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2.

14 Bioreactor Engineering
J-J Zhong, Shanghai Jiao Tong University, Shanghai, China
© 2011 Elsevier B.V. All rights reserved.

2.14.1 Introduction 166


2.14.2 Design and Types of Bioreactors 167
2.14.2.1 Fundamental Design Principles 167
2.14.2.2 Stirred-Tank Bioreactors 167
2.14.2.3 Pneumatically Agitated Bioreactors 167
2.14.2.4 Membrane Bioreactors 168
2.14.2.5 Fixed-Bed Bioreactors 169
2.14.2.6 Fluidized-Bed Bioreactors 169
2.14.2.7 Wave Bioreactors 169
2.14.3 Effects of Process Parameters on Biological Performances 170
2.14.3.1 Temperature 170
2.14.3.2 pH 171
2.14.3.3 Mixing 171
2.14.3.4 Oxygen Transfer 171
2.14.3.5 Shear Force 172
2.14.4 Bioreactor Operation Strategy 172
2.14.4.1 Fed-Batch Culture 172
2.14.4.2 Continuous and Semicontinuous Culture 173
2.14.4.3 Perfusion Culture 173
2.14.5 Industrial Applications of Bioreactors 173
2.14.5.1 Bioreactor Scale-Up 173
2.14.5.2 Multiscale Study of Industrial Bioreactors and Bioprocesses 173
2.14.5.3 Measurement of Parameters in Industrial-Scale Bioreactors 174
2.14.5.4 Modeling and Simulation 174
2.14.6 Trends in Bioreactor Engineering 174
2.14.6.1 Microbioreactor 174
2.14.6.2 Cell as a Super Bioreactor 175
2.14.6.3 Plant and Animal as Powerful Protein-Producing Bioreactors 176
Acknowledgment 176
References 176

Glossary bioreactor operation strategy The strategy that aims to


bioreactor As the core of bioprocess, it is a vessel in which improve the cost effectiveness of bioprocesses by
a biological reaction or change takes place and an providing a high volumetric productivity, which includes
optimum external environment is provided to meet the multistage batch, fed-batch, single- or multistage
needs of the biological reaction system so that a high yield continuous (chemostat), semicontinuous (draw-and-fill
of the bioprocess is achieved; bioreactor is also called or repeated batch), and perfusion (chemostat with cell
fermentor in fermentation industry. retention) cultivations; in order to develop an efficient
bioreactor engineering This covers an extensive area, bioreactor operation, cellular metabolism must be
being a systematic science-based approach to studying considered together with the flow profile and the mass
bioreactor, in which two distinct bodies of knowledge, transfer characteristics of the bioreactor because they
that is, molecular biology and process engineering, are closely interact with each other.
involved. shear sensitivity This reflects the sensitiveness of a
biological system This includes enzyme, microorganism, biological system to shear force from excessive agitation
animal cell, plant cell, and tissue, on which intensive and sparging, over which a cell or enzyme is caused with
studies, such as cell growth, metabolism, genetic damage and/or malfunction such as abnormal metabolic
expressions, metabolic manipulation, and bioreaction and physiological responses; compared to microorganisms,
pathways, are needed to understand the cells’ requirement animal and plant cell cultures and filamentous fungal
on their physical and chemical environment. fermentations are generally considered shear sensitive.

