1
Fermentation Pilot Plant
Yujiro Harada, Kuniaki Sakata, Seiji Sato
and Shinsaku Takayama
PROLOGUE (by Yujiro Harada)
The rapid development of biotechnology has impacted diverse sectors
of the economy over the last several years. The industries most affected are
the agricultural, fine chemical, food processing, marine, and pharmaceuti-
cal. In order for current biotechnology research to continue revolutionizing
industries, new processes must be developed to transform current research
into viable market products. Specifically, attention must be directed toward
the industrial processes of cultivation of cells, tissues, and microorganisms.
Although several such processes already exist (e.g., r-DNA and cell fusion),
more are needed and it is not even obvious which of the existing processes
is best.
To develop the most cost efficient process, scale-up data must be
collected by repeating experiments at the bench and pilot scale level. These
datamust be extensive. Unfortunately, the collection is far more difficult than
it would be in the chemical and petrochemical industries. The nature of
working with living material makes contamination cobonplace and repro-
ducibility of data difficult to achieve. Such problems quickly distort the
relevant scale-up factors.
In this chapter, three research scientists from Kyowa Kogyo Co. Ltd.
have addressed the problems of experimentation and pilot scale-up for
I
2 Fermentation and Biochemical Engineering Handbook
microorganisms, mammalian cells, plant cells, and tissue. It is our sincere
hope that the reader will find this chapter helpful in determining the best
conditions for cultivation and the collection of scale-up data. Hopehlly, this
knowledge will, in turn, facilitate the transformation of worthwhle research
programs into commercially viable processes.
1.0 MICROBIAL FERMENTATION (by Kuniaki Sakato)
Chemical engineers are still faced with problems regarding scale-up
and microbial contamination in the fermentation by aerobic submerged
cultures. Despite many advances in biochemical engineering to address these
problems, the problems nevertheless persist. Recently, many advances have
been made in the area of recombinant DNA, which themselves have spun off
new and lucrative fields in the production of plant and animal pharmaceuti-
cals. A careful study of this technology is therefore necessary, not only for
the implementation of efficient fermentation processes, but also for compli-
ance with official regulatory bodies.
There are several major topics to consider in scaling up laboratory
processes to the industrial level. In general, scale-up is accomplished for a
discrete system through laboratory and pilot scale operations. The steps
involved can be broken down into seven topics that require some elaboration:
1. Strain improvements
2. Optimization of medium composition and cultural condi-
3. Oxygen supply required by cells to achieve the proper
tions such as pH and temperature
metabolic activities
4. Selection of an operative mode for culture process
5. Measurement of rheological properties of cultural broth
6. Modelling and formulation of process control strategies
7. Manufacturing sensors, bioreactors, and other peripheral
equipment
Items 1 and 2 should be determined in the laboratory using shake flasks
or small jar fermenters. Items 3-7 are usually determined in the pilot plant.
The importance ofthe pilot plant is, however, not limited to steps 3-7. The
pilot plant also provides the cultured broths needed for downstream
Fermentation Pilot Plant 3
processing and can generate information to determine the optimal cost
structure in manufacturing and energy consumption as well as the testing of
various raw materials in the medium.
1.1 Fermentation Pilot Plant
Microorganisms such as bacteria, yeast, fungi, or actinomycete have
manufactured amino acids, nucleic acids, enzymes, organic acids, alcohols
and physiologically active substances on an industrial scale. The “New
Biotechnology” is making it increasingly possible to use recombinant DNA
techniques to produce many kinds of physiologically active substances such
as interferons, insulin, and salmon growth hormone which now only exist in
small amounts in plants and animals.
This section will discuss the general problems that arise in pilot plant,
fermentation and scale-up. The section will focus on three main topics: (i)
bioreactors and culture techniques, (ii) the application of computer and
sensing technologies to fermentation, and (iii) the scale-up itself.
1.2 Bioreactors and Culture Techniques for Microbial Processes
Current bioreactors aregrouped into either culture vessels and reactors
using biocatalysts (e.g., immobilized enzymes/microorganisms) or plant and
animal tissues. The latter is sometimes used to mean the bioreactor.
Table 1 shows a number of aerobic fermentation systems which are
schematically classified into (i) internal mechanical agitation reactors, (ii)
external circulation reactors, and (iii) bubble column and air-lift loop
reactors. This classification is based on both agitation and aeration as it
relates to oxygen supply. In this table, reactor 1 is often used at the industrial
level and reactors (a)2, (b)2, (c)2, and (c)3, can be fitted with draught tubes
to improve both mixing and oxygen supply efficiencies.
Culture techniques can be classified into batch, fed-batch, and con-
tinuous operation (Table 2). In batch processes, all the nutrients required for
cell growth and product formation are present in the medium prior to
cultivation. Oxygen is supplied by aeration. The cessation ofgrowth reflects
the exhaustion of the limiting substrate in the medium. For fed-batch
processes, the usual fed-batch and the repeated fed-batch operations are
listed in Table 2.
A fed-batch operation is that operation in which one or more nutrients
are added continuously or intermittently to the initial medium after the start
of cultivation or from the halfway point through the batch process. Details
4 Fermentation and Biochemical Engineering Handbook
of fed-batch operation are summarized in Table 3. In the table the fed-batch
operation is divided into two basic models, one without feedback control and
the other with feedback control. Fed-batch processes have been utilized to
avoid substrate inhibition, glucose effect, and catabolite repression, as well
as for auxotrophic mutants.
Table 1. Classification of Aerobic Fermentation Systems
(a) Internal mechanical agitation reactors
1. Turbine-stimng installation
2. Stirred vessel with draft tube
3. Stirred vessel with suction tube
(b) External circulation reactors
1. Water jet aerator
2. Forced water jet aerator
3. Recycling aerator with fritted disc
(c) Bubble column and air-loop reactors
1. Bubble column with fritted disc
2. Bubble column with a draft tube for gyration flow
3. Air lift reactor
4. Pressure cycle reactor
5. Sieve plate cascade system
Table 2. Classification of Fermentation Processes
1. Batch process
2. Fed-batch process (semi-batch process)
3.
4.
