Biochemical Engineering Journal 34 (2007) 185–192

Alkaline protease production by submerged fermentation in stirred

tank reactor using Bacillus licheniformis NCIM-2042:
Effect of aeration and agitation regimes
Ravichandra Potumarthi, Subhakar Ch., Annapurna Jetty ∗
Bioengineering and Environmental Centre, Indian Institute of Chemical Technology (CSIR), Tarnaka, Hyderabad 500007, India
Received 1 August 2006; received in revised form 2 November 2006; accepted 4 December 2006

Abstract
The effects of aeration and agitation on alkaline protease production and protease yield were studied by submerged fermentation in a batch STR
using Bacillus licheniformis NCIM-2042. The agitation rate varied in the range of 200–400 rpm at each airflow rates of 1–3 vvm. The maximum
protease production was found on third day (72 h) and then decreased during fourth and fifth day of operation in all batches of STR operation.
The best combination of airflow and agitation rate for the present system was at 3 vvm of airflow rate and 200 rpm of agitation rate on the basis
of maximum specific protease production rate. Maximum specific protease production of 102 U/mg DC was observed on third day at 3 vvm and
200 rpm. The effects of casein concentrations on protease production, viscosity changes and oxygen transfer rate were studied. The increase in
casein concentration has resulted in increased viscosity of the fermentation broth with increase in time of bioreactor operation, which in turn
resulted in decreased oxygen mass transfer coefficient with reduced oxygen transfer rates.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Stirred tank reactor; Protease production; Yield; Viscosity; Volumetric oxygen mass transfer coefficient; Limiting substrate

1. Introduction amount of extracellular protease have been exploited commer-

Alkaline proteases are one of the most widely studied group
of enzymes because of their wide use in many industrial appli-
cations such as food, pharmaceutical and leather [1] and with
two-third of share in detergent industry alone [2]. Microbial
proteases are gaining more importance than conventional chem-
icals that cleave peptides because of the cheaper production cost
and use of renewable resources. Microbial proteases can be pro-
duced from bacteria, fungi and yeast using many processes like
solid-state fermentation, submerged fermentation [1–3]. Bacte-
ria of the genus Bacillus are active producers of extracellular
alkaline proteases. Currently, large portions of commercially
available alkaline proteases are derived from Bacillus strains
[1,3–5]. Although protease production is an inherent property
of all organisms, only those microbes that produce a substantial
Abbreviations: STR, stirred tank reactor; rpm, revolution per minute; vvm,
volume of air per volume of media per minute; DO, dissolved oxygen; OTR,
oxygen transfer rate; DCW, dry cell weight
∗ Corresponding author. Tel.: +91 40 27160123x2663; fax: +91 40 27193159.
E-mail address: annapurna@iictnet.org (A. Jetty).
cially [6].
It is well known that extracellular protease production
by microorganisms in bioreactors is greatly influenced by
media components, physical factors such as, aeration, agi-
tation, temperature, inoculum density, dissolved oxygen and
incubation time [5,7]. Industrial fermentation is moving away
from traditional and largely empirical operation towards
knowledge based and better-controlled process. The rational
design and optimization of the latter requires the understanding
of production kinetics. Although, there have been a number
of studies on protease production by Bacillus species, little
information on kinetic analysis of the protease production
process is available in literature [8–11]. Mixing is important
in the microbial synthesis of protease enzyme in free cell
bioreactor and can be imparted by means of aeration and
agitation. It is also largely dependent on higher oxygen mass
transfer and lesser shear forces on microorganisms. For aerobic
fermentation, oxygen transfer is a key variable and is a function
of aeration and agitation. Therefore, it is necessary to establish
optimum combination of airflow and agitation for maximum
yield. The aim of present work was to analyze the effects of

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2006.12.003
186 R. Potumarthi et al. / Biochemical Engineering Journal 34 (2007) 185–192
Nomenclature
C* saturated oxygen concentration in the media
(mg/l)
CL concentration of dissolved oxygen at any given
time (mg/l)
kLa volumetric liquid side oxygen mass transfer coef-
ficient (h−1 )
Pg Power requirement for gassed STR (W)
Yp/s yield of protease with respect to substrate (U/g)
Yp/x specific product formation (SPF) of protease with
respect to biomass (U/mg DC)
Yx/s yield of biomass with respect to substrate (mg/g)
airflow rate and agitation rate on protease production in a lab
scale bioreactor to identify the optimum combination of airflow
rate in order to process and to identify optimum combination of
airflow and agitation parameters that control protease produc-
tion. Further experiments were conducted to study the effect of
levels of limiting substrate concentration on maximum protease
production and effects of viscosity on volumetric oxygen mass
transfer coefficient. Oxygen transfer rate in the aerobic fermen-
tation process is also dependent on the broth rheology most
importantly variations in the viscosity with time of bioreactor
operation.
