Enzyme and Microbial Technology 39 (2006) 11451151

Optimization of nutrient feed concentration and addition time for production of poly(-hydroxybutyrate)
Shilpi Khanna, Ashok Kumar Srivastava
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India Received 26 August 2005; received in revised form 23 February 2006; accepted 23 February 2006

Abstract PHB is an intracellular storage compound produced by many organisms under conditions of nutrient limitation (N, P, etc.) when carbon source is present in excess. Model-based fed-batch cultivation is one of the techniques to induce desired nutrient limitations intelligently for over production of such partially growth-associated polymeric products. In the present study, batch kinetics of PHB synthesis by Wautersia eutropha was established in a bioreactor. Batch kinetics featured a biomass concentration of 19.68 g/l with a PHB content of 10.89 g/l in 60 h. A mathematical model was then developed, which adequately described the batch kinetics. The model equations were extrapolated to fed-batch cultivation by incorporating the dilution terms due to fresh nutrient (carbon and nitrogen) feeding. Model-based fed-batch fermentation was conducted by feeding nitrogen (7 g/l) from 2030 h at the rate of 70 ml/h and fructose (300 g/l) from 2434 h at the rate of 80 ml/h constantly which yielded 32 g/l biomass containing 14 g/l PHB in 50 h. The productivity was 1.5-times greater than that obtained in batch increasing from 0.18 to 0.28 g/l-h. The model proved to be highly instrumental in the design of suitable fed-batch cultivation. 2006 Elsevier Inc. All rights reserved.
Keywords: Wautersia eutropha; Mathematical model; PHB; Model-based fed-batch cultivation

1. Introduction Poly(-hydroxybutyrate) (PHB) is an intracellular carbon and energy storage material accumulated by many microorganisms under unfavorable growth conditions such as limitation of N, P, S, Mg, or O2 and excess of carbon source [1,2]. PHB is biodegradable thermoplastic polyester that can be considered analogous to many conventional petrochemical-derived plastics currently in use [3,4]. Wautersia eutropha (earlier called as Ralstonia eutropha) is the most widely used microorganism for the production of PHB since it is able to accumulate large amounts of PHB (up to 80% of dry cell weight) [5,6]. Fed-batch fermentation is the most common method to achieve a high cell density and induce desired nutrient limitation, which are often necessary for high yield and productivity of the nal product in certain cultivations. In literature studies, nutrients were usu-

Abbreviations: PHB, poly(-hydroxybutyrate); SSWR, sum of squares of weighed residues Corresponding author. Tel.: +91 11 26596109; fax: +91 11 26582282. E-mail address: ashokiitd@hotmail.com (A.K. Srivastava). 0141-0229/$ see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.02.023

ally fed in the reactor intermittently or constantly in response to dissolved oxygen (DO) [7] or pH [8] as a feedback parameter. In these approaches, however, substrate(s) (N and/or P) concentration in the culture broth could not be precisely maintained due to the nature of indirect estimation. However, for efcient production of PHB, non-limiting concentration of carbon source and limiting concentration of nitrogen are needed [2]. In order to identify the appropriate feed time, its concentration and addition rate mathematical model-based cultivation approach is very useful. There have been some previous reports on modeling of PHB fermentation, which feature the characteristic kinetic behavior of the micro-organism [911]. A mathematical model was proposed [10] wherein growth rate of the culture was described by combination of Monod and Sigmoidal kinetics. Later on, N/C ratio was indicated to be more comprehensive term reecting the true impact on growth and product formation by W. eutropha. Mulchandani et al. [11] carried out substrate inhibition studies and proposed another model incorporating the term for N/C inhibition [11]. Raje and Srivastava [12] later on modied the earlier proposed model structures by taking into account a term for N/C inhibition in the growth of the active component of the

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Nomenclature dP/dt dR/dt dS/dt F Ks K1 K2 ms n P qp qs R Sm S1 S2 V Wj X YR/S YP/S rate of product formation (g/l-h) rate of biomass formation (g/l-h) rate of substrate consumption (g/l-h) total feed rate (ml/h) saturation constant (g/l) kinetic parameter (g/g) in Eq. (5) kinetic parameter (g/g h) in Eq. (5) maintenance coefcient (g/g h) exponent PHB concentration (g/l) specic product formation rate (g/g biomass h) specic substrate consumption rate (g/g biomass h) residual biomass (g/l) maximum value of substrate at which specic growth rate is zero fructose concentration (g/l) nitrogen concentration (g/l) volume of the reactor (l) weight of each variable (usually the maximum value of each variable) biomass concentration (g/l) yield of biomass with respect to substrate (g/g) yield of product with respect to substrate (g/g)

