kinetics of proteinase production in constant fed- batch bioreactor

2nd Mercosur Congress on Chemical Engineering 4 Mercosur Congress on Process Systems Engineering th

KINETICS OF PROTEINASE PRODUCTION IN CONSTANT FEDBATCH BIOREACTOR Irapuan O. Pinheiro1, Claudemir S. Costa1, Fábio H.P.C. Oliveira1, Glauciane D. Santos1, Ana M. Souto-Maior1* 1

Departamento de Antibióticos - Universidade Federal de Pernambuco

Abstract. The genus Bacillus is used extensively for production of industrial enzymes. B. firmus produces an alkaline protease compatible to be used as an additive in detergents. As in other Bacilli, proteinase in B. firmus is synthesized in response to the depletion of nutrients. In this work, the kinetics of growth and alkaline proteinase production were investigated in a constant glucose fed-batch bioreactor. The experiments were performed in a Bioflo III bioreactor with 4 liters working-volume using a chemically defined fermentation medium. A Watson-Marlow pump was used to continuously feed the bioreactor with a constant feed rate (F=0.02 L.h-1) after an initial growth phase in batch mode. A highly concentrated glucose solution was used to feed the reactor (600 g.L-1) so that volume variation was less than 10%. The specific growth rate, µ, varied in the range of 0.25-0.01 h-1. The specific production rate showed a maximum value at µ around 0.15 h-1, and the production was halted at µ= 0.05 h-1. Growth was modeled using Monod equation, and the yield on biomass, Ysx, and the maintenance coefficient, m, were considered constants. With the estimated kinetics parameters, an equation was obtained which predicts an optimal feed profile to maximize specific production rate. The results have indicated that stimulus for protease production may be

related not only to the level of nutrient concentration, but also to concentration changes Keywords: Fed-Batch, Proteinase and Feed Profile.

1. Introduction The genus Bacillus is used extensively for production of industrial enzymes. Bacillus proteases are the most important industrial enzymes from an economic point of view. Alkaline proteases are robust enzymes with considerable industrial potential in detergents, leather processing, silver recovery, medical purposes, food processing, feeds and chemical industrial, as well as waste treatment. The use of Bacillus serine proteases in the manufacture of detergents is by far the major application of these enzymes, and alkalophilic Bacillus proteases have been found particularly suitable for use in detergent formulations (Horikoshi, 1996; Kumar and Takagi, 1999). Alkalophilic Bacilli are very common in nature and may be isolated from most soil samples. Some of these microorganisms can grow at pH values above 10 and the proteases are active and stable up to pH 12 (Horikoshi and Akiba, 1982; Souto-Maior et al., 1997). B. firmus var. arosia is an alkalophilic strain that produces an alkaline protease compatible to be used as an additive in detergents (Villandsen and. Vestberg, 1976). As in other Bacilli, protease in this microorganism is synthesized in response to the depletion of nutrients. In synthetic growth medium, extracelullar protease production by this microorganism is derepressed when growth is stressed by low concentrations of the nitrogen source, the carbon source or both sources (Souto-Maior, 1991). Enzyme production can be successfully achieved using constant fed-batch bioreactor to keep one or both substrates at low levels inside the bioreactor (SoutoMaior, 1994; Marques, 1998). As with B. licheniformis (Frankena et al. 1993), the relationship between specific protease production rate, qP, and specific growth rate, µ, shows a maximum production at an intermediate value of µ (Souto-Maior and Simões, 2000). Fed-batch cultivation techniques have been extensively used in industry for enzyme production since it enables the combination of high biomass concentration with low specific growth rate. The fed-batch bioreactor __________________________________________

*

To whom all correspondence should be addressed. Address: Departamento de Antibióticos, UFPE, 50.670-901, Recife - PE – Brazil E-mail: [email protected]

