Optimisation of Itaconic Acid

Production with Membrane

Systems
N. Nemestóthy, L. Tóth, G. Tóth, P. Bakonyi, L. Gubicza, K. Belafi-Bako,
Research Institute on Bioengineering, Membrane Technology and Energetics,
University of Pannonia
Veszprém, Hungary
 Production of Oragnic Acids
Organic acids are produced commercially either by
chemical synthesis or fermentation. However,
fermentation processes are the most commonly
used method.
All organic acids of tricarboxylic acid cycle can be
produced in high yields in microbiological
Pilot scale processes.
Levulinic acid
Acrylic acid
Adipic acid
Itaconic acid
Malic acid Lab development
Fumaric acid
Butyric acid
Glucaric ascid
Glycolic acid
Dodecaneolic acid
Propionic acid
Sebacic acid

Portfolio and stage of development of different organic acids produced via

microbial fermentation. Alonso et al. Crit Rev Biotechnol, 2015; 35(4): 497–513
 Itaconic Acid

Frederike C. et al Bioresource Technology, 137,2013,

Amina Ahmed El-Imam Biodivers Biopros Dev 2014, 1

 Aims
Optimisation Parameter
of identification
fermentation

Integrated
system Membrane
processes
test
Aspergillus terreus
No toxin production Integrated
system
Low growth rate desing
pH, aeration, substrate, biomass
Optimization of Fermentation Process
Seed broth Concentrations Production broth Concentrations [g/l]
[g/l]

Glucose Glucose 120

10
KH2PO4 0,1
NaCl 20 NH4NO3 3
Potato 40 MgSO4·7H2O 1
Dextrose agar CaCl2·2H2O 5
Agar FeCl3·6H2O 1,67·10-3
15
ZnSO4·7H2O 8·10-3
CuSO4·7H2O 15·10-3

Sartorius Biostat B plus 2 LAMBDA Minifor

 Effect of pH 50

control pH 40

IA [g/l]
1 Non controlled 3.0 30

20
2 Continuous 3.0
10
3 Continuous 2.5
0
4 Continuous 4.0
0 24 48 72 96 120 144 168 192 216 240 264 288 312
5 One step 3.0  2.5 Time [h]
6 Continuous 3.0  2.5
1. 2. 3. 4. 5. 6.
5.00 4.45

41.4 42.98 3.83

45 4.00

Productivity [g/l*d]
Final IA concentration[g/l]

35 3.00 2.77
26.28
2.16
25 19.98
2.00
13.99 1.34
15
5.1 1.00 0.50
5
0.00
-5 1. 2. 3. 4. 5. 6. 7.
Nr. 1. 2. 3. 4. 5. 6. 7.
Nr.
 Nr.

1
2
RPM

150
150
Aeration

2 L/min
2 L/min +5% O2
Aeration
3 150 2 L/min + 50% O2
4 150 2 L/min + 100% O2
5 0 2 L/min
6 150 0 L/min
7 600 2 L/min
8 1200 2 L/min

80
1
7

IA concentration [g/l]
70
Productivity [g L/d]

6 60 2
5 50 3
4 40
3 30 4
2 20 5
1 10
6
0 0
1 2 3 4 5 6 7 8 0 2 4 6 8 10 12 14 7
Nr. time [d] 8
 Substrate / Biomass
Nr. Glucose [g/l] Biomass [g/l]

1 80 1.5

2 120 1.5
3 180 1.5
4 250 1.5
5 120 3
6 120 15

50

IA concentration [g/l]
4 40 1
Productivity [g L/d]

2
3 30
3
2 20
4
1 10 5
0 0 6
1 2 3 4 5 6 0 2 4 6 8 10 12 14
Nr. time [d]
 Fitted Surface; Variable: Productivity
6 Factor Screening Design; MS Residual=1.439308
DV: Productivity

Effects Pareto Chart of Standardized Effects; Variable: Productivity

6 Factor Screening Design; MS Residual=1.439308
DV: Productivity

Profiles for Predicted

>8 Values and Desirability
pH pH control Aeration O2 < 8 RPM Glucose Biomass Desirability
10.000 <6 (3)Aeration O2 3.318824
<4
<2
<0 1.
6.3000

