ELSEVIER

Biochemical

Engineering

Journal 2 ( 1998)

1 l-21

Biochemical Engineering Journal

A structured model for penicillin production on mixed substrates

G.C. Paul, M.T. Syddall, C.A. Kent, C.R. Thomas *
Centre for Bioprocess Engineering, School of Chemical Received Engineering, 23 January The University

of Birmingham,
1998

Edgbaston,

Birmingham,

B15 2lT,

UK

1998; accepted 7 May

Abstract
A structured kinetic modelpreviouslydeveloped to describe the growth,differentiation, andpenicillinproduction of Penicillium chryysogenum has been enhanced andextended in orderto apply it to a mixedcarbonsource fermentation. Thefilamentous hyphae aredividedinto

four distinctregions onthebasis of theiractivitiesandthephysiological structure (i.e., vacuolation)of thehyphalcompartments: viz., actively growing(mainly apical)regions, non-growing or penicillinproducing regions, vacuoles, anddegenerated or metabolically inactiveregions. A simpleapproach is takento give quantitativedescriptions of hyphal extension, branchformation,vacuolation anddifferentiation.The fermentation medium contained glucose andlactose monohydrate asthe maincarbonsources. The source of the lactose waswhey powder used in excess in theinoculum medium, whilstglucose wasfedcontinuously throughout thefermentation. Lactose, adisaccharide, is hydrolysed to two monosaccharides, glucose and gaiactose, when the residual glucose concentration in the medium drops to a very low level. The utilisationof glucose andthat of galactose following the hydrolysisof lactosewereobserved to occursimultaneously. This allowedthe assumption of simplelactoseutilisationkineticsin which lactosehydrolysiscould be considered asproducingan equivalentamountof glucose. The model hasbeen used for successful predictions of fed-batch penicillinfermentations usinganindustrial P. chyogenum strain under different glucose feedrates.Quantitativeinformation on proportions of thehyphalregions wasobtained from image analysis measurements andtheparameters of themodelwereidentified. Whentheglucose feedrateto theproduction culturewasswitched between ahighand a low value, the modelsuccessfully predicted the dynamicchanges of differentiationandthe resulting penicillinproduction caused by the
variations in the nutrient conditions. The use of image analysis to characterise differentiation as a basis for structured modelling of the penicillin fermentation appears to he very powerful, and such models have great potential for use in process simulation and control of antibiotic

fermentations.
Keywords: Structured

0 1998ElsevierScience S.A. All rightsreserved.

model: Differentiation; Vacuolation; Penicillin production: Penicillium chrysogenum; Image analysis

1. Introduction The commercial production of penicillin is from Penicilchrysogenum, a filamentous microorganismconsisting of a multicompartment structure of morphologically heterogeneoushyphae. The physiology and differentiation of the compartmentschange along the length of the hyphae and during fermentation. Biochemical andmorphological aspects of P. chrysogenum growth, morphology, differentiation and penicillin production have been discussed in several papers [ l-71. Increasing understanding and the availability of experimentaldata allow successful process modelsto be built which incorporate previously unmeasurable biological variables[8]. There are many structured models of the penicillin fermentation basedon someunderstandingof P. chrysogenum
lium * Correspondingauthor.Tel.: E-mail: c.r.thomas@bham.ac.uk +44 1214145355;Fax: +44 1214145324:

physiology 19-121. Although these models involve many parametersand kinetic constants,they are indispensableif the extremely complex nature of the microorganisms is to be taken into account, andthey ought to have considerable practical value. Recently, Paul and Thomas [ 121 developedsuch a model using data acquired by image analysis. The model considereddifferentiation of the hyphae into apical (actively growing) regions, non-growing or penicillin producing regions, vacuoles, and degenerated or metabolically inactive regions. The model was verified on a range of simple fedbatch penicillin fermentationsusing an industrial P. chtyogeaum strain I 12). However, to apply this model to real industrial fermentations, it will be necessaryto incorporate further states.In particular, one ought to allow for the complex substrate mixtures commonly used in fed-batch fermentations. The work reported here is of a development of the structured model of Paul and Thomas [ 121. The part of the model describing hyphal vacuolation and degenerationkinetics has

1369-703X/98/$ - see front matter 0 PUS1369-703X(98)00012-6

1998 Elsevier

Science S.A. All rights reserved

12

G.C.

Paul

et al. /Biochemical

Engineering

Journal

2 (1998)

11-21

been simplified in order to improve the computational speed of simulations and to make the mathematical analysis more tractable. This simplified model has been expanded to incorporate mixed carbon sources containing glucose and lactose. This latter medium ingredient is significant for the study of the penicillin process when an industrial-type complex medium is used. The other essential nutrients (phenylacetic acid, nitrogen and oxygen) were assumed to be in excess during the whole fermentation. The different physiological states of the biomass were obtained by image analysis measurements [5,6] and the model has been tested using data from several fed-batch penicillin fermentations.