2. Materials and methods
2.1. Microorganism and seed culture
The protease producing Bacillus licheniformis NCIM-2042
was procured from NCL, Pune, India. The microorganism was
grown on nutrient agar slants at 35 + 2 ◦ C and pH 7.5; subcul-
tured using medium having composition (g/l) peptone, 5; beef
extract, 3; yeast extract, 2; NaCl, 5; agar, 15; pH 7.2. Cul-
ture growing on solid media was transferred into liquid broth
having composition as defined above except agar. Submerged
cultivation of B. licheniformis NCIM-2042 was carried out in
250 ml Erlenmeyer flasks with 100 ml of production medium
having composition (g/l) casein, 10; malt extract, 10; polypep-
tone, 10; Na2 CO3 , 10; pH, 9.5. The flasks were incubated for
120 h at 35 + 2 ◦ C and 150 rpm. The culture was centrifuged at
10,000 rpm for 10 min at 4 ◦ C and the supernatant obtained was
tested for protease production (data not shown).
2.2. Bioreactor operation
2.2.1. Effect of different airflow and agitation rates on
protease production
The experiments were carried out in a lab scale 1.5 l biore-
actor (B-Braun Biostat) with 1 l working volume, fixed with
two-stage rushton type impeller of 50 mm diameter. In the initial
phase, STR was operated to optimize airflow rate and agitation
rate for the production of proteases. Three levels of airflow rates:
1, 2 and 3 vvm were studied and at each airflow rate three dif-
ferent agitation rates, such as 200, 300 and 400 were tested by
inoculating the batch STR with 10% inoculum and cultivating
for 5 days at 35 + 2 ◦ C. Estimation of carbohydrates and pro-
tease production was carried out at every 24 h interval. Power
requirement for each batch was calculated [12].
2.2.2. Effect of casein concentration on the viscosity of
fermentation broth
After determining the optimum levels of airflow and agitation
rates with respect to protease production in the first phase, four
experiments E1, E2, E3 and E4 were conducted in batch mode,
at different initial concentrations of casein in the levels of (g/l)
20, 30, 40 and 50, respectively, for the maximum production of
protease in the second phase of STR operation. The 2-day-old
inoculum grown in 250 ml flask was used as seed to the reactor
(10% of the working volume). Each batch was cultivated for
5 days at constant pH of 9.5 and temperature at 35 + 2 ◦ C and
during the operation at every 24 h protease production, carbo-
hydrates, DO, viscosity and volumetric oxygen mass transfer
coefficient were estimated and recorded. Simple Monod growth
kinetic studies were done in batch STR using casein as limiting
substrate.
2.2.3. Determination of volumetric oxygen mass transfer
coefficient (kLa ) and oxygen transfer rate (OTR) in the
bioreactor
2.2.3.1. Determination of kLa by dynamic gassing out method
[12]. This method is simple one, based upon the dynamic oxy-
gen balance in a batch culture, which has the following form.
dCL
= kLa (C∗ − CL ) − QO2 X (1)
dt
where QO2 is the rate of oxygen consumption per unit mass of
cells (mm O2 /g h).
