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Application of thin-layer chromatography-flame ionization detection (TLC-FID) to total lipid quantitation in mycolic-acid synthesizing Rhodococcus and Williamsia species

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Nahar, A, Baker, AL ORCID: 0000-0001-5455-2271, Nichols, DS ORCID: 0000-0002-8066-3132, Bowman, JP ORCID: 0000-0002-4528-9333 and Britz, ML 2020 , 'Application of thin-layer chromatography-flame ionization detection (TLC-FID) to total lipid quantitation in mycolic-acid synthesizing Rhodococcus and Williamsia species' , International Journal of Molecular Sciences, vol. 21, no. 5 , pp. 1-19 , doi: 10.3390/ijms21051670.

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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.

Item Type: Article
Authors/Creators:Nahar, A and Baker, AL and Nichols, DS and Bowman, JP and Britz, ML
Keywords: TLC-FID, Iatroscan rods, NMR, lipid extraction, mycolic acids, Williamsia, Rhodococcus, Antarctic bacteria, lipid analysis, new methods
Journal or Publication Title: International Journal of Molecular Sciences
Publisher: MDPI
ISSN: 1661-6596
DOI / ID Number: 10.3390/ijms21051670
Copyright Information:

Copyright 2020 The Authors. Licensed under Creative Commons Attribution 4.0 International (CC BY 4.0) https://creativecommons.org/licenses/by/4.0/

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