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224

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

Growth of Alcaligenes eutrophus strain H16. Eur. J. Appl. Microbiol. BiotechnoL 11, 8.1

8.3 8.3.1
System

Fed Batch Reactors Variable Volume Fermentation (VARVOL and VARVOLD)

Semi-continuous or fed batch cultivation of micro-organisms is common in the fermentation industries. The fed batch fermenter mode is shown in Fig. 1 and was also presented in the example FEDBAT. In this procedure a substrate feed stream is added continuously to the reactor. After the tank is full or the biomass concentration is too high, the medium can be partially emptied, and the filling process repeated. Since the variables, volume, substrate and biomass concentration change with time, simulation techniques are useful in analyzing this operation. This example demonstrates the use of dimensionless equations.

Figure 1. Filling and emptying sequences in a fed batch fermenter.

Model
The balances are as follows: Volume,
dv

dT = FO

Substrate,
Biological Reaction Engineering, Second Edition. I. J. Dunn, E. Heinzle, J. Ingham, J. E. Pfenosil Copyright 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30759-1

8.3 Fed Batch Reactors

225

- = F0S0 Biomass,

d(VX) dt = rx
The kinetics are

rx = MX
|LimS

** - (Ks + S) and rx rs = -Y
The dilution rate is defined as

In order to simplify the equations and to present the results more generally, the model is written in dimensionless form. Defining the dimensionless variables:
v

V X

X< =

s
F =

- _ JL
Mm

tft' = t

226

Expanding the derivatives gives

d(V S) = V dS + S dV and d(V X) = V dX + X dV

dV'

Biomass
dX' dt'

Substrate
dS'
dt

= (l-S)D-jiX

The Monod equation is:

KS +S

In Fig. 2 a computer solution shows the approach to and attainment of the quasi-steady state of the dimensionless fed-batch model.

227

Figure 2. Dynamic simulation results for a fed batch culture.

Programs
The program VARVOL is based on the model equations with normal dimensions. The program VARVOLD is based on the dimensionless equations as derived above. Both are on the CD-ROM.

Nomenclature Symbols
D F KS r S V

Dilution rate Flow rate Saturation constant Reaction rate Substrate concentration Reactor volume

228 X Y

Indices
0 f m S X

Refers Refers Refers Refers Refers Refers

to feed and initial values to final to maximum to substrate to biomass to dimensionless variables

Dimensionless Variables
Dimensionless flow rate Dimensionless saturation constant Dimensionless substrate concentration Dimensionless volume Dimensionless biomass concentration Dimensionless time Dimensionless specific growth rate

S' V X1 t'

Exercises

8.3 Fed Batch Reactors

229

Results
During the quasi-steady state, \l becomes equal to D, and this requires that S must decrease steadily in order to maintain the quasi-steady state as the volume increases (Fig. 3). Increasing flow rates from 0.01 to 1.0 causes a delay in the onset of linear growth and causes the final biomass levels to be higher (Fig. 4).
Run 1:105 steps in 0 seconds
4.5

Figure 3. Fed batch concentration and growth rate profiles, showing quasi-steady state.
Run 7:105 steps in 0 seconds

2.5

C/>

10

230

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

Figure 4. Influence of flow rate on growth. Flow rate increase from 0.01 to 1.0.

References
Dunn, I.J., and Mor, J.R. (1975) Variable Volume Continuous Cultivation. Biotechnol. Bioeng. 17, 1805. Keller, R., and Dunn, I.J. (1978) Computer Simulation of the Biomass Production Rate of Cyclic Fed Batch Continuous Culture. J. AppL Chem. Biotechnol. 28, 784.

8.3.2
System

Penicillin Fermentation Using Elemental Balancing (PENFERM)

This example is based on the publication of Heijnen et al. (1979), and encompasses all the principles of elemental balancing, rate equation formulation, material balancing and computer simulation. A fed batch process for the production of penicillin as shown in Fig. 1 is considered with continuous feeding of glucose. Ammonia, sulfuric acid and o-phosphoric acid are the sources of nitrogen, sulfur and phosphorous respectively. Ophosphoric acid is sufficiently present in the medium and is not fed. Oxygen and carbon dioxide are exchanged by the organism. The product of the hydrolysis of penicillin, penicilloic acid, is also considered, thus taking the slow hydrolysis of penicillin-G during the process into account.

