This document summarizes research optimizing zeaxanthin production using immobilized Flavobacterium sp. cells in a fluidized bed bioreactor. The researchers conducted experiments to determine the best conditions for zeaxanthin production, including culture medium composition and bioreactor parameters. An orthogonal experimental design was used to evaluate factors like aeration, pH, pellet size, and carbon-nitrogen ratio. Maximum zeaxanthin production of 3.16 g/L was achieved under optimal conditions, approximately 10 times greater than previous reports. Dimensionless numbers were also correlated to describe process behavior and enable scale-up.

1. Optimization of zeaxanthin production by immobilized Flavobacterium sp. cells in
fluidized bed bioreactor
E. M. ESCAMILLA1, M. Aguilar Torres, V1, T. D. C. Flores2, and I. Torres Pacheco3.
(1) Chemical Engineering Dept., Instituto Tecnológico de Celaya, Ave. Tecnológico y Antonio García Cubas S/N, Celaya, 38010, Mexico, (2) Chemical Engineering Department, Instituto Tecnológico de Celaya, Ave. Tecnológico y Antonio García Cubas
S/N, Celaya, Gto., 38010, (3) Biotecnología, Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Km. 6 Carr. Celaya-San Miguel de Allende, Celaya, Gto., 38010, Mexico
INTRODUCTION
The use of natural and artificial pigments represents a market has on the economy of Mexico and the world. The
pigments market is important to consider the intensity of color, because this is the first exposure to the consumer
about the product that is applied to the pigment (e.g. food), followed by the smell and taste. In the past 50 years, the
development of science and food technology have broadened the landscape of research on natural pigments, so they
have discovered several substances that can perform functions beneficial to food. The natural pigments are a vast
range of chemicals that vary with their method of production and point of origin. Among these features carotenoids
belonging to the group of xanthophylls, which come from yellow corn, corn gluten, dried leaf meal and marigold
flowers. Carotenoids are hydrocarbons with or without oxygen in the molecule that give them different properties of
pigment. The major carotenoids in food are lutein and zeaxanthin. These carotenoids generate tones of golden yellow
yolk and pigment. Zeaxanthin is produced by microorganisms, Flavobacterium sp strain being more productive with
Zeaxanthin (Asker. 2007). In this work, we developed a correlation based on engineering parameters that govern the
process in the production of zeaxanthin and lay the foundations for the escalation of the industrial levels.
OBJECTIVE
Zeaxanthin optimize production in a fluidized bed reactor with recirculation for a possible scale up.
Specific
• Apply an orthogonal experimental design to select the factors that affect the process.
• Achieve the best conditions for the flow in the recirculation reactor.
• Get maximum production of zeaxanthin.
• Model cell growth and production of Zeaxanthin in the process.
•Determine the engineering parameters through relationships with design characteristics of transport properties using
dimensionless numbers
RESULTS AND DISCUSSION
CONCLUSIONS
 Experimental orthogonal L18 design, allowed to find the best composition of culture medium, which a
concentration of zeaxantina of 2,46 g/l was obtained; in shake flask a, before never obtained according to the
reports of Literature.
 Optimal conditions of fermentation process in fluidized bioreactor let us to obtain the maximum yield of
zeaxantina production, whose value was 3.16g/l, and is approximately 10 times greater to the value reported at the
moment in literature.
 The analysis of variance showed that air flow , saline solution and pH were the parameters that more influed in
the zeaxanthin biosyntehes.
