][43] (fatty acid methyl ester) tests generally the accepted methods of classification by microbiologists. Only recently has mass spectrometry combined both protein and lipid analysis into one mass spectrometric technique.…”

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry

“…In addition, related methods for the identification of fatty acids from bacterial lipids, either directly, by FAB MS (e.g. [5]), or after derivatisation to the methyl esters, by gas chromatography (GC) MS (see for example [18]), are not considered. Also, broad studies on highly specific compounds not explicitly used for typing, e.g.…”