Biochemical Engineering Journal 49 (2010) 95–103

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Kinetics of hyaluronic acid production by Streptococcus zooepidemicus

considering the effect of glucose
Mashitah Mat Don ∗ , Noor Fazliani Shoparwe
School of Chemical Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Seberang Perai South, Penang, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: An unstructured model of hyaluronic acid (HA) fermentation by Streptococcus zooepidemicus considering
Received 25 June 2009 the effect of glucose was proposed and validated. Experiments were performed in a glucose concentration
Received in revised form range of 10–60 g l−1 in a 2 l bioreactor of batch mode. Three different models, namely, the Logistic equa-
26 November 2009
tions for cell growth, the Logistic incorporated Leudeking–Piret-like equation for glucose consumption,
Accepted 5 December 2009
and the Logistic incorporated Leudeking–Piret equation with time delay, t, for HA productions were
proposed. The kinetic parameters were estimated by fitting the experimental data to the models. Simula-
tion was made using the estimated kinetics parameter values and was compared with the experimental
Keywords:
Hyaluronic acid
data. For glucose inhibition, S. zooepidemicus tolerated up to 40 g l−1 glucose. Beyond this concentration,
Stirred tank bioreactor cell growth was inhibited. The Han and Levenspiel model and the Teissier-type model gave the best fit
Kinetics for all the systems studied with R2 of 0.997 and 0.985, respectively.
Mathematical models © 2009 Elsevier B.V. All rights reserved.
Submerged
Biopolymer

1. Introduction
Hyaluronic acid (HA), a polysaccharide, is a high-value biopoly-
mer with a wide variety of medical and cosmetic applications.
HA belongs to a family of glycosaminoglycan, also known as
mucopolysaccharide. This polymer is comprised of d-glucuronic
acid (GlcUA) and N-acetylglucosamine (GlcNAc) linked by a ␤(1-
3)-glycosidic bond, with the disaccharide repeating units linked by
␤(1-4)-glycosidic bonds [1,2]. Conventionally, it has been extracted
from animal tissues such as rooster combs and bovine vitreous
humor [3]. However due to limited tissue sources, risks of viral
infection and high cost, HA production from microbial sources
through the fermentation process has received increased attention,
especially when using the gram-positive bacterium Streptococcus
zooepidemicus [3–5].
S. zooepidemicus is a catalase-negative, facultative anaerobe but
is also aerotolerant [6]. In sheep blood agar plates, colonies of these
␤-hemolytic bacteria will produce a clear zone with HA identified
as mucoid or slimy translucent layer surrounding bacteria colonies.
Under the microscope, these non-sporulating and non-motile bac-
teria appear as spherical or ovoid cells that are typically arranged
in pairs or chains surrounded by an extensive extracellular capsule
[5]. The capsule is composed of HA polymers identical to that found
∗ Corresponding author. Tel.: +60 4 5996468; fax: +60 4 5941013.
E-mail addresses: chmashitah@eng.usm.my, ita56@hotmail.com (M.M. Don).
in mammalian connective tissues [7]. The capsule may also protect
the bacteria against reactive oxides released by white cells to fight
off the bacteria [8].
Batch fermentation is a standard method for HA production,
and several studies have been conducted regarding the optimal
culture condition for HA production [7,9–12]. However, the prin-
cipal disadvantages of batch HA fermentation is the inhibition of
substrate and by-product concentration, a conventional property
of batch fermentation [9]. Ding and Tan [14] reported that the
production of aimed metabolites was limited due to the strong inhi-
bition of substrates under high concentrations. Shuler and Kargi
[13] previously stated that a high substrate or product concen-
trations and the presence of inhibitory substances in the medium
inhibit growth.
As reviewed by other researchers, the majority of growth, sub-
strate consumption and product formation models in the literature
involved Monod-like relationships which are taken into account
for the inhibition kinetics of many fermentation processes [15,16].
Subsequently, the Logistic model and Luedeking–Piret equations
considered by Jian et al. [17] have been developed to interpret the
experimental observations of cell growth and the formation of the
desired metabolic products. The use of several models that involved
more than one substrate state variable has also been reported on
HA production by S. zooepidemicus. Cooney et al. [18] proposed a
structured model based on two compartments for HA fermentation
under anaerobic and aerobic conditions. In addition, Richard and
Margaritis [19] proposed an empirical model, whereas Huang et al.