REFERENCES
ABSTRACT
The current trend in the food industry has taken up the use of natural products such as nutraceuticals, additives,
flavourings and others. A special case is the use of pigments such as carotenoids, which are used for human and
animal consumption. As is the Zeaxanthin, which is the main pigment of yellow corn and is used mainly as a pigment
in the skin of poultry and egg yolk. The most appropriate process for the production of Zeaxanthin is by submerged
fermentation using Flavobacterium sp. The process for producing Zeaxanthin required determining the engineering
parameters for design and scaling of industrial equipment, in this paper we pose the optimization of engineering
parameters obtained correlated with dimensionless numbers such as are the Reynolds, Sherwood and Schmidt. The
kinetics of the growth of Flavobacterium sp, was obtained by the application of Gompertz model with two parameters
and showed a good correlation with our model: To determine the most important factors of the process we used an
orthogonal experimental design. The factors analyzed were: Recycling, Aeration, pellet size and Carbon - Nitrogen. The
variance analysis showed the most relevant factors: recirculation, aeration, Pellet size and finally the carbon – nitrogen
respectively. So through an unrestricted minimization is defined that the optimal production of Zeaxanthin during the
experiment was 1.2506 mg / ml with an aeration of 1vvm, carbon-nitrogen ratio of 1 :1, recirculation rate of 40 ml / min
and pellet diameter 3mm. Achieving a correlation with experimental data determined by the Quasi-Newton numerical
method. Well, was the best fit to the experimental data obtained during fermentation and In addition curves were
developed describing the behaviour of the process, by means of dimensionless numbers.
MATERIAL AND METHODS
Conditioning and
conservation of
Flavobacterium ssp
Growth of Flavobacterium sp. and
Zeaxanthin production kinetics
Using the optimal culture media
Extraction, Purification,
Characterization and
Quantification of secondary
metabolite (zeaxanthin)
Culture medium
optimization
Experimental design
Fig. 1. Zeaxanthin production process in shake
flask
Inmmobilization process
Figure. 3 Orthogonal L8 (27) experiment design for zeaxanthin production with immobilized
Flavobacterium spp in a fluidized bioreactor
H 2 SO 4 NaOH
H 2 O in
H 2 O outlet
pH
Temperat
ure
sensor
Samplin
g point
Air outlet
Sampling
point
Steel mesh
Biocatalysts
Pouros glass
filter difussor
Gas in
F Description Leves
J
Air flow a 3
vvm
5
vvm
K
pH 7.2 5.2
L
Temperature 30 °
C
27
° C
M
Pellet
diameter
3.0
mm
1.0
mm
N
Inoculum -
Medium ratio
10% 5 %
O
Lihgt No Yes
P
Saline
solution
(4.5g/l NaCl)
No
add
Add
H 2 SO 4 NaOH
H 2 O in
H 2 O outlet
pH
Temperat
ure
sensor
Samplin
g point
Air outlet
Sampling
point
Biocatali
Pouros glass
filter difussor
Gas in
F Description Leves
J
Air flow a 3
vvm
5
vvm
K
pH 7.2 5.2
L
Temperature 30 °
C
27
° C
M
Pellet
diameter
3.0
mm
1.0
mm
N
Inoculum -
Medium ratio
10% 5 %
O
Lihgt No Yes
P
Saline
solution
(4.5g/l NaCl)
No
add
Add
FF DescriptionDescription LevesLeves
JJ
Air flow aAir flow a 3
vvm
3
vvm
5
vvm
5
vvm
KK
pHpH 7.27.2 5.25.2
LL
TemperatureTemperature 30 °
C
30 °
C
27
° C
27
° C
MM
Pellet
diameter
Pellet
diameter
3.0
mm
3.0
mm
1.0
mm
1.0
mm
NN
Inoculum -
Medium ratio
Inoculum -
Medium ratio
10%10% 5 %5 %
OO
LihgtLihgt NoNo YesYes
PP
Saline
solution
(4.5g/l NaCl)
Saline
solution
(4.5g/l NaCl)
No
add
No
add
AddAdd
 Under sterilized conditions a mixture (1:1) of Flavobacterium sp
suspension and poligaracturonic acid solution (8% w/v) was
prepared Centrifugated cells were mixtured with poligalacturonic
acid at 8% solution.
