LAMBDA MINIFOR LaboratoryFermentor - Bioreactor
Operation Manual
LAMBDA CZ s.r.o.
Lozibky 1
CZ-61400 Brno
Czech Republic
LAMBDA Laboratory Instruments
Sihlbruggstrasse 105
CH-6340 Baar
Switzerland
Hotline: +420 603 274 677
Email: support@lambda-instruments.com
Tel.: +41 444 50 20 71
1
Table of Contents
1
2
Delivery check and Inspection ............................................................................................. 8
1.1
Check the boxes and the equipment ....................................................................................... 8
1.2
Verification of the Delivery note ............................................................................................... 8
Introduction ............................................................................................................................... 8
2.1
MINIFOR design concept ............................................................................................................ 8
2.1.1 MINIFOR Advanced and Start-up Kit ...................................................................................................... 9
2.1.2 LAMBDA Base Control Unit .................................................................................................................... 11
2.1.3 MINFOR Vessels ...................................................................................................................................... 11
2.1.4 Agitation unit ............................................................................................................................................. 13
2.1.5 Temperature and pH probe .................................................................................................................... 13
2.1.6 pH control .................................................................................................................................................. 13
2.1.7 Temperature control................................................................................................................................. 14
2.1.8 Aeration and pO2 Control ........................................................................................................................ 14
2.1.9 REDOX Potential measurement ............................................................................................................ 16
2.1.10
Sampling device ................................................................................................................................... 16
2.1.11
Antifoam Control .................................................................................................................................. 16
2.1.12
Outgas condenser ............................................................................................................................... 17
2.1.13
Parameter “X” ....................................................................................................................................... 18
2.1.14
Quadruple plug box ............................................................................................................................. 18
2.2
Lambda MINIFOR Fermentation Modes / Setup / Options ............................................... 18
2.2.1
2.2.2
2.2.3
2.3
Safety Instructions ..................................................................................................................... 22
2.3.1
2.3.2
2.3.3
2.3.4
2.3.5
2.3.6
3
Batch Fermentations ................................................................................................................................ 20
Variable Volume Fed-Batch .................................................................................................................... 20
Continuous Fermentation ........................................................................................................................ 21
Glass vessel handling .............................................................................................................................. 22
LAMBDA Double Lock connectors ........................................................................................................ 23
Power connection of additional instruments......................................................................................... 23
Sterilization ................................................................................................................................................ 24
Pressure .................................................................................................................................................... 24
Dangerous reagents and microbes ....................................................................................................... 24
Installation and Set-up (based on MINIFOR 1L Advanced Kit) .................................. 25
3.1
Essential things for MINIFOR Setup and Installation - OVERVIEW............................... 25
3.2
Get ready with the LAMBDA Base Control Unit ................................................................. 25
3.3
Holders and Support Rods for LAMBDA Peristaltic Pumps ........................................... 26
3.4
Setting up of the Fermentation Vessel ................................................................................. 27
3.4.1
3.4.2
3.5
Agitation Unit and Sparger....................................................................................................... 33
3.5.1
3.6
Setting up the 7L reactor vessel ............................................................................................................ 30
Set-up of the 7L MINIFOR vessel with liquid O-ring ........................................................................... 31
0.3L Vessel and minimal working volume: Butterfly-shaped stirrer disc – no sparging................. 37
Air-Input ........................................................................................................................................ 38
3.6.1
Surface aeration ....................................................................................................................................... 40
3.7
pO2 probe and setup .................................................................................................................. 41
3.8
pH and temperature probe setup ........................................................................................... 44
3.9
Pressure Limiting Security Valve ........................................................................................... 47
3.10
Assembly of Sampling and Addition devices ..................................................................... 49
3.10.1
3.10.2
Quadruple sampling and addition ports ............................................................................................ 49
Preparation of LAMBDA PEEK double seal tubing connectors .................................................... 50
2
3.10.3
3.10.4
3.10.5
3.10.6
3.11
Preparation of Inoculation device (Septum) ..................................................................................... 52
Preparation of the sampling device ................................................................................................... 53
Preparation of storage bottles ............................................................................................................ 54
Mounting of sampling and addition devices ..................................................................................... 56
Medium cooling device ............................................................................................................. 57
3.11.1
3.11.2
3.12
Cooling loop .......................................................................................................................................... 58
Electronic Peltier cooling finger ......................................................................................................... 60
Outgas condenser and air outlet filter .................................................................................. 62
3.12.1
3.12.2
3.13
Glass outgas condenser with air outlet filter .................................................................................... 63
Assembly of Electronic outgas condenser ....................................................................................... 65
Scale module installation for continuous mode of operation ........................................ 67
3.13.1
3.13.2
3.13.3
3.13.4
3.14
Adapt the measuring range for your working volume / mass ........................................................ 67
Connection of the weighing module to the MINIFOR fermenter-bioreactor ................................ 68
Range selection ................................................................................................................................... 68
Connection of the addition or withdrawal pump .............................................................................. 69
Antifoam control system (ANTIFO & DOZITO) Installation ............................................. 70
3.14.1
3.14.2
3.14.3
3.15
Setting up ANTIFO foam detector and controller ............................................................................ 70
Preparation of DOZITO, the Miniature Syringe pump .................................................................... 71
Connecting ANTIFO and DOZITO .................................................................................................... 75
Cable Connections ..................................................................................................................... 76
3.15.1
3.15.2
3.15.3
3.15.4
3.15.5
3.15.6
3.15.7
3.15.8
3.15.9
3.15.10
3.15.11
3.15.12
3.15.13
3.15.14
3.16
Peristaltic Pump Installation.................................................................................................... 88
3.16.1
3.16.2
3.16.3
3.16.4
3.16.5
3.16.6
4
Installation of tubing ............................................................................................................................. 89
Setting up of the flow rate ................................................................................................................... 90
Choosing flow direction ....................................................................................................................... 91
Fast filling or emptying the line .......................................................................................................... 92
Connection of Peristaltic pumps ........................................................................................................ 92
Placement of the Peristaltic Pumps .................................................................................................. 93
Display and control panel .................................................................................................... 94
4.1
Keys................................................................................................................................................ 95
4.1.1
4.1.2
4.1.3
Function keys (C, R) ................................................................................................................................ 95
Navigation Keys (Arrow Keys)................................................................................................................ 96
Number Keys ............................................................................................................................................ 97
4.2
Modes (states) of MINIFOR ...................................................................................................... 98
4.3
SET-point and ALARM............................................................................................................... 99
4.3.1
4.3.2
4.3.3
5
Overview of all MINIFOR connections .............................................................................................. 76
Agitation Unit ........................................................................................................................................ 78
pH and temperature probe ................................................................................................................. 78
Peltier Cooling finger for medium ...................................................................................................... 79
Peristaltic Pump and Massflow .......................................................................................................... 80
pO2 probe .............................................................................................................................................. 81
Gas flow inlet ........................................................................................................................................ 82
Gas flow outlet ...................................................................................................................................... 83
Parameter X.......................................................................................................................................... 84
ANTIFO and DOZITO ......................................................................................................................... 84
Grounding the MINIFOR ..................................................................................................................... 85
PC connection ...................................................................................................................................... 86
Alarm ..................................................................................................................................................... 87
Main power supply ............................................................................................................................... 87
Setting the temperature ......................................................................................................................... 100
Setting the pH, pO2, Air-flow, stirrer, X................................................................................................ 100
Alert (de)activation ................................................................................................................................. 101
Calibrations ........................................................................................................................... 102
5.1
Stirrer, Temperature, Flow rate: No calibration ................................................................ 102
5.2
pH probe calibration (gauging) ............................................................................................. 102
3
5.2.1
5.2.2
5.2.3
5.3
pO2 probe calibration (gauging) ........................................................................................... 107
5.3.1
5.3.2
5.3.3
5.3.4
5.3.5
5.3.6
5.3.7
5.4
General introduction to the X-channel................................................................................................. 118
Calibration of Weighing module ........................................................................................................... 118
Setting up of the pump regulation ........................................................................................................ 121
Antifoam system ....................................................................................................................... 122
5.6.1
5.6.2
6
Volumetric Calibration ........................................................................................................................... 116
Calibration by weight ............................................................................................................................. 117
X-channel calibration (gauging): Example Weighing module ...................................... 118
5.5.1
5.5.2
5.5.3
5.6
List of consumables ............................................................................................................................... 108
Inspection of the sensor ........................................................................................................................ 108
Polarization.............................................................................................................................................. 109
Oxygen saturation in water ................................................................................................................... 109
Calibration of pO2 ................................................................................................................................... 111
Messages / Errors .................................................................................................................................. 114
Crosscheck the pO2 calibration ............................................................................................................ 114
Pump flow calibration.............................................................................................................. 116
5.4.1
5.4.2
5.5
List of consumables ............................................................................................................................... 102
Step by step Calibration of the pH ....................................................................................................... 103
Crosscheck the pH calibration.............................................................................................................. 105
Sensitivity adjustment in ANTIFO ........................................................................................................ 122
Setting up of volume step in DOZITO ................................................................................................. 123
PC Connection & Fermentation Software ..................................................................... 125
6.1
FNet easy to use fermentation software............................................................................. 125
6.1.1
6.1.2
6.1.3
6.2
SIAM Industrial fermentation software ............................................................................... 126
6.2.1
6.2.2
6.2.3
6.3
7
Well Sophisticated Software ................................................................................................................. 126
Devices analysed using SIAM .............................................................................................................. 127
Applications ............................................................................................................................................. 127
MINI-4-gas automatic gas-mixing module software ........................................................ 128
6.3.1
6.3.2
6.4
Ready to use software ........................................................................................................................... 125
Data archiving ......................................................................................................................................... 125
Process Control ...................................................................................................................................... 126
Types of gas-mix systems .................................................................................................................... 128
Process Control ...................................................................................................................................... 128
PC Connection .......................................................................................................................... 128
Sterilization & Reinstallation ............................................................................................ 129
7.1
Preparation for Sterilization ................................................................................................... 129
7.2
Sterilization of storage bottles and pump lines................................................................ 132
7.3
Sterilization of pH-temperature probe................................................................................. 133
7.4
Checklist before sterilization ................................................................................................. 134
7.5
Autoclave space and Sterilization Cycle ............................................................................ 135
7.6
Re-installation............................................................................................................................ 135
7.7
Sterility test ................................................................................................................................ 136
8
Inoculation ............................................................................................................................. 136
8.1
Checklist before inoculation.................................................................................................. 137
8.2
Inoculation process ................................................................................................................. 138
9
Sterile Sampling ................................................................................................................... 139
10
Maintenance ...................................................................................................................... 142
10.1
Overpressure security valve maintenance ........................................................................ 142
4
10.2
pH Probe maintenance and storage .................................................................................... 142
10.2.1
10.2.2
10.3
Cleaning pH probe ............................................................................................................................. 143
pH probe storage ............................................................................................................................... 143
pO2 probe maintenance and storage ................................................................................... 144
10.3.1
pO2 membrane cleaning and maintenance .................................................................................... 144
10.4
Cleaning the reaction vessel ................................................................................................. 145
10.5
Sterile inlet and exhaust filter maintenance ...................................................................... 145
11
Troubleshooting ............................................................................................................... 145
12
Technical Specification .................................................................................................. 147
13
CE Declaration .................................................................................................................. 149
14
Warranty ............................................................................................................................. 149
5
Copyright Notice
LAMBDA CZ s.r.o.