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may be operated in a variety of ways by regulating the feed rate using either feedforward or feedback control (Yamane and Shimizu, 1984). The use of the concentration of the fed substrate to direct control the addition rate of further substrate is an ideal operation, as the substrate directly affects the physiological state of the microorganism, and unforeseen deterioration of the cultivation is minimized. However, the lack of reliable, sensitive and sterilizable sensors, suitable for use in industrial processes, has limited the use of this technique. In the operation with indirect feedback control, other feedback parameters can be correlated with the course of the fermentation process. When feedforward control is used, the feed rate is changed during the operation according to a pre-determined profile, determined from a mathematical model of the process. Fed-batch bioreactor in which the feed rate is kept constant are referred to as constant fed-batch reactor. When the culture is increased exponentially with time, the culture is known as an exponentially fed-batch culture. In this work, the production of alkaline protease by B. firmus var. arosia was investigated in fed-batch cultivations with constant and exponential pre-determined feed profiles. The exponentially-fed-batch technique enables the specific growth rate of the microorganism to be kept at a constant value in high-cell-density cultivations. Potentially it could be a good option for industrial production of extracellular protease, as the specific growth rate might be kept at the value were specific production rate was maximized.

2. Material and Methods 2.1 Microorganism. Bacillus firmus var. arosia NCIB 10557 was used in this work. The strain was kept at 5º C on nutrient agar slopes and subcultured every three months. To adjust the pH to around 10, Na2CO3 was added to the preservation medium. 2.2 Medium. Cultivation medium consisted of (per liter of distilled water): glucose, 15 g; urea, 2 g; yeast extract, 5 g; Na2CO3, 10 g; MgSO4·7H2O, 1 g; CaCl2·2H2O, 1g; FeSO4·H2O, 100 mg; ZnSO4·7H2O, 10 mg; CuSO4·H2O, 8.8 mg; MnSO4·H2O, 7.6 mg. 2.3 Bioreactor and Cultivation Conditions. The cultivations were performed in a 4-liter working-volume bioreactor (BiofloIII, New Brunswick Scientific). Temperature, aeration rate and agitation were kept at 37º C, 1.5 vvm and 1500 rpm, respectively. The initial pH was adjusted to 10, but not regulated afterwards. The cultivations consisted of two phases: batch, for biomass build-up, and fed-batch, for enzyme production. After an initial growth phase in batch mode, highly concentrated glucose solution was used to feed the bioreactor, so that volume variation could be negleted. For the exponential feed profile, a microcomputer-controlled, predefined, open-loop regulation was used to implement the feed rate setpoint. The microcomputer was connected to a peristaltic pump (Watson-Marlow 101U) through an 8-bit digital/analogic interface (Souto-Maior, 1993), and the programming language was

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Visual Basic 5. Experimental feed profile was determined using a digital balance, where the feed vessel was placed. Analytical Methods. Cell mass was determined as dry-weight by drying the cells to constant weight, at 80 °C for 24 hours, after filtering a 10-mL sample through a Millipore filter paper and washing with 0.01M HCl. The procedure of washing the cells with acid solution was adopted to minimize retention of precipitates on the filter paper. Protease activity was determined by monitoring the amount of aromatic aminoacids released by casein. The method of Kunitz (1947), as modified by Hagihara et al. (1958), was used for the assay. The results were expressed as µg (tyrosine).min-1.mL-1 (same as U.mL-1) using a calibration curve, which was set up using an Ltyrosine standard solution in 0.2 M HCl. Glucose and urea concentrations were assayed utilizing GOLD ANALISA enzymatic kits (glucose-PP and urea UV-PP, respectively).

3. Results and Discussion 3.1 Constant Fed-batch Cultivation Protease is synthesized by B. firmus var. arosia in response to limitation of carbon and nitrogen sources. Figure 1 presents the results obtained for growth and protease production during glucose constant fed-batch cultivation. During the batch stage, the pH decreased from 10 to 9, range adequate for the microorganism growth (Souto-Maior et al., 1997). After 5 hours of batch cultivation, when an increase in pH from 8,0 to 8,5 was observed (indicative of glucose depletion), a highly concentrated glucose solution (SF = 600 g.L-1) was fed to the bioreactor at a constant flow rate (F = 0.02 L.h-1). A highly concentrated urea solution was also fed to the reactor to keep urea in non-limiting concentration levels throughout the process. During the fed-batch stage, the pH remained approximately constant at a value around 9. Protease concentration reached a maximum value (~5000 U.mL-1) at 9 hours of fed-batch cultivation, while growth proceeded up to 23 hours, when 25 g.L-1 biomass was reached.

