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2 Basic Bioreactor Concepts

2 1 Information for Bioreactor Modelling

Both phy sical and biological in form ation are required in the design andinterpretation of biological reactor performance, as indicated in Fig. 2.1.

Physical factors that affect the general hydrodynamic environment of thebioreactor include such parameters as liquid flow pattern and circulation time,air distribution efficiency and gas holdup volume, oxygen mass transfer rates,intensity of mixing and the effects of shear. These factors are affected by thebioreactor geometry and that of the agitator (agitator speed, effect of baffles)and by physical property effects, such as liquid viscosity and interfacial tension.Both can have a large effect o n gas bubb le size and a correspond ing effect on

both liquid an d gas phase hydro dyn am ics. The biokinetic input involves suchfactors as cell g row th rate, cell produ ctivity and substrate uptake rate. Often thisinformation may come from laboratory data, obtained under conditions whichare often far removed from those actually existing in the large scale bioreactor.

Although shown as separate inputs in Fig. 2.1, there are, in fact, considerableinteractions between th e bioreactor hydrodynamic conditions and the cellbiokinetics, morphology and physiology, and one of the arts of modelling is tomake proper allowance for such effects. Thus in the large scale bioreactor,some cells may suffer local starvation of essential nutrients owing to acombination of long liquid circulation time and an inadequate rate of nutrient

supply, caused by inadequate mixing or inefficient mass transfer. Agitation andshear effects can affect cell morphology an d hence liquid viscosity, wh ich willalso vary with cell density. This means that the processes of cell growth affectthe bioreactor hydrodynamics in a very complex and interactive manner.Changes in the cell physiology, such that the cell processes are switched fromproduction of fu rther biomass to that of a secondary metabolite or product, canalso be affected by selective limitation on the quantity and rate of supply ofsome essential nutrient in the medium. This can in turn be influenced by thebioreactor hy dro dy na m ics and also by the mode of the operation of thebioreactor.

The overall problem is therefore very complex, but as seen in Figure 2.1,when all the information is combined successfully in a realistic and wellfounded Bioreactor Model the results obtained can be quite impressive and

Biological Reaction Engineering Second Edition I. J. Du nn, E. Heinzle, J. Ing ham , J. E. Pfenosil

Copyright © 2003 W I L E Y - V C H Verlag GmbH Co. KGaA, W einhe im

ISBN: 3-527-30759-1

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56 2 Basic Bioreactor Concepts

may enable such factors as cell and product production rates, productselectivities, optimum process control and process optimization to bedetermined with some considerable degree of confidence.

Physical As pects

flow patterns, residence

time, m ass transfer)

Biokinetics

order, inhibition,pH,

temperature)

Production rate

Selectivity

Control

Figure 2 1 Information fo r bioreactor modelling.

Bioreactor Operation

The rates of cell growth and product formation are, in the main, dependent onthe conc entration levels of nutrients and products within the bioreactor. Theconce ntration dependen cies of the reaction or prod uction rate are often quitesimple, but may also be very complex. The magnitude of the rates, however,depend upo n the level of conc entrations, and it will be seen that con cen trationlevels within the bioreactor depend very muc h on its type and mode of

operation. Differing modes of operation for the bioreactor can therefore leadto differing rates of cell growth, to differing rates of product formation andhence to substantially differing productivities.

Generally, the various types of bioreactor can be classified as either stirredtank or tubular and column devices and according to the mode of operation asbatch, semi-continuous or continuous operation.

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2.2 Bioreactor Operation 57

2 2 1 Batch Op eration

Most industrial bioreactors are operated under batch conditions. In this, thebioreactor is first charged with med ium, inoculated with cells, and the cells areallowed to grow for a sufficient time, such that the cells achieve the requiredcell density or optimum product concentrations. The bioreactor contents aredischarged, and the bioreactor is prepared for a fresh charge of medium.Operation is thus characterized by three periods of time: the filling period, thecell growth and cell production period and the final emptying period asdepicted in Fig. 2.2. It is only during the reacting period, that the bioreactor isproductive. During the period of cell growth, strictly speaking, no additionalmaterial is either added to or removed from the bioreactor, apart from minoradjustments needed for control of pH or foam, small additions of essentialprecursors, the removal of samples and, of course, a continuous supply of airneeded for aerobic fermentation. Concentrations of biomass, cell nutrients andcell products thus change continuously with respect to time, as the variousconstituents are either produced or consumed during the time course of thefermentation, as seen in Fig. 2.3.

