Simulation of Penicillin Production in Fed-Batch Cultivations Using a Morphologically Structured Model

Teresa C. Zangirolami,1 Claus L. Johansen,2 Jens Nielsen,3 Sten Bay Jrgensen1

1 Chemical Engineering Department, Technical University of Denmark, DK-2800, Lyngby, Denmark 2 Unilever Research Laboratorium, NL-3133 AT, Vlaardingen, The Netherlands 3 Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark

Received 1 August 1996; accepted 19 December 1996

Abstract: A mathematical model is formulated to describe trends in biomass and penicillin formation as well as substrate consumption for fed-batch cultivations. The biomass is structured into three morphological compartments, and glucose and corn steep liquor are considered as substrates for growth. Penicillin formation is assumed to take place in the subapical compartment and in the growing region of the hyphal compartment. Furthermore, it is inhibited by glucose. Model parameters are estimated using an evolutionary algorithm and fitting the model to a standard fed-batch cultivation. The model is validated on experimental data from three different fedbatch cultivations, including two repeated fed-batch cultivations. The model predictions show good agreement with the measurements of biomass and pencillin concentrations for all fed-batch cultivations. 1997 John Wiley &
Sons, Inc. Biotechnol Bioeng 56: 593604, 1997.

Keywords: structured model; fed-batch cultivation; repeated fed-batch cultivations; penicillin production; Penicillium chrysogenum

INTRODUCTION Penicillin production is one of the most important industrial cultivation processes. Traditionally it is carried out in a fed-batch mode, and the development of mathematical models for the process as a whole is crucial for gaining understanding of the mycelial physiology as well as for process optimization and control. There have been many attempts at modeling penicillin production in fed-batch cultivations. The unstructured model developed by Bajpai and Reu (1980, 1981) has become the reference model for other unstructured models with different levels of complexity (Heijnen et al., 1979; Nicola et al., 1991; Menezes et al., 1994; Tiller et al., 1994). In some of these improved versions, the contribution of the complex nitrogen source for

Correspondence to: Jens Nielsen Contract grant sponsor: CAPES, Brazil Contract grant number: DBE6351/94, PROC 1488/94

the biomass formation is taken into account and the biotic phase has been divided into two different cell types: active (metabolizing) and inactive (nonmetabolizing) biomass (Menezes et al., 1994; Tiller et al., 1994). Despite these enhancements, the extended models fail to provide a good description of the process, presumably due the neglect of inevitable cellular differentiation observed in cultivations of filamentous fungi. In fact, Paul and Thomas (1996) demonstrated that penicillin formation is related to cellular differentiation during growth. Recent measurements of different cell forms during fed-batch cultures showed that penicillin production is correlated with the fraction of subapical cells in the mycelium. As early as 1970, Megee et al. (1970) proposed a morphologically structured model describing the growth and product formation of Aspergillus awamori. The main drawback of the Megee model is the large number of parameters, which makes its identification and validation difficult. Later Nestaas and Wang (1983) used a simplified version of the model of Megee et al. (1970) to implement a control strategy for penicillin production. The kinetic expressions proposed for differentiation did not include the influence of a limiting substrate which severely restricts its applicability. Furthermore, in order to fit experimental data, different parameter values had to be applied for the different phases of the fed-batch cultivations. Recently Paul and Thomas (1996) formulated a very comprehensive mathematical morphological model describing growth, differentiation, and penicillin production in Penicillium chrysogenum. The model is structured into four compartments and contains five metamorphosis reactions. Penicillin production is assumed to occur only at the nongrowing region. The model was validated by comparing the simulation results with experimental data from four different fed-batch cultivations. For all cases, the model fits the biomass concentration and penicillin concentration quite well. However, the model is rather complex and has a large

