Chemical Engineering Science 54 (1999) 3137}3142

Modelling and optimization of the cephalosporin C production

bioprocess in a fed-batch bioreactor with invert sugar
as substrate
A.J.G. Cruz *, A.S. Silva , M.L.G.C. Araujo
, R.C. Giordano , C.O. Hokka
Departamento de Engenharia Qun& mica da Universidade Federal de SaJ o Carlos, Cx.Postal 676, CEP 13565-905, SaJ o Carlos, SP, Brazil

Departamento de Tecnologia, Instituto de Qun& mica - UNESP, Campus de Araraquara, Cx. Postal 355, CEP 14801-970, Araraquara, SP, Brazil

Abstract

Cephalosporin C production process optimization was studied based on four experiments carried out in an agitated and aerated
tank fermentor operated as a fed-batch reactor. The microorganism Cephalosporium acremonium ATCC 48272 (C-10) was cultivated
in a synthetic medium containing glucose as major carbon and energy source. The additional medium contained a hydrolyzed sucrose
solution as the main carbon and energy source and it was added after the glucose depletion. By manipulating the supplementary feed
rate, it was possible to increase antibiotic production. A mathematical model to represent the fed-batch production process was
developed. It was observed that the model was applicable under di!erent operation conditions, showing that optimization studies can
be made based on this model. 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Cephalosporin C; b-lactam; Cephalosporium acremonium; Invert sugar; Fed-batch; Bioprocess optimization

1. Introduction improvement is due to this aspect or to the methionine

Cephalosporin C is a b-lactam antibiotic synthe-
sized as a secondary metabolite by strains of a strictly
aerobic fungus Cephalosporium acremonium, usually
in aerated and agitated tanks. This natural antibiotic
is modi"ed through chemical or enzymatic methods
to produce di!erent semi-synthetic cephalosporins,
which are of major importance in the pharmaceutical
market.
The most recent works reporting cephalosporin C pro-
duction process deal with media containing two carbon
sources, glucose and sucrose (Chu and Constantinides,
1987; Vicik et al., 1990; Basak et al., 1995). Vicik et al.
(1990) worked with fed-batch process where glucose and
methionine were added slowly. The yield improvement
obtained by them is explained by the combined addition
of sucrose and methionine. However, sucrose is intrinsi-
cally a slowly metabolized sugar, and it is not clear if that
*Corresponding author. e-mail: ajgcruz@power.ufscar.br. Fax
55 16 260 8266
feeding.
The production process of cephalosporin C in batch-
type bioreactor shows two distinct phases: during the
"rst phase, an easily metabolizable carbon source like
glucose is quickly consumed and most of the growth
takes place; while in second phase, a slowly metab-
olizable carbon source like sucrose is assimilated and the
antibiotic synthesis commences. This is a typical diauxic
e!ect of the consumption of two carbohydrates. This
phenomenon was also observed in the beginning of peni-
cillin G production process by Soltero and Johnson
(1953) and Hosler and Johnson (1953), utilizing glucose
and lactose as carbon sources. They proposed replace-
ment of the slowly metabolized sugar, lactose, by less
expensive glucose or sucrose added at a controlled feed
rate. In the cephalosporin C production process the slow
metabolism of the sucrose may be explained by the low
activity of the enzyme responsible for the hydrolysis of
this sugar during the process (Vicik et al., 1990; Machado
et al., 1997), and this provides good conditions for pro-
duction of high antibiotic concentrations in the conven-
tional batch process.

0009-2509/99/$ - see front matter  1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 0 9 - 2 5 0 9 ( 9 8 ) 0 0 3 6 4 - 9
3138 A.J.G. Cruz et al./Chemical Engineering Science 54 (1999) 3137}3142
Indeed the papers of Chu and Constantinides (1987)
and Basak et al. (1995) deal with production optimization
in a batch bioreactor utilizing glucose and sucrose con-
taining media. Vicik et al. (1990) work with fed-batch
process where sucrose and methionine are added slowly.
However, sucrose is already a slowly metabolizable
sugar, and the improvement obtained by them should be
explained by methionine addition.
In this work, studies regarding the production of
cephalosporin C by strains of C. acremonium ATCC
48272 were carried out in a stirred aerated tank reactor
(5 l working volume), utilizing a synthetic medium.
The "rst run was carried out as a batch process using
glucose and sucrose as main carbon source. The results
allowed the conditions of the carbon source feed rate
in the fed-batch bioreactor to be de"ned, based on
the sucrose hydrolysis reaction rate observed in the
batch process. The other runs were carried out as fed-
batch processes employing glucose in the initial batch
phase and hydrolyzed sucrose (invert sugar) in the
supplementary medium with di!erent feed rates. Invert
sugar is a rather unexpensive raw material and its feed
at a controlled rate can yield high cephalosporin C
productivity.
An unstructured mathematical model was developed
to represent the fed-batch production process, resulting
in a useful tool for studies in enhancing yield of cephalos-
porin C.
2. Materials and methods
2.1. Microorganism
Cephalosporium acremonium strain ATCC 48272 was
employed throughout this work.
2.2. Media and culture conditions
C. acremonium cultures were maintained on agar slants
as proposed by Shen et al. (1986). Cells from a slant were
suspended in 5 ml sterilized salt solution (NaCl 0.9%).
This solution was transferred to the seed medium in
250 ml shake #asks (10% in volume) and incubated for
48 h at 250 rpm and 263C. After spore germination an
aliquot was transferred to inoculum medium and was
grown for 24 h in the same conditions. The seed and the
inoculum medium (Demain et al., 1963) contain (in g/l):
glucose (30.0), oleic acid (1.5), DL-methionine (3.0), am-
monium acetate (8.8), KH PO (2.3), K HPO (5.8),
   