12 8RI

2. Materials

and methods

An industrial strain of P. chr)lsogenum (SmithKline Beecham Pharmaceuticals, Worthing, UK) was used. The inoculum culture was grown in a 2 1shake flask ( inoculum volume 0.5 1) using a complex medium [ 131 containing whey powder and corn-steep liquor (Sigma, Poole, UK). A 32-h-old vegetative culture was inoculated in a 6 1 (working volume 5 1) fermenter (Life Science Laboratories, Luton, UK) using a complex medium [ 41. The initial volume after inoculation was 4.0 1. The agitation speed was started at 800 pm and increased to 1000 rpm after 20 h of the fermentation. The temperature was controlled at 25C, pH at 6.5 and aeration at 1 vvm. Various protocols for the addition of glucose (50% w/v) solution were implemented via a computer controlled peristaltic pump (Watson Marlow, Cornwall, UK). Glucose feeding protocols used in fermentations Rl-R4 are presented in Fig. 1. 5%( w/v) phenylacetic acid (PAA) neutralised with sodium hydroxide at pH 7.1 and supplemented with 5%(w/v) ammonium sulphate was added continuously to keep the residual PAA concentrations below 0.2 g/l from inoculation to 20 h, and between 0.45 and 0.60 g/l from 20 h to the end of the fermentation. This was done to avoid the toxic effect of PAA on biomass growth [ 141. All the operating variables were monitored and controlled via the SETCIM data management and control system (AspenTech, MA, USA). Cell dry weight was measured according to the method of Paul et al. [ 61. Analysis of glucose, lactose, PAA and penicillin G concentrations was performed by two on-line HPLC systems (Model 1050, Hewlett Packard, Waldbronn, Germany). Cell-free samples from the fermenter were pumped out continuously through a sterilisable membrane sampling system (ABC, Puchheim, Germany) and passed through a 300 ~1 flow cell built in-house. The flow cell was mounted on a Gilson 232XL on-line column switching device (Gilson, Villiers-le-Bel, France) fitted with two injection ports and connected to the Hewlett-Packard pump modules. The first system, fitted with a Spherisorb S5C8 200 X 4.6 mm column (Hichrom, Reading, UK) and a UV detector was used to monitor PAA, penicillin G and other minor compounds ( such

20

40

60

80

100

120

140

160

Time (h)
Fig. I. Protocols tations R144. for the addition of 50% w/v glucose solution for femen-

as penicillin G degradation compounds and PAA oxidisation compounds). The second system, fitted with a Bio-Rad fermentation monitoring column ( 100 X 7.8 mm column, Cat. No. 1250155, Bio-Rad Laboratories, Hertfordshire, UK) and a RI detector gave concentration measurements of glucose, lactose, galactose, lactic acid and some other minor organic acid compounds. The injection sequences were programmed to give simultaneous sample analysis. Analysis of results, including integration, quantification and report generation was customised using Hewlett-Packard ChemStation application software. The final report was transferred to the SETCIM process computer ( Micro-Vax 3 100 model 40, Digital Equipment, MA, USA) via a RS 232 link. The concentration of PAA in the medium was controlled using a peristaltic pump. A Quantimet 570 (Leica Cambridge, Cambridge, UK) was used for automatic characterisation of hyphal differentiation of P. chlysogenum fermentation samples. The detailed image analysis method and the estimation of the volumes and weights of the differentiated regions have been published previously [ 61.

3. Model description The details of the earlier model have been described in Paul and Thomas [ 121. This section will summarise theoverall structure and provide the kinetic expressions associated with each reaction scheme. The part of the model incorporating the lactose metabolism will be described in full, with

G.C. Paul et al. /Biochemical

Engineering

Journal

2 (1998)

11-21

13

experimental data from the penicillin supporting fermentations. In submerged fermentations the hyphae can be divided into four distinct regions on the basis of the activity and structure of the cells [ 121:
Non-growmg

Growing

tips, A0

Vacuote. A* repion, A,

Actively growing (mainly apical) regions Non-growing cytoplasm Vacuoles (small vacuoles in the non-growing regions) Degenerated or metabolically inactive regions

Autolysed region. Aq

this paper, the biomass states are represented by Ai (where = 0 represents the growing regions; i = 1, the non-growing regions; i = 2, the vacuoles in the non-growing regions; and i = 3, the degenerated regions), the dry weight concentrations corresponding to these regions by a, and the volumetric concentrations by ui. The dry weight contributions of vacuoles were assumed to be negligible. Total biomass (represented by A,, and its concentration by a,) is obtained by a summation of the growing, non-growing and degenerated regions (A,,, A,, and A,). Other states: autolysed biomass, glucose, lactose monohydrate and penicillin G are represented by Ad, S,, Sz and P, respectively and their concentrations (all in g/l) by a4, s,, s2 and p. respectively. The schematic representation of the four hyphal regions is shown in Fig. 2 and the overall reaction scheme is given in Fig. 3. In the model, glucose and lactose are the main carbon sources limiting the growth, differentiation and penicillin production. The influences of nitrogen, oxygen and PAA were not considered because they were kept in excess during the fermentations to avoid limitations by these compounds. Although, the supply of sufficient amounts of oxygen might become a critical problem at high biomass concentrations, particularly at large scales, the mode1 reported here was tested (at the 5-1 scale) with dissolved oxygen levels held above 50% saturation. Squires [ 151 and Konig et al. [ 161 reported critical dissolved oxygen concentrations for penicillin production of around 5-lo%, whilst Vardar and Lilly [ 171 observed this critical value to be at the higher level of 30%. A very recent study by Henriksen et al. [ 181 with a steadystate P. chrysogenum culture reported that the specific productivity of penicillin remained unchanged at dissolved oxygen concentrations above 23% whilst a drastic drop was observed just below this level. No significant influence on the growth and respiration was observed with dissolved oxygen concentrations between 7-134%. This suggests that a minimum dissolved oxygen level of 50% in all the experiments presented here was high enough to prevent limitation of growth, differentiation and penicillin production. The kinetics of branching, extension, differentiation, vacuolation, degeneration and penicillin production are briefly described below. The full details can be found in Paul and Thomas [ 121. The mechanisms for branch formation and hyphal extension closely follow the concepts of Megee et al. 1191.
In i

