Kinetics is a study that deals with rate of the reaction. It is based on the
mechanisms of any processes-physical, chemical or biological. The chemical kinetics
deals with how the rate of reaction is dependent upon the concentration of the reactants.
In a biochemical reaction, kinetics is a little complicated. A living cell, during
fermentation or any biological processes, require essential nutrients and suitable
environment to survive. Hence a biochemical reaction proceeds with the intervention of
microbial cells in a liquid medium containing nutritive sources under conditions like pH,
temperature. The cells multiply and grow, consuming a part of substrate and other
necessary components from the fermentation broth. Cell growth is associated with two
Certain parameters/ phenomena that determine cell population kinetics are the
characteristics of culture broth, nutrients and substrates used for growth and production
of metabolites, cell to cell heterogeneity, microbial cells of different ages manifesting
metabolic activities and characterization of biochemical pathway (Figure 5.1). These
complex parameters make it difficult to formulate a simple kinetic model for cellular
activities (Bailley and Ollis, 1986). A kinetic model describes the behavior of the cellular
processes through possible mathematical equations and it serves to be a very effective
tool to test and eliminate the extremities. Different kinetic models have been proposed in
several research works for microbial growth, substrate utilisation and product formation
in various fermentation modes.
accumulates various products as the result of metabolic activities. According to number
of components used in microbial representations and considering cells as heterogenous
mass or wholly as average cell clusters, different perspective are put forward for cell
population kinetic representations (Figure 5.2).
Structured model is one perspective in which the microbial cells are considered as
multicomponent systems. When cell population is treated as one component system, it is
referred to as unstructured model. The rate of reaction depends only on the parameters
inside the fermenter. These models only contain growth, substrate consumption and
product formation kinetics. Cell to cell heterogeneity and discrete cells compose a multi
component entity, constituting ultimately a segregated perspective. An unsegregated view
considers average cellular properties. In actual cellular cases, the situation is a structured
and segregated one. In balanced growth of cells, the metabolic activities are coordinated
in such a way that the average cell composition is not influenced by increase in cell mass.
In such cases, models that do not consider the multi component nature of cells are
necessary. Hence unsegregated, unstructured model is the most idealized situation when
analyzing the growth of cells (Bailley and Ollis, 1986).
Figure 5.2 Different perspectives for cell population kinetic representation (Adapted
from Bailley and Ollis,1986)
ୢୗ ଵ ୢ୶ ଵ ୢ
ൌ െ െ ୶
ୢ୲ ଢ଼୶Ȁୱ ୢ୲ ଢ଼୮Ȁୱ ୢ୲
!
where Yx/s is the yield coefficient for biomass with respect to substrate consumed and Yp/s
is the yield coefficient for product formed with respect to the substrate consumption.
ୟୱୱ୭ୡୣ୪୪୭୰୫ୣୢ ୶ ୶ି୶బ
୶Ȁୱ ൌ ൌ ൌ
ୟୱୱ୭ୱ୳ୠୱ୲୰ୟ୲ୣୡ୭୬ୱ୳୫ୣୢ ୱ ୱబ ିୱ
" !
ୟୱୱ୭୮୰୭ୢ୳ୡ୲୭୰୫ୣୢ ିబ
Ȁୱ ൌ ൌ ൌ
ୟୱୱ୭ୱ୳ୠୱ୲୰ୟ୲ୣୡ୭୬ୱ୳୫ୣୢ ୱ ୱబ ିୱ
# !
If the amount of carbon sources used for product formation and maintenance
constants are neglected when modeling substrate consumption rate, the model becomes,
ୢୗ ଵ ୢ୶
ൌ െ ............................................. (5.4)
ୢ୲ ଢ଼౮Ȁ౩ ୢ୲
ଵ ୢ୶
െ ൌ െ
ଢ଼౮Ȁ౩ ୢ୲
!
