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Volume 1 Number 1 January-June 2011, pp. 27-33, International Science Press (India)

Thadikamala Sathish1* and Reddy Shetty Prakasham2

Dept of Civil and Environmental Engineering Center, University of Windsor, Windsor, Ontario, Canada. 2 Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Hyderabad 500 607, India.

Abstract: L-Glutaminase production by Bacillus subtilis RSP-GLU was carried out in a lab scale stirred reactor (STR). An unstructured mathematical model was employed to understand the information on relationship between bacterialgrowth and L-glutaminase production. Influence of substrate concentration for enhanced enzyme production was studied. Also effect of different aeration (0.5-2 vvm) and agitation rates (50 to 200 rpm) in a stirred tank reactor was examined. The biomass growth and enzyme production titers were altered with the change in substrate concentration. A maximum enzyme production of 323 U ml1 was achieved in the medium having low concentrations of L-glutamine (1.0 %). Both aeration and agitation significantly affected L-glutaminase production as well as biomass formation. Keywords: L-Glutaminase, Stirred Tank Reactor, Aeration, Agitation, Mathematical model, L-glutamine.

1. INTRODUCTION In current days, amidases a class of amine transferring enzymes are gaining importance in market, based on their wide variety of applications in various pharmaceutical and chemical industry use [1]. Among various amidases L-asparaginase and L-glutaminase are popular in market, based on their therapeutic applications. L-glutaminase (EC is an important enzyme present in all living cells which is a responsible for the nitrogen metabolism [2-5]. L-glutaminase converts the L-glutamine to L-glutamate and ammonia, L-glutamate and ammonia play a vital role to synthesis of nucleotides and enzymes. Unlike normal cells, tumor cells cannot synthesis the L-glutaminase they depends on the external source from blood. The use of L-glutaminase deprives neoplasms for essential amino acid and causes selective death of glutamine-dependent tumor cells by depriving of L-glutamine [6-8]. Apart from the cancer therapy it is also used in the treatment of HIV ref. L-glutaminase is used as an analytical agent for determination of L-glutamine in the
* Corresponding Author:, Telephone: +15192533000 Ext- 2516 Telefax: +15192533000

media [9,10]. It is also used as biosensing agent [11,12]. L-glutaminase is used as flavor-enhancing agent in the soya sauce preparation [13-16]. Based on its gamma glutamyl transfer reactions, it is usedin chemical industries for the production of forte chemicals such as threonine which is a major aroma component in coffee and tea [17]. Industrial fermentation is moving away from traditional methods and largely empirical operation towards knowledge based and better-controlled processes [18,19]. The rational design and optimization of the latter requires the understanding of production kinetics. A few reports are available on the production of L-glutaminaseatlab scale reactor level. Best of our knowledge studies on Bacillus subtilis for L-glutaminaseproduction in reactors are not yet reported in the scientific literature. Optimization of the inducer concentration is one of the most critical stages in the development of an efficient and economic bioprocess for L-glutaminase production [1,13]. From bench scale production, it was observed that mixing was important in microbial synthesis of L-glutaminase enzyme [1]. In free cell bioreactor, mixingcan be imparted by means of aeration and agitation. Mixing is also largely