165
166 Bioreactors – Design

2.14.1 Introduction

Bioreactor is a vessel in which a biological reaction or change takes place. The biological systems involved include enzymes,
microorganisms, animal cells, plant cells, and tissues. The bioreactor is a place where an optimum external environment is provided
to meet the needs of the biological reaction system so that a high yield of the bioprocess is achieved. To design an appropriate
bioreactor for a particular bioprocess, intensive studies on the biological system, such as cell growth, metabolism, genetic
manipulation, and protein or other product expression, are needed to understand the cells’ requirement on their physical and
chemical environment. Various bioreactor types and configurations have thus been exploited and developed along with the
advances in the understanding of biological systems. In addition, it is necessary to control the bioreactor operating parameters in
order to favor the desired functions of the living cells or enzymes. Dissolved oxygen (DO) concentration, pH, temperature, mixing,
and supplementation of nutrients need to be controlled and optimized. Because two distinct bodies of knowledge, that is,
molecular biology and process engineering, are involved and bioreactor is the core of bioprocess, a systematic science-based
approach to studying bioreactor is needed and the term ‘bioreactor engineering’ becomes more appropriate than ‘bioreactor’ or
‘fermentor’. Figure 1 shows a simplified schematic process and the scope of bioreactor engineering.
As shown in Figure 1, the bioreactor actually is the core of biological processes. To consider a bioreactor system, the final
objective of this biological process must be identified, which is often determined by the market demand for a certain product or
beneficial biotransformation process. Because of the rapid advances in recombinant DNA technology and genome sequencing, the
same product or biological process may be achieved by different biological systems: microorganisms, plant cells, animal cells, or
enzymes. Their genetic expressions, metabolic manipulation, and bioreaction pathways all need to be understood. The next step is
to identify the medium requirements for the efficient performance of the chosen biological system. The media design and
optimization can be based on basic knowledge of stoichiometry and experimental data, including monitoring the composition
changes of the media, intermediates, products, and nutrients. Stoichiometric calculations provide quantitative relationships
between yields of biomass and product synthesis and maintenance requirement and energy production. Complementing stoichio-
metric data for the design of a bioreactor, a kinetics study will reveal the biological reaction rates, including cell growth, substrate
consumption, product synthesis, and byproduct formation rates. Many enzymatic reactions are involved, and inhibitions caused by
products, byproducts, or even substrate at high concentrations are often observed. On the other hand, the physical environment
directly affects the biological performance. Shear stress, mass transfer, mixing, pH, and temperature are all interrelated and can
influence biological reactions. In addition, equally important factors to be considered are downstream process requirements. With
this understanding of the biological system and its requirements on its physical and chemical environment, a proper bioreactor type

Product identification and the general requirement of the whole bioprocess

Biological reaction system identification

Genetic expression and metabolic manipulation, pathway identification

Stoichiometry and medium Physical environment requirements


design, kinetics studies (oxygen transfer, shear, mixing, temperature, pH)

Bioreactor type selection (biological requirement, upstream


constraints, downstream constraints)

Bioreactor operation mode selection Bioreactor characterization (hydrodynamics,


(batch, fed-batch, continuous, mass and heat transfer, mixing, power
perfusion) consumption)

Bioreactor system design and scale-up including control and support system

Integration of bioreactor system into the whole bioprocess

Figure 1 Schematic representation of the process and the scope of bioreactor engineering.
Bioreactor Engineering 167

can be selected. Among the bioreactor types available for a certain bioprocess, it is important to have a balanced consideration of
many factors, including oxygen transfer, mixing, shear, operational stability and reliability, scale-up, and cost. The chosen bioreactor
should be further characterized and the operational mode should be optimized. The bioreactor characteristics and operational
mode also greatly affect the biological performances. An efficient bioreactor system relies greatly on its control and support systems.
No matter how important the bioreactor system is, it must be closely and efficiently integrated into the whole production system.
Therefore, other process requirements and constraints should also be considered.
Bioreactor engineering covers an extensive area. This article briefly describes a variety of bioreactor systems, with an overview of
advances in their design, control, and application.

2.14.2 Design and Types of Bioreactors

In general, most biological reaction systems can be classified into two main groups: suspension systems and immobilization
systems. Stirred-tank, airlift, and bubble-column bioreactors are mainly used for suspension cultures; membrane, packed-bed,
and fluidized-bed bioreactors are mainly used for cultivating attached cells or immobilized enzymatic reactions. Obviously,
there are some bioreactors that can be applied in both of these two categories. For example, with the appropriate carriers, the
immobilized cells or enzymes on carriers can be suspended in stirred-tank bioreactors (STRs) or airlift/bubble-column
bioreactors.

