INT J CURR SCI 2011, 1: 1-10

RESEARCH ARTICLE

Modeling and simulation of a structured model for analysis of hyaluronic acid fermentation
by Streptococcus zooepidemicus under aerobic and anaerobic batch conditions
Chelliah V. Navin* and Philomena George
Department of Biotechnology, Karunya University, Coimbatore - 641 114, India
*Corresponding author: Present address: E-mail: navincv@yahoo.com; Phone: +1-312-813-9880
Biological and Agricultural engineering, Louisiana State University, Baton Rouge, LA-70803, USA
Abstract
Hyaluronic acid (HA) is a linear high molecular weight glycosaminoglycan polymer made up of repeating units of
(14) N-Acetylglucosamine and (13) D - glucuronic acid. Producing large quantities of high molecular weight polymer with
very low dispersity of molecular size is seen as a niche in a huge hyaluronic acid world market and microbial derived product
could effectively fit into it. This study is to understand the model and simulate the aerobic and anaerobic batch fermentation using
MATLAB R2007a and to make more sense of the existing data and improve the production which is one of the widely used
methods to describe the process mathematically and construct an abstract model. By simulating it and varying appropriate
parameters one can predict the nature of the fermentation and the total yield of the polymer. The studies were performed on
Streptococcus zooepidemicus under aerobic and anaerobic batch conditions. In case of aerobic and anaerobic batch fermentation
the rate constants of the reactions were varied by increasing it by 10 and 100 % and also decreasing it by 10 and 50 % and the
initial carbon/nitrogen ratio were varied in various proportions.
Keywords: hyaluronic acid, matlab, aerobic batch condition, anaerobic batch condition
Received: 19th September; Revised: 29th September; Accepted: 04th October; IJCS New Liberty Group 2011
molecular weight Hyaluronic Acid in the order of 3 MDa,

Introduction
Hyaluronic acid (HA) is a linear high molecular

with its high viscoelasticity, length (Armstrong, 1997) and

weight glycosaminoglycan polymer made up of repeating

high affinity to cells is used in healing damaged cartilages

units of (14) N-Acetylglucosamine and (13) D -

(Uzuki and Sawai, 2001), treating osteoarthritis (Asari et al.,

glucuronic acid and can reach a length of more than 20,000

1998; Adam and Ghosh, 2001) or as adjuvants for

disaccharide units (Weismann and Meyer, 1954). The

ophthalmic vehicles (Saettone et al., 1991).

physiological concentration of the polymer in the synovial

fluid is about 2 - 3 mg/mL (Balazs

Hyaluronic acid also occurs as a capsular

et.al., 1967) and is

polysaccharide in many microbial species and is widespread

found to be polydisperse with its range of molecular weight

in Lancefield group A and C Streptococci species.

being 10 - 10 Da (Shimada and Matsumura, 1975). It has

Streptococcus equi sub sp. zooepidemicus is the widely used

been extensively studied and used since its isolation, first

microorganism for study and commercial production of

from the vitreous humor of the eye in 1934 (Meyer and

hyaluronic acid (Kim et al., 1996; Armstrong, 1997; Liu

Palmer, 1934) mainly due to its high viscoelasticity and

et al., 2008a).The microbial source of hyaluronic acid

water-holding capability.

production is devoid of problems of viral contaminations and

hyaluronic

acid

depends

The biological function of

on

its

length.

Low

proteoglycans complexation and enjoys possibility of

molecularpolymers are used in embryogenesis and cancer

limitless production making it the subject of intensive

treatment, while high molecular weight polymers are used

research over the past few decades (O'Regan et al., 1994;

for space filling and therapeutic applications. The high

Ruffing and Chen, 2006). A thorough comprehensive

Navin and George, 2011

compartmental model describing the hyaluronic acid batch

and fed batch process has been developed (Cooney et al.,

4.29

1999). The paper considered the aerobic and anaerobic

fermentation of Streptococcus zooepidemicus strain on

4.30

glucose and yeast extract as the carbon and nitrogen source

Aerobic Condition

4.31
4.5
4.32

4.6
4.7

4.33
4.8

4.34

4.9

4.35

4.10
4.36

Anaerobic Condition
4.1

4.37

4.2
4.3

4.38

4.4
Aerobic batch culture

4.39
4.23

4.40
Fitted Parameters

4.24

4.25

4.26

4.27

4.28

Navin and George, 2011

Initial Conditions

Initial Condition
Aerobic Batch Studies
Providing aeration to the batch cultures has been a useful
tool to obtain higher energy yields as also tilt the production
profile from homolactic to mixed acid fermentation from the
paper by Cooney et al. (1999). Literature is available on the
useful role played by aeration in enhancing the concentration
Anaerobic batch culture