Lozíbky 1
CZ-61400 Brno
Czech Republic
© LAMBDA Laboratory Instruments
All Rights Reserved.
LAMBDA may modify the equipment and the document without any further notice.
Reproduction, adaptation without any prior written permission from LAMBDA
Laboratory Instruments is prohibited.
Disclaimer Notice
LAMBDA CZ s.r.o. reserves the right to change information in this document without
notice. Updates to the information in this document reflect our commitment to
continuing product development and improvement.
6
Symbols and signs used in the manual:
Tips and tricks message allows learning more about the easy handling of
the equipment.
Attention message provides safety instructions, safeguards for you and the
equipment. If not followed, it may lead to the damage of the instrument and
personal injury.
Thumbs up messages indicate the things that are recommended to be
done.
Thumbs down messages forbids the things not to be done with the
MINIFOR fermentor-bioreactor.
Background information provides you with the most essential information
about the instrument.
Bold
Texts found in “bold” emphasis keywords or phrases.
7
1 Delivery check and Inspection
1.1
Check the boxes and the equipment
After receiving the order, inspect the boxes carefully and also the culture vessel and the other parts
made of glass for any damages that may have occurred during shipping.
In case of damages, please report to your local purchasing department immediately or send us
your claim note. Please note that we can help in claiming the insurance, only when the problem is
reported to us within 2 weeks after dispatch.
1.2
Verification of the Delivery note
Kindly check the delivery note and verify that you have received everything and nothing is missing.
If any part of the order found missing, fails to operate or even damaged during shipping, report to
us immediately.
2 Introduction
2.1
MINIFOR design concept
LAMBDA MINIFOR fermentor-bioreactor, designed for bench-top surfaces in laboratories is
suitable for volumes from as low as 35 ml to 6.5 l.
LAMBDA MINIFOR fermentor-bioreactor has been designed such that several of these can be
placed side by side and are well suited for the optimization of bio-transformations and growth
parameters of the culture. LAMBDA MINIFOR takes up the minimum amount of space on the
laboratory benches and allows having good access to all parts of the unit.
MINIFOR can be used either to work independently or it can be connected directly with a PC to
control it remotely and for extensive data treatment, regulation and storage.
Figure 2.1-1 MINIFOR 1L Advanced Kit, front view
Figure 2.1-2 MINIFOR 1L Advanced Kit, side view
8
Several innovations and new ideas have been introduced,
For many years, the whole glass vessels have been used in cell cultures and proven to
maintain perfect sterility, we have used this idea in our fermentor / bioreactor.
The agitation system is based on up-and-down movement. The agitator system has a
motor with a membrane ensuring sterility and efficient mixing without forming a vortex
(thus no baffles are needed). The bio-mimicking 'fish-tail' stirring discs offer maximum
efficiency without cutting edges; they mix the cells gently and produce less foam.
The culture is heated by 'sun-like' radiation with a gold reflector and a parabolic radiator
under the vessel to maintain the temperature precisely.
Compact unit, without a casing tower, it is possible to measure and control six
parameters with the use of its modern microprocessor stored at the front of the
instrument.
2.1.1 MINIFOR Advanced and Start-up Kit
LAMBDA MINIFOR fermentor-bioreactor system includes the Advanced and Start-up kit.
Table 1 Short overview of LAMBDA MINIFOR ADVANCED KIT and MINIFOR START-UP KIT
MINIFOR
Advanced Kit
Start-up Kit
Reactor scale
Laboratory bench-top fermentor and bioreactor
Reactor design
Stirred Tank
Operation mode
batch, fed-batch*, continuous*, (perfusion*)
Parameter control
stirrer speed, temperature, pH, Dissolved Oxygen,
airflow, free parameter, level control for continuous
mode.
* Optional Parameter control *
Microprocessor
With internal software, display and keypad
Agitation
Automatic stirrer controller
0, 0.1, 0.2, …., 19.9, 20 Hz
min. 0 Hz ( = 0 rpm)
max. 20 Hz ( = 1200 rpm)
Measurement
whole range 0 - 20 Hz
Temperature
Automatic temperature control
from 5 °C over RT to 70 °C
Measurement with Pt100
from 0 to 99.9 °C in 0.1 °C steps
IR (infrared) heater
Cooling loop
pH
Automatic pH controller
weight control for continuous mode, antifoam,
pCO2, conductivity, REDOX, LUMO (light)
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
9
0 - 13
Measurement with pH probe
0 -13
Acid pump (PRECIFLOW)
Base pump (PRECIFLOW)
Holders and rods for the pumps
x
x
x
x
x
* optional
* optional
* optional
x
* optional
x
x
x
* optional
x
x
x
x
x
x
x
x
x
x
x
* optional
x
* optional
x
* optional
Feed and harvest pump: PRECIFLOW /
MULTIFLOW / HiFLOW / MAXIFLOW / VIT-FIT / VITFIT HP, pump INTEGRATORS
* optional
* optional
WEIGHING MODULE for continuous mode
Automatic Antifoam-system (ANTIFO & DOZITO)
* optional
* optional
* optional
* optional
O2-enrichment / 4-gas-mix: controller and
massflows MASSFLOW 500 /MASSFLOW 5000
* optional
* optional
Air compressor AEROSILENTO
REDOX probe, controller, pump / MASSFLOW
PELTIER COOLER for medium / off-gas
* optional
* optional
* optional
* optional
* optional
* optional
LUMO light and controller for PBR
* optional
* optional
x
x
Storage vessels and lines for acid and for base
DO (pO2)
Automatic DO controller (proportional)
0 - 25 mg Oxygen/L (in 0.1 mg steps)
Measurement with DO probe
0 - 25 mg Oxygen/L (in 0.1 mg steps)
Airflow
Automatic aeration controller (proportional)
0 - 5 L/min in 0.01 L/min steps
Measurement with internal MASSFLOW
0 - 5 L/min in 0.01 L/min steps
Free Parameter
Automatic controller, socket, display
Alerts
For high and low values of each parameter
Overpressure valve
Sampling device
Cooling device for outgas/exit gas
PC-software (FNet, SIAM), Laptop
Operation manual and video
Warranty
2 years
LAMBDA MINIFOR is the most flexible fermentor-bioreactor in the market. Initially working with the
Start-up kit or even with the Advanced Kit, the system can be upgraded according to the project
requirements. For example: changing vessels, adding Pumps and MASSFLOWS, ANTIFO &
DOZITO, etc.
10
2.1.2 LAMBDA Base Control Unit
LAMBDA knows that the biological systems are complex and more the parameters are controlled,
the better. Therefore we supply the base control unit that involves measurement of parameters and
regulation loops (°C, pH, pO2, air flow rate, stirring and another selectable channel X, which
can be used for any other controller. It is ready to accept a scale module for continuous
fermentations) for precise control of the parameters.
All processes are controlled digitally by two microprocessors. The modern technology allows a
much better control than it was used before. Modern technology allows us to gather all the control
loops in a small space at relatively low cost and we have used it to make our customer benefit a lot
from it, instead of selling each and every module at a high cost.
The base control unit contains the control panel that is used for the display and control of the
parameters.
2.1.3 MINIFOR Vessels
MINIFOR can be used to cultivate the minimal working volume of 35 ml with the precise control of
6 parameters!
It has the largest volume range available on the market, because it is the only instrument that can
be used for volumes ranging from 35 ml to over 6 l. The base unit will remain the same but the
working vessels can be exchanged according to the project requirements. The speed of signal
traffic is higher and the regulation is more precise in each and every base unit.
LAMBDA designed and created ports on the sides of the vessels. The access is much better and
global overview is very good. The innovative form of the LAMBDA vessel eliminates the expensive
head plate completely. The threaded central screw cap allows an easy mounting and opening with
just one hand movement.
11
Table 2
MINIFOR Vessel Overview
Vessel type
0.3L
0.4L
1L
3L
7L
Minimum (L)
0.035
0.15
0.3
0.5
1.0
Maximum (L)
0.4
0.45
1.7
3.0
6.5
Height (cm)
34
22
34
37
50
Diameter (cm)
22
23
25
34
30
6
8
8
8
8
22
22
22
22
Working volume
Vessel dimension
for autoclaving
Ports
No. of side necks
≈ traditional ports
2.1.3.1 LAMBDA Liquid O-ring for 7L vessels
Standard O-rings have several disagreeable properties.
If any dirt or filaments are closed under the O-ring, then microbes will have a large tunnel to
get into the vessel.
Large O-rings require large compression forces to be perfectly tight. The screwing of the
connection surfaces must be done perfectly and regularly in order to eliminate the higher
compression on one side and lower compression on the other side. The resulting
mechanical forces may have a high stress on glass parts, especially during the sterilization.
Frequent replacement of large O-rings may represent high recurrent costs.
Therefore, for the large 7 L vessels, LAMBDA proposes a new solution to the above mentioned
problems, the liquid O-ring:
The liquid O-ring consists of a new partial hardening silicone resin filled with the purest
quartz powder of extremely fine (fume like) consistency.