1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2009.12.001
96 M.M. Don, N.F. Shoparwe / Biochemical Engineering Journal 49 (2010) 95–103

Na2 HPO4 ·12H2 O 1.5 and MgSO4 ·7H2 O 0.5. The medium was
Nomenclature prepared and autoclaved at 121 ◦ C for 20 min. Glucose solu-
tion was autoclaved separately to avoid caramelisation, and
Notations
mixed aseptically with the other components upon cooling
OD optical density
[22].
O2 oxygen
HA hyaluronic acid
HAMW hyaluronic acid molecular weight 2.3. Cell suspension preparation
HPLC high performance liquid chromatography
mM millimolar Cell suspension for the inoculum was prepared by inoculating
rpm rotation per minute a stock culture of Streptococcus zooepidemicus onto SBA-plates and
SBA sheep blood agar incubating overnight at 37 ◦ C. The colonies formed were punched
R2 correlation coefficient (dimensionless) using a sterile cork borer to obtain five round disks 0.85 cm in
Ks Monod saturation constant (dimensionless) diameter. The disks were then put into a sampling bottle con-
Ki inhibition constant (dimensionless) taining 50 ml of sterile distilled water. The sampling bottle was
ms maintenance coefficient (gglucose gbiomass −1 h−1 ) then vortexed for 3 min so that the cells were evenly distributed
t time delay (h) [22].
t time (h)
0 specific growth rate (h−1 ) 2.4. Inoculum preparation
max maximum specific growth rate (h−1 )
S substrate concentration (g l−1 ) The seed culture or inoculum was prepared by inoculating 15 ml
S0 substrate concentration at time 0 (g l−1 ) of cell suspension into a 500 ml Erlenmayer flask containing 135 ml
T temperature (◦ C) of the fermentation medium. The flask was incubated in a rotary
X biomass concentration (g l−1 ) shaker at 37 ◦ C, 250 rpm for 8 h. The inoculum was standardized
Xt biomass concentration at time t (h) by measuring the absorbance (optical density) at 600 nm using a
X0 biomass concentration at time 0 (h) spectrophotometer (Thermo Spectonic, Genesys 20). An inoculum
Xm maximum biomass concentration (g l−1 ) with an optical density within 0.6–0.9 was used to inoculate the
YHA/glu HA yield per glucose consumed (gHA gglu −1 ) fermentation medium.
YX/glu biomass yield per glucose consumed (gcell gglu −1 )
YP/X product yield per biomass produced 2.5. Fermentation
(gproduct gbiomass −1 )
YX/S biomass yield per substrate consumed Batch fermentation was carried out in a 2 l bioreactor (B-
(gbiomass gsubstrate −1 ) Braun, Biotech International, Germany) with a working volume
of 1.5 l. The initial glucose concentration in the medium was
varied from 10 to 60 g l−1 . The pH was maintained at 7.0 ± 0.1
[20] proposed a delayed growth-associated model for HA produc- using 3 M NaOH and 3 M HCl. The agitation speed and tempera-
tion. Recently, Liu et al. [21] reported a two-stage culture strategy ture were set at 300 rpm, 37 ◦ C and aeration was maintained at
model to enhance HA production in either a batch or fed-batch cul- 2.0 l min−1 .
ture. The batch culture had a higher specific HA synthesis rate, while
the fed-batch culture had a higher specific cell growth rate. The 2.6. Analytical procedure
enhanced HA production by this model resulted from the decreased
inhibition of cell growth and the increased transformation rate of Samples were withdrawn at regular time intervals and analyzed
sucrose to HA. for cell, glucose and product concentrations. Cell concentration
In this study, mathematical models that described growth, sub- was determined by measuring the optical density (OD) at 600 nm
strate utilization and inhibition, and HA production were proposed, using a spectrophotometer (Thermo Spectonic, Genesys 20) and
taking into account the effect of the initial glucose concentration the dry cell weight method [21]. A correlation between dry cell
on the kinetic parameters. To achieve this goal, experiments were weight and OD600 was established. Glucose and HA concentra-
performed at an initial glucose concentration range of 10–60 g l−1 tions were analyzed by high performance liquid chromatography
in the batch fermentation. (HPLC) (Model: Perkin Elmer) system equipped with an ultra-
hydrogel linear column (7.8 mm × 300 mm) and a guard column
2. Materials and methods (6 mm × 40 mm) (Waters Associates, Japan), and detected by refrac-
tive index detector. A hyaluronic acid standard was prepared using
2.1. Microorganism Streptococcal HA (Sigma, H-9390), and HA concentrations were
determined using the method as described in previous research
Streptococcus equi subsp. zooepidemicus ATCC 39920 was [9,22,23].
obtained from the American Type Culture Collection (Rockville, For the molecular weight of HA, gel permeation chromatography
MD) as a freeze-dried culture in ampoules. The strains were main- (GPC) equipment consisting of a pump connected to a 2414 refrac-
tained by weekly transfer on sheep blood agar (SBA) and stored tive index (RI) detector and 2695 separation module were used.
at 4 ◦ C after being incubated at 37 ◦ C for 24 h. Monthly subculture For best resolution, the gel permeation chromatography (GPC) was
ensured the availability of sufficient stock cultures for the experi- equipped with an Ultrahydrogel Linear (7.8 mm × 300 mm) and
mental processes. Ultrahydrogel 2000 column. The mobile phase used was degassed
with 0.1 M NaNO3 and was operated at a flow rate of 0.8 ml min−1
2.2. Fermentation medium at ambient temperature. Hyaluronic acid standards of 0.68, 1.6
and 1.8 MDa were prepared and tested. All data was acquired and
The composition of the medium used comprised the fol- processed using the Millennium GPC software as attached to the
lowing (g l−1 ): glucose (10–60), yeast extract 10, KH2 PO4 0.5, equipment.
 M.M. Don, N.F. Shoparwe / Biochemical Engineering Journal 49 (2010) 95–103 97