ଵ
ൌ െ ሺ୲ െ ሻ
ଢ଼౮Ȁ౩
$ !
The growth of microbial cells include four major phases namely lag phase,
exponential phase, stationary phase and the decline phase, which had been previously
described in chapter II. An emphasis on the exponential or logarithmic phase had been
given in this particular work, since the production of the product, exopolysaccharide, is
found maximum and directly proportional to the microbial cell growth.
Various models had been used for studying growth kinetics. The models are being
mentioned under this topic.
The simplest cell growth model is the Malthusian model commonly known as
exponential law. In a batch fermentation operated under ideal conditions the
concentration and weight of biomass increases proportionally with increase in time.
Hence it could be said that all the cells have the same probability to multiply. Thus the
overall rate of biomass formation is proportional to the biomass itself. For a batch system,
this is equivalent to
ୢ୶
ൌ Ɋ .................................... (5.7)
ୢ୲
where x is the mass of cells per unit volume, µ is the proportionality constant known as
'specific growth rate' (h-1) and t is the time (h).
Here as the time increases, cell mass will also increase. On integrating (1) gives
The demerit of this model is that it ignores the substrate needed for its growth and
that the resources are constrained.
Contois model is another unstructured model that depends upon substrate and cell
concentration which is given as
ୱ
Ɋ ൌ Ɋ୫ୟ୶
୶ାୱ
& !
Moser model relies upon the growth rate and substrate concnetration and the two
parameter model is depicted as
ିଵ
Ɋ ൌ Ɋ୫ୟ୶ ൫ͳ ୱ ି ൯ ' !
Tessier model is another growth kinetic model that also rely upon the substrate
concentration and is explained by,
ୱ
Ɋ ൌ Ɋ୫ୟ୶ ൫ͳ െ ି ൗୱ ൯ .......................... (5.11)
Moser and Tessier model render algebraic solution of the growth equations much
more difficult than the Monod model.
%
5.2.5 Verlhurst model
Verlhurst kinetic model depends on the cell concentration. This model has two
kinetic constants of maximum specific growth rate (µmax) and maximum cell
concentration (xmax). Verlhurst model is expressed by the following equation
ஜౣ౮
Ɋ ൌ Ɋ୫ୟ୶ െ
୶ౣ౮
" !
Andrew model is proposed by the following equation for the growth dependent
which incorporate substrate inhibition.
ஜౣ౮
Ɋ ൌ ౩ൗ ୱ
............... (5.13)
ቂቀଵି ୱቁቀଵା ൗ౩ ቁቃ
The commonly used unstructured kinetic models to evaluate cell growth are
Monod and Logistic models.
The concepts in microbial growth kinetics have been mainly analysed by semi-
empirical model proposed by Monod (1949). The Monod model introduces the concept
of a growth controlling substrate, called limiting substrate which indicates that the
microbial growth rate rely upon the actual concentration of a particular metabolite. There
exists a relationship between the exhaustion of the limiting substrate and the end of
growth, hence the Monod model may be considered deterministic. The relationship is
described as,
ஜౣ౮ ୱ
Ɋ ൌ ............................................. (5.14)
౩ ାୱ
where µmax is the maximum growth rate achievable and Ks is the limiting substrate
concentration when the specific growth rate is equal to the half the maximum specific
growth rate when µ = µmax/2.
(
5.2.8 Logistic model
The model which could be appropriate for the present research is the logistic
model. Verlhurst in 1844 and Pearl and Reed in 1920 contributed to a theory which
included an inhibiting factor to population growth. Assuming that inhibition is
proportional to x2, they used
ୢ୶ ୶
ൌ ሺͳ െ ሻ
ୢ୲ ୶౩
* !