[15] was proposed which not only adequately described the observed experimental data well, but was also highly sensitive to nutrient limitations and inhibitions. Thus, it was possible to use this model to computer simulate and execute desirable nutrient limitations in alternative modes of reactor operations. In a model-based optimization approach, ofine computer simulations are done to predict the nutrient (nitrogen/fructose) feeding strategies for desired biomass and/or PHB accumulation. One such suitable feeding strategy of fed-batch cultivation for high productivity was devised, implemented and compared with the simulation results to demonstrate the applicability of modelbased optimization approach to a peculiar microbial cultivation which features high PHB accumulation under very specic nutrient conditions.
2. Materials and methods 2.1. Microorganism and media
Media used for cultivation of W. eutropha (R. eutropha NRRL B14690) in the present study was as follows: urea1.0 g/l; CSL0.5 g/l; KH2 PO4 1.5 g/l; Na2 HPO4 4.0 g/l; MgSO4 7H2 O0.51 g/l; CaCl2 0.02 g/l; trace metal solution10 ml/l [16]. The trace metal solution consisted of: 1.3 mg/l ZnSO4 7H2 O, 0.2 mg/l FeSO4 7H2 O, 0.6 mg/l (NH4 )6 Mo7 O24 4H2 O and 0.6 mg/l H3 BO3 . Fructose was used as carbon source in concentration of 40 and 10 g/l for production media and inoculum development, respectively. Fructose, urea, Na2 HPO4 and salt solutions were sterilized separately at 121 C and then aseptically reconstituted at room temperature prior to inoculation. The pH of the resulting broth was adjusted to 7.0 with 2N NaOH/2N HCl.

Greek letters constant in Eq. (8) constant in Eq. (8) ij difference between the model and experimental values specic growth rate (h1 ) m maximum specic growth rate (h1 )

2.2. Culture conditions

Seed culture was prepared in 1 l Erlenmeyer ask containing 200 ml media with 10 g/l fructose agitated at 150 rpm at 30 C for 24 h. Batch cultivation was carried out at 30 C in a 7 l, LF-1523 Bioengineering (Bioengineering AG, Switzerland) bioreactor containing 4 l media. The reactor was equipped with two conventional turbine-type impellers and four bafes. The reactor was sterilized in situ at 121 C for 20 min, cooled and then inoculated with 5% (v/v) inoculum. Culture pH was maintained at 7.0 by automatic addition of 2N NaOH/2N HCl through a pH-mV controller M 7832N. Dissolved oxygen concentration was maintained at 30% saturation value by manually adjusting the speed of the agitator as well as ow rate of sterile air. The dissolved oxygen concentration in the fermentor was measured using an in situ Ingold DO probe (Ingold, Switzerland). Samples were withdrawn at an interval of 34 h. Residual biomass (R) was described as total dry cell weight minus PHB.

biomass. They also incorporated a term for product inhibition and a term which would act as a nitrogen switch for production of PHB. Patwardhan and Srivastava [13] then re-identied the same model for their experimental data and improved the biomass and PHB production by implementing the fed-batch nutrient feeding strategies. Shahhosseini [14] extrapolated the Mulchandani et al. [11] model structure for fed-batch cultivation. Two model simulated nutrient feed rates for fed-batch cultivation were designed and implemented by them which resulted in a 50 and 150% increase in PHB productivity, respectively, as compared to batch. However, the nal maximum PHB content obtained in fed-batch cultivation was only 7.5 g/l. Since, tting of the experimental data in the form of a mathematical model can tremendously reduce the number of trial and error experiments for improving the PHB productivity, batch experimental data were tted [15] to two literature reported models (Mulchandani et al. and Raje and Srivastava). However, both the model structures were not able to explain the kinetics of growth and product formation of the experimental data [15]. To overcome these deciencies, a simple kinetic model

2.3. Fed-batch cultivation

The culture conditions were same as explained above. The reactor was run as batch till 20 h after which nitrogen feeding (7 g/l) was started at a rate of 70 ml/h using peristaltic pumps (Watson-Marlow 101U, England). Feeding of nitrogen was carried out in the form of urea. The amount of urea equivalent to 7 g/l nitrogen was used as stock solution. Fructose feeding (300 g/l) was started at a rate of 80 ml/h at 30 h. Both nutrient feeding(s) were carried out for 10 h. Samples were withdrawn at regular intervals of 34 h.