2.7. Proposed kinetics model Simplifiying Eq. (6) gives:

1
 X0 Xm e0 t

2.7.1. Microbial growth S = S0 − − X0
YX/S Xm − X0 + X0 e0 t
During fermentation processes, microorganism growth
Xm mS

Xm − X0 + X0 e0 t

becomes very complex. In order to demonstrate the inhibitory
− ln (9)
effect, the Logistic equation, a substrate independent method was 0 Xm
used as an alternative empirical function [24]. Logistic equations
So, the final equation for substrate utilization can be written as:
are a set of equations that characterize growth in terms of carrying
capacity [13] and the equation is utilized to describe the kinetics X0 Xm e0 t
S = S0 −
of several polysaccharides fermentation systems [25]. It can be YX/S (Xm − X0 + X0 e0 t )
described as in Eq. (1):
X0 Xm mS Xm − X0 + X0 e0 t
dX
 X
 +
YX/S
−
m
ln
Xm
(10)
= 0 X 1− (1)
dt Xm
Eq. (10) represents a nonlinear relationship between substrate
where Xm is the maximum cell biomass concentration that can be concentrations (S) and fermentation time (t). The parameters YX/S
obtained with a particular cultivation system (strain, medium and and ms can subsequently be estimated using a variety of nonlinear
cultivation conditions) corresponding to the maximum carrying schemes as suggested by Razvi et al. [26].
capacity, and (1 − (X/Xm )) represents the unused carrying capac-
ity [26]. The integrated form of Eq. (1) using X = X0 (at t = 0) gives 2.7.3. Product formation
a sigmoidal variation of (X) as a function of (t), which may present Product formation can be described using the Leudeking–Piret
both the exponential and the stationary phase (Eq. (2)). kinetics equation [15]. The rate of product formation has been
X0 Xm e0 t shown to depend upon both the instantaneous biomass concen-
X = (2) tration, X, and growth rate dX/dt, in a linear fashion, such as:
Xm − X0 + X0 e0 t

Eq. (2) can also be generated by assuming that a toxin is generated dP dX

=˛ + ˇX (11)
as a by-product of the growth limit. dt dt
where the ˛ and ˇ values are empirical constants that may vary
2.7.2. Substrate consumption with the fermentation condition, as well as with the microbial
Glucose was used as a substrate for growth and HA production strain, as shown in Table 1. These kinetic expressions have been
by S. zooepidemicus. A generalized Logistic mass balance on glucose proven to be extremely useful and versatile in fitting the prod-
consumption was incorporated to the Leudeking–Piret-like equa- uct formation data from different types of fermentations, including
tion (Eq. (3)) [15], where S (g l−1 ) is the substrate concentration, those of biopolymers [27,28].
YX/S (gcell biomass gglucose −1 ) is the maximum yield coefficients, and Huang et al. [20] stated that HA concentration increased propor-
ms (gglucose gcell biomass −1 h−1 ) is the maintenance coefficient. tionally to the increase in biomass during the fermentation process.
This occurred especially during the exponential phase of the cell
dS 1 dX growth. A significant relationship was found between the specific
− = + mS X (3)
dt YX/S dt HA production rate and the specific growth rate, which had been
expected earlier with the growth-associated product formation.
Integration of Eq. (3) leads to: Thus Eq. (11) can be written as:
S t t dP dX
1 = YP/X (12)
− dS = dX + mS Xdt (4) dt dt
YX/S
S0 0 0
However, the formation of HA in a culture broth was found to be
very low during the lag phase. This is due to the introduction of t,
Thus, Eq. (4) can be solved as: a parameter of the lag time, to describe the delay of HA production
correlated to cell growth [20]. Thus Eq. (12) can be modified and
t solved using integrated form given as:
1
− [S − So ] = [X]t0 + mS Xdt (5)
YX/S P 
t−t
0 dX
dP = YP/X dt (13)
dt
Substituting Eq. (2) into Eq. (5), and integrating yields the fol-
0 0−t
lowing equation:
  Solve Eq. (13) gives:
1 X0 Xm e0 t X0 Xm e0
− [S − S0 ] = − P = YP/X [X]t−t (14)
YX/S Xm − X0 + X0 e 0
 t Xm − X0 + X0 e0 0−t

