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Materials Today: Proceedings 3 (2016) 34443449 www.materialstoday.com/proceedings

International Conference on Advances in Bioprocess Engineering and Technology 2016

(ICABET 2016)

Studies on the effect of agitation and aeration for the improved

protease production by Bacillus licheniformis NCIM-2042
Apurba Dey*a, Biswanath Bhuniab and Subhasish Duttaa
*a
Department of Biotechnology, National Institute of Technology, Durgapur -713209, India
b
Department of Bio Engineering, National Institute of Technology, Agartala, -799046, India

Abstract

The present research work deals with the effect of agitation and aeration for protease production using Bacillus licheniformis
NCIM-2042. The experiments were carried out in a lab scale 2.2 l bioreactor with 2 l working volume at 37 oC for 84 hour.
Determination of kLa was estimated by dynamic gassing out method. The value of k La was found to be 41, 54 and 56 1/h at 1, 2
and 3 vvm of airflow rate respectively. The maximum protease production was found to be 382.46 U at optimum flow rate of 2
vvm and agitation speed of 180 rpm.

2015 Elsevier Ltd. All rights reserved.

Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in
Bioprocess Engineering and Technology 2016.

Keywords:Protease; agitation; aeration

* Corresponding author

Tel No: 0343-2546-397, Fax No: 0343-2547375, Email: apurbadey.bt@gmail.com

2214-7853 2015 Elsevier Ltd. All rights reserved.

Selection and Peer-review under responsibility of the Committee Members of International Conference on Advances in Bioprocess Engineering
and Technology 2016.
 A..Dey et al. / Materials Today: Proceedings 3 (2016) 34443449 3445

1. Introduction

Proteases are obtained through a wide variety of sources such as plants, animals, and microorganisms. Alkaline
protease is one of the most important groups of industrial enzymes, being extensively used in leather, food,
pharmaceutical, textile, organic chemical synthesis, waste water treatment and other industries [1, 2]. They hold a
major share of the enzyme market with two-third share alone in detergent industry [3-5]. Although there are many
microbial sources available for producing proteases, only few are recognized as commercial producers. Of these,
genus of Bacillus sp. are considered an important commercial protease producer [6, 7]. Large scale production of
protease can only fulfil the demand and usefulness of the proteases in the industry. In industry, microbial enzyme
production was carried out through fermentative process which offers a great deal of advantage in terms of reducing
the process cost and the raw material utility [8]. In Fermentation process the metabolism and product production
pattern of each microorganism depend mainly on their fermentative, nutritional, physiological and genetic nature [9-
12]. Exploitation of such microbial metabolism by regulating the critical fermentation parameters helps in
commercial economic production of the required enzyme [13, 14]. Temperature and pH are key environmental
parameters that affect the fermentation process. Hence, careful kinetics studies are required to monitor the growth of
microorganisms and alkaline protease production on various levels of substrates, different temperature and pH [15,
16]. Mixing has been found to be important in the microbial production of protease enzyme in bioreactor which can
be controlled by means of aeration and agitation. It is obvious that protease production also largely dependent on
higher oxygen mass transfer and lesser shear forces on microorganisms. For aerobic fermentation, oxygen transfer is
a crucial variable which is the function of aeration and agitation. Therefore, it is required to establish optimum
combination of airflow and agitation for maximum protease synthesis.

The aim of present work was to determine the effects of airflow rate and agitation rate on extracellular protease
production 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 production.

2. Material and methods

2.1. Microorganism and seed culture

The microorganism used in the present study was a strain of Bacillus licheniformis NCIM-2042 procured from
National Chemical Laboratory, Pune, India. The microorganism was grown on nutrient agar slants at 37 oC and pH
7.4. It was maintained by regular sub-culturing on nutrient agar slants at pH 7.4. The culture was revived by adding
a loop full of pure culture into 50 ml of sterile nutrient broth (pH 7.4).