Rearranging Eq. (1) yields.
QO2 X + (dCL /dt)
CL = C∗ − (2)
kLa
The air supply is turned off at a certain time during fermen-
tation and the variation of CL with time is followed with the
aid of a DO probe. Since the term kLa (C* − CL ) becomes zero
when air is turned off, the CL value decreases linearly with time
according to Eq. (1). The slope of the CL versus time curve yields
a value for QO2 X. The air is then turned on and the increase in
DO with time is followed. Having determined the QO2 X value,
CL is plotted against (QO2 X + dCL /dt). The slope of this plot is
equal to the reciprocal of kLa .
2.2.4. Determination of OTR in batch bioreactor operation
[13]
Oxygen transfer rate for stirred tank reactor is given by fol-
lowing Eq. (3)
OTR = kLa (C∗ − CL ) (3)
 R. Potumarthi et al. / Biochemical Engineering Journal 34 (2007) 185–192
2.3. Analytical methods
2.3.1. Protease assay
Protease activity was determined by modified method [14]
using casein as substrate. Fifty microliters of crude protein was
added to 450 ␮l of substrate solution (1%, v/v, casein with
50 mM Tris–HCl buffer; pH 8.0) and incubated at 30 ◦ C for
30 min independently with respective controls. The reaction
was stopped by adding 750 ␮l of 5% TCA mixture (5% TCA,
9% Na–acetate, 9% acetic acid) followed by 30 min holding at
room temperature followed by centrifugation at 10,000 rpm for
15 min. The absorbance of supernatant was measured at 280 nm.
One unit of enzyme activity was defined as the amount of enzyme
which releases 1 ␮mol of tyrosine per minute under the assay
conditions. The amount of tyrosine was determined from the
tyrosine standard curve.
2.3.2. Protein assay
The protein content of the enzyme preparation was estimated
by Lowry et al.’s method [15].
2.3.3. Carbohydrate estimation
Total carbohydrate content was determined according to the
phenol–sulfuric acid method [16].
2.3.4. Estimation of biomass
Two milliliter sample was collected in a pre-weighed eppen-
dorf tube and centrifuged at 5000 rpm for 10 min. Supernatant
was discarded and the pellet was washed thrice with sterile dis-
tilled water, followed by drying the pellets at 95 ◦ C till constant
weight and expressed in DCW (mg/ml).
2.3.5. Viscosity of the broth
The viscosity of the broth sample was measured by using a
Cannon–Fenske type viscometer (Fisher Scientific, Pittsburgh,
PA, USA). All the data analyzed and presented was the average
of three estimations.
2.3.6. DO estimation
Polarographic type, METTLER TOLEDO, >98% response
in less than 90 s.
3. Results and discussions
3.1. Optimization of airflow rates and agitation rates
The effect of temperature (in the range of 25–45 ◦ C) on
extracellular protease enzyme production by B. licheniformis
NCIM-2042 in shake flask was studied. It was found that
35 + 2 ◦ C is optimum for the maximum production of protease.
Therefore, all the reactor studies by B. licheniformis NCIM-2042
were carried out at 35 ± 2 ◦ C as constant temperature.
The batch STR was run with 10% inoculum for 5 days
at 35 ± 2 ◦ C and tested the effects of airflow rates and agita-
tion rates on protease production. Three different airflow rates
1–3 vvm were tested and, at each airflow rate, three agitation
187
rates 200, 300 and 400 rpm were studied. Fig. 1 shows the pro-
tease production in STR under different cultivation conditions.
From Fig. 1a–i, it is evident that each batch of the STR has
resulted in different levels of protease production. However, the
trend of production and/or formation of protease and biomass
growth (DCW, g/l) were similar in all batches irrespective of air-
flow and agitation rates. But the variations in concentrations of
protease production indicate the effect of airflow and agitation
rates.