Highly v/acuolated compartment

Fig. 2. Schematic representation of differentiation mycelium in submerged fermentation.

of a P. chryogenum

Hydrolysis b

Vacuolation
Autolysis b

Fig. 3. The reaction scheme for the overall process. Glucose and lactose are the limiting substrates for growth, differentiation and penicillin production. Oxygen, nitrogen and phenylacetic acid are kept in excess throughout the fermentation.

3. I. Kinetic expressions 3. I. I. Branch formation A new tip, A,, is produced by the appearance of a branch from the non-growing regions, A,. The branch formation occurs in response to the substrate concentration, and the rate expression for branch formation is given by the following kinetic expression: r -- wOalsl bra- K('fs,

(1)

where h is the rate constant (h - ) , a, is the concentration of A, (g DW/l), si is the concentration of glucose (g S, /l), and K, is the saturation constant (g S, /l) . 3.i.2. Extension When an apical cell, Ao, is produced the hypha extends in length by utilising substrate. Simultaneously non-growing

14

G. C. Paul et al. /Biochemical

Engineering

Journal

2 (1998)

1 l-21

regions, A,, are formed. Thus, the first differentiation occurs as a result of extension. The growth of this process is described by the following expression:

concentration of vacuoles (cm3/1) and p3 the density of the degenerated regions (g DW/cm3). It waspreviously shown that in the heavily vacuolatedregions,the volume proportion of the large vacuoles was approximately 50% of the compartments [ 61; this lead to the factor 2 appearingin Eq. (5). 3.1.6. Autolysis The regions in the hyphae containing large vacuoles can either be autolysed or fragmented by shear forces [7,20] and the rate of lossof materialsby this processis assumed to be first order, proportional to the concentration of the degenerated regions:
rd,4=&a 1

where CL,is the rate constant (h- ), a, is the concentration of A0 (g DWA), and K= is the saturation constant (g S1/I). 3.1.3. Differentiation from A0 to A, This differentiation of A0 to A, responds to the substrate concentration in the medium. The kinetic expression for this is given below. The detailed explanation for the chosen form can be found in Paul and Thomas [ 121 and Megee et al. [ 191: r d-ylao

(6)

K,+s,

(3)

where y, is the maximum rate of substrateutilisation (g S,/ h) for this differentiation process.This equationimplies that differentiation is inhibited by the presence of high concentration of substrate,but at very low substrateconcentrations (e.g., during the production phase) the differentiation might be adequatelydescribedby first order kinetics in a,. 3.1.4. Vacuolation Differentiation of non-growing regions,A,, by the formation of vacuoles (AZ) and further growth of vacuolesby this continuing processare assumed to occur becauseof limitations of the substrates. The reaction mechanism for vacuole formation and growth is describedby Eq. (4) :
rd.2=p1Ic+p2u2

where pa is the first order rate constant (h- ) and a3 is the concentration of the degeneratedregions (g DWA). This accountsfor the total biomass lost by autolysis and fragmentation. No recycling of autolysed materialsis considered. 3. I. 7. Penicillin production Penicillin production is assumed to occur at regions containing non-growing cytoplasm in the presenceof substrate. Excesssubstratecan inhibit penicillin production. Introducing substrateinhibition kinetics of the type used by Bajpai and Reuss[ 21,221, the following expressioncan be obtained for the rate of penicillin production:
lIpLlcPcS1

%= K,+s,(l+s,/K,)

(7)

(4)

where ,u, is the first order rate constantfor vacuole formation (h- ), ulc the volumetric concentration of the non-growing cytoplasm ( cm3/1, total regions-vacuoles+ell walls), b the first order rate constantfor vacuole growth (h- ) and v2 is the volumetric concentration of vacuoles ( cm3/1). This is a simplified expressionof the vacuolation kinetics compared to that in the previous model [ 121, which was basedon a population balance of vacuoles. This simplification is suggestedto improve the computationalspeedof the model simulation, and in recognition of the poor physiological knowledge underpinning the previous kinetic expression. 3.1.5. Degeneration When vacuoles increase in size under the condition of nutrient limitation in the medium, they produce regionswith little or no cytoplasmleft in the compartments. Compartments with large vacuolesare degenerated and do not contribute to penicillin biosynthesis. The generation of these regions causes a lossof non-growing cytoplasm (A, ) as indicatedin Fig. 3. In the model, first order kinetics in A2 are assumed to describethis degenerationprocess: r,,3=P3(2u2)p3 (5)

where pp is the specific penicillin production rate (hh), vlc isthe volumetric concentrationof thenon-growing cytoplasm ( cm3/1) in the non-growing regions,enclosed by andexcluding the cell walls, Kp is the saturationconstant for substrate limitation for product formation (g S,A), and K, is the saturation constantfor substrate inhibition for product formation (g S,/l). 3.1.8. Penicillin hydrolysis Penicillin hydrolysis is considered to follow first order kinetics in penicillin concentration in the broth:
rh=phP

