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

Abstract

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.

FTIR spectroscopy as a unified method for simultaneous analysis of intra- and extracellular metabolites in high-throughput screening of microbial bioprocesses

“…However, it should be noted that our study has covered one order of magnitude higher range of glucose concentration (up to 80 g L −1 glucose) compared to the one in the above mentioned study [ 27 ]. The only previous reported HTS measurement of glucose, where similarly high concentrations of glucose were measured in an artificial set of model solutions (and not an actual cultivation medium), has resulted with very large errors of prediction [ 28 ].…”

“…The analysis is usually achieved by depositing biomass samples on a multi-well IR-light-transparent microplate [ 7 – 10 , 24 – 26 ]. Several studies have shown a high correlation of HTS FTIR spectroscopy with traditional analytical techniques, such as GC, HPLC, and biochemical assays, for different types of bioprocesses [ 7 , 9 , 27 , 28 ]. However, the majority of studies have been focused on quantitative analysis of biomass, while high-throughput studies of substrates and extracellular metabolites had very limited scope [ 27 , 28 ].…”

“…Several studies have shown a high correlation of HTS FTIR spectroscopy with traditional analytical techniques, such as GC, HPLC, and biochemical assays, for different types of bioprocesses [ 7 , 9 , 27 , 28 ]. However, the majority of studies have been focused on quantitative analysis of biomass, while high-throughput studies of substrates and extracellular metabolites had very limited scope [ 27 , 28 ]. Thus, the application of FTIR spectroscopy in high-throughput screening of microorganisms has not been fully explored, despite the fact that it can perform high-throughput multi-analyte quantification of both intra- and extracellular metabolites and substrate consumption.…”

Effects of light and nitrogen availability on photosynthetic efficiency and fatty acid content of three original benthic diatom strains

“…Absorbance spectra were collected between 4 000 cm -1 and 700 cm -1 with 30 scans and averaged. The spectra were analyzed by the integral ratios method [50]. The lipid signature was associated with the ester bond (Eb) signal (~1740 cm -1 ), whereas the carbohydrate signature was associated with the C-O-C signal of the polysaccharides (~1200–900 cm -1 ) [37] and the protein signature associated with the amide II band (~1540 cm -1 ) of the N-H of the amides associated with the proteins.…”

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

“…For that purpose, numerous approaches have illustrated the effect of the microalgae culture conditions on the molecular determinants that trigger lipids production [11][12][13] and the physiological modifications associated. [14][15][16][17] However, it is still difficult to understand the relationship between the metabolic fate of the carbon, the fine chemical characterization of the accumulated lipids, and the physiological adaptation of the microalgae during the biosynthesis accumulating lipids in the form of droplets.…”