Based on the maximum protease production at 340 U/ml
(Fig. 1), 300 rpm was found to be the optimum agitation speed
with 2 vvm of airflow rate. During the cultivation period, pro-
tease production and biomass growth in batch STR has been
found to be negatively affected by variations in agitation rates
beyond 300 rpm, whereas at an agitation speed less than 300 rpm
and at all airflow rates (1–3 vvm), protease production was in the
range of 223–290 U/ml at the end of 72 h of reactor operation.
The trend of protease production was similar in all batches
of STR operation, showing a decline in protease concentration
after 72 h. Besides this, it is obvious from the given protease
production and biomass growth data, that mixing is crucial for
better oxygen and nutrient transfer rate during entire period of
operation. However, mixing by means of only agitation was
found to inhibit protease production at rpm’s above 300, whereas
at all airflow rates, the protease production was high except
at higher agitation rates. The decrease in protease production
was observed from Fig. 1a–i after 72 h of reactor operation,
even though there was an increase in the biomass concentration.
This phenomenon could be attributed to the protein inactivation
beyond 72 h [17].
Specific product formation of protease (SPF), U/mg DC, were
calculated with respect to dry cell weight (DCW, mg/ml) of
biomass in the reactor for all batches for 72 h of operation and
tabulated in Table 1. A maximum SPF of 102 U/mg DC was
recorded at 3 vvm and 200 rpm during 72 h of bioreactor oper-
ation. Whereas, maximum protease production was observed at
300 rpm and 2 vvm (Fig. 1e). Fig. 2 shows the effect of airflow
and agitation rates on SPF. The trend of SPF was increasing
in the case of 1 and 2 vvm of airflow rates and at all agitation
rates. The reverse trend was observed at 3 vvm of airflow rate.
Maximum SPF of 102 U/mg DC was observed at 200 rpm and
3 vvm. The kLa values were estimated by dynamic gassing out
method for all batches and the data are reported in Table 1.
During the kLa estimation, by dynamic gassing out method, the
typical DO concentrations before the aeration stopped was in the
range of 5–3.5 mg/l and the DO concentrations before reaera-
tion was 2–1.5 mg/l for all experiments. This pattern of DO data
shows that a sufficient difference in DO concentration was avail-
able for kLa estimation by dynamic gassing out method. Also the
DO concentration before the reaeration did not decrease below
the critical DO concentration, which in turn did not affect the
microbial activity before reaeration. The critical DO concen-
tration for the strain used in the present studies was found to
be 1.5 mg/l. The characteristics of DO probe are: polarographic
type, METTLER TOLEDO, >98% response in less than 90 s.
The volumetric oxygen mass transfer coefficient was fol-
lowed an increasing trend with the increase in agitation and air-
188 R. Potumarthi et al. / Biochemical Engineering Journal 34 (2007) 185–192

Fig. 1. Production of protease in STR under different airflow and agitation rates. (a) 1 vvm and 200 rpm; (b) 1 vvm and 300 rpm; (c) 1 vvm and 400 rpm; (d) 2 vvm
and 200 rpm; (e) 2 vvm and 300 rpm; (f) 2 vvm and 400 rpm; (g) 3 vvm and 200 rpm; (h) 3 vvm and 300 rpm; (i) vvm and 400 rpm. () Protease production (U/ml);
() DCW (mg/ml).

Table 1
Protease production formation in batch STR: effects of airflow and agitation rates (at the end of 72 h of bioreactor operation)
Airflow

1 vvm 2 vvm 3 vvm

200 rpma 300 rpma 400 rpma 200 rpma 300 rpma 400 rpma 200 rpma 300 rpma 400 rpma

Yp/x 74 79 80 72 73 87 102 93 89
Pg 0.0224 0.0534 0.0891 0.0226 0.0537 0.0896 0.0229 0.0541 0.0912
kLa 41 54 56 54 61 62 62 64 66
a Agitation.