(8)

where ~~ is the first order penicillin hydrolysis constant (h- ) andp is the penicillin concentration (g P/l). 3.1.9. Lactoseconversion The metabolismof mixed substrates is poorly understood in fungi. It is assumed here that the processes are analogous to those occurring in bacteria, particularly Escherichia coli [ 23 1. With growth of E. coli on a mediumcontaining glucose and lactose,there are two distinct phases of substrateutilisation. Glucose is metabolised first, and when the glucose concentration becomes very low, B-galactosidase is synthesised and growth continues on lactose.Considering Fig. 4, a similar kind of substrate utilisation is observable in P, chrysogenum.

wherem is the first order ratecontant (h- ), vZthe volumetric

G. C. Paul et al. /Biochemical

Engineering

Journal

2 (1998)

I l-21

15

3.2. Generalised

balance

equations

The kinetic expressions describedin Eqs. ( 1)-( 9) areused to produce generalised balanceEqs. ( lo)-( 18) for the fedbatch penicillin fermentation. These incorporate substrate accumulation, broth dilution by feed (F,) and precursor (F,,) additions, and filtered sample removal for on-line HPLC analysis (H,). Growing regions:

da,--rb,O-ld,!dt
0 20 40 60 80 100

(Ff+Fa,-H,)ao
V

Non-growing regions:
Time (h)
Fig. 4. Typical profiles for glucose and lactose monohydrate concentrations in a fed-batch penicillin fermentation (Rl). The utilisation of lactose started when the residual glucose concentration dropped to a low level. Due to the carbon limited growth, neither glucose nor galactose (one of the hydrolysed components of lactose) were detected in the medium after 32 h of the fermentation.

!&- -e.]
dt

(F,+Fa,-H,b,
-rb,0+r,.l-r,.3-

Vacuoles:

dl;,dt
-r,,2-rda31P2-

(Ff+Fap-Hr)UZ
V

(12)

Degenerated regions: The source of lactose monohydrate in the fermentation medium waswhey powder usedin excessasa carbon source in the inoculum medium.The concentrationof lactosemonohydrate after inoculation in the fermenter varied, typically in the range of 5 to 6 g/l. Lactose, a disaccharide,is hydrolysed to two monosaccharides, glucoseand galactose,by pgalactosidase.The total weight of glucose and galactose equalsthe weight of lactosemonohydrate utilised. The utilisation of thesetwo monosaccharides following the hydrolysis of lactose monohydrate appeared to occur simultaneously (i.e., without any preferential utilisation of thesetwo substrates)in the fermentations.This allows simple lactose utilisation kinetics to be assumed, in which lactosehydrolysis can be considered as a process producing an equivalent amount of glucose, when the residualglucoseconcentration drops to a very low level. The activity of the enzyme responsible for lactose hydrolysis is assumed proportional to the amountof active biomass, i.e., the combinedamountof growing (A,) and non-growing regions (A,) in the broth. The following competitive substrate inhibition kinetic expression is appropriate to describethe rate of conversion of lactoseto an equivalent amount of glucose, given the assumptions above:

da,--ld.3
dt

(F,+Fa,-H,)a3
-rd.4-

(13)

Autolysed biomass:

da,--ld,4dt Penicillin:

(F,+Fa,-Hrh
V

(14)

dp -z
dr

rp - r, -

(F,+F,,)p

(15)

Glucose:

ds,---Lyorb,o-~,r,,,dt
-cxprp-

mOaOS1

K,+s,

mlwvl

K,+s, --y
+r,2

(16)

(F,+F&,
V

+ FGP

Lactose: ds, -=-r dt s2

-

(F~+Fap)sz
V

(17)

Fermenter volume: dV
- =F,+F,,-H, dt

k%(ao+a,)
%= (K,,+s,)(l+s,lK,,)

(9)

(18)

where pL is the rate constant for lactoseconversion (h- ), s, the concentration of glucose (g S, /l), s2the concentration of lactosemonohydrate (g S,/l), KsL the saturationconcentration of lactosemonohydratelimiting the conversion (g Sz/ 1), and K,, the saturation constant for glucose inhibition lactoseof conversion (g S,/I).

where V is the fermenter volume (I), s, the concentration of glucosein the feed (g S, /l), cu,the stoichiometriccoefficient for glucose conversion to A, (g S,/g A,), CX~ the stoichiometric coefficient for conversion of glucose to A, (g S, /g A, ) , (Y,,the stoichiometric conversionof glucoseto penicillin G (g S, /g P), m, the maintenancecoefficient for A, (g S, /g A,lh) and m, the maintenancecoefficient for A,, (g S,/g

16

G.C. Paul et al. /Biochemical

Engineering

Journal

2 (1998)

11-21

A ,,/h). It is to be noted here that the maintenancerequirement terms used in Eq. (16) are glucose concentration dependent [ 10,121. 4. Estimation of model parameters

