OPTIMIZATION OF ZEAXANTHIN PRODUCTION BY IMMOBILIZED FLAVOBACTERIUM SP. CELLS IN FLUIDIZED BED BIOREACTOR

N. Valadez1 and E. M. Escamilla2

1Chemical Faculty, Autonomous University of Querétaro, University Center, Cerro de las Campanas. C.P. 76010. Querétaro, Qro., México. 2*Chemical Department, Technological Institute of Celaya, Av. Tecnológico y A. García Cubas S/N, C.P.

38010, Celaya Gto., México. Fax: + 52-461-17744. e-mail address: eleazar@iqcelaya.itc.mx

Bioprocesos y Biocatálisis: Biorreactores y Bioprocesos. Nuevas configuraciones y aplicaciones.

More than 600 carotenoid structures are known and only 8 of them have been used [1]. Carotenoids are natural pigments synthesized by plants and microorganisms as hydrocarbons (carotenes) and their oxygenated derivatives or oxycarotenoids (xanthophylls). The bacteria Flavobacterium, synthesize Zeaxanthin (90-95%)[2]. Xanthophylls are responsible for the colour, especially Zeaxanthin (orange-yellow pigment). Furthermore, there is evidence that xanthophylls, especially Zeaxanthin and Lutein, have a relevant function in preventing cardiovascular and ophthalmological diseases, as well as different types of cancer [1]. The microbiological production of carotenoids has not been optimized to obtain amounts of production of pigments and recovery of carotenoids that lower costs of the production.

The main objective of this work was to investigate a better experimental condition to increase the Zeaxanthin production process using an orthogonal experimental design and using immobilized Flavobacterium sp. ATCC 21588 cells in a shake tank and fluidized bioreactor.

Flavobacterium sp. was grown on tripticasein soy agar. The colonies were then inoculated Erlenmeyer flasks (500 ml) that contained 250 ml of minimal media, pH 7.2, 27oC and 250 rpm for 56 h. The nutrients and concentrations studied were: Glucose, Corn steep liquor, ZnSO4, NH4Cl, KH2PO4, MgSO4, FeSO4, MnSO4 and CoCl2. For the cell immobilization, under sterile conditions a mixture (1:1) of Flavobacterium sp. suspension and poligaracturonic acid solution (8% w/v) was prepared. After soaking for 3 h the pellets of 1 and 3 mm ( 0.13) were selected for the production of Zeaxanthin. An orthogonal experimental design L8 (34) was used to investigate effects of temperature, pH, air flow, natural light, diameter of pellet, NaCl addition and inoculum concentration on Zeaxanthin production. Two 3.5 l bioreactors (CRODE, Mexico) were used in a random sequence for 56 h at 27°C. 8095 ( 845) pellets were added per batch and each pellet had a wet weight of 1.15 10-4 g cells so that approximately a total of 0.93 g of cells was added. Zeaxanthin production started increasing rapidly when biomass was in the stationary phase and glucose and NH4

+-N kept constant (around 25 h), with a maximum production in 50 h of cultivation (Fig. 1).

Figure 1. Dynamics of Zeaxanthin (g l-1), biomass (g l-1), glucose (g l-1), NH4+ (g l-1),

PO4-3 and Mg+2 during the cultivation process.

The best results were obtained with a combination of low air flux and high pH (Fig.2A). Zeaxanthin production was also greatly affected by the addition of sodium chloride. Response surface analysis showed maximum Zeaxanthin production with pH increased (Fig. 2B). The response surface of Zeaxanthin production for changes in temperature and pH showed a maximum production at 27°C and pH 7.2 (Fig.2C).

Figure 2. A) Response surface of air flux and pH in Zeaxanthin production (g l-1).B) Response surface of Zeaxanthin production (g l-1) for changes of pH and saline solution. C) Response surface of Zeaxanthin production (g l-1) for changes of temperature and pH.

Theoretically, the maximum Zeaxanthin production can be obtained with the following conditions: C: N of 2.0, air flux 3wm, NaCl 4.5 g l-1and pH 7.2. Zeaxanthin production was calculated with a predictive equation using the values of a maximum production:

Y = Yt + ( A – Yt ) + (B – Yt) + (C – Yt) + (D – Yt )Where Y: Estimated Zeaxanthin production, Yt: Experimental mean of Zeaxanthin, A: (C: N ratio), B: (air flux), C: (NaCl), and D: (pH). The equation predicts a concentration of 3.18 g L-1.Zeaxanthin production was from 1.25 to 2.96 g l-1 in stirred flasks, using 10 g l-1 dextrose, 5 g l-1 corn steep liquor, 5 g l-1 NH4Cl, 8 g l-1 KH2PO4, 1.5 g l-1 MgSO4.7H2O, 0.05M ZnSO4.7H2O, 0.05M FeSO4.7H2O, 0.05M CoCl2.6H2O, 0.01M MnSO4.7H2O and 4.5 g l-1

NaCl.References[1] Johnson, E.A. and Schrueder, W.A. 1995. Microbial Carotenoids. Adv. Biochem. Eng.

Biotechnol. 53: 119-178. [2] Sandmann, G., Albrecht, M., Schnurr, G., Knörzer, O. and Böger, P. 1999. The

Biotechnological Potencial and Design of Novel Carotenoids by Gene Combination in Escherichia coli. TIBTECH. 17: 233-239.

0

2

4

6

8

10

12

0 10 20 30 40 50 60

Fermentation time (h)

Con

cent

ratio

n (g

l -1)

0

0,3

0,6

0,9

1,2

1,5

1,8

Con

cent

ratio

n (g

l -1)

Dextrose

NH4+

PO4-3

Zeaxanthin

Biomass

Mg+2

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 + 0,334X 2+3,5*X*Y -14,718Y 2

N aCl (Y )

pH (X)

Zeax

anth

in

(Z)

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 -8,6 65X 2 -3*X*Y -18,048Y 2

pH (Y ) Tem perature(X)

Zeax

anth

in

(z)

99,39 100,78 102,171 103,561 104,951 106,341 107,732 109,122 110,512 111,902 above

z= 4 8 ,6 3 7 + 1 8 ,67 5 * p H + 6 6 ,1 2 * A F - 4 ,8 9 2 p H2 - 3 .5 * p H * A F - 2 2 ,0 4 * A F2

Zeax

anth

in (z

)

p H