Fe(NH ) (SO ) ) 6H O (0.16), CaCO (2.0); pH 7.0$
   
0.1. Micronutrients were provided by adding a salt solu-
tion (50 ml/l), with the following composition (in g/l):
Na SO (16.2), MgSO ) 7H O (7.68), CaCl ) 2H O
     
(7.68), MnSO ) H O (0.64), ZnSO ) 7H O (0.64),
   
CuSO ) 5H O (0.004).
 
A stirred-tank reactor (5 l working volume) was used
with 10% inoculum for the production of cephalosporin
C. The fermentation medium of the simple batch process
contained (in g/l) glucose (27.0), sucrose (36.0), KH PO
 
(1.8), K HPO (2.97) and the same amounts of the
 
remaining components as the precultures (seed and in-
oculum medium). For the fed-batch process, the com-
position of the initial batch medium was the same as used
in the conventional batch, excluding sucrose. After de-
pletion of glucose, supplementary medium was added
employing a peristaltic pump at established #ow rates.
The supplementary medium provided to the process con-
tained the same components as the initial batch, plus
hydrolyzed sucrose. The sucrose was hydrolyzed at 403C
in a bu!er solution of 10\ M sodium acetate, pH 4.5
(Ribeiro, 1989), utilizing invertase enzyme (Novo
Ferment).
The fermentor was operated at 263C, with automatic
control of the dissolved oxygen, kept at 27% of its satura-
tion value by manipulating the agitation speed which
started at 250 rpm. The air #ow rate was maintained at
3.0 l/min throughout the experimental runs.
2.3. Analysis
The cell-mass concentration was evaluated as dry
weight at 1053C, in g/l. Glucose was measured utilizing
enzymatic GOD-PAP method. Cephalosporin C titers
were determined by an agar di!usion bioassay (Claridge
and Johnson, 1962) employing Alcaligenes faecalis ATCC
8750.
3. Mathematical model
3.1. Stoichiometric equations
The kinetic model was based on the stoichiometric and
rate equations proposed by Chu and Constantinides
(1987), Araujo et al. (1996), and Cruz et al. (1998). These
equations represent the main stages of variation in con-
centration of cell, limiting substrates and product. The
other nutrients are considered to be in excess. Some
modi"cations were made to explain the complex mecha-
nism of the cephalosporin C formation.
In this model, it was assumed that the total biomass
was composed of cells X , submitted to catabolite repres-

sion by glucose, S , during the growth phase, and cells

X , derepressed after glucose depletion and able to pro-

duce high amounts of the enzyme complex responsible
for the antibiotic synthesis (Eqs. (1) and (3)). When the
glucose concentration falls below a certain critical value,
C , cells X are transformed into cells X (Eq. (2)). As
  
pointed out by Chu and Constantinides (1987) these
cells are able to produce enzyme(s) of the b-lactam
biosynthetic pathway that are crucial to cephalosporin
 A.J.G. Cruz et al./Chemical Engineering Science 54 (1999) 3137}3142 3139

C synthesis, and they can also consume sucrose, a slowly The "rst and the second terms in Eqs. (7) and (8)
metabolized sugar. represent growth of cells X and X on glucose as carbon
 
Concerning product formation it was assumed to be source (S ) and the transformation of repressed into

regulated by the reaction catalyzed by the enzyme, derepressed cells, respectively. Contois model (Bailey and
E (Eq. (4)). Eqs. (5) and (6) represent product and enzyme Ollis, 1986) for speci"c growth rate and glucose con-
degradation, respectively, sumption rate was assumed. The third term is a dilution
term. Eq. (9) represents the glucose consumption rate,
S #X P(1#Y )X , (1)
    assuming that this substrate is used only for growth by
cells X , and for growth and maintenance by cells X .
I2 X &

X &
IB D , (2)  
   The terms > and m are pseudo-stoichiometric and the

maintenance coe$cient, respectively, while b is a rate
S #X P(1#Y )X #E, (3) constant.
   