F i l l ing Reacting Emptying Cleaning

Figure 2.2. Periods of operation for batch reactors.

concentration

ubstrate

biomass

product

time

Figure 2.3. Concentration-t ime profi les during batchwise operation.

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2 Basic Bioreactor Concepts

During the reaction period, there are changes in substrate and productconce ntration with time, and the other time periods are effectively lost as

regards production.Since there is no flow in or out of the bioreactor, du ring norm al operation,

the biomass and substrate balances both take the form,

(Rate of accumulation within the reactor) = (Rate of production)

This will be expressed in more quantitative terms in Ch. 4.

Batch reactors thus have the following characteristics:

1) Tim e-variant reaction conditions2) Disco ntinuous production3) Downtime fo r cleaning and filling

2 .2 .2 Semicontinuous or Fed Batch Operation

In sem i-con tinuou s or fed batch operation, add itional substrate is fed into the

bioreactor, thus prolonging operation by providing an additional continuoussupply of nutrients to the cells. No material is removed from the reactor, apartfrom normal sampling, and therefore the total quantity of material within thereactor will increase as a function of time. However if the feed is highlyconcentrated, then the reactor volume will not change much and can beregarded as essentially constant.

Figure 2.4. Fed batch bioreactor configuration.

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Semi-continuous operation shares the same characteristics as pure batchoperation, in that concentration levels generally change with time and that somedowntime occurs during the initial charging and final discharge period at the

end of the process.The ability to m anipu late conc entration levels within the bioreactor by an

appropriate controlled feeding strategy confers a high degree of flexibility tofed batch or semi-continuous operation, since differing concentration levels canbe utilized to manipulate the rates of reaction. In Fig. 2.4, both the volumetricfeeding rate, F, and the feed substrate concentration S Q may be constant or may

vary with time, giving the p ossibility of such feeding strategies as:

1. Slow constant feeding, wh ich can be shown to result in linear growthof the total cell biomass.

2. Exponen tial feeding to maintain constant substrate concentration and,resulting in unlimited, exponential cell growth.

3. Feedback control of the feed rate, based on m on itorin g some keycomponent concentration.

The important characteristics of fed batch operation are therefore as follows:

1. Extension of batch growth or product production by additionalsubstrate feeding.

2. Possibility of operating with separate conditions fo r growth andproduction phases.

3. Con trol possibilities on feeding policies.

4. Developm ent of high biomass and product concentration.

For fed-batch operation, th e cell balance follows th e same form as for batchoperation, but since additional substrate feeding to the reactor now occurs, thesubstrate balance takes the form:

Rate

<* | Substrate \ Substrate > |accumulat ion = f d̂ [n) _ consumption

V of substrate rate )

Under controlled conditions, in which th e substrate concentration is maintainedconstant or kept small, the accumulation term in the above equation will also be

small, with th e result that the feed rate of substrate into the reactor will balanceth e rate of consumption by reaction.

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60 2 Basic B ioreactor Concepts

One other balance equation, however, is also necessary, i.e. the total massbalance,

f Rate of accum ulation of ̂ Mass flow rate of feed ^V mass in the reactor

= V to the reactor )

which for constant density conditions reduces to

(Rate of change of volume) = (Volumetric rate of feeding)

Further extensions of fed batch operation are possible, such as the cyclic orrepeated fe d batch, which involves changing volume with a filling andemptying period. The changing reactor concentrations repeat themselves with

each cycle. This operation has similarities w ith con tinuo us operation andapproaches most closely to continuous operation, when the amount withdrawnis small and the cycle time is short. The simulation examples FEDBAT, Sec.8.1.3 and in Sec. 8.3 (VARVOL, PENFERM, PENOXY, ETHFERM , REPFED)allow detailed investigations of fed batch performance to be made on thecomputer .

2 2 3 Continuous Operation

In continuous operation fresh medium is added continuously to the bioreactor,while at the same time depleted medium is continuously removed. The rates ofaddition and removal are such that the volume of the reactor contents ismaintained constant. The depleted material, of course, contains any productsthat have been excreted by the cells and, in the case of suspended-cell culture,also contains effluent cells from the bioreactor.

Continuous reactors are of two main types, as indicated in Fig. 2.5, and thesemay be considered either as discrete stages, as in the co ntinuous, stirred-tank

bioreactor, or as differential devices, as represented by the continuous tubularor column reactor.

Continuous tank bioreactor Continuous tubular bioreactor

Figure 2.5. The two main types of co ntinu ous reactors.