as well as molecular weight of hyaluronic acid. One theory

4.11
4.12
4.13

proposed for this increase is the shielding effect of the

polymer from the reactive oxygen species formed in the
aerated culture broth (Cooney et al., 1999).
Studies in the lab have found that increase in aeration
increases the ATP levels via Acetate formation, mainly by

4.14
4.15
4.16

the activation of NADH oxidases. The energy affect of the

process has not been incorporated in the model equations.
The non-incorporation of the energy factor is critical and due
to this absence we may have a skewed result. This could
mainly be due to the resource allocation towards another

4.17
4.18

product acetate that indicates a lower yield in all the

variables. The overall comparison of the trend is very
insightful on the role played by various factors under aerated

4.19

conditions. Table 1 shows the comparison of the yield

coefficients

4.20
4.21

obtained

aerobic

and

anaerobic

cultivations.
Table 1. Production rates of HA for anaerobic and aerobic
conditions with their maximum titer

4.22
Fitted Parameter

under

Aerobic

Max
titer

Anaerobic

Max
titer

Y(HA/XT)(gHA/gX)

0.12

1.86

0.68

1.76

Y(HA/S) (gHA/gS)

0.01

2.15

0.05

2.55

Y(HA/SN)(gHA/gSN)

0.63

1.35

0.10

1.35

Y(XT/S) (gXT/gS)

0.15

19.9

0.07

19.9

Y(XT/SN) (gHA/gX)

5.20

15.12

0.14

17.9

Navin and George, 2011

The overall production of hyaluronic acid and biomass

increase in the rate of formation of Hyaluronic Acid when

formation maybe affected severely by the effect of H2O2 and

compared with the initial control condition. This has been

other reactive oxygen species. The critical levels of these

enhanced by the conversion of glucose and nitrogen to the

species would play an important role in aerated batch

formation of hyaluronic acid. The pathway to the formation

studies. Figures 1 to 3 are the simulated results of the aerated

of lactic acid which has to take place is reduced much when

batch studies. Under the levels of H2O2 mentioned there is

compared to the initial condition. The reduction in the

no serious decrease in the biomass or hyaluronic acid

formation of lactic acid leads to the formation of hyaluronic

formation. Similar to the anaerobic batch studies, the

acid in a positive manner.

changes in the variables like the rates of the reactions and

Table 2. Changes in the K values for all rates in aerobic

the Carbon to Nitrogen ratios have been simulated. The

condition

changes have been developed within experimentally

Unmodified

manipulative conditions.

value

10%

100%

10%

50%

increase increase decrease decrease

Fig. 1. Simulated result of aerobic batch fermentation

k1

9.7

10.67

19.4

8.73

4.85

describing hydrogen peroxide and biomass kinetics

k2

1.9

2.09

3.8

1.71

0.95

k3

0.21

0.23

0.42

0.18

0.10

k4

1.15

1.26

2.3

1.03

0.57

k5

0.05

0.05

0.10

0.04

0.02

Aerobic Batch Growth

4

Units: k1 =
0.05

Biomass

Hydrogen Peroxide

0.1

gXa-1 h-1

gGL gXa-1 h-1

k4 =

gXa gXa-1 h-1

k2=
k5 =

gGL gXa-1

-1

k3 = gN

gN gXa-1 h-1

Change in k2
The reaction rate r2 details the underlying process

Hydrogen Peroxide

in the formation of acetic acid and carbon dioxide from the

Biomass
0

glucose. A large increase in the k2 value led to a dramatic

0
9

Time h

decrease in the hyaluronic acid yield per cell, indicating that

Changes in the rate constants

there is a lowering of hyaluronic acid yield when the glucose

As described earlier for the anaerobic conditions, changing

is being used up for acetate formation. When k2 is increased

the rates of the reactions can only be achieved by varying the

by 10%, we can see an increase in the rate of formation of

levels of their respective rate constants. Five reaction rates,

lactate but this increase is decreased with further increase in

r1, r2, r3,r4 and r5 are described in the model and varying

the k2 values. Small to large decrease in the reaction rates

their rates can only be done by varying the levels of their

led to an increase of about 450% of YX/S. Decrease in the

rate constants, k1, k2, k3,k4 and k5 respectively (details

formation of acetate dramatically improves the biomass

provided in Materials and methods). Table 2 provides the

yield per substrate.

change in the rate constants calculated by varying them by a

Change in k3

10% and 100% increase and also by a 10% and 100%

Varying the values of k3 implies that the rate of

decrease.

conversion of substrates to form the active biomass (X A).