It fills the cavities between the joining surfaces, is completely gas tight and also does not generate
any stress on the glass.
It will either cure in several hours at room temperature or within minutes in the autoclave during
sterilization. The liquid O-ring forms a large surface joint and not just a few mm with a large contact
surface to cover as in the case of standard O-rings.
Moreover, this liquid O-ring paste can be reused many times.
12
2.1.4 Agitation unit
The LAMBDA agitation unit is included in each MINIFOR kit.
LAMBDA uses a non-rotational agitation system with up and down movement. This agitation
system shows a new possibility to achieve an easy and perfect sterility, maintained only with an
expensive magnetic coupling. The problems caused by the magnetic coupling are totally eliminated
here and combined with the agitation system used.
The vibromixer: a strong motor moves one or more stirring discs up and down.
The frequency of agitation is controlled by a microprocessor. The stirrer range is 0 – 20 Hz and can
be chosen in steps of 0.1 Hz.
The stirrer frequency as well as the amount and type of stirrer discs can be choosed according to
the applications.
Available stirrer discs are:
- Standard fish-tail stirrer discs: for mammalian cells, plant cells, fungi, bacteria, yeast. The
softest agitation and biomimetic 'fish-tails' ensures uniform mixing throughout the
fermentation / bioreaction.
- Optional metal stirrer discs: for viscous medium
- Butterfly stirrer discs for low working volume (for example: 35 ml)
2.1.5 Temperature and pH probe
The temperature probe (Pt100) is incorporated with the pH probe. The pH/Pt100 probe is included
in each MINIFOR kit.
The Pt 100 is used for
- Automatic temperature correction for the corresponding pH measurement
- Measurement of the temperature of the fermentation medium / cell broth
- Automatic temperature control of the fermentation medium / cell broth.
The pH probe is used for
- Measurement of the pH.
2.1.6 pH control
Each MINIFOR kit contains the microprocessor for the automatic temperature and pH control.
The MINIFOR ADVANCED KIT includes 2 PRECIFLOW peristaltic pumps, lines and storage
vessels for an automatic pH control.
For an automatic pH control, the microprocessor compares the actual pH value with the set-point.
According to the difference of those values, the acid or base will be added to the medium.
Liquid base and/or liquid acid are added automatically by the LAMBDA peristaltic pumps according
to the difference between set-point and actual measurement. Thus, it is not done by the on/off but
over the whole speed range of the pump.
Instead liquid acid, it is also possible to add gases like: CO2 for the automatic pH control. Then, it is
necessary to disconnect the peristaltic pump for acid and connect the optional MASSFLOW gas
controller for the automatic pH control.
13
2.1.7 Temperature control
Heating blocks as well as silicone heating pads have their well-known disadvantages.
-
-
Their efficiency is limited by the quality of the contact between the heating surface and the
vessel walls. The transmission of heat occurs only by diffusion, which is slow especially
when the temperature gradient is small.
Further disadvantage is that the view into the vessel is restricted and it is not possible to
illuminate cultures of algae from outside.
An even worse criterion is the overheating of cell culture, which occurs when the level of
the culture liquid is below the upper part of the pad.
Another major disadvantage is that heating clamps and heating mats prevent natural
cooling of the vessel and a complementary cooling is then required. This increases both the
cost and complexity of the system. Even a small damage in heating mats can lead to
electrocution.
To eliminate all the above mentioned problems in using the heating blocks and pads, LAMBDA
invented an IR radiation heater which is placed under the bottom of the vessel.
The heating spiral is placed in a gilded parabolic reflector, which concentrates the energy at the
bottom of the vessel. The heat rays optimally heat the medium from underneath. This creates a
natural upwards convection even without any mechanical agitation and without creating any hot
spots at any medium volume in the vessel. About a half of the heat is adsorbed by the glass
bottom and about a half of the energy is absorbed directly in the medium. It is not possible to
imagine a softer sun-like way of heating.
The change of temperature is much faster and its setting and adjustment is more precise and
simple to handle.
The temperature is measured with the Pt100 which is incorporated with the pH probe (please refer
2.1.5 Temperature and pH probe).
If it is necessary to work with temperatures lower than 4°C above ambient temperatures or if
cooling is needed due to the exothermic reaction, then the range of temperature control can be
shifted by the addition of the optional cooling system.
The MINIFOR kit includes the cooling loop which allows the circulating cooling liquid (for
example: tap water or cooling liquid from the thermostatic water bath) to maintain the temperature.
Or it is also possible to get the electronic PELTIER Cooling device for the working volumes up to
3L
2.1.8 Aeration and pO2 Control
LAMBDA MASSFLOW controller system is specially designed to use with LAMBDA laboratory
bioreactors and fermenters for the measurement and control of air and other gases.
A high quality laminar mass flow sensor measures the flow rate. The result appears on the digital
display of the base control unit. The mass flow cell has a very low pressure drop and a linearity
error less than ±3% reading. The repeatability is better than ±0. 5% reading.
The flow rate is regulated by a special proprietary proportional needle valve controlled by a
microprocessor. The flow rate can be programmed and volume can be calculated.
Initially the parameter needs to be chosen for the control:
14
-
Automatic flow-control [L/min] of one gas (mix)
or
Automatic control of pO2 (DO, dissolved Oxygen) [Please refer chapter 5.3]
2.1.8.1 Air flow
Before using the gas-flow (L/min), it’s a must to branch the pressured gas in terms of 0.05.
Each MINIFOR kit contains an internal MASSFLOW: 0 – 5000 ml/min, control in 10 ml/min steps.
The proportional valve for the gas supply opens according to the set-point.
The internal MASSFLOW of MINIFOR can be used for one stream of gas or one stream of gasmix.
For gas mix and O2 enrichment, optional external MASSFLOWs can be used:
- External MASSLFOW 5000: 0 – 5000 ml/min, control in 10 ml/min steps
- External MASSFLOW 500: 0 – 500 ml/min, control in 1 ml/min steps
2.1.8.2 pO2 (DO = dissolved Oxygen) probe
By adding air/O2, the internal MASSFLOW (0-5000 ml/min, control in 10 ml/min steps) can also be
used for automatic pO2 control.
Furthermore, the pO2 probe (OD probe = dissolved Oxygen probe) is required.
The MINIFOR ADVANCED KIT is equipped with the DO probe. It is possible to get the pO2 probe
to be used with the MINIFOR Start-up Kit, if needed.
Technical properties of the DO probe:
• pO2 probe can be sterilized up to 130°C.
• Short response time, less than 1 min. to 95 % of end signal
• Wide measuring range 0-25 mg dissolved oxygen/l
• Automatic temperature compensation
• Process pressure up to 3 bars
• Polarization time less than 2 hours
LAMBDA has developed an oxygen probe using a completely non-metal body made of a new, very
resistant material, PEEK, which has a similar chemical resistance to PTFE, but it is mechanically
much more stable.
The electrode is of a Clark type with a large Pt cathode and Ag/Cl reference anode. The
membrane is made from glass reinforced with a thin layer of PTFE. The PTFE is better than
silicone membranes because much less deposits form on the Teflon surface than on any other
material.
The membrane is almost fully protected and only a very small opening above the cathode is free.
The probability of damaging the membrane is therefore greatly reduced.
Principle of operation: The PTFE membrane is permeable to gases and will not let any other
dissolved substance to pass through. By selection of the right polarization voltage, the oxygen,
which diffuses through the membrane, is reduced on the cathode and the electric current
proportional to the oxygen concentration is generated. This signal is measured and transformed to
the concentration of oxygen dissolved in the medium.
15
2.1.9 REDOX Potential measurement
The measurement of the RedOX potential for the anaerobic culturing can be done with the REDOX
probe and the control unit.
REDOX probe is an optional tool that can be obtained if needed.
The measurement of Red-Ox potential is done by using a sterilizable combined pH/temperature
probe with an additional Pt electrode fixed on its glass body. This probe is connected to the
MINIFOR fermenter-bioreactor in the same way as that of the standard pH probe.
LAMBDA REDOX allows the measurement of the Red-Ox potential and the digital transfer of the
data to the PC with the help of the fermentation software SIAM.
The control unit displays the measured RedOx potential in terms of mV.
The output RedOx signal can be received at the “PUMP” socket at the rear of the MINIFOR
laboratory fermentor-bioreactor base unit.
2.1.10
Sampling device
The MINIFOR sampling device is included in each MINIFOR ADVANCED KIT.
To decrease the risk of contamination during sample withdrawal, LAMBDA supplies an easy to use
sampling device. It consists of a glass trap with three inlet/outlet tubes. Out of which one tube is
connected with the sampling port, second has to be connected with the air filter and the third is the
outlet that is used for the sample withdrawal.
Samples can be taken from the vessel using one of the cannula from the quadruple sampling
assembly (the one with the longest needle).
If needed, the MINIFOR standard sampling device can be replaced by any other sampling device
(like the standard in your laboratory).
2.1.11
Antifoam Control
The foam level control system is an optional tool and not included in the MINIFOR Start-up and
Advanced Kit.
The LAMBDA MINIFOR laboratory bioreactor-fermenter can be equipped with a novel foam
detector and control system:
ANTIFO and DOZITO
2.1.11.1
DOZITO = miniature syringe pump for antifoam addition
The space around fermenter/bioreactor vessel is very precious and the amount of antifoam
substance used in fermentation and cell culture processes is usually only a few ml.
For this reason, LAMBDA has developed probably the world’s smallest syringe pump system. A
special frequently sterilizable 5 ml glass syringe is used for the addition of anti-foam agent.
16
The magnetic holder of DOZITO can be placed in any convenient place on the upper cover plate of
the MINIFOR fermenter/bioreactor base control unit casing.
The volume of added anti-foam liquid can be varied from a dozen micro-litres to about 0.3 ml.
Specifications:
Pushing force: more than 20 N
Step length: adjustable from 0 to 4 mm
Protection: electronic in case of blocking
Dimensions: 3.6 x 2.7 x 12.7 cm (L x W x D)
Weight: 230 g
2.1.11.2
ANTIFO = foam detection system and controller of DOZITO
The presence of foam in the reactor vessel is detected by the measurement of electrical
conductivity.