2.2. Chemicals and reagents

Glucose (Sigma, USA), Starch (Sigma, USA), Bradford Reagent (Sigma, USA), Phenyl methyl sulfonyl fluoride
(PMSF), Bovine serum albumin (BSA), (Himedia, India), Trichloroacetic acid (Merck, India), soybean meal
(Carbon 20% and Nitrogen 8%) (Himedia, India) and Casein (Himedia, India) were used in this study. All other
chemicals used were of analytical grade and commercially available in India.

2.3. Enzyme assay and determination of protein concentration

Protease activity was determined by Folin and Ciocalteu method [17, 18]. 200 l of the protease broth was added to
the reaction mixture, containing 0.65% (w/v) casein in 800 l of 50 mM phosphate buffer (pH 9). The mixture was
incubated at 75oC for 10 min. The reaction was stopped by the addition of 1 ml of 5% (w/v) trichloroacetic acid
(TCA). This was followed by centrifugation at 10,000 g for 15 min. The supernatant was analyzed by the Folin-
Ciocalteu reagent. One unit of protease activity is defined as the amount of enzyme that liberated 1g tyrosine per
3446 A..Dey et al. / Materials Today: Proceedings 3 (2016) 34443449

min per ml of protease broth. Protein concentration was determined by the method of Bradford using bovine serum
albumin (BSA) as the standard [19]. All experiments were done in triplicate.

2.4. Estimation of starch in media

The starch analysis was determined by method of Englyst et al. [20]. The fermentation broth was centrifuged at
10,000 g for 10 min at 4 oC. The cell pellet was discarded and the supernatant was used for residual starch
determination. 1 ml of supernatant was mixed with 1 ml of 4 M KOH in test tube. One milliliter from each tube was
then added to 11 ml of 0.5 M acetic acid and mixed. Three ml of amyloglucosidase (50 units/ml) were added and the
tubes placed in a water bath (70 C) for 30 min. The tubes were then boiled for 10 min and allowed to equilibrate at
room temperature. The pH was adjusted by adding 0.4 ml of 6 M KOH and the tubes mixed and centrifuged (1500 x
g) to obtain a clear supernatant. The amount of glucose in the supernatant was then assayed using glucose oxidase
and peroxidase method [21].

2.5. Bioreactor operation

The experiments were carried out in a 2.2 l bioreactor (New Brunswick, USA) with 2 l working volume. A 2% fresh
culture (OD550 0.2) was inoculated in fermentor, containing media at pH-7.4. The impeller speed was kept at 180
rpm for 4 days at 37 oC. The dissolved oxygen was maintained above 30%. The culture was centrifuged at 10,000g
for 10 min at 4oC. Samples were withdrawn periodically at an interval of 6 h and analyzed for protease production,
residual starch and biomass concentration. Other fermentation parameters, such as temperature, pH, dissolved
oxygen and airflow were controlled in the bioreactor.

2.5.1. Effect of different airflow and agitation rate on protease production

The experiments were carried out in a lab scale 2.2 l bioreactor (New Brunswick, USA) with 2 l working volume. A
2% fresh culture (OD550 0.2) was inoculated in fermentor, containing optimized media (g/l); starch, 30.8; soybean
meal, 78.89; MgSO4, 0.5 and NaCl, 5.3 at pH-7.4. The impeller speed was initially adjusted to 180 rpm and
compressed sterile air was sparged at three levels of airflow rates: 1, 2 and 3 vvm cultivating for 4 days at 37 oC.
Further, the impeller speed was varied with 150 and 210 rpm. The dissolved oxygen was not allowed to fall below a
fixed set point of 30%. The culture was centrifuged at 10,000 g for 10 min at 4 oC. Samples were withdrawn
periodically at an interval of 6 h and analyzed for protease production, residual starch and biomass estimation. Other
fermentation parameters, such as temperature, pH, dissolved oxygen and airflow were continuously monitored
automatically.

