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.

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.

Biochemical, physiological and behavioural markers in the endobenthic bivalve Scrobicularia plana as tools for the assessment of estuarine sediment quality.

The aim of this study was to link the responses at different levels of biological organisation of the endobenthic bivalve Scrobicularia plana differentially exposed to anthropogenic pressure. Clams were collected in April 2008 from three estuaries along a pollution gradient (Goyen < Loire < Seine). Biomarkers of defence (metallothionein concentration and glutathione-S-transferase activity) were activated in the Loire and the Seine. Biomarkers of damage revealed neurotoxicity (decreased AChE activity) and impairment of digestive enzyme activities (cellulase or amylase) in these estuaries. The highest lactate dehydrogenase activity was registered in the Loire estuary, in parallel with enhanced levels of vanadium (a metal present in petroleum), likely as a consequence of a small oil spill that occurred one month before the sampling collection. Physiological biomarkers (energy reserves as glycogen, lipids and proteins, condition and gonado-somatic indices) showed a few intersite differences. However, the median size was significantly lower in clams exposed to direct (chemicals) or indirect (available food) effects in the most contaminated site. Burrowing behaviour was disturbed in clams from both of the Loire and Seine estuaries, a response probably due to physiological impairment rather than to avoidance of contaminated sediment. The activation of defence mechanisms towards metals (metallothionein) and other classes of contaminants (the biotransformation enzyme glutathione-S-transferase) do not ensure a total protection since a number of impairments were observed at the infra-organismal (AChE and digestive enzyme activities) and individual (burrowing behaviour) levels in relation to the degree of anthropogenic pressure. However, even in the most contaminated estuary (Seine), historical records do not show a consistent decrease of S. plana populations.