2.14.2.1 Fundamental Design Principles


The basic function of a bioreactor is to provide optimum conditions for cell physiology and metabolism by regulating various
chemical and/or physical factors. The design and selection of each type of bioreactors are unique but there are some basic
fundamental principles. In general, the main criteria for designing a bioreactor should consider adequate oxygen transfer, low
shear stress, and adequate mixing [1, 2].
In considering the reactor design and selection, nutrients must be effectively provided to the cells, and waste products must be
removed. Cell growth and product formation kinetics should be assessed so that an optimal environmental condition can be
defined and an operational mode can be determined. Transport phenomena, including mixing, shear force, and oxygen transfer,
should be studied in order to define the criteria for bioreactor design and scale-up. Operating parameters, such as temperature, pH,
DO concentration, and substrate concentrations, should be easy to control and monitor. In addition, the bioreactor should be as
simple and inexpensive as possible and it should easily operate free of contamination with microorganisms. In the biopharma-
ceutical industry, bioreactor design and selection should also consider current good manufacture practice (cGMP) compliance. Most
often, it is difficult or impossible to meet all the requirements, and so some compromise must be made. For example, it is very
important to give a balanced consideration between mixing and mass transfer requirements and the shear sensitivity of cells in the
design of large-scale bioreactor systems [3].

2.14.2.2 Stirred-Tank Bioreactors


STRs are one of the most conventional bioreactors. Due to their advantages such as easy scale-up, good fluid mixing and oxygen
transfer ability, alternative impellers, and easy compliance with cGMP requirements, STRs are commonly used. However, this type
of bioreactor also has several limitations, such as high power consumption, high shear, and the concerns about sealing and stability
of shafts in tall bioreactors. Compared to microorganisms, both animal cells and plant cells are shear sensitive, which results in
remarkable efforts to modify and optimize the impeller system in order to balance mixing and mass transfer requirement and
potential damage by hydrodynamic force. Numerous modifications of conventional STRs have been made by developing new
impeller designs (Figure 2) [4–8]. A number of researchers have studied the hydrodynamics of multiple impeller systems [9–11].
Several biopharmaceutical manufacturers have successfully implemented STRs at a 10 000–20 000-l scale for large-scale animal cell
cultures (Table 1) [12, 13].

2.14.2.3 Pneumatically Agitated Bioreactors


Pneumatic bioreactor is a type of gas–liquid dispersion reactor consisting of a cylindrical vessel, where compressed air or gas
mixture is usually introduced at the bottom of the vessel through nozzles, perforated plates, or a ring sparger, for aeration,
mixing, and fluid circulation, without moving mechanical parts. There are two main types of pneumatically agitated
bioreactors: airlift and bubble-column bioreactors, which are generally of low shear stress and simple design and construction.
They consist of a main body; air-bubbling device; steam generator for sterilization; air inlet, air vent system; various control
system for monitoring temperature, oxygen, and pH; and pipeline systems for transportation of steam, air, medium, and
product masses.
Airlift bioreactors (Figure 3) have some advantages over STRs, such as distributing shear stress more gently as not having a mixer
or impeller and easy to construct and scale-up with low cost. However, the lack of an impeller also brings some disadvantages such
as poor fluid mixing for highly viscous culture compared to STR and serious foaming under high aeration. Airlift bioreactors with
168 Bioreactors – Design

(a) (b) (c)


Gas out Motor

Impeller

(f) 1 2
3
Gas in
4
(d) Fluorescent lamp (e)
10
Probe 5
w
h
9

8 d

Probes
6
7

d
Air D

Figure 2 Schematic diagram of stirred-tank bioreactors (STRs) with different impellers: (a) STR with turbine impeller; (b) gate paddle bioreactor with a
spiral sparger; (c) large flat-bladed impeller bioreactor; (d) internally illuminated bioreactor; (e) helical ribbon impeller bioreactor; (f) centrifugal impeller
bioreactor. 1, stirrer; 2, gas in; 3, head plate; 4, shaft; 5, measuring points for liquid velocity; 6, sparger; 7, blade; 8, draft tube; 9, DO probe; and 10,
rotating pan.