3. Result and Discussion

3.1Effect of airflow rate

3.1.1. Determination of volumetric oxygen mass transfer coefficient (kLa)

Determination of kLa was estimated by dynamic gassing out method. The value of different kinetic parameters are
measured and reported in Table 1. From the Table 1 it is observed that the value of k La increases with increase in air
flow rates. The value of kLa is found to be 41, 54 and 56 1/h at 1, 2 and 3 vvm of airflow rate respectively.
 A..Dey et al. / Materials Today: Proceedings 3 (2016) 34443449 3447

3.1.2. Optimization of air flow and agitation rate

The batch STR was run with 2% inoculum for 4 days at 37 0C and tested the effects of airflow rates on protease
production. Three different airflow rates viz. 1, 2 and 3 vvm were tested. Fig. 1 shows the biomass and protease
production in STR under different conditions of airflow rates.
Table 1. Effects of airflow rates on protease production in batch STR at the end of 84 h of bioreactor operation

Parameter Air flow rate (vvm)

1 vvm 2 vvm 3 vvm

kLa (1/h) 42.55 56.49 62.5

Yp/x (Ul/g) 11.17 12.09 11.25

C* 7.344 7.888 8.228

Qo2 9.27 9.42 9.90

From Fig. 1, it is evident that different levels of biomass and protease production are found at each batch of the
STR. However, the trend of biomass growth (DCW, g/l) and production of protease (U) are similar in all batches
irrespective of airflow. But the variations in concentrations of protease production indicate the effect of airflow on
bacterial growth. The maximum protease production was found to be 382.46 U at 2 vvm (Fig. 1). During the
cultivation period, protease production in batch STR is fairly constant by variations in airflow rates beyond 2 vvm
but biomass production is found to be increased at 3 vvm.

It also indicates that the specific product formation (SPF) of protease with respect to the biomass concentration is
dependent on aeration rates (Table 1). At each condition of airflow rates in STR, production of protease is different;
therefore this parameter could act as metabolic regulating parameters in the bioreactor for the maximum SPF. The
calculation of SPF yields will help in determining the optimum airflow rate. There are several reports [22-24] where
the oxygen transfer rates are dependent on aeration rate during the batch STR operation with free cells of B.
licheniformis [22], which in turn results in maximum protease yields [23, 24].
3448 A..Dey et al. / Materials Today: Proceedings 3 (2016) 34443449

Fig. 1. Production of Biomass and protease in STR under different airflow. (a) 1 vvm; (b) 2 vvm and (c) 3 vvm.

The optimal airflow rate is found on the basis of maximum SPF is 2 vvm. Most of the industrial fermentation
processes for proteases use bacterial species, being sensitive for proper oxygen mass transfer, which is required for
the optimal cell mass production or product formation [25]. 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 [23, 24]. Further, agitation speed was varied at 150 and 210 rpm. However, maximum extracellular
protease production is obtained in presence of xylan from Beech wood, as a complex carbon source, producing
382.46 U (U) which is the maximum yield in comparison with the other agitation speed. This is demonstrated by
fact that, agitation raises the oxygen mass transfer which is required for aerobic fermentation. But at higher agitation
friction force is developed which leads to decrease microbial growth [26].

The trend of protease production is similar in all batches of STR operation. The decrease in protease
production is observed from Fig. 1 after 84 h of reactor operation, even though there is an increase in the biomass
 A..Dey et al. / Materials Today: Proceedings 3 (2016) 34443449 3449

concentration. This phenomenon may be attributed to the protease auto-degradation beyond 84 h [27]. Termination
of STR operation during maximum production period, i.e. 84 h could be economical. The scale-up of aerobic
cultures from shake 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.

Therefore, oxygen transfer rate can be considered as one of the important parameter for maximal production of
microbial protease in batch STR operation and can be controlled by means of both aeration and agitation for better
product formation [22, 23, 28]. Many researchers have reported the agitation speeds between the range of 150300
rpm for protease production using similar types of strains [29, 30].

4.Conclusion

Mixing is very crucial phenomenon for the maximum production of extracellular protease and it could be
achieved by optimum combination of aeration and agitation. However, it was noticed that cell may disrupt due to
higher stirring speeds in the reactor. High agitation creats vortex which may result in poor mass transfer of oxygen
during aerobic fermentation. Therefore, it is required to provide optimum combination of aeration and agitation
during batch bioreactor operation. From the present investigation, it is concluded that agitation and aeration have a
significant effect on protease production.

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