Substituting Eq. (2) into Eq. (14) gives:

t
X0 Xm e0 t  X0 Xm e0 t
t−t
+ mS dt (6) P = YP/X (15)
Xm − X0 + X0 e0 t Xm − X0 + X0 e0 t 0−t
0

To solve the integration part in Eq. (6), we define: Table 1

Empirical constants of product formation.
T = em t (7) Parameter Product formation pattern

and differentiated Eq. (7) gives: If, ˛ =/ 0 and ˇ = 0, Product formation is growth associated
If, ˛ =/ 0 and ˇ =/ 0 Product formation is mixed-growth-associated
dT = 0 e0 t dt (8) If, ˛ = 0 and ˇ =
/ 0 Product formation is non-growth-associated
98 M.M. Don, N.F. Shoparwe / Biochemical Engineering Journal 49 (2010) 95–103
Solving Eq. (15) yields the product formation, P as:
 X0 Xm eo (t−t) X0 Xm e−0 t
 fermentation kinetics. By fitting the experimental data to this equa-
P = YP/X − (16)
Xm − X0 + X0 e0 (t−t) Xm − X0 + X0 e−0 t
This equation also represents a nonlinear relationship between (P)
and (t) with time delay (t) when it was combined with the ana-
lytical solution of the Logistic equation, namely, Eq. (2).
2.8. Model parameters estimation
Kinetic equations which describe the activity of a microorgan-
ism on a particular substrate are crucial in understanding various
phenomena in biotechnological processes [29,30]. It is important
to note that most kinetic models and their integrated forms are
nonlinear. This makes parameter estimation relatively difficult
[31]. Although some of these models can be linearized, their use
is limited because they transform the error associated with the
dependent variable, making it normally distributed and therefore
giving rise to inaccurate parameter estimations. Hence, the non-
linear least-squares regression is often used to estimate kinetic
parameters from nonlinear expressions [30]. The parameter esti-
mates obtained from the linearized kinetics expressions can be
used as initial estimates in the iterative nonlinear least-squares
regression using the Levenberg–Marquardt method [31].
In this study, the Polymath® Version 5.1 (CACHE Corpn., USA)
was employed sequentially in order to estimate the value of
the parameters within the nonlinear model equations (i.e. Eqs.
(2), (10) and (16) from the experimental results. The parameters
were evaluated using the nonlinear least square method of the
Levenberg–Marquardt optimization, which was used to minimize
the residual sum of squares. Each experiment was repeated twice,
and the results were reported as averaged values.
2.9. Model validation
Model validation is possibly the most important step in the
model building sequence [32]. In order to validate the model, the
kinetic parameter values obtained from Section 2.8 were used to
simulate the profiles of cell growth, substrate utilization and prod-
uct formation with other batch experimental data for various initial
glucose concentrations. Simulation of the batch fermentation for
each initial glucose concentrations in the bioreactor were per-
formed using ordinary differential method of Polymath® Version
5.1 (CACHE Corpn., USA) software. A set of ordinary differential
equation (ODE) was solved using the Runge-Kutta Fehlberg (RKF45)
algorithm. The profiles from simulation of the models and experi-
mental data were then evaluated using mean squares error (MSE).
This performance criterion was chosen because they are easy to
identify and have convenient mathematical properties. The MSE
value can be calculated by the sum squares errors divided by the
length of actual data period [32]:
NT
(y
I=1 i
− fi )2
MSE (%) = 100
nt
where fi , yi , and nt are the model data, experimental data and length
of actual data period, respectively.
3. Results and discussion
3.1. Microbial growth
To investigate the behavior of S. zooepidemicus growth in aero-
bic batch fermentation, the fermentation medium supplemented
with varying initial concentrations of glucose (10–60 g l−1 ) was
chosen. The most popular kinetic expression for microbial growth,
the Logistic equation (Eq. (2)) was fitted to represent the batch HA
tion, the values of parameters as shown in Table 2 were obtained.
The data showed that the specific growth rate,  increased along
with the increase in glucose concentration from 10 to 40 g l−1 .
Beyond these values, the 0 values decreased gradually. The
optimum 0 was 1.14 h−1 corresponding to the initial glucose con-
centration of 40 g l−1 .
Fig. 1 shows the experimental and theoretical prediction pro-
files on the growth of the tested strain during HA fermentation. A
classical sigmoidal growth trend for the S. zooepidemicus cells, in
which an initial lag phase (∼2 h) varied in duration, was observed.
This was followed by the exponential growth phase, the stationary
phase and the death phase, accordingly. Sikyta [33] reported that
the length of the lag phase could be affected by several factors: (1)
composition of medium, (2) type and age of the strains, (3) num-
ber of cells, and (4) physical factors such as temperature and pH.
As shown in Fig. 1, the cell growth of this tested strain was very
low in the lag phase. This is in agreement with the results of Huang
et al. [20] who reported that the growth and formation of HA in
the culture broth were found to be very low during the lag phase
due to the adaptation of bacterial cells to the new environmental
condition.
After a lag phase, the cells entered an exponential phase either
for a lower or higher glucose level. It started at 2 h and continued
up to 8 h. Once the limiting nutrient glucose started to decrease,
the cells biomass stopped increasing exponentially, but it still con-
tinued to increase due to the presence of other components that
accumulated in the culture media. Cheng et al. [34] reported that
yeast extract was the best supplement for N source in HA fermenta-
tion and provided convenient growth factors for most streptococci.
Furthermore, the salt component of the medium, usually sodium or
potassium phosphate and a form of magnesium salt, was necessary
for the activation of hyaluronate synthase [22,35], thus stimulating
the significant growth of the tested strain. Eventually, a stationary
phase was reached, which started at 8 or 9 h and lasted until 12 h.
During this phase, the cell concentration was almost constant or
had little variations along that period of time. The predicted max-
imum cell biomass obtained from the Logistic model for each 10,
(17)
Fig. 1. Fitting of the experimental data to the model describing cell growth over
time at 10–60 g l−1 of initial glucose concentrations.
 M.M. Don, N.F. Shoparwe / Biochemical Engineering Journal 49 (2010) 95–103 99