Eq. (10) represents the concentration of the rate-limit-
# P,
R&
(4) ing enzyme(s), which is linked to the growth of cells X .

The coe$cient a was assumed to relate productivity to
glucose mass feed rate (= ). It was adjusted empirically
PPD ,

(5) +1
to the actual experimental data. The term k is the
#
EPD , (6) decomposition rate of the enzyme.

It was postulated that cephalosporin C formation was
where total cell mass (X) includes both cells types X and
 regulated by the reaction catalyzed by the enzyme
X ; D can be regarded as dead cells and D and D are
    (Eq. (4)). However, its rate is much higher than that of the
products of degradation of antibiotic (P) and enzyme (E), enzyme production. Therefore, the rate of product forma-
respectively. R can be considered a reagent in excess in tion is controlled by the rate of enzyme production
the fermentation medium, probably the precursor mol- (Eq. (11)). The k term is the decomposition rate of
ecule of cephalosporin C. F
product.

3.2. Balance equations

4. Results and discussion
The mathematical model based on the mass balance of
the components in a fed-batch reactor is represented by 4.1.Batch fermentation
the following di!erential equations:
dCx k Cs k Cx Q Cx The "rst run was carried out as a batch process using
"   Cx ! 2  ! KQ  (7) glucose and sucrose as main carbon sources. The results
dt k Cx #Cs  k #Cx <
V     for a 144 h process is shown in Fig. 1. The diauxic e!ect
dCx k Cs k Cx can be observed in this picture, when glucose is preferen-
"   Cx # 2 
dt k Cx #Cs  k #Cx tially consumed, and high growth rate takes place. After
V    
Q Cx
!k Cx ! KQ  (8)
B  <
dCs 1 k Cs Cs
"!   Cx!mCx 
dt > k Cx#Cs k#Cs
 V  
Q (C !Cs )
# KQ KQ  , (9)
<
dC k Cs Q C
#"a   Cx !k E! KQ # , (10)
dt k Cx#Cs  # <
V 
dC dC C Q C
N"b # Cx N !k C ! KQ N , (11)
dt dt  k#C F N <
N
Cx"Cx #Cx (12)
 
<"< #Q t - , (13)
  

Fig. 1. Experimental data and simulation results of cephalosporin
where < is the initial batch volume, Q the volumetric
 KQ C batch production process by C. acremonium in a stirred-aerated tank
feed rate and t } the fed-batch time of process. reactor.


3140 A.J.G. Cruz et al./Chemical Engineering Science 54 (1999) 3137}3142
its depletion, sucrose starts to be metabolized at a slower
rate. During this phase, cephalosporin C begins to be
formed. The results obtained allowed the conditions of
the carbon source feed rate in the fed-batch bioreactor to
be de"ned, in terms of the sucrose hydrolysis reaction
rate observed in the batch process.
In the other runs, the fermentor was operated in fed-
batch mode, employing glucose in the initial phase and
hydrolyzed sucrose in the supplementary medium, fed at
the same rate as in the batch process, and at three other
rates.
4.2. Fed-batch experiments
All fed-batch runs are illustrated in Fig. 2. Analysis of
the results regarding cell concentration shows that there
is a good reproducibility of the maximum values ob-
tained, very close to that of the biomass from the batch
process, about 15 g cell/l. Due to supplementation of the
medium the cell concentration of the fed-batch fermenta-
tion remained practically constant and around its
Fig. 2. Experimental data and "t of the kinetic model proposed to growth and glucose consumption phase from the four experiments carried out.
maximum value during the whole production phase. De-
pletion of glucose and subsequent onset of the idiophase
in the fed-batch runs were anticipated in relation to the
simple batch fermentation. Addition of supplementary
medium was started after 37 h fermentation at all #ow
rates investigated. Analysis of glucose and fructose con-
centrations in the fermentation medium, during the sup-
plementation stage of these sugars, indicated values close
to zero, with no signi"cant di!erences in the four condi-
tions investigated.
Regarding parameter estimation, a nonlinear regres-
sion (Marquardt, 1963) was applied. The model is com-
posed of "ve di!erential equations, "ve state variables
and 11 parameters. Three of "ve state variables were
measured: total cell (Cx), glucose (Cs ) and cephalos-

