USER MANUAL

Autoclavable Bioreactor 2 - 7 liter CONTENTS

October 1994

TABLE OF CONTENTS

Chapter Description Page

1 Hardware specification 1-1

1.1 General 1-1
1.2 The reactors 1-2
1.3 The head plates 1-7
1.4 The stirrer assemblies 1-9

2 Mixing and aeration 2-1

2.1 Mixing 2-1
2.2 Aeration 2-6
2.3 Literature 2-9

3 Operation 3-1
3.1 General 3-1
3.2 Preparing for sterilization 3-1
3.3 Sterilization 3-2
3.4 Installation 3-2
3.5 Preparing for operation 3-2
3.6 Inoculation 3-3
3.7 Actions during fermentation 3-3
3.8 Pasteurization 3-3

4 Maintenance 4-1
4.1 The reactor 4-1
4.2 The stirrer assembly 4-2

5 Auxiliaries 5-1
5.1 Sampling 5-1
5.2 Aeration 5-6
5.3 Addition 5-9
5.4 Mixing 5-11
5.5 Heat exchangers 5-14
5.6 Sensor holders 5-15
5.7 Blind stoppers 5-16

6 Drawings 6-1
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HARDWARE SPECIFICATION Autoclavable Bioreactor 2 - 7 l ter
October 1994

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Autoclavable Bioreactor 2 - 7 liter MIXING AND AERATION
October 1994

CHAPTER 2

MIXING AND AERATION

For an optimum performance of any biological system, it is necessary to keep the environment
of the micro-organisms at optimal conditions. Apart from temperature and medium
composition, the two most important factors that effect this environment are the degree of
mixing and aeration.

2.1 MIXING:

The aim of mixing is to obtain uniform conditions in the working volume of the bioreactor,
in order to obtain an optimal mass transfer, to avoid gradients of any of the medium
components, and to keep the microcarriers or cells in suspension. In a normally used stirred
tank reactor, mixing is accomplished by the impeller. The resulting flow pattern is a function
of the impeller configuration, agitation speed, the geometry of the system and gas inflow rate
employed.

One aspect of mixing is preventing the cells or microcarriers to settle. In order to achieve this,
the fluid velocity must at least be equal to the settling velocity of the particles (vsett), which can
be calculated according to Stokes' law (Cherry and Papoutsakis, 1986):

vsett = dp2
(
s -
f)
g/(18
) where: dp = particle diameter (m),

s = particle density (kg m-3),

f = medium density (kg m-3),
g = gravitational constant (m s-2)
 = medium viscosity (N m s-1).

Stokes' law only holds for particles whose Reynolds numbers are smaller than 1 (which holds
for all normally used biological systems). As the biomass should be homogeneously
distributed throughout the reactor, not only must the particles be lifted from the bottom of the
vessel but they must also be transported through the whole volume of the reactor.

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Generally, the minimum required stirring speed to achieve homogeneous suspension, Nhs, is
much greater than that to lift the particles from the bottom of the reactor. Buurman et al.
(1986) also derived a simple Froude relationship to define homogeneity in a stirred tank
reactor (STR), based on the assumption of fluctuating velocity being proportional to the
circulation velocity:

f
Nhs2
Di2/(g
(
s -
f)
dp) = constant

where: Di = diameter of the impeller (m)

which implies that:

Nhs  1/Di

The parameter used to describe the medium homogeneity aspect of mixing is the mixing time,
which is in fact also the characteristic time for mixing. The mixing time, tm, is often expressed
in the liquid circulation time, tc. In the case of a stirred tank tm equals 4 times tc (Voncken,
1966).
The liquid circulation time in STRs with a Rushton impeller can be calculated according to
(Oosterhuis, 1984):

tc = V/(0.75
N
Di3) where: V = working volume of the reactor (m3),

N = rotation speed of the impeller (s-1),

If the geometry of the system has to be taken into account in ungassed STRs the following
correlation for the mixing time can be used:

tm = 1.2/N
(D/Di)3
(H/D)
Np-1/3
(Di/HI)2/3
where: D = diameter of the reactor (m),
H = liquid height in the reactor (m),
Np = impeller power number (-),
HI = height of impeller blade (m).

The impeller power number is a constant for a given system and related to the power input by
the stirrer (Ps) in the following way:

Ps = Np

f
N3
Di5

The mixing time in a gassed STR is, as a rule of thumb, twice as high as in an ungassed STR.
The resulting mixing time in ADI's autoclavable bioreactors for mammalian cell and bacterial
culture under average operating conditions (ungassed) is given in the following table.