flow rates in all batches. Therefore, mixing is important for max-

imal production of microbial protease by optimizing the mixing
rates in batch STR operation and can be imparted by means
of both aeration and agitation for better oxygen mass transfer
rate for better product formation [4,18,19]. Again, higher stir-
ring rates may cause shear effects on microbial cells resulting in
reduced biomass concentration. There should be an optimum
balance between the aeration (oxygen tension) and agitation
(shear) to have maximum cell growth and SPF. This could be
achieved by optimizing the combinations of airflow and agitation
rates that are important for maximal SPF. Many researchers have
reported the agitation speeds between the range of 150–300 rpm
Fig. 2. Effect of airflow rate and agitation rate on specific product formation for protease production using similar types of strains
rate. [10,20–22].
 R. Potumarthi et al. / Biochemical Engineering Journal 34 (2007) 185–192
As the impeller speed increases the power requirement for
the gassed STR also increased (Table 1). The power required
at 300 rpm and 2 vvm was 0.0537 W, but it was 0.0229 W for
200 rpm and 3 vvm. At this stage maximum SPF could be the
better data for scaling up the process economically. It also
indicates that the specific product formation of protease with
respect to the biomass concentration is dependent on aeration
and agitation rates. At each combination of airflow and agitation
rates in STR, production of protease was different; therefore
these parameters could act as metabolic regulating parameters
in the bioreactor for the maximum SPF. The calculation of SPF
yields will help in determining the optimum combination of air-
flow and agitation rates. The oxygen transfer rates are dependent
on aeration and agitation rates during the batch STR operation
with free cells of B. licheniformis [19], which in turn results
in maximum protease yields [4,18,23]. Termination of STR
operation during maximum production period, i.e. third day
or 72 h could be economical. The scale-up of aerobic cultures
from flask to lab scale bioreactor and from lab scale bioreactor
to pilot plant, and subsequently to industrial level have been
generally based on the volumetric oxygen transfer coefficient.
From the results it is evident that as the agitation rate (shear rate)
increases the SPF was also increased (Fig. 2 and Table 1) with
the increase in kLa values. Table 1 also shows that the power
requirement of gassed STR increased with the increase in shear
rate. At 1 vvm of airflow rate, as the agitation rate increased
from 200, 300 and 400 rpm the kLa values also increased to 41,
54 and 56 h−1 along with increase in power requirements of
0.0224, 0.0534 and 0.081 W, respectively. Similar trend was
observed at 2 and 3 vvm of airflow rate at 200, 300 and 400 rpm
of agitation rates. The optimal airflow rate and agitation rate
found on the basis of maximum SPF is 3 vvm and 200 rpm,
respectively. The power requirement at this combination of
airflow and agitation rate was 0.0229 W with the respective kLa
of 62 h−1 .
For large-scale systems, power consumption during aeration
and agitation is a significant fraction of the total operating
cost. To increase agitation rate, the power of the impeller
motor must be increased [24]. The increase in agitation rate
produces higher shear stress in the broth, which may cause
a decrease in the growth of shear-sensitive microorganisms.
Most of the industrial fermentation processes for proteases use
bacterial species, being sensitive for shear forces; the bacterial
culture need lesser mechanical agitation and at the same
time requires proper oxygen mass transfer. The aeration and
agitation altogether are used to enhance oxygen transfer from
gas (air) to liquid medium for the optimal cell mass production
or product formation [25]. This could be done, by optimizing
the airflow and agitation rates simultaneously rather optimizing
individually.
Current literature indicates that low agitation can cause a
drastic reduction in the protease production by Bacillus sp.