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A s show n later, these two differing forms of continuous reactor operation havequite different operational characteristics. Bo th how ever are characterized byth e fact that after a short transient period, during which conditions within the

bioreactor change with time, the bioreactor w ill then achieve a steady state. Thism eans that operating conditions, both with in the bioreactor and at thebioreactor outlet, then remain constant, as shown in Fig. 2.6.

ConcentrationStartupperiod

Steady state

time

Figure 2.6. Startup of a continuous reactor.

Continuous reactors, however, have found little use as biological reactors on aproduction scale, although there are a few im portant exam ples (Id's single-cellprotein air lift process, w astew ater treatment and the isomerization of corn sugarto fructose syrup). Frequent use is made of continuous reactors in thelaboratory fo r studying the kinetics of organism growth and for enzymereaction kinetics. This is because the resulting form of the balance equation,

leads to an easy method for the determination of reaction rate, as discussed inCh. 4.The behavior of the two differing forms of continuous reactor, are best

characterized by their typical concentration profiles, as shown in Fig. 2.7. Inthis case, S is the concentration of any given reactant consumed, and P is theconcentration of any given product.

S o

Cone.

TankS o

Cone.

Tube

distance distance

Figure 2.7. Profiles of substrate and product in steady state continuo us tank and tubu lar

reactors.

As seen, th e concentrations in a perfectly mixed tank are uniform, th roug hou t

the whole of the reaction vessel contents and are therefore identical to theconcentration of the effluent stream. In a tubular reactor the reactantconcentration varies continuously, falling from a high value at the inlet to the

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lowest concentration at the reactor outlet. The product concentration rises frominlet to outlet. These differences arise because in the tank reactor the enteringfeed is continuo usly being mixed with the reactor bulk contents and therefore

being diluted by the tank conten ts. The feed to the tubula r reactor, however, isnot subject to m ixing and is transfo rm ed only by reaction, as material mov esdown th e reactor.

No real situation will exactly correspo nd to the above idealized cases ofperfect mixing or zero mixing (plug flow), although the actual behavior oftanks an d tubes tends in the limit towards the corresponding idealized model.The characteristics of continuous operation are as follows:

1. Steady state after an initial start-up period (usually)2. N o variation of concen trations with time

3. Co nstan t reaction rate4. Ease of balancing to determine kinetics5. N o dow n-time for cleaning, filling, etc.

The balance equations at steady state for a well-mixed tank reactor have theform

0 = (Input) - (Output) + (Production)

since at steady-state the rate of accumulation and therefore the rate of change iszero.

This equation predicts that the reaction rate causes a depletion of substratefrom the feed condition to the outlet, (the product will increase) and that therate of production can be obtained from this simple balance:

(Rate of production) = (Rate of output) - (Rate of input)

For a non well-mixed reactor such as a tubular or column reactor, steady-stateimplies the same non-transient conditions, but now concentrations also varywith position. The same situation also applies to the case of a series of well-mixed tanks.

The balance form is then:0 = (Rate of input) - (Rate of output) + (Overall Rate of Production)

Here the overall rate of reaction is obtained by summing or integrating overevery part of the reactor volume.

The concentration characteristics of a tubular reactor, as shown in Fig. 2.7,are well app roxim ated by a series of tank reactors. Referrin g to Fig. 2.8, andmoving downstream along the reactor cascade, the substrate concentrationdecreases stepwise from tank to tank, while the product concentration increases

in a similar stepwise manner. As the number of tanks in the cascade increases,so the performance becomes more and more similar to that of a tubular reactor.In the case of a reaction, whose rate of reaction increases with increasing

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substrate conc entration S, the mu ltiple tank config uration or a tubu lar reactorwould thus have a kinetic advantage over that of a single tank. The same is true,in the case of product inhibition kinetics, in which the rate would be lowered by

high product concentration, P. Substrate inhibition systems would be runpreferably in single tanks, however, since then the substrate concentration isalways at its lowest value.

Cone

distance

Figure 2.8. Stirred tanks in series and their concentration profiles.

A calculation of the tank volume or residence time requirement involves theformulation of the tank balance equations, as before and then the application ofthe equations, successively from tank to tank such that the effluent from thepreceding tank is the feed of the next and so on. Tank s-in-series bioreactoroperations are illustrated by the sim ulation ex amples TW OSTAG E, S TA GEDand DEACTENZ in Sec. 8.4.

2 .2 .4 Summary and Comparison

The operating characteristics of the various reactor modes are sum m arized inTable 2.1.