2.4. Analytical methods

Optical density (OD) of the cell suspension at 600 nm was measured by UVIKON 930 Spectrophotometer (Kontron Instuments, USA). Biomass was estimated from an OD versus concentration (g/l) correlation, established a priori. The supernatant obtained by centrifugation (Sorvall RC5B Centrifuge) of the culture broth at 10,000 rpm for 10 min (at 4 C) was used for residual substrate analysis. The rest of the estimation methods were same as described earlier [15].

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gration program based on the RungeKutta method of fourth order. The optimization program for the direct search of the minimum of the multivariable function was based on the original method of Rosenbrock [19]. The minimization criteria used was:
n m

SSWR =
i=1 j =1

2 ij
2 Wj

(1)

where: SSWR represents sum of the square of weighed residues. i and j are the number of experimental data points and number of variables, respectively. n and m are the total number of experimental data points and total number of variables, respectively. Wj is the weight of each variable (usually the maximum value of each variable). ij is the difference between the model and experimental value (ymodel yexpt ). Model consisted of the following equations. Biomass (X) was structured as having two components: X=R+P (2)

Fig. 1. Batch fermentation kinetics by W. eutropha. Comparison between experimental data (points) and simulation results (solid line) ( ) = fructose; ( ) = PHB; ( ) = total biomass; () = nitrogen.

3. Results and discussion 3.1. Cultivation in 7 l bioreactor Two batch experiments were carried out in a 7 l, LF-1523 Bioengineering (Bioengineering AG, Switzerland) bioreactor and the average values of the biomass, PHB and residual nutrients are shown in Fig. 1. W. eutropha was grown in nitrogen limited batch cultures. After a lag phase of approximately 10 h, the biomass increased to 19.7 g/l in 60 h. In this period, 0.49 g/l nitrogen and approximately 30 g/l of fructose was metabolized. The synthesis of PHB started after 15 h, although a small amount was produced during the growth phase also. The nal concentration obtained at the end of the fermentation was 10.9 g/l. The yield (YP/S ) of PHB to fructose was 0.36 g/g and productivity of 0.18 g/l-h was obtained. 3.2. Modelling Development of the mathematical model was based on the following assumptions: I. Biomass (X) is composed of two components: a. The catalytically active component consists of proteins and nucleic acids (R) b. Product PHB (P) is an inert component II. Nitrogen is the limiting nutrient affecting the PHB production in a complex manner. 3.3. Batch model Average values of different process variables (biomass, nitrogen, PHB and fructose) at different time points were calculated and used for identication of model parameters. For optimal estimation of model parameters, a non-linear regression technique assisted by a computer program [17,18] was used to minimize the deviations between the model predictions and the experimental batch results. For the calculation of the model parameters, the system of differential equations was solved using an inte-

The specic growth rate () of the microorganism was expressed as a function of two limiting nutrient (fructose (S1 ) and nitrogen (S2 )) concentration by the sigmoidal relationship [10]: = m (S1 )n1 (KS1 )n1 + (S1 )n1 (S2 )n2 (KS2 )n2 + (S2 )n2 (3)

where Ks is the saturation constant, m the maximum specic growth rate and n a constant. Presence of a nutrient at high concentration has been shown to decrease the overall value of specic growth rate () [15]. Therefore, a term of substrate inhibition (with respect to fructose and nitrogen) was added to Eq. (3) to obtain nal biomass growth rate equation (4). Thus, the overall differential equation for biomass growth in the reactor was: = 1 dR R dt (S1 )n1 (KS1 )n1 + (S1 )n1 S1 Sm1
a1

= m

(S2 )n2 (KS2 )n2 + (S2 )n2 S2 Sm2

a2

(4)

where Sm1 and Sm2 are the maximum fructose and nitrogen concentrations at which complete inhibition ( = 0) occurs, and a1 and a2 are exponents. In the present model, inhibition term in the specic growth rate equation was expressed as a function of carbon and nitrogen concentration independent of each other as opposed to C/N ratio mentioned in the earlier works [11,12]. This was done because it is difcult to maintain a particular C/N ratio throughout the experiment, as it would require the feeding of both the nutrients continuously. Moreover, for PHB production nitrogen limitation