Change in k1

When we increased the value of k3 by 10%, we observed a

The reaction rate r1 denotes the conversion of the carbon

huge decrease in YHA/X by about 90%. A decrease in the rate

source towards lactate formation. The mechanism of action

by 10% obtained an increase of 20% which decreased on

seems to be similar to that observed under anaerobic

further decrease of k3 value. Like in the case of anaerobic

cultivation. When k1 is decreased by 50%, we check an

batch growth the interaction is complex and not simple to

Navin and George, 2011

dissect. A 450 % increase in the total biomass yield (YX/S)

The effect on YHA/S is hardly increased in any of the

was obtained by even a 10% increase in the reaction rate.

simulations. Altering the values seems to more dramatically

Change in k4

alter the YX/S and YX/SN values. The results are in much

Varying the values of k3 implies that the rate of conversion

contradiction to that obtained under anaerobic batch culture.

of active biomass (XA) to inactive biomass (XG) has been

However a slight increase in hyaluronic acid yield per

manipulated. As seen in the case of anaerobic culture,

biomass of about 20% was obtained under conditions of

manipulation of this enzymatic step does not dramatically

depleted nitrogen or when SN was halved. Here again, like in

improve the yield of hyaluronic acid (YHA/X). When we

anaerobic culture, maintaining nitrogen under limiting

increase the value of k4 to 10%, we can see an increase in

conditions may induce higher concentration of hyaluronic

the value of hyaluronic acid production rate to the biomass

acid per cell.

production rate. The interesting fact in this process is that the

Fig. 3. Simulated result of anaerobic batch fermentation

YHA/X changes stays greater than the YLA/X on a 10 %

describing glucose and lactic acid kinetics

increase in the k4 value.

Anaerobic Batch Growth

20

Fig. 2. Simulated result of anaerobic batch fermentation

describing nitrogen, biomass and hyaluronic acid kinetics

15

Glucose
Lactic Acid

Anaerobic Batch Growth

concentration g/l

2.5

10

concentration g/l

0
1.5

-5

Total Biomass
Hyaluronic Acid
Nitrogen

0.5

5
Time h

5
Time h

10

Anaerobic batch studies

10

Change in k5
The changes in k5 value affect the formation of hyaluronic
acid. As expected, an increase or decrease in the k5 values
corresponds to a similar increase or decrease in the
hyaluronic acid production as evident by the values of
YHA/X, YHA/S and YHA/SN. Interestingly the YLA/X and YLA/S
remained unaltered indicating some kind of decoupling of
the two processes.
Changing the C/N ratio
Altering the C/N values offers another method of changing
the input parameters. The partitioning of the carbon and
nitrogen source in to the various products affects the product
yields and can bring about large variation in the bioprocess
operation. Table 8 summarizes the percentage changes
obtained on varying the S/SN values.

The principle difference between anaerobic and aerobic

batch studies other than the influence of dissolved oxygen is
the absence of H2O2 and acetate production. H2O2 is known
to be toxic to biomass formation and this may have a
limiting effect on this catalase negative strain. The formation
of acetate theoretically leads to higher ATP yields when
compared to homolactic fermentation producing only lactic
acid (Chong et al., 2005). Anaerobic batch is an example of
homolactic

fermentation

and

aeration

switches

the

fermentation profile into a mixed acid form which is taken

from the paper by Cooney et al. (1999). Figures 2 and 3
provide a view of the simulated result for anaerobic batch
culture. As expected we see a fall in the specific growth rate
value with the drop in the glucose levels. From the simulated
result it is clear that the limiting nutrient in this batch culture
has been Glucose. The concomitant drop in the specific

Navin and George, 2011

growth rate leads to a flattening of the biomass, Hyaluronic

the underlying process. The following are the results

Acid and Lactic acid (LA) concentration as is the

obtained by varying the input parameters within reasonable

characteristic profile of a batch reactor run (ref for Batch

experimentally feasible levels.

reactor profile).