Table I List of model parameters penicillin fermentations Parameters PC Pe Pa @P CLh PI. CL1 P2 IL3 f% ml YI % a, % &I. KS1 4 KP Ku K K, K2 The parameter Table 2 Estimates Rl

and their

values obtained

from

two fed-batch

R2 0.027 + 0.001 0.398 * 0.004 0.050 i 0.003 0.0380 + 0.0005 0.0031 f 0.0002 0.245 k 0.023 0.025 + 0.001 0.0025* l.Oe-4 0.052 + 0.002 0.0252 If 0.0003 0.0275 f 0.0008 0.0107 *o.o001 1 X6+0.05 2.00 f 0.05 0.83 kO.04 0.102 f 0.003 0.0012~0.0002 0.415 f 0.005 0.048 f 0.002 0.0348 f 0.0002 0.117+0.003 0.106*0.003 0.287 lir 0.003

The model as represented by Eqs. ( lo)-( 18) contains 23 parameters and 9 states (A,, Al, A,, A,, A,, S,, S,, P and V) . Total biomass (A,) is obtained by a summation of the physiological states A,, A, and A,. The dry weight contribution of vacuoles (A2) is assumed to be negligible as stated previously. The parameter estimation was performed using Simusolv (Dow Chemical, Midland, USA) simulation, optimisation and parameter estimation software installed on a DEC Alpha workstation (Model 250 41266, Digital Equipment, MA, USA). The parameter estimates and the uncertainty in the estimates were obtained by maximising the log of the likelihood function as the non-linear objective function. In the log likelihood method the Gaussian probability distribution function was used as the error model, i.e., the experimental errors were assumed to be normally distributed. The objective function was solved by the generalised-reducedgradient (GRG) method. The GRG algorithm implemented in Simusolv (referred to as GRG2) uses an optimisation code following some improvements proposed by Lasdon and Waren [24]. The model equations were integrated numerically using the method of Gear. The estimates of model parameters were obtained from two sets of experimental data, 1~1 and R2. Fermentation Rl was carried out at constant glucose feed rate and R2 at a constant feed rate during the rapid growth phase and a pulsed square-wave feed rate during the production phase (see Fig. 1). In using the Simusolv parameter estimation method, a set of initial parameter estimates should be provided. Most of these were chosen from previous work [ 121 and the others were chosen based on experimental experience and subsequently refined through successive parameter estimation. The estimation of the whole set of parameters was considered necessary primarily due to the use of a different fermentation method from that used previously, and the incorporation of lactose utilisation into the model. Table I gives the parameter values estimated for these two data sets. In the estimation process it was also decided to obtain estimates of the initial conditions of the hyphal differentiation states (A,,, A,, A, and A3). These are listed in Table 2. Normally A,-A, would be measured by image analysis [ 61. Table 2 suggests that a consistent inoculation protocol would allow the use of estimated initial values of these states, It could then be possible to use the model (with predetermined parameters) on fermentations without the need for image analysis. This is discussed further below. 5. Results and discussion Figs. 5 and 6 show the model fits for the reference fermentation Rl, comparing simulation results obtained using the

0.033 f 0.001 0.409 f 0.007 0.044 * 0.009 0.0287 f 0.0003 0.0028 f 0.000 I 0.24 I f 0.005 0.019+0.001 0.0020 f 2.0e - 5 0.054 f 0.002 0.0256 f 0.001 I 0.0242 f 0.0009 0.0122 *0.0004 1.85 f 0.04 1.83 + 0.06 0.85 k 0.02 0.086 f 0.00 1 0.0011 * 0.0003 0.366 +O.OS 1 0.041+_0.006 0.0352 f 0.0005 0.079 + 0.002 0.09 1 f 0.003 0.302 f 0.008

values are listed with their standard deviations.

of initial differentiation states

states from fermentations RI 0.15+0.003 0.7 1 IO.02 0.12rtO.003 0.02 f 0.05

Rl and R2 R2 0.18rtO.002 0.8OiO.05 0.15 * 0.01 0.00 f 0.0 I

Differentiation A0 A, 4 4

Rl parameter values from Table 1 with experimental data. The glucose feed rate was kept constant throughout this run. Fig. 5 shows the time profiles of glucose, lactose monohydrate and, penicillin G concentrations and Fig. 6 the corresponding four hyphal differentiation states and the total biomass concentration. The model fits the experimental data extremely well. The competitive substrate inhibition kinetic form chosen in the model is clearly successful in describing lactose utilisation in the penicillin fermentation. Model fitting was examined further on fermentation R2 in which the feed rate of glucose was pulsed in a square wave during the production phase (Figs. 7 and 8). The R2 parameter values estimated using this fermentation are given in Table 1. This provided a good test of how the model would represent fermentations with rapid transients of the substrate feeding. From Figs. 7 and 8, it can be seen that the agreement between the experimental data and model simulation remains very good. Changes in the penicillin titre caused by the repeated switching of the glucose feed rate were matched well by the model, and there was reasonable agreement for

G.C. Paul et al. /Biochemical

Engineering

Journal

2 (1998)

II-21

17

00 8

20

10

60

80

IW

120

140

Time(h)