Repeated fed-batch process (cyclic fed-batch process)
Repeated fed-batch process (semi-continuous process or cyclic
batch process)
5. Continuous process
Fermentation Pilot Plant 5
Table 3. Classification of Fed-Batch Processes in Fermentation
1. Without feedback control
a. Intermittent fed-batch
b. Constant rate fed-batch
c. Exponentially fed-batch
d. Optimized fed-batch
2. With feedback control
a. Indirect control
b. Direct control
Setpoint control (constant value control)
Program Control
Optimal control
The continuous operations of Table 2 are elaborated in Table 3 as
three types of operations. In a chemostat without feedback control, the feed
medium containing all the nutrients is continuously fed at a constant rate
(dilution rate) and the cultured broth is simultaneously removed from the
fermenter at the same rate. A typical chemostat is shown in Fig. 1. The
chemostat is quite useful in the optimization of media formulation and to
investigate the physiological state of the microorganism. A turbidostat with
feedback control is a continuous process to maintain the cell concentration
at a constant level by controlling the medium feeding rate. A nutristut with
feedback control is a cultivation technique to maintain a nutrient concentra-
tion at a constant level. A phuuxostut is an extended nutristat which
maintains the pH value of the medium in the fermenter at a preset value.
6 Fermentation and Biochemical Engineering Handbook
Figure 1 is an example of chemostat equipment that we call a single-stage
continuous culture. Typical homogeneous continuous culture systems are
shown in Fig. 2.
Table 4. Classification of continuous fermentation processes
1. Without feedback control
a. Chemostat
2. With feedback control
a. Turbidostat
b. Nutristat
c. Phauxostat
F
i
Med i urn
reservoir
Air
Motor
1
Broth
re servo i r
(a1Single-stage continuous culture
system
f
(bltevel controller
Figure 1. Chemostat System. V: Operation volume. F: Feedrate ofmedium.
Sf: Concentration of limiting substrate.
Fermentation Pilot Plant
L
r I 11 1
I
I
mM -
0
7
-
_N_v
m
(a)Single-stage continuous operat ion
Figure 2. Homogeneous systems for continuous fermentation.
8 Fermentation and Biochemical Engineering Handbook
1.3 Application of Computer Control and Sensing Technologies for
Fermentation Process
The application of direct digital control of fermentation processes
began in the 1960’s. Since then, many corporations have developed
computer-aided fermentation in both pilot and commercial plants. Unfortu-
nately, these proprietary processes have almost never been published, due to
corporate secrecy. Nevertheless, recent advances in computer and sensing
technologies do provide us with a great deal of information on fermentation.
This information can be used to design optimal and adaptive process controls.
In commercial plants, programmable logic controllers and process
computers enable both process automation and labor-savings. The present
and likely future uses of computer applications to fermentation processes in
pilot and industrial plants are summarized in Table 5. In the table, open
circles indicate items that have already been discussed in other reports while
the open triangles are those topics to be elaborated here.
Table 5. Computer Applications to Fermentation Plants
Sequence control
Feedback control
Data acquisition
Estimation of
state variables
Advanced control
Optimized Control
Modelling
Scheduling
Pilot Scale
Present Future
Production Scale
Present Future
A few
cases
A A
Fermentation Pilot Plant 9
The acquisition of data and the estimation of state parameters on
commercial scales will undoubtedly become increasingly significant.
Unfortunately, the advanced control involving adaptive and optimized
controls have not yet been sufficiently investigated in either the pilot or
industrial scale.
Adaptive control is of great importance for self-optimization of
fermentation processes, even on a commercial scale, because in ordinary
fermentation the process includes several variables regarding culture condi-
tions and raw materials. We are sometimes faced with difficulties in the
mathematical modelling of fermentation processes because of the complex
reaction kinetics involving cellular metabolism. The knowledge-based
controls using fuzzy theory or neural networks have been found very useful
for what we call the “black box” processes. Although the complexity of the
process and the number of control parameters make control problems in
fermentation very difficult to solve, the solution of adaptive optimization
strategies is worthwhile and can contribute greatly to total profits. In order
to establish such investigations, many fermentation corporations have been
building pilot fermentation systems that consist of highly instrumented
fermenters coupled to a distributed hierarchical computer network for on-and
off-line data acquisition, data analysis, control and modelling. An example
of the hierarchical computer system that is shown in Fig. 3 has become as
common in the installation of large fermentation plants as it is elsewhere in
the chemical industry. Figure 4 shows the details of a computer communi-
cation network and hardware.
As seen in Fig. 3, the system is mainly divided into three different
functional levels. The first level has the YEWPACK package instrumenta-
tion systems (Yokogawa Electric Corporation, Tokyo), which may consist of
an operator’s console (UOPC or UOPS) and several field control units
(UFCU or UFCH) which are used mainly for on-line measurement, alarm,
sequence control, and various types of proportional-integral-derivative (PID)
controls. Each of the field control units interfaces directly with input/output
signals from the instruments of fermenters via program controllers and signal
conditioners. In the second level, YEWMAC line computer systems
(Yokogawa Electric Corporation, Tokyo) are dedicated to the acquisition,
storage, and analysis of data as well as to documentation, graphics, optimi-
zation, and advanced control. A line computer and several line controllers
constitute a YEWMAC. The line controller also governs the local area
network formed with some lower level process computers using the BSC
multipoint system. On the third level, a mainframe computer is reserved for
modelling, development of advanced control, and the building of a data base.
IO Fermentation and Biochemical Engineering Handbook
DATA HIOHWAY
Finally, the mainframe computer communicates with a company computer
via a data highway. This is used for decision-making, planning, and other
managerial functions. The lower level computer, shown as the first level
in Fig. 3, is directly interfaced to some highly-instrumented fermenters.
Figure 5 illustrates a brand new fermenter for fed-batch operation. Control
is originally confined to pH, temperature, defoaming, air flow rate, agitation
speed, back pressure, and medium feed rate, Analog signals from various
sensors are sent to a multiplexer and ND converters. After the computer
stores the data and analyzes it on the basis of algorithms, the computer
sends the control signals to the corresponding controllers to control the
fermentation process.
level m
MAINFRAME COMPUTER
Figure 3. Configuration of distributed hierarchical computer system for fermentation
pilot plant.
Optimi zit ion
VEWMAC 300
LINE COMPUTOR
tevei II
Data aquisition
LINE CONTROLLER
3600-M$A
Optimization
Sophisticated control
I
!
UFCU
Fermentation Pilot Plant 11
Mainframe Computer
Printer YEWMAC 300
Line Computer
Main Memory: ROY l6KB tRAY 1YB
Hard Disk: IOYB. FDD: 1YB
Linb controller 3610C
1 Main Ybmory: ROM 16 KB
CRT 111
BSC multi-point ryrtom
UOPC (UOPS) :Operator console.