The important bioreactor operating parameters will depend on the mode ofoperation. In batch operation, concentration levels can be varied by adjustmentof the initial values, whereas in continuous an d semi-continuous operation, the

concen tration levels depend on the feed rate and feed co ncentration. A sindicated p reviously , the m anne r in w hich the bioreactor is operated cantherefore give rise to different concentration levels and therefore differingproductivities. The consequent concentration profiles depend, of course, on thereaction kinetics, which express the rate of reaction as a function of theconcentrations of reactants and products.

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Table 2.1. Summary of reactor modes.Mode of operation A dvantag es Disadvantages

Batch Equipm ent simple. Suitable Do w ntime fo r loading andfor small produc tion. cleaning. Reaction

conditions change withtime.

Co ntinuo us Provides high production. Requires flow control.Better product quality due Culture may be unstableto constant conditions. over long periods.

Good fo r kinetic studies.

Fed batch Control of enviro nm ental Requires feeding strategy toconditions, e.g. substrate obtain desired

concentration. concentrations.

Table 2.2 lists the main operating parameters for the three differing modes ofbioreactor operation.

Table 2.2. Operating variables for batch and continuous bioreactors.

Batch Con tinuous Sem icontinuous

Initial m edium composition Inlet m edium Feed and initial substrateand inoculum com position comp osition

Tem perature, pressure Tem perature, pressure Tem perature, pressure

pH if controlled pH if controlled pH if controlled

Reaction time Liquid flow rate Liquid flow rate

(residence time) (residence tim e)Aeration rate

A eration rate Feeding rate and controlStirring rate pro gra m

Stirring rateAeration rate

Stirring rate

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The foregoing discussion of the varying characteristics of the different reactortypes and their concentration profiles allows a qualitative comparison of thevolume requirements for the different typ es of reaction, according to the

particular kinetics. For this it is first necessary to consider the qualitative natureof the basic forms of kinetic relationship: zero order, first order, product andsubstrate inhibition. The detailed quantitative treatment of these kinetic forms isdealt with in Ch. 3.

The rate of a zero order reaction is independent of concentration. M an ybioreactions at high substrate concentrations follow zero order kinetics and aretherefore insensitive to concentration and to the effects of concentrationgradients. From th e kinetic viewpoint, therefore, any reactor type would beequally suitable.

First order reaction rates are directly proportional to concentration.

Bioreactions at low concentration are generally first order, and this would favoroperation in either a batch or a tubular/column type reactor. This is becausereactant concentrations in such reactors are generally high overall and henc ethe overall rates of reaction are also consequently high. Hence the reactorvolume required for a given duty would generally be small. (In the case of abatch reactor, this of course neglects the time lost for filling, em ptyin g andcleaning.)

A reaction with substrate inhibition wo uld be best run in a tank at lowsubstrate concentration, since the concentration wo uld be low throu gho ut thewhole of the tank contents. Conversely, produ ct inh ibition wo uld be m orepronounced in tank reactors, since product concentration would be at itshighest. In this case, a tubular type reactor or batch reactor would be preferred.

Table 2.3. Kinetic considerations fo r reactor choice.

ReactionKinetics

Zero order

First order

Substrateinhibition

Productinhibition

Production

triggered byshift inenvironment

Batch Tank

OK

Best

L ow initialconcentration

Best

OK for temp-

erature-shift

ContinuousTanks-in-Series orTubularOK

Best

L ow tankconcentrations

Best

Possible

ContinuousSingle Tank

OK

Low con-version onlyBest

Low con-version only

Not suitable

Fed Batch

L ow con-

versionOK

Best

Low con-version only

Best fo r con-

centration-shift

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Table 2.3 compares the performance of batch tanks, continuous tubular ortanks-in-series reactors and single continuous tank reactors. A s discussed in

Sec. 4.2.1, batch tank concentration-time profiles are exactly analogous to thesteady state concentration-distance profiles obtained in continuo us tub ula rreactors. In terms of performance, therefore, the batch reactor would be thesame as a tube, wh en compared on the basis of equal batch time in the tank andtime of passage through the tube. Tanks-in-series reactors, as shown in Fig. 2.8,involve step wise gradients, wh ich in the limit are very similar to those ofcontinuous tubular reactors, hence, making their performance similar to that ofa tubular reactor. Owing to the high degree of mixing which leads to a uniformconcentration, the performance of the single continuous stirred tank reactor isvery different to that of the other reactor types. An exact quantitative

comparison can be made using th e mass balance equations developed in Ch. 4fo r each reactor type.

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