1997 John Wiley & Sons, Inc.

CCC 0006-3592/97/060593-12

number of parameters, several biomass compartments, and model states. Besides, the contribution of corn steep liquor (CSL) to the biomass formation is not explicitly accounted for. In the simplified morphologically structured model formulated by Nielsen (1993), the hypha is divided into three morphological forms (apical, subapical, and hyphal compartments). The model comprises three metamorphosis reactions as well as growth reactions for apical and subapical compartments. The result is a flexible model containing only eight parameters with well defined physical meaning. The model is capable of representing morphological changes of four different filamentous microorganisms. This article investigates the applicability of Nielsens morphologically structured model (1993) to describe penicillin production in fed-batch cultivations. It is assumed that penicillin production takes place in the subapical and part of the hyphal compartments and the specific penicillin production rate is represented by Haldane kinetics. In addition, corn steep liquor is also taken into account as a substrate for growth. Considering the complexity of the process, the final result is a relatively simple model containing eight states and 14 parameters. The predictive power of the model is illustrated by comparison of simulation results to experimental data from four different fed-batch cultivations, including two repeated fed-batch fermentations. In the following, a detailed description of the model is presented, accompanied by an outline of the methodology used for parameter estimation, model validation and sensitivity analysis, as well as a discussion of simulated results.

is called the subapical compartment (Zs) (Fiddy and Trinci, 1976). Cells further away from the tip contain large vacuoles. These cells do not participate directly in the tip extension process, but they are believed to be of importance in creating a sufficient intracellular pressure that ensures transport of protoplasm toward the tip section. This part of the hyphal element is referred to as the hyphal compartment (Zh). The transition from active subapical cells to completely vacuolated hyphal cells takes place gradually. Therefore, the hyphal cells located in the vicinity of the subapical compartment belong to a transition state and, to some extent, they are assumed to still retain the same metabolic activity and growth ability exhibited in the subapical compartment. Figure 1 shows the position of the three compartments and the transition zone in an enlarged picture of the main hypha.

Metamorphosis Reactions Three metamorphosis reactions are considered in the model: branching, tip extension, and differentiation (Nielsen, 1992, 1993, 1995). When a tip extends some apical cell mass becomes subapical cell mass and the net result of the tip extension is the formation of subapical cells from apical cells. Branching is the mechanism by which new apical compartments are formed and it is mainly observed in the subapical compartment. When the cell age increases, the cell becomes more and more vacuolated. These vacuolated cells have a completely different metabolism than the actively growing apical and subapical cells and eventually do not contribute to the overall growth process. Differentiation represents the sum of processes by which vacuolated hyphal cells are formed from the subapical cells. The kinetic expressions for branching (u1) [Eq. (1)] and tip extension (u2) [Eq. (2)] are considered to be first order in the morphological form that disappears. The kinetic expression for differentiation (u3) [eq. (3)] is also first order in Zs, and it is furthermore assumed to be inhibited by the total substrate at high concentrations. The total substrate concentration ST is

MODEL DESCRIPTION In the model described here, glucose is regarded as the only limiting substrate for growth and penicillin production. The other key nutrients such as phenoxyacetic acid and oxygen are assumed to be in excess during the whole cultivation. Although the supply of sufficient amounts of oxygen may become a critical problem at high biomass concentrations, as long as the dissolved oxygen concentration is kept above 45% of the saturation value, oxygen limitation on both biomass and product formation is unlikely to occur.

Morphologically Structured Model The morphological structure of the model is described in detail in Nielsen (1993, 1995) and is therefore only summarized here. The hyphae are divided into three cell types: apical, subapical, and hyphal. Active growth (e.g., uptake of substrates and formation of biomass) occurs mainly in the apical and subapical compartments. The apical compartment (Za) is defined as the part of the hyphal element between the tip and the first septum. The cells just behind the apical compartment have an intracellular composition very similar to the apical cells and this part of the hyphal element

Figure 1. Division of the hyphal element into three compartments: apical, subapical, and hyphal. Septa in the subapical compartment and the transition zone are also shown.

