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2

Basic Bioreactor Concepts

2.1 Information for Bioreactor Modelling


Both physical and biological information are required in the design and interpretation of biological reactor performance, as indicated in Fig. 2.1. Physical factors that affect the general hydrodynamic environment of the bioreactor 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 the bioreactor 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 on gas bubble size and a corresponding effect on both liquid and gas phase hydrodynamics. The biokinetic input involves such factors as cell growth rate, cell productivity and substrate uptake rate. Often this information may come from laboratory data, obtained under conditions which are 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, considerable interactions between the bioreactor hydrodynamic conditions and the cell biokinetics, morphology and physiology, and one of the arts of modelling is to make proper allowance for such effects. Thus in the large scale bioreactor, some cells may suffer local starvation of essential nutrients owing to a combination of long liquid circulation time and an inadequate rate of nutrient supply, caused by inadequate mixing or inefficient mass transfer. Agitation and shear effects can affect cell morphology and hence liquid viscosity, which will also vary with cell density. This means that the processes of cell growth affect the bioreactor hydrodynamics in a very complex and interactive manner. Changes in the cell physiology, such that the cell processes are switched from production of further biomass to that of a secondary metabolite or product, can also be affected by selective limitation on the quantity and rate of supply of some essential nutrient in the medium. This can in turn be influenced by the bioreactor hydrodynamics and also by the mode of the operation of the bioreactor. 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 well founded Bioreactor Model, the results obtained can be quite impressive and
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

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may enable such factors as cell and product production rates, product selectivities, optimum process control and process optimization to be determined with some considerable degree of confidence.
Physical Aspects (flow patterns, residence time, mass transfer) Biokinetics (order, inhibition,pH, temperature)

Production rate Selectivity Control Figure 2.1. Information for bioreactor modelling.

2.2

Bioreactor Operation

The rates of cell growth and product formation are, in the main, dependent on the concentration levels of nutrients and products within the bioreactor. The concentration dependencies of the reaction or production rate are often quite simple, but may also be very complex. The magnitude of the rates, however, depend upon the level of concentrations, and it will be seen that concentration levels within the bioreactor depend very much on its type and mode of operation. Differing modes of operation for the bioreactor can therefore lead to differing rates of cell growth, to differing rates of product formation and hence to substantially differing productivities. Generally, the various types of bioreactor can be classified as either stirred tank or tubular and column devices and according to the mode of operation as batch, semi-continuous or continuous operation.

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2.2.1 Batch Operation


Most industrial bioreactors are operated under batch conditions. In this, the bioreactor is first charged with medium, inoculated with cells, and the cells are allowed to grow for a sufficient time, such that the cells achieve the required cell density or optimum product concentrations. The bioreactor contents are discharged, and the bioreactor is prepared for a fresh charge of medium. Operation is thus characterized by three periods of time: the filling period, the cell growth and cell production period and the final emptying period as depicted in Fig. 2.2. It is only during the reacting period, that the bioreactor is productive. During the period of cell growth, strictly speaking, no additional material is either added to or removed from the bioreactor, apart from minor adjustments needed for control of pH or foam, small additions of essential precursors, the removal of samples and, of course, a continuous supply of air needed for aerobic fermentation. Concentrations of biomass, cell nutrients and cell products thus change continuously with respect to time, as the various constituents are either produced or consumed during the time course of the fermentation, as seen in Fig. 2.3.

Filling

Reacting

Emptying

Cleaning

Figure 2.2. Periods of operation for batch reactors.

concentration

ubstrate

biomass

product

time Figure 2.3. Concentration-time profiles during batchwise operation.

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During the reaction period, there are changes in substrate and product concentration with time, and the other time periods are effectively lost as regards production. Since there is no flow in or out of the bioreactor, during normal 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) 2) 3) Time-variant reaction conditions Discontinuous production Downtime for cleaning and filling

2.2.2 Semicontinuous or Fed Batch Operation


In semi-continuous or fed batch operation, additional substrate is fed into the bioreactor, thus prolonging operation by providing an additional continuous supply of nutrients to the cells. No material is removed from the reactor, apart from normal sampling, and therefore the total quantity of material within the reactor will increase as a function of time. However if the feed is highly concentrated, then the reactor volume will not change much and can be regarded as essentially constant.

Figure 2.4. Fed batch bioreactor configuration.