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Applikon Dependable Instruments Autoclavable Bioreactors: Mixing times

Reactor configuration Mammalian cell culture Bacterial culture
N=100rpm, Marine imp. N=1000rpm, Rushton imp.
Reactor Impeller Reactor Liquid Impeller Impeller Mixing Impeller Impeller Mixing

volume diameter diameter height power height time power height time

V (l) Di (mm) Dr (mm) H (mm) no Np HI (mm) tm (sec) no Np HI (mm) tm (sec)

1 45 96 150 3 28 1.3 6 11 0.2

2 45 115 125 3 28 1.6
2 45 105 175 12 11 0.2
3 45 130 200 3 28 3.2 12 11 0.4
5 60 170 200 3 37 2.8 12 15 0.3
7 60 170 300 6 37 3.4 18 15 0.4
15 74 222 365 6 45 4.3 18 15 0.5
20 74 222 550 9 45 5.7 18 19 0.8

Measurements of the mixing time in a two and three liter reactor have shown to comply well
with these correlations (Kakes and Oosterhuis, 1990). It is obvious from this table that the
mixing times are very small in relation to the characteristic times that are to be expected for
the metabolism (e.g. oxygen uptake rate, substrate consumption rate) of the micro-organisms.
Therefore the medium can be looked upon as being ideally mixed at all times during
fermentation.

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As the bubbles are coalescing behind the impeller and broken up by vortices created by the
impeller, the gas will be homogenised in a similar way as the medium and its components. The
influence of gas feed on this mechanism is illustrated in the figure below (Warmoeskerken,
1986). Despite this mixing of the gas phase, the cavities behind the impeller blades will
decrease the power input to the reactor and thus decrease the oxygen transfer rate to the
reactor medium (see chapter 2.2: aeration).

Cavity shape behind Rushton type impellers for small, medium and high gas feed rates.

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MIXING AND AERATION Autoclavable Bioreactor 2 - 7 l ter
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2.2 AERATION:

Gas-liquid mass transfer in cell culture systems is governed by the solubility of the gas in the
liquid medium (Bliem and Katinger, 1988), its molecular diffusivity and the driving force of
the gas (which vary with temperature and pressure), and may be described by the expression:

OTR = kla
(O* - Ol)

where: OTR = oxygen transfer rate,
kl = liquid phase mass transfer coeff.,
a = gas-liquid interfacial area per unit liquid volume,
O* = liquid phase oxygen concentration in equilibrium with the
bulk gas phase (at 1 atm and 37C: 0.18 mmol O2/l).
Ol = actual oxygen concentration of the liquid phase.

The term kla represents the volumetric overall mass transfer coefficient and is one of the most
common parameters used to describe the efficiency of an aeration system. Bliem and Katinger
(1988) compared the typical efficiency of some forms of aeration which are given in the
following table.

Efficiencies of different aeration systems

Typical oxygen transfer rates kl a (s-1) Aeration system
under culture conditions, using air
(cm s-1)
10-6 10-6 static liquid surface
aeration.
10-6 - 10-3 10-6 - 10-3 stirred liquid surface
aeration.
10-5 - 8 * 10-5 10-4 - 10-2 silastic membrane
aeration.
5 * 10-5 - 3 * 10-4 10-4 - 3 * 10-4 dynamic wire mesh
(with agitation).
< 7 * 10-3 sparged and stirred
liquid for cell culture.
10-2 - 7 * 10-1 sparged and stirred
liquid for microbial
culture.

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October 1994

The kla-values are strongly influenced by the medium composition. For example, the addition
of silicone antifoam can cause the kla to decrease drastically whereas the addition of salts to
the medium will increase the kla (Lavery and Nienow, 1987; Bliem and Katinger, 1988).

For a mammalian cell culture to a density of 5
109 cells/l (a typical batch culture), the oxygen
requirement is in the range of 5-50 ml O2/l
h (at 37C and atmospheric pressure). Therefore,
the required kla for a typical batch culture operation is likely to be 0.5-5
10-4 s-1. Static liquid
surface aeration is insufficient even for these low requirements (see table on previous page).
For high density cultivation, as reached in long-term perfusion systems, the required kla may
increase to as much as 0.1-1
10-2 s-1; this oxygen requirement can only be supplied by
sparging.

Sparging is the most efficient way to obtain high kla values. Most experimental data for kla
values can be estimated by the correlations of Van 't Riet (1979) and Henzler (1982) (adapted
for temperature difference):

for coalescing media:

kla = (1.022(T-20))
0.026
(Pg/V)0.4
vs0 5

for non-coalescing media:

kla = (1.022(T-20))
0.016
(Pg/V)0.7
vs0.2

where: T = temperature (oC),

Pg = the gassed power input by impeller (N m s-1),
V = volume (m3),
vs = superficial gas velocity (m s-1).