UFCU (UFCH) :Fib Id contro I un i 1.
UCIA-2: RS232C conmunication
i ntbrf IC(.
Figure 4. Local area network for pilot fermentation plant.
12 Fermentation and Biochemical Engineering Handbook
coo I
wate
-.
Ag i
Tor
wat
Autosampler Biosensors
Air flow rate
-
0
-
APM. torque
PHlC
DIC
Figure 5. Highly instrumented pilot fermentor for fed-batch operations.
Fermentation Pilot Plant 13
Sensing in the fermentation area tends to lack the standard of reliability
common to the chemical industry. Steam sterilization to achieve aseptic
needs in fermentation is crucial for most sensors such as specific enzyme
sensors. The various sensors that can be used in fermentation are summarized
in Table 6. As in the chemical industry, almost all the physical measurements
can be monitored on-line using sensors, although an accurate measurement
device, such as a flow meter, is not yet available. The chemical sensors listed
in Table 6 reflect the measurement of extracellular environmental conditions.
The concentration of various compounds in the media are currently deter-
mined off-line following a manual sampling operation except for dissolved
gas and exhaust gas concentration. Exhaust gas analysis can provide
significant information about the respiratory activity which is closely related
to cellular metabolism and cell growth, This analysis is what is called
gateway sensor and is shown schematically in Fig. 6.
Table 6. Sensors for Fermentation Processes
Physical Chemical
Temperature
Pressure
Shaft speed
Heat transfer rate
Heat production rate
Foam
Gas flow rate
Liquid Flow Rate*
Broth volume or weight
Turbidity *
Rheology or viscosity*
Biochemical
Viable cell concentration*
NAD/NADH level*
ATP/ADP/AMP/level*
Enzyme Activity'
Broth composition*
PH
OW
Ionic strength
Gaseous 0, concentration
Gaseous CO, concentration
Dissolved 0, concentration
Dissolved CO, concentration
Carbon source concentration
Nitrogen source concentration*
Metabolic product concentration*
Minor metal concentration*
Nutrient concentration*
*Reliable sensors are not available.
I4 Fermentation and Biochemical Engineering Handbook
I I
Figure 6. Estimation of metabolic parameters using gateway sensor.
The data analysis scheme of Fig. 6 includes the steady-state oxygen
balance method and the carbon balancing method. In addition, the system
can provide the oxygen supply conditions that relate to volumetric oxygen
transfer coefficient &a), oxidation-reduction potential (OW) and degree of
oxygen saturation Qo2X/(Qo2X),,. For the data analysis scheme of Fig. 6,
the most significant advance in the fermentation field has been the develop-
ment of steam sterilization, dissolved oxygen electrodes and the application
ofmass spectrometry to the exhaust gas analysis. Dissolved oxygen probes
can be classified as either potentiometric (galvanic) or amperometric (po-
larographic). These electrodes are covered with agas-permeable membrane;
an electrolyte is included between the membrane and the cathode. It should
be noted that these probes can measure the oxygen tension but not the
concentration. The signal from both models of electrodes often drifts with
time for long continuous measurements. Calibration then becomes difficult
because of possible contamination. Most commercial probes have a vent to
balance the pressure between the inside and outside ofthe probe. Often, the
broth and electrolyte mix through the vent causing signal drift and rapid
reduction in probe life. Therefore, fiber-optic chemical sensors such as pH,
dissolved oxygen and carbon dioxide electrodes which need pressure com-
pensation interference by medium components, drift and so on. This type of
sensor is based on the interaction of light with a selective indicator at the
Fermentation Pilot Plant 15
waveguide surface of optical fiber. Fiber-optic sensors do not suffer from
electromagnetic interferences. Also, these can be miniaturized and multi-
plexed, internally calibrated, steam-sterilized and can transmit light over
long distances with actually no signal loss as well as no delayed time of the
response. At present, a key factor for these sensors is to avoid the
photodecomposition of the dyes during longtime measurements. Generally,
the majority ofmeasurements on oxygen uptake (Qo2X) have been made with
a paramagnetic oxygen analyzer while those on carbon dioxide evolution rate
(QcozX) have been made with an infrared carbon dioxide analyzer.
Gateway sensors have become quite widespread in use in fermentation
processes at both the pilot and plant levels. The sample’s gas has to be dried
by passing through a condenser prior to the exhaust gas analysis to avoid the
influence of water vapor on the analyzers. Except for bakers’ yeast
production, few studies have been reported documenting the application of
the steady-state oxygen balance method to the process control of fermentation
processes in pilot and production plants. Recently the industrial use of this
method has been published for the fed-batch process of glutathione fermen-
tation. Based on the overall oxygen uptake rate Q0,XV and the exit ethanol
concentration, the feed-fonvardfeedback control system of sugar feed rate
has been developed to successfully attain the maximum accumulation of
glutathione in the broth on the production scale (Fig. 7). In the figure, the
feed-forward control of sugar cane molasses feeding was made with total
oxygen uptake rate Q0,XV and the sugar supply model which is based on the
oxygen balance for both sugar and ethanol consumptions. In this system,
oxygen, carbon dioxide and ethanol in outlet gas were measured on-line with
a paramagnetic oxygen analyzer and two infrared gas analyzers as “gate
way” sensors for a 120-kl production fermenter. Oxygen and ethanol
concentration in outlet gas at the pilot level was continuously monitored with
the sensor system consisting of two semiconductors. For the feedback
control, a PID controller was used to compensate for a deviation, e, from a
present ethanol concentration, ESet, calculated by the ethanol consumption
rate model. Based on the deviation e, a deviation &from the set-point feed
rate Fcan be calculated as shown in Fig. 7. The performance of this system
was found to be very good using a YEWPACK Package Instrumentation
System (Yokogawa Electric Corporation, Tokyo) and a 120-kl production
fermenter (Fig. 8). The results, an average of 40% improvement of
glutathione accumulation in the broth was attained, were compared with a
conventionally exponential feeding of sugar cane molasses.
16 Fermentation and Biochemical Engineering Handbook
1
1
OUTPUT TO FEED PUYP
Total 02 uptake rate
Cu I ture vo I ume
-
'x' Feed rate
*
I-
v
Ethanol consumption
mode I**
Set-point of E
at 8 time t
F.;..1- Dev irt ion
L
Figure 7. Configuration of process control system for glutathione fermentation.