Productivity
.5
3.1875
2.6499
(6)Biomass -1.81808
0.
.07500

-4.000
(1)pH -1.35433

Desirability
.41363
(4)RPM -1.33031
Observed vs. Predicted Values
6 factors at two levels; MS Residual=1.439308
DV: Productivity
2.5
2.9688
4. 0.
.125
1. 0. 100. 150. 1200. 80.
129.38
8
250. 1.5 15. (5)Glucose -1.2869
7

6
(2)pH control .7859918
5
Predicted Values

3 p=.05
2 Standardized Effect Estimate (Absolute Value)
1

-1
-1 0 1 2 3 4 5 6 7
Observed Values
 Aeration by membranes
• Bubble free environment
• High mass transfer
• Selectivity
• Support ‚  immobilization

• PDMS, PES, PVDF

 Results with PDMS tubes
• PDMS 8 m, od 2.2 mm, 550 cm2

60

50
IA concentration [g/l]

40
1 bar air
30
3 bar air
20 1 bar O2
3 bar O2
10

0
0 5 10 15
time [d]
 MF / Biomass
• PVDF 0,22 µM 150 cm2 AT/V vs. V/A
2000000
• Flat sheet, TMP 2 bar 1500000

At/V [s/m]
1000000
y = 377,714,078.18x + 645,495.89
500000 R² = 0.97
0
0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035
V/A [1/m]

0.07
0.06
0.05
permeate V [L]

0.04
0.03 𝑚
0.02 𝛼 = 1,0465 ∗ 1017
0.01 𝑘𝑔
0
0 1000 2000 3000 4000 5000 6000
time [s]
MF reverse flow
50

IA concentration [g/l]
40

30
control
20
80 g/l glucose
Product 10
120 g/l glucose
remmoval
0
0 5 10 15
Substrate time [d]
Filtration
solution
4

Productivity [g L/d]
3

2
Regeneration
1

0
control 80 g/l 120 g/l
glucose glucose
 Electrodyalisis
Fumasep FKS (PA) 7
6
Fumasep FAA (PEEK) 5

IA c[g/L]
0,5 M Na2SO4 4
3
3 stack 180 cm2 2
1
0
0 2000 4000 6000 8000
t[sec]

5.00

4.00
glucose c[g/L]

3.00

2.00

1.00

0.00
0 time[min] 40
20 60 80
 Combined system

MF with PVDF
membrane Electrodyalisis

pH Control
Aeration by
PDMS
membrane MF with PVDF
membrane
regeneration
 Fitted Surface; Variable: productivity
3 factors at two levels; MS Residual=.011025
DV: productivity

Synergic effect ?
45
40
Observed vs. Predicted Values

35 3 factors at two levels; MS Residual=.011025 > 4.1

IA concentration [g/l]

DV: productivity < 4.1

5.0
<4
< 3.9
30 4.8
< 3.8
< 3.7
4.6

25 4.4

120 g/l glucose

Predicted Values
4.2

20 4.0

120 g/l glucose ED 3.8

15 Pareto3.6Chart of Standardized Effects; Variable: productivity

120 g/l glucose ED + pH 3 factors at two levels; MS Residual=.011025
3.4

10 3.2 DV: productivity

3.0

5 3.0 3.2 3.4 3.6 3.8 4.0 4.2

Observed Values
4.4 4.6 4.8 5.0 5.2

0
(2)MF biomass retention -9.08915
0 5 10 15
time [d]

(1)PDMS aeration 3.

(3)ED IA removal .2699746

p=.05
Standardized Effect Estimate (Absolute Value)
 Conclusions
• With fermentation optimization productivity can be increased 3.3 6.2
g/L day
• Most important parameter is the oxygen supply
• PDMS membrane is capable to maintain Aspergillus terreus
• After an MF electrodyalisis can remove IA

• A combined membrane system can have different optimum parameters

than a stirred bioreactor
• Bipolar ED is needed for pH control
8th International Scientific Conference

PERMEA 2019,
the Membrane Science and
Technology Conference of Visegrád Countries
 Thank you for the attention

Acknowledgement
The János Bolyai Research Scholarship of the Hungarian Academy of
Sciences and the Hungarian Scientific Research Fund (NKFIH K- 119940
project). is duly acknowledged for the support.