where x0 is the initial biomass concentration (gL-1) and t is the time (h). The logistic
curve is sigmoidal and leads to a stationary population of size, xs +
, -
The type of kinetic description that may be employed for product formation by
cell population parallel those used to describe cell population growth. The simplest types
of product formation kinetics arise where there is a simple stoichiometric connection
between product formation and substrate utilization or cell growth. The rate of production
of the product parallels the rate of cell growth. Substrate concentration falls continuously
as fermentation proceeds. Such product formation kinetics is sometimes called as growth
associated. In many fermentation, involving secondary metabolite, significant product
formation does not occur until relatively late in a batch cultivation, that is, production
happens in the late exponential phase or during the stationary phase. Production rate is
proportional to cell concentration rather than growth rate. This kind of kinetics is referred
to as non growth associated model.
)
A typical and widely used product kinetic model is Luedeking- Piret model
(1959), which is an unstructured approach contributed to both growth and non growth
associated phenomena for product formation. This model was originally developed for
the formation of lactic acid by Lactobacillus delbruckeii and the equation is given as
rfp = fx . . . . . . . . . / / / / / / / / / / / / / 0 1 / 2 1 3
where rfp is the rate of product formation, rfx is the rate of biomass formation and 4 and 5
are the kinetic constants of Luedeking- Piret model. This two-parameter expression has
proven extremely useful and versatile in fitting product formation data from different
fermentations. This is an expected kinetic form when the product is the result of energy
yielding metabolism.
According to this model, the product formation rate depends linearly upon the
growth rate and the cell concentration
ୢ ୢ୶
ൌ Ƚ Ⱦ ...................................... (5.16)
ୢ୲ ୢ୲
The Luedeking- 6 7 8 9 : ; 7 < 9 : 7 = > ? 8 ? @ 9 : 9 8 A B C ? < D E D 9 > 9 < D F > G < ? < D H ? 8 I J 7 : K : K 9
L L
ౚౌ
ቀ ቁୱ୲ୟ୲୧୭୬ୟ୰୷୮୦ୟୱୣ
ౚ౪
Ⱦൌ ................................ (5.17)
୶౩
ܻȀ௫ ,
L L
ୟୱୱ୭୮୰୭ୢ୳ୡ୲୭୰୫ୣୢ ିబ
Ȁ୶ ൌ ൌ ൌ ................... (5.18)
ୟୱୱ୭ୡୣ୪୪୭୰୫ୣୢ ୶ ୶ି୶బ
୶ ୶
ሺሻ െ ሺͲሻ െ Ⱦ ቀ ౩ ቁቂͳ െ బ ൫ͳ െ ୩୲ ൯ቃ ൌ Ƚሺ୲ െ ሻ .............. (5.19)
୩ ୶ ౩
L
Cell growth, substrate utilisation and product formation were examined and
simulated with the experimental data of EPS yield by B.subtilis and P.fluorescens, using
cane molasses and rice bran respectively. The logistic equation was used for the cellular
growth kinetic study and Luedeking Piret model for substrate consumption and product
formation studies. The simulation of the experiment was carried out using MATLAB
(v.7.10.0.0499, The Mathworks, USA) software. The MATLAB program is listed in
Appendix I and II. The kinetic parameter 'k' of logistic model was obtained using curve
fitting (cftool) tool kit of the same software and the high R2 values represented that the
equation fit the experiment (Sivaprakash et. al., 2011a). Using the obtained 'k' values for
each biological system, the kinetic constants of Luedeking- Piret model, α and β, were
evaluated (Table 5.1). The model was analysed using MS- Excel by graphical method.
The yield coefficient Yx/s, for substrate utilisation rate, was also obtained by graphical
method. Predicted values of this model, which were consistent with the observed values,
were obtained by solving the differential equations by Runge K F : : ? T A < F @ 9 8 7 = ? O
integration using ODE23 solver, in same software, which is given in Appendix III
(Sivaprakash et. al., 2011b). The theoretical and predicted values obtained are tabulated
in Table 5.2 and Table 5.3.