*The feed rate F can be calculated from the oxygen balance for sugar and ethanol
consumption in the broth.
**The optimal ethanol consumption profile is obtained for a constant consumption rate.
Fermentation
Pilot
Plant
17
(lW/6fl)
HS3
0
0
0
0
0
0
N
-
0 8
3 B a
0
M
0
0 * 0 N
0
c4 c
0, E
C,
v) v) aJ V 0 L
v .I- n
18 Fermentation and Biochemical Engineering Handbook
Recent research using mass spectrometry has made it possible to
almost continuously measure not only oxygen and carbon dioxide concentra-
tions but also many other volatiles at the same time. The increased reliability,
freedom of calibration, and rapid analysis with a mass spectrometer has
allowed the accurate on-line evaluation of steady-state variables in Fig. 8 for
process control and scale-up. Figure 9 shows schematically the instrumen-
tation system using amembrane on the inlet side for analyzing the exhaust gas
from the fermenter. In Fig. 9, the left part is the gas sampling system that
consists of a knockout pot, preventing the broth from flowing into the mass
spectrometer, a filter and a pump, for sampling.
81'
hi Levti switch SUS fllttr SV4 t i
--.
sv I
--
i sv3
-
F I ow mt tar
Dtmbrant -
!
!
1
1 Knockout pot
Bypass
sv2
Drain
c c
VtcuuD indicator
I
P Mars spactrawttr i
MSP - 300 j
W!
Punp 0-100
Pump 0- ,100 svII B
Figure 9. Schematic representation of analytical system for outlet gas from fermenter.
(SVJ solenoid valve; (NVJ needle valve; (Thy) thermistor.
Fermentation Pilot Plant 19
As shown in the right side of Fig. 9, a quadrupole mass spectrometer,
MSG 300, with a gas-tight ion source, secondary electron multiplier,
direction detector, and a turbo-molecular pump (TURBOVAC 150) is
equipped with a membrane inlet (all from Nippon Shinku, Tokyo). The
resolution scale is 300. Mass spectrometry can also be used for the
measurement of dissolved gases in a liquid phase using a steam sterilizable
membrane probe. Recently, the application of the mass spectrometer to
fermentation processes has increased markedly.
A laser turbidimeter has been developed for the on-line measurement
of cell concentration, which is correlated to the turbidity ofthe cultured broth.
However, the application of this turbidimeter to the continuous monitoring of
cell growth might be limited to the lower range of cell concentration even in
the highly transparent broths compared to the production media containing
solid materials such as cane sugar molasses and corn steep liquor.
As indicated in Table 6, the biochemical sensor can be used for
intracellular activities, which are closely related to the level of key interme-
diates such as NAD/NADH and ATP/ADP/AMP. Only one sensor for
monitoring on-lineNADH on the intracellular level is commercially available
(BioChemTechnology, Malvern, PA). Thefluorometer sensor can measure
continuously the culture fluorescence, which is based on the fluorescence of
NADH at an emission wavelength of 460 nm when excited with light at 360
nm. The sensor response corresponds to the number of viable cells in the
lower range of the cell concentration. It should be especially noted that the
sensor reflects the metabolic state of microorganisms.
The most attractive sensors now being developed are the Fourier
transform infrared spectrometer (FTIR) and the near-infrared (NIR) spec-
trometer for the on-line measurement of composition changes in complex
media during cultivation. The FTIRmeasurements are based on the type and
quantities of infrared radiation that a molecule absorbs. The NIR measure-
ments are based on the absorption spectra following the multi-regression
analyses. These sensors are not yet available for fermentation processes.
1.4 Scale-up
The supply of oxygen by aeration-agitation conditions are closely
related to the following parameters:
1. Gadliquid interfacial area
2. Bubble retention time (“hold-up”)
3. Thickness of liquid film at the gashquid interface
20 Fermentation and Biochemical Engineering Handbook
Based on these three parameters, the four scale-up methods have been
investigated keeping each parameter constant from laboratory to industrial
scale. The parameters for scale-up are the following:
1. Volumetric oxygen transfer coefficient (ha)
2. Power consumption volume
3. Impeller tip velocity
4. Mixingtime
Even for the simple stirred, aerated fermenter, there is no one single
solution for the scale-up of aeration-agitation which can be applied with high
probability of success for all fermentation processes. Scale-up methods
based on aeration efficiency (ha) or power consumptiodunit volume have
become the standard practice in the fermentation field.
Scale-up based on impeller tip velocity may be applicable to the case
where an organism sensitive to mechanical damage was employed with
culture broths showing non-Newtonian viscosity. Furthermore, scale-up
based on constant mixing time cannot be applied in practice because of the
lack of any correlation between mixing time and aeration efficiency. It might
be interesting and more useful to obtain information on either mixing time or
impeller tip velocity in non-Newtonian viscous systems.
The degree of oxygen saturation Qoz/(Qo2),, and oxidation-reduction
potential (OW) have already been found to be very effective for the scale-up
of fermentation processes for amino acids, nucleic acids, and coenzyme Qlo.
The successful scale-up of many aerobic fermentations suggests that the
dissolved oxygen concentration level can be regarded as an oxygen. Mea-
surements using conventional dissolved oxygen probes are not always
adequate to detect the dissolved oxygen level below 0.01 atm. Even 0.0 1 atm
is rather high compared to the critical dissolved oxygen level for most
bacterial respirations. Due to the lower detection limit of dissolved oxygen
probes, oxidation-reduction potential (OW) was introduced as an oxygen
supply index, which is closely connected to the degree of oxygen saturation.
The OW value Eh in a non-biological system at a constant temperature
is given in the following equation:
Eh = 454.7 - 59.1 + lOg(PL)
where
Fermentation Pilot Plant 21
PL = the dissolved oxygen tension = (atm)
E,, = the potential vs hydrogen electrode
In microbial culture systems, the ORP value E can be expressed as follows:
where
ED0 = the dissolved oxygen
EpH = the pH
Et = the temperature
Emd = the medium
E,, = all metabolic activity to the whole ORP E
For most aerobic fermentations at constant pH and temperature, Eq.
(2) can be simplified to the following,
As a result, we can generally use the culture ORP to evaluate the dissolved
oxygen probe.