[4,10,17–21] This could be due to lower availability of dis-
solved oxygen with low mixing rates. As dissolved oxygen is the
rate-limiting factor because of its low solubility in the aqueous
solutions, it affects the cell growth and yield of products in the
aerobic fermentation [4,18].
189
Table 2
Protease production kinetics: effects of substrate concentration (at the end of
72nd of bioreactor operation)
Experiment Yx/s (mg/g) Yp/s (U/g) Yp/x (U/mg DC) OTR (mg/l h)
E1 0.375 44 119 59.8
E2 0.188 15 84 42.42
E3 0.323 15 46 38.4
E4 0.2953 12 40 34.32
3.2. Effect of limiting substrate concentration on protease
production
From the results of earlier shake flask studies (unpublished) it
has been observed that casein was acting as limiting nutrient for
the protease production and growth by B. licheniformis. There-
fore, the experiments were conducted in batch STR at different
casein concentrations for protease production using previously
optimized agitation and airflow rates based on maximum SPF,
i.e. 200 rpm and 3 vvm.
The initial concentrations of casein used in the production
medium of E1, E2, E3 and E4 were 20, 30, 40 and 50 g/l,
respectively. The initial concentrations of carbohydrates in the
fermentation broth for E1, E2, E3 and E4 was estimated at
0 h and found to be 25.2, 33.6, 36.2 and 52.4 g/l, respectively.
Further, during the fermentation carbohydrates were estimated
at every 24 h interval for entire period of fermentation pro-
cess and the same was represented in Fig. 3. The variations
in DCW, carbohydrate utilization and protease production dur-
ing the course of fermentation is shown in Fig. 3. It is evident
from Fig. 3 that during batch STR operation (E1–E4), the trend
of carbohydrate utilization, biomass formation (DCW) and pro-
tease production was similar with maximum protease production
during 72 h.
As the initial carbohydrate concentration in the STR
increases, the protease production rate decreases. Maximum
production (E3) of 320 U/ml, was observed at 36.2 g/l of carbo-
hydrate concentration and a further increase in the carbohydrate
concentration of 52.2 g/l inhibited the product formation rate
by recording a low protease production of 180 U/ml on third
day. Higher concentration of casein (E4) also inhibited the
growth and biomass yields with a low DCW of 5.6 mg/ml on
third day, which was very less when compared with E1, where
a maximum DCW was observed at 8.2 g/l on third day. Higher
concentrations of casein resulted in inhibition of cell growth,
which in turn affected the protease production. The effects of
casein concentration on protease production in batch STR could
be best studied by calculating the specific product formation of
protease with respect to DCW, and the yields of biomass and
protease with respect to carbohydrates concentration for the 72 h
of operation. The results for all batches (E1–E4) were tabulated
in Table 2. From Table 2 it is evident that maximum SPF was
119 U/mg DC (E1) during 72 h of bioreactor operation. But, in
the case of E3 and E4, the SPF was drastically reduced to 46 and
40 U/mg DC, respectively, during the 72 h of STR operation. By
observing the trend of SPF, it was evident that the increase in
concentration of casein had shown significant effect on protease
190 R. Potumarthi et al. / Biochemical Engineering Journal 34 (2007) 185–192
Fig. 3. Production of protease in STR at different initial carbohydrate con-
centrations. (E1) 25.2 g/l carbohydrate; (E2) 33 g/l carbohydrate; (E3) 36.2 g/l
carbohydrate; (E4) 52.2 g/l carbohydrate. () Carbohydrate concentration (g/l);
(䊉) DCW (mg/ml); () protease production (U/ml).
yields beyond 20 g/l. Similarly the effects of limiting substrate
concentrations on yield of protease were calculated (Table 2).
Maximum protease yields recorded was 44 U/g substrate (E1)
and the lowest was 12 U/g substrate (E4) during 72 h of STR
operation. Further, yield of biomass with respect to the limiting
substrate concentration was calculated for third day of operation
(Table 2). It can be inferred from the results that, the concentra-
tion of limiting substrate had significant effect on the protease
production and biomass growth and the substrate was clearly
acted as growth limiting factor as reported by previous reports
[26,27].