At very low aeration rates Pg will be equal to the ungassed power input Ps (Lavery and
Nienow, 1987) but at higher aeration rates the gassed power input has to be calculated
according to:

Pg  0.5
Ps = 0.5
Np

f
N3
Di5

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The theoretical kla values from these correlation for ADI's autoclavable reactors are given in
the table below. Measurements of kla values on a two and three liter scale have shown that the
oxygen transfer capacity that can be reached in practice is upto 10 times higher (Kakes and
Oosterhuis, 1990). Sparging, however, is known to cause inadmissible shear stress for
mammalian cells, will increase the problem of foam formation in bacterial as well as
mammalian cultures and is, therefore, limited in its applications.

Applikon Dependable Instruments Autoclavable Bioreactors: Theoretical

oxygen transfer rates.
Reactor configuration Mammalian cell culture, Bacterial culture, N=1000
N=100 rpm, Marine rpm, Rushton
impellers, Aeration = impellers, Aeration = 1
0.05 vvm. vvm.
Reactor Impeller Reactor Impeller Oxygen transfer Impeller Oxygen transfer

volume diameter diameter power coalesc. non coal power coalesc. non coal
-1 -1 -1
V (l) Di (mm) Dr (mm) no Np kla (h ) kla (h ) no Np kla (h ) kla (h-1)

1 45 96 3 1 18 6 7 33

2 45 115 3 1 12

2 45 105 12 8 37

3 45 130 3 1 9 12 7 27

5 60 170 3 2 18 12 10 52

7 60 170 6 2 24 18 12 59

15 74 222 6 3 31 18 16 75

20 74 222 9 4 36 18 16 65

Since the required kla value and admitted aeration flow vary widely from system to system,
various ways are used to introduce a gas flow into the system.
For mammalian cell cultures mostly an (oxygen enriched) air overlay is used in combination
with an axially pumping impeller. In order to get a large surface area and thus improve the
oxygen transfer, reactors with a low H/D value (usually  1) are used for this application.
Fermentations that require both a low stirrer speed and gas flow need a sintered steel sparger
in order to create small bubbles and thus a large area for oxygen transfer.
High gas flow rates can be reached with open pipes, or pipes with several relatively large
holes.

All options are available for every ADI autoclavable bioreactor.

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October 1994

2.3 LITERATURE:

Bliem, R. and Katinger, H. (1988) Tibtech, 6, 190-195 and 224-230.

Buurman, C., Resoort, G. and Plaschkes, A. (1986) Chem. Eng. Sci., 41, 2865.
Cherry, R.S. and Papoutsakis, E.T. (1986) Bioproc. Eng., 1, 29-41.
Henzler, H.J. (1982) Chem. Ing. Techn., 54, 461.
Kakes, E. and Oosterhuis, N.M.G. (1990) A poster presentation on the Dutch Congress on
Biotechnology.
Lavery, M. and Nienow, A.W. (1987) Biotechnol. Bioeng., 30, 368-373.
Nienow, A.W., Wisdom, D.J. and Middleton, J.C. (1977) 2nd Eur. Conf. on Mixing,
30-3, Cambridge, England, Paper F1.
Oosterhuis, N.M.G. (1984) Ph.D. Thesis, T.U. Delft, The Netherlands.
Van 't Riet, K. (1979) Ind. Eng. Chem. Proc. Des. Dev., 18, 367-375.
Voncken, R.M. (1966) Ph.D. Thesis, T.U.Delft, The Netherlands.
Warmoeskerken, M.M.C.G. (1986) Ph.D. Thesis, T.U. Delft, The Netherlands.

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Autoclavable Bioreactor 2 - 7 liter OPERATION
October 1994

CHAPTER 3

OPERATION
3.1 GENERAL:

When unpacking the equipment, verify if there is any transport damage and if the reactor is
complete (the way you ordered it). Clean all parts with 70% ethanol to remove dust or dirt
from shipping. When assembling the reactor, make sure not to damage the threaded ports;
always screw in the auxiliaries straight and by hand. Do not use tools to tighten the auxiliaries
in the head plate.
Make sure that an O-ring is present between the auxiliary and the head plate in order to ensure
sterility.