An example using the OW as a scale-up parameter has been reported
for the coenzyme Qlo fermentation using Rhodopseudomonas spheroides. In
this case, coenzyme Qlo production occurred under a limited oxygen supply
where the dissolved oxygen level in the broth was below a detection limit of
conventional dissolved oxygen probes. Therefore, the oxidation-reduction
potential (OW) was used as a scale-up parameter representing the dissolved
oxygen level. As a result, the maximum coenzyme Qlo production was
attained, being kept the minimum ORP around 200 mV in the last phase of
culture (Fig. 10).
In the scale-up of ordinary aerobic processes, oxygen transfer condi-
tions have been adjusted to the maximum oxygen requirement of the
fermentation beer during the whole culture period. However, the excess
oxygen supply occurs in the early growth due to the lower cell concentration
under these conditions. It should be noted that such excess supply of oxygen
sometimes has the harmhl effect of bioproducts formation. In other words,
the oxygen supply should be altered according to the oxygen requirements of
microorganisms in various culture phases.
22 Fermentation and Biochemical Engineering Handbook
Process time(h)
Figure 10. Coenzyme Ql0 fermentationunder an optimal aeration-agitation conditionusing
30 liter jar fermenter and the constant rate fed-batch culture. DCW dry cell weight, OW:
oxidation-reduction potential.
1.5 Bioreactors for Recombinant DNA Technology
There are many microorganisms used widely in industry today that
have been manipulated through recombinant DNA technology. To assure
safety in the manufacture of amino acids, enzymes, biopharmaceuticals such
as interferons, and other chemicals using altered microorganisms, guidelines
have existed for their industrial application. More than 3,000 experiments
using recombinant DNA technology have been made in Japan, while the
industrial applications are around 500. In most ofthe OECD countries, large-
scale fermentation processes can be regarded as those including cultured
broths over 10 liters. Organizations which have pilot plants employing
recombinant DNA organisms must evaluate the safety of the microorganism
and process based on the safety of a recipient microorganism and assign it to
one of the following categories: GILSP (Good Industrial Large-Scale Prac-
tice), Categories 1, 2, and 3 or a special category.
This classification is quoted from Guideline forIndusfrialApplication
ofRecombinant DNA Technology which has been published by the Ministry
of International Trade and Industry in Japan. This guideline can be applied
Fermentation Pilot Plant 23
to the manufacturing of chemicals. There are also two major guidelines for
pharmaceuticals and foods by the Ministry of Health and Welfare, and for the
agricultural and marine field by the Ministry of Agricultural, Forestry and
Fishery.
Regulatory guidelines for industrial applications of recombinant DNA
technology, even though there are differences in each country, are primarily
based on “Recombinant DNA Safety Considerations” following the “Recom-
mendation of the Council,” which have been recommended to the member
nations of OECD in 1986.
GILSP (Good Industrial Large-Scale Practice). A recipient organism
should be nonpathogenic, should not include such organisms as pathogenic
viruses, phages, and plasmids; it should also have a long-term and safe history
of industrial uses, or have environmental limitations that allow optimum
growth in an industrial setting, but limited survival without adverse
consequences in the environment.
Category 1. A nonpathogenic recipient organism which is not
included in the above GILSP.
Category 2. A recipient organism having undeniable pathogenicity to
humans that might cause infection when directly handled. However, the
infection will probably not result in a serious outbreak in cases where
effective preventive and therapeutic methods are known.
Category 3. A recipient organism capable of resulting in disease and
not included in Category 2 above. It shall be carefully handled, but there are
known effective preventive and therapeutic methods for said disease. A
recipient organism which, whether directly handled or not, might be
significantly ham&l to human health and result in a disease for which no
effective preventive nor therapeutic method is known, shall be assigned a
classification separate from Category 3 and treated in a special manner.
Based on the Category mentioned above, the organization should take
account of “Physical Containment.” Physical containment involves three
elements of containment: equipment, operating practiceshechniques, and
facilities. Physical containment at each Category for the GILSP level is given
in “Guideline for Industrial Application of Recombinant DNA Technology”
in Japan. Using appropriate equipment, safe operating procedures, and
facility design, personnel and the external environment can be protected from
microorganisms modified by recombinant DNA technology. For an update
on the latest safety guidelines for recombinant DNA technology, see the 1987
report issued by the National Academy of Science, U. S .A.
24 Fermentation and Biochemical Engineering Handbook
References (Section 1)
1. Aiba, S., Humphery, A. E., and Mills, N. F., Biochemical Engineen'ng (2nd
ed.), Academic Press, New York (1 973)
2. Banks, G. T., Scale-up of fermentation process, Topics in Enzyme and
Fermentation Technology, 3: 170 (1 979)
3. Blanch, H. W., and Bhabaraju, S. M., Non-Newtonian Fermentation Broths:
Rheology and Mass Transfer, Biotechnol. Bioeng., 28:745 (1 976)
4. Committee on the Introduction of Genetically Engineered Organisms into
the Environment, in Introduction OfRecombinantDNA-Engineered Organ-
isms into the Environment: Key Issues, National Academy of Science,
Washington (1 987)
5. Heinzle,E.,Kramer,H.,andDunn,I. J., Stateanalysisoffermentationusing
a mass spectrometer with membrane probe, Biotechnol. Bioeng, 27:238,
(1 985)
6. Humphrey, A. E., Algorithmic monitors for computer control of fermenta-
tions, Horizons ofBiochemica1 Engineering, (S. Aiba, ed.), p. 203, Tokyo
Press, Tokyo (1 987)
7. Kenny, J. F., and White, C. A., Principles ofhnobilization ofEnzymes, in
Handbook of Enzyme Biotechnology (2nd ed.), p. 147, Ellis Howood,
Chichester (1 985)
8. Konstantinov, K. B. and Yoshida, T., Knowledge-based control of fermen-
tation processes, Biotechnol. Bioeng., 39:479486 (1 992)
9. Martin G. A., and Hempfling, W. P., A method for the regulation of
microbial population density during continuous culture at high growth rates,
Arch. Microbiol., 107:4147 (1976)
10. Organization for Economic Co-operations and Development: Recombinant
DNA Safety Considerations - Safety Considerations for Industrial, Agricul-
tural Environmental Applications of Organisms derived by Recombinant
DNA Techniques, OECD, Paris (1 986)
1 1. Organization for Economic Co-operations andDevelopment: Recommenda-
tion of the Council - Concerning Safety Considerations for Applications of
Recombinant DNA Organisms in Industry, Agriculture and Environment,
OECD, Paris (1 986)
12. Rolf, M. J., and Lim, H. C., Adaptive on-line optimization for continuous
bioreactors, Chem. Eng. Commun., 29:229 (1984)
13. Sakato, K., and Tanaka, H., Advanced Control of Gluthathione Fermenta-
tion Process, Biotechnol. Bioeng., 40:904 (1 992)
14. Sakato, K., Tanaka, H., Shibata, S., and Kuratsu, Y., Agitation-aeration
studies on coenzyme Q10 production using Rhodopseudomonas Spheroides,
Biotechnol. Appl. Biochem., 16: 19 (1 992)
15. Wang, N. S., and Stephanopoulus, G. N., Computer applications to fermen-
tation processes in CRC Critical Reviews, Biotechnology, 2: 1 (1984)
Fermentation Pilot Plant 25
Tempest, D. W. and Wouters, J. T. M., Properties and performance of
microorganisms in chemostat culture, Enme Microb. Technol., 3:283
(1981)
Yamane, T. and Shimizu, S., Fed-batch techniques in microbial processes,
Ad. Biochem. Eng., 30:148 (1984)
Venetka, I. A., and Walt, D. A., Fiber-optic sensor for continuous monitoring
of fermentation pH, Bioflechnology, 1 1 :726-729 (1 993)
MAMMALIAN CELL CULTURE SYSTEM (bu Seijo Sato)
Introduction
The large-scale production of mammalian cell culture has become one
of the most important technologies since the advent of genetic engineering in
1975. Interest in mammalian cell culture intensified with the development of
interferons.[’] Suddenly, large amounts of human fibroblasts[2] and lympho-
cyte cells[3] were needed to run clinical trials and laboratory tests on the so-
called “miracle drugs.” The demand for large scale reactors and systems
resulted in rapid gains in the technology. At the same time, culture media,
microcamerd4] and hollow-fiber membraned5] were also being improved.