231

Glucose

Carbon dioxide

232

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

Table 1. Component properties and rate designations. Compound Chemical formula Mol wt. Enthalpy Conversion (Daltons) (kcal/mol) rate (mol/h) Glucose 180 - 303 C6H1206 Rl Mycelium 24.52 28.1 R2 CHi.64Oo.52No. 16 So.0046P<).0054 C16H1804N2S Penicillin 334 - 115 R3 C16H2005N2S R4 352 - 183 Penicilloic acid Oxygen 02 32 0 R5 CO2 Carbon Dioxide 44 -94 R6 NH3 Ammonia 17 - 19 R8 H2SO4 R9 Sulfuric Acid 98 - 194 Phosphoric Acid H3PO3 98 - 319 RIO Phenylacetic Acid C8H802 136 -69 RH H2O Water 18 -68 Rl2

Model

a) Elemental Balancing
Knowing the composition of all chemical substances and the biomass mycelium (Table 1) allows the following steady state balances of the elements in terms of mol/h: For carbon

6 RI + R6 + 16 R3 + 8 RH + 16 RH + R2 = 0
For oxygen

6 RI + 2 R5 + 2 R6 + R12 + 4 R3 + 4 R9 + 4 R10 + 2 RH + 5 R4 + 0.52 R2 = 0

For nitrogen 0.16R 2 + 2 R 3 + 2R 4 + R8 = 0 For sulfur

0.00 46 R2 + R3 + R4 + R9 = 0

8.3 Fed Batch Reactors

233

For hydrogen
12 RI + 1.64 R2 + 18 R3 + 20 R4 + 3 R8 + 2 R9 + 3 RIO + 8 R n + 2 R12 = 0

For phosphorus 0.0054 R2 + RIO = 0 A steady state enthalpy balance gives the following
- 303 RI - 28.1 R2 - 115 R3 - 183 R4 - 94 R6 - 19 R8 - 194 R9 -

- 3 1 9 R i o - 6 9 R n -68Ri 2 + rH = 0 where TH is the rate of heat of production (kcal/h). A total of 12 unknowns (Ri through R6, Rg through Ri 2 and TH) are involved with a total of 7 equations (6 elemental balances and one heat balance). The five additional equations are provided by five reaction kinetic relationships. The remaining rates can be expressed in terms of these basic kinetic equations. From the carbon balance
- R6 = 6 RI + R2 + 16 R3 + 16 R4 + 8 RH

From the nitrogen balance - R 8 = 0.16R2 + 2R 3 + 2R 4 From the sulfur balance - R9 = 0.0046 R2 + R3 + R4 From the phosphor balance - R i o = 0.0054 R2 From the hydrogen and nitrogen balances
- R5 = -6 RI - 1.044 R2 - 18.5 R3 - 18.5 R4 - 9 R n

From the enthalpy balance

rH = - 669 RI - 110.1 R2 - 1961 R3 - 1961 R4 - 955 RH

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8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

To complete the model, equations for glucose uptake rate (-Ri), biomass formation rate (R2), rate of penicillin formation (Rs), precursor consumption rate (-Rn), and rate of penicillin hydrolysis (R4) must be known. Note that the reaction rates are defined with respect to total broth weight, since the process is the fed-batch type and broth weight is variable with respect to time.

