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.