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Biotechnol. Prog.

2001, 17, 669675 669

Study and Mathematical Modeling of the Production of Propionic


Acid by Propionibacterium acidipropionici Immobilized in a Stirred
Tank Fermentor
Cristina Coronado, J. Enrique Botello,* and Fabiola Herrera
Instituto Tecnologico de Celaya, Dep. de Ingeniera Qumica, Ave. Tecnologico y Antonio Garca Cubas,
C.P. 38010, Celaya Gto. Mexico

A mathematical model was developed that describes production of propionic acid by


fermentation of sweet whey with Propionibacterium acidipropionici immobilized in
calcium polygalacturonate beads in a fermentor-type stirred tank. This mathematical
model is constituted by a partial differential equations system, which fits consumption,
production, growth and internal diffusion rates in the support. Fermentation was
experimentally studied with free cells and immobilized cells, effective diffusivities of
lactose and propionic acid were estimated in the support, and typical parameters of
the model were obtained by nonlinear regression of the experimental data. The variance
analysis shows that the combination of max and Kd parameters is the source of
variation most significative, also they were found to be the most sensitive parameters
of the model. Finally, an effectiveness factor was calculated in order to assess the
effect of mass transfer on the overall reaction rate observed.

Introduction mathematical modeling is very important for several


different good reasons: to discover new strategies to
Propionic acid is produced by anaerobic fermentation control and design process, to make important corrections
of a variety of carbon sources with propionic bacteria (1- in the conventional process, to understand the essential
4). Propionic acid is used in the manufacture of herbi- qualitative features, and many others (13-16). The
cides, chemical intermediates, artificial fruit flavors, development of mathematical models with immobilized
cellulose acetate propionate, and preservatives for food, reactors seeks to provide a rational base for process
animal feed, and grain (4). Sweet whey used in this work design strategies. Additionally, it seeks to understand
is a byproduct from the cheese-making industry and phenomena occurring inside the reactor, which leads to
contains approximately 5% lactose and less than 0.05% the identification of limiting stages on which to focus
proteins. Sweet whey is not very suitable for animal or improvement effort (17). There have been extensive
human feeding because of its low protein content, and studies of cells and enzymes immobilized with math-
its high biological oxygen demand, 50 g/L, makes its ematical models to study influence of substrate or product
disposal a costly pollution problem. There have been on activity and biomass spatial profiles, determination
extensive studies of using whey as a fermentation of kinetics expressions and parameters, and the study
substrate to produce a wide range of products (5). of external mass transfer resistance and intraparticle
Conventional fermentation technologies for propionic difussion resistance (15-20).
acid production from glucose and lactose are limited by The goal of this study is to obtain a mathematical
low reactor productivity (<1 g/Lh), low product yield model for propionic acid fermentation of sweet whey with
(<50% w/w), and low product concentration (<40 g/L) (6). Propionibacterium acidipropionici CDBB-1048 immobi-
In the propionic fermentation, propionic acid has an lized on calcium polygalacturonate beads in a stirred
inhibitory effect on cell growth, which leads to low fermentor tank. The appropriate equation was utilized
consumption and production rates. An alternative is the to obtain a kinetic intrinsic expression of propionic
use of systems with immobilized cells (7-9) and extrac- fermentation with immobilized cells and to analyze the
tive fermentations (6, 10). In systems with immobilized effect of intraparticle diffusion resistance of lactose and
cells, higher reaction rates are achieved as a result of propionic acid on growth, consumption, and production
the high cell density in the supporting material. It is also rates. The model can to be a powerful tool in tasks of
possible to reach high dilution rates and high produc- design, scale-up, and optimization of this process. The
tivities without culture washout. Another advantage of aim of the experimental stage is to obtain kinetic data
such systems is that they reduce separation costs of the of fermentation with free cells, diffusion kinetic data, and
product stream (11). kinetic data of fermentation with immobilized cells,
Bailey (12) presents the past accomplishments and which in turn can be used as a basis for designing and
future opportunities of mathematical modeling in bio- validating the mathematical models.
technology processes and biochemical engineering. The
Materials and Methods
* Ph: +52 (4) 6117575, ext 209. Fax: +52 (4) 6117744. E-mail: Microorganism and Culture Medium. The micro-
botelloenrique@hotmail.com. organism used was P. acidipropionici CDBB-1048. The
10.1021/bp010059p CCC: $20.00 2001 American Chemical Society and American Institute of Chemical Engineers
Published on Web 07/10/2001
670 Biotechnol. Prog., 2001, Vol. 17, No. 4

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

In the support beads: PaI



) RD (
2PaI 2 PaI
R2
+
R R
+ )
( )
CaI 2CaI 2 CaI
) + (7) SaIXBaIXBI0 KP XBaIXBI0
R2 R R YP/S YS/X1 + 2 (15)
KS + S0SaI KP + S0P S0
In the culture medium:
In the fermentation broth:
dCaL VI CaI
) -3 0 1
R2
dR (8) dSaL VI 1 2SaI
d VL
d
) -3
VL 0
R

dR -

Initial and boundary conditions:


3
VI 1 2
VL 0
R 1YS/X (
SaIXBaIXBI0 KP
KS + S0SaI KP + S0P
+

)
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

SI Table 1. Model Parameters


SaI ) (31) parameter free cells immobilized cells
S0
max 0.1305 1/h 0.1861 1/h
SL KS 32.5000 g/L 5.3425 g/L
SaL ) (32) KP 4.4218 g/L 32.0000 g/L
S0 Kd 1.69 10-3 1/h 0.1090 g/L
m 0.0000 1/h 0.0000 1/h
PI YS/X 6.9055 g/g 2.7725 g/g
PaI ) (33) YP/S 0.3067 g/g 0.6034 g/g
S0
metabolic orientation toward production in the system
PL with immobilized cells is higher than that in free cells.
PaL ) (34)
S0 Table 1 shows the parameter value of the kinetic model
of free cells fermentation estimated by nonlinear regres-
XBI sion. Figure 1 shows the experimental data and the
XBaI ) (35) curves that represent the solutions to eqs 1-3 with the
XBI0
estimated parameters. The solution data have a variance
with respect to the experimental data due to a lack of fit
Solution of the Models and Estimation of Param- of 1.9930, which was estimated according to the proce-
eters. The equations of the kinetic model with free cells dure proposed by Constantinides (24), and the experi-
(eqs 1-3) were solved numerically using the fourth order mental data have a variance due to an experimental error
Runge-Kutta method (24). Differential equation systems of 2.0458. These variances are not significantly different,
of the diffusion (eqs 4-12), and diffusion reaction eqs 13- for a confidence level of 95%. Therefore, it is reliable to
35 were solved using the method of lines with discreti- conclude that the model presents an adequate adjustment
zation of derivatives in the radial coordinate in finite of the experimental data model parameters in free cells.
differences, solving the resulting system of ordinary Figure 2 shows the experimental diffusion data and
differential equations in time also with the Runge-Kutta the curves that represent the solutions to eqs 7-12 with
method (24). The solution to those equations is composed the estimated effective diffusivities of lactose and pro-
of concentration radial profiles and the concentrations pionic acid in the calcium polygalacturonate beads. It can
in the culture medium (variables with subindexes I and be observed that diffusion phenomenon is faster than
L, respectively). Estimation of typical parameters for the kinetic processes of consumption and production shown
model was done by nonlinear regression (25), considering in Figure 1. A priori mass transfer resistance has not a
as reference the experimental data obtained in various big effect. Effective diffusivities in the calcium poly-
experiments. galacturonate beads estimated for lactose and propionic
acid are 2.19 10-6 and 3.55 10-6 m2/h, respectively.
Results and Discussion The values obtained are very close to molecular diffu-
The concentration of propionic acid in the fermentation sivities in water reported in the literature (26). This is
with free cells was 10.30 g/L with a productivity of 0.17 similar to the results reported by others researchers, who
g/Lh, whereas with immobilized cells the values were have found that effective diffusivities for these supports
18.61 g/L and 0.31 g/Lh, respectively. This indicates that are very close to molecular diffusivities (27, 28). The

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

parameters of Table 1 and the effective diffusivities


estimated above were incorporated to the diffusion-
reaction model in order to solve it. Important differences
were found between the estimated data and the experi-
mental data for lactose and propionic acid; similarly,
experimental consumption and production rates were
markedly superior to those predicted by the model. On
the other hand, the amount of immobilized biomass
measured experimentally in the beads showed little
growth, whereas the model predicts an important growth.
The kinetic parameters of the model were re-estimated,
taking as reference the same experimental data for
fermentation with immobilized cells. The new parameter
values are shown also in Table 1, and these have
differences compared to the values with free cells. This
matches results of previous work, which mentioned that
the values of the kinetic parameters for fermentations
with immobilized cells differ significantly from the values
obtained with free cells (28,29). This is due to the fact
that the conditions to which cells are exposed in the
support microenvironment are significantly different
from those in a liquid medium. It is also important to
note that in both columns the parameter m is neglectable,
which indicates that, in the free-cells system as in
immobilized cells, propionic acid produced is associated
with cell growth. Moreover, the yield factor from lactose
to propionic acid (YP/S) in the system with immobilized
cells is higher than that in free cells, which indicate
metabolic orientation toward production.
Figure 3 shows the experimental data of fermentation
with immobilized cells and the curves of the solution of
the diffusion-reaction model. In this figure, the amount
of estimated biomass shows no major changes, as it first
increases and later decreases with slight slopes. This
might indicate that inside the beads a balance is estab-
lished between the biomass growth and death rates. The
data of the solution with respect to experimental data
have a variance due to a lack of fit of 1.8773, whereas
the experimental data have a variance of 1.5027; both
Figure 2. Diffusion of lactose and propionic acid form culture are nonsignificantly different, for a confidence level of
media to support beads immersed inside. S0 ) 50.75 g/L, P0 ) 95%. The effect of the parameters on the variance due to
8.20 g/L. Experimental and computed data. a lack of fit was studied by numerical simulation in two

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

Table 2. Analysis of Sensitivity Greek Symbols


parameter, Par (S + P)/Par S/Par P/Par max maximum specific biomass growth rate (1/h)
max 3780.5 3441.6 338.91 adimensional time
Kd -533.98 -1079.6 545.63
YS/X 57.549 54.758 2.7817 Subscripts
YP/S -54.891 -50.643 -4.2487
a adimensional
KS -8.5541 -10.143 1.5892
KP 1.7339 1.8787 -0.1447 0 initial condition
I refers to the support
factorial designs 26-2 (30), and the study levels of L refers to the culture medium
parameters were fixed at (10% and (5% of optimum
values. The variance analysis shows that the combination
of max and Kd is the source of variation most significant. References and Notes
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