Fig. 5. Model fits of glucose, lactose monohydrate, and penicillin trations and glucose feed rate for fermentation Rl

G concen-

the other model states. The discrepancies between the measured vacuolation (AZ) and degenerated regions (A,), and the model predictions (Fig. 8) was expected owing to the great simplification of this part of the model compared to the previous version [ 121. It is worth mentioning that lack of detailed knowledge about vacuole formation and vacuole growth makes it difficult to choose an exact form of these kinetics. The incorporation of a more complex mathematical expression, as suggested previously [ 121, may give a better fit but may not necessarily reflect the underlying physiology. Until further studies reveal the mechanism of this process, it has been decided to use a simple form (i.e., first order kinetics) in the model. Nevertheless, this simple approach still provides a reasonable representation of these two states (A2 and A,) by the model and an excellent representation of the remaining six concentration states. In both cases (fermentations Rl and R2), the model describes the lactose and glucose utilisation remarkably well. The approach of using simple competitive substrateinhibition kinetics in the model was validated by experimental observations using on-line HPLC to quantify the substrates. Online HPLC is clearly a powerful tool for this sort of study. In principle, its use would allow incorporation of the utilisation kinetics of minor substrates such as lactic acid (typically around 1 to 2 g/l in the complex medium used in this work). However, this was deemed undesirable in practice, to avoid further model complexity and the addition of extra parameters. Nevertheless, the present success opens up the possibil-

20

40

60

SO

loo

120

140

Time(h)

Fig. 6. Model fits of the biomass states of fermentation experimental data and lines for model fits.

Rl. Symbols

are for

ity of further modelling to accommodate other mixed substrates found in industrial complex media. The parameters obtained by tuning the model with experimental data for fermentations Rl and R2 are presented in Table 1. In most cases, corresponding parameters estimated from each run have similar values and agree within the confidence limits. This implies that the model structure appropriately describes the penicillin G fermentation and is able to cope with disparate feeding profiles. The values of the yield and maintenance parameters obtained from each experiment are within the ranges suggested in the literature [ 1,2,2 1,221. These are common parameters used to characterise a fermentation process and without appropriate values the model may not provide an adequate description of substrate utilisation, biomass growth and product formation. The penicillin hydrolysis rate constant is within the range reported by other workers [9,21,22]. The values for the saturation constants (K,,, K,, K,, and K2) are similar to those proposed by Nielsen [ 111. The parameters associated with the penicillin production kinetics (b, Kp and K,) are expected to be dependent on strain as well as on the medium. The values of the other parameters are difficult to compare directly with previous studies due to differences in model structure.

18

G.C. Paul et al. /Biochemical

Engineering

Journal

2 (1998)

I l-21

2-1
0 M 40 60 S IW iZ0 140 160

Time(h) Fig. 7. Model fits of glucose, lactose monohydrate, and penicillin trations and glucose feed rate for fermentation R2.

G concen-

20

40

60

so

100

120

140

160

Model simulations are compared with data for fermentations R3 and R4 in Figs. 9 and 10, respectively. Fermentation R3 was performed at a constant glucose feed rate throughout the run and R4 at a constant feed rate during the rapid growth phase and a pulsed square wave feed rate during the production phase. In each case both sets of parameter values estimated from experimental data of Rl and R2 (see Table 1) were used to make predictions. Additionally, the simulations used the initial estimates for differentiation states A,-A, from fermentations Rl and R2 (see Table 2). Fig. 9 shows good agreement between the experimental data for total biomass, glucose, lactose and penicillin G, and the corresponding predictions using either set of parameter values. The simulations for fermentation R4 presented in Fig. 10 demonstrate the excellent predictive power of the model. In this case, the glucose feed rate during the production phase was pulsed in a square wave, giving a very variable behaviour throughout the fermentation. Reasonably good predictions to the experimental data are evident, even for the very stringent test of this fermentation, and even without access to measured initial values of the states A,-A,. On the basis of the graphs shown in Figs. 9 and 10, it is not possible to say which of the two sets of parameter estimates (shown in Table 1) gives the better predictions; the two sets of parameter values are quite similar. This suggests that it may be possible to estimate the model parameters and initial A,-A, values using image analysis measurements on just a few test fermentations. Provided

Time (h) Fig. 8. Model fits of the biomass states of fermentation experimental data and lines for model fits.

R2. Symbols

are for

the inoculation protocols lead to consistent operation, it could then be possible to forgo further use of image analysis. Although the incorporation of mixed carbon sources (i.e., glucose and lactose) in the model was remarkably successful, the effects of other fermentation conditions such as dissolved oxygen concentration and agitation speed were not included. These might well affect the parameter values and possibly the structure of the model, and remain to be investigated. The mechanisms for vacuolation, degeneration and fragmentation (or autolysis) rely on simple first-order kinetics. The simple form is justified due to the lack of adequate knowledge of vacuolation and of how the vacuolation affects hyphal degeneration and fragmentation and/or autolysis. Recent work did indicate a relationship between vacuolation and fragmentation [20]. Detailed experimental data under a range of operating conditions (particularly agitation speed) may be necessary to determine the mechanistic basis of this process. Alternatively, it may be possible to incorporate into the model the energy dissipation/circulation time function recently proposed by J&ten et al. [7,25,26] for correlating mycelial fragmentation with agitation conditions.