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taken to be the extracellular glucose concentration plus the concentration of nutrients in corn steep liquor converted into glucose equivalents according to Eq. (4). Branching: Zs Za Tip extension: Za Zs Differentiation: Zs Zh u3 = ku3 Zs ST Ku3 + 1 (3) (4) u2 ku2 Za (2) u1 ku1 Zs (1)

Notice that fragmentation, cell lysis, degeneration, and mutation are not considered in this model. According to Paul et al. (1994), there is a link between vacuolation and fragmentation. Because vacuolation takes place mainly at the hyphal compartment, it will be more susceptible to breakage under the shear caused by mechanical forces. Thus, fragmentation will affect the total hyphal lengths and the mean number of tips per mycelium, but the fractions of apical, subapical, or hyphal cells in the total biomass will remain unchanged. Fragmentation will lead to some cell lysis. However, since there is no experimental evidence to support cell lysis, it is not considered in the model. Product Formation The formation of penicillin from glucose and phenoxyacetic acid can be expressed by the simplified stoichiometric equation represented below. The stoichiometric coefficients are in (C mol glucose)/(C mol penicillin) and (C mol phenoxyacetic acid)/(C mol penicillin). The elemental composition for penicillin is CH1.1875O0.3125N1/8S1/16 (Nielsen, 1995). 0.5SGLU + 0.5SPHOX P (12)

ST SGLU + CSL SCSL

where CSL is a conversion factor for the nutrients in corn steep liquor. Growth Reactions Simplified stoichiometric expressions for apical and subapical compartments as well as for the active fraction of the hyphal compartment (fh) are formulated in Eqs. (5), (6), and (7). The stoichiometric coefficients 1a, 1s, and 1h are in (C mol glucose)/(C mol biomass). The elemental composition for biomass is CH1.7O0.55N0.16 (Christensen, 1992). Apical compartment: 1a SGLU X + (1a 1)CO2 Subapical compartment: 1s SGLU X + (1s 1)CO2 Active fraction of the hyphal compartment: 1h SGLU X + (1h 1)CO2 (7) (6) (5)

Penicillin production is assumed to occur at the subapical compartment and at the growing part of the hyphal compartment as a consequence of differentiation. In addition, the presence of excess glucose can inhibit penicillin formation. This effect is expressed by Haldane type kinetics. rP = k2 SGLU
2 SGLU SGLU + K2 + KI

Zs + fhZh

(13)

The growth of apical, subapical, and the active fraction fh of hyphal cells is described by Monod kinetics [Eqs. (8), (9), and (10), respectively]. a = ka s = ks h = kh
ST ST + K s ST ST + K s ST ST + Ks

(8) (9) (10)

For all experiments considered in this work, the measurements of penicillin V and hydrolysis products were pooled into one fraction called total penicillin V equivalents. Thus, the hydrolysis reaction does not need to be included, but the model of Christensen et al. (1992) could easily be applied in combination with the present model to describe the profile of both penicillin V and penicilloic acid. Consumption of Glucose and Corn Steep Liquor Corn steep liquor is the major nitrogen source present in penicillin cultivations. It contains a large number of nutrients such as free amino acids (around 40% in weight), proteins, and vitamins, as well as lactate in high concentrations (Johansen, 1993; Nielsen, 1995). Due to this complex composition, it is difficult to specify each of the components contributing to growth. In this model, the fraction of corn steep liquor utilized for growth is converted into glucose equivalents by the conversion factor CSL. Thus, the total amount of substrate available for biomass formation can be expressed by Eq. (4). According to Johansen (1993), after 30 h of cultivation the amino acids content in corn steep liquor is almost depleted. This time period coincides with the sharp initial

The specific growth rate of the total biomass is given by aZa + SZS + fhhZh (11)

Since the cells located at the transition zone (the apical and the subapical cells) are fairly similar in composition and function, it is reasonable to assume the same growth kinetics for them. Thus, in expressions (5), (6), and (7) the stoichiometric coefficients 1a, 1s, and 1h will be taken to be equal (1a 1s 1h 1). For the same reason, the rate constants in the expressions for the specific growth rate [Eqs. (8), (9), and (10)] are assumed to be the same (ka ks kh k).