porin C (C ) concentrations. Parameter estimation was
N
carried out using data from runs 1 to 4 for cell and
glucose concentrations. The estimated parameters were
k , k , > and m. The parameters k and k are related
 V  2 
to morphological di!erentiation present by this microor-
ganism, and the adopted value were that used by Cruz
 A.J.G. Cruz et al./Chemical Engineering Science 54 (1999) 3137}3142
Table 1
Estimated values of parameters
Maximum speci"c growth rate
Contois constant
Death rate constant
Decomposition rate of enzyme
Decomposition rate of product
Yield coe$cient
Maintenance coe$cient
Kinetic constants (morphological di!erentiation rate)
Constant
Enzyme-linked production formation
These parameters were estimated using a nonlinear regression with 95% con"dence limits.
Fig. 3. Simulation results and experimental data of cephalosporin C at
four di!erent glucose mass feed rates.
et al. (1998). The death constant was set zero. Parameters
k , k and b were determined by "tting the experimental
F #
data. The parameter k was set at a small value to avoid
negative concentrations. Table 1 shows the set of para-
meters estimated to assess the proposed model. The in-
itial values adopted were those used by Cruz et al. (1998).
The resulting di!erential equations were solved by the
DDASSL algorithm (Petzold, 1989). The program was
written in FORTRAN and run through the PC 486
computer.
Information on kinetic parameter for cephalosporin
C production is very scarce in the literature. Growth rate
and substrate consumption rate parameters, k and

> were estimated by Araujo et al. (1996) for this same

strain in batch experiments as being 0.0645 h\
and 0.655 g/g, respectively. However, these data were
obtained in a medium containing glucose and sucrose. In
the present experiments, during the growth phase, only
glucose was present. During the production phase invert
sugar (glucose plus fructose) was added. Chu and
Constantinides (1987) present an estimated value equal
to 0.02217 g/g h and Araujo et al. (1996) obtained
0.036 g/g h. However, these values refer to sucrose con-
sumption. In this work the coe$cient m is related to
invert sugar consumption rate. It should be stressed that
3141
k 0.0917$0.0017 (h\)

k 0.00513$0.0384 (gS /gX )
V  
k 0.0
B
k 0.01 (h\)
#
k 0.0001 (h\)
F
> 0.5840$0.0185 (gX /gS )
  
m 0.01217$0.00630 (gS /gX .h)
 
k k 6.0 (gX /l.h) 0.01 (gX /l)
2   
k 0.0001 (dimensionless)
b 0.010 (L/g)
Fig. 4. E!ect of mass #ow rate on the product concentration, according
to the model.
in fed-batch process the substrate concentration is kept
very low (near zero) and process behavior is controlled
by the substrate feed rate rather than by kinetics itself.
This fact accounts for the very large con"dence region of
parameters k and m.
V
An empirical relation between glucose mass feed rate
(= ) and a coe$cient was represented by equation:
+1
a"1.64#31.5458e\ 5+1\  (12)
Fig. 2 depicts the comparison between experimental and
simulated results for cell mass and glucose concentration
in the four experiments. Glucose consumption shows
a similar behavior in the four runs. Regarding biomass
concentration, runs 1}3 present reproducible values
about 15 g/l. Due to the addition of supplementary me-
dium, cell concentration remained practically constant
during the entire fed-batch phase. In run 4, cell concen-
tration remains at about 12 g/l during the fed-batch
phase showing that in this case a carbon source limita-
tion occurred. However, cephalosporin C yield was still
satisfactory (see Fig. 3) due to the absence of glucose
repression.
Calculated values obtained by simulating the process
are plotted in Fig. 3 together with experimental points.
3142 A.J.G. Cruz et al./Chemical Engineering Science 54 (1999) 3137}3142
As can be observed the model "ts very well in each of the
four conditions employed.
Fig. 4 illustrates the process simulation in a search for
an optimum mass #ow rate in order to "nd a maximum
product concentration, where the four experimental re-
sults are indicated in the "gure.
According to the model there is an optimum feed rate
that will promote a higher antibiotic production.
5. Conclusions
It was evident from the results that the fed-batch
fermentation favored maintenance of higher antibiotic
production, by promoting minimization of catabolite
repression. An empirical relation between antibiotic pro-
duction and glucose feed rate was established in this
work. The proposed model "tted the experimental data
adequately and gave further information allowing pro-
cess optimization.
Acknowledgements
The authors gratefully acknowledge the "nancial sup-
port from FAPESP (Proc. 96/5918-9, 96/3170-7,
96/05908-3). The English review by Timothy Roberts is
also acknowledged.
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