Table 2
Kinetic parameters values for growth, substrate consumption and product formation at different initial glucose concentrations.

Parameter estimation Initial glucose concentration (g l−1 )

10 20 30 40 50 60

Cell biomass
X0 (g l−1 ) 0.036 0.022 0.029 0.036 0.052 0.048
Xm (g l−1 ) 1.86 2.05 2.21 2.24 2.17 1.94
o (h−1 ) 0.61 0.84 1.06 1.14 1.00 0.86
R2 (correlation coefficient) 0.997 0.996 0.998 0.998 0.995 0.974
 (variance) 2.40 × 10−3 4.68 × 10−3 4.25 × 10−3 3.15 × 10−3 5.67 × 10−3 2.59 × 10−2
Glucose consumption
S0 (g l−1 ) 10.27 20.26 30.23 41.44 51.24 61.21
ms (gglucose gcellbiomass −1 h−1 ) 0.04 0.51 0.65 1.56 0.99 0.86
YX/S (gcellbiomass gglucose −1 ) 0.16 0.22 0.30 0.37 0.34 0.32
R2 (correlation coefficient) 0.998 0.997 0.974 0.996 0.985 0.980
 (variance) 1.97 × 10−1 2.73 × 10−1 6.84 × 10−1 6.60 × 10−1 7.42 × 10−1 7.56 × 10−1
HA production
YP/X (gHA gglucose −1 ) 0.310 0.310 0.934 0.958 0.889 0.572
t (h) 0.49 0.87 0.86 0.90 1.40 2.16
R2 (correlation coefficient) 0.991 0.996 0.996 0.998 0.994 0.907
 (variance) 8.97 × 10−4 1.47 × 10−3 3.14 × 10−3 1.85 × 10−3 5.07 × 10−3 1.11 × 10−2