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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. Present work was aimed to study the effect of levels of inducer substrate concentration on maximizing L-glutaminase production, analyze the effects of airflow and agitation rates on Lglutaminase production in a lab scale bioreactor. 2. MATERIALS AND METHODS 2.1 Microorganism and Inoculum Preparation Isolated Bacillus subtilis RSP-GLU (MTCC No: 9727; GenbankAccession No. AM990996) was used in this study. The culture was maintained on glutamine medium (pH -7.0) slants consisting of (g L1) L-glutamine-5, K2HPO4 1, KH2PO4 0.1, MgSO4 1, NaCl 0.5, yeast extract 0.5 and agar 20. Inoculated slants were incubated at 35 oC for 24 h for microbial growth and were stored at 4 oC in a refrigerator for further use. The inoculum wasgrown in a 250 ml conical flasks containing 100 ml of medium (pH 7.0) by incubating at 35 oC and at 120 rpm in an orbital shaker (LabTech LSI 3016 R). All experiments were performed in triplicate and average values are reported in the present study. 2.2 Effect of L-glutamine Concentration on L-glutaminase Production All the experiments were carried out in a lab scale 1.5 L bioreactor (B-Braun Biostat) with 1 L working volume, fixed with two-stage rushton type impeller of 50mm diameter. In order to study the effect of substrate concentration on biomass and L-glutaminase production, various concentrations of L-glutamine was tested. L-glutamine in the levels of 0.5 to 2.0 % were varied at aeration 1 vvm, agitation 100 rpm, pH 7.1, inoculum concentration 2% and temperature 37 o C. Each experiment was carried out until the biomass reaches the stationary phase. Estimation of biomass and L-glutaminase activity was carried out for every 1 hrs. A growth kinetic model was developed to explain the Bacillus subtilis RSP-GLU growth in batch STR using L-glutamine as a limiting substrate. 2.3 Effect of Aeration and Agitation on L-glutaminase Production In order to study the influence of aeration and agitation on L-glutaminase production in STR, the reactor was initially operated at various airflow rates (0.5 to 2vvm) and at optimum aeration rate four different agitation rates

(50 to 200) were tested by inoculating the batch STR with 2% inoculum and cultivating for upto 16 hr at 37 oC. Samples were withdrawn at every 1 hr to estimate biomass and L-glutaminase activity.The oxygen mass transfer coefficient (kLa) and oxygen transfer rate (OTR) in the bioreactor was calculated according to Potumarthi et al [19]. 2.4 Measurement of Enzyme Activity and Biomass L-glutaminase activity was determined using L-glutamine as substrate and the product, ammonia released during the reaction was measured by using Nesselers reagent according to method of Imadaet al. [20]. One unit of enzyme activity was defined as the amount of enzyme that liberates 1 M of ammonia per ml per min under optimal assay conditions.Initially biomass was estimated by measuring optical density (OD) of liquid samples at 600nm, later it was converted to dry weight by interpreting OD into a standard graph between OD vs dry weight of Bacillus subtilis RSP-GLU. 2.5 Determination of the Kinetic Parameters A nonlinear least-squares (Levenberg-Marquardt) method was used to fit the developed model and estimate the parameters (specific growth rate and lag period). The error between the observed and predicted values was minimized by adjusting the number of iterations in the system. Statistica 6.0 (StatSoft, Inc. 2001) was used to estimate and evaluate the significance of the parameters estimated by the adjustment of the experimental values to the proposed mathematical model. 3. RESULTS AND DISCUSSION 3.1 Development of Pragmatic Equations for Growth and Production Kinetics for L-glutaminase Production Most of the growth processes are explained in terms of Monod or logistic equations (unstructured models). Logistic equation is simple for calculation of fermentation parameters related to biological and geometrical significance using sigmoid profiles independent of substrate concentration[18]. The sigmoidal growth pattern of Bacillus subtilis RSP-GLU was analyzed using logistic equation for its variation against time during log phase. In order to develop logistic equation the following assumptions were made. 1. L-Glutamine act as both carbon and nitrogen source and it is the only limiting factor during fermentation.