Recent advances in genetic engineering have once again generated
interest in the large scale cultivation of mammalian cells. Through genetic
engineering the mass production of cells derived from proteins and peptides
has real possibilities. Mammalian cells are not only useful proteins and
peptides for genetic engineering, but also serve as competent hosts capable
of producing proteins containing sugar chains, large molecular proteins and
complex proteins consisting of subunits and variegated proteins, such as
monoclonal antibodies. Since monoclonal antibodies cannot be produced by
bacterial hosts, mammalian cells must be used. Therefore, the demand for
large scale production of high-density mammalian cells will most certainly
increase.
Hopefhlly, industry wdl respond quickly to develop new methods to meet
this growing demand as it has done in the past for industrial microbiology.
2.2 Culture Media
Since a mammalian cell culture medium was first prepared by Earle et
a1.[6] many different kinds ofbasal media have been established. For example,
26 Fermentation and Biochemical Engineering Handbook
there are Eagle's minimum essential medium (MEM),n Duldecco's modified
MEM (DME),L8] 199 RPMI-1640,[10] L-15,[11] Hum F-10 and
Hum F-12,[l2] DM-160 and DM-170, etc.[I3] The MIT group[14] created the
High-GEM Wgh Growth Enhancement Medium) in which fructose replaces
glucose as the energy source to achieve a 3- to 4-fold decrease in the
accumulation of lactic acid. These basal media are now commercially
available.
In order to generate useful proteins in very small amounts, the serum-
free or chemically defined media are more useful than media containing
serum. Yamane et al.[15] detected that the effective substances in albumin
were oleic acid and linoleic acid; he then tried to formulate a serum-free
medium containing those fatty acids as RITC-media. Barnes and Sato[l6I
hypothesized that the role of serum is not to supply nutrients for cells, but to
supply hormones and growth factors. They then made up different kinds of
serum-free media containing either peptide hormones or growth factors. The
additive growth factors used for serum substituents were PDGF (platelet
derived growth factor),[17] EGF (epidermal growth factor),["] FGF (fibro-
blast growth factor),[l9I IGF-I,[201 IGF-II[211 (insulin-like growth factor I, 11,
or somatomedins), NGF (nerve growth factor),[22] TGF,[231[24] (transforming
growth factor -a, -p). IL-2[251 or TCGFrZSl (interleukin 2 or T-cell growth
factor), IL-3 (interleukin-3 or muti-CSF),[261 IL-4[271 or BCGF- 1 (interleukin-
4 or B-cell growth factor-1), IL-6[28] or MGF (interleukin-6 or myeloma
growth factor), M-, GM-, G-CSF[291 (macrophage-, macrophage-granulo-
cyte-, granulocyte-colony stimulating factor), Epo (erythropoietin),[30] etc.
The way to create a serum-free culture is to adapt the cells to the serum-
free medium. In our laboratory, we tried to adapt a human lymphoblastoid
cell line, Namalwa, from a medium containing 10% serum to serum-free. We
were able to adapt Namalwa cell to a ITPSG serum-free medium which
contained insulin, transferrin, sodium pyruvate, selenious acid and galactose
in RPMI-1640.[311 In the case of cell adaptation for production of autocrine
growth factor, we were able to grow the cell line in serum- and protein-free
media as well as in K562-K1 (T 1) which produces an autocrine growth factor,
LGF- 1 (leukemia derived growth factor- 1).[32]
2.3 Microcarrier Culture and General Control Parameters
The method for animal cell culture is chosen according to whether the
cell type is anchorage dependent or independent. For anchorage dependent
cells, the cells must adhere to suitable material such as a plastic or glass dish
or plate. As shown in Table 7, several types of culture methods were
Fermentation Pilot Plant 27
developed for cell adherent substrates such as glass, plastic, ceramic and
synthetic resins. Adherent reactors were made up to expand the cell adherent
surfaces such as roller bottle, plastic bag, multidish, multi-tray, multi-plate,
spiral-film, glass-beads propagator,[33] Gyr~gen[~~] and so on. In 1967, van
Welzel demonstrated the feasibility of growing cells on Sephadex or DEAE-
cellulose beads kept in suspension by The drawback for the
anchoragedependent cells has been overcome by the development of the
microcarrier culture method. Using the microcarrier culture systems and
anchoragedependent cells, it is now possible to apply the suspension culture
method on a commercial scale.r5I
Table 7. Available Materials and Methods For Cell culture.