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S. Khanna, A.K. Srivastava / Enzyme and Microbial Technology 39 (2006) 11451151 Table 1 Model parameters obtained through simulation of batch fermentation Model parameters (units) m KS1 (g l1 ) n1 (Dimensionless) KS2 (g l1 ) n2 (Dimensionless) (g g1 ) (g g1 h1 ) YS2 (g g1 ) mS2 (g g1 h1 ) K1 (g g1 ) K2 Sm1 Sm2 a1 a2 (h1 ) Value 0.302 22.836 3.5938 0.234 2.213 0.48 0.0348 16.7 0.0000045 0.008 0.034 90.11 10.11 3.19 0.97

is needed when PHB accumulation phase starts. Thus, feeding of nitrogen cannot be carried out during this stage. Thus, inhibition was expressed as a function of both the nutrients and their lumped effect on specic growth rate was ensured by taking both terms in the growth rate equation. Product formation was observed in both growth and stationary phases. At high nitrogen concentration product accumulation was low while at low nitrogen concentrations large amounts of product was accumulated. Hence, rate of product formation (qP ) was represented to contain growth (K1 ) and non-growth (K2 ) associated components as described below: qP = 1 dP = K1 + K2 R dt (5)

Fructose utilization rate was assumed to be due to growth of R, formation of P and maintenance (mS1 , proportional to R). The rate can be represented by the following equation: dS1 1 = dt YR/S1 dR dt 1 YP/S1 dP mS1 R dt (6)

esis of a zero mean deviation of the model and experimental data for batch kinetics. The mean residual of each variable j was calculated as follows: j = 1 n
n

YR/S is the yield of biomass with respect to fructose, YP/S is the yield of product with respect to fructose. From Eqs. (4) and (5), it follows: dS1 = dt K1 1 + YR/S1 YP/S1 dR + dt K2 + mS1 YP/S1 R (7)

ij
i=1

Thus, fructose consumption rate (qS1 ) was given by, qS1 = where: = = 1 YR/S1 + K1 YP/S1 (9) (10) 1 dS1 = ( + ) R dt (8)

where n is the total number of experimental data points and ij the difference between the experimental value of a variable and its corresponding model simulation value. The variance of the error of a residual (Sj ) was then estimated as follows: Sj = 1 n1
n

(ij j )2
i=1

where m is the number of variables. The value of the statistics dened as () was calculated. = (n m)n (n 1)m
m j =1

K2 + mS1 YP/S1

2 j Sj

Nitrogen is consumed due to the growth of the catalytically active biomass (R) and for the maintenance functions (mS2 ) of the cell. The specic nitrogen uptake rate (qS2 ), is represented by the following equation: qS2 = 1 dS2 = + mS2 R dt YX/S2 (11)

YX/S2 is the yield of biomass with respect to nitrogen. The values of the optimized parameters (Table 1) were found out by minimizing the difference between model predictions and experimental data at all data points and all process variables. The model Eqs. (2)(11) described above were simulated on the computer using the optimal values of the model parameters. A comparison of the model simulation (smooth line) and experimental data (points) is shown in Fig. 1. The agreement between the model simulation and the experimental data (points) is clearly reected. To further evaluate the degree of reliability of the model, a method recommended by Bard [20] was used to test the hypoth-

The statistics has the F(m,nm) distribution. Its value was calculated as 5.1, which was less than the F(4,11) value (obtained from F tables) for 99% condence for the whole experimental set. This made it possible to accept the hypothesis of zero mean deviation between experimental data and the model thus establishing the validity of the model. It was of interest to further extend the applicability of the proposed model to the literature data. Therefore, applicability of the proposed model was examined to represent the batch kinetics of W. eutropha reported by Mulchandani et al. [11]. The simulation results are presented in Fig. 2. The proposed model adequately described the experimental observation of growth and product formation of W. eutropha as reported by Mulchandani et al. [11]. This further reinforced the validity of the proposed model structure. It was observed that the proposed model helped in providing a mathematical description to the observed experimental batch kinetics. The growth rate of the culture could be explained as a function of both inhibition and limitation by the major nutrients.

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Fig. 2. The comparison of experimental data from Mulchandani et al. [11] and simulated values obtained through the proposed model. Dots represent the experimental data of Mulchandani et al. [11] and the smooth lines represent the simulated values obtained through the present model.