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increase in the biomass concentration typically observed in fed-batch penicillin cultivations. Tiller et al. (1994) included corn steep liquor in the model by adding another expression for the specific growth rate as a function of corn steep liquor concentration. However, the specific growth rate is found to be the same in a defined and in a complex medium (Nielsen and Krabben, 1995). To account for the quick assimilation of the individual nutrients in corn steep liquor, the uptake rates of these nutrients are assumed to be proportional to the relative amount of nutrients, which is similar to the approach used by Menezes et al. (1994). With all the nutrients of corn steep liquor pooled together, this gives only two substrates: glucose and the pool of nutrients in corn steep liquor [Eqs. (14), (15)]. The uptake rate of each substrate is taken to be proportional to its relative amount. The expression for the specific rate of glucose uptake also includes the consumption of glucose for maintenance and product formation [second and third terms in Eq. (14), respectively]. rGLU = 1 rCSL = 1 SGLU + ms + 2rP SGLU + CSL SCSL SCSL SGLU + CSL SCSL (14)

the fermentor (F), withdrawal of manual samples (FP sp), continuous on-line sampling (Fols), evaporation and medium carryover by the air flow, and addition of base and acid for pH control. The latter two factors tend to compensate each other and they were omitted from the volume balance shown in Eq. (19). For repeated fed-batch cultivations, the drain of cultivation medium (FP drain) and the addition of ) must also be taken into account. fresh medium (FP rep dV P P P = F Fsp Fols Fdrain + Frep dt Total biomass dX = X dt (19)

P F + Frep V

Fols X+X V

(20)

The second term in Eq. (20) reflects the dilution of biomass concentration due to the additions to the bioreactor. During on-line sampling, filtrated medium flows from the bioreactor and the last term represents the biomass retention caused by the on-line sampling. Penicillin V production

(15)

dP = rPX dt Glucose consumption dSGLU = dt

P F + Frep V

(21)

Differential Balances All kinetic expressions related previously are combined with the differential balance equations for fed-batch bioreactors in Eqs. (16)(23).

P F Frep SF + Srep V V

P F + Frep V

SGLU rGLUX (22)

Morphological Compartments
The formation rate of each morphological form is determined by the metamorphosis reactions and by the growthassociated reactions for each form. Because the concentrations of morphological forms are given as fractional concentrations of total biomass, the last term in Eqs. (16), (17), and (18) represents the dilution effect due to biomass growth. Apical cells: dZa = u1 u2 + Zaa Za dt Subapical cells: dZs = u2 u1 u3 + Zss Zs dt Hyphal cells: dZh = u3 + fhZhh Zh dt (18) (17) (16)

Consumption of the nutrients pool in corn steep liquor

P dSCSL Frep = S dt V CSLrep

P F + Frep V

SCSL rCSLX

(23)

The second right-hand side terms in Eqs. (21), (22), and (23) represent the dilution effect caused by the additions to the bioreactor. The first terms in Eqs. (22) and (23) reflect the mass flow of substrate added to the bioreactor due to the feed and the addition of fresh medium in repeated fed-batch cultivations. MODEL IDENTIFICATION Material and Methods The experimental data used in this work were from four fed-batch cultivations carried out by Johansen (1993). The microorganism used was a high yielding strain of P. chrysogenum donated by Novo Nordisk A/S, Denmark. All cultivations were carried out in a standard 41-L Chemap bioreactor with a working volume of 25 L. The experimental conditions are reported in detail elsewhere (Johansen, 1993; Jrgensen et al., 1995a,b; Nielsen, 1995) and are summarized in Table I. Experiment FB023 was performed as a standard fed-batch cultivation and it was used for parameter estimation. Runs FB022 and FB027 were started as normal fed-batch cultivations. After 147 h for FB022 and 144 and

Reactor Volume
In fed-batch cultures the total volume changes mainly due to the following factors: feed of glucose and other nutrients to