Changes in the rate constants

As explained earlier, comparison of changes induced by

The reaction rates are characteristic of the cellular metabolic

varying the input parameters is crucial and investigative of

state and are tightly regulated for its optimal performance.

Table 8. Changes in the initial condition of glucose and nitrogen ratios in aerobic conditions
2S & 1SN
Y(HA/XT)
(gHA/gX)
Y(HA/S) (gHA/gS)
Y(HA/SN)
(gHA/gSN)
Y(XT/S) (gXT/gS)
Y(XT/SN)
(gHA/gX)
Y(LA/XT)
(gLA/gXT)
Y(LA/S) (gLA/gS)

1/2S & 1SN

1S & 2SN

1S & 1/2SN

2S & 2SN

1/2S & 1/2SN

-67.30

-13.47

-91.84

25.43

-93.65

22.39

-16.68

0.64

-31.55

-50.59

-65.35

-5.13

66.65

-49.71

-65.79

-1.20

-65.40

-5.18

154.85

16.25

738.04

-60.57

445.15

-22.53

409.51

-41.87

318.91

-21.23

445.02

-22.52

-62.40

-13.43

-92.38

43.88

-83.69

40.34

-4.22

0.63

-36.21

-43.34

-11.10

8.72

Recent developments in gene manipulation, enzyme

processes which depend on the availability of glucose to a

engineering as well as addition of inhibitors or activators

large extent. Only when k1 is decreased by 50% there was a

have made it possible to vary the rates of the intracellular

significant increase in the YHA/S value. Lactate formation

processes. Four reaction rates, r1, r2, r3 and r4 are described

decreased and thus the available glucose was more readily

in the model and varying their rates can only be done by

available for consumption by hyaluronic acid process and

varying the levels of their rate constants, k1, k2, k3 and k4

biomass formation. A small increase in YHA/X, YHA/S and

respectively. The change in the rate constants calculated by

YHA/SN over the yield coefficients involving biomass is

varying them by a 10% and 100% increase and also by a

indicative of the slightly higher diversion of glucose towards

10% and 100% decrease.

hyaluronic acid synthesis than biomass formation. All other

Table 9. Changes in the K values for all rates in anaerobic

increases did not produce a significantly higher yield.

condition

Change in k2

Unmodified 10%
100%
value
increase increase
11
12.1
22
k1

10%
decrease
9.9

50%
decrease
5.5

k2

0.21

0.23

0.42

0.18

0.10

k3

1.15

1.26

2.3

1.03

0.57

k4

0.04

0.05

0.09

0.04

0.02

Units: k1 = gGL gXa-1 h-1

gXa h-1

k2 = gN gXa-1 h-1

Increase in k2 is an increase in the rate of formation of the

independent active component (XA), (rA). But its decrease by
10% slightly enhanced the yield coefficient YHA/S and
YHA/SN and its further decrease decreased the same yield
coefficients by about 80%. This covertly implies that there
must be a tradeoff between the improving production of
hyaluronic acid and enhancing cell growth. The YHA/X,

k3 = gXa

k4 = gN gXa-1 h-1

Change in k1
Varying in k1 influences the change in the rate of glucose
consumption and thus may have an impact on the other

however increased by about 20% indicating that there is a

higher portioning of substrate towards hyaluronic acid
formation. Influencing the increase of r2 may not be very
practicable for an objective of enhancing hyaluronic acid
production.

Navin and George, 2011

Change in k3

achieved by maintain lower carbon levels and much higher

The rate r3 indicates the conversion of the active biomass

nitrogen levels. Hence, for better hyaluronic acid production

(XA) towards inactive or structural biomass component (XG).

per cell, maintaining higher carbon source levels was found

In general there was not a significant change in YHA/S but

to be the best strategy.

there was a profound change observed when k3 was raised

Table 10. Changes in the initial condition of glucose and

by 100%. The increase led to a dramatic drop in the yield

nitrogen ratios in anaerobic conditions

coefficients of all parameters. Although this increase did

2S 1/2S
and
and
1SN 1SN

1S 1S
2S
1/2S
and
and
and
and
2SN 1/2SN 2 SN 1/2SN
% Change

0.64

0.42

-1.07

0.99

-3.51

-3.50 2.71

7.68

-14.46

6.10

-9.49

93.01 -48.61

71.14
46.18

6.11

-9.51

-4.13 4.91

7.17

5.07

-6.24

91.77 -47.53

72.97
46.40

5.07

-6.23

lead to a decrease of YHA/X of 30% indicating that less

hyaluronic acid were produced per cell, the decrease was
observed for YX/S and YX/SN. Decreasing the rate in fact
improved the overall yield YX/S, providing evidence that the
structural component did not contribute much to the biomass
formation. All this seems to indicate that there is a complex
phenomena involved in this partitioning of the substrate
towards the formation of the structural component of the cell
which affects the overall process.