Instead of an additional antifoam probe, two cannulas (long and small) from the quadruple
sampling port of the LAMBDA MINIFOR fermenter vessel are used. No additional ports need to
be used for the anti-foam detection and control.
Specification:
Power supply: 12 V DC, 2W (from the pump socket of the MINIFOR fermenter)
Conductivity range: 1 k Ohm to 0.5 M Ohm
Frequency: 4 kHz AC
Measuring voltage: 100 mV AC
Dimensions: 3 x 3 x 16 cm (L x W x D)
Weight: 220 g
2.1.11.3
Automatic prevention of antifoam over-dosage
The addition of too much antifoam agent is detrimental for the oxygen transfer from the air bubbles
into the culture medium. The excessive addition of antifoam liquid (anti-foam over-dosage) is
avoided by the introduction of a waiting interval of about 20 seconds after the first antifoam agent
addition.
The second addition of antifoam agent occurs only when the foam did not disappear during the
elapsed waiting interval time. Additionally, the anti-foam dose can also be controlled in the steps of
volume by the LAMBDA DOZITO miniature syringe pump.
2.1.12
Outgas condenser
The outgas condenser prevents the condensation of water on the output filter and the resulting
blocking of the outgas air flow. Condensed water flows back into the vessel.
This flow-back is important, particularly when working with low volumes for weeks, otherwise the
medium would become concentrated and the working volume would decrease.
2.1.12.1
Outgas condenser with cooling liquid
A glass out-gas condenser that can be connected with a cooling liquid (cooling water) is included in
each MINIFOR Kit.
17
2.1.12.2
Electronic outgas condenser
The electronic outgas condenser is an optional tool. It is not included in the MINIFOR Advanced
and Start-up kit.
As an alternative to water-cooled exhaust air condensers, LAMBDA offers a Peltier-based outgas
condenser with an electronically controlled operating temperature down to 5 °C.
This results in a better removal of moisture from the outgas stream than the water cooled
condensers.
Such an electronic condensing system also allows a more efficient and constant cooling without
any cooling water!
2.1.13
Parameter “X”
The parameter “X” allows the control of any other parameter apart from °C, pH, pO2, airflow rate
and stirring. It is an optional parameter control to be utilized when needed.
Usually, for the continuous mode of fermentation, the weighing module (chemostat) can be used
as parameter “X” for controlling the addition or removal of the substrate from the reaction vessel.
It is possible to control the conductivity probe, redox potential and also the CO2 probe as
parameter “X”.
2.1.14
Quadruple plug box
The quadruple plug box is an optional tool that can be used with the MINIFOR. It is not included in
the standard kits LAMBDA MINIFOR ADVANCED KIT or LAMBDA MINIFOR START-UP KIT.
The quadruple plug box consists of four additional 8-pole connectors (two in the rear and two in the
front) that can be connected and controlled by the MINIFOR fermentor-bioreactor.
If additional instruments are needed to be used with the MINIFOR, then the quadruple plug box
helps to extend the connection for the addition of up to four additional LAMBDA instruments (e.g.
pumps, mass flow gas flow controller, weighing module, antifoam controller, etc.)
This plug box provides a power supply and RS-485 line connections.
Up to two quadruple plug boxes can be connected together to further extend the number of
available connection sockets.
2.2
LAMBDA MINIFOR Fermentation Modes / Setup / Options
LAMBDA MINIFOR can be used for batch, fed-batch and continuous mode of operation.
In the following, a short overview of the tools that are needed for the different modes of operations
will be explained:
18
Table 3
Fermentation modes: batch, fed-batch & continuous mode
Batch
Fed-Batch
Continuous
= LAMBDA MINIFOR kit (Advanced or Start-up)
= LAMBDA MINIFOR kit (Advanced or Start-up) + 1 Feed-Pump
= LAMBDA MINIFOR kit (Advanced or Start-up) + 1 Feed-Pump + 1 Harvest Pump
+ 1 weighing module (Speed of Feed-Pump = Speed of Harvest-Pump = growth rate)
Batch
MINIFOR
(Advanced or
Start-up Kit)
Fed-Batch
MINIFOR
(Advanced or
Start-up Kit)
Feed-Pump
(Peristaltic Pump)
MINIFOR kit
(Advanced or
Start-up Kit)
Feed-Pump
(Peristaltic Pump)
Continuous
Harvest-Pump
(Peristaltic Pump)
Weighing Module
19
2.2.1 Batch Fermentations
Table 4
Fermentation mode: Batch = LAMBDA MINIFOR kit (Advanced or Start-up)
Batch Mode
MINIFOR
(Advanced or
Start-up Kit)
-
-
-
A conventional mode of fermentation in a closed system is batch fermentation.
The procedure of incubation, after the inoculation with micro-organisms to a sterile nutrient
solution, is carried out. Nothing is added to the fermentation process except acid or base to control
and maintain the pH. And air (O2) is controlled in the case of aerobic micro-organisms.
There is a general constant change of the composition of the culture medium / biomass
concentration as a result of the metabolism of the cells.
2.2.2 Variable Volume Fed-Batch
Table 5
Fermentation mode: Fed-Batch = LAMBDA MINIFOR kit (Advanced or Start-up) + 1 FeedPump
Fed-Batch Mode
MINIFOR
(Advanced or
Start-up Kit)
Feed-Pump
(Peristaltic Pump)
An enhancement to closed batch process where all of the substrate is added at the beginning of
the fermentation is the fed-batch fermentation.
This is where the substrate is added in small proportions during the fermentation process. These
increments are initially small concentrations of the nutrient solution at the beginning of the process
and during the production phase in small doses.
There are 2 basic approaches to this mode of fermentation. These are:
- Constant Volume Fed-Batch Culture (Fixed Volume Fed Batch)
- Variable Volume Fed-Batch
Due to the substrate feed, the volume changes with respect to the fermentation time.
20
Options:
- Medium used in the batch mode can be added.
- Addition of the same concentration of the limiting substrate solution used in the initial medium.
- Addition at a reduced rate of a concentrated limiting substrate solution is used.
2.2.3 Continuous Fermentation
Table 6
Continuous mode = LAMBDA MINIFOR kit (Advanced or Start-up) + 1 Feed-Pump + 1
Harvest Pump
Continuous + 1 Weighing module (speed of feed pump = speed of harvest pump = growth rate)
Continuous
Mode
MINIFOR kit
(Advanced or
Start-up Kit)
Feed-Pump
(Peristaltic Pump)
Harvest-Pump
(Peristaltic Pump)
Weighing Module
The fermentation mode in which there is an open system set up is the continuous fermentation
mode.
An equal quantity of the converted nutrient solution with micro-organisms is removed from the
bioreactor vessel and a sterile nutrient solution is added to the reactant vessel continuously.
Two terms are used in the case of a homogeneously mixed bioreactor. The first term, known as
chemostat, in the steady state, the concentration of one substrate is adjusted accordingly to
control cell growth. The second term, known as turbidistat, the biomass concentration is
monitored, cell growth is kept constant and the rate of feed of the nutrient solution is adjusted
appropriately.
2.2.3.1 Weighing module
Our goal was to develop an extremely compact, precise and easy to use device, which could be
used for continuous fermentation and cell cultures. The scale module is an optional accessory for
the LAMBDA MINIFOR laboratory fermenters and bioreactors. It allows keeping the amount of
medium constant, independent of the extent of aeration, foaming and stirring.
This is a much more precise method for the control of medium volume than a low cost overflow
tubes or similar devices which control only the level of the culture surface. Therefore, it allows an
exact evaluation and calculation of culture parameters.
21
2.3
Safety Instructions
Do follow the safety instructions and precautions while handling the equipment for your own safety
and also for the safety of your equipment. It minimizes the potential risks that are caused during
the usage.
Follow your laboratory safety precautions!
2.3.1 Glass vessel handling
Glass is still the best material for bioreactor vessels. It does not leak out heavy metal ions like steel
or not polymerized chemicals like plastic materials. Therefore, without any compromise LAMBDA
supplies only whole glass vessels.
Glass, however, is fragile, so handle it with all necessary care, especially when you wash vessels.
Pay particular attention that glass parts and vessels can be broken, if not
handled with care.
Protect the glass surface from scratches made by silica or other hard
materials.
Always use the safety over-pressure valve on the vessel! This will prevent a
pressure increase in case of the blocking of the output filter in case of excessive
foaming.
During autoclaving, leave always a ventilation opening for pressure
compensation!
Do not screw the screw caps with too much force! It is not necessary and
the glass threads may break.
Never heat an empty reactor! The heat radiation is absorbed by the glass walls
and their temperature would increase to such an extent, that thermal dilatation
would lead to a vessel burst.
During autoclaving, never fill more than 2/3 of the vessel volume with liquid!
Use brushes with detergent to remove dirt and acidic solutions (acetic acid,
citric acid or hydrochloric acid for salt deposits).
Always place a metal washer under the screw-cap to reduce the necessary
screw force!
To facilitate the insertion of the multiple-seal silicone stoppers into the
glass necks (e.g. probes, cooling loop, cooler, tubing etc.), you can wet them
with a few drops of distilled water. They will then slide in and out easily.
For removing the stoppers and probes you can also add a drop or two of
distilled water between the stopper and the glass wall. Move them from side to
side while at the same time pulling them out.
22
It is highly recommended to place an intermediary vessel between the
condenser outlet and the output gas filter. A small amount of convenient
antifoam solution can be placed on the bottom of this bottle. This will destroy the
foam should it enter this additional vessel and increase the protection of the
output filter against clogging.
All glass components are predestined to get broken at some time. If breakage
is limited to side necks, generally the vessel can be repaired by a glassblower.
LAMBDA keeps also a stock of threaded necks for repairs. Broken vessels can
also be sent to us. Please, contact us for further information.
2.3.1.1 Special precautions for handling 0.3L vessel
The 0.3L vessels are the smallest and the jacketed reactor vessels of the MINIFOR fermentorbioreactor. Special safety precautions and tips would help to master safe working with the
minimum working volume reactor vessels.
The small jacketed 0.3L reactors must be used only with the vessel jacket
completely filled with water. The hose connectors of the jacket are closed with
silicone tubing.