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Table I. Initial corn steep liquor concentration (SCSL) in the medium, glucose concentration in the feed (SF) and feed profiles for fed batch cultivations FB023, FB022, FB027 and FB028. (Data from Johansen9) Feed Rate Profile Cultivation FB023 Characteristics Standard fed-batch Initial SCSL in the medium: 200 g/l (80 g/l of nutrients) SF 450 g/l Repeated fed-batch Initial SCSL in the medium: 200 g/l (80 g/l of nutrients) SF 450 g/l Srep 3 g/l Repeated fed-batch Initial SCSL in the medium: 100 g/l (40 g/l of nutrients) SF 450 g/l Srep 3 g/l Period (h) 06 654 54240 06 650 50147 148173 173264 05 548 48144 144168 168286 286310 310406 05 553 53110 Feed rate (l/h) 0 0.061 0.076 0 0.059 0.076 0.059 0.076 0 0.059 0.076 0.059 0.076 0.059 0.076 0 0.059 0.069

FB022

FB027

FB028

Fed-batch supplemented with a solution of amino acids Initial SCSL in the medium: 100 g/l (40 g/l of nutrients) SF 450 g/l

286 h for FB027, approximately 40% of the bioreactor content was drained and an equal amount of fresh medium was added. Cultivation FB028 was performed as a standard fedbatch fermentation, but the amino acids directly involved in penicillin biosynthesis (cysteine, valine and -aminoadipic acid) were added to the bioreactor in a separate feed. Simulation and Parameter Estimation The simulation and parameter estimation were performed in a MATLAB/SIMULINK environment. This approach was already successfully employed for identification of complex biochemical models by Pisarra et al. (1995). The evolutionary parameter estimation method is outlined in detail by Schmidt et al. (1995). The model represented in Eqs. (1)(23) was implemented in C code. After compilation using a Watcom 32 bit C compiler, the model equations were integrated by Gears method for stiff systems. The inputs to the model consisted of the flows contributing to the volume changes. The substrate feed flow rate was implemented as a step change whereas the on-line sampling flow rate was included as a constant continuous flow. The intermittent flows (manual sampling, drainage of cultivation medium, and addition of fresh medium) were implemented as pulses of determined duration and intensity. For parameter estimation, the experimental measurements of biomass, penicillin, and substrate concentrations from the standard cultivation FB023 were compared to the simulation data generated for each set of parameters. Optimization of the parameter vector was accomplished by random perturbations of the parameter val-

ues and subsequent selection of the parameter that improved the objective function (sum of the squared deviation between model predictions and experimental results). From the 14 parameters originally present in the model, only six were estimated by the procedure described above (see Table II). The values of ku1, ku2, and 1 were obtained from Nielsen (1992). The values of ku3 and Ku3 were modified from the ones proposed by Nielsen (1992) in order to fit the repeated fed-batch cultivations. For the same reason, fh was taken to be 0.13. The value for the rate constant k was chosen as 0.14, which is within the range reported (Menezes
Table II. Model parameters and reported values. FB023 FB022 FB027 2.3000 0.7000 0.1900 20.0000 0.1300 2.2000 0.6858

Parameter ku1 (h1) ku2 (h1) ku3 (h1) Ku3 (l/g glucose) fh (g active Zh/g Zh) 1 (g glucose/g DW) 2 (g glucose/g penicillin)

FB028 2.3000 0.7000 0.1900 20.0000 0.1300 2.2000 0.6858 0.4200 0.1400 0.0015 0.0281 1.77 0.03 0.0132 0.0101

Source Nielsen (1992) Nielsen (1992) Attributed Attributed Attributed Nielsen (1992) Calculated [eq. (12)] Estimated Attributed Estimated Estimated Estimated Estimated Estimated

CSL (g glucose/g CSL) 0.42 0.01 k (h1) 0.1400 Ks (g glucose/l) 0.0015 0.0004 0.0281 0.0008 ms (h1) k2 (h1) 1.35 0.02 K2 (g glucose/l) 0.0132 0.0002 K1 (g glucose/l) 0.0101 0.0002