Y(HA/XT)
(gHA/gX)
Y(HA/S)
(gHA/gS)
Y(HA/SN)
(gHA/gSN)
Y(XT/S)
(gXT/gS)
Y(XT/SN)
(gHA/gX)

-2.06

-13.56

Change in k4
The rate r4 directly influences the rate of

Conclusions

hyaluronic acid production from both glucose and nitrogen

Firstly, on comparison between anaerobic and aerobic

sources. Increase in the rates does increase the amount of

batch cultivation, a slightly higher concentration of biomass

hyaluronic acid produced per cell and its decrease indicates

and hyaluronic acid was produced under aerobic mode

the reverse process happening. However the impact of this

indicating the superiority of the aerobic fermentation

diversion of substrates towards hyaluronic acid formation

process. This is inspite of the limitation of absence of

seems to be quite less on YX/S and YX/SN. This again provides

model equations describing the energy yields within the

inference that substrate diversion towards biomass formation

system. Maintaining lower levels of H2O2 was found to be

may be less coupled with that of the hyaluronic acid process.

a good strategy in aerobic batch cultivations. Secondly,

Improving the Hyaluronic Acid conversion rate is thus

manipulating of reaction rates, by varying the respective

another route for providing a huge increase in the hyaluronic

rate constants is another way to predict conditions that can

acid yield.

improve productivity. All changes were done within

Changing the C/N Ratios

experimentally feasible limits. In both aerobic and

In these anaerobic conditions, the ratio of Carbon

anaerobic batch cultures, reduction of rates for reactions

to nitrogen could play a role in partitioning the resources

synthesizing by products like lactate or acetate, has the

towards biomass or hyaluronic acid formation. It was thus

largest effect on YHA/X and YHA/S. Another possible

thought that varying the ratios of S/SN may have an impact

manipulation of the reaction rate can be done on the

on the hyaluronic acid production (Table 10). Although the

enzymes synthesizing the polymer. However, both the steps

interaction was complex and not easily explainable, the

require a more complete understanding of the enzymes

striking aspect of the changes were that whenever the

involved in the pathway and a precise targeting of the rate

nitrogen source (SN) was kept low or limiting and carbon

limiting step to enhance the overall reaction rates.

source (S) higher, the YHA/SN and YX/SN was substantially

higher. Also for getting better Y(HA/X) albeit very slightly,
this was the best strategy. Improving the YHA/S could only be

Thirdly, in batch studies, variation of C/N ratios is done to

try understanding and improving the process. Observing the

Navin and George, 2011

simulated results indicated that maintaining lower levels of

production. Appl Microbiol Biotechnol 66: 341-

nitrogen when compared to the carbon source improved the

351.

YHA/X although the overall yield of Hyaluronic Acid may

Blank LM, McLaughin RL, Nielsen LK (2004). Stable

be lower. This is an important find, as it indicates that more

production of hyaluronic acid in Streptococcus

amount of hyaluronic acid is produced per cell and this may

zooepidemicus chemostats operated at high dilution

improve the molecular weight of the polymer Cooney et.al.

rate. Biotechnol Bioeng 90: 685 - 693.

(1999).

Cho SP, Kim HG, Kim JG, Kim TH, Park SY (2004).
Extraction method for high purity hyaluronic acid

Acknowledgements

from rooster comb. WO Pat: KR20040022760.

Authors are grateful to Prof.K.B.Ramachandran, Department

Chong BF, Blank LM, McLaughin R, Nielsen LK, (2005),

of Biotechnology, Indian Institute of Technology Madras,

Microbial

Chennai, India for providing laboratory facilities. Authors

Microbiol Biotechnol 66: 341 351.

are also thankful to Mr.Jagannath, Houston, USA.

acid

production.

Appl

Cooney MJ, Goh LT, Lee PL, Johns MR (1999). Structured

model based analysis and control of the hyaluronic

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