While using the small jacketed 0.3L vessel, the initial temperature may slightly
overshoot after the first start-up of MINIFOR. It is therefore advisable to set the
temperature about 4°C lower than the final working temperature. After the
temperature approaches this temperature, set the final working temperature. This will
shorten the time taken for temperature stabilization. If time is not critical, you
may just set the desired temperature and wait until the system equilibrates
automatically.
The vessel jackets of the 0.3 L vessels are not intended for cooling but only for
buffering the temperature variations. It can, however, be used with a circulating
thermostatic bath or for rapid cool-down, if required for special reasons. In this
case, inactivate the heating on the MINIFOR by setting the temperature to a low
value (e.g. 10°C).
Otherwise follow the general safety construction for handing glass reactor vessels, refer the
previous 2.3.1 Glass vessel handling!
2.3.2 LAMBDA Double Lock connectors
The LAMBDA DOUBLE LOCK connection system allows safe and easy connection of tubing to
the vessel. It is made of PEEK. PEEK is a new material similar to PTFE in its extreme chemical
resistance and very high melting point (350°C). Therefore, it can be even flamed. PEEK has a
much higher mechanical stability and does not “flow” like PTFE. For its superior qualities, it has
been selected by LAMBDA despite its very high price.
2.3.3 Power connection of additional instruments
When connected to the MINIFOR system and its RS-485 line, the pumps and
external MASSFLOW gas flow controllers MUST NOT be powered by any
external power supply. This is because the used 12V voltage of the external
23
power supply and the MINIFOR power supply may not be exactly the same and
the current may flow from one instrument to the other. This could generate
problems and damage the instruments. If such a connection is inevitable a diode
in the power supply line would be needed.
Also follow all safety rules of your laboratory and common safety rules while handling electrical
equipment!
2.3.4 Sterilization
Never autoclave any cable or the MINIFOR console or other electronic devices,
otherwise they will be destroyed!
Never use overpressure on the vessel, otherwise it will be destroyed! Therefore
make sure, that you leave open at least one line (no tube clamps!) which leads to the
headspace (not medium) for pressure balance. Usually, the off-gas line is the best
for it.
See also the special safety rules for the vessels in chapter 2.2.1 and sub chapters
for handling the glass vessels!
For autoclaving use slow cooling down / pressure balance as it is usually
programmed for “liquid sterilization”
2.3.5 Pressure
Never overpressure the vessel, otherwise it will be destroyed!
Refer the special safety rules for sterilization on chapter 2.3.4
2.3.6 Dangerous reagents and microbes
While handling corrosives and other dangerous reagents put on goggles and gloves
and follow your laboratory safety instructions!
While handling pathogens and dangerous microbes apply the necessary safety
precautions according to the law / your laboratory zone!
While handling pathogens and dangerous microbes apply the necessary safety
precautions according to the law / your laboratory zone!
Do not use HCl as an acid for your fermentation as far as your process allows others
like H3PO4 or H2SO4.
24
3 Installation and Set-up (based on MINIFOR 1L Advanced Kit)
3.1 Essential things for MINIFOR Setup and Installation - OVERVIEW
Make the workplace ready, to start the MINIFOR Installation.
Table area
22 x 40cm (W x D)
Power supply
Mains 100-240 V AC/50-60Hz, 510W, CE
conform
Ambient Temperature
0 – 40 °C
Relative humidity
0 - 90 % RH (non-condensing)
Supply of Air or Oxygen (Aerobic Culture)
0.05 – 0.2 MPa
Supply of N2 or other gas (Anaerobic Culture)
0.05 – 0.2 MPa
pH calibration
Standard solutions,
2 Buffers according your calibration range
(standard pH 7.0 and pH 4.0)
pO2 calibration
Standard gases: Air for maximum, N2 for
minimum
Control gel for pO2 = 0 mg/L
Sterilization of the vessel and other needed
parts
Optional: Maintaining reduced temperature with
cooling loop
Autoclave
Cooling water circulation or a thermo stabilized
water bath circulation
Please refer to the safety precautions in the chapter 2.3
3.2 Get ready with the LAMBDA Base Control Unit
Initially for starting the installation process, get ready with the LAMBDA base control unit on which
the other parts need to be fixed for working. The base control unit includes pH and temperature
probe connectors, IR heating coil, MASSFLOW and other microprocessors that control all the
parameters that need to be measured and controlled.
25
Place the base control unit on the work place
where the complete installation of MINIFOR
Bioreactor will be made.
Figure 3.2-1 The front view of the LAMBDA MINIFOR
Base control Unit.
3.3 Holders and Support Rods for LAMBDA Peristaltic Pumps
For the Advanced Kit (which includes two LAMBDA PRECIFLOW Peristaltic Pumps), a setup for
placing the pumps and sample bottles has to be made in the MINIFOR base unit which involves
the easy and controlled addition of, for example: acid, base, medium, nutrients, minerals, etc. into
the working reaction vessel.
Figure 3.3-1 As a first step, insert the support rods in
the present basic control unit as shown. Rotate it
clockwise for fixing.
Figure 3.3-2 Further fix the inserted support rod tightly
with the supplied spanner.
Figure 3.3-3 Insert the second support rod and fix it.
So now both the support rods are in place.
Figure 3.3-4 Fix the second support rod tightly with the
spanner like before.
26
Figure 3.3-5 The adjustable support plate that needs
to be inserted on the support rods. (N.B: In case, the
LAMBDA Peristaltic pumps are used, for example:
controlled addition of acid and base to maintain the
pH).
Figure 3.3-6 Insert the adjustable support plate on one
of the fixed support rods and fix it tightly using the
screws provided for tightening. The support plate can
be used for supporting, for example, a LAMBDA
Peristaltic Pump.
3.4 Setting up of the Fermentation Vessel
The fixing of the support rods and support plate for peristaltic pump has to be followed by the
installation of the holders for fermentation vessel and sterile sampling device.
Figure 3.4-1 Reaction vessel holder and the sterile
sampling device holder.
Figure 3.4-2 Insert the holder of sterile sampling
device into the reaction vessel holder.
Figure 3.4-3 Position the sterile sampling device
holder (approximately near the top) on the reaction
vessel holder, so that there is sufficient place for the
sampling device and also for the sampling
vessel/tubes to be placed underneath.
Figure 3.4-4 After inserting the sterile sampling device
holder and positioning it, fix the corresponding screws
as shown with the given key to make it hold firmly.
27
Figure 3.4-5 Also tighten the other screw like before,
as shown in the figure. (N.B. If you will need to use the
sterile sample device)
Figure 3.4-6 The vessel reaction holder with the sterile
sampling device holder needs to be fixed on the
MINIFOR base control unit.
Place the reaction vessel holder in the 2 desired slots
on either side of the sun-like heating element.
Figure 3.4-7 Deeply insert the reaction vessel holder
into the desired slots.
Figure 3.4-8 The port displayed (bigger i.e larger in the
diameter port) is the quadruple port for sterile sampling
and also for the addition of acid, base.
Figure 3.4-9 The port next to the quadruple port is the
port usually defined for pO2 Probe. The port just
opposite to pO2 probe is the port defined for pH probe.
These ports for the probes are specially designed
(placed nearly on the circumference of the vessel
while the other ports are somewhat placed inside) to
avoid the interference with the agitation unit.
Figure 3.4-10 Adjust the deeply inserted reaction
vessel holder around the reaction vessel so that it is
supported well. This may be a little stiff, so please be
careful.
28
Figure 3.4-11 While supporting the reaction vessel
holder with one hand, tighten around the slots of the
reaction vessel holder with the given key.
Figure 3.4-12 Tighten the slots also on the other side
so that it's secure. (N.B. If you will need to use the
sterile sample device you can attach the holder to e.g.
the right-hand support)
The above installation guidance resembles the installation of reaction vessel holder for 0.4L, 1L
and 3L vessels. The reaction holder for 0.3L and 7L will be different from this type of reaction
holder.
Figure 3.4-13 Adjustable lateral reactor holder for 0.3L
reactor.
Figure 3.4-14 Adjustable lateral reactor holder for 7L
reactor.
Figure 3.4-15 Insert the adjustable reaction holders on
each slot of the MINIFOR base control unit and tighten
the slots with the provided key.
Figure 3.4-16 Place the reaction vessel on the base
unit with which the distinct side arms for pH and pO2
probes are supported by the lateral reaction holders.
The elastic silicone ring helps to affix the vessel with
the reaction holder.
29
Figure 3.4-17 Place the elastic silicone ring on the
distinct side arms of the pH and pO2 probe.
Figure 3.4-18 Pull the elastic silicone ring and affix the
side arms of the vessel with the lateral reaction holder.
Figure 3.4-19 The reaction vessel mounted on the
base unit and made intact with the help of the lateral
holder and elastic silicone ring.
Never heat an empty reactor! The heat radiation is absorbed by the glass walls
and their temperature would increase to such an extent, that thermal dilatation would
lead to a vessel burst.
The small jacketed 0.3L reactors must be used only with the vessel jacket
completely filled with water. The hose connectors of the jacket should be closed
(e.g.) with silicone tubing.
The best range for the automatic temperature control is between 4°C above
ambient temperature and 60°C. Higher temperature you may reach by isolating the
vessel with flannel and aluminium foil.
3.4.1 Setting up the 7L reactor vessel
The 7L MINIFOR vessel consists of two glass parts that needs to be fixed together. In the
following, the preparation of the liquid O-ring and setting up of the vessel will be explained.
3.4.1.1 Preparation of LAMBDA Liquid O-ring for the 7L MINIFOR vessel
The liquid O-ring consists of a new partially hardening silicone resin filled with the purest quartz
powder of extremely fine (fume like) consistency.
30
Figure 3.4-20 LAMBDA Liquid O-ring ingredients:
Solution A, Solution B, Aerosil
Table 7 Preparation of LAMBDA liquid O-ring
Ingredient
Preparation
Parts
(weight or volume)
Proposed
1
2g
1
2g
Solution A
1.
Mix solution A with solution B.
Solution B
2.
AEROSIL
powder
Add AEROSIL in such amount until
the mixture is no longer free flowing
and with a consistency similar to
toothpaste is reached
3.4.2 Set-up of the 7L MINIFOR vessel with liquid O-ring
Figure 3.4-21 Get ready with the reaction vessel,
Liquid O-ring ingredients and the closing belt.
