BIOENERGY AND BIOFUELS Metabolic flux analysis model for optimizing xylose conversion into ethanol by the natural C5-fermenting yeast Candida shehatae Carine Bideaux 1,2,3 & Julie Montheard 1,2,3 & Xavier Cameleyre 1,2,3 & Carole Molina-Jouve 1,2,3 & Sandrine Alfenore 1,2,3 Received: 27 February 2015 /Revised: 17 September 2015 /Accepted: 13 October 2015 # Springer-Verlag Berlin Heidelberg 2015 Abstract A metabolic flux analysis (MFA) model was devel- oped to optimize the xylose conversion into ethanol using Candida shehatae strain. This metabolic model was compartmented and constructed with xylose as carbon sub- strate 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 compo- sition and thus production were integrated considering the major biochemical synthesis reactions from monomers to each constitutive macromolecule (i.e., proteins, lipids, polysaccha- rides, nucleic acids). The construction of the model resulted into a 122-linear equation system to be resolved. A first ex- periment 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 Cmol ethanol /Cmol xylose for an oxygen requirement of 0.33 mol oxygen /mol xylose 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 Cmol ethanol /Cmol xylose ) was obtained for an oxy- gen supply of 0.47 mol oxygen /mol xylose . Keywords Candida shehatae metabolism . Metabolic flux analysis . Ethanol production . Ratio oxygen/xylose Introduction Due to the importance of biobased alternatives for fuel substitution, ethanol from lignocellulosic biomass has been intensely studied over the last years. While the conversion of C6 sugars like glucose has been broadly mastered, C5 conversion in particular of xylose is still a scientific and technical challenge. The C5 fraction in lignocellulosic materials can represent a significant pro- portion in the range 10–40 % (w/w) (Singh and Mishra 1995). In order to maximize sugar conversion into etha- nol, two strategies exist: one based on engineering ge- netically modified strains (mainly in Saccharomyces cerevisiae) to convert both C6 and C5 sugars and the other based on the use of naturally C5-fermenting yeasts like those of the Pichia or Candida genera. Candida shehatae yeast has been described as a poten- tial converter of xylose into ethanol (Toivola et al. 1984). Studies have shown that oxygen strongly impacts the ca- pacities of the strain to produce ethanol, but its role is not entirely understood (Du Preez and van der Walt 1983; Sánchez et al. 1997). The oxygen supply affects xylose Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7085-0) contains supplementary material, which is available to authorized users. * Carine Bideaux bideaux@insa-toulouse.fr 1 Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France 2 INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France 3 CNRS, UMR5504, F-31400 Toulouse, France Appl Microbiol Biotechnol DOI 10.1007/s00253-015-7085-0