27

6000 Batch Fed-Batch

5000

21

4000

15

Biomass Urea pH Glucose Protease

-1

12 9

2000

6

1000

3 0

3000

-1

18

Protease (U.mL )

-1

Biomass (g.L ), Urea (g.L ), -1 pH, Glucose (g.L )

24

0

3

6

9

12

15

18

21

24

27

0 30

Time (h) Fig. 1. Time course of cell growth, substrate consumption, protease production and pH variation

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during batch and constant fed-batch cultivation of Bacillus firmus var. arosia

Figure 2 shows the variation of specific growth rate and specific protease production rate during the fedbatch stage. Specific growth rate decreased throughout the process from 0.226 h-1 to 0.007 h-1. Specific production rate showed a maximum at around 2.5 hours of fed-batch cultivation, and degradation occurred at 9 hours, which resumed after 15 hours. The relationship between specific production rate and specific growth rate is presented in Figure 3. Maximum qP (~84 U.g-1.h-1) occurred at around µ = 0.15 h-1, and qP = 0 at around µ = 0.05 h-1. These results corroborate previous data obtained at lower cell densities using molasses-based medium (Marques, 1998).

0.25

100 µ

qp

80

0.20

20

-1

µ (h )

-1

40

-1

0.15

qp (U.g .h )

60

0.10 0 -20

0.05

-40 0.00

0

2

4

6

8 10 12 14 16 18 20 22 24

Time (h) Fig. 2. Time course of specific growth rate, µ, and specific protease production rate, qP, during constant fed-batch cultivation of B. firmus var. arosia

120 100

2

qP = -146,25 + 3173,98 µ -10803,25 µ

80

qP (h-1)

60 40 20 0 -20

Experimental Fit

-40 -60 0,00

0,05

0,10

0,15

0,20

0,25

-1

µ (h )

Fig. 3. Relationship between specific protease production rate, qP, and specific growth rate, µ, during constant fed-batch cultivation of B. firmus var. arosia

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3.2 Modeling of Constant Fed-Batch Bioreactor. For the fed-batch bioreactor, it was assumed that dV/dt ≈ 0, since a highly concentrated glucose solution was fed to the bioreactor. It was also assumed that dS/dt ≈ 0, since glucose was maintained at very low concentrations throughout the production phase. Mass balance led to the equations:

µ=

rx 1 dX = X X dt

qs =

qp =

rS DSF − m.X = X X

rp X

=

1 dP X X

(Eq.1)

(Eq.2)

(Eq.3)

where X= biomass concentration (g.L-1); S= glucose (limiting substrate) concentration (g.L-1); SF= glucose concentration in the feed solution; P= protease concentration (U. L-1); V= volume (L); F= flow rate (L.h-1); D= F/V (h-1); rx= growth rate (g.L-1.h-1); µ= specific growth rate (h-1); rS= glucose consumption rate (g.L-1.h-1); qS= specific consumption rate (g.g-1.h-1); rp= protease production rate (U.L-1.h-1); qp= specific protease production rate (U.g-1.h-1). Considering that the maintenance coefficient “m” is constant and that q s = µ Y where Y is the constant yield coefficient, µX = (DSF − m.X )Y

(Eq. 4)

Consequently,

dX = (DSF − m.X )Y dt

(Eq. 5)

The solution of Eq. (5) above is

X( t ) =

D.SF .Y  D.SF .Y  +  X0 −  exp(−mYt ) mY  mY 

(Eq. 6)

Fitting Eq. (6) to the experimental results (Fig. 4), the yield and the maintenance coefficients obtained are 0.099 and 0.671 h-1, respectively. From the experimental results for qp and µ (Fig. 3), a quadratic relation between these two specific velocities was used.

