Bioprocess and Biosystems Engineering 24 (2002) 273279 DOI 10.

1007/s004490100263

Understanding the bioreactor

n G. Lide

Abstract Analysis of bioreactors is central for successful design and operation of biotechnical processes. The bioreactor should provide optimum conditions, with respect to temperature, pH and substrate condition, for example, besides its basic function of containment. The ability to control the substrate concentration is an important function of the bioreactor. The substrate concentration can be subject to spatial variation advertently or inadvertently and may also change with time in batch or fed-batch operation. The cellular metabolism will depend on local concentrations in the reactor, as well as on the physiological status of the cell. In order to understand the bioreactor operation, cellular metabolism must be considered together with the ow prole and the mass transfer characteristics of the bioreactor. Some fundamental aspects of bioreactor operation for yeast and bacterial cultivations are discussed in this short review. Gradients

Keywords Bioreactor analysis, Fed-batch control,

List of symbols cj concentration of compound j (mol/m3) R product formation rate (mol/h) r volumetric product formation rate (mol/m3 h) t time (h) u input process variables V reactor volume (m3) x length coordinate (m) z state vector of environmental variables aj stoichiometric coefcient for j G exchange coefcient (m2/s) q density (kg/m3) rc turbulent Schmidt number, i.e. leff/qDeff leff effective turbulent viscosity (kg/m s) m velocity (m/s)

Received and accepted: 19 June 2001 Published online: 26 September 2001 Springer-Verlag 2001 n G. Lide Chemical Engineering II, Lund University, P.O. Box 124, 221 00 Lund, Sweden E-mail: Gunnar.liden@chemeng.lth.se Tel.: +46 462220862 Fax: +46 46149156

Introduction The bioreactor can, with some justication, be claimed to be the heart of any industrial biotechnical process. In the words of Cooney [1]; ``The continued success of biotechnology depends signicantly on the development of bioreactors, which represents the focal point for interaction between the life scientist and the process engineer''. However, while the progress in molecular microbiology has been very apparent in the past decade shown not least by the emergence of several ``ome-tagged'' disciplines the bioreactor itself has (seemingly) undergone only moderate changes in the past 40 years. One reason for this could be that the design and operation of bioreactors are already more or less fully understood. An alternative explanation, however, is that the operation of a large scale bioreactor is in fact so complex, that it has been difcult to approach the fundamental problems at all, a view expressed in [2] for example. The archetypical large-scale fermentation process today is a fed-batch process with a high nal density based on as cheap a substrate as possible. The fed-batch process was originally developed in the 1910s for baker's yeast production [3]. The reason behind the signicant improvement of biomass yield in fed-batch operation compared to batch operation is that glucose repression and overow metabolism in the yeast Saccharomyces cerevisiae leading to ethanol formation can be avoided in the former [4]. Overow metabolism is by no means limited to S. cerevisiae, but is a rather common phenomena shown by several other microorganisms such as Escherichia coli, an industrially very important host for recombinant protein production. In E. coli, overow metabolism results in the formation of acetate [5]. Overow metabolism is one important reason for using a fed-batch operation. Another reason for fed-batch operation is to minimize inhibition effects. These can be caused by the main carbon source itself or other compounds or impurities in the feed medium. Understanding inhibition effects may be very crucial for designing a successful operating strategy [6]. A fed-batch operation may also be called for by limitations in the mass or heat transfer in the reactor. The overall oxygen or heat transfer rates may set a limit for the maximum permissible carbon source feed rate. It is not uncommon that the maximum feed rate is determined by overow metabolism in the beginning of fed-batch operation, but limited by mass or heat transfer towards the end of operation. In large bioreactors spatial inhomogeneities, e.g. with respect to energy dissipation and concentration of oxygen

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and/or principal carbon source, will most likely occur. These inhomogeneities depend on the ow eld, the position and mode of substrate addition and the reaction kinetics, i.e. the cellular metabolism. Developed tools in computational uid dynamics (CFD) have improved the possibilities to resolve macroscopic ow pattern. However, cellular physiology must be considered together with the physical transport phenomena. Early experimental studies concerning gradients merely showed the existence or absence of gradients in large-scale bioreactors. Subsequently, overall effects on product yields were studied, and more recently also space-resolved intracellular measurements of mRNA-levels, for example, have been made in large-scale bioreactors [7]. In this short review, the basic tasks of the bioreactor and some fundamental problems of bioreactor operation are discussed.

Basic tasks of the bioreactor There exist a great variety of bioreactors, both commercial and natural. Examples of the latter include ponds, calf stomachs and termite guts [1, 8]. Most commercial bioreactors and some natural fall into one of the following categories; unstirred vessels, stirred vessels, bubble columns, airlift reactors, membrane reactors, uidized beds, or packed beds. Division into different categories may also be based on the mode of agitation (internal mechanical or external pumps) or the prevailing continuous phase (gas or liquid) [9]. From a commercial point of view, the task of the bioreactor is simply to minimize production costs. This includes achieving a high product yield, a high productivity and increasingly important a high reproducibility. This overall task can be decomposed into a number of subfunctions in different ways (a good analysis is given in [10]). A typical summary is given in Table 1. One may note that the basic tasks are not orthogonal, but rather highly collinear. Several of the tasks, e.g. temperature control, suspension and dispersion, are for instance helped by stirring. With respect to gasliquid mass transfer, focus is normally put on oxygen transfer. Obtaining a high oxygen transfer rate while maintaining sterility was in fact one of the critical questions in the early development of the penicillin process in the 1940s. One could even argue that in solving this problem, the entire scientic discipline of biochemical engineering emerged [11]. Many good reviews are available on this subject and the overall mass transfer characteristics of the bioreactor will, despite its
Table 1. Basic tasks of the bioreactor

importance, not be further discussed here (for a recent review see [12]). Another important factor is the transfer of product gases predominantly carbon dioxide out of the reactor, since these may be inhibitory to the microorganism [13]. The importance of mammalian cell cultures is increasing. In mammalian cell cultures shear rates are highly signicant, which in turn lead to problems with control of oxygen tension. Due to the slow growth the risks of contamination are also much greater. Innovative bioreactor designs for these kinds of cultures have recently been introduced, e.g. the spinning basket design. Also, for growth of plant cells, shear sensitivity may lead to problems with sufcient oxygen transfer [14] so the proper design of the impellers is important [15]. In short, the bioreactor should provide an optimum environment for the desired microbial reactions. In line with the ``ome-clature'' of current biotechnology (i.e. genome, transcriptome, proteome, metabolome), one could introduce the term ``envirome'' for the state vector containing all relevant extracellular variables, e.g. concentration of all chemical compounds and all relevant physical variables (temperature, shear rate, local velocity, turbulence intensity and so on). The interactions between the ``envirome'' and the other ``omes'' are schematically shown in Fig. 1. One may look upon the bioreactor as an envirome generator. The task of the biochemical engineer is thus to control the ``envirome'' such that the process is optimized. This should be accomplished by using only a

Function

Fig. 1. Schematic representation of interactions between the cellular environment (``the envirome'') and the cellular metabolism. The Containment (ensurance of sterility) biochemical synthetic route (or the information ow) from the Introduction of gaseous reactants (e.g. oxygen) genome to the metabolome is shown as unbroken arrows. Possible Introduction of liquid reactants (e.g. carbon source) interactions in that route (e.g. transcription factors or effectors binding to the genome, enzymatic feed-back loops, or poisoning Removal of gaseous products (e.g. carbon dioxide) Control of the physical environment (e.g. temperature, shear rate, effects) are shown by dashed arrows. The metabolome interacts with the envirome via excretion or uptake of metabolites. However, the pH) metabolome does not dene the envirome in the same manner as the Suspension (e.g cells, particulate matter) genome denes the transcriptome. The arrow between the metaboDispersion (two-phase systems) lome and the envirome is dashed to indicate this difference

n: Understanding the bioreactor G. Lide

very limited number of input variables, which furthermore operation have indeed been reported, e.g. [16]. The interest in implementation of periodic operation has so far are subject to severe constraints. been very limited, however, and here we only consider fedbatch operation. To be able to make any use of Eqs. (3) Optimizing bioreactor operation and (4) it is necessary to dene the relevant state vector z and to quantitatively describe the relation between r and z. Steady-state operation The dimensionality of z is of course large, however, kinetic The optimization problem to be solved in a continuous expressions (valid within a limited range of operation) process can be expressed by Eq. (1). may sometimes be obtained by considering only one max u u R u 1 element in the state vector; the concentration of a ``limiting'' substrate. The functional dependence of r where on z can then be investigated and an empirical or Z Z Z semi-empirical model can be derived. A typical example rzx; udV 2 R would be a Monod type kinetic equation, including v perhaps a substrate or product inhibition term. With The control vector (u) consists of variables that can be known parameters, the variational problem can then be solved and a feed prole, e.g. for substrate or inducer controlled, such as temperature set-point, pH set-point, addition, can be calculated. Chae et al. [17] studied two substrate feed rate, and stirrer rate, whereas the state different constructs for production of chloramphenicovector (z) contains all relevant state variables in the lacetyltransferase (CAT) based on the inducers isopropyl reactor. In chemostat operation, the problem is thus to b-D-thiogalactopyranoside (IPTG) and arabinose. By nd an operating point, u*, such that the state-vector z (the ``envirome vector'') within the range dened by the fusing the CAT protein with the green uorescent protein (GFP), they were able to get on-line data for protein domain of u, is optimal for the process. production, although the lag time of cyclization of the GFP protein had to be considered [18, 19]. Open-loop optimal Dynamic operation In fed-batch operation, the problem is more difcult, since feed proles for the inducer addition were calculated for the control vector u is now a time-dependent function. The both IPTG and arabinose. The authors also discussed the potential of using the GFP uorescence for feed-back optimization problem is therefore transformed into a control of inducer addition. variational problem. Control strategies need not be model based (Table 2). Z Sometimes, reliable models are simply not yet available F u R u t d t 3 and other methods must then be used. Altenbach-Rehm t et al. [20] made an empirical optimization of the addition prole of the inducer IPTG for heterologous protein with production in E. coli. A exible feed rate prole described Z Z Z by a double sigmoid function with a ve-parameter model was used and parameters were optimized using a sorzut ; xdV 4 R called genetic algorithm. The resultant activity of the v model enzyme (GDP- mannose pyrophosphorylase) was The problem is now to nd the function u*(t) which more than doubled after modication of the inducer maximizes the functional F(u). In principle, a continuous addition prole. process can be also operated in transient mode by, e.g. Another strategy for fed-batch control is to use a periodic changes of inlet medium composition or strategy based on acquired physiological knowledge, temperature. Possible improvements by using periodic which is not necessarily formalized mathematically [21].

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Table 2. Optimization strategy of introduction of substrate or inducer in fed-batch fermentations

Principle of optimization Empirical Physiological

Comment No mechanistic model is available. Statistical methods can be applied to nd optimum u No mathematical model is necessarily available. However, physiological reasoning suggests that one or several measurable variables should be kept constant or below a treshhold value A model based on a possible mechanism is available. Theoretical analysis can give the optimum u

Recent examples Altenbach-Rehm et al. [20] kesson et al. [22]; Taherzadeh et al. [24] A

Model based

Chae et al. [17]

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Examples of physiological control are to hold a substrate concentration at or below a threshhold value, to maintain a specic growth rate at or below a critical value, or to operate at a particular value of the respiratory quotient. One signicant advantage of this kind of control strategy is that it is often readily amenable to feed-back control implementation. An interesting variant of physiological control is the so-called probing control [22, 23]. In probing control, the physiological status is periodically probed by making perturbations in the substrate ow. By monitoring the response in dissolved oxygen tension, for instance, it is possible to determine if overow metabolism occurs and hence to lower the feed rate. A similar control strategy was used in [24] for feedrate control of inhibiting hydrolyzates in fed-batch fermentation using S. cerevisiae. Some of the toxic furan compounds were shown to be converted by yeast cells [25]. The rate of addition of hydrolyzate was controlled by measuring the response in the carbon dioxide evolution rate after step changes in feed-rate.

  @ cj @ @ @ cj vi cj C aj r @t @ xi @ xi @ xi C leff qrc

5 6

The problem of gradients In large-scale aerated reactors there will almost certainly be gradients present, both with respect to oxygen and, in the case of fed-batch or continuous operation, also with respect to the limiting substrate. This means that the reaction rate must be known locally (cf. Eqs. 2, 4). What is even more problematic is that r will not only depend on the local value of z, but also on the history of the cell (as discussed in [26]). This is due to the fact that changes in extracellular conditions may trigger regulatory phenomena, e.g. gene expression turn-on or turn-off. Obviously, this makes it exceedingly difcult to model the kinetics of product formation during dynamic conditions and the problem must be approached in a different way. One suggestion is to consider the four questions given below.
(1) What does the ow pattern look like in the reactor, and is this likely to lead to large gradients? (2) Will the gradients inuence process performance in a negative way? (3) What is the probable mechanism behind negative effects? (4) How should the negative effects be minimized? The straightforward method of determining presence of gradients is of course to measure concentrations locally in a large-scale reactor [27, 28, 29]. However, for practical reasons only a few discrete points can be examined, and the picture obtained will therefore be rather incomplete. A different option is to use mathematical modelling of the ow pattern. Computational calculations can be made at different levels of sophistication, from a low degree of discretisation using compartment models up to fully edged computational uid dynamic calculations (CFD) of the ow eld [26]. For a one-phase ow, the concentration eld for a substance, j, can in principle be calculated from Eq. (5) in combination with solving the velocity eld from the NavierStokes equations.

(The summation convention has been used above for repeated indicies.) Since the ow is turbulent in most bioreactors, the effective turbulent diffusivity must be obtained from a model, e.g. the Kolmogorov k model. The turbulence model in itself can be questioned, but equally difcult for reasons discussed previously is to nd an expression for the sink term (i.e. the reaction term). In practice, one has to resort to an expression based on steady-state kinetics. Calculated concentration proles therefore may or may not be fully accurate. Another very important complication is the fact that most bioreactors are aerated and therefore a turbulent two-phase ow needs to be modelled. The success in terms of quantitative agreement between experimental data (e.g. from laser Doppler anemometry) and simulation results from such modelling has so far been limited. CFD should therefore still be regarded mainly as a qualitative, but very helpful tool, which can give clues to the probability of severe concentration gradients. The inuence on process performance has to be experimentally addressed. Although measurements at large scales have been made, most experimental studies have been performed using scaled-down systems. An overview of some selected studies is shown in Table 3. Primarily, the effects of oxygen or glucose gradients have been investigated, since these commonly occur. The two most often studied microbial systems have been Baker's yeast (S. cerevisiae) and E. coli. Both these organisms are of large industrial importance and, furthermore, both exhibit overow metabolism and anaerobic metabolism. They are therefore sensitive to both glucose excess and to oxygen limitation (in fact, not very attractive features for organisms used in large-scale production). Simulations of gradients in a scale-down system have been done by step-change experiments in small-scale stirred tank reactors (STR), or by connecting two small reactors. Step-change experiments in one reactor can be used to study dynamic effects of a sudden increase in glucose concentration or aerobic/anaerobic transitions [38, 39]. A very rapid mixing of glucose can be obtained in a small STR. However, the oxygen transfer rate is not sufciently high to enable studies of fast dynamics related to aerobic/anaerobic transitions. A combination of a STR and a plug ow reactor may in this case be a better option [35]. The a priori expectation is that gradients are likely to have a negative effect on process performance, which is also reported for biomass yield (cf. Table 3). However, not all results show decreased performance in the large-scale process. The leavening capacity of Baker's yeast was found to be higher in both a scaled-down model system and a large-scale process compared to an ideally mixed system [36]. Also the stability of a heterologous protein was found to increase in a non-ideally mixed system [37].

n: Understanding the bioreactor G. Lide Table 3. Overview of some selected investigations concerning effects of substrate gradients in bioreactors

System studied Effects of oxygen gradients on gluconic acid production by Gluconobacter oxydans Effect of oxygen gradients in Baker's yeast production Effect of glucose gradients in Baker's yeast production Effects of oxygen gradients on recombinant protein production in E. coli Effect of glucose gradients in Baker's yeast production

Experimental approach Step-change experiments in one bioreactor (1.6 l); Two connected lab scale bioreactors (working volume 1.6 and 0.6 l) one operating aerobically and the other anaerobically Step change experiments in one bioreactor; Continuous cultivation in two connected bioreactors (1.5 and 3 l volumes) Lab-scale bioreactor (2 l) with a recirculation loop. Simulation of log-normal circulation times achieved by controlling substrate addition into the recirculation loop Monte Carlo simulation used for switching aeration on/off in a 2 l bioreactor Two fermenter system, stirred tank reactor (15 l) in series with a tubular reactor (0.8 l); large-scale (215 m3) bubble column Large-scale (30 m3) and a scaled down reactor system stirred tank reactor (15 l) in series with a tubular reactor (0.8 l). Monitoring of mRNA levels at different positions Two fermenter system STR+PFR (fed batch) Pilot scale (3 m3)

Findings Gluconic acid production corresponded to time spent aerobically

Reference Oosterhuis et al. [30]

Effects of glucose gradients on physiology of E. coli

Biomass yield decreased and formation of acetic acid increased with increasing circulation time No measureable effect on biomass yield from the glucose gradients. Some acetic acid formation at long circulation times Plasmid number was lower in cells during simulated large scale conditions Biomass yield decreased about 7% compared to ideal mixed reference system. Gassing power of produced yeast increased 1025% compared to an ideal mixed system Highest level of stress in substrate entrance zone. Transcriptional activation occurred within about 10 s Glucose-induced oxygen limitation indicated by formate formation. Cell lysis increased in SDR compared to well mixed control experiments, but degradation of rhGH monomer decreased

Sweere et al. [31, 32]

277
Namdev et al. [33]

Namdev et al. [34] George et al. [35, 36]

Schweder et al. [7]

Effect of glucose gradients in production of human growth hormone (hGH) in E. coli

Bylund et al. [37]

There are several different mechanistic reasons behind the effects of gradients. There will be direct mass action effects if the cells are constitutively equipped for both aerobic and anaerobic metabolism. Ethanol formation in S. cerevisiae will, for example, occur very rapidly if oxygen is depleted. Also, the intracellular response in NADH levels to a high glucose concentration has been found to occur within a few seconds [40]. Furthermore, oxygen and glucose trigger several regulatory responses. As discussed by Konz et al. [41], the regulatory response time will be determined by the rate of mRNA formation and the rate of translation. In a study by Schweder et al. [7], seven different mRNA levels in E. coli were studied in a scaleddown system. It was found that the transcription of several genes, such as the proU gene, which is involved in osmoregulation, responded within 15 s of exposure to high glucose concentration. The rate of protein synthesis is probably somewhat lower. The peptide elongation rate has been estimated to be between 1316 amino acids per second in E. coli [40]. For this reason, a single ``dip'' in oxygen concentration of short duration may not result in the formation of a regulated protein. However, the effects of repeated depletions occurring for a cell circulating in a large-scale bioreactor are difcult to predict.

How can the problems of gradients be avoided? One method of avoiding potential problems is of course to avoid creating the gradients. Use of multiple substrate inlets is here often preferable to increasing the stirrer rate. In the long run, it may also be worthwhile to use metabolic engineering to decrease the sensitivity of the production organisms to the gradients. Tsai et al. [42] expressed haemoglobin from Vitreoscilla in E. coli. The constructed strains could thus ``store'' oxygen for use in times of oxygen starvation and a decreased excretion of fermentation products was indeed reported. Other studies have focused on the overow metabolism in E. coli. Reductions of acetate formation have been achieved by, e.g. heterologous expression of acetolactate synthase from Bacillus subtilis [43], or by mutation of the pyruvate kinase or the phosphotransferase system [44]. In the rst approach the ux to acetate was diverted into acetoin, whereas in the other strategy the maximum specic growth rate was reduced, thereby avoiding overow metabolism.

Final remarks It is certainly not simple to analyse the bioreactor. An increased usage of CFD modelling will be needed to obtain a better description of ow patterns and

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concentration gradients, and will help in the design of less gradient-prone substrate inlet systems. However, important problems regarding modelling of turbulence and multiphase ow remain to be solved. Cellular physiology needs to be studied at process conditions, i.e. using high-density cultivations and industrial media. Tools developed in the eld of functional genomics must be utilised to get an insight into responses on transcript, protein and metabolite level, and the question of dynamic responses must be both theoretically and experimentally addressed. New approaches in kinetic modelling are required in which also the transcriptional and translational levels are included. By combining novel experimental and computational tools, it will certainly be possible to design smarter production systems for the biotechnology industry of tomorrow.
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