List of figures

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List of figures

Figure 2.1.Sacharomyces cerevisiae cell. Taken from Murtey and Ramasamy

(2016). ... 25

Figure 2.2. Example of a metabolic pathway involving catabolism and anabolism in the pentose phosphate pathway. Glucose is phosphorylated to Glucose-6-P, process of adding a phosphate group (anabolism), thus requiring energy. Then, Glucose-6-P is reduced to ribose-5-P (catabolism). Finally, ribose-5-P is used for building nucleic acids (anabolism), which are fundamental components in biomass composition. ... 27

Figure 2.3. General scheme of S. cerevisiae metabolism. Link between anabolism and catabolism adapted from Stephanopoulos (1998). ... 28

Figure 2.4. Glycolysis pathway. ... 29

Figure 2.5 TCA cycle and glyoxylate cycle (reactions in dash lines) in S. cerevisiae. ... 30

Figure 2.6 Ethanol and acetate production in the fermentation pathway. ... 31

Figure 2.7 Formation of glycerol in the fermentative pathway. ... 32

Figure 2.8 Pentose phosphate pathway. ... 32

Figure 2.9. Interconversion of alpha-ketoglutarate, ammonia, glutamate and glutamine in the CNM. ... 33

Figure 2.10. Scheme of amino acid biosynthesis in S. cervesiae. Taken from Feldmann (2012). ... 34

Figure 2.11. Amino acids metabolism ... 35

Figure 2.12 Biomass composition of S. cerevisiae ... 36

Figure 4.1 Schematic representation of a bioreactor ... 41

Figure 4.2 Bioreactor in batch mode. ... 42

Figure 4.3. Bioreactor in fed-batch mode ... 42

Figure 4.4 Bioreactor in continuous mode ... 43

Figure 4.5 Bioreactor in perfusion mode ... 43

Figure 4.6 Typical S. cerevisiae growth curve ... 45

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Figure 4.8 Different types of model complexity ... 47

Figure 4.9 A general representation of modelling bioprocesses. a)In microscopic modelling, a general macroscopic reaction squeme represents the transformation of the subtrates in products without taking into account the internal metabolism of the cell b) In microscopic modelling, the substrates enter the cell and are converted into metabolic products via intracellular metabolites. ... 52

Figure 4.10 Reconstruction of a microscopic model. ... 53

Figure 4.11 Description of the Stoichemtric matrix S ∈ ℝ 9 × 𝟔 for glycolysis.. 55

Figure 4.12. DFBA in a fed-batch culture with substrate concentration (𝜉𝑠), metabolic product concentration (𝜉𝑝), and biomass concentration (X), 𝑣𝜉𝑠 is the vector of substrate specific uptake rates and 𝑓𝑆(𝜉𝑠,𝜉𝑝) is a vector function of concentrations 𝜉𝑠 and 𝜉𝑝. Adapted from Henson and Hanly (2014) ... 63

Figure 4.13.Local and global minima for an objectibe cost function ... 66

Figure 5.1. Ethanol time profile selected for the 4 experiments. ... 72

Figure 5.2. Culture medium feeding profile used for the 4 experiments. ... 72

Figure 5.3. Measurements of experiments: Exp. 1: low nitrogen condition - Exp. 2: high nitrogen condition - Exp. 3: intermediate nitrogen condition - Exp. 4: starvation condition. ... 74

Figure 5.4. Metabolic network of S. cervevisie ... 80

Figure.5. Schematic representation of “overflow metabolism” introduced by Sonnleitner and Käppeli (1986): a.) Respiration of glucose for cell growth without “saturation” of the maximun respiratory capacity (grey ring). b.) Respiration of glucose reaches the maximum respiratory capacity with biomass production (at its maximum) but without ethanol production. c.) Glucose consumption exceeds maximum respiratory capacity, and the surplus is used in the fermentation pathway for ethanol production... 83

Figure 5.6. Comparison between Richelle, Fickers and Bogaerts model simulation and measurements of the 4 experiments – Exp. 1: low nitrogen condition – Exp. 2: high nitrogen condition – Exp. 3: intermediate nitrogen condition – Exp. 4: starvation condition. ... 85

Figure 6.1. Measured concentrations of glucose, biomass, ammonium, ethanol (with 95 % confidence intervals) and their corresponding smoothing splines (in blue) for the different experiments. ... 91

Figure 6.2. External input (glucose, ammonium) and output (ethanol, biomass) fluxes based on smoothing splines and mass balances. ... 92

Figure 6.3. MFA-in-out for Exp 1: maximum (blue) and minimum (red) flux profiles using glucose (v1), ammonium (v48) and ethanol (v79) as external metabolites. Units of the fluxes are c-mol g-1_h-1_{. ... 94}

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Figure 6.5. MFA-in-out for Exp 3: maximum (blue) and minimum (red) flux profiles using glucose (v1), ammonium (v48) and ethanol (v79) as external

metabolites. Units of the fluxes are c-mol g-1_h-1_{. ... 96}

Figure 6.6. MFA-in-out for Exp 4: maximum (blue) and minimum (red) flux profiles using glucose (v1), ammonium (v48) and ethanol (v79) as external

Figure 6.7. Upper bound for biomass flux (𝑣79𝑚𝑎𝑥) corresponding to the MFA-in-out problem in blue and specific growth rate from the measurements in green (vX): a. Exp 1, b. Exp 2, c. Exp 3, and d. Exp 4. ... 98

Figure 6.8. FBA-in-out for Exp 1: maximum (blue) and minimum (red) fluc profiles using glucose (v1), ammonium (v48) and ethanol (v79) as external

Figure 6.12. Representation of glucose overflow metabolism. a. Respiration: glucose is fully oxidized. b. Respiro-fermentation: overflow metabolism with glucose excess and ethanol production. c. Ethanol and nearly zero glucose are oxidized. ... 108 Figure 6.13. Optimal biomass growth flux (in blue) predicted with FBA-in and

measured 𝑣79𝑜𝑝𝑡 specific growth rate (vX, in green):a. Exp 1, b. Exp 2, c. Exp

3, and d. Exp 4 ... 111 Figure 6.14. Maximum (in blue) and minimum (in red) admissible values of ethanol production flux (v68) predicted with FBA-in and measured ethanol

specific production rate (vE, in green): a. Exp 1, b. Exp 2, c. Exp 3, and d. Exp

4. ... 112

Figure 6.15. FBA-in for Exp 1 using glucose (v1), ammonium (v48) as external

metabolites: maximum (blue) and minimum (red) flux profiles. Units of the fluxes are c-mol g-1_h-1_{. ... 113}

metabolites: maximum (blue) and minimum (red) flux profiles. Units of the fluxes are c-mol g-1_h-1_{. ... 114}