G.C. Paul et al. /Biochemical

Engineering

Journal

2 (1998)

11-21

12

Fig. 9. Model predictions of fed-batch fermentation R3 using parameters estimated fromfermentationsR1 andR2. Thefeedrate wasconstant throughout the fermentation. Symbols are for experimental data, solid lines represent simulation using R 1 parameters, and broken lines represent simulation using R2 parameters.

Fig. 10. Model predictions of fed-batch fermentation R4 using parameters estimated from fermentations Rl and R2. The feed rate was constant throughout the fermentation. Symbols are for experimental data, solid lines represent simulation using R 1 parameters, and broken lines represent simulation using R2 parameters.

6. Conclusions The model presented here gives a quantitative description of penicillin fermentations utilising mixed substrates, ascommonly used in industry. The competitive substrate inhibition kinetic describing the utilisation of glucose and lactose appeared to be remarkably successful. The model describes the physiological structure of fungal biomass and has been validated against states measured using image analysis. The capability of the model in predicting the dynamic behaviour of fed-batch penicillin fermentations is likely to be the consequence of its structure, and the accurate and reliable methods used to quantify the model parameters. The model has potential use in process simulation and optimisation of penicillin production, at least in cases where oxygen limitation is not severe. However, parameter estimation would be needed for each industrial medium and strain, and the model might need further development to allow for yet more complex industrial fermentations.

7. Nomenclature

Differentiated regions of hyphae i = 0: growing regions i = 1: non-growing regions i = 2: vacuoles i = 3: degenerated regions i = 4: autolysed biomass i = t: total biomass A,, Non-growing cytoplasm a, Concentrations of the differentiated regions (i = 0, 1, 2,3,4, t; see Ai for meaning of i values) (g DW/l) a,, Concentration of non-growing cytoplasm (g DW/l) Far, Precursor (PAA) addition rate (l/h) Ff Feed rate (l/h) H, Sample removal rate for on-line HPLC (l/h) K0 Saturation constant for branch formation (g S,/l) K, Saturation constant for hyphal extension (g S1/l)

Ai

20

G.C. Paul etal. /Biochemical

Engineering

Journal

2 (1998)

II-21

Kl
K2 Kl

KP KS,

K SL
m. ml

P P
rb.O rd,l

rd,2 ld.3

ld.4 re,, rh P rs2

Sl Xl s2 s2 Sf I

V Li

"IC

Saturation constant for A, to A r differentiation (g S, / 1) Saturation constant for A, maintenance (g S, 11) Glucose inhibition constant for penicillin production (g S/l) Saturation constant penicillin production (g S, /l) Glucose inhibition constant for lactose monohydrate conversion (g S,/l) Saturation constant for lactose monohydrate conversion (g S,/l) Maintenance coefficient for A0 (g S, /g A,lh) Maintenance coefficient for A, (g S, /g A, /h) Penicillin G Penicillin G concentration (g P/l) Rate of branch formation (g A,lh) Rate of formation of A, by differentiation of A, (g A, / hf Rate of vacuolation ( cm3 A,/h) Rate of formation of A3 by degeneration of A 1 ( g A, / h) Rate of loss of A, by autolysis (g A,lh) Rate of formation of Al by hyphal extension (g A, /h) Rate of loss of penicillin G by hydrolysis (g P/h) Rate of penicillin G production (g P/h) Rate of formation of glucose by conversion of lactose monohydrate (g S, /h) Glucose Glucose concentration (g S, /I) Lactose monohydrate Lactose monohydrate concentration (g S,/l) Concentration of glucose in the feed (g S, /l) Fermentation time (h) Fermenter volume (L) Volume concentrations of the differentiated regions ( cm3/1) (i = 0, 1,2, 3,4, t; see Ai for meaning of i values) Volume concentration of non-growing cytoplasm ( cm3/1)

PI pc

Density of the differentiated regions (g A,lcm3) (i = 0, 1,2, 3, t; see Aj for meaning of i values) Density of the cytoplasmic regions ( g/cm3)

Acknowledgements This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC), UK. The authors thank SmithKline Beecham Pharmaceuticals (Worthing, UK) for providing a Penicillium chrysogenum strain for this research. Ms. S. Wijesinghe, University of Birmingham, UK, is thanked for assisting with the fermentations.