Figure 3.4-22 Take equal parts of solution A and
solution B in a non-glass beaker. For example: 2g of
Solution ‘A’ and 2g of Solution ‘B’. Aerosil powder is
added in such an amount until the mixture is no more
flowing.
31
Figure 3.4-23 Transfer the prepared liquid O-ring into
a syringe for an easy application of the mixture in the
vessel.
Figure 3.4-24 Apply the mixture on the whole
circumference of the lower vessel part’s brim like a
“spaghetti”.
Figure 3.4-25 Lower part of the vessel with the liquid
O-ring.
Figure 3.4-26 Apply a small amount of silicone oil on
the vessel upper part’s brim for easy removal of the
parts after completing the experiment.
Figure 3.4-27 Place the upper part onto the lower
vessel part.
The paste fills any cavity between the joining surfaces
and is completely gas tight and also does not generate
any stress in the glass.
Figure 3.4-28 Fix them together with the segmented
vessel closing belt.
The mixture will cure either during staying at room
temperature in several hours or within minutes in the
autoclave during sterilization.
The liquid o-ring forms a large surface joint and not just a few mm large contact as it
is the case with standard o-rings.
32
Liquid O-ring can be reused many times. The polymerized silicone joint can be
removed manually and cleaned, for example: with alcohol or acetone or it can also
just be wiped away with a piece of paper.
3.5 Agitation Unit and Sparger
The next step is to mount the agitation unit along with the air sparger on the reaction vessel for all
the sterile applications.
Axis of thread is 60º (do not force)
During setting up the stirring axis, pay attention that you do not touch the tips of
the pH or pO2 probes.
Therefore, it is highly recommended to mount the stirrer axes first and then the
probes.
If you are going to use Minifor without stirring then put the stirrer set-point to 0
(zero).
With the optional automation software SIAM, you can create tools to change the
stirrer frequency according the pO2 value. That may be useful during O2-limited
phases (with maximum aeration) of automatic standard fed-batch.
Figure 3.5-1 Self-cleaning micro sparger along with
the assembling parts.
Figure 3.5-2 Sparger has to be assembled with the
given parts like shown.
33
Figure 3.5-3 The items displayed needs to be
connected together for LAMBDA PEEk double-seal
tubing connector.
Figure 3.5-4 Insert the tubing into the double-seal
PEEK fitting-part.
Figure 3.5-5 Allow the tubing to freely flow on the
other side of the double-seal PEEK fitting-part.
Figure 3.5-6 The double-seal conical insert has to be
inserted into the tubing.
Figure 3.5-7 Insert the double-seal conical insert into
the tubing which is freely flowing on the other side of
the PEEK fitting.
Figure 3.5-8 The resulting double-seal conical insert
with the tubing should be like in figure i.e. half the
portion of the conical insert can be found inside the
tubing.
Figure 3.5-9 Take a spanner and the double seal
conical insert with the tubing.
Press the conical insert with the tubing against the
spanner to make it inserted totally into the tubing.
Figure 3.5-10 Picture shows the prepared PEEK
double-seal tubing connector.
34
Figure 3.5-11 Displayed is the agitator assembly. The
membrane visualized by holding in the hand is the
silicone sterility membrane.
Figure 3.5-12 Hold the silicone sterile membrane so
that the convex surface of membrane is outside (as
depicted in figure) and concave inside.
Figure 3.5-13 Insert the sparger pipe on the concave
(curved inwards) surface of the sterility membrane.
The silicone sterility membrane needs to be fitted
properly as shown.
Figure 3.5-14 The mobile cock for magnetic coupling
has to be fixed at the threaded end of the sparger pipe.
Screw up the mobile cock for magnetic coupling at the
threaded end of the sparger pipe.
Figure 3.5-15 Securely tighten the nut at the concave
side of the silicone sterility membrane which in-turn
firmly holds the mobile cock with the membrane.
Figure 3.5-16 Place the head of the agitation unit
above the prepared mobile cock with silicone sterility
membrane in the sparger pipe.
35
Figure 3.5-17 The head of the agitation unit has to be
placed in such a way that the air input tubing holder
intended for the double-seal tubing connector is
showing through the opening.
Figure 3.5-18 Insert the prepared double-seal tubing
connector into the mobile cock through the head of
agitation unit. Screw the double-seal tubing connector
clockwise to tighten it.
(ATTN: Axis of thread is 60º (please do not force it))
Figure 3.5-19 Ensure that the large silicon sterility
membrane is fitted correctly and firmly in the designed
grooves on the head of the agitation unit.
Figure 3.5-20 Start inserting the fish tails in the
sparger pipe. This depends on the reaction volume /
preferences (usual distance 5-6 cm.
N.B.: can be adapted in number of fish-tails or
distance).
Figure 3.5-21 Fasten the fish tails on the sparger pipe
with one of the keys supplied.
(E.g. the screw piece can be facing down for all fish
tails)
Figure 3.5-22 After fixing the fish tails securely and
distributed evenly on the sparger pipe, the selfcleaning micro-sparger has to be added at the end of
it.
36
Figure 3.5-23 The key tightening of the self-cleaning
microsparger at the end of the sparger pipe.
Figure 3.5-24 Place the large central screw cap for
head fixing over the head of the agitation unit as
shown.
Figure 3.5-25 Place the agitation unit with the fish tails
discs and microsparger into the vessel and fix the
central screw cap on the reaction vessel.
Figure 3.5-26 Ensure that the large central screw cap
for head fixing is securely tightened to the rim of the
reaction vessel. See that the mobile PEEK cock tubing
for air input is positioned as in the figure.
3.5.1 0.3L Vessel and minimal working volume: Butterfly-shaped stirrer disc –
no sparging.
If the 0.3L vessel with minimal volume is used, a special stirrer disk is needed: the butterfly-shaped
disk. It allows the insertion of the probes into the minimal working volume.
Figure 3.5-27 Butterfly-shaped stirrer disc for the 0.3L
vessels.
Figure 3.5-28 Butterfly shaped stirrer disc mounted on
the sparger pipe instead of the self cleaning
microsparger for 0.3L vessel.
37
If you mount the butterfly-shaped stirrer disc instead of the self-cleaning microsparger, then do not apply sparging. Therefore clamp the air tubing between airinlet-filter and the sparging tube. See section 3.6.1 Surface aeration.
Implement a y-piece for surface aeration or for sparging through a cannula
Use the MINIFOR automatic pO2 control or automatic gas flow control only with
surface aeration for minimal volumes in the 0.3L vessels
Do not use the MINIFOR automatic pO2 control with sparging for the minimal
volumes in the 0.3L vessels.
The internal MASSFLOW delivers at a minimum 100ml gas / min. Therefore, while
using minimal working volumes in your 0.3L vessel, replace the sparging by surface
aeration.
For the automatic pO2 control of the minimal working volumes in the 0.3L vessels,
you may add an optional external MASSFLOW 500 and use the optional SIAM PCsoftware “PID controller”.
In case, you would like to apply sparging in the minimal volume of your 0.3L vessel,
you may use a cannula for it.
3.6 Air-Input
Always use the safety over-pressure valve on the vessel! This will prevent a
pressure increase in-case of the blocking of the output filter during excessive
foaming. (Refer chapter 3.9).
This will ensure that you have the proper filtering from your air input into your
fermentation / bioreaction
To make the sliding easy you may wet the tip of the probe by distilled water.
Figure 3.6-1 Take the free end of the air-input tubing
(double seal connector tubing attached with mobile
cock) from the head of agitation unit as shown.
Figure 3.6-2 Connect the air-input tubing from the
agitation unit to one end of the air-input filter.
38
Figure 3.6-3 If the insertion of tubing into the air-inlet
filter is found hard to be done. Then use this provided
tubing connector for the connection of air-inlet tubing
and air-inlet filter.
Figure 3.6-4 Picture shows the connection of the airinlet filter with the tubing from the head of the agitation
unit using the tubing connector.
Figure 3.6-5 Connect a small piece of tubing (cut as
desired) to the shown free end of the air-input filter.
Figure 3.6-6 With the other end of the tubing from the
air-input filter, connect it to the air outlet on the base
control unit as shown.
Insert the tubing deeply till the bottom of the air outlet.
Figure 3.6-7 The safety lock is provided for the air
input. This protects the air-input filter for liquids
entering the air-output / air-input line).
Figure 3.6-8 Secure the safety lock tight on the tubing
just before the air-input filter.
(N.B. Cut tubing as desired)
To facilitate the insertion of tubing, you can wet them with a few drops of
distilled water. They will then slide in and out easily.
39
3.6.1 Surface aeration
The aeration into the headspace (surface aeration) instead of sparging is mainly used for aerobe
culturing in small working volumes or for any other aerobe culture with a slow consumption of
dissolved O2. It is also possible to use the surface aeration for the diffusion of gases in stem cell
culture.
Figure 3.6-9 Insert the tubing with the filter at the air
outlet in the MINIFOR base unit.
Figure 3.6-10 Add a Y-piece into the aeration tubing.
Figure 3.6-11 Connect the tubing with the filter from Ypiece to the sparger.
Figure 3.6-12 Insert another tubing with the filter to the
Y-piece for surface sparging.
Figure 3.6-13 Connect the tubing for surface aeration
to the hollow small cannula in the quadruple sampling
port that stays in the head space of the MINIFOR
above the medium surface.
Figure 3.6-14 Secure the tubing to the sparger with a
lock when surface sparging is used.
40
3.7 pO2 probe and setup
The pO2 probe is included in the MINIFOR Advanced kit or delivered as an option for the MINIFOR
Start-up kit.
The pO2 probe has a slightly larger diameter than the pH probe. It is therefore
important to use the coloured open stopper for the pO2 probe and any open
transparent stopper for the pH probe.
The female plugs of cables cannot be cleaned and must be kept absolutely clean.
The signal of the pO2 probe is of very high impedance. Any dirt, salt solution or other
contamination can negatively affect the precision of the measurement.
During the set-up of the stirring axis, pay attention that you do not touch the tips of
the pH or pO2 probes.
Do not clean the female plug of the probe cable! (It must be kept absolutely
clean)
Never put any cable into the autoclave for sterilization! This is similar for all
sterilizable probes.