b) Formulation of the Kinetic Equations

Substrate (Glucose) Uptake Rate: A MONOD type equation for the uptake of sugar by P. Chrysogenum is used. -QlCiM2 Biomass Formation Rate: A linear relationship between the glucose consumption rate and growth rate of biomass is assumed. Hence,
1 - Rl = y^ ^2 + m M2

where Y2 is the maximum growth yield and m is the maintenance rate factor (mol glucose/mol mycelial biomass h). Some sugar is used in the formation of the product. Hence,
- Rl = Yj R2 + m M2 + YJ (R3 + R*)

where 3 is the conversion yield for glucose to penicillin (mol penicillin/mol glucose). The total rate of biomass formation equals the net rate of formation, corrected for the amount transformed to penicilloic acid. Therefore, R2 = - Y 2 R i - Y 2 m M 2 - yf (Rs + 4) Precursor Conversion Rate It is assumed that the precursor is only used for penicillin synthesis. Thus

- R l l = R 3 + R4
where - RH is the precursor consumption rate.

8.3 Fed Batch Reactors

235

Rate of Penicillin Synthesis The specific rate of penicillin synthesis is assumed not to be a function of specific growth rate. So that
R 3 = Q3 M2 - R4

where Q3 is the maximum specific rate of penicillin synthesis (mol/mol h), Equation for the Rate of Penicillin Hydrolysis The hydrolysis of penicillin takes place by a first-order reaction.

R 4 = K 3 M3

c) Balance Equations
Total Mass Balance The individual feed rates of glucose, sulfuric acid and ammonia are adjusted to equal their molar consumption rates. Water lost by evaporation is neglected. The change in mass due to gas uptake and production is neglected. The mass flow rates are calculated from the molecular weights, the uptake rates and the mass ratio compositions. Feed rate of glucose stream (kg/h)
F

l =

180 500

F 2.78

where F = mol glucose /h. Feed rate of NH3 stream (kg/h) F8 = R 8 25Q Feed rate of [2804 stream (kg/h)
18 F9 - R95QO R9 ~ 2.55

= T4JT

dG F

"dT = Tn

R9

235"

Rg

TTTT

236

Glucose
~3T = R l + F

dM4

~ar = R2
dM3 .

dM2

The concentrations in mol/kg are as follows:

Tr
M3

M2

c4 =
where the masses MI, M2, M3 and M4 are in mol units.

M4

d) Metabolism Relations
The various metabolic relationships are given from Specific growth rate for cells
R2

* = Ml
Respiration quotient
R

Q = R7

Re

OUR = -R 5

CO2 production rate

CPR = R6

Fraction of N2 in mycelium

8.3 Fed Batch Reactors

R2

237

f 2 = 0.16 R|
N2 fraction in penicillin

f3 = 1-F 2
Fraction of sulfur used for mycelium

R2 f 4 = 0.046 R|
Sulfur fraction used for penicillin

f5 = 1 - F4
Fraction of glucose for cell growth
=
R2

Fraction of glucose for penicillin

S3 = - Y3 R!
Fraction of glucose for maintenance

R3 + R4

M2 g4 = -M R^-

Program
The Madonna program covers a fermentation time of 200 h starting from the initial conditions of 5500 mol glucose, 4000 mol biomass, 0 mol penicillin and 0.001 mol penicilloic acid in an initial broth weight of IxlO 5 kg. The program is on the CD-ROM.

Nomenclature Symbols
a, b C CPR F Flow rate variables Component concentration Carbon dioxide production rate Feed rate various mol/kg mol/h kg/h

238

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

h fl
f5 G 82 g3 g4
Kl

K3 M m OUR Q R RQ rq Y

Fraction of nitrogen in mycelium Nitrogen fraction in penicillin Fraction of sulfur used for mycelium Fraction of sulfur used for penicillin Mass in reactor Fraction of glucose for cell growth Fraction of glucose for penicillin Fraction glucose for maintenance Saturation constant Hydrolysis rate constant Mass of individual components Maintenance rate factor Oxygen uptake rate Maximum specific rates Conversion Respiration quotient Heat production rate Respiratory quotient Yield coefficient Specific growth rate

kg

mol/kg 1/h mol mol/(mol h) mol/h mol/(mol h) mol/h kcal/h 1/h

Indices
0 1 2 3 4 5 6 8 9 10 11 12
initial glucose biomass penicillin penicilloic acid oxygen carbon dioxide ammonia sulfuric acid phosphoric acid phenylacetic acid water