Fig. 4. Initial saturation oxygen concentration (mg/l) and viscosity (kg/m s).
3.2.1. Effect of viscosity of fermentation broth on OTR
Mass transfer from the gas to the liquid phase has been iden-
tified as an important rate limiting process and is dependent
on viscosity of the fermentation broth. The culture viscosity
was found to be changing with increase in time of STR opera-
tional period throughout the cultivation indicating that the initial
concentration of casein had significant effect on the culture vis-
cosity and the fermentation broth followed non-Newtonian fluid
rheology. Fig. 4 shows the initial viscosity of the fermentation
broth and saturated dissolved oxygen concentration at 0 h with
increased levels of initial casein concentrations of 20, 30, 40
and 50 g/l. The effect of initial viscosity on the kLa was small
during 24 h of bioreactor operation, but as the time of bioreactor
operation increased the viscosity also increased in all the four
batches, which in turn affected the kLa of the system. The initial
concentration of casein also affected the viscosity of fermen-
tation broth as shown in Fig. 5. From Fig. 5 it is evident that
the effect of initial casein concentration is significant on spe-
cific product formation rate and oxygen transfer rate. At 20 g/l
of casein concentration the maximum SPF of 119 U/mg DC was
reordered and further it was decreased with the increase in casein
concentration. When the initial concentration of casein varied in
the levels of 20, 30, 40 and 50 g/l, the viscosity varied between
0.018, 0.021, 0.027 and 0.031 kg/m s, respectively, at the end of
24 h of operation. At the end of 72 h of operation the viscosity
of the batches, E1, E2, E3 and E4 was 0.031, 0.038, 0.043 and
Fig. 5. Effect of initial casein concentration on specific product formation and
oxygen transfer rate.
 R. Potumarthi et al. / Biochemical Engineering Journal 34 (2007) 185–192
Fig. 6. Variation in volumetric oxygen mass transfer coefficient and viscos-
ity with time during the operation of bioreactor. () kLa (h−1 ); () viscosity
(kg/m s).
0.056 kg/m s, respectively. As the viscosity of the fermentation
broth increased with the time of bioreactor operation, the volu-
metric oxygen mass transfer coefficient was decreased (Fig. 6).
The kLa values for E1, E2, E3 and E4 on 24 h were 62, 59, 52
and 49 h−1 and the values on 72 h was 46, 42, 32 and 26 h−1 ,
respectively. The OTR at the end of 72 h of bioreactor operation
was 59.8, 42.42, 38.4 and 34.32 mg/l h for E1, E2, E3 and E4,
respectively. As seen from the results, though the initial casein
concentration was affected, the OTR during all the batch opera-
tions (E1–E4), the DO concentration was not reduced to below
the critical oxygen concentration during fermentation.
This indicated that the initial concentration of casein sig-
nificantly affected the viscosity of the fermentation broth
throughout the experimentation which in turn affected the volu-
metric oxygen mass transfer coefficient, ultimately resulting in
lower protease production rates. As reported, for protease pro-
duction by B. licheniformis, oxygen transfer regulates protease
191
production and byproduct formations during the fermentation
and the change in the oxygen transfer rate influenced the fluxes
of the main pathways and consequently changed the product and
byproduct formation profiles throughout the fermentation pro-
cess [18,28]. Since, casein acts as carbon and nitrogen source for
the metabolism of Bacillus sp. [29,30], higher concentrations of
casein may lead to repression in extracellular protease produc-
tion and changes in metabolic functions. The strains of Bacillus
in particular B. licheniformis and Bacillus subtilis are more prone
to PGA synthesis during the fermentation with small variations
in environmental conditions [28]. The metabolic pathway of the
Bacillus sp. is more complex and the ability of bacilli to excrete
other enzymes, amino acids and organic acids as byproducts is
dependent on the micro environmental conditions around them
[18]. The reduction in the kLa values was probably due to the
reduction in the surface area of the bubbles caused by the vis-
cous forces generated in the fermentation broth by the formation
of PGA. With an increase in the viscosity, the resistances to the
mass transfer increases.
