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culture medium was prepared with deproteinized and Variation of its concentration is followed through time
enriched sweet whey. The composition was (g/L) pow- sampling and analysis of samples of the solution. The
dered sweet whey, 67.0 (Land OLakes, Inc.); sodium goal is to establish the value of effective diffusivity that
citrate, 2.0 (J. T. Baker); ammonium phosphate acid, 2.0 causes the solution of the diffusion model presented below
(J. T. Baker); magnesium sulfate heptahydrate, 0.1 (J. to coincide with the experimental data obtained. In these
T. Baker); manganese sulfate monohydrate, 0.05 (J. T. experiments 79 mL of support beads were suspended in
Baker); and yeast extract, 2.5 (Bioxon). The medium pH 200 mL of culture medium at a temperature of 35 C and
was maintained by the addition of concentrated am- a pH of 5.5.
monium hydroxide. Fermentation with Immobilized Cells. In the batch
Cell Immobilization. P. acidipropionici was grown fermentation with immobilized cells, the reactor was
in dilution bottles with culture medium with 6.0 g/L of loaded with 1500 mL of culture medium after being
yeast extract for 60 h. The microorganism was harvested sterilized and cooled to 35 C. Next, 250 mL of beads that
by centrifugation, to obtain a suspension with 14.0 g/L had been prepared the previous day were added and
of biomass. The immobilization technique used was refrigerated in the calcium chloride solution. The reactor
entrapment on calcium polygalacturonate beads (21). was bubbled with nitrogen for 20 min, and the pH was
This suspension was mixed with a solution of ethanol and maintained at 5.5 with addition of concentrated am-
polygalacturonic acid (Sigma) at 4% v/v and 8% w/v, monium hydroxide. Samples were taken every 4 h to
respectively, with a pH of 8.0. The resulting suspension determine lactose and propionic acid.
was dropped through a 0.3 mm hypodermic needle into
a sterile solution of 0.2 M calcium chloride (J. T. Baker) Modeling
and 0.01 M sodium tetraborate (J. T. Baker). The sup-
port beads obtained are uniform with an average diam- Kinetic with Free Cells. Equations 1-3 describe the
eter of 2.8 mm, and an initial biomass load of 7.0 g/L of kinetic model proposed for propionic fermentation with
support. free cells. In eq 1, biomass growth is represented by
Fermentor. The fermentation was done in a stainless Monods equation modified by an inhibition term of
steel stirred tank reactor with a capacity of 2.2 L, which propionic acid and a death term (see Notation). In eq 2
has a concentric cuirass through which water is recal- the first term on the right represents lactose consumption
culated to maintain a constant temperature in the associated to growth biomass estimated by YS/X (yield
fermentation broth. Agitation in the reactor is done with factor), whereas the second is a nonassociated consump-
an egg-shaped magnetic stir bar of 6.5 cm that spins at tion term, which is characterized by a specific mainte-
300 rpm. In the reactors lid there are five circular holes nance constant m (1/h). Equation 3 represents propionic
where the following are adapted: a diving pipe for acid production rate that is directly associated to lactose
nitrogen bubbling, a pipe for dosage of ammonium consumption rate by the yield factor YP/S.
hydroxide as neutralizing agent, a pipe for sampling, a
hatchway for gas exit, and another for the pH and dXB SXB KP
temperature sensor. ) max - KdXB (1)
dt KS + S KP + P
Analytical Methods. Biomass was quantified as dry
weight correlated to the optical density of the 1/16 diluted
( )
samples determined in an Espectronic spectrophotometer SXB KP
dS
at 650 nm. Lactose concentration was measured with the ) -YS/X max - mXB (2)
spectrophotometric method of dinitrosalicylic acid DNS dt KS + S KP + P
(22). Acid propionic concentration was determined by gas
[ ( ) ]
chromatography, in a Perkin-Elmer Autosystem XL GC,
with a flame ionization detector (hydrogen/air), and with dP SXB KP
) YP/S YS/X max + mXB (3)
an Alltech Econocap column Ec-100 15 m 0.53 mm dt KS + S KP + P
1.2 m at 250, 150, and 275 C in the injector, the column,
and the detector, respectively. Nitrogen was used as the Diffusion Kinetic. The diffusion model of a substance
mobile phase at 3 mL/min. from the culture medium perfectly mixed to support
Fermentation with Free Cells. The operation condi- beads is represented in eqs 4-12. The premises of these
tions and the broth composition utilized in the fermenta- models that were considered are (a) initial substance
tions with free and immobilized cells were determined concentration in the supports beads is zero, (b) the
as optimum for the propionic fermentation with im- solution is so perfectly agitated that substance concentra-
mobilized cells by Herrera (23). In the batch fermentation tion in the solution is considered to the same as that in
with free cells the reactor was filled with 1800 mL of the surface of the support beads, (c) internal mass
culture medium, which was sterilized together with its transference is diffusional and is characterized by effec-
accessories for 20 min at 121 C. The reactor was cooled tive diffusivities, and (d) mass transport occurs only in a
until it reached the operation temperature of 35 C, and radial direction, generating symmetrically radial con-
200 mL of inoculum of Propionibacterium acidipropionici centration profiles with a maximum or a minimum in
CDBB-1048 was added with 36 h of growth at 33 C. The the bead center.