Exercises

8.3 Fed Batch Reactors

239

Results
The results of Fig. 2 show the substrate MI to pass through a maximum, while the penicillin M2 develops linearly, for this constant feeding situation. Increasing the feeding linearly with time (F = 500 + 5* time) gave the results in Fig. 3, where it is seen that maintenance accounts for about 70 % of glucose consumption at the end of the fermentation.
Run 1:215 steps in 0 seconds

20

40

60

80

100

120

140

160

180

200

TIME

Figure 2. Penicillin fed batch fermentation with total masses of glucose (M]) and biomass (M2).

240

0.9-, 0.80.7-

0.6-

r- 0.40.30.20.1-

020 60 80

100

120

TIME

Figure 3. Linear increase of feeding with time F = 500 + 5*T.

Reference
Heijnen, J., Roels, J. A., and Stouthamer, A.H. (1979). Application of Balancing Methods in Modeling the Penicillin Fermentation. Biotechnol. and Bioeng., 21, 2175-2201.

8.3.3
System

Ethanol Fed Batch Diauxic Fermentation (ETHFERM)

Yeast exhibits diauxic behavior with respect to the glucose and ethanol in the medium as alternative substrates. In addition, the glucose effect, when glucose levels are high, will cause fermentation, instead of respirative oxidation, to take place, such that the biomass yields are much reduced (Fig. 1). In this example the constant a designates the fraction of respiring biomass and (1 - a) the fraction of biomass that ferments. The rates of the process are controlled by three enzymes.

8.3 Fed Batch Reactors

24 1

^^
Glucose ^^^*Figure 1. Pathways of aerobic ethanol fermentation.

C02 + X

Ethanol + X

Model
The rates of the processes are as follows: Respirative oxidation on glucose,
R, =

Glu+K sl

Fermentation to ethanol,
R2 = K2 (1 - a) X Glu + KS2

Conversion of ethanol to biomass,

Enzyme activation for the transformation of ethanol to biomass is assumed to involve an initial concentration of starting enzyme EQ, which is converted to enzyme 2 and which catalyzes growth on ethanol through an intermediate enzyme EI. Thus, the production rate of enzyme EI is inhibited strongly by glucose,
R4 = - -rXEo K S4 +Glu 3

and the production rate of enzyme 2 controlling the conversion of biomass to ethanol depends on EI, R5 = K 5 X E i The mass balances for the biomass, substrates and enzymes are those for a fed batch with variable volume.

242

For the total mass balance with constant density,

dt
The component balances are written by separating the accumulation term, noting that
-

d(VC) _ VdC CdV _ VdC - + - dt dt dt dt

Thus,
dt
f

^o
dt

E0Q
V

f -*-.-*

Program
Note that the program on the CD-ROM is formulated in terms of C-mol for the biomass. This is defined as the formula weight written in terms of one C atom, thus for yeast CHL667Oo.5No.i67-

8.3 Fed Batch Reactors

243

Nomenclature

Symbols
C

E EtOU Glu K Q R V X Y a

Component concentration Enzyme concentration Ethanol concentration Substrate feed concentration Rate constants Feed flow rate Reaction rate Reactor volume Biomass concentration Yield coefficient Fraction of respiring biomass

Indices
0 1 2 3 4 5

1 2 3 4 5

Exercises

244

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

Results
Seen in Fig. 3 are the simulation results giving the concentrations (glucose, ethanol and biomass) during the fed batch process. In Fig. 4 the maximum in ethanol concentration as a function of feedrate is given from a Parameter Plot.
Run 1: 605 steps in 0.0167 seconds

30

25

60

245

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Figure 4. Influence of flowrate on the maximum ethanol concentration.

Reference
This example was contributed by C. Niklasson, Dept. of Chemical Reaction Engineering, Chalmers University of Technology, S - 41296 Goteborg, Sweden.