Table 1 Large-scale processes of animal cells

Cell line Scale (l) Reactor Product

BHK 21 10 000 Agitated tank Foot and mouth disease vaccine


CHO 10 000 Agitated tank tPA
Namalwa cells 8000 Agitated tank Lymphoblastoid interferon
Bowes melanoma 7000 Agitated tank/microcarrier tPA
Murine hybridoma 2000 Airlift mAbs
Vero cells 1000 Agitated tank/microcarrriers Killed polio vaccine
Murine hybridomas 1000 Stirred-tank mAbs against cell-surface
antigens of
Adenocarcinomas
BHK 500 Agitated tank/perfusion Factor VIII

Adapted from Kretzmer G (2002) Applied Microbiology and Biotechnology 59: 135–142.

various configurations have been constructed for use in a variety of fermentation processes, cell cultures, and biological wastewater
treatment. The airlift bioreactor is the second type that is well documented and characterized, but is less so than the STR.

2.14.2.4 Membrane Bioreactors


Membrane bioreactors, which utilize specialized membranes to retain cells in the reactors, are designed for in situ separation of cells
from medium and integrate production and separation into a single step. The main advantages of membrane bioreactors are high
cell density, high volumetric productivity, and low shear stress. However, the disadvantages such as poor cell viability, poor process
stability (membrane fouling and clogging), product inhomogeneity, and diffusion gradients limit large-scale applications. Despite
the weaknesses, many membrane configurations have been tested (such as flat sheet and rotating bioreactors), and, among them,
the hollow-fiber configuration is a particularly interesting one. Membrane reactors have been used for an enormous variety of
applications in four major areas: biocatalysis, fermentation, cell cultures, and wastewater and waste gas treatments.
Bioreactor Engineering 169

(a) (b) (c)

Liquid
circulation

Riser

Dowmcomer
Draft
tube

Air Air Air

(d) (e) (f)

Air Air Air


Figure 3 Airlift and bubble-column bioreactors. (a) Airlift with internal loop; (b–d) airlift with external loop; (e) bubble column; (f) slanted-bottom bubble
column.

2.14.2.5 Fixed-Bed Bioreactors


Fixed (packed)-bed reactors are one of the most frequently employed types of bioreactors for immobilization systems. This type of
bioreactor has the advantages of simplicity of operation and high reaction rates. Enzymes or cells are immobilized in appropriate
carriers, which are packed in the fixed reactors, resulting in high solid–liquid specific interfacial contact areas, and the velocity of
liquid creeping over the static solid particles substantially alleviates the film resistance to mass transfer. The major disadvantages of
the fixed-bed bioreactor are their relatively poor mass and heat transfer coefficients due to low liquid velocities. For aerobic
biological systems, efficient gas–liquid contact and carbon dioxide removal are very critical. A fixed-bed reactor often accumulates
stagnant gas pockets, causing gas flooding and producing poor liquid distribution. It has therefore not been widely used in aerobic
microbial fermentation processes.

2.14.2.6 Fluidized-Bed Bioreactors


The fluidized-bed reactor is another widely employed bioreactor for immobilization systems. A fluidized-bed reactor can provide a
degree of mixing intermediate between the two extremes of the packed-bed reactor and the STR. On the one hand, the upward
movement of fluid carries immobilized cells upward; the particles rise until the force of gravity causes the particles to fall.
Fluidization is achieved by the combined upward and downward movement of particles. Compared to the fixed-bed reactor, the
major advantages of the fluidized-bed bioreactor include the following: The system is homogeneous and easier to monitor and
control the operating parameters; good mixing is achieved; higher mass transfer and heat transfer rates are expected; and scale-up
can be achieved without increasing concentration gradients. However, there are also operational difficulties. The major problem is
that it is not easy to predict the back-mixing and fluidization patterns. The application of the fluidized-bed reactor with
immobilized cells has been primarily achieved in wastewater treatment. Fluidized reactors have also been employed for microcarrier
cultures.