20, 30, 40, 50, and 60 g l−1 of the initial glucose concentration was nance coefficient (ms ) can range from as little as 0.02 to as high
1.86, 2.05, 2.21, 2.24, 2.17, and 1.94 g l−1 , respectively (Table 2). The as 4.0 gsubstrate gcell −1 h−1 , depending on the environmental condi-
highest cell biomass was obtained (2.24 g l−1 ) at the initial glucose tions surrounding the cell and on its rate of growth. This clearly
concentration of 40 g l−1 . The predicted results also gave a relatively showed that the Logistic incorporated Leudeking–Piret-like equa-
good agreement with the experimental data, with R2 values above tion was capable of predicting the experimental results of glucose
0.9 (Table 2). This showed that the proposed Logistic model was consumption with a good amount of accuracy (>90%) for each of
sufficient to describe both the exponential and stationary phase the glucose concentration tested.
of S. zooepidemicus growth in a batch culture. According to Char-
alampopoulus, Vazquez and Pandiella [36], a number of models 3.3. Product formation
had been successfully used to describe the sigmoidal curves of bac-
terial growth, including the Logistic model which fitted cell growth For HA production, the experimental data obtained from batch
over time and took into account growth inhibition in the stationary fermentation studies with different glucose concentration were
phase of growth. fitted to Eq. (16). The results showed that HA fermentation by
S. zooepidemicus with glucose as a substrate was highly growth-
associated even though a lag time appeared in the profiles. The
3.2. Substrate consumption fitting results were satisfactory and gave good agreement to the
model used, with correlation coefficient (R2 ) values above 0.9
The rate of glucose consumption was mainly a function of three (Fig. 3). The parameter estimation for this model (Table 2) showed
factors: growth rate, HA production and rate of substrate uptake
for cell maintenance (ms ). The experimental data were fitted to Eq.
(10). The parameter values of the glucose uptake model for each ini-
tial glucose concentration are shown in Table 2. Fig. 2 shows that
the fitting results were satisfactory and gave good agreement to
the model used, with correlation coefficient (R2 ) values above 0.9.
As can be seen from Fig. 2 and the predicted values of yield coef-
ficient (YX/S ) in Table 2, the yield significantly increased with the
increase in glucose concentration from 10 to 40 g l−1 . Beyond this,
an opposite trend was observed, suggesting that a significant inhi-
bition would have occurred for the glucose concentration higher
than 50 g l−1 .
Generally, when energy and carbon sources were in excess,
microorganisms tend to dispense the excess energy and carbon
through the formation of storage compounds or extracellular prod-
ucts. Some of the products, such as acetic acid, lactic acid, hydrogen
peroxide, and ethanol, are toxic to cell growth [9,21,22]. Hence, a
maintenance coefficient (ms ) was used to describe the specific rate
of substrate uptake for cellular maintenance, which was found to
vary for each initial glucose concentration tested (Table 2). Zheng
et al. [37] reported that the maintenance coefficient (ms ) might
vary with the specific growth rate, , particularly in aerobic cul-
ture and energy sufficient conditions. Shuler and Kargi [13] later
stated that cellular maintenance represented energy expenditures
necessary to repair damaged cellular components, to transfer some
nutrients and products in and out of the cells, for motility, and
to adjust the osmolarity of the cells at interior volume. Sinclair Fig. 2. Fitting of the experimental data to the model describing glucose consumption
and Kristiansen [38] also reported that the values of the mainte- over time at 10–60 g l−1 of initial glucose concentrations.
100 M.M. Don, N.F. Shoparwe / Biochemical Engineering Journal 49 (2010) 95–103

Fig. 4. Effect of initial glucose concentration on the molecular weight of HA (HAMW )

produced by S. zooepidemicus in a batch culture (conditions: temp. 37 ◦ C, pH 7.0,
aeration rate 2 l min−1 , agitation 300 rpm).

