Xavier Cameleyre

Minimization of Glycerol Production during the High-Performance Fed-Batch Ethanolic Fermentation Process in Saccharomyces cerevisiae, Using a Metabolic Model as a Prediction Tool

ABSTRACT On the basis of knowledge of the biological role of glycerol in the redox balance of Saccharomyces cerevisiae, a fermentation strategy was defined to reduce the surplus formation of NADH, responsible for glycerol synthesis. A metabolic model was used to predict the operating conditions that would reduce glycerol production during ethanol fermentation. Experimental validation of the simulation results was done by monitoring the inlet substrate feeding during fed-batch S. cerevisiae cultivation in order to maintain the respiratory quotient (RQ) (defined as the CO2 production to O2 consumption ratio) value between 4 and 5. Compared to previous fermentations without glucose monitoring, the final glycerol concentration was successfully decreased. Although RQ-controlled fermentation led to a lower maximum specific ethanol production rate, it was possible to reach a high level of ethanol production: 85 g · liter−1 with 1.7 g · liter−1 glycerol in 30 h. We showed here that by using a metabolic model as a tool in prediction, it was possible to reduce glycerol production in a very high-performance ethanolic fermentation process.

In situ rheometry of concentrated cellulose fibre suspensions and relationships with enzymatic hydrolysis

This work combines physical and biochemical analyses to scrutinize liquefaction and saccharification of complex lignocellulose materials. A multilevel analysis (macroscopic: rheology, microscopic: particle size and morphology and molecular: sugar product) was conducted at the lab-scale with three matrices: microcrystalline cellulose (MCC), Whatman paper (WP) and extruded paper-pulp (PP). A methodology to determine on-line viscosity is proposed and validated using the concept of Metzner and Otto (1957) and Rieger and Novaks (1973). The substrate suspensions exhibited a shear-thinning behaviour with respect to the power law. A structured rheological model was established to account for the suspension viscosity as a function of shear rate and substrate concentration. The critical volume fractions indicate the transition between diluted, semi-diluted and concentrated regimes. The enzymatic hydrolysis was performed with various solid contents: MCC 273.6 gdm/L, WP 56.0 gdm/L, PP 35.1 gdm/L. During hydrolysis, the suspension viscosity decreased rapidly. The fibre diameter decreased two fold within 2 h of starting hydrolysis whereas limited bioconversion was obtained (10-15%).

Assessment of Candida shehatae viability by flow cytometry and fluorescent probes.

Quantification of different physiological states of Candida shehatae cells was performed by flow cytometry associated with two fluorescent probes. Propidium iodide and carboxyfluorescein diacetate acetoxymethyl ester fluorescent dyes were chosen based on data from the literature. A staining procedure, developed from the previous works was applied to the yeast. Then, the protocol was improved to fit with fermentation constraints such as no physiological interference between the staining procedure and the cells, shortest preparation time and small amounts of dyes. From this optimisation, propidium iodide was included in the sample at 8 mg/L whereas carboxyfluorescein was first diluted in Pluronic® agent and used at 3mg/L, samples were incubated for 10 min at 40°C. Repeatability and accuracy were evaluated to validate this flow cytometry procedure for viability determination.