Application of Thin-Layer Chromatography-Flame Ionization Detection (TLC-FID) to Total Lipid Quantitation in Mycolic-Acid Synthesizing Rhodococcus and Williamsia Species

Abstract

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

Conjugated linoleic acid ameliorates hepatic steatosis by modulating intestinal permeability and gut microbiota in ob/ob mice

“…Total liver lipids were extracted using a mixture of methanol and chloroform (1:2, vol/vol) and resolved in a mixture of hexane: diethyl ether: acetic acid (70: 30: 0.2, by vol) for quantitative lipid class analyses in the thin-layer chromatography flame ionization detection system as previously described ( 22 ). Phospholipids (PL) were separated into classes by high-performance liquid chromatography ( 23 ).…”

Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species

“…Major MA components (>20% relative abundance) in strain 1135 contained 52-58 carbons with three double bonds and 54-58 carbons with 2 to 4 double bonds in strain 1138. MA detected under the growth conditions used for the latter stain also included components with 60 carbons and up to five double bonds in the bound MA fraction, while prior analysis of MA separated by thin layer chromatography followed by flame ionization-thermal conductivity/MS/MS showed minor components with smaller carbon chains (31-44 carbons, 0 to 5 double bonds) [41]. Multiple isomers of all MA major components were detected (Table S2).…”

Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications

“…While the determination of the FA profile by gas chromatography combined with mass spectrometry (GCMS) is commonly used to assess cell membrane composition [20,21], headgroup profile investigation has often been neglected, possibly missing out valuable information. The identification of lipid headgroups is usually performed by thin-layer chromatography [22][23][24][25][26], but solution 31 P NMR can be used to identify and quantify cell membrane phospholipids, although it has rarely been applied to bacterial samples [27][28][29][30][31][32][33][34][35][36][37]. More recently, liquid chromatography combined with mass spectrometry (LCMS) provided a much more powerful approach [38][39][40][41][42][43], allowing to determine both headgroup and FA composition with very high sensitivity and resolution.…”