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et al., 1994). Exactly the same set of parameters estimated for FB023 was used in the simulation of FB022 and FB027. For cultivation FB028 the rate constant for the penicillin production reaction was estimated again in order to cope with the changes in the penicillin kinetics caused by the addition of the three precursor amino acids. It is not possible to directly compare the estimated values for the parameters with the reported ones because most of the parameters depend on the strain of P. chrysogenum as well as on the model structure. However, some of the differences are worthy of mention. The value calculated for 2 is lower than the reported range (0.831.2) (Bajpai and Reu, 1981; Menezes et al., 1994; Paul and Thomas, 1996; Tiller et al., 1994) due to inclusion of phenoxyacetic acid in the stoichiometric equation for penicillin prodution [Eq. (12)]. The value estimated for k2 (FB023) is significantly higher than the reported range (0.0030.015) (Menezes et al., 1994). However, it is important to note that the fraction of the subapical compartment plus the active part of the hyphal compartment is a multiplicative factor in the expression for penicillin production rate. If the value of k2 is multiplied by (Zs + fhZh) after 100 h of cultivation, it is reduced to about 0.18 h1. RESULTS AND DISCUSSION Figure 2 shows experimental data and simulation results for the standard cultivation FB023 used for parameter estimation. The model predictions fit the data for biomass and penicillin concentration very well. In Figure 2a the three distinct growth phases characteristically observed when fedbatch cultivations are carried out on a complex medium are clearly delineated by the model. A rapid growth phase took place during the first 30 h. The initial sharp increase in the biomass concentration coincided with the simulated decrease of the nutrients concentration in corn steep liquor (Fig. 2b), indicating that they were the main support for the growth during the early stage. When these nutrients were depleted, a linear growth phase was initiated. This phase lasted until approximately 150 h of cultivation, and at that point the biomass concentration reached about 45 g/L. At high biomass concentrations, the feed of substrate only allowed very slow growth. The model was not successful in predicting the changes in glucose concentration as can be observed in Figure 2b. The simulated dynamics of glucose accumulation and consumption when the feed is started was faster than experimentally observed. In the production phase, the estimated glucose concentration was approximately 5 104 g/L, which is 10 times lower than the expected value. By decreasing the value of k to 0.13, a better agreement for glucose concentration in the production phase would be achieved; however, an amplified initial overshoot would be obtained. Similar problems were detected in the simulation of glucose concentration for FB022, FB027, and FB028 (Figs. 4b, 5b, 6b, respectively). This deviation is likely to be a consequence of a too

Figure 2. Experimental data and simulation results for standard cultivation FB023.

simple description of the glucose uptake and catabolism in the model, especially the description of the transients taking place in the presence of free amino acids until the depletion of this source. To improve the fit for glucose, a more detailed modeling of glucose uptake as well as modifications in the morphological structure, which would require experimental determination of the morphological compartments in fed-batch cultivations, could be attempted. Furthemore, the misfit of the glucose data may be explained by two other effects: the role of oxygen as substrate was ignored; and initially there were some pellets in the culture (Nielsen et al., 1995) and it may have resulted in a nonhomogeneous population of cells. In Figure 3 the simulated time profiles for the morphological compartments Za, Zs, and Zh are represented. When the glucose concentration decreased and stabilized at a low value, the differentiation processes were no longer inhibited, leading to an increase in Zh and a simultaneous decrease in Za and Zs. Similar trends for the time profiles of the morphological forms were observed in FB022, FB027, and FB028 (data not shown). Unfortunately it was not possible to experimentally quantify the fractions of cell compartments during any of the fed-batch cultivations. Image

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Figure 3. Simulated time profiles for the morphological forms.

analysis measurements of cells cultivated on solid media showed that at poor growth conditions the biomass was characterized by long hyphae with few branch points, which corresponds to a high fraction of the hyphal compartment (Nielsen, 1995). However, the hyphae characteristics in fedbatch cultivations were quite different. Due to fragmentation the total hyphal length and the mean number of tips remained approximately constant in FB027 and FB028 (Johansen, 1993). The measurements of biomass and penicillin concentrations in FB022 are shown in Figure 4a together with model simulations. The simulated biomass and penicillin concentrations showed good agreement with the experimental values almost throughout the whole cultivation. The overpro-

Figure 4. Experimental data and simulation results for repeated fed-batch cultivation FB022. Penicillin was monitored on-line using an FIA-system with a biosensor (Carlsen et al., 1993).