Modeling the Effect of L-Glutamine, Aeration and Agitation Regimes on Production of L-Glutaminase



Bacillus subtilis. RSP-GLU growth and metabolism is dependent on the glutamine availability in the fermentation medium. L-Glutaminase production is extracellular in nature and is associated with the biomass growth.


glutaminase production, various concentrations of initial L-glutamine was tested. In this experiment L-glutamine concentration was varied from 0.5 to 2.0 % and other parameters kept constant (aeration at 1 vvm, agitation 100 rpm, pH 7.1 and temperature 37oC). It was observed that the initial L-glutamine concentration effects the growth of microorganismas well as L-glutaminase production. From figure 1 it was observed that up to 1.00 % glutamine concentration, growth of the organism increased and beyond this concentration the growth was decreased. It was evident that rise of L-glutamine from 1.0 % to 1.5 % resulted 8.0% growth reduction. Doubling the initial concentration (from 1.00 % to 2.00 %) decreased 13.0% of the bacteria growth. From the results obtained, it was observed that, initial substrate concentration affects the tmax. At low substrate concentration thetmax was observed to be lower than substrate at higher concentrations. The tmax was observed to be 11 and 12 hrs at 0.50 % and 0.75% Lglutamine concentrations respectively. At 1.00 % substrate concentration the tmax was observed to be 13 hrs.

On the basis of above assumptions, the following logistic equation was used to analyze the microbial growth.

dX X = max 1 X dt X max


At the beginning of the fermentation when t = 0, the inoculum is considered as the initial concentration where X = X0. After integration of the equation (1) between time interval t0 and t, the explicit form of biomass (X) as a function of time (t) is
X= X max 1 + exp [ 2 + max ( X t ) ]



Xmax = maximum biomass max = specific growth rate max = lag phase

3.2 Production Kinetics The kinetics of L-glutaminase production was studied based on the Luedeking and Piret model. This model has a great utility with modifications which allow the incorporation of other effects in the description and specifications of the metabolites produced by the microbes. According to the Luedeking and Piret equation the rate of L-glutaminase production is
X max P= 1 + X max 1 e X 0 X0

max .t

Figure 1: Effect of L-glutamine Concentration on Isolated Bacillus subtilis RSP-GLU Growth in STR

The equations 2 and 3 are used to the describe the kinetic parameters of the L-glutaminase production and Bacillus subtilis RSP-GlU growth in the rest of all experiments. 3.3 Effect of L-glutamine Concentration on Growth and Enzyme Production In the present study L-glutamine was used as a sole carbon and nitrogen source for L-glutaminase production by isolated Bacillus subtilis RSP-GLU. In order to study the effect of substrate concentration on biomass and L-

Table 1 depicts the growth and production kinetic parameters of isolated Bacillus subtilis RSP-GLU.It was observed that increasing the L-glutamine concentration from 1.50 % to 2.0%, the lag-phase period was increased from 3.3 to 3.5 hrs. It was noticed that the specific growth rate of Bacillus subtilis RSP-GLU was increasedalong with the increase of substrate concentration up to 1.50 %. The highest max (0.7263 h1) was observed with the 1.50 % glutamine concentration.

Table 1 Kinetic Parameters of the L-glutaminase Production by Bacillus subtilis RSP-GLU at Various L-glutamine Concentrations Parameter tmax (hrs) Xmax ( g L1) max (h1) vmax (g L1 h1) x (h) td (h) Pmax (U ml1) Yx/s Yp/s Yp/x L-Glutamine concentration (%, w/v) 0.5 11.00 0.6862 0.6032 0.1035 3.1 1.1486 255 386.44 0.6238 232.71 373.03 0.75 12.00 0.6911 0.6354 0.1097 3.3 1.0906 296 443.60 0.6282 269.99 429.72 1.0 13.00 0.7791 0.6835 0.1331 3.3 1.0138 339 448.72 0.7083 309.08 436.36 1.5 13.00 0.7172 0.7263 0.1302 3.3 0.9541 323.90 465.15 0.6520 294.45 451.61 2.0 13.00 0.6731 0.7027 0.1182 3.5 0.9861 303.99 465.85 0.6119 276.35 451.56

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of the oxygen limitation problem, a sufficient supply of oxygen is necessary for microbialmetabolism [19]. Keeping this is in the view the aeration experiments were conducted by supplying the air from 0.5 to 2.0 vvm by keeping the agitation at 100 rpm and L-glutamine concentration at 1.00 %.