It is highly recommended to mount the stirrer axis first and then the probes.
Place the tip of the probe about 1 cm from the edge of the closest stirring disc.
This will ensure a good exchange of the solution flowing to the membrane.
At the same time it will help to displace air bubbles, which may form accidentally on
the membrane.
The contacts of the probes / plugs must be kept clean. (Prevent over-boiling at the
end of sterilization).
To make the sliding easy you may wet the tip of the probe with distilled water.
The contacts on the probe can be cleaned with distilled water and wiped off by clean
paper towels.
Measurement of pO2 during the experiment is easy and can be read in the display. Adjustments,
range (minimum and maximum settings), alarm settings, current readings can be controlled and
modulated on the MINIFOR Base Control Unit.
41
Figure 3.7-1 Before going on with the pO2 probe
installation, ensure the presence of stoppers for blind,
the pH and pO2 (DO) probes (3 types), washers, open
screw cap (small) to the reaction vessel.
Figure 3.7-2 Put off the protective cap from the
membrane side of the probe. (Protection cap can be
removed easily when washed with warm water)
Figure 3.7-3 Use the coloured open stopper for the
pO2 probe as shown.
Figure 3.7-4 Please be careful not to touch this part of
the probe.
Figure 3.7-5 Insert the coloured rubber stopper for the
pO2 (DO) probe on the right side of the port which is
next to the sampling port (larger in diameter)
Figure 3.7-6 Make sure the washer is placed on to the
rubber stopper.
Figure 3.7-7 Place the black screw cap loosely on to
the stopper and washer.
Figure 3.7-8 Moisten the pO2 (DO) probe with deionized water to ease insertion of probe.
42
Figure 3.7-9 Insert the probe through the fixed
coloured stopper and screw cap.
Then, push the probe carefully into the port and adjust
the height of the probe to be immersed in the medium
Figure 3.7-10 Tightly secure the black screw cap
placed on the top of the coloured stopper.
Figure 3.7-11 pO2 (DO) probe connector have to be
connected between the pO2 probe and the pO2 (DO)
probe socket in the base control unit.
Adjust the sleeve of the pO2 (DO) probe connector by
pushing it up as shown.
Figure 3.7-12 Place and adjust the pO2 (DO) probe
connector by rotating to find the position for exact
locking of grooves with the pO2 (DO) probe.
Figure 3.7-13 After fixing the pO2 (DO) probe and
connector exactly in locking position, push down the
sleeve of the pO2 (DO) probe connector and secure it
sufficiently with screw fitting.
Figure 3.7-14 Remove the protection cap by pressing
and unlock by rotating from the pO2 (DO) probe socket
on the base control unit.
43
Figure 3.7-15 Secure the pO2 (DO) probe connector to
the socket by rotating and pressing to lock it together.
To facilitate the insertion of the probe and the multiple-seal silicone stoppers
into the glass necks, you can wet them with a few drops of distilled water. They
will then slide in and out easily.
Always place a metal washer under the screw-cap to reduce the necessary
screw force!
Do not screw the screw caps with too much force! It is not necessary and the
glass threads may break.
3.8 pH and temperature probe setup
The temperature probe is incorporated with the pH probe and it is included in both the start-up and
advanced MINIFOR kit.
The pH probe has a slightly smaller diameter than the pO2 probe. It is therefore
important to use the coloured open stopper for the pO2 probe and any open
transparent stopper for the pH probe.
The female plugs of cables cannot be cleaned and must be kept absolutely
clean.
During the setup of the stirring axis, pay attention that you do not touch the tips of
the pH or pO2 probes.
Do not clean the female plug of the probe cable! (It must be kept absolutely
clean)
Never add any cable into the autoclave for sterilization! This is similar for all
sterilizable probes.
It is highly recommended to mount the stirrer axis first and then the probes.
The contacts of the probes / plugs must be kept clean. (Prevent over-boiling at the
end of sterilization).
To make the sliding easy you may wet the tip of the probe by distilled water.
44
The signal of the pH probe is of very high impedance. Any dirt, salt solution or
other contamination can negatively affect the precision of the measurement.
The contacts on the probe can be cleaned with distilled water and wiped off by clean
paper towels.
If a new pH probe is used: Remove the protection cap from the tip of the electrode and rinse the
electrode with deionised water. Carefully remove the silicone protection from the diaphragm. If
necessary shake the probe gently to bring the solution into the tip part of the glass electrode.
Place the probe for at least 24 hours into the buffer solution pH 6-7. (This conditions the glass layer
of the electrode and stabilizes the signal.) Before measurements and calibration rinse the probe
with distilled water and remove the last drop with filter paper. Wait for the stabilization of both pH
and temperature readings.
Refer the leaflet “InPro® 325X, InPro® 325X (ISM), InPro® 325X i” provided along with the pH probe
for further information.
Measurement of pH during the experiment is easy and can be read in the display. Adjustments,
range (minimum and maximum settings), alarm settings, current readings can be controlled and
modulated on the MINIFOR Base Control Unit.
Figure 3.8-1 pH probe can be installed in the ports
with any of the white open stoppers provided as
shown.
Figure 3.8-2 Care should be taken not to touch the
sensor part of the probe.
Figure 3.8-3 Place any one of the provided white open
stopper on the port which is opposite to the pO2 probe
port.
Figure 3.8-4 Make sure that the washer is placed on
the white open stopper.
45
Figure 3.8-5 Place the black screw cap loosely on the
white open stopper and washer.
Figure 3.8-6 For smooth insertion of the pH probe on
the port, moisten it well with the distilled water.
Figure 3.8-7 Carefully insert the pH probe on the preprepared port with the stopper, washer and black
screw cap.
Figure 3.8-8 Tightly secure the black screw cap, after
insertion of the pH probe.
Figure 3.8-9 Take the pH probe connector from the
base control unit of the MINIFOR Bioreactor. (Black
cable from the base unit)
Figure 3.8-10 Remove the black protection cap on the
sensor (top part) of the cable and red protective cap
from the pH probe.
46
Figure 3.8-11 Connect the pH probe connector (black)
cable from the MINIFOR base control unit with the pH
probe. Rotate and adjust the connector cable for exact
locking of grooves.
Figure 3.8-12 After locking the grooves of the pH
probe and connector cable, tighten the connection by
screwing the threaded lock on the connector cable by
rotating as shown.
Always place a metal washer under the screw-cap to reduce the necessary
screw force!
Do not screw the screw caps with too much force! It is not necessary and the
glass threads may break.
3.9 Pressure Limiting Security Valve
Always use the safety over-pressure valve on the vessel!
The use of the delivered pressure limiting security valve is imperative! This will
prevent a pressure increase in the case of the blocking of the output filter and in the
case of excessive foaming.
Maximum input pressure must not exceed 0.2 MPa even for a short period of
time! If this happens, the air tubing inside the MINIFOR fermenter will burst and must
have to be replaced!
The safety valve must be perfectly cleaned and the silicone tubing must be
replaced after each overpressure event. Otherwise, the tubing may prevent the
correct operation of the over-pressure security valve!
While other systems use rupture discs and get contaminated, the LAMBDA overpressure valve prevents contamination and the run can be usually saved from
being contaminated.
Glass is the optimal material for laboratory bioreactors and fermenters. Glass is inert and does not
release undesired substances into the medium. Unfortunately, glass is breakable and glass culture
vessels do not support high pressures. Under normal conditions there is no problem, because
internal pressures in the vessels are low, since the used air (out gas) escapes through the output
air filter.
47
However, when the output filter is blocked (e.g. by too long reuse of the same air filter or by
penetration of liquid or foam into the filter) then the real pressure in the vessel can be equal to the
)
gas input pressure* . This may be dangerous with glass vessels. Glass suffers progressively during
many sterilization runs and its surface may be scratched accidentally by sand, rings etc. All these
effects will lower the final pressure resistance of glass vessels.
)
* Recommended input air pressure is 0.1 MPa
When the pressure inside the vessel approaches an overpressure of about 0.1 MPa, the valve
starts to leak. As the pressure increases the leak stream is bigger and bigger and the pressure is
kept in a safe range. The whistle of escaping air can usually be heard. This should attract the
attention of the user that something is wrong. At this moment, the input air pressure must be
decreased and the output filter has to be exchanged or a new one needs to be installed.
Overpressure valve assembly and maintenance:
Over-pressure security valve components: Stainless steel cannula with LAMBDA PEEK connector,
double-conical tubing insert and overpressure security valve.
The over-pressure safety valve consists of a threaded tube with perpendicular holes covered by a
5 mm long piece of common silicone tubing external diameter 6 mm, internal diameter 4 mm and
wall thickness 1 mm. When a critical pressure is attained, the tubing extends and air escapes.
Figure 3.9-1 Insert one end of the double-conical
tubing insert to the over pressure security valve.
Figure 3.9-2 Insert the other end with the stainless
steel cannula with LAMBDA PEEK connector.
Figure 3.9-3 Insert the assembled over pressure
limiting security valve to the rubber stopper.
Figure 3.9-4 Insert the stopper with the pressure
limiting security valve into the port. (Probably the
Pressure limiting security valve can be placed beside
the pH probe port on either side)
48
Figure 3.9-5 Ensure that the washer is placed on the
stopper with the security valve.
Figure 3.9-6 At last, place the black screw cap and
tighten it securely by rotating clockwise.
3.10 Assembly of Sampling and Addition devices
Sampling and addition device includes the sterile sampling device and the quadruple port for taking
samples and addition of acid, base or buffers, minerals, nutrients, inoculum into the sterilized
medium, etc. respectively.
3.10.1
Quadruple sampling and addition ports
Quadruple sampling and addition ports include the fours cannulas or needles with the with female
LAMBDA double-seal connection. The cannulas used can be of stainless steel (Refer Figure
3.101) or it can be customized with PTFE tubing instead of stainless steel cannulas (Refer Figure
3.102).
Double-seal connection ensures a tight connection and no means of contamination through the
ports during the addition of the nutrients, etc and even while sampling.
LAMBDA DOUBLE LOCK connection is made of PEEK. PEEK is a new material similar to PTFE in
its extreme chemical resistance and very high melting point (350°C). It can be even flamed. PEEK
has a much higher mechanical stability and does not “flow” like PTFE. For its superior qualities, it
has been selected by LAMBDA despite of its high price.