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duction of penicillin after 230 h of cultivation was not followed by the model and might have been caused by experimental errors in the determination of the penicillin concentration. The model also describes the experimental data for biomass and penicillin concentration in the cultivation FB027 remarkably well (Fig. 5a). Here the model shows its predictive capacity by following the dynamics of the three consecutive fed-batch cultivations, where the feed rate was changed 7 times. Notice also the qualitatively correct description of the glucose concentration at the beginning of each addition of fresh medium. At the end of the third repeat, the model predictions for penicillin concentration are higher than the experimental data. This loss of productivity was also verified in glucose limited continuous culture after 200 h, and it is attributed to degeneration of the strain (Christensen, 1992; Nielsen, 1995). The results for fermentation FB028 presented in Figure 6

demonstrate the predictive power of the proposed model. In FB028 the influence of the precursor amino acids of penicillin biosynthesis was investigated. Despite the alterations in the cultivation conditions, only one parameter had to be reestimated for FB028. When the new value of the rate constant for the penicillin formation reaction was used together with the same parameters estimated for FB023, a very good fit between simulation results and experimental data for biomass and penicillin concentrations was obtained for FB028. Indeed, the value of k2 estimated from FB028 was higher than the original value estimated from FB023 (see Table II). It corresponds well with the experimental verification that the specific rate of penicillin production increased due to the addition of the amino acids solution (Johansen, 1993; Nielsen, 1995). The influence of the nutrients pool in corn steep liquor on the initial growth is clear when the changes in the biomass concentration for FB028 (Fig. 6a) are compared to the cor-

Figure 5. Experimental data and simulation results for repeated fed-batch cultivation FB027.

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Figure 6. Experimental data and simulation results for fed-batch cultivation FB028.

responding changes in FB023 (Fig. 2a). The initial concentration of corn steep liquor in FB023 was 200 g/L, and at the end of the rapid growth phase the biomass concentration was over 20 g/L. In FB028 the initial concentration of corn steep liquor was only 100 g/L, and the biomass concentration at the end of the rapid growth phase reached approximately 15 g/L. The sensitivity of the model states to changes in the different parameters was investigated according to the method proposed by Holmberg (1982). The original model was extended to include the parameter sensitivity functions and integrated again. The time profiles for the relative sensitivities are plotted in Figure 7ad. The relative values are obtained by multiplying the sensitivity functions (Yp) by the corresponding parameter (p). In this way it is possible to compare the influence of the different parameters on a variable. The results obtained for different variables show common features. In the batch phase, the concentration of biomass, glucose, and nutrients in corn steep liquor (Fig.

7a,b,d) are predominantly sensitive to k, , and CSL. In the same way, the morphological states Za, Zs, and Zh are mainly sensitive to the rate constants of metamorphosis reactions (ku1, ku2, and ku3) and growth reaction (k) (data not shown). On the other hand, in the production phase the relative sensitivities do not follow a common pattern and each variable is specifically more sensitive to different parameter sets. Since experimental data for biomass, penicillin and glucose concentration were used for parameter estimation, it is interesting to analyse which parameters are separately identifiable under these experimental conditions. From the relative sensitivities with respect to X (Fig. 7a) it can be concluded that CSL and ms are both distinguishable, the former just after the batch phase is started and the latter at the end of the cultivation. From Figure 7b, where the relative sensitivities for SGLU are represented, one can observe that only Ks is identifiable at the end of the cultivation. The analysis of the relative sensitivities with respect to P (Fig. 7c) is more complicated. The sensitivity functions of