From figure 2 it was observed that production of L-glutaminaseis influenced by the substrate concentration. It was observed that at 1.00 % substrate concentration highest amounts of L-glutaminase production (323 U ml1) was noticed. Above and below this concentration (1.0 % L-glutamine) decreased enzyme production was noticed.

Figure 3: Effect of Aerate on Bacillus subtilis RSP-GLU Growth in STR

Figure 2: Effect of L-glutamine Concentration on L-glutaminase Production by Bacillus subtilis RSP-GLU in STR

Figure 4: Effect of Aerate on L-glutaminase Production by Bacillus subtilis RSP-GLU in STR

3.4 Influence of Aeration on Biomass and L-glutaminase Production Aeration in aerobic fermentations is extremely important since growth and production can be seriously affected by the dissolved oxygen concentration [19]. Rheological properties of the culture may also play crucial roles in fermentation. For example, the highly viscous nature of bacterial liquid cultures can significantly obstruct the oxygen transfer. Therefore, in order to minimize the effect

Figure 3 & 4 shows the biomass and production profiles at various aeration rates. The highest yield of L-glutamiinase (372 U ml1) was obtained with aeration rate of 1.5 vvm. Whereas further increase or decrease in aeration rate at values over 1.5 vvm decreased Lglutaminase production (Fig. 4). At this aeration the biomass was observed to be 0.8094 g L1(Fig. 3). The maximum concentrations of L-glutaminase at aeration rates of 0.5, 1 and 2 vvm were 321, 339 and 351 U ml1, respectively.

Modeling the Effect of L-Glutamine, Aeration and Agitation Regimes on Production of L-Glutaminase Table 2 Various Growth and L-glutaminase Production Kinetic Parameters at Different Aeration Rates in STR Parameter 0.5 tmax (hrs) Xmax ( g L1) max (h1) vmax (g L1 h1) x (h) td (h) Pmax (U ml1) Yx/s Yp/s Yp/x kL a 15.00 0.7365 0.6608 0.1216 3.8057 1.0485 321.94 455.07 0.6695 292.67 437.12 27.82 Aeration rate (vvm) 1 13.00 0.7791 0.7223 0.1407 3.2036 0.9593 339.99 448.42 0.7083 309.08 436.36 32.58 1.5 12.00 0.8094 0.8700 0.1760 3.0056 0.7964 372.71 438.15 0.7358 338.82 460.42 35.68 2 12.00 0.8202 0.9327 0.1912 2.8025 0.7429 351.26 409.83 0.7456 319.32 428.24 41.25


hour and agitation speed of 150 rpm (Fig 5). At a higher stirrer speed of 200 rpm, biomass production indicated a lower value (0.6812 g L1). The maximum L-glutaminase production (414U ml1) was obtained at 12th hour from the RSP-GLU culture with a relatively mild agitation rate of 150 rpm (Fig 6). The maximum production of Lglutaminase in cultures at 50, 100, and 200 rpm were 283, 372 and 263 U ml1, respectively.

The growth and production kinetic data at different aeration rates are represented in Table 2. The yield of Lglutaminase from substrate (YP/S) and the specific growth rate of the cells () at 1.5 vvm were 338 and 0.87 h1, respectively. The kLa value is increased along the aeration increases. This result suggests that maintenance of high DO levels is important for both cell growth and Lglutaminase production by Bacillus subtilis RSP-GLU. These results are in accordance with the Martin and Bailey [21] and Pferrerle et al [22] for the production of secondary metabolites from A. campestris NRRL2334 and Streptosporangium sp. respectively. 3.5 Influence of Agitation Speed on Biomass and L-glutaminase Production in STR Among other factors having an impact on the operating conditions during fermentation in bioreactors is agitation and mixing. Agitation is important for uniform mixing of the medium components within the fermenter (dispersion of cells and nutrients) as well as mass transfer phenomena (e.g., oxygen transfer rates) [19]. At low agitation speed the mixing may either be inadequate, leading to cell sedimentation while at high speed cell shearing may been enhanced. To determine the optimum rotational speed, Bacillus subtilis RSP-GLU cells were cultivated under different agitation speeds in the STR. A set of batch experiments were carried out in a 1.5 L STR at various agitation speeds in the levels of 50, 100, 150, and 200 rpm. Biomass production was enhanced as agitation speed increased from 50 to 150 rpm at 1.5 vvm of airflow rate. The highest production of biomass 0.8366 g L1 was obtained at 12th