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Semi-continuous operation shares the same characteristics as pure batch operation, in that concentration levels generally change with time and that some downtime occurs during the initial charging and final discharge period at the end of the process. The ability to manipulate concentration levels within the bioreactor by an appropriate controlled feeding strategy confers a high degree of flexibility to fed batch or semi-continuous operation, since differing concentration levels can be utilized to manipulate the rates of reaction. In Fig. 2.4, both the volumetric feeding rate, F, and the feed substrate concentration SQ, may be constant or may vary with time, giving the possibility of such feeding strategies as: 1. 2. 3. Slow constant feeding, which can be shown to result in linear growth of the total cell biomass. Exponential feeding to maintain constant substrate concentration and, resulting in unlimited, exponential cell growth. Feedback control of the feed rate, based on monitoring some key component concentration.

The important characteristics of fed batch operation are therefore as follows: 1. 2. 3. 4. Extension of batch growth or product production by additional substrate feeding. Possibility of operating with separate conditions for growth and production phases. Control possibilities on feeding policies. Development of high biomass and product concentration.

For fed-batch operation, the cell balance follows the same form as for batch operation, but since additional substrate feeding to the reactor now occurs, the substrate balance takes the form:
Rate <* "| accumulation V of substrate J

( Substrate \ ( Substrate >| (f^d [n) _ consumption \ rate )

Under controlled conditions, in which the substrate concentration is maintained constant or kept small, the accumulation term in the above equation will also be small, with the result that the feed rate of substrate into the reactor will balance the rate of consumption by reaction.

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One other balance equation, however, is also necessary, i.e. the total mass balance, f Rate of accumulation of ^ V mass in the reactor /
=

( Mass flow rate of feed ^ 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 or repeated fed batch, which involves changing volume with a filling and emptying period. The changing reactor concentrations repeat themselves with each cycle. This operation has similarities with continuous operation and approaches most closely to continuous operation, when the amount withdrawn is 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 the computer.

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 of addition and removal are such that the volume of the reactor contents is maintained constant. The depleted material, of course, contains any products that 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 these may be considered either as discrete stages, as in the continuous, stirred-tank bioreactor, or as differential devices, as represented by the continuous tubular or column reactor.

Continuous tank bioreactor

Continuous tubular bioreactor

Figure 2.5. The two main types of continuous reactors.

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As shown later, these two differing forms of continuous reactor operation have quite different operational characteristics. Both however are characterized by the fact that after a short transient period, during which conditions within the bioreactor change with time, the bioreactor will then achieve a steady state. This means that operating conditions, both within the bioreactor and at the bioreactor outlet, then remain constant, as shown in Fig. 2.6.
Concentration Startup period Steady state

time Figure 2.6. Startup of a continuous reactor.

Continuous reactors, however, have found little use as biological reactors on a production scale, although there are a few important examples (Id's single-cell protein air lift process, wastewater treatment and the isomerization of corn sugar to fructose syrup). Frequent use is made of continuous reactors in the laboratory for studying the kinetics of organism growth and for enzyme reaction kinetics. This is because the resulting form of the balance equation, leads to an easy method for the determination of reaction rate, as discussed in Ch. 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. In this case, S is the concentration of any given reactant consumed, and P is the concentration of any given product.
So
Cone. Tank

So
Cone.

Tube

distance

distance

Figure 2.7. Profiles of substrate and product in steady state continuous tank and tubular reactors.

As seen, the concentrations in a perfectly mixed tank are uniform, throughout the whole of the reaction vessel contents and are therefore identical to the concentration of the effluent stream. In a tubular reactor the reactant concentration 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 from inlet to outlet. These differences arise because in the tank reactor the entering feed is continuously being mixed with the reactor bulk contents and therefore being diluted by the tank contents. The feed to the tubular reactor, however, is not subject to mixing and is transformed only by reaction, as material moves down the reactor. No real situation will exactly correspond to the above idealized cases of perfect mixing or zero mixing (plug flow), although the actual behavior of tanks and tubes tends in the limit towards the corresponding idealized model. The characteristics of continuous operation are as follows: 1. 2. 3. 4. 5. Steady state after an initial start-up period (usually) No variation of concentrations with time Constant reaction rate Ease of balancing to determine kinetics No down-time for cleaning, filling, etc.

The balance equations at steady state for a well-mixed tank reactor have the form 0 = (Input) - (Output) + (Production) since at steady-state the rate of accumulation and therefore the rate of change is zero. This equation predicts that the reaction rate causes a depletion of substrate from the feed condition to the outlet, (the product will increase) and that the rate 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-state implies the same non-transient conditions, but now concentrations also vary with position. The same situation also applies to the case of a series of wellmixed 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 over every part of the reactor volume. The concentration characteristics of a tubular reactor, as shown in Fig. 2.7, are well approximated by a series of tank reactors. Referring to Fig. 2.8, and moving downstream along the reactor cascade, the substrate concentration decreases 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 concentration S, the multiple tank configuration or a tubular reactor would 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 run preferably in single tanks, however, since then the substrate concentration is always 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 the formulation of the tank balance equations, as before and then the application of the equations, successively from tank to tank such that the effluent from the preceding tank is the feed of the next and so on. Tanks-in-series bioreactor operations are illustrated by the simulation examples TWOSTAGE, STAGED and DEACTENZ in Sec. 8.4.

2.2.4 Summary and Comparison


The operating characteristics of the various reactor modes are summarized in Table 2.1. The important bioreactor operating parameters will depend on the mode of operation. In batch operation, concentration levels can be varied by adjustment of the initial values, whereas in continuous and semi-continuous operation, the concentration levels depend on the feed rate and feed concentration. As indicated previously, the manner in which the bioreactor is operated can therefore give rise to different concentration levels and therefore differing productivities. The consequent concentration profiles depend, of course, on the reaction kinetics, which express the rate of reaction as a function of the concentrations of reactants and products.

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Table 2.1. Summary of reactor modes. Mode of operation Advantages Batch

Disadvantages

Equipment simple. Suitable Downtime for loading and for small production. cleaning. Reaction conditions change with time. Provides high production. Better product quality due to constant conditions. Good for kinetic studies. Control of environmental conditions, e.g. substrate concentration. Requires flow control. Culture may be unstable over long periods. Requires feeding strategy to obtain desired concentrations.

Continuous

Fed batch

Table 2.2 lists the main operating parameters for the three differing modes of bioreactor operation. Table 2.2. Operating variables for batch and continuous bioreactors. Batch Continuous Semicontinuous Initial medium composition Inlet medium and inoculum composition Temperature, pressure pH if controlled Reaction time Aeration rate Aeration rate Stirring rate Stirring rate Aeration rate Stirring rate Feeding rate and control program Feed and initial substrate composition

Temperature, pressure Temperature, pressure pH if controlled Liquid flow rate (residence time) pH if controlled Liquid flow rate (residence time)

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The foregoing discussion of the varying characteristics of the different reactor types and their concentration profiles allows a qualitative comparison of the volume requirements for the different types of reaction, according to the particular kinetics. For this it is first necessary to consider the qualitative nature of the basic forms of kinetic relationship: zero order, first order, product and substrate inhibition. The detailed quantitative treatment of these kinetic forms is dealt with in Ch. 3. The rate of a zero order reaction is independent of concentration. Many bioreactions at high substrate concentrations follow zero order kinetics and are therefore insensitive to concentration and to the effects of concentration gradients. From the kinetic viewpoint, therefore, any reactor type would be equally suitable. First order reaction rates are directly proportional to concentration. Bioreactions at low concentration are generally first order, and this would favor operation in either a batch or a tubular/column type reactor. This is because reactant concentrations in such reactors are generally high overall and hence the overall rates of reaction are also consequently high. Hence the reactor volume required for a given duty would generally be small. (In the case of a batch reactor, this of course neglects the time lost for filling, emptying and cleaning.) A reaction with substrate inhibition would be best run in a tank at low substrate concentration, since the concentration would be low throughout the whole of the tank contents. Conversely, product inhibition would be more pronounced in tank reactors, since product concentration would be at its highest. In this case, a tubular type reactor or batch reactor would be preferred. Table 2.3. Kinetic considerations for reactor choice. Continuous Reaction Batch Tank Tanks-inContinuous Kinetics Series or Single Tank Tubular Zero order OK OK OK First order Substrate inhibition Product inhibition Production triggered by shift in environment Best Low initial concentration Best Best Low tank concentrations Best
Low conversion only Best Low conversion only

Fed Batch
Low conversion OK

Best
Low conversion only

OK for temp- Possible erature-shift

Not suitable

Best for concentrationshift

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Table 2.3 compares the performance of batch tanks, continuous tubular or tanks-in-series reactors and single continuous tank reactors. As discussed in Sec. 4.2.1, batch tank concentration-time profiles are exactly analogous to the steady state concentration-distance profiles obtained in continuous tubular reactors. In terms of performance, therefore, the batch reactor would be the same as a tube, when compared on the basis of equal batch time in the tank and time of passage through the tube. Tanks-in-series reactors, as shown in Fig. 2.8, involve step wise gradients, which in the limit are very similar to those of continuous tubular reactors, hence, making their performance similar to that of a tubular reactor. Owing to the high degree of mixing which leads to a uniform concentration, the performance of the single continuous stirred tank reactor is very different to that of the other reactor types. An exact quantitative comparison can be made using the mass balance equations developed in Ch. 4 for each reactor type.