Conditions which reduced the HAMW in the presence of excess glu-

cose could be due to the availability and channeling of common
Fig. 3. Fitting of the experimental data to the model describing HA production over resources into other competing metabolic pathways. Mausolf [44]
time at 10–60 g l−1 of initial glucose concentrations. reported that when the activated sugar monomers UDP-GlcUA and
UDP-GlcNAc were present at high concentrations, HA chain elon-
that the values of YP/X increased along with the increase in substrate gation is thought to persist. However, according to Jagannath and
concentration up to 40 g l−1 . However, with further increase in the Ramachandran [45], the nature, levels and complexity of the car-
initial glucose concentration, the values of YP/X gradually decreased. bon source could also alter the strength of the glycolytic process
This indicated that HA production has been inhibited as the glucose and regulate the HA molecular weight. Nevertheless, the most fre-
concentration was increased from 50 to 60 g l−1 . quently reported HAMW were in the range of 1 × 106 –2.5 × 106 Da
The lag time t, which described the time delay of HA produc- with initial glucose concentration of 10–60 g l−1 in a batch culture.
tion correlated to cell growth, varied from 0.49 to 2.16 h, which is in Above this level the HAMW is considered high [10,42,45,46].
the same range of magnitude to the value of t = 0.75 h previously
reported [20]. Nickel et al. [39] stated that a 56-kDa ectoprotein 3.4. Model validation
kinase from the plasma membrane of group C streptococci does
not shed its HA to the medium simultaneous to cell growth; rather, In order to prove the reliability of the microbial growth, sub-
it needs a lag time for HA to appear in the medium, whose function strate utilization and product formation models for different initial
is to regulate the formation and shedding of HA. During the expo- glucose concentration, the parameters estimated in Table 2 as
nential phase, the cell biomass and HA concentrations increased described in Section 2.8 were used to simulate the model using
exponentially. According to Almeida and Oliver [40], the percent- 4th/5th order Runge-Kutta method. Comparison between the sim-
age of capsulated Streptococcus cells (S. uberis) increased from as ulated and experimental values were made and only one response
low as 10% to over 90% during the exponential phase, but the num- curve was shown in Fig. 5, but the models have been checked
bers fell at the end of the batch culture. In another study which used for R2 and variance () for all the studied substrate concentra-
phase contrast microscopy with India ink preparation, Van De Rijn tion during the entire course of fermentation. The error analysis
[41] found that the largest capsule appeared at the mid-exponential
growth phase. Armstrong and Johns [42], for their part, reported
that the HA concentration increased in the broth after the cessa-
tion of growth due to glucose exhaustion. This is due to the release
of HA capsules from the cells during the HA biosynthesis.
Glucose is the primary carbon and energy source, and its effect
on the molecular weight of hyaluronic acid (HAMW ) produced
by S. zooepidemicus was carried out at standard conditions. Only
values obtained at the 10 h of fermentation were considered as
maximum HA concentrations were detected for most of the glu-
cose levels tested. As can be seen in Fig. 4, the HAMW reached its
maximum value of (2.52 ± 0.001) × 106 Da at initial glucose concen-
tration of 40 g l−1 , and then decreased with an increasing glucose
concentration. Similar as the observation of Armstrong and Johns
[42], the HAMW increased by 17–21% when the initial glucose
level was doubled, and reduced as the level increased to 60 g l−1 .
According to Armstrong [43], microorganisms were able to detect
changes in environmental osmotic pressure and may response by
Fig. 5. Validation of the experimental data and simulation values for biomass, glu-
altering their metabolism. Glucose above 40 g l−1 represents an cose consumption and HA production at 40 g l−1 glucose concentration (() biomass,
environment of higher osmotic pressure than at 10, 20 and 30 g l−1 . () glucose consumption, (䊉) HA production and (–) simulated values)).
 M.M. Don, N.F. Shoparwe / Biochemical Engineering Journal 49 (2010) 95–103 101

Table 3
Mean squares error (MSE) values of microbial growth, substrate consumption and
HA production at different glucose concentrations.

MSE (%) Initial glucose concentration (g l−1 )

10 20 30 40 50 60

Cell biomass 0.550 0.899 0.287 0.445 0.607 0.618

Glucose consumption 8.505 6.883 7.810 9.205 9.434 6.593
HA production 0.241 0.322 0.472 0.354 0.589 0.507

between experimental and simulated values was also determined

using mean squares error (MSE) and shown in Table 3. The value
of error for each initial glucose concentration was less than 10%. It
implies that the model proposed adequately represents the real
process, and it can be used to describe the batch profile of HA
fermentation by S. zooepidemicus.

3.5. Comparison with the substrate inhibition model

Based on results of Sections 3.1–3.3, it is evident that at higher

initial glucose concentration, inhibition effect was seen for the
Streptococcus cells growth. The specific growth rate increased along
with increase in substrate concentration from 10 to 40 g l−1 . Above
this, a reverse trend was observed. In most biotechnological pro-
cesses, higher concentrations of substrates or products often lead
to inhibitory effects which result in the poor utilization of the
substrate [47]. These effects also decrease both the cell growth
and product yield. The literature provided several expressions
which relate the specific growth rate to the substrate concentra-
tion [15–17,19,24,25]. Aside from substrate limitation, inhibition
by substrate was likewise quite often found in the biotechnology
process. This has also been examined by many authors [29,48–51].
When the substrate inhibited the microbial growth, the origi-
nal Monod model became unsatisfactory. In this case, the Monod
derivatives that provided correction for substrate inhibition (by
incorporating the inhibition, Ki ) can be used to describe the kinetic
growth of the fermentation process.
Several substrate inhibition kinetic models (Eqs. (18)–(24))
were tested and compared in this work (Table 4). All the models
were fitted to the experimental data with varying initial glucose
concentrations. In order to determine the model kinetic parame-
ters and fitting constants, values of specific growth rate, 0 of the
Logistic model (Table 2) were used to simulate the models using a
nonlinear regression technique. The estimated values for max , KS ,
Ki , S0 , m and n as returned by the fitting algorithm are shown in
Table 5, where Ks is the Monod half saturation constant, Ki is the
substrate inhibition concentration, S0 is the maximum substrate
inhibition concentration at which no growth was observed, and n
Fig. 6. Comparison of the experimental data and simulations from substrate inhi-
bition models at different initial glucose concentrations.
and m are the constants which accounted for the relation between
 and S, respectively.
It is clear that growth models that incorporate the substrate
inhibition parameter gave better fits to the experimental data with
R2 > 0.9. Among the models that took into account the substrate
inhibition factor, the Han and Lenvenspiel type model showed
the best fit of the experimental data (R2 = 0.997), as compared to
Teisser-type (R2 = 0.985) model. The graphical outputs showing the
fits of the experimental data by the models are shown in Fig. 6.
According to Annuar et al. [52], the R2 (correlation coefficient) is
frequently used to judge whether the model represents correctly
the data, implying that, if the correlation coefficient is close to
one, then the regression model is correct. However, many exam-
ples exist where the correlation coefficients are close enough to
one but the model is still not appropriate. Hence, the mean square
error (MSE) was used with R2 for the comparison of various inhi-
bition models representing the same dependent variable used in
this study. A model with small MSE represents the data more accu-