2.14.2.7 Wave Bioreactors


Wave reactors (Figure 4) [14] have emerged and been applied in recent years, which can generate a wave motion by mechanically
rocking of a culture-containing bag back and forth. These waves provide mixing and mass transfer, resulting in a suitable
environment for suspension culture of both plant and animal cells. Several cell lines, including Chinese hamster ovary (CHO)
170 Bioreactors – Design

5 6

Inlet air
Exhaust
4

Rocking
motion

3 2 1
Figure 4 Schematic of disposable wave bioreactor: 1, base; 2, pivot; 3, cell culture media in bag; 4, inflated plastic bag, which forms a disposable
cultivation chamber; 5, exhaust vent filter; 6, inlet air filter [14].

cells, NS0 cells, hybridoma cells, insect cells, as well as anchorage-dependent cells using microcarriers, have been tested in the
system. In addition, the reactor uses a presterilized, plastic disposable chamber, providing the ultimate ease in operation and
protection against cross-contamination. The chamber is placed on a special rocking platform and facilitates the compliance of cGMP
regulations. Recently, such wave bioreactor systems have been scaled up to a capacity of 1000 l, and a cell density as high as 107 cells
ml−1 was achieved [14, 15].

2.14.3 Effects of Process Parameters on Biological Performances

The analysis of bioreactors is central to the successful design and operation of biotechnical processes. The main objective of
bioreactor selection, design, and control is to provide the optimal environment for a biological reaction system. The bioreactor
should provide optimum conditions (e.g., temperature, pH, oxygen transfer, mixing, and substrate concentration), in addition
to its basic function of containment. For example, the ability to control the substrate concentration is an important function of
the bioreactor. The substrate concentration can be subjected to spatial variation – advertently or inadvertently – and may also
change with time in batch or fed-batch operation. Cellular metabolism depends on local concentrations in the reactor, as well
as on the physiological status of the cell [16]. In order to understand bioreactor operation, cellular metabolism must be
considered together with the flow profile and the mass transfer characteristics of the bioreactor because they closely interact with
each other.

2.14.3.1 Temperature
Temperature is one of the most critical parameters to be closely controlled in a bioreactor. Microorganisms are often classified
according to their growth temperature as either thermophiles (growth temperature: >50 °C), mesophiles (growth temperature: from
20 to 50 °C), or psychrophiles (growth temperature: <20 °C) [17]. Regardless of the microorganism type, microorganisms always
have a quite narrow optimal temperature range for growth. If grown at a temperature below the optimum, growth occurs slowly
resulting in a reduced rate of cellular production and product synthesis. On the other hand, if the growth temperature is too high,
not only will death occur, but protein expression or metabolite synthesis will also be seriously affected, lowering product yield or
affecting product quality.
The effect of temperature on chemical or enzymatic reactions is typically modeled using the Arrhenius equation. This has also
been used to describe the effect of temperature on the specific cell growth or cell death of the microbial system [17]. The cell growth
rate increases when the temperature is increased toward the optimum. When the temperature exceeds the optimum, the growth rate
decreases and thermal death occurs. The net cell growth rate is proposed as follows:

dX
¼ ðμ−kd ÞX ½1%
dt
where X and t are cell concentration and time, µ and kd are cell growth and cell death rate, respectively. Both µ and kd can be
expressed as functions of temperature following the Arrhenius equation:

μ ¼ A ⋅e ð − Ea = RT Þ ½2%

kd ¼ A′ ⋅ e ð − Ed = RT Þ ½3%
Bioreactor Engineering 171

where Ea and Ed are activation energies for cell growth and thermal death. Equation 2 represents the increase in specific growth rate
with temperature, and the value of Ea is typically in the range of 10–20 kcal mol−1. Equation 3 represents the thermal death rate,
which is substantially increased with temperature as Ed is much higher than Ea. Ed is typically in the range of 60–80 kcal mol−1 [17].
In a conventional microbial fermentation process, once the optimal temperature is determined, it will normally be maintained
throughout the whole fermentation process. This, however, may not always be the case for mammalian cell culture processes. In
mammalian cell culture process, a large portion of the protein product is synthesized during the postgrowth phase. Since the cell
viability drops quickly after the cell density approaches maximum, the cultivation of cells at reduced temperatures has been
proposed to improve batch culture performances. It has been consistently reported that a decrease in cultivation temperature leads
to prolonged culture viability. However, a culture temperature below 37 °C normally inhibits cell growth. A concept of two stages
is therefore proposed: a growth phase and a production phase. During the first stage, the temperature favoring cell proliferation
(e.g., 37 °C) is used to obtain a high cell density. In the second stage, temperature is reduced in order to decelerate the drop in cell
viability. This strategy, however, is not straightforward as temperature is also a very critical parameter for protein synthesis. The effect
of reduced temperature on heterologous protein production of mammalian cells varied among different studies. An optimal
temperature exists for each individual cell culture process.