References
R.C. Righelato, A.P.J. Trinci. S.J. Pitt, A. Peat, The influence of maintenance energy and growth rate on metabolic activity, morphology and conidiation of Penicillium chry~ogenum, J. Gen. Microbial. SO(1968) 399412. [21 D.D.Y. Ryu, J. Hospodka, Quantitative physiology of Penicillium chryogenum in penicillin fermentation, Biotechnol. Bioeng. 22 ( 1980) 289-298. 131 J.C. van Suijdam, B. Metz, Influence of engineering variables upon the morphology of filamentous molds, Biotechnol. Bioeng. 23 ( 198 I) 111-148. and control through L41 D.G. Mou, C.L. Cooney, Growth monitoring computer-aided on-line mass balancing in a fed-batch penicillin fermentation, Biotechnol. Bioeng. 25 ( 1983) 225-255. G.C. Paul, CA. Kent, C.R. Thomas, Quantitative characterization of vacuolization in Penicillium chrysogenum using automatic image analysis, Trans. IChemB 70 ( 1992) 13-20 (Part C). I61 G.C. Paul, CA. Kent, CR. Thomas, Image analysis forcharacterizing differentiation of PetziciNium chysogenum, Trans. IChemB 72 (1994) 95-105 (Part C). [71 P. J&ten, G.C. Paul, A.W. Nienow, C.R. Thomas, Dependence of Penicillium chrysogenum growth, morphology, vacuolation and productivity in fed-batch fermentations on impeller type and agitation intensity, Biotechnol. Bioeng., in press. and Process Per[81 K. Schiigerl (Ed.), Relation Between Morphology formance, Advances in Biochemical Engineering/Biotechnology, Vol. 60, Springer-Verlag, Berlin, 1998. 191 E. Nestaas, D.I.C. Wang, Computer control of the penicillin fermentation using a filtration probe in conjunction with a structured process model, Biotechnol. Bioeng. 25 ( 1983) 781-796. [IO1 J.W. Cagney, V.K. Chittur, H.C. Lim, Use of filtration measurements for estimation of cellular activity in penicillin production, Biotechnol. Bioeng. Sym. Ser. 4 (1984) 619634. structured model describing the 1111 J. Nielsen, A simple morphologically growth of filamentous microorganisms, Biotechnol. Bioeng. 41 ( 1993) 715-727. 1121 G.C. Paul, C.R. Thomas, A structured model for hyphal differentiation and penicillin production using Penicillium chrysogenum, Biotechnol. Bioeng. 51 ( 1996) 558-572. I131 SW. Queener, R.W. Swartz, Penicillins: biosynthesis and semisynthetic, Economic Microbiology, Vol. 3, Academic Press, New York, 1979, pp. 35-123. [I41 J. Moller, R. Hiddessen, J. Niehoff, K. Schiigerl. On-line high-performance liquid chromatography for monitoring fermentation proc esses for penicillin production, Anal. Chim. Acta 190 ( 1986) 195203.

Greek symbols Stoichiometric coefficient for A, (g S, /g A,) Stoichiometric coefficient for A, (g S, /g A, ) Stoichiometric coefficient for penicillin G (g S, /g P) Rate of substrate utilisation for differentiation of A, to A, (g&/h) Rate constant for branch formation (h- ) Rate constant for vacuole formation (h-l) Rate constant for vacuole growth (h-l) Rate constant for hyphal degeneration (h- ) Autolysis rate constant (he- ) Rate constant for hyphal extension (hh ) Penicillin hydrolysis constant (h- ) Rate constant for lactose monohydrate conversion (h-l) Penicillin production rate constant (h- )

G.C. Paul et al. /Biochemical R.W. Squires, Regulation of the penicillin fermentation by means of a submerged oxygen-sensitive electrode, Dev. Ind. Microbial. 13 (1972) 128-135. 1161 B. Konig, Ch. Seewald, K. Schiigerl, Process engineering investigations of penicillin production, Eur. J. Appl. Microbial. Biotechnol. 12 (1981) 205-211. 1171 F. Vardar, M.D. Lilly. Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations. Eur. J. Appl. Microbial. Biotechnol. 14 ( 1982) 203-211. 1181 C.M. Hemiksen, J. Nielsen, J. Viladsen, Influence of the dissolved oxygen concentration on the penicillin biosynthetic pathway in steadystate cultures of Penicillium chrysogenum, Biotechnol. Prog. 13 (1997) 776-782. H.M. Tsuchiya, Differ1191 R.D. Megee, S. Kinoshita, A.G. Fredrikson, entiation and product formation in molds, Biotechnol. Bioeng. 12 (1970) 771-801. 1201 G.C. Paul, CA. Kent, C.R. Thomas, Hyphal vacuolation and fragmentation in Penicillium chrysogcnum, Biotechnol. Bioeng. 44 ( 1994) 655-660. [I51

Engineering

Journal

2 (1998)

11-21

21

r-7-11R.K. [=I
I231 I241

I251

(261

Bajpai. M. Reuss, A mechanistic model for penicillin production, J. Appl. Chem. Technol. 30 (1980) 332-344. R.K. Bajpai, M. Reuss, Evaluation of feeding strategies in carbon regulated secondary metabolite production through mathematical modelling, Biotechnol. Bioeng. 23 ( 1981) 717-738. H.R. Horton, L.A. Moran, R.O. Ochs, J.D. Rawn, K.G. Scrimgeour, Principles of Biochemistry, 2nd edn., Prentice-Hall, NJ. 1996. L.S. Lasdon. A.D. Waren, Generalized reduced gradient software for linearly and non-linearly constrained problems, Design and Implementation of Optimization Sotware, Sithoff and Noordhoff, Netherlands, 1978, pp. 335-362. P. Jiisten, G.C. Paul, A.W. Nienow, CR. Thomas, Dependence of mycelial morphology on impeller type and agitation intensity, Biotechnol. Bioeng. 52 ( 1996) 672-684. P. Jiisten, G.C. Paul, A.W. Nienow, C.R. Thomas, A mathematical model for agitation-induced fragmentation of Penicillium chrysogenum. Bioprocess Eng. 18 (1998) 7-16.