Table 4
Substrate inhibition kinetic models used in this study.

Names of model Substrate inhibition kinetic models References


Andrew  = max S (18) Andrew [46]
(K S + S) 1 + S/Ki
 n  
S S
Han and Lenvenspiel  = max 1− (19) Han and Levenspiel [43]
S• S + KS (1 − (S/S • ))
m

max S
Aiba = exp−S/Ki (20) Aiba et al. [44]
KS + S
max S
Competitive substrate inhibition = (21) Shuler and Kargi [13]
KS (1 + (S/Ki )) + S
max S
Non-competitive substrate inhibition (if Ki > Ks ) = (22) Shuler and Kargi [13]
KS + S + (S 2 /Ki )
S
Edward  = max (23) Edward [16]
S + KS + (S 2 /Ki )(1 + S/KS )
Teissier-type substrate inhibition  = max [exp(−S/Ki ) − exp(−S/KS )] (24) Edward [16]
102 M.M. Don, N.F. Shoparwe / Biochemical Engineering Journal 49 (2010) 95–103

Table 5
Estimated parameter values of substrate inhibition models as returned by the numerical calculations for the batch fermentation of S. zooepidemicus at different initial glucose
concentration.

Parameter Model

Andrew Han and Aiba Competitive Non-competitive Edward Teissier-type

model Lenvenspiel model substrate substrate model model
model inhibition model inhibition model

Equations (18) (19) (20) (21) (22) (23) (24)

Ks 1.70 40.54 9.08 7.80 11.10 21.20 41.40
Ki 101.99 – 86.86 103.84 131.32 128.50 55.00
max 1.14 2.19 1.92 1.26 1.56 1.95 2.90
m – 0.77 – – – – –
n – 0.48 – – – – –
S• – 71.92 – – – – –
Correlation coefficient) (R2 ) 0.710 0.997 0.802 0.914 0.935 0.614 0.985
Variance () 8.84 × 10−2 4.21 × 10−4 5.63 × 10−2 1.96 × 10−2 1.47 × 10−2 3.39 × 10−3 9.31 × 10−3
Means square error (MSE) (%) 5.53 0.18 1.90 1.19 0.76 21.54 0.57

rately than the models with larger MSE. It is also found that the Han
and Lenvenspiel type model, and the Teisser-type model both have
much lower MSE at 0.18% and 0.57%, respectively.
As indicated by the inhibition constant Ki on cell growth
(Table 5), glucose provided a stronger inhibition which predomi-
nantly corresponded to the culture of 50 and 60 g l−1 initial glucose
concentrations (Fig. 6). According to a few researchers, glucose
above a concentration of 50 g l−1 may inhibit growth due to the
reduction in water activity [22,23,42,53]. Being a monosaccharide,
glucose’s osmotic pressure will be higher; hence, it could cause
more substrate inhibition [22]. In fact, Cooney et al. [18] reported
that, at a high glucose concentration, lactic acid bacteria such as
S. zooepidemicus would produce large quantities of lactate from
the catabolism of glucose (75–85%). Consequently, growth will be
inhibited.
4. Conclusion
The results clearly showed that the proposed unstructured
mathematical model satisfactorily predicted the cell growth, sub-
strate utilization, and HA production for the fermentation with
an initial substrate concentration of 10–60 g l−1 . For the substrate
inhibition, the Han and Levenspiel model and the Teissier-type
model gave the best fit for all the systems studied with R2 of 0.997
and 0.985, respectively. All the models tested not only adequately
described the relationship between the principal state variables or
quantitatively explained the behavior of a system, but could also
be used for the design, optimal control, and economic analysis of
HA fermentation processes.
Acknowledgements
The authors would like to thank the Malaysian Ministry of
Higher Education for its financial support via the Fundamental
Research Grant Scheme (FRGS: Acc Number: 6070018).
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