Figure 5: Effect of Agitation Speed on Bacillus subtilis RSPGLU Growth in STR

Figure 6: Effect of Agitation Speeds on L-glutaminase Production by Bacillus subtilis RSP-GLU in STR

Agitation results in a better mixing of the culture broth, allowing it to maintain a satisfactory supply of nutrients to the cells, while it facilitates the removal of gases and other byproducts of catabolism from the microenvironment of the cells. Pfefferle et al [22] showed a 2 fold increase in secondary metabolite formation by increasing the stirrer speed from 500 rpm to 750 rpm using Streptosporangium. However Guo et al [23] reported that during the gentamicin fermentation process, mycelial morphology has


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a close relationship with gentamicin. Hypha could be hurt mechanically and shortened by high shear, which resulted in the decrease of the production. Agitation rate not only affects the oxygen availability, but it also exerts influence on the availability of other nutrients in the medium. Low L-glutaminase production at higher agitation rates might be attributed to the effect of shear stress on Bacillus subtilis RSP-GLU cells. Agitation rate of 150 rpm has been observed optimum for the production of L-glutaminase by Bacillus subtilis RSP-GLU. High agitation rates create shear forces that may affect microbial growth in some other ways, including morphological changes of the culture, cell damage and formation of metabolites [24]. The growth kinetic data of RSP-GLU with different agitation rates are illustrated in Table 3. The yield of L-glutaminase production from substrate (YP/S) and the specific growth rate of the cells () at 150 rpm were 376 and 0.6353 h1, respectively.
Table 3 Various Kinetic Parameters of Bacillus subtilis RSP-GLU at Various Agitation Speeds Parameter 50 tmax (hrs) Xmax ( g L1) max (h1) vmax (g L1 h1) x (h) td (h) Pmax (U ml1) Yx/s Yp/s Yp/x kL a 15.00 0.6881 0.6079 0.1045 4.1268 1.1399 283.53 412.35 0.6255 257.75 412.04 29.68 Agitation speed (rpm) 100 12.00 0.8094 0.8700 0.1760 3.0056 0.7964 372.71 438.15 0.7358 338.82 460.42 35.94 150 12.00 0.8366 0.6353 0.1328 2.4599 1.0907 414.53 516.92 0.7606 376.84 495.45 43.51 200 15.00 0.6812 0.5685 0.0968 4.5435 1.2188 263.20 398.29 0.6193 239.27 386.32 45.73

vmax x dp/dt

: : : :

Pmax Y X/S Yp/s Yp/x

: : : :

Maximum growth rate. (gL1h1) Growth lag phase (h) protease rate production dimensions Luedeking and Piret parameter (growthassociated constant for L-glutaminase production) Maximum L-glutaminase production (U ml1) Yield factor for biomass formation on Lglutamine Yield factor for L-glutaminase formation on L-glutamine Yield factor for L-glutaminase formation with respect to biomass References

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dX/dt X0 X t tmax td Xmax max

: : : : : : : :

4. NOMENCLATURE Growth rate. (g.L1.h1) Initial biomass. (g.L1) Biomass. (g.L1) Time. (h) time at the Xmax Doubling time (h) Maximum biomass. (g.L1) Specific maximum growth rate (biomass production per unit of biomass and time) (h1)

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