Composition and Quantitation of Microalgal Lipids by ERETIC 1H NMR Method

Abstract

Accurate characterization of biomass constituents is a crucial aspect of research in the biotechnological application of natural products. Here we report an efficient, fast and reproducible method for the identification and quantitation of fatty acids and complex lipids (triacylglycerols, glycolipids, phospholipids) in microalgae under investigation for the development of functional health products (probiotics, food ingredients, drugs, etc.) or third generation biofuels. The procedure consists of extraction of the biological matrix by modified Folch method and direct analysis of the resulting material by proton nuclear magnetic resonance (1H NMR). The protocol uses a reference electronic signal as external standard (ERETIC method) and allows assessment of total lipid content, saturation degree and class distribution in both high throughput screening of algal collection and metabolic analysis during genetic or culturing studies. As proof of concept, the methodology was applied to the analysis of three microalgal species (Thalassiosira weissflogii, Cyclotella cryptica and Nannochloropsis salina) which drastically differ for the qualitative and quantitative composition of their fatty acid-based lipids.

Determination of lipid content of oleaginous microalgal biomass by NMR spectroscopic and GC–MS techniques

“…These signals were more intense in ClMe extracts than in cyclohexane extracts. The presence of these signals was also indicated by the 1 H NMR spectral analyses of these extracts [16,20,25,35]. The region from 170 to 220 ppm did not show signals due to aldehyde (CH=O) and keto (C=O) groups, although signals of very weak intensity were clearly observed in the 7-11-ppm region in the 1 H NMR spectra.…”

“…Singlecomponent standards such as triglyceride oleate (TGO), triglyceride stearate, or triglyceride palmitate have been used to study the fatty acid composition of algal extracts. The matrix effect has not been taken into account when single-component standards have been used for quantitative analyses of lipid content in algal extracts [16,23,31]. If matrix effects are not considered, the results may be misleading owing to overlapping chemical shift regions or resemblance with the matching structures in complex matrices containing a multicomponent system of TG, FFA, and polar lipids as observed in the present studies.…”

“…The determination of lipid content in algal biomass is done by methods based on solvent extraction followed by gravimetric lipid quantification [3,4,6,9,27], fat cellular staining by fluorescence microscopy using a lipid stain such as Nile red [19], solid-state time-domain NMR spectroscopy for direct estimation [15], liquid-state NMR spectroscopy [16,23,25,26,31], Fourier transform IR spectroscopy with statistical correlation [28], and fatty acid methyl ester (FAME) conversion determined by GC-MS [30,34]. Specifically, high-pressure LC can be used to quantify different types of lipids in algal extracts, e.g., monoglycerides, diglycerides, and TG [21].…”

comparison of oil extraction methods for algae by NMR and Chromatographic techniques

“…Detailed chemical composition of algae biomass is essential to understand the chemistry of neutral and polar lipids to explore their biodiesel and high value product potential as well as help in the prediction of quality, yield and physio-chemical properties of the algae derived fuels. Therefore, analytical methods based on NMR [7,8,14,24,[29][30][31][32][33][34][35][36][37][38][39] , GC/GC-MS [7,8,40,41] , IR [24,42] and HPLC [43] techniques are used to characterize and determine the neutral lipids content and their fatty acids composition of the algal oils. The importance of solvent polarity in the extraction of lipids and their composition, fatty acid profile, and yield has been reported from critical analyses of extracted oils obtained by different solvents by GC-MS and gravimetric methods [21] .…”

Comprehensive multiphase NMR: a promising technology to study plants in their native state

“…One such method, namely Electronic REference To access In vivo Concentrations (ERETIC), which uses electronic references for quantification, have yielded significant success in accurately determining analyte concentration for 1D NMR spectra. [53] This method used an artificial signal generated from a calibrant that can be used for quantification purposes. Therefore, this type of processing method can be applicable for in vivo quantification NMR studies of plants in the foreseeable future because ERETIC eliminates the potential spectral or chemical interferences that arise from internal standards.…”