Revealing Lipid Body Formation and its Subcellular Reorganization in Oleaginous Microalgae Using Correlative Optical Microscopy and Infrared Nanospectroscopy.

The purpose of this work is to develop an integrated imaging approach to characterize without labeling at the sub-cellular level the formation of lipid body droplets (LBs) in microalgae undergoing nitrogen starvation. First conventional optical microscopy approaches, gas chromatography, and turbidimetry measurements allowed to monitor the biomass and the total lipid content in the oleaginous microalgae Parachlorella kesslerii during the starvation process. Then a local analysis of the LBs was proposed using an innovative infrared nanospectroscopy technique called atomic force microscopy-based infrared spectroscopy (AFM-IR). This label-free technique assessed the formation of LBs and allowed to look into the LB composition thanks to the acquisition of local infrared spectra. Last correlative measurements using fluorescence microscopy and AFM-IR were performed to investigate the subcellular reorganization of LB and the chloroplasts.

Characterization of an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC-MS system: application to Arthrospira platensis.

We developed an easy-to-use method for the routine analysis of the central metabolism using an affordable low-resolution GC-MS system run in SIM mode. The profiling approach was optimized for the derivatization protocol of some 60 targeted metabolites. The performance of two silylation reagents (MSTFA and BSTFA) that allowed the comprehensive derivatization of 42 key intermediary metabolites of the 60 initially targeted (organic acids, phosphate derivatives, monosaccharides and amino acids) was measured. The experimental results unequivocally showed that the MSTFA reagent met mandatory criteria including ease of handling (a very simple one-step protocol was developed), comprehensiveness of derivatization (the 42 compounds covered the extended metabolic pathways of the central carbon metabolism, with a coverage percentage ranging from 17% for the worst to 90% for the best result), optimized response coefficient of the whole derivatives (median value greater than the others by one order of magnitude) and repeatability of the protocol (RSD value below 25% for the whole procedure). When tested in real conditions (cyanobacteria polar extract), the experimental results showed that the profiling methodology was adequately repeatable (RSD = 35%) to ensure quantification results comparable with much more sensitive analytical techniques (capillary electrophoresis/mass spectrometry and liquid chromatography/triple quadrupole mass spectrometry system), while needing only about twice the quantity of biomass. Graphical abstract Schematic overview of an easy-to-use profiling method for the routine analysis of the central metabolism using a low-resolution GC-MS system.

Influence of physical and chemical properties of HTSXT-FTIR samples on the quality of prediction models developed to determine absolute concentrations of total proteins, carbohydrates and triglycerides: a preliminary study on the determination of their absolute concentrations in fresh microalgal biomass.

Absolute concentrations of total macromolecules (triglycerides, proteins and carbohydrates) in microorganisms can be rapidly measured by FTIR spectroscopy, but caution is needed to avoid non-specific experimental bias. Here, we assess the limits within which this approach can be used on model solutions of macromolecules of interest. We used the Bruker HTSXT-FTIR system. Our results show that the solid deposits obtained after the sampling procedure present physical and chemical properties that influence the quality of the absolute concentration prediction models (univariate and multivariate). The accuracy of the models was degraded by a factor of 2 or 3 outside the recommended concentration interval of 0.5-35 µg spot(-1). Change occurred notably in the sample hydrogen bond network, which could, however, be controlled using an internal probe (pseudohalide anion). We also demonstrate that for aqueous solutions, accurate prediction of total carbohydrate quantities (in glucose equivalent) could not be made unless a constant amount of protein was added to the model solution (BSA). The results of the prediction model for more complex solutions, here with two components: glucose and BSA, were very encouraging, suggesting that this FTIR approach could be used as a rapid quantification method for mixtures of molecules of interest, provided the limits of use of the HTSXT-FTIR method are precisely known and respected. This last finding opens the way to direct quantification of total molecules of interest in more complex matrices.

Unravelling the matrix effect of fresh sampled cells for in vivo unbiased FTIR determination of the absolute concentration of total lipid content of microalgae.

Over the past years, the substitution of the classical biochemical quantification techniques by Fourier transform infrared (FTIR) spectroscopy has been widely studied on microalgae because of its tremendous application potential for bioprocess monitoring. In the present work, mandatory aspects that have never been approached by FTIR end-users working onto fresh biomass were assessed. We demonstrated first that fresh cells' FTIR spectra main characteristics could be severely and unspecifically altered when the properties of the sampled biomass were not monitored. Microscopy indicated that important cell reorganization could occur when diminishing the cells density of the sample. Molecular probing approach suggested that such a modification could provoke an alteration of the hydrogen-bonding network of the sample. The sample heterogeneity was found to impact also the shape and intensity of the recorded FTIR bands, participating then to a matrix effect uncharacterized until now. In the second part of our study, we selected FTIR spectra not influenced by this matrix effect and the corresponding accurate calibration data obtained by the whole cell analytical procedure to elaborate an optimized total lipid quantification PLS-R model. Results demonstrated that our strategy could provide a small volume sampling (1 mL of fresh culture), rapid (within minutes), robust (physiological condition independent), and accurate (as accurate as the reference method could be) FTIR absolute quantification method to determine the fresh microalgae intracellular total lipid content. To validate our unbiased FTIR approach, a photobioprocess monitoring pipeline was developed and allowed assessing the effect of light attenuation on total lipid production by the marine microalga Nannochloropsis oculata.