The developed model is a very simple, yet useful tool for understanding the behavior of microbial system W. eutropha. 3.4. Fed-batch model The model was further extrapolated to the fed batch cultivation by taking into account the dilution due to the nutrient(s) feeding (Table 2). This was then used for simulation of several fed batch strategies for improvement in the productivity. Model-based fed batch cultivations would help in providing a more logical approach to fed batch cultivations and would minimize the experimentation to increase the yield and productivity of PHB accumulation. The fed-batch model equations are given below: dV = F1 + F2 = F dt dR F = R R dt V dS1 F1 S01 FS1 = [ + ]R + dt V V dS2 F2 S02 R FS2 + mS2 R + = dt YX/S2 V V dP dt = [K1 R + K2 R] F P V (12) (13) (14) (15) (16)

F1 and F2 are fructose and nitrogen feed rate, respectively. F is the total feed rate. V is the volume of the reactor. In batch cultivation, nitrogen was totally exhausted in 20 h; however, 30 g/l of fructose was still available. A fed-batch was therefore designed (using the mathematical model) to suppleTable 2 Comparative table for batch and fed batch cultivation Cultivation strategy Biomass (g/l) Pred Batch Fed-batch 21.2 38.8 Exp 19.7 32 PHB (g/l) Pred 12.6 17.7 Exp 10.9 14 4.0 5.5

ment the nitrogen concentration and enhance the biomass accumulation. Objective of developing a nutrient feeding strategy was to rst increase the biomass accumulation by supplementing nitrogen and in the later stages when the reactor had high biomass, then induce conditions for PHB accumulation. A simple constant feeding of nitrogen was attempted, starting from the time when nitrogen concentration approached almost zero level (20 h) in batch fermentation. The feeding of nitrogen was done to increase the biomass so that PHB accumulation was induced in higher amount of biomass. 7 g/l of nitrogen was fed at a constant rate of 70 ml/h from 20 h so that its limitation could be removed. However, this led to a decrease in the concentration of fructose at around 24 h. Therefore, fructose feeding (80 ml/h) of 300 g/l was also initiated from 24 h for a period of 10 h. The feeding of nitrogen was stopped at 30 h. Further, simulation indicated that feeding of fructose any further than this led to a decrease in the nal biomass accumulation. This was also associated with a decrease in the product formation. To avoid this the reactor was operated as batch after 34 h (for the next 16 h) so that the excess fructose accumulated in the reactor could be converted to PHB. The batch operation after 34 h also prevented unnecessary wastage of nutrients towards the end of fermentation. The fed-batch simulation model indicated an accumulation of 38.6 g/l biomass with a PHB content of 17.9 g/l in 50 h. Therefore, this fed-batch cultivation strategy was found interesting and was experimentally implemented. The effect of feeding of fructose and nitrogen on batch fermentation kinetics is shown in Fig. 3. The fermentation was completed in 50 h with a biomass and PHB concentration of 32 and 14 g/l, respectively. The polymer content of the cell was 44%. PHB production started after 34 h when nitrogen had become limiting. The rate of PHB formation during this period was 0.48 h1 . An overall PHB productivity of 0.28 g/l-h was obtained towards the end of the fermentation. The strategy resulted in a higher biomass production rate of 3.5 g/h as opposed to 1.3 g/h in batch cultivation. There was a 2-fold increase in the PHB production rate increasing from 0.7 g/h in batch cultivation to 1.5 g/h in fed-batch cultivation. In order to nd the validity of the fed batch model, the hypothesis of a zero mean deviation of the model and experimental data for fed-batch kinetics was carried out. The statistics has the F(m,nm) distribution. Its value was calculated as 8.8, which was less than the F(4,6) value (obtained from F tables) for 99% condence for the whole experimental set. This made it possible to accept the hypothesis of zero mean deviation between experimental data and the model, thus establishing the validity of the model for fed-batch cultivation.

Final volume (l)

Biomass prod. rate (g/h) Pred 1.4 4.3 Exp 1.3 3.5

PHB prod. rate (g/h) Pred 0.8 1.9 Exp 0.7 1.5

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Fig. 3. Fed-batch fermentation with constant nitrogen and fructose feeding. Comparison between model predictions (solid line) and experimental data (points) ( ) = fructose; ( ) = PHB; ( ) = total biomass; () = nitrogen). Nitrogen feeding (7 g/l) was carried out during 2030 h at the rate of 70 ml/h. Fructose feeding (300 g/l) was carried out during 2434 h at the rate of 80 ml/h. Batch fermentation (3550 h).