% %
B.subtilis 0.09126 0.9829 10.22 6.63 0.5 0.0808 3.05
P.fluorescens 0.08422 0.9852 5.5 5.45 0.6 0.114 7.47
Q R R
The high R2 and prediction values denote that the model is highly suitable for
exopolysaccharide production with a minimal error of 4.52 % and 9.91% from Bacillus
subtilis and Pseudomonas fluorescens. Figures 5.3 5.4, 5.5, 5.6, 5.7 and 5.8, illustrate the
experimental and predicted values for each variable S
Table 5.2 Experimental and predicted values of cell mass concentration, substrate
utilization and product formation of B.subtilis
Q R U
14
12
4
Experimental
2 Simulated
0
0 20 40 60 80 100 120
Time (h)
Figure 5.3 Comparative chart showing experimental and predicted values of cell
concentration of B.subtilis
1.6
Experimental
1.4
Simulated
Substrate Consumption (g/L)
1.2
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120
Time (h)
Q R V
7
Product (g/L)
4
2 Experimental
Simulated
1
0
0 20 40 60 80 100 120
Time (h)
Table 5.2 Experimental and predicted values of cell mass concentration, substrate
utilization and product formation of Pseudomonas fluorescens
Figure 5.6 Comparative chart showing experimental and predicted values of cell
concentration of P.fluorescens
2
1.8
Experimental
Substrate Consumption (g/L)
1.6 Simulated
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 10 20 30 40 50 60 70
Time (h)
Figure 5.7 Comparative chart showing experimental and predicted values of cell
substrate consumption of P.fluorescens
Q U X
6
Product (g/L)
4
0
0 10 20 30 40 50 60 70
Time (h)
The utilization rate was well predicted and found to be the most suited model
for the exopolysaccharide production from cane molasses and rice bran respectively with
a minimal error of 6.63% and 5.45% for Bacillus subtilis and Pseudomonas fluorescens
respectively. Similarly, the predicted and theoretical values were found to be in
agreement with the experimental data of the exopolysaccharide formation kinetics with a
minimum error of 3.05% and 7.47% for Bacillus subtilis and Pseudomonas fluorescens
respectively.
The overall trend of the simulated or the predicted values, obtained using the
implemented models in accordance with the experimental data for B.subtilis and
P.fluorescens is depicted in Figure 5.9. The studies confirmed that the Logistic and
Luedeking Piret models befitted accurately and explained adequately the mechanisms of
cell growth, substrate consumption and product formation in B.subtilis and P.
fluorescens.
Q U Q
14 2
1.8
12
Product (g/L) 1.6
10
1.2
8
1
Cell mass (g/L)
6
0.8
4 0.6
0.4
2
0.2
0 0
0 20 40 60 80 100 120
Time (h)
Figure 5.9 Overall experimental and predicted values of Logistic and Luedeking
Piret models for Bacillus subtilis and Pseudomonas fluorescens (cell mass of B.
subtilis, experimental and predicted ; product formation by B. subtilis,
experimental and predicted ; substrate consumed by B. subtilis,
experimental and predicted ; cell mass of P. fluorescens, experimental
and predicted ; product formation by P. fluorescens experimental and
predicted ; substrate consumed by P. fluorescens experimental and
predicted)
The viscosity measurements were performed for the cultures after 24 h till 120h to
check their viscosity. The viscosities and relative viscositites were found to be constant
after certain period (Fig.5.10). In case of B.subtilis, the relative viscosity was constant
after 72h, whereas for culture broth of P.fluorescens, the relative viscosity stayed the
same after 48h. The trends of both cultures exhibited a Newtonian behaviour.
Q U Y
1.02
1
0.98
Relative viscosity
0.96
0.94
0.92
0.9
B.subtilis
0.88
P.fluorescens
0.86
0.84
0 20 40 60 80 100 120
Time (h)
Figure 5.10 Relative viscosity of the two culture filtrate with EPS
Q U Z