8.3.4
System

Repeated Fed Batch Culture (REPFED)

A single cycle of a repeated fed batch fermentation is shown in Fig. 1. In this operation a substrate is added continuously to the reactor. After the tank is full, the culture is partially emptied, and the filling process is repeated to start the next fed batch. The operating variables are initial volume, final volume, substrate feed concentration and flow rates of filling and emptying.

246

Figure 1. One cycle of a repeated fed batch.

Model
The equations are the same as given in the example FEDBAT (Section 8.1.3), where the balances for substrate and biomass are written in terms of masses, instead of concentrations. The only difference is that an outlet stream is considered here to empty the fermenter at the end of the production period.

Program
Since in a Madonna program, the initial conditions cannot be reset, an outlet stream is added. The inlet and outlet streams are controlled by conditional statements as shown below. The full program is on the CD-ROM.
{Statements to switch the feed and emptying streams) Fin=if time> = 10 then Flin else 0 {batch start up} Flin= if time> = 33 then 0.5 else if time> = 32 then 0 else if time> = 21 then 0.5 else if time> = 20 then 0 else 0.5 Fout= if time>=33 then 0 else if time>=32 then 5.39 else if time> = 21 then 0 else if time> = 20 then 5.39 else 0

8.3 Fed Batch Reactors

247

Nomenclature Symbols
D F Kl and K2 KS P S X V V 0 VX VS Y |i Dilution rate Flow rate Product kinetic constants Saturation constant Product concentration Substrate concentration Biomass concentration Reactor volume Initial volume of liquid Biomass in reactor Substrate in reactor Yield coefficient Specific growth rate

1/h m3/h various kg/m3 g/m3 kg/m3 kg/m3 m3 m3 kg kg kg/kg 1/h

Indices
S X
0 (zero) initial Refers Refers Refers Refers Refers Refers to substrate to biomass to initial and inlet values to initial values to inlet to exit

in out

Exercises

248

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

Results
Shown below are results of a simulation with three filling cycles.

60 -|

.-80

50-

40-

30-

20-

10-

A pr^Ti /- 70 | "vsi| , go / I / I /"( / .50 / I ' J -40 f I / \ / I / i /-V / I/ I / ^\ \l XV ,* \ -10

--*'"-'
0 5 10 15 20 25 30 35 40

0-

L
45

-Q
50

TIME

Figure 2. Masses of substrate and biomass during filling and emptying cycles.

8.3 Fed Batch Reactors

249
Run 1: 5004 steps in 0.35 seconds

10

Figure 3. Concentrations of product, substrate and biomass during filling and emptying cycles. The volume is also shown.

References
Dunn, I.J., Mor, J.R., (1975) Variable Volume Continuous Cultivation Biotechnol. Bioeng. 17, 1805. Keller, R., Dunn, I.J. (1978) Computer Simulation of the Biomass Production Rate of Cyclic Fed Batch Continuous Culture J. AppL Chem. Biotechnol. 28, 784.

8.3.5
System

Repeated Medium Replacement Culture (REPLCUL)

Slow-growing animal and plant cell cultures require certain growth factors and hormones which begin to limit growth after a period of time. To avoid this, part of the entire culture is replaced with fresh medium. A single cycle of repeated replacement culture is shown in Fig. 1. In this procedure part of the medium volume (with cells) is removed after a certain replacement time and replaced with fresh medium. Each cycle operates as a constant volume batch in

250

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

which the concentration of substrate decreases, while that of biomass increases. The operating variables are replacement volume, replacement time, and substrate concentration in the replacement medium. The initial conditions for each cycle are determined by the final values in the previous cycle and the replacement volume and concentration.

VX

VS

Initial Conditions Figure 1. One cycle for medium replacement culture.

Model
The equations are those of batch culture, where for convenience the total masses are used. dVS = r

"dT

sV

dvx

Monod kinetics is used. The effective starting conditions for each batch can be calculated using the final conditions of the previous cycle from the volume replaced, VR? and the total volume, V, by the equations,

* VR f=
VX = (1 - ) VXF

251

VS= ( l - f ) V S F + V R S 0 where f is the volume fraction replaced.