5


q P = a + bµ + cµ 2

(Eq. 7)

25 EXPERIMENT FIT

-1 Biomass (g.L )

20 15 10 5 0

Biomass = 30,26521 - 24,14773.exp (-0.06651. t)

0

5

10

15

20

25

Time (h) Fig. 4. Growth curve fitting

From Eq. (3), Eq. (4) and Eq. (6),

(DSF Y − m.Y.X ) dP = a.X + b.(DSF Y − m.Y.X ) + c. dt X

2

(Eq. 8)

where X is given by Eq.(6). Solving Eq. (8),

P( t ) = P0 +

a D SF t  a DSF   + − b + cYm . X 0 − .(exp(−mYt) − 1) + m m   mY 

  1 (exp(−mYt) − 1) X 0 − DSF   DSF cY ln1 + m    X0

(Eq. 9)

Figure 5 shows the time course of protease production during constant fed-batch cultivation and the curve fit

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5000

Protease (U.mL-1)

4000

3000

2000

Fit Experimental

1000

0 0

5

10

15

20

25

Time (h)

Fig. 5. Time course of protease production during constant fed-batch cultivation of B. firmus var. arosia

3.3 Exponentially Fed-Batch Bioreactor.

With the estimated kinetics parameters above, an equation was obtained to predict an optimal feed profile to maximize specific production rate. Considering µ* the “ideal” specific growth rate correspondent to the maximum specific production rate, qPMAX, the mass balance leads to the equations:

dX = µ *X dt

(Eq.10)

dS µ*X = DSF −mX dt Y

(Eq.11)

(

( ) )X

dP = a + b µ* + c µ* dt

2

(Eq.12)

Solving Eq. (10)

X( t ) = X 0 exp(µ* t )

(Eq.13)

From Eq. (11) and using Eq. (13) an equation is obtained which predicts an optimal feed profile to maximize specific production rate.

F=

 X 0 V  µ*  + m  exp(µ* t ) SF  Y 

(Eq.14)

where the initial flow rate is given by

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F0 =

 X 0 V  µ*  + m  SF  Y 

(Eq.15)

The solution of the Eq. (12) gives the predict profile for protease production.

(

( ) ) Xµ (exp(µ t) − 1)

P = P0 + a + b µ * + c µ *

2

*

0 *

(Eq.16)

In Figure 6, the results of fed-batch cultivation using this feed profile are presented, where µ*= 0.158 h-1 and X0 = 5 gL-1 were assumed. Batch phase lasted approximately 5 hours, after which a highly concentrated glucose solution (613 gL-1) was started to be fed to the bioreactor. As with the constant-fed-batch, the pH remained around 9 throughout the process. Protease accumulation showed initially an exponential profile, which changed and started to decline after around 3.5 hours of fed-batch cultivation, reaching around 2.400 U.mL-1 at 8.5 hours, which corresponds to half of the amount obtained during constant fed-batch. According to Eq. (13), the estimated biomass profile was described by the following equation: X = 5. exp(0.158. t)

(Eq.17)

However, growth showed an exponential profile (Fig. 7), but with µ*= 0.124 h-1, and the experimental initial biomass concentration was X0 = 6.0 gL-1. In Figure 8, the variation of specific protease production rate is shown.

-1

3000

18

Biomass Urea Protease

15

Glucose pH

2500 2000

12 1500 9 1000

6

500

3 0

-1

Fed-Batch

Protease (U.mL )

Batch

-1

Biomass (g.L ), Urea (g.L ), -1 pH, Glucose (g.L )

21

0

2

4

6

8

10

12

0 14

Time (h)

Fig. 6. Time course of cell growth, substrate consumption, protease production and pH variation during batch and exponentially fed-batch cultivation of B. firmus var. arosia

8


18

Biomass (g.L-1)

16

X = 6.274 exp(0.124 t) 2 R = 0.996

14 12 10 8 6

0

1

2

3

4

5

6

7

8

9

Time (h) Fig. 7. Exponential growth of B. firmus var. arosia during exponentiallyfed-batch cultivation. Predefined and experimental values for µ* were µ* = 0.158 h-1 and µ* = 0.121 h-1, respectively.