In the quadruple sampling and addition port assembly, 3 cannulas with the female LAMBDA
double-seal connection are connected with the PEEK double seal tubing connectors that are
meant for the addition of acid, base, medium, nutrients, buffers, etc. and sampling. A special
septum is used for the cannula that is used for the inoculation.
In the following, the preparation of PEEK double seal tubing connectors, septum for inoculation,
sterile sampling device and also the storage bottles that are used for having acid, base, medium,
etc. for adding into the working vessel through the port.
To make the sliding easy you may wet the tip of the tubing and metal parts with
distilled water.
49
Figure 3.10-1 Quadruple sampling and addition ports
with the stainless steel cannulas.
3.10.2
Figure 3.10-2 Quadruple sampling and addition ports
customized with PTFE tubing instead of stainless steel
cannulas.
Preparation of LAMBDA PEEK double seal tubing connectors
LAMBDA PEEK double-seal tubing connector has to be prepared, like it has been prepared for the
installation of agitation unit, to ensure proper closure of the sampling and addition ports.
Now at this stage, the double-seal tubing connectors are prepared for the sterile connection of the
respective cannula in the quadruple sampling port with the storage bottle with acid, base, buffers,
medium, etc. So, the tubing should have the PEEK double-seal tubing connectors at both the ends
of the tubing that needs to be connected with the quadruple sampling port.
To make the sliding easy you may wet the tip of the tubing and metal parts with
distilled water.
Figure 3.10-3 The items displayed needs to be
connected together for LAMBDA PEEk double-seal
tubing connector.
Figure 3.10-4 Insert the tubing into the double-seal
PEEK fitting-part.
50
Figure 3.10-5 Allow the tubing to freely flow on the
other side of the double-seal PEEK fitting-part.
Figure 3.10-6 The double-seal conical insert has to be
inserted into the tubing.
Figure 3.10-7 Insert the double-seal conical insert into
the tubing which is freely flowing on the other side of
the PEEK fitting.
Figure 3.10-8 The resulting double-seal conical insert
with the tubing should be like in figure i.e. half the
portion of the conical insert can be found inside the
tubing.
Figure 3.10-9 Take a spanner and the double seal
conical insert with the tubing.
Press the conical insert with the tubing against the
spanner to make it inserted totally into the tubing.
Figure 3.10-10 Picture shows the prepared PEEK
double-seal tubing connector.
51
3.10.3
Preparation of Inoculation device (Septum)
Septum is the inoculation device that will be connected with one of the cannulas in the quadruple
sampling device.
Figure 3.10-11 Parts need to be connected together to
form the inoculation device.
From left to right; 1. The tubular septum containing the
double seal conical insert for tubing inside;
2. The septum adaptor body with a white septum Oring for septum adaptor body;
3. The septum protection cap containing the sealing
disc.
Figure 3.10-12 The tubular septum (which is sealed at
one end) should be fitted over the double seal conical
insert with the use of, for example, a hard ruler leaving
part of the tubular septum free and exposed as shown.
Figure 3.10-13 The prepared double seal conical
insert with the tubular septum has to be inserted into
the septum adaptor body.
Figure 3.10-14 Note that the sealed end of the tubular
septum should be fitted into the septum adaptor body
(on the other side of white septum O-ring) such as
shown.
Figure 3.10-15 Fix the septum protection cap on the
threaded side of the white septum O-ring.
Figure 3.10-16 Complete set for inoculation. The free
end with the double seal conical insert has to be
inserted on one of the cannulas or needles from the
quadruple sampling port.
52
3.10.4
Preparation of the sampling device
The sampling device is included in each MINIFOR ADVANCED KIT.
Keep the lines (tubing) between the MINIFOR and the sampling device as short
as possible.
(Since the back-flushing is risky for contamination, you will have to do a washing of
the line before taking the sample. The shorter the line, the minimum working volume
you will lose)
To make the sliding easy you may wet the tip of the tubing and glass parts with
distilled water.
Figure 3.10-17 Schematic representation of LAMBDA
Sterile sampling device.
Figure 3.10-18 Pictorial representation of the
LAMBDA Sterile Sampling Device.
As depicted in the Figure 3.1017 and 3.1018, the sterile sampling device is made of glass with
3 provisions for inlet and outlet connections.
- The vertical provision of the glass device which is adjacent to the side provision arm needs to
be connected with the Bioreactor for sampling.
- The device has to be connected by the PEEK double seal conical insert tubing with one of the
cannulas of the quadruple sampling assembly (the one with the longest needle).
- Clamp the tubing between bioreactor and sampling device before the autoclave!
- A sterilizable air-filter (small ventilation filter 25 mm, 0.2 µm pore size) has to be connected to
the side arm by means of short silicone tubing.
- Silicone tubing which is 10 – 20 cm long needs to be connected to the output arm provision
on the glass device i.e. the vertical arm opposite to the inlet sampling connection with the
Bioreactor (on the lowest part of the device).
- Everything has to be sterilized in autoclave with the vessel.
- Most important is that all tubing must be clamped immediately after sterilization.
- Sampling device has to be fixed in the sampling device holder after sterilization.
53
Figure 3.10-19 Silicone tubing which is 10 - 20 cm
long needs connected to the output arm provision on
the glass device i.e. the vertical arm opposite to the
inlet sampling connection with the Bioreactor (on the
lowest part of the device)
Figure 3.10-20 A sterilizable air-filter (small ventilation
filter 25 mm, 0.2 µm pore size) connected to the side
arm by means of a short silicone tubing.
Figure 3.10-21 The tubing with the PEEK double-seal
insert needs to be connected with the sterile sampling
device. (Refer 3.10.2 Preparation of LAMBDA PEEK
double seal connectors) Inlet provision for connecting
the PEEK double-seal tubing has a provision tube
deep inside the device glass part. (Please refer the
picture)
You may replace the MINIFOR standard sampling device by any other device
(as is standard in your laboratory) with the use of a tubing connection.
3.10.5
Preparation of storage bottles
Storage bottles are the stock bottles used for the addition of acid, base or buffers, medium,
nutrients, minerals, etc. by means of automatic pumping into the working vessel.
54
Figure 3.10-22 Reagent bottle 250ml with pipe
(cannula with the female LAMBDA double-seal
connection) and Luer-Lock fitting.
Table 8
Storage bottle: ports and purpose
Amount
Needle
Purpose
What has to be installed on it
1
Long
Pumping liquid out of the bottle to
the bioreactor (liquid)
1
Short
Ventilation
(gas)
LAMBDA double-seal connector (for
preparation, please refer 3.10.2) and tubing
leading through the pump on to the
quadruple port of the fermentor vessel.
Gas filter with 0.2µm pore diameter,
autoclavable
The filters have to be well fixed on the needle. This may depend on the supplier.
If the filter and the needle do not fit, then insert the silicone tubing between the
needle and the filter. Fix it with autoclavable cable binders.
Wet tubing can be mounted well on the needles –pass water through the tubing
before mounting. Furthermore, the water will be helpful during sterilization since the
vapour ensures heat transfer. However, make sure that your filter stays dry.
Do not use HCl as acid for your fermentation and bioreactions as far as your
process allows others like H3PO4 or H2SO4.
3.10.5.1
Line between storage bottles and MINIFOR vessel
The storage bottles along with the silicone tubing can be sterilized together with the fermentor /
bioreactor vessel.
All lines between storage bottles and MINIFOR vessel should be kept as short as
possible for easier handling, less contamination and long term usage. You should
also take into account the additional length needed for pump head length and/ or
welding line.
55
LAMBDA peristaltic pumps (head) work best with the silicone tubing. Tube welding
machine, may need other tubing which is too hard for LAMBDA peristaltic pumps.
For the line (between storage bottle and MINIFOR vessel) you can use a different
types of tubing which are connected with an autoclavable tubing connector.
In case, autoclavable tubing connectors are used, fix the tubing with a cable binder.
(For metal connectors, do not use the cable binder since the silicon tubing will be
melted on to the metal after autoclaving).
If you need to know the quantity of liquid added without the use of a balance or if you
want to measure the flow rate, then the LAMBDA pump speed / flow rate has to be
calibrated.
However, the inner diameter of the silicon tubing may change during sterilization.
The first sterilization has the biggest impact on the material. Use only AUTOCLAVED
tubing for preparing the lines between storage bottles and MINIFOR vessels.
3.10.6
Mounting of sampling and addition devices
To make the sliding easy you may wet silicon / glass / metal with distilled water.
After the preparation of the quadruple sampling assembly, PEEK double-seal tubing connector,
inoculation device and the sterile sampling device, everything have to be mounted in its place in
order to make it a complete set-up.
Figure 3.10-23 Insert the quadruple sampling
assembly into the port which is larger in diameter and
adjacent to pO2 probe set-up.
Fix and tightly secure it with the black screw cap.
Figure 3.10-24 Place the sterile sampling device on
the sterile sampling device holder (Refer 3.4 Setting up
of fermentation Vessel) and tighten it with the sidescrew.
56
Figure 3.10-25 Remove the double-seal tubing
protection closure on the longest cannula of the
quadruple sampling port as shown.
Figure 3.10-26 Take the PEEK double-seal tubing
from the sterile sampling device and connect it with the
longest cannula on the quadruple sampling port.
Figure 3.10-27 Take the prepared tubing with the
PEEK double-seal tubing connectors at both the ends
of the tubing (Refer 10.3.2 Preparation of PEEK
double-seal tubing connectors).
Figure 3.10-28 Connect one end of the prepared
tubing with the PEEK double seal tubing connector to
the short cannula in the quadruple sampling port.
Figure 3.10-29 Connect the other end of the prepared
tubing with PEEK double seal tubing connector to the
long needle of the storage bottle.
Figure 3.10-30 Stock bottles can be conveniently kept
at the back of the MINIFOR base control unit using the
magnetic bottle holder.
3.11 Medium cooling device
LAMBDA MINIFOR offers two different ways to cool down the medium:
- Cooling loop: for cooling with cooling liquid (delivered with each MINIFOR KIT)
- Peltier cooling finger based on peltier cell: thermoelectro based cooling, without cooling liquid.
(Optional device for up to 3...
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