Program
The program as shown on the CD-ROM makes use of the PULSE function to vary the biomass and substrate concentrations corresponding to the replacement of a fraction F of the culture medium. The time for each batch is the value of INTERVAL.

Nomenclature Symbols
D f KS

s
v

X V

vx vs
VR Y

Dilution rate Fraction of volume replaced Saturation constant Substrate concentration Biomass concentration Reactor volume Initial volume of liquid Biomass in reactor Substrate in reactor Volume replaced Yield coefficient Specific growth rate

1/h

1/h

Indices
F S X 0

Refers Refers Refers Refers

to final values at end of the cycle to substrate to biomass to initial and inlet values

252

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

Exercises

Results
Fig. 2 shows how the biomass increases, until after six cycles the time profiles become almost identical.
TIME= 19.29 X = 1.26

10

20

30

40

50 TIME

60

70

90

100

Figure 2. Oscillations of biomass and substrate concentrations with replacement cycles for Interval 10 and F=0.8

253

8.3.6

Penicillin Production in a Fed Batch Fermenter (PENOXY)

A fed batch process is considered for the production of penicillin, as described by Muttzall (1), The original model was altered to include oxygen transfer and the influence of oxygen on the growth kinetics.

Figure I. Fed batch reactor showing nomenclature.

Model
As explained in the example FEDBAT the balances are: Total mass

dt
Biomass: d(MassX) = Vr-X dt Substrate: d(MassS) = FSf+Vrs dt Product:

254

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna d(MassP) _. = V fp p

dt

Dissolved oxygen, neglecting the content of the inlet stream is calculated from d(MassO) = K L a*(O sat -0) + Vr 0 dt The influence of biomass concentration approximated here by KX+X The concentrations are calculated from
1\. """""""""""""^ ,

on

the

oxygen

transfer is

_MassX V

MassS , V

MassP , V

MassO V

The growth kinetics take into account the oxygen influence

o
The substrate uptake kinetics includes that amount used for growth, for product and for maintenance
J*o ~ Jft o _/V

V Y

XS

V" Y

PS

Product production involves two terms whose constants are turned on and off according to the value of |ii, as seen in the program.

=-TT Y xo

8.3 Fed Batch Reactors

255

Program
The program is on the CD-ROM.

Nomenclature

Symbols
F KLa Ko KS KX Mass mo ms
Osat

Sf V
'max YPS

YXO YXS
H-max

Feed flowrate Oxygen transfer coeff. Monod constant for oxygen Monod constant for glucose Constant for biomass effect on Component mass Maintenance coeff. for oxygen Maintenance coeff. for glucose Saturation for oxygen Feed cone, of glucose Volume Maximum volume Yield product to substrate Yield biomass to oxygen Yield biomass to substrate max.specific growth rate

m3/h 1/h kg/m 3 kg/m 3 kg/m3 kg kg O/kg X h kg S/kg X h kg/m3 kg/m3 m3 m3 kg/kg kg/kg kg/kg 1/h

Exercises

256

8 Simulation Examples of Biological Reaction Processes Using Berkeley Madonna

II!

References
K. Mutzall, "Modellierung von Bioprozesses", Behr's Verlag, 1994. Program and model developed by Reto Mueller, ETH Zurich.

Results
Run 1: 2023 steps in 0.117 seconds 0.008

20

40

60

80

100

120

140

160

180

200

8.4 Continuous Reactors

257
Run 3: 2021 steps in 0.15 seconds -0.008

120

0.007
100

80

! I i I

0.006 0.005 0.004 O -0.003 -0.002

- 60

40

: I Li I I

20

-0.001
0 0 20 40 60 80 100 120 140 160 180 200

TIME Figure 3. Influence of initial KLa value from 100 to 160 h"^ on the S and O profiles.

8.4
8.4.1
System

Continuous Reactors