70

0.30

µ

50

qP

40

0.15

30 20

0.10

-1

-1

µ (h )

-1

0.20

qP (U.g .h )

60

0.25

10 0.05 0.00

0 0

1

2

3

4

5

6

7

8

9

-10

Time (h) Fig.8. Time course of specific growth rate, µ, and specific protease production rate, qP, during constant fed-batch cultivation of B. firmus var. arosia

Despite the fact that growth showed an exponential profile, as predicted by the model, protease production did not follow the expected optimum profile. As with constant fed-batch cultivation, a maximum was observed, which occurred around 3.5 h of exponentially-fed-batch fermentation, and, at 8 hours, qP was nearly zero.

4. Conclusions Production of protease by B. firmus var. arosia in constant fed-batch cultivation showed higher productivity than in cultivation using predefined exponential feed profile. The highest specific productivity is observed in 9


both cases in the beginning of the fed-batch stages, less than 4 hours after the initial batch phase ended. Timedependent decrease in specific productivity is similar to that observed for savinase-producing B. clausii (Christiansen et al., 2003) during fed-batch cultivation with linear and exponential profiles. Keeping µ constant did not assure an optimal qP as predicted by mathematical model. This indicates that stimulus for protease production may be related not only to the level of nutrient concentration, but also to concentration changes.

References Christiansen, T.; Michaelsen, S.; Wümpelmann, M.; Nielsen, J. (2003), Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations. Biotechnology and Bioengineering, 83(3), p. 344-352. Frankena, J.; Koningstein, G. M.; Van Verseveld, H.; Stouthamer, A. H. (1986), Effect of different limitations in chemostat cultures on growth and production of exocellular protease by Bacillus lincheniformis. Applied Microbiol. Biotechnol. 24, p. 106-112. Kumar, C. G. and Takagi, H. (1999). Microbial Alkaline Proteases: from a bioindustrial viewpoint. Biotechnology Advances. 17, 561–94.

Kunitz, M. (1947), Crystalline soybean trypsin inhibitor. Journal of General Physiology, vol. 30, p. 291-310. Hagihara, B.; Matsubara, H.; Nakai, M; Okunuki, K. C. Cristalline bacterial proteinase. I. Preparation of crystalline proteinase of Bacillus subtilis. Journal of Biochemistry, v. 45, p. 185-194, 1958. Horikoshi, K. & Akiba, T. (1982), Alkalophilic Microorganisms: A New Microbial World. Japan Scientific Societies Press, Tokyo. Horikoshi, K. (1996), Alkaliphiles: from an industrial point of view. FEMS Microbiology Reviews, 18, p. 259-270. Marques, O. M. (1998), Produção de proteinase por Bacillus firmus em meio de melaço. MSc. Thesis, Universidade Federal de Pernambuco. Souto-Maior, A. M. (1991), Proteinase production from an alkalophilic Bacillus especies. PhD Thesis, University of Manchester. Souto-Maior, A. M. (1993). Controle de biorreator via microcomputador. Anais do ENZITEC-93, pp. 67-68, Rio de Janeiro. Souto-Maior, A. M. (1994),. Proteinase production from an alkalophilic Bacillus species. Arquivos de Biologia e Tecnologia. v. 37(2), p. 247-255. Souto-Maior, A. M.; Marques, O. M.; Pontual, R. S. (1997), Effect of the initial pH on the growth of a proteinase-producing alkalophilic Bacillus species. Arquivos de Biologia e. Tecnologia, v. 40(3), p. 616-623. Souto-Maior, A. M. & Simões, D. A. (2000), Uso e produção de proteases microbianas alcalinas. Revista Brasileira de Engenharia Química, v. 19, p. 10-14. Villadsen, K. J. S. & Vestberg, K. P. (1976), U.S. Patent 3 960 665. Yamane, T. And Shimizu, S. (1984), Fed-batch techniques in microbial processes. In Advances in Biochemical Engineering/Biotechnology (A. Fiechter, ed.), vol. 30, pp. 147-194. Spring-Verlag, Berlin.

Acknowledgments The authors are grateful to “Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE)”, for the financial support, and to “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)”, for the postdoctoral scholarship to I. O. Pinheiro. 10