Metabolic flux analysis model for optimizing xylose conversion into ethanol by the natural C5-fermenting yeast Candida shehatae.

A metabolic flux analysis (MFA) model was developed to optimize the xylose conversion into ethanol using Candida shehatae strain. This metabolic model was compartmented and constructed with xylose as carbon substrate integrating the enzymatic duality of the first step of xylose degradation via an algebraic coefficient. The model included the pentose phosphate pathway, glycolysis, synthesis of major metabolites like ethanol, acetic acid and glycerol, the tricarboxylic acid cycle as well as the respiratory chain, the cofactor balance, and the maintenance. The biomass composition and thus production were integrated considering the major biochemical synthesis reactions from monomers to each constitutive macromolecule (i.e., proteins, lipids, polysaccharides, nucleic acids). The construction of the model resulted into a 122-linear equation system to be resolved. A first experiment allowed was to verify the accuracy of the model by comparing calculated and experimental data. The metabolic model was utilized to determine the theoretical yield taking into account oxido-reductive balance and to optimize ethanol production. The maximal theoretical yield was calculated at 0.62 Cmolethanol/Cmolxylose for an oxygen requirement of 0.33 moloxygen/molxylose linked to the cofactors of the xylose reductase. Cultivations in chemostat mode allowed the fine tuning of both xylose and oxygen uptakes and showed that lower was the oxygen/xylose ratio, higher was the ethanol production yield. The best experimental ethanol production yield (0.51 Cmolethanol/Cmolxylose) was obtained for an oxygen supply of 0.47 moloxygen/molxylose.

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.