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