Anchored-------Flat plate _---__----- --Solid single trays and dishes
--Multi-plate
--Multi-tray
--Mu 1 t i-di sh
--Spiral film
--Gyrogen
--Ultrafiltration membrane
--Hollow fiber
Cylinder & tubes-- --Roller bottle
Membrane _____-____ --Dialysis membrane
Suspended-------Microcarrier-------- --Polymer beads
--Glass beads[36]
Microcapsule--------- Sodium alginate gel
Soluble polymer------Serum (Serum albumin)
--Methylcellulose
--Pluronic F 68 (Pepo1 B188)
--Polyethyleneglycol
--Polyvinylpyrrolidone
The most important factor in this method is the selection of a suitable
microcarrier for the cells. Microcarriers are made of materials such as
dextran, polyacrylamide, polystyrene cellulose, gelatin and glass. They are
coated with collagen or the negative charge of dimethylaminoethyl,
diethylaminopropyl and trimethyl-2-hydroxyaminopropyl groups as shown
in Table 8.
28 Fermentation and Biochemical Engineering Handbook
Table 8. Microcarriers
Hollow
glass
Co i lagen
Celabin
CUI lure
treated
KHS Glass 1.04 100-150 385
Pus ion
Growth
Bioglas ISolohilI /Class
Class
-
I-
7Z-T- Supplier
I
8 i oca r r i r B i a-Rad
I
Superbeads Flow Labs
I
Cylodex I Pharmacia
i
Material S.C. Size
( P)
Poly-acrylamide 1. 04 120-180
Dextran - 135-205
,
Poly-acrylamide 1.03 131-220
s. A.
(cm/g)
5000
-
-
5000-
6000
-
6000
Cytodex 2 Pharmacia Dcxtran
I/ I l.O4
Langen
141-198 5500
-
I-
I-
40-50
(L:80-400)
DE-53 1Wlialman 1 Micro-cellulose I - I 40-50 1
1. 04
I
Cytodex 3 Pharrnacia Dextran
(L:80-4001
+
I I I I I
CIJSS beads Whatman Glass 1.02- 150-210 -
I
CoI lagen
235-115
Fermentation Pilot Plant 29
In scaling up batch culture systems, certain fundamental laws of
microbial cell systems can be applied to mammalian cells where the suspen-
sion cultures contain the anchoragedependent cells. This is not the case with
animal cells which are sensitive to the effects of heavy metal ion concentra-
tion, shear force of impeller agitation or air sparging, and are dependent on
serum or growth factors. For these reasons, the materials for construction of
fermenters are 3 16 low carbon stainless steel, silicone and teflon. Different
agitation systems such as marine-blade impeller types, vibromixer and air-lift
are recommended to mitigate the shear stress. The maximum cell growth for
large scale cell suspension using ajar fermenter is governed by several critical
parameters listed in Table 9.
Table 9. Critical Parameters of General Cell Culture
1) Chemical parameters:
Decrease of general critical nutrients:
Increase of inhibitory metabolites:
Oxidation-reduction potential:
glutamine and glucose
ammonium ions and lactic acid (PH control)
gas sparging, chemically by adding cysteine,
ascorbic acid and sodium thioglycollate, etc.
2) Physical parameters:
Decrease of dissolved oxygen:
aeration volume, agitation speed and oxygen
contents of gas phase
optimum condition control.
control of additional ion concentration etc.
Temperature and pressure:
Osmotic pressure:
3) Physiological parameters:
Cell viability:
Cell density:
contamination of cytotixic compounds
increase of inhibitory metabolites and chalone
like substance, ratio of fresh medium and cell
adhesive surface
cell density and induction conditions, etc.
Product concentration:
30 Fermentation and Biochemical Engineering Handbook
For each parameter, the pH, DO (dissolved oxygen), ORP (oxidation-
reduction potential), temperature, agitation speed, culture volume and pres-
sure can be measured with sensors located in the fermenter. The output of the
individual sensors is accepted by the computer forthe on-line, continuous and
real-time data analysis. Information stored in the computer control system
then regulates the gas flow valves and the motors to the feed pumps. A model
of a computer control system is shown in Fig. 11. The computer control
systems, like the batch systems for mammalian cell culture, seem to level out
at a maximum cell density of lo6 celldml. It may be impossible for the batch
culture method to solve the several limiting factors (Table 10) that set into
high density culture where the levels are less than lo7 cell~/ml.[~~]
impe I
- voliime-
- temp. -
- pll -
--om -
- I)O -
- DCO -
le rmen le I controller
recorder
El
Figure 11. General control system of batch fermenter.
Fermentation Pilot Plant 31
Table 10. Limiting Factors of High Density Cell Cultivation[37]
Limiting factors:
Physical factors: Shear force
Physiological factors: Accumulation of chalone-like substances
Contact inhibition
Chemical factors: Exhaustion of nutrient
Decrease of dissolved oxygen
Accumulation of ammonium ion
Accumulation of lactate
2.4 Perfusion Culture Systems as a New High Density Culture
Technology
In monolayer cultures, Knazeck et al.[36] have shown that an artificial
capillary system can maintain high density cells using perfusion culture. The
artificial capillary system is very important when cell densities approach
those of in vivo values obtained via in vitro culture systems. Perfusion culture
systems are continuous culture systems that are modelled after in vivo blood
flow systems. In perfusion culture systems, a continuous flow of fresh
medium supplies nutrients and dissolved oxygen to the cultivating cells.
Inhibitory metabolites such as ammonium ions, methylglyoxal, lactate and
high molecular chalone-like substances are then removed automatically. If
the cells cultivated under continuous flow conditions can be held in the
fermenter membranes, filters, etc., then the cells can grow into high density
by the “concentrating culture.” Thus, these perfusion culture systems may
be able to solve some of the limiting factors associated with high density cell
growth such as the mouse ascites level.
The perfusion culture systems are classified into two types by static and
dynamic methods as shown in Fig. 12.
32 Fermentation and Biochemical Engineering Handbook
Hollow fiber Ceramic opticore Membrof erm
Suspension & perfusion culture system
Membrane
'dialysis
517
Membra ne
agitator
Rotating
fi 1 ter
Sed imen ta ti on
column
Figure 12. Static maintenance culture systems. Static maintenance type: hollow
ceramic ~pticell,[~~] mernbrofe~m,[~~] static maintenance systems.[41] Suspension culture
type: membrane dialysis,[42] rotating filter,[431[44] membrane agitator,[45] sedimentation
column
The most important technique for perfusion culture methods is to
separate the concentrated cells and conditioned medium from the suspended
culture broth. As noted above, the separation methods chiefly used are
filtration with tubular and flat membranes as well as ceramic macroporous
filters. These membrane reactors can be employed for both anchorage-
dependent and suspensiongrowing cells. Static maintenance type systems are
commercially available for disposable reactors, and small size unit reactors
from 80 ml to 1 liter are used for continuous production of monoclonal
antibodies with hybridoma cells. The maintainable cell densities are about
107-108 cells/ ml which is essentially mouse ascites level. However, in these
systems, the cell numbers cannot be counted directly because the cells adhere
to membranes or hollow fibers. Therefore, the measurement of cell density
must use indirect methods. Such indirect methods include the assaying ofthe
quantities of glucose consumption and the accumulation of lactate. The
parameters of scale-up have not yet been established for these static methods.