reactor was bubbled for 20 min with nitrogen, and then
the run was started. pH was kept constant at 5.5 by C
addition of concentrated ammonium hydroxide. Samples Ca ) (4)
C0
were taken every 5 h to determine biomass, lactose, and
propionic acid. DECt
Diffusion Experiments. Lactose and propionic acid ) (5)
effective diffusivities in the calcium polygalacturonate R20
were estimated with the technique used by Melick et al.
r
(17). With this technique, support beads are suspended R) (6)
in an agitated solution of the substance in question. Rb
Biotechnol. Prog., 2001, Vol. 17, No. 4 671
)
CaL( ) 0) ) CaL0 (9)
XBaIXBI0
2 dR (16)
CaI( ) 0, 0 e R e 1) ) 0 (10) S0
dPaL VI 1 2PaI
CaI( g 0, R ) 1) ) CaL (11)
d
) -3
VL 0
R
dR +
CaI
(R ) 0) ) 0 (12) 3
VI 1 2
VL 0 R Y P/S 1YS/X (SaIXBaIXBI0 KP
KS + S0SaI KP + S0P
+
)
R
XBaIXBI0
Model variables (see Notation) were adimensionalized 2 dR (17)
S0
according to the variables defined in eqs 4-6. In eq 7,
the term on the left represents accumulation of C in a
Initial and borderline conditions:
differential element of the sphere, whereas the right side
represents flow mass entering and leaving the differential
element, which are ruled by concentration radial gradient SaI( ) 0, 0 e R e 1) ) 1 (18)
and effective diffusivity. Similarly, in eq 8, the term on
the left represents accumulation of C in the culture SaL( ) 0) ) 1 (19)
medium, which is equal to the amount of C accumulated
in all the beads through time represents for the term on SaI( g 0, R ) 1) ) SaL( g 0) (20)
the right. Finally, initial and boundary conditions con-
gruent with the premises of the model proposed are
presented in eqs 9-12. SaI
Fermentation Kinetic with Immobilized Cells. ( g 0, R ) 0) ) 0 (21)
R
The fermentation model with free cells proposed above
was used as a kinetic intrinsic expression in order to
PaI( ) 0, 0 e R e 1) ) PaL (22)
design a diffusion and reaction model in a stirred
fermentor tank in batch operation with immobilized cells
in beads, as proposed by Nakasaky et al. (20). The model PaL( ) 0) ) 0 (23)
consists of two sets of equations; the first regulates the
growth biomass rate, consumption of lactose rate, and PaI( g 0, R ) 1) ) PaL( g 0) (24)
propionic acid production rate, as well as its concentra-
tion radial distributions, and its variation through time
inside the support beads (eqs 13-15). The second set PaI
regulates the dynamic behavior of lactose and propionic ( g 0, R ) 0) ) 0 (25)
R
acid concentrations in the culture medium (eqs 16 and
17). In addition to the assumptions made in the diffusion XBaI( ) 0, 0 e R e 1) ) 1 (26)
model, initial distribution of cells in the support beads
is assumed to be uniform. The model also assumes that
there is no cell escape toward the culture medium, that Auxiliar equations:
the beads are completely lactose saturated, and that the
initial content of propionic acid in the beads and the
DEP
RD ) (27)
culture medium is zero. Rd is defined by eq 27, and 1, DES
2, and 3 are constants defined in eqs 28, 29, and 30,
respectively. In the beads: R20max
1 ) (28)
XBaI S0SaIXBaI KP DES
) 1 - 3XBaI (13)
KS + S0SaI KP + S0PaI
R20m
( )
SaI 2
SaI 2 SaI 2 ) (29)
DES
) + -
R2 R R
SaIXBaIXBI0 KP XBaIXBI0 R20KD
YS/X1 - 2 (14) 3 ) (30)
KS + S0SaI KP + S0P S0 DES
672 Biotechnol. Prog., 2001, Vol. 17, No. 4
Figure 1. Kinetic of the propionic fermentation with free cells at 35 C and pH 5.5. S0 ) 56.45 g/L, XB0 ) 0.57 g/L. Experimental
and computed data.
Biotechnol. Prog., 2001, Vol. 17, No. 4 673
Figure 3. Kinetic of the propionic fermentation with immobilized cells at 35 C and pH 5.5. S0 ) 37.17 g/L, XBI0 ) 7.0 g/L.
Experimental and computed data.
674 Biotechnol. Prog., 2001, Vol. 17, No. 4
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ington, DC, 1992; pp 260-276. for Ethanol Production. Biotechnol. Bioeng. 1987, 30, 836-
(20) Nakasaki, K.; Murai, T.; Akiyama, T. Dynamic Modeling 843.
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tation. Biotechnol. Bioeng. 1989, 33, 1317-1323. nation of Mass Transfer Limitation in Immobilized Cell
(21) Montes, M. C.; Magana I. P. -Dehydrogenation of Steroids Reactor. Biotechnol. Bioeng. 1989, 34, 320-336.
by Arthrobacter simplex Immobilized in Calcium Polygalac-
turonate Beads. J. Ind. Microbiol. 1991, 8, 259-264. (29) Moynihan, H. L. Urea Hydrolysis by Immobilized Urease
(22) Miller, G. L. Anal. Chem. 1959, 31, 426. in Fixed Bed Reactor: Analysis and Kinetic Parameter
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