Lipid analysis of Neochloris oleoabundans by liquid state NMR

Abstract

This study is an evaluation of liquid state NMR as a tool for analyzing the lipid composition of algal cultures used for biodiesel production. To demonstrate the viability of this approach, (13)C NMR was used to analyze the lipid composition of intact cells of the algal species, Neochloris oleoabundans (UTEX #1185). Two cultures were used in this study. One culture was "healthy" and grown in conventional media, whereas the other culture was "nitrogen-starved" and grown in media that lacked nitrate. Triglyceride was determined to be present in both cultures by comparing the algal NMR spectra with published chemical shifts for a wide range of lipids and with a spectrum obtained from a triglyceride standard (glyceryl trioleate). In addition, it is shown that (1) the signal-to-noise ratio of the approximately 29.5 ppm methylene peak is indicative of the lipid content and (2) the nitrogen-starved culture contained a greater lipid content than the healthy culture, as expected. Furthermore, the nitrogen-starved culture produced spectra that primarily contained the characteristic peaks of triglyceride (at approximately 61.8 and approximately 68.9 ppm), whereas the healthy culture produced spectra that contained several additional peaks in the glycerol region, likely resulting from the presence of monoglyceride and diglyceride. Finally, potential interferences are evaluated (including the analysis of phospholipids via (31)P NMR) to assess the specificity of the acquired spectra to triglyceride. These results indicate that NMR is a useful diagnostic tool for selectively identifying lipids in algae, with particular relevance to biodiesel production.

Effect of Various Pretreatment for Extracting Intracellular Lipid fromNannochloropsis oculataunder Nitrogen Replete and Depleted Conditions

“…This indicated that nitrogen was essential in synthesis of chlorophyll for photosynthesis which also enhanced growth of microalgae. Similar result was also found in Beal et al [18] which showed that nitrogen starved algal paste was yellow green in color and healthy sample was dark green. Figure 2 shows that nitrogen starved cells contained more lipid droplets with increased cell size than the normal cells.…”

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

“…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.…”

“…From the detailed NMR spectral interpretation of solvent extracts of algal biomasses and GC-MS analyses, we summarize that algal extracts are composed of the following components: As a general practice reported in the literature, the fat or glyceride content in algal or vegetable oils is determined by use of a single-component external reference such as TGO, triglyceride stearate, or triglyceride palmitate [16,23,31]. This approach might not be appropriate in the case of algal oils for the following reasons:…”

“…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].…”

Monitoring of Microalgal Processes

“…NMR spectroscopy has been used to determine the lipid contents of lyophilized biomass [228] and of wet biomass [229]. From the perspective of this review, the most interesting application so far has been the use of liquid state 1 H-NMR for noninvasive quantification of the lipid (triacylglyceride, TAG) content within live cultures [166].…”