The mixture was forced through a multi-needle template (gauge
21 for 3 mm beads) with a peristaltic pump (CRODE, Mexico)
flowing at 10 ml min-1 and droplets were collected in a sterile 3.5
% CaCl2 solution
Fig 2. Bioreactos and zeaxanthin production
3R,3R’---caroten-3,3’-diol
OH
HO
ZEAXANTHIN
VARIABLESa
Experiment
No.
A
Molar
B
g/l
C
g/l
D
g/l
E
g/l
F
g/l
G
Molar
H
Molar
Zeaxanthin
(mg/ml)
8c
0.01 30 5 5 5 1 0.1 0.01 2.426
11 0.05 10 5 1 3 2 0.1 0.05 0.359
7 0.01 30 3 3 3 2 0.05 0.1 1.059
13 0.05 20 3 3 8 1 0.1 0.05 1.834
10 0.05 10 3 5 8 1.5 0.05 0.01 0.912
16 0.05 30 3 5 5 2 0.01 0.05 1.247
3 0.01 10 7 5 8 2 0.1 0.1 0.160
14 0.05 20 5 5 3 1.5 0.01 0.1 0.463
5 0.01 20 5 3 8 2 0.01 0.01 0.543
9 0.01 30 7 1 8 1.5 0.01 0.05 0.936
17 0.05 30 5 1 8 1 0.05 0.1 0.726
1 0.01 10 3 1 3 1 0.01 0.01 0.725
15 0.05 20 7 1 5 2 0.05 0.01 1.370
2 0.01 10 5 3 5 1.5 0.05 0.05 1.058
12 0.05 10 7 3 5 1 0.01 0.1 0.566
6 0.01 20 7 5 3 1 0.05 0.05 0.357
4 0.01 20 3 1 5 1.5 0.1 0.1 1.599
18 0.05 30 7 3 3 1.5 0.1 0.01 0.549
Table 1. Experimental results for zeaxanthin production in shake flask
Media=.938306 Sigma=.584378 MS Error=.076189 df=2
(Las líneas Discontínuas Indican ±2*Error Estándar)
Zeaxanthinconcentration
0,5
0,6
0,7
0,8
0,9
1,0
1,1
1,2
1,3
1,4
ZN DEX LM NH4 PO4 MG FE-CO MN
Table 2. Optimal levels for the culture medium for the zeaxanthin
production in shake flask
Table 3. Analysis of variance for zeaxanthin production in shake flask
Factor SS DF MS F pa
Zn 0.6793 1 0.6793 8.9167 0.0962
Dextrose 0.9264 2 0.4632 6.0796 0.1412
Corn
steep
liquor
0.9306 2 0.4653 6.1071 0.1407
NH4 1.4203 2 0.7101 9.3208 0.0969
PO4 0.1522 2 0.0761 0.9991 0.5002
Mg 0.4698 2 0.2349 3.0829 0.2449
Fe-Co 0.1448 2 0.0724 0.9501 0.5127
Mn 0.9298 2 0.4649 6.1016 0.1408
Residual 0.1524 2 0.0763
Media = 0.993850 Sigma = 0.756319
FACTOR Level Effect size Standard
error
A) zinc; 0.05 M 2 0.1943 0.112686
B) Dextrose; 10 g/l 1 0.1657 0. 112686
C) Corn step liquor; 5 g/l 2 0.3114 0.11269
D) NH4 5 g/l 3 0.3923 0. 11269
E) PO4;8 g/l 3 0.1132 0. 11269
F) Magnesio; 1.5 g/l 2 0.2220 0 .11269
G) Fe and Cobalt; 0.5 M 2 0.0953 0 .11269
H) Manganese 0.01 M 1 0.286244 0.112686
Zeaxanthin estimation g/lb
2.718728
Table 4 Optimal condictions for zeaxanthin production in shake
flask
Microscopical photographic of
Flavobacterium ssp
FACTO RS
Experim ent
No. Replicateb
J
VVM
K
pH
L
O
C
M
m m
N
%
O
No or
yes
P
No or
yes
Zeax.c
(m g/m l)
2 1 5 5.2 30 3.0 5 yes no 1.25
13 2 5 7.2 27 1.0 10 yes no 1.23
9 2 3 5.2 27 1.0 5 no no 2.27