This strategy resulted in high PHB production with a very simple feeding prole, i.e., with just constant feeding of nitrogen and fructose. This was considerable improvement than that obtained in batch (0.18 g/l-h). In earlier studies, Shahhosseini [14] had used a model-based approach for fed-batch cultivation. Although, a 50 and 150% increase in PHB productivity was achieved in two fed-batch strategies as compared to batch but the nal maximum PHB content obtained in fed-batch cultivation was only 7.5 g/l. In the literature studies, Patwardhan and Srivastava obtained 7 g/l PHB in 44 h, corresponding to 16% polymer content in fed-batch cultivation [13]. A two-stage cultivation method was used to produce PHB using R. eutropha ATCC 17697 (equivalent to NRRL B 14690 used in the present study) [21]. Biomass and PHB accumulation of 22.9 and 12.6 g/l were obtained in 83 h leading to productivity of 0.15 g/l-h. Moreover, cultivation was carried out under autotrophic conditions (where a mixture of CO2 , H2 and O2 was used for PHB accumulation). In autotrophic fermentations there can be a possibility of spontaneous detonation. To prevent this oxygen concentration in gas phase has to be kept below 6.9% (v/v), which could result in very low oxygen transfer rate and result in oxygen availability problem to growing culture. A constant nutrient feeding strategy similar to the one used in the present study has been reported in the studies carried out by Grothe and Chisti on Alcaligenes latus [22]. After the rst experiment, an accumulation of carbon and nitrogen sources was found by the end of the fermentation. Thus, a second experiment was carried out, wherein the time of feeding and concentration of feed was changed. Although, this resulted in a higher productivity than the rst experiment but at the expense of time and valuable nutrients, which could have been prevented, had they adopted model-based fed-batch strategy. During the two experiments a productivity of 0.31 and 1.15 g/l-h has been reported but only the feeding time was considered while calculating the productivity. A high productivity of

2.42 g/l-h was obtained with fed-batch fermentation using nitrogen limitation [23]. Here, glucose concentration was maintained between 0 and 20 g/l using on-line enzymatic glucose analyzer. Although a high productivity (2.42 g/l-h) was obtained but in a large-scale cultivation it is difcult to use such enzymatic sensors for control of substrate concentration. All the fed-batch studies mentioned above focused on maintenance of either pH, DO or substrate concentration, which were used a feed back parameter for deciding the feeding rate of the substrate. However, a model-based approach, wherein the developed mathematical model accounts for the inhibition and limitation has not been greatly explored. This helps in generating feeding strategies wherein the feeding rates, time and concentration can be decided beforehand and then implemented for high PHB production. In the present study, this different approach was used to optimize the feeding rates of limiting nutrients. The proposed batch model was extrapolated to fedbatch by incorporating dilution terms arising due to feeding of nitrogen (as urea) and fructose. Simulation strategies featuring better productivity as compared to batch studies were experimentally implemented. Ideally on-line measurement of process variables and feed back control is desirable for better results from model-based process optimization but this is cumbersome, as it requires measurement of substrate and product concentrations. Measurement of product concentration is rather difcult particularly when the product is intracellular in nature. Therefore, in the absence of appropriate sensors in process industries, model-based ofine and feed forward optimizations are rather simple and very useful in improving the process productivity.

4. Conclusions Batch kinetics of the PHB synthesis was established in a 7 l bioreactor under controlled conditions of pH (7.0) and DO (30%). It resulted in a total biomass content of 19.68 g/l with a productivity of 0.18 g/l-h. A mathematical model was proposed for the synthesis of PHB by W. eutropha and model parameters were identied using the batch experimental results. The model successfully simulated the observed batch kinetics. The applicability of the developed mathematical model was demonstrated for the computer simulation of nutrient feed strategies for enhanced PHB production and accumulation in the cell population in fed-batch cultivation. A feeding strategy was designed wherein nitrogen feeding (7 g/l) at the rate of 70 ml/h was carried out for a period of 10 h starting from 20 h. Thereafter, fructose feeding (300 g/l) was carried out at the rate of 80 ml/h initiating from 24 h and continuing till 34 h. These feeding rates and feeding time ensured presence of excess carbon and nitrogen during the growth phase of the culture and limiting nitrogen and excess fructose during production phase of the culture. After implementation a total biomass of 32 g/l with a PHB content of 14 g/l (productivity, 0.28 g/l-h) was obtained in 50 h with this strain of W. eutropha. Productivity greater than batch cultivation (0.18 g/l-h) carried out with the same culture was obtained with the model-based fed-batch cultivation.

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Acknowledgement One of the authors (Ms. Shilpi Khanna) is thankful to the Council of Scientic and Industrial Research (CSIR), Govt. of India, New Delhi, for providing fellowship during the work done. References
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