Fermentation Pilot Plant 33
Tolbert et al.[43] and Himmelfarb et al.14] have obtained high density
cell growth using a rotating filter perfusion culture system. Lehmann et al.[451
used an agitator of hollow fiber unit for both perfusion and aeration. In our
laboratory, constructed a membrane dialysis fermenter using a flat
dialysis membrane. The small size system is well-suited for the cultivation
of normal lymphocytes (Lymphokine actived killer cells).[48] These cells are
employed in adoptive immunotherapy due to their high activities for thirty or
more days and their acceptance by the reactor cells.
To eliminate the use of a membrane and a filter, we have also tried to
make a perfusion culture system using a sedimentation COIU~~.[~~]
2.5 Sedimentation Column Perfusion Systems
We have developed several new perfusion systems which do not use
filtration methods for cell propagation. When the flow rate of the continuous
supplying medium is minimized, for example, when it is 1 to 3 times its
working volume per day, the system has the ability to separate the suspended
cells from the supernatant fluid. This is accomplished by means of an internal
cell-sedimentation column in which the cells settle by gravity. The shape and
length ofthe column are sufficient to ensure complete separation ofcells from
the medium. Cells remain in culture whereas the effluent medium is
continuously withdrawn at a rate less than that of the cell sedimentation
velocity. We experimented with several shapes for the sedimentation column
and found that the cone and two jacketed types work best.
With the cone for a continuous flow rate of perfusion, the flow rate in
the column is inversely proportional to the square of the radius of the cone at
any given position. If the ratio of the radii of the inlet and outlet is 1 : 10 and
the flow rate of the outlet is 1/100 of the inlet flow rate, then the separation
efficiency of the supernatant fluid and suspended cells are improved. As
shown in Fig. 13, the jacket type sedimentary system allows easy control of
the temperature for separating the static supernatant from the cells. This
jacket method was applied to an air-lift fermenter since it had not been done
in an air-lift perfusion culture. According to Katinger et al.,i4’I air-lift
methods have smaller shear forces than impeller type agitation. However, in
pefision culture, comparable maximum cell densities were obtained using
all three types of fermenters.
34 Fermentation and Biochemical Engineering Handbook
CONE TYPE
IN WT
--A OUT
7
AIR-LIFT TYPE
I
_--
If
AIR- INLET
Figure 13. Sedimentation column perfusion system.
2.6 High Density Culture Using a Perfusion Culture System with
Sedimentation Column
The specific standard methods of a new perfusion culture will now be
described for growth and maintenance of mammalian cells in suspension
cultures at high density. The biofermenter was used for high density culture
ofNamalwa cells with serum-free medium as the model. In 1980, the parent
Namalwa cells were obtained from Mr. F. Klein of Frederick Cancer
Research Center, Frederick, Maryland, U. S. A. In our laboratories, we were
able to adapt the cells to a serum and albumin-free medium and named the
cells KJM- 1. ITPSG and ITPSG + F68 used a serum-free medium containing
insulin, 3 g/d; Transferrin, 5 g/ml; sodium pyruvate, 5 mM; selenious acid,
1.25 x lO-'M; galactose, 1 mg/ml; and/or Pluronic F 68 (Pepo1 B-188) 0.1
mg/ml; in WMI-1640 basal medium.
Fermentation Pilot Plant 35
The biofermenter BF-F5OO system consisted of a 1.5 1 culture vessel,
2 1 medium reservoir and effluent bottle (2 1 glass vessels) for fresh and
expended media which were connected to the perfusion (culture) vessel by a
peristaltic pump. As shown in Fig. 14, the fermenter systems have a conical
shape sedimentation column in the center of the fermenter, and an impeller on
the bottom of the sedimentation column. The Namalwa cells, KJM-1, were
cultivated by continuous cultivation in the biofermenter. In Fig. 15, the
culture has been inoculated at 1 to 2 x lo6 cells/ml with an initial flow rate
of approximately 10 mvh, sufficient to support the population growth. At
densities of 7 x lo6 - 1.5 x lo7 cells/ml, we have used a nutrient flow rate of
1340 mvh using ITPSG and ITPSG-F68 serum-free media. The flow rate of
fresh media was increased step-wise from 240 to 960 mud in proportion to
the increase in cell density. This resulted in an increase of 4 to 10 fold in cell
density compared to the conventional batch culture systems. This system was
then scaled up to a 45 1 SUS316L unit mounted on an auto-sterilization
sequence system with a medium reservoir and an effluent vessel of 90 1 each.
The system was agitated from below by a magnet impeller and was
controlled and analyzed using a personal computer system. The system is
shown in Fig. 17. In the 45 1 pehsion fermenter, we were able to obtain high
density cell growth and duplicate the results of the small scale fermenter
system.
2.7 Acknowledgment
This work was supported by finds obtained through the Research and
Development Project of Basic Technologies for Future Industries from the
Ministry of International Trade and Industry of Japan
36 Fermentation and Biochemical Engineering Handbook
I II
1, Fresh medium vessel
2. Feeding pump
3. Effluent pump
4. Effluent vessel
5. Sensor 14. Sampling line
6. Feeding line 15. Dectector
7. Effluent line 16. Recorder
8. Air line 17. Stirrer
9. Sedimentation column
10. Impeller
11. Air inlet
12. Air outlet
13. Sampling system
Figure 14. Continuous culture system.
Fermentation Pilot Plant 37
20
10
a5
- 3
0
0,
2i
s
2
1
O.!
100
%
90
p 80
3
$ 70
60
.CI
s
t
Viable cell density
\
'. fl
P
200
100
50
:
20
10
5
' 40
9
. 30 2
2
' 20 g
10
Llhr
Figure 15. High density Namalwa cell culture in serum-free medium
38 Fermentation and Biochemical Engineering Handbook
Figure 16. A 45L SUS 316L Unit.
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Fermentation Pilot Plant 39
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