3.2 PREPARING FOR STERILIZATION:

Fill the vessel with culture medium, do not exceed the total volume that is specified in chapter
1 (hardware specification). Be sure to leave enough volume for additions after sterilization
(e.g. inoculum, separately sterilized nutrients, etc.). Fasten the six mill nuts crosswise by hand.
Verify the functioning of the electrodes (refill electrolyte of the pH electrode). Insert the Level
probe as far as possible into the vessel and fasten it (after sterilization, the position of this
probe can be adjusted, upward to the desired height).
Verify the mounting of all nipples and other auxiliaries. Make connections for liquid
additions, air in and air out with silicone tubing or other suitable sterilizable material. Use
appropriate filters for air in and air out. To avoid wetting of the inlet filter during sterilization,
use a clamp to close the tubing between the head plate and the filter. Close all other
connections (except the air out) air-tight with a hose and a hose clamp. Close all open tubing
ends with cotton and cover the ends with sterilizable foil or paper.
Make sure that the vessel is not completely closed, since pressure differences during
sterilization may damage the reactor or the probes. Use the air outlet filter to maintain pressure
equilibrium in- and outside the reactor. The heat exchanger should be empty during
sterilization.

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OPERATION Autoclavable Bioreactor 2 - 7 l ter
October 1994

3.3 STERILIZATION:

The bioreactor with all accessories (except the stirrer motor) can be placed in an autoclave.
The space, needed in the autoclave can be derived from the drawings that are enclosed in the
English copy of this manual.

The autoclave should stay at 121oC for at least 20 minutes in order to kill all organisms and
thermo-resistant spores. After sterilization, let the autoclave cool down without opening it,
until the temperature is below 90oC. When the temperature in the autoclave has dropped below
90oC, it can be opened to allow it to cool down further. This cooling procedure should be
performed to avoid low pressure in any part of the reactor system. Low pressure in tubing
might result in contamination when the tube is connected to a peripheral device. Low pressure
in a pH-electrode may result in sudden boiling of the electrolyte.

Note:
In case of a jacketed reactor, the sterilization interval might need to be increased, since the
empty jacket has a poor heat transfer capacity.
If the medium cannot stand a longer interval, you can fill the jacket with water to improve the
heat transfer (connect and close the lower tube fitting, do not close the upper one).

3.4 INSTALLATION:

Put the reactor as near as possible to the control equipment (the Bio Processor or Bio
Controller) and connect all electrodes to this equipment. Connect the heat exchanger to the
thermo circulator; hook up the air inlet pipe to the flow console and, if present, connect
cooling water to the condenser. Connect all sterile fluids (acid, base, etc.) that have to be
added to the medium, aseptically to the inlet pipes.
To improve heat transfer between the thermometer pocket and the Pt100-sensor, fill the
thermometer pocket with water or silicone oil. This will decrease the dead time of the sensor
and will make your temperature control more accurate.
When operating at higher temperature, silicone oil has the advantage of a lower vapour
pressure (less evaporation).

3.5 PREPARING FOR OPERATION:

After connecting all cables and tubing, adjust the setpoints of the controllers to the desired
value (temperature, pH, DO2, etc.).
Switch on the thermo circulator, stirrer motor, acid and/or base pumps and gas flow.
When temperature, pH etc. have reached their setpoint (and are stabilized), the bioreactor is
ready for inoculation.

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Autoclavable Bioreactor 2 - 7 liter MAINTENANCE
October 1994

CHAPTER 4

MAINTENANCE

4.1 THE REACTOR AND HEAD PLATE:

After finishing the fermentation process, the glass and stainless steel parts should be cleaned
thoroughly.
Use hot water, 70% ethanol or other suitable cleaners to clean all parts. Never use abrasive
materials to clean the metal parts.

After cleaning, dry the parts and reassemble the reactor. Take care not to damage or forget any
O-rings, since this can cause contamination during the next run.

When the reactor is used frequently, it is advised to replace the O-rings of the auxiliaries twice
a year.

Note:
Make sure that the pH-electrode is cleaned thoroughly before it is stored; refer to the user
manual of this electrode.

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October 1994

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Autoclavable Bioreactor 2 - 7 liter DRAWINGS
October 1994

CHAPTER 6

DRAWINGS

The following drawings are enclosed:

D3002-1b.12.006 Bioreactor 2l dished H/D = 1.5

D3002-1 .12.013 Bioreactor 2l jacketed H/D = 1.5
D3003-1 .12.018 Bioreactor 3l flat bottom
D3003-1 .12.017 Bioreactor 3l dished bottom
D3003-1 .12.019 Bioreactor 3l jacketed
D3005-1a.12.001 Bioreactor 5l dished bottom
D3005-1a.12.007 Bioreactor 5l jacketed
D3007-1a.12.014 Bioreactor 7l dished bottom
D3007-1a.12.015 Bioreactor 7l jacketed
D3002-1a.06.005 Stirrer assembly lipseal 2 - 5 liter
D3007-1a.06.004 Stirrer assembly lipseal 7 liter
D3002-1 .06.006 Stirrer ass. magn. coupled 2 - 5 liter
D3007-1 .06.003 Stirrer ass. magn. coupled 7 liter

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