The change in viscosity of the system is because, during the
production of proteases by Bacillus sp. in aerobic fermentation,
formation of a water-soluble, viscous slime material containing
d-glutamic acid and l-glutamic acid residues is common [28].
This polyglutamic acid (PGA) is polymerized via amide link-
ages between the ␣-amino and ␥-carboxylic groups of the amino
acid residues accumulation of polyglutamic acid. However, the
synthesis of even small amounts of PGA can be a problem in the
fermentation industry, most notably in the production of extra-
cellular enzymes from bacilli, where PGA accumulation causes
increased viscosity of the fermentation broth, reduced enzyme
yield, uncontrollable foaming, and complications in product
recovery [31]. In the metabolism of aerobic B. licheniformis
during lag phase it tries to adjust to the new environmental con-
ditions in the bioreactor and the molecular oxygen concentration
acts as required environmental condition and in the log phase
of growth, molecular oxygen concentration (i.e. O2 ) is critical
for the multiplication of cells. During the production phase, that
is after 24 h of fermentation for extracellular enzyme produc-
tion by Bacillus sp. accumulation of by products like PGA [28]
results in variations of rheological properties of the fermenta-
tion broth. Hence, the viscosity effects are more significant in
the production phase than in the initial 24 h of bioreactor oper-
ation. Therefore, it could be concluded that the initial viscosity
of the fermentation broth had less effects in the initial hours of
operation.
The effect of non-Newtonian fermentation broth viscosity on
mass transfer of oxygen to the broth from bulk gas phase and then
to culture has been gaining attention in bioprocess engineering
studies and it is an important factor at industrial level production.
4. Conclusion
Mixing is very crucial for the maximum productivity in
microbial fermentations and it could be achieved by means of
aeration and agitation. But agitation at higher stirring speeds
may cause disruption of free cells in the reactor by shear forces
and formation of vortex which may result in poor mass transfer
192 R. Potumarthi et al. / Biochemical Engineering Journal 34 (2007) 185–192
(oxygen/substrate). Therefore, it is important to provide opti-
mum combination of aeration and agitation in free cell batch
bioreactor operation. From the results of present investigation,
it could be concluded that agitation and aeration have a sig-
nificant effect on protease production. The maximum SPF of
102.04 U/mg DC was observed at 200 rpm and 3 vvm of agita-
tion and airflow rates, respectively. Further the concentration of
casein, which is a limiting substrate for B. licheniformis in the
production medium, when the initial concentration varied in the
levels of 20, 30, 40 and 50 g/l has shown a considerable effect
on protease production. At 20 g/l of initial casein concentration,
the SPF of 119 U/mg DC was observed. The increase in casein
concentration has resulted in increase in viscosity of the fer-
mentation broth as the time of bioreactor operation increased.
With the variation of initial casein concentration on the levels
of 20, 30, 40 and 50 g/l, the viscosity of the fermentation broth
was increased to 0.031, 0.038, 0.043 and 0.056 kg/m s, respec-
tively, at the end of 72 h of bioreactor operation. The increased
viscosity affected the oxygen transfer rate in bioreactor. The
OTR variation was 59.8, 42.42, 38.4 and 34.32 mg/l h when the
levels of initial casein concentration varied at 20, 30, 40 and
50 g/l, respectively, at the end of 72 h of bioreactor operation.
As the concentration of limiting substrate increased, the protease
production was found to decrease.
Acknowledgement
Authors are thankful to Dr. J.S. Yadav, Director, Indian
Institute of Chemical Technology, Hyderabad, India, for his
encouragement.
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