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Figure 7. Time relative parameter sensitivities for (a) X, (b) SGLU, (c) P, and (d) SCSL for a fed-batch cultivation (parameter values shown in Table II). 602 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 56, NO. 6, DECEMBER 20, 1997

k2, K2, ms, and Ks are almost linearly related and the parameters are not completely distinguishable. Notice that the remaining parameter in the penicillin formation kinetics (KI) does not have a significant influence on P. This means that it is not easy to identify the kinetic parameters of penicillin production and different combinations of these parameters could yield the same fit. CONCLUSION The model gives a good quantitative description of penicillin production in fed-batch cultivations. The inclusion of corn steep liquor in the model contributed to an improvement in the biomass prediction. All growth phases were clearly identified and the rapid growth during the initial phase could be related to the consumption of the nutrients in corn steep liquor. The model also provided very good predictions for penicillin concentration. The limited number of states and parameters as well as the demonstrated predictive strength enable the model to be used for control strategies, design of estimators, etc.
We are grateful to Pedro Pissara and Karsten Schimdt who helped in setting up the method for simulation and parameter estimation. The first author also gratefully acknowledges the financial support of CAPES, Brazil (Grant DBE6351/94, PROC 1488/94).

V Ypi Za Zh Zs 1 2 CSL a, s, h

volume (L) sensitivity function for parameter i apical compartment or fraction of apical compartment (g/g DW) hyphal compartment or fraction of hyphal compartment (g/g DW) subapical compartment or fraction of subapical compartment (g/g DW) stoichiometric coefficient for glucose (biomass formation) (g glucose/g DW) stoichiometric coefficient for glucose (penicillin formation) (g glucose/g penicillin) conversion factor (g glucose/g CSL) total specific growth rate (g/g DW h) specific growth rate for apical, subapical, and hyphal cells, respectively (g/g DW h)

References
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NOMENCLATURE
FP sp Fols FP drain FP rep F fh k k2 K2 KI Ks ku1 ku2 ku3 Ku3 ms pi rCSL rGLU rp SCSL SF SGLU SPhOX Srep ST u1 u2 u3 pulse of manual sample (L/h) volumetric feed flow rate for on-line sampling (L/h) pulse of medium drained from the bioreactor (L/h) pulse of fresh medium added to the bioreactor (L/h) volumetric feed flow rate (L/h) active part of the hyphal compartment rate constant for growth reactions (h1) rate constant for penicillin production reaction (h1) saturation constant for penicillin production reaction (g glucose/L) inhibition constant for penicillin production reaction (g glucose/L) saturation constant for growth reactions (g glucose/L) rate constant for branching reaction (h1) rate constant for tip extension reaction (h1) rate constant for differentiation reaction (h1) saturation constant for differentiation reaction (L/g glucose) maintenance coefficient (h1) value of parameter i specific rate of uptake of nutrients in corn steep liquor (g/g DW h) specific rate of glucose uptake (g/g DW h) specific rate of penicillin production (g/g DW h) concentration of nutrients in corn steep liquor (g/L) concentration of glucose in the feed to the bioreactor (g/L) concentration of glucose (g/L) concentration of phenoxyacetic acid concentration of glucose added to the bioreactor in repeated fed-batch cultivations (g/L) total substrate concentration (g/L) branching reaction (h1) tip extension reaction (h1 differentiation reaction (h1)

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fragmentation in Penicillium chrysogenum. Biotechnol. Bioeng. 44: 655660. Pissarra, P. N., Nielsen, J., Bazin, M. D. 1995. Pathway kinetics and metabolic control analysis of a high-yielding strain of Penicillium chrysogenum. Biotechnol. Bioeng. to appear. Schmidt, K., Pissara, P. N., Nielsen, J. 1995. A practical guide to nonlinear optimization in biochemical research using evolutionary strategies: Implementation in the Matlab/Simulink environment. Proceedings of the Nordic Matlab Conference, Stockholm, Sweden. Tiller, V., Meyerhoff, J., Sziele, D., Schu gerl, Bellgardt, K. H. 1994. Segregated mathematical model for fed-batch cultivation of a high producing strain of Penicillium chrysogenum. J. Biotechnol. 34: 119131.

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