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