12 2 3 7.2 30 3.0 10 no no 1.78
8 1 3 7.2 30 1.0 5 yes yes 2.76
16 2 3 7.2 30 1.0 5 yes yes 2.85
14 2 5 7.2 27 3.0 5 no yes 1.31
1 1 3 5.2 27 1.0 5 no yes 2.29
10 2 5 5.2 30 3.0 5 yes yes 1.32
15 2 5 5.2 30 1.0 10 no yes 2.3
7 1 5 5.2 30 1.0 10 no yes 2.3
4 1 3 7.2 30 3.0 10 no no 1.4
6 1 5 7.2 27 3.0 5 no yes 1.33
11d
2 3 5.2 27 3.0 10 yes yes 3.21
5 1 5 7.2 27 1.0 10 yes no 1.25
3 1 3 5.2 27 1.0 10 yes yes 2.98
0
5
10
15
20
25
30
35
0 10 20 30 40 50 60
Fermentation time (h)
Concentration(g/l)
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
Dextrose
Biomass
pH
Zeaxanthin
Table 5. Experimental results for the zeaxanthin production in a fluidized
bioreactor
Factors SS DF MS F p
Air flow 2.441 1 2.441 28.07 0.0007
pH 1.569 1 1.569 18.03 0.0028
Temperature 0.052 1 0.052 0.595 0.4626
Pellet diameter 0.174 1 0.174 2.004 0.1945
Inoculum/medium
ratio
0.268 1 0.268 3.079 0.1173
Natural light 0.047 1 0.047 0.544 0.4818
NaCl 1.723 1 1.722 19.81 0.0021
Residual 0.696 8 0.087
Table 6. Analysis of variance for zeaxanthin production
In fluidized bioreactor.
Mean = 1.92687 Sigma = 0.681652
FACTOR Nivel Effect size Standard Error
Air flow 1 .390625 .147452
pH 2 .313125 .147452
Temperature 2 .056875 .147452
Pellet diameter 2 .104375 .147452
Inoculum/médium
ratio
1 .129375 .147452
Natural light 2 .054375 .147452
Na Cl 2 .328125 .147452
zeaxanthin ( g/l) 3.303758
Table 7. Optimal levels for zeaxanthin production in fludized bioreactor.
99,39
100,78
102,171
103,561
104,951
106,341
107,732
109,122
110,512
111,902
above
z=48,637+18,675*pH+66,12*AF-4,892pH1*pH*AF-22,04*pH
2
Zeaxanthin(g/l)
Air flow
(vvm
) pH
Figure 5 Responce Surface of air flow versus pH in
the zeaxanthin production in fluidized bioreactor
Figure 4. Dynamic of zeaxanthin production by
Flavobacterium ssp.
100,517
101,726
102,936
104,145
105,355
106,564
107,773
108,983
110,192
111,402
above
z=74,481-3,752*x+43,655*y+0,334*x*x+3,5*x*y-14,718*y*y
Saline solution(x) pH(y)
Zeaxanthin(g/l)(z)
Figure 6. Responce surface of Sodium cloride versus
pH in the zeaxanthin production in fluidized bioreactor
99,363
100,727
102,09
103,454
104,817
106,181
107,544
108,907
110,271
111,634
above
z=43,074+29,996*x+61,144*y-8,665*x*x-3*x*y-18,048*y*y
pH(x) Temperature(y)
Zeaxanthin(g/l)(z)
Figure 7. Responce surface of temperature versus pH in
the zeaxanthin production in fluidized bioreactor
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Culture media with zeaxanthin and
immobilized Flavobacterium ssp