2.14.3.2 pH
Different biological systems have different optimal pH ranges. Most microorganisms grow best between pH 5 and 7. During
fermentation, pH can change. As the cells grow, metabolites are released into the medium; substrate consumption also causes pH
change. A number of researchers have investigated the effect of pH on the growth kinetics of microorganisms, enzymatic activities,
and product synthesis.
In animal cell culture processes, culture pH is often controlled by the addition of an alkaline reagent, such as NaHCO3 or NaOH,
to neutralize the acidic effects of lactate and CO2 production during cell growth. Another scheme for pH control in animal cell
culture process is CO2 addition. CO2 is added to a sodium bicarbonate-containing medium in order to control the pH. In general,
using CO2 to control pH is simple and efficient. However, it may cause the following problems: in high-cell-density cell cultures, a
high rate of CO2 production will limit controllability by CO2 addition, CO2 sparging can decrease the oxygen supply and upset DO
control, and, in the case of high lactate concentrations or during periods of rapid lactate production, the limited buffering capacity
of the bicarbonate system may become inadequate. Animal cells are more sensitive to changes in pH than microorganisms. A small
change in culture pH can greatly influence the cell growth, metabolism, and production synthesis.

2.14.3.3 Mixing
In bioreactors, adequate mixing is essential in order to ensure the adequate supply of nutrients and to prevent the accumulation of
toxic metabolites. For a bioreactor designed for a suspension system, mixing time is a critical parameter to be studied and evaluated.
The fluid hydrodynamics, fluid rheology, impeller type, power input, and vessel size can all influence the mixing conditions.
Generally, the following equation can be used to describe the effects of different parameters on the mixing time in an STR [18]:
!d Va P "
tm ¼ f ; N; η; ρ; ; ; εT ½4%
D V Pa
where tm is the mixing time (s), d is the stirrer diameter (m), D is the bioreactor diameter (m), N is the impeller rotational speed
(rpm), V is the medium volume (m3), Va is the volumetric air flow rate (m3 s−1), P is the power consumption for mixing nonaerated
broth (W), Pa is the power consumption for mixing aerated broth (W), η is the apparent viscosity (cP), ρ is the density (kg m−3), and
εT is the energy dissipated (W m−3).
It was found that the presence of biomass significantly reduced the mixing efficiency, even at low broth viscosity levels. The
magnitude of this effect depends on the type of biomass and its concentration and morphology. The mixing time increases in the
following order: fungal free mycelia > fungal pellets > yeasts > bacteria. At the same concentration and under the same operational
conditions, the mixing time for fungal cell suspensions was significantly higher due to their high viscosity and non-Newtonian behavior.
In fermentation or cell culture processes, mixing has often been evaluated in terms of biological performance, such as cell growth
rate and productivity. The control of temperature, pH, and substrate concentration depends on good mixing in the bioreactor.
Although it is easy to maintain a homogeneous condition in a small-scale reactor, mixing often becomes one of the constraints
during scale-up. In large-scale bioreactors, poor mixing often leads to undesirable concentration gradients and a decrease in mass
transfer efficiency. In shear-sensitive biological systems, such as animal and plant cell cultures and filamentous fungal fermentation,
mixing cannot be enhanced simply by increasing agitation intensity because excessive agitation can cause mechanical damage to
living cells. There are numerous reports on the effect of mixing on biological performance in the literature.

2.14.3.4 Oxygen Transfer


Oxygen transfer is always a concern in aerobic biological systems. Most nutrients required for cellular growth and metabolism are
highly soluble in water; sufficient and timely supply of these nutrients can be achieved in a well-mixed bioreactor. However, oxygen
transfer often becomes a limiting step to the optimal performance of biological systems and also for scale-up because oxygen is only
172 Bioreactors – Design

sparingly soluble in aqueous solutions. When the supply of oxygen is limited, both cell growth and product formation can be
severely affected. For example, it was reported that ceasing aeration in the medium during penicillin fermentation for just a few
minutes seriously impacted the ability of the cells to produce the antibiotic [19]. In a well-mixed suspension system, the oxygen
mass balance is written as

dCo
¼ kL aðC&o −Co Þ −Qo X ½5%
dt
where kLa, C*,
o Co, and Qo are the volumetric mass transfer coefficient, saturated oxygen concentration, the oxygen concentration in
the liquid, and the specific oxygen uptake rate, respectively.
At steady state, the above equation can be solved to obtain the oxygen concentration in the liquid:

Qo X
Co ¼ C&o − ½6%
kL a
Since C* o is constant at a fixed air pressure, Co is determined by three factors: the specific oxygen uptake rate Qo, which is
determined by the biological system, cell concentration X, and the volumetric mass transfer coefficient, kLa. For a given
biological system (bacteria, yeast, animal or plant cells), a serious shortage of oxygen can be expected at a high cell density.
Aggravating this problem, high cell density often causes the oxygen transfer coefficient to deteriorate. Since kLa is so important in
supplying oxygen to the medium, a very critical aspect of bioreactor design is to achieve a sufficiently high oxygen transfer
coefficient, kLa, which is affected by many factors, including the geometrical and operational characteristics of the reactor vessel,
agitation speed, aeration rate, fluid hydrodynamics, media composition, cell type, morphology and concentration, and bioca-
talyst properties. It was estimated that oxygen transfer management accounts for about 15–20% of all operating costs for aerobic
fermentation [20].
There are numerous reports studying the effects of oxygen concentration or oxygen transfer on microbial fermentation. Although
the oxygen consumption of plant and animal cells is lower than that of microorganisms, limitation in oxygen transfer is also often a
constraining factor for cell cultures at high cell density. Maintaining a suitable oxygen concentration in the culture broth is equally
important. The optimal DO concentration may be different for cell growth and product formation.

2.14.3.5 Shear Force


A couple of parameters are related to the shear-induced cell death, such as shear stress, shear time, power dissipation, and
the growth phase of the cells. Sparging can also cause the shear damage, which can occur at different locations in a
bioreactor: Bubble-generation zone at the sparger; the rising zone through the bulk liquid; and the surface of the suspension
(either be covered with foam layer or free of foam). A possibility to overcome the problem of the rising air bubbles is
bubble-free aeration using membranes for indirect aeration, in which the supply of oxygen is diffusion-controlled and no
bubbles arise.
For shear-sensitive cell cultures, reducing the shear stress intensity by decreasing the agitation speed of the impeller is a general
solution. However, this can also bring inadequate mixing and may conflict with enhancing oxygen and heat transfer rates in a high-
viscosity cell culture broth. Furthermore, at high biomass concentrations, low agitation rates can also enhance the clumping of cells
into cell aggregates of various sizes. However, appropriate bioreactor design and control can minimize shear damage from agitation
and aeration.

2.14.4 Bioreactor Operation Strategy

An effective bioreactor operating strategy should provide a high volumetric productivity, which means more products can be formed
per unit time per unit reactor volume. A major disadvantage of standard batch processes is that significant amounts of time are taken
up by the sterilization of system and medium, filling, emptying, and cleaning of the system. Thus, to improve the cost effectiveness
of cell culture processes, various operational modes such as multistage batch, fed-batch, single- or multistage continuous (chemo-
stat), semicontinuous (draw-and-fill or repeated batch), and perfusion (chemostat with cell retention) cultivations have been
applied or developed so far.

2.14.4.1 Fed-Batch Culture


Generally, the final product concentration is primarily affected by the specific productivity of cells and integrated cell growth. To
overcome nutrient limitation, fed-batch processes have been widely practiced which involves the addition of one or more nutrients
continuously or intermittently to the initial medium after the start of cultivation, or from a certain point during the batch process.
Fed-batch cultures are currently used for most cell culture processes, especially for intracellular products of cell cultures that are
stored within the cells. Fed-batch processes are also common in animal cell cultures and have been operated at scales up to 25 000-l
working volume for large-scale antibody production [21]. A comparison of different feeding modes is presented in Table 2; among
these, the most popular and successfully used ones are fed batch and perfusion.