Profiling whole microalgal cells by high-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy

Abstract

Microalgae are a rich source of high value compounds such as carbohydrates, lipids, proteins and bioactive compounds. In particular, microalgae have been identified as a potentially important resource for carboncapture and as a feedstock for green biofuels. Successful cultivation of microalgae can occur under a variety of nutrient and environmental conditions with each condition producing a unique distribution of compounds. In order to steer the cultivation towards a particular distribution of compounds, rapid and accurate methods for compound identification are required. Current methods for determining the absolute quantity of each component are time consuming and arduous making cultivation optimization impractical. High-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy offers a robust and rapid screening method capable of ascertaining the absolute quantity of each component with minimal sample manipulation. Sample preparation consists of harvested, centrifuged and freeze-dried whole-cell Nannochloropsis granulata from large-scale photobioreactors being accurately weighed and rehydrated with deuterium oxide and placed in an HR-MAS rotor. One-dimensional HR-MAS NMR spectra were recorded under quantitative conditions to determine the lipid and carbohydrate profile of the microalgae. The total time per sample for preparation, data acquisition and analysis was approximately 1 h. Changes in resonance profiles corresponding to varying proportions of saturated and polyunsaturated fatty acids were correlated to the time of harvest. In addition, standard two dimensional experiments were used to identify the major carbohydrate components. HR-MAS NMR spectroscopy has been used to profile the lipid and carbohydrate content of N. granulata and we have begun to establish methodologies for quality analysis/quality control for cultivation of various microalgal strains.

In-cell NMR: Why and how?

“…1 H and 13 C HR-MAS NMR also proved to be valuable for analyzing the whole-cell metabolic profile of algae in one single experiment [998][999][1000], which would usually have been carried out from separate water-soluble fractions and lipophilic extracts. Such a convenient profiling method of intact cells by HR-MAS NMR was presented as well suited to the analysis of algal content for nutritional and bio-fuel applications [960,[1001][1002][1003] and the identification of marine microorganisms [1004,1005]. Recent approaches favor flow-cell 1 H NMR in line with bioreactors to monitor the global lipid content of live microalgae cultures in real-time [960,961], also using benchtop NMR spectrometers [962,963].…”

Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State ¹H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

“…Our NMR measurements are in agreement with those of Davey et al [ 4 ], who found that the lipid content of algae cultures measured via liquid-state NMR was less than that measured via fatty acid methyl ester derivation followed by gas chromatography (FAME-GC), which can quantitate both TAGs and ordered lipids such as membrane phospholipids. Use of semisolid NMR techniques such as high-resolution magic-angle spinning (HR-MAS) or intermolecular multiple-quantum coherence (iMQC) would facilitate quantitation of these ordered lipid molecules for improved calibration of BODIPY fluorescence [ 15 , 46 , 51 ]. However, the excellent correlation that we measure between BODIPY fluorescence and TAG content by liquid-state NMR suggests that the cellular concentration of these ordered lipids is unaffected by nitrogen limitation, with additional lipid accumulation mostly in the form of TAGs which can be quantitated via liquid-state NMR.…”

“…Nuclear magnetic resonance (NMR) spectroscopy is capable of determining the composition of complex biological mixtures with minimal sample preparation that generally avoids the complexity of chromatographic separation or chemical derivatization [ 35 ]. NMR spectroscopy can interrogate the interior of intact cells, enabling characterization of lipids and other metabolites within whole algae cells [ 14 , 15 , 36 , 37 ]. Of particular interest to this study, 1 H NMR spectroscopy has been used to quantitate intracellular TAG deposits within intact cells in biological tissues [ 38 ].…”

Magnetic Resonance Technologies: Molecules to Medicine

“…[99] In addition samples can be in solution or semi-solid states. [100] Relatively simple protocols have been developed that use a single certified external standard to calibrate the instrument. From the calibrated system, other samples can be rapidly quantitated.…”

“…Macro molecular assemblies on-theother-hand can be examined for profiling and quantitation of algae lipid content and polysaccharides or metabolites. [100]…”