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Optimization of the citric acid production by Aspergillus niger
through a metabolic flux balance model
Published by
Daniel V Guebel and Nester V Darias
Studied and Presented by
Alaa Kububja and Meteab Al-Otaibi
Introduction
• Leading source of citric acid is aspergillus niger fermentation
• Several efforts made to develop dynamical models
• The acid producing stage, Idiophase,
Paper Objective
Based on metabolic flux analysis, a mathematical model is developed aiming for:
• Better understanding and description of the process of citric acid production
• Helping in the design of the best genetic strategies leading to the optimization of citric acid production rate
Idiophase Stages
• Early Idiophase1 mol glucose + 1.5 mol O2 3.81g biomass + 0.62
mol citric acid + 0.76 mol CO2 + 0.37 mol polyols
• Medium Idiophase1 mol glucose + 2.4 mol O2 1.54g biomass + 0.74
mol citric acid + 1.33 mol CO2 + 0.05 mol polyols
• Late Idiophase1 mol glucose + 3.9 mol O2 + 0.42 mol polyols
0.86 mol citric acid + 2.41 mol CO2
Model Development
• Hypothesis: existence of a close energetic coupling between the citric acid production and the intracellular pH regulation (due to strong acidic conditions for A. niger, extracellular pH =2)
• Focusing at the idiophase stage (80-220 hrs culture time) when the growth drops and acid production becomes the main cellular activity
Model Development (cont.)
• Establishing the transient idiophase nature by stoichiometric analysis
• Computing the main intracellular fluxes by application of material and physiological constraints at culture time 120 hrs.
Determination of the rate of GABA cycle
dNH4+(out)/dt+dNH4
+(c) /dt+dNH4+(m)/dt=0 (1)
dNH4+(out)/dt=Vgen(out)(NH4
+)-Vuptake(c)(NH4+)=0 (2)
dNH4+(c) / dt = Vuptake(c)(NH4
+) - Vdiss(NH4+ (c))
+ Vcatab(AA(c)) - Vuptake(m)(NH4+) = 0 (3)
dNH4+(m)/dt = Vuptake(m)(NH4
+)-Vdiss(c)(NH4+ (m))
– R14 + Vcatab(AA(m)) = 0 (4)By summation of (2)-(4)
R14 = (Vcatab(AA) + Vgen(out)(NH4+ ))
– (Vdiss(NH4+ (c)) + Vdiss(NH4
+ (m)) = 0 (5)
Pyruvate(c) + (1/3) H2O CO2 + (2/3)NADH2(m) + (1/3) NADH2(c) + (1/3) FADH2(m) +(1/3) H+(m) R14
Determination of the rate of GABA cycle (cont.)
dNH3(c) /dt = Vdiss(NH4+ (c))
+ Vleak(NH3(m)) - Vleak(NH3(c)) = 0 (6)
dNH3(out) /dt = Vleak(NH3(c))
- Vgen(NH4(out)) = 0 (7)
dNH3(m)/dt = Vdiss(NH4+ (c))
+ Vleak(NH3(m)) = 0 (8)
From (6) to (8), we obtain:
Vleak(NH3(c)) = Vdiss(NH4+ (c))
+ Vleak(NH4+(c)) =0 (9)
By substituting (9) in (5)
R14 = Vcatab(AA) (10)
Effects on A. niger idiophase synthesis to changes in metabolic processes
Process Perturb-
ation
Expected Response (%)
Magnitude Citrate Synthesis
(%) Available Carbon
Available ATP
Direct Citrate
Excretion
Net Effect
Glucose
uptake
+100 +140 +280 -93 +47
Glycerol-P phosphatase
-100 +5.4 Null -3.6 +1.8
R5P Reductase
-100 +1.9 Null -1.3 +0.6
Results
• Citric productivity would be increased in 45% by
• Increase citric acid synthesis rate through:– Increasing glucose uptake– Decreasing biosynthesis rate of by products
(polyols)– Decreasing